07 Anxiety and Stress Disorders

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VII ANXIETY AND STRESS DISORDER

DENNIS CHARNEY
The chapters in this section reflect the advances that have been made in our understanding of the molecular neurobiology and neural circuits underlying fear and anxiety states and the potential for badly needed advances in the diagnosis, pathophysiology, and treatment of anxiety disorders. Many readers will be surprised by the data cited in the Kessler and Greenberg chapter on the economic burden of anxiety and stress disorders. Recent surveys demonstrate that anxiety and stress disorders are the most commonly occurring of all mental disorders. Anxiety disorders are temporally the primary disorders in many people with a lifetime history of any mental disorder. Indeed the combined occurrence of high lifetime prevalence with an early age at onset and high chronicity makes anxiety disorders unique. The chapter by Stein and Lang reviews in detail the course of anxiety and stress disorders over the lifetime. One point they make that is especially important to emphasize is the critical need for further research in childhood anxiety disorders. The presence of an anxiety disorder in childhood or adolescence is a predictor of the persistence into adulthood of not only anxiety disorders but other psychiatric disorders as well, particularly depression. It is not known if early effective treatment of childhood and adolescent anxiety disorders will prevent the development of psychiatric disorders later in life. Important insights into the pathogenesis of anxiety disorders come from preclinical investigations reviewed by Davis in his chapter. The identification of the brain structures and neural circuits involved in the generation of fear and anxiety-related behaviors are most noteworthy. The delineation of specific neural pathways mediating conditioned and unconditioned fear can logically guide the design and conduct of clinical studies investigating the neurobiological mechanisms of anxiety disorders such as generalized anxiety disor-
Dennis Charney: Mood and Anxiety Disorder Research Program, National Institute of Mental Health, Bethesda, Maryland.
der (unconditional fear) and phobic disorders (conditioned fear). Increased knowledge of the neural mechanisms and neurotransmitters involved in extinction may offer novel therapeutic approaches. If we can discover pharmacologic methods to enhance extinction, more effective drug treatments for conditioned fear or anxiety may be found. There is evidence of extinction being mediated by -aminobutyric acid (GABA) release. This suggests the possibility of augmenting extinction-based psychotherapy with GABA agonist drug treatment. Determination of the genetic contribution to anxiety disorders is critically important to progress in understanding etiology and improving treatment. Merikangas and Pine review the evidence that the major subtypes of the anxiety disorders aggregate in families. However, the magnitude of heritability is relatively moderate, indicating a strong environmental contribution to etiology. Unfortunately, thus far linkage and association studies in anxiety disorders have not been fruitful. Future studies should determine if components of anxiety syndromes are controlled by specific genes. The revolution occurring in genomics research as a consequence of sequencing the human genome and the identification of over 112 million single nucleotide polymorphisms (SNPs) should make discovery of anxiety-related disease genes a reality. Strategies that are complementary to linkage analyses and utilize data from linkage studies are indicated. Such approaches couple the genotyping of candidate functional SNPs with linkage and equilibrium mapping in chromosomal regions implicated in linkage studies. In parallel with the genetic studies, enhanced efforts to identify endophenotypic biological vulnerability markers are indicated. The studies cited in the chapter on temperament, anxiety sensitivity, autonomic reactivity, psychophysiologic function, ventilatory function, neurochemical, and neuroendocrine factors are good examples of this approach. Bakshi and Kalin point out in their chapter the advan-
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tages of using putative animal endophenotypes of stress and anxiety to identify genetic abnormalities associated with anxiety disorders. Rodent and nonhuman primate studies of mother-infant interactions are particularly compelling, given the important clinical implications if these interactions are found to be a critical factor in future fearful disposition. Targeted mutations leading to anxiety-like endophenotypes in transgenic mice have suggested roles for serotonin receptor subtype 1A (5-HT1A), corticotropinreleasing hormone (CRH), GABA, neuropeptide Y, cholecystokinin, and substance P neural systems in the generation on anxiety-fear behaviors. These studies provide clues for the discovery of new medications and pharmacogenomic approaches to treatment. Accelerated drug development efforts focusing on corticotropin-releasing factor 1 (CRF-1) receptor antagonists and benzodiazepine agonists with an anxioselective subunit profile are indicated. The feasibility of pharmacogenomic investigations designed to evaluate the relationships among functional polymorphisms of the 5-HT1A receptor, benzodiazepine receptor, and GABA synthesis enzymes and therapeutic responses to specific drugs should be explored. It is imperative that these findings from the preclinical studies of anxiety and fear states be translated into increased knowledge of the neural circuits and associated neural mechanisms that can account for the signs and symptoms in patients with anxiety disorders. Rauch and Shin reviewed the neuroimaging findings relevant to anxiety and stress disorders. Their chapter emphasizes the areas of congruence between animal studies and clinical neuroimaging investigations. For example, imaging studies in healthy subjects support a role for the amygdala in fear conditioning and the frontal cortex in extinction. In the imaging studies of patients with anxiety disorders, progress has been made in identifying specific neural circuits. Functional relationships among the amygdala, hippocampus, and medial prefrontal cortex have been reported in patients with posttraumatic stress disorder (PTSD). The imaging findings in patients with obsessive-compulsive disorder (OCD) are consistent with pathology in cortico-striatal-thalamo-cortical circuitry. Unfortunately, a critical gap in knowledge exists regarding the relevant neural circuits involved in panic disorder, social anxiety disorder, and generalized anxiety disorder. The neurochemical systems associated with anxiety and fear circuits are reviewed in the chapter by Charney and Drevets. As predicted from the preclinical studies, abnormalities in norepinephrine, benzodiazepine, glucocorticoid, and CRH systems have been identified in patients with anxiety disorders. However, most of the findings reviewed should be deemed preliminary, and they require replication. None of
the reported neurobiological distinctions between patients and controls is robust enough to be of diagnostic relevance. Tallman, Cassella, and Kehne review the mechanism of action of anxiolytic drugs and the status of new and novel therapeutic agents. They highlight the therapeutic potential and current status of CRH antagonist drug development. They also note the potential of developing targets for the CRF-2 receptor and other peptides, such as vasoactive intestinal peptide (VIP), involved in the regulation of stress. In regard to benzodiazepine drug development, they note an ideal drug might have limited effects on the 1 subtype with increased responsiveness at 2 and 3 subunits. Glutamate receptor agonists and modulators are proposed as viable targets for anxiety disorders. For example, point mutations in the glycine-binding site of the NR1 subunit result in mice that have reduced glycine affinity and an anxiolytic profile. Group II metabotrophic glutamate agonists are in early clinical development for the treatment of anxiety disorders. Other novel drug targets include antagonists of AMPA receptors and antagonists of strychnine-sensitive glycine site, both of which show anxiolytic profiles in animal models. Ultimately, the goal of research on the neurobiological underpinnings of anxiety disorders is to lead to more effective, more rapidly acting treatments, to achieve a more complete response, and to be able to predict responses to specific treatments. In their review, Gorman, Kent, and Coplan highlight the extremely broad spectrum of action of norepinephrine and serotonin transport inhibitors in anxiety disorders. These drugs are limited by a delayed onset and incomplete response in many patients. This suggests that norepinephrine and serotonin have broad modulatory effects on other neuronal systems, which are more primarily related to the pathogenesis of anxiety disorders. In summary, a reading of these chapters reveals that there have not been fundamental advances in our ability to diagnose anxiety disorders based on known etiology. The mainstay of medication treatment continues to be classes of medications that have existed for decades. There are major gaps in our knowledge of anxiety disorders in children. The mechanisms responsible for the occurrence of anxiety disorders in childhood and adolescence leading to increased risk for other psychiatric disorders in adulthood are unknown. However, the potential for progress is great. A multidisciplinary team approach utilizing the findings from preclinical investigations on neural circuitry, neurochemistry, and genetics to inform clinical investigations of genetic vulnerability, environmental risk factors, neuroimaging, pharmacogenomics, and novel drug design and testing will be a pathway to discovery.
Neuropsychopharmacology: The Fifth Generation of Progress. Edited by Kenneth L. Davis, Dennis Charney, Joseph T. Coyle, and Charles Nemeroff. American College of Neuropsychopharmacology 2002.
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ANXIETY AND STRESS DISORDERS: COURSE OVER THE LIFETIME
MURRAY B. STEIN ARIEL J. LANG
This chapter traces the course and trajectory of anxiety disorders across the life span. The chapter discusses these disorders in three age groups: (a) childhood, (b) young adulthood to middle age, and (c) older adulthood.
ANXIETY AND STRESS DISORDERS IN CHILDHOOD Temperamental and Environmental Precursors There are a number of factors that relate to the development of anxiety in general. Although this remains a controversial area, current evidence suggests that anxiety does not appear to be specifically heritable; what clusters in families is a more general predisposition to mood or anxiety disorders. Evidence of this comes from twin studies (1) as well as from studies of the incidence of disorders in the families of anxious patients (2). Specific phobia, however, may be an exception to this general heritability; family members with specific phobias tend to be associated with increased risk only for specific phobias (3). Any biological predisposition is likely moderated by environmental factors. For example, Beidel and Turner (2) found that parental psychopathology was a risk factor for the development of disorder only among the lower socioeconomic status (STS) portion of their sample. It has been suggested that environmental factors play a significant role in the manifestation of specific psychopathology (1). Anxiety in particular is believed to be related to a combination of negative affect, a sense of lack of control over situations or environments, and attentional self-focus. Early experiences of being unable to influence or control situations, therefore, may lead to the development of anxiety (4). Specific patterns within the family may lead to increased
Murray B. Stein and Ariel J. Lang: Department of Psychiatry, University of CaliforniaSan Diego, San Diego, California.
risk of development of childhood anxiety. Expressed emotion (EE), which is an interactional style composed of emotional overinvolvement and high levels of criticism, has been associated with an increased likelihood of childhood anxiety disorders (5) and with long-term functioning in those with anxiety disorders (6). Parents may also influence a childs anxiety by providing positive (e.g., the child gets attention) or negative (e.g., the child is allowed to avoid anxiety-provoking situations) reinforcement for expressions of anxiety, by providing inadequate affection and by excessive control/ overprotection (4). Can it be said that some people are born anxious? The answer is a qualified yes. The last decade or so of research by Jerome Kagan and colleagues has led to the identification of a temperament, behavioral inhibition (BI), that appears to be related to the subsequent onset of anxiety disorders. BI involves reacting to unfamiliar or novel situations with behavioral restraint and physiologic arousal (5). When confronted with an unfamiliar person or object, a BI child will withdraw, cling, be reluctant to interact, show emotional distress, and stop other activities. BI has also been associated with physiologic differences, such as high and stable heart rate, increased salivary cortisol and urinary catecholamines, pupillary dilation, and laryngeal muscle tension (7). These findings have led to the hypothesis that BI is related to a low threshold for arousal in the amygdala and hypothalamus (8). This characteristic, which appears to be present in 10% to 15% of children, has been identified in children as young as 14 months, has been shown to persist throughout childhood (9), and is more commonly found in offspring of anxious parents (8). The inhibited temperament has been associated with risk of developing an anxiety disorder, most commonly social phobia (10). Some have suggested that childhood anxiety and depression are so closely related that they are best considered as part of the same construct, often referred to as internalizing disorder. This lack of differentiation appears to be characteristic of younger children, with increased specificity developing over time (11,12). At least by middle childhood, there is support for the set of anxiety-related diagnoses in the
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Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). Spence (12) conducted a confirmatory factor analysis with data from children of 8 to 12 years. The best model included six correlated factorspanic-agoraphobia, social phobia, separation anxiety, obsessive-compulsive problems, generalized anxiety, and fear of physical injury (including dogs, dentists, heights, doctors)and a single higher-order factor reflecting overall anxiety. There is a clear need for further research in childhood anxiety. Estimates of prevalence and recovery vary widely because of a lack of standardization of criteria, assessment instruments, and methodology (e.g., clinician rating vs. selfreport, child vs. parent or teacher report). A few general conclusions can be drawn about childhood internalizing disorders. Internalizing symptoms appear to remain fairly stable over time (13,14). Among boys, internalizing symptoms are not only predictive of later internalizing symptoms but also of subsequent externalizing problems (15). Although there may be high rates of recovery associated with a particular anxiety disorder, children who recover are at increased risk of developing other psychiatric diagnoses, most commonly other anxiety disorders or depressive disorders (16). Similarly, the presence of an anxiety or depressive disorder [overanxious disorder (OAD)/generalized anxiety disorder (GAD), panic, major depression] in adolescence is nonspecifically predictive of anxious or depressive disorders in adulthood (17). The remainder of this section discusses disorder-specific information, including clinical manifestations, prevalence, and course. Generalized Anxiety Disorder (GAD) GAD is characterized by excessive anxiety or worry, which is difficult to control and is accompanied by symptoms of tension and physiologic arousal (18). The GAD diagnosis is currently used in place of overanxious disorder (OAD), which was eliminated with the publication of the DSM-IV. Children and adolescents with GAD most frequently worry about future events, personal safety, and social evaluation, and often present with multiple somatic complaints, such as headaches and stomachaches (19). There is little information about the prevalence of GAD in children and adolescents because the diagnosis of OAD was used previously. Prevalence estimates of OAD tended to be quite variable, 2% to 19% (19), and often very high, partially because functional impairment was not necessary for the diagnosis (20). Recent estimates of the prevalence of GAD are in the range of 2.1% to 10.8% (21). It appears to be more prevalent in older children and in girls (19). Onset typically falls between 10.8 and 13.4 years of age (4). Information about course is not yet available for the GAD diagnosis, but some extrapolation from OAD is possible. Cohen et al. (22) looked at three different statistical measures of persistence. They found that OAD was very
stable in a subset of children and younger adolescents (ages 11 to 16), but that there was a substantial amount of new onset among older adolescents (ages 17 to 19). Their conclusion was that the disorder may be trait-like for those who exhibit symptoms early and that the development of the disorder in others may be triggered in adolescence. Cantwell and Baker (23) also found considerable stability; 25% of children who had been diagnosed with OAD approximately 4 years earlier had recovered (although the group size, eight, limits the usefulness of this estimate). In contrast, Last et al. (16) reported that 80% of their OAD sample had recovered 3 to 4 years later; however, 25% had developed another anxiety disorder and 25% had developed a depressive disorder. GAD/OAD is a frequently co-occurring disorder. Of those with a primary diagnosis of OAD, there is often an additional diagnosis of separation anxiety disorder (37% to 44%), social phobia (4% to 57%), simple phobia (9% to 43%) or a depressive disorder (1% to 69%) (24). There are a number of potential reasons for the high rates of comorbidity, including true covariation of distinct disorders, the presence of a single underlying construct, overlap of diagnostic categories, and measurement error (12,25). Obsessive-Compulsive Disorder (OCD) OCD is recognizable by the presence of obsessions and compulsions, which are distressing or cause marked interference in ones life (18). Among young children, the disorder often presents with excessive and rigid ritualized behavior. For children in general, compulsions alone are more common than obsessions alone (26). The most common compulsions include washing/cleaning, repeating/redoing, and checking, and the most common obsessions include germs/contaminants and fear of harm to the self or to another (26). OCD symptoms change over time in 90% of children (4). OCD is relatively uncommon, with estimated prevalences of approximately 0.3% for children and 0.35% to 1.9% for adolescents (27,28). Mean age of onset is approximately 10 years. Information about the course of OCD is variable and may be best described as chronic but fluctuating (4). Leonard et al. (6) followed children and adolescents who had been part of National Institute of Mental Health (NIMH) treatment studies. During the 2- to 7-year followup period, the patients on average received two different modalities of treatment (medication, behavioral therapy, other individual therapy, and family therapy), with 96% having had additional psychopharmacologic treatment and 46% some form of behavioral therapy. In spite of ongoing treatment, at follow-up 43% met the criteria for OCD, 18% had subclinical OCD, and 28% had OC features. Of the 11% who were symptom-free, only three (of 54) patients had no symptoms and were not on current medications. However, treatment was associated with decreased functional impairment. Last et al. (16), on the other hand, found
Chapter 60: Anxiety and Stress Disorders: Course over the Lifetime
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a 75% recovery rate in their sample. Other estimates of continued OCD at follow-up (1.5 to 7 years) range from 31% to 68% (16). Comorbidity is a frequent problem with OCD. The most common co-occurring conditions include other anxiety disorders (38%), tic disorders (24% to 30%), mood disorders (26% to 29%), and specific developmental disabilities (24%) (26). Leonard et al. (6) found that both a lifetime history of tic disorder and current affective disorder at baseline were associated with poorer outcome. Panic Disorder (with and without Agoraphobia) (PD) PD involves recurrent and unexpected panic attacks, which cause significant distress for the affected individual. This frequently leads to avoidance of various situations for fear of developing symptoms and being unable to escape (18). Young children tend to articulate their panic-related fears in a different way than do adolescents or adults by virtue of their developmental level; they are more likely to express concerns about sudden somatic symptoms and less likely to describe fears of dying, losing control, or going crazy (4). PD is uncommonly reported in children, to the point that there has been some debate as to whether it exists before puberty. Evidence of the existence of PD in children comes from both retrospective reports of adults with PD and from case reports (29). There is no epidemiologic study to date, and it is likely that many cases go undetected because of the predominance of somatic symptoms in presentation. Prevalence of PD in adolescents is low, 0.7% girls and 0.4% boys (0.6 overall) according to one large high school sample (28). The rate of panic attacks is expectedly higher, 11.6% for at least one full attack and an additional 3.2% for at least one limited symptom attack (30). PD appears to be two to three times more common in females (29). Hayward et al. (31) examined the relationship between the occurrence of panic attacks and sexual development in girls and found a positive relationship. There were no reported panic attacks among the least sexually mature girls, and a rate of 8% among those who were most developed. The authors proposed a number of theories for this association, including hormonal changes, psychosocial factors, and emergence of the ability to think abstractly, but further work is necessary to draw any definitive conclusions. In the one study of the course of early PD, 30% continued to have PD and 30% had another psychiatric disorder 3 to 4 years later, but the generalizablity of this result is questionable because of the small size of the study population (ten) (16). Retrospective reports suggest that earlier onset is associated with poorer outcomes, including greater functional impairment and increased incidence of alcohol abuse and suicidality (32). Available information suggests that there is a high rate of comorbidity in adolescents with PD, particularly with affective disorders; again, this should
be interpreted with caution because it is based on a single, small (N 28) study of adolescents (33). Posttraumatic Stress Disorder (PTSD) To meet the criteria for a diagnosis of PTSD, a person must have been exposed to a traumatic event and as a result is exhibiting symptoms of reexperiencing, numbing/avoidance, and arousal (18). A recent confirmatory factor analytic study supported the presence of these three basic clusters of symptoms in children and adolescents, although it found that arousal is often manifested as general somatic complaints (34). PTSD presentations that are specific to children include reenactment of the trauma in play, physical attempts (e.g., covering eyes or ears) to avoid memories of the trauma, reduced interest in activities, behavioral regression (e.g., thumb-sucking, enuresis), and sleep disturbance (35). Diagnosis of PTSD depends on exposure to a traumatic stressor. Each year, 6% to 7% of Americans are exposed to traumatic events (36), but the incidence is much greater in certain subpopulations. For example, studies of urban youth report exposure rates of up to 75% (37). Not everyone who is exposed to trauma goes on to develop PTSD. Estimates vary tremendously depending on the type of trauma and the elapsed time between the event and assessment. In Saigh et al.s (37) review of the literature, they found PTSD prevalence among exposed youth to be 0% to 70.8% for crimerelated events, 8.3% to 75% for war, and 0% to 95% for disasters. Overall, it appears that exposed children may be somewhat more likely to develop PTSD than are exposed adults (36). PTSD is more common in those who have been exposed to more severe trauma (34). The course of PTSD in children over the short- or longterm has not been well studied, but it appears that prognostically important factors are whether the trauma involves a single occurrence or is repeated, and whether it involves abuse. Although the evidence is not entirely consistent, it appears that a single exposure is less likely to lead to longterm symptomatology (36). Comorbidity is generally high, particularly with other anxiety disorders (7.7% to 41.6%) and affective disorders (16.7% to 85%), but there is also substantial co-occurrence of attention-deficit/hyperactivity disorder (5.8% to 34.6%), conduct disorder (0% to 26.9%), and oppositional defiant disorder (25%) (37). Additional disorders may be integrally related to the trauma, such as fears about safety of the self or loved ones or grief about loss (35). Other psychopathology may also be a function of other factors, such as a disrupted or disorganized childhood or engagement in risky behaviors, which increase the risk for both psychopathology and traumatic exposure (38). Separation Anxiety Disorder (SAD) SAD is the only current anxiety disorder that is uniquely diagnosed in children and adolescents. The hallmark feature
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of this disorder is excessive concern about separation from attachment figures. This is frequently manifested as distress at separation and excessive worry that harm will befall the attachment figure or that some negative event will lead to separation (18). These children frequently avoid going to school, fear being left alone or sleeping alone, and exhibit a panic-like physiologic response to separation (32). Prevalence estimates for SAD are 2.0% to 5.4% for children and 1.3% to 4.6% for adolescents, with some evidence of higher rates in girls, those of lower SES, and those with less educated parents (21,32). The onset of SAD is usually early and associated with a major stressor (4). Of nine children with SAD followed by Cantwell and Baker (23), only one was still diagnosable 4 to 5 years later; this was the highest rate of recovery of any of the disorders that they followed. Similarly, Last et al. (16) found an approximately 96% recovery rate among their 24 children and adolescents with SAD, although 25% had developed another disorder, most commonly depressive, 3 to 4 years later. SAD frequently co-occurs with other disorders, most often other anxiety disorders (OAD, 23% to 33%; specific phobias, 12.5% to 27%; social phobia, 8%) (24) or a depressive disorder (approximately one-third) (32). SAD has been suggested to be a childhood manifestation of PD. Evidence that has been cited in support of this idea includes the symptomatic similarity between a panic attack and the response to separation in a child with SAD; the frequency of a history of SAD in panic patients; the clustering of SAD, PD, and depressive disorders in families; and the similarities in effective pharmacologic treatments for the two conditions (3941). Documented cases of panic episodes unrelated to separation, however, argue against this hypothesis (29). Nonetheless, SAD appears to be a risk factor for the later development of PD, at least among females (4).
Social Phobia Social phobia involves marked and persistent fear of one or more performance or social situations in which the person is exposed to unfamiliar people or to possible scrutiny by others (18). The anxious response in such situations is associated with cognitions involving concerns about being humiliated or embarrassed. Childhood social phobia is associated with significant impairment and distress, and frequently leads to extensive phobic avoidance and deficient social skill development (43). Although there are few good epidemiologic studies of social phobia in childhood, data from community studies in adolescents suggest that it is quite common (1% to 2%), with a noticeable jump in prevalence rates sometime between ages 12 to 13 and ages 14 to 17 (44). One longitudinal study suggested that many cases of social phobia in childhood remit within 3 to 4 years (86.4%) (16). However, when social phobia is present in adolescence, it is a strong predictor of social phobia in adulthood (17). These data, taken together, suggest that social phobia in childhood may be a more transitory phenomenon than social phobia in adolescence. If these findings are confirmed in future studies, they may suggest critical developmental time frames during which preventative efforts may be applied.
ANXIETY AND STRESS DISORDERS IN YOUNG TO MIDDLE-AGE ADULTS Epidemiology Several epidemiologic studies have documented the high rate of anxiety disorders among adults in the general population. In reports from the Epidemiologic Catchment Area (ECA) Study, anxiety disorders were found to occur as a lifetime diagnosis in 14.6% of the adult U.S. population aged 18 years or older (45). More recently, the National Comorbidity Survey (NCS) found that 24.9% of adults in the age group 15 to 54 years had a lifetime anxiety disorder diagnosis (46). The two studies used somewhat different sampling methods, and different diagnostic interviews, probably therein explaining at least some of the variance in rates between studies (47). Comorbidity Comorbidity among the anxiety, mood, and substance use disorders is extensive (47). For example, two-thirds of persons in the community with generalized anxiety disorder in a 12-month period also had major depressive disorder during that time frame (48). In a clinical sample of 85 patients with major depression, 29% met criteria for a current anxiety disorder and 34% had at least one anxiety disorder during their lives (49). What is particularly noteworthy about this relationship
Specific Phobia A specific phobia is diagnosed if a child consistently displays significant and excessive fear in response to a specific object or situation (18). The most common fears among children are heights, small animals, doctors/dentists, dark, loud noises, and thunder/lightening (19). The prevalence of this disorder is in the range of 0.3% to 9.1%, with somewhat higher rates in girls and younger children (19,21,42). Unlike the other anxiety disorders reviewed here, children with specific phobias remain a fairly distinct group. Last et al. (16) found that those with specific phobias were least likely to recover within 3 to 4 years (69.2%) but also were least likely to show onset of a different disorder within the follow-up period. Similarly, Pine et al. (17) found that simple phobias in adolescence were related only to adult simple phobias.
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is the temporal sequencing of disorders. Certain anxiety disorders, social phobia in particular (which has a median onset of between 13 and 15 years of age), almost inevitably begin prior to the onset of the mood or substance use disorder (50,51). In one study of depressed patients, social phobia was the most common lifetime anxiety disorder (occurring in 15% of cases) followed closely by panic disorder with agoraphobia (in 12%) (49). Social phobia occurred on average 2 years prior to the onset of major depressive disorder in these patients. Similar findings have emerged from community studies (52), suggesting that particular anxiety disorders such as social phobia may be considered risk factors for the subsequent development of major depression. It remains to be established what the mechanisms might be for this observed relationship. Does being socially anxious lead to increased isolation or decreased self-worth, thereby leading to an increase in subsequent major depression? Is social phobia merely the earliest manifestation of an anxiety-mood disorder diathesis? These questions will only be answered with future research that focuses broadly on psychosocial and biological vulnerabilities for anxiety and mood disorders. Another interesting aspect of the anxiety-depression link lies in the relationship between major depressive disorder (MDD) and PTSD. Extensive comorbidity between PTSD and MDD is the norm in studies of various traumatized groups, including persons exposed to combat (53,54), disasters (55), and intimate partner violence (56). Community studies also demonstrate strong ties between these two disorders, with approximately 35% to 50% of cases of PTSD in the general population being comorbid with MDD (57). Studies that have examined the temporal association between major depression and PTSD have posited several causal pathways. It has been observed that preexisting major depressive disorder increases an individuals risk for PTSD following exposure to traumatic events (58,59). The converse has also been observed, namely that preexisting PTSD is a risk factor for the later development of MDD (58,60). The mechanisms for this apparent reciprocal risk have yet to be explained, but might involve a general vulnerability to stress that can result in major depression (61) or PTSD in susceptible individuals. Functioning, Quality of Life, and Cost to Society Data have been collected in the past several years that highlight the disability and reduced quality of life associated with anxiety disorders in young and middle-aged adults. Studies in clinical samples of patients with PD, PTSD, OCD, and social phobia all depict these as serious conditions that rob individuals of enjoyment and pleasure and impair functioning in multiple domains (62). Epidemiologic studies, where the range of severity is expected to be wider and where many milder cases are expected to be seen,
also provide persuasive evidence of the seriousness of anxiety disorders (52,63,64). The annual cost of anxiety disorders in the United States was estimated at $42.3 billion in 1990, or $1,542 per sufferer (65). Other than simple phobia, all anxiety disorders analyzed were associated with impairment in workplace performance. These observations, gleaned from a variety of clinical and nonclinical perspectives, portray anxiety disorders in adults as serious mental disorders worthy (and in need) of greater societal willingness to develop and apply better interventions to prevent or mitigate their impact on the lives of individuals. ANXIETY AND STRESS DISORDERS IN OLDER ADULTS Although anxiety is among the most prevalent of psychiatric disorders in the elderly, research in this area has lagged far behind that of depression and dementia (66). But in the past few years, several important studies have been conducted that provide novel information about the prevalence, features, and course of anxiety disorders in older adults. Epidemiology Whereas it had previously been believed that anxiety disorders decline in prevalence with age, several possible explanations for this finding have been put forward. It has been suggested that this might be an artifact of measurement error, owing to differences in the way older individuals report anxiety (67). Previous epidemiologic studies may also have underestimated the prevalence of anxiety disorders in the elderly by limiting participation to community-dwelling older adults, who may have lower rates of anxiety disorder than those living in institutions (68). Fortunately, data have recently become available from a new community survey that provides a more accurate and detailed perspective on anxiety disorders in older adults (69). The Longitudinal Aging Study Amsterdam (LASA) is based on a random sample of 3,107 older adults (ages 55 to 85), stratified for age and sex. The overall prevalence of anxiety disorders in the community was estimated at 10.2%. GAD was most common in a 6-month time period (7.3%), followed by social phobia (3.1%), PD (1.0%), and OCD (0.6%). For comparison purposes, it is noteworthy that the 6-month prevalence of major depression in the same study was 2.0%. Thus, anxiety disorders were far more common than depressive disorders in the elderly, underscoring the point made earlier that it is surprising that the elderly have received so little attention in the clinical and research literature to date. This study also examined vulnerability factors for anxiety disorders in older adults (69). Many of the vulnerability factors for anxiety disorders in younger adults are common to older adults (e.g., female sex, lower levels of education),
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but several unique risk factors were also encountered (e.g., having suffered extreme experiences during World War II). These investigators were also able to show that current stresses commonly experienced by older people (e.g., recent losses in the family and chronic physical illness) also played a part in the onset or exacerbation of anxiety disorders. Current life stresses, then, should be evaluated as possible contributors not only to depression, but also to anxiety in the elderly. Comorbidity Comorbidity patterns of older adults with anxiety disorders are remarkably similar to those of younger adults. In the LASA, 48% of those with MDD also met criteria for anxiety disorders, whereas 26% of those with anxiety disorders also met criteria for MDD (70). The entity known as anxious depression warrants special mention in this context. Although definitions vary, anxious depression usually refers to MDD with accompanying anxiety. Anxious depression is a particularly common presentation in the elderly (66). Although anxious depression is frequently severe and impairing, its outcome is no worse than nonanxious depression when treated appropriately (71). Special Features of Anxiety Disorders in the Elderly The fact that medical illness becomes more common with increasing age can put a special twist on the presentation and origins of certain anxiety disorders in the elderly. First and foremost, it must be recognized that many medical illnesses (e.g., thyroid disease, chronic obstructive pulmonary disease, and stroke, to name just a few) may be associated with de novo anxiety symptoms or with the exacerbation of a preexisting anxiety disorder (66). Most new anxiety disorders in older life are either GAD or agoraphobia, whereas most other anxiety syndromes seen in the elderly (e.g., PD and OCD) reflect recurrence or worsening of an anxiety disorder that had its onset earlier in life (72). Agoraphobia in older adults is usually a different phenomenon, with different etiology, from agoraphobia in younger adults. In younger adults, agoraphobia is almost always a complication of PD (73)the individual comes to avoid situations that are associated with the possible occurrence of panic or difficulty escaping should a panic attack occur. In the elderly, the new onset of agoraphobia is rarely associated with spontaneous panic attacks, but instead is a maladaptive reaction to some form of medical illness experience that renders the individual fearful of being unable to function safely away from home (66). An example is an elderly woman who breaks her hip, and even after it has satisfactorily healed, is afraid to maneuver without help and therefore avoids leaving the house alone.
SUMMARY Anxiety disorders span the full range of human existence from childhood to old age, though symptoms may vary considerably owing to developmental differences and related factors. Anxiety disorders in children are often transitory phenomena, with the majority showing remission by adolescence or early adulthood. Yet, in a minority, extremely shy and fearful temperament in childhood can merge almost imperceptibly into social phobic and panic disorders in adolescence. Anxiety disorders in youth appear to be a risk factor for the subsequent development of major depression (and, although less certain, possibly also substance use disorders) in late adolescence and young adulthood. By adulthood, comorbidity is the rule, with most individuals experiencing multiple anxiety disorders, or concurrent mood and anxiety disorders. For the most part, anxiety disorders are chronic, and these persist from young adulthood into old age. But even in later life, new onset of anxiety disorders can occur, often in the context of medical illness or other sources of life stress.
ACKNOWLEDGMENTS Dr. Stein has received research support from the following companies: Bristol-Myers Squibb; Eli Lilly and Company; Forrest Laboratories; Hoffman-LaRoche Pharmaceuticals; Novartis; Parke-Davis; Pfizer; SmithKline Beecham; and Solvay Pharmaceuticals. He is currently or has been in the past a consultant for Forrest Laboratories; Hoffmann-La Roche Pharmaceuticals; Janssen Research Foundation; SmithKline Beecham and Solvay Pharmaceuticals. Finally, he receives or has received speaking honoraria from BristolMyers Squibb; Eli Lilly and Company; Hoffmann-La Roche Pharmaceuticals; Pfizer; Pharmacia & Upjohn; SmithKline Beecham and Solvay Pharmaceuticals.
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61. Kendler KS, Neale MC, Kessler RC, et al. Causal relationship between stressful life events and the onset of major depression. Am J Psychiatry 1999;156:837841. 62. Mendlowicz MV, Stein MB. Quality of life in individuals with anxiety disorders. Am J Psychiatry 2000;157:669682. 63. Markowitz JS, Weissman MM, Ouellette R. Quality of life in panic disorder. Arch Gen Psychiatry 1989;46:984992. 64. Stein MB, Kean Y. Disability and quality of life in social phobia. Am J Psychiatry 2000;157:16061613. 65. Greenberg PE, Sisitsky T, Kessler RC, et al. The economic burden of anxiety disorders in the 1990s. J Clin Psychiatry 1999;60: 427435. 66. Flint AJ. Anxiety disorders in late life. Can Fam Physician 1999; 45:26722679. 67. Stanley MA, Beck JG, Zebb BJ. Psychometric properties of four anxiety measures in older adults. Behav Res Ther 1996;34: 827838. 68. Kogan JN, Edelstein BA and McKee DR. Assessment of anxiety in older adult: current status. J Anxiety Disord 2000;14:109 132. 69. Beekman ATF, Bremmer MA, Deeg DJH, et al. Anxiety disorders in later life: report from Longitudinal Aging Study Amsterdam. Int J Geriatr Psychiatry 2000;13:717726. 70. Beekman ATF, de Beurs E, van Balkom AJ, et al. Anxiety and depression in later life: co-occurrence and communality of risk factors. Am J Psychiatry 2000;157:8995. 71. Flint AJ, Rifat SL. Two-year outcome of elderly patients with anxious depression. Psychiatr Res 1997;66:2331. 72. Flint AJ. Epidemiology and comorbidity of anxiety disorders in the elderly. Am J Psychiatry 1994;151:640649. 73. Horwath E, Lish JD, Johnson J, et al. Agoraphobia without panic: clinical reappraisal of an epidemiologic finding. Am J Psychiatry 1993;150:14961501.
50. Stein MB, Chavira DA. Subtypes of social phobia and comorbidity with depression and other anxiety disorders. J Affect Disord 1998;50:S11S16. 51. Kessler RC, Stang P, Wittchen HU, et al. Lifetime comorbidities between social phobia and mood disorders in the U.S. National Comorbidity Survey. Psychol Med 1999;29:555567. 52. Wittchen HU, Stein MB, Kessler RC. Social fears and social phobia in a community sample of adolescents and young adults: prevalence, risk factors, and co-morbidity. Psychol Med 1999;29: 309323. 53. Shalev AY, Freedman S, Peri T, et al. Prospective study of posttraumatic stress disorder and depression following trauma. Am J Psychiatry 1998;155:630637. 54. Southwick SM, Yehuda R, Giller ELJ. Characterization of depression in war-related posttraumatic stress disorder. Am J Psychiatry 1991;148:179183. 55. Green BL, Lindy JD. Post-traumatic stress disorder in victims of disasters. Psychiatr Clin North Am 1994;17:301309. 56. Stein MB, Kennedy C. Major depressive and posttraumatic stress disorder comorbidity in female victims of intimate partner violence. J Affect Disord (in press). 57. Kessler RC, Sonnega A, Bromet E, et al. Posttraumatic stress disorder in the National Comorbidity Survey. Arch Gen Psychiatry 1995;52:10481060. 58. Breslau N, Davis GC, Peterson EL, et al. Psychiatric sequelae of posttraumatic stress disorder in women. Arch Gen Psychiatry 1997;54:8187. 59. Bromet E, Sonnega A, Kessler RC. Risk factors for DSM-III-R posttraumatic stress disorder: findings from the National Comorbidity Survey. Am J Epidemiol 1998;147:353361. 60. Mellman TA, Randolph CA, Brawman-Mintzer O, et al. Phenomenology and course of psychiatric disorders associated with combat-related posttraumatic stress disorder. Am J Psychiatry 1992;149:15681574.
61
GENETIC AND OTHER VULNERABILITY FACTORS FOR ANXIETY AND STRESS DISORDERS
KATHLEEN R. MERIKANGAS DANIEL PINE
Despite dramatic advances in our understanding of genetics and neurobiology, the etiology of the anxiety disorders is still relatively unknown. To date, there remain no pathognomonic markers with which a presumptive diagnosis of an anxiety disorder may be made. This highlights the importance of the empirical epidemiologic approach to investigating the definitions and risk factors for the expression of anxiety across the life course. Anxiety disorders are developmental conditions that often emerge during childhood and follow varied developmental trajectories (1,2). Research on early-life vulnerability factors that predict the trajectory of anxiety symptoms across development holds promise for elucidating mechanistic pathways in anxiety. In evaluating the risk factors for the development of anxiety disorders, there are several issues requiring consideration. First, there is substantial overlap between the anxiety disorders and other psychiatric disorders both concomitantly and longitudinally. Second, manifestations of anxiety change substantially across the life course, particularly during childhood and adolescence. Therefore, a developmental perspective is essential in evaluating links between risk factors and anxiety disorders. Third, the assessment of anxiety requires evaluation of the context in which the individual experiences anxiety as well as the subjective response to anxiety-inducing situations. As such, anxiety becomes a disorder when there is a mismatch between inherent threat posed by a particular stimulus or situation and the cognitive or somatic response. Research on vulnerability factors has undergone a relatively marked transformation in recent years, due to conceptual changes in causal theories of mental disorders. Such conceptual changes are reflected in three major themes that organize current research on vulnerability factors in anxiety.
First, although studies through the early 1990s often emphasized the role of one or another particular risk factor, more recent studies emphasize the manner in which multiple risk factors might interact to cause mental syndromes, including anxiety, as part of a mechanistic pathway. For example, although dysregulation in fear conditioning has been linked to anxiety for more than two decades (3), such dysregulation is now viewed as part of a larger chain of intrinsic and extrinsic events that may ultimately culminate in an anxiety disorder (4). Second, as a corollary to this view, vulnerability markers are now conceptualized as tied to families of anxiety disorders, as opposed to specific conditions. This change in perspective follows the observation that validators of individual mental syndromes related to differential course, familial aggregation, or psychophysiology relate more closely to families of disorders than to particular disorders. Third, marked advances over the past 20 years in neuroscience have stimulated a closer integration of basic and clinical work on vulnerability markers in anxiety disorders. Progress in elucidating neural circuits related to anxiety has facilitated research on vulnerability markers for anxiety disorders that integrates data from basic and clinical science. This chapter examines the major risk factors for the development of anxiety disorders across the life span. Particular attention is paid to the specificity of vulnerability factors and to developmental differences in expression of the disorders themselves. MAGNITUDE AND DEMOGRAPHIC RISK FACTORS Magnitude of Anxiety in General Population The anxiety disorders are the most common psychiatric disorders both in the United States and elsewhere (2,5,6). The
Kathleen R. Merikangas and Daniel Pine: National Institute of Mental Health, Bethesda, Md.
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results of the two large-scale community-based surveys of psychiatric disorders of adults in the United States, the Epidemiological Catchment Area study (ECA) (2) and the National Comorbidity Study (NCS) (5), reveal that the total prevalence rates of anxiety disorders are greater than those of the affective disorders, behavior disorders, and substance use disorders. Phobias tend to be the most common anxiety disorder, whereas panic disorder is fairly rare in the general population. There is substantial overlap both cross-sectionally and longitudinally between the anxiety disorders and other disorders, as well as between the subtypes of anxiety disorders themselves (6). On average, there is a threefold increased risk of having a second disorder compared to that of manifesting an anxiety disorder alone across the lifetime. Comorbidity between anxiety disorders and other psychiatric disorders has been demonstrated in both clinical and community samples. Anxiety disorders are most strongly associated with affective disorders and with substance use disorders (6), though they are generally associated with all other major classes of disorders including depression, disruptive behaviors, eating disorders, and substance use. Comorbidity between anxiety disorders and other disorders in the Diagnostic and Statistical Manual of Mental Disorders, third edition revised or fourth edition (DSM-IIIR or -IV) may be even more common in adolescents than in adults (7). A review of comorbidity of anxiety and depression by Brady and Kendall (8) suggests that anxiety and depression may be part of a developmental sequence in which anxiety is expressed earlier in life than depression. Although the association between anxiety and depression is quite consistent, the evidence of links between anxiety disorders and behavior problems is inconclusive. Sex Differences in Anxiety Disorders Similar to the affective disorders, females tend to exhibit greater rates of anxiety disorders, though there is some vari-
ability according to specific subtypes. Table 61.1 presents the sex-specific lifetime rates of the major subtypes of anxiety disorders assessed in the ECA and NCS (5,6). Although the magnitude of the rates of anxiety disorders varies substantially between the two studies, the sex ratio is strikingly similar: women have an approximately twofold elevation in lifetime rates of panic, generalized anxiety disorder, agoraphobia, and simple phobia than men in both studies. In contrast, there is a nearly equal sex ratio for the lifetime prevalence of social phobia. There is increasing evidence from community-based studies that anxiety symptoms and disorders are also the most common problems in childhood and adolescence as well. The rates of anxiety disorders in community or schoolbased surveys of children and adolescents as defined by contemporary diagnostic criteria range from 0.1% to 13.3% in males and 0.4% to 28.6% in females (9). Similar to the sex ratio for adults, girls tend to have more of all subtypes of anxiety disorders, irrespective of the age composition of the sample. For example, in a recent epidemiologic study, females compared to males had greater rates of current anxiety disorders (i.e., 12.2% vs. 8.5%), past anxiety disorders (5.2% vs. 2.7%), as well as anxiety symptom scores on a dimensional rating (mean 1.9 vs. 0.9) (10). Nevertheless, Lewinsohn et al. (10) reported that despite the greater rates of anxiety in girls across all ages, there was no difference between boys and girls in the average age at onset of anxiety (mean for girls 8.0 3.9; mean for boys 8.5 3.8).
Age-Specific Patterns of Expression of Anxiety Disorders Retrospective reports of adults with anxiety disorders suggest that the onset of anxiety disorders generally occurs in childhood or adolescence. Although there is substantial
TABLE 61.1. SEX-SPECIFIC LIFETIME PREVALENCE RATES OF ANXIETY DISORDERS IN COMMUNITY SURVEYS IN THE UNITED STATES
Epidemiologic Catchment Area Study (5) Males Anxiety disorders Anxiety disorders, total Generalized anxiety disorder Panic disorder Phobic disorders Agoraphobia without panic Simple phobia Social phobia Females Sex Ratio (F:M) Males National Comorbidity Survey (6) Females Sex Ratio (F:M)
1.8 4.3 0.9 3.2 7.8 2.5
10.3 6.8 2.0 7.9 14.5 2.9
5.7 1.6 2.2 2.5 1.9 1.2
19.2 3.6 2.0 3.5 6.7 11.1
30.5 6.6 5.0 7.0 15.7 15.5
1.6 1.8 2.5 2.0 2.3 1.4
Chapter 61: Genetic and Other Vulnerability Factors for Anxiety and Stress Disorders
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variation across studies, the results of prospective community-based research reveal differential peak periods of onset of specific subtypes of anxiety: separation anxiety and specific phobias in middle childhood (i.e., ages 7 to 9); overanxious disorder in late childhood (i.e., 10 to 13); social phobia in middle adolescence (i.e., 15 to 16); and panic attacks, sometimes progressing to panic disorder, in late adolescence (i.e., 17 to 18) (1,1114). Anxiety disorders, particularly the phobias, tend to persist across the life course. However, there are major differences among the anxiety subtypes in terms of specificity and chronicity. Whereas the phobic states tend to be fairly stable and nonprogressive, generalized anxiety and panic tend to be less specific and less stable over time (1,15,16). Several follow-up studies of children and adolescents have shown that anxiety symptoms and disorders in general tend to exhibit some stability, but with substantial switching across categories of anxiety disorders over time (17,18). A recent 8-year follow-up study of a community sample of youth ages 9 to 18 at study entry provides compelling evidence of the stability of the subtypes of anxiety disorders (17). The stability of both social phobia and simple phobia was highly specific over time, whereas overanxious disorder was associated with major depression, social phobia, and generalized anxiety in early adulthood. Epidemiologic surveys of adults reveal that the female preponderance of anxiety disorders is present across all stages of life but is most pronounced throughout early and mid-adulthood. The rates of anxiety disorders in males are also rather constant throughout adult life, whereas the rates in females peak in the fourth and fifth decades of life and decrease thereafter. The increased rates in females are present across all ages and do not diminish as the rates of anxiety decrease in late life. The importance of pure anxiety disorders in late life was described by Beekman et al. (19), who found different risk factors for anxiety disorders than for either depression or comorbid anxiety and depression in a community sample of adults over age 55.
Social Class and Ethnicity Rates of anxiety disorders in general are greater among those at lower levels of socioeconomic status (20). Several community studies have yielded greater rates of anxiety disorders, particularly phobic disorders, among African-Americans (5). With respect to children, Compton et al. (14) found that Caucasian children were more likely to report symptoms of social phobia, whereas African-American children had more separation anxiety symptoms. Pine et al. (1) reported that phobias were greater among those at lower levels of social class. The reasons for ethnic and social class differences have not yet been evaluated systematically; however, both methodologic factors as well as differences in exposure to stressors have been advanced as possible explanations. FAMILIAL AND GENETIC FACTORS The familial aggregation of all of the major subtypes of anxiety disorders has been well established (21). As reviewed below, the results of more than a dozen controlled family studies of probands with specific subtypes of anxiety disorders converge in demonstrating a 3- to 5-fold increased risk of anxiety disorders among first-degree relatives of affected probands compared to controls. The importance of the role of genetic factors in the familial clustering of anxiety has been demonstrated by numerous twin studies of anxiety symptoms and disorders (22,23). However, the relatively moderate magnitude of heritability also strongly implicates environmental etiologic factors. Table 61.2 summarizes the results of family and twin studies of anxiety disorders. Review of Family and Twin Studies of Anxiety Disorders in Adults Panic Disorder Of the subtypes of anxiety, panic disorder is the anxiety syndrome that has been shown to have the strongest degree of familial aggregation. A recent review of family studies of
TABLE 61.2. SUMMARY OF FAMILY AND TWIN STUDIES OF ANXIETY DISORDERS

Type of Study Family Comparison Rel of probands vs. rel of controls Number of Studies 13 panic 4 social phobia 3 general anxiety 3 OCD 3 panic 4 phobias 1 OCD Average Relative Risk 5.4 3.1 4.3 3.5 2.4 2.6 4.9 Range (4.217.8) (2.59.7) (2.75.6) (1.05.1) (2.22.5) (1.49.5)
Twin
MZ vs. DZ
DZ, dizygotic; MZ, monozygotic; OCD, obsessive-compulsive disorder.
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panic disorder by Gorwood et al. (24) cited 13 studies that included 3,700 relatives of 780 probands with panic disorder compared to 3,400 relatives of 720 controls. The lifetime prevalence of panic was 10.7% among relatives of panic probands compared to 1.4% among relatives of controls, yielding a relative risk of 6.8. In addition, early-onset panic, panic associated with childhood separation anxiety, or panic associated with respiratory symptoms has each been shown to have a higher familial loading than other varieties of panic disorder (25). Although there has been some inconsistency reported by twin studies of panic disorder (26), two studies applying modern diagnostic criteria demonstrated considerably higher rates for monozygotic compared to dizygotic twins (27,28). Furthermore, current estimates derived from the Virginia Twin Registry show panic disorder to have the highest heritability of all anxiety disorders at 44% (29). Phobic Disorders Though there are far fewer controlled family and twin studies of the other anxiety subtypes, all of the phobic states (i.e., specific phobia, agoraphobia) have also been shown to be familial (30-33; see refs 3436 for reviews). The average relative risk of phobic disorders in the relatives of phobics is 3.1. Stein et al. (33) found that the familial aggregation of social phobia could be attributed to the generalized subtype of social phobia. Data from the Virginia Twin Study report the estimated total heritability for phobias to be 35% (29,37). Generalized Anxiety Disorder There is also evidence of both the familial aggregation and heritability of generalized anxiety disorder in a limited number of studies. The average familial odds ratio is approximately 5 (32,38), and the heritability was 0.32 among female twin pairs (37). Obsessive-Compulsive Disorder Likewise, there are also very few controlled family studies of obsessive-compulsive disorder. Two of the three studies (39,40) reported familial relative risks of 3 to 4, whereas Black et al. (41) found no evidence for familial aggregation. Nestadt et al. (40) found that both the age of onset and obsessions were associated with greater familiality. Twin studies have yielded weak evidence for heritability of obsessive compulsive disorder (4244). Linkage and Association Studies Based on indirect evidence implicating the adrenergic system in panic disorder (45), several linkage studies have investigated the role of mutations in adrenergic receptor loci
on chromosomes 4, 5, or 10 (46), but without success. Other work has similarly excluded linkage with -aminobutyric acid receptor A (GABAA) genes (47). Reports from a genomic survey of panic disorder using 600 markers have not yielded evidence of linkage (48). Similarly, linkage studies have excluded the possibility that panic disorder was due to mutations in adrenergic receptor loci on chromosomes 4, 5, or 10 (46), and other work has similarly excluded linkage with GABAA receptor genes (47). Recent reports from a genomic survey of panic disorder using 600 markers have not yielded evidence of linkage (48). Family Studies and Phenotypic Definitions The lack of success in identifying specific genes for anxiety disorders is not surprising given their complexity. Similar to several other psychiatric disorders, the anxiety disorders are complicated by etiologic and phenotypic heterogeneity, a lack of valid diagnostic thresholds, unclear boundaries between discrete anxiety subtypes, and comorbidity with other forms of psychopathology. Impediments to estimating genetic influences in youth are demonstrated by dramatic differences in heritability according to the informant regarding child psychopathology. For example, Eaves et al. (49) found that the heritability of both separation anxiety and overanxious disorder was far greater for parent-reported rather than child-reported disorder. The family study approach, particularly when employed with systematic community-based samples, is one of the most powerful strategies to minimize heterogeneity because etiologic factors for the development of a particular disorder can be assumed to be relatively homotypic within families. There is a dearth of studies that have employed withinfamily designs to examine either phenotypic expression or some of the putative biological factors underlying the major anxiety disorders. For example, both Perna et al. (50,51) and Coryell (52) have shown that healthy relatives of probands with panic disorder have increased sensitivity to CO2 challenge, suggesting that CO2 sensitivity may be a promising trait marker for the development of panic, as described below. Smoller and Tsuang (36) discuss the value of family and twin studies in identifying phenotypes for genetic studies. Both family and twin studies have been used to examine sources of overlap within the anxiety disorders, and between the anxiety disorders and other syndromes including depression, eating disorders, and substance abuse. Fyer et al. (31, 53) have demonstrated the independence of familial aggregation of panic and phobias. With respect to comorbidity, whereas panic disorder, generalized anxiety, and depression have been shown to share common familial and genetic liability (23,54,55), there is substantial evidence for the independent etiology of anxiety disorders and substance use disorders (36,55,56). Similar results have emerged from
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studies of symptoms of anxiety and depression in youth in which both anxiety and depression was found to result from common genetic diathesis (57,58). In a comprehensive consideration of what may be inherited, Marks (59) reviews the components of anxiety that have been investigated in both human and animal studies. Evidence from twin studies has indicated that somatic manifestations of anxiety may lie under some degree of genetic control. These studies demonstrate that physiologic responses, such as pulse, respiration rate, and galvanic skin response, are more alike in monozygotic than in dizygotic twin pairs. Furthermore, twin studies of personality factors have shown high heritability of anxiety reaction. Finally, the results of animal studies have suggested that anxiety or emotionality is under genetic control. Selective breeding experiments with mammals have demonstrated that emotional activity analogous to anxiety is controlled by multiple genes (59). These findings suggest that anxiety and fear states are highly heterogeneous and that future studies need to investigate the extent to which the components of anxiety result from common versus unique genetic factors and the role of environmental factors, either biologic or social, in either potentiating or suppressing their expression. High-Risk Studies of Anxiety Disorders Given the early age of onset for anxiety disorders, studies of children of parents with anxiety have become an increasingly important source of information on the premorbid risk factors and early forms of expression of anxiety. Increased rates of anxiety symptoms and disorders among offspring of parents with anxiety disorders have been demon-
strated by Turner et al. (60), Biederman et al. (61), Sylvester et al. (62), Last et al. (63), Warner et al. (64), Beidel and Turner (65), Beidel (66), Capps et al. (67), Merikangas et al. (68), Unnewehr et al. (69), and Warner et al. (70). Table 61.3 presents the risk of anxiety disorders among offspring of parents with anxiety disorders compared to controls averages 3.5 (range 1.3 to 13.3), suggesting specificity of parent-child concordance within broad subtypes of anxiety disorders. However, similar to studies of adults that show common familial and genetic risk factors for anxiety and depression (27,71,72), studies in children have also revealed a lack of specificity with respect to depression (60,64,65,73). Studies that employed a comparison group of parent probands with depressive disorders have shown that rates of anxiety disorders are also increased among the offspring of these parents (60,62,65,70); conversely, offspring of parents with anxiety disorders and depression have elevated rates of depression when compared to those of controls (62) or to offspring of anxiety-disordered parents without depression (61). Similar findings emerged from the family study by Last et al. (63), who found an increase in rates of major depression among the adult relatives of children with anxiety. Weissman et al. (74) have even suggested that childhood anxiety represents one of the earliest manifestations of familial risk for depression. These findings are usually interpreted as providing evidence for age-specific expression of common risk factors for anxiety in childhood and depression with or without comorbid anxiety in adulthood. The high rates of anxiety disorders among offspring of parents with anxiety suggest that there may be underlying psychological or biological vulnerability factors for anxiety disorders in general, which may already manifest in children
TABLE 61.3. CONTROLLED HIGH-RISK STUDIES OF ANXIETY

Sample Study Author (year) Sylvester et al., 1987 (73) Turner et al., 1987 (60) Capps et al., 1996 (67) Warner et al., 1995 (64) Beidel et al., 1997 (65) Merikangas et al., 1998 (68) Unnewehr et al., 1998 (69) Panic Agoraphobia/OCD Agoraphobia Panic/MDD Anxiety + depression Panic/social phobia Panic Anxiety MDD Dysthymia Agoraphobia/Panic Panic Anxiety Alcohol or drugs Simple Proband Other Other Early-onset MDD MDD Substance + anxiety Spouse No Dx Not evaluated Not evaluated Dx No Dx Dx Not evaluated Offspring N 91 43 43 145 129 192 87 Age 717 712 824 629 712 717 515 Relative Risk 13.3 4.8 1.3 4.0 2.0 9.2
Dx, diagnosis; MDD, major depressive disorder.
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prior to puberty. Previous research has shown that children at risk for anxiety disorders throughout life are characterized by behavioral inhibition (75), autonomic reactivity (66,76), somatic symptoms (60,77), social fears (60,62), enhanced startle reflex (76), and respiratory sensitivity (160). Empirical research on each of these domains of risk is reviewed in the next section.
VULNERABILITY MARKERS The current section reviews recent studies on vulnerability markers in anxiety disorders. This includes data on temperamental factors and biological profiles. The first section reviews evidence regarding individual-level vulnerability factors, whereas the subsequent section examines data linking exogenous or environmental factors with risk for anxiety. As noted above, both sets of vulnerability markers operate within complex causal chains involving multiple interacting risk factors. Moreover, in such complex chains, the boundary between intrinsic and exogenous risk factors can become blurred. For example, the effects of exogenous factors, including life events; social rearing experiences, such as trauma or parental nurturance; and factors such as the use of illicit substances may operate through effects on intrinsic factors, such as the regulation of neural systems that monitor danger. Intrinsic, individual-oriented vulnerability markers for anxiety disorders can be conceived across a range of perspectives, focusing on increasingly more specified biological systems. At the most complex or global level, specific temperamental or personality characteristics, such as neuroticism, harm avoidance, and behavioral inhibition have been linked to risk for anxiety. At a more specified level, vulnerability can be modeled through the assessment of cognitive function, in the form of attention and memory, or peripheral physiologic function, as reflected in autonomic reactivity profiles, changes in the startle reflex, or changes in ventilatory control. These cognitive and physiologic functions, in turn, reflect functional aspects of neurochemical or neuroanatomic systems that are presumably homologous with systems linked to fear and anxiety across a range of mammalian species. Data from humans at each of these levels is reviewed within the context of research on fear and anxiety in other species. Temperament/Personality Behavioral Inhibition One of the earliest indicators of vulnerability to the development of anxiety is behavioral inhibition, characterized by increased physiologic reactivity or behavioral withdrawal in the face of novel stimuli or challenging situations (79). Behavioral inhibition may be a manifestation of a biological
predisposition characterized by both overt behavioral (e.g., cessation of play, latency to interact in the presence of unfamiliar objects and people) and physiologic indicators (e.g., low heart rate variability, accelerated heart rate, increased salivary cortisol level, pupillary dilation, increased cortisol level). There is an increased frequency of behavioral inhibition among children of parents with anxiety disorders compared to those of normal controls (61,62,65,75,8082). Few studies have evaluated the differences in manifest inhibition and approach/avoidance in both clinical and nonclinical samples, leaving gaps in the conceptualization of the construct of inhibition. Some studies have shown that there is more stability of behavioral inhibition across early childhood among girls than among boys (83). The expression of behavioral inhibition studied prospectively may reveal patterns of anxiety symptomatology similar to those endorsed in adult populations. In a prospective study of a large community cohort of subjects from age 3 months to 13 years, Prior et al. (84) found that maternal ratings of persistent shyness and shyness in late childhood were associated with the development of anxiety disorders in adolescence. Anxiety Sensitivity Anxiety sensitivity is another potential sensitive and specific trait marker for the development of anxiety disorders (85). Anxiety sensitivity is characterized by beliefs that anxiety sensations are indicative of harmful physiologic, psychological, or social consequences (e.g., fainting or an impending heart attack). The misinterpretation of bodily cues that characterizes anxiety sensitivity may lead to a self-perpetuating fear of fear cycle. Thus, the fear of benign arousal sensations produces anxiety, which in turn increases the frequency and intensity of physiologic sensations, and subsequently fuels apprehension regarding the significance of these sensations. This process may ultimately result in a fullblown panic attack. Anxiety sensitivity is thought to represent a stable traitlike factor that is qualitatively different from general fear and anxiety (86). It has been proposed that anxiety sensitivity may interact with environmental experiences (e.g., hearing misinformation about the negative outcome of certain bodily sensations) to shape beliefs about the dangers of anxiety sensations. Thus, anxiety sensitivity may be involved in the development of certain anxiety disorders, particularly panic disorder (87,88). Of particular interest is the finding of the specificity of anxiety sensitivity with respect to development of anxiety disorders but not depression in a nonclinical sample (88). Likewise, Pollock et al. (89) reported that anxiety sensitivity appears to be specific to anxiety, as it did not contribute unique variance above self-rated anxiety symptoms in the prediction of depressive symptoms. Anxiety sensitivity has been shown to be under genetic (90) and familial influence; anxiety sensitivity was found to
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constitute a potential premorbid marker for the development of anxiety disorders in high-risk but not low-risk youth (89). Prospective studies of youth have also demonstrated the prognostic significance of anxiety sensitivity in predicting the development of anxiety disorders. Based on the results of a 5-year prospective study of adolescents, Hayward et al. (91) concluded that anxiety sensitivity appeared to be a specific risk factor for the development of panic attacks in adolescents. These findings from prospective research, particularly the specificity with respect to anxiety, together with the importance of genetic and familial liability suggest that anxiety sensitivity is an important vulnerability factor that should be examined in future studies. Comorbid Disorders Psychiatric The magnitude of comorbidity in adults and adolescents with anxiety suggests that investigation of the role of other disorders in enhancing the risk of the initial development and persistence of anxiety disorders over time may be fruitful. The difficulty in dating onset of specific disorders, particularly from retrospective data, diminishes our ability to determine the temporal relations between disorders. Nevertheless, some prospective studies have examined the links between anxiety disorders and earlier expression of other forms of psychopathology. For example, whereas some studies suggest that childhood depression may presage the onset of panic attacks, the results of a fairly large prospective study suggest a bilateral temporal association between panic attacks and depression (91). Other disorders that may enhance the risk of development of anxiety disorders include eating disorders (92), depression, and substance use and abuse. With respect to substance use disorders, Rao et al. (93) found that anxiety disorders may comprise a mediator of the link between depression and the subsequent development of substance use disorders in a clinical sample. The potential mechanisms through which anxiety may be associated with smoking in adolescents were examined by Patton et al. (94), who found that both anxiety and depression were associated with smoking initiation through increased susceptibility to peer influences. Conversely, some research suggests that substance use may trigger anxiety disorders in susceptible youth. For example, a prospective study of a community sample revealed that posttraumatic stress disorder (PTSD) may be triggered by substance abuse in about 50% of the cases (95). Similarly, Johnson et al. (96) found that adolescent smoking predicted adult onset of panic attacks, panic disorder, and agoraphobia (96). Thus, although comorbidity between anxiety and both depression and substance problems is quite common in children and adolescents, further research on the mechanisms for links between specific disorders both across and within genders is necessary.
Medical Symptoms/Disorders Several studies have also suggested that there is an association between childhood medical conditions and the subsequent development of anxiety. Kagan et al. (97) reported an association between allergic symptoms, particularly hay fever, and inhibited temperament in young children. In a retrospective review of pre- and perinatal and early childhood risk factors for different forms of psychiatric disorders in adolescence and early adulthood, Allen et al. (98) found that anxiety disorders in adolescents were associated specifically with illness during the first year of life, particularly high fever. Likewise, Allen and Matthews (102) reported that adolescents and young adults with anxiety disorders were more likely to have suffered from infections during early childhood than others. The prevalence of high fevers in childhood along with other diseases associated with immune system were also elevated among offspring of parents with anxiety disorders in the Yale High Risk Study (76). Kagan (101) proposed that the high levels of cortisol associated with anxiety may lead to immunologic sensitivity to environmental stimuli. Taylor et al. (99) reported that immunologic diseases and infections were specifically associated with emotional disorders because children with developmental or behavioral disorders had no elevation in infections or allergic diseases. On the other hand, Cohen et al. (100) suggest that such medical problems show stronger associations with depressive as opposed to anxiety disorders during adolescence. These findings suggest that it may be fruitful to examine links between immunologic function and the development of anxiety disorders. Prospective studies have revealed that the anxiety disorders may comprise risk factors for the development of some cardiovascular and neurologic diseases. Haines et al. (103) reported that phobic anxiety was associated with ischemic heart disease, particularly fatal ischemic events. Bovasso and Eaton (104) employed cardiac and respiratory symptoms and illness to subtype panic attacks and their association with depression in a large community-based sample. They found that respiratory panic attacks were associated with the subsequent risk of myocardial infarction. Likewise, phobic disorder is strongly associated with migraine, with the onset of phobias predating that of migraine (105,106). The results of both family studies and prospective cohort studies suggest that there may be a subtype of migraine with shared liability for anxiety and depression (105).
Autonomic Reactivity Reactions to threatening stimuli among various organisms, including primates and lower mammals, involve changes in the autonomic nervous system. These changes can be detected through an analysis of time series for heart rate, heart period variability, blood pressure, and catecholamine levels.
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There is a long history of research in this area, and much of the initial work concerned the assessment of physiologic changes associated with acute anxiety states. Hence, acute episodes of anxiety, both in the laboratory and in natural settings, are typically characterized by acute changes in heart rate, blood pressure, and heart period variability (107). These changes result from coordinated changes in the parasympathetic and sympathetic innervation of the cardiovascular system. More recent work on physiologic changes during acute anxiety states has attempted to identify specific physiologic patterns associated with one or another emotion. The identification of such emotion-specific patterns may provide insights on emotion-specific patterns of brain activity. For example, some forms of anxiety, such as acute panic, may be characterized by marked parasympathetic withdrawal in the face of sympathetic enhancement. Other emotions, such as anger, may be characterized by a distinct physiologic finger print, reflecting the involvement of distinct brain systems across emotions (108110). In general, consistent associations are found across development between acute anxiety states and changes in peripheral autonomic indices, including heart rate, blood pressure, or heart period variability. As a result, some suggest that perturbations in autonomic regulation may index an underlying vulnerability to develop anxiety disorders. This underlying vulnerability is thought to relate to the functioning of particular neural circuits within the brain that exert effects on both subjective internal states and physiologic activity. Potentially relevant neural circuits have been identified through basic science studies on the neural basis of fear and anxiety. Despite consistent evidence of an association between acute anxiety states and changes in autonomic physiology, the degree to which such changes index vulnerability for anxiety, as opposed to the acute state of anxiety, remains unclear. If such changes in autonomic physiology primarily reflect downstream manifestations of relatively high degrees of acute fear, they would provide limited advantages as vulnerability markers. On the other hand, at least some of the underlying autonomic abnormalities in panic disorder persist after remission and may be independent of the current state. This suggests that changes in autonomic physiology may mirror subtle person-specific differences in brain processes related to the processing of risk or to the experience of fear. As such, autonomic indices might index vulnerability in a fashion that is more sensitive than indices derived through self-report measures. A series of recent studies provide preliminary evidence consistent with this possibility. Autonomic physiologic profiles have been studied among individuals who face high risk for anxiety disorders. Physiologic profiles have been tied to at least three indicators of risk: temperamental factors, family history, and traumatic events. In terms of temperamental factors, Kagan (111)
noted the relationship between behavioral inhibition, which predicts later anxiety, and a distinct autonomic physiology profile. Children with behavioral inhibition exhibit an autonomic physiology characteristic of the profile found during acute anxiety. Specifically, behaviorally inhibited children exhibit under conditions of novelty a shift from parasympathetic to sympathetic control of the cardiovascular system, manifest as an increase in heart rate and a reduction in highfrequency components of the heart period variability power spectrum. Such abnormalities in autonomic physiology are viewed as downstream reflections of perturbations based within the limbic system. In terms of family history, Bellodi et al. (112) found similar temperamental and physiologic abnormalities among children of parents with panic disorder. Such data are consistent with other studies finding high rates of behavioral inhibition among offspring of patients with anxiety disorders. Finally, in terms of traumatic events, physiologic reactions to an acute stress may index underlying vulnerability to develop anxiety states. Consistent with this possibility, Shalev et al. (113) found that enhanced cardiovascular activity in the emergency room immediately following a motor vehicle accident predicted the development of PTSD. Taken together, available data clearly delineate associations between acute anxiety and autonomic physiology profiles, but the implications of this work for the study of risk remain unclear. Moreover, the underlying assumption in this work posits an effect of perturbations in brain systems on both autonomic physiology and anxiety symptoms. As such, more work is also needed relating brain function to autonomic physiology.
Psychophysiologic Function Research on fear conditioning has facilitated an integration of basic and clinical work on vulnerability for anxiety. Fear conditioning develops following the pairing of a neutral conditioned stimulus (CS ), such as a tone or a light, and an aversive unconditioned stimulus (UCS), such as a shock, a loud noise, or an air puff. Across a range of mammalian species, including humans, fear conditioning results from changes in a relatively simple neural circuit that involves distinct amygdala nuclei, including the basolateral and central nucleus. Basic science research on the role of this circuit in learned fears has also called attention to the role played by related but relatively distinct neural circuits in the responses to other forms of danger. For example, reactions to intrinsically dangerous contexts, such as a brightly lit room for a rodent, involve a relatively extended period of vigilance. These reactions may more intimately involve the basolateral nucleus and the bed nucleus of the stria terminalis than the central nucleus of the amygdala. Such reactions in animals may model worry in humans as characteristically found in many anxiety disorders (114).
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Similarly, acute reactions to intrinsically dangerous stimuli often involve rapid changes in behavior designed to facilitate escape or defense. Such reactions in animals may involve the hypothalamus and lower brainstem structures; such reactions in animals may be model acute panic in humans. Work in neuroscience delineating circuits involved in mammals response to danger has stimulated a series of studies on risk for anxiety in humans. Much of this work quantifies physiologic reactions to innate and learned fears with the goal of comparing physiology across high- and lowanxiety groups. Based on skin conductance data, Eysenck and Eysenck (115) suggested that abnormal habituation of conditioned fear responses confers risk for anxiety. Similarly, Raine et al. (116) suggest that deficiencies in learned fear, as modeled by skin conductance, relates to low anxiety and high risk for chronic behavior problems. However, due to methodologic advantages, more recent studies rely on startle as a physiologic index of activity in brain circuits tied to fear. Most importantly, it has been possible to map circuits that regulate startle in more precise detail, relative to circuits that regulate skin conductance or other indicators of autonomic response, such as heart rate. Cross-species parallels in startle regulation facilitate integration of basic and clinical work (76,114,117124). For example, molecular genetic studies on fear conditioning in mice generate specific hypotheses on the genetics of risk human disorders (125131). Fear-relevant stimuli in animals may potentiate startle through effects on genes in limbic structures, such as the amygdala, that are involved in fear conditioning. In adult humans, distinct stimuli effect startle across emotional disorders, but abnormal startle in some form is seen in many disorders, including phobias (132), PTSD (122124), depression (133,134), and panic disorder (135). Moreover, there is some evidence that startle specifically indexes risk for anxiety. Three studies found startle abnormalities in children born to adults with an array of anxiety disorders (76,119), and a fourth study found startle abnormalities in inhibited children, who face high risk for anxiety disorders (111). In a high-risk study of offspring of parents with anxiety disorders compared to psychiatric and normal controls, the startle reflex and its potentiation by aversive states was used as a possible vulnerability marker to anxiety disorders in adolescent offspring of parents with anxiety disorders (122). Startle was found to discriminate between children at highand low-risk for anxiety disorders, as well as to discriminate between children at risk for anxiety compared to those at risk for alcoholism. However, different abnormalities in startle amplitude for high-risk males and females were observed. Startle levels were elevated among high-risk females, whereas high-risk males exhibited greater magnitude of startle potentiation during aversive anticipation. Two possible explanations for the gender differences in the high-risk groups were suggested by the authors: (a) differential sensitivity among males and females to explicit threat versus the
broader contextual stimuli that are mediated by different neurobiologic pathways, and (b) different developmental levels in males and females in which the vulnerability to anxiety may be physiologically expressed earlier in females. Nevertheless, more work in this area is needed, given inconsistencies across genders and across conditions under which startle is most discriminatory (76). These data are also consistent with the findings of Watson et al. (136). Overall, the data suggest that startle indices may provide an important window for assessing dysfunction in limbic circuits broadly related to mood and anxiety regulation. Ventilatory Function As in the area of autonomic physiology, a wealth of research delineates associations between respiratory perturbation and acute anxiety. This association has been most convincingly demonstrated in panic disorder, where various forms of respiratory stimulation, including lactate infusion (137) and CO2 inhalation, consistently produce high degrees of anxiety and more pronounced perturbations in respiratory physiologic parameters. Of note, these associations extend beyond the specific diagnosis of panic disorder, because enhanced sensitivity to respiratory perturbation is also found in conditions that exhibit strong familial or phenomenologic associations with panic disorder, including limited symptom panic attacks; certain forms of situational phobias; childhood anxiety disorders, particularly separation anxiety disorder; and high ratings on anxiety sensitivity scales. Compared to the work on autonomic physiology, a larger body of research implicates abnormalities in respiration in risk or vulnerability for anxiety. At least four sets of findings suggest that respiratory indices index risk for anxiety, independent of any association between current state and respiratory function. First, asymptomatic adult relatives of patients with panic disorder consistently exhibit enhanced subjective sensitivity to respiratory stimulation, in the form of exogenously inhaled CO2 (51,138,139). Second, among patients with panic disorder, stronger family loading is found in panic patients with evidence of respiratory dysregulation, as opposed to those with no sign of respiratory dysregulation (51,140). Third, respiratory indices linked to panic disorder are strongly heritable, raising questions on the potential shared genetic vulnerability for panic attacks and respiratory dysregulation. Fourth, Pine et al. (78) reported increased carbon dioxide sensitivity in children with anxiety disorders. Such data are also consistent with work on respiratory disease (141) and smoking (96,142), which suggest that abnormalities in respiration predispose to later anxiety. Based on this work, abnormalities in respiration appear to provide some information on the vulnerability for anxiety states that are related to acute panic. Despite the consistency of findings in this area, a number of questions remain. The most consistent data emerge for
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subjective indices of respiratory sensitivity, manifest as a tendency to report dyspnea during stress or during respiratory stimulation. The mechanisms that contribute to such enhanced sensitivity remain poorly specified. At a cognitive level, such hypersensitivity may result from an overall sensitivity to somatic sensations, consistent with data linking high degrees of anxiety sensitivity to future panic attacks (143). On the other hand, enhanced sensitivity to respiratory sensations appears more closely tied to panic attacks than sensitivity to other somatic factors; the tie between anxiety sensitivity and respiratory sensitivity also appears relatively weak in some studies. At the physiologic level, such hypersensitivity may result from perturbations in brain systems involved in respiratory regulation or primary as opposed to learned fear states. Unfortunately, the precise role of fear systems in both respiratory regulation and human anxiety states also remains poorly specified. Neurochemical and Neurohormonal Factors As reviewed in other sections of this book, extensive data document associations between alterations in various neurochemical factors and ongoing anxiety disorders. This includes data on the serotoninergic, noradrenergic, and GABAergic systems. Moreover, there is some evidence to implicate neurochemical alterations in the causal chain contributing to anxiety disorders. In animal models, genetic manipulations of serotoninergic receptors, the serotonin reputake transporter gene, and components of the GABA complex each produce behavioral and physiologic effects reminiscent of clinical anxiety states. Similarly, clinical studies find that acute pharmacologic manipulations in these neurochemical systems produce concomitant change in acute anxiety. For example, the inverse GABA agonist flumazenil precipitates anxiety in patients with panic disorder, whereas GABA agonists are potent treatments for various forms of anxiety. These findings are consistent with evidence of a deficiency in GABAergic modulation among adults with anxiety disorders. Similarly, manipulations of the serotoninergic system, either through tryptophan depletion or treatment with medications, also produce both acute and more chronic changes in anxiety. Finally, manipulations of the noradrenergic system produce similar changes in both children and adults. Sallee et al. (144) found that the 2-agonist yohimbine elevated selectively anxiety symptoms and was associated with blunting of growth hormone in children with anxiety disorders. Interestingly, as with the response of children to CO2 inhalation (160), the response to yohimbine appeared particularly abnormal in children with separation anxiety disorder. However, evidence of perturbed noradrenergic function in children with depression or facing high familial risk for depression (145) suggest that these findings may not be specific to anxiety but rather may relate to broad risk for mood and anxiety disorders.
Despite the consistency of these findings relating neurochemical factors to anxiety, relatively few studies have examined the manner in which individual differences in neurochemical function predict vulnerability to anxiety. There is evidence from studies in adult patients that some of these neurochemical abnormalities persist after remission. For example, much like symptomatic patients, remitted patients with panic disorder exhibit abnormal secretory profiles in terms of the growth hormone and the hypothalamic-pituitary-adrenal (HPA) axis. These neurohormonal abnormalities are thought to reflect trait-related abnormalities in neurochemical systems involved in neurohormonal regulation. Finally, there have been numerous studies of patients and at-risk relatives using lactate challenge to induce anxiety (146148). The limited information provided on neural pathways by this provocation test limits its value in informing the pathophysiology of anxiety disorders. Although these studies raise the possibility that risk for anxiety may result at least partially from underlying neurochemical abnormalities, other studies are needed to confirm this possibility. For example, there are almost no studies of neurochemical function in high-risk youth, a key source of information regarding the underlying role of biological parameters in the development of anxiety disorders. One exception is the study of Reichler et al. (77), who assessed several biological factors in their high-risk study of panic disorder including lactate metabolism, mitral valve prolapse, urinary catecholamines, and monoamine oxidase. Although none of these parameters discriminated high-risk from lowrisk youth, the lack of differences may have been attributable in part to low statistical power. Likewise, very few studies have compared neurochemical function in asymptomatic relatives of patients with and without anxiety disorders. Similarly, no studies have examined family loading for anxiety disorders in patients stratified in terms of their neurochemical functioning. Beyond this work examining monamine systems influence on neurohormonal regulation and vulnerability for anxiety, a relatively extensive body of work examines the precise relationship between anxiety and HPA axis regulation. Corticotropin-releasing factor (CRF) represents a key neuropeptide in the regulation of this system. CRF infusions in animals produce behavioral and physiologic effects in animals that bear similarities to human anxiety states. Similarly, genetic manipulations that alter CRF produce similar effects. As such, this work suggests that an underlying dysregulation in the HPA axis, possibly centrally involving CRF, may contribute to vulnerability for anxiety. Consistent with basic science studies, clinical research notes a relationship between acute anxiety states and alterations in HPA axis function. For example, a variety of acute stressors induce consistent elevations of cortisol; patients with PTSD exhibit multiple signs of HPA axis dysregulation; multiple
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other anxiety disorders exhibit other signs of HPA axis dysregulation. Vigilance/Attention Studies of the association between attention regulation and anxiety have revealed that adults with anxiety disorders exhibit enhanced vigilance for threat cues, as indexed by effects of fear-related words or pictures on reaction times. These effects have been attributed to amygdala influences on attention allocation (149153). Enhanced attentional bias in acute anxiety represents a particularly robust finding, noted in more than 20 studies using various paradigms across virtually all anxiety disorders. These effects appear particularly robust in two paradigms, the emotional Stroop and the dotprobe tests. From a theoretical perspective, this enhanced bias is considered a vulnerability marker that antedates the developmental of anxiety disorders among adults. Consistent with this possibility, an enhanced bias for threat cues is found early in the course of anxiety disorders, particularly among children with anxiety disorders. On the other hand, this enhanced bias is generally not found in remitted patients (153), and studies have yet to document enhanced bias for threat cues in at-risk but asymptomatic individuals.
ENVIRONMENTAL EXPOSURES Perinatal Exposures There is virtually no evidence that either prenatal factors or delivery complications comprise risk factors for the development of anxiety disorders. The results of three studies that retrospectively assessed perinatal events converged in linking such exposures to behavioral outcomes, but not to subsequent anxiety. For example, Allen et al. (98) found that children who suffered from a variety of exposures ranging from prenatal substance use to postnatal injuries were more likely to develop behavior disorders, particularly attention deficit disorder and conduct problems, but not anxiety disorders. Likewise, the results of the Yale High-Risk Study yielded no association between pre- and perinatal risk factors and the subsequent development of anxiety disorders (76). Life Events/Stressors The role of life experiences in the etiology of anxiety states, particularly phobias and panic disorder, has been widely studied (154157). Life events have often been designated a causal role in the onset of phobias, which are linked inherently to particular events or objects. More broadly, life experiences that to some extent threaten ones notion of safety and security in the world are often at least retrospectively perceived to trigger or precipitate the onset of anxiety disor-
ders. In evaluating the evidence on the causal role of life experiences, it is critical to consider separately the subtypes of anxiety disorders. Although it is likely that life stress may exacerbate phobic and generalized anxiety states, Marks (59) concludes that phobic states resulting from exposure are far more rare than those that emerge with no apparent exposure. In contrast, posttraumatic stress disorder (PTSD) is defined as a sequela of a catastrophic life event. The major impediment to evaluation of the causal role of life events in anxiety (or depression) is the retrospective nature of most research addressing this issue. For example, Lteif and Mavissakalian (158) found that patients with panic or agoraphobia exhibited an increased tendency to report life events in general; this suggests that studies that limit assessment of life events to those preceding onset of a disorder may be misleading because they fail to provide comparison for the time period of onset. Moreover, stressful life events may interact with other risk factors such as family history of depression in precipitating episodes of panic (159). In one of the few prospective studies, Pine et al. (160) did demonstrate a predictive relationship between life events during adolescence and both depressive as well as generalized anxiety disorder symptoms. Interestingly, the association with anxiety was limited to females, consistent with differential vulnerability to stress across genders. In terms of specific environmental risk factors, there has been abundant literature on the role of parenting in enhancing vulnerability to anxiety disorders. Based on Bowlbys (161) theory that anxiety is a response to disruption in the mother-child relationship, it has been postulated that maternal overprotection is related to anxiety, particularly separation anxiety. Using the Parental Bonding Instrument of Parker et al. (162), several studies of clinical samples have found that adult patients with anxiety disorders recall their parents as less caring and more overprotective than did controls (163). These findings have been supported in nonclinical samples as well (164,165). However, all of these studies caution that a causal link cannot be established because of the lack of independent assessment of parent behaviors and offspring anxiety. Another parental behavior that may enhance risk of anxiety in offspring is parental sensitization of anxiety through enhancing cognitive awareness of the child to specific events and situations such as bodily functions, social disapproval, the importance of routines, and necessity for personal safety (164). Bennet and Stirling (164) found that subjects with anxiety disorders and those with high trait anxiety reported greater maternal and paternal overprotection and increased maternal sensitization to anxiety stimuli than controls. Another feature of the parental relationship that has received widespread attention in recent research has been exposure to severe childhood trauma through either separation or abuse (161,166). There is increasing animal research on the impact of early adverse experiences on brain systems and subsequent development (167,168). Pynoos et al. (169)
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present a comprehensive developmental life-trajectory model for evaluating the effects of childhood traumatic stress and anxiety disorders. They propose different avenues by which dangerous circumstances, childhood traumatic experiences, and PTSD can intersect with other anxiety disorders across the life span. The developmental perspective is critical in light of different levels of neural response to experience at different stages of development (170). SUMMARY AND FUTURE DIRECTIONS FOR RESEARCH ON ANXIETY VULNERABILITY Despite the rich array of constructs associated with anxiety (Table 61.4), our ability to predict those who will suffer from anxiety disorders in adulthood is severely limited. The principles of multifinality (i.e., many outcomes of the same risk factor) and equifinality (i.e., diverse risk profiles leading to the same endpoint) apply to many of the risk pathways investigated herein (171). Although we distinguish between intrinsic and extrinsic risk factors for the development of anxiety disorders, there is increasing evidence that there is a bidirectional association between the factors subsumed under these two domains. Only a small proportion of those with known vulnerability factors truly develop anxiety disorders in adulthood, despite the vast majority of those with adulthood anxiety reporting onset in childhood and early adolescence. The major impediments to identifying specific risk factors for anxiety are exclusive reliance on retrospective data, blurred boundaries between normal and pathologic anxiety, difficulty distinguishing between risk factors and early manifestations of anxiety, limited interdisciplinary conceptualization of models of risk and pathogenesis, lack of evidence
of the specificity of risk factors with respect to anxiety disorders or subtypes thereof, and limited tools for direct measurement of brain function. Moreover, many of the risk factors have been shown to operate differently according to gender and age, as well as the specific subtype of anxiety. Elucidation of the different risk profiles will provide valuable information on classification, etiology, treatment, and prevention. Future research should do the following: Establish more accurate and developmentally sensitive methods of assessment of anxiety, with a focus on developing objective measures of the components of anxiety. Apply within-family design to minimize etiologic heterogeneity and to refine diagnostic boundaries and thresholds. Investigate specificity of putative markers with respect to other psychiatric disorders and the longitudinal stability of specific subtypes of anxiety disorders. Develop research on hormonally mediated neurobiological function in order to understand gender differences predisposing women to experience decreased resiliency to fear-provoking stimuli. Examine mechanisms for associations between panic attacks with extrinsic exposures (i.e., substance use), developmental periods (i.e. pubertal development), and cessation in later life.
ACKNOWLEDGMENTS This work was supported primarily by grant DA05348 and in part by grants AA07080, AA09978, DA09055, MH36197, Research Scientist Development Awards K02 DA00293 (to Dr. Merikangas), from Alcohol, Drug Abuse, and the Mental Health Administration of the United States Public Health Service.
TABLE 61.4. VULNERABILITY FACTORS FOR ANXIETY DISORDERS

Individual Genetic factors Temperament Behavioral inhibition Anxiety sensitivity Preexisting psychiatric/Medical disorder Autonomic reactivity Respiratory sensitivity Neurobiological factors Neuroendocrine factors Exogenous Exposure to stress Drug use Parenting Modeling Sensitization Life events
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ANIMAL MODELS AND ENDOPHENOTYPES OF ANXIETY AND STRESS DISORDERS
VAISHALI P. BAKSHI NED H. KALIN
ANIMAL MODELS OF PSYCHIATRIC ILLNESS Animal Models: Types Of Validity An important criterion for developing animal models to study psychopathology involves establishing the validity of the model as a true representation of the process being studied. Generally, three types of validity are applied to animal models: face validity, construct validity, and predictive validity (13). Face validity refers to the outward similarity in appearance between the model and the illness. Construct validity, on the other hand, does not exclusively involve outward tangible signs of the modeled illness. Rather, it refers to the internal mechanism or state that underlies the illness. Finally, predictive validity refers to the ability of the animal model to identify therapeutic treatments for the illness. It should be noted that the different types of validity can be independent of each other; an animal model can possess predictive and construct validity without possessing face validity. Ideally, an animal model should possess both construct and predictive validity so that it may be used to understand the mechanisms and etiology of the disorder and also to identify promising treatments for the disorder. Endophenotype Approach Species differences in the manifestation of a particular internal state can cloud the usefulness of face validity in animal models. In addition, when considering a complex psychiatric illness, it is likely that several different symptom clusters contribute to the final pathologic condition; these different sets of symptoms may have different underlying substrates and thus may be ameliorated by different treatments. There-
Vaishali P. Bakshi and Ned H. Kalin: Department of Psychiatry, University of Wisconsin at Madison, Wisconsin Psychiatric Institute and Clinics, Madison, Wisconsin.
fore, it is difficult to come up with an animal model for an illness that meets the aforementioned criteria and also models the pathologic syndrome in its entirety. An alternative approach that has been used involves the modeling of discrete symptom clusters and physiologic alterations rather than the whole syndrome, with the assumption that what causes the symptoms contributes mechanistically to the illness. This general approach has involved the use of endophenotypes that may be related to a particular psychiatric disorder. The term endophenotype refers to a set of behavioral and/or physiologic characteristics that accompany a basic process that is altered in relation to the illness that is being studied (4). It is important to note that this more narrowly defined endophenotype approach does not necessarily have to capture specific symptoms that are a part of the clinical diagnosis, but rather may focus on a core process or function that is abnormal in the clinical population under study and that is thought to be related to the manifestation of the illness. For example, in the case of anxiety-related disorders, investigators have focused on studying the genetic, physiologic, and neurochemical correlates of fearful or anxious endophenotypes because a core aspect of anxiety-related disorders involves the aberrant expression of fearful responses to neutral or mildly stressful contexts (5). Thus, by identifying animals that display fearful endophenotypes, it is possible to study the neural substrates that contribute to this basic process that may underlie the development and expression of anxiety-related psychopathology. Using endophenotypes that are based on core and basic processes rather than the entire illness offers certain advantages. Because the whole illness is not being modeled, the endophenotype approach affords greater possibility for construct and predictive validity in the model, and can incorporate species-specific manifestations of the core process being modeled. This approach may also make screening for genetic abnormalities associated with the disorder more fruitful, because the genetic factors associated with a very discrete
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process (which could be mediated by a small number of genes) rather than an entire syndrome (which is likely caused by a complex set of interactions between multiple genes) is being studied (4,6). Moreover, heterogeneity within a diagnostic category could potentially dilute the strength of a sample population (i.e., not all patients with anxiety disorders are identical in their clinical presentation), and diminish the chances of identifying genes that contribute to the illness. Ideally, one might be able to generate several different endophenotypes for a particular disorder, and then study the genetic underpinnings of each of these separate core processes in order to identify a set of genes that might be implicated in that particular disorder. The definition and use of endophenotypes in animal models of psychiatric illness is a developing area. This chapter presents some promising candidates of animal models of fearful and anxious endophenotypes, and outlines some of the preliminary genetic factors that have been identified to contribute to the manifestation of these endophenotypes. PUTATIVE ANIMAL ENDOPHENOTYPES OF STRESS AND ANXIETY: STUDIES OF FEARFUL TEMPERAMENT Defensive Behaviors In an attempt to understand the basic neural mechanisms underlying psychiatric conditions involving fear and anxiety, several groups have focused on identifying the neural substrates of defensive behaviors in animals. Defensive behaviors are exhibited by a wide array of species including rats, nonhuman primates, and humans in response to perceived threats from the environment, and are essential components of an organisms behavioral repertoire that ensure its protection and survival. Because organisms display defensive behaviors in reaction to threat, it is thought that the aberrant expression of defensive behaviors may represent a good example of a fearful endophenotype that would have relevance to stress and anxiety-related disorders. Although the specific behavioral responses that compose defensive behaviors are dependent on the environmental context and vary from species to species, a common element that unites this cross-species phenomenon is that defensive behaviors represent an organisms behavioral response to fear. Because defensive behaviors are expressed in response to an immediate threat, they characteristically supersede and interrupt the expression of other normal homeostatic behaviors such as feeding and reproduction that may be ongoing at the time of the perceived threat (711). One defensive response pattern expressed by many species is to inhibit all body movements and assume an immobile or freezing posture. This phenomenon of behavioral inhibition is effective in preventing detection and attack by predators (12,13), and may have special relevance for understanding psychopathology. In nonhuman primates, defensive behaviors are com-
posed of a constellation of responses that include vocalizations, freezing, fleeing, or defensive hostility and aggression. The particular set of responses that is emitted depends on, among other variables, the nature of the perceived threat (14,15). Studies of defensive behaviors in rhesus monkeys may provide valuable information that could aid in the understanding of fear and anxiety-related psychopathology in humans, because extreme fearful or defensive responses occur in dispositionally fearful humans who have an increased risk to develop psychopathology (16). Psychiatric illnesses such as anxiety disorders and depression might involve the aberrant expression of defensive behaviors. In other words, pathologic anxiety could be conceptualized as the inappropriate expression of defensive or fearrelated behaviors, consisting of either an exaggerated or overly fearful response to an appropriate context, or a fearful response to an inappropriate or neutral context. Although appropriate levels of defensive behaviors in response to environmental threats are adaptive and ensure survival, the overly intense or context-inappropriate display of fearrelated defensive behaviors may represent a liability that interferes with normal behavior and would likely contribute to certain forms of fear-related psychopathology. Thus, inappropriate or exaggerated expression of defensive behaviors may represent an important animal endophenotype of anxiety. An understanding of the specific neural substrates underlying the expression and regulation of defensive behaviors may therefore ultimately shed insight into the processes that become dysregulated in stress-related psychopathology. In defining animal endophenotypes relevant to anxiety, specific symptoms of a particular type of anxiety disorder are not being modeled, but rather the general phenomenon of hyperreactivity to mildly stressful stimuli is studied. The approach of modeling anxiety by studying defensive behaviors in animals has been described previously for rodent models (17,18). In the following sections, both primate and rodent analogues of stress hyperresponsiveness are described, with a particular emphasis on models of either the overly intense but context-appropriate expression of defensive behaviors or the normal but context inappropriate expression of defensive behaviors. Initially, various behavioral paradigms that have been used to measure an animals level of defensive behavior are described, and subsequently, specific examples of fearful endophenotypes that have been identified using these tests are discussed. Measuring Defensive Behaviors in Nonhuman Primates: Human Intruder Paradigm One laboratory paradigm that has been developed to identify animals with fearful dispositions characterizes monkeys fearful behavioral responses to a human intruder. In the human intruder paradigm (HIP), the monkey is placed by itself in a test cage where it remains for 30 to 40 minutes
Chapter 62: Animal Models and Endophenotypes of Anxiety and Stress Disorders
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while its behavior is recorded on videotape. A human intruder then enters the test area, representing a potential predatorial threat to the animal (14,15,19). The test session consists of three consecutive brief conditions: alone (A, animal left alone in cage); no eye contact (NEC, animal presented with the facial profile of a human standing 2.5 m away); stare (ST, animal presented with a human who faces it and engages it in direct eye contact). Typically, animals respond to the A condition by increasing their levels of locomotion and by emitting frequent coo vocalizations, which have been likened to the human cry and function to signal the infants location and facilitate maternal retrieval (20,21). The NEC condition causes a reduction in cooing and an increase in behavioral inhibition, which functions to help the monkey remain inconspicuous in the face of a predator and is often manifested as hiding behind the food bin and freezing. The ST condition elicits aggressive (openmouth threats, lunges, cage shaking, barking vocalizations) and submissive (lip smacking, fear-grimacing) behaviors that represent adaptive responses to the perceived threat of the staring experimenter. The different test conditions (A, NEC, ST) reliably elicit responses in young or adult laboratory-reared monkeys or in feral animals (14,19). Moreover, these context-specific defensive responses are not dependent on the gender of the intruder, and can also be elicited by showing the animal a videotape of the intruder (Kalin et al., unpublished data). Behavioral Tests Used to Measure Fearful Endophenotype in Rodents To identify fearful endophenotypes in rodents, a variety of behavioral paradigms have been employed. All of the paradigms in some manner provide an assessment of the rodents level of defensive behavior, which is essentially thought to be an index of its level of fearfulness or anxiousness. The behavioral tests measure one of four general categories of stress-related behavior: approach-avoidance conflicts, conditioned fear, aggression, and punished responding conflicts. Detailed descriptions and protocols for these tests can be found in a recent review by File and colleagues (22). Approach-Avoidance Conflicts Briefly, all of the paradigms presented in this section measure the animals ratio of approach versus avoidance behaviors by presenting a choice between an environment that is safe (usually a dark, enclosed, small space) and an environment that seems novel but risky (usually bright, wide open, large spaces). The entries into and amount of time spent in the safe environment relative to the risky environment are used as an index of the animals stress level (an increase in exploratory behaviors toward and into the risky environment indicate a relatively low level of stress). A number of
paradigms including the elevated plus maze (composed of safer closed, dark arms versus riskier open bright arms), the open field (consisting of a darker wall-bordered peripheral portion versus a brighter open center section), a light-dark transition box (consisting of an exploratorium divided into two halves, one that is dark and one that is bright), and a defensive withdrawal apparatus (composed of a small dark chamber that is inside of a brightly lit open field) have been frequently used and validated as paradigms that are sensitive to detecting shifts in an animals approach-avoidancebased conflict (23). Conditioned Fear Behavioral tests that measure conditioned fear utilize basic principles of Skinnerian conditioning. Two frequently used paradigms to assess fear conditioning are conditioned freezing and fear-potentiated startle. Conditioned freezing is evaluated using a two-step procedure. First, during the training or conditioning phase, a stressful unconditioned stimulus (UCS, such as a foot shock) that elicits freezing is paired with a neutral stimulus that subsequently becomes a conditioned stimulus (CS). On the test day, the amount of freezing in response to the CS is assessed; animals that have not undergone the CS-UCS pairing do not normally freeze when the CS is presented, but animals that have learned to associate the CS with a foot shock show marked levels of freezing simply in response to this stimulus. The CS can either be a context (i.e., the environment in which the shock is delivered) or a discrete cue (e.g., a tone or light). The level of conditioned freezing is thought to correspond to the level of fear or anxiety that the animal is experiencing due to anticipation of a threat (24). In the case of fearpotentiated startle, the unconditioned startle response to a sudden stimulus (e.g., a loud noise burst) is measured in the presence and in the absence of a CS that has been paired previously with shock. The startle response is markedly increased when the startling stimulus occurs in the presence of the CS; this relative increase in startle magnitude is quantified, and serves as an index of the level of fear (thought to be elicited by a discrete cue as the CS) or anxiety (thought to be elicited by a contextual CS) that the animal may be experiencing (25). Aggression and Social Behavior Aggressive behaviors are emitted as part of the behavioral repertoire an animal displays when it encounters a threatening situation. The study of defensive aggressive behaviors has been summarized and reviewed by a number of investigators (2628). Briefly, aggressive behaviors can be studied using a resident intruder paradigm, in which the offensive/ agonistic responses (e.g., upright postures, attacks) of a male resident or the defensive responses (e.g., submissive posture, flight, freezing) of a male intruder are measured. Other
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stress-related paradigms involve the study of affiliative behaviors and include the social interaction test in which approach toward and contact between two rats is measured (e.g., sniffing or grooming each other). Punished Responding Conflict The basic principle of punished responding tests is to present the animal with a situation in which a particular behavioral response results in both a rewarding outcome and an aversive outcome. The extent to which the animal exhibits the behavioral response during the conflict schedule is used as an index of its level of stress. For example, in the classic Geller-Seifter conflict test (29), rats are trained to press a lever for a food reward. Gradually, the bar press is also paired with a mild foot shock, and a stable rate of responding is established under the conflict schedule. Drugs can then be administered and evaluated for their ability to increase responding under the punished schedule. For example, benzodiazepines have been found to increase bar-pressing during the conflict schedule, putatively by decreasing the stress or anxiety induced by the aversive stimulus. Similarly, in the Vogel punished drinking paradigm (30), thirsty rats with access to a water bottle are periodically given mild electric shocks through the spout of the bottle; the extent to which licking is decreased is used as an index of stress. INDIVIDUAL DIFFERENCES IN DEFENSIVE BEHAVIORS: NATURALLY OCCURRING FEARFUL ENDOPHENOTYPES Primates Several lines of evidence support the notion that an individuals level of defensive responding is a relatively stable trait characteristic (which in part may be derived from the nature of early postnatal maternal interactions, see below). Extreme individual differences detected early in life may be predictive of future psychopathology. For example, extremely inhibited children are at greater risk to develop anxiety and depressive disorders and are more likely to have parents that suffer from anxiety disorders (3134). Moreover, behavioral inhibition in childhood (based on retrospective self-reports) is highly associated with anxiety in adulthood (35). Some of the physiologic correlates that have been observed in extremely inhibited children are elevated levels of the stressrelated hormone cortisol (36) and greater sympathetic nervous system activity (37). In nonhuman primates, individual differences in defensive behaviors have been studied in an attempt to elucidate the neuroendocrine and neurobiological concomitants of extreme behavioral inhibition and to characterize a primate analogue of an anxiety-related endophenotype. Marked individual differences among rhesus monkeys have been noted with regard to the intensity of context-
specific defensive responses. These defensive responses have been characterized using the HIP (see previous section). For example, some monkeys tend to coo frequently during the A condition (in which the animal is isolated), whereas other same-aged animals engage in little or no cooing. Large individual differences have also been observed in the duration of NEC-induced freezing (in the presence of a human profile) and ST-induced hostility (in response to direct eye contact with the human intruder). Some animals freeze the entire length of the test period, whereas at the other extreme some never freeze and act relatively undisturbed by the human intruder. These individual differences in fear-related responses seen in the laboratory are similar to those that have been observed in rhesus monkeys who inhabit Cayo Santiago, a 45-acre island with approximately 1,000 freeranging monkeys (Kalin et al., unpublished data). Importantly, it has been found that monkeys individual differences in defensive responses are relatively stable over time, suggesting that the intensity of defensive behavior that is displayed reflects a trait rather than a state characteristic. It was initially demonstrated that the duration of NECinduced freezing behavior remained stable in 12 animals tested twice with an interval of 4 months (r .94). Using a larger sample size, the stability of NEC-induced freezing was confirmed; ST-induced hostility was also found to be relatively stable (Kalin et al., unpublished data). Interestingly, significant correlations between the magnitude of the different types of defensive responses were not observed within an animal. Thus, monkeys that exhibited extreme levels of NEC-induced freezing did not necessarily display extreme levels of ST-induced hostility. This lack of correlation between different types of defensive responses suggests that cooing, freezing, and defensive hostility represent different and somewhat unrelated characteristics of animals defensive styles. Pharmacologic data also support this notion. For example, manipulations of the opiate system affect A (alone condition)induced cooing without affecting threat-induced freezing or hostility. Conversely, benzodiazepines reduce the threat-related behaviors, but have little effect on A-induced cooing (14). Finally, to identify some of the mechanisms underlying these individual differences in defensive responding, the relationships between the stress-related hormone cortisol or asymmetric frontal EEG activity and individual differences in fearful behavior were examined. Thus, in 28 motherinfant pairs, it was found that in both mothers and infants freezing duration was significantly and positively correlated with baseline (nonstressed) cortisol levels (38). These data are consistent with findings from human studies demonstrating that extremely inhibited children have elevated levels of salivary cortisol (36,37), and is also consistent with findings in rodents that corticosterone (the rodent analogue of cortisol) is required for rat pups to develop the ability to freeze when threatened (39). Extremely fearful monkeys (as identified by the HIP) also exhibit characteristic EEG patterns. In adult humans,
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asymmetric right frontal brain activity has been associated with negative emotional responses (40). Our studies in rhesus monkeys have demonstrated similarities in this measure between monkeys and humans (41). Thus, it has been found that dispositionally fearful monkeys have extreme right frontal brain activity, paralleling the pattern of extreme right frontal activity in humans who suffer from anxiety-related disorders. In addition, it was found that individual differences in asymmetric frontal activity in nonhuman primates in the 4- to 8-Hz range are a stable characteristic of an animal (41,42). Furthermore, a significant positive correlation between relative right asymmetric frontal activity and basal cortisol levels in 50 one-year-old animals was found. As predicted, the more right frontal an animal was, the higher was its cortisol level. An extreme groups analysis revealed that extreme right compared to extreme left frontal animals had greater cortisol concentrations as well as increased defensive responses, such as freezing and hostility. The association between extreme right frontal activity and increased cortisol appeared to be long-lasting because the right frontal animals continued to demonstrate elevated cortisol levels at 3 years of age. These results are the first to link individual differences in asymmetric frontal activity with circulating levels of cortisol. This finding is important because both factors have been independently associated with fearful temperamental styles. It has recently been found that cerebrospinal fluid (CSF) levels of corticotropin-releasing hormone (CRH), a peptide that mediates stress responses, are significantly elevated in monkeys that display exaggerated defensive responses to threatening stimuli (5). As stated before, these extreme individual differences in defensive behaviors are stable over time. Moreover, it was found that CSF CRH levels are also stable over time in rhesus monkeys. Finally, when comparing monkeys with extreme right frontal activity (that display exaggerated fearful responses) to those with extreme left frontal activity (that display low levels of fearful behaviors), the right frontal group was found to consistently have increased CSF CRH levels over a period of 4 years (5). Thus, it appears that extreme fearful behavioral responses in nonhuman primates are associated with increased levels of stress hormones such as cortisol and brain CRH, and also with extreme right frontal brain activity versus left frontal brain activity, a profile that has been found in humans suffering from stress-related psychopathology (43). Taken together, these findings suggest that in primates, a fearful endophenotype can be conceptualized as a constellation of hormonal, electrophysiologic, and behavioral characteristics. Studying species-specific defensive behaviors and their neuroendocrine and physiologic correlates offers a powerful approach for identifying animal correlates of anxiety. Rodents Extreme individual differences in the expression of stressrelated defensive behaviors have also been noted in rodent
species. The examination of naturally occurring genetic variations with regard to stress reactivity may have important implications for the elucidation of individual differences in sensitivity to stressful situations. One example of naturally occurring individual differences comes from the study of different rodent strains with regard to their level of stress-like behavioral responding to environmental stimuli. Because of the important role of the CRH system in regulating defensive behaviors induced by stressful or threatening situations, attention has been focused on identifying rat or mouse strains that display differential stress reactivity and different baseline levels of CRH gene expression. For example, it has been found that baseline levels of CRH messenger RNA (mRNA) are significantly higher in the amygdala of fawn-hooded rats compared to either Sprague-Dawleys or Wistars (44,45). Fawn-hooded rats have also been reported to exhibit exaggerated behavioral responses to stress such as enhanced freezing, leading to the suggestion that this strain may have utility as a model for endogenous stress-related CRH overexpression and anxiety. Strain differences, which essentially reflect differential genetic makeups, have also been found to influence the effects of acute environmental stressors on regulating CRH system gene expression. Thus, the stress of whole-body restraint produces a much larger increase in CRH mRNA levels within the hypothalamus of Fisher rats than in Wistars or Sprague-Dawleys (46,47). Similarly, the spontaneously hypertensive and borderline hypertensive strains of rats have increased basal and stressinduced levels of hypothalamic CRH mRNA compared to the Wistar and Sprague-Dawley strains (4850). In mice, it has been shown that the BALB/c strain is hyperresponsive to a variety of stressors compared to the C57BL/6 strain; BALB/c mice exhibit significantly higher avoidance of aversive areas in a light-dark transition test and an open field (51,52). These mice also show high levels of neophobia (53). Recent genetic mapping studies in these strains have revealed that these behavioral differences may be associated with differential levels of -aminobutyric acid receptor A (GABAA) expression between the strains. For example, it has been found that BALB/c mice have significantly lower levels of benzodiazepine binding sites in the amygdala compared to C57BL/6 mice (54). As described below, alterations in the expression of GABAA receptors have been found to lead to increased anxiety-like behaviors in genetically modified mice (see CRH System Transgenic Mice). Taken together, these findings indicate that different rodent strains, as a consequence of their distinct genetic makeups, display different baseline levels of gene expression within various systems that are known to regulate the expression of stress-induced defensive behaviors. The study of various rodent strains may thus help to identify the neurogenetic differences that contribute to individual differences in stress susceptibility, and thereby further characterize the interaction between genes and environmental
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conditions in the etiology of anxiety. Although such information is useful, it remains to be determined whether or not the specific genetic differences identified above actually underlie the different behavioral effects. It is probable that a number of genes in addition to those described above are differentially expressed across different rodent strains. Which other genes differ across strains, and of these, which ones contribute to the behavioral profile? It is also unclear whether the differential gene expression patterns are the cause or the result of the different phenotypes observed in the separate strains. Future studies in which behavioral phenotypes are assessed after the application of novel gene targeting techniques to selectively disrupt or restore gene function in these rodent strains will aid in clarifying these issues. MATERNAL DEPRIVATION: AN ENVIRONMENTAL MANIPULATION THAT CAN LEAD TO FEARFUL ENDOPHENOTYPES IN PRIMATES AND RODENTS Converging lines of evidence from a number of species point to the importance of the early postnatal period, and in particular the bond between mother and infant, in the development of normal defensive behaviors and the putative emotional states underlying these behaviors. It has been observed that children who were placed in nurseries that lacked adequate social stimulation developed a syndrome of protest, despair, and detachment that may be analogous to an increase in defensive responses (55). Furthermore, recent reports suggest that children reared without appropriate nurturance can display neuroendocrinologic alterations and may develop long-term behavioral and emotional difficulties including an increased risk for stress-related psychiatric illness (56,57). Perhaps the most significant environmental factor during the early development of mammals is the interaction between the infant and its mother. As described above, separation of an infant from its mother during this early developmental phase represents a significant stressor that markedly and negatively affects the subsequent emotional development of the infant (55,57). In fact, disruption of normal attachment behavior at critical developmental phases can, in a number of species, lead to marked and persistent disturbances in behaviors and brain systems that are thought to participate in the regulation of fear-related responses; this disruption may ultimately contribute to an individuals propensity to develop exaggerated or inappropriate defensive responses. Altered maternal-infant interactions can lead to anxiety endophenotypes in nonhuman primates and rodents, thus identifying an environmental manipulation that can be used to create animal models of increased stress-related functioning. Indeed, a large body of work in monkeys and rats indicates that a number of deleterious and long-lasting effects
are produced as a result of separating infants from their mothers prior to weaning. The notion that perturbations in the early postnatal environment might have enduring neuroendocrine, neurochemical, and behavioral effects was originally put forth several decades ago by Levine (58). It has since been demonstrated that a likely source of these alterations is a disruption of the interaction between mothers and pups (59,60). Nonhuman Primates The classic studies by Harlow and colleagues (20,61,62) of the effects of maternal separation in primates found that in addition to life-supporting nourishment, physical contact and comfort are necessary for primates normal social and emotional development. During the first months of life, the attachment between mother and infant is intense, and as a consequence the infant remains in close proximity to its mother (61,63). Long-term maternal separation can result in profound alterations in stress-related behavioral responses in the separated offspring. Monkeys that have been separated from their mothers for prolonged periods during this time exhibit symptoms of enhanced defensive or fear-related behavioral responses into adulthood and appear socially withdrawn, a phenomenon that has led to the suggestion that the behavioral and neuroendocrine sequelae of maternal separation might provide a model for some of the dysfunction that is observed in anxiety disorders and depression (6468). Furthermore, neuroendocrine studies in rhesus monkeys indicate that an infants stress hormone levels are negatively correlated with the number of offspring the mother had, suggesting that when mothers are less experienced, cortisol levels in their (early born) infants are high; elevated cortisol levels also correspond to increased fearful behavioral responses in the infants (38). Cortisol has been found to play an important role in mediating the development of defensive responses (69); thus, factors that were expected to affect infant primate cortisol concentrations were examined. It was found that maternal cortisol levels were moderately correlated with those of their infants (38). Interestingly, it was also found that maternal parity was negatively correlated with infant cortisol levels such that the current infants of mothers that previously had more offspring were likely to have lower cortisol levels. This finding indicates that a mothers past infant rearing and/or pregnancy experience may contribute to individual differences in infant baseline cortisol levels, and provides further support for the notion that the mother-infant interactions may be a critical factor in determining the future fearful disposition of the offspring (38). Although the precise mechanism for this interaction remains to be determined, it is likely that mothers with little rearing experience would interact differently with their infants than mothers with more experience. Evidence for the notion that long-lasting dysregulation
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of the CRH system may in part underlie the harmful consequences of early developmental stressors has been provided in a study of nonhuman primates that were exposed to adverse rearing conditions during infancy. Coplan and colleagues (70,71) found that CSF levels of CRH are basally and chronically elevated in adult bonnet macaques whose mothers were exposed for 3 months to an unpredictable variable foraging demand (VFD), in comparison to mothers confronted with either a high but predictable or low but predictable foraging demand. Infants reared by VFD-exposed mothers have been found to subsequently display abnormal affiliative social behaviors in adulthood (72). These findings are consistent with the recent results from this lab that indicate that CSF CRH levels are elevated in dispositionally fearful monkeys, and that this CRH elevation is a stable trait-like characteristic of fearful endophenotype (5). Rats These aforementioned findings in nonhuman primates support the notion that mother-infant interactions may be a critical factor in determining the future fearful disposition of the offspring. Maternal separation has also been found to produce long-term changes in defensive behaviors into adulthood in rats. Using the maternal separation paradigm in rats, investigators have also been able to begin to elucidate some of the alterations in gene expression that take place in response to this early life stressor. Interestingly, the nature of the separation determines the direction of the long-term changes, as has been reviewed in detail recently (7375). Thus, brief periods of separation (3 to 15 minutes per bout, once a day, for roughly 2 weeks) from the mother result in a profile indicative of diminished anxiety, whereas more protracted separations (3 hours or more) have the opposite effect, resulting in increased stresslike responses. In an elegant series of studies by Plotsky and Meaney (76), the long-term effects of these different types of maternal separation have been described, and the behavioral and neuroendocrine mechanisms underlying these long-term effects have been characterized. It was initially found that rat pups that underwent very short periods of separation (termed handling) from their mothers had decreased basal levels of hypothalamic CRH mRNA and median eminence CRH immunoreactivity as adults compared to undisturbed control rats. As adults, these handled pups also displayed significantly lower elevations of stress-induced corticosterone levels and blunted CRH release from the median eminence relative to controls. It has since been found that the mechanism underlying this reduction in stress-related functioning in handled rat pups involves the type of maternal behavior that is displayed after the pups are returned to the mother (77), confirming earlier hypotheses that maternal behavior is the critical component in the developmental milieu of the infant (58). A brief removal of rat pups from the dam results in a significant
increase in the amount of licking, grooming, and archedback nursing (LG-ABN) that the mother lavishes on the pups when they are returned; the total amount of time spent nursing and contacting the offspring is not affected, but rather the quality of the interaction between mother and pup is altered. In nonseparated pups, individual differences in LG-ABN predict hypothalamic-pituitary-adrenal (HPA) axis responsivity in adulthood such that mothers that engage in high levels of LG-ABN have offspring that, as adults, show reduced HPA axis activation in response to stress and have decreased levels of CRH mRNA in the paraventricular nucleus (PVN) of the hypothalamus (77). Pups that are born to mothers that naturally exhibit high levels of LGABN grow up into adults that display low-anxietylike behaviors (increased exploration of novel environments) and compared to lowLG-ABN offspring, have decreased levels of CRH receptors in brain regions such as the locus coeruleus that are thought to mediate stress responses (78). Taken together, these findings indicate that increased nurturing physical contact from the mother can lead to a toned-down stress-responsive system in the offspring. In contrast, longer periods of maternal separation seem to have the opposite effect on stress-related functioning later in life. Rat pups that are separated from the mother for 3 hours or longer (investigators have often used a 24-hour separation) show in adulthood increased CRH system gene expression, exaggerated HPA axis responses to stress, and increased stress-like behaviors in paradigms such as the elevated plus maze (76,79,80). Other intense stressors such as an endotoxin insult during the perinatal stage are also able to produce marked elevations in basal CRH gene expression and lead to an exaggerated stress-induced HPA axis response in adulthood (81). It has accordingly been hypothesized that the perinatal environment plays a critical role in programming or setting the animals stress coping system (perhaps through alterations in CRH system gene expression) for the remainder of its life (7375). Maternally separated rats also show alterations in other systems that are known to regulate stress-related behaviors and that are consistent with an increased fearful endophenotype. For example, maternal separation increases the release of norepinephrine into the PVN of the hypothalamus in response to restraint stress; stress-induced plasma adrenocorticotropic hormone (ACTH) levels were also elevated in maternally deprived rats (82). Early life stressors such as maternal separation may therefore play an important role in determining the eventual stress-related endophenotype that is exhibited in adulthood. Moreover, the aforementioned studies provide an example of how the animal endophenotype approach can be applied to investigating molecular correlates of anxiety-related conditions. It should be mentioned that prenatal stress can also produce alterations in indices of stress-induced responding in adulthood. For example, in rats, disturbing the prenatal environment by stressing the mother can lead to increases in
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CRH gene expression in the fetal PVN, increases in CRH content in the amygdala of adult offspring, and potentiation of stress-like behavioral responses in these rats whose mothers had undergone stress during pregnancy (8385). These findings further support the notion that mother-infant interactions may be a critical factor in determining the future fearful disposition of the offspring.
lation of stress-related fearful endophenotypes, it should be kept in mind that there are a number of important caveats regarding the interpretation of findings from transgenic animal studies (described at the end of this section). As outlined below, alterations of discrete genes within each of these systems results in an anxiety-like murine endophenotype, characterized by the increased expression of certain aspects of rodent defensive behaviors. The CRH System CRH and the related endogenous peptide agonist urocortin (95) bind to the two cloned CRH receptors, designated CRH1 and CRH2 (9698), and to the CRH binding protein (CRH-BP) (99). The CRH-BP has been postulated to function as an endogenous buffer for the actions of the CRH family of ligands at their receptors (100). CRH, its receptors, and its binding-protein are expressed in key structures of the HPA axis, and thereby participate in mounting the neuroendocrine response to environmental perturbations. The various elements of the CRH system are widely and heterogeneously expressed in cortical, limbic, and brainstem structures and these regions are thought to regulate behavioral responses to stress. CRH System Transgenic Mice Given that central infusion of CRH results in enhanced fear-related defensive behaviors, CRH-overexpressing mice were predicted to display increased stress-like behavioral responses. Indeed, these mice have been found to have several behavioral effects associated with acute CRH administration. CRH overexpressing mice exhibit a profile that is consistent with increased levels of stress, such as reduced baseline and stress-induced exploration of a novel environment, and decreased activity and time spent in the open arms of an elevated plus maze (101,102). These effects are potently blocked by administration of the CRH receptor antagonist -helical CRH. CRH transgenic mice also show a profound decrease in sexual behaviors and significant deficits in learning; higher order functions such as these are typically abolished when a situation is found threatening and defensive behaviors are recruited (103,104). Thus, CRH overexpressing transgenic mice may represent a genetically engineered model of a murine anxiety-like endophenotype. Consistent with the notion that heightened CRH transmission elicits stress-like behaviors is the finding that CRHBP knockout mice show increased stress-like behaviors. CRH-BP knockout mice displayed decreases in open arm entries and open arm time in an elevated plus maze, and showed a decrease in the number of exits from a safe box in a defensive withdrawal/open field paradigm (105). These results indicate a heightened level of neophobia in these mice. Moreover, CRH-BP knockouts have reduced body weight gain over several weeks (105,106), which is also syn-
TARGETED MUTATIONS LEADING TO ANXIETY-LIKE ENDOPHENOTYPES: STUDIES OF TRANSGENIC MICE A perhaps more direct approach for studying the genetic underpinnings of a particular animal endophenotype is to characterize the change in an organisms interaction with its environment following either overexpression or underexpression of a particular gene product. Transgenic and knockout mice are thus now widely used in the ongoing effort to understand the contributions of specific genes to psychopathology. The detailed methodology for the generation of these animals and their use in neuroscience research has been reviewed (86). Briefly, genetic alterations are introduced in the embryonic stage such that the mouse develops with the mutation, thereby putatively providing a model for congenital abnormalities that may contribute to anomalous functioning and the expression of a particular endophenotype. Using this strategy, a variety of components within the CRH, serotonin (5-hydroxytryptamine, 5-HT), and GABA systems have been successfully targeted and studied for their roles in mediating stress-related behavioral effects (8789). It should be noted that in addition to the aforementioned systems, there are several other important central regulators of stress and anxiety-related processes. The norepinephrine system has long been implicated in the modulation of anxiety states. Several recent reviews detail the preclinical and clinical evidence for the involvement of norepinephrine (NE) in anxiety-related disorders such as panic and posttraumatic stress disorder (90,91). Indeed, -adrenergic receptor antagonists and 2-receptor agonists are effective in the treatment of certain anxiety-related symptoms in humans. In addition, recent preclinical evidence indicates that a variety of central nervous system (CNS) peptides including cholecystokinin (CCK), neuropeptide Y (NPY), and substance P/neurokinins participate in the regulation of anxiety-like behaviors (9294). Although these peptide and neurotransmitter systems undoubtedly play a role in stress- and anxiety-related behaviors, the focus of the following section will be the CRH, 5-HT, and GABA systems because these three systems are perhaps the most thoroughly studied with transgenic models. Although the transgenic/knockout approach has provided valuable new information about the genetic regu-
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tonic with increased basal CRH activity. The behavioral profile of CRH-BP knockouts is similar to that which is seen with exogenous CRH administration (107), and supports the notion that removal of the CRH-BP may lead to increased basal stress responses due to increased CRH tone. Thus, as with the CRH-overexpressing mice, CRH-BP knockouts may represent another rodent anxiety-like endophenotype. In terms of the predictive validity of these models, CRH receptor antagonists have been found to block the stress-like behavioral profile observed in these animals; one recent clinical study indicates that CRH1 receptor antagonists may indeed prove to be effective anxiolytics or antidepressants (108). Interestingly, deletion of the CRH gene does not appear to decrease stress-like behaviors, as might be predicted from the aforementioned work. Although certain endocrinologic deficits are observed in CRH knockout mice, stress-related behavioral function in these animals remains relatively unaffected as assessed by multiple stress-related paradigms (109114). This sparing of normal stress responsivity may be due to compensatory increases in the expression of other CRH system ligands such as urocortin. Deletion of the CRH1 receptor gene, however, does appear to consistently result in a putative reduction in anxiety (115117). For example, CRH1 knockout mice show increased exploration of the open arms on an elevated plus maze and spend more time in the brightly lit compartment of a dark-light transition box than do wild-type controls. Moreover, CRH1 knockout mice appear to be immune to the anxiogenic effects of ethanol withdrawal (117). Studies of CRH2 receptor knockout mice, on the other hand, indicate that these mice display a less consistent behavioral profile than the CRH1 knockout mice (118120). Part of the behavioral profile of CRH2 knockouts is suggestive of increased stresslike responding, but other aspects of the behavioral profile indicate either no alteration of stress-related responding (118,119), or a decrease in anxiety-like behaviors (120). The observed increases in anxiety-like behaviors in these genetically altered mice may be due to increased levels of brain CRH and/or urocortin; in two of the three studies, an elevation of baseline CRH or urocortin mRNA levels in the CNS was seen in CRH2 knockout mice (118,119). Thus, the endophenotype displayed by CRH2 knockout mice may actually be indirectly due to a compensatory alteration induced by the mutation rather than simply due to a lack of CRH2 receptor expression. It should be noted that acute blockade of CRH2 receptors results in a decrease in stress-induced defensive behaviors; thus the behavioral profile of these animals is opposite to that of mice that are missing the CRH2 receptor (121,122). Thus, the timing of the gene deletion may critically influence the nature of the behavioral phenotype that ensues. Future studies utilizing novel inducible-knockout technologies may help in clarifying the developmental versus acute role of various genes
in the development of anxiety-related endophenotypes (123). Clinically Effective CRH System Drugs for StressRelated Disorders A large body of preclinical literature indicates that CRH is a critical modulator of stress and anxiety-like behaviors in nonhuman primates and rodents (107,124). Based on the ability of CRH1 receptor-selective antagonists to block many of the behavioral effects of stress or CRH administration, these antagonists have been proposed as potentially therapeutic agents for the treatment of stress-related psychiatric conditions including anxiety and depression (125). Pharmacologic analysis of stress-induced primate defensive responses has also revealed that the CRH system is a critical modulator of this index of anxiety-related behavior. For example, administration of CRH into the cerebral ventricles of nonhuman primates results in a constellation of behavioral responses that closely resemble the defensive responses that are exhibited upon presentation of a stressor (124). Consistent with the notion that increased levels of CRH are associated with increased anxiety-like responding are the recent findings that small-molecule CRH1 receptor antagonists block the expression of some behavioral, physiologic, and neuroendocrine responses to stressors in rhesus monkeys (126,127). The first report of an open-label clinical trial with a CRH1 antagonist was recently published, and revealed a significant effect of this compound in ameliorating symptoms of depression and anxiety (108). Although further research is needed to firmly establish the utility of CRH1 antagonists as psychotherapeutic agents and also to determine the possible side effects associated with their use, these preliminary data support the notion that these compounds represent an important new class of drugs that may offer great promise for the treatment of illnesses associated with increased anxiety and stress. The 5-HT System Serotonin is a member of the monoamine family of transmitters that also include dopamine and norepinephrine. As is typical for the monoamines, cell bodies for this neurotransmitter are found in discrete nuclei within the midbrain (dorsal and medial raphe nuclei) and send widespread 5-HTcontaining projections throughout the brain (128). 5-HT produces its effects through at least 15 different 5HT receptors that are differentially distributed throughout the CNS; the principal mode of 5-HT inactivation is cellular reuptake via terminal transporter proteins (129). The 5HT system has long been implicated in the regulation of mood states and anxiety, and selective serotonin reuptake inhibitors (SSRIs) constitute a major class of antidepressants that have anxiolytic effects. As outlined below, 5-HT transmission also plays a critical role in the regulation of anxiety-
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like behaviors. Given the plethora of 5-HT receptors and the paucity of highly selective ligands for these multiple target sites, several investigators have employed murine gene targeting strategies to elucidate the roles of specific 5-HT receptors in the regulation of stress and anxiety. 5-HT System Transgenic Mice Studies of targeted gene deletions within the 5-HT system have revealed an important role for this system in the regulation of stress and anxiety-related behaviors in mice. The behavioral sequelae of disrupting 5-HT receptor gene expression have been elegantly summarized in several review articles (89,130132). Perhaps the best-characterized 5-HT mutant mice are the 5-HT1A and 5-HT1B receptor knockouts. Mice with a mutation in the 5-HT1A receptor gene have been found to display increased stress-like behaviors in multiple tests of approach-avoidance conflicts. These animals show decreased entries into and time spent in the more aversive region in paradigms such as the open field, elevated plus maze, and the elevated zero maze; thus 5-HT1A knockout mice avoid the center of an open field, the open arms of a plus maze, and the unenclosed regions of a zero maze (133135). It is worth noting that this increased anxiety pattern of results was found consistently across three different research labs, indicating its robustness and reproducibility. Consistent with this profile is the finding that these mice also exhibit decreased activity in the presence of and approach toward a novel object (135). This increase in stress-like responding is not accompanied by changes in overall locomotor activity or motor and spatial coordination, as assessed in photocell cages and a RotaRod apparatus. Curiously, 5-HT1A knockout mice display increased mobility in response to an acute stressor such as forced swimming or tail suspension (133135). Taken together, these findings indicate that 5-HT1A knockout mice may represent another animal endophenotype of increased anxiety. The constitutive knockout of the 5-HT1A receptor does not seem to lead to compensatory alterations in the expression of serotonin or its transporter, or to changes in catecholamine levels in several brain regions (135). Interestingly, a recent report indicates that this mutation alters GABA system expression and function (136). It has been found that anxiety-like behaviors in 5-HT1A knockout mice are relatively unaffected by benzodiazepine treatment. Analysis of brain tissue from these animals indicates that GABAA receptor binding is reduced and that the expression of 1 and 2 subunits of the GABAA receptor are decreased in the amygdala. The anxiolytic actions of benzodiazepines may in part be mediated by GABAA receptors within the amygdala; the profile of results in 5-HT1A knockout mice has led to the intriguing speculation that the anxiety-like endophenotype in these mice may actually in part derive from a decrease in the expression and function of the GABAA recep-
tor (136). This proposed mechanism is consistent with the increase in stress-related behaviors that are seen in certain transgenic mice with mutations in the GABAA receptor (see below). Further work is necessary to determine the precise mechanisms through which the developmental interruption of 5-HT1A gene expression results in the observed anxietylike endophenotype. In contrast to 5-HT1A knockout mice, mice that lack the 5-HT1B receptor show decreased anxiety-like behaviors in several tests of approach-avoidance conflicts. 5-HT1B knockout mice spend more time in the center of an open field and more readily explore novel objects than their wildtype controls; this profile is opposite from that of 5-HT1A knockout mice, and is suggestive of diminished neophobia (89,137). Consistent with this pattern of results is the finding that as pups, 5-HT1B mice emit fewer ultrasonic vocalizations when separated from their mothers; separationinduced vocalizations are thought to provide a measure of anxiety and distress in pups (138,139). It is interesting to note, however, that no changes in contextual or cue-induced conditioned freezing are observed in 5-HT1B mutant mice, suggesting that approach-avoidance conflicts and conditioned fear may be differentially modulated by the 5-HT system. The other main behavioral effect of constitutive 5-HT1B receptor deletion is a marked increase in aggressive behavior (89,140,141). Given that aggressive behaviors represent an important part of an organisms response to threat, 5-HT1B knockout mice may also provide valuable information on the neural and genetic factors associated with stress and anxiety-related functioning (89,142). It should be noted that mice with null mutations of other 5-HT receptor subtypes have also been generated, but these animals have not been found to display as robust an anxietyrelated behavioral profile as the 5-HT1A or 5-HT1B knockout mice. It has been found that 5-HT5A receptor knockout mice show increased exploratory activity in the presence of novelty, but do not differ from wild-type controls with regard to avoidance behaviors from an aversive environment such as the open arms of a plus maze, or the center of an open field (143). These knockout mice also do not respond differently from control subjects in tests of startle reactivity or in burying a probe that delivered a brief electric shock. Thus, these animals appear to have yet a different behavioral profile from that of the 5-HT1A or 5-HT1B knockout mice. An initial report indicates that 5-HT6 receptor deficient mice may exhibit increased avoidance of aversive environments; although these preliminary findings are interesting, further work is needed to fully characterize the phenotype of these mutant mice (144,145). Mice lacking either the 5-HT2A or 5-HT2C receptors have also been created; to the best of our knowledge, the stress-related behavioral functioning of these animals has yet to be reported (146,147).
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Clinically Effective 5-HT System Drugs for StressRelated Disorders As mentioned above, one of the most commonly prescribed and effective classes of drugs that is used in the treatment of depression and anxiety is the SSRIs, which block the reuptake of 5-HT by its transporter and thereby increase serotoninergic transmission. Based on the findings of preclinical studies including those obtained from 5-HT receptor knockout mice, 5-HT1A agonists have been developed for the treatment of anxiety. The clinical utility of this class of compounds, however, remains to be determined. As these transgenic approaches develop and become more refined, they will undoubtedly aid in clarifying the roles of the many other 5-HT receptor subtypes in processes related to stress and anxiety and will aid in drug development. The GABA System The primary inhibitory neurotransmitter in the CNS is GABA; GABA-synthesizing cells are distributed throughout the brain (128). The actions of GABA are mediated by two major classes of receptors, GABAA and GABAB, both of which modulate the activity of ion channels. The principal mode of inactivation of GABA transmission is the presynaptic reuptake of GABA by its transporter protein. Although both types of GABA receptors are widely distributed through the CNS, several important differences exist between the two. Relevant to psychopharmacology is the finding that traditional anxiolytics (benzodiazepines) do not bind to GABAB receptors, but rather mediate their effects through GABAA receptors. GABAA receptors consist of a chloride channel formed by the pentameric arrangement of at least 18 different protein subunits ( 16, 14, 1-3, , , , 13), thus allowing for considerable heterogeneity of the GABAA receptor isoforms (148). Typically, benzodiazepine-responsive GABAA receptors consist of , , and subunits; in addition to the benzodiazepine site, these receptors also contain distinct sites for the binding of GABA, barbiturates, and ethanol. These various regions act as allosteric regulators of GABA-induced chloride channel opening. Although psychotherapeutic effects such as anxiolysis are achieved through facilitation of GABA transmission at this receptor, drugs that act as GABAA receptor agonists also produce several deleterious side effects. The extent to which differences in GABAA receptor subunit composition might contribute to possible dissociations between the beneficial and negative effects of these compounds is currently being investigated. GABA System Transgenic Mice The synthesis of GABA is regulated by two isoforms of the enzyme glutamate decarboxylase (GAD), GAD67, and the
shorter form GAD65 (149). Whereas GAD67 is thought to maintain basal GABA levels, GAD65 is thought to regulate the synthesis of GABA at nerve terminals in response to high GABA demand (150). Given the important role of GABA in inhibitory neurotransmission associated with anxiolysis, several investigators have evaluated the behavioral profile of genetically altered mice that lack the GAD65 gene. Two separate groups have reported that GAD65 / mice display an increase in stress-like behaviors in numerous behavioral paradigms (151,152). GAD65 knockout mice had fewer entries into and time spent in the center of an open field or the open areas of an elevated zero maze (similar to an elevated plus maze), indicating that they were more avoidant of inherently aversive areas. Similarly, these mice had lower levels of activity in the bright portion of a lightdark transition box. It should be mentioned that GAD65 / mice also displayed an elevation in the occurrence of spontaneous and stress-induced seizures, and that these mice had a dramatically increased mortality rate starting at 4 to 5 weeks after birth (151). Thus, although the behavioral profile of GAD65 knockout mice is suggestive of increased anxiety-like responses, it is possible that these effects are secondary to the occurrence of seizures and to the factors leading to early lethality. The usefulness of this knockout as a model for anxiety-related deficits may therefore be limited. Given that benzodiazepines and barbiturates act as positive modulators of GABA transmission at the GABAA receptor by enhancing GABA-induced chloride channel opening, it is of interest to note that GAD65 / mice were not sensitive to the effects of either benzodiazepines or barbiturates, but did respond to the direct GABAA agonist muscimol, which binds directly to the GABA site of the GABAA receptor and increases opening of the chloride channel in the absence of GABA (152). This pharmacologic profile is consistent with the finding that GABA synthesis is blocked by the GAD65 null mutation, but that GABAA receptor binding is unaffected by this change. Furthermore, this mutation does not seem to alter the functioning of GABA receptors because direct agonists stimulate the receptor but indirect modulators of GABA do not. In an attempt to delineate the roles of the various GABAA receptor subunits in the regulation of stress- and anxietyrelated behaviors, investigators have generated mutant mice with alterations in the expression of specific GABAA receptor subunits. It was initially reported that deletion of the 2 subunit led to a selective (94%) reduction in the expression of benzodiazepine sites in the CNS without alterations in the level of GABA sites or changes in the expression of other GABAA receptor subunits (153). Thus, 2 knockout mice possessed functional GABAA receptors that responded normally to GABA site ligands or barbiturates, but did not respond to benzodiazepines; these findings led to the conclusion that the 2 subunit is not necessary for the formation of functional GABAA receptors, but is required to create
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the benzodiazepine-responsive site of those receptors. Mice that were homozygous for the mutation, however, did not live past weaning in this study. In mice carrying only one copy of the functional 2 gene, a 20% reduction in benzodiazepine sites was observed, but these mice did not show overt developmental deficits. In a recent study, a detailed characterization of the behavioral profile of these animals was carried out. Heterozygotes displayed a decrease in the number of entries into and amount of time spent in the open arms of an elevated plus maze and the bright compartment of a light-dark box. These animals also exhibited a decrease in the exploration of novel areas, and an increase in certain forms of fear conditioning that are thought to be mediated by the hippocampus. Finally, 2 heterozygotes were found to react to partially conditioned stimuli (only weakly paired with aversive consequences) as if they were full and potent predictors of threat; compared to wild-types, which showed low levels of defensive behaviors to the partially conditioned stimulus, heterozygotes displayed high levels of conditioned freezing to the partial conditioned stimulus that were identical to those displayed by all animals in response to the full conditioned stimulus. This profile has been proposed to be a model for the tendency to interpret neutral situations as threatening that is seen in anxiety patients. Taken together, the results from this extensive behavioral profile indicate that 2 / mice have increased neophobia and stress-like responses and may thus provide a model for increased anxiety-like behaviors (154156). Interestingly, all of the elevations in stress-like behaviors in 2 heterozygotes were blocked by the benzodiazepine diazepam, suggesting that this animal model may also have good predictive validity for identifying clinically effective anxiolytics. It is also extremely important to mention the 1 subunit transgenic mice, whose behavioral profiles have been thoroughly and insightfully reviewed in recent articles (157, 158). In these mice, a single amino acid is altered (histidine replaced by arginine at the 101 position of the peptide) in the 1 subunit of the GABAA receptor complex. This subtle change does not produce any overt alterations in baseline responses to stress in the genetically altered mice; these animals behave similarly to wild-type controls in tests such as the elevated plus maze and the fear-potentiated startle paradigm, a measure of conditioned fear (159,160). Thus, under drug-free, normal conditions, these animals do not display a behavioral pattern that is consistent with an anxiety-like endophenotype. When these mice are treated with conventional benzodiazepines, however, they react very differently to the drug than their wild-type counterparts. Mice with the mutation in the 1 subunit display a normal reduction of stress-induced anxiety-like behaviors after benzodiazepine treatment, but fail to display some of the more deleterious side effects associated with this class of drugs such as sedation, amnesia, and ataxia. These results indicate that the anxiolytic effects of benzodiazepines can be sepa-
rated from the negative side effects of these compounds, and that the 1 subunit of the GABAA receptor is likely to mediate some of these potentially harmful properties of benzodiazepines. Interestingly, McKernan and colleagues (160) demonstrate that a novel benzodiazepine-site ligand that binds to GABAA receptors containing 2, 3, or 5 subunits but avoids receptors with the 1 subunit produces a behavioral profile that is identical to that of the 1 subunit knockout mice; in normal mice, this compound decreases murine anxiety-like behaviors without eliciting sedation or ataxia (160). Clinically Effective GABA System Drugs for Stress-Related Disorders As stated above, the most widely used GABA system-based drugs for the treatment of anxiety are the benzodiazepines, which facilitate GABA transmission through the GABAA receptor. As outlined in the previous section, the search for novel compounds that may act selectively at specific GABAA subunits is ongoing, with the ultimate hope of discovering ligands that produce anxiolysis but do not cause some of the serious side effects that are commonly associated with benzodiazepines. As demonstrated by McKernan and colleagues (160), drugs that selectively target certain GABAA receptor subunits may hold great promise for the treatment of anxiety without harmful side effects. This development would represent a major breakthrough in the pharmacotherapy of anxiety-related disorders. The use of targeted genetic alterations in identifying the roles of various GABAA subunits will undoubtedly aid in this effort to create designer drugs for the treatment of anxiety (158). General Issues and Caveats of Transgenic Animal Studies As mentioned above, mice carrying certain mutations within either the CRH, the 5-HT, or the GABA system display an anxiety-like endophenotype. It appears that these genetically engineered mouse models also have some predictive validity; the stress-like endophenotype observed in at least two of the aforementioned models is normalized by administration of a clinically effective antianxiety agent that acts within the system that was genetically targeted. It remains to be determined, however, the extent to which these genetically altered models serve to identify potential antianxiety agents from different chemical classes. For example, do benzodiazepines reduce stress-like effects of CRH overexpressers? The extent to which the stress-like endophenotype in these animals is altered by compounds that act on systems that were not directly targeted by the genetic mutation will aid in determining the generalizability and utility of these models as predictors of novel anxiolytic agents. If one assumes that these animals provide a model of inherent trait-like anxiety, they can serve as a powerful tool for
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screening new potential anxiolytics. These models do provide a sound approach to study the long-term effects of congenital abnormalities in these neurotransmitter and neuropeptide systems. Several broad issues should be considered when interpreting studies utilizing genetically altered mice. Generally, the hypotheses regarding the behavioral profiles of transgenic mice are based on earlier findings from psychopharmacologic studies. For example, within the CRH field, the prediction that CRH overexpressers would display increased anxiety-like behaviors was based on the observation that CRH administration produces stress-like behaviors in rodents and primates (107,124). When the outcome of the transgenic studies agrees with the psychopharmacologybased prediction, the findings are taken as a confirmation of that hypothesized mechanism of action. When the outcome of the transgenic studies disagrees with the predicted phenotype, however, concerns about possible developmental confounds are raised. One of the most commonly cited drawbacks of the transgenic/knockout strategy is that the gene of interest is altered from the embryonic stage, therefore possibly influencing other genes involved in the normal development of the animal. Thus, it is difficult to tease apart the effects of under- or overexpression of that gene on the endpoints under study from effects due to compensatory or downstream developmental changes that may have occurred as a result of the mutation (86,87,161). Therefore, the transgenic/knockout approach provides an excellent method for modeling a congenital abnormality that leads to a disease state, but this approach may be less useful for identifying the discrete functions of a specific gene product because of the problems of interpretation that arise from the developmental confound. Indeed, with regard to all of the studies discussed in this section on genetically altered mice, it will be important in future studies to delineate the compensatory alterations that occur in response to the congenital mutation, and that may indirectly contribute to the adult endophenotypes that are reported for these animals. Future studies utilizing novel inducible-knockout strategies will circumvent the developmental issue; inducible knockouts may thus become a valuable tool for exploring the functions of discrete gene products for which no selective ligands are available (123). It should also be noted that there is a large literature concerning the use of antisense oligonucleotide infusions to knock down the expression of particular gene products that may be related to fearful endophenotypes. The antisense oligonucleotide approach, however, has been plagued with a number of issues regarding toxicity, and may therefore not represent the optimal method for studying gene function in vivo (162). FUTURE DIRECTIONS Although the studies summarized in this chapter have contributed a great deal of knowledge about some of the genetic
contributions to the development of stress and anxiety-like endophenotypes in animals, further information is needed to understand the precise nature of geneenvironment interactions in stress regulation. It is likely that a particular stressor results in alterations of gene expression in myriad systems and that the overall response to stress involves the coordination of gene activation and/or suppression within these various systems. Novel high-throughput technologies have recently been developed that enable the expression of thousands of genes to be assayed at once. Gene chips and DNA arrays are two powerful new tools for analyzing complex multilocus genetic interactions associated with a particular environmental perturbation or disease state (163, 164). This approach and its application to psychiatry research have been discussed comprehensively in a recent review article (165). Briefly, gene chip and DNA array technology involve the hybridization of gene transcripts from a tissue sample onto a glass slide or filter that contains up to 10,000 different nucleotide sequences. The amount and pattern of the signal hybridized to the array are then assessed; this method thus permits a rapid analysis of changes in the expression of multiple genes. This technology can also be used to identify single nucleotide polymorphisms in a particular gene by comparing the hybridization patterns of samples from different candidate populations on chips that contain multiple copies of the gene of interest, each copy differing from the previous one by just one base in the sequence. Theoretically, depending on the size of the gene, it would be possible to carry out a base-by-base examination of the entire gene on a single gene chip. However, it is important to realize that although a broad approach can be taken with this technology, it may not be sensitive enough to detect small but functionally important changes in gene expression. This technology can be applied to preclinial and clinical questions regarding the complex genetic control of stress and anxiety by examining event-related gene expression changes and also baseline differences in gene sequences (polymorphisms) that might contribute to differential stress responsivity (165). This technique, along with the recent completion of the Human Genome Project, not only raises the potential to simultaneously profile multiple gene expression systems at once, but also holds great promise for the identification of completely novel genes in stress regulation and anxiety. A greater challenge, however, is the elucidation of the functional role of these new genes in processes related to stress and anxiety. Given this daunting task, methods for more specific and long-term gene targeting will increasingly gain importance in neuroscience research aimed at uncovering genetic dysregulation relating to psychopathology. One technique that is likely to be helpful is that of virally mediated gene transfer. In this method, a gene of interest is cloned into viral vector (with most of the viral genome removed to reduce toxicity and infection) and the modified vector is then infused into a particular brain region using
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63
NEUROBIOLOGICAL BASIS OF ANXIETY DISORDERS
DENNIS S. CHARNEY WAYNE C. DREVETS
The 1990s witnessed tremendous progress in the acquisition of knowledge about the molecular, cellular, and anatomic correlates of fear and anxiety. Advances in neuropharmacology and molecular biology have enabled elucidation of multiple chemical neurotransmitter systems that play roles in fear and anxiety behavior. The anatomic circuits where these transmitters participate in mediating and modulating fear and anxiety are also being illuminated through improvements in neurotoxic techniques, which have enhanced the selectivity of lesion analyses in experimental animals, and by advances in neuroimaging technology, which have permitted mapping of the neurophysiologic correlates of emotion in humans. The findings of these investigations have informed the design and interpretation of clinical neuroscience approaches aimed at investigating how dysfunction within these neurochemical and anatomic systems may result in psychiatric conditions such as panic, posttraumatic stress, and phobic disorders. This chapter reviews the preclinical and clinical data regarding the neural mechanisms underlying normal and pathologic anxiety and discusses their implications for guiding development of novel treatments for anxiety disorders.
NEUROANATOMIC CIRCUITS SUPPORTING FEAR AND ANXIETY Fear and anxiety normally comprise adaptive responses to threat or stress. These emotional-behavioral sets may arise in response to exteroceptive visual, auditory, olfactory, or somatosensory stimuli or to interoceptive input through the viscera and the endocrine and autonomic nervous systems. Anxiety may also be produced by cognitive processes me-
diating the anticipation, interpretation, or recollection of perceived stressors and threats. Emotional processing in general can be divided into evaluative, expressive, and experiential components (1). Evaluation of the emotional salience of a stimulus involves appraisal of its valence (e.g., appetitive versus aversive), its relationship with previous conditioning and behavioral reinforcement experiences, and the context in which it arises (2,3). Emotional expression conveys the range of behavioral, endocrine, and autonomic manifestations of the emotional response, whereas emotional experience describes the subjective feeling accompanying the response. To optimize their capacity for guiding behavior, all these aspects of emotional processing are modulated by complex neurobiological systems that prevent them from becoming persistent, excessive, inappropriate to reinforcement contingencies, or otherwise maladaptive. The emotional processes pertaining to fear and anxiety that have been most extensively studied (largely because of their amenability to experimental manipulation) have involved pavlovian fear conditioning and fear-potentiated startle (4,5). These types of fear learning have been shown to comprise experience-dependent forms of neural plasticity in an extended anatomic network that centers around the critical involvement of the amygdala (1,6). The structures that function in concert with the amygdala during fear learning include other mesiotemporal cortical structures, the sensory thalamus and cortices, the orbital and medial prefrontal cortex (mPFC), the anterior insula, the hypothalamus, and multiple brainstem nuclei (1,5,7). Much of this network appears to participate in the general process of associating a conditioned stimulus (CS) or operant behavior with an emotionally salient unconditioned stimulus (US) (see Fig. 63.1 on p. 905) (5,811). Role of the Amygdala in Fear Learning and Expression The anatomic systems supporting fear learning are organized to permit both rapid responses to simple perceptual
Dennis S. Charney: Mood and Anxiety Disorder Research Program, National Institute of Mental Health, Bethesda, Maryland. W. C. Drevets: Section on Mood and Anxiety Disorders Imaging, Molecular Imaging Branch, National Institute of Mental Health, Bethesda, Maryland.
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elements of potentially threatening stimuli and longerlatency responses to more highly processed information about complex sensory stimuli and environmental contexts. The former processes depend on monosynaptic projections from the sensory thalamus to the amygdala, whereas the latter involve projections from sensory association cortices and mesiotemporal cortical structures to the amygdala (1, 12). These neural networks also respond to visceral input received both directly through the nucleus paragigantocellularis and the nucleus tractus solitarius (NTS) of the vagus nerve and indirectly through the locus ceruleus (LC), the anterior insula, and the infralimbic and prelimbic cortices (4,7,13). Finally, neural activity within the amygdala is modulated by cortisol, norepinephrine (NE), and other neurotransmitters and by mnemonic input related to previous conditioning and reinforcement experiences conveyed by projections from mesiotemporal and prefrontal cortical structures (1418). The lateral nucleus of the amygdala (LA) comprises the primary sensory interface of the amygdala and receives synaptic input representing sensory information from the sensory thalamus and cortex (4). Single neurons within the LA are responsive to auditory, visual, and somatic stimuli, thus enabling the LA to serve as a locus of convergence for information about CS and US (19). Olfactory input, in contrast, directly projects to the periamygdaloid cortex from the olfactory bulb through the olfactory tract (20). The olfactory tract also sends projections to the pyriform cortex and the entorhinal cortex, areas with reciprocal connections to the amygdala (20). Although the periamygdaloid cortex neurons project to deeper amygdaloid nuclei, the specific pathways conveying olfactory information through the amygdala have not been delineated. In addition to its role in conditioning to explicit sensory stimuli, the amygdala is involved in the development of emotional responses to environmental context. The projections from the hippocampal formation to the amygdala through the fornix have been specifically implicated in spatial contextual conditioning (21,22). Thus, lesioning these projections specifically prevents fear conditioning to the chamber or the position within a maze in which aversive stimulation previously occurred (2225). Other structures that participate in the modulation of contextual fear include the rostral perirhinal cortex and the ventrolateral PFC/ anterior (agranular) insula. Lesions of the latter regions reduce fear reactivity to contextual stimuli, but they do not affect CS acquisition or response extinction (26). In contrast, lesions placed in the rostral perirhinal cortex after fear conditioning interfere with the expression of conditioned fear responses elicited by visual and auditory stimuli when these stimuli are presented in contexts that differ from the initial conditioning context (27). Notably, genetic studies in mice identified a quantitative trait locus for contextual conditioning (28,29) that was associated with mouse emotionality in another study (30), although the molecular genetic, neu-
rochemical, and functional anatomic correlates of this trait have not been established. The projections from sensory thalamus to the LA are thought to support rapid conditioning to simple visual and auditory features, presumably accounting for fear responses below the level of conscious awareness (31). Thus, lesioning the auditory cortex before conditioning does not prevent conditioning to single auditory tones. In contrast, projections to the LA from the primary sensory and sensory association cortices appear to be essential for some aspects of conditioned responding to more complex sensory stimuli (4, 32). These relationships are modality specific. For example, disruption of the projections from the auditory thalamus and auditory cortex to the LA specifically prevents acquisition of fear conditioning to auditory stimuli and fear-conditioned responses to previous auditory CSs (3335). After sensory input enters the LA, the neural representation of the stimulus is distributed in parallel to various amygdaloid nuclei, where it may be modulated by diverse functional systems, such as those mediating memories from past experiences or knowledge about ongoing homeostatic states (36). The most extensive extranuclear projections of the LA are composed of reciprocal projections to the basal and accessory basal nuclei and the central nucleus of the amygdala (CE) (37,38). Lesions of either the LA or the CEbut not of other amygdala nucleidisrupt fear conditioning to a tone CS, a finding suggesting that this direct projection from LA to CE is sufficient to mediate conditioning to simple sensory features (4). The projections from LA to the basal amygdaloid nuclei also participate in forming long-lasting memory traces for fear conditioning (2,15,39). Functional inactivation of the lateral and basal amygdaloid nuclei before pavlovian fear conditioning interferes with acquisition of learning, whereas inactivation immediately after conditioning has no effect on memory consolidation (40). The basal nuclei have widespread intranuclear connections and also project to other amygdalar nuclei, including the CE and the LA (41). They also share extensive, reciprocal projections with the orbital and mPFC (43). The basal nuclei are thus anatomically positioned to modulate neuronal responses in both the LA and the PFC (42,43). The plasticity within the amygdala that constitutes memory for conditioning experiences has been shown to involve long-term potentiationlike associative processes (6). Plasticity related to fear learning also occurs in cortical areas, presumably making possible the establishment of explicit or declarative memories about the fear-related event through interactions with the medial temporal lobe memory system (44,45). The influence of the amygdala on cortically based memories has been most clearly characterized with respect to late plastic components of the auditory cortex neuronal responses to a CS. Single-unit recordings during fear conditioning indicate that some auditory cortex neurons, which before conditioning did not respond to the CS tone, develop
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late-conditioned responses (i.e., 500 to 1,500 milliseconds after CS onset) that anticipate the US and show extinctionresistant memory storage (46). These late-conditioned auditory cortical neuronal responses take more trials to learn and respond more slowly than LA neurons within trials, and their late development is prevented by amygdala lesions. Thus, whereas rapid conditioning of fear responses to potentially dangerous stimuli depends on plasticity in the amygdala, learning involving higher cognitive (i.e., mnemonic and attentional) processing of fear experiences may depend on plasticity involving cortical neurons that is influenced by neural transmission from the amygdala to the cortex. Other auditory cortex neurons show an early (less than 50 milliseconds of stimulus onset) plastic component during fear conditioning, in which the preexisting electrophysiologic responses of auditory cortex neurons to the CS become enhanced by conditioning (46). This short-latency plasticity within the auditory cortex appears to depend on input from the auditory thalamus and is unaffected by amygdala lesions. Nevertheless, such short-latency responses are extinguished more quickly (during repeated exposure to the CS alone) in animals with amygdala lesions, a finding implying that the amygdala is involved in preventing extinction of these responses. In human neuroimaging studies, hemodynamic activity in the amygdala increases during initial exposures to fearconditioned stimuli (47,48). However, during repeated, unreinforced exposures to the same stimulus, single-trial functional magnetic resonance imaging (fMRI) studies show that this initial elevation of hemodynamic activity attenuates and subsequently decreases to less than baseline (47). This observation suggests that synaptic input into the amygdala may be actively reduced during the extinction process (49), although the level at which this suppression of afferent synaptic activity into (or within) the amygdala is being suppressed during nonreinforced exposures to the CS has not been established. Activation of the amygdala during an emotional event enhances the strength of long-term memory for emotional stimuli represented in other cortical memory circuits as well (16,50,51). These circuits presumably involve the medial temporal lobe memory system, which has extensive anatomic connections with the amygdala and presumably provides a neuroanatomic substrate for the interaction between storage and explicit recall of affectively salient memories (16). For example, as healthy humans read stories, the magnitude of physiologic activation in the amygdala correlates both with the negative emotional intensity and with the subsequent recall performance of the storys content (52, 53). Physiologic activity in the amygdala and the hippocampus measured during memory encoding reportedly correlates with enhanced episodic memory for pleasant as well as aversive visual stimuli (54), and the amygdalas role in modulating emotional memory may depend more generally
on the degree of arousal or the behavioral salience associated with verbally conveyed information (9,16). Human neuroimaging and electrophysiologic and lesion analysis studies have also demonstrated that the amygdala is involved in the recall of emotional or arousing memories (4,53,55). In humans, bursts of electroencephalographic activity have been recorded in the amygdala during recollection of specific emotional events (56). Moreover, electrical stimulation of the amygdala can evoke emotional experiences (especially fear or anxiety) (57) and the recollection of emotionally charged life events from remote memory (58). Role of the Amygdala in Organizing Emotional Expression The amygdaloid output nuclei, especially the CE, receive convergent information from multiple amygdala regions and generate behavioral responses that are thought to reflect the sum of neuronal activity produced by different amygdaloid nuclei (36). The CE comprises the interface between the amygdala and the motor, autonomic, and neuroendocrine systems involved in expressing fear behavior (4,5). The CE projects to nuclei in the hypothalamus, midbrain, and medulla that mediate the neuroendocrine, autonomic, and behavioral responses associated with fear and anxiety. For example, the amygdala facilitates stress-related corticotropin-releasing hormone (CRH) release by both intrinsic CRH-containing neurons and bisynaptic (double -aminobutyric acidergic [GABAergic]) anatomic projections to the paraventricular nucleus (PVN) of the hypothalamus (59). Electrical stimulation of the CE produces responses similar to those elicited by fear-conditioned stimuli (60,61), and lesions of the CE prevent the expression of fear responses of various types (4,62,63). In contrast, lesioning of specific structures efferent to the CE, such as the lateral hypothalamus or periaqueductal gray (PAG), produces selective deficits in cardiovascular or somatomotor behavioral fear responses, respectively (1,64). The amygdala also sends projections to the thalamus, the nucleus accumbens, the ventromedial caudate, and parts of the ventral putamen that participate in organizing motor responses to threatening stimuli (65). For example, activation of the amygdalar projections to the ventral striatum arrests goal-directed behavior in experimental animals (66), a finding suggesting a possible neural mechanism for the cessation of motivated or reward-directed behavior during anxiety and panic. The amygdala may also influence motor behavior by projections through the hypothalamus and PAG (1). For example, in experimental animals, stimulation of the lateral PAG produces defensive behaviors, sympathetic autonomic arousal, and hypoalgesia, whereas stimulation of the ventrolateral PAG produces social withdrawal and behavioral quiescence, as in response to deep injury or visceral pain (67).
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Other Roles of the Amygdala in Fear Processing The amygdala also appears to play important roles in mediating innate fear and in processing affective elements of social interactions (68). Amygdala lesions cause rats to lose their fear of cats and monkeys to lose their fear of snakes (reviewed in ref. 4). In monkeys, amygdala lesions reduce aggression as well as fear and cause animals to become more submissive to dominant animals (69). In humans, blood flow increases in the amygdala as subjects view faces expressing fear or sadness (70,71), and amygdala lesions impair the ability to recognize fear or sadness in facial expression (55, 72) and fear and anger in spoken language (73). Bed Nucleus of the Stria Terminalis: Hypothesized Role in Anxiety The hypothalamic and brainstem structures that mediate the expression of emotional behavior can also be activated directly by the bed nucleus of the stria terminalis (BNST) (5). Anxiety-like responses elicited either by exposure to a threatening environment for several minutes or by intraventricular administration of CRH appear to be specifically mediated by the BNST, rather than the CE (5). This system is thus hypothesized to play a role in mediating anxiety during exposure to less explicit, or less well defined, sensory cues or to contexts that occur over a longer duration. Other Temporal Cortical Structures The perirhinal cortex shares reciprocal anatomic connections with the amygdala (74), and it is thought to play a role in conveying information about complex visual stimuli to the amygdala during presentation of fear-conditioned visual stimuli. Lesions of the anterior perirhinal cortex, the basolateral nucleus of the amygdala, or the CE can each completely eliminate fear-potentiated startle during exposure to some conditioned visual stimuli (75,76). In contrast, complete removal of the entire visual cortex, insular cortex, mPFC, and posterior perirhinal cortex produces no significant effect on the magnitude of fear-potentiated startle, and lesions of the frontal cortex only partly attenuate fear-potentiated startle. The perirhinal cortex receives input regarding conditioned visual stimuli from the lateral geniculate nucleus, and lesions of this structure can also block fear-potentiated startle (77). Finally, the anterior perirhinal cortex receives afferent projections from the visual cortices as well as from the anterior cingulate cortex (ACC), the infralimbic cortex, and the parietal cortex (74), structures implicated in modulating behavioral responses to fear-conditioned stimuli. The temporopolar cortex has been implicated in modulating autonomic aspects of emotional responses and in processing emotionally provocative visual stimuli. Electrical stimulation of various sites within the temporopolar cortex
can alter a variety of autonomic responses (reviewed in ref. 1). In humans with simple phobias or posttraumatic stress disorder (PTSD), physiologic activity increases in the anterior temporopolar cortex during experimentally induced exacerbations of anxiety involving visual exposure to phobic stimuli or word scripts describing traumatic events, respectively (78,79). Blood flow also increases in the anterior temporopolar cortex of healthy humans during exposure to emotionally provocative visual stimuli, whether the stimuli convey sad, disgusting, or happy content, relative both to conditions involving exposure to emotionally neutral visual stimuli and to conditions in which corresponding emotional states are elicited by recall of autobiographic information (80,81). Portions of the temporopolar cortex may thus function as sensory association areas that participate in evaluating the emotional salience of actual or anticipated stimuli and in modulating autonomic responses to such stimuli. Neuroendocrine and Autonomic Responses during Fear or Stress The peripheral hormonal and autonomic responses to threat mediated by the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic and parasympathetic autonomic nervous systems also play adaptive roles in responding to threat or stress (5). Stimulation of the lateral nucleus of the hypothalamus by afferent projections from the CE of the amygdala, the BNST, or the ventral striatum (82) activates the sympathetic system and produces increases in blood pressure and heart rate, sweating, piloerection, and pupillary dilation. Stress stimulates release of CRH from the PVN of the hypothalamus and amygdala. The CRH secretion from the PVN, in turn, increases peripheral adrenocorticotropic hormone (ACTH) levels, and this stimulates the adrenal glands to secrete cortisol. The ACC, anterior insula, and posterior orbital cortex send anatomic projections to the hypothalamus that participate in modulating or inhibiting cardiovascular and endocrine responses to threat and stress (1,43,83). The vagus and splanchnic nerves constitute the major efferent projections of the parasympathetic nervous system to the viscera. The vagal nuclei receive afferent projections from the lateral hypothalamus, the PVN, the LC, the amygdala, the infralimbic cortex, and the prelimbic portion of the ACC (43,84). The splanchnic nerves receive afferent connections from the LC. The innervation of the parasympathetic nervous system from these limbic structures is thought to mediate visceral symptoms associated with anxiety, such as gastrointestinal and genitourinary disturbances (Fig. 63.1). Role of Prefrontal Cortical Structures in Modulating Fear and Anxiety Behavior Multiple areas of the medial and orbital PFC appear to play roles in modulating anxiety and other emotional behaviors.
FIGURE 63.1. The innervation of the parasympathetic nervous system from limbic structures is thought to mediate visceral symptoms associated with anxiety.
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These PFC structures are thought to participate in interpreting the higher-order significance of experiential stimuli, in modifying behavioral responses based on competing reward versus punishment contingencies, and in predicting social outcomes of behavioral responses to emotional events (8, 11,85,86). These areas share extensive, reciprocal projections with the amygdala, through which the amygdala can modulate PFC neuronal activity and the PFC can modulate amygdala-mediated responses to emotionally salient stimuli (17,18,42,43). Areas within the orbital and mPFC and the anterior insula also participate in modulating peripheral responses to stress, including heart rate, blood pressure, and glucocorticoid secretion (13,17,43,87). The neuronal activities within these areas are, in turn, modulated by various neurotransmitter systems that are activated in response to stressors and threats. For example, the noradrenergic, dopaminergic, and serotonergic systems play roles in enhancing vigilance, modulating goal-directed behavior, and facilitating decision making about probabilities of punishment versus reward by modulating neuronal activity in the PFC (86,8890). Medial Prefrontal Cortex The mPFC areas implicated in anxiety and fear-related behavior in humans and experimental animals include the infralimbic cortex, the ACC located ventral (subgenual) and anterior (pregenual) to the genu of the corpus callosum, and a more dorsal mPFC region that extends from the rostral ACC (BA 24, 32) toward the frontal pole (91). The reciprocal projections between the amygdala and the mPFC are hypothesized to play critical roles in attenuating fear responses and extinguishing behavioral responses to fearconditioned stimuli that are no longer reinforced (17,18). Lesions of the ACC in rats resulted in enhanced freezing to a fear-conditioned tone, a finding suggesting that this mPFC region may be involved in fear reduction (17). In addition, neurons in the rat prelimbic cortex (thought to be homologous to subgenual PFC) reduce their spontaneous firing activity in the presence of a conditioned, aversive tone to an extent that is inversely proportional to the magnitude of fear (42). This suppression of prelimbic cortex neuronal firing activity is inversely correlated with increases in amygdala neuronal activity. Finally, lesions of the infralimbic cortex specifically interfere with the recall of extinction processes after long delays between the acquisition of extinction learning and reexposure to the initial CS (18). Extinction does not appear to occur by erasing memory traces of the CS-US association, but rather by new learning through which the behavioral response to the CS is actively inhibited (31). In humans, the pregenual ACC shows areas of elevated hemodynamic activity during a variety of anxiety states elicited in healthy or anxiety-disordered subjects (reviewed in ref. 49). Electrical stimulation of this region elicits fear,
panic, or a sense of foreboding in humans and vocalization in experimental animals (reviewed in ref. 7). Nevertheless, physiologic activity also increases in the ACC during the generation of positive emotions in healthy humans (92,93) and during depressive episodes in some subtypes of major depressive disorder (MDD) (94,95). The subgenual ACC has been implicated in healthy sadness, MDD, mania, and PTSD (90,96,97). In patients with familial unipolar and bipolar depression, reductions in cerebral blood flow (CBF) and metabolism were associated with left-lateralized reductions in the volume of the corresponding cortex (96,98,99). The subgenual PFC activity shows a mood state dependency in which the metabolism is higher in the depressed than the remitted phase of MDD, consistent with the findings that blood flow increases in this region in healthy, nondepressed humans during experimentally induced sadness (85,100,101) and in persons with PTSD during internally generated imagery of past trauma (97). Both the subgenual and the pregenual ACC share reciprocal anatomic connections with areas implicated in emotional behavior such as the posterior orbital cortex, amygdala, hypothalamus, nucleus accumbens, PAG, ventral tegmental area (VTA), raphe, LC, and NTS (Fig. 63.1) (102,103). Humans with mPFC lesions that include the pregenual and subgenual ACC show abnormal autonomic responses to emotionally provocative stimuli, inability to experience emotion related to concepts, and inability to use information regarding the probability of aversive social consequences versus reward in guiding social behavior (104). In rats, bilateral or right-lateralized lesions of the ventral mPFC composed of infralimbic, prelimbic, and ACC cortices attenuate corticosterone secretion, sympathetic autonomic responses, and gastric stress disorders during restraint stress or exposure to fear-conditioned stimuli (17,83,105). In contrast, left-sided lesions of this cortical strip increase sympathetic arousal and corticosterone responses to restraint stress (105). Finally, the ventral ACC contains glucocorticoid receptors that, when stimulated, inhibit stress-induced corticosterone release in rats (87). Physiologic activity also increases in more dorsal mPFC areas in healthy humans as they perform tasks that elicit emotional responses or require emotional evaluations (81, 106,107). During anxious anticipation of an electrical shock, CBF increases in the rostral mPFC (vicinity of anterior BA24, BA32, and rostral BA9), and the magnitude of CBF correlates inversely with changes in anxiety ratings and heart rate (107). In rats, lesions of the rostral mPFC result in exaggerated heart rate responses to fear-conditioned stimuli, and stimulation of these sites attenuates defensive behavior and cardiovascular responses evoked by amygdala stimulation (83). In primates, whereas BA24 and 32 have extensive reciprocal connections with the amygdala through which they may modulate emotional expression, the BA9 cortex has only sparse projections to the amygdala. Nevertheless, all three areas send extensive efferent projections to the PAG and the hypothalamus through which cardiovascu-
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lar responses associated with emotional behavior can be modulated (43,108). In the depressed phase of MDD and bipolar disorder, metabolic activity is abnormal in the dorsomedial and dorsal anterolateral PFC (in the vicinity of rostral BA9) (91,109). Postmortem studies of these regions have shown abnormal reductions in the size of glia and neurons in MDD (110). Given the preclinical and neuroimaging evidence presented earlier, indicating that this area may modulate anxiety, it may be hypothesized that dysfunction of this mPFC area contributes to the development of anxiety symptoms in mood disorders. Orbital and Anterior Insular Cortex Other areas of the PFC that are implicated in studies of fear or anxiety in human and nonhuman primates are the posterior and lateral orbital cortex, the anterior (agranular) insula, and the ventrolateral PFC (1,43). Physiologic activity increases in these areas during experimentally induced anxiety states in healthy subjects and in subjects with obsessive-compulsive disorder (OCD), simple phobia, and panic disorder (PD) (49,111). (See Chapter 65) The baseline metabolic activity is also abnormally elevated in these regions in unmedicated study subjects with primary MDD (91) and OCD (112) scanned while resting with eyes closed. The elevated activity in these areas in both MDD and OCD appears state dependent, and effective antidepressant or antiobsessional treatment results in decreases in CBF and metabolism in the medicated-improved relative to the unmedicated-symptomatic phase (112114). A complex relationship exists between anxiety-depressive symptoms and physiologic activity in the orbital cortex and the ventrolateral PFC. In MDD, whereas CBF and metabolism increase in these areas in the depressed relative to the remitted phase, the magnitude of these measures correlates inversely with ratings of depressive ideation and severity (115,116). Similarly, posterior orbital cortex flow increases in OCD and animal phobic subjects during exposure to phobic stimuli and in healthy subjects during induced sadness, but this change in CBF correlates inversely with changes in obsessive thinking, anxiety, and sadness, respectively (114,117,118). These data appear consistent with electrophysiologic and lesion analysis data showing that the orbital cortex participates in modulating behavioral and visceral responses associated with fearful, defensive, and reward-directed behavior as reinforcement contingencies change. Nearly one-half of the orbital cortex pyramidal neurons alter their firing rates during the delay period between stimulus and response, and this firing activity relates to the presence or absence of reinforcement (11). These cells are thought to play roles in extinguishing unreinforced responses to aversive or appetitive stimuli (7,11,66). The posterior and lateral orbital cortex and the amygdala send projections to each other and to
overlapping portions of the striatum, hypothalamus, and PAG through which these structures modulate each others neural transmission (Fig. 63.1) (42,66,108,119). For example, the defensive behaviors and cardiovascular responses evoked by electrical stimulation of the amygdala are attenuated or ablated by concomitant stimulation of orbital sites, which, when stimulated alone, exert no autonomic effects (120). Humans with orbital cortex lesions show impaired performance on tasks requiring application of information related to punishment or reward, perseverate in behavioral strategies that are unreinforced, and exhibit difficulty in shifting intellectual strategies in response to changing task demands (11,121). Likewise, monkeys with surgical lesions of the lateral orbital cortex and ventrolateral PFC demonstrate perseverative interference, characterized by difficulty in learning to withhold prepotent responses to nonreinforced stimuli as reinforcement contingencies change (122). Activation of the orbital cortex during anxiety or obsessional states may thus reflect endogenous attempts to attenuate emotional expression or to interrupt unreinforced aversive thought and emotion (91). Conversely, dysfunction of the orbital cortex may contribute to pathologic anxiety and obsessional states by impairing the ability to inhibit nonreinforced or maladaptive emotional, cognitive, and behavioral responses to social interactions and sensory or visceral stimuli. Posterior Cingulate Cortex Many functional imaging studies report that exposure to aversive stimuli of various types increases physiologic activity in the retrosplenial cortex and other portions of the posterior cingulate gyrus (reviewed in ref. 123). Posterior cingulate cortical flow and metabolism have also been found abnormally elevated in some studies of depressed subjects with MDD (reviewed in ref. 91). In contrast, Mayberg et al. reported that script-driven sadness resulted in decreased posterior cingulate activity in healthy subjects, and flow was decreased in depressed relative to remitted subjects with MDD, findings raising the possibility that this large region is functionally heterogenous with respect to emotional behavior (101). The posterior cingulate cortex appears to serve as a sensory association cortex and may participate in processing the affective salience of sensory stimuli. The posterior cingulate cortex sends a major anatomic projection to the ACC, through which it may relay such information into the limbic circuitry (124). FUNCTIONAL ANATOMIC CORRELATES OF SPECIFIC ANXIETY DISORDERS Neuroimaging studies have assessed neurophysiologic abnormalities in anxiety-disordered samples in the baseline,
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resting condition and during symptom provocation. These data converge with those obtained from studies of healthy subjects and of experimental animals to implicate the limbic, paralimbic, and sensory association areas reviewed earlier in the functional anatomy of emotional behavior. Nevertheless, the results of most of the imaging studies reviewed herein await replication, and the data they provide do not clearly establish whether differences between anxiety-disordered and control subjects reflect physiologic correlates of anxiety symptoms or traitlike biological abnormalities underlying the vulnerability to anxiety syndromes.
Panic Disorder The baseline state in PD is characterized by mild to moderate levels of chronic anxiety (termed anticipatory anxiety). In this state, abnormalities of CBF and glucose metabolism have been reported in the vicinity of the hippocampus and parahippocampal gyrus. Reiman et al. initially reported an abnormal resting asymmetry (left less than right) of blood flow and oxygen metabolism in a region of interest placed over the parahippocampal gyrus (125). Nordahl et al. similarly found that glucose metabolism measured over the hippocampus-parahippocampal gyrus was asymmetric and concluded that this abnormality reflected an abnormal metabolic elevation on the right side (126). Bisaga et al. also found abnormal metabolism in this vicinity, but with the opposite laterality (i.e., elevated metabolism in the left hippocampal-parahippocampal area) in lactate-sensitive PD study subjects relative to healthy controls (127). In contrast, De Cristofaro et al. reported that resting perfusion, measured using single photon emission computed tomography (SPECT) and [99mTc]hexamethylpropyleneamineoxime (HMPAO), was abnormally decreased in the hippocampus, bilaterally, in lactate-sensitive PD study subjects relative to controls (128). Each of these studies employed region-of-interest based approaches that were incapable of localizing the center of mass of the abnormality in this region. Reanalysis of some of these data using a voxel-by-voxel approach suggested that the abnormal radioactivity in the vicinity of the mesiotemporal cortex may actually reflect elevated metabolism in the adjacent midbrain (111). This midbrain region, which may reflect the lateral PAG, has been implicated in lactate-induced panic (129), other acute anxiety states (130), and animal models of panic attacks (67). Study subjects with PD have also been imaged during panic elicited using a variety of chemical challenges. Panic attacks induced by intravenous sodium lactate infusion were associated with regional CBF increases in the anterior insula, the anteromedial cerebellum, and the midbrain (129); areas of increased CBF may also exist in the temporal polar cortex, but these findings were confounded by corresponding in-
creases in the adjacent facial muscles during severe anxiety (115). Blood flow also increased in these regions in animal phobic subjects during exposure to phobic stimuli and in healthy subjects during the threat of a painful electrical shock, findings suggesting that these CBF changes reflect the neurophysiologic correlates of fear processing in general (111,130). Consistent with this hypothesis, anxiety attacks induced in healthy humans using cholecystokinin tetrapeptide (CCK-4) were also associated with CBF increases in the insular-amygdala region and the anteromedial cerebellum (131). Indirect evidence suggests that the neurophysiologic responses in the PFC during panicogen challenge may differ between PD subjects and healthy controls. For example, panic attacks induced using CCK-4 were associated with CBF increases in the ACC in healthy humans (131), but flow did not significantly change in the ACC in subjects with PD during lactate-induced panic (129). The ACC was also a region where flow significantly increased in healthy subjects but not in subjects with PD during fenfluramine challenge in a study in which fenfluramine induced panic attacks in 56% of subjects with PD but in only 11% of control subjects (132). Finally, Cameron et al. found that normalized medial frontal CBF increased in healthy controls after yohimbine administration (i.e., after normalizing to remove effects on whole brain CBF) (133), whereas Woods et al. found that the relative prefrontal cortical flow was decreased in PD relative to control subjects following yohimbine challenge (134). Structural MRI studies have begun to investigate whether morphometric or morphologic abnormalities may exist in PD. Ontiveros et al. reported qualitative abnormalities of temporal lobe structure in PD (135), although these findings have not been replicated. Vythilingam et al. reported that hippocampal volume did not differ between PD and healthy control subjects (136).
Phobias In simple animal phobias, phobic anxiety was imaged by acquiring blood flow scans during exposures to the feared animal. During the initial fearful scans, flow increased in the lateral orbital-anterior insular cortex, bilaterally, the pregenual ACC, and the anteromedial cerebellum (78,111), areas where CBF also increases in other anxiety states (see earlier). During the development of habituation to phobic stimuli, the magnitude of the hemodynamic responses to the phobic stimulus diminished in the anterior insula and the medial cerebellum, but it increased in the left posterior orbital cortex in an area where flow had not changed during exposures that preceded habituation (117). The magnitude of the CBF increase in this latter region was inversely correlated with the corresponding changes in heart rate and anxi-

TABLE 63.1. EVIDENCE OF ALTERED CATECHOLAMINERGIC FUNCTION IN ANXIETY DISORDERS
PTSD Increased resting heart rate and blood pressure Increased heart rate and blood pressure response to traumatic reminders/panic attacks Increased resting urinary NE and E Increased resting plasma NE or MHPG Increased plasma NE with traumatic reminders/panic attacks Increased orthostatic heart rate response to exercise Decreased binding to platelet 2 receptors Decrease in basal and stimulated activity of cAMP Decrease in platelet MAO activity Increased symptoms, heart rate and plasma MHPG with yohimbine noradrenergic challenge Differential brain metabolic response to yohimbine +/ +++ + + + + +/ + ++ + Panic Disorder +/ ++ +/ +/ + +/ + NS +++ +
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regarding context may be involved in the pathogenesis of phobias (21). Posttraumatic Stress Disorder PTSD is hypothesized to involve the emotional-learning circuitry associated with the amygdala, because the traumatic event constitutes a fear-conditioning experience, and subsequent exposure to sensory, contextual, or mnemonic stimuli that recall aspects of the event elicits psychological distress and sympathetic arousal. Potentially consistent with this expectation, some studies demonstrated activation of the amygdala as patients with PTSD listened to auditory scripts describing the traumatic event (79) or to combat sounds (in combat-related PTSD) (138) or generated imagery related to the traumatic event without sensory cues (139). However, other studies found no significant changes in amygdala CBF as patients with PTSD listened to scripts describing the traumatic event or viewed trauma-related pictures, and studies comparing CBF responses with traumarelated stimuli have not shown significant differences in the amygdala between patients with PTSD and traumamatched, non-PTSD control subjects (97,139141). The extent to which these negative findings reflect limitations in statistical sensitivity or in positron emission tomography (PET) temporal resolution must be addressed in provocation studies involving larger subject samples and employing fMRI instead of PET. In this regard, it is noteworthy that a preliminary fMRI study found exaggerated hemodynamic changes in the amygdala in patients with PTSD relative to trauma-matched, non-PTSD control subjects during exposure to pictures of fearful faces presented using a backwardmasking technique (142). If replicated, this finding may suggest that the emotional dysregulation associated with PTSD may involve amygdalar responses to emotional stimuli of various types. Other limbic and paralimbic cortical structures have also been implicated in provocation studies of PTSD. In both patients with PTSD and trauma-matched, non-PTSD control subjects, CBF increases in the posterior orbital cortex, anterior insula, and temporopolar cortex during exposure to trauma-related stimuli, but these changes have generally not differentiated PTSD and control samples (79,139,140). In contrast, the pattern of CBF changes elicited in the mPFC by traumatic stimuli may differ between PTSD and control subjects. During exposure to trauma-related sensory stimuli, flow decreased in the left (97,140) but increased in the right pregenual ACC in PTSD (79,138), a finding potentially consistent with the evidence reviewed earlier that the role of the mPFC in emotional behavior is lateralized (105). However, CBF in the right pregenual ACC increased significantly more in non-PTSD, trauma-matched control subjects than in patients with PTSD (139). Moreover, in the infralimbic cortex, CBF decreased in patients with combatrelated PTSD but increased in combat-matched, non-PTSD
, One or more studies did not support this finding (with no positive studies), or the majority of studies do not support this finding; +/, an equal number of studies support this finding and do not support this finding; +, at least one study supports this finding and no studies do not support the finding, or the majority of studies support the finding; ++, two or more studies support this finding, and no studies do not support the finding; +++, three or more studies support this finding, and no studies do not support the finding; cAMP, cyclic adenosine 3, 5-monophosphate; E, epinephrine; MAO, monoamine oxidase; MHPG, 3-methoxy-4-hydroxyphenylglycol; NE, norepinephrine; NS, not studied; PTSD, posttraumatic stress disorder.
ety ratings. As discussed earlier, the posterior orbital cortex was a site where CBF increased in subjects with OCD during exposure to phobic stimuli, with the increase in flow inversely correlated with obsessional ratings (114). In social anxiety disorder, an aversive conditioning paradigm (in which the US was an aversive odor and the CS was a picture of a human face) showed that hemodynamic activity decreased in the amygdala and the hippocampus during presentations of the CS in healthy controls, but it increased in social phobic subjects (137). Interpretation of these data was confounded by the problem that both human faces and aversively CSs normally activate the amygdala, so it remained unclear which of the stimuli produced abnormal responses in social phobia. Nevertheless, these data appear conceptually intriguing, given the role of hippocampalamygdalar projections in mediating contextual fear and the possibility that deficits in the transmission of information
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control subjects during exposure to combat-related visual and auditory stimuli (141). Given evidence that the ACC and the infralimbic cortex play roles in extinguishing fear-conditioned responses (17, 18), the observation that patients with PTSD fail to activate these structures to a similar extent as traumatized, nonPTSD control subjects during exposure to traumatic cues (139,141) suggests that neural processes mediating extinction to trauma-related stimuli may be impaired in PTSD. Compatible with this hypothesis, PTSD samples have been shown to acquire de novo conditioned responses more readily and to extinguish them more slowly than control samples (143,144). Such an impairment could conceivably be related to the vulnerability to developing PTSD, because PTSD occurs in only 5% to 20% of individuals exposed to similar traumatic events. Structural MRI studies of PTSD have identified subtle reductions in the volume of the hippocampus in PTSD samples relative to healthy or traumatized, non-PTSD control samples (145-148). Although limitations existed in these studies in the matching of alcohol use or abuse between PTSD and control samples, the reductions in hippocampal volume did not correlate with the extent of alcohol exposure in the PTSD samples, and no volumetric differences were found between PTSD and control samples in the amygdala, entire temporal lobe, caudate, whole brain, or lateral ventricles. Although the magnitude of the reduction in hippocampal volume only ranged from 5% to 12% in the PTSD samples relative to trauma-matched controls, these abnormalities were associated with short-term memory deficits in some studies (145,147). It remains unclear whether the difference in hippocampal volume may reflect a result of the chronic stress associated with PTSD (e.g., from sustained exposure to elevated glucocorticoid concentrations) or a biological antecedent that may confer risk for developing PTSD (149,150). Obsessive-Compulsive Disorder The anatomic circuits involved in the production of obsessions and compulsions have been elucidated by converging evidence from functional neuroimaging studies of OCD, analysis of lesions resulting in obsessive-compulsive symptoms, and observations regarding the neurosurgical interventions that ameliorate OCD (113,114,151). PET studies of OCD have shown that resting CBF and glucose metabolism are abnormally increased in the orbital cortex and the caudate nucleus bilaterally in primary OCD (reviewed in ref. 112). With symptom provocation by exposure to relevant phobic stimuli (e.g., skin contact with contaminated objects for patients with OCD who have germ phobias), flow increased further in the orbital cortex, ACC, caudate, putamen, and thalamus (114). During effective pharmacotherapy, orbital metabolism decreased toward normal, and both drug treatment and behavioral therapy were associated
with a reduction of caudate metabolism (112). The baseline areas of hypermetabolism in the orbital cortex and the caudate may thus reflect physiologic concomitants of obsessive thoughts or chronic anxiety, and, conversely, the reduction in caudate metabolism associated with effective (but not ineffective) treatment may reflect a physiologic correlate of symptom resolution rather than a primary mechanism of treatment. Based on the evidence reviewed earlier from electrophysiologic and lesion analysis studies indicating that the orbital cortex participates in the correction of behavioral responses that become inappropriate as reinforcement contingencies change, posterior orbital areas may be specifically activated as an endogenous attempt to interrupt patterns of nonreinforced thought and behavior in OCD (11,91). Compatible with this hypothesis, the posterior orbital cortex CBF increases during symptom provocation in OCD, but the magnitude of this increase correlates inversely with the corresponding rise in obsession ratings (r 0.83) (114). In contrast, flow also increases in an area of the right anterior orbital cortex implicated in a variety of types of mnemonic processing, and the change in CBF in this region correlates positively with changes in obsession ratings (114,152). The neurologic conditions associated with the development of secondary obsessions and compulsions also provide evidence that dysfunction within circuits formed by the basal ganglia and the PFC may be related to the pathogenesis of OCD. Such conditions involve lesions of the globus pallidus and the adjacent putamen: Sydenham chorea (a poststreptococcal autoimmune disorder associated with neuronal atrophy in the caudate and putamen), Tourette syndrome (an idiopathic syndrome characterized by motoric and phonic tics that may have a genetic relationship with OCD), chronic motor tic disorder, and lesions of the ventromedial PFC (151154). Several of these conditions are associated with complex motor tics (repetitive, coordinated, involuntary movements occurring in patterned sequences in a spontaneous and transient manner). It is conceivable that complex tics and obsessive thoughts may reflect homologous, aberrant neural processes manifested within the motor and cognitive-behavioral domains, respectively, because of their origination in distinct portions of the corticalstriatal-pallidal-thalamic circuitry (113,155). In contrast to the regional metabolic abnormalities found in primary OCD, imaging studies of obsessive-compulsive syndromes arising in the setting of Tourette syndrome or basal ganglia lesions have not found elevated blood flow and metabolism in the caudate and in some cases have found reduced metabolism in the orbital cortex in such subjects relative to controls (111,151). The differences in the functional anatomic correlates of primary versus secondary OCD are consistent with a neural model in which dysfunction arising at various points within the ventral prefrontal cortical-striatal-pallidal-thalamic circuitry may result in pathologic obsessions and compulsions. This circuitry ap-
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pears to be generally involved in organizing internally guided behavior toward a reward, switching of response strategies, habit formation, and stereotypic behavior (66, 155). These circuits have also been implicated in the pathophysiology of MDD, another illness in which intrusive, distressing thoughts recur to an extent that the ability to switch to goal-oriented, rewarding cognitive-behavioral sets is impaired (91). Although MDD and OCD appear distinct in their course, prognosis, genetics, and neurochemical concomitants, substantial comorbidity exists across these syndromes. Major depressive episodes occur in about one-half of patients with OCD, pathologic obsessions can arise in primary MDD, and the pharmacologic interventions that ameliorate OCD can also effectively treat MDD. Moreover, the neurosurgical procedures that are effective at reducing both obsessive-compulsive and depressive symptoms in intractable cases of OCD and MDD interrupt white matter tracts carrying neural projections between the frontal lobe, the basal ganglia, and the thalamus (155). The clinical comorbidity across these two disorders may thus reflect involvement of an overlapping neural circuitry by otherwise distinct pathophysiologic processes.
Role of the Central Noradrenergic System in Fear and Anxiety Exposure to stressful stimuli of various types increases central noradrenergic function. Thus, exposure to fear-conditioned stimuli, immobilization stress, foot shock, or tail pinch increases NE turnover in the LC, the hypothalamus, the hippocampus, the amygdala, and the cerebral cortex (156). The firing activity of LC neurons also increases during exposure to fear-conditioned stimuli and other stressors or threats (157159). For example, the firing activity of NE neurons in the cat LC increases two- to threefold during confrontation with a dog or an aggressive cat, but it remains unchanged during exposure to other novel stimuli or to nonaggressive cats (160). However, repeated exposure to severe stressors from which the animal cannot escape results in the behavioral pattern termed learned helplessness, which is associated with depletion of NE, possibly reflecting a point at which NE synthesis cannot keep pace with NE release (161,162). Acquisition of fear-conditioned responses requires an intact central noradrenergic system, a finding suggesting that NE release plays a critical role in fear learning (157,163, 164). For at least some types of emotional learning, memory consolidation depends on noradrenergic stimulation of and 1-adrenoreceptors in the basolateral nucleus of the amygdala (15). The activation of NE release in such models may, in turn, depend on effects of stress hormones on noradrenergic neurons (15). The responsiveness of LC neurons to future novel stressors can be enhanced by chronic exposure to some stressful experiences. In rats, the amount of NE synthesized and released in the hippocampus and the mPFC in response to a novel stressor or to local depolarization is increased after repeated exposure to chronic cold stress (165167). This effect may result from a stress-mediated alteration in the sensitivity of presynaptic 2-adrenoreceptors, which inhibit NE synthesis and release. In the native state, administration of the 2-adrenoreceptor antagonists, idazoxan or yohimbine, increases the electrophysiologic response of LC neurons to stressful stimuli (without altering their basal firing rates) and increases NE release and synthesis, whereas administration of the 2-adrenoreceptor agonist, clonidine, decreases NE release and synthesis (167,168). In chronically cold-stressed rats, idazoxan administration produces a greater increase in NE release and synthesis, and clonidine administration produces a blunted attenuation of NE release and synthesis relative to naive rats (167). Consistent with these observations, Torda et al. found that cold immobilization stress decreases the 2-adrenoreceptor density in the hippocampus and the amygdala (169). The effect of chronic stress on noradrenergic responses to subsequent, novel stressors may constitute a form of behavioral sensitization, a process by which single or repeated exposures to aversive stimuli or pharmacologic agents can
NEUROCHEMICAL BASIS OF FEAR AND ANXIETY The neuroanatomic circuits that support fear and anxiety behavior are modulated by a variety of chemical neurotransmitter systems. These include the peptidergic neurotransmitters, CRH, neuropeptide Y (NPY), and substance P, the monoaminergic transmitters, NE, serotonin (5-hydroxytryptamine or 5-HT), and dopamine (DA), and the amino acid transmitters, GABA and glutamate. The neurotransmitter systems that have been best studied in association with responses to stress or threat involve the HPA axis and the central noradrenergic system. These neurochemical systems subserve important adaptive functions in preparing the organism for responding to threat or stress, by increasing vigilance, modulating memory, mobilizing energy stores, and elevating cardiovascular function. Nevertheless, these biological responses to threat and stress can become maladaptive if they are chronically or inappropriately activated. Additional neurochemical systems that play important roles in modulating stress responses and emotional behavior include the central GABAergic, serotonergic, dopaminergic, opiate, and NPY systems. The preclinical and clinical literature regarding these neurochemical concomitants of stress and fear and their potential relevance to the pathophysiology of anxiety disorders are reviewed in the following sections.
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increase the behavioral sensitivity to subsequent stressors (reviewed in ref. 170). Such phenomena are hypothesized to account for clinical observations that patients with anxiety disorders report experiencing exaggerated sensitivity to psychosocial stress. Neural models for the pathogenesis of anxiety disorders built on sensitization phenomena thus hold that repeated exposure to traumatic stress comprises a risk factor for the subsequent development of anxiety disorders, particularly PTSD. Noradrenergic Function in Anxiety Disorders The recurrent symptoms of anxiety disorders, such as panic attacks, insomnia, exaggerated startle, and chronic sympathetic autonomic arousal, may conceivably reflect elevated noradrenergic function (171173). Patients with PTSD and PD show evidence of heightened peripheral sympathetic nervous system arousal that, because of the correlation between peripheral sympathetic activity and central noradrenergic function, is compatible with the hypothesis of increased central NE activity in these disorders (174,175). Moreover, patients with PD, PTSD, and phobic disorders report that their hyperarousal symptoms and intrusive memories are attenuated by alcohol, benzodiazepines (BZDs), and opiates, agents known to decrease LC neuronal firing activity, but are exacerbated by cocaine, which increases LC neuronal firing. The risk of abuse of these substances appears increased in patients with anxiety disorders, a finding raising the possibility that such patients are selfmedicating anxiety symptoms with these agents. It remains unclear, however, whether alterations in noradrenergic function play a primary, etiologic role in the pathogenesis of anxiety disorders, or instead reflect secondary, compensatory changes in response to disorders in other systems. PD has been specifically associated with elevations of 2adrenoreceptor sensitivity and nocturnal urinary NE excretion (176), although -adrenoreceptor function, baseline heart rate and blood pressure, and other measures reflecting central NE secretion have not been consistently altered in PD (see Table 63.1) (177). Altered 2-adrenoreceptor sensitivity is evidenced by findings that administration of the 2-adrenoreceptor agonist, clonidine, results in greater hypotension and larger reductions in plasma 3-methoxy-4hydroxyphenylethylene glycol (MHPG) in PD relative to control subjects (178181). In addition, administration of the 2-adrenoreceptor antagonist, yohimbine (which stimulates NE release by antagonizing presynaptic 2-adrenoreceptors) produces exaggerated anxiogenic and cardiovascular responses and enhanced plasma MHPG and cortisol increases in PD relative to control subjects (133,172,173, 182186). Finally, yohimbine administration resulted in reduced relative frontal cortex flow in patients with PD that did not occur in control subjects, as measured with SPECT and [99mTc]HMPAO (134); it remains unclear, however,
whether this difference reflected a differential physiologic sensitivity to yohimbine or an effect of greater anxiety in the patients with PD, because all the patients with PD but only one control subject developed increased anxiety in response to yohimbine. The sensitivity of 2-adrenoreceptors also appears increased in PTSD. Patients with combat-related PTSD show increased behavioral, chemical, and cardiovascular responses to yohimbine, relative to healthy controls (187189). Considerable evidence also indicates that noradrenergic function is abnormal in PTSD (see Table 63.1). Women with PTSD secondary to childhood sexual abuse showed elevated 24-hour urinary excretion of catecholamines and cortisol (190). In addition, menbut not womenwith PTSD resulting from a motor vehicle accident exhibited elevated urinary levels of epinephrine, NE, and cortisol 1 month after the accident and still had higher epinephrine levels 5 months later (191). Similarly, maltreated children with PTSD excreted greater amounts of urinary DA, NE, and cortisol over 24 hours than controls, with the urinary catecholamine and cortisol output positively correlated with the duration of PTSD trauma and the severity of PTSD symptoms (192). Exposure to traumatic reminders (e.g., combat films or sounds) produced greater increases in plasma, epinephrine, NE, and cortisol in patients with PTSD than in control subjects (191,193,194), although baseline concentrations of catecholamines are not consistently altered in combat-related PTSD (188,189). Geracioti et al. found that cerebrospinal fluid (CSF) NE concentrations are abnormally elevated in PTSD (195). Finally, platelet 2-adrenoreceptor density (196), platelet basal adenosine, isoproterenol, forskolin-stimulated cyclic adenosine monophosphate signal transduction (197), and basal platelet monoamine oxidase activity (198) were decreased in PTSD, findings hypothesized to reflect compensatory responses to chronically elevated NE release. In study subjects with specific phobias, plasma NE and epinephrine concentrations, heart rate, blood pressure, and subjective anxiety ratings increase in response to exposure to phobic stimuli (199). Subjects with social anxiety disorder show greater increases in plasma NE during orthostatic challenge than healthy subjects or those with PD (200).The growth hormone response to intravenous clonidine (a marker of central 2-adrenoreceptor function) is blunted in social anxiety disorder (201), although the density of lymphocyte -adrenoreceptors has not differed between social anxietydisordered and control samples (202) (Table 63.1). Finally, Gerra et al. reported that, plasma NE concentrations increased to a greater extent in male peripubertal patients with generalized anxiety disorder than in controls in response to a psychological stress test (203). However, the
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pretest baseline NE concentrations did not differ between the anxious and control subjects. Hypothalamic-Pituitary-Adrenal Axis and Corticotropin-Releasing Hormone Exposure to acute stress of various types results in release of CRH, ACTH, and cortisol. This HPA-axis activation during acute stress can produce a transient elevation of the plasma cortisol concentration and partial resistance to feedback inhibition of cortisol release that persists during and shortly after the duration of the stressful stimulus. This phenomenon may involve a rapid down-regulation of glucocorticoid receptors, because elevated glucocorticoid levels such as those elicited by acute stress decrease the number of hippocampal glucocorticoid receptors, with a resulting increase in corticosterone secretion and feedback resistance (204). After stress termination, as glucocorticoid levels decrease (presumably because the limbic drive on CRH release diminishes), glucocorticoid-receptor density increases, and feedback sensitivity normalizes (204). During some types of chronic stress, adaptive changes in ACTH and corticosterone secretion occur such that the plasma ACTH and corticosterone concentrations achieved are lower than those seen in response to acute stress (205). In contrast, other types of chronic stress are associated with enhanced corticosterone secretion in rats (206). Moreover, Dallman and Jones showed that the experience of prior stress can result in augmented corticosterone responses to subsequent stress exposure (207). The factors that determine whether adaptation or sensitization of glucocorticoid activity occurs after chronic stress remain poorly understood. Some stressors experienced within critical periods of neurodevelopment exert long-term effects on HPA-axis function. In rats exposed to either severe prenatal (in utero) stress or early maternal deprivation stress (208,209), the plasma concentrations of corticosterone achieved in response to subsequent stressors are increased, and this tendency to show exaggerated glucocorticoid responses to stress persists into adulthood. Early postnatal adverse experiences such as maternal separation are associated with long-lasting alterations in the basal concentrations of hypothalamic CRH mRNA, hippocampal glucocorticoid-receptor mRNA, median eminence CRH, and in the magnitude of stress-induced CRH, corticosterone, and ACTH release (210212). In nonhuman primates, adverse early experiences induced by variable maternal foraging requirements reportedly result in alterations in juvenile and adult social behavior, such that animals are more timid, less socially interactive, and more subordinate (213). Adult monkeys who were raised in such a maternal environment are also hyperresponsive to yohimbine and have elevated CRH concentrations and decreased cortisol levels in the CSF, findings that parallel those in humans with PTSD (213).
Conversely, positive early-life experiences during critical developmental periods may have beneficial long-term consequences on the ability to mount adaptive responses to stress or threat. For example, daily postnatal handling of rat pups by human experimenters within the first few weeks of life has been shown to produce persistent (throughout life) increases in the density of type II glucocorticoid receptors. This increase was associated with enhanced feedback sensitivity to glucocorticoid exposure and reduced glucocorticoid-mediated hippocampal damage in later life (214, 215). These effects are hypothesized to comprise a type of stress inoculation induced by the mothers repeated licking of the pups after they were handled by humans. Taken together with the data reviewed in the preceding paragraph, these data indicate that a high degree of plasticity exists in stress-responsive neural systems during the prenatal and early postnatal periods that programs future biological responses to stressful stimuli (210). Regional differences in the regulation of CRH function by glucocorticoid-receptor stimulation and stress may play major roles in the mediation of fear and anxiety (216). The feedback inhibition of CRH function by glucocorticoids (to suppress HPA-axis activity) occurs at the level of the PVN of the hypothalamus, where systemically administered glucocorticoids reduce CRH expression, and the anterior pituitary, where glucocorticoids decrease CRH receptor expression (217220). The regulation of CRH receptor mRNA expression shows a regional specificity that becomes altered when stress occurs concomitantly with elevated glucocorticoid concentrations. After both short-term and long-term corticosterone (CORT) administration, the CRH receptor RNA expression decreases in the PVN and the anterior pituitary (219). However, after acute or repeated immobilization stress sufficient to produce a large increase in plasma CORT levels, the CRH mRNA expression decreases in the anterior pituitary, but increases in the PVN. In contrast, neither CORT administration nor restraint stress alters the CRH receptor expression in the CE of the amygdala or the BNST. Furthermore, CRH secretion is not constrained by glucocorticoids in the CE or the lateral BNST, and CRH mRNA expression increases in these areas during systemic CORT administration (217,218,220). It is thus conceivable that the positive feedback of glucocorticoids on extrahypothalamic CRH function in the amygdala or the BNST may contribute to the production of anxiety symptoms (216, 221). Another level through which the CRH-glucocorticoid system maintains homeostasis and provides mechanisms for modulating mechanism over stress or anxiety responses involves functional differences between CRH-receptor subtypes. The CRH1 and CRH2 receptors appear to play reciprocal roles in mediating stress responsiveness and anxiety-like behaviors (221). Mice genetically deficient in CRH1-receptor expression exhibit diminished anxiety and
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stress responses to threat or stress (222,223). In contrast, mice deficient in CRH2 receptors display heightened anxiety in response to stress (224,225). The affinity of CRH is higher for CRH1 than CRH2 receptors, a finding consistent with evidence that CRH elicits anxiogenic effects either when exogenously administered to native animals or when endogenously released in mice genetically altered to overexpress CRH (221). Also consistent with the hypothesis that CRH1-receptor stimulation facilitates anxiety responses, oral administration of the CRH1-receptor antagonist, antalarmin, inhibits the behavioral, sympathetic autonomic, and neuroendocrine responses (i.e., attenuating increases in the CSF CRH concentration and in the pituitary-adrenal and adrenal-medullary activity) to acute social stress in monkeys (226). Regional differences in the anatomic distribution of CRH1 and CRH2 receptors likely play a role in balancing facilitatory versus modulatory effects of CRH-receptor stimulation on stress responses. In monkeys, the CRH1-receptor density is high in most amygdaloid nuclei, the cingulate cortex, the PFC, the insular cortex, the parietal cortex, the dentate gyrus, and the entorhinal cortex, and it is moderate in the CE and the LC. The CRH2-receptor density is high in the cingulate cortex, the mPFC, the CE, the CA-1 region of the hippocampus, and the PVN and supraoptic nucleus of the hypothalamus. An important avenue of future research will involve assessments of the homeostatic balance between CRH1- and CRH2-receptor systems in anxiety disorders. HPA-Axis Function and CRH Release in Anxiety Disorders The anxiety disorder for which abnormalities of CRH or HPA-axis function has been most commonly reported is PTSD. Nevertheless, the nature of such abnormalities has been inconsistent across studies, because basal plasma or 24-hour urine cortisol concentrations have been reported to be abnormally decreased (227229), not different (230, 231), or abnormally increased (190,192,232,233) in PTSD samples relative to healthy or trauma-matched control samples. Differences across these studies may reflect effects of gender, age of illness onset (i.e., childhood versus adult), trauma type or duration, or physiologic variation relative to illness phase. For example, Hawk et al. showed that 24hour urine cortisol concentrations were elevated in males but not females with PTSD, and that this abnormality in males was evident at 1 month but not 6 months after the traumatic event (191). The HPA-axis responses to behavioral or pharmacologic challenge have also been assessed in PTSD. During provocation of PTSD symptoms by exposure to combat sounds, the changes in plasma cortisol and ACTH concentrations did not differ between patients with combat-related PTSD and either healthy or combat-matched, non-PTSD control subjects (232). In response to dexamethasone administra-
tion, cortisol suppression was found to be normal (234) or enhanced (228,235,236) in PTSD, with the latter result particularly found in response to low-dose (0.25 and 0.5 mg) dexamethasone. Yehuda et al. also observed that patients with PTSD have an increased density of glucocorticoid receptors on peripheral lymphocytes (228). This finding, together with the observations that patients with PTSD show hypersensitivity to low-dose dexamethasone, led Yehuda et al. to hypothesize that an increase in hypothalamic glucocorticoid-receptor function results in enhanced feedback sensitivity to cortisol, leading to decreased peripheral cortisol levels (237). Preliminary data suggest that a reduced cortisol response after trauma exposure may predict PTSD development, a finding raising the possibility that enhanced feedback sensitivity to cortisol may arise acutely or may even antedate illness onset in some patients with PTSD (229, 238). The central release of CRH in PTSD was examined in two studies of CSF concentrations, both of which found abnormally increased in chronic, combat-related PTSD (239,240). Potentially consistent with this observation, PTSD samples show a blunted ACTH response to CRH relative to control samples (241,242). Although these observations would appear most consistent with findings that basal cortisol secretion and excretion are abnormally increased in PTSD (190,192,232,233), they do not clearly contradict the findings of normal or reduced peripheral cortisol concentrations in PTSD because hypothalamic and extrahypothalamic CRH secretion are independently regulated (216). Nevertheless, the studies that either identified reductions or were unable to identify elevations in peripheral cortisol concentrations in PTSD present a challenge to the hypothesis that the reduced hippocampal volume found in MRI studies of PTSD (reviewed earlier) are accounted for by cortisol hypersecretion (150). This hypothesis may still be reconciled with the peripheral cortisol measures associated with chronic PTSD if the cortisol secretion was elevated near the time of the stressor (191,243). Longitudinal studies in male patients who developed PTSD after motor vehicle accidents suggest that cortisol secretion is elevated 1 month after the trauma, but it is normal when measured 6 months after the trauma (191). In rats, the atrophy of pyramidal cell apical dendrites that occurs in response to stress-induced corticosterone secretion is reversible when the exposure to elevated glucocorticoid concentrations is terminated early, but it can become irreversible if the elevated corticosterone concentration persists beyond a critical time period (149). Hippocampal damage may thus conceivably occur in PTSD during a period of excessive cortisol secretion that follows the traumatic event and is prolonged enough so that hippocampal neuronal atrophy becomes irreversible. An alternative hypothesis for the reduction of hippocampal volume in PTSD, however, is that this abnormality antedates the

TABLE 63.2. EVIDENCE OF ALTERATIONS IN CRF-HPA AXIS FUNCTION IN ANXIETY DISORDERSa
PTSD Alteration in urinary cortisol Altered plasma cortisol with 24-hour sampling Supersuppression with DST Blunted ACTH response to CRF Elevated CRF in CSF Increased lymphocyte glucocorticoid receptors +/ + (dec.)
a
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Functional Interactions among Noradrenergic, HPA, and CRH Systems Coordinated functional interactions between the HPA axis and the noradrenergic systems play major roles in producing adaptive responses to stress, anxiety, or fear. The secretion of CRH increases LC neuronal firing activity and results in enhanced NE release in a variety of cortical and subcortical regions (252,253). Conversely, NE release stimulates CRH secretion in the PVN (the nucleus containing most of the CRH-synthesizing neurons in the hypothalamus). During chronic stress in particular, the LC is the brainstem noradrenergic nucleus that appears preferentially to mediate NE release in the PVN (254). Conversely, as CRH release in the PVN stimulates ACTH secretion from the pituitary and thereby increases cortisol secretion from the adrenal glands, the rise in plasma cortisol concentrations acts through a negative feedback pathway to decrease both CRH and NE synthesis at the level of the PVN. Glucocorticoid-mediated inhibition of NE-induced CRH stimulation may be evident primarily during stress, rather than under resting conditions, as an adaptive response that restrains stress-induced neuroendocrine and cardiovascular effects mediated by the PVN (254). NE, cortisol, and CRH thus appear tightly linked as a functional system that offers a homeostatic mechanism for responding to stress. A clinical phenomenon of anxiety disorders that may be specifically regulated by interactions between NE and glucocorticoid secretion involves the acquisition and consolidation of traumatic memories. A characteristic feature of PTSD and PD is that memories of the traumatic experience or the original panic attack, respectively, persist for decades and are recalled in response to multiple stimuli or stressors. In experimental animals, alterations of both brain catecholamine and glucocorticoid levels affect the consolidation and retrieval of emotional memories (50,51). Glucocorticoids influence memory storage by activation of glucocorticoid receptors in the hippocampus, whereas NE effects are mediated in part through -adrenoreceptor stimulation in the amygdala (255). In humans, adrenocortical suppression blocks the memory-enhancing effects of amphetamine and epinephrine (256), and propranolol impairs memory for an emotionally provocative story, but not for an emotionally neutral story (257). These data suggest that the acute release of glucocorticoids and NE in response to trauma may modulate the encoding of traumatic memories. It is conceivable that long-term alterations in these systems may account for memory distortions seen in PTSD, such as the memory fragmentation, hypermnesia, and deficits in declarative memory. Central Benzodiazepine-GABAReceptor System Several lines of preclinical and clinical evidence have established that BZD-receptor agonists exert anxiolytic effects
Panic Disorder +/ + (inc.)/ +/ NS
++b ++ ++ ++
a Findings of decreased urinary cortisol in older male combat veterans and holocaust survivors and increased cortisol in younger female abuse survivors may be explainable by differences in gender, age, trauma type, developmental epoch at the time of the trauma, or timing within illness course. b Pertains specifically to response to low-dose dexamethasone (0.25 and 0.5 mg). , One or more studies did not support this finding (with no positive studies), or the majority of studies do not support this finding; +/, an equal number of studies support this finding and do not support this finding; +, atleast one study supports this finding and no studies do not, or the majority of studies support the finding; ++, two or more studies support this finding, and no studies do not support the finding; +++, three or more studies support this finding, and no studies do not; ACTH, adrenocorticotropic hormone; CRF, corticotropin-releasing factor; CSF, cerebrospinal fluid; dec., decrease; DST, dihydrostreptomycin; HPA, hypothalamic pituitary adrenal axis; inc., increase; NS, not studied; PTSD, posttraumatic stress disorder.
development of PTSD and may comprise a risk factor for developing PTSD in response to traumatic stress. In PD, the results of studies examining CRH-receptor and HPA-axis function have been less consistent (Table 63.2). Elevated plasma cortisol levels were reported in one study (244), but not in another (245), and the results of studies assessing urinary free cortisol have been similarly inconsistent (177,246). In a study of 24-hour secretion of ACTH and cortisol, PD subjects had subtle elevations of nocturnal cortisol secretion and greater amplitude of ultraradian secretory episodes relative to control subjects (247), but these findings await replication. Both normal and elevated rates of cortisol nonsuppression after dexamethasone administration have been reported in PD (248). After combined dexamethasone-CRH challenge, the HPAaxis response was higher in PD subjects than in healthy controls, but the magnitude of this abnormality was less than that seen in depressed samples (249,250). The ACTH response to CRH was blunted in some studies (249,250), but not in others (250), in PD relative to control samples, although CSF levels of CRH did not differ between PD and control samples (251). The extent to which pathophysiologic heterogeneity within PD samples may account for the inconsistency of these findings remains unclear.
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and have suggested that BZD-receptor function may be altered in anxiety disorders. Central BZD receptors are expressed are present throughout the brain, but they are most densely concentrated in the cortical gray matter. The BZD and GABAA receptors form parts of the same macromolecular complex, and although they constitute distinct binding sites, they are functionally coupled and regulate each other in an allosteric manner (258). Central BZD-receptor agonists potentiate and prolong the synaptic actions of the inhibitory neurotransmitter, GABA, by increasing the frequency of GABA-mediated chloride channel openings (258, 259). Microinjection of BZD-receptor agonists in limbic and brainstem regions such as the amygdala and the PAG exert antianxiety effects in animal models of anxiety and fear (260). Conversely, administration of BZD-receptor inverse agonists, such as -carboline-3-carboxylic acid ethylester, produces behaviors and increases in heart rate, blood pressure, plasma cortisol, and catecholamines similar to those seen in anxiety and stress (261,262), effects that can be blocked by administration of BZD-receptor agonists (263). Transgenic mouse studies have identified behavioral roles for specific GABAA-receptor subunits. The anxiolytic action of diazepam appears absent in mice with 2 subunit point mutations, but it is present in mice with 1 or 3 subunit point mutations (264,265). These data suggest that the anxiolytic effect of BZD agonists is at least partly mediated by the GABAA-receptor 2 subunit, which is largely expressed in the limbic system, but not by the 3 subunit, which is predominately expressed in the reticular activating system, or the 1 subunit, which is implicated in mediating the sedative, amnestic, and anticonvulsive effects of BZDs (265,266). These findings hold clear implications for investigations of the pathophysiology of anxiety disorders and for the development of anxioselective BZD-receptor agonists. Some other agents with anxiolytic effects appear to modulate the function of the GABAA/BZD-receptorchloride ionophore complex by mechanisms distinct from those of the BZD agonists. The neurosteroid, allopregnenolone, exerts antianxiety effects in conflict paradigms that serve as putative animal models of anxiety. The anticonflict effects of allopregnenolone are reversed by either isopropylbicyclophosphate, which binds at the picrotoxinin site on the GABAA receptors, or RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5- ]-[1,4]benzodiazepine-3-carboxylate), a BZD-receptor inverse agonist that inhibits GABAA-activated chloride flux in neuronal membranes. In contrast, administration of the BZD-receptor antagonist flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl6-oxo-4H-imidazo[1,5- ]-[1,4]benzodiazepine-3- carboxylate) does not block allopregnenolones anxiolytic-like effects, a finding indicating that allopregnenolone does not bind at the BZD site. Allopregnenolone may thus exert anxiolytic-like effects by stimulating the chloride channel in GABAA receptors by binding at the picrotoxinin site or at a site specific for RO15-4513. The antianxiety effects of antidepressant drugs with pri-
mary effects on monoamine reuptake may also be partly mediated through a GABAergic mechanism. These agents are effective for the treatment of a spectrum of anxiety disorders including social anxiety disorder, generalized anxiety disorder, PD, and PTSD. One of the multiple secondary effects of these agents involves potentiation of GABAergic function. For example, in rats, the effective dose of phenelzine (15 mg/kg) on the elevated plus maze administered produces a more than twofold increase in whole-brain level GABA concentrations, whereas an ineffective dose of phenelzine (5.1 mg/kg) does not significantly alter GABA levels (267). Moreover, the N-acetylated metabolite of phenelzine, N-2-acetylphenelzine, which potently inhibits monoamine oxidase but does not change whole-brain GABA concentrations, does not produce anxiolytic effects in the elevated plus-maze test (267). Phenelzines anxiolytic effects in the plus-maze model may thus depend on elevating brain GABA concentrations, in contrast to the mechanism of the classic BZDs, which instead increase the affinity of GABAA receptors for GABA.
Effects of Stress on Benzodiazepine-GABAA Receptors BZD- and GABA-receptor function can be altered by exposure to stress in some brain regions. In experimental animals exposed to inescapable stress in the form of cold swim or foot shock, the BZD-receptor binding decreases in the frontal cortex, with less consistent reductions occurring in the hippocampus and hypothalamus, but no changes in the occipital cortex, striatum, midbrain, thalamus, cerebellum, or pons (268). Chronic stress in the form of repeated foot shock or cold water swim resulted in decreased BZD-receptor binding in the frontal cortex and hippocampus, and possibly in the cerebellum, midbrain, and striatum, but not in the occipital cortex or pons (268270). These reductions in BZD-receptor binding were associated with deficits in maze escape behaviors that may have reflected alterations in mnemonic processing (269,270). Some of these stress effects may be mediated by glucocorticoids, because chronic exposure to stress levels of CORT alters mRNA levels of multiple GABAA-receptor subunits (271). Consistent with the effects of chronic stress on BZD-receptor expression, the Maudsley genetically fearful rat strain shows decreased BZD-receptor density relative to other rats in several brain structures including the hippocampus (272). Stressors arising early in life may also influence the development of the GABAergic system. In rats, early-life adverse experiences such as maternal separation result in decreased GABAA-receptor concentrations in the LC and the NTS, reduced BZD-receptor sites in the LC, the NTS, the frontal cortex, and the CE and the LA of the amygdala, and reduced mRNA levels for the 2 subunit of the GABAA-receptor complex in the LC, the NTS, and the amygdala (273). The extent to which these developmental responses to early-life
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stress may alter the expression of fear and anxiety in adulthood remains unclear. Benzodiazepine-GABAReceptor Function in Anxiety Disorders The central BZD receptor has been implicated in anxiety disorders on the basis of the anxiolytic and anxiogenic properties of BZD agonists and inverse agonists, respectively, and by the evidence that the BZD-receptor sensitivity to BZD agonists is reduced in some anxiety-disordered subjects (21,274,275). Hypotheses advanced regarding the role of GABAA-BZDreceptor function in anxiety disorders have proposed either that changes in the GABAA-BZD macromolecular complex conformation or that alterations in the concentration or properties of an endogenous ligand account for the pathologic anxiety symptoms seen in anxiety disorders. However, these hypotheses have not been conclusively tested by in vivo or postmortem studies of anxietydisordered humans. In PD, oral (276) and intravenous (274) administration of the BZD-receptor antagonist, flumazenil, produces panic attacks and increases anticipatory anxiety in some subjects with PD, but not in healthy controls. In addition, the sensitivity to the effects of diazepam on saccadic eye movement velocity is abnormally reduced in PD, a finding implying that the functional sensitivity of the GABAA-BZD supramolecular complex is attenuated in brainstem regions controlling saccadic eye movements (275). Subjects with PD also show abnormally reduced sensitivity to the suppressant effects of diazepam on plasma NE, epinephrine, and heart rate (see Table 63.3 on p. 920) (277). Receptor imaging studies using PET and SPECT have assessed central BZD-receptor binding in anxiety disorders. SPECT studies have reported reduced uptake of the selective BZD-receptor radioligand, [123I]iomazenil, in the frontal (278280), temporal (278,279), and occipital (278) cortices in subjects with PD relative to control subjects. However, interpretation of these results was limited by the absence of medication-free PD study subjects and of healthy controls (278,279) or by the dependence on nonquantitative methods for estimating BZD-receptor binding. A SPECT-iomazenil study that quantitated BZD-receptor binding by derivation of distribution volumes found reduced binding in the left hippocampus and precuneus in unmedicated PD relative to healthy control samples and reported an inverse correlation between panic anxiety ratings and frontal cortex iomazenil binding (281). Another SPECT-iomazenil study reported lower distribution volumes for BZD receptors in the dorsomedial PFC in PTSD relative to control samples (281a). These findings appeared consistent with the evidence cited earlier that stress downregulates BZD-receptor binding in the frontal cortex and the hippocampus of experimental animals. Central BZD-receptor binding has also been assessed in PD using PET and [11C]flumazenil. Malizia et al. reported a
global reduction in BZD site binding in seven study subjects with PD relative to eight healthy controls, with the most prominent decreases evident in the right orbitofrontal cortex and the right insula (areas consistently activated during normal anxiety processing) (282). In contrast, Abadie et al. found no differences in the Bmax, Kd or bound/free values for [11C]flumazenil in any brain region in ten unmedicated PD study subjects relative to healthy controls (283).
Dopaminergic System Acute stress increases DA release and turnover in multiple brain areas. The dopaminergic projections to the mPFC appear particularly sensitive to stress, because brief or lowintensity stressors (e.g., exposure to fear-conditioned stimuli) increase DA release and turnover in the mPFC in the absence of corresponding changes in other mesotelencephalic dopaminergic projections (284). For example, in rats, low-intensity electric foot shock increases tyrosine hydroxylase activity and DA turnover in the mPFC, but not in the nucleus accumbens or the caudate-putamen (285). In contrast, stress of greater intensity or longer duration additionally enhances DA release and metabolism in other areas as well (285). The regional sensitivity to stress appears to follow a pattern in which dopaminergic projections to the mPFC are more sensitive to stress than the mesoaccumbens and nigrostriatal projections, and the mesoaccumbens dopaminergic projections are more sensitive to stress than the nigrostriatal projections (284). Thus far, there is little evidence that dopaminergic dysfunction plays a primary role in the pathophysiology of human anxiety disorders. In PD, Roy-Byrne et al. found a higher plasma concentration of the DA metabolite, homovanillic acid (HVA), in patients with high levels of anxiety and frequent panic attacks relative to controls (286). Patients with PD were also shown to have a greater growth hormone response to the DA-receptor agonist, apomorphine, than depressed controls (287). However, Eriksson et al. found no evidence of alterations in the CSF HVA concentrations in patients with PD or for correlations between CSF HVA and anxiety severity or panic attack frequency (288). In addition, genetic studies examining associations between PD and gene polymorphisms for the DA D4 receptor and the DA transporter have produced negative results (289). In social phobia, two preliminary SPECT imaging studies involving small subject samples reported abnormal reductions in DA-receptor binding. Tiihonen et al. found a significant reduction in -CIT binding in the striatum in social phobic relative to healthy control samples (290), presumably reflecting a reduction in DA-transporter binding. Schneier et al. reported reduced uptake of the DA D2/D3receptor radioligand, [123I]IBZM, in social phobic subjects relative to healthy control subjects (291). Both findings await replication.
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Serotonergic System Exposure to various stressors including restraint stress, tail shock, tail pinch, and high-level (but not low-level) foot shock results in increased 5-HT turnover in the mPFC, nucleus accumbens, amygdala, and lateral hypothalamus in experimental animals (285). During exposure to fear-conditioned stimuli, the 5-HT turnover in the mPFC appears particularly sensitive to the severity of stress, increasing as the aversiveness of the US and the magnitude of the conditioned fear behavioral response increases (285). However, exposure to repeated electric shocks sufficient to produce learned helplessness is associated with reduced in vivo release of 5-HT in the frontal cortex (292), a finding possibly reflecting a state in which 5-HT synthesis is outpaced by release. Preadministration of BZD-receptor agonists or tricyclic antidepressant drugs prevents stress-induced reductions in 5-HT release and interferes with the acquisition of learned helplessness, whereas infusion of 5-HT into the frontal cortex after stress exposure reverses learned-helplessness behavior (292,293). Finally, administration of 5-HTreceptor antagonists produces behavioral deficits resembling those of the learned helplessness seen after inescapable shock during animal stress models that do not ordinarily result in learned helplessness (293). The effect of stress in activating 5-HT turnover may stimulate both anxiogenic and anxiolytic pathways within the forebrain, depending on the region involved and the 5-HTreceptor subtype that is predominantly stimulated. For example, microinjection of 5-HT into the amygdala appears to enhance conditioned fear, whereas 5-HT injection into the PAG inhibits unconditioned fear (260). Graeff et al. hypothesized that the serotonergic innervation of the amygdala and the hippocampus mediates anxiogenic effects by 5-HT2A receptor stimulation (260), whereas serotonergic innervation of hippocampal 5-HT1A receptors suppresses formation of new CS-US associations and provides resilience to aversive events. Potentially compatible with this hypothesis, 5-HT1A receptor knockout mice exhibit behaviors consistent with increased anxiety and fear, and longterm administration of 5-HT1A receptor partial agonists exerts anxiolytic effects in generalized anxiety disorder (295). Notably, stress and glucocorticoids exert major effects on the genetic expression of 5-HT1A and 5-HT2A receptors. Postsynaptic 5-HT1A receptor gene expression is under tonic inhibition by adrenal steroids in the hippocampus and possibly other regions where mineralocorticoid receptors are expressed (reviewed in ref. 296). Thus, 5-HT1A receptor density and mRNA levels decrease in response to chronic stress or CORT administration and increase after adrenalectomy (296299). The stress-induced down-regulation of 5-HT1A receptor expression is prevented by adrenalectomy, a finding showing the importance of circulating adrenal steroids in mediating this effect (296). Although both
mineralocorticoid-receptor stimulation and glucocorticoidreceptor stimulation are involved in mediating this effect, the former is most potent, and 5-HT1A mRNA levels markedly decrease within hours of mineralocorticoid-receptor stimulation (296). Conversely, 5-HT2A receptor expression is up-regulated during chronic stress and CORT administration, and it is down-regulated in response to adrenalectomy (298,300). In view of evidence that 5-HT1A and 5-HT2A receptors may play reciprocal roles in mediating anxiety, it is conceivable that these corticosteroid mediated effects on 5-HT1A and 5-HT2A expression may be relevant to the pathophysiology of anxiety. Serotonergic Function in Anxiety Disorders The literature regarding serotonergic function in anxiety disorders is in disagreement (see Table 63.3). In PD, platelet 5-HT uptake has been reported to be abnormally elevated (301), normal (302), or abnormally reduced (303). Platelet imipramine binding (to a site related to the 5-HT transporter site), did not differ in PD relative to control samples (304,305). Another study reported reduced concentrations of circulating 5-HT in PD relative to control samples (306), although this finding has not been replicated. Pharmacologic challenge studies involving 5-HT have been similarly unable to establish a primary role for 5-HT in the pathophysiology in PD. Neuroendocrine responses to challenge with the 5-HT precursors, L-tryptophan and 5-hydroxytryptophan (5-HTP), did not differentiate PD study subjects from healthy controls (307,308). Moreover, tryptophan depletion did not prove anxiogenic in unmedicated PD study subjects (309). Nevertheless, challenge with the 5-HT releasing agent, fenfluramine, produced greater increases in anxiety, plasma prolactin, and cortisol in PD compared with control subjects (131,310). Fenfluramine challenge also resulted in reduced CBF in the left posterior parietal-superior temporal cortex in PD study subjects relative to healthy controls (131), although it was unclear whether this abnormality reflected an abnormality of serotonergic function or a physiologic correlate of fenfluramineinduced anxiety, because more PD study subjects (56%) developed panic attacks than did control subjects (11%). Preliminary data regarding the sensitivity of specific 5HTreceptor subtypes appear more promising, particularly because the elevation of plasma ACTH and cortisol and the hypothermic responses to the 5-HT1A partial agonist, ipsapirone, were blunted in PD relative to healthy control samples (311). Finally, increases in anxiety and plasma cortisol in PD relative to control samples have been reported after oral (312), but not intravenous, administration of the 5-HT2 receptor agonist, m-chloromethylpiperazine (mCPP) (313). Samples with combat-related PTSD have been shown to have decreased paroxetine binding in platelets relative to controls, a finding suggesting alterations in the 5-HT trans-
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porter (314). Southwick et al. observed that a subgroup (five of 14 subjects) with PTSD experienced panic anxiety and flashbacks after mCPP challenge (189). Thus, a subgroup of patients with PTSD may have abnormal sensitivity to serotonergic provocation. Cholecystokinin CCK is an anxiogenic neuropeptide present in both the brain and the gastrointestinal tract. CCK-containing neurons are found in high density in the cerebral cortex, amygdala, hippocampus, midbrain PAG, substantia nigra, and raphe. Iontophoretic administration of CCK has depolarizing effects on pyramidal neurons and stimulates action potential formation in the dentate gyrus of the hippocampus (reviewed in ref. 315). The CCK-receptor agonist, CCK-4, is anxiogenic in a variety of animal models of anxiety, whereas CCK-receptor antagonists exert anxiolytic effects in the same models (315). CCK has important functional interactions with other systems implicated in anxiety and fear (noradrenergic, dopaminergic, BZD). For example, the panicogenic effect of CCK-4 in PD is attenuated by administration of the adrenoreceptor antagonist, propranolol, and by long-term imipramine treatment, which down-regulates -adrenoreceptors (316). Study subjects with PD or PTSD are more sensitive to the anxiogenic effects of CCK-4 than are control subjects (317,318). For example, Strohle et al. found that of 24 PD study subjects tested, 15 experienced a panic attack after CCK-4 administration (319). Although the mechanism underlying the enhanced sensitivity to CCK-4 has not been elucidated, it is noteworthy that CSF concentrations of CCK are lower in PD study subjects than in healthy controls (320). The neuroendocrine effects associated with CCK-4 induced panic appear to differ between PD and PTSD. In PTSD, CCK-4induced panic was associated with a lower ACTH response in the PTSD study subjects than in healthy controls, and cortisol concentrations increased in both the PTSD and control groups (318). The elevation in the cortisol concentrations attenuated more rapidly in the PTSD group than in the control group. In contrast to the findings in PTSD, ACTH secretion was higher in subjects with PD who developed panic attacks in response to CCK-4 than in those who did not, although even the latter subjects showed brief, less pronounced increases in ACTH concentrations (319). Neither PD subgroup showed significant changes in the plasma cortisol concentration after CCK-4 administration. The elevation of ACTH concentrations suggested that CRH secretion increases in CCK-4induced panic in PD (consistent with preclinical evidence regarding the role of CRH in stress and anxiety and the interaction of CRH and CCK in modulating anxiety) (221).
The CCK receptors are classified into CCK-A and CCKB subtypes. Kennedy et al. reported a significant association between PD and a single nucleotide polymorphism found in the coding region of the CCK-Breceptor gene (321). In contrast, genetic polymorphisms for the CCK-Areceptor gene and the CCK-pre-pro hormone genes showed no association with PD (321). If confirmed by replication, these data would suggest that a CCK-Breceptor gene variation may be involved in the pathogenesis of PD. Pande et al. assessed the efficacy of the selective CCKBreceptor antagonist, CI-988, for preventing panic attacks in PD (322). No differences in the rate of panic attacks were seen between the active drug and placebo treatment groups. Nevertheless, because of the limited bioavailability of oral CI-988, studies involving this drug may not have sufficiently tested the hypothesis that CCK-Breceptor antagonism produces antipanic effects in PD.
Other Neuropeptides Opioid Peptides Acute, uncontrollable shock increases secretion of opiate peptides and decreases -opiatereceptor density (323, 324). The elevation of opioid peptide secretion may contribute to the analgesia observed after uncontrollable stress and exposure to fear-conditioned stimuli (325). This analgesic effect shows evidence of sensitization, because subsequent exposure to less intense shock in rats previously exposed to uncontrollable shock also results in analgesia (326). Potentially consistent with these data, Pitman et al. found that patients with PTSD showed reduced pain sensitivity compared with veterans without PTSD after exposure to a combat film (327), an effect that was reversed by the opiate antagonist naloxone (a finding suggesting mediation by endogenous opiate release during symptom provocation). In the baseline state, the CSF -endorphin levels were abnormally elevated in PTSD relative to control samples (328). However, Hoffman et al. found lower morning and evening plasma -endorphin levels in a PTSD group versus healthy control samples (329). Another study found no differences in plasma methionine-enkephalin concentrations between PTSD subjects and control subjects, although this compounds degradation half-life was higher in the PTSD group (330). During opiate administration, Bremner et al. reported that some patients with combat-related PTSD experience an attenuation of their hyperarousal symptoms (331). Because preclinical studies in experimental animals have shown that opiates potently suppress central and peripheral noradrenergic activity, these data appear compatible with the hypothesis that some PTSD symptoms are mediated by noradrenergic hyperactivity (discussed earlier). Conversely, during opiate withdrawal noradrenergic activity increases, and it
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TABLE 63.3. EVIDENCE OF ALTERATION IN OTHER NEUROTRANSMITTER SYSTEMS IN ANXIETY DISORDERS
PTSD Benzodiazepine Increased symptomatology with benzodiazepine antagonist Decreased number of benzodiazepine receptors using SPECT-iomazenil or PET-flumazenil binding Opiate Naloxone-reversible analgesia Reduced plasma -endorphin Elevated levels of CSF -endorphin Serotonin Decreased serotonin reuptake site binding in platelets Decreased serotonin transmitter in platelets Blunted endocrine response to 5-HT1A probe Altered serotonin effect on cAMP in platelets (5-HT1A probe) Increased anxiogenic responses to 5-HT agonists Thyroid Increased baseline indices of thyroid function Increased TSH response to TRH Somatostatin Increased somatostatin levels at baseline in CSF Cholecystokinin Increased anxiogenic responses to CCK agonists Panic Disorder ++ +++/
has been noted that some symptoms of PTSD resemble those of opiate withdrawal (170). Neuropeptide Y NPY administered in low doses intraventricularly attenuates experimentally induced anxiety in a variety of animal models (332). Consistent with these data, transgenic rats that overexpress hippocampal NPY show behavioral insensitivity to restraint stress and absent fear suppression of behavior in a punished drinking task (333). In healthy humans subjected to uncontrollable stress during military training exercises, plasma NPY levels increased to a greater extent in persons rated as having greater stress resilience (334). During stress exposure, the NPY plasma levels were positively correlated with plasma cortisol concentrations and behavioral performance, and they were negatively correlated with dissociative symptoms (334). In humans with PD, plasma NPY concentrations were abnormally elevated, and this finding, given NPYs putative anxiolytic effects, may reflect an adaptive response to anxiety symptoms (335). In contrast, patients with combat-related PTSD had lower plasma NPY concentrations both at baseline and in response to yohimbine challenge than healthy controls (336). In the PTSD group, the baseline NPY levels were inversely correlated with PTSD and panic symptoms and with yohimbine-induced increases in MHPG and systolic blood pressure (336). If this finding proves reproducible, it suggests that a deficit in endogenous NPY secretion may be involved in the generation of anxiety and sympathetic autonomic symptoms in PTSD. Thyrotropin-Releasing Hormone and the Thyroid Axis In the early twentieth century, Graves described cases in which thyroid hormone hypersecretion was associated with anxiety, palpitations, breathing difficulties, and rapid heart rate in persons recently exposed to traumatic stress. Nevertheless, systematic epidemiologic studies of the relationship between stress and thyroid disease have not been conducted. Although few studies have looked at thyroid function in anxiety disorders, Mason et al. found elevated levels of triiodothyronine in patients with combat-related PTSD (337) (Table 63.3), a finding consistent with evidence that stress results in long-lasting elevations of thyroid hormone secretion (338). Respiratory System Dysfunction in Panic Disorder Associations between respiratory perturbation and acute anxiety have been demonstrated in PD, in which various forms of respiratory stimulation consistently produce panic
+ + + ++ + + + + +
NS NS +/ +/ + NS +/
+++
, One or more studies did not support this finding (with no positive studies), or the majority of studies do not support this finding; +/, an equal number of studies support this finding and do not support this finding; +, at least one study supports this finding and no studies do not support the finding, or the majority of studies support the finding; ++, two or more studies support this finding, and no studies do not support the finding; +++, three or more studies support this finding, and no studies do not support the finding; +++/, three or more studies support this finding, and one study does not support the finding; cAMP, cyclic adenosine 3, 5-monophosphate; CCK, cholecystokinin; CSF, cerebrospinal fluid; NS, not studied; PTSD, posttraumatic stress disorder; SPECT, single photon emission computed tomography; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone.
anxiety and alterations in parameters of respiratory physiology (339342). The most straightforward forms of respiratory stimulation that produce panic anxiety produce elevations of carbon dioxide pressure (hypercapnia). Thus, panic attacks can be consistently induced in patients with PD by rebreathing air, inhaling 5% to 7% carbon dioxide in air (343,344), or inhaling a single deep breath of 35% carbon dioxide (345,346). Other panicogenic chemical challenges have also been hypothesized to induce anxiogenic effects
921
through respiratory stimulation (340,341,347). Although the panicogenic mechanism of intravenous administration of sodium lactate remains unclear, it may also involve respiratory stimulation (339,340). The evidence that respiratory parameters index risk for panic anxiety includes data showing the following: (a) asymptomatic adult relatives of patients with PD have abnormally increased sensitivity to respiratory stimulation by carbon dioxide inhalation; (b) among PD samples, stronger family loading for PD is found among persons with evidence of respiratory dysregulation; and (c) the respiratory indices associated with PD are heritable, a finding suggesting a shared genetic vulnerability for panic attacks and respiratory dysregulation (reviewed in Chapter 61). Nevertheless, these data partly depend on subjective ratings of dyspnea during stress or respiratory stimulation, and the mechanisms underlying this sensitivity remain unclear. One possibility is that this hypersensitivity reflects an overall sensitivity to somatic sensations, because high degrees of anxiety sensitivity are linked to future panic attacks (348). The associations between respiratory perturbation and acute anxiety are not specific to PD. Exaggerated sensitivity to respiratory perturbation has also been reported in anxiety-disordered patients with some simple phobias, limited symptom panic attacks, childhood separation anxiety disorder, or limited-symptom anxiety attacks and in nonpsychiatrically ill subjects with high ratings on anxiety sensitivity scales. (See Chapter 61) For example, children with separation anxiety disorder exhibit greater changes in somatic symptoms during carbon dioxide inhalation that positively correlate with increases in respiratory rate, tidal volume, minute ventilation, end-tidal carbon dioxide pressure, and irregularity in respiratory rate during room-air breathing (349). CONCLUDING REMARKS The inconsistency in the results of biological investigations of anxiety disorders highlights the importance of addressing the neurobiological heterogeneity inherent within criteriabased, psychiatric diagnoses. Understanding this heterogeneity will be facilitated by the continued development and application of genetic, neuroimaging, and neurochemical approaches that can refine anxiety disorder phenotypes and can elucidate the genotypes associated with these disorders. Application of these experimental approaches will also facilitate research aimed at elucidating the mechanisms of antianxiety therapies. The knowledge reviewed herein regarding the neurobiology of fear and anxiety already suggests themes along which the development of new therapeutic approaches can be organized. In general, anxiolytic treatments appear to inhibit neuronal activity in the structures mediating fear expression and behavioral sensitization and facilitate endog-
enous mechanisms for modulating the neural transmission of information about aversive stimuli and responses to such stimuli. Novel treatments being developed to exploit the former type of mechanisms include pharmacologic agents that selectively target subcortical and brainstem pathways supporting specific components of emotional expression (e.g., CRH-receptor antagonists). In contrast, nonpharmacologic treatments for anxiety may augment the brains systems for modulating anxiety responses, by facilitating the extinction of putative fear-conditioned responses or directing the reinterpretation of anxiety-related thoughts and somatic sensations (so they produce less subjective distress). Informed by increasingly detailed knowledge about the pathophysiology of specific anxiety disorders and the neural pathways involved in anxiety and fear processing, the development of therapeutic strategies that combine both types of approaches may ultimately provide the optimal means for reducing the morbidity of anxiety disorders.
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64
NEURAL CIRCUITRY OF ANXIETY AND STRESS DISORDERS
MICHAEL DAVIS
BRAIN SYSTEMS IN THE GENERATION OF FEAR AND ANXIETY Role of the Amygdala Many data now indicate that the amygdala, along with its many efferent connections, is critically involved in emotion. Although the amygdala complex is generally defined by several distinct groups of cells, including the lateral, basal, accessory basal central, medial, and cortical nuclei, new data indicate that it is more useful to think of the amygdala as the basolateral amygdala (Bla) and to think of its several target areas as parts of a broader network that subserve more specialized functions (Fig. 64.1). The Bla receives sensory information from the thalamus, cortex (169), and ventral hippocampus (54) and then activates or modulates synaptic transmission in target areas appropriate for the reinforcement signal with which the sensory information has been associated. This involves both positive and negative associations. However, because most of the literature on the amygdala has analyzed the role of the Bla and its adjacent target, the central nucleus of the amygdala (CeA), in aversive conditioning, this work serves as the main focus of this chapter. Brief summaries of the role of Bla outputs to other targets shown in Fig. 64.1 follow. Because the periaqueductal gray (PAG) has received considerable attention in the study of defensive behavior and the hippocampus in the study of contextual fear conditioning, these data are reviewed next. Finally, brain systems and neurotransmitters involved in the inhibition of fear are reviewed, given the clinical significance of this information. Basolateral Nucleus of the Amygdala to CeA or BNST Pathway as It Relates to Conditioned and Unconditioned Fear Figure 64.1 shows that the Bla projects directly to the CeA, as well as to a related structure, the lateral division of the
bed nucleus of the stria terminalis (BNST), to form part of the lateral extended amygdala (6). Figure 64.2 summarizes work done in many different laboratories indicating that the CeA and BNST have direct projections to various anatomic areas that may be expected to be involved in many of the symptoms of fear or anxiety (65). The CeA and BNST have been grouped together because fibers from the Bla that project to the BNST pass through the CeA and cells in the lateral division of the CeA project to the BNST. Thus, many effects previously attributed to the CeA may really depend on the BNST. Autonomic and Hormonal Measures Anatomically, the CeA and the BNST are well situated to mediate the various components of the fear response. Both structures send prominent projections to areas such as the lateral hypothalamus, which is involved in activation of the sympathetic autonomic nervous system seen during fear and anxiety (155). Direct projections to the dorsal motor nucleus of the vagus, nucleus of the solitary tract, and ventrolateral medulla may be involved in lateral extended amygdala modulation of heart rate and blood pressure, which are known to be regulated by these brainstem nuclei (222). Projections to the parabrachial nucleus may be involved in respiratory (as well as cardiovascular changes) during fear, because electrical stimulation and lesions of this nucleus are known to alter various measures of respiration. Indirect projections of the CeA to the paraventricular nucleus through the BNST and preoptic area may mediate the prominent neuroendocrine responses to fearful or stressful stimuli. Attention and Vigilance Projections from the lateral extended amygdala to the ventral tegmental area may mediate stress-induced increases in dopamine metabolites in the prefrontal cortex (101). Direct projections to the dendritic field of the locus ceruleus or indirect projections through the paragigantocellularis nucleus may mediate the increase in firing rates of cells in the locus ceruleus in the presence of a fearful stimulus. Direct
Michael Davis: Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia.
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FIGURE 64.1. Schematic diagram of the outputs of the basolateral amygdala to various target areas and how these connections may be involved in fear and anxiety.
projections to the lateral dorsal tegmental nucleus and parabrachial nuclei, which have cholinergic neurons that project to the thalamus, may mediate increases in synaptic transmission in thalamic sensory relay neurons during states of fear. This cholinergic activation, along with increases in thalamic transmission accompanying activation of the locus ceruleus, may thus lead to increased vigilance and superior signal detection in a state of fear or anxiety. As emphasized by Kapp et al. (141), in addition to its direct connections to the hypothalamus and brainstem, the CeA has the potential for indirect widespread effects on the cortex through its projections to cholinergic neurons that project to the cortex. The rapid development of conditioned bradycardia during pavlovian aversive conditioning, critically dependent on the amygdala, may reflect a general increase in attention. Motor Behavior Release of norepinephrine onto motor neurons by lateral extended amygdala activation of the locus ceruleus, or through projections to serotonin containing raphe neurons, could lead to enhanced motor performance during a state of fear, because both norepinephrine and serotonin facilitate excitation of motor neurons. Direct projections to the nu-
cleus reticularis pontis caudalis, as well as indirect projections to this nucleus through the central gray, probably are involved in fear potentiation of the startle reflex. Direct projections to the lateral tegmental field, including parts of the trigeminal and facial motor nuclei, may mediate some of the facial expressions of fear as well as potentiation of the eyeblink reflex. The lateral extended amygdala also projects to regions of the central gray that appear to be a critical part of a general defense system and that have been implicated in conditioned fear in certain behavioral tests including freezing, sonic and ultrasonic vocalization, and stress-induced hypalgesia (20,33,78,103,121,155). Elicitation of Fear Responses by Electrical or Chemical Stimulation of the Extended Amygdala Electrical stimulation or abnormal electrical activation of the amygdala (i.e., by temporal lobe seizures) can produce a complex pattern of behavioral and autonomic changes that, taken together, highly resembles a state of fear. Autonomic and Hormonal Measures As outlined by Gloor: The most common affect produced by temporal lobe epileptic discharge is fear. . . . It arises out
FIGURE 64.2. Schematic diagram of the outputs of the central nucleus of the amygdala and the lateral division of the bed nucleus of the stria terminalis to various target areas and how these connections may be related to specific aspects of fear and anxiety. BNST, bed nucleus of the stria terminalis; CER, conditioned emotional response; EEG, electroencephlographic; N, nucleus.
Chapter 64: Neural Circuitry of Anxiety and Stress Disorders
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of the blue. Ictal fear may range from mild anxiety to intense terror. It is frequently, but not invariably, associated with a rising epigastric sensation, palpitation, mydriasis, and pallor and may be associated with a fearful hallucination, a frightful memory flashback, or both (98). In humans, electrical stimulation of the amygdala elicits feelings of fear or anxiety as well as autonomic reactions indicative of fear (57,99). Although other emotional reactions occasionally are produced, the major reaction is one of fear or apprehension. Electrical stimulation of the CeA or chemical activation by the cholinergic agonist carbachol or the neurotransmitter glutamate produces prominent cardiovascular effects that depend on the species, site of stimulation, and state of the animal. CeA stimulation can also produce gastric ulceration and can increase gastric acid, and these features can be associated with chronic fear or anxiety. It can also alter respiration, a prominent symptom of fear, especially in panic disorder. Using very small infusion cannulas and very low doses, Sanders and Shekhar found increases in blood pressure and heart rate when the -aminobutyric acidA (GABAA) antagonist bicuculline was infused into the Bla but not the CeA (215). Local infusion of N-methyl-D-aspartate (NMDA) or AMPA into the basolateral nucleus also increased blood pressure and heart rate (230). Repeated infusion of initially subthreshold doses of bicuculline into the anterior basolateral nucleus led to a priming effect in which increases in heart rate and blood pressure were observed after three to five infusions (216). This change in threshold lasted at least 6 weeks and could not be ascribed to mechanical damage or generalized seizure activity based on EEG measurements. Similar changes in excitability were produced by repetitive infusion of very low doses of corticotropin-releasing hormone (CRH) or the related peptide, urocortin (210). Once primed, these animals exhibited behavioral and cardiovascular responses to intravenous sodium lactate, a panic-inducing treatment in certain types of psychiatric patients. In general, electrical stimulation of the amygdala causes an increase in plasma levels of corticosterone. The effect of electrical stimulation appears to depend on both norepinephrine and serotonin in the paraventricular nucleus. Depletion of these transmitters through local infusions of 6-hydroxydopamine or 5,7-DHT, or local infusion of the norepinephrine or serotonin antagonists prazosin or ketanserin, into the paraventricular nucleus attenuated the effects of electrical stimulation (80). Attention and Vigilance Studies in several species indicate that electrical stimulation of the CeA increases attention or processes associated with increased attention. For example, stimulation of sites in the CeA that produce bradycardia (142) also produce low-voltage fast EEG activity (140). In fact, an attention or orienting reflex was the most common response elicited by electrical
stimulation of the amygdala (16,241). These and other observations led Kapp et al. to hypothesize that the central nucleus and its associated structures function, at least in part, in the acquisition of an increased state of nonspecific attention or arousal manifested in a variety of CRs which function to enhance sensory processing. This mechanism is rapidly acquired, perhaps via an inherent plasticity within the nucleus and associated structures in situations of uncertainty but of potential import; for example, when a neutral stimulus (CS) precedes either a positive or negative reinforcing, unexpected event (US) (141). Electrical stimulation of the amygdala can also activate cholinergic cells that are involved in arousal-like effects depending on the state of sleep and perhaps the species. Motor Behavior Electrical or chemical stimulation of the CeA produces a reduction of prepotent, ongoing behavior, a critical component in several animal models such as freezing, the operant conflict test, the conditioned emotional response, and the social interaction test. Electrical stimulation of the amygdala also elicits jaw movements and activation of facial motoneurons, which may mimic components of the facial expressions seen during the fear reaction. These motor effects may be indicative of a more general effect of amygdala stimulation, namely, that of modulating brainstem reflexes such as the massenteric, baroreceptor nictitating membrane, eyeblink, and the startle reflex. Summary of the Effects of Stimulation of the Amygdala Viewed in this way, the pattern of behaviors seen during fear may result from activation of a single area of the brain (the extended amygdala), which then projects to various target areas that themselves are critical for each of the specific symptoms of fear (the expression of fear), as well as the experience of fear. Moreover, it must be assumed that all these connections are already formed in an adult organism, because electrical stimulation produces these effects in the absence of prior explicit fear conditioning. Thus, much of the complex behavioral pattern seen during a state of conditioned fear has already been hard wired during evolution. For a formerly neutral stimulus to produce the constellation of behavioral effects used to define a state of fear or anxiety, it is only necessary for that stimulus to activate the amygdala, which, in turn, will produce the complex pattern of behavioral changes by virtue of its innate connections to different brain target sites. Viewed in this way, plasticity during fear conditioning probably results from a change in synaptic inputs before or in the Bla (173,192,204), rather than from a change in its efferent target areas. The ability to produce long-term potentiation (LTP) in the Bla (55, 56,58,91,129,226) that can lead to an increase in responsiveness to a physiologic stimulus (203) and the finding
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TABLE 64.1. EFFECTS OF LESIONS OF THE AMYGDALA ON CONDITIONED FEAR

Method Aspiration (pre) Aspiration (pre) Electrolytic Ibotenic acid (pre) Ibotenic acid (Ce) Electrolytic (MG) Cooling (pre) Electrolytic (pre) NMDA (pre or post) Species Human Human Rabbit Rabbit Rat Cat Rat Rat Site AC AC Ce Ce Ce MG Ce L C Effect of Lesion Decrease galvanic skin response during classic fear conditioning unilateral lesions Decrease galvanic skin response during classic fear conditioning Decrease bradycardia to cue paired with shock Decrease bradycardia to cue paired with shock Decrease blood pressure rise to cue paired with shock (Ce, unilateral, MG, contralateral) Decrease bradycardia, respiratory increases and blood pressure changes to cue paired with shock Decrease blood pressure rise during classic conditioning Decrease secretion of corticosterone and defecation to cue paired with shock; decrease rise in dopamine, serotonin, or norepinephrine metabolites in prefrontal cortex to cue paired with shock Decrease freezing to cue paired with shock Decrease freezing to context paired with shock Decrease freezing to cue paired with shock Decrease freezing to cues paired with shock; decrease shock probe avoidance Decrease freezing to cue paired with shock (Ce, unilateral; MG, contralateral), some damage to L and Bla Reference 153 26 139 168 137 259 154,206 101
Electrolytic (pre) Ibotenic acid (pre) Electrolytic (pre) Radiofrequency (pre) Ibotenic acid (amygdala) M (medial geniculate) (pre) NMDA (pre or post)
Rat Rat Rat Rat Rat
Ce Ce and Bla L AC Ce, MG
157 118 154 30 137
Rat
Bla
NMDA (pre and post)
Rat
Bla
Lidocaine (pre or pretesting or both) Muscimol (pre or post) NMDA (pre or post) Ibotenic acid (pre) Electrolytic (pre) Quinolinic acid or 6-OHDA (pre) NMDA (pre) Ibotenic acid (pre) Quinolinic acid (pre) NMDA (pre or post) NMDA (post) Ibotenic acid (post) Ibotenic acid (pre)
Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat
Ce and Bla Bla Bla Ce not Bla Ce not Bla Bla AC Ce, not Bla Bla not Ce Bla Bla Ce Ce and Bla
Decrease acquisition of freezing to odor or context or expression of both when lesions made 1 or 15 days after conditioning Decrease acquisition or expression of freezing to context or cue paired with shock even when lesions made 1 month after training Decrease expression of freezing to context paired with shock; weaker effect when inactivation given before training Decrease freezing when given either before testing or training but not immediately after training Decrease freezing or high-frequency vocalizations to shock or cues paired with shock Anticonflict (licking), decrease effects of restraint stress on plus maze Anticonflict effect Decrease reduction in licking during conditioned emotional response test Decrease avoidance of water spout paired with shock but not with quinine Decrease disruption of bar pressing to cue paired with shock; could still avoid shock bar Decrease avoidance of shocked bar, no reduction of disruption of bar pressing to cue paired with shock Decrease expression or acquisition of fear-potentiated startle to visual conditioned stimulus Decrease expression of fear-potentiated startle even when lesions made 1 month after training Decrease expression of fear-potentiated startle to visual or auditory conditioned stimulus Decrease expression of hypoalgesia to context paired with shock (formalin test)
61
166
119 183 101 175 225,256 224 48 146 146 214 159 52 118
AC, amygdala complex; Bla, basolateral complex; central nucleus; MG, medial geniculate; post, posttraining; pre, pretraining.
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that local infusion of NMDA antagonists into the amygdala block the acquisition of fear conditioning (65) are consistent with this hypothesis.
Effects of Lesions of the Amygdala in Humans and Nonhuman Primates In nonhuman primates (160,184,205) and in humans (9, 117), cells have been found in the amygdala that respond selectively to faces or direction of gaze (42). In humans, removal of the amygdala has been associated with an impairment of memory for faces (4,138,240,257) and deficits in recognition of emotion in peoples faces and interpretation of gaze angle (41,50,257). In a very rare case involving bilateral calcification confined to the amygdala (Urbach-Wiethe disease), patient SM046 could not identify the emotion of fear in pictures of human faces and could not draw a fearful face, even though other emotions such as happy, sad, angry, and disgusted were identified and drawn within the normal range. The deficit in recognizing facial expressions of fear
Effects of Lesions of the Amygdala on Conditioned and Unconditioned Fear in Rodents and Other Species Many studies in rodents and other species indicate that lesions of the Bla or CeA block many different measures of conditioned fear, as well as unconditioned fear. Tables 64.1 and 64.2 show selected examples of such studies in animals, which have been extensively reviewed elsewhere (64,65). More recent studies in humans also point to the amygdala in fear and anxiety.
TABLE 64.2. EFFECTS OF LESIONS OF THE AMYGDALA ON UNCONDITIONED FEAR OR STRESS

Method Electrolytic Ibotenic acid Radiofrequency Electrolytic (pretraining but not posttraining) Radiofrequency Radiofrequency Electrolytic Ibotenic acid Electrolytic Electrolytic Electrolytic Electrolytic Electrolytic Electrolytic Radiofrequency Radiofrequency Species Rat Rat Rat Rat Site Ce Ce ** Ce Effect of Lesion Decrease secretion of ACTH to immobilization stress Decrease rise corticosterone to immobilization stress Decrease the compensatory hypersecretion of ACTH that normally occurs following adrenalectomy Decrease secretion of corticosterone and prolactin to shock; no effect on epinephrine, norepinephrine Reference 23,24 242 7,8 207
Rat Rat Rat Rat Rat Wild Rat Rat Many species Wild Rat Rat Rat Rat
Ce Ce Ce Ce Ce CO, Ce CO, Me Ce AC CO, Ce CO, Me Ce Ce Ce, L and Bla L or Ce Ce, not Bla Ce not Bla or Me Ce
Decrease ulceration produced by restraint Decrease ulceration produced by shock stress Decrease gastric ulcers to water restraint No effect on ulcers to water restraint Decrease noise-elicited hypertension Decrease emotionality in measured in terms of flight and defensive behaviors Increase the number of contacts a rat will make with a sedated cat General taming effect Decrease emotionality in measured in terms of flight and defensive behaviors Increase the number of contacts a rat will make with a sedated cat Decrease jump withdrawal sign in morphine dependent rats after intraperitoned naloxone Decrease analgesia produced by exposure to cat or shock (tail flick test) Decrease loud noise-induced hypoalgesia (tail fick test) Decrease analgesic effects of systemic flumazenil (hot plate test) Decrease morphine (low dose) induced antinociception (formalin or tail flick tests) Decrease morphine induced antinociception (tail flick test)
122,123 124 59 59 88 144,145 30 For review, see 100 144,145 30 51 85
Electrolytic Electrolytic NMDA
Rat Rat Rat
28 106 164,165
Lidocaine
Rat
165
AC, amygdala complex; Bla, basolateral complex; Ce, central nucleus; Me, medial nucleus.
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only seemed to occur after bilateral amygdala damage (3). This patient and two others also tended to view even the most threatening faces as trustworthy and approachable (2). A more detailed evaluation of patient SM046 showed that she correctly identified valence (e.g., pleasant versus unpleasant) in faces displaying happy, surprised, afraid, angry, disgusted, or sad emotion, but she was highly abnormal in rating the level of arousal to the afraid, angry, disgusted, and sad faces (1). Another patient (SP) with extensive bilateral amygdala damage also showed a major deficit in her ability to rate levels of fear in human faces, yet was perfectly normal in generating a fearful facial expression in comparison with neurologically normal subjects, based on the ratings of three judges (13). Patients with unilateral (153) or bilateral (26) lesions of the amygdala also have been reported to have deficits in classic fear conditioning using the galvanic skin response as a measure of fear. In monkeys, removal of the amygdala decreases reactivity to sensory stimuli measured with the galvanic skin response (17,18). Effects of Local Infusion of Drugs into the Amygdala on Measures of Fear and Anxiety If the amygdala is critically involved in fear and anxiety, then drugs that reduce fear or anxiety clinically may well act within the amygdala. It is also probable that certain neurotransmitters within the amygdala may be involved in fear and anxiety. In fact, many studies indicate that local infusions of GABA or GABA agonists, benzodiazepines, CRH antagonists, opiate agonists, neuropeptide Y, dopamine antagonists, or glutamate antagonists decrease measures of fear and anxiety in several animal species. Table 64.3 gives selected examples of some of these studies, which have been extensively reviewed (65). Conversely, local infusions of GABA antagonists, CRH or CRH analogues, vasopressin, thyrotropin-releasing hormone, opiate antagonists, cholecystokinin (CCK) or CCK analogues tend to have anxiogenic effects. Table 64.4 shows selected examples of such studies that also have been reviewed (65). In summary, connections between the Bla and the CeA or BNST are critically involved in various autonomic and motor responses seen during a state of fear or anxiety. However, it is also the case that connections between the basolateral nucleus and other target areas are involved in emotional behavior. Basolateral Nucleus of the Amygdala to Dorsal Striatum Pathway as It Relates to Avoidance or Escape from Aversive Events As emphasized by McGaugh, Packard, and others, the amygdala modulates memory in various tasks such as inhibitory avoidance and motor or spatial learning (49,170,171, 185,186). For example, posttraining intracaudate injections
of amphetamine enhanced memory in a visible platform water maze task but had no effect in the hidden platform, spatially guided task (185,186). Conversely, posttraining intrahippocampal infusion of amphetamine enhanced memory in the hidden platform water maze task but not in the visible platform task. However, posttraining intraamygdala injections of amphetamine enhanced memory in both water maze tasks (185,186). Moreover, preretention intrahippocampal lidocaine injections blocked expression of the memory-enhancing effects of posttraining intrahippocampal amphetamine injections in the hidden platform task, and preretention intracaudate lidocaine injections blocked expression of the memory-enhancing effects of posttraining intracaudate amphetamine injections in the visible platform task. However, preretention intraamygdala lidocaine injections did not block the memory-enhancing effect of posttraining intraamygdala amphetamine injections on either task. Finally, in the hidden platform task, posttraining intrahippocampal, but not intracaudate, lidocaine injections blocked the memory-enhancing effects of posttraining intraamygdala the visible platform task, posttraining intracaudate, but not intrahippocampal, lidocaine injections blocked the memoryenhancing effects of posttraining intraamygdala amphetamine. The findings indicate a double dissociation between the roles of the hippocampus and caudate-putamen in memory and suggest that the amygdala exerts a modulatory influence on both the hippocampal and caudate-putamen memory systems. Perhaps similarly, lesions of the CeA block freezing but not escape to a tone previously paired with shock, whereas lesions of the basal nucleus of the Bla have just the opposite effect (10). However, lesions of the lateral nucleus, which receive sensory information required by both measures, block both freezing and escape. Lesions of the Bla, but not the CeA, also block avoidance of a bar associated with shock (146). It is possible that basolateral outputs to the dorsal or the ventral striatum mediate the escape behavior, given the importance of the striatum in several measures of escape or avoidance learning. However, combined, unilateral lesions of each structure on opposite sides of the brain would be required to evaluate whether this results from serial transmission from the basolateral nucleus to the striatum. Basolateral Nucleus of the Amygdala to Hippocampus Pathway as It Relates to Avoidance or Escape from Aversive Events As mentioned earlier, posttraining intrahippocampal as well as intraamygdala injections of amphetamine selectively enhance memory in a hidden platform water maze task (185, 186). Posttraining infusion of norepinephrine into the basolateral nucleus enhanced retention in the hidden platform water maze task, whereas posttraining infusion of propranolol had the opposite effect (113). These results suggest that

TABLE 64.3. EFFECTS OF NEUROTRANSMITTER AGONISTS INFUSED INTO THE AMYGDALA ON FEAR AND ANXIETY
Substance GABA or chlordiazepoxide GABA or benzodiazepines Benzodiazepines Midazolam Diazepam Diazepam Muscimol Muscimol Species Rat Rat Rat Rat Rat Mice Rat Rat Site Ce Bla Ce Bla Ce or Bla AC Bla Bla Effect of Substance Infused Decrease stress-induced gastric ulcers Increase punished responding in operant conflict test (anticonflict effect) Increase punished responding in operant conflict test (anticonflict effect) More time on open arms in plus-maze, no effect on shock probe avoidance Decrease freezing to footshock More time in light side in light-dark box test (anxiolytic effect) Anxiolytic effect in the social interaction test; no effect in Ce Increase punished responding in operant conflict test (anticonflict effect); no effect in Ce Block noise-elicited increase in tryptophan hydroxylase in cortex Anxiolytic effect (plus maze) in socially defeated rat Anxiolytic effect in plus maze during ethanol withdrawal in ethanol-dependent rats; no effect in plus maze in nondependent rats Decrease behavioral effects of opiate withdrawal Anxiolytic effect in the plus maze in rats that previously experienced defeat stress Decrease duration of freezing to an initial shock treatment or to re-exposure to shock box 24 h later No effect on grooming and exploration activity under stress-free conditions Decrease stress-induced gastric ulcers, prevented by 6-OHDA or clozapine Block acquisition of conditioned bradycardia Anxiolytic effect in social interaction test Anxiolytic effect in social interaction test, blocked by Y-1 antagonist Anxiolytic effects in conflict test; NPY-Y2 agonist much less potent Decrease stress-induced bradycardia and immobility responses Decrease expression of fear-potentiated startle Decrease acquisition and expression of freezing to tone or context; not due to state-dependent learning Blocks expression of fear-potentiated startle (visual, auditory CS) Blocks expression of fear-potentiated startle (visual CS) Block facilitation of eyeblink conditioning by prior stress when given prior to stressor session Anxiolytic effect in social interaction test Decrease naloxone precipitated withdrawal signs in morphine-dependent rats
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Reference 232 105,126,189, 218,239 225,234 188 120,258 60 216 218
a-CRH a-CRH a-CRH
Rat Rat Rat
Ce Ce Ce
35 116 194
a-CRH CRH receptor antisense a-CRH
Rat Rat Rat
Ce Ce Ce
115 161 233
a-CRH Enkephalin analogue Opiate agonists Morphine Neuropeptide Y Neuropeptide Y1 agonist Oxytocin SCH 23390 SCH 23390
Rat Rat Rb Rat Rat Rat Rat Rat Rat
Ce Ce Ce Ce Bla, not Ce Ce Ce AC AC
247 195,196,198 89,90 83 213 114 209 151 107
CNQX NBQX AP5
Rat Rat Rat
Bla Bla or Ce Bla
149 243 227
AP5 or CNQX CNQX
Rat Rat
Bla Ce
212 235
AC, amygdala complex; Bla, basolateral complex; Ce, central nucleus.
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TABLE 64.4. EFFECTS OF NEUROTRANSMITTER ANTAGONISTS INFUSED INTO THE AMYGDALA ON FEAR AND ANXIETY
Substance Bicuculline, picrotoxin Bicuculline Species Rat Rat Site Bla Bla Effect of Substance Infused Anxiogenic effects in the social interaction test; repeated infusion led to sensitization Anxiogenic effects in social interaction, blocked by either NMDA or non-NMDA antagonists into the amygdala Increases in blood pressure, heart rate, and locomotor activity; bigger effect with repeated infusions Increases in blood pressure, heart rate; blocked by infusion of either NMDA or non-NMDA antagonists into the amygdala Increases in blood pressure, heart rate blocked by either NMDA or non-NMDA antagonists infused into Bla or the dorsomedial hypothalamus Increase heart rate; effect blocked by a-CRH into Ce Increase in blood pressure, heart rate, and plasma catecholamines After repeated subthreshold doses get increase in blood pressure to systemic lactate Increased grooming and exploration in animals tested under stress-free conditions (i.e., in the home cage) Increase defensive burying Anxiogenic effect in plus maze, sensitization with repeated subthreshold doses; now get behavioral and cardiovascular effects to systemic lactate Increased stress-induced bradycardia and immobility responses in rats bred for low rates of avoidance behavior but not the more aggressive rats that show high avoidance rates Bradycardia (low doses) or tachycardia and release of corticosterone (high dose); tachycardia blocked by oxytocin antagonist Immobility, seizures second infusion Immobility in rats bred for low rates of avoidance but not bred for high avoidance rates Increase stress-induced gastric ulcers Increase stress-induced gastric ulcers, blocked by muscarinic or benzodiazepine agonists Increase gastric contractility, blocked by vagotomy Produce gastric lesions and stimulated acid secretion No effect on gastric secretion, whereas large effect after infusion into dorsal vagal complex or nucleus ambiguus Increase stress-induced gastric ulcers Elicit certain signs of withdrawal (depending on site) in morphine-dependent rats (unilateral) Place aversion to context where injections given to morphine-dependent rats Weak withdrawal signs in morphine-dependent rats Facilitation of the startle reflex Anxiogenic effect in plus maze but not clear because significant decrease in overall activity Increase acoustic startle, blocked by CCK B antagonist that also blocked effect of pentagastrin (ICV) Reference 216 211
Bicuculline (un) Bicuculline (un)
Rat Rat
Bla not Ce Bla
215,216 211
Bicuculline NMDA, AMPA (un) CRH CRH, TRH, or CGRP Urocortin or CRH CRH
Rat
Bla
229,230
Rat Rat Rat Rat
Ce Ce Bla Ce, not Bla Ce Bla
248 43 210 247,250
CRH CRH or Urocortin Vasopressin
Rat Rat
249 210
Rat
Ce
209
Vasopressin
Rat
Ce
208
Vasopressin Vasopressin TRH TRH or physostigmine TRH analogue TRH TRH analogue
Rat Rat Rat Rat Rat Rat Rat
Ce Ce Ce Ce Ce Ce AC
251 209 196,198 197 182 125 136
Naloxone Naloxone Methyl naloxonium Methyl naloxonium Yohimbine CCK analogues Pentagastrin
Rat Rat Rat Rat Rat Rat Rat
Ce AC AC AC Ce AC AC
196,198 51 231 163 82 27 86
AC, amygdala complex; Bla, basolateral complex; Ce, central nucleus; un, unanesthetized.
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the amygdala exerts a modulatory influence on hippocampal-dependent memory systems, presumably by direct projections from the basolateral nucleus of the amygdala, perhaps by modulation of LTP in the hippocampus. Lesions (131), NMDA antagonists (132), or local anesthetics (134) infused into the Bla decrease LTP in the dentate gyrus of the hippocampus. Conversely, high-frequency stimulation of the amygdala facilitates induction of LTP in the dentate gyrus (130,133). However, combined, unilateral lesions of each structure on opposite sides of the brain would be required to evaluate whether this results from serial transmission from the basolateral nucleus to the hippocampus. Basolateral Nucleus of the Amygdala to Frontal Cortex Pathway as It Relates to Emotion Importance of the Bla in US Representation After pavlovian conditioning, presentation of a conditioned stimulus (CS) elicits some neural representation of the unconditioned stimulus (US) with which it was paired. For example, the sound of a refrigerator door opening or an electric can opener may bring the family cat into the kitchen in expectation of dinner. Several studies suggest that the Bla, perhaps by connections with cortical areas such as the perirhinal cortex (93), is critical for these US representations based on studies using a procedure called US devaluation. In these experiments, a neutral stimulus (e.g., a light) is first paired with food so a conditioned response can be measured. Some animals then have the food paired with something that makes them sick (US devaluation). After such treatment, these animals show a reduction in the conditioned response to the light compared with animals that did not experience US devaluation. This result suggests that, after conditioning, animals have a representation of the value of a reinforcement that is elicited by the cue paired with that US. When that representation is changed, then the behavior elicited by the cue also is changed in the same direction. Lesions of the basolateral, but not the CeA, block US devaluation (112). In a related paradigm, rats are trained to be fearful of a weak shock in the presence of a tone. When this is followed by presentation of a stronger shock, without further tone-shock pairing, more freezing occurs to the tone. Temporary inactivation of the Bla during this inflation procedure blocks this effect when testing subsequently occurs with a normal, unlesioned, amygdala (5). Second-order conditioning also depends on a US representation elicited by a CS. In this procedure, cue 1 is paired with a particular US (e.g., shock or food), and cue 2 is paired with cue 1. After such training, cue 2 elicits a similar behavior as that elicited by cue 1, depending on the US with which cue 1 was paired. Thus, it may elicit approach behavior if cue 1 was formerly paired with food and avoidance if cue 1 was paired with shock. This indicates that cue 1 elicits a representation of the US that then becomes associated with cue 2. Lesions of the Bla, but not the CeA,
block second-order conditioning (72,73,112), as do local infusions of NMDA antagonists into the Bla (92). Importance of the Bla Projection to the Frontal Cortex in Using US Representations to Guide Behavior Converging evidence now suggests that the connection between the Bla and the prefrontal cortex is critically involved in the way in which a US representation (e.g., very good, pretty good, very bad, pretty bad) guides approach or avoidance behavior. Patients with late- or early-onset lesions of the orbital regions of the prefrontal cortex fail to use important information to guide their actions and decision making (14,25,63). For example, on a gambling task, they choose high, immediate reward associated with long-term loss rather than low, immediate reward associated with positive long-term gains. They also show severe deficits in social behavior and make poor life decisions. Studies using single-unit recording techniques in rats indicate that cells in both the Bla and the orbitofrontal cortex fire differentially to an odor, depending on whether the odor predicts a positive (e.g., sucrose) or negative (e.g., quinine) US. These differential responses emerge before the development of consistent approach or avoidance behavior elicited by that odor (220). Many cells in the Bla reverse their firing pattern during reversal training (i.e., the cue that used to predict sucrose now predicts quinine and vice versa) (221), although this has not always been observed (217). In contrast, many fewer cells in the orbitofrontal cortex showed selectivity before the behavioral criterion was reached, and many fewer reversed their selectivity during reversal training (221). These investigators suggest that cells in the Bla encode the associative significance of cues, whereas cells in the orbitofrontal cortex are active when that information, relayed from the Bla, is required to guide choice behavior. Taken together, these data suggest that the connection between the Bla and the frontal cortex may be involved in determining choice behavior based on how an expected US is represented in memory. The necessity for communication between the amygdala and the frontal cortex was shown in monkeys using a disconnection approach in which the amygdala on one side of the brain and the frontal cortex on the other side were lesioned together (22). Because the reciprocal connections between the two structures are ipsilateral, this procedure completely eliminated activity of the network connections while preserving partial function of each structure. Using this approach in rhesus monkeys, Baxter et al. found a decrease in US devaluation after unilateral neurotoxic lesions of the basolateral nucleus in combination with unilateral aspiration of orbital prefrontal cortex (22). These monkeys continued to approach a food on which they had recently been satiated, whereas control monkeys consistently switched to the other food.
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Neuroimaging Studies of the Amygdala in Humans As reviewed by Whalen (244), neuroimaging studies in normal human subjects have shown activation of the amygdala by presentation of biologically relevant sensory stimuli that probably induce strong negative emotional states. For example, the functional magnetic resonance imaging (fMRI) signal intensity within the amygdala is greater when subjects view graphic photographs of negative material (e.g., mutilated human bodies) compared with when they view neutral pictures (135). Positron emission tomography metabolic activity within the amygdala increased to negative material presented by film clips (199), and the amount of amygdala activity during film clips predicted later recall (47). In addition, fMRI signal intensities in humans during classic fear conditioning increased in response to stimuli that predicted an aversive event (45,150,179). Amygdala activation also seems to be greater during presentations of fearful faces compared with neutral facial expressions (40,180), happy facial expressions (180,246), or when subjects looked at a fixation point on an otherwise blank screen (246). Whalen et al. used a backward masking technique in which very brief presentations of fearful and happy facial expressions (33 milliseconds) were followed immediately by presentations (167 milliseconds) of neutral faces (245). Most study subjects reported seeing neutral expressionless faces, but not any afraid or smiling faces. Nonetheless, the amygdala demonstrated greater fMRI signal intensity to masked fearful faces compared with masked happy faces. In addition, subjects reported that these masked stimuli did not induce any noticeable changes in their state of emotional arousal. As suggested by Whalen (244), this study offers preliminary support for the notion that the amygdala constantly monitors the environment for such signals. More than functioning primarily for the production of strong emotional states, the amygdala would be poised to modulate the moment-to-moment vigilance level of an organism. Role of the Periaqueductal (Central) Gray Outputs from the CeA to the ventral central gray appear to mediate several components of the fear response including freezing, conditioned analgesia, and fear-related vocalizations, but, surprisingly, maybe not cessation of operant behavior (11). More dorsal regions of the PAG play a role in active defensive responses (33), depending on whether the threat is distal or proximal (31,71). Fanselow (79), for example, showed that dorsal, but not ventral, PAG lesions eliminate activity bursts elicited by foot shock, whereas ventral, but not dorsal, PAG lesions diminish freezing responses elicited by cues previously paired with foot shock. Fanselow suggested that these stimuli (i.e., foot shock versus stimuli predicting foot shock) access different points on a preda-
tory imminence continuum in which proximal threats activate the dorsal PAG to generate active defensive behaviors. More distal threats activate the ventral PAG and generate passive or preparatory defensive behaviors such as freezing and analgesia. From a similar perspective, Deakin and Graeff et al. proposed that moderately threatening stimuli inhibit the dorsal PAG (66,104), but this inhibition is overcome with more extreme danger, thus allowing active defense or panic behaviors to emerge. Results from stimulation studies have suggested an anatomic division of function within PAG. In particular, Depaulis and colleagues showed that chemical or electrical stimulation of PAG regions lateral to the aqueduct produces active behaviors such as forward avoidance, defensive aggression, and cardiovascular activation (67,68), whereas stimulation of more ventral regions of the PAG elicits passive responses such as behavioral arrest and decreased cardiovascular output (21,69). Electrical stimulation of the dorsal PAG in humans produces a pattern of cardiovascular effects that resemble those seen during a natural panic attack, and patients often experience fear, anxiety, and the desire to terminate stimulation (162). Exposure of rats to a cat or high-frequency vocalizations of conspecifics that often signal a predator in the environment increases neuronal firing in the dorsal PAG inferred from an increase in the immediate early gene c-fos (162). Based on these and other data, several investigators have suggested that the dorsal PAG may be involved in panic attacks in humans, perhaps resulting from a dysregulation of various transmitters systems within this structure (162). The dorsal PAG has heavy innervation of the panicogenic peptide CCK, which has been shown to excite the majority of cells in this region. CCK antagonists functionally decrease the effects of electrical stimulation of the dorsal PAG, as does elevating serotonin, perhaps relevant to the use of serotonin reuptake inhibitors in the treatment of panic disorder. Whether these effects depend on connections between the amygdala and the PAG or whether they represent examples where the PAG can function autonomously remains to be determined. Role of the Hippocampus in Contextual Fear Conditioning Rats given cue-shock pairings learn to be afraid of the cue as well as the place where cue-shock pairings occurred (context conditioning). In 1992, two seminal articles reported that the hippocampus was necessary for context but not explicit cue conditioning (148,190). Both studies found that electrolytic lesions of the dorsal hippocampus blocked freezing in the presence of a fearful context but not in the presence of a cue paired with shock in that context. Kim and Fanselow found such effects when lesions were made shortly after training (1 or 7 days) but not 28 days later (148), whereas Phillips and LeDoux used pretraining lesions (190).
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Although the role of the hippocampus in contextual fear conditioning had been discovered earlier using a place aversion measure (223), these articles were more influential because they integrated the well-known role of the hippocampus in spatial learning with a simple, yet powerful measure of classic fear conditioning. Contextual freezing was quickly adopted by investigators interested in the role of hippocampal LTP in learning because contextual fear conditioning was rapid and long lasting, like LTP, and it was easy to measure without complex or expensive equipment. The idea was that the hippocampus was required to form a representation of the context and that this representation was then associated with shock, perhaps in the amygdala. The hippocampus was not needed when an explicit cue, such as a tone, was used because this could be relayed directly to the amygdala without having to be processed by the hippocampus. As attractive as this hypothesis is, there are problems with concluding that the hippocampus is involved in all forms of contextual conditioning (96,97,202). Hippocampal lesions often produce substantial behavioral activation, which may interact with the expression of freezing and lead to a disruption of the freezing response itself, rather than of contextual fear. In fact, hippocampal lesions disrupt not only conditioned freezing responses, but also unconditioned freezing responses, such as the response elicited by a rat when confronted by a cat (32,34,147). The finding that hippocampus lesions did not block freezing to an explicit cue makes this competing response interpretation more difficult to accept, but some studies have found that hippocampal lesions disrupt freezing to an explicit cue (167). However, increases in activity cannot account for disruption of contextual freezing by hippocampal lesions in all instances. In an elegantly designed study, Anagnostaras et al. showed that hippocampal lesions disrupted freezing to a context that had been paired with shock shortly before surgery (12). In the same subjects, however, freezing to a second context, that had been paired with shock 28 days preoperatively, was not impaired. Thus, the freezing deficit to the recently conditioned context could not have resulted from an inability to freeze. Although pretraining electrolytic lesions of the dorsal hippocampus (167) block contextual fear conditioning, neurotoxic lesions fail to do so (97,167,202), as does local infusion of muscimol (128). To explain this difference, Maren et al. suggested that rats with damage to cells in the hippocampus pick out salient explicit cues in the context and use these as elemental cues for fear conditioning (167). However, these investigators suggested that rats with electrolytic lesions do not do this because the lesion disrupts fibers that connect the ventral subiculum to the nucleus accumbens, which decreases exploration and thus sampling of the context to pick out salient explicit cues to associate with shock. In fact, experiments found a blockade of the acquisition but not the expression of contextual fear conditioning measured by freezing using infusion into the nucleus
accumbens of a local anesthetic (108). This effect did not occur using tone-shock pairings, even using a weaker trace conditioning procedure that produced relatively low levels of freezing to the tone. Another possibility is that fibers from the dorsal to the ventral hippocampus are important in these anterograde amnestic effects of electrolytic lesions of the dorsal hippocampus because neurotoxic lesions of either the entire hippocampus (102,202) or just the ventral hippocampus blocked contextual freezing, whereas neurotoxic lesions of the dorsal hippocampus again failed to block contextual conditioning (202). However, in contrast to the hypothesis that contextual fear conditioning involves processes similar to spatial learning, lesions of the ventral hippocampus did not block but instead actually facilitated spatial learning in a water maze task (202). As these investigators concluded, these data directly contradict the widely held notion that spatial and contextual forms of learning are essentially different manifestations of the same basic underlying process (202). Because these lesions also impaired freezing to a tone, these authors suggested that the ventral lesions disrupted freezing by increasing activity. Because all these studies have relied on freezing as the measure of conditioned fear, it is important to assess the effects of hippocampal lesions on other behavioral or autonomic responses associated with fear. If hippocampal lesions disrupted multiple measures of contextual fear, it would provide further support for the hippocampal theory of context conditioning. However, posttraining hippocampal lesions were found not to disrupt context-specific potentiated startle, even though context-elicited freezing was disrupted in the same animals (174). This could not be explained by an excitatory effect of hippocampal lesions on startle amplitude itself (96). In contrast, lesions of the CeA completely blocked both freezing and startle. In another experimental design (95), lesions of the dorsal hippocampus failed to block a phenomenon called contextual blocking, whereby prior contextual fear conditioning retards subsequent cue conditioning. However, as in other studies, freezing to the fearful context was blocked by hippocampal lesions. These data, along with several other reports in the literature (96,97,202), severely limit the general impression that the hippocampus is required for contextual fear conditioning. However, it does seem to be involved in certain situations, so further work is needed to predict those occasions in which it is and is not involved. BRAIN SYSTEMS IN THE INHIBITION OR SUPPRESSION OF FEAR AND ANXIETY Extinction The inability to suppress unwanted fear memories or irrational worry is a major problem in many psychiatric disorders, yet very little is known about brain systems involved
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in the inhibition of fear. One way to study this important problem is to analyze brain systems involved in extinction, defined as a reduction in conditioned fear when the CS is presented many times in the absence of the US. Although such a procedure can decrease the conditioned response, this does not result from an erasure of the original fear memory. Instead, something new is learned that overcomes or competes with the original fear memory. For example, an extinguished conditioned response can return with the passage of time (spontaneous recovery, 187), after a subsequent stressor (reinstatement, 201), or when testing occurs in a different context (renewal, 36). Such results indicate that extinction (but see discussion in ref. 74) may involve a form of active inhibition that is fragile compared with conditioned fear itself.
Brain Areas in Extinction or Conditioned Inhibition Sensory Cortex Assuming that extinction results from active inhibition (see earlier), one could expect that lesions of various brain areas would disrupt either the development or expression of extinction. LeDoux, Romanski, and Xagoraris reported that rats given ablations of visual cortex before lightfoot shock pairings failed to show extinction of lick suppression relative to sham controls over days (156). In a similar study employing heart rate conditioning in the rabbit, Teich et al. showed that although bilateral lesions of either auditory or visual cortex did not disrupt acquisition of fear conditioning to a tone CS, auditory cortex lesions, but not visual cortex lesions, blocked extinction of conditioned heart rate responses to the tone (237). Based on known anatomic connections between sensory cortex and thalamic structures, the authors of both experiments concluded that, during extinction, sensory cortices exert a modality specific inhibition of the thalamic structures important for the performance of conditioned responses. However, my colleague and I found no effect of complete visual cortex lesions on extinction of fear-potentiated startle using a visual CS when the lesions were made either before light-shock pairings or after light-shock pairings and extinction (77). Although there were procedural differences between these reports, the conclusion that sensory cortex is universally involved in extinction of conditioned fear is not supported. Frontal Cortex Rats with lesions of the ventral medial prefrontal cortex made before fear conditioning required more days to reach an extinction criterion using an auditory CS and freezing as the measure of fear (178). However, in these same animals, extinction of conditioned fear to contextual cues was not impaired. In an extensive series of experiments, my colleagues and I found normal rates of extinction to both explicit and contextual cues after total removal of the ventral medial prefrontal cortex using both freezing and fear-potentiated startle as measures of conditioned fear (94). Because the lesions in the study by Morgan et al. were performed before fear conditioning (178), the apparent blockade of extinction after ventral medial prefrontal cortex lesions may have resulted from an increase in the strength of original fear conditioning, rather than from interfering with the process of extinction. Although the lesions and shams groups did not differ significantly in their level of freezing before the extinction sessions, freezing to explicit cues often becomes maximal after a very few training trials, so ceiling effects may well have been operating. Because extinction rate can be a more sensitive index of the strength of original
Conditioned Inhibition In a conditioned inhibition procedure, cue 1 predicts food or shock, and a compound stimulus (cue 1 plus cue 2) predicts the absence of these USs. There is general agreement that conditioned inhibition, closely related to extinction, does involve active inhibition. In fact, it has been argued that extinction is a special case of conditioned inhibition (38). The summation test is the basic method for observing conditioned inhibition (200). In this procedure, the putative conditioned inhibitor (e.g., a light) is presented in compound with an excitatory CS (e.g., a tone). If the combination produces a decrease in the conditioned response below the level observed when the CS is presented alone, then that stimulus is said to act as a conditioned inhibitor. When the conditioned inhibitor is removed, excitation returns to its original level. Various control procedures indicate that a stimulus trained in this way is in fact acting by inhibition. Because psychotherapy often involves procedures to rid patients of unwanted fear memories, a behavioral analysis of extinction or conditioned inhibition has certain clinical implications, as suggested by Bouton and Swartzentruber (39). As they pointed out, performance after extinction is inherently unstable (39). Phenomena such as spontaneous recovery and reinstatement may explain why conditioned fears and phobias in humans sometimes seem to return spontaneously without any obvious cause. The renewal effect may explain why fears reduced successfully in the therapists office reappear when the patient returns home or to work. If a drug is used as an adjunct to therapy, renewal of fear could occur when the fearful stimulus is encountered in the absence of the drug. In fact, animal experiments show that when benzodiazepines are given during extinction, fear of the CS returns when testing occurs in the absence of the drug (37).
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conditioning than the terminal level of performance before the initiation of extinction (15), the slower rate of extinction in the lesioned animals may have reflected a stronger degree of original learning. Although these authors do not believe their effects can be explained in this way (177), the finding that the lesions had no effect on the rate of extinction of context conditioning, which clearly was not at the ceiling of the freezing scale, is consistent with this interpretation. Similarly, we did not find any effect of pretraining ventral prefrontal cortex lesions on extinction of contextual fearpotentiated startle or freezing (94). In addition, we did not find any effect of ventral medial prefrontal cortex lesions on extinction when lesions were made after fear conditioning but before extinction (94). Morgan and LeDoux also found no effect on the rate of extinction when ventral prefrontal cortex lesions were made after fear conditioning, but before extinction (176). If the frontal cortex is required for the development of extinction or for the inhibition of fear after extinction, one would expect lesions to block the development of extinction, irrespective of whether the lesions were made before or after the initial phase of fear conditioning. Similarly complex effects on extinction have been reported regarding depletion of dopamine in the prefrontal cortex (181). Preconditioning lesions of dopamine terminals in the medial prefrontal cortex retarded the rate of extinction when a 0.8-mA shock was used but not when a 0.4-mA shock was used. Inspection of the results strongly suggests that the 0.8-mA group was at the ceiling of the measurement scale at the beginning of the extinction session, whereas the 0.4-mA group was not. Conversely, 6-hydroxydopamine lesions of the frontal cortex substantially retarded extinction after 0.8-mA tone-shock pairings, even when the lesions were made after training (181). Thus, it is possible that dopamine levels in the prefrontal cortex are important for extinction when conditioning has produced high, but not more moderate, levels of fear, although further studies using posttraining lesions are required to verify this. Quirk et al. found that pretraining lesions of the ventral medial prefrontal cortex did not block the development of conditioned freezing or the rate of within session extinction (191). However, the lesioned rats showed much more spontaneous recovery measured 24 hours later. Similar results were found in rats given systemic administration of an NMDA antagonist (193). In contrast, we found no change in the rate or final level of extinction, measured with fearpotentiated startle and freezing, when extinction was assessed over 18 daily sessions using a small number of CS presentations each day (94). There also were no differences in the degree of spontaneous recovery measured 5 days later or in shock-induced reinstatement measured 24 hours after a single foot shock. Hence, the findings of Quirk et al. may depend critically on the use of a relatively small amount extinction training (191,193). Moreover, their lesions were
generally more ventral than ours, and this may have contributed to the differences. Clearly, more work needs to be done to examine the limits of the role of the prefrontal cortex in extinction of conditioned fear, given the clinical importance of these data. Hippocampus Although a complete review of the hippocampal literature on extinction is beyond the scope of this chapter, this brain area has received a great deal of experimental attention and was once widely believed to be involved in extinction. Theories of extinction confront the problem of designing a mechanism capable of discriminating occasions of reinforcement from nonreinforcement. Douglas suggested that the hippocampus is a nonreinforcement detector providing the organism with the means to tune out information that is of no motivational consequence (70). It is possible that the hippocampus recognizes that the CS is no longer followed by the US and inhibits relevant sensory or conditioned response production centers. Various conditioning paradigms have been used to assess the role of the hippocampus in extinction, including the rabbit nictitating membrane response (29,219,228), conditioned heart rate (44), and conditioned suppression (152). Although some of these experiments have found that hippocampal lesions attenuate extinction (219), others have found no effect (228), and still another has shown facilitated extinction (152). Because extinction is context specific (see earlier), one could expect that lesions of the hippocampus would disrupt this contextual control of extinction. However, direct tests of this hypothesis have not found a disruption of context specific extinction using pretraining lesions. Hence, neither fimbria-fornix lesions (252) nor excitotoxic lesions of the entire hippocampus (87) had any effect on the rate of extinction or on renewal of conditioned fear, although both types of lesions disrupted reinstatement. In contrast, large hippocampal lesions were not found to disrupt reinstatement of appetitively conditioned behavior (84). Overall, therefore, the role of the hippocampus in extinction remains uncertain. Neurotransmitters in Extinction Role of NMDA Receptors in Extinction Local infusion of NMDA antagonists into the amygdala blocked the development of extinction measured with fearpotentiated startle (76). Intraamygdala infusion of AP5 also blocked extinction using an auditory CS and freezing as a measure of conditioned fear (158). Perhaps similarly, systemic administration of the NMDA antagonist MK-801 fully blocked extinction of conditioned analgesia, a reliable
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measure of conditioned fear (62), and this effect could not be explained by state-dependent context extinction. A similar blockade of extinction was reported using a lick-suppression paradigm (19), as well as extinction of the rabbit nictitating membrane preparation (143). Taken together, these data indicate that NMDA antagonists can block the development of extinction measured on subsequent test sessions. This may even occur under conditions in which the antagonist does not block the development of short-term extinction. Thus, systemic injection of the NMDA antagonist CPP before extinction blocked the expression of conditioned freezing by about 40% but did not block the development of extinction. However, the CPP group showed substantial recovery of conditioned freezing measured 24 hours later, a finding suggesting that CPP blocked the long-term development of extinction. (193). Interestingly, these investigators found a similar effect with preconditioning lesions of the ventral prefrontal cortex (191), although the connection between these two sets of data remains to be made. Role of GABA in Extinction Several studies have suggested that GABA agonists given before nonreinforced CS presentations interfere with the development of extinction (37,74). However, many of these effects may be attributable to state-dependent learning rather than to a blockade of learning during nonreinforced CS exposure. For example, Bouton, using lick suppression as a measure of fear, showed a blockade of extinction when rats were given chordiazepoxide during nonreinforced CS presentations and were then tested in the absence of the drug (37). However, when chordiazepoxide also was given before testing, extinction was still evident. This finding suggests that the benzodiazepine did not actually block the learning that was occurring during extinction, but, instead, the change in drug state between extinction and testing was the factor that produced renewal (36). Interestingly, a series of experiments by Harris and Westbrook found similar effects on excitatory conditioning. For example, rats given fear conditioning after injection with benzodiazepines showed an impairment in conditioned freezing measured 24 hours later in the same context compared with rats trained under the drug but tested in a different context (111) or rats given a stressor before testing (109). Thus, the benzodiazepines did not actually prevent original learning, but instead produced a state during conditioning that interfered with retrieval during testing. Hence, it would seem that GABA agonists do not directly interfere with either excitatory or inhibitory learning, but, instead, act on processes that are important for retrieval of prior learning. However, if extinction is a form of active inhibition, it is possible that GABA may be one of the neurotransmitters necessary for the expression of extinction. In fact, in an elegant set of experiments, Harris and Westbrook provided evidence that extinction is mediated by
GABA release (110). Pretraining or pretesting administration of the inverse agonist FG 7142, which decreases GABA transmission, blocked both development and expression of extinction to an auditory CS paired with foot shock, using freezing as a measure. This effect could not be ascribed to state dependency or to a ceiling effect. Pretest administration of FG 7142 reinstated freezing when assessed in the context where extinction took place, but not in a novel context, which itself reinstated freezing, and the two effects were not additive statistically. However, the disruption of extinction by FG 7142 was not complete, a finding leaving open the possibility that other mechanisms and neurotransmitters also may be involved. Role of Adrenocorticotropic Hormone and Vasopressin in Extinction Work by DeWied, Van Wiersima, Izquierdo, and Richardson and their co-workers indicated that administration of various peptides such as adrenocorticotropic hormone or vasopressin either before or after extinction training attenuates subsequent extinction performance (for review see ref. 74). Neural Systems in Conditioned Inhibition Using fluorodeoxyglucose autoradiography to measure neural activity, Mcintosh and Gonzalez-Lima compared regionspecific activity in parallel auditory pathways in two groups presented with a tone-light compound (172). In both groups, the tone was a fear-eliciting CS. The groups differed with respect to the significance of the light, which had been trained as a conditioned inhibitor in one group and as a neutral stimulus in the other. Thus, the experiment allowed for an analysis of whether a conditioned inhibitor modulates activity in sensory areas normally activated by an auditory fear-eliciting CS. Interestingly, in only one area, the ventral medial geniculate nucleus, was there a significant difference in activation between the two groups. This structure showed less activation in the conditioned inhibition group, a finding suggesting that a conditioned inhibitor may act at this locus in the auditory pathway to inhibit conditioned fear normally produced by an auditory CS. We found normal conditioned inhibition of fear-potentiated startle, using a visual excitatory stimulus and an auditory conditioned inhibitor after lesions of either the medial prefrontal cortex (94) or the nucleus accumbens (75). In addition, local infusion into the nucleus accumbens of either amphetamine or glutamate antagonists did not alter the magnitude of conditioned inhibition, as they alter responding to conditioned reinforcers trained in an operant situation (46,236). In an appetitive learning situation, Holland et al. reported that lesions of the hippocampus appeared to block
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feature negative conditional discrimination (127), a phenomenon closely related to conditioned inhibition. Several studies suggested that the lateral septum may play an important role in conditioned inhibition. Using pavlovian discriminative fear conditioning, single-unit firing rates in the dorsal lateral septal nucleus increased in the presence of a conditioned inhibitor and decreased in the presence of a conditioned excitor (238,254). This finding was not seen when recordings were made in the medial septal nucleus (253). More recently, Yadin and Thomas reported that stimulation of the same area of dorsolateral septal nucleus inhibited restraint stress-induced ulcers (255). Using c-fos mRNA as a measured of neuronal activation, we found a unique increase in c-fos in a ventral part of the lateral septum, the so-called septohypothalamic nucleus, when a conditioned inhibitor of fear was presented (53). Curiously, however, lesions of the lateral septal nucleus did not block the expression of conditioned inhibition in preliminary pilot studies, although further work certainly is required to evaluate the role of the lateral septum, perhaps using acute inactivation techniques rather than lesions. One study suggests that the dorsal central gray may play an important role in conditioned inhibition of fear. Fendt reported that posttest infusions of 5 ng of picrotoxin (a GABA chloride channel blocker) into the dorsal central gray, but not the lateral or ventrolateral central gray, reduced the expression of conditioned inhibition without affecting the expression of conditioned fear (81). Although this result is complicated by the finding that neither 2.5-ng doses nor 10-ng doses affected conditioned inhibition, it raises the intriguing possibility that a conditioned inhibitor of fear releases GABA into the dorsal central gray. Alternatively, because low doses of picrotoxin would be expected to activate the dorsal central gray by removing tonic inhibition, these results could be interpreted as indicating that the dorsal central gray is involved in inhibiting an unknown brain structure mediating conditioned inhibition (81). Given the prominent role of the central gray in the expression of fear (162), more work is needed to investigate the role of the dorsal central gray in conditioned inhibition of fear. REFERENCES
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STRUCTURAL AND FUNCTIONAL IMAGING OF ANXIETY AND STRESS DISORDERS
SCOTT L. RAUCH LISA M. SHIN
GENERAL PRINCIPLES Neuroimaging research has emerged as a powerful force in shaping neurobiological models of psychiatric disorders. In this chapter, neuroimaging findings pertaining to anxiety and stress disorders are reviewed. This review necessarily extends previous ones that we have written, together with our colleagues, on this same and related topics (97,98,103). Anxiety and Stress Disorders Whereas anxiety disorders comprise a discrete category within the current version of the Diagnostic and Statistical Manual (2), the concept of stress disorders is less well defined. Although growing evidence suggests that stress may play a role across a variety of psychiatric disorders (such as major depression), posttraumatic stress disorder (PTSD) and acute stress disorder remain the only conditions for which exposure to traumatic stress is explicitly acknowledged as an etiologic factor and a criterion for diagnosis. In this chapter, we survey the imaging data pertinent to models of PTSD and other anxiety disorders, including obsessive-compulsive disorder (OCD), specific phobias (SpP), social phobia (SoP; also called social anxiety disorder) and panic disorder (PD). In general, patients with anxiety disorders suffer either exaggerated fear responses to relatively innocuous stimuli (e.g., phobias) or spontaneous fear responses in the absence of true threat (e.g., PD). Thus, it is important to consider the mediating neuroanatomy of normal threat assessment
and the fear response. In fact, contemporary models focus on these systems as candidate neural substrates for the anxiety disorders. Relevant Neuroanatomy The limbic system plays an important role in mediating human emotional states, including anxiety. Anterior paralimbic cortex (i.e., posterior medial orbitofrontal, anterior temporal, anterior cingulate, and insular cortex) links cortical regions subserving higher level cognition and sensory processing with deep limbic structures, such as the amygdala and the hippocampus (81). Modern models of threat assessment and the normal fear response have focused on the role of the amygdala (1,75). The amygdala is positioned to receive input regarding the environment both directly and, thus, rapidly from the thalamus as well as from sensory cortex. The amygdala appears to serve several related functions, including preliminary threat assessment; facilitation of fight-or-flight responses; facilitation of additional information acquisition; and enhancement of arousal and plasticity, so the organism can learn from the current experience to guide responses optimally in future similar situations. Conversely, several brain areas provide important feedback to the amygdala (1,75): medial frontal cortex (e.g., anterior cingulate and orbitofrontal cortex) may provide critical top-down governance over the amygdala, thus enabling attenuation of the fear response once danger has passed or when the meaning of a potentially threatening stimulus has changed; the hippocampus provides information about the context of a situation (based on information retrieved from explicit memory stores); and corticostriatothalamic circuits mediate gating at the level of the thalamus and thereby regulate the flow of incoming information that reaches the amygdala. Finally, neuromodulators influence the activity within each of these various brain areas, as well as the interactions
Scott L. Rauch: Department of Psychiatry, Harvard Medical School; Departments of Psychiatry and Radiology, Massachusetts General Hospital, Boston, Massachusetts. Lisa M. Shin: Department of Psychiatry, Harvard Medical School; Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts; Department of Psychology, Tufts University, Medford, Massachusetts.
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among the nodes of the entire system outlined earlier. Ascending projections from the raphe nuclei (serotonin) and the locus ceruleus (norepinephrine), as well as widespread local -aminobutyric acidergic (GABAergic) neurons, are perhaps most relevant to the physiology of anxiety (30,65, 114). These transmitter systems likely serve as the principal substrates for contemporary anxiolytic medications, including serotonergic reuptake inhibitors, monoamine oxidase inhibitors, other antidepressant medications, and benzodiazepines. Neuroimaging Methods Morphometric magnetic resonance imaging (mMRI) methods can be used to test hypotheses regarding abnormalities in the size or shape of particular brain structures. Functional imaging methods include positron emission tomography (PET) with tracers that measure blood flow (e.g., oxygen15labeled carbon dioxide) or glucose metabolism (i.e., fluorine-18labeled fluorodeoxyglucose [FDG]), single photon emission tomography (SPECT) with tracers that measure correlates of blood flow (e.g., technetium-99 labeled hexamethylpropylene amine oxime [TcHMPAO]), and functional MRI (fMRI) to measure blood oxygenation leveldependent (BOLD) signal changes. Each of these techniques yields maps that reflect regional brain activity. Functional imaging methods can be applied in the context of various experimental paradigms. In neutral state paradigms, subjects are studied during a nominal resting state or while they perform a nonspecific continuous task. Thus, between-group comparisons are made to test hypotheses regarding group differences in regional brain activity, without particular attention to state variables. In treatment paradigms, subjects are scanned in the context of a treatment protocol. In pre/posttreatment studies, subjects are scanned both before and after a trial. Then, within-group comparisons are made to test hypotheses regarding changes in brain activity profiles associated with symptomatic improvement. Alternatively, correlational analyses can be performed to identify pretreatment brain activity characteristics that predict good or poor treatment response. In symptom provocation paradigms, subjects are scanned during a symptomatic state (after having their symptoms intentionally induced) as well as during control conditions. Within-group comparisons can be made to test hypotheses regarding the mediating anatomy of the symptomatic state; group-by-condition interactions can be sought to distinguish responses in patient versus control groups. Behavioral and pharmacologic challenges can be used to induce symptoms. In some cases, when symptomatic states occur spontaneously, experiments are designed to capture these events without the need for provocation or induction per se. In cognitive activation paradigms, subjects are studied while they perform specially designed cognitive-behavioral tasks. This approach is intended to increase sensitivity by employing tasks that specifically
activate brain systems of interest. Again, group-by-condition interactions are sought to test the functional responsivity or integrity of specific brain systems in patients versus control subjects. Imaging studies of neurochemistry have employed PET and SPECT methods in conjunction with radiolabeled high-affinity ligands. In this way, regional receptor number and or affinity can be characterized in vivo (i.e., receptorcharacterization studies). Other approaches include the use of magnetic resonance spectroscopy (MRS) to measure the regional relative concentration of select MRS-visible compounds. For instance, MRS can be used to measure the compound N-acetylaspartate (NAA), which is a purported marker of healthy neuronal density. These various neuroimaging techniques should be viewed as complementary. Convergent findings across paradigms and laboratories yield the most cohesive and compelling models of pathophysiology.
RELEVANT NEUROIMAGING FINDINGS IN HEALTHY SUBJECTS Anxiety and Other Negative Emotional States Behaviorally Induced Fear and Anxiety Fischer and colleagues used PET to study regional cerebral blood flow (rCBF) in bank officials while they viewed security camera videotape of a robbery that they had experienced previously (46). Watching the robbery video was associated with rCBF increases in orbitofrontal cortex, visual cortex, and posterior cingulate gyrus; rCBF decreases occurred in Brocas area, left angular gyrus, operculum, and secondary somatosensory cortex. Paradiso et al. studied rCBF in healthy elderly subjects who viewed emotionally evocative film clips; a fear/disgust versus neutral comparison revealed activation in orbitofrontal cortex, among other areas (93). Similarly, Kimbrell et al. studied induced emotional states in healthy adults (67); relative to a neutral condition, an anxiety condition was associated with rCBF increases in left anterior cingulate and left temporal pole, whereas rCBF decreases were found in nonparalimbic frontal cortical regions. Liotti et al. used PET and autobiographic memory scripts to examine the neural correlates of anxiety and sadness in healthy women (77); relative to a neutral condition, an anxiety condition was associated with rCBF increases in insular, orbitofrontal, and anterior temporal cortex and rCBF decreases in parahippocampal gyri. Pharmacologically Induced Fear and Anxiety Benkelfat et al. examined rCBF changes associated with the administration of cholecystokinin tetrapeptide versus saline in healthy persons (10). Administration of cholecystokinin
Chapter 65: Structural and Functional Imaging of Anxiety and Stress Disorders
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tetrapeptide was accompanied by increased heart rate and panic symptoms and rCBF increases in right cerebellar vermis, left anterior cingulate gyrus, bilateral claustrum-insulaamygdala, and bilateral temporal poles. However, further analyses suggested that the apparent temporal pole activations were attributable to extracranial artifacts of jaw muscle contraction. Ketter et al. used PET to study rCBF changes during procaine versus saline administration in healthy persons (66). In the procaine versus saline contrast, rCBF increases occurred in amygdala and anterior paralimbic structures, including anterior cingulate gyrus, insular cortex, and orbitofrontal cortex. Blood flow in left amygdala was positively correlated with fear intensity and was negatively correlated with euphoria intensity. Similar results were reported by Servan-Schreiber et al. (126). Other Negative Emotions Several studies examining rCBF associated with behaviorally induced sadness in healthy subjects have implicated both anterior paralimbic and nonparalimbic frontal cortical areas (4,54,55,77,94). Some studies of behaviorally induced sadness have also found rCBF changes within the amygdala (71, 120,121). Of note, studies of other behaviorally induced negative emotions, including anger (39,67) and guilt (127), have likewise found activation of anterior paralimbic cortical territories. Processing Unpleasant, Arousing, or Threat-Related Stimuli In functional imaging studies of responses to unpleasant pictures (73), several studies have found amygdala activation when contrasted with a neutral (63,72) or pleasant picture (92) comparison condition. In a separate study, Lane et al. reported that anterior paralimbic regions were activated when study subjects attended to the emotions evoked by the pictures rather than when subjects attended to physical attributes of the depicted scenes (70). Functional imaging studies have also demonstrated a correlation between amygdala activity during encoding of emotionally arousing pictures or film clips and subjects subsequent memory of them (29,59). Several functional imaging studies have shown greater amygdala responses to overtly presented fearful human facial expressions in comparison with neutral or happy faces (14, 86). Whalen et al. used fMRI and a technique called backward masking to study amygdala responses to emotional faces in the absence of explicit knowledge (142). Although subjects were unaware of seeing the masked emotional faces, a comparison of the masked fear and masked happy conditions yielded activation in the amygdala bilaterally. Habituation The term habituation refers to a decrement in responses over repeated presentations of a stimulus. Measures of habi-
tuation can be obtained peripherally (e.g., skin conductance) or more centrally (e.g., rCBF or fMRI BOLD signal). For example, declining fMRI BOLD signal within the amygdala has been observed in response to repeated presentations of fearful faces, regardless of whether subjects are aware the stimuli are present (14,142). A few studies have directly examined the neural correlates of habituation. Fischer et al. used PET to study rCBF changes over repeated presentation of videotaped scenes in healthy women (45). In separate scanning conditions, subjects watched two repeated videotaped presentations of neutral park scenes and snakes. From the first to the second presentation of the videotapes, rCBF decreased in bilateral secondary visual cortex and right medial temporal cortex, including amygdala and hippocampus. In an fMRI study, Fischer et al. also found response decrements over repeated presentations of human face stimuli in amygdala and hippocampus, as well as thalamus, and prefrontal, inferior temporal, and posterior cingulate cortex (47). Similar results have also been reported by Wright et al. (146). Conditioning and Extinction Fear conditioning involves the presentation of a neutral stimulus (i.e., a conditioned stimulus [CS]), such as a tone, that is temporally paired with an aversive stimulus (i.e., the unconditioned stimulus [US]), such as a shock. After repeated presentations of the CS and US, the CS alone begins to elicit fear-related autonomic changes, such as skin conductance responses. Subsequently, over repeated presentations of the CS without the US, fear responses decline, and this process is referred to as extinction. Existing research suggests that the amygdala plays a critical role in fear conditioning (27,35,74,75,141), and the medial prefrontal cortex may play a critical role in the process of extinction (56,83, 84). Fredrikson and Furmark and their colleagues used PET to study healthy subjects who viewed a videotape of snakes (CS) both before and after the video was paired with shock (US) (49,52). The findings revealed a significant correlation between rCBF changes in right amygdala and electrodermal activity changes. Hugdahl et al. used PET to compare patterns of blood flow before and after classic conditioning in healthy male study subjects, by employing a paradigm in which a tone (CS) was paired with brief shock (US) (62). Extinction was associated with widespread activations in right prefrontal, including orbitofrontal, cortex, as well as left occipital and superior frontal cortex. In a different conditioning paradigm, Morris et al. showed study subjects pictures of faces that were previously paired with an aversive burst of white noise (CS ) and faces that were never paired with the noise (CS ) (85). A comparison of the CS versus CS conditions yielded activation in right thalamus, orbitofrontal cortex, and superior frontal gyrus. There was a positive correlation between
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activation in thalamus and in right amygdala, orbitofrontal cortex, and basal forebrain. Morris and colleagues subsequently used PET and backward masking techniques to study rCBF responses to conditioned face stimuli with and without awareness in healthy male subjects (87). When all CS conditions were compared with all CS conditions, bilateral activation in amygdala was observed. Right amygdala activation was found in the condition in which subjects were aware; left amygdala activation was found in the condition in which subjects were unaware of the emotionally expressive face stimuli. In a single-trial fMRI study, LaBar et al. examined amygdala activation during both acquisition and extinction in a mixed-gender cohort (69). In the acquisition condition, a colored shape (CS ) was paired with a shock (US), whereas a different colored shape (CS ) was never paired with shock. No shocks were delivered during the extinction condition. Comparing CS with CS trials revealed activation in periamygdaloid cortex and amygdala during early acquisition and early extinction trials, respectively. Activation in both these regions declined over time. Buchel and colleagues also used a single-trial fMRI to study the neural correlates of fear conditioning in healthy subjects (26). Study subjects were scanned during an acquisition phase in which two neutral faces (CS ) were presented with a loud tone (US) and two other neutral faces were presented alone (CS ). To disambiguate the effects of the CS and US, the US was not presented on half of the CS trials (i.e., CS unpaired). The critical comparison, CS unpaired versus CS , revealed activation in anterior cingulate, bilateral insular, parietal, supplementary motor, and premotor cortex. A time by event type interaction revealed that fMRI signal in amygdala decreased over time in the CS unpaired condition relative to the CS condition. Similar results were reported by Buchel et al. in a trace conditioning study, in which a temporal gap occurs between the offset of the CS and onset of the US (28). These researchers also found conditioningrelated hippocampal activation that declined over time. Summary Taken together, functional imaging studies in healthy human subjects extend findings from animal research. Normal anxiety and fear reactions are associated with increased activity in limbic and paralimbic regions, whereas other territories of heteromodal association cortex exhibit decreased activity. However, similar patterns of limbic and paralimbic activation may be observed in association with other emotional states, and hence this general profile should not be taken as specific to anxiety or fear. Exposures to unpleasant, arousing, or threat-related stimuli often produce detectable amygdala responses, which can be associated with enhanced memory. Additional paralimbic recruitment may be related to a persons attention to his or her emotional state. Habituation can be observed in widely distributed brain regions,
including limbic, paralimbic, and sensory areas. Consistent with animal data, human imaging results point to a role for the amygdala in fear conditioning and for the frontal cortex in extinction. The accessory role of the hippocampus in these processes remains less well defined. POSTTRAUMATIC STRESS DISORDER Amygdalocentric Neurocircuitry Model We previously presented a neurocircuitry model of PTSD that emphasizes the central role of the amygdala and its interactions with the hippocampus, medial prefrontal cortex, and other heteromodal cortical areas purported to mediate higher cognitive functions (103). Briefly, this model hypothesizes hyperresponsivity within the amygdala to threat-related stimuli, with inadequate top-down governance over the amygdala by medial prefrontal cortex, specifically, the affective division of anterior cingulate cortex (142), and the hippocampus. Amygdala hyperresponsivity mediates symptoms of hyperarousal and explains the indelible quality of the emotional memory for the traumatic event; inadequate influence by the anterior cingulate cortex underlies deficits of habituation, and decreased hippocampal function underlies deficits in identifying safe contexts, as well as accompanying explicit memory difficulties (21). Further, we propose that in threatening situations, patients with PTSD exhibit an exaggerated reallocation of resources to regions that mediate fight-or-flight responses and away from widespread heteromodal cortical areas, as a neural substrate for dissociation. Structural Imaging Findings mMRI studies have reported smaller hippocampal volumes in veterans with PTSD than in comparison subjects. Bremner and colleagues (21) found that right hippocampal volumes were 8% smaller in 26 veterans with PTSD than in 22 civilians without PTSD. In addition, the PTSD group exhibited poorer performance on a standard measure of verbal memory, and their percent retention scores on this test were directly correlated with right hippocampal volume (i.e., lower scores were associated with smaller right hippocampal volumes). Gurvits and colleagues (58) used mMRI to study seven Vietnam combat veterans with PTSD, seven Vietnam combat veterans without PTSD, and eight nonveterans without PTSD. These investigators found significantly smaller hippocampal volumes bilaterally for the PTSD group in comparison with both control groups. Across the 14 veterans, hippocampal volume was inversely correlated with extent of combat exposure and PTSD symptom severity. Similar hippocampal volumetric differences also have been reported in mMRI studies of PTSD resulting from childhood abuse. Bremner and colleagues (22) found 12%
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smaller left hippocampal volumes in 17 adult survivors of childhood abuse with PTSD than in 21 nonabused comparison subjects. Stein and colleagues (134) found 5% smaller left hippocampal volumes in 21 adult survivors of childhood sexual abuse (most of whom had PTSD) than in 21 nonabused comparison subjects. Furthermore, total hippocampal volume was smaller in abused subjects with high PTSD symptom severity than in those with low PTSD symptom severity. In contrast to these results, DeBellis et al. (36) failed to find decreased hippocampal volumes in 44 maltreated children and adolescents with PTSD, compared with 61 nonabused healthy subjects. However, the PTSD group had smaller intracranial and cerebral volumes than did the comparison group. Taken together, the results of structural neuroimaging studies of adult samples suggest that PTSD is associated with reduced hippocampal volume, which, in turn, may be associated with cognitive deficits and PTSD symptom severity. Although the extent of traumatic exposure may be correlated with hippocampal volume, it appears that differences between PTSD and control groups cannot be explained by traumatic exposure alone (58). The results of DeBellis et al. (36) suggest that hippocampal volumetric differences between groups may not be evident in samples of children and adolescents or in samples of persons with relatively recent traumatic exposures. Stress, Glucocorticoids, and the Hippocampus In this review about neuroimaging of anxiety and stress disorders, it is worth elaborating on the potential relationship between stress and hippocampal findings in PTSD. Animal research has provided evidence that stress is associated with damage to the hippocampus (16). For example, sustained, fatal social stress in vervet monkeys was associated with degeneration of neurons in the CA3 region of the hippocampus (139); chronic restraint stress in rats was associated with atrophy of apical dendrites of hippocampal CA3 pyramidal neurons (140); and exposure to cold water immersion stress in rats was related to structural damage to hippocampus (CA3 and CA2 fields) and deceased local CBF in hippocampus (42). The effect of stress on hippocampus appears to be mediated by glucocorticoid hormones. Exposure to glucocorticoids is associated with hippocampal damage in both rats and primates. For example, Sapolsky et al. reported that chronic exposure to corticosterone in rats led to a loss of neurons in the CA3 region of the hippocampus (115). Woolley and colleagues found that daily corticosterone injections decreased dendritic branching and length in the CA3 region of the rat hippocampus (145). In a study of primates, Sapolsky et al. reported that chronic exposure to cortisol (through steroid-secreting pellets stereotactically implanted in hippocampus) was related to neuronal shrink-
age and dendritic atrophy in the CA3 region (117). Moreover, chronic stress during development is capable of inhibiting normal cellular proliferation within the hippocampus, a process mediated by glucocorticoids and glutamatergic transmission by an N-methyl-D-aspartate receptordependent excitatory pathway (57). Clinical research has also revealed decreased hippocampal volumes in humans with elevated cortisol levels resulting from Cushings syndrome (130); furthermore, in these patients, a treatment-related reduction of cortisol levels is associated with increased hippocampal volumes (131). High cortisol levels and decreased hippocampal volumes have also been found in patients with major depressive disorder (25). The hippocampus is also involved in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis, and lesions to hippocampus appear to increase the release of glucocorticoids during stress (43,60); this, in turn, may further damage the hippocampus (116). Although these findings may have great relevance to anxiety and stress disorders, the picture is complicated by the finding that cortisol levels are characteristically reduced, rather than elevated, in PTSD (147). One parsimonious theory suggests that patients with PTSD suffer hypersensitivity to glucocorticoids, resulting in both reduced levels of cortisol (because of accentuated feedback inhibition) and reduced hippocampal volume (147). Functional Imaging Findings Semple and colleagues used PET to study six patients with combat-related PTSD and comorbid substance abuse versus seven normal control subjects (125). rCBF was measured in three conditions: resting state, an auditory continuous performance task, and a word generation task. Compared with the control group, the PTSD group exhibited greater rCBF during both task conditions within orbitofrontal cortex. Rauch and colleagues studied a mixed-gender cohort of eight subjects with PTSD, using PET and a script-driven imagery method for inducing symptoms (104). In the provoked versus control condition, patients exhibited increased rCBF within anterior cingulate cortex, right orbitofrontal, insular, anterior temporal and visual cortex, and right amygdala. rCBF decreases occurred within left inferior frontal (Brocas area) and left middle temporal cortex. Interpretations of this initial study, with regard to the pathophysiology of PTSD, were limited by the absence of a comparison group. Using a similar paradigm and a comparison group, Shin and colleagues studied eight women with childhood sexual abuserelated PTSD and eight matched trauma-exposed control subjects without PTSD (129). In the traumatic versus neutral comparison, both groups exhibited anterior paralimbic activation. However, a group-by-condition interaction revealed that the control group manifested a
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significantly greater rCBF increase within anterior cingulate cortex than did the PTSD group, whereas the PTSD group showed significantly greater rCBF increases within anterior temporal and orbitofrontal cortex. Bremner and colleagues (20) also used script-driven imagery and PET to study rCBF in ten female survivors of childhood sexual abuse with PTSD and 12 without PTSD. Consistent with the findings of Shin et al. (129), Bremner and colleagues (20) reported relatively attenuated recruitment of anterior cingulate cortex in the PTSD group. Bremner and colleagues (23) studied rCBF responses to trauma-related pictures and sounds in ten Vietnam veterans with PTSD and in ten veterans without PTSD. In the combat versus neutral comparison, the PTSD group exhibited rCBF decreases in medial prefrontal cortex (subcallosal gyrus) and anterior cingulate cortex. Liberzon and colleagues (76) used SPECT to study rCBF in 14 Vietnam veterans with PTSD, 11 veteran control subjects, and 14 healthy nonveterans. In separate scanning sessions, subjects listened to combat sounds and white noise. In the combat sounds versus white noise comparison, all three groups showed activation in anterior cingulate/medial prefrontal cortex, but only the PTSD group exhibited activation in the left amygdaloid region. Bremner et al. (17) used PET to examine the effect of yohimbine challenge on glucose metabolic rates in ten combat veterans with PTSD and in ten nonveteran subjects without PTSD. Yohimbine administration was associated with increased anxiety and panic symptoms , as well as widespread decreases in cerebral glucose metabolism in the PTSD group. Shin and colleagues studied seven patients with combatrelated PTSD and seven matched trauma-exposed control subjects without PTSD in the context of a PET cognitive activation paradigm (128). Subjects were required to make judgments about pictures from three categories: neutral, general negative, and combat-related. Subjects performed two types of tasks: one involved responding while actually seeing the pictures (perception), and another involved responding while recalling the pictures (imagery). In the combat imagery versus control conditions, the PTSD group exhibited rCBF increases in right amygdala and ventral anterior cingulate gyrus and rCBF decreases in left inferior frontal gyrus (Brocas area). Using another cognitive activation paradigm, Rauch et al. studied the functional integrity of the amygdala in eight combat veterans with PTSD and eight healthy combat veterans (108). During fMRI, subjects viewed fearful and happy faces temporally masked by neutral faces. Healthy persons are typically aware of seeing only the neutral faces, although they show amygdala activation to the masked fearful faces (142). Rauch and colleagues found greater amygdala activation to masked fearful faces in persons with PTSD than in control subjects (108). Furthermore, the magnitude of amygdala activation was correlated with PTSD severity.
These results suggest that PTSD may be characterized by amygdala hyperresponsivity to general threat-related stimuli, consistent with our neurocircuitry model of PTSD. Imaging Studies of Neurochemistry Schuff and colleagues used mMRI and MRS to study seven veterans with PTSD and seven nonveteran control subjects (123). Although these investigators found a nonsignificantly smaller (6%) right hippocampus in the PTSD group by mMRI, they found an 18% reduction in right hippocampal NAA by MRS, a finding suggestive of reduced density or viability of neurons in this region. DeBellis and colleagues used MRS to study NAA/creatine ratios in 11 maltreated children and adolescents with PTSD and 11 healthy comparison subjects without histories of maltreatment (37). The PTSD group had lower NAA/ creatine ratios in pregenual anterior cingulate gyrus. This result is consistent with those of symptom provocation PET studies (20,23,129), which have reported failure to activate anterior cingulate in PTSD. Bremner et al. (18) used SPECT and [123I]iomazenil to study benzodiazepine-receptor binding in 13 veterans with PTSD and 13 nonveterans without PTSD. These investigators found decreased benzodiazepine-receptor binding in prefrontal cortex in the PTSD group, relative to the control group. Summary Taken together, data from neuroimaging studies are consistent with the current neurocircuitry model of PTSD that emphasizes the functional relationship among the amygdala, hippocampus, and medial prefrontal cortex. Hippocampal volumes and NAA levels appear to be decreased in persons with PTSD. These findings dovetail with animal research that points to a relationship among stress, HPA axis function, and cell viability within the hippocampus. Functionally, in comparison with control subjects, patients with PTSD exhibit the following: (a) greater activation within orbitofrontal cortex, anterior temporal poles, and the amygdala; (b) diminished activation in anterior cingulate and medial prefrontal cortex, as well as reduced NAA/creatine ratios in pregenual anterior cingulate; and (c) decreased activation within widespread areas that are associated with higher cognitive functions, such as Brocas area and dorsolateral prefrontal cortex.
OBSESSIVE-COMPULSIVE DISORDER Corticostriatal Model One current neuroanatomic model of OCD focuses on corticostriatothalamocortical circuitry (98,106). According to
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this model, the primary disorder lies within the striatum (specifically, the caudate nucleus). This leads to inefficient gating at the level of the thalamus, which results in hyperactivity within orbitofrontal cortex (corresponding to the intrusive thoughts) and hyperactivity within anterior cingulate cortex (corresponding to anxiety, in a nonspecific manner). Compulsions are conceptualized as repetitive behaviors that are performed to recruit the inefficient striatum ultimately to achieve thalamic gating and hence to neutralize the unwanted thoughts and anxiety. Structural Imaging Findings The results of several mMRI investigations of OCD have suggested volumetric abnormalities involving the caudate nucleus, although the nature of the observed abnormalities has been somewhat inconsistent. Scarone et al. (119) studied a mixed-gender cohort of 20 patients with OCD versus 16 matched controls and found increased right caudate volume in the OCD group. Robinson et al. (111) studied a mixedgender cohort of 26 patients with OCD versus 26 matched controls and found bilaterally decreased caudate volumes in the OCD group. Jenike et al. (64) studied an all-female cohort of ten patients with OCD versus matched controls and found trends toward a rightward shift in caudate volume (p .06) as well as overall reduced caudate volume (p .10) in the OCD group. Aylward et al. (3) studied a mixed-gender cohort of 24 patients with OCD versus 21 matched controls and found no significant differences in striatal volumes. Rosenberg et al. (112) studied 19 treatment-naive pediatric subjects with OCD and 19 casematched psychiatrically healthy comparison subjects. These investigators found reduced striatal volumes in the OCD group and an inverse correlation between striatal volume and OCD symptom severity. Functional Imaging Findings Neutral state paradigms employing PET and SPECT have most consistently indicated that patients with OCD exhibit increased regional brain activity within orbitofrontal and anterior cingulate cortex, in comparison with neurologically normal control subjects (6,7,78,89,113,136). Observed differences in regional activity within the caudate nucleus have been less consistent (6,113). Pre/posttreatment studies have reported treatment-related attenuation of abnormal regional brain activity within orbitofrontal cortex, anterior cingulate cortex, and caudate nucleus (8,9,61,95,124,137). In addition, both pharmacologic and behavioral therapies appear to be associated with similar brain activity changes (8,124). Some treatment studies have also reported that lower pretreatment glucose metabolic rates in orbitofrontal cortex predict a better response to serotonergic reuptake inhibitors (24,118,136). Symptom provocation studies employing PET (80,99)
as well as functional MRI (15) have also most consistently shown increased brain activity within anterior-lateral orbitofrontal cortex, anterior cingulate cortex, and caudate nucleus during the OCD symptomatic state. Cognitive activation studies using PET and fMRI have probed the functional integrity of the striatum in OCD (102,105). In these studies, patients with OCD perform an implicit (i.e., nonconscious) learning paradigm shown reliably to recruit striatum in healthy individuals (101,107). In both studies, patients with OCD failed to recruit striatum normally and instead activated medial temporal regions typically associated with conscious information processing.
Imaging Studies of Neurochemistry Several MRS studies have been performed to measure NAA concentrations in patients with OCD versus healthy comparison subjects. Ebert and colleagues (41) found reduced relative NAA levels in right striatum and anterior cingulate cortex in 12 patients with OCD in comparison with six healthy control subjects. Bartha and colleagues (5) found lower left striatal NAA concentrations in 13 patients with OCD than in 13 matched control subjects. MRS has also been used to demonstrate elevated glutamatergic concentrations within the striatum of a child with OCD (82). Glutamate is the principal transmitter mediating frontostriatal communication. Interestingly, elevated striatal glutamate levels were attenuated toward normal after successful pharmacotherapy. These findings suggest that orbitofrontal hyperactivity in OCD may be mirrored by elevated glutamate at the site of orbitofrontal ramifications in striatum, and treatment-related attenuation of orbitofrontal activity may be accompanied by decreased glutamate concentration within the striatum.
Summary Taken together, these neuroimaging findings are consistent with disorders in corticostriatothalamocortical circuitry. Consistent with the hypothesis of a primary abnormality in the striatum, MRI and MRS studies of OCD have shown reduced striatal volumes and reduced striatal NAA, respectively. PET studies have revealed hyperactivity within orbitofrontal cortex, and the magnitude of this hyperactivity predicts response to treatment. In addition, in neurologically normal persons, the performance of repetitive motor routines does facilitate striatal recruitment in the service of thalamic gating, whereas this pattern is not readily demonstrated in patients with OCD. These imaging data further support the working model of striatal pathology and striatothalamic inefficiency, together with orbitofrontal hyperactivity.
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SOCIAL AND SPECIFIC PHOBIAS Neuroanatomic Models Currently, there are no cohesive neuroanatomically based models for the phobias (53,132). One possibility is that phobias are learned and hence reflect another example of fear conditioning to specific stimuli or situations. Alternatively, phobias may represent the product of dysregulated systems for detecting potentially threatening stimuli or situations. For instance, if humans have evolved a neural network specifically designed to assess social cues for threatening content, and another to assess threat from small animals, these may represent the neural substrates for the pathophysiology underlying phobias. Structural Imaging Findings Given the high prevalence of phobias and the relative ease with which medication-free phobic subjects without significant comorbidities can be recruited, it is striking how few imaging studies have been conducted in this arena. Potts et al. used mMRI to examine volumetric measures of total cerebrum, caudate, putamen, and thalamus in 22 patients with SoP and in 22 matched healthy control subjects (96). The groups did not significantly differ on any of these measures. Functional Imaging Findings Studies of SpP to date have principally employed PET symptom provocation paradigms and have reported somewhat inconsistent results. Mountz and colleagues found that persons with small-animal phobia exhibited increased heart rates, respiratory rates, and subjective reports of anxiety during exposure to phobic stimuli; however, no changes in rCBF measurements were observed (88). Wik and colleagues studied six patients with snake phobias during exposure to videotapes of neutral, generally aversive, and snake-related scenes (143). During the phobic condition, they found significant rCBF increases in secondary visual cortex and rCBF decreases in prefrontal cortex, posterior cingulate cortex, anterior temporopolar cortex, and hippocampus. These findings were similar to those of two other studies of phobia from the same laboratory (50,51). Using in vivo exposure and PET, Rauch and colleagues studied rCBF in seven persons with a variety of small animal phobias (100). In the provoked versus control condition, patients with phobias exhibited rCBF increases within multiple anterior paralimbic territories (i.e., right anterior cingulate, right anterior temporal pole, left posterior orbitofrontal cortex, and left insular cortex), left somatosensory cortex, and left thalamus. Whereas one neutral-state SPECT study of patients with SoP and healthy control subjects found no significant between-group differences in rCBF (133), more recent cogni-
tive activation studies performed in conjunction with fMRI have yielded more informative results. Birbaumer et al. (11) used fMRI to study seven patients with SoP and five healthy control subjects during exposure to slides of neutral human faces or aversive odors. In comparison with the control group, the SoP group exhibited hyperresponsivity within the amygdala that was specific to the human face stimuli. In a follow-up study, Schneider et al. used fMRI to study 12 patients with SoP and 12 healthy control subjects (122). The researchers used a classic conditioning paradigm in which neutral face stimuli were the conditioned stimuli and odors (negative odor and odorless air) served as the unconditioned stimuli. In response to conditioned stimuli associated with the negative odor, the SoP group displayed signal increases within amygdala and hippocampus, whereas healthy comparison subjects displayed signal decreases in these regions. Imaging Studies of Neurochemistry Davidson et al. (34) used MRS to study NAA in 20 patients with SoP and in 20 healthy control subjects. Relative to the control group, the SoP group exhibited decreased NAA in white matter and cortical and subcortical gray matter (e.g., caudate and thalamus). Tiihonen et al. used SPECT and I-123labeled -CIT to measure the density of dopamine reuptake sites in 11 patients with SoP and in 28 healthy comparison subjects (138). They found significantly reduced striatal dopamine reuptake binding site density in the SoP versus control group. Summary Although relatively few neuroimaging studies of SpP have been conducted, findings from existing research suggest activation of anterior paralimbic regions and sensory cortex corresponding to stimulus inflow associated with a symptomatic state. Although such results are consistent with a hypersensitive system for assessment of or response to specific threat-related cues, they do not provide clear anatomic substrates for the pathophysiology of SpP. Cognitive activation neuroimaging studies of SoP reveal exaggerated responsivity of medial temporal lobe structures to human face stimuli; this hyperresponsivity may reflect a neural substrate for social anxiety in SoP.
PANIC DISORDER Neuroanatomic Models Neurobiological models of PD have emphasized a wide range of disparate elements (31). Satisfactory models of PD must account for spontaneous panic attacks, which are a
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defining feature of PD. It is possible that spontaneous panic corresponds to a normal physiologic anxiety response that is mediated by intact fear-anxiety circuits but, owing to homeostatic deficits, occurs in inappropriate, threat-free situations. This is consistent with theories of hypersensitivity to carbon dioxide at the level of the brainstem (i.e., the suffocation false alarm model), as well as theories regarding fundamental monoaminergic dysregulation. Another possibility is that panic attacks emerge in the context of what should be minor anxiety episodes because of failures in the systems responsible for limiting such normal responses; hippocampal deficits may underlie such a failure to inhibit anxiety responses. Finally, panic episodes described as spontaneous (i.e., without identifiable precipitants) could reflect anxiety responses to stimuli that are not processed at the conscious (i.e., explicit) level, but instead, recruit anxiety circuitry without awareness (i.e., implicitly). There is strong evidence that the amygdala can be recruited into action in the absence of awareness that a threat-related stimulus has been presented (142). By this model, PD may be characterized by fundamental amygdala hyperresponsivity to subtle environmental cues, triggering full-scale threat-related responses in the absence of conscious awareness. Structural Imaging Findings Fontaine et al. (48) published a qualitative MRI study involving 31 patients with PD and 20 matched healthy control subjects. The frequency of gross structural abnormalities was higher in the PD group (40%) than in the control group (10%); the most striking focal findings in the PD group involved abnormal signal or asymmetric atrophy of the right temporal lobe. Functional Imaging Findings In an initial PET neutral-state study, Reiman and colleagues studied 16 patients with PD and 25 normal control subjects (109). In the subset of patients who were vulnerable to lactate-induced panic (n 8), the investigators found abnormally low left/right ratios of parahippocampal blood flow. DeCristofaro et al. used SPECT to measure rCBF at rest in seven treatment-naive patients with PD and in five age-matched healthy control subjects (38). Relative to the control group, the PD group exhibited elevated rCBF in left occipital cortex and reduced rCBF in the hippocampal area bilaterally. Nordahl et al. used PET and FDG to measure regional cerebral metabolic rate glucose (rCMRglc) in 12 patients with PD and 30 normal control subjects during an auditory continuous performance task (90). The investigators found that the PD group exhibited a lower left/right hippocampal ratio. In a follow-up experiment (91), these investigators used PET-FDG methods to study imipraminetreated subjects with PD and found a rightward shift in symmetry of rCMRglc within hippocampus and posterior
inferior frontal cortex. In comparison with the untreated group, the imipramine-treated group exhibited rCMRglc decreases in posterior orbital frontal cortex. Bisaga et al. (12) used PET and FDG to study a cohort of six women with PD and six matched control subjects. In contrast to previous studies, the PD subjects displayed elevated rCMRglc in the left hippocampus and parahippocampal area. The literature contains three symptom provocation studies of PD, all of which have employed pharmacologic challenges. Stewart et al. used SPECT and the xenon inhalation method to measure CBF during lactate infusion in ten patients with PD and in five healthy control subjects (135). The patients with PD who experienced lactate-induced panic attacks (n 6) displayed global cortical CBF decreases. Woods et al. used SPECT and yohimbine infusions to study six patients with PD and six normal control subjects (144). In the PD group, yohimbine administration increased anxiety and decreased rCBF in bilateral frontal cortex. In a PET study, Reiman et al. measured rCBF during lactate infusions in 17 patients with PD and in 15 normal control subjects (110). The eight patients who suffered lactate-induced panic episodes exhibited rCBF increases in bilateral temporopolar cortex and bilateral insular cortex/ claustrum/putamen. Healthy control subjects and patients with PD who did not experience lactate-induced panic attacks did not exhibit such rCBF changes. Of note, the temporopolar findings were subsequently questioned as possibly reflecting extracranial artifacts from muscular contractions (40,10). In a symptom capture case report, Fischer and colleagues (44) found that a spontaneous panic attack was associated with rCBF decreases in right orbitofrontal, prelimbic (area 25), anterior cingulate, and anterior temporal cortex. Imaging Studies of Neurochemistry Dager and colleagues used MRS to measure brain lactate levels during hyperventilation in seven treatment-responsive patients with PD and in seven healthy comparison subjects (32). The PD group showed a significantly greater rise in brain lactate in response to the same level of hyperventilation. Dager et al. also used MRS to measure brain lactate levels during lactate infusions in 15 patients with PD and in ten healthy comparison subjects (33). The PD group exhibited a significantly greater brain lactate level during lactate infusion, a finding consistent with the interpretation of reduced clearance, rather than higher production, of lactate in PD. Three studies have used SPECT and [123I]iomazenil to measure benzodiazepine-receptor binding in PD. Kuikka et al. (68) studied 17 subjects with PD and 17 matched healthy comparison subjects and found that the PD group exhibited a greater left/right ratio in benzodiazepine-receptor uptake that was most prominent in prefrontal cortex. Brandt et al. (13) studied 12 medication-naive patients with PD and nine
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matched healthy control subjects and found that the PD group exhibited significantly elevated benzodiazepine-receptor binding within right supraorbital frontal cortex, as well as a trend toward elevated binding in the right temporal cortex. Bremner et al. (19) included 13 patients with PD and 16 healthy comparison subjects and found that the PD group showed decreased benzodiazepine-receptor binding in left hippocampus and precuneus. Using PET and carbon-11labeled flumazenil, Malizia et al. studied seven patients with PD and eight healthy comparison subjects (79). These investigators found that the PD group exhibited a global reduction in benzodiazepine binding that was most pronounced in right orbitofrontal and right insular cortex. Summary Resting state neuroimaging studies have suggested abnormal hippocampal activity in PD. Symptom provocation studies have revealed reduced activity in widespread cortical regions, including prefrontal cortex, during symptomatic states. MRS studies have reported greater brain lactate levels in response to hyperventilation and lactate infusions. Finally, receptor-binding studies of PD suggest widespread abnormalities in the GABAergic/benzodiazepine system. Consistent with prevailing neurobiological models of PD, it is possible that fundamental abnormalities in monoaminergic neurotransmitter systems, originating in the brainstem, underlie the abnormalities of metabolism, hemodynamics, and chemistry found in widespread territories of cortex. Further, regional abnormalities within the medial temporal lobes provide some support for theories regarding hippocampal or amygdala dysfunction in PD. CONCLUSIONS AND FUTURE DIRECTIONS Neuroimaging research is helping to advance neurobiological models of anxiety and stress disorders. At the current early stage of this scientific enterprise, there are hints of commonalities across anxiety disorders as well as leads regarding disorder-specific features. Beyond the need for a general expansion of the existing database, it will be critical to explore the specificity of initial findings by conducting studies with psychiatric comparison groups in addition to healthy control subjects. This is of particular relevance to the concept of stress disorders, in which common etiologic factors or vulnerability factors may have corresponding pathophysiologic profiles that are independent of our current diagnostic scheme. For instance, the relationship between early or chronic life stress and hippocampal structure and function may well span anxiety, mood, and even psychotic disorders. In this light, longitudinal and developmental studies may be of particular importance in elucidating the neural correlates and consequences of stress.
Similarly, genetic studies in animals and humans will benefit from neuroimaging methods that can illuminate the bidirectional link from behavior to brain structure, function, and chemistry. For instance, research regarding the heritability of anxious temperament may be enhanced by using extended phenotypes of conditionability or distributed brain function within amygdala, hippocampus, and medial frontal cortex. In fact, the gamut of existing animal and human experimental paradigms with relevance to anxiety and stress disorders provides a promising context for advancing integrated models across scales and neuroscientific modes of inquiry. As such integrated models evolve, targets for new and improved neuropsychopharmacotherapies are destined to emerge. Indeed, neuroimaging is likely to play a role not only in conceptually motivating but also in discovering and testing such new therapies as part of the next generation of progress in this domain. REFERENCES
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CURRENT AND EMERGING THERAPEUTICS OF ANXIETY AND STRESS DISORDERS
JACK M. GORMAN JUSTINE M. KENT JEREMY D. COPLAN
During the 1960s and 1970s the concept of pharmacologic dissection became popular as a putative method for differentiating among different categories of psychiatric illness. At the time, it was widely held that anxiety disorders, but not depression, respond to benzodiazepines, whereas depression, but not anxiety disorders, responds to antidepressants. Panic disorder was held to be the one exception, responding only to antidepressants. Alprazolam, selective serotonin reuptake inhibitors (SSRIs), and buspirone had not yet been tested. On the basis of these observations, it was asserted that anxiety and depression are clearly distinct categories of illness. Thirty years later we find that the situation has changed dramatically. The first inconsistency with the notion of pharmacologic dissection was the clear finding that panic disorder does indeed respond to benzodiazepines. For a while, alprazolam and then clonazepam were regarded by some authorities and clinicians as first-line therapies for panic disorder, replacing tricyclics and monoamine oxidase inhibitors. An even more potent challenge, however, has come from the evidence not only that anxiety disorders respond to antidepressants but also that antidepressants work better than benzodiazepines for most of them. As this chapter discusses, antidepressants are now considered the appropriate pharmacologic intervention for panic disorder, social anxiety disorder, posttraumatic stress disorder (PTSD), and generalized anxiety disorder (GAD). The latter case is particularly interesting. Once considered the sole domain of benzodiazepines, GAD was then shown to be responsive to a drug in an entirely new category, with no relationship whatsoever to the benzodiazepine receptor or -aminobutyric acid (GABA)buspirone. At about the same time evidence
began to accumulate from just a few studies that GAD might also respond to antidepressants. This evidence was largely ignored, and pharmaceutical companies were advised to stay away from GAD, a condition supposedly so placeboresponsive that no drug would ever be shown effective in large clinical trials. To the contrary, venlafaxine is now approved by the Food and Drug Administration (FDA) for the treatment of GAD, and there is also evidence for the efficacy of paroxetine. At this point, rather than dissecting among the anxiety disorders or between anxiety disorders and depression, pharmacologic grounds might lead one to assume that these conditions are variants of each other. This also would probably be an oversimplification. Anxiety and depression are different and we can make distinctions among the anxiety disorders. Nevertheless, the finding that antidepressants are so ubiquitously effective across categories raises interesting questions and challenges. We review here the evidence for responsiveness of four anxiety disorders to medication. PANIC DISORDER Panic disorder (PD) has a reported lifetime prevalence of between 1.5% and 3.5% (1,2), is highly comorbid with major depression, and is associated in its own right with significant impairment in psychosocial functioning independent of depressive symptomatology. In the Epidemiologic Catchment Area study, subjective reports of patients with PD indicate that approximately one-third experience poor physical and emotional health, rates comparable with major depression (2). Historical Notes
Jack M. Gorman, Justine M. Kent, and Jeremy D. Coplan: Columbia University, New York State Psychiatric Institute, New York, New York.
Recognized as a distinct disorder that could be distinguished from the general diagnosis of anxious neurosis, in part
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through the pharmacologic dissection work of Klein and Fink (3,4) in the 1960s, PD was first categorized as a discrete diagnostic entity in the Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III), in 1980. Despite some minor changes in diagnostic criteria in the third edition revised (DSM-III-R) and the fourth edition (DSM-IV), primarily involving the number and frequency of attacks required, the major criteria remain essentially the same. The key triad of symptoms are (a) the occurrence of spontaneous panic attacks; (b) the presence of anticipatory anxiety; and (c) the presence of phobic avoidance, resulting in some degree of functional impairment. The pharmacologic treatment of PD has evolved dramatically since the heterocyclic antidepressants were first established as possessing powerful antipanic properties in the early 1960s (4). Throughout the 1970s and 1980s, the heterocyclic antidepressants continued to be the mainstay of pharmacologic treatment of PD, with the monoamine oxidase inhibitors (MAOIs) used primarily in patients who failed trials of heterocyclic antidepressants. The high potency benzodiazepines were increasingly prescribed as both primary and adjunct treatments throughout this same time period. With the introduction of the SSRIs in the United States in the late 1980s and early 1990s for the treatment of depression, this class of drug began being used in the treatment of PD with promising results. In the late 1990s, several large-scale, controlled trials established the SSRIs to be effective and safe treatments for PD, thus supplanting the heterocyclic antidepressants and benzodiazepines as first-line treatment. Although the serotonin, norepinephrine, and GABA systems remain the traditional targets for the majority of antipanic medications, widely different classes of drugs targeting an array of neurochemical systems are now being explored as potential treatments for PD. Heterocyclic Antidepressants Numerous controlled trials have confirmed the efficacy of the heterocyclic antidepressants, since the initial observations of Klein (4), in both the acute and long-term treatment of PD. In general, heterocyclics with greatest serotoninergic reuptake inhibition effect, such as imipramine and clomipramine, appear to be most effective in the treatment of PD (57). By far the best studied of this class of antidepressants is imipramine, which due to its well-established efficacy has been generally accepted as the gold standard of PD treatment (8). In the Cross-National Collaborative Panic Study, more than 1,000 patients in 14 countries were randomized into a study comparing imipramine, alprazolam, and placebo (9). At the studys end, imipramine and alprazolam were found to have comparable efficacy, and both were significantly superior to placebo on most outcome measures. A positive doseresponse relationship between imipramine levels and clinical improvement has been reported, with plasma levels of 140 mg/mL associated with the
greatest improvement in panic symptoms (10). Although several other of the heterocyclic antidepressants have been used in the treatment of PD (amitryptyline, desipramine, nortriptyline, clomipramine), far less controlled data are available supporting their efficacy (11,12). Although effective, side effects often limit the use of this class of medication in the treatment of PD. This is particularly true in the case of clomipramine, which due to its anticholinergic and antihistaminergic effects can be difficult for patients to tolerate (13). The use of the heterocyclic antidepressants is often limited by the presence of comorbid medical conditions such as cardiac disease and glaucoma. Lethality in overdose is another concern given the reported high rates of suicide in this population when depression is comorbid (1416). Although the SSRIs are often touted as offering a more tolerable side-effect profile than the heterocyclics, the sideeffect burden of imipramine has recently been shown to be comparable to, although different in nature from, that of the SSRIs, with most side effects (with the exception of dry mouth, sweating, and constipation) not persisting beyond the first few weeks of treatment (17).
Monoamine Oxidase Inhibitors Like the heterocyclic antidepressants, the MAOIs, are clearly established to be effective in the treatment of PD, yet have yielded to newer antidepressants with similar antipanic efficacies but less drugdrug and dietary interactions. Among the MAOIs, phenelzine is the best studied in PD, and its efficacy in the acute treatment of PD is supported by several studies (1820). Dietary restrictions, lethality in overdose, and drug interaction concerns limit the widespread use of the traditional MAOIs. Stemming from these concerns, the reversible inhibitors of MAOI (RIMAs) were developed and have demonstrably fewer drugdrug and dietary interactions. These include moclobemide and brofaromine, neither of which is currently marketed in the United States, but are used extensively throughout Europe and other parts of the world. The efficacy of moclobemide has been shown in the treatment of PD in placebo-controlled studies (21), and moclobemide has been found to be comparable in efficacy to clomipramine (21) and fluoxetine (22). Moclobemide has also been shown to be as effective as fluoxetine in maintenance treatment of PD (23). Brofaromine was shown to be comparable to fluvoxamine (24) and clomipramine (25) in small randomized, double-blind studies lacking a placebo. In a placebo-controlled study of the efficacy of brofaromine, 30 patients with PD (DSM-III-R definition) were treated for 12 weeks. Although there was no significant reduction in the number of panic attacks for those patients treated with brofaromine, patients demonstrated clinical improvement on several other measures, including agoraphobic avoidance (26).
Chapter 66: Current and Emerging Therapeutics of Anxiety and Stress Disorders
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Benzodiazepines The high-potency benzodiazepines, another mainstay of treatment for PD, have been shown to be effective, well tolerated, and safe. In the absence of comorbid substance abuse, concerns about abuse potential have proven largely unfounded in this population (2730). Among the benzodiazepines, alprazolam and clonazepam are labeled for the treatment of panic disorder and have been shown in numerous, controlled trials to be effective treatments (3136). Clonazepam, with a long half-life of 20 to 50 hours, allows fewer doses per day than the short-acting alprazolam, and may reduce the likelihood of rebound symptoms between doses. The benzodiazepines have repeatedly been shown to offer an early advantage in the treatment of anxiety by providing almost immediate relief of anxiety-related somatic symptoms such as muscle tension and insomnia (27,37,38). However, in the long term, antidepressants may offer the advantage of better targeting and relief of psychic symptoms of anxiety (37), and provide the added benefit of treating associated depressive symptoms. Discontinuing long-term pharmacotherapy with benzodiazepines can be difficult, with as many as a third of patients with PD being unable to discontinue use due to dependence/withdrawal (27). Thus, despite their efficacy and safety, many clinicians remain concerned about the risk of dependence (39). Selective Serotonin Reuptake Inhibitors (SSRIs) Among the antidepressants currently used in the treatment of PD, the SSRIs have become first-line treatments (40,41). Following in the wake of numerous promising open and controlled trials, several large, multicenter, placebo-controlled studies involving hundreds of subjects each have demonstrated the efficacy of the SSRIs in the treatment of PD (Table 66.1) (4250). Both sertraline and paroxetine are labeled in the United States for the treatment of PD, and citalopram is approved in several European countries for this indication. Although fluvoxamine has not been studied in clinical trials on the scale of the other SSRIs, it has been shown to be efficacious in smaller ( 100 subjects), randomized, placebo-controlled studies (25,44). Despite their established efficacy in the treatment of PD, there are certain problems inherent in prescribing the SSRIs in patients with PD. Of primary concern on the initiation of treatment is the commonly observed anxiogenic effect, which, despite being dose-dependent, can nonetheless make the initial several days of treatment a challenge for patients. Initiating treatment with a dose of one-fourth to one-half that of the normal starting dose may greatly reduce the patients feelings of restlessness and increased anxiety. The delayed onset of anxiolytic action contributes to making the period of SSRI initiation difficult for patients with PD. Other effects such as sexual dysfunction and weight gain
with long-term use can make these drugs as problematic as earlier classes for some patients. Overall, though, the sideeffect burden associated with SSRI treatment has been shown to be more tolerable for most patients than the heterocyclics and benzodiazepines (51). Because the SSRIs have been associated with a discontinuation syndrome characterized by anxiety, tremor, dizziness, paresthesias, nausea, and other symptoms when abruptly stopped, these medications should be tapered over a few weeks, if possible, to minimize discontinuation symptoms (38).
Newer Antidepressants Among the newer antidepressants, several have demonstrated promise in PD. Venlafaxine, a serotonin-norepinephrine reuptake inhibitor, has shown efficacy (on some measures) in a small, placebo-controlled study (52). Nefazodone, a weak serotonin-norepinephrine reuptake inhibitor with serotonin receptor subtype 2C (5-HT2C) antagonist properties, has been shown to reduce anxiety in depressed patients with comorbid PD (53). Mirtazapine enhances both noradrenergic and serotoninergic neurotransmission without reuptake inhibition. Results of an open study involving ten patients suggested that mirtazapine might be effective in the treatment of PD (54). More recently, in a double-blind randomized trial comparing mirtazapine and fluoxetine in the treatment of PD, both drugs showed comparable efficacy on the primary outcome measures and on most secondary outcome measures (55). Adverse events differed between treatments, with weight gain occurring more frequently in those patients receiving mirtazapine, whereas nausea and paresthesias occurred more often in those receiving fluoxetine.
Anticonvulsants Among the anticonvulsants being used in the treatment of PD are valproate and carbamazepine, and the newest anticonvulsants gabapentin, lamotrigine, pregabalin, and vigabatrin. Valproate has shown promise in several open trials (5658), and one small placebo-controlled study (59). It may be particularly effective when mood instability is comorbid (60). There is far less support for the use of carbamazepine in the treatment of PD, with uncontrolled studies in patients with PD with EEG abnormalities demonstrating some benefit from carbamazepine treatment (58). However, the only controlled trial of carbamazepine in a small number of PD patients (N 14) did not report a significant difference for carbamazepine versus placebo in reducing panic attack frequency (61). Gabapentin has shown promise (62) and is recognized as having a benign side-effect profile. Lamotrigine, pregabalin, and vigabatrin are currently under investigation.
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TABLE 66.1. EFFICACY OF THE SSRIs IN THE ACUTE TREATMENT OF PANIC DISORDER BASED ON LARGE-SCALE, PLACEBO-CONTROLLED STUDIES
SSRI Fluoxetine Investigators Michelson et al., 1998 Study Design Multicenter, 10-week study of 243 patients Dose Range Fixed dose: 10 or 20 mg/day Outcome 20-mg dose was most effective, demonstrating significant change versus placebo on panic attack frequency, CGI improvement scores, Hamilton Anxiety and Depression scores, phobic symptoms, and functional impairment as measured by the Sheehan Disability Scale (family life and social life); the two fluoxetine groups did not differ from placebo in the number of patients who were panic-free at endpoint A significantly greater percentage of patients on fluoxetine were panic-free at endpoint (42%, versus 28% on placebo); significant change versus placebo were found for the CGI Severity, HAM-A, State Anxiety Inventory, and Sheenhan Disability Scale (work and social impairment) Fluvoxamine was superior to placebo and cognitive therapy at endpoint as measured by the Clinical Anxiety Scale and CGI Severity and Improvement scales; fluvoxamine was superior to placebo as measured by a greater reduction in mean panic attack severity, and number of panic-free patients; fluvoxamine-treated patients demonstrated significant reductions versus placebo on several measures of the Sheehan Disability Scale (work, social/leisure) Number of patients with at least 50% reduction in panic attacks was significantly greater in the paroxetine-treated than placebo group, as was number of patients who had only one or no panic attacks during the final study week. The paroxetine group had significantly greater mean reductions in HAM-A and CGI scores versus placebo 40-mg group demonstrated the greatest effects, with significant improvement versus placebo on measures of reduction in number of panic attacks, intensity of attacks, CGI Severity and Improvement scores, phobic fear score, HAM-A score, and MADRS Significant decreases versus placebo at endpoint in number of panic attacks, CGI Improvement and Severity scales, PDSS scores; sertraline also demonstrated superiority on improvement in quality of life scores All three doses of sertraline were superior to placebo on frequency of panic attacks, CGI Improvement, and change in panic burden (attack frequency X severity), with no consistent dose-response effect (continued)
Michelson et al., 2000
Multicenter, 12-week study of 180 patients
Flexible dose: 2060 mg/day (mean dose = 20 mg)
Fluvoxamine
Black et al., 1993
Multicenter, 8-week study of 75 patients randomized to either fluvoxamine, cognitive therapy, or placebo
Flexible dose: up to 300 mg/day (mean dose = 230 mg)
Paroxetine
Oehrberg et al., 1995
Multicenter, 12-week study of 120 patientsall received cognitive therapy
Flexible dose: 2060 mg/day
Ballenger et al., 1998
Fixed dose: 10, 20, or 40 mg/day
Sertraline
Pollack et al., 1998
Flexible dose: 50200 mg
Sheikh et al., 2000
Pooled data from two 12-week studies with a total of 322 patients
Fixed dose: 50, 100, or 200 mg/day
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TABLE 66.1. (continued)

SSRI Citalopram Investigators Wade et al., 1997 Study Design Multicenter, 8-week comparative study with clomipramine and placebo of 475 patients Dose Range Fixed dose: citalopram 10 or 15 mg/day, 20 or 30 mg/day, 40 or 60 mg/day; clomipramine 60 or 90 mg/day Outcome Patients treated with citalopram 20/30 or 40/60 mg/day were comparable to clomipramine and superior to placebo as measured by the number of patients panic-free in the final week of treatment, and by mean reduction in HAM-A total and psychic subscale, and the MADRS; only the 40/60-mg/day dose demonstrated superiority to placebo, along with clomipramine, on the HAM-A somatic subscale; on the Physicians Global Improvement Scale and Patients Global Improvement Scale, the 20/30-mg dose exhibited greater effects; however, both the 20/30- and 40/60-mg/day doses were superior to placebo
CGI, Clinical Global Impression; HAM, Hamilton Anxiety Rating Scale; MADRS, Montgomery-Ashberg Depression Rating Scale; SSRI, selective serotonin reuptake inhibitor.
Beta-Blockers Although the beta-blockers are more commonly used in the treatment of performance anxiety and as adjunctive treatment in PTSD, a small number of open studies suggest they may be effective in the treatment of PD (63), although they are not considered a first-line treatment. Future Directions Several classes of drugs, although initially viewed as promising, have shown limited efficacy in the treatment of PD. These include buspirone, bupropion, ondansetron, and the cholecystokinin (CCK) antagonists (6467). A number of new classes of drugs are being studied, including the benzodiazepine partial agonists such as abecarnil and pagaclone, and the corticotropin-releasing hormone (CRH) inhibitors. Experimental agents showing promise in panic-like models in rodents include the group II metabotropic glutamate receptor agonist LY354740 (68) and drugs acting at the neuropeptide receptors, including neuropeptide-Y agonists and neurokinin substance P antagonists (69). In summary, the expansion of the antipanic armamentarium suggests that, as with many of the psychiatric disorders, there is no single effective treatment of PD. Among the most commonly prescribed classes of drugs for the treatment of PD [benzodiazepines, SSRIs, tricyclic antidepressants (TCAs), and MAOIs], there are probably no major differences in treatment efficacy, with most reported differences in efficacy between classes probably attributable to differences in study design and samples (27). The use of antidepressants, however, and particularly the SSRIs, has supplanted the long-term use of benzodiazepines for this
disorder. Antidepressant use has two main advantages over the benzodiazepines: (a) it provides antidepressant benefits in a population highly susceptible to depressive symptomatology and comorbid major depression (70), and (b) it eliminates the difficulties associated with withdrawal symptoms upon benzodiazepine discontinuation. In the case of comparable efficacy, medication choice is based on factors such as latency to onset of therapeutic action, safety, and the individual side-effect profiles of each medication. In this regard, the SSRIs are currently considered first-line treatment for PD, demonstrating comparable efficacy and superior tolerability to other treatment classes. Combination therapies are frequently used for treatment resistance, and an approach of prescribing a benzodiazepine at the initiation of treatment with an SSRI, and later tapering it, has proved to be popular with clinicians and has recently been demonstrated to be advantageous in the early stages of treatment over an SSRI alone (71).
GENERALIZED ANXIETY DISORDER The diagnostic criteria for generalized anxiety disorder (GAD) have evolved over the past two decades, undergoing substantial revision to the current definition emphasizing excessive, unrealistic worry as the cardinal feature of this disorder. Defined in 1980 in DSM-III as a disorder of continuous or persistent worry symptoms of at least 1 months duration, the diagnosis of GAD was reframed in DSM-IIIR to require symptoms extended for 6 months or longer, and an emphasis on unrealistic worry was stressed. With DSM-IV, GAD was defined as excessive and persistent
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worry, accompanied by three or more physical or psychological symptoms of anxiety, persisting 6 months or longer. Because of the shift in diagnostic criteria and required duration of symptoms over the years, comparison of pharmacologic treatment studies performed prior to the introduction of DSM-III-R is difficult. In comparison with panic disorder, PTSD, and obsessive-compulsive disorder (OCD), there are fewer publications devoted to GAD overall, and only a limited number of published controlled clinical medication trials. Several reasons have been proposed for this deficiency, including underrepresentation in clinical settings and a view of GAD as a mild disorder (72). In reality, GAD is one of the most commonly diagnosed anxiety disorders, with a lifetime prevalence of 4.1% to 6.6% (73,74), which is often chronic (75), and associated with significant compromise in functioning (76,77). There remains substantial controversy surrounding the validity of GAD as a primary disorder, as opposed to a comorbid condition or a prodromal/residual phase of another disorder (78,79). Findings from epidemiologic studies of GAD suggest that current comorbidity with other disorders is as high as 58% to 65% (73,76), and lifetime comorbidity rates are between 80% and 90% (76,80). Non-comorbid, pure GAD lifetime prevalence was found to be only 0.5% in the National Comorbidity Survey (76). Overall, the sum of studies examining quality of life issues support the idea that non-comorbid GAD is relatively rare, but is associated with significant impairment in its own right (81,82). Historical Notes Prior to the introduction of the benzodiazepines, the main agents available for the treatment of anxiety were the tricyclic antidepressants (doxepin, imipramine, amitryptyline), antihistamines (diphenhydramine, hydroxyzine), barbiturates (mephobarbital), and the sedative antianxiety agent meprobamate (Milltown). The development of the benzodiazepines in the mid-1950s led to the introduction of chlordiazepoxide (Librium) in 1959, and ushered in an era of benzodiazepine use in the treatment of a wide range of anxiety symptoms related to anxiety and mood disorders, psychosis, and alcohol withdrawal. Greater tolerability and the superior safety profiles of the benzodiazepines resulted in a sharp decline in the use of barbiturates for anxiety disorders (83). The benzodiazepines have remained a common treatment choice for GAD throughout the past two decades. However, concerns about dependence and withdrawal, short-term memory impairment, interactions with alcohol, and psychomotor impairment have resulted in increased interest in alternative medications. The introduction of drugs such as buspirone (1986), SSRIs (from 1980 on), and the serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine (1994) have broadened the available treatment armamentarium for GAD significantly.
Tricyclic Antidepressants Tricyclic antidepressants (TCAs) have been in use in the treatment of GAD for many decades; however, data supporting their efficacy from controlled clinical trials are extremely limited. Imipramine is the only TCA shown to be effective in placebo-controlled trials of GAD patients without comorbid depression (84,85). In comparator trials, imipramine has been shown to have clinical efficacy comparable to the benzodiazepines (84,86,87). Benzodiazepines Five benzodiazepines (alprazolam, chlordiazepoxide, clorazepate, diazepam, and lorazepam) are currently labeled as treatments for anxiety (GAD) (88). Clonazepam and alprazolam are labeled specifically for the treatment of PD. There are a limited number of clinical trials demonstrating the efficacy of benzodiazepines in the treatment of GAD in its current (DSM-IV) definition (89,90); however, benzodiazepines have been shown effective in controlled studies using DSM-III criteria for GAD (91). Buspirone Buspirone is a serotonin receptor subtype 1A (5-HT1A) partial agonist with anxiolytic properties. In a metaanalysis of placebo-controlled comparator trials with benzodiazepines, buspirone showed comparable efficacy to the benzodiazepines in eight trials in 735 patients meeting DSM-III criteria (1 months duration of illness) for GAD (92). In a metaanalysis of eight placebo-controlled studies in over 500 GAD patients with coexisting depressive symptoms, buspirone demonstrated significant superiority to placebo (93). Prior recent treatment with a benzodiazepine ( 1 month) has been shown to predict poor response to subsequent buspirone treatment in GAD (92). This may be due to a combination of factors including the presence of benzodiazepine withdrawal, exacerbation of benzodiazepine discontinuation symptoms by buspirone and its metabolite a-(2-pyridinyl)-piperazine via enhancement of noradrenergic activity (94), and psychological factors such as patient expectations. SSRIs The first published study of a medication with significant serotonin reuptake inhibition properties in GAD involved a small, open-label trial of clomipramine (95). The suggestion of efficacy in this study, along with the success of clomipramine in treating other anxiety disorders, raised interest in pharmacologic agents for GAD that target the serotoninergic system. Following several years later, the first comparison trial of an SSRI in the treatment of GAD was published by Rocca and colleagues (89). Treatment efficacy of paroxetine was compared with the tricyclic imipramine and the
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benzodiazepine 2-chlordesmethyldiazepam in 81 subjects with GAD. Of the 63 patients who completed the randomized, 8-week study, 68% of the paroxetine group, 72% of the imipramine group, and 55% of the 2-chlordesmethyldiazepam group were judged to be responders as measured by a 50% or more decrease in Hamilton Anxiety Rating Scale (HAM-A) scores. The greatest improvement during the first 2 weeks occurred in the group receiving the benzodiazepine, as expected by the early relief of physical anxiety symptoms and insomnia provided by this class of medication. However, from the fourth week forward, the paroxetine and imipramine groups demonstrated superior benefits, particularly in the area of psychic symptoms of anxiety. More recently, the efficacy of paroxetine was demonstrated in a large, fixed-dose study of more than 500 patients with a DSM-IV diagnosis of GAD without major depression (96). Patients were randomized to receive paroxetine 20 mg/ day, paroxetine 40 mg/day, or placebo for 8 weeks. Patients receiving both doses of paroxetine demonstrated significant differences in the primary outcome measure, reduction in HAM-A score, versus placebo, with 68% on 20 mg paroxetine and 81% on 40 mg paroxetine rated as responders based on a Clinical Global Impression (CGI-I) score of 1 or 2, versus 52% on placebo. Venlafaxine Venlafaxine is an inhibitor of SNRI. Venlafaxine has recently been demonstrated in humans, using peripheral measures, to have primarily 5-HT reuptake inhibition properties at low doses (75 mg/day), with increasing norepinephrine (NE) reuptake inhibition properties at higher doses (375 mg/day) (97). Shown to be effective in the treatment of anxiety symptoms associated with major depression (98,99), the extended release (XR) form of venlafaxine has been shown to be effective in the treatment of GAD (DSM-IV criteria) in several placebo-controlled studies (100,101). In a placebo-controlled multicenter comparator trial, 405 patients with GAD were randomized to receive venlafaxine XR (75 or 150 mg/day), buspirone (30 mg/day), or placebo for 8 weeks. For the 365 patients for whom efficacy measures were obtained, there was no significant difference between groups in improvement on the primary outcome measure, the HAM-A. However, both doses of venlafaxine were shown to be superior to placebo in improving HAMA psychic anxiety and anxious mood scores at the endpoint (week 8), and venlafaxine demonstrated superiority to placebo and buspirone on the CGI-S at the same time point. More robust efficacy findings for venlafaxine were reported in a recent large, multicenter trial, involving 377 outpatients with GAD without comorbid depression (101). Patients were randomly assigned to receive either placebo or venlafaxine XR at one of three doses (75, 150, or 225 mg/day) for 8 weeks. Of the 349 patients included in the efficacy analysis, those receiving 225 mg/day demonstrated signifi-
cant improvement across seven of the eight outcome measures, and the 225-mg/day group was the only group to show significant improvement in scores on both of the CGI subscales (severity, global improvement) versus placebo. Other Agents (Trazodone, Nefazodone, Anticonvulsants, Partial GABA Agonists) In a randomized, placebo-controlled comparator trial of trazodone, diazepam, and imipramine in the treatment of 230 patients with GAD, trazodone was found to be superior to placebo yet somewhat less effective than diazepam and imipramine at the studys endpoint (84). The antidepressant nefazodone, which antagonizes the 5-HT2C receptor and inhibits the reuptake of both serotonin and NE, has shown promise in the treatment of GAD in an open trial (102). Anticonvulsants such as valproate and carbamazepine have been used in the treatment of GAD; however, evidence of their efficacy is primarily anecdotal, as there are no controlled clinical trials for either of these medications in the treatment of GAD (103). Other agents such as the partial GABA agonist abecarnil have not demonstrated significant efficacy versus placebo in GAD (101). In summary, although the benzodiazepines have been the mainstay of pharmacotherapy for GAD since their introduction, significant concerns regarding their long-term use in GAD have fueled the search for other effective treatments. Given the chronic nature of GAD, medications such as buspirone, and the antidepressants venlafaxine and paroxetine, which have fewer effects on cognitive and psychomotor function, now represent first-line therapies for GAD.
SOCIAL PHOBIA Social phobia (SP) (or social anxiety disorder) is reported to be the most common anxiety disorder with a 1-year prevalence of 7% to 8% and a lifetime prevalence of 13% to 14% in patients aged 15 to 54 years. Social anxiety disorder can be classified into two subtypesdiscrete and generalized. Level of disability with SP can be high, and 70% to 80% of patients have comorbid psychiatric disorders, particularly depression and substance abuse (104). Given the high degree of burden of illness in SP, its treatment has become a major priority. Historical Notes Liebowitz et al. (105) noted that SP, like atypical depression (106), had a specific responsivity to the MAOIs, whereas TCAs, although effective for PD and typical major depression, were not effective for SP (107). The efficacy of the MAOIs, which block reuptake of dopamine in addition to NE and serotonin, prompted speculation about a poten-
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tial dopaminergic component to the neurobiology of SP. Several open clinical studies have attempted to utilize the dopamine component concept in phamacotherapy with some success, e.g., seligiline (108) and pergolide (109). However, as data accumulated, other systems were also implicated, and the pharmacologic dissection approach seemed less applicable (see above). Positive results with the highpotency benzodiazepine clonazepam (110) suggested a GABAergic component. However, the suitability of benzodiazepines for long-term treatment of a chronic condition such as social anxiety disorder has been questioned. In addition, the benzodiazepines are ineffective against comorbid depression. RIMAs (Reversible Inhibitor of Monoamine Oxidase A) Although phenelzine demonstrated efficacy, the need for dietary restrictions severely limited its use. Moclobemide is a RIMA with a much lower propensity to induce hypertensive crises and has a more favorable side-effect profile. Moclobemide had been reported to have efficacy in early studies in the treatment of social phobia (111). However, conflicting results have subsequently been reported in placebo-controlled trials. Some studies have shown moclobemide to be more effective than placebo, whereas two recent, large, randomized placebo-controlled trials conducted in the United States have reported less robust results (112,113). Brofaromine, another drug in the RIMA class, may still hold promise. The safety and efficacy of brofaromine were examined in a multicenter trial of 102 outpatients with SP (114). Brofaromine produced a significantly greater change from baseline in Liebowitz Social Anxiety Scale (LSAS) scores compared with placebo. SSRIs Based on clinical evidence, SSRIs are the first-line treatment in social anxiety disorder (115). The most extensive database for the treatment of social anxiety disorder exists for the SSRI paroxetine. Several large, multicenter, placebo-controlled trials have been completed on three different continents (116119). In all cited studies, a significantly greater proportion of patients responded to paroxetine treatment compared with placebo. Paroxetine is currently the only SSRI licensed for use in this condition in the United States. The SSRIs are particularly attractive agents due to their favorable tolerance and safety profile, although typical SSRI side effects may nevertheless be problematic. Despite promising open studies with fluvoxamine, fluoxetine, and citalopram (120,121) only fluvoxamine has been tested under double-blind, randomized, placebo-controlled trial conditions (122). Like paroxetine, fluvoxamine yielded efficacy data superior to placebo. A report on a multicenter sertraline trial was pending at the time of this writing.
As is the case with PD, buspirone does not appear effective for SP as monotherapy in placebo-controlled doubleblind studies (123). It may, however, have a role in augmentation of the SSRIs. Serotonin/Norepinephrine Reuptake Inhibitors One open label study (124) aimed to evaluate the clinical response to venlafaxine in SP in 12 patients who were nonresponders to SSRIs, and to assess how the response could be influenced by Axis II comorbidity with avoidant personality disorder (APD). The duration of the study was 15 weeks using an open flexible-dosing regimen in individuals with or without concomitant APD. Venlafaxine improved SP and/or APD symptomatology, as demonstrated by decreasing LSAS total scores. Similar favorable open-labeled results have been reported for nefazodone (125,126). Placebo-controlled studies are warranted. Anticonvulsants A randomized, double-blind, placebo-controlled, parallelgroup study was conducted to evaluate the efficacy and safety of gabapentin in relieving the symptoms of social phobia. A significant reduction in the symptoms of social phobia was observed among patients on gabapentin compared with those on placebo as evaluated by clinician- and patient-rated scales (127). Adverse events were consistent with the known side-effect profile of gabapentin. The efficacy of other novel anticonvulsants remains to be investigated, although encouraging results have been reported for the gabapentin-like compound, pregabalin (128). POSTTRAUMATIC STRESS DISORDER Despite the high prevalence, chronicity, and associated comorbidity of PTSD in the community, relatively few placebo-controlled studies have evaluated the efficacy of pharmacotherapy for this disorder. The symptom overlap between PTSD and other pharmacotherapy-responsive disorders has suggested that pharmacotherapy might be effective. Nevertheless, in those placebo-controlled trials investigating the pharmacotherapy of PTSD that have been carried out, statistically significant efficacy for the treatment being studied has traditionally been inconsistent. One of the key methodologic limitations has been the fact that most studies have been conducted with war veterans, who are likely to constitute a more treatment-refractory population. SSRIs More recently, a total of 187 civilian outpatients with DSMIII-R PTSD (73% were women, and 61.5% experienced
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physical or sexual assault) were treated with the SSRI sertraline in a placebo-controlled design (129). Sertraline effectively diminished symptoms of PTSD of moderate to marked severity in comparison to placebo. Using a conservative last-observation-carried-forward analysis, treatment with sertraline resulted in a responder rate of 53% at the studys endpoint compared with 32% for placebo (p .008). Sertraline is the first medication approved by the FDA for the treatment of PTSD. Similar positive results have been reported for the SSRI fluoxetine in civilian populations (130,131). In a study by van der Kolk et al. (132), fluoxetine was found to be most effective in the nonveteran versus veteran portion of his study sample. Although published placebo-controlled data for paroxetine are not available, Marshall et al. (133) have argued that this particular SSRI may have specific advantages because of its relatively low activating properties. Direct comparative studies are lacking. Combat Veteran PTSD Among combat veterans, PTSD is a highly prevalent and often chronic disorder, persisting in as many as 15% of Vietnam veterans for at least 20 years (134). Treatment response in veterans with combat-related PTSD has been disappointing. Although anxiolytics, anticonvulsants, antipsychotics, and antidepressants, including SSRIs, have been tried, none has been consistently associated with improvement in all primary symptom domains (i.e., intrusive recollections, avoidance/numbing, and hyperarousal). In an open study using nefazodone, at mean daily doses of 430 mg (range, 200 to 600 mg/day), 19 treatment-refractory PTSD patients demonstrated benefit after 12 weeks (134). Doubleblind placebo controlled studies would be of interest. The efficacy of the antidepressant drug bupropion in the treatment of male combat veterans with chronic PTSD was investigated in an open-label study of 6 weeks duration (135). Improvement was seen in hyperarousal symptoms but was less significant than the change in depressive symptoms. Mirtazapine, a novel drug with both noradrenergic and serotoninergic properties, may be effective in individuals who demonstrate intolerance to side effects of, or a limited response to, SSRIs. Three of six severely refractory PTSD patients treated with mirtazapine were assessed as responders in a pilot study (136). Case reports have suggested benefit for refractory patients treated with venlafaxine (137) and risperidone (138). Raskind et al. (139) reported that the 1-adrenergic antagonist prazosin ameliorated combat nightmares in a small sample of veterans with PTSD. Monoamine Oxidase Inhibitors Traditional MAOIs have shown efficacy in the treatment of PTSD, but their use is limited by serious drug and food interactions. Moclobemide, a RIMA, is relatively free of
these limitations and is therefore potentially useful in the treatment of PTSD. Moclobemide was highly effective in an open-labeled design (140). However, in a double-blind, randomized, placebo-controlled, multicenter study, brofaromine, also a RIMA, failed to surpass efficacy levels seen with placebo. Thus, the role for RIMAs remains unclear at this time. Anticonvulsants Despite a long-recognized role for anticonvulsants in the treatment of PTSD (141), few placebo-controlled studies have been reported. An open study of divalproex reported favorable results (142). In a placebo-controlled study, patients treated with lamotrigine showed improvement on reexperiencing and avoidance/numbing symptoms compared to placebo-treated patients (143). The authors concluded that lamotrigine may be effective as a primary psychopharmacologic treatment in both combat and civilian PTSD and could also be considered as an adjunct to antidepressant therapy used in the treatment of PTSD. Further large-sample, double-blind, placebo-controlled trials are warranted. Summary and Future Directions Current management of PTSD is well summarized by Davidson et al. (144). Clearly, there are many challenges associated with the treatment of PTSD. Different patients with PTSD may not respond to pharmacotherapy in the same manner, and it is unclear whether this is related to gender, trauma type, or other factors. Antidepressants, particularly the SSRIs, are currently the form of pharmacotherapy for patients with PTSD with greatest support in the literature. Psychosocial techniques, such as cognitive-behavioral therapy or stress inoculation training, are effective and may be considered as adjunctive therapy with medication. Larger placebo-controlled studies for many different classes of medications would be desirable in moving the field forward. In addition, carefully conducted polypharmacy in which drug interactions are well understood may well be necessary for more difficult cases. In a review by Shalev and colleagues (145), a synthesis of findings in PTSD studies is provided. Most studies explored a single treatment modality (e.g., pharmacologic, behavioral). The cumulated evidence from these studies suggests that several treatment protocols reduce PTSD symptoms and improve the patients quality of life. The magnitude of the results, however, was often limited, and remission was rarely achieved. Given the shortcoming of unidimensional treatment of PTSD, it was suggested by the authors that combining biological, psychological, and psychosocial treatment yields the best results. A host of novel compounds show promise for the treatment of PTSD (146). Such classes of compounds include corticotropin-releasing factor antagonists, neuropeptide-Y
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enhancers, antiadrenergic compounds, drugs that downregulate glucocorticoid receptors, more specific serotoninergic agents, agents that normalize opioid function, substance P antagonists, N-methyl-D-aspartate facilitators, glutamatergic antagonists, and antikindling/antisensitization anticonvulsants. CONCLUSION Antidepressants are the logical first choice for most patients with anxiety disorders, based on their efficacy and tolerability. Although maintaining a role, the use of benzodiazepines for first-line or long-term therapy is now less likely. Does this mean that anxiety disorders are a variant of depression? Certainly, anxiety disorders and depression are highly comorbid. Untreated, the majority of patients with anxiety disorders eventually develop depression, whereas a large fraction of depressed patients suffer from clinically significant comorbid anxiety, if not an overt syndromal anxiety disorder. Pharmacologic dissection is clearly perilous, leaving us prone to inferences based on limited knowledge. Most of the antidepressants that successfully treat anxiety disorders manipulate the reuptake of serotonin, norepinephrine, or both. Altering the brain circuits through these modulatory neurotransmitters in turn has wide-ranging effects on many other systems in the brain. The release of CRH from extrahypothalamic sites like the amygdala is only one such system. Hence, there may be a common denominator among anxiety disorders and between anxiety disorders and depression at one or more points in these complex circuits. The observation, then, that antidepressants work for the four anxiety disorders discussed in this chapter warrants, in our opinion, only the following inference: it is highly likely that some substrate of the serotonin and norepinephrine systems is malfunctioning in several anxiety disorders and depression. This could be the locus of a common genetic or environmental vulnerability to both categories of illness. Although it will not likely tell us that anxiety and depression are fundamentally the same thing, the search for such common substrates and vulnerabilities, suggested but not guaranteed by the psychopharmacologic findings, is likely to be very illuminating. ACKNOWLEDGMENTS Dr. Gorman has received research support from Pfizer, Eli Lilly, the National Institute of Mental Health, and NARSAD. In addition, he has been a consultant and received honoraria from a number of pharmaceutical companies, including Pfizer, Eli Lilly, Bristol-Myers Squibb, Wyeth-Ayerst, SmithKline Beecham, Astra Zeneca, Janssen, Organon, Forrest, Parke-Davis, Lundbeck, Solvay, Merck,
Sanofi-Synthelabo, and Aventis. Dr. Kent has served as a consultant for Bristol-Myers Squibb and SmithKline Beecham. Dr. Coplan has received research support from SmithKline Beecham, Eli Lilly, and Janssen. In addition, he has served on a speakers bureau and/or an advisory board for the following companies: SmithKline Beecham, WyethAyerst, and Bristol-Myers Squibb. REFERENCES
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67
THE ECONOMIC BURDEN OF ANXIETY AND STRESS DISORDERS
RONALD C. KESSLER PAUL E. GREENBERG
No society can afford to guarantee universal health insurance coverage for treatment of all illnesses for all of its citizens. The number of illnesses is simply too large and the costs of treatment too great for such a guarantee even in the most economically advantaged societies. Resource allocation rules are consequently needed (1). The most widely accepted of these rules emphasizes cost-effectiveness. According to this rule, medical interventions are appropriate only if their expected benefits clearly exceed the sum of their direct costs and their expected risks (2). The difficulty in implementing this decision rule is that no obvious comparability exists between the single metric in which the costs of treatment are usually defined (i.e., dollars) and the many different metrics in which the benefits of treatment can be defined (e.g., physical pain, discomfort, psychological distress, and role impairment). To create transformations across these different metrics to allow for comparisons of costs and benefits on a single metric, a number of strategies have been developed, such as assessments of willingness to pay, time trade-off, standard gamble, and other utility or quasi-utility measures (3). In addition, a special interest has evolved in the indirect economic costs of illness and the benefits of treatment in terms of sickness absence and disability from work. The costs of these role impairments can be more easily assessed than the costs of other adverse effects of illness and represent the cost-benefit trade-off to purchasers of employer-sponsored health insurance plans (4). The most ambitious effort to date to evaluate the costs of illness in terms of role impairments and disabilities is the World Health Organization (WHO) Global Burden of Disease (GBD) Study, an initiative designed to generate a rank ordering of the diseases that create the greatest societal burdens in terms of impairment and disability (5). The over-
Ronald C. Kessler: Department of Health Care Policy, Harvard Medical School, Boston, Massachusetts. Paul E. Greenberg: Analysis Group, Cambridge, Massachusetts.
arching goal of GBD is to help health policy planners priori

tize disorder-specific resource allocation decisions. GBD fo
cuses on economic costs of illness using a metric known
as the disability-adjusted life year (DALY) (6), a weighted
composite that combines expected years of lost life with
expected years of decreased functioning due to a particular
disease (or constellation of comorbid diseases).
The first generation of GBD estimates suggest that men-
tal disorders, as a group, are the most costly diseases in the
world and that major depression, in particular, is the single
most costly disease among people in the middle years of life in terms of overall DALYs (5). Although the GBD rated mood disorders as considerably more costly than anxiety or stress disorders, focused cost-of-illness studies carried out subsequent to the publication of these estimates strongly suggest that the GBD underestimated the costs of anxiety and stress disorders and that the true costs of anxiety disor ders are actually quite comparable to the costs of mood disorders (7,8). Three reasons for the underestimation of the costs of anxiety and stress disorders in the GBD are worthy of note. The evidence to support all three of them is reviewed in this chapter. First, the epidemiologic studies used in GBD underestimated the prevalences of anxiety disorders. Sec ond, the estimated effects of specific diseases on functioning were based on the judgments of experts rather than on ob jective evaluations of actual impairments in representative samples of people with the diseases. These judgments underestimated the impairments due to anxiety disorders. Third, comorbidities were ignored in making GBD cost estimates. As shown below, a consideration of comorbidities is critical
in assessing the costs of anxiety disorders.
This chapter reviews available evidence on the economic
burdens of anxiety and stress disorders. By focusing on eight
factors that lead to the high societal costs of these disorders, we present evidence on the three sources of GBD underesti mation listed above. These eight factors are as follows. First, anxiety and stress disorders are among the most commonly occurring of all chronic diseases. Second, the prevalences of
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these disorders are increasing in recent cohorts in many countries. Third, these disorders have much earlier ages of onset than other commonly occurring chronic conditions. Fourth, anxiety and stress disorders are usually very chronic. Fifth, early-onset anxiety and stress disorders have a wide range of adverse effects on secondary outcomes, such as teen childbearing, marital stability, and educational attainment that have substantial economic implications. Sixth, these disorders are often associated with substantial impairments in role functioning. Seventh, anxiety and stress disorders are highly comorbid and usually temporally primary. Some of the disorders that are temporally secondary to anxiety and stress disorders, such as ulcers and substance abuse, have adverse economic effects that should be considered in part among the costs of anxiety and stress disorders. Eighth, despite the fact that effective treatments are available, only a minority of people with anxiety and stress disorders receives these treatments. Furthermore, those who receive these treatments usually do so only after many of the adverse effects of the disorders have occurred, making it very diffi cult to reverse the economic impacts of having had the disor ders even with successful treatments. Based on all these fac tors, anxiety and stress disorders have to be considered among the most costly of all chronic physical and mental disorders. PREVALENCES A new generation of psychiatric epidemiologic surveys, which began with the Epidemiologic Catchment Area (ECA) Study in the early 1980s (9), has dramatically increased our knowledge about the general population preva lences and correlates of anxiety disorders. The ECA Study was the first psychiatric epidemiologic study to use a fully structured research diagnostic interview designed specifi cally for use by lay interviewers to operationalize the criteria of a wide range of mental disorders. This interview, known as the Diagnostic Interview Schedule (DIS) (10), was used throughout the 1980s and early 1990s to carry out parallel epidemiologic surveys in a number of countries (11,12). The DIS was also used as the basis for an elaborated interview developed by the WHO and known as the Composite International Diagnostic Interview (CIDI) (13). The CIDI was designed to generate diagnoses according to the defini tions and criteria of both the International Classification of Diseases (ICD) and Diagnostic and Statistical Manual of Mental Disorders (DSM) systems. WHO auspices resulted in over a dozen large-scale, general-population CIDI surveys being carried out around the world over the past decade. Comparative analysis of these data has been facilitated by the creation of the WHO International Consortium in Psy chiatric Epidemiology (ICPE) (14), which is currently coor dinating national CIDI surveys in 25 countries around the world, with a combined sample size of over 150,000 re
spondents, as part of the WHO World Mental Health 2000 (WMH2000) Initiative (15). The DIS and CIDI surveys show that anxiety and stress disorders are the most commonly occurring of all mental disorders. Clear illustration can be found in a recent report based on the results of six CIDI surveys carried out in Latin America, North America, and Europe (16). These surveys found that the lifetime prevalences of DSM third edition revised (III-R) anxiety disorders were as high as 25%, whereas prevalences in the year before the survey were as high as 17%. These prevalences were higher than those of any other class of mental disorders in the vast majority of the surveys. (The exceptions were a survey of adolescents in Germany and of residents of a large catchment area in Mexico City. In both of these surveys, substance use disor ders were more common than anxiety disorders in the 12 months before the interview.) It was noted above that the epidemiologic data available to the GBD researchers, which came from the DIS surveys carried out in the 1980s, underestimated the prevalence of anxiety and stress disorders. Three of the most prevalent and seriously impairing anxiety disorders were involved in this underestimation: generalized anxiety disorder (GAD), social phobia, and posttraumatic stress disorder (PTSD). The reasons for the underestimations differ from one of these disorders to the next. In the case of GAD, prevalence was underestimated in the early DIS surveys due to the fact that the excessively unrealistic criterion in the DSM-III was operationalized by requiring that respondents endorse a statement that they worried about things that were not really serious or about things that were not likely to happen. This requirement is overly restrictive in two ways. First, there is no requirement in DSM that people with GAD have insight into their worries being excessive or unrealistic. Although they must be aware that they worry more than other people do, they can perceive others as worrying too little rather than themselves as worrying too much. Second, even in the presence of a recognition that their worrying is excessive, there is no requirement in DSM that the worries of people with GAD must be exclusively focused on things that are not important or unlikely to happen. Indeed, the heteroge neous worries that are characteristic of most people with GAD (e.g., excessive concerns about job stability, how the children are going to turn out, neighborhood safety, global warming, etc.) often focus on serious matters that have nontrivial probabilities of occurring. The restrictive assessment in the DIS led to the estimate that only about 3% of the population meet criteria for GAD at any time in their lives (17). Early CIDI surveys followed this same method of assessment and yielded similar preva lence estimates (18,19). Subsequent CIDI surveys expanded the assessment of excessive worry in GAD by asking re spondents if there was ever a time in their lives when they were worriers or when they worried a lot more than most other people in their same situation, without requiring that
Chapter 67: The Economic Burden of Anxiety and Stress Disorders
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the worry be exclusively about things that are not serious or not likely to happen. Prevalence estimates were found to be considerably higher when this modification was intro duced (20). In addition, these new studies investigated the implica tions of the requirements in the DSM-IV and ICD-10 that the worry in GAD persists for a minimum of 6 months and found that this requirement might be too restrictive. In particular, many people with chronic excessive worry report having fairly short episodes, each of which lasts for several weeks or months, that continue in a chronic intermittent course for many years. Such individuals are currently ex cluded from a diagnosis of GAD and, because of their high comorbidity with depression, are classified as being depressed even though their most prominent symptoms are often associated with anxiety rather than depression. The new WHO WMH2000 Initiative is investigating this mat ter in some detail in an effort to evaluate whether the classifi cation rules for GAD or mixed anxiety-depression should be modified to take these cases into consideration. In the case of social phobia, the underestimation in the early DIS surveys was due to the fact that all phobias were assessed in a single question that presented respondents with a long checklist of feared situations and asked them if they ever had unreasonably strong fears of these situations. In addition to being mixed in with a number of specific fears, only five social phobic situations, all involving performance fears, were included in the ECA list. This method of assessment led to the estimate that only 2.7% of the population meet criteria for social phobia at any time in their lives (21). Subsequent surveys that used the CIDI corrected this problem by screening for social phobia with a separate, longer list of social fears (both inter actional and performance). These later surveys consistently found social phobia to be much higher than in the DIS surveys, with lifetime prevalences as high as 13% (18) and current prevalences as high as 8% (22). Posttraumatic stress disorder was also wildly underesti mated in the early DIS surveys. This seems to have been a result of including only a single extremely long and complex screening question for PTSD in the first version of the DIS. This question began with a statement that many people live through events that are outside the range of usual human experience, such as combat in a war or sexual assault, and that people who experience these events often have bad emotional reactions such as nightmares, flashbacks, and changes in mood. Respondents were then asked if they ever had such an event that caused such reactions and, if so, to tell the interviewer what this event was. Subsequent debriefing showed that this question was too complex for many respondents, that the absence of a detailed event list interfered with effective memory search, and that the re quirement that the respondent describe the event out loud rather than give a yes or no response to event-specific ques tions led to underreporting of embarrassing events (23).
Assessments of PTSD in epidemiologic surveys that used the DIS led to the estimate that only about 1% of the United States population meet criteria for this disorder at any time in their life (2426). Subsequent surveys that used the CIDI modified the assessment of PTSD by including a detailed traumatic event checklist and by asking respon dents to give separate yes or no reports for whether each of these events ever occurred to them. In some CIDI surveys, a visual checklist was used that aimed at making it easier for respondents to report embarrassing events (e.g., Did event number five on the list ever occur to you? rather than Were you ever raped?). CIDI PTSD symptom assessment proceeded very much along the same lines as the DIS after documenting that trauma exposure had occurred. Yet the prevalence estimates obtained in the CIDI surveys were dra matically higher than in the DIS surveys, with lifetime prev alences as high as 12.2% (23,27). It should also be noted that psychiatric epidemiologic surveys have not, up to now, attempted to assess either DSM acute stress disorder (a short-term disorder that occurs in reaction to traumatic stress) or adjustment disorder (a disor der that occurs in reaction to nontraumatic stress). This is important because epidemiologic surveys that include as sessments of current nonspecific psychological distress typi cally find that a high proportion of the respondents who report clinically significant current distress in the anxietymood spectrum do not meet criteria for any of the anxiety or mood disorders typically assessed in these surveys (which usually include GAD, panic disorder, phobia, PTSD, obses sive-compulsive disorder, major depression, dysthymia, and mania). Given the extremely high prevalences of exposure to stressful events found in surveys of stress exposure (28), it is plausible to think that many of these people have a diagnosis of either acute stress disorder or adjustment disor der. The new WHO WMH2000 surveys mentioned earlier in this chapter are investigating this possibility by evaluating the link between stress and clinically significant nonspecific psychological distress among respondents who do not meet criteria for other anxiety or mood disorders. Taken together, these results suggest that the combined prevalences of all anxiety and stress disorders make these among the most commonly occurring classes of seriously impairing chronic conditions. A rough comparison is pro vided by the recently completed Midlife Development in the U.S. (MIDUS) survey carried out by the John D. and Catherine T. MacArthur Foundation. In this survey, paral lel assessments were made of commonly occurring physical and mental disorders, along with assessments of the effects of these disorders on day-to-day functioning (29). As in most other health surveys of chronic physical conditions, of which a great many exist (e.g., 30,31), the significantly impairing physical disorders with the highest reported prev alences in the year before interview were back problems (20.3%), arthritis (19.4%), hypertension (18.2%), and sea sonal allergies (15.7%). However, past health surveys of
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chronic physical conditions have seldom assessed emotional disorders along with these physical disorders. In doing so, the MIDUS survey found that 16.4% of respondents reported an anxiety or stress disorder exclusive of either major or minor depression, and that an additional 14.1% of re spondents reported major or minor depression. These find ings make anxiety-stress the fourth most commonly occurring impairing class of chronic disorders in the general population and major or minor depression the sixth most commonly occurring class of such disorders.
Increases for panic, specific phobia, agoraphobia, and obses sive-compulsive disorder, in comparison, have been more modest. Although these studies have not investigated either acute stress disorder or adjustment disorder, separate evi dence of secular increases in exposure to traumatic stress is consistent with the likelihood that the prevalences of these disorders have also been on the rise (33).
AGE AT ONSET The discussion up to now has not clearly distinguished between lifetime and recent prevalences. This is an important distinction because the societal burden of a disorder is largely associated with its prevalence at a point in time. The latter, in turn, is a complex function of lifetime prevalence, age at onset, and chronicity. The comparatively high recent prevalence of anxiety-stress disorders found in the MIDUS survey indicates that the combined effects of these three components are strong. This is true, in part, because anxiety and stress disorders occur to a high proportion of the popu lation at some point in the course of life. It is also true because these disorders have comparatively early ages at onset and high rates of chronicity. We focus first on age at onset. Retrospective reports about age at onset are routinely collected in epidemiologic surveys and used to estimate syn thetic onset distributions. Figure 67.1 presents KaplanMeier curves that show these onset distributions for any anxiety disorders in six countries the ICPE surveyed (16). The median age at onset of anxiety disorders in these surveys is less than 15 years of age. The only commonly occurring chronic physical disorder that has a similar age-at-onset dis tribution is hay fever. All other commonly occurring chronic physical disorders that have been shown to have an effect on role functioning have median ages at onset that occur much later, in some cases decades later, than anxiety disorders. Other mental disorders, including depression, substance use disorders, oppositional-defiant disorder, conduct disorder, and attention-deficit hyperactivity disorder, also have comparatively early ages at onset, although anxiety disorders are the temporally primary disorders in the vast majority of people with a lifetime history of any mental disorder (34). No information is available, in comparison, on age at onset of acute stress disorders or adjustment disor ders.
COHORT EFFECTS In addition to anxiety and stress disorders having great im portance because they are very common, they are also becoming increasingly prevalent over time. An illustration of this finding is presented in Table 67.1, taken from ICPE surveys carried out in six countries (16). These results are based on synthetic cohort analyses using retrospective ageat-onset reports to evaluate intercohort differences in lifetime risk of anxiety disorders over a period of four decades. The data are clear in showing that the relative odds of having an anxiety disorder have steadily increased over this period in all six countries. More detailed analyses of these and other data show that the increased prevalences of anxiety disorders are more pro nounced than the increased prevalences of other mental dis orders and that the apparent cohort effects for some other disorders, such as major depression, are largely due to increases in secondary disorders associated with primary anxi ety (32). Furthermore, the increasing prevalences within the anxiety disorders have been found to be especially pro nounced for GAD, generalized social phobia, and PTSD.
TABLE 67.1. THE EFFECTS (ODDS RATIOS) OF COHORT IN PREDICTING LIFETIME ANXIETY DISORDERS IN SIX COUNTRIESa
Age Group 1824 Brazil Canada Mexico Netherlands Turkey United States
a
2534 3.1* 1.7* 2.0 1.8* 1.7* 1.4
3544 1.8* 1.4* 2.0 1.5* 1.3 1.1
4554 1.0 1.0 1.0 1.0 1.0 1.0
23 64.4* 20.7* 2.3 88.4* 18.0* 27.9*
3.3* 1.9* 2.1 2.2* 1.8* 1.8*
CHRONICITY Although psychiatric epidemiologic surveys typically are cross-sectional, making it impossible to track illness course, indirect assessments of chronicity in these surveys have been carried out by comparing the ratios of current prevalence
Results are based on discrete-time survival analysis.

*Significant at the .05 level, two-sided test.
From WHO International Consortium of Psychiatric Epidemiology:
cross-national comparisons of the prevalences and correlates of
mental disorders: an ICPE study. Bull WHO 2000;78:420, with
permission.
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Cumulative Probability of Lifetime Disorder
Age
FIGURE 67.1. Age-at-onset distributions for any anxiety disorders in six countries. (Modified from WHO ICPE. Cross-national comparisons of the prevalences and correlates of mental disorders: an ICPE study. Bull WHO 2000;78:418, with permission.)
to lifetime prevalence in subsamples of respondents with specific lifetime mental disorders. Results clearly suggest that anxiety disorders are the most chronic of all mental disorders (35). This indirect evidence is consistent with the results of longitudinal studies carried out in clinical samples, which uniformly show that anxiety disorders are typically very chronic (3638). It is noteworthy that this high chron icity is not greater than that found among a number of impairing physical disorders, such as arthritis, asthma, and diabetes. However, the combined occurrence of high lifetime prevalence with early age at onset and high chronicity makes anxiety disorders unique. The one chronic physical disorder with comparable lifetime prevalence and early onset, hay fever, is active for only a few weeks each year. No systematic data exist on the chronicity of adjustment disorders, although epidemiologic data showing that PTSD is often a very persistent disorder (23,39) are consistent with the possibility that the same may be true for adjustment disorders.
ADVERSE EFFECTS ON SECONDARY OUTCOMES Virtually all cost-of-illness studies focus on the effects of prevalent disorders on current role functioning, taking cur-
rent roles as givens. The question implicitly addressed by these studies is whether it is in the financial interests of employers to invest in employee health care. Would the increased direct costs of treatment be offset by decreased indirect costs in such things as sickness absence, poor work performance, and accidents? This important question is dis cussed below. However, even when the focus is on narrow financial costs, the preceding is not the only question of importance in evaluating the societal costs of illness. Equally, if not more, important from a societal perspective is the question of whether the human capital potential of the individual is adversely affected by illness. Specifically, what difference does the existence of a particular chronic condition make to the individuals lifetime profile of pro ductivity? There is good evidence that anxiety disorders have longterm effects of this sort that are not captured in analyses of current role functioning. Both vital statistics (see Table 292A, Trend C in ref. 40) and prospective epidemiologic surveys (41) show that anxiety is associated with elevated risk of early death. Epidemiologic data also show that anxi ety is associated with elevated risk of subsequent unemploy ment (42,43). Clinical experience also suggests that anxiety is associated with more subtle decrements in role performance. It is com mon for patients with chronic GAD or PTSD, for example,
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to work at low-paying jobs because they are unable to cope with the stresses of higher paying jobs. This would be con sidered a cost of illness from the societal perspective, but not from the perspective of the employer. Very little scientific evidence exists regarding opportunity costs of this sort. The most sustained examination of these costs was carried out in a series of reports from the National Comorbidity Survey (NCS) in which retrospective reports about the ages at onset of individual mental disorders were used to define timevarying predictors of subsequent transitions in educational attainment (44), teen childbearing (45), marital timing and stability (46), and earnings (42,43). The results clearly show that mental disorders, in general, and anxiety disorders, in particular, are associated with significantly elevated risks of several different life course events that have important adverse financial implications. In terms of standardized (for sociodemographics) odds ratios, NCS respondents with some early-onset anxiety disorders had 40% elevated odds of high school and college failure, 30% elevated odds of teenage childbearing, 60% elevated odds of marital instabil ity, and 150% elevated odds of current unemployment at the time of interview. It is important to recognize that this constellation of adverse individual life course consequencesespecially school failure coupled with teen childbearing and marital instabilitymakes up the core components of welfare dependency. The costs of public assistance to single mothers with dependent children are paid by all taxpayers rather than by the welfare recipients themselves. For this reason, the component of welfare dependency costs explained by early-onset anxiety disorders should be considered a societal cost of anxiety. A number of innovative welfare-to-work programs are currently being carried out in response to wel fare reform legislation in the United States (e.g., 47,48). Interestingly, early reports on these programs suggest that their success hinges on the mental health of welfare recipi ents (49). EFFECTS ON CURRENT ROLE FUNCTIONING As noted in the previous subsection, a number of cost-ofillness studies have evaluated the effects of chronic condi tions on work role functioning. Most of these studies focus on physical disorders (e.g., 31). Most of those concerned with mental disorders focus on depression (e.g., 50). A small number of recent studies examined the effects of anxiety disorders on work functioning and found that these effects are quite substantial. These findings are an important ele ment in the argument that anxiety disorders are among the most costly of all chronic conditions. One of these studies, based on the NCS, examined the effects of individual mental disorders on work loss (missing a full day of work) and work cutback (either missing part
of a day or working less efficiently than usual) during the month prior to the interview (51). Each of the six anxiety disorders evaluated in that study (GAD, panic disorder, spe cific phobia, social phobia, agoraphobia, and PTSD) had significant effects on work-cutback days, from a high of 4.9 days per month associated with PTSD to a low of 1.1 associated with social phobia. None of the six was signifi cantly associated with work-loss days, implying that anxiety influences work largely by affecting the quality of perfor mance on days at work rather than by reducing the amount of time spent at work. The MIDUS survey yielded information that is even more interesting because it assessed both mental and physi cal disorders. Gross bivariate analyses showed that two men tal disorders, both anxiety disorders, were among the top five of all chronic conditions in terms of average per capita number of past month work impairment days. These top five included GAD (6.0 work impairment days per month), thyroid disease (5.8 days), tuberculosis (5.4 days), varicose veins (5.4 days), and panic disorder (5.3 days). Furthermore, multivariate analyses controlling for age, gender, and other sociodemographic factors found that the same two anxiety disorders were among the top six in terms of unique effects on work impairment (29). Calculating the salaryequivalent magnitude of these effects, using self-reported salaries and partialing out the effects of other comorbid mental and physical disorders, led to the estimate that the excess absenteeism and lost productivity directly associated with anxiety disorders is approximately $4.1 billion per year in the United States (8). PSYCHIATRIC COMORBIDITY A number of studies in both treatment samples (52) and general population samples (35) document high rates of psychiatric comorbidity among people with anxiety disor ders. Illustrative results from the NCS are reported in Table 67.2. Shown here are odds ratios between anxiety disorders and other mental disorders both for lifetime comorbidities and for comorbidities of disorders that were active in the 6 months prior to the interview. As the latter odds ratios are generally larger than the former, there must be comor bidities between the persistence of anxiety disorders and the persistence of other disorders. Several different possible explanations exist for these comorbidities. One is that prior history of other mental disorders might be associated, either as a risk factor or as a marker, with risk of the subsequent onset and persistence of anxiety disorders. The other is that anxiety disorders might be associated with the subsequent onset and persis tence of other mental disorders. As briefly mentioned above, epidemiologic studies have found that the latter possibility is more consistent with the data. Comorbid anxiety disor-

TABLE 67.2. COMORBIDITIES (ODDS RATIOS) BETWEEN DSM-III-R ANXIETY DISORDERS AND OTHER MENTAL DISORDERS ASSESSED IN THE NATIONAL COMORBIDITY SURVEY
Panic Disorder Lifetime comorbidities Major depression Dysthymia Mania Alcohol abuse/dependence Drug abuse/dependence Nonaffective psychosis Six-month comorbidities Major depression Dysthymia Mania Alcohol abuse/dependence Drug abuse/dependence Nonaffective psychosisa Phobias GAD PTSD
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6.6 4.8 10.4 1.6 3.0 20.1 14.4 12.2 15.8 1.4 3.9
4.1 3.0 7.9 1.7 2.2 4.7 6.4 4.4 13.4 2.3 3.9
9.4 12.5 9.6 2.0 2.9 15.0 17.8 21.5 10.4 2.7 5.0
5.2 4.9 6.2 1.7 3.2 9.4 7.1 7.4 9.4 2.2 2.9
All values on table are significant at the .05 level, two-sided test.
DSM-III-R, Diagnostic and Statistical Manual of Mental Disorders, third editionrevised; GAD,
generalized anxiety disorder; PTSD, posttraumatic stress disorder.
a Diagnostic hierarchy rules were suppressed in defining the disorders. Six-month nonaffective
psychosis (NAP) was too rare to calculate odds-ratios with any of the anxiety disorders.
From Tsuang MT, Tohen M, Zahner GEP, eds. Textbook in psychiatric epidemiology. New York: Wiley,
1995:181, 183, with permission.
ders are usually temporally primary to other comorbid men tal disorders (34). In addition, survival analyses show that temporally primary anxiety disorders are powerful predic tors of the subsequent onset and course of other mental disorders (35). In addition, panic disorder (53) and PTSD (54) are powerful predictors of suicidal behaviors. It is not clear from these results that anxiety disorders are causal. Another possibility is that anxiety disorders are early outcomes of other causal factors, either environmental or genetic, that cause both anxiety disorders and the other mental disorders with which anxiety disorders are comorbid. To the extent that anxiety disorders are causal, the adverse effects of mental disorders that are secondary to anxiety disorders should be counted among the adverse conse quences of anxiety disorders. A good case in point is second ary substance use disorders. Epidemiologic data show that early-onset anxiety disorders are significant predictors of subsequent substance use disorders, most likely mediated by self-medication (55). If these associations are causal, simulations suggest that the early intervention and success ful treatment of anxiety disorders would prevent as many as one-fourth of all substance use disorders in the U.S. (56). The component of the costs of substance use disorders due to prior anxiety disorders, therefore, should be counted among the costs of anxiety in a comprehensive evaluation. Whether anxiety disorders are causal and to what extent is an especially important issue in the case of depression, as many comparative cost-of-illness studies, including the
WHO GBD study, suggest that depression is the most costly of all mental disorders (5). Yet epidemiologic data show that close to half of all cases of depression are second ary to one or more preexisting anxiety disorders (57). This priority of anxiety over depression is never taken into con sideration in evaluating the costs of depression. Indeed, in those few instances where anxiety-depression comorbidity is considered, diagnostic hierarchy rules typically specify that the depression should be considered primary even though epidemiologic evidence consistently shows that anx iety is usually temporally primary. The rationale for this hierarchy of depression over anxiety is usually that the impairments associated with such cases is thought to be due to the depression rather than to the anxiety (58), but available evidence argues against this claim. A good case in point involves comorbidity between GAD and depression. The results in Table 67.3, taken from two U.S. national surveys, the NCS and the MIDUS, show that GAD without major depression is as important as major depression without GAD in leading to impairments in role functioning (20). Further analysis of these data showed that impairment is considerably higher among people with com orbid GAD and major depression than those with either GAD alone or major depression alone. Coupling the fact that GAD is temporally primary in the majority of these cases with the fact that GAD without major depression is associated with impairments equal to those of major depres-
988
TABLE 67.3. THE EFFECTS (ODDS RATIOS) OF 12-MONTH GENERALIZED ANXIETY DISORDER (GAD) WITHOUT MAJOR DEPRESSION (MD) AND MAJOR DEPRESSION WITHOUT GAD IN PREDICTING IMPAIRMENTS IN TWO U.S. NATIONAL SURVEYS, CONTROLLING FOR SOCIODEMOGRAPHICS AND OTHER 12-MONTH DSM-III-R DISORDERSa
GAD Without Major Depression (MD) Survey 1 Fair/poor perceived mental health High work impairment High social impairment 6.0* Survey 2 4.8* Major Depression (MD) Without GAD Survey 1 3.3* Survey 2 5.2* GAD Without MD vs. MD Without GAD Survey 1 1.6 Survey 2 0.8
3.5 2.5*
3.5 1.2
3.5* 2.0*
8.5* 1.6*
0.9 1.5
0.5 1.0
a The two surveys indicated here are the National Comorbidity Survey (Survey 1) (59) and the Midlife
Development in the U.S. Survey (Survey 2) (19). Results are based on separate regression equations
evaluating the effect of either GAD or MD in predicting one of the impairment measures in one of the
samples controlling for sociodemographic variables (age, gender, education, race-ethnicity,
employment status, marital status, and urbanicity) and other 12-month DSM-III-R disorders. Models in
the first two columns evaluate the effect of 12-month GAD on the subsample of respondents who did
not have 12-month major depression. Models in the middle two columns evaluate the effect of
12-month major depression on the subsample of respondents who did not have 12-month GAD.
Models in the last two columns evaluate the relative impairments of GAD without MD versus MD
without GAD in analyses that are confined to respondents in those two subsamples.
From Kessler RC, DuPont RL, Berglund P, et al. Impairment in pure and comorbid generalized anxiety
disorder and major depression in two national surveys. American Journal of Psychiatry 1999;
156:19151923, with permission.
sion without GAD, argues that this interactive effect is due at least as much to GAD as to major depression. PHYSICAL COMORBIDITY Although it has not been as extensively studied, evidence from clinical samples suggests that anxiety disorders have significant comorbidities with certain chronic physical dis orders (60,61). Table 67.4 presents nationally representative general
The results are odds ratios for the relationships between the 12-month prevalences of the DSM-III-R anxiety disorders assessed in the NCS and selected physical disorders assessed in the NCS chronic conditions checklist. As shown in the table, all but one of the odds ratios are greater than 1.0, indicating a positive relationship, and half are statistically significant at the .05 level. The NCS did not obtain information about age at onset for these physical disorders, making it impossible to examine whether anxiety disorders are temporally primary. In some cases, such as the strong association of some anxiety disor-
TABLE 67.4. COMORBIDITIES (ODDS RATIOS) BETWEEN 12-MONTH PREVALENCES OF DSM-III-R ANXIETY DISORDERS AND CHRONIC PHYSICAL DISORDERS IN THE NATIONAL COMORBIDITY SURVEY
GAD Arthritis Asthma Hypertension Kidney/liver disease Ulcer 1.7 1.9 1.5 2.0 3.1* Panic Disorder 2.1* 2.1* 2.2* 1.4 2.7* Simple Phobia 1.4 2.0* 1.5* 3.5* 2.7* Social Phobia 1.2 1.4 1.0 1.2 2.7* Agoraphobia 1.5 1.2 2.2* 2.3 2.9* PTSD 2.0* 1.7* 1.6 1.9 2.0*
989
ders with ulcers, the most plausible interpretation is that anxiety had a causal impact on the subsequent onset of the physical condition. In other cases, it is equally plausible that the physical condition helped promote the subsequent onset of anxiety. It is also possible that bidirectional causal influ ences were at work or that common causes led to both conditions. The eventual resolution of this uncertainty is important for an evaluation of the costs of anxiety disorders, as both the direct treatment costs and the indirect costs of physical disorders that are partly caused by anxiety should be included in evaluating the overall societal costs of anxiety. Comorbidities of anxiety with physical disorders are also important because of evidence that anxiety disorders reduce the quality of life of patients with physical disorders (62) and complicate the expression and course of physical disease (63). The most plausible explanation for these findings is that anxiety heightens sensitivity about both physical signs and symptoms and adequacy of treatment. This possibility is consistent with the finding that adjunctive treatments for comorbid anxiety often increase adherence to physical disorder treatment regimens (64). MENTAL HEALTH TREATMENT Effective psychological (65) and pharmacologic (66) thera pies exist for the treatment of most anxiety disorders. The indirect costs of anxiety disorders would consequently be expected to decline if a high proportion of people with these disorders sought treatment. However, a substantial part of the adverse effects of anxiety disorders are associated with secondary effects that occur early in life (e.g., teen childbear
ing, school failure), so it is important that treatment occur early in the course of the anxiety disorder. As anxiety disor ders have early ages of onset, initial treatment must occur during childhood or adolescence to be maximally effective in preventing adverse effects. We are aware of only two epidemiologic studies that investigated speed of initial treatment contact after first onset of anxiety disorders (67,68). These studies considered three anxiety disorders: GAD, panic disorder, and phobias. Both studies found that only a small proportion of people with childhood-onset or adolescent-onset anxiety disorders seek treatment prior to adulthood. Median delays between first onset and initial treatment contact were found to be more than a decade for some anxiety disorders. Furthermore, delays were found to be inversely related to age at onset. Because of these delays, only a minority of people with active anxiety disorders receives treatment in a given year. This is shown clearly in Table 67.5, which presents nationally representative U.S. data from the NCS on seeking professional help for DSM-III-R anxiety disorders during the 12 months prior to the survey. Only about one out of every four people with an anxiety disorder sought any type of treatment and only 13.3% received any type of mental health specialty treatment during this year. INAPPROPRIATE USE OF GENERAL MEDICAL SERVICES Although anxiety disorders typically are not treated, it is a great irony that people with anxiety disorders are often high
TABLE 67.5. 12-MONTH PREVALENCE OF PROFESSIONAL HELP-SEEKING IN SEPARATE SERVICE SECTORS IN THE NATIONAL COMORBIDITY SURVEY, BY 12-MONTH DSM-III-R ANXIETY DISORDER
Help-Seeking in Health Care Sectorsa Specialty Mental Disorders % 19.8 24.3 12.5 11.3 15.7 22.3 13.3 (SE) (3.5) (4.4) (1.5) (1.7) (3.2) (3.4) (1.4) % 31.8 35.2 16.4 15.3 24.9 28.2 18.7 Help-Seeking in Other Sectorsa
General Medical Disorder Generalized anxiety disorder Panic disorder Simple phobia Social phobia Agoraphobia Posttraumatic stress disorder Any % 18.6 21.5 8.5 5.9 13.6 12.5 9.0 (SE) (3.8) (5.1) (1.4) (0.9) (3.6) (2.4) (1.3)
Any (SE) (4.8) (5.6) (1.7) (1.9) (4.7) (3.6) (1.7)
Human Services % 10.8 21.0 10.6 8.0 12.5 16.3 9.6 (SE) (2.1) (4.2) (1.8) (1.3) (3.1) (2.3) (0.9)
Self-Help % 11.0 12.5 8.1 7.0 9.2 11.8 8.2 (SE) (3.0) (4.1) (1.3) (1.5) (2.9) (2.9) (1.0)
Any Help-Seekinga % 38.7 46.4 25.7 23.0 33.2 38.3 26.5 (SE) (4.3) (6.6) (2.3) (2.2) (5.0) (4.2) (2.0)
SE, standard error.

a Prevalence estimates are percentaged by rows. For example, in the first row of numbers, 18.6% is the percent of people with generalized
anxiety disorder who used general medical services, not the percent of people using general medical services who carried a diagnosis of
generalized anxiety disorder.
American Journal of Psychiatry 1999;156:117, with permission.
990
utilizers of primary care services. Indeed, people with un treated anxiety disorders make up a large proportion of the people who overuse primary care for only vaguely defined physical complaints (69,70). A recent anxiety disorders costof-illness study estimated that unnecessary medical care costs represented the largest single component of the cost of anxiety disorders in the U.S., equal to $23 billion per year (8). There is good reason to believe that aggressive screening and outreach efforts in primary care could detect these people with untreated anxiety, channel them into ap propriate treatment, and possibly have a major offset effect in reducing unnecessary primary care costs. Interventions to evaluate the magnitude of this offset effect are currently under way. OVERALL COSTS There have been two recent attempts to estimate the total annual cost of anxiety disorders in the U.S. The first, carried out by DuPont et al. (7) in 1996, estimated that the annual cost of anxiety disorders is $47 billion, whereas the second, carried out by Greenberg et al. (8) in 1999, estimated that this cost is $42 billion. These estimates are quite comparable to the annual cost of depression, which has been estimated to be between $44 billion (71) and $53 billion (72). The rough equivalence to the cost of depression is important because, as noted in the introduction, depression is generally considered the most costly of all physical or mental disorders among people in the early to middle years of life (5). The true societal costs of anxiety disorders, however, are actually a great deal larger than these estimates suggest, as the estimates were based only on a limited set of cost compo nents. The components included direct psychiatric treat ment costs, unnecessary medical treatment costs, work per formance costs in terms of sickness absence and workcutback days, and mortality costs (evaluated as lost earnings potential). The major excluded costs were long-term oppor tunity costs (i.e., excess unemployment and underemploy ment) and costs associated with comorbidity. The first of these two excluded costs is likely to be in excess of $2,000 per year for each person with a lifetime history of anxiety disorder (42), which is equivalent to an annual cost of more than $100 billion in the total U.S. population. The second of the excluded costs is impossible to calculate with currently available data, but would have to include substantial compo nents of the costs conventionally attributed to depression, alcohol, and drug abuse, and the many other mental and physical disorders with lifetime prevalences and courses that are influenced by the prior existence of anxiety disorders. DISCUSSION Anxiety disorders are unique among all chronic conditions, both physical and mental, in having a combination of very
high prevalence, early age at onset, high chronicity, and substantial role impairment. Although our knowledge about the comparative costs of different illnesses is too primitive to make precise comparisons, this conjunction of factors arguably makes anxiety disorders one of the most costly classes of illness in existence. Increased treatment is the key to reducing these costs. Although an increase in treatment will add to direct costs, the fact that available treatments are effective and that the adverse effects of anxiety are chronic means that the costs of effective treatment can be amortized over many years. The fact that most anxiety disorders have childhood or adolescent onsets means that early outreach and treatment could be carried out in collaboration with schools. Unfortu nately, as most people with anxiety delay initial contact with the treatment system for many years and usually present for treatment only after the onset of secondary comorbid disorders, little is known about the long-term effects of early treatment of pure childhood and adolescent anxiety disor ders. Demonstration projects and long-term follow-up stud ies are needed to evaluate these effects and to target opportu nities for incremental cost-effectiveness associated with refinements in diagnosis and treatment. Although the outcomes of such studies are uncertain, it is difficult to think of another disorder where an investment in early interven tion has as great a potential for long-term societal benefits.
ACKNOWLEDGMENTS Preparation of this chapter was supported, in part, by U.S. Public Health Service grants K05 MH00507, R01 MH46376, R01 MH49098, and RO1 MH52861, W.T. Grant Foundation grant 90135190, and an unrestricted grant from the Anxiety Disorders Association of America (ADAA). The authors appreciate the helpful comments of Naomi Breslau, Evelyn Bromet, Kathleen Merikangas, Bedirhan Ustun, and Uli Wittchen on an earlier version of this manu script. Dr. Kessler receives research support from Pfizer, SmithKline Beecham, and Wyeth-Ayerst, Inc.
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59. Kessler RC. The National Comorbidity Survey of the United States. Int Rev Psychiatry 1994;6:365376. 60. Sherbourne CD, Jackson CA, Meredith LS, et al. Prevalence of comorbid anxiety disorders in primary care outpatients. Arch Fam Med 1996;5(1):2734. 61. Zaubler TS, Katon W. Panic disorder and medical comorbidity: a review of the medical and psychiatric literature. Bull Menninger Clin 1996;60:A12A38. 62. Sherbourne CD, Wells KB, Meredith LS, et al. Comorbid anxiety disorder and the functioning and well-being of chronically ill patients of general medical providers. Arch Gen Psychiatry 1996; 53(10):889895. 63. Stoudemire A. Psychological factors affecting medical conditions: summary. In: Stoudemire A, ed. Psychological factors affecting med ical conditions. Washington, DC: American Psychiatric Press, 1995:187192. 64. Meichenbaum D, Turk DC. Facilitating treatment adherence: a practitioners guidebook. New York: Plenum Press, 1987. 65. Barlow DH. Anxiety and its disorders. New York: Guilford, 1988. 66. Haskett RF, Desmet A, Salinas EO. Venlofaxine XR in the treat ment of anxiety. Acta Psychiatr Scand 1999; in press. 67. Kessler RC, Olfson M, Berglund PA. Patterns and predictors of treatment contact after first onset of psychiatric disorders. Am J Psychiatry 1998;155:6269. 68. Olfson M, Kessler RC, Berglund PA, et al. Psychiatric disorder onset and first treatment contact in the United States and On tario. Am J Psychiatry 1998;155:14151422. 69. Katon W, Von Korff M, Lin E, et al. Distressed high utilizers of medical care: DSM-III-R diagnoses and treatment needs. Gen Hosp Psychiatry 1990;12(6):35562. 70. Manning WG Jr., Wells KB. Effects of psychological distress and psychological well-being on use of medical services. Med Care 1992;30:541553. 71. Greenberg PE, Stiglin LE, Finkelstein SN, et al. The economic burden of depression in 1990. J Clin Psychiatry 1993;54: 405418. 72. Greenberg PE, Kessler RC, Nells TL, et al. Depression in the workplace: an economic perspective. In: Feighner JP, Boyer WF, eds. Selective serotonin re-uptake inhibitors: advances in basic research and clinical practice. New York: Wiley, 1996:327363.
46. Kessler RC, Walters EE, Forthofer MS. The social consequences of psychiatric disorders: III. Probability of marital stability. Am J Psychiatry 1998;155:10921096. 47. Friedlander D, Burtless G. Five years after: the long-term effects of welfare-to-work programs. New York: Russell Sage Foundation, 1996. 48. Gueron JM, Pauly E. From welfare to work. New York: Russell Sage Foundation, 1991. 49. Danzinger SK, Corcoran M, Danzinger SH, et al. Barriers to the employment of welfare recipients [discussion paper]. Madison, WI: University of Wisconsin-Madison; Institute for Research on Poverty, 1999. Available at http://www.ssc.wisc.edu/irp/dplist.htm. 50. Mintz J, Mintz LI, Arruda MJ, et al. Treatments of depression and the functional capacity to work. Arch Gen Psychiatry 1992; 49:761768. 51. Kessler RC, Frank RG. The impact of psychiatric disorders on work loss days. Psychol Med 1997;27:861873. 52. DiNardo PA, Barlow DH. Syndrome and symptom co-occur rence in the anxiety disorders. In: Maser JD, Cloninger CT, eds. Comorbidity of mood and anxiety disorders. Washington, DC: American Psychiatric Press, 1990:205230. 53. Weissman MM, Klerman GL, Markowitz JS, et al. Suicidal idea tion and suicide attempts in panic disorder and attacks. N Engl J Med 1989;321:12091214. 54. Kessler RC, Borges G, Walters EE. Prevalence and risk factors of lifetime suicide attempts in the National Comorbidity Survey. Arch Gen Psychiatry 1999;56:617626. 55. Kessler RC, Crum RM., Warner LA, et al. The lifetime co occurrence of DSM-III-R alcohol abuse and dependence with other psychiatric disorders in the National Comorbidity Survey. Arch Gen Psychiatry 1997;54:313321. 56. Kessler RC, Aguilar-Gaxiola S, Andrade L, et al. [Mental-substance comorbidities in the ICPE surveys]. Psychiatria Fennica 2001;32(suppl. 2):6280. 57. Kessler RC, Nelson CB, McGonagle KA, et al. Comorbidity of DSM-III-R major depressive disorder in the general population: results from the US National Comorbidity Survey. Br J Psychiatry 1996;168(suppl 30):1730. 58. Roy-Byrne PP. Generalized anxiety and mixed anxiety-depres sion: association with disability and health care utilization. J Clin Psychiatry 1996;57:8691.
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MECHANISM OF ACTION OF ANXIOLYTICS
JOHN F. TALLMAN JAMES CASSELLA JOHN KEHNE
Drugs to reduce anxiety have been used by human beings for thousands of years. One of the first anxiolytics and one that continues to be used by humans is ethanol. A detailed description of ethanols action may be found in Chapter 100. A number of other drugs including the barbiturates and the carbamates (meprobamate) were used in the first half of the 20th century and some continue to be used today. This chapter focuses on current drugs that are used for the treatment of anxiety and approaches that are currently under investigation.
CORTICOTROPIN-RELEASING FACTOR (CRF) Corticotropin-releasing factor (CRF) is a 41 amino acid peptide that plays an important role in mediating the bodys physiologic and behavioral responses to stress (1). Figure 68.1 illustrates that this role of CRF may be mediated by multiple sites of action. As a secretagogue, CRF stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary. In addition, CRF plays a neurotransmitter or neuromodulatory role through neurons and receptors distributed in diverse brain regions (2). CRF neurons, localized in the hypothalamic periventricular nucleus, are a major mediator of stress-induced activation of the hypothalamicpituitary-adrenal (HPA) axis, whereas pathways innervating limbic and cortical areas are thought to mediate the behavioral effects of CRF. There is a large body of both preclinical and clinical literature implicating a key role of CRF in affective disorders such as anxiety and depression. A significant clinical literature suggests that dysfunctions of CRF in its role as a hormone in the HPA axis or as a neurotransmitter in the brain may contribute to the etiology of a variety of psychiatric
conditions, including anxiety and depression (3). The link between CRF and depression is particularly strong, as numerous clinical studies have demonstrated that depressed patients show elevated cerebrospinal fluid (CSF) levels of CRF, elevated plasma cortisol, and a blunted ACTH response following intravenous CRF. Successful antidepressant treatment was shown to have a normalizing effect on CRF levels. A role of CRF in anxiety disorders has also been postulated, though the clinical evidence is not as strong as it is for depression (4). Preclinical studies have demonstrated that CRF administered exogenously into the central nervous system (CNS) can produce behaviors indicative of anxiety and depression, for example, heightened startle responses, anxiogenic behaviors on the elevated plus maze, decreased food consumption, and altered sleep patterns. The anxiogenic effects of CRF are not blocked by adrenalectomy, suggesting that they are centrally mediated effects occurring independently of the HPA axis (5). Other studies strengthening the link between CRF and anxiety include recent work by Kalin et al. (6) demonstrating that a fearful phenotype in monkeys is associated with increased pituitary-adrenal activity and increased brain CRF levels. Other studies have shown that exposure to early postnatal separation stress in rat pups results in elevated levels of CRF messenger RNA (mRNA) in brain regions including the paraventricular nucleus (PVN) and the central nucleus of the amygdala (7,8). Molecular Mechanism of Action A substantial scientific effort has been directed toward characterizing the molecular biology of CRF pathways (9). Perrin and Vale (9) first isolated CRF and identified it as a secretagogue for ACTH in primary cultures of rat pituitary cells. CRF activity is shared by two nonmammalian peptides, sauvagine and urotensin I, which share a 50% homology with CRF, and by a new mammalian peptide, urocortin, which has a 45% sequence homology.
John F. Tallman, James Cassella, and John Kehne: Neurogen Corporation, Branford, Connecticut.
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FIGURE 68.1. The role of corticotropin-releasing factor.
CRF acts through two Gs-protein coupled receptors, the CRF-1 and CRF-2 receptor subtypes (9,10). CRF-1 receptors show homology to a number of other neuropeptide receptors, including vasointestinal peptide (VIP) and calcitonin. Three splice variants of the CRF-2 receptor subtype, the CRF-2 , CRF-2 , and CRF-2 , and two splice variants of the CRF-1 receptor, have been identified (9). Molecular characterization studies have demonstrated that there is approximately a 70% sequence homology between CRF-1 and CRF-2 receptor subtypes. Cloning of the human CRF-2a gene revealed that it is 94% identical to the rat CRF-2 receptor and 70% identical to the human CRF-1 receptor. There is currently no evidence of the existence of the CRF2 receptor in humans. CRF-1 and CRF-2 receptors have different pharmacology and different localizations in the brain and periphery. In situ hybridization and receptor autoradiography techniques been used to map the relative distributions of CRF-1 and CRF-2 receptors in the rat brain (11,12). High expression of CRF-1 receptors was seen in the pituitary, and in a number of brain regions including the PVN of the hypothalamus, cerebral cortex, olfactory bulb, cerebellar cortex, and basolateral and medial amygdala. In contrast, high densities of CRF-2 are found in more circumscribed regions, including the lateral septum, ventromedial nucleus of the thalamus, and choroid plexus. Moderate densities of CRF-2 receptors were reported for the medial amygdala and dorsal raphe nucleus. Further characterization has indicated that the CRF-2 splice variant accounts for the brain localization of CRF-2 receptors, whereas the CRF-2 accounts for choroid plexus. Urocortin, rather than CRF, most closely maps to CRF-2 receptors, leading to the suggestion that it may be the endogenous ligand for CRF-2 receptors. Recent work has shown that the distribution of CRF-2 receptors may differ significantly in the nonhuman primate brain relative to the rodent brain such that the CRF-2 subtype may play a more significant role than previously thought (13). CRF receptors utilize 3,5-cyclic adenosine monophosphate (cAMP) as a second messenger in the pituitary and brain and can be regulated by chronic activation. Thus,
desensitization following exposure to CRF has been demonstrated both in vitro (14) and in vivo (15). Furthermore, chronic stress can down-regulate CRF receptors and decrease CRF-stimulated cAMP production in multiple brain areas (16,17). Down-regulation of pituitary CRF receptors following adrenalectomy presumably results from decreased ACTH mediated inhibitory feedback, which produces excess CRF stimulation. There are a number of pharmacologic agents available for dissecting the functional significance of CRF-1 and CRF-2 receptors. Much work has been carried out using the peptide antagonists -helical-CRF (941) and D-Phe CRF (1241). However, these compounds have shortcomings in that they do not penetrate the CNS and therefore have to be administered intracerebrally. Furthermore, they do not discriminate between CRF receptor subtypes and therefore do not allow a determination of their relative contributions to behavior. More recently, the development of selective, nonpeptidic antagonists of the CRF-1 receptor such as CP 154,526 (18) have provided important pharmacologic tools for the analysis of CRF-1 receptor function. Mutation studies have demonstrated that peptide and nonpeptide antagonists bind to different domains of the CRF-1 receptor (9). To date, selective CRF-2 antagonists have not been described, though recently, nonpeptide dual antagonists of the CRF-1 and CRF-2 receptors have been described (19). In addition to CRF receptor subtypes, another potential target for pharmacologic manipulation of CRF is the CRF binding protein (CRF-BP), a 322 amino acid peptide. CRFBP is a specific carrier for CRF and related peptides found in human plasma and brain. CRF-BP is thought to be a modulator of CRF activity. The CRF-BP is found in high densities in the rat amygdala, cortex, and bed nucleus of the stria terminalis. Genetically Altered Mouse Models Studies utilizing transgenic and knockout mouse models have provided important information with regard to the contribution of CRF and CRF receptor subtypes to processes including energy balance, emotionality, cognition, and drug dependence (20). This chapter focuses on the evidence implicating CRF and CRF receptors in anxiety states. Overexpression of CRF in transgenic mice produced anxiogenic effects using either the black-white box test (21) or the elevated plus maze (22). The latter effect was reversed by central administration of the CRF receptor antagonist -helical CRF, but not by adrenalectomy, supporting the role of central CRF pathways independent of the HPA axis (22). Studies using antisense directed against CRF in rats have produced evidence of anxiolytic activity (23). Finally, overexpression of CRF-BP is anxiolytic, whereas binding protein knockout mice (in which free CRF levels are elevated) display an anxiogenic phenotype in the elevated plus
Chapter 68: Mechanism of Action of Anxiolytics
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maze (24). These data generally support the link between CRF and anxiety. More recently, several studies have highlighted the importance of the CRF-1 receptor subtype in anxiety. CRF1 knockout mice demonstrated a diminished anxiogenic response on the elevated plus maze and decreased ACTH and corticosterone responses to restraint stress (25). Similar findings were reported by Timpl et al. (26), using the blackwhite box anxiety paradigm. Furthermore, inactivation of the CRF-1 receptor with an antisense oligonucleotide was shown to reduce the anxiogenic effect of intraventricularly administered CRF (23). Liebsch et al. (27) provided evidence of anatomic localization by showing anxiolytic activity from CRF-1 antisense that was chronically infused into the central nucleus of the amygdala, an area of the limbic system shown by Michael Davis, Joe LeDoux, and others to be important in mediating fear and anxiety processes. Finally, CRF-2 knockout mice show anxiety-like behavior and are hypersensitive to stress (28), indicating that the CRF-2 receptor has an opposite functional role to that of the CRF-1 receptor. Thus, it could be argued that CRF-2 agonists, rather than antagonists, might be potentially useful as anxiolytic agents. Another potential use for CRF antagonists is in the treatment of drug abuse. Several lines of evidence suggest that during the period of withdrawal from drugs of abuse such as ethanol, morphine, and cocaine, there is an activation of central CRF pathways. Anxiety is among the many physical symptoms of drug withdrawal, and given the link that has been made between CRF and anxiety, it is not surprising that CRF-1 receptor knockout mice demonstrated decreased anxiety responses during withdrawal from alcohol (26). Current Drugs in Development A number of nonpeptidic, small-molecule compounds that show high selectivity for the CRF-1 receptor have been proposed for the treatment of depression, anxiety, and stress disorders (2931). These include CP-154,526 (Pfizer), and a methylated analogue, antalarmin (Pfizer); SC 241 (Dupont); NBI 30775 (aka R-121919; Janssen-Neurocrine); and CRA 1000 and CRA 1001 (Taisho Pharmaceuticals). An extensive preclinical literature has investigated potential anxiolytic effects of these compounds. Studies using CP154,526 have demonstrated anxiolytic-like effects in some (3234) but not all (33,34) preclinical anxiolytic paradigms evaluated. Griebel et al. (33) proposed that high-stress conditions may be required to demonstrate efficacy of CRF-1 receptor antagonists. CP-154,526 produces an anxiolytic effect in the separation-induced vocalization assay (35), an animal model of anxiety in which a preweaning rat pup separated from its litter emits a series of ultrasonic vocalizations that can be dose-dependently suppressed by either benzodiazepine or nonbenzodiazepine anxiolytics (36).
Of the compounds listed above, the compound discovered by Neurocrine Biosciences and licensed by Janssen Pharmaceuticals, R-121919, has proceeded the furthest in clinical evaluation. A recent report (37) describes results from a phase II, open-label, dose-escalating trial in which 20 severely depressed (Hamilton Depression Score 25) patients were administered R-121919 in one of two dose ranges: 5 to 40 mg, or 40 to 80 mg. In the low-dose group, 50% of the patients responded positively to treatment as indicated by a reduction in the Hamilton Depression Score of at least 50%, and 20% were remitters (score 8). In the middle-dose group, 80% responders and 60% remitters were reported. In addition, no significant untoward side effects were reported, and basal or stress-induced levels of ACTH or cortisol where unaffected, suggesting that chronic blockade of the HPA axis might not necessarily produce untoward side effects. Although these preliminary data are promising, it is important to bear in mind that they were gathered using an open label design without placebo control. Firm conclusions regarding the efficacy and safety of CRF-1 antagonists in depression and anxiety will require more rigorous double-blind, placebo-controlled trials. R121919 is no longer under development because of reported elevations in liver enzymes; however, Neurocrine Biosciences has announced that further candidates are being pursued for clinical evaluation. As mentioned previously, selective CRF-2 antagonists have not been described, though recently, nonpeptide dual antagonists of CRF-1 and CRF-2 receptors have been described (19). As there is contradictory evidence regarding the role of CRF-2 receptors in mediating anxiety, careful preclinical and clinical evaluation of these compounds will be needed to validate the contribution of CRF-2 receptors.
Future Drugs and Directions As indicated above, a number of drug companies have dedicated significant efforts to identifying potent and selective CRF-1 receptor antagonists suitable for clinical development. To date, no compounds have completed phase II evaluation. Clearly, a challenge for the future will be to achieve this milestone and, in the process, validate with carefully executed clinical trials the concept that CRF-1 receptor antagonists are novel anxiolytics and/or antidepressants. Also, given increasing evidence of the importance of CRF-2 receptors in the human brain, significant efforts should be dedicated to evaluating this target for anxiety. It will be of interest to determine if agonists, rather than antagonists, of the CRF-2 receptor have anxiolytic profiles. Finally, the prospect of identifying additional CRF receptor subtypes, as well as other receptors for peptides, such as VIP, that are involved in the regulation of stress, provides fertile ground for future investigations.
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GABA A RECEPTOR MODULATORS (BENZODIAZEPINES AND RELATED DRUGS) A majority of the synapses in the mammalian CNS use the amino acids l-glutamic acid, glycine, or -aminobutyric acid (GABA) for signaling. GABA is formed by the decarboxylation of l-glutamate, stored in neurons, and released, and its action is terminated by reuptake; GABAs action mimics the naturally occurring inhibitory transmission in the mammalian nervous system. Because of these findings, it has been accepted for over 20 years that GABA fulfills the characteristics of a neurotransmitter (38). Along with l-glutamate, acetylcholine, and serotonin, GABA possesses two different types of receptor conserved across different species and phyla that control both excitation and inhibition. Molecular biological studies of the receptors causing these effects have indicated that GABAs effects on ionic transmission (ionotropic) and metabolism (metabotropic) are mediated by proteins in two different superfamilies. The first superfamily (GABAA receptors) is a set of ligand-gated ion channels (ligand-gated superfamily) that convey GABAs effects on fast synaptic transmission (39). When a GABAA receptor is activated, an ion channel is opened (gated) and this allows chloride to enter the cell; the usual result of chloride entry is a slowing of neuronal activity through hyperpolarization of the cell membrane potential. The second superfamily (GABAB) is slower, mediating GABAs action on intracellular effectors through a seven transmembrane spanning receptor (serpentine superfamily) that modulates the action of certain guanine nucleotide binding proteins (G proteins) (40). Through their activity on other effector systems, G proteins can change second messenger levels, altering signal transduction and gene expression, or open ion channels that are dependent on the G-protein subunit activities (41). Both excitatory and inhibitory activities are possible on a time scale that is longer than GABAA receptor mediated events. There is extensive heterogeneity in the structure of the GABAA receptor members of the ligand-gated superfamily. These receptors are the targets of a number of widely used and prescribed drugs for sleep, anxiety, seizure disorders, and cognitive enhancement; they may also contribute to mediating the effects of ethanol on the body. Structure and Molecular Pharmacology of GABA A Receptors It is well established that the GABAA receptors possess binding sites for the neurotransmitter GABA, as well as allosteric modulatory sites for benzodiazepines, barbiturates, neurosteroids, anesthetics, and convulsants (4244). The initial cloning of complementary DNAs (cDNAs) coding for the subunits of GABAA receptors indicated that the chloride channel gated by GABA is intrinsic to the structure of the receptor and that each of the binding sites also possesses
specific requirements for subunit composition (45,46). At present, almost 20 different cDNAs have been identified and classified into six classes based upon sequence homology. Cloned from vertebrates, there are six , four , four , one , one , and two subunits and some splice variants; the subunits share a basic motif where the amino acids span the membrane four times. This four transmembrane spanning motif is shared with subunits that form other receptor members of the ligand-gated superfamily (39). Extensive mutagenesis and structural examination has been carried out with the GABA and acetylcholine family of receptors (47,48). Acetylcholine receptors have been shown to possess a pentameric subunit structure with a heterogeneous subunit composition; evidence for this conclusion has been obtained through the use of monoclonal antibodies and through direct electron microscopic visualization of the densely packed receptor in the Torpedo eel. Similar electron microscopic analysis of GABAA receptors has been carried out (49). It is thought that the native GABAA receptors also possess such a pentameric structure with general composition of 2 , 2 , and one subunit forming the majority of the GABAA receptors in vertebrates. Evidence of this has been more circumstantial, generated by molecular biological and pharmacologic inferences, described below, and by the behavior of solubilized recombinant complexes on sucrose gradient centrifugation in the presence and absence of different subunit specific antibodies (50). The natural receptor in endogenous tissue appears to be also pentameric (51). Evidence from studies of acetylcholine receptors has also indicated that the second transmembrane spanning sequence forms the actual ion channel of acetylcholine receptors, and that mutations of amino acids at the inner (cellular) side of the membrane are responsible for the ability of specific cation ions to pass through the channel pore (48). The ionic selectivity can be changed by altering the charge of some of these specific amino acids, and the acetylcholine receptor can be forced to gate chloride, rather than sodium, by such changes. Thus, a relatively firm case for the involvement of this spanning region in the formation of the ion channel can be made. Because the core ion pore is highly conserved among the large number of GABAA receptor subtypes, a number of drugs that interact nonspecifically with all the members of the GABAA receptor family were identified in the past. These include anesthetic barbiturates, picrotoxin, neurosteroids, and some organic insecticides (4244). More recently, because the ion channel shows little variation between GABAA receptor subtypes, it has not been as active a target for pharmaceutical discovery as the convenient allosteric modulatory site for benzodiazepines, drugs discovered by chance almost 40 years ago. Originally, two subunits of the GABAA receptor family were cloned and, when expressed in oocytes, were capable of forming a receptor that would gate chloride in response to GABA (45). At that time, some responses were seen to barbiturates, toxins, and benzodiazepines. It is now known
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that a full response to the benzodiazepines requires the incorporation of a third subunit, the subunit (52). One of the major forms of native GABAA receptor in vertebrates probably has the structure 1 2 2, most likely in a 2:2:1 stoichiometry. The 1 subunit contains the major site that is photoaffinity labeled with the benzodiazepine 3H-flunitrazepam at His 101 (53). For the functional modulation of GABAergic activity by benzodiazepines, in addition to the subunit, a subunit must be incorporated into the complex. Thus, there is reasonable evidence that benzodiazepines and related drugs stimulate GABA activity without opening channels directly; depending on their ability to potentiate GABA activity, they are called full or partial agonists. The binding site for these drugs incorporates a binding site composed of components of both and . Other drugs (called inverse agonists) may occupy the same site to negatively modulate the action of GABA, such as -carboline derivatives. Yet a third class of compounds exist, drugs such as flumazenil, may occupy the site as antagonists of both agonist and inverse agonists. By themselves, these antagonists have no affect on GABAergic activity and are behaviorally silent. The important allosteric modulatory effects of drugs at the benzodiazepine site were recognized early and the distribution of activities at different receptor subtypes has been an area of intense pharmacologic discovery for many years (39,4244). The details of some of these findings are described later. Because the expression of an or subunit by itself does not form a functional receptor (54), but expression of both together constitute a functional GABAA receptor, the binding sites for GABA could be associated with the combination of the two subunits. Systematic mutagenesis of and subunits have identified a number of amino acids on both subunits that appear to contribute to the ability of GABA to bind to the GABAA receptor and modulate chloride conductance (53). Thus, the GABA binding sites are also complex, composed of a binding pocket that is made up of amino acids from both subunits; there are two GABA binding sites per receptor [located at the two identical (or homologous in the case of heterogeneous mixtures of subunits) interfaces], and electrophysiologic studies indicate that both must be occupied for the chloride channel to open. Perhaps the most interesting aspect of these mutagenesis studies is the observation that the GABA binding site (two per complex) and the benzodiazepine binding site (one site per complex) are structurally related to one another; homologous amino acids that contribute to binding in each case are found at similar positions in each subunit. They represent in the case of the GABA binding site interfaces and in the case of the benzodiazepine binding site the interface (Fig. 68.2). By binding to the benzodiazepine binding site, the drugs clearly give a positive allosteric signal to the receptor and/or to the GABA binding sites; this is reflected in a change in affinity at a low-affinity GABA
binding site (43). This signal is analogous to the signal that a single GABA molecule binding to one of the GABA binding sites gives to the receptor and second GABA binding site. The original finding relating GABA and benzodiazepines allosterically showed evidence of a similar signal being transmitted from the occupied GABA binding site to the benzodiazepine site, resulting in a higher affinity state at the benzodiazepine binding site in the presence of GABA at its binding site (55). This type of pseudosymmetric binding site is characteristic of allosteric protein binding interactions and is found also in acetylcholine and in glycine receptors (46,56,57). The theoretical details of such interactions allow one to rationalize how a set of drugs like benzodiazepines could stabilize a channel open state but not be able to open the channel directly. Variations in these binding sites that are dependent on different , , and subunit amino acid sequences, particularly and as the components of the benzodiazepine binding site, underlie the heterogeneity of GABAA receptors and point to the possibilities for GABAA receptor subtypespecific drugs. Genomic Organization of GABA A Receptor Subunits The natural association of compositions of GABAA receptors is also underscored by the genomic organization of the subunits. There is a cluster of the genes coding for 1, 2, and 2 subunits on chromosome 5 and similar clusters of other subunits on other chromosomes such as 4 and 15 (58, 59). They point to the phylogenetic age of GABAA receptors, and similarities in organization point to the possibility that ancestral GABAA receptor gene cluster duplications spawned some of the different clusters coding for unique GABAA receptor isoforms, whereas mutations in individual subunits may have created additional diversity. Such an organization also points to probable coordinated control of subunit production and many interesting aspects of cellular and brain regional regulation of expression yet to be examined. Regional diversity also can mean functional diversity and drugs that affect certain aspects of behavior, but not others. Functional Activities of Therapeutics at Individual Recombinant Constructs One reason for the diversity of subtypes of GABAA receptors is that this is how neurons integrate information and change the behavioral status of the animal. Whereas GABAA receptors are found on many neurons, particularly local interneurons, some GABAA receptor subtypes are selectively localized to specific brain regions, specific cell layers within those regions, and even specific parts of cells (6062). Each subtype has a unique set of electrophysiologic and pharmacologic properties. A number of factors can determine the response properties of GABAA receptor subtypes. The most
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FIGURE 68.2.
-Aminobutyric acid (GABA) binding site
interfaces.
important determinant is subunit composition. Secondarily, these receptor subtypes can have unique profiles of modulation driven by membrane potential, ion gradients, biochemical conditions, and, last but not least, drugs. Attempts have been made to categorize GABAA receptors pharmacologically in terms of responses to specific drugs that interact with the benzodiazepine binding site. Thus, in the original nomenclature, two subtypes of GABAA receptor were described (63). They were called type 1 and type II receptors and were defined in terms of differential affinity for CL 218,872 and a number of other compounds. We now know that type I GABAA receptors contain the composition 1 x 2 and are responsive to CL 218,872, a related
compound zaleplon, and zolpidem (64). Both zaleplon and zolpidem are marketed hypnotics. Type II receptors are a mixed heterogeneous class containing 2 x 2, 3 x 2, and 5 x 2. 4- and 6-containing x 2 subunit combinations are generally agreed to constitute a separate category of GABAA receptors due to their lowered affinity for classic benzodiazepine site ligands (65). For example, flumazenil has a very low affinity for these 4- and 6-containing GABAA receptor recombinant constructs compared to 1-, 2-, 3-, or 5-containing GABAA receptors (100 nM versus 0.5 nM). The differences in amino acids between the subunits pointed to the mutagenesis studies described above to delineate GABA and benzodiazepine binding sites.
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Thus, affinity differences in the benzodiazepine binding site have been a primary method for differentiating GABAA receptor subtypes. In contrast to the affinity models of benzodiazepine binding sites, it is not clear that a simple molecular biologically based efficacy model will emerge to simplify our understanding of 1-, 2-, 3-, or 5-containing GABAA receptors. The GABAA receptor subtypes with their benzodiazepine receptor sites are an example of a unique situation in biology. Compounds have been synthesized that can allosterically modulate GABA response over a wide range through this site. Modulation of GABA responses has spanned the range of 700% increase in response amplitude to inhibition as great as 60%. Control of efficacy by drugs with subunit specificity can be achieved. One approach of drug companies has been to develop drugs that increase GABA responses less than 100% and develop selectivity for some subunits without increasing responses at other subunit combinations. These have been called subtype-selective partial agonists and will probably represent the next generation of GABAergic modulators to enter the clinic and become drugs. A number of studies suggest, by circumstantial evidence such as message distribution, genomic localization, and biochemical study, that the major subtype combinations in brain are 1 2 21, 2 3 21, 3 3 21, and 5 3 21. 1 has been implicated in sedation by virtue of the fact that zolpidem, a marketed hypnotic under the trade name Ambien, is a type I ( 1) selective compound and can cause cognitive deficits (see next subsection). An ideal anxiolytic drug might have limited effects on this subtype while increasing responses at 2- and 3-containing subtypes, as they are located in the limbic parts of the brain directly implicated in generation and reduction of anxiety. The full examination of subtype selective drugs in humans is in the near future, and it will be interesting to see how these hypotheses fare in clinical trials. Involvement of GABA A Receptors in Human Disease and Transgenics The extensive investigation of the amino acids involved in the binding of GABA and benzodiazepines allows a specific and elegant approach to be made to the in vivo investigation of the involvement of GABAA receptor subtypes with specific neural pathways and specific behavioral activities. This approach can be made either through the examination of chromosomal deletions, the use of specific knockouts of subunit genes, or knock-ins of particular point mutations. These techniques naturally complement the development of drugs with specific activities at GABAA receptor subtypes. Some naturally occurring chromosomal deletions of particular GABAA receptor subunits showed phenotypes of craniofacial deficits, mental retardation, and epilepsy. Deletion of large areas of human chromosome 15, containing 5, 3,
and 3 subunit genes, results in this phenotype, which causes a human genetic disorder called Prater Willi/Angelman syndrome (66). In a targeted study in mice, animals with the specific deletion of the 3 subunit shows a similar syndrome with cleft palate and neurologic abnormalities. These studies point to the importance of certain GABAA receptors in neuronal development (67,68). It is also clear that the deletion of an entire subunit does not result in the rescue of function by substitution of other subunits (69). Other rare genetic disorders of GABAA receptor function are likely to emerge as our knowledge of the genomic basis of neurologic disorders evolve. A very elegant approach, based on the molecular biological studies described above, has been taken to examine the significance of GABAA receptor subtypes by replacing an important amino acid for benzodiazepine binding (histidine) found in 1, 2, 3, and 5 with an arginine characteristic of 4 and 6. Through this conservative mutation, GABA sensitivity is retained, so the receptors function normally, but drug sensitivity is lost (70,71). From these studies, it appears that the 1-containing subtypes are important in mediating the anticonvulsant, sedative, and amnestic effects of benzodiazepines, but to a smaller degree the muscle relaxant and anxiolytic effects. These animals are still susceptible to the development of tolerance to the sedative effects. In the near future, we may learn the consequence of deletion of the specific benzodiazepine modulatory sites from the other subtypes. Thus, from a mechanistic point of view this class of drugs has a well-defined mode of action. SEROTONIN RECEPTOR MODULATORS AND REUPTAKE INHIBITORS Preclinical Studies Serotonin has long been viewed as a neurotransmitter involved in regulating emotional states. Of the 14 or so mammalian serotonin receptor subtypes that have been described in the literature, at least four have been implicated in anxiety in various animal models (72). As reported by Lucki (72) the original hypothesis implicating serotonin in anxiety surfaced from observations that reduced levels of serotonin can produce anxiolytic effects. One of the receptor subtypes implicated in anxiety is the serotonin 1A receptor subtype (5HT1A), which is an autoreceptor located presynaptically on serotonin neurons. When stimulated, this receptor inhibits the synthesis and secretion of serotonin. The 5-HT1Areceptor agonist buspirone exhibits anxiolytic effects in animals and was approved by the Food and Drug Administration (FDA) in 1986 for human generalized anxiety disorder. Other serotonin receptors potentially involved in anxiety include the 5-HT2A, 5-HT2C, and 5-HT3 receptors. Antagonists for the 5-HT2Areceptor, like ritanserin, exhibit anxiolytic effects in some animal models (73,74). Likewise, blockade of the 5-HT2C receptor produces anxiolytic effects in
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animals (75) and prevents the anxiogenic effects of m-CPP (76). Finally, the 5-HT3receptor antagonist ondansetron was reported to be anxiolytic in some animal models (77). Recent advances in molecular biology has led to the development of serotonin receptor gene knockout methodology, which generates mice lacking the 5-HT1A receptor, allowing for the evaluation of this receptor subtype in a variety of measurable behaviors. Ramboz et al. (78) reported results consistent with the 5-HT1A agonists data cited above. Mice lacking this receptor displayed less exploratory activity in an open field and more anxious behavior than the wild types in the elevated plus maze. According to the serotonin hypothesis of anxiety (79), removing the negative feedback control of 5-HT with the 5-HT1A receptor knockout animals should result in increased levels of 5-HT in the synaptic cleft, which would be expected to lead to the anxiogenic behavior. However, Ramboz et al. (78) reported normal levels of 5-HT, which confuses the issues related to anxiety modulation and serotonin levels. As David Julius (80) points out, the interpretation of standard gene knockout experimentation is complicated by the possibility of long-term development changes and this is true with the 5HT1Aknockout animal. So despite the apparent consistency between the 5-HT1A knockout animal and 5-HT1A agonist studies in terms of the behavioral outcomes of each manipulation, the exact role of the 5-HT1Areceptor in anxiety is not absolutely clear at this time. Clinical Studies In 1986, the FDA approved the 5-HT1A partial agonist for generalized anxiety disorder. This drug was the first to challenge the benzodiazepines for this patient group and was generally perceived as an improvement because of the lack of benzodiazepine side effects. The efficacy of buspirone, however, was not the same as that of the benzodiazepines in terms of its delayed onset of action, and it is generally accepted that when buspirone offers clinical benefit to generalized anxiety disorder (GAD) patients, it takes 3 to 4 weeks to match the efficacy of benzodiazepines such as diazepam and alprazolam (81). The 5-HT1A partial agonist properties of buspirone are believed to account for its clinical effects, but it should be noted that the drug is also a D2 antagonist and is extensively metabolized. One of the major metabolites, 1-pyrimidinylpiperazine (1-PP), may contribute to the pharmacologic activity of buspirone (82). In a double-blind, placebo-controlled study of buspirone in GAD patients (83), the drug was reported to be as efficacious as lorazepam at the end of a 4-week treatment period. After the drugs were discontinued, however, the lorazepamtreated patients worsened whereas the buspirone-treated subjects maintained clinical improvement. Thus, there continues to be evidence that buspirone is effective in GAD. The development of selective serotonin reuptake inhibitors (SSRIs) in the 1980s and 1990s widely expanded the
treatment for depressive disorders, and these drugs (fluoxetine, sertraline, venlafaxin, paroxetine) have recently made inroads in treating anxiety disorders such as panic, obsessivecompulsive disorder, social phobia, and GAD. Successful treatment of GAD with a class of drugs working through the serotoninergic system will come from the SSRIs (84). Obsessive-compulsive disorder (OCD) is a chronic, disabling anxiety disorder. In a review of the diagnosis and treatment of OCD, Goodman (85) states that the backbone of pharmacologic treatment for OCD is a 10- to 12-week trial with an SSRI in adequate doses. It is clear from a review of the role of the 5-HT1A receptor (86) in OCD that partial agonists such as buspirone are generally ineffective in treating OCD. The authors also note that in studying the potential to augment efficacy of the standard OCD medication, buspirone was not different from placebo as an augmenting agent. Drugs that work through other serotonin receptor subtypes also appear to be ineffective in treating OCD. Thus, drugs modifying the 5-HT1A, 5-HT1D, and 5-HT3 receptors appear ineffective in treating OCD symptoms and rule out a critical involvement of these receptor subtypes in OCD (87,88). In the past, tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors, as well as high potency benzodiazepines, have been used to treat patients with panic disorder. The SSRIs have also been added to the list of effective agents for the disorder. In reviewing the pharmacotherapy of panic disorder, den Boer (89) notes that antidepressants are more effective than benzodiazepines in reducing associated depressive symptomatology and are at least as effective for improving anxiety, agoraphobia, and overall impairment. Bell and Nutt (90) remark that SSRIs improve 60% to 70% of panic patients, a similar percentage to those seen with the TCAs. Like OCD, panic disorder is well treated by SSRIs but does not appear to be effectively treated by receptor specific compounds. Copland et al. (86) reviewed the role of 5HT1Adrugs such as buspirone in panic disorder and reported that buspirone does not significantly treat panic in several well-controlled studies. Using the 5-HT1A receptor agonist flesinoxan, van Vliet et al. (91) reported a worsening of symptoms in panic patients treated with high doses of the drug. It has also been reported that the 5-HT2A/2C antagonist ritanserin had no effects on panic attacks or phobic avoidance, and a similar negative finding has been reported with the 5-HT3 antagonist ondansetron. NEUROKININ RECEPTOR ANTAGONISTS Rationale There is an extensive literature demonstrating that the peptide tachykinins such as substance P and their associated receptors have a widespread distribution in the brain, spinal cord, and periphery, and may play important roles in
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chronic pain and inflammation processes (9296). In addition, anatomic and physiologic evidence has also indicated that these peptides may affect limbic structures that are involved in the regulation of mood and affect, such as the amygdala, hypothalamus, and periaqueductal gray (97). This notion is supported by early positive clinical findings using a selective neurokinin-1 (NK-1) antagonist for the treatment of depression and anxiety (98). Molecular Mechanism of Action Tachykinins collectively refer to small peptides that include substance P (SP), neurokinin A (NK-A), and neurokinin B (NK-B). These peptides show preferential affinity for three receptors, designated NK-1, NK-2, and NK-3, respectively, which are members of the seven-transmembrane, G-proteincoupled family. Of these three receptors, NK-1 and NK-3 are found in the brain, whereas NK-2 is primarily localized peripherally in smooth muscle of the respiratory, urinary, and gastrointestinal tracts. Neurokinin receptors are localized in a number of different brain areas that are implicated in anxiety, including the amygdala, hypothalamus, and locus coeruleus. Studies assessing the effects of direct administration of neurokinin agonists such as substance P into the nervous system are complicated by the findings that, depending on factors such as the site and dose, opposite effects on behavior may be achieved. Current Drugs in Development Numerous NK-1 antagonists have been described in the literature, including MK-869 (Merck) and an analogue, L760,735 (Merck), SR140333 (Sanofi), CP-122,721 (Pfizer), RP67580 (Rhone-Poulenc), FK-888 (Fujisawa), SDZ NKT 343 (Novartis), and PD 154075 (Parke-Davis). NK-1 antagonists have been reported to demonstrate anxiolytic effects in animal models such as social interaction (99), though these effects are not consistently seen across all compounds (34). Researchers from Merck have reported that vocalizations elicited by maternal separation in guinea pigs are robustly blocked by NK-1 antagonists such as MK-869, an effect that is shared by a range of antidepressant and anxiolytic agents (98). Of the compounds listed above, the primary indication has been for the treatment of conditions such as pain, chemotherapy-induced emesis, and migraine (94). MK869, which progressed to phase III trials for emesis, has also been evaluated in a phase II depression trial in which it was reported that, in addition to showing a significant antidepressant effect, MK-869 also showed significant anxiolytic activity that emerged over the course of the 6-week study (98). The data supported the conclusion that NK-1 antagonists might be useful for the treatment of depression and anxiety. Further development of MK-869 for depression
was discontinued, but these early clinical data will undoubtedly lead to further clinical evaluation of NK-1 antagonists. NK-2 antagonists include SR48968 (Sanofi). Preclinical studies have shown that NK-2 antagonists such as GR159897 and SR48968 have also demonstrated activity in social interaction and exploration anxiolytic models, and activity has been reported in the marmoset monkey using the human threat test. Good therapeutic ratios were described for these agents. NK-3 antagonists described in the literature include osnetant (Sanofi-Synthelabo), talnetant, PD-161182, and PD-157672 (Parke-Davis). The latter two have been designated for the treatment of anxiety disorders, though there have been no reports of clinical trials with any NK-3 antagonist for this indication. It should be noted that preclinical data described to date are sparse, and there is some suggestion that NK-3 agonism may produce an anxiolytic profile. Thus, intraventricular administration of the NK-3 agonist senktide produced anxiolytic effects in mice that could be blocked by administration of the NK-3 antagonist SR 142801, and SR 142801 was found to have some anxiogenic activity (100). Future Drugs and Directions Further depression and anxiety clinical trials with centrally active NK-1 antagonists are needed to provide further validation of the role of NK-1 receptors in treating depression and anxiety disorders. In addition, further assessment of the role of NK-2 and NK-3 subtypes is needed to determine the possible relevance, if any, of these receptor subtypes.
GLUTAMATE RECEPTOR AGONISTS AND MODULATORS Rationale Glutamate is the major mediator of excitatory neurotransmission in the CNS. Despite this ubiquity, the elucidation of numerous glutamate receptor subtypes with differential localizations in the brain, and the development of selective pharmacologic agents, has led to the realization that glutamate receptors might be viable targets for a number of different neurologic and psychiatric disorders, including anxiety and depression (101103). Molecular Mechanism of Action The molecular biology of glutamate receptors has been the subject of numerous reviews (101,102). Glutamate receptors are classified as either ionotropic or metabotropic. Ionotropic receptors, which mediate fast synaptic transmission, are coupled to cation-specific ion channels and bind the agonists N-methyl-D-aspartate (NMDA), -amino-3-hy-
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droxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainic acid (KA). These receptors gate both voltage-dependent and voltage-independent currents carried by Na , K , and Ca . NMDA receptors, which are selectively activated by NMDA, form a receptor/channel complex that is allosterically regulated by several sites. Receptor activation results in depolarization and Ca influx. Allosteric binding sites include a strychnine-insensitive glycine site, a polyamine site, a zinc site, and a channel site that binds agents such as MK-801 or phencyclidine to block channel opening. The NMDA receptor has been cloned and has two families of subunits, the NR1, with seven splice variants (1A1G), and NR2, with four splice variants (2A2D). NR1 receptors possess the receptor/ion channel complex, whereas the NR2 receptors lack the ion channel and appear to be modulatory. NR2 receptors, however, can form functional heteromeric channels by combining with NR1 subtypes. NMDA antagonists include competitive antagonists at the NMDA receptor such as AP5, CPPene, and CGS 19755; noncompetitive antagonists at the ion channel site such as MK-801 and phencyclidine; and noncompetitive antagonists that bind to the glycine site such as 5,7-dichlorokynurenic acid, L689,560, ACEA 1021, and MDL 105,519 (104). Non-NMDA receptors refer to the class of ionotropic glutamate receptors activated by kainic acid or AMPA, agonists that activate voltage-independent channels that gate a depolarizing current mediated primarily by Na ions. There are four AMPA subunits (GluR1GluR4) and five KA subunits (GluR5GluR7 and KA-1KA-2). Functional channels can be produced by homomeric expression of GluR5 or GluR6 subunits, or by heteromeric expression of KA-1 or KA-2 with GluR5 or GluR6. KA sites are found in the hippocampus, cortex, and thalamus, whereas AMPA receptors are additionally localized in septal and cerebellar sites. Pharmacologic agents for blocking non-NMDA receptors include the competitive antagonists CNQX, DNQX, and NBQX, and the noncompetitive antagonist GYKI 52466. Metabotropic glutamate receptors (mGluRs) mediate slower synaptic transmission and utilize phosphatidyl inositol (PI) and cAMP as second messengers. Opposite effects on cAMP may be mediated by either Gi or Gs stimulation. Agonists include a conformationally restricted glutamate analogue, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD). Metabotropic receptors have been classified into three subgroups: group I (mGluR1, mGluR5), which stimulate PI hydrolysis; and two groups that inhibit adenylate cyclase, group II (mGluR2, mGluR3) and group III (mGluR4, mGluR6). Metabotropic receptors are widely distributed throughout the brain, in areas such as the hippocampus, cerebellum, thalamus, olfactory bulb, and striatum, though the precise distribution varies considerably between groups. In addition to the agonist trans-ACPD, other agonists that are more specific for receptor subtypes include
LY354740 (group II), L-AP4 (group III), and L-CCG-I (mGluR2). Pharmacologic agents for antagonizing metabotropic glutamate receptors are currently limited. Genetically Altered Mouse Models Site-directed mutagenesis studies have indicated that point mutations in the glycine binding site of the NR1 subunit result in mice that have reduced glycine affinity and have an anxiolytic profile as seen by decreased natural aversion to an exposed environment (105). These data supported other pharmacologic lines of evidence (see below), indicating that blockade of the glycine site can have anxiolytic actions. Current Drugs in Development LY354740 is an orally active group II metabotropic receptor agonist (106) currently in clinical development. Preclinically, the compound has anxiolytic activity in fear-potentiated startle (107), the elevated plus maze (107), conflict testing (108), the four-plate test (108), and in a lactateinduced panic attack model (109). LY354740 has also been shown to decrease withdrawal signs seen during naloxoneprecipitated morphine withdrawal (110). Future Drugs and Directions Competitive and noncompetitive NMDA antagonists have primarily undergone clinical evaluation for the treatment of stroke and trauma (103). Unfortunately, clinical development for many of these compounds was halted because of severe side effects, including psychotic-like symptoms. The potential for these side effects has been a major deterrent for using NMDA antagonists for the treatment of psychiatric disorders. Although there are currently no drugs reported to be in development, preclinical studies have suggested that selective antagonists of the strychnine-sensitive glycine site can have anxiolytic properties with reduced side-effect potential relative to competitive and noncompetitive NMDA antagonists such as AP5 and MK-801 (36,111). Recent work has supported such a profile (112,113). Antagonists of AMPA receptors have also been proposed to have anxiolytic actions in preclinical models. Thus, LY326325 was shown to have anxiolytic activity in the elevated plus maze and in a conflict test (punished drinking) in rats (114), and CNQX injected directly into the periaqueductal gray produced anxiolytic effects on the elevated plus maze (115). The antagonists CNQX and GYKI 52466 were also able to block the anxiogenic responses produced by bicuculline injected into the basolateral amygdala (116). CCKB ANTAGONISTS Cholecystokinin (CCK) is a peptide found extensively both in the gut (where it was originally identified) and in the
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brain (117). CCK exists in multiple forms, the most predominant of which is CCK octapeptide (CCK8) and, to a lesser extent, CCK tetrapeptide (CCK4) (118). CCK is colocalized with a number of different neurotransmitters, including serotonin, dopamine, GABA, substance P, neuropeptide Y, and VIP. CCK-like immunoreactivity has been demonstrated in anatomic regions that include the amygdala, cerebral cortex, hippocampus, striatum, hypothalamus, and spinal cord (119). There are two subtypes of CCK receptor, CCKA (sulfated CCK) and CCKB (unsulfated CCK) (120). CCKA receptors are localized in the nucleus accumbens, posterior hypothalamus, and area postrema. CCKB receptors are localized in cortex, olfactory bulb, nucleus accumbens, and other brain areas (121). A number of selective antagonists for the CCKB receptor have been synthesized, including LY288513 (Lilly), PD 135158 (Parke-Davis), L-365,260 (Merck), and CI-988. Griebel (34) reviewed the extensive literature on the effects of CCK antagonists in models of anxiety and concluded that the results were contradictory and did not lead to a clear conclusion. L-365,260 and CI-988 have been evaluated clinically in panic disorder, but neither of these agents had significant anxiolytic effects (122124).
4. 5. 6.
7.
8.
9. 10. 11.
CONCLUSION Many different neurotransmitter receptor systems have been shown to modulate anxiety and possess anxiolytic effects. This is not surprising because many of these transmitters control anatomic circuitry important in anxiety. The next generation of marketed anxiolytics will be determined more by their side-effect profile than by their anxiolytic activity. It will be interesting to see which of the mechanisms described in this chapter will provide the most useful anxiolytics in human populations.
12. 13.
14.
15. 16.
ACKNOWLEDGMENT Drs. Tallman, Cassella, and Kehne are all full-time employees of Neurogen Corporation.
17. 18.
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VII ANXIETY AND STRESS DISORDER

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

29. 30. 31. 32. 33. 34.

35. 36. 37.

38. 39. 40. 41. 42. 43. 44. 45. 46.

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

1.8 4.3 0.9 3.2 7.8 2.5

10.3 6.8 2.0 7.9 14.5 2.9

5.7 1.6 2.2 2.5 1.9 1.2

19.2 3.6 2.0 3.5 6.7 11.1

30.5 6.6 5.0 7.0 15.7 15.5

1.6 1.8 2.5 2.0 2.3 1.4

TABLE 61.2. SUMMARY OF FAMILY AND TWIN STUDIES OF ANXIETY DISORDERS

DZ, dizygotic; MZ, monozygotic; OCD, obsessive-compulsive disorder.

Neuropsychopharmacology: The Fifth Generation of Progress

TABLE 61.3. CONTROLLED HIGH-RISK STUDIES OF ANXIETY

Dx, diagnosis; MDD, major depressive disorder.

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

TABLE 61.4. VULNERABILITY FACTORS FOR ANXIETY DISORDERS

27. 28. 29.

10. 11. 12.

30. 31. 32. 33. 34. 35. 36. 37. 38.

15. 16. 17. 18.

39. 40. 41. 42.

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50. 51. 52. 53. 54. 55. 56.

57. 58. 59. 60. 61.

63. 64. 65. 66. 67.

114. 115. 116.

Neuropsychopharmacology: The Fifth Generation of Progress

136. 137. 138. 139. 140.

155. 156. 157. 158. 159.

160. 161. 162. 163. 164. 165.

166. 167. 168. 169.

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

29. 30. 31.

Neuropsychopharmacology: The Fifth Generation of Progress

92. 93. 94. 95. 96. 97.

120. 121. 122.

123. 124. 125.

Neuropsychopharmacology: The Fifth Generation of Progress

154. 155. 156. 157. 158. 159. 160. 161.

Neuropsychopharmacology: The Fifth Generation of Progress

Chapter 63: Neurobiological Basis of Anxiety Disorders

Neuropsychopharmacology: The Fifth Generation of Progress

Neuropsychopharmacology: The Fifth Generation of Progress

Chapter 63: Neurobiological Basis of Anxiety Disorders

Neuropsychopharmacology: The Fifth Generation of Progress

Chapter 63: Neurobiological Basis of Anxiety Disorders