1521-0081/66/4/10021032$25.00 http://dx.doi.org/10.1124/pr.114.

009126
PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:10021032, October 2014
Copyright 2014 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: LESLIE A. MORROW

Targeting the Modulation of Neural Circuitry for the

Treatment of Anxiety Disorders
David H. Farb and Marcia H. Ratner
Laboratory of Molecular Neurobiology, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,
Boston, Massachusetts

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
II. Types of Anxiety Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
III. Brain-Imaging Studies: Assessment of Brain Activation in Patients with Anxiety Disorders . . 1005

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A. Functional Magnetic Resonance Imaging as a Method for Biomarker Identification. . . . . . . 1006
B. Positron Emission Tomography: Advances and Caveats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
C. Single Photon Emission Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008
D. Magnetic Resonance Spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
E. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010
IV. Neural Systems and the Neurochemical Basis of Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
A. Neural Systems Underlying Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
1. A Connectome Hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
2. Optogenetics and Electrophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012
B. Type A GABA Receptor and Serotonin Receptor Structure/Roles in Anxiety . . . . . . . . . . . . . . 1013
1. GABA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014
2. Serotonin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1016
V. Neuropsychopharmacology of Anxiety Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018
A. Positive Allosteric Modulation of GABAergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018
1. Benzodiazepines and Related Modulators.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018
2. Natural Product Modulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019
3. Increasing GABA Levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020
B. Modulators of Glutamatergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020
1. Inhibitors of Glutamatergic Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020
2. Potentiators of Glutamatergic Activity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021
3. Neurosteroids as Modulators of Glutamatergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . 1021

AbstractAnxiety disorders are a major public
health concern. Here, we examine the familiar area of
anxiolysis in the context of a systems-level understanding
that will hopefully lead to revealing an underlying
pharmacological connectome. The introduction of
benzodiazepines nearly half a century ago markedly
improved the treatment of anxiety disorders. These
agents reduce anxiety rapidly by allosterically enhancing
the postsynaptic actions of GABA at inhibitory type A
GABA receptors but side effects limit their use in chronic
anxiety disorders. Selective serotonin reuptake inhibitors
and serotonin/norepinephrine reuptake inhibitors have
emerged as an effective first-line alternative treatment of
such anxiety disorders. However, many individuals are
not responsive and side effects can be limiting. Research
into a relatively new class of agents known as neurosteroids
has revealed novel modulatory sites and mechanisms of
action that are providing insights into the pathophysiology
of certain anxiety disorders, potentially bridging the gap
between the GABAergic and serotonergic circuits
underlying anxiety. However, translating the pharmacological
activity of compounds targeted to specific receptor
subtypes in rodent models of anxiety to effective
therapeutics in human anxiety has not been entirely
successful. Since modulating any one of several broad
classes of receptor targets can produce anxiolysis, we
posit that a systems-level discovery platform combined
with an individualized medicine approach based on
noninvasive brain imaging would substantially advance
the development of more effective therapeutics.

Address correspondence to: Dr. David H. Farb, Laboratory of Molecular Neurobiology, Department of Pharmacology and Experimental
Therapeutics, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118. E-mail: dfarb@bu.edu
dx.doi.org/10.1124/pr.114.009126.

1002
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis 1003

C. Dopaminergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021

D. Epigenetics of Anxiolysis: Histone Deacetylase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021
E. Modulators of Serotonergic and Noradrenergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
F. Neuroactive Steroids as Neuromodulators of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026

I. Introduction use in some patients, including delays in producing

Anxiety is a recognized symptom of many psychiatric
disorders, including generalized anxiety disorder (GAD),
social anxiety disorder (SAD), obsessive-compulsive
disorder (OCD), and post-traumatic stress disorder
(PTSD). Although anxiety is a commonly reported clinical
manifestation, it can be severely debilitating if left
untreated. According to the Epidemiologic Catchment
Area Study, the prevalence of anxiety disorders is about
7% in the adult population of the United States (Regier
et al., 1990, 1998). Studies looking at the incidence of
anxiety disorders indicate that about 18% of adults in the
United States may seek treatment for anxiety in any
given year (Kessler et al., 2005b). Women are more likely
than men to be diagnosed with an anxiety disorder but
how much of this sex difference is due to socioeconomic
factors has not been established. That said, the need for
a unified theory that will aid the development of targeted
anxiolytics could not be more urgent with the emergence
of PTSD as a major global public health challenge faced
after more than a decade of war.
The importance of an improved approach to treating
anxiety is highlighted by the inconsistent results seen
with the class of drugs considered to be the contempo-
rary first-line treatment: selective serotonin reuptake
inhibitors (SSRIs) and serotonin and norepinephrine
reuptake inhibitors (SNRIs). Although these agents
have been shown to be beneficial for the treatment of
certain anxiety disorders, not all patients achieve an
adequate clinical therapeutic response. The existence
of nonresponders indicates that SSRIs and SNRIs are
not the fail-safe solution to treating anxiety clinicians
have been looking for. In addition, SSRIs and SNRIs
are associated with complications that can limit their
the desired clinical reduction in anxiety or even po-
tential worsening of the anxiety (Ravindran and Stein,
2010; Sertraline, 2012; Venlafaxine, 2012; Fluoxetine,
2013), not to mention the risk for the SSRI discontin-
uation syndrome in noncompliant patient populations.
Furthermore, some SSRIs (e.g., citalopram) are not
suitable for patients with heart problems (Wang and
Pae, 2013).
Another class of drugs, benzodiazepine (BZD) anxi-
olytics, has played a central role in the pharmacologic
management of anxiety disorders for about 50 years.
Although not as widely prescribed as in the past, these
compounds nevertheless remain an effective alterna-
tive to SSRIs. That said, the nonselective BZDs are not
devoid of side effects, which can include drowsiness,
ataxia, impairment of cognition, as well as the risk of
dependence and withdrawal symptoms, including in-
creased anxiety, tremors, and seizures (Noyes et al.,
1988; Bennett et al., 1998; Yamawaki, 1999; Allgulander
et al., 2003). The identification of subtype-selective
GABA receptorpositive allosteric modulators, such as
zolpidem, which have proven to be effective in the
treatment of patients with insomnia while producing
fewer untoward side effects than nonselective BZDs,
suggested that similar drugs could be developed for anx-
iety disorders. Unfortunately, although subunit-preferring
compounds are effective for insomnia, they are not yet
proven to be any more beneficial for the management
of anxiety disorders than the classic BZDs, such as
diazepam (Farkas et al., 2013).
Another alternative to enhancing inhibitory neuro-
transmission mediated by GABA is modulating excit-
atory glutamatergic neurotransmission mediated by

ABBREVIATIONS: 5-HT, serotonin; BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; BOLD, blood oxygen leveldependent; BZD,
benzodiazepine; BZDR, benzodiazepine receptor; CBT, cognitive behavioral therapy; CL 218872, 3-methyl-6-[-3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]
pyridazine; CNS, central nervous system; Co 3-0593, 3b-ethenyl-3a-hydroxy-5a-pregnan-20-one; DSM, Diagnostic and Statistical Manual of Mental Disorders;
fMRI, functional magnetic resonance imaging; GABAAR, type A GABA receptor; GAD, generalized anxiety disorder; GPCR, G proteincoupled receptor; HPA,
hypothalamic-pituitary-adrenocortical; K36, 5,7,29-trihydroxy-6,8-dimethoxyflavone; L655,708, ethyl(13aS)-7-methoxy-9-oxo-11,12,13,13a-tetrahydro-9H-
imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate; MRK-409, 7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,6-difluorophenyl)-1,2,4-triazolo
[4,3-b]pyridazine; MRS, magnetic resonance spectroscopy; NMDA, N-methyl-D-aspartate; OCD, obsessive-compulsive disorder; PET, positron emission
tomography; PKA, protein kinase A; PTSD, post-traumatic stress disorder; rCBF, regional cerebral blood flow; Ro 15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-
6-oxo-4H-imidazo-1,4-benzodiazepine-3-carboxylate; Ro 16-6028, (13aS)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]
benzodiazepine-1-carboxylic acid 1,1-dimethylethyl ester; Ro 19-8022, (R)-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)carbonyl]-2-pyrrolidine-
methanol; RU 28362, 11b,17b-dihydroxy-6,21-dimethyl-17a-pregna-4,6-trien-20yn-3-one; RY80, ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-
imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate,[ethyl-3H]; SAD, social anxiety disorder; SERT, serotonin transporter; SL651498, 6-fluoro-9-methyl-
2-phenyl-4-(pyrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one; SNRI, serotonin/norepinephrine reuptake inhibitor; SPECT, single photon
emission tomography; SSRI, selective serotonin reuptake inhibitor; TPA023B, 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-
1,2,4-triazolo[4,3-b]pyridazine; VPA, valproic acid; VRE, virtual reality exposure.
1004 Farb and Ratner
N-methyl-D-aspartate (NMDA) receptors. Although NMDA
receptors offer a promising therapeutic target for
treating anxiety disorders, the efficacy of NMDA re-
ceptor modulators developed to date also remains limited
(Swanson et al., 2005; Barkus et al., 2010).
Collectively, these findings reveal a clear need for an
improved understanding of the neural systems underly-
ing anxiety as well as a more effective approach to de-
veloping anxiolytic agents. We believe that three recent
developmentsthe use of noninvasive imaging in human
subjects to identify biomarkers implicated in neurologic
disorders, and a convergence of pharmacologic effects in
human populations of drugs that augment GABAergic
tone or serotonergic tone, combined with the develop-
ment of systems-based approaches to drug discovery
offer a promising path forward. Systems-based drug
discovery differs from traditional target-based discovery
in that it utilizes an understanding of complex systems to
search for potential therapeutic agents, as opposed to
identifying agents based solely on interactions with
a single target (Desbiens and Farb, 2012).
Research into neuroactive steroids recently revealed
an important role for these agents in the pathophys-
iology of anxiety, both influencing and being influenced
by the GABAergic, serotonergic, and other neural sys-
tems associated with anxiety and demonstrates both
the complexity of the problem and why focusing on
a specific target without regard for the entire neural
network may actually be counterproductive. This re-
view aims to consider the basic molecular mechanisms
by which neurotransmission can be modulated across
various neural systems implicated in anxiety disor-
ders, as well as to parse out how best to approach the
treatment of specific anxiety disorders based on the
neural networks involved in each, with the ultimate
goal of developing a systems-based approach to the
discovery of novel anxiolytics.
Our understanding of anxiolytics starts with the
recognition that at least in some cases, therapeutics
appear to effectively interact with specific receptors
to modulate apparently simple and conserved neural
systems implicated in hypervigilance and fear in both
humans and animals. A closer look at the problem of
anxiety in humans reveals that complex cognitive
processes such as executive function mediated by the
frontal lobes also play a role in how subjects respond to
environmental stimuli and that a single drug target
may not be the most effective approach in the treat-
ment of all types of anxiety. A growing body of research
is now also shedding light on the multiple genetic,
environmental, and social factors that interact in the
development of anxiety disorders (Lpine, 2002; Stein
et al., 2002, 2014; Pigott, 2003; Gelernter et al., 2004;
Hettema et al., 2005; Porcelli et al., 2012). In addition,
it is increasingly evident that a diverse array of
neurotransmitters and their respective receptors and
transporters, including those for GABA, serotonin
(5-HT), and NMDA, play critical roles in the develop-
ment of anxiety disorders (Swanson et al., 2005; Barkus
et al., 2010; Graeff and Zangrossi, 2010). An increased
understanding of the neural systems underlying a path-
ologic response to environmental stimuli that do not
induce anxiety in healthy subjects therefore begins with
the development of connectivity maps that in turn may
lead to more individualized approaches to the pharma-
cological management of various anxiety disorders.
II. Types of Anxiety Disorders
A comprehensive review of anxiety disorders must begin
with a definition of the problem. Anxiety per se is generally
considered to be synonymous with acute nervousness,
fretfulness, and apprehension. Although the words fear,
anxiety, and worry can sometimes be used interchange-
ably, these terms generally represent distinct emotional
responses that can be distinguished clinically by intensity
and duration. Among healthy individuals, the magnitude
of this type of emotional response is proportional to the
specificity and intensity of the stimulus. The specificity of
the stimulus is determined by its relevance to a particular
individual based on his or her current circumstance
combined with past life experience with that particular
stimulus (i.e., individual sensitivity). For example, expo-
sure to a stimulus that has previously been paired with an
adverse life event will produce a different response than
will exposure to a novel stimulus. Likewise, fear, fright,
flight, and panic are more intense and acute reactions than
trepidation, apprehension, and worry and normally occur
in close chronological and physical relation to an exposure
to the appropriate stimuli. For example, an individual may
feel slightly nervous and fretful while waiting to meet
a blind date, or may be apprehensive about his or her
ability to perform a novel task in public, but fearful of
encountering an armed robber while walking down a dark
alley alone at night. All of these responses are typically
brief and resolve spontaneously with termination of ex-
posure to the respective stimuli.
Fear, an intense fight-or-flight response that occurs
in reaction to a real or perceived threat, is a survival
response that is in contrast with panic disorder, which
is a fight-or-flight response in the absence of any identifiable
danger, therefore appearing to be a pathologic condition.
Because these symptoms occur spontaneously and without
warning, persons with panic disorder will typically avoid
situations that their past experience suggests can trigger
the onset of an attack. Patients who have difficulty
assigning a specific trigger for their attacks may become
socially withdrawn.
Worry is synonymous with a more persistent concern
or preoccupation with the unknown outcome of future
life events. Worrying can be controlled so as to permit
normal functioning in social and occupational settings.
By contrast, worrying such as that which occurs among
patients with GAD is associated with persistent concern
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
about routine life experiences, such as job performance or
personal relationships, and is severe enough to interfere
with the afflicted individuals ability to concentrate and
perform activities of daily living.
The clinical anxiety disorders recognized in the fifth
edition of the Diagnostic and Statistical Manual of
Mental Disorders (DSM-V), include the following: GAD,
OCD, panic disorder, acute and chronic PTSD, and the
various phobias, including agoraphobia, social phobia,
and specific phobia (e.g., fear of flying) (American Psychi-
atric Association, 2013). Each of these recognized clinical
anxiety disorders is unique with respect to the intensity
and duration of the anxiety experienced as well as the type
of stimuli that can induce it.
A systems-level approach to the development of
anxiolytic agents starts with a recognition of the range
of anxiety disorders from which patients can suffer,
including GAD, SAD, panic disorder, various phobias
(e.g., fear of flying), and medication-induced anxiety
disorder (DSM-V; American Psychiatric Association,
2013). In addition, disorders that fall under their own
category in the DSM-V, such as acute and chronic PTSD,
include clinical manifestations, such as hypervigilance,
exaggerated startle response, and fear as a component of
their diagnostic criteria. Likewise, OCD includes fear of
some dreaded event or situation among its diagnostic
criteria. Each of these disorders is unique with respect to
the intensity and duration of the symptoms experienced
by the patient as well as the type of stimuli that can
induce these. For example, persons with panic disorder
experience recurrent, discrete, and typically brief periods
of intense anxiety referred to as uncued panic attacks
in the absence of any identifiable source of danger. This
anxiety is associated with somatic symptoms, such as
shortness of breath, chest pain, lightheadedness, and
sweating. Persons with panic disorder often have per-
sistent concerns about the causes or consequences of
having a panic attack. By contrast, cued anxiety attacks
and phobias are defined as those in which the sufferer
experiences debilitating symptoms in response to a
clearly defined object or situation. In the cases of
phobias, exposure to the phobic stimulus produces an
immediate onset of increased anxiety that is severe
enough to interfere with the individuals ability to function
normally in social or occupational settings even when the
person recognizes that his or her own anxiety is excessive
or unreasonable.
The onset of an anxiety disorder may be insidious or
may follow an identifiable life event. For example,
acute stress disorder is characterized by symptoms
that occur during the first month after exposure to an
extremely stressful event that evokes intense fear and/or
a sense of helplessness, such as being in an automobile
accident or witnessing a murder. Symptoms of acute
stress disorder include increased arousal, avoidance of
places and events that remind one of the traumatic
event, and recurrent intrusive memories or dreams
1005
about the event. Although severe enough to interfere
with activities of daily living, these symptoms typically
resolve spontaneously within about 4 weeks after the
event. By contrast, chronic PTSD is characterized by
similar symptoms that persist for more than 1 month
and, in many cases, can last for years after exposure to
a traumatic event. These types of anxiety disorders
respond well to treatments that combine virtual reality
exposure (VRE) therapy with pharmacologic interven-
tions (Difede et al., 2014).
Unlike incident-evoked anxiety disorders, GAD is
characterized by at least 6 months of persistent and
excessive apprehension and worry in response to a
plethora of different stimuli, including such routine life
events as job performance, health of family members,
or simply being on time for appointments. The onset of
GAD is typically insidious and is often not associated
with a readily identifiable precipitating event. Individ-
uals with GAD can be differentiated from those with
nonpathological anxiety by two main criteria: 1) they
find it difficult to control their worrying, and 2) this
perseverative thought process interferes with their
ability to function in social and occupational settings.
The clinical manifestations of OCD are unique among
the disorders that include fear and anxiety as a clinical
manifestation in that the fear is about what will happen
if the individual does not perform recurrent thoughts,
impulses, or compulsions (e.g., repetitive hand washing)
that the individual recognizes to be unreasonable and
unrelated to real-life problems but nevertheless finds
extremely difficult to stop. These symptoms cause distress,
and thus, the affected individual may avoid situations
that promote the obsession. For example, a person who
obsesses about personal contamination may not leave
the house to prevent placing himself in a situation
where he might have to shake hands.
III. Brain-Imaging Studies: Assessment of Brain
Activation in Patients with Anxiety Disorders
Although a range of clinical diagnostic criteria and
assessment tools (e.g., Hamilton Anxiety Rating Scales
and the Minnesota Multiphasic Personality Inventory)
have historically been used in the differential diagnosis
of anxiety disorders, recent research has also shown that
various neuroimaging technologies can be employed to
identify common and unique brain regions associated
with the different disorders. Neuroimaging studies can
be used to measure differences in regional cerebral blood
flow (rCBF) and for identifying significant changes in
binding to specific receptor subtypes and changes in
uptake by transporters implicated in these disorders.
Although it is premature to say that silver bullets
targeting specific type A GABA receptor (GABAAR)
subtypes or serotonin transporters (SERTs) within
particular brain regions are on the horizon, it seems
likely that critical advances in brain imaging and
1006 Farb and Ratner
subunit subtype analysis are improving the chances
for optimizing therapeutic approaches to patients
with anxiety disorders.
A. Functional Magnetic Resonance Imaging as
a Method for Biomarker Identification
It is natural to wonder whether serotonergic neurons
in the dorsal and median raphe nuclei that project to
corticolimbic circuits may be controlled by GABAergic,
serotonergic, and neurosteroid modulators. A noninva-
sive approach for assessing the elements of a potential
anxiety connectome is to evaluate the pharmacologic ef-
fects of anxiolytics on specific brain regions by determining
changes in rCBF associated with anxiety-inducing
stimulithese change in response to different pharmaco-
logic interventions. Blood oxygen leveldependent (BOLD)
functional magnetic resonance imaging (fMRI) can also
be used to differentiate between types of anxiety, such
as between anxious apprehension (which involves verbal
rumination and worry) and anxious arousal (which in-
volves intense fear or panic or both) (Engels et al., 2007).
An fMRI study of healthy subjects with no history
of psychiatric illness or treatment revealed that those
who also tested positive for a regulatory variant
(5-HTTLPR) in the SERT gene (SLC6A4), which was
previously associated with reduced transcription of the
transporter, had greater amygdaloid reactivity in re-
sponse to presentation of fearful and angry faces
independent of patient sex. These findings suggest
that the amygdala can be biased for reactivity based
on genetics and that this may represent a susceptibility
factor for a pathologic response to stressful life ex-
periences (Hariri et al., 2005). The observation that
SSRIs promote downregulation of SERTs (Mirza et al.,
2007) also implicates dysregulation of serotoninergic
neurotransmission in etiology of certain anxiety disor-
ders. This polymorphism has also been associated with
reduced responsiveness to SSRI treatment (Zanardi
et al., 2001).
The potential for an anxiolytic effect of 5-HT via
attenuation of amygdala activation was shown in a
study of healthy subjects treated with the SSRI citalopram
for 7 days. Compared with control subjects, the fMRIs of
individuals treated with citalopram showed a bilateral
reduction in activation of the amygdala, hippocampus,
and medial prefrontal cortex in response to presentation of
threatening stimuli (Harmer et al., 2006).
Evaluation of fMRI responses to the presentation of
pleasant or unpleasant words while monitoring left
versus right hemisphere activity indicates that the left
side (inferior frontal gyrus) is more active in patients
with anxious apprehension, consistent with the fact
that this type of anxiety is more verbally mediated.
These findings may seem to be discordant with data
supporting a left-sided bias in response to pleasant
emotion, but positive emotional content preferentially
activates different regions of the left hemisphere: the
left frontal region (dorsolateral prefrontal cortex) (Engels
et al., 2007).
Results from fMRI imaging studies support the clinical
impression that GAD patients overreact to both pathology-
specific and nonspecific cues and that treatment-induced
reduction of anxiety attenuates the response to both types
of cues (Hoehn-Saric et al., 2004). In a small cohort of six
patients, subjects were initially studied using fMRI
while each listened to verbal descriptions of a personal
worry or a neutral statement before treatment with
citalopram and again after 7 weeks of treatment. Reduced
BOLD responses to worry statements were observed
in prefrontal regions, the striatum, and insula and
paralimbic regions. In addition, contrasts before and after
treatment revealed reductions in the differential response
that existed between worry and neutral statements.
Overall reduction of the BOLD response was most
prominent during neutral statements, particularly in
the left hemisphere. These findings are consistent with
the suggestion that frontolimbic structural connectivity
underlies the preservative thoughts as well as the
emotional disturbances seen in patients with GAD
(Tromp et al., 2012).
An fMRI study of 21 patients (8 males and 13 females)
with generalized social phobia, characterized by an
exaggerated and pervasive fear and avoidance of
scrutiny by others, suggested that activation of the
amygdala in response to presentation of angry and
fearful faces was attenuated relative to baseline after
12 weeks of treatment with the SSRI sertraline (Phan
et al., 2013). All patients received a stable dose (100150
mg/day) at the time of testing and had been titrated
based on their clinical response to treatment. At testing,
14 of 21 patients were deemed to be responsive to
sertraline treatment, whereas 7 were considered non-
responders. All treated patients (clinical responders and
nonresponders) were included in the study but blood
levels of sertraline were not measured. A post hoc
analysis looking at differences between responders and
nonresponders indicated that the attenuation from
pretreatment to post-treatment in amygdala reactivity
to fearful faces was significant in responders (P = 0.01)
but not in nonresponders (P = 0.22), as was attenuation
in ventromedial prefrontal cortex reactivity to angry
faces (responders, P = 0.02; nonresponders, P = 0.29).
Activation of the amygdala in all of the patients before
and after treatment was also compared with that of
19 healthy controls (10 males and 9 females). Consistent
with reports from other studies, activation of the amygdala
in response to fearful faces before treatment was found to
be greater in the anxiety patients than in control patients;
importantly, this difference was abolished by treatment
with sertraline. By contrast, activation of the ventromedial
prefrontal cortex in response to angry faces before
treatment was found to be significantly lower in anxiety
patients than in control patients, and this difference
was also abolished by sertraline treatment.
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
The effects of chronic paroxetine treatment (20 mg/day)
versus placebo have also been measured in patients with
SAD using fMRI, along with performance of several tasks
including a public-speaking task, public exposition of a
recorded performance task, and an emotional face-
processing task (Gimnez et al., 2014). Paroxetine
treatment was associated with reduced activation in
the insula, thalamus, and anterior cingulate cortex at
rest and during performance of the public exposition
of a recorded performance task. By contrast, during
the emotional face-processing task, paroxetine treat-
ment was associated with an increase in activation of
the right amygdala and the insula bilaterally.
Collectively, these findings point to a role for corticolimbic
circuits, including the amygdala and medial prefrontal
cortex in SAD, and suggest that reductions in the level of
activation in these brain regions may prove to be a useful
biologic marker for the efficacy of novel anxiolytics.
B. Positron Emission Tomography: Advances
and Caveats
rCBF can also be measured using positron emission
tomography (PET) scans. Studies of rCBF using PET
suggest that paralimbic structures together with the
right inferior frontal cortex and subcortical nuclei mediate
symptoms across three different anxiety disorders: OCD,
simple phobia, and PTSD (Rauch et al., 1997). Oxygen
15labeled carbon dioxide was used as a tracer to measure
rCBF in 23 right-handed adult subjects (8 men and 15
women) meeting criteria for one of these three disorders
and relative rCBF measured in the context of subjects
specific symptom provocation paradigms (e.g., exposure of
a patient with OCD to a contaminated object). Overall,
analysis of pooled imaging data revealed activation in the
right inferior frontal cortex and right posterior medial
orbitofrontal cortex, and bilateral activation in the insula,
lentiform nuclei, and brainstem during symptomatic
versus control conditions.
The limitations of measuring changes in rCBF as a
measure of SSRI efficacy in the treatment of anxiety
disorders is hampered by a deficiency of knowledge
about the underlying neural circuitry, or functional
connectome, that gives rise to feelings of anxiety and
pharmacologically induced anxiolysis. For example, when
PET techniques were used to assess the effects of SSRI
treatment in patients with SAD, the SSRI-treated subjects
showed a selective deactivation of the left lateral
amygdala not associated with a reduction in anxiety
status. By contrast, the left basomedial/basolateral
and right ventrolateral regions of the amygdala were
deactivated in both SSRI-treated and placebo patients
who showed improvement in anxiety symptoms (Faria
et al., 2012) (Fig. 1). This latter deactivation was not
observed in nonresponders (i.e., subjects who showed no
reduction in anxiety scores) irrespective of treatment
group, suggesting that this change was mediated by a
reduction in anxiety unrelated to SSRI treatment. Thus,
1007
Fig. 1. PET scans of subjects with SAD who were treated with either an
SSRI or placebo for a minimum of 6 weeks does not identify a brain region
modulated by drug and associated with anxiolysis. Scans were made
while subjects performed a public-speaking task. Subjects were divided
into two groups based on clinical response to treatment (responders
versus nonresponders). An attenuation of rCBF that was independent of
treatment was observed in the left basomedial/basolateral and right
ventrolateral amygdala of responders. Red indicates the site of reduced
activation irrespective of treatment (placebo versus SSRI) seen in responders;
blue indicates a purely pharmacodynamic nonaxiolytic effect on rCBF that
was seen in both responders and nonresponders treated with an SSRI;
and yellow indicates an effect related to repeated testing. Reprinted with
permission from Faria et al. (2012). L, left; R, right.
SSRI treatment was no more effective than placebo for
treating SAD, perhaps due to the difficulty of separating
anxiolysis from a placebo effect in conjunction with
a small sample size. Unfortunately, the subjects were
not tested for the 5-HTTLPR polymorphism in the 5-HT
transporter gene (associated with reduced responsive-
ness to SSRI treatment; Zanardi et al., 2001), further
confounding interpretation of the results.
An advantage of PET versus fMRI as a clinical re-
search tool can be exemplified by a study that observed
decreased flumazenil binding in specific brain regions in
medication-free persons diagnosed with panic disorder
(Malizia et al., 1998). Global reduction in BZD site binding
throughout the brain were found in patients with panic
disorder (n = 7) compared with controls (n = 8) (Fig. 2A).
In addition, several loci thought to be essential in the
regulation of anxiety (right orbitofrontal cortex and right
insula) were found to have the largest regional decrease
in binding. These results are consistent with previous
clinical psychopharmacologic evidence of involvement
of the GABAAR (Hasler et al., 2008) using PET with
[11C]flumazenil in patients with panic disorder compared
with healthy controls, which showed reduced binding
potential across regions of the frontal, temporal, and
parietal cortices, particularly the dorsal anterolateral
prefrontal cortex.
Dynamic three-dimensional PET scans show that the
BZD-GABAAR may play a role in the pathophysiology
of PTSD, a finding that is consistent with previous
animal and clinical pharmacological studies (Geuze et al.,
2008). In this study, radiolabeled [11C]flumazenil was
1008 Farb and Ratner

Fig. 2. The BZD binding site on the GABAAR plays a role in the pathophysiology of PTSD. (A) Results of PET studies using radiolabeled
[11C]flumazenil to study BZDR kinetics in vivo. Axial images of middle brain (12 mm above the anterior commissureposterior commissure plane)
showing median volume of distribution map for controls (left) and patients with panic disorder (right). Note the generalized decrease in volume of
distribution affecting the thalami and the heads of the caudate nucleus among patients with panic disorder. Reprinted with permission from Malizia
et al. (1998). (B) PET scan images highlighting those areas that showed decreased binding of [11C]flumazenil in drug-nave subjects with PTSD (n = 9)
compared with controls (n = 7). Reduced binding was observed in the cortex, hippocampus, and thalamus. Reprinted with permission from Geuze et al.
(2008). CN, caudate nucleus; FC, frontal cortex; Ins, insula; L, left; OC, occipital cortex; R, right; TC, temporal cortex; Th, thalamus.

used as a tracer in nine drug-nave male veterans with
deployment-related PTSD and seven male veterans without
PTSD. After a scan of 60 minutes, reduced [11C]flumazenil
binding was observed throughout the cortex, hippocampus,
and thalamus of the PTSD patients (Fig. 2B).
PET techniques have also proven valuable in evaluating
variations in the distribution of specific subtypes of the
GABAAR within the human brain and the roles of these
subunits in mediating different functions. Competition
studies in vivo in the rat revealed that [11C]Ro 15-4513
(ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1,4-
benzodiazepine-3-carboxylate) uptake was reduced to
nonspecific levels only by drugs that have affinity for
the a5 subtype, such as flunitrazepam, RY80 (ethyl-8-
acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]
[1, 4]benzodiazepine-3-carboxylate,[ethyl-3H]), Ro 15-4513,
and L655,708 [ethyl(13aS)-7-methoxy-9-oxo-11,12,13,13a-
tetrahydro-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-
1-carboxylate], but not by the a1-selective agonist zolpidem.
[11C]Ro 15-4513 uptake was relatively greater in limbic
areas compared with [11C]flumazenil, but lower in the
occipital cortex and cerebellum (Lingford-Hughes et al.,
2002). The authors concluded that [11C]Ro 15-4513 PET
labels the GABAAR containing the a5 subunit in vivo in
limbic structures and can be used to further explore the
functional role of this subunit in humans. This technique
was used to characterize BZD recognition site occupancy
in the human brain associated with administration
of several a2-selective compounds, such as TPA023B
[7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-
3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine] and
MRK-409 [7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-
3-ylmethoxy)-3-(2,6-difluorophenyl)-1,2,4-triazolo[4,
3-b]pyridazine] (Fig. 3) (Atack et al., 2006b, 2011a,b; Van
Laere et al., 2008).
C. Single Photon Emission Computed Tomography
A single photon emission computed tomography (SPECT)
study using 123I-labeled 2b-carbomethoxy-3b-(4-iodophenyl)-
N-(3-fluoropropyl)-nortropane revealed an increase in
dopamine transporter binding in the striatum of SAD
subjects treated with escitalopram for 12 weeks
(Warwick et al., 2012). These subjects also showed a
decrease in anxiety scale scores associated with treat-
ment, but the scores were not correlated with changes
in dopamine transporter binding. The scores remained
relatively high after therapy, suggesting that treatment
may not have been completely successful for all par-
ticipants. These subjects were also not stratified as
responders versus nonresponders, thus limiting the
interpretation of the data.
As revealed by SPECT scans using [123I]iomazenil,
patients with panic disorder exhibit decreased benzo-
diazepine receptor (BZDR) binding in the left hippo-
campus and precuneus relative to controls (Bremner
et al., 2000). Further comparisons of patients with
panic disorder who either did or did not have a panic
attack at the time of the scan revealed a decrease in
BZDR binding in the prefrontal cortex among patients
who had a panic attack. These findings suggest that
BZDR function in the prefrontal cortex appears to be
involved in changes in state-related panic anxiety.
These findings also suggest a role for brain regions
implicated in episodic memory function, such as the
precuneus. This in turn may be amenable to cognitive
behavioral therapy (CBT) and targeting by pharmacologic
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis 1009

Fig. 3. PET studies comparing TPA023B identify BZD binding sites in the human brain. (A) Comparison of the structures of TPA023B (Atack et al.,
2011a), TPA023 (Atack et al., 2006b), and MRK-409 (Atack et al., 2011b). (B) Pseudocolor images on the left show the inhibition of [11C]flumazenil
binding produced by a single 1.5-mg oral dose of TPA023B in PET scans performed 1 hour before and 5 and 24 hours after dosing. TPA023B has
prolonged occupancy at the BZD binding site of GABAARs in the human brain. The right graph shows occupancy plotted as a function of the
corresponding plasma TPA023B concentrations. Modified with permission from Van Laere et al. (2008).

interventions that can enhance extinction of salient
memories that trigger panic attacks. The results indicate
that the neuronal circuits implicated in panic attacks
(i.e., the precuneus) are distinctly different from those
implicated in GAD (i.e., more involvement of the frontal
lobe).
In another study of depressed patients with or without
panic disorder (N = 18), radiolabeled iomazenil SPECT
scans demonstrated that those patients with panic disorder
had a significant decrease (P , 0.05) in the regional activity
index in the lateral inferior temporal lobes (right and left),
medial inferior temporal lobes (left), and inferior frontal
lobes (right and left) after 2 hours (Kaschka et al., 1995).
The findings may be due to either regional blood flow
differences or BZDR effects, but the former hypothesis is
confirmed to some extent by similar findings in the scans
acquired after 10 minutes. Only hypoactivity in the left
lateral temporal region seemed to be independent of the
reduced blood flow seen in panic disorders.
A SPECT study in patients with social phobia found
that citalopram therapy decreased activity in the left
cingulum, the anterior and lateral part of the left temporal
cortex, and the anterior, lateral, and posterior part of the
left midfrontal cortex (Van der Linden et al., 2000).
This broad engagement of cortical areas in anxiety
disorders and anxiolysis may be the consequence of a
primary abnormality or may be a cause of the disorders.
In some ways, similar to the phenomenon of kinetic
ambiguity common to the study of enzyme mechanisms
and bio-organic chemistry, systems ambiguity presents
a conundrum for the pharmacologist seeking to translate
target-based pharmacology into pharmacotherapeutic
effects based on a mechanistic understanding of an
anxiolysis connectome.
D. Magnetic Resonance Spectroscopy
Magnetic resonance spectroscopy (MRS) allows for
the noninvasive monitoring of levels of GABA as well
as other neurochemicals in the human brain (Goddard
et al., 2001; Strawn et al., 2013; Zwanzger et al., 2013).
MRS has been used to measure GABA levels in pa-
tients with PTSD, social anxiety, and panic disorder
(Ham et al., 2007; Pollack et al., 2008; Rosso et al.,
2014). A study of 14 unmedicated patients with panic
disorder and 14 healthy control subjects matched for
age and sex revealed a 22% mean reduction of GABA in
the occipital lobe of 12 of 14 subjects compared with
controls. There were, however, no significant correla-
tions between GABA levels in the occipital lobe and
any measures of illness or state anxiety (Goddard et al.,
2004). In a study of subjects with PTSD, MRS mea-
surement of GABA levels in the insula revealed that
patients had 30% less GABA than healthy controls. This
finding could not be accounted for by age or sex. This
1010 Farb and Ratner

difference could also not be accounted for by the difference
in the percent gray matter versus white matter propor-
tions within the voxel of interest. In addition there was a
significant negative correlation between State-Trait
Anxiety Inventory scores and GABA levels (Rosso et al.,
2014) (Fig. 4).
MRS can also be used to study acute drug-induced
changes in brain glutamate levels. Experimental pharma-
cologic induction of panic has been used to elucidate the
neurobiological correlates of panic attacks. Cholecystokinin-
tetrapeptide-4induced panic attack was studied using
MRS and was found to be associated with an increase in
glutamate plus glutamine/creatine ratios in the anterior
cingulate cortex (Zwanzger et al., 2013). This suggests that
a chemically induced (i.e., by cholecystokinin-tetrapeptide-4)
panic attack alters the glutamatergic system in the anterior
cingulate cortex and may be involved in the pathogenesis
of panic attack. Given the activation of precuneus in salient
episodic memoryinduced panic attack, it is tempting to
speculate that a targetable downstream network involving
the anterior cingulate cortex susceptible to glutamatergic
pharmacotherapy could be an effective approach toward
treating panic attack.
By contrast, in patients with PTSD, positive modulators
of glutamatergic transmission (e.g., D-cycloserine and
acamprosate; see section V.B) have been used in combi-
nation with behavioral therapy to extinguish salient
memories associated with PTSD.
E. Overview
Taken as a whole, brain-imaging studies implicate
the amygdala, prefrontal, insular, limbic, paralimbic,
and occipital cortices as well as the basal ganglia more
often than other regions in pathologic anxiety. Given
the role that the insula plays in integrating sensory,
emotional, and cognitive information, it is certainly not
surprising to find changes in the level of activation in
this brain region. Given that the insula spans from the
prefrontal cortex to the posterior parietotemporal lobes
and thereby creates extensive connections between the
lateral prefrontal cortex, ventromedial prefrontal cortex,
orbitofrontal cortex, cingulate, amygdala, bed nucleus
of the stria terminalis (BNST), and ventral striatum, it
seems reasonable to think of the insula as a node in the
anxiety connectome.
These neuroimaging findings also reveal some evi-
dence of lateralization and localization based on how the
anxiety-inducing stimulus is presented (e.g., verbally
versus visually), suggesting a path forward for differen-
tiating among types of anxiety and tailoring treatment

Fig. 4. MRS studies showing that brain GABA levels in the right insula are lower in patients with PTSD. (A) T1-weighted images depicting voxel
placement as well as difference edited and 68-millisecond spectra from the ACC (A and B) and insula (C and D) of a representative subject. (B)
Associations of GABA/Cr in right anterior insula with state anxiety (A) and trait anxiety (B) in the whole sample. Circles correspond to PTSD patients
(asterisk indicates the single medicated patient) and triangles are control subjects. All fitted metabolite areas were normalized to total creatine
(creatine and phosphocreatine combined). ACC, anterior cingulate cortex; Cho, choline; Cr, creatine; NAA, N-acetylaspartate. Reprinted with
permission from Rosso et al. (2014).
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
appropriately based on systems-level analysis of data.
For example, a meta-analysis of neuroimaging studies
related to emotional processing in SAD, PTSD, and
specific phobia suggests that common brain regions
(hyperactivation of the amygdala and insula) play a role
in these anxiety disorders that can be triggered by
specific stimuli. In addition, the results revealed that
hypoactivation in the dorsal and rostral anterior cingulate
cortices and the ventromedial prefrontal cortex may play a
role in the dysregulation of emotions associated with
PTSD (Etkin and Wager, 2007).
Human imaging studies in which both PET and
fMRI are used to study biomarkers in the same cohort
may be the most suitable for identifying relationships
between individual variability in receptor binding and
brain region activation in response to treatments (although
studies employing one technology are also useful). This
dual-technology approach was used by Fisher et al. (2006)
to study 5-HT1A autoreceptor binding in relation to
amygdala activation and by Rhodes et al. (2007) to
study the association between 5-HT transporter binding
and amygdala activation. Kobiella et al. (2011) took this
approach one step further and studied 5-HT transporter
polymorphisms in relation to amygdala region activation,
further demonstrating the unique power of multimodal
neuroimaging studies.
Nonhuman animal imaging using experimental mod-
els of anxiety are also helping to parse out inferences
concerning the possible differences and similarities
underlying various anxiety disorders. One MRS study
using a chronic stressinduced depression model revealed
a reduction in brain levels of N-acetyl-aspartate, total
creatine, and choline-containing compounds in tree shrews
exposed to psychosocial stress. In this study, the voxel
of interest was the forebrain including parasagittal neo-
cortex, subjacent white matter, and portions of subcortical
forebrain structures, including striatum (Czh et al.,
2001). Postmortem analysis showed reduced cell pro-
liferation in the dentate gyrus and a decreased GluR2
expression in the prefrontal cortex. Concomitant admin-
istration of a novel compound combining partial dopamine
D2 receptor agonism with SSRI activity prevented these
changes, suggesting that combining dopamine D2 receptor
agonism with SSRI activity may serve as a novel treat-
ment of those anxiety disorders presenting with comorbid
depression (Michael-Titus et al., 2008). In another study
using fluorodeoxyglucose-PET, three specific character-
istics of the anxious phenotype identified in rhesus
monkey (hypothalamic-pituitary-adrenal activity, freezing
behavior, and expressive vocalizations) were associated
with a common elevation of activity in the central nucleus
of the amygdala and the anterior hippocampus (Shackman
et al., 2013).
However, can these types of imaging data produce
useful information for real-life therapy in human
patients with a range of anxiety disorders? In patients
with SAD, fMRI was used to measure brain activation
1011
prior to intervention with CBT and showed that changes
in regional activation in response to angry versus neutral
faces or emotional versus neutral scenes are an effective
predictor of response to CBT (Doehrmann et al., 2013).
IV. Neural Systems and the Neurochemical Basis
of Anxiety
In the following sections, we explore the neurochemical
components of the brain, placing these findings in the
context of a systems-level approach to therapeutics based
on the real-time differences in rCBF demonstrated by
functional neuroimaging studies and changes in brain
levels of GABA and glutamate revealed by MRS.
A. Neural Systems Underlying Anxiety
1. A Connectome Hypothesis. Anxiety is a complex
emotional response that involves multiple neurotrans-
mitters acting at various receptors within the limbic
system (i.e., hippocampus, parahippocampal gyrus, cin-
gulate gyrus, hypothalamus, and amygdala) and within
the paralimbic regions of the cerebral cortex such as the
orbital and medial prefrontal cortex (Rauch et al., 1997;
Simpson et al., 2000; Pape and Stork, 2003; Phillips
et al., 2003; Sah et al., 2003; Yilmazer-Hanke et al., 2003;
Zwanzger et al., 2003; Kang-Park et al., 2004; Phan et al.,
2004). From a neural systems perspective, fear and
anxiety seem to be controlled by two major circuits: those
involved in our most primitive innate responses to simple
and overt threatening stimuli, and those involved in
moderating responses to more complex situations. The
prefrontal cortex appears to be the brain region most
often implicated in the latter.
The prefrontal cortex in particular is implicated in
attenuation of responses to anxiety-inducing stimuli.
Lesions to the ventrolateral prefrontal cortex have
been associated with increased anxiety in nonhuman
primates (Agustn-Pavn et al., 2012). It has been
proposed that the amygdala is hyper-responsive to
threatening stimuli, whereas the response of the
ventromedial prefrontal cortex is a blunted in patients
with anxiety disorders, resulting in inadequate regu-
lation of their responses to anxiety-inducing stimuli
(Kent and Rauch, 2003; Rauch et al., 2003). This
hypothesis is supported in part by functional neuro-
imaging studies showing that individuals with GAD
have an attenuated discriminatory response pattern in
the ventromedial prefrontal cortex when presented with
threatening versus nonthreatening stimuli (Greenberg
et al., 2013).
The amygdala, which receives afferent inputs from
the sensory cortex and sends its efferent projections to
the hypothalamus, periaqueductal gray matter, locus
ceruleus, raphe nuclei, and ventral tegmental area
(Sah et al., 2003), appears to be primarily involved in
the encoding and consolidation of emotionally charged
memories (Isenberg et al., 1999; Roozendaal et al.,
1012 Farb and Ratner
2001; Sah et al., 2003; Pelletier and Par, 2004). The
basolateral amygdala (BLA), which is composed of the
lateral, basomedial, and basolateral nuclei, plays a
central role in this process and sends projections to the
medial temporal lobe and nucleus accumbens and
septal nuclei via the stria terminalis (Roozendaal
et al., 2001; Pelletier and Par, 2004). Lesions of the
BLA or its major efferent pathway, the stria terminalis,
interfere with the encoding and consolidation of mem-
ories induced by emotional arousal (McGaugh, 2000;
Roozendaal et al., 2001). In addition, the effects of
glucocorticoids such as RU 28362 (11b,17b-dihydroxy-
6,21-dimethyl-17a-pregna-4,6-trien-20yn-3-one) on memory
consolidation are dependent on an intact BLA nucleus and
its efferent fibers that run through the stria terminalis
(Roozendaal et al., 2001).
The subunit composition and pharmacological prop-
erties of GABAARs vary widely between and within
brain regions. For example, receptors containing the a2
and a5 subunits, which show pharmacologic profiles
similar to the classic BZD II or type 2 receptors, are
found in higher concentrations in the amygdala and
hippocampus, whereas receptors containing a6 subunits,
which are insensitive to BZD anxiolytics, are localized
to the cerebellum (Wieland et al., 1992; McKernan and
Whiting, 1996; Whiting et al., 1999).
The neuroanatomic distribution of 5-HT receptors is
complex, as would be expected based on the pharma-
cological effects of receptor agonists, antagonists, and
SSRIs. The limbic system is critical for the generation
of motivational and affective states, with the limbic
forebrain containing high levels of 5-HT (an evolutionarily
ancient neurotransmitter that has functional ortholog
receptors even in the Drosophila fruit fly) (Nichols et al.,
2002). The 5-HT fibers that project into the forebrain
originate for the most part from the median raphe nucleus
and the dorsal raphe nucleus. These projections are
heterogeneous in terms of morphology and electro-
physiology, representing yet another level of intricacy
that could influence treatment outcomes (Hensler, 2006).
The hippocampus is well known to be involved in
the formation of new memories. Neurogenesis in the
hippocampal dentate gyrus has been implicated in the
formation of both spatial memory and anxiety disor-
ders, as well as in the action of SSRIs. The temporal
behavior of dentate gyrus granule cells with respect to
theta rhythm activity is different between rats with
normal and impaired levels of neurogenesis, suggest-
ing that immature neurons could distinctly affect the
temporal dynamics of hippocampal encoding (Rangel
et al., 2013). Serotonergic antidepressants can reverse
the established state of neuronal maturation in adult
hippocampal cells, and upregulation of 5-HT4 receptor
mediated signaling may play a role in this process
(Kobayashi et al., 2010). The role of dentate granule
cell hypoactivity in anxiety and in the mechanism of
action of SSRIs was recently suggested based on the
observation that challenge-induced hypoactivation of
dentate gyrus neurons in mice with high trait anxiety
is normalized by fluoxetine treatment (Sah et al., 2012).
Differences in the intrinsic physiology and synaptic
inhibition between the mature and immature cell pop-
ulations could separate the activity of differently aged
neurons in a temporal coding regimen, influencing
downstream CA3 neuron integration of information
conveyed by young and mature granule cells. In line
with this hypothesis, fluoxetine treatment induces active
somatic membrane properties in a manner reflective of
immature granule cells (Kobayashi et al., 2010). These
changes do not seem to be explained by an increase in
newly minted immature neurons and may be character-
ized as dematuration of mature granule cells. These
observations suggest that normalization of hippocampal
hypoactivity may serve as a neurobiological marker of
anxiolytic effectiveness and successful remission.
2. Optogenetics and Electrophysiology. Optogenetic tech-
niques have been used to further elucidate the role of the
amygdala in anxiety. Tye et al. (2011) used viral trans-
fections to study the role of BLAcentrolateral nucleus
pathway in the expression of anxiety. Illumination of
channel rhodopsins expressed in BLA terminals located at
the central nucleus of the amygdala was shown to induce an
acute reversible anxiolytic effect. Selective illumination of
specific BLA terminals produces a behavioral response
opposite that induced by activation of all glutamatergic
BLA terminals suggesting that multiple subpopulations
or projections of BLA neurons can act in opposition (e.g.,
direct excitation of centromedial nucleus along with feed-
forward inhibition of centromedial nucleus) (Fig. 5).
Subsequent studies demonstrated that bilateral
optogenetic inhibition of BLA axon terminals in the
ventral hippocampus reduces anxiety-related behaviors in
mice, suggesting that BLA inputs to the ventral hippo-
campus may play a role in modulating basal levels of
anxiety (Felix-Ortiz et al., 2013). A reduction of hippo-
campal theta activity has also been implicated in the
anxiolysis induced by various classes of anxiolytics, in-
cluding buspirone (McNaughton et al., 2007; Chee et al.,
2014). A reduction of hippocampal theta activity has also
been implicated in the anxiolysis induced by various
classes of anxiolytics, including buspirone (McNaughton
et al., 2007; Chee et al., 2014). McNaughton et al. (2013)
proposed that conflict-specific, right frontal activation
recorded noninvasively with surface electrodes is a read-
ily obtainable noninvasive human functional homolog
of rodent hippocampal-cortical activity that may also
be useful as an objective biologic marker of anxiolytic
efficacy.
An optogenetics approach has also been used to
elucidate the role of the dentate gyrus in the processes
of learning and memory, as well as the pathophysiol-
ogy of anxiety (Kheirbek et al., 2013). Specifically,
granule cells in the ventral dentate gyrus suppress
innate anxiety but do not affect contextual learning,
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
Fig. 5. Projection-specific excitation induced by illumination of BLA terminals
in the CeA induces acute reversible anxiolysis in mice. (A) Illustrations showing
where illumination was directed in the channelrhodopsin-2 (ChR2):BLA-CeA,
eYFP:BLA-CeA, and ChR2:BLA (somata) mice. The mice received 5-millisecond
light pulses at 20 Hz for all light-on conditions. (B) Representative paths
in the elevated plus maze during light-on and light-off conditions. (C)
Histograms showing increased probability of open arm entries during light
on phase in ChR2:BLA-CeA mice. (D and E) Open field test data demonstrating
that ChR2:BLA-CeA mice spend more time in center with light on than do
eYFP:BLA-CeA and ChR2:BLA (somata) controls (P , 0.00001). CeA, central
amygdala; CeL, centrolateral nucleus; CeM, centromedial nucleus; eYFP,
enhanced yellow fluorescent protein. Reprinted from Tye et al. (2011).
whereas those in the dorsal region control the encoding
of contextual fear memories but not their retrieval. As
noted, findings from this study suggest that distur-
bances in pattern separation may underlie anxiety
disorders in some patients, and therapies targeting the
excitability of the ventral dentate gyrus may offer
a beneficial approach in this population. These kinds of
techniques have also born fruit in animal models as
well, potentially allowing for translation of data from
animal models to studies in humans. The connection
between hippocampal-mediated learning and anxiety
1013
was further elucidated using optogenetic techniques,
showing that hippocampal dentate gyrus neurons
activated during fear learning in mice can be reac-
tivated later using light stimulation, producing an
increased freezing response outside of the original
context (Liu et al., 2012).
Optogenetics combined with in vivo electrophysio-
logical recordings may further elucidate activity in
the neural networks implicated in anxiety disorders.
Optogenetic inhibition of the BNST in mice increased
exploration of open spaces in the elevated plus maze test
and open field test, whereas stimulation of the BNST
with the excitatory channelrhodopsin-2 was associated
with increased anxiety in both tests. To elucidate the
neural circuitry between the BLA and the anterodorsal
BNST, microdrive electrode arrays containing stereotrodes
surrounding a fiber-optic were implanted in the anterodorsal
BNST of mice expressing channelrhodopsin-2 in the
BLA. Excitation of the glutamatergic BLA terminals
was associated with increased activity of anterodorsal
BNST neurons. Conversely, optically stimulating in-
puts to the oval nucleus of the BNST induced a net
inhibition of neural activity in the BLA, suggesting
that the oval nucleus and anterodorsal BNST play
antagonistic roles in modulating anxiety (Kim et al.,
2013). These findings are consistent with observations
that the extended amygdala plays an important role in
the neural circuitry implicated in anxiety.
The BNST occupies a central position in the neural
circuitry regulating the HPA axis response to stress
and thus plays an important role in regulating the
neuroendocrine and behavioral responses to stress (Henke,
1984; Dunn, 1987; Fernandes et al., 2002; Walker et al.,
2009). The release of neuroactive steroids in this brain
region may represent an endogenous homeostatic mech-
anism for modulating anxiety and restoring normal
GABAergic neurotransmission and HPA axis activity
after exposure to stress-inducing stimuli (Patchev et al.,
1994; Barbaccia et al., 1996; Fernandes et al., 2002).
Stressors that activate the BNST also activate seroto-
nergic systems, and changes in 5-HT receptor expression
in this region were suggested to play a role in pathologic
responses to stress (Guo et al., 2009; Hazra et al., 2012).
Thus, modulation of the activity of specific 5-HT receptor
subtypes in this region may be a novel approach to
treating anxiety disorders.
B. Type A GABA Receptor and Serotonin Receptor
Structure/Roles in Anxiety
A disturbance of the nuanced integration of excit-
atory and inhibitory transmission can occur on a local
network level that ultimately affects interconnected
domains of larger-order structures linking to the outside
world. Unraveling this will undoubtedly involve the
action of networks serving as low-pass as well as high-pass
filters, all of which are based on the integrated action of
multiple transmitters at their respective receptors.
1014
1. GABA. There is general agreement that GABA-
mediated inhibitory neurotransmission plays a partic-
ularly important role in the modulation of emotional
responses to fear-inducing stimuli (Davis and Myers,
2002; Stork et al., 2002, 2003; Walker et al., 2003), and
the concept of positive and negative allosteric modulation
of fast postsynaptic GABAergic inhibitory neurotrans-
mission by BZDs was central to anxiolytic pharmacology
for decades (Choi et al., 1977; Macdonald and Barker,
1978; Chan and Farb, 1985). GABAARs are located
primarily in the postsynaptic membrane at synaptic and
extrasynaptic sites and mediate the majority of fast
postsynaptic inhibitory neurotransmission in the adult
central nervous system (CNS) (Rabow et al., 1995;
Barnard et al., 1998; Ben-Ari, 2002). The action of
GABA on GABAARs can modulate both the frequency
and duration of chloride channel openings to maintain
the membrane near its resting voltage (Macdonald et al.,
1989; Twyman et al., 1989).
The GABAAR is a member of the cys-loop superfamily
of receptors, which includes the nicotinic acetylcholine
receptors, the glycine receptors, and the 5-HT3 receptors.
These receptors all have a characteristic loop formed by
a disulfide bond between two cysteine residues (Sieghart
and Sperk, 2002). The GABAAR is a pentameric complex,
which can be composed from the members of eight
recognized families of glycoprotein subunits (a, b, g, d, ,
p, u, and r) (Fig. 6). Several of these subunit families are
known to include multiple isoforms (e.g., a1a6 and
g1g 3), giving rise to at least 19 distinct subunits that,
when combined, permit the assembly of a large number
of receptor subtypes with variable affinities for GABAAR
modulators (Barnard et al., 1998; Smith et al., 2001).
Each of these receptor subunits has an extracellular
domain, which serves as a ligand-binding site, and four
transmembrane domains, which are involved in chloride
ion channel structure (Fig. 7).
The majority of GABAARs are composed of two a
subunits, two b subunits, and a single g subunit (Pritchett
and Seeburg, 1991; Chang et al., 1996; Graham et al.,
1996; Tretter et al., 1997; Wingrove et al., 2002), with the
GABA binding site located on the a/b subunit (Casalotti
Fig. 6. Schematic representation of GABAAR and the GABAergic synapse. (A) Top view of GABA and BZD binding sites. (B) Side view. Adapted from
Berezhnoy et al. (2007).
Farb and Ratner
et al., 1986). Residues that contribute to the GABA
binding site are F64 on the a subunit and Y157, T160, and
Y205 located on the b subunit (Amin and Weiss, 1993;
Amin et al., 1997; Baur and Sigel, 2003). Substitution of
any of these four amino acids decreases the sensitivity
of the GABAAR to activation by GABA and muscimol.
However, these same amino acid substitutions do not alter
GABAAR activation by pentobarbital, indicating that
these three compounds act at distinct binding sites (Amin
and Weiss, 1993). A large fraction of GABAARs have a
subunit composition of a1b2g 2, and possess many of the
pharmacologic properties associated with the classic BZD
I or type 1 receptors.
Compounds that affect GABAAR-mediated responses
fall into three categories: agonists, antagonists, and
modulators. Agonists, such as muscimol, directly and
specifically interact with the GABA recognition site on
the GABAAR to induce a response. Competitive antag-
onists, such as bicuculline, or noncompetitive antago-
nists, such as picrotoxin, bind to the receptor and block
GABA from activating a response via its recognition
site. Allosteric modulators bind at modulatory sites and
increase, decrease, or null modulate the binding of
GABA to the GABAAR, thereby enhancing, inhibiting,
or null modulating the ensuing response to GABA (Farb
et al., 1984; Chan and Farb, 1985). Chlordiazepoxide
and diazepam were the first demonstrated therapeutics
to act as positive allosteric modulators at the GABAAR
(Braestrup and Squires, 1977; Choi et al., 1977; Mhler
and Okada, 1977; Macdonald and Barker, 1978; Gallager
and Tallman, 1983; Ferrero et al., 1984). These com-
pounds appear to impart a conformational change in the
transmembrane receptor that alters its affinity for GABA
and GABA agonists (Czajkowski and Farb, 1986; Lavoie
and Twyman, 1996; Boileau and Czajkowski, 1999).
Activation of GABAARs with the agonist muscimol also
increases the affinity for the receptor of positive allosteric
modulators [e.g., diazepam and the triazolopyridine CL
218872 (3-methyl-6-[-3-(trifluoromethyl)phenyl]-1,2,4-
triazolo[4,3-b]pyridazine)], indicating that this rela-
tionship is also reciprocal (Chiu and Rosenberg, 1979;
Lippa et al., 1979; Villiger, 1984). Sensitivity to BZDs
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis 1015

Fig. 7. Molecular docking and transmembrane structure of the GABAAR. (A and B) The GABAAR interface (A) and structures (B) of BZD binding site
ligands used in the study. Panel A shows the homology model of a1 and g 2 subunits of the GABAAR with BZD binding site loops highlighted in yellow;
loop F is green. Dashed lines indicate limits of cell membrane. Figure courtesy of Dr. Cynthia Czajkowski. Also see Hanson and Czajkowski (2008).

appears to be mediated primarily by the g 2 and a1, a2,
a3, and a5 subunits (Brooks-Kayal and Pritchett, 1993;
Boileau and Czajkowski, 1999).
Dissimilar compounds influence GABAARs differently.
For example, although BZDs and barbiturates both act
on GABAARs, electrophysiological studies suggest that
the actual mechanism of action is different. These studies
indicate that diazepam increases the duration of GABA-
induced channel openings or burst by only 9.3%, but
increases the number of bursts by 102.5%. In contrast,
phenobarbital increases burst duration by 81.9%, but
increases the number of bursts by only 6.3% (Twyman
et al., 1989). Although the mechanism of receptor
modulation has not been fully elucidated, BZDs appear
to enhance single-channel burst frequencies by increas-
ing the affinity of GABA for its binding site, whereas
barbiturates appear to slow receptor deactivation by
stabilizing the interaction between GABA and the
GABAAR (Mathers, 1985; Twyman et al., 1989; Rabow
et al., 1995; Steinbach and Akk, 2001). The binding of
BZDs to the allosteric BZD site increases the affinity of
the receptor for GABA and enhances GABAergic neuro-
transmission by increasing the number of chloride
channels opened at a given synaptic concentration of
GABA, which in turn hyperpolarizes the cell membrane.
The a subunit appears to be intimately related to
GABAAR sensitivity to the prototypical BZDs and
particularly to diazepam, playing an important role
in drug binding and concentration response relation-
ships (Pritchett et al., 1989; Pritchett and Seeburg,
1990; Wafford et al., 1993; Smith et al., 2001) via
interacting with the a/g interface (Buhr and Sigel,
1997). The g subunit contributes to receptor affinity for
specific ligands as well as the single-channel character-
istics (Herb et al., 1992; Gnther et al., 1995; Benke
et al., 1996).
GABAAR subtypes that are modulated by BZDs can
be divided into those that are diazepam sensitive and
those that are not (Lo et al., 1982). BZDRs that recog-
nize the classic 5-phenyl-1,4-BZDs (e.g., diazepam and
flunitrazepam) are referred to as diazepam-sensitive
receptors, whereas those that do not recognize these
ligands are referred to as diazepam-insensitive recep-
tors (Malminiemi and Korpi, 1989; Hadingham et al.,
1996; Knoflach et al., 1996). It was originally thought that
there were just two subtypes of BZD-sensitive GABAARs.
The first group, type 1 receptors that are enriched in the
cerebellum and globus pallidus, display a higher affinity
for the triazolopyridine CL 218872 and the b-carboline
[3H]propyl-b-carboline-3-carboxylate. By contrast, type II
1016 Farb and Ratner
receptors, which are found in the superficial layer of the
superior colliculus, striatum, and the dentate gyrus of
the hippocampus, show low affinity for CL 218872 and
[3H]propyl-b-carboline-3-carboxylate (Braestrup and
Squires, 1977, 1978; Braestrup and Nielsen, 1981;
Young et al., 1981). It has since been shown that there
are multiple classes of BZDRs, reflecting differences
in GABAAR subunit compositions such as substitution
of the g3 and g2 subunits.
Overall, it appears that sedation and amnesia associated
with classic BZD actions are mediated primarily via a1
subunitcontaining GABAARs in rodents, whereas re-
duction of vigilance is mediated primarily via the a2 and
a3 subunits (Lw et al., 2000; McKernan et al., 2000; Dias
et al., 2005), with activation of the g subunit also playing a
role in BZD efficacy (Rudolph et al., 1999). The a2 subunit
is largely expressed in limbic structures implicated in
anxiety and fear, including the amygdala and hippocam-
pus. Staining intensities of the a2 subunit are highest in
the accessory olfactory bulb, molecular layer of the dentate,
hippocampal area CA3, central and lateral amygdaloid
nuclei, septum, striatum, accumbens, and hypothalamus.
The lateral septum has a high concentration of a2
subunits and has been shown to participate in the
regulation various visceral and somatic functions as
well as in modulation of anxiety, aggression, and fear
responses in rodent models (Pesold and Treit, 1996;
Gavioli et al., 1999). Neurons in this region are also
not sensitive to zolpidem-induced enhancement of
GABAergic inhibition, which has been associated with
activation of receptors containing the a1 subunit (Duncan
et al., 1995). Cat odor exposure induces Fos expres-
sion in the posterior accessory olfactory bulb, which
has also been shown to contain a high concentration
of the a2 subunit (McGregor et al., 2004). Cat odor
induction of Fos expression is also seen in the main
olfactory bulb and midazolam inhibited Fos expres-
sion in this region. Midazolam also inhibited Fos
expression in key limbic regions involved in pheromone
transduction (medial amygdala and BNST) and de-
fensive behavior (prelimbic cortex, lateral septum, lateral
and medial preoptic areas, and dorsal premammillary
nucleus).
H101R mice in which the a2 subunit was rendered
insensitive to diazepam by a knock-in point mutation
(His101Arg) show reduced sensitivity to the anxio-
lytic effects of diazepam. This mutation was not
associated with any overt phenotypic changes and the
animals expressed all subunits tested (a1, a2, a3, b2/3,
and g 2) at normal levels and distribution. After ad-
ministration of diazepam, the a2 knock-in mice did not
show the increase in exploratory behavior (as mea-
sured by the amount of time spent and the number of
entries into the open arms) on the elevated plus maze
that is typically seen in wild-type mice. This difference
in behavior could not be attributed to motor impair-
ment because the motor activity in the enclosed arms
was similar in a2 H101R and wild-type mice irrespective
of the treatment (Lw et al., 2000).
A subsequent study looked at the behavior of H101R
mice in a conditioned emotional response task (Morris
et al., 2006). In these experiments, lever pressing for
food on a variable interval schedule of reinforcement
was suppressed by delivery of a foot shock paired
with a conditioned stimulus tone or light. During the
conditioned emotional response test session, wild-type
and H101R mice were pretreated with diazepam
(0, 0.5, 1, and 2 mg/kg) 30 minutes prior to testing.
The wild-type mice showed an increase in lever pressing
during treatment with diazepam, whereas H101R mice
were resistant to the anxiolytic effects of diazepam.
These observations naturally suggest that develop-
ment of compounds that are selective for those receptor
subtypes implicated in anxiety may prove to be extremely
fruitful in the management of anxiety disorders. To date,
efforts to fine-tune the binding affinity or functional
efficacy of compounds that display selectivity or prefer-
ence for specific receptor subtypes remain promising
in rodent experimental animal systems, yet elusive
in human clinical trials (Ballenger et al., 1991, 1992;
Rudolph et al., 1999; Lw et al., 2000; Sandford et al.,
2001; Atack, 2003; Lippa et al., 2005; Rudolph and
Mhler, 2006; Atack et al., 2011a,b; Olivier et al., 2013).
Additional consideration for targeting therapeutics,
which take advantage of the rapid rates of GABAAR
turnover and the nonlysosomal pathway for degradation
(Borden et al., 1984; Borden and Farb, 1988), may also
yield greater selectivity and reduced adverse events.
Further research directed along a neural-systems level
may well resolve the drug discovery puzzle at hand if
the receptor subtype and selective modulatory thera-
peutic can be effectively aligned for the treatment of
specific anxiety disorder subtypes.
2. Serotonin. The role of 5-HT in the pathophysiol-
ogy of anxiety disorders has become increasingly well
established, and agents that inhibit the reuptake of
this neurotransmitter now represent important com-
ponents of current approaches to anxiolysis (Graeff and
Zangrossi, 2010; Sertraline, 2012; Venlafaxine, 2012;
Fluoxetine, 2013). 5-HT is a monoamine whose postsynaptic
action is terminated via 5-HT reuptake into the presynaptic
nerve terminal via a 5-HT transporter. The efficacy of SSRIs
is thus based on augmenting the concentration of 5-HT in
the synaptic cleft by inhibiting its clearance (Ressler and
Nemeroff, 2000; Nichols and Nichols, 2008).
Effects of 5-HT are mediated by as many as 14 receptor
subtypes, including 13 G proteincoupled receptors
(GPCRs) and a single ligand-gated ion channel. These
receptors are divided into seven distinct classes (5-HT1
to 5-HT7) (Fig. 8); however, postgenomic modifications
can produce at least 20 additional GPCRs, leaving more
than 30 5-HT receptors that signal through G proteins.
In addition, activation may result in variable effects,
depending on the brain region in which the receptors
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
Fig. 8. Model of a serotonergic neuron and synapse. 5-HT1A receptors can
function both as autoreceptors on the serotonergic cell body and as postsynaptic
receptors. Most 5-HT receptors are GPCRs and are located postsynaptically to
serotonergic neurons. The 5-HT3 receptor is the exception because it is a ligand-
gated ion channel. AC, adenylyl cyclase; DAG, diacylglycerol; GIRK, G protein
coupled inwardly rectifying potassium channel; IP3, inositol trisphosphate;
PLC, phospholipase C; TPH, tryptophan hydroxylase. Adapted from Gray and
Roth (2007).
are expressed and on individual differences in respon-
siveness mediated by genetic polymorphisms (Raymond
et al., 2001; Hoyer et al., 2002; Gray and Roth, 2007).
Although it is tempting to conceptualize the actions
of 5-HT at its receptor sites as being either inhibitory
or excitatory, the complexity of the signal transduction
pathways activated ultimately may confound such
an appealing target- and hypothesis-driven approach.
That said, 5-HT receptors generally have the following
functions (Polter and Li, 2011): 1) 5-HT1 receptors inhibit
adenyl cyclase, reduce cyclic AMP, and inhibit protein
kinase A (PKA); 5-HT1A receptors are the prototypical
5-HT1 receptors; 2) 5-HT2 receptors increase inositol
trisphosphate, increase intracellular calcium levels, increase
1017
diacylglycerol, and activate PKA; 5-HT2A receptors are the
prototypical type 5-HT2 receptors; 3) 5-HT3 receptors are
ligand-gated cation channels; 4) 5-HT4 receptors activate
adenyl cyclase, increase cyclic AMP, and activate PKA; 5)
5-HT5 receptors inhibit adenyl cyclase, reduce cyclic AMP,
and inhibit PKA; and 6) 5-HT6 and 5-HT7 receptors activate
adenyl cyclase, increase cyclic AMP, and activate PKA.
The complex distribution and functionality of these
receptor subtypes contributes to their unique ability to
modulate emotional responses and other bodily func-
tions via the systems into which they feed forward.
The 5-HT3 receptor is a ligand-gated ion channel. The
remaining subtypes are GPCRs with the latter also
being defined as a type A family, rhodopsin-like receptors.
Evidence suggests that most, if not all, of the 5-HT GPCRs
undergo dimerization, and that dimerization may be
necessary for proper functioning of the receptor (Nichols
and Nichols, 2008). For example, when inactive 5-HT2C
receptors are coexpressed with a wild-type 5-HT2C re-
ceptor, the inactive receptor is able to inhibit the
activity of the wild-type receptor through the formation
of a nonfunctional heterodimer, suggesting that di-
merization is essential for receptor function (Herrick-
Davis et al., 2005). Studies to date suggest that the
5-HT receptors primarily related to anxiety and mood
are 5-HT1A, 5-HT2A, and 5-HT2C (Toth, 2003; Van Oekelen
et al., 2003; Mato et al., 2010).
Interestingly, the roles of these receptor subtypes
may vary depending on the form of anxiety involved, as
outlined in a review by Graeff and Zangrossi (2010).
The authors suggest that SSRIs improve symptoms
in patients with panic disorder by enhancing the
responsiveness of 5-HT1A and 5-HT2A receptors in the
dorsal periaqueductal gray matter in the midbrain, but
improve symptoms in patients with generalized anxi-
ety through the desensitization of 5-HT2C receptors and,
perhaps, through stimulation of 5-HT1A receptors in the
forebrain.
Understanding the effects of 5-HT in anxiety disorders
requires attention to variations in transporter reuptake
subtypes that may potentially influence the clinical
response to SSRIs. Genetic polymorphisms of the 5-HT
transporter gene promoter (5-HTTLPR) have been
associated with efficacy of antidepressants (Porcelli
et al., 2012), as well as with anxiety. For example,
a functional polymorphism of the 59-flanking region
of the 5-HT transporter gene (SLC6A4) is associated
with differences in anxiety-related personality traits
(Heils et al., 1996; Lesch et al., 1996). A 43-bp deletion/
insertion in 5-HTTLPR is associated with long L
(16 repeats) and short S (14 repeats) alleles (Heils
et al., 1996). There appear to be ethnic differences in the
prevalence of these polymorphisms as well, with Cau-
casians having about 22% S/LG alleles and Asians having
about 60% S/LG alleles (Hu et al., 2006). Gene/environment
interactions may therefore account for susceptibility
to anxiety disorders and for patient response to
1018 Farb and Ratner
pharmacological treatment with SSRIs and SNRIs (Lesch
et al., 1996; Nakamura et al., 2000; Hu et al., 2006;
Klumpers et al., 2012). The L(A) allele is overtransmitted
by 2-fold to patients with OCD (Hu et al., 2006), whereas
subjects with at least one short allele exhibit greater
fear-potentiated startle than do subjects with the long
allele (Klumpers et al., 2012).
Another genetic factor implicated in the clinical re-
sponse to SSRIs is a polymorphism in the gene that codes
for RGS2, a protein involved in neuronal GPCR responses
to neurotransmitters including 5-HT. Stein et al. (2014)
found that a mutation that reduces expression of RGS2
is associated with a poor clinical response to sertraline
treatment.
V. Neuropsychopharmacology of
Anxiety Disorders
Different combinations of subunits comprise the
GABAA, NMDA, and 5-HT receptors, providing tre-
mendous variability in affinity for endogenous and
exogenous ligands and thus for the ability of the receptor
to modulate neuronal function (Smith et al., 2001;
Nichols and Nichols, 2008). Below, we review a range
of established and investigational anxiolytic agents,
outlining the impact of selective modulation of receptor
subtypes where data are available. The pharmacology of
drug action is inherently the result of the net outcome of
systems-level actions of the neuroactive agent. Therefore,
it must be considered that, although subtype-preferring
compounds exist, highly selective therapeutics have not
yet been developed.
A. Positive Allosteric Modulation of
GABAergic Transmission
1. Benzodiazepines and Related Modulators.
Although the actions of BZDs on GABAARs can reduce
anxiety, this therapeutic effect is not without several
adverse side effects, including drowsiness and ataxia
as well as the risk of dependence and withdrawal reactions
(Noyes et al., 1988; Bennett et al., 1998; Yamawaki, 1999;
Allgulander et al., 2003). Because of these untoward effects,
the use of BZDs for long-term treatment of chronic anxiety
disorders such as GAD is undesirable (Allgulander et al.,
2003). As indicated previously, side effects and efficacy of
anxiolytic effects may be mediated through different
a subunits (Lw et al., 2000; McKernan et al., 2000;
Dias et al., 2005), providing one impetus for research
into the development of more selective compounds.
Partial positive allosteric modulators have a lower
intrinsic ability to induce a modulatory effect at given
fractional occupancy as compared with full allosteric
positive modulators and represent an approach to
developing anxiolytics with fewer side effects and toxicity
(Atack, 2003). The relationship between fractional re-
ceptor occupancy and fractional effect can be assessed by
determining the percentage of inhibition of flumazenil
binding to GABAARs and potentiation of GABA-evoked
chloride currents. Potentiation of GABA-gated currents
by 25% occurs at about 35% receptor occupancy for
diazepam and about 45% for triazolam (Facklam
et al., 1992b). The benzoquinolizinone Ro 19-8022
[(R)-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-
1-yl)carbonyl]-2-pyrrolidine-methanol], a BZDR partial
allosteric modulator, produced 25% potentiation when
receptor occupancy reached about 95%. However, the
imidazobenzodiazepinone bretazenil, or Ro 16-6028
[(13aS)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H-
imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylic
acid 1,1-dimethylethyl ester], which also acts as a partial
allosteric positive modulator, did not produce 25%
potentiation even at 100% receptor occupancy. These
in vitro observations can be related to in vivo responses
in animal models, which indicate that bretazenil does
not protect against seizures as well as the full allosteric
positive modulator diazepam (Brouillet et al., 1991;
Haefely et al., 1992). By contrast, most studies in humans
and animals indicate that bretazenil can provide anxio-
lytic effects with less sedation and motor impairments
than diazepam (Facklam et al., 1992a; Cole and Rodgers,
1993; Busto et al., 1994). However, there is an increase in
symptoms of sedation and deficits in certain measures of
attention and executive function (digit symbol) among
subjects receiving 0.5 mg bretazenil (van Steveninck et al.,
1996).
Although most compounds possessing the b-carboline
structure are anxiogenic, the b-carboline abecarnil
(isopropyl-6-benzyloxy-4-methoxymethyl-b-carboline-3-
carboxylate) produces anxiolytic effects without sub-
stantial sedation (Stephens et al., 1990; Ballenger
et al., 1992). Abecarnil exhibits high affinity for the BZD
recognition site, although no differences in affinities
were observed for recombinant receptors expressed in
human embryonic kidney cells containing a1, a2, or a3
subunits (Hadingham et al., 1993). Another study shows
that abecarnil only partially inhibits diazepam-insensitive
binding of [3H]Ro 15-4513 in the cerebellum, suggesting
that this site can be differentiated into abecarnil-sensitive
and abecarnil-insensitive components (Mehta and Shank,
1995).
Electrophysiological studies indicate that abecarnil
potentiates GABA-induced chloride currents less than
full allosteric modulators. Interestingly, abecarnil is
most effective with the fewest side effects at the lowest
dose examined in a double-blind, placebo-controlled
clinical trial for treatment of GAD (Ballenger et al.,
1991, 1992). However, higher concentrations (.7.5 mg/day)
of abecarnil were associated with reports of symptoms and
signs of CNS depression including somnolence, difficulty
concentrating, and dizziness. These observations suggest
that efficacy alone may not account for all of the adverse
side effects of BZDs.
The cyclopyrrolone pagoclone, another compound
believed to act as a partial positive allosteric modulator
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
of the GABAAR, has also been investigated for its
anxiolytic effects. A randomized double-blind study of
patients suffering from panic attacks found that this
compound provides anxiolytic effects without the typical
side effects associated with BZDs (Sandford et al., 2001),
but it has not been approved for use in the treatment of
anxiety disorders (Bateson, 2003; Atack et al., 2006a; de
Wit et al., 2006).
Agents such as imidazenil and SL651498 [6-fluoro-9-
methyl-2-phenyl-4-(pyrolidin-1-yl-carbonyl)-2,9-dihydro-
1H-pyrido[3,4-b]indol-1-one], which have a relatively
higher affinity for specific GABAAR subunits, such as
the a2 subunit, also appear to produce reduced mea-
sures of vigilance (a model for anxiolytic effects in
human) with fewer side effects than the classic BZDs
(Sieghart, 1994; Griebel et al., 2001; Williams and Akabas,
2001; Collins et al., 2002; Licata et al., 2005). This is
consistent with the hypothesis that the a2 subunit
containing receptor subtype, which is largely expressed
in the limbic system, may be an important contributor
to the subtle positive modulation of GABAergic in-
hibition necessary to induce a reduction in vigilance
in rodents and possibly induce anxiolysis in humans
without undesirable side effects (Pritchett et al., 1989;
Pritchett and Seeburg, 1990; Lw et al., 2000; Kaufmann
et al., 2003; Licata et al., 2005). Yet bridging the gap
between convincing results of a therapeutic candidate
in animal models for anxiety and its beneficial clinical
utility in humans remains an elusive if not daunting
objective.
Research into the allosteric modulatory effects of com-
pounds such as zolpidem, which act on the BZD site but
do not possess the 7-member ring associated with the
structure of the classic BZDs, has yielded compounds with
a preference for specific GABAAR subunits (Ghiani et al.,
1994; Serra et al., 1994; Drover, 2004). For example,
zaleplon, zolpidem, and zopiclone exhibit a modest
preference of up to 6-fold for GABAARs containing a1
subunits compared with a2 or a3 subunits using model
systems (Drover, 2004), and are effective sedative/
hypnotic agents reported to produce less tolerance
and dependence than diazepam (Follesa et al., 2002).
Compounds that do not act at GABAARs containing
a1 subunits may be useful as nonsedating anxiolytics.
Whereas zolpidem and zaleplon appear to lack signif-
icant anxiolytic effects and seem to also not have any
active metabolites, zopiclone is metabolized in vivo
to the active metabolite (S)-desmethylzopiclone, which
has been shown to produce anxiolysis with fewer neg-
ative effects on locomotor activity than diazepam and
alprazolam, suggesting that structurally related com-
pounds may be useful in the treatment of anxiety
disorders (Carlson et al., 2001; Fleck 2002).
Ocinaplon (pyrazolo[1,5-a]-pyrimidine) is a novel
pyrazolopyrimidine that potentiates GABAergic neuro-
transmission, increases the response of rats and primates
in measures of anxiety (conflict test), and attenuates
1019
symptoms among human subjects with GAD as demon-
strated by scores on the Hamilton Anxiety Rating Scales
(Lippa et al., 2005). Electrophysiological studies indicate
that ocinaplon has lower potency and efficacy than
diazepam. However, the potentiation of GABA-gated
currents in recombinant receptors expressed in Xenopus
oocytes observed in this study indicates that ocinaplon
has greater efficacy at a1 subunits than at a2 subunits,
a finding that on the surface appears to be in conflict
with the hypothesis that anxiolysis is mediated by a2
subunitcontaining GABAARs.
A compound with comparable binding affinity but
different efficacies at the various receptor subtypes
can exert its effects predominantly at those receptor
subtypes implicated in anxiolysis, rather than having
nonspecific effects and/or producing untoward side
effects such as sedation (Atack, 2003). Interestingly,
although ocinaplon shows selectivity for modulating a1
subunitcontaining GABAARs, competitive binding
assays using native GABAAR membrane preparations
derived from rat cerebellum (wherein a1 subunits are
highly expressed) indicate that this compound is also much
less potent at inhibiting the binding of flunitrazepam
than is diazepam (Lippa et al., 2005). Since the clinical
potency of a drug depends on its binding affinity for the
target and its efficacy of signal transduction, it is plausible
that lower receptor occupancy may contribute to the
ability of ocinaplon to produce anxiolysis without inducing
sedation.
Alternatively, some positive modulation of GABAARs
containing a1 subunits may be necessary to effectively
induce anxiolysis in the human nervous system com-
pared with mouse models, wherein even mild dysreg-
ulation of higher-order cognitive processes mediated by
the frontal lobes has been implicated in controlling
threat assessment and anxiety (Cha et al., 2014). Ir-
respective of which of these hypotheses or others turns
out to be the most valid, the development of this promising
compound was halted due to reports of elevated liver
enzymes in just a few subjects, underscoring the inherent
difficulties associated with bringing a novel anxiolytic to
market even using a well defined target.
SL651498 is a pyridoindole that preclinical studies
suggest acts at the BZD binding site to produce anxiolytic
effects qualitatively similar to those of BZDs with re-
duced ataxia or sedation. This agent is a full positive
modulator at recombinant rat GABAARs containing a2 or
a3 subunits (approximately 95% enhancement) and a
partial positive modulator at receptors containing a1 or
a5 subunits (approximately 45% enhancement). Binding-
affinity assays indicate that SL651498 does not differen-
tiate between a1 (17 nM), a2 (73 nM), and a3 (83 nM)
subtypecontaining GABAARs (Griebel et al., 2001),
indicating that binding affinity does not contribute to
the anxiolytic actions of this drug.
2. Natural Product Modulators. Some natural products
may also provide new platforms for discovery. For example,
1020 Farb and Ratner
chrysin, one of the first flavonoids shown to interact
with the BZD binding site, initiated the search for
natural anxiolytics among traditional medicinal herbs
(Medina et al., 1989, 1990, 1997; Wolfman et al., 1994,
1996). A number of other naturally occurring flavonoids
and their derivatives, such as K36 (5,7,29-trihydroxy-6,8-
dimethoxyflavone) (Huen et al., 2003), hispidulin (Kavvadias
et al., 2004), wogonin (Hui et al., 2000, 2002), and related
compounds (Nielsen et al., 1988; Hberlein et al., 1994;
Wolfman et al., 1996; Goutman et al., 2003), are
allosteric modulators at GABAARs. In a mouse study,
wogonin (7.530 mg/kg p.o.) was found to produce an
anxiolytic response similar to diazepam in the elevated
plus maze without accompanying sedation (Hui et al.,
2002). K36, a naturally occurring flavonoid, increases
entries onto the open arms of the elevated plus maze
without changing locomotor activity, sedation, myorelax-
ation, or motor incoordination in mice (Huen et al., 2003).
3. Increasing GABA Levels. On the basis of all of
these results, it would be expected that increasing
synaptic concentrations of GABA, either by inhibiting
its reuptake or degradation, could be another approach
to enhancing GABAergic neurotransmission and re-
ducing anxiety disorders. GABA-transaminase inhib-
itors, such as vigabatrin (g-vinyl-GABA) and valproic
acid (VPA) (Rando et al., 1981; Gram, 1988), increase
synaptic concentrations of GABA by preventing catab-
olism of GABA (Zwanzger et al., 2001a,b; Nemeroff,
2003). Vigabatrin raises levels of GABA in nerve terminals
and inhibits striatal dopamine release (Kushner et al.,
1997). However, reports of psychosis and depression
associated with vigabatrin suggest that this particular
compound may have significant clinical limitations
(Levinson and Devinsky, 1999; Ashton and Young, 2003).
In retrospect, elevating GABA in a way that is pro-
portionate to its endogenous levels might be expected to
activate the natural distribution of relevant receptor
subtypes nonselectively, thus increasing the probability
of side effects. A more nuanced, neural systemslevel
approach might be capable of modulating anxiety in
a similar fashion to the effective SSRIs.
B. Modulators of Glutamatergic Transmission
A delicate balance between excitatory and inhibitory
neurotransmission is essential to modulation of anxi-
ety and fear responses. Not surprisingly, disturbances
of glutamatergic as well as GABAergic neurotransmis-
sion have been implicated in anxiety disorders (Jardim
et al., 2005).
1. Inhibitors of Glutamatergic Activity. Glutamatergic
neurotransmission in the lateral amygdala is essential
to the acquisition and extinction of fear-related memories
and behavioral responses associated with re-exposure to
fear-inducing stimuli (Sajdyk and Shekhar, 1997). When
a neutral stimulus is paired with an adverse stimulus
such as presentation of a tone paired with delivery of
a foot shock, the previously neutral stimulus is soon able
to elicit a fear response independently. NMDA receptor
antagonists such as D,L-2-amino-5-phosphonopentanoic
acid, which prevent induction of long-term potentiation,
have also been shown to block the acquisition but not
the expression of conditioned fear responses in rodents
(Miserendino et al., 1990; Falls et al., 1992). As pre-
viously mentioned, the prefrontal cortex appears to be
the brain region most often implicated in attenuation of
anxiety.
Memantine is another NMDA receptor antagonist
that is currently approved for the treatment of Alzheimers
disease, but there is some evidence that it may also have
anxiolytic properties. A study in mice found that high
stable doses of memantine (10, 30, and 100 mg/kg
per day) produced some improvements in measures of
anxiety, including reductions in isolation-induced aggres-
sion, escape latency (hidden platform), and wall swimming
tendency in the Morris water maze test (Minkeviciene
et al., 2008). In addition, several case studies in humans
have shown some benefit for memantine in reducing
symptoms associated with PTSD, SAD, and GAD
(Battista et al., 2007; Schwartz et al., 2012). For example,
in a small study of four patients with combat exposure
induced PTSD, treatment with memantine led to consis-
tent improvements in memory as well as depressive and
hyperarousal symptoms (Battista et al., 2007).
Another study showed that acamprosate, an NMDA
and metabotropic glutamate 5 receptor modulator, was
effective as a therapeutic for idiopathic and chemical
withdrawalinduced anxiety disorders (Kotlinska and
Bochenski, 2008; Hertzman et al., 2009; Schwartz et al.,
2010; Paucha-Poniewiera and Pilc, 2012; Koltunowska
et al., 2013). Acamprosate seems to be a safe and ef-
fective therapeutic option for patients presenting with
comorbid alcoholism. These findings were initially
not entirely surprising given that enhancing inhibitory
neurotransmission is a well established method of treating
anxiety; thus, it logically followed that if acamprosate
attenuated excitatory neurotransmission, it could yield
a similar clinical outcome (Berton et al., 1998). However,
subsequent research has revealed both enhancement as
well as inhibition of network synaptic elements underlying
glutamatergic and GABAergic transmission, further em-
phasizing the need for a larger-scale understanding of
drug action in the context of connectivity.
There is also evidence that riluzole, which reduces
symptoms of GAD (Pittenger et al., 2008), may exert
its effects by inhibiting voltage-gated sodium channels
and enhancing astrocytic uptake of extracellular gluta-
mate (Urbani and Belluzzi, 2000; Wang et al., 2004). In
an open-label study of GAD patients, 8 of 15 patients
treated with riluzole had remission of anxiety over a
period of 8 weeks, with a median time to response of
2.5 weeks (Mathew et al., 2005). Thus, therapeutics that
inhibit glutamatergic neurotransmission (e.g., riluzole)
may prove to be more useful in treating GAD and the
hyperarousal seen in PTSD.
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
2. Potentiators of Glutamatergic Activity. Admin-
istration of D -cycloserine, a partial NMDA receptor
agonist, has been shown to facilitate extinction of learned
fear responses in rodents and of phobias, SAD, and PTSD
among patients undergoing behavioral exposure therapy
(Walker et al., 2002; Ressler et al., 2004; Woods and
Bouton, 2006; Mao et al., 2008; Aupperle et al., 2009;
Difede et al., 2014).
A randomized, double-blind, placebo-controlled clinical
trial examining the efficacy of a combination of D-cycloserine
or placebo and exposure therapy for treatment of SAD
revealed that short-term dosing of partial NMDA receptor
agonists may also be effective in treating this disorder
(Hofmann et al., 2006). This compound seems to be most
effective for treating anxiety disorders that respond to
exposure therapy in which learning not to fear the
stimulus facilitates improvement in symptom measures.
In a similar study, Difede and colleagues (2014) compared
the effectiveness of VRE therapy alone versus the
combined treatment with D-cycloserine plus VRE, and
found that D-cycloserine augmented the therapeutic re-
sponse to VRE, suggesting that negative memories may
be extinguished using cognitive enhancers. Overall, these
observations suggest that compounds that increase
glutamatergic neurotransmission are useful in the treat-
ment of certain types of anxiety disorders (e.g., specific
phobias) and PTSD wherein re-exposure to a specific
salient stimuli can trigger a pathologic response that
interferes with activities of daily living (Heresco-Levy
et al., 2002; Difede et al., 2014).
3. Neurosteroids as Modulators of Glutamatergic
Transmission. The observation that stress-induced
anxiety changes glutamatergic neurotransmission (Masneuf
et al., 2014) raises the possibility that endogenous
mechanisms for modulating the balance of excitatory
to inhibitory neurotransmission could underlie the
genesis of anxiety. Neurosteroids such as pregnanolone
sulfate, which enhances cognition, positively modulates
NMDAR function, and stimulates NMDAR trafficking to
the cell surface (Wu et al., 1991; Kostakis et al., 2013),
may represent one such endogenous mechanism for
linking fearful stimuli and the emotional response
subject to therapeutic manipulation (Plescia et al.,
2013). Neuroactive steroids, such as pregnanolone
sulfate and pregnanolone hemisuccinate, which in-
hibit glutamatergic neurotransmission by negatively
modulating NMDAR function (Park-Chung et al.,
1994) and may also exhibit mixed function by serving
as both a negative modulator of the NMDAR and
a prodrug for pregnanolone (a positive modulator
of GABAAR) delivery across the bloodbrain barrier,
may also represent a novel platform for discovery
(Weaver et al., 1997, 2000).
C. Dopaminergic Transmission
In a recent study by Zweifel and colleagues (2011),
cue conditioning was found to be significantly impaired
1021
in knockout mice lacking functional NMDA receptors
on dopaminergic neurons of the ventral tegmental
area. This impairment was associated with development
of a GAD-like phenotype, suggesting that dopaminergic
neurotransmission plays a role in this anxiety disorder.
Interestingly, increased anxiety is among the symptoms
associated with dopamine agonist withdrawal in humans
(Nirenberg, 2013). The interplay between the glutamatergic
and dopaminergic systems is well studied in drug ad-
diction, wherein motivation for drug seeking is mediated,
in part, by this relationship. Nucleus accumbens and pre-
frontal cortex dopaminergic and glutamatergic systems
have been implicated in anxiety disorders and addiction
(Ahmadi et al., 2013) and it has been suggested that the
D2/D3 receptor agonist quinpirole may induce compulsive
checking behavior in rodents by enhancing dopaminergic
activity (Alkhatib et al., 2013). The role of motivation in
OCD is well established and plays a key role in the ability
of an individual to override his or her pathologic responses
to anxiety-inducing stimuli; it is not uncommon for pa-
tients with OCD and phobias to temporarily overcome
their fears to avoid life-threatening events.
In contrast with the D2/D3 receptor agonist quinpirole,
the anxiolytic buspirone has been shown to block D3
receptors in primates (Kim et al., 2014). However, the
role that this mechanism plays in attenuating anxiety
is not entirely clear since the compound also acts at
serotonin and adrenergic receptors (Skolnick et al.,
1984).
D. Epigenetics of Anxiolysis: Histone
Deacetylase Inhibitors
Combining histone deacetylase inhibitors with CBT
can also facilitate extinction of learned fear responses in
humans and animals (Heinrichs et al., 2013; Kuriyama
et al., 2013). Administration of VPA at a dose of 400 mg/kg
to human subjects was shown to augment acquisition
and delayed offline consolidation of extinction training
(Kuriyama et al., 2013). The unexpected effect of VPA
on acquisition could be due to in part to an anxiolytic
effect associated with enhanced GABAergic neurotrans-
mission mediated by inhibition of GABA transaminase
by VPA at this dose.
VPA seems to be rather nonspecific in terms of mech-
anisms of action, at least at present. For example, in
addition to inhibition of histone deacetylases, VPA also
increases levels of brain-derived neurotrophic factor
and cAMP response element-binding protein in the
hippocampus and amygdala (Jeon et al., 2006; Casu
et al., 2007; Jornada et al., 2010). A case of familial
bipolar disorder with comorbid anxiety has been
studied as part of the National Institute of Mental
Health Human Genetics Initiative and was found to be
associated with the genes that code for the extracellu-
lar signal-regulated kinase/mitogen-activated protein
kinase and cAMP response element-binding protein
regulated intracellular signaling pathways (Kerner
1022 Farb and Ratner
et al., 2013). Although further research in this area is
needed, these findings suggest that behavioral modifi-
cation therapy could be combined with drugs that
modulate gene expression to facilitate extinction of the
fear-inducing stimuli associated with specific phobias
and PTSD.
E. Modulators of Serotonergic and
Noradrenergic Transmission
Several studies reveal the potential benefits of SSRIs
over BZD pharmacotherapy of certain types of anxiety.
For example, it has been shown that patients who
present with comorbid symptoms of depression and
anxiety may respond better to SSRIs, whereas those
who suffer from anxiety alone may respond better to
BZDs. In addition, patients suffering from GAD re-
spond well to BZDs, whereas those with PTSD and
OCD have a relatively better clinical response to SSRIs
(Rickels and Rynn, 2002; Varia and Rauscher, 2002;
Blanco et al., 2003; Davidson, 2003).
Paroxetine was the first SSRI to receive approval
from the US Food and Drug Administration for the
treatment of anxiety disorders and was superior to
placebo in short-term clinical trials among patients
with GAD (Allgulander et al., 2003). The SSRI fluoxetine
is widely used for the treatment of patients who are
comorbid for depression and anxiety (Cassano et al.,
2004). It seems unclear whether the anxiolytic effects of
fluoxetine may paradoxically be due entirely to enhance-
ment of GABAergic tone at the neural-systems level.
Electrophysiological results based on recombinant
GABAARs transiently expressed in mammalian cells
reveal that fluoxetine increases the response of these
receptors to submaximal GABA concentrations, although
it does not alter the maximum current amplitude
(Robinson et al., 2003), and this result is of uncertain
relevance to the physiologic effects of SSRIs.
Although data are less abundant for SNRIs, several
clinical trials have established the efficacy of venlafaxine
in patients with anxiety (Liebowitz et al., 2005; Stein
et al., 2005). In one double-blind study of adult patients
with generalized SAD (N = 271, intent to treat), venlafaxine
extended release produced significant improvements in the
Liebowitz Social Anxiety Scale total score, Clinical Global
Impressions-Severity of Illness scale, and the Social Phobia
Inventory, compared with placebo over 12 weeks (Liebowitz
et al., 2005). Similarly positive results were seen over
6 months (Stein et al., 2005).
A recent meta-analysis compared nine drugs for the
treatment of GAD in the United Kingdom (duloxetine,
escitalopram, fluoxetine, lorazepam, paroxetine, pregabalin,
sertraline, tiagabine, and venlafaxine). The investigators
found that fluoxetine was ranked first for response and
remission (probability of 62.9 and 60.6%, respectively),
whereas sertraline was ranked first for tolerability (49.3%)
(Baldwin et al., 2011). These findings are generally in line
with current practice, in which SSRIs are often the
first-line treatment of a range of anxiety disorders. An-
other review in the United States assessed data from
placebo-controlled clinical trials of BZDs, azapirones,
antidepressants, and anticonvulsant and antipsychotic
drugs, and concluded that patients with general anxiety
disorder should receive either an SSRI or SNRI as first-
line treatment (Reinhold et al., 2011).
F. Neuroactive Steroids as Neuromodulators
of Transmission
Neuroactive steroid modulation of GABAergic neu-
rotransmission in the central amygdala has been
implicated in anxiety (Wang et al., 2007). There is some
evidence that fluoxetine increases brain levels of the
neurosteroid allopregnanolone, a positive allosteric mod-
ulator of GABAARs (Matsumoto et al., 1999), and it has
been suggested that the anxiolytic effects of fluoxetine
and possibly other SSRIs (e.g., paroxetine) are related to
this effect on allopregnanolone (Uzunova et al., 1998;
Griffin and Mellon, 1999; Khisti and Chopde, 2000). This
hypothesis is supported by a recent study in a mouse
model of social isolation, which found that an SSRI-
induced increase in allopregnanolone significantly con-
tributes to the anxiolytic effects of SSRI treatment
(Pinna, 2010). It is therefore of interest to take a closer
look at the mechanisms of action and modulatory sites of
neuroactive steroids, such as allopregnanolone.
The search for an endogenous system of new targets
for modulation has been intensive, and neuroactive
steroid-based options offer some promise. Selye (1970)
first demonstrated that endogenous steroids and their
metabolites can produce rapid effects on CNS activity,
including modulation of GABAergic neurotransmission
associated with anxiety (Lambert et al., 2001, 2003;
Yang et al., 2002).
The potential effects of neuroactive steroids on the
development of anxiety disorders can be observed during
puberty, a developmental stage associated with increased
levels of reproductive hormones and onset of many
psychiatric disorders, including GAD, social anxiety,
and panic attacks (Hayward and Sanborn, 2002; Kessler
et al., 2005a; Merikangas et al., 2009, 2010; Reardon
et al., 2009; Van Oort et al., 2009). Anxiety symptoms
have been found to increase from middle to late
adolescence (Van Oort et al., 2009), with a particularly
high prevalence of all anxiety disorders reported among
adolescent girls (Merikangas et al., 2009; Leikanger
et al., 2012; Legerstee et al., 2013).
Of particular interest in this setting are the neuroac-
tive steroids allopregnanolone (in humans and rats) and
pregnanolone (in humans only), metabolites of the
reproductive hormone progesterone that are also pro-
duced in the brain in response to stress (Purdy et al.,
1991; Grdler et al., 2001). Allopregnanolone decreases
in the cerebral spinal fluid of women with PTSD
(Rasmusson et al., 2006), but increases in depressed
patients receiving SSRIs (Uzunova et al., 1998). Moreover,
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis 1023

administration of sodium lactate and cholecystokinin-
tetrapeptide to persons with panic disorderinduced
attacks decreases plasma concentrations of both
pregnanolone and allopregnanolone, and increases the
concentration of the functional antagonistic isomer
3b,5a-tetrahydroprogesterone (Strhle et al., 2003).
Together these findings suggest that changes in endoge-
nous brain levels of neuroactive steroids associated with
age, sex, stress, and administration of SSRIs may play
a role in the onset and clinical response to treatment in
certain anxiety disorders.
Supporting this hypothesis are findings related to
the effects of allopregnanolone on gonadotrophin hormone-
releasing hormone, the primary chemical messenger
implicated in the onset of puberty and sexual matura-
tion (Moguilevsky and Wuttke, 2001). Allopregnanolone
has been found to suppress the release of hypothalamic
gonadotrophin hormone-releasing hormone via alloste-
ric modulation of GABAARs (Calogero et al., 1998; Sim
et al., 2001). Although this inhibition is increased by
allopregnanolone administration before puberty and in
adulthood, it is paradoxically reduced during puberty,
leading to increased excitability of pyramidal cells in
hippocampal region CA1. This effect appears to be due
in part to inhibition of a4-containing GABAARs, which
are expressed at higher levels than normal in the CA1
region of the hippocampus during puberty.
It has also been shown that GABAARs of the a4b2d
subtype, which have a d subunit instead of a g subunit,
play a role in tonic inhibition in areas such as the dentate
gyrus and cortex (Wisden et al., 1992). GABAAR-mediated
conductance is normally inhibitory; however, the reversal
potential of GABAAR-mediated postsynaptic current in
dentate gyrus granule cells is positive to the resting
membrane potential, making membrane hyperpolar-
ization of GABAARs unlikely. Inhibition of shunting
appears to play a role in overcoming this process, such
that nonhyperpolarizing inhibitory conductance re-
duces the depolarizing effect of postsynaptic poten-
tials by decreasing proximal membrane resistance
(Staley and Mody, 1992).
In the dentate gyrus and cortex, the GABAergic
current is inward (i.e., chloride flux is outward) (Staley
and Mody, 1992; Gulledge and Stuart, 2003); thus,
inhibition in these areas is enhanced by allopregnanolone.
However, in the CA1 region, the current is normally
outward (Alger and Nicoll, 1982); thus, increased
expression of a4b2d GABA receptors paradoxically
results in allopregnanolone attenuating rather than
enhancing inhibition. The reduction in current generated
by allopregnanolone at a4b2d GABAAR is dependent
upon the presence of arginine 353 in the intracellular
loop of a4, where it may serve as a chloride modulatory
site (Shen et al., 2007). This polarity-dependent decrease
in inhibition mediated by allopregnanolone may have
important implications for how we approach the treat-
ment of anxiety disorders in the future.
The modulation of GABAergic neurotransmission by
neuroactive steroids is mediated by interactions with
allosteric sites on GABAAR (Lambert et al., 2001,
2003; Yang et al., 2002; Hosie et al., 2006). The in-
teraction of certain neuroactive steroids with GABAAR
is stereoselective, suggesting that the binding sites
for these compounds are of a specific dimension and
shape (Harrison and Simmonds, 1984; Park-Chung et al.,
1994; Covey et al., 2000). There is also evidence that the
transmembrane domains play a role in neuroactive
steroidmediated modulation of GABAARs (Hosie et al.,
2006) (Fig. 9), and that the effects of neuroactive steroids on
GABAARs may be associated with its actions at d subunit
containing receptors (Mihalek et al., 1999, 2001; Spigelman
et al., 2003; Stell et al., 2003). Although changes in these
receptors have not been associated with alterations in
behaviors considered indicative of vigilance or possibly

Fig. 9. Neurosteroid activity at the GABAAR is determined by a-subunit M1 domain residues. (A) The M1 through the end of M2 murine a1 and b2
subunits were replaced with the corresponding sequence from the resistance to dieldrin (RDL) subunit, forming the chimeras aR and bR, respectively,
which were previously shown to have very low sensitivity to potentiation and to lack direct activation by micromolar concentrations of neurosteroids.
Polar residues present only in a1 (blue) are in bold, with Thr236 and Gln241 (red) highlighted. The transmembrane domains are boxed. The
pharmacology of receptors containing the modified a subunit was then compared with that of wild-type receptors using whole-cell recordings from
human embryonic kidney cells. (B) THDOC concentrationresponse curves for direct activation (red) and for potentiation (blue) of EC10 (concentration
causing 10% of maximal response) GABA currents expressed as a percentage of the maximum GABA response, at wild-type a1b2g 2 (squares) and
chimeric aRb2g 2 (circles) receptors showing that potentiation and direct activation by THDOC and allopregnanolone is abolished on receptors
incorporating aR. (C) Structures of ALLOP and THDOC. ALLOP, allopregnanolone; THDOC, tetrahydrodeoxycorticosterone. Reprinted with
permission from Hosie et al. (2006).
1024 Farb and Ratner

anxiety in animals (Mihalek et al., 1999), evidence
strongly suggests that a change in subunit subtype from
a1bxg 2 to a4bxg2 increases receptor desensitization and
decreases GABAergic inhibition, a process that may
play a role in epilepsy (Roberts et al., 2005, 2006; Lund
et al., 2008; Grabenstatter et al., 2012). Could such
a change in subunit composition also underlie increased
anxiety and serve as a biomarker to identify patients
resistant to existing anxiolytic therapeutics? Could
switching of receptor subunit subtypesperhaps involving
the a4 subunit, which is insensitive to BZD positive
modulatorsbe a control point in anxiolysis as in epilepsy?
Both allopregnanolone and pregnenolone have been
implicated in the prevalence of anxiety disorders in
men and women (Le Melldo and Baker, 2004), and a
range of preclinical studies suggest potential mechanistic
rationales for the anxiolytic effects of these neuroactive
steroids. It has been shown that allopregnanolone
exhibits anticonflict effects pharmacologically at 8 mg/kg
in rats (Brot et al., 1997), and that it attenuates certain
effects of caffeine in rats indicative of vigilance or anxiety
(Jain et al., 2005). Pregnanolone potentiates electro-
physiological responses to and binding of muscimol to
GABA A Rs (Majewska et al., 1988; Wu et al., 1993;
Park-Chung et al., 1994), and exhibits sedative hypnotic
properties via its likely interaction with GABAARs
(Wang et al., 2001). Overall, these observations suggest
that allopregnanolone and pregnanolone act as endoge-
nous modulators of GABAergic neurotransmission in
vivo (Lambert et al., 2001, 2003; Yang et al., 2002;
Strhle et al., 2003). In support of the hypothesis that
allopregnanolone and pregnanolone have anxiolytic
properties, two small studies suggest that the plasma
concentrations of pregnenolone sulfate, a precursor of
these steroids, is lower in males with generalized social
phobia or GAD (Semeniuk et al., 2001; Heydari and Le
Melldo, 2002). Administration of pregnenolone is asso-
ciated with an increase in allopreganolone and reduced
activity in the amygdala and insula, as well as a reduction
in self-reported anxiety (Sripada et al., 2013) (Fig. 10).
Because they have poor bioavailability and can
potentially be metabolized to hormonally active ste-
roids, endogenously occurring neuroactive steroids
have limited therapeutic use in patients. Synthetic
analogs of neuroactive steroids, which are more re-
sistant to metabolism and better able to cross the
bloodbrain barrier, are now being investigated for
potential use as anxiolytics, anesthetics, and anticon-
vulsants (Weaver et al., 1997; Gasior et al., 1999). One
synthetic neurosteroid, pregnanolone hemisuccinate,
produces sedation in mice and rats (Weaver et al.,
1997), raising the possibility that it acts either by
inhibiting the NMDA receptor or by crossing the blood
brain barrier and undergoing metabolism to pregnanolone.
Synthetic neurosteroids bearing a hemisuccinate group
are more resistant to hydrolysis than the correspond-
ing sulfate esters and are partly unionized at physiologic
pH, allowing ready passage across the blood-brain
barrier.
Ganaxolone (3-hydroxy-3-methyl-5-pregnane-20-one),
an orally active synthetic analog of allopregnanolone, is
a positive allosteric modulator of GABAAR that has
shown promising basic and early stage clinical outcomes
and is currently under investigation as a novel treat-
ment of epilepsy and PTSD (Carter et al., 1997; Gasior
et al., 1999; Reddy and Rogawski, 2012). Another

Fig. 10. Functional MRI studies showing that administration of PREG (400 mg) is associated with a reduction in activity in regions associated with
generation of negative emotion. (A) Compared with placebo, pregnenolone administration decreased activation in right amygdala and right insula. (B)
Compared with placebo, pregnenolone administration increased dorsal medial prefrontal cortex activation during appraisal. The percent signal change
is displayed next to each figure. PBO, placebo; PREG, pregnenolone administration group. Reprinted with permission from Sripada et al. (2013).
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
synthetic analog of allopregnanolone, Co 3-0593 (3b-ethenyl-
3a-hydroxy-5a-pregnan-20-one), was found to have anxi-
olytic effects comparable to BZDs after both subcutane-
ous and oral administration in rodents. An absence of
tolerance to this drug is suggested by the fact that its
effects were maintained even after chronic administration
(Wieland et al., 1997).
Although these data suggest that anxiety disorders
may be amenable to treatment with synthetic neuro-
steroids or through modulation of the synthesis of
endogenous neuroactive steroids, the more compelling
value of these agents to date has been the insights they
have provided into the pathophysiology of anxiety, both
influencing and being influenced by the GABAergic,
serotonergic, and other neural systems in a range of
anxiety disorders.
VI. Conclusions
Personalized medicine is now a familiar concept, not
only in the scientific literature but also in the popular
media. The concept is so familiar, in fact, that some are
now questioning whether the term has outlived its
usefulness, implying both too much and too little: too
much in the sense that unique treatments are not being
developed for every individual patient, and too little in
the sense that personalized medicine is what clinicians
already aspire to every time they treat a patient
(Katsnelson, 2013). Regardless of the term we use to
describe this aspiration, it is certainly true that the more
we are able to tailor treatments to a patients individual
profile, the greater the chance of clinical success. In the
cases of mental disorders such as anxiety or depression,
the idea of individualized approaches to therapy take on
even greater urgency, given the uniquely personal and
complex mental processes that underlie the disorders.
We believe that an adequate response to these
challenges will require a systems-based approach to
research. Although traditional target-based discovery
has shown success in identifying new drugs against
previously validated targets, the reductionist nature of
this approach often leads to late-stage failure when the
drug is assessed in increasingly complex systems. By
contrast, a systems-based approach may speed the drug
discovery process by focusing less on drug interactions
with single targets and more on systems-level assays
representative of the disease (Desbiens and Farb, 2012).
A pharmacologic connectome is emerging based
upon which disorder-specific novel therapeutic agents
for anxiety can be developed (Fig. 11). Functional
neuroimaging studies are revealing the brain regions
and nuclei implicated in specific anxiety disorders and
are providing important clues about which pharmaco-
logical interventions are the most appropriate for which
disorders. The use of drugs that enhance extinction is
gaining acceptance for the treatment of specific phobias
and the intrusive memories associated with PTSD
1025
Fig. 11. A pharmacologic connectome that depicts how specific brain
regions and nuclei respond to therapeutic agents via connectivity maps
activated in anxiety is beginning to be assembled as the human brain is
deconstructed. Episodic memories associated with anxiety-inducing events
are stored in the parietal lobes (precuneus). The amygdala assigns an
appropriate emotional-response tag to a stimulus, such as a smiling or an
angry face. The frontal lobes in turn provide for the top-down regulation
of emotional responses to these same stimuli. The cingulate and insula
form the major association pathways between the various regions involved
in anxiety, whereas the BNST connects the amygdala to the HPA axis,
which in turn regulates how the endocrine system responds to the stimulus.
In anxiety disorders that are triggered by a specific stimulus (e.g., phobias
and PTSD), the amygdaloid-mediated response to the stimuli is highly
memory-dependent. In disorders of increased apprehension and persever-
ative cognition (e.g., GAD), the frontal lobe and top-down modulation
of amygdaloid nucleus function would be altered.
(Rothbaum et al., 2014). Compounds that inhibit excit-
atory neurotransmission or enhance inhibitory neuro-
transmission are proving to be more effective for treating
GAD and the hypervigilance associated with PTSD
(Mathew et al., 2005). At the same time, we are beginning
to understand that because of genetic polymorphisms,
some patients may not respond to SSRIs and SNRIs
(Zanardi et al., 2001). The opportunity to apply this
growing body of knowledge to the development of novel
therapeutics for anxiety disorders has not been greater.
Neuroactive steroids, which modulate various neuro-
transmitter pathways and are a locus for the neural
crosstalk involved in anxiety disorders, may prove to be an
important tool for advancing a systems-based approach to
drug development. The unique place of neuroactive
steroids in this setting is given further support by
a growing body of evidence showing that these agents
are important for both improving the survival of neurons
and increasing the number of newly formed neurons
(Charalampopoulos et al., 2008).
1026 Farb and Ratner
The results reviewed in this article suggest that
current research into brain activation and therapeutic
targeting of GABA, 5-HT, and NMDA receptor subunits
has not produced a silver bullet for treating anxiety that
optimizes efficacy and reduces risk of adverse events.
Moreover, true personalized medicine in patients suffer-
ing from various anxiety disorders would require a
comprehensive and multifaceted approach beyond anal-
ysis of neurologic activation. This could include assess-
ment of patient demographics, clinical presentation,
environmental factors, family history, relevant genetic
polymorphisms, and biomarkers, as well as treatment
options encompassing not only pharmacotherapy but
also psychotherapeutic approaches such as CBT and
behavioral activation therapy. Nevertheless, research
into the brain regions and variations in receptor
subunits engaged in anxiety provides reason for opti-
mism that the pathophysiology underlying this complex
mosaic of perceptions will lead to treatments better
tailored to the individual needs of patients suffering
from a range of anxiety disorders based on results from
noninvasive brain imaging and improved therapeutics.
Acknowledgements
The authors thank Dr. Cynthia Czajkowski for Fig. 7 and Dr. Conor
C. Smith for artwork in Fig. 8.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Farb, Ratner.
References
Ahmadi H, Nasehi M, Rostami P, and Zarrindast MR (2013) Involvement of the
nucleus accumbens shell dopaminergic system in prelimbic NMDA-induced
anxiolytic-like behaviors. Neuropharmacology 71:112123.
Alger BE and Nicoll RA (1982) Pharmacological evidence for two kinds of GABA
receptor on rat hippocampal pyramidal cells studied in vitro. J Physiol 328:
125141.
Alkhatib AH, Dvorkin-Gheva A, and Szechtman H (2013) Quinpirole and 8-OH-DPAT
induce compulsive checking behavior in male rats by acting on different functional
parts of an OCD neurocircuit. Behav Pharmacol 24:6573.
Allgulander C, Bandelow B, Hollander E, Montgomery SA, Nutt DJ, Okasha A,
Pollack MH, Stein DJ, and Swinson RP; World Council of Anxiety (2003) WCA
recommendations for the long-term treatment of generalized anxiety disorder.
CNS Spectr 8 (Suppl 1)5361.
American Psychiatric Association (2013) Diagnostic and Statistical Manual of Mental
Disorders, Fifth Edition (DSM-5), American Psychiatric Association, Washington,
DC.
Amin J, Brooks-Kayal A, and Weiss DS (1997) Two tyrosine residues on the alpha
subunit are crucial for benzodiazepine binding and allosteric modulation of
gamma-aminobutyric acidA receptors. Mol Pharmacol 51:833841.
Amin J and Weiss DS (1993) GABAA receptor needs two homologous domains of the
beta-subunit for activation by GABA but not by pentobarbital. Nature 366:
565569.
Ashton H and Young AH (2003) GABA-ergic drugs: exit stage left, enter stage right.
J Psychopharmacol 17:174178.
Atack JR (2003) Anxioselective compounds acting at the GABA(A) receptor benzo-
diazepine binding site. Curr Drug Targets CNS Neurol Disord 2:213232.
Atack JR, Hallett DJ, Tye S, Wafford KA, Ryan C, Sanabria-Bohrquez SM, Eng WS,
Gibson RE, Burns HD, and Dawson GR, et al. (2011a) Preclinical and clinical
pharmacology of TPA023B, a GABAA receptor α2/α3 subtype-
selective partial agonist. J Psychopharmacol 25:329344.
Atack JR, Pike A, Marshall G, Stanley J, Lincoln R, Cook SM, Lewis RT, Blackaby
WP, Goodacre SC, and McKernan RM, et al. (2006a) The in vivo properties of
pagoclone in rat are most likely mediated by 59-hydroxy pagoclone. Neurophar-
macology 50:677689.
Atack JR, Wafford KA, Street LJ, Dawson GR, Tye S, Van Laere K, Bormans G,
Sanabria-Bohrquez SM, De Lepeleire I, and de Hoon JN, et al. (2011b) MRK-409
(MK-0343), a GABAA receptor subtype-selective partial agonist, is a non-sedating
anxiolytic in preclinical species but causes sedation in humans. J Psychopharmacol
25:314328.
Atack JR, Wafford KA, Tye SJ, Cook SM, Sohal B, Pike A, Sur C, Melillo D, Bristow L,
and Bromidge F, et al. (2006b) TPA023 [7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-
3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective
for alpha2- and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in
rodents and primates. J Pharmacol Exp Ther 316:410422.
Agustn-Pavn C, Braesicke K, Shiba Y, Santangelo AM, Mikheenko Y, Cockroft G,
Asma F, Clarke H, Man MS, and Roberts AC (2012) Lesions of ventrolateral pre-
frontal or anterior orbitofrontal cortex in primates heighten negative emotion. Biol
Psychiatry 72:266272.
Aupperle RL, Hale LR, Chambers RJ, Cain SE, Barth FX, Sharp SC, Denney DR,
and Savage CR (2009) An fMRI study examining effects of acute D-cycloserine
during symptom provocation in spider phobia. CNS Spectr 14:556571.
Baldwin D, Woods R, Lawson R, and Taylor D (2011) Efficacy of drug treatments for
generalised anxiety disorder: systematic review and meta-analysis. BMJ 342:
d1199.
Ballenger JC, McDonald S, Noyes R, Rickels K, Sussman N, Woods S, Patin J,
and Singer J (1991) The first double-blind, placebo-controlled trial of a partial
benzodiazepine agonist abecarnil (ZK 112-119) in generalized anxiety disorder.
Psychopharmacol Bull 27:171179.
Ballenger JC, McDonald S, Noyes R Jr, Rickels K, Woods SW, Patin J, Lydiard B,
Garvey M, Cook B, and Goodard AW, et al. (1992) The first double-blind, placebo-
controlled trial of a partial benzodiazepine agonist, abecarnil (ZK 112-119), in
generalized anxiety disorder. Adv Biochem Psychopharmacol 47:431447.
Barbaccia ML, Roscetti G, Trabucchi M, Mostallino MC, Concas A, Purdy RH,
and Biggio G (1996) Time-dependent changes in rat brain neuroactive steroid
concentrations and GABAA receptor function after acute stress. Neuroendocrinol-
ogy 63:166172.
Barkus C, McHugh SB, Sprengel R, Seeburg PH, Rawlins JN, and Bannerman DM
(2010) Hippocampal NMDA receptors and anxiety: at the interface between cog-
nition and emotion. Eur J Pharmacol 626:4956.
Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G, Braestrup C,
Bateson AN, and Langer SZ (1998) International Union of Pharmacology. XV.
Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of
subunit structure and receptor function. Pharmacol Rev 50:291313.
Bateson A (2003) Pagoclone Indevus. Curr Opin Investig Drugs 4:9195.
Battista MA, Hierholzer R, Khouzam HR, Barlow A, and OToole S (2007) Pilot trial
of memantine in the treatment of posttraumatic stress disorder. Psychiatry 70:
167174.
Baur R and Sigel E (2003) On high- and low-affinity agonist sites in GABAA recep-
tors. J Neurochem 87:325332.
Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the
nurture. Nat Rev Neurosci 3:728739.
Benke D, Honer M, Michel C, and Mohler H (1996) GABAA receptor subtypes dif-
ferentiated by their gamma-subunit variants: prevalence, pharmacology and sub-
unit architecture. Neuropharmacology 35:14131423.
Bennett JA, Moioffer M, Stanton SP, Dwight M, and Keck PE Jr (1998) A risk-benefit
assessment of pharmacological treatments for panic disorder. Drug Saf 18:
419430.
Berezhnoy D, Gravielle MC, and Farb DH (2007) Pharmacology of the GABA-A
receptor, in Handbook of Contemporary Neuropharmacology (Sibley D, Kuhar
M, Hanin I, and Skolnick P eds), pp 465568, John Wiley & Sons, Inc., Hoboken,
NJ.
Berton F, Francesconi WG, Madamba SG, Zieglgnsberger W, and Siggins GR (1998)
Acamprosate enhances N-methyl-D-apartate receptor-mediated neurotransmis-
sion but inhibits presynaptic GABA(B) receptors in nucleus accumbens neurons.
Alcohol Clin Exp Res 22:183191.
Blanco C, Schneier FR, Schmidt A, Blanco-Jerez CR, Marshall RD, Snchez-Lacay A,
and Liebowitz MR (2003) Pharmacological treatment of social anxiety disorder:
a meta-analysis. Depress Anxiety 18:2940.
Boileau AJ and Czajkowski C (1999) Identification of transduction elements for
benzodiazepine modulation of the GABA(A) receptor: three residues are required
for allosteric coupling. J Neurosci 19:1021310220.
Borden LA, Czajkowski C, Chan CY, and Farb DH (1984) Benzodiazepine receptor
synthesis and degradation by neurons in culture. Science 226:857860.
Borden LA and Farb DH (1988) Mechanism of gamma-aminobutyric acid/
benzodiazepine receptor turnover in neuronal cells: evidence for nonlysosomal
degradation. Mol Pharmacol 34:354362.
Braestrup C and Nielsen M (1981) [3H]Propyl beta-carboline-3-carboxylate as a se-
lective radioligand for the BZ1 benzodiazepine receptor subclass. J Neurochem 37:
333341.
Braestrup C and Squires RF (1977) Specific benzodiazepine receptors in rat brain
characterized by high-affinity (3H)diazepam binding. Proc Natl Acad Sci USA 74:
38053809.
Braestrup C and Squires RF (1978) Pharmacological characterization of benzodiaz-
epine receptors in the brain. Eur J Pharmacol 48:263270.
Bremner JD, Innis RB, White T, Fujita M, Silbersweig D, Goddard AW, Staib L,
Stern E, Cappiello A, and Woods S, et al. (2000) SPECT [I-123]iomazenil mea-
surement of the benzodiazepine receptor in panic disorder. Biol Psychiatry 47:
96106.
Brooks-Kayal AR and Pritchett DB (1993) Developmental changes in human gamma-
aminobutyric acidA receptor subunit composition. Ann Neurol 34:687693.
Brot MD, Akwa Y, Purdy RH, Koob GF, and Britton KT (1997) The anxiolytic-like
effects of the neurosteroid allopregnanolone: interactions with GABA(A) receptors.
Eur J Pharmacol 325:17.
Brouillet E, Chavoix C, Bottlaender M, Khalili-Varasteh M, Hantraye P, Fournier D,
Dodd RH, and Mazire M (1991) In vivo bidirectional modulatory effect of benzo-
diazepine receptor ligands on GABAergic transmission evaluated by positron
emission tomography in non-human primates. Brain Res 557:167176.
Buhr A and Sigel E (1997) A point mutation in the gamma2 subunit of gamma-
aminobutyric acid type A receptors results in altered benzodiazepine binding site
specificity. Proc Natl Acad Sci USA 94:88248829.
Busto U, Kaplan HL, Zawertailo L, and Sellers EM (1994) Pharmacologic effects and
abuse liability of bretazenil, diazepam, and alprazolam in humans. Clin Pharmacol
Ther 55:451463.
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
Calogero AE, Palumbo MA, Bosboom AM, Burrello N, Ferrara E, Palumbo G, Petraglia
F, and DAgata R (1998) The neuroactive steroid allopregnanolone suppresses hy-
pothalamic gonadotropin-releasing hormone release through a mechanism mediated
by the gamma-aminobutyric acidA receptor. J Endocrinol 158:121125.
Carlson JN, Haskew R, Wacker J, Maisonneuve IM, Glick SD, and Jerussi TP (2001)
Sedative and anxiolytic effects of zopiclones enantiomers and metabolite. Eur J
Pharmacol 415:181189.
Carter RB, Wood PL, Wieland S, Hawkinson JE, Belelli D, Lambert JJ, White HS,
Wolf HH, Mirsadeghi S, and Tahir SH, et al. (1997) Characterization of the anti-
convulsant properties of ganaxolone (CCD 1042; 3a-hydroxy-3b-methyl-5a-
pregnan-20-one), a selective, high-affinity, steroid modulator of the g-aminobutyric
acid(A) receptor. J Pharmacol Exp Ther 280:12841295.
Casalotti SO, Stephenson FA, and Barnard EA (1986) Separate subunits for agonist
and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer. J
Biol Chem 261:1501315016.
Cassano P, Soares CN, Cohen LS, Lyster AK, and Fava M (2004) Sex- and age-
related differences in major depressive disorder with comorbid anxiety treated
with fluoxetine. Arch Women Ment Health 7:167171.
Casu MA, Sanna A, Spada GP, Falzoi M, Mongeau R, and Pani L (2007) Effects of
acute and chronic valproate treatments on p-CREB levels in the rat amygdala and
nucleus accumbens. Brain Res 1141:1524.
Cha J, Greenberg T, Carlson JM, Dedora DJ, Hajcak G, and Mujica-Parodi LR (2014)
Circuit-wide structural and functional measures predict ventromedial prefrontal
cortex fear generalization: implications for generalized anxiety disorder. J Neurosci
34:40434053.
Charalampopoulos I, Remboutsika E, Margioris AN, and Gravanis A (2008) Neurosteroids
as modulators of neurogenesis and neuronal survival. Trends Endocrinol Metab 19:
300307.
Chan CY and Farb DH (1985) Modulation of neurotransmitter action: control of
the gamma-aminobutyric acid response through the benzodiazepine receptor.
J Neurosci 5:23652373.
Chang Y, Wang R, Barot S, and Weiss DS (1996) Stoichiometry of a recombinant
GABAA receptor. J Neurosci 16:54155424.
Chee SS, Menard JL, and Dringenberg HC (2014) Behavioral anxiolysis without
reduction of hippocampal theta frequency after histamine application in the lateral
septum of rats. Hippocampus 24:615627.
Chiu TH and Rosenberg HC (1979) GABA receptor-mediated modulation of
3H-diazepam binding in rat cortex. Eur J Pharmacol 56:337345.
Choi DW, Farb DH, and Fischbach GD (1977) Chlordiazepoxide selectively augments
GABA action in spinal cord cell cultures. Nature 269:342344.
Cole JC and Rodgers RJ (1993) An ethological analysis of the effects of chlordiaz-
epoxide and bretazenil (Ro 16-6028) in the murine elevated plus-maze. Behav
Pharmacol 4:573580.
Collins I, Moyes C, Davey WB, Rowley M, Bromidge FA, Quirk K, Atack JR,
McKernan RM, Thompson SA, and Wafford K, et al. (2002) 3-Heteroaryl-2-
pyridones: benzodiazepine site ligands with functional delectivity for alpha 2/alpha
3-subtypes of human GABA(A) receptor-ion channels. J Med Chem 45:18871900.
Covey DF, Nathan D, Kalkbrenner M, Nilsson KR, Hu Y, Zorumski CF, and Evers AS
(2000) Enantioselectivity of pregnanolone-induced gamma-aminobutyric acid(A)
receptor modulation and anesthesia. J Pharmacol Exp Ther 293:10091016.
Czajkowski C and Farb DH (1986) Transmembrane topology and subcellular distri-
bution of the benzodiazepine receptor. J Neurosci 6:28572863.
Czh B, Michaelis T, Watanabe T, Frahm J, de Biurrun G, van Kampen M, Bartolomucci
A, and Fuchs E (2001) Stress-induced changes in cerebral metabolites, hippocampal
volume, and cell proliferation are prevented by antidepressant treatment with
tianeptine. Proc Natl Acad Sci USA 98:1279612801.
Davidson JR (2003) Pharmacotherapy of social phobia. Acta Psychiatr Scand Suppl
417:6571.
Davis M and Myers KM (2002) The role of glutamate and gamma-aminobutyric acid
in fear extinction: clinical implications for exposure therapy. Biol Psychiatry 52:
9981007.
de Wit H, Vicini L, Haig GM, Hunt T, and Feltner D (2006) Evaluation of the abuse
potential of pagoclone, a partial GABAA agonist. J Clin Psychopharmacol 26:
268273.
Desbiens S and Farb DH (2012) Current needs for new therapeutic agents and dis-
covery strategies a systems pharmacology approach, in Development of Thera-
peutic Agents (Gad S ed) vol 416, pp 124, John Wiley & Sons, Inc., Hoboken, NJ.
Dias R, Sheppard WF, Fradley RL, Garrett EM, Stanley JL, Tye SJ, Goodacre S,
Lincoln RJ, Cook SM, and Conley R, et al. (2005) Evidence for a significant role of
alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzo-
diazepines. J Neurosci 25:1068210688.
Difede J, Cukor J, Wyka K, Olden M, Hoffman H, Lee FS, and Altemus M (2014)
D-cycloserine augmentation of exposure therapy for post-traumatic stress disorder:
a pilot randomized clinical trial. Neuropsychopharmacology 39:10521058.
Doehrmann O, Ghosh SS, Polli FE, Reynolds GO, Horn F, Keshavan A, Triantafyllou
C, Saygin ZM, Whitfield-Gabrieli S, and Hofmann SG, et al. (2013) Predicting
treatment response in social anxiety disorder from functional magnetic resonance
imaging. JAMA Psychiatry 70:8797.
Drover DR (2004) Comparative pharmacokinetics and pharmacodynamics of short-
acting hypnosedatives: zaleplon, zolpidem and zopiclone. Clin Pharmacokinet 43:
227238.
Duncan GE, Breese GR, Criswell HE, McCown TJ, Herbert JS, Devaud LL,
and Morrow AL (1995) Distribution of [3H]zolpidem binding sites in relation to
messenger RNA encoding the alpha 1, beta 2 and gamma 2 subunits of GABAA
receptors in rat brain. Neuroscience 64:11131128.
Dunn JD (1987) Plasma corticosterone responses to electrical stimulation of the bed
nucleus of the stria terminalis. Brain Res 407:327331.
Engels AS, Heller W, Mohanty A, Herrington JD, Banich MT, Webb AG, and Miller
GA (2007) Specificity of regional brain activity in anxiety types during emotion
processing. Psychophysiology 44:352363.
1027
Etkin A and Wager TD (2007) Functional neuroimaging of anxiety: a meta-analysis of
emotional processing in PTSD, social anxiety disorder, and specific phobia. Am J
Psychiatry 164:14761488.
Facklam M, Schoch P, Bonetti EP, Jenck F, Martin JR, Moreau JL, and Haefely WE
(1992a) Relationship between benzodiazepine receptor occupancy and functional
effects in vivo of four ligands of differing intrinsic efficacies. J Pharmacol Exp Ther
261:11131121.
Facklam M, Schoch P, and Haefely WE (1992b) Relationship between benzodiazepine
receptor occupancy and potentiation of gamma-aminobutyric acid-stimulated
chloride flux in vitro of four ligands of differing intrinsic efficacies. J Pharmacol
Exp Ther 261:11061112.
Falls WA, Miserendino MJ, and Davis M (1992) Extinction of fear-potentiated startle:
blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci 12:
854863.
Farb DH, Borden LA, Chan CY, Czajkowski CM, Gibbs TT, and Schiller GD (1984)
Modulation of neuronal function through benzodiazepine receptors: biochemical
and electrophysiological studies of neurons in primary monolayer cell culture. Ann
N Y Acad Sci 435:131.
Faria V, Appel L, hs F, Linnman C, Pissiota A, Frans , Bani M, Bettica P, Pich
EM, and Jacobsson E, et al. (2012) Amygdala subregions tied to SSRI and placebo
response in patients with social anxiety disorder. Neuropsychopharmacology 37:
22222232.
Farkas RH, Unger EF, and Temple R (2013) Zolpidem and driving impairment
identifying persons at risk. N Engl J Med 369:689691.
Felix-Ortiz AC, Beyeler A, Seo C, Leppla CA, Wildes CP, and Tye KM (2013) BLA to
vHPC inputs modulate anxiety-related behaviors. Neuron 79:658664.
Fernandes GA, Perks P, Cox NK, Lightman SL, Ingram CD, and Shanks N (2002)
Habituation and cross-sensitization of stress-induced hypothalamic-pituitary-
adrenal activity: effect of lesions in the paraventricular nucleus of the thalamus
or bed nuclei of the stria terminalis. J Neuroendocrinol 14:593602.
Ferrero P, Guidotti A, and Costa E (1984) Increase in the Bmax of gamma-
aminobutyric acid-A recognition sites in brain regions of mice receiving diazepam.
Proc Natl Acad Sci USA 81:22472251.
Fisher PM, Meltzer CC, Ziolko SK, Price JC, Moses-Kolko EL, Berga SL, and Hariri
AR (2006) Capacity for 5-HT1A-mediated autoregulation predicts amygdala re-
activity. Nat Neurosci 9:13621363.
Fleck MW (2002) Molecular actions of (S)-desmethylzopiclone (SEP-174559), an an-
xiolytic metabolite of zopiclone. J Pharmacol Exp Ther 302:612618.
Fluoxetine (2013) Prescribing information. Eli Lilly and Company, Indianapolis, IN.
Follesa P, Mancuso L, Biggio F, Cagetti E, Franco M, Trapani G, and Biggio G (2002)
Changes in GABA(A) receptor gene expression induced by withdrawal of, but not
by long-term exposure to, zaleplon or zolpidem. Neuropharmacology 42:191198.
Gallager DW and Tallman JF (1983) Consequences of benzodiazepine receptor oc-
cupancy. Neuropharmacology 22:14931498.
Gasior M, Carter RB, and Witkin JM (1999) Neuroactive steroids: potential therapeutic
use in neurological and psychiatric disorders. Trends Pharmacol Sci 20:107112.
Gavioli EC, Canteras NS, and De Lima TC (1999) Anxiogenic-like effect induced by
substance P injected into the lateral septal nucleus. Neuroreport 10:33993403.
Gelernter J, Page GP, Stein MB, and Woods SW (2004) Genome-wide linkage scan for
loci predisposing to social phobia: evidence for a chromosome 16 risk locus. Am J
Psychiatry 161:5966.
Geuze E, van Berckel BN, Lammertsma AA, Boellaard R, de Kloet CS, Vermetten E,
and Westenberg HG (2008) Reduced GABAA benzodiazepine receptor binding in
veterans with post-traumatic stress disorder. Mol Psychiatry 13:7483, 3.
Ghiani CA, Serra M, Motzo C, Giusti P, Cuccheddu T, Porceddu ML, and Biggio G
(1994) Chronic administration of an anticonvulsant dose of imidazenil fails to in-
duce tolerance of GABAA receptor function in mice. Eur J Pharmacol 254:299302.
Gimnez M, Ortiz H, Soriano-Mas C, Lpez-Sol M, Farr M, Deus J, Martn-Santos
R, Fernandes S, Fina P, and Bani M, et al. (2014) Functional effects of chronic
paroxetine versus placebo on the fear, stress and anxiety brain circuit in Social
Anxiety Disorder: initial validation of an imaging protocol for drug discovery. Eur
Neuropsychopharmacol 24:105116.
Grdler SS, Straneva PA, Light KC, Pedersen CA, and Morrow AL (2001)
Allopregnanolone levels and reactivity to mental stress in premenstrual dysphoric
disorder. Biol Psychiatry 49:788797.
Goddard AW, Mason GF, Almai A, Rothman DL, Behar KL, Petroff OA, Charney DS,
and Krystal JH (2001) Reductions in occipital cortex GABA levels in panic disorder
detected with 1h-magnetic resonance spectroscopy. Arch Gen Psychiatry 58:556561.
Goddard AW, Mason GF, Appel M, Rothman DL, Gueorguieva R, Behar KL,
and Krystal JH (2004) Impaired GABA neuronal response to acute benzodiazepine
administration in panic disorder. Am J Psychiatry 161:21862193.
Goutman JD, Waxemberg MD, Doate-Oliver F, Pomata PE, and Calvo DJ (2003)
Flavonoid modulation of ionic currents mediated by GABA(A) and GABA(C)
receptors. Eur J Pharmacol 461:7987.
Grabenstatter HL, Russek SJ, and Brooks-Kayal AR (2012) Molecular pathways
controlling inhibitory receptor expression. Epilepsia 53 (Suppl 9):7178.
Graeff FG and Zangrossi H Jr (2010) The dual role of serotonin in defense and the
mode of action of antidepressants on generalized anxiety and panic disorders. Cent
Nerv Syst Agents Med Chem 10:207217.
Graham D, Faure C, Besnard F, and Langer SZ (1996) Pharmacological profile of
benzodiazepine site ligands with recombinant GABAA receptor subtypes. Eur
Neuropsychopharmacol 6:119125.
Gram L (1988) Experimental studies and controlled clinical testing of valproate and
vigabatrin. Acta Neurol Scand 78:241270.
Gray JA and Roth BL (2007) Serotonin systems, in Handbook of Contemporary
Neuropharmacology (Sibley D, Kuhar M, Hanin I, and Skolnick P eds), pp 257298,
John Wiley & Sons, Inc., Hoboken, NJ.
Greenberg T, Carlson JM, Cha J, Hajcak G, and Mujica-Parodi LR (2013) Ventro-
medial prefrontal cortex reactivity is altered in generalized anxiety disorder during
fear generalization. Depress Anxiety 30:242250.
1028 Farb and Ratner
Griebel G, Perrault G, Simiand J, Cohen C, Granger P, Decobert M, Franon D,
Avenet P, Depoortere H, and Tan S, et al. (2001) SL651498: an anxioselective
compound with functional selectivity for alpha2- and alpha3-containing gamma-
aminobutyric acid(A) (GABA(A)) receptors. J Pharmacol Exp Ther 298:753768.
Griffin LD and Mellon SH (1999) Selective serotonin reuptake inhibitors directly
alter activity of neurosteroidogenic enzymes. Proc Natl Acad Sci USA 96:
1351213517.
Gulledge AT and Stuart GJ (2003) Excitatory actions of GABA in the cortex. Neuron
37:299309.
Gnther U, Benson J, Benke D, Fritschy JM, Reyes G, Knoflach F, Crestani F, Aguzzi
A, Arigoni M, and Lang Y, et al. (1995) Benzodiazepine-insensitive mice generated
by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid
type A receptors. Proc Natl Acad Sci USA 92:77497753.
Guo JD, Hammack SE, Hazra R, Levita L, and Rainnie DG (2009) Bi-directional
modulation of bed nucleus of stria terminalis neurons by 5-HT: molecular ex-
pression and functional properties of excitatory 5-HT receptor subtypes. Neuro-
science 164:17761793.
Hberlein H, Tschiersch KP, and Schfer HL (1994) Flavonoids from Leptospermum
scoparium with affinity to the benzodiazepine receptor characterized by structure
activity relationships and in vivo studies of a plant extract. Pharmazie 49:912922.
Hadingham KL, Garrett EM, Wafford KA, Bain C, Heavens RP, Sirinathsinghji DJ,
and Whiting PJ (1996) Cloning of cDNAs encoding the human g-aminobutyric acid
type A receptor a 6 subunit and characterization of the pharmacology of a 6-containing
receptors. Mol Pharmacol 49:253259.
Hadingham KL, Wingrove P, Le Bourdelles B, Palmer KJ, Ragan CI, and Whiting PJ
(1993) Cloning of cDNA sequences encoding human alpha 2 and alpha 3 gamma-
aminobutyric acidA receptor subunits and characterization of the benzodiazepine
pharmacology of recombinant alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing
human gamma-aminobutyric acidA receptors. Mol Pharmacol 43:970975.
Haefely W, Facklam M, Schoch P, Martin JR, Bonetti EP, Moreau JL, Jenck F,
and Richards JG (1992) Partial agonists of benzodiazepine receptors for treatment
of epilepsy, sleep and anxiety disorders, in GABAergic Synaptic Transmission
(Biggio G, Concas A, and Costa E eds) pp 379394, Raven Press, NY.
Ham BJ, Sung Y, Kim N, Kim SJ, Kim JE, Kim DJ, Lee JY, Kim JH, Yoon SJ,
and Lyoo IK (2007) Decreased GABA levels in anterior cingulate and basal ganglia
in medicated subjects with panic disorder: a proton magnetic resonance spectros-
copy (1H-MRS) study. Prog Neuropsychopharmacol Biol Psychiatry 31:403411.
Hanson SM and Czajkowski C (2008) Structural mechanisms underlying benzodi-
azepine modulation of the GABA(A) receptor. J Neurosci 28:34903499.
Hariri AR, Drabant EM, Munoz KE, Kolachana BS, Mattay VS, Egan MF,
and Weinberger DR (2005) A susceptibility gene for affective disorders and the
response of the human amygdala. Arch Gen Psychiatry 62:146152.
Harmer CJ, Mackay CE, Reid CB, Cowen PJ, and Goodwin GM (2006) Antidepres-
sant drug treatment modifies the neural processing of nonconscious threat cues.
Biol Psychiatry 59:816820.
Harrison NL and Simmonds MA (1984) Modulation of the GABA receptor complex by
a steroid anaesthetic. Brain Res 323:287292.
Hasler G, Nugent AC, Carlson PJ, Carson RE, Geraci M, and Drevets WC (2008)
Altered cerebral gamma-aminobutyric acid type A-benzodiazepine receptor binding
in panic disorder determined by [11C]flumazenil positron emission tomography.
Arch Gen Psychiatry 65:11661175.
Hayward C and Sanborn K (2002) Puberty and the emergence of gender differences
in psychopathology. J Adolesc Health 30:4958.
Hazra R, Guo JD, Dabrowska J, and Rainnie DG (2012) Differential distribution of
serotonin receptor subtypes in BNST(ALG) neurons: modulation by unpredictable
shock stress. Neuroscience 225:921.
Heils A, Teufel A, Petri S, Stber G, Riederer P, Bengel D, and Lesch KP (1996)
Allelic variation of human serotonin transporter gene expression. J Neurochem 66:
26212624.
Heinrichs SC, Leite-Morris KA, Rasmusson AM, and Kaplan GB (2013) Repeated
valproate treatment facilitates fear extinction under specific stimulus conditions.
Neurosci Lett 552:108113.
Henke PG (1984) The bed nucleus of the stria terminalis and immobilization-stress:
unit activity, escape behaviour, and gastric pathology in rats. Behav Brain Res 11:
3545.
Hensler JG (2006) Serotonergic modulation of the limbic system. Neurosci Biobehav
Rev 30:203214.
Herb A, Wisden W, Lddens H, Puia G, Vicini S, and Seeburg PH (1992) The third
g subunit of the g-aminobutyric acid type A receptor family. Proc Natl Acad Sci
USA 89:14331437.
Heresco-Levy U, Kremer I, Javitt DC, Goichman R, Reshef A, Blanaru M, and Cohen
T (2002) Pilot-controlled trial of D-cycloserine for the treatment of post-traumatic
stress disorder. Int J Neuropsychopharmacol 5:301307.
Herrick-Davis K, Grinde E, Harrigan TJ, and Mazurkiewicz JE (2005) Inhibition of
serotonin 5-hydroxytryptamine2c receptor function through heterodimerization:
receptor dimers bind two molecules of ligand and one G-protein. J Biol Chem 280:
4014440151.
Hertzman M, Patt IS, and Spielman LA (2009) Open-label trial of acamprosate as
a treatment for anxiety. Prim Care Companion J Clin Psychiatry 11:267.
Hettema JM, Prescott CA, Myers JM, Neale MC, and Kendler KS (2005) The
structure of genetic and environmental risk factors for anxiety disorders in men
and women. Arch Gen Psychiatry 62:182189.
Heydari B and Le Melldo JM (2002) Low pregnenolone sulphate plasma concen-
trations in patients with generalized social phobia. Psychol Med 32:929933.
Hoehn-Saric R, Schlund MW, and Wong SH (2004) Effects of citalopram on worry and
brain activation in patients with generalized anxiety disorder. Psychiatry Res 131:
1121.
Hofmann SG, Meuret AE, Smits JA, Simon NM, Pollack MH, Eisenmenger K, Shiekh
M, and Otto MW (2006) Augmentation of exposure therapy with D-cycloserine for
social anxiety disorder. Arch Gen Psychiatry 63:298304.
Hosie AM, Wilkins ME, da Silva HM, and Smart TG (2006) Endogenous neuro-
steroids regulate GABAA receptors through two discrete transmembrane sites.
Nature 444:486489.
Hoyer D, Hannon JP, and Martin GR (2002) Molecular, pharmacological and func-
tional diversity of 5-HT receptors. Pharmacol Biochem Behav 71:533554.
Hu XZ, Lipsky RH, Zhu G, Akhtar LA, Taubman J, Greenberg BD, Xu K, Arnold PD,
Richter MA, and Kennedy JL, et al. (2006) Serotonin transporter promoter gain-of-
function genotypes are linked to obsessive-compulsive disorder. Am J Hum Genet
78:815826.
Huen MS, Hui KM, Leung JW, Sigel E, Baur R, Wong JT, and Xue H (2003) Natu-
rally occurring 29-hydroxyl-substituted flavonoids as high-affinity benzodiazepine
site ligands. Biochem Pharmacol 66:23972407.
Hui KM, Huen MS, Wang HY, Zheng H, Sigel E, Baur R, Ren H, Li ZW, Wong JT,
and Xue H (2002) Anxiolytic effect of wogonin, a benzodiazepine receptor ligand
isolated from Scutellaria baicalensis Georgi. Biochem Pharmacol 64:14151424.
Hui KM, Wang XH, and Xue H (2000) Interaction of flavones from the roots of
Scutellaria baicalensis with the benzodiazepine site. Planta Med 66:9193.
Isenberg N, Silbersweig D, Engelien A, Emmerich S, Malavade K, Beattie B, Leon
AC, and Stern E (1999) Linguistic threat activates the human amygdala. Proc Natl
Acad Sci USA 96:1045610459.
Jain NS, Hirani K, and Chopde CT (2005) Reversal of caffeine-induced anxiety by
neurosteroid 3-alpha-hydroxy-5-alpha-pregnane-20-one in rats. Neuropharmacology
48:627638.
Jardim MC, Aguiar DC, Moreira FA, and Guimares FS (2005) Role of glutamate
ionotropic and benzodiazepine receptors in the ventromedial hypothalamic nucleus
on anxiety. Pharmacol Biochem Behav 82:182189.
Jeon GS, Park SH, Lee KJ, Lee MS, Chun BG, and Shin KH (2006) Valproate pre-
vents MK801-induced changes in brain-derived neurotrophic factor mRNA in the
rat brain. Eur J Pharmacol 545:142146.
Jornada LK, Moretti M, Valvassori SS, Ferreira CL, Padilha PT, Arent CO, Fries GR,
Kapczinski F, and Quevedo J (2010) Effects of mood stabilizers on hippocampus
and amygdala BDNF levels in an animal model of mania induced by ouabain.
J Psychiatr Res 44:506510.
Kang-Park MH, Wilson WA, and Moore SD (2004) Differential actions of diazepam
and zolpidem in basolateral and central amygdala nuclei. Neuropharmacology 46:
19.
Kaschka W, Feistel H, and Ebert D (1995) Reduced benzodiazepine receptor binding
in panic disorders measured by iomazenil SPECT. J Psychiatr Res 29:427434.
Katsnelson A (2013) Momentum grows to make personalized medicine more
precise. Nat Med 19:249.
Kaufmann WA, Humpel C, Alheid GF, and Marksteiner J (2003) Compartmentation
of alpha 1 and alpha 2 GABA(A) receptor subunits within rat extended amygdala:
implications for benzodiazepine action. Brain Res 964:9199.
Kavvadias D, Sand P, Youdim KA, Qaiser MZ, Rice-Evans C, Baur R, Sigel E, Rausch
WD, Riederer P, and Schreier P (2004) The flavone hispidulin, a benzodiazepine
receptor ligand with positive allosteric properties, traverses the blood-brain barrier
and exhibits anticonvulsive effects. Br J Pharmacol 142:811820.
Kent JM and Rauch SL (2003) Neurocircuitry of anxiety disorders. Curr Psychiatry
Rep 5:266273.
Kerner B, Rao AR, Christensen B, Dandekar S, Yourshaw M, and Nelson SF (2013)
Rare genomic variants link bipolar disorder with anxiety disorders to CREB-
regulated intracellular signaling pathways. Front Psychiatry 4:154.
Kessler RC, Berglund P, Demler O, Jin R, Merikangas KR, and Walters EE (2005a)
Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the
National Comorbidity Survey Replication. Arch Gen Psychiatry 62:593602.
Kessler RC, Chiu WT, Demler O, Merikangas KR, and Walters EE (2005b) Preva-
lence, severity, and comorbidity of 12-month DSM-IV disorders in the National
Comorbidity Survey Replication. Arch Gen Psychiatry 62:617627.
Kheirbek MA, Drew LJ, Burghardt NS, Costantini DO, Tannenholz L, Ahmari SE,
Zeng H, Fenton AA, and Hen R (2013) Differential control of learning and anxiety
along the dorsoventral axis of the dentate gyrus. Neuron 77:955968.
Khisti RT and Chopde CT (2000) Serotonergic agents modulate antidepressant-like
effect of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in mice. Brain
Res 865:291300.
Kim SW, Fowler JS, Skolnick P, Muench L, Kang Y, Shea C, Logan J, Kim D, Carter
P, and King P, et al. (2014) Therapeutic doses of buspirone block D3 receptors in
the living primate brain. Int J Neuropsychopharmacol 17:12571267.
Kim SY, Adhikari A, Lee SY, Marshel JH, Kim CK, Mallory CS, Lo M, Pak S, Mattis
J, and Lim BK, et al. (2013) Diverging neural pathways assemble a behavioural
state from separable features in anxiety. Nature 496:219223.
Klumpers F, Heitland I, Oosting RS, Kenemans JL, and Baas JM (2012) Genetic
variation in serotonin transporter function affects human fear expression indexed
by fear-potentiated startle. Biol Psychol 89:277282.
Knoflach F, Benke D, Wang Y, Scheurer L, Lddens H, Hamilton BJ, Carter DB,
Mohler H, and Benson JA (1996) Pharmacological modulation of the diazepam-
insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2
gamma 2 and alpha 6 beta 2 gamma 2. Mol Pharmacol 50:12531261.
Kobayashi K, Ikeda Y, Sakai A, Yamasaki N, Haneda E, Miyakawa T, and Suzuki H
(2010) Reversal of hippocampal neuronal maturation by serotonergic anti-
depressants. Proc Natl Acad Sci USA 107:84348439.
Kobiella A, Reimold M, Ulshfer DE, Ikonomidou VN, Vollmert C, Vollstdt-Klein S,
Rietschel M, Reischl G, Heinz A, and Smolka MN (2011) How the serotonin trans-
porter 5-HTTLPR polymorphism influences amygdala function: the roles of in vivo
serotonin transporter expression and amygdala structure. Transl Psychiatr 1:e37.
Koltunowska D, Gibula-Bruzda E, and Kotlinska JH (2013) The influence of
ionotropic and metabotropic glutamate receptor ligands on anxiety-like effect of
amphetamine withdrawal in rats. Prog Neuropsychopharmacol Biol Psychiatry 45:
242249.
Kostakis E, Smith C, Jang MK, Martin SC, Richards KG, Russek SJ, Gibbs TT,
and Farb DH (2013) The neuroactive steroid pregnenolone sulfate stimulates
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
trafficking of functional N-methyl D-aspartate receptors to the cell surface via
a noncanonical, G protein, and Ca21-dependent mechanism. Mol Pharmacol 84:
261274.
Kotlinska J and Bochenski M (2008) The influence of various glutamate receptors
antagonists on anxiety-like effect of ethanol withdrawal in a plus-maze test in rats.
Eur J Pharmacol 598:5763.
Kuriyama K, Honma M, Yoshiike T, and Kim Y (2013) Valproic acid but not
D-cycloserine facilitates sleep-dependent offline learning of extinction and habit-
uation of conditioned fear in humans. Neuropharmacology 64:424431.
Kushner SA, Dewey SL, and Kornetsky C (1997) Gamma-vinyl GABA attenuates
cocaine-induced lowering of brain stimulation reward thresholds. Psychopharma-
cology (Berl) 133:383388.
Lambert JJ, Belelli D, Harney SC, Peters JA, and Frenguelli BG (2001) Modulation
of native and recombinant GABA(A) receptors by endogenous and synthetic neu-
roactive steroids. Brain Res Brain Res Rev 37:6880.
Lambert JJ, Belelli D, Peden DR, Vardy AW, and Peters JA (2003) Neurosteroid
modulation of GABAA receptors. Prog Neurobiol 71:6780.
Lavoie AM and Twyman RE (1996) Direct evidence for diazepam modulation of
GABAA receptor microscopic affinity. Neuropharmacology 35:13831392.
Le Melldo JM and Baker G (2004) Role of progesterone and other neuroactive ste-
roids in anxiety disorders. Expert Rev Neurother 4:851860.
Legerstee JS, Verhulst FC, Robbers SC, Ormel J, Oldehinkel AJ, and van Oort FV
(2013) Gender-specific developmental trajectories of anxiety during adolescence:
determinants and outcomes. The TRAILS study. J Can Acad Child Adolesc
Psychiatry 22:2634.
Leikanger E, Ingul JM, and Larsson B (2012) Sex and age-related anxiety
in a community sample of Norwegian adolescents. Scand J Psychol 53:
150157.
Lpine JP (2002) The epidemiology of anxiety disorders: prevalence and societal
costs. J Clin Psychiatry 63 (Suppl 14):48.
Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Mller
CR, Hamer DH, and Murphy DL (1996) Association of anxiety-related traits with
a polymorphism in the serotonin transporter gene regulatory region. Science 274:
15271531.
Levinson DF and Devinsky O (1999) Psychiatric adverse events during vigabatrin
therapy. Neurology 53:15031511.
Licata SC, Platt DM, Cook JM, Sarma PV, Griebel G, and Rowlett JK (2005)
Contribution of GABAA receptor subtypes to the anxiolytic-like, motor, and
discriminative stimulus effects of benzodiazepines: studies with the functionally
selective ligand SL651498 [6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-
carbonyl)-2,9-dihydro-1H-pyridol[3,4-b]indol-1-one]. J Pharmacol Exp Ther
313:11181125.
Liebowitz MR, Mangano RM, Bradwejn J, and Asnis G; SAD Study Group (2005) A
randomized controlled trial of venlafaxine extended release in generalized social
anxiety disorder. J Clin Psychiatry 66:238247.
Lingford-Hughes A, Hume SP, Feeney A, Hirani E, Osman S, Cunningham VJ, Pike
VW, Brooks DJ, and Nutt DJ (2002) Imaging the GABA-benzodiazepine receptor
subtype containing the alpha5-subunit in vivo with [11C]Ro15 4513 positron
emission tomography. J Cereb Blood Flow Metab 22:878889.
Lippa A, Czobor P, Stark J, Beer B, Kostakis E, Gravielle M, Bandyopadhyay S,
Russek SJ, Gibbs TT, and Farb DH, et al. (2005) Selective anxiolysis produced
by ocinaplon, a GABA(A) receptor modulator. Proc Natl Acad Sci USA 102:
73807385.
Lippa AS, Coupet J, Greenblatt EN, Klepner CA, and Beer B (1979) A synthetic non-
benzodiazepine ligand for benzodiazepine receptors: a probe for investigating
neuronal substrates of anxiety. Pharmacol Biochem Behav 11:99106.
Liu X, Ramirez S, Pang PT, Puryear CB, Govindarajan A, Deisseroth K,
and Tonegawa S (2012) Optogenetic stimulation of a hippocampal engram acti-
vates fear memory recall. Nature 484:381385.
Lo MM, Strittmatter SM, and Snyder SH (1982) Physical separation and charac-
terization of two types of benzodiazepine receptors. Proc Natl Acad Sci USA 79:
680684.
Lw K, Crestani F, Keist R, Benke D, Brnig I, Benson JA, Fritschy JM, Rlicke T,
Bluethmann H, and Mhler H, et al. (2000) Molecular and neuronal substrate for
the selective attenuation of anxiety. Science 290:131134.
Lund IV, Hu Y, Raol YH, Benham RS, Faris R, Russek SJ, and Brooks-Kayal AR
(2008) BDNF selectively regulates GABAA receptor transcription by activation of
the JAK/STAT pathway. Sci Signal 1:ra9.
Macdonald RL and Barker JL (1978) Different actions of anticonvulsant and anes-
thetic barbiturates revealed by use of cultured mammalian neurons. Science 200:
775777.
Macdonald RL, Rogers CJ, and Twyman RE (1989) Kinetic properties of the GABAA
receptor main conductance state of mouse spinal cord neurones in culture.
J Physiol 410:479499.
Majewska MD, Mienville JM, and Vicini S (1988) Neurosteroid pregnenolone sulfate
antagonizes electrophysiological responses to GABA in neurons. Neurosci Lett 90:
279284.
Malizia AL, Cunningham VJ, Bell CJ, Liddle PF, Jones T, and Nutt DJ (1998)
Decreased brain GABA(A)-benzodiazepine receptor binding in panic disorder:
preliminary results from a quantitative PET study. Arch Gen Psychiatry 55:
715720.
Malminiemi O and Korpi ER (1989) Diazepam-insensitive [3H]Ro 15-4513 binding in
intact cultured cerebellar granule cells. Eur J Pharmacol 169:5360.
Mao SC, Lin HC, and Gean PW (2008) Augmentation of fear extinction by
D-cycloserine is blocked by proteasome inhibitors. Neuropsychopharmacology 33:
30853095.
Masneuf S, Lowery-Gionta E, Colacicco G, Pleil KE, Li C, Crowley N, Flynn S,
Holmes A, and Kash T (2014) Glutamatergic mechanisms associated with stress-
induced amygdala excitability and anxiety-related behavior. Neuropsychopharmacology
85:190197.
1029
Mathers DA (1985) Spontaneous and GABA-induced single channel currents in cul-
tured murine spinal cord neurons. Can J Physiol Pharmacol 63:12281233.
Mathew SJ, Amiel JM, Coplan JD, Fitterling HA, Sackeim HA, and Gorman JM
(2005) Open-label trial of riluzole in generalized anxiety disorder. Am J Psychiatry
162:23792381.
Mato S, Vidal R, Castro E, Daz A, Pazos A, and Valdizn EM (2010) Long-term
fluoxetine treatment modulates cannabinoid type 1 receptor-mediated inhibition of
adenylyl cyclase in the rat prefrontal cortex through 5-hydroxytryptamine 1A
receptor-dependent mechanisms. Mol Pharmacol 77:424434.
Matsumoto K, Uzunova V, Pinna G, Taki K, Uzunov DP, Watanabe H, Mienville JM,
Guidotti A, and Costa E (1999) Permissive role of brain allopregnanolone content
in the regulation of pentobarbital-induced righting reflex loss. Neuropharmacology
38:955963.
McGaugh JL (2000) Memorya century of consolidation. Science 287:248251.
McGregor IS, Hargreaves GA, Apfelbach R, and Hunt GE (2004) Neural correlates of
cat odor-induced anxiety in rats: region-specific effects of the benzodiazepine
midazolam. J Neurosci 24:41344144.
McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S,
Myers J, Cook G, and Ferris P, et al. (2000) Sedative but not anxiolytic properties
of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype. Nat
Neurosci 3:587592.
McKernan RM and Whiting PJ (1996) Which GABAA-receptor subtypes really occur
in the brain? Trends Neurosci 19:139143.
McNaughton N, Kocsis B, and Hajs M (2007) Elicited hippocampal theta rhythm:
a screen for anxiolytic and procognitive drugs through changes in hippocampal
function? Behav Pharmacol 18:329346.
McNaughton N, Swart C, Neo P, Bates V, and Glue P (2013) Anti-anxiety drugs
reduce conflict-specific thetaa possible human anxiety-specific biomarker.
J Affect Disord 148:104111.
Medina JH, Paladini AC, Wolfman C, Levi de Stein M, Calvo D, Diaz LE, and Pea C
(1990) Chrysin (5,7-di-OH-flavone), a naturally-occurring ligand for benzodiaze-
pine receptors, with anticonvulsant properties. Biochem Pharmacol 40:22272231.
Medina JH, Pea C, Levi de Stein M, Wolfman C, and Paladini AC (1989)
Benzodiazepine-like molecules, as well as other ligands for the brain benzodiaze-
pine receptors, are relatively common constituents of plants. Biochem Biophys Res
Commun 165:547553.
Medina JH, Viola H, Wolfman C, Marder M, Wasowski C, Calvo D, and Paladini AC
(1997) Overviewflavonoids: a new family of benzodiazepine receptor ligands.
Neurochem Res 22:419425.
Mehta AK and Shank RP (1995) Interaction of abecarnil, bretazenil, and RO 19-8022
with diazepam-sensitive and -insensitive benzodiazepine sites in the rat cerebel-
lum and cerebral cortex. Life Sci 57:22152222.
Merikangas KR, He JP, Brody D, Fisher PW, Bourdon K, and Koretz DS (2010)
Prevalence and treatment of mental disorders among US children in the 2001-2004
NHANES. Pediatrics 125:7581.
Merikangas KR, Nakamura EF, and Kessler RC (2009) Epidemiology of mental
disorders in children and adolescents. Dialogues Clin Neurosci 11:720.
Michael-Titus AT, Albert M, Michael GJ, Michaelis T, Watanabe T, Frahm J,
Pudovkina O, van der Hart MG, Hesselink MB, and Fuchs E, et al. (2008)
SONU20176289, a compound combining partial dopamine D(2) receptor agonism
with specific serotonin reuptake inhibitor activity, affects neuroplasticity in an
animal model for depression. Eur J Pharmacol 598:4350.
Mihalek RM, Banerjee PK, Korpi ER, Quinlan JJ, Firestone LL, Mi ZP, Lagenaur C,
Tretter V, Sieghart W, and Anagnostaras SG, et al. (1999) Attenuated sensitivity to
neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit
knockout mice. Proc Natl Acad Sci USA 96:1290512910.
Mihalek RM, Bowers BJ, Wehner JM, Kralic JE, VanDoren MJ, Morrow AL,
and Homanics GE (2001) GABA(A)-receptor delta subunit knockout mice have mul-
tiple defects in behavioral responses to ethanol. Alcohol Clin Exp Res 25:17081718.
Minkeviciene R, Banerjee P, and Tanila H (2008) Cognition-enhancing and anxiolytic
effects of memantine. Neuropharmacology 54:10791085.
Mirza NR, Nielsen E, and Troelsen KB (2007) Serotonin transporter density and
anxiolytic-like effects of antidepressants in mice. Prog Neuropsychopharmacol Biol
Psychiatry 31:858866.
Miserendino MJ, Sananes CB, Melia KR, and Davis M (1990) Blocking of acquisition
but not expression of conditioned fear-potentiated startle by NMDA antagonists in
the amygdala. Nature 345:716718.
Moguilevsky JA and Wuttke W (2001) Changes in the control of gonadotrophin secre-
tion by neurotransmitters during sexual development in rats. Exp Clin Endocrinol
Diabetes 109:188195.
Mhler H and Okada T (1977) Benzodiazepine receptor: demonstration in the central
nervous system. Science 198:849851.
Morris HV, Dawson GR, Reynolds DS, Atack JR, and Stephens DN (2006) Both
alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties of
benzodiazepine site ligands in the conditioned emotional response paradigm. Eur J
Neurosci 23:24952504.
Nakamura M, Ueno S, Sano A, and Tanabe H (2000) The human serotonin trans-
porter gene linked polymorphism (5-HTTLPR) shows ten novel allelic variants. Mol
Psychiatry 5:3238.
Nemeroff CB (2003) Anxiolytics: past, present, and future agents. J Clin Psychiatry
64 (Suppl 3):36.
Nichols CD, Ronesi J, Pratt W, and Sanders-Bush E (2002) Hallucinogens and
Drosophila: linking serotonin receptor activation to behavior. Neuroscience 115:
979984.
Nichols DE and Nichols CD (2008) Serotonin receptors. Chem Rev 108:16141641.
Nielsen M, Frkjaer S, and Braestrup C (1988) High affinity of the naturally-
occurring biflavonoid, amentoflavon, to brain benzodiazepine receptors in vitro.
Biochem Pharmacol 37:32853287.
Nirenberg MJ (2013) Dopamine agonist withdrawal syndrome: implications for pa-
tient care. Drugs Aging 30:587592.
1030 Farb and Ratner
Noyes R Jr, DuPont RL Jr, Pecknold JC, Rifkin A, Rubin RT, Swinson RP, Ballenger
JC, and Burrows GD (1988) Alprazolam in panic disorder and agoraphobia: results
from a multicenter trial. II. Patient acceptance, side effects, and safety. Arch Gen
Psychiatry 45:423428.
Olivier JD, Vinkers CH, and Olivier B (2013) The role of the serotonergic and GABA
system in translational approaches in drug discovery for anxiety disorders. Front
Pharmacol 4:74.
Paucha-Poniewiera A and Pilc A (2012) Involvement of mGlu5 and NMDA receptors
in the antidepressant-like effect of acamprosate in the tail suspension test. Prog
Neuropsychopharmacol Biol Psychiatry 39:102106.
Pape HC and Stork O (2003) Genes and mechanisms in the amygdala involved in the
formation of fear memory. Ann N Y Acad Sci 985:92105.
Park-Chung M, Wu FS, and Farb DH (1994) 3 alpha-Hydroxy-5 beta-pregnan-20-one
sulfate: a negative modulator of the NMDA-induced current in cultured neurons.
Mol Pharmacol 46:146150.
Patchev VK, Shoaib M, Holsboer F, and Almeida OF (1994) The neurosteroid tet-
rahydroprogesterone counteracts corticotropin-releasing hormone-induced anxiety
and alters the release and gene expression of corticotropin-releasing hormone in
the rat hypothalamus. Neuroscience 62:265271.
Pelletier JG and Par D (2004) Role of amygdala oscillations in the consolidation of
emotional memories. Biol Psychiatry 55:559562.
Pesold C and Treit D (1996) The neuroanatomical specificity of the anxiolytic effects
of intra-septal infusions of midazolam. Brain Res 710:161168.
Phan KL, Coccaro EF, Angstadt M, Kreger KJ, Mayberg HS, Liberzon I, and Stein
MB (2013) Corticolimbic brain reactivity to social signals of threat before and after
sertraline treatment in generalized social phobia. Biol Psychiatry 73:329336.
Phan KL, Wager TD, Taylor SF, and Liberzon I (2004) Functional neuroimaging
studies of human emotions. CNS Spectr 9:258266.
Phillips ML, Drevets WC, Rauch SL, and Lane R (2003) Neurobiology of emotion
perception I: The neural basis of normal emotion perception. Biol Psychiatry 54:
504514.
Pigott TA (2003) Anxiety disorders in women. Psychiatr Clin North Am 26:621672,
vivii.
Pinna G (2010) In a mouse model relevant for post-traumatic stress disorder, selec-
tive brain steroidogenic stimulants (SBSS) improve behavioral deficits by nor-
malizing allopregnanolone biosynthesis. Behav Pharmacol 21:438450.
Pittenger C, Coric V, Banasr M, Bloch M, Krystal JH, and Sanacora G (2008) Riluzole
in the treatment of mood and anxiety disorders. CNS Drugs 22:761786.
Plescia F, Marino RA, Cannizzaro E, Brancato A, and Cannizzaro C (2013) The role of
pregnenolone sulphate in spatial orientation-acquisition and retention: an in-
terplay between cognitive potentiation and mood regulation. Behav Processes 99:
130137.
Pollack MH, Jensen JE, Simon NM, Kaufman RE, and Renshaw PF (2008) High-field
MRS study of GABA, glutamate and glutamine in social anxiety disorder: response
to treatment with levetiracetam. Prog Neuropsychopharmacol Biol Psychiatry 32:
739743.
Polter AM and Li X (2011) Glycogen Synthase Kinase-3 is an Intermediate Modulator
of Serotonin Neurotransmission. Front Mol Neurosci 4:31.
Porcelli S, Fabbri C, and Serretti A (2012) Meta-analysis of serotonin transporter
gene promoter polymorphism (5-HTTLPR) association with antidepressant effi-
cacy. Eur Neuropsychopharmacol 22:239258.
Pritchett DB, Lddens H, and Seeburg PH (1989) Type I and type II GABAA-
benzodiazepine receptors produced in transfected cells. Science 245:13891392.
Pritchett DB and Seeburg PH (1990) Gamma-aminobutyric acidA receptor alpha 5-
subunit creates novel type II benzodiazepine receptor pharmacology. J Neurochem
54:18021804.
Pritchett DB and Seeburg PH (1991) gamma-Aminobutyric acid type A receptor point
mutation increases the affinity of compounds for the benzodiazepine site. Proc Natl
Acad Sci USA 88:14211425.
Purdy RH, Morrow AL, Moore PH Jr, and Paul SM (1991) Stress-induced elevations
of gamma-aminobutyric acid type A receptor-active steroids in the rat brain. Proc
Natl Acad Sci USA 88:45534557.
Rabow LE, Russek SJ, and Farb DH (1995) From ion currents to genomic analysis:
recent advances in GABAA receptor research. Synapse 21:189274.
Rando RR, Bangerter FW, and Farb DH (1981) The inactivation of gamma-
aminobutyric acid transaminase in dissociated neuronal cultures from spinal
cord. J Neurochem 36:985990.
Rangel LM, Quinn LK, Chiba AA, Gage FH, and Aimone JB (2013) A hypothesis for
temporal coding of young and mature granule cells. Front Neurosci 7:75.
Rasmusson AM, Pinna G, Paliwal P, Weisman D, Gottschalk C, Charney D, Krystal
J, and Guidotti A (2006) Decreased cerebrospinal fluid allopregnanolone levels in
women with posttraumatic stress disorder. Biol Psychiatry 60:704713.
Rauch SL, Savage CR, Alpert NM, Fischman AJ, and Jenike MA (1997) The func-
tional neuroanatomy of anxiety: a study of three disorders using positron emission
tomography and symptom provocation. Biol Psychiatry 42:446452.
Rauch SL, Shin LM, and Wright CI (2003) Neuroimaging studies of amygdala
function in anxiety disorders. Ann N Y Acad Sci 985:389410.
Ravindran LN and Stein MB (2010) The pharmacologic treatment of anxiety dis-
orders: a review of progress. J Clin Psychiatry 71:839854.
Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal
JS, and Garnovskaya MN (2001) Multiplicity of mechanisms of serotonin receptor
signal transduction. Pharmacol Ther 92:179212.
Reardon LE, Leen-Feldner EW, and Hayward C (2009) A critical review of the em-
pirical literature on the relation between anxiety and puberty. Clin Psychol Rev 29:
123.
Reddy DS and Rogawski MA (2012) Neurosteroids endogenous regulators of sei-
zure susceptibility and role in the treatment of epilepsy, in Jaspers Basic Mech-
anisms of the Epilepsies (Noebels JL, Avoli M, Rogawski MA, Olsen RW,
and Delgado-Escueta AV eds) 4th ed, pp 9841002, Little, Brown and Company,
Boston, MA.
Regier DA, Narrow WE, and Rae DS (1990) The epidemiology of anxiety disorders:
the Epidemiologic Catchment Area (ECA) experience. J Psychiatr Res 24 (Suppl 2):
314.
Regier DA, Rae DS, Narrow WE, Kaelber CT, and Schatzberg AF (1998) Prevalence
of anxiety disorders and their comorbidity with mood and addictive disorders. Br J
Psychiatry Suppl (34):2428.
Reinhold JA, Mandos LA, Rickels K, and Lohoff FW (2011) Pharmacological
treatment of generalized anxiety disorder. Expert Opin Pharmacother 12:
24572467.
Ressler KJ and Nemeroff CB (2000) Role of serotonergic and noradrenergic systems
in the pathophysiology of depression and anxiety disorders. Depress Anxiety 12
(Suppl 1):219.
Ressler KJ, Rothbaum BO, Tannenbaum L, Anderson P, Graap K, Zimand E, Hodges
L, and Davis M (2004) Cognitive enhancers as adjuncts to psychotherapy: use of
D-cycloserine in phobic individuals to facilitate extinction of fear. Arch Gen
Psychiatry 61:11361144.
Rhodes RA, Murthy NV, Dresner MA, Selvaraj S, Stavrakakis N, Babar S, Cowen PJ,
and Grasby PM (2007) Human 5-HT transporter availability predicts amygdala
reactivity in vivo. J Neurosci 27:92339237.
Rickels K and Rynn M (2002) Pharmacotherapy of generalized anxiety disorder.
J Clin Psychiatry 63 (Suppl 14):916.
Roberts DS, Hu Y, Lund IV, Brooks-Kayal AR, and Russek SJ (2006) Brain-derived
neurotrophic factor (BDNF)-induced synthesis of early growth response factor 3
(Egr3) controls the levels of type A GABA receptor alpha 4 subunits in hippocampal
neurons. J Biol Chem 281:2943129435.
Roberts DS, Raol YH, Bandyopadhyay S, Lund IV, Budreck EC, Passini MA,
Wolfe JH, Brooks-Kayal AR, and Russek SJ (2005) Egr3 stimulation of
GABRA4 promoter activity as a mechanism for seizure-induced up-regulation
of GABA(A) receptor alpha4 subunit expression. Proc Natl Acad Sci USA 102:
1189411899.
Robinson RT, Drafts BC, and Fisher JL (2003) Fluoxetine increases GABA(A) re-
ceptor activity through a novel modulatory site. J Pharmacol Exp Ther 304:
978984.
Roozendaal B, de Quervain DJ, Ferry B, Setlow B, and McGaugh JL (2001) Baso-
lateral amygdala-nucleus accumbens interactions in mediating glucocorticoid en-
hancement of memory consolidation. J Neurosci 21:25182525.
Rosso IM, Weiner MR, Crowley DJ, Silveri MM, Rauch SL, and Jensen JE (2014)
Insula and anterior cingulate GABA levels in posttraumatic stress disorder: pre-
liminary findings using magnetic resonance spectroscopy. Depress Anxiety 31:
115123.
Rothbaum BO, Price M, Jovanovic T, Norrholm SD, Gerardi M, Dunlop B, Davis M,
Bradley B, Duncan EJ, and Rizzo A, et al. (2014) A randomized, double-blind
evaluation of D-cycloserine or alprazolam combined with virtual reality exposure
therapy for posttraumatic stress disorder in Iraq and Afghanistan War veterans.
Am J Psychiatry 171:640648.
Rudolph U, Crestani F, Benke D, Brnig I, Benson JA, Fritschy JM, Martin JR,
Bluethmann H, and Mhler H (1999) Benzodiazepine actions mediated by specific
gamma-aminobutyric acid(A) receptor subtypes. Nature 401:796800.
Rudolph U and Mhler H (2006) GABA-based therapeutic approaches: GABAA re-
ceptor subtype functions. Curr Opin Pharmacol 6:1823.
Sah A, Schmuckermair C, Sartori SB, Gaburro S, Kandasamy M, Irschick R,
Klimaschewski L, Landgraf R, Aigner L, and Singewald N (2012) Anxiety- rather
than depression-like behavior is associated with adult neurogenesis in a female
mouse model of higher trait anxiety- and comorbid depression-like behavior.
Transl Psychiatr 2:e171.
Sah P, Faber ES, Lopez De Armentia M, and Power J (2003) The amygdaloid com-
plex: anatomy and physiology. Physiol Rev 83:803834.
Sajdyk TJ and Shekhar A (1997) Excitatory amino acid receptors in the basolateral
amygdala regulate anxiety responses in the social interaction test. Brain Res 764:
262264.
Sandford JJ, Forshall S, Bell C, Argyropoulos S, Rich A, DOrlando KJ, Gammans
RE, and Nutt DJ (2001) Crossover trial of pagoclone and placebo in patients with
DSM-IV panic disorder. J Psychopharmacol 15:205208.
Schwartz TL, Siddiqui UA, and Raza S (2012) Memantine as an augmentation
therapy for anxiety disorders. Case Report Psychiatry 2012:749796.
Schwartz TL, Siddiqui UA, Raza S, and Costello A (2010) Acamprosate calcium as
augmentation therapy for anxiety disorders. Ann Pharmacother 44:19301932.
Selye H (1970) Adaptive steroids: retrospect and prospect. Perspect Biol Med 13:
343363.
Semeniuk T, Jhangri GS, and Le Melldo JM (2001) Neuroactive steroid levels in
patients with generalized anxiety disorder. J Neuropsychiatry Clin Neurosci 13:
396398.
Serra M, Ghiani CA, Motzo C, Cuccheddu T, Floris S, Giusti P, and Biggio G (1994)
Imidazenil, a new partial agonist of benzodiazepine receptors, reverses the in-
hibitory action of isoniazid and stress on gamma-aminobutyric acidA receptor
function. J Pharmacol Exp Ther 269:3238.
Sertraline (2012) Prescribing information. Pfizer/Roehrig, New York.
Shackman AJ, Fox AS, Oler JA, Shelton SE, Davidson RJ, and Kalin NH (2013)
Neural mechanisms underlying heterogeneity in the presentation of anxious
temperament. Proc Natl Acad Sci USA 110:61456150.
Shen H, Gong QH, Aoki C, Yuan M, Ruderman Y, Dattilo M, Williams K, and Smith
SS (2007) Reversal of neurosteroid effects at alpha4beta2delta GABAA receptors
triggers anxiety at puberty. Nat Neurosci 10:469477.
Sieghart W (1994) Pharmacology of benzodiazepine receptors: an update. J Psychi-
atry Neurosci 19:2429.
Sieghart W and Sperk G (2002) Subunit composition, distribution and function of
GABA(A) receptor subtypes. Curr Top Med Chem 2:795816.
Sim JA, Skynner MJ, and Herbison AE (2001) Direct regulation of postnatal GnRH
neurons by the progesterone derivative allopregnanolone in the mouse. Endocri-
nology 142:44484453.
 Targeted Modulation of Neural Circuits Implicated in Anxiolysis
Simpson JR, Ongr D, Akbudak E, Conturo TE, Ollinger JM, Snyder AZ, Gusnard
DA, and Raichle ME (2000) The emotional modulation of cognitive processing: an
fMRI study. J Cogn Neurosci 12 (Suppl 2):157170.
Skolnick P, Paul SM, and Weissman BA (1984) Preclinical pharmacology of buspirone
hydrochloride. Pharmacotherapy 4:308314.
Smith AJ, Alder L, Silk J, Adkins C, Fletcher AE, Scales T, Kerby J, Marshall G,
Wafford KA, and McKernan RM, et al. (2001) Effect of alpha subunit on allosteric
modulation of ion channel function in stably expressed human recombinant
gamma-aminobutyric acid(A) receptors determined using (36)Cl ion flux. Mol
Pharmacol 59:11081118.
Spigelman I, Li Z, Liang J, Cagetti E, Samzadeh S, Mihalek RM, Homanics GE,
and Olsen RW (2003) Reduced inhibition and sensitivity to neurosteroids in hip-
pocampus of mice lacking the GABA(A) receptor delta subunit. J Neurophysiol 90:
903910.
Sripada RK, Marx CE, King AP, Rampton JC, Ho SS, and Liberzon I (2013)
Allopregnanolone elevations following pregnenolone administration are associated
with enhanced activation of emotion regulation neurocircuits. Biol Psychiatry 73:
10451053.
Staley KJ and Mody I (1992) Shunting of excitatory input to dentate gyrus granule
cells by a depolarizing GABAA receptor-mediated postsynaptic conductance.
J Neurophysiol 68:197212.
Stein MB, Jang KL, Taylor S, Vernon PA, and Livesley WJ (2002) Genetic and
environmental influences on trauma exposure and posttraumatic stress disorder
symptoms: a twin study. Am J Psychiatry 159:16751681.
Stein MB, Keshaviah A, Haddad SA, Van Ameringen M, Simon NM, Pollack MH,
and Smoller JW (2014) Influence of RGS2 on sertraline treatment for social anxiety
disorder. Neuropsychopharmacology 39:13401346.
Stein MB, Pollack MH, Bystritsky A, Kelsey JE, and Mangano RM (2005) Efficacy of
low and higher dose extended-release venlafaxine in generalized social anxiety
disorder: a 6-month randomized controlled trial. Psychopharmacology (Berl) 177:
280288.
Steinbach JH and Akk G (2001) Modulation of GABA(A) receptor channel gating by
pentobarbital. J Physiol 537:715733.
Stell BM, Brickley SG, Tang CY, Farrant M, and Mody I (2003) Neuroactive steroids
reduce neuronal excitability by selectively enhancing tonic inhibition mediated by
delta subunit-containing GABAA receptors. Proc Natl Acad Sci USA 100:
1443914444.
Stephens DN, Schneider HH, Kehr W, Andrews JS, Rettig KJ, Turski L, Schmiechen
R, Turner JD, Jensen LH, and Petersen EN, et al. (1990) Abecarnil, a metabolically
stable, anxioselective beta-carboline acting at benzodiazepine receptors. J Phar-
macol Exp Ther 253:334343.
Stork O, Ji FY, and Obata K (2002) Reduction of extracellular GABA in the mouse
amygdala during and following confrontation with a conditioned fear stimulus.
Neurosci Lett 327:138142.
Stork O, Yamanaka H, Stork S, Kume N, and Obata K (2003) Altered conditioned
fear behavior in glutamate decarboxylase 65 null mutant mice. Genes Brain Behav
2:6570.
Strawn JR, Chu WJ, Whitsel RM, Weber WA, Norris MM, Adler CM, Eliassen JC,
Phan KL, Strakowski SM, and DelBello MP (2013) A pilot study of anterior cin-
gulate cortex neurochemistry in adolescents with generalized anxiety disorder.
Neuropsychobiology 67:224229.
Strhle A, Romeo E, di Michele F, Pasini A, Hermann B, Gajewsky G, Holsboer F,
and Rupprecht R (2003) Induced panic attacks shift gamma-aminobutyric acid type
A receptor modulatory neuroactive steroid composition in patients with panic
disorder: preliminary results. Arch Gen Psychiatry 60:161168.
Swanson CJ, Bures M, Johnson MP, Linden AM, Monn JA, and Schoepp DD (2005)
Metabotropic glutamate receptors as novel targets for anxiety and stress disorders.
Nat Rev Drug Discov 4:131144.
Toth M (2003) 5-HT1A receptor knockout mouse as a genetic model of anxiety. Eur J
Pharmacol 463:177184.
Tretter V, Ehya N, Fuchs K, and Sieghart W (1997) Stoichiometry and assembly of
a recombinant GABAA receptor subtype. J Neurosci 17:27282737.
Tromp DP, Grupe DW, Oathes DJ, McFarlin DR, Hernandez PJ, Kral TR, Lee JE,
Adams M, Alexander AL, and Nitschke JB (2012) Reduced structural connectivity
of a major frontolimbic pathway in generalized anxiety disorder. Arch Gen Psy-
chiatry 69:925934.
Twyman RE, Rogers CJ, and Macdonald RL (1989) Differential regulation of gamma-
aminobutyric acid receptor channels by diazepam and phenobarbital. Ann Neurol
25:213220.
Tye KM, Prakash R, Kim SY, Fenno LE, Grosenick L, Zarabi H, Thompson KR,
Gradinaru V, Ramakrishnan C, and Deisseroth K (2011) Amygdala circuitry me-
diating reversible and bidirectional control of anxiety. Nature 471:358362.
Urbani A and Belluzzi O (2000) Riluzole inhibits the persistent sodium current in
mammalian CNS neurons. Eur J Neurosci 12:35673574.
Uzunova V, Sheline Y, Davis JM, Rasmusson A, Uzunov DP, Costa E, and Guidotti A
(1998) Increase in the cerebrospinal fluid content of neurosteroids in patients with
unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc Natl
Acad Sci USA 95:32393244.
Van der Linden G, van Heerden B, Warwick J, Wessels C, van Kradenburg J, Zungu-
Dirwayi N, and Stein DJ (2000) Functional brain imaging and pharmacotherapy in
social phobia: single photon emission computed tomography before and after
treatment with the selective serotonin reuptake inhibitor citalopram. Prog
Neuropsychopharmacol Biol Psychiatry 24:419438.
Van Laere K, Bormans G, Sanabria-Bohrquez SM, de Groot T, Dupont P, De
Lepeleire I, de Hoon J, Mortelmans L, Hargreaves RJ, and Atack JR, et al. (2008)
In vivo characterization and dynamic receptor occupancy imaging of TPA023B, an
alpha 2/alpha 3/alpha 5 subtype selective gamma-aminobutyric acid-a partial
agonist. Biol Psychiatry 64:153161.
Van Oekelen D, Luyten WH, and Leysen JE (2003) 5-HT2A and 5-HT2C receptors
and their atypical regulation properties. Life Sci 72:24292449.
1031
Van Oort FV, Greaves-Lord K, Verhulst FC, Ormel J, and Huizink AC (2009) The
developmental course of anxiety symptoms during adolescence: the TRAILS study.
J Child Psychol Psychiatry 50:12091217.
van Steveninck AL, Gieschke R, Schoemaker RC, Roncari G, Tuk B, Pieters MS,
Breimer DD, and Cohen AF (1996) Pharmacokinetic and pharmacodynamic
interactions of bretazenil and diazepam with alcohol. Br J Clin Pharmacol 41:
565573.
Varia I and Rauscher F (2002) Treatment of generalized anxiety disorder with cit-
alopram. Int Clin Psychopharmacol 17:103107.
Venlafaxine (2012) Prescribing information. Pfizer/Wyeth Pharmaceuticals, Inc.,
Philadelphia.
Villiger JW (1984) CL 218,872 binding to benzodiazepine receptors in rat spinal cord:
modulation by gamma-aminobutyric acid and evidence for receptor heterogeneity.
J Neurochem 43:903905.
Wafford KA, Whiting PJ, and Kemp JA (1993) Differences in affinity and efficacy of
benzodiazepine receptor ligands at recombinant gamma-aminobutyric acidA re-
ceptor subtypes. Mol Pharmacol 43:240244.
Walker DL, Miles LA, and Davis M (2009) Selective participation of the bed nucleus
of the stria terminalis and CRF in sustained anxiety-like versus phasic fear-like
responses. Prog Neuropsychopharmacol Biol Psychiatry 33:12911308.
Walker DL, Ressler KJ, Lu KT, and Davis M (2002) Facilitation of conditioned fear
extinction by systemic administration or intra-amygdala infusions of D-cycloserine
as assessed with fear-potentiated startle in rats. J Neurosci 22:23432351.
Walker DL, Toufexis DJ, and Davis M (2003) Role of the bed nucleus of the stria
terminalis versus the amygdala in fear, stress, and anxiety. Eur J Pharmacol 463:
199216.
Wang C, Marx CE, Morrow AL, Wilson WA, and Moore SD (2007) Neurosteroid
modulation of GABAergic neurotransmission in the central amygdala: a role for
NMDA receptors. Neurosci Lett 415:118123.
Wang MD, Zhu D, Bckstrm T, and Wahlstrm G (2001) The interaction between
ethanol and pregnanolone at induction of anaesthesia investigated with a thresh-
old method in male rats. Br J Pharmacol 134:13931402.
Wang SJ, Wang KY, and Wang WC (2004) Mechanisms underlying the riluzole in-
hibition of glutamate release from rat cerebral cortex nerve terminals (synapto-
somes). Neuroscience 125:191201.
Wang SM and Pae CU (2013) How much to worry about the FDA warning in the use
of citalopram? Expert Rev Neurother 13:883886.
Warwick JM, Carey PD, Cassimjee N, Lochner C, Hemmings S, Moolman-Smook H,
Beetge E, Dupont P, and Stein DJ (2012) Dopamine transporter binding in social
anxiety disorder: the effect of treatment with escitalopram. Metab Brain Dis 27:
151158.
Weaver CE, Land MB, Purdy RH, Richards KG, Gibbs TT, and Farb DH (2000)
Geometry and charge determine pharmacological effects of steroids on N-methyl-
D-aspartate receptor-induced Ca(2+) accumulation and cell death. J Pharmacol
Exp Ther 293:747754.
Weaver CE Jr, Marek P, Park-Chung M, Tam SW, and Farb DH (1997) Neuro-
protective activity of a new class of steroidal inhibitors of the N-methyl-D-
aspartate receptor. Proc Natl Acad Sci USA 94:1045010454.
Whiting PJ, Bonnert TP, McKernan RM, Farrar S, Le Bourdells B, Heavens RP,
Smith DW, Hewson L, Rigby MR, and Sirinathsinghji DJ, et al. (1999) Molecular
and functional diversity of the expanding GABA-A receptor gene family. Ann N Y
Acad Sci 868:645653.
Wieland HA, Lddens H, and Seeburg PH (1992) A single histidine in GABAA
receptors is essential for benzodiazepine agonist binding. J Biol Chem 267:
14261429.
Wieland S, Belluzzi J, Hawkinson JE, Hogenkamp D, Upasani R, Stein L, Wood PL,
Gee KW, and Lan NC (1997) Anxiolytic and anticonvulsant activity of a synthetic
neuroactive steroid Co 3-0593. Psychopharmacology (Berl) 134:4654.
Williams DB and Akabas MH (2001) Evidence for distinct conformations of the two
alpha 1 subunits in diazepam-bound GABA(A) receptors. Neuropharmacology 41:
539545.
Wingrove PB, Safo P, Wheat L, Thompson SA, Wafford KA, and Whiting PJ (2002)
Mechanism of alpha-subunit selectivity of benzodiazepine pharmacology at
gamma-aminobutyric acid type A receptors. Eur J Pharmacol 437:3139.
Wisden W, Laurie DJ, Monyer H, and Seeburg PH (1992) The distribution of 13
GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon,
mesencephalon. J Neurosci 12:10401062.
Wolfman C, Viola H, Marder M, Wasowski C, Ardenghi P, Izquierdo I, Paladini AC,
and Medina JH (1996) Anxioselective properties of 6,39-dinitroflavone, a high-
affinity benzodiazepine receptor ligand. Eur J Pharmacol 318:2330.
Wolfman C, Viola H, Paladini A, Dajas F, and Medina JH (1994) Possible anxiolytic
effects of chrysin, a central benzodiazepine receptor ligand isolated from Passiflora
coerulea. Pharmacol Biochem Behav 47:14.
Woods AM and Bouton ME (2006) D-cycloserine facilitates extinction but does not
eliminate renewal of the conditioned emotional response. Behav Neurosci 120:
11591162.
Wu FS, Gibbs TT, and Farb DH (1991) Pregnenolone sulfate: a positive allosteric
modulator at the N-methyl-D-aspartate receptor. Mol Pharmacol 40:333336.
Wu FS, Gibbs TT, and Farb DH (1993) Dual activation of GABAA and glycine
receptors by beta-alanine: inverse modulation by progesterone and 5 alpha-
pregnan-3 alpha-ol-20-one. Eur J Pharmacol 246:239246.
Yamawaki S (1999) The use and development of anxiolytics in Japan. Eur Neuro-
psychopharmacol 9 (Suppl 6):S413S419.
Yang P, Jones BL, and Henderson LP (2002) Mechanisms of anabolic androgenic
steroid modulation of alpha(1)beta(3)gamma(2L) GABA(A) receptors. Neurophar-
macology 43:619633.
Yilmazer-Hanke DM, Roskoden T, Zilles K, and Schwegler H (2003) Anxiety-related
behavior and densities of glutamate, GABAA, acetylcholine and serotonin
receptors in the amygdala of seven inbred mouse strains. Behav Brain Res 145:
145159.
1032 Farb and Ratner
Young WS 3rd, Niehoff D, Kuhar MJ, Beer B, and Lippa AS (1981) Multiple benzo-
diazepine receptor localization by light microscopic radiohistochemistry. J Phar-
macol Exp Ther 216:425430.
Zanardi R, Serretti A, Rossini D, Franchini L, Cusin C, Lattuada E, Dotoli D,
and Smeraldi E (2001) Factors affecting fluvoxamine antidepressant activity: in-
fluence of pindolol and 5-HTTLPR in delusional and nondelusional depression. Biol
Psychiatry 50:323330.
Zwanzger P, Baghai T, Boerner RJ, Mller HJ, and Rupprecht R (2001a) Anxiolytic
effects of vigabatrin in panic disorder. J Clin Psychopharmacol 21:539540.
Zwanzger P, Baghai TC, Schuele C, Strhle A, Padberg F, Kathmann N, Schwarz M,
Mller HJ, and Rupprecht R (2001b) Vigabatrin decreases cholecystokinin-tetrapeptide
(CCK-4) induced panic in healthy volunteers. Neuropsychopharmacology 25:699703.
Zwanzger P, Eser D, Padberg F, Baghai TC, Schule C, Rtzer F, Ella R, Mller HJ,
and Rupprecht R (2003) Effects of tiagabine on cholecystokinin-tetrapeptide (CCK-
4)-induced anxiety in healthy volunteers. Depress Anxiety 18:140143.
Zwanzger P, Zavorotnyy M, Gencheva E, Diemer J, Kugel H, Heindel W, Ruland T,
Ohrmann P, Arolt V, and Domschke K, et al. (2013) Acute shift in glutamate
concentrations following experimentally induced panic with cholecystokinin tet-
rapeptidea 3T-MRS study in healthy subjects. Neuropsychopharmacology 38:
16481654.
Zweifel LS, Fadok JP, Argilli E, Garelick MG, Jones GL, Dickerson TM, Allen JM,
Mizumori SJ, Bonci A, and Palmiter RD (2011) Activation of dopamine neurons is
critical for aversive conditioning and prevention of generalized anxiety. Nat Neu-
rosci 14:620626.