ORIGINAL ARTICLES Dopamine D2 Receptor Availability and AmphetamineInduced Dopamine Release in Unipolar Depression

Ramin V. Parsey, Maria A. Oquendo, Yolanda Zea-Ponce, Janine Rodenhiser, Lawrence S. Kegeles, Mali Pratap, Thomas B. Cooper, Ronald Van Heertum, J. John Mann, and Marc Laruelle
Background: Reduced dopaminergic transmission has been implicated in the pathophysiology of major depression. The aim of the present study was to measure striatal D2 receptor availability and amphetamineinduced dopamine release in nonpsychotic, unmedicated, unipolar patients during an episode of major depression. Methods: The striatal equilibrium specific to nonspecific partition coefficient (V3) of the D2 receptor antagonist [123I]IBZM was measured with single photon emission computerized tomography before and after amphetamine administration in 9 depressed subjects and 10 matched healthy control subjects. Results: No significant differences were observed in preamphetamine D2 receptor availability between depressed patients (0.73 0.08) and control subjects (0.78 0.10, p .23). Amphetamine-induced reduction in [123I]IBZM V3 (V3) was similar in depressed patients (9.8 5.5%) and control subjects (7.8 2.5%, p .32). Amphetamine induced a transient improvement in symptomatology in depressed patients, but this improvement did not correlate with [123I]IBZM V 3 . Conclusions: This study did not replicate previously reported alterations in striatal D2 receptor density in depressed patients and suggests that stimulant-induced dopamine release is not altered in major depression. Biol Psychiatry 2001;50:313322 2001 Society of Biological Psychiatry Key Words: Dopamine, depression, amphetamine, D2 receptors, SPECT.

Introduction
large body of evidence supports the involvement of serotonergic and noradrenergic functions in major depression. In addition, alterations in dopaminergic transmission have been suspected to play a role in the pathophysiology of this condition (Randrup et al 1975). The critical role of dopamine in brain reward systems, the reports of low cerebrospinal fluid homovanillic acid levels in depressed patients, the association of major depression with Parkinsons disease, and the enhancement of dopaminergic activity by several antidepressant treatments suggest that a deficiency of dopaminergic function might be associated with major depression (for review see Brown and Gershon 1993; Diehl and Gershon 1992; Kapur and Mann 1992; Willner et al 1992). More recently, brain imaging techniques have been used to directly assess the in vivo availability of dopamine D2 receptors in patients with major depression. Four studies compared the binding of the single photon emission computerized tomography (SPECT) radiotracer [123I]IBZM, a selective D2/D3 antagonist (Kung et al 1988), in patients with major depression and control subjects. Two of the four studies reported higher [123I]IBZM specific binding in the striatum of depressed subjects compared to control subjects (DHaenen and Bossuyt 1994; Shah et al 1997), whereas two studies reported no changes (Ebert et al 1996; Klimke et al 1999). Because these studies were performed with a semi-quantitative method, in which the outcome measures can be affected by between-subject differences in plasma clearance or regional blood-flow (Carson et al 1993; Laruelle et al 1994a), such factors potentially account for discrepant results. The in vivo binding of [123I]IBZM is affected by competition with endogenous dopamine (Laruelle et al 1997a), and it has been proposed that increased [123I]IBZM binding in depression might be due to decreased synaptic dopamine concentration and subsequent up-regulation of D2 receptors (DHaenen and Bossuyt 1994). Recently, several groups have demonstrated that,
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From the Departments of Psychiatry (RVP, MAO, YZ-P, JR, LSK, MP, JJM, ML) and Radiology (RVH, JJM, ML), Columbia University College of Physicians and Surgeons; and Division of Brain Imaging, Departments of Neuroscience and Analytical Psychopharmacology, New York State Psychiatric Institute (TBC), New York, New York. Address reprint requests to Ramin V. Parsey, New York State Psychiatric Institute, 1051 Riverside Dr., Unit # 342, New York, NY 10032. Received June 22, 2000; revised December 27, 2000; accepted January 4, 2001.

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under specific conditions, in vivo neuroreceptor binding techniques can also be used to measure acute fluctuations in the concentration of endogenous transmitters in the vicinity of neuroreceptors (for review see Laruelle 2000). Specifically, the decrease in the availability of D2 receptors to [123I]IBZM binding following acute amphetamine administration has been validated as an indirect measure of the change in synaptic dopamine concentration induced by the challenge: amphetamine-induced decreases in [123I]IBZM binding were correlated with increased extracellular dopamine measured with microdialysis, and dopamine depletion blunted the effect of amphetamine on [123I]IBZM binding (Laruelle et al 1997b). Similar results have been reported with the positron emission tomography radioligand [11C]raclopride (Breier et al 1997). Using this technique, psychostimulant-induced dopamine release has been shown to be increased in untreated patients with schizophrenia (Abi-Dargham et al 1998; Breier et al 1997; Laruelle et al 1996, 1999), decreased in detoxified cocaine abusers (Malison et al 1999; Volkow et al 1997b) and normal in euthymic bipolar subjects (Anand et al 2000). Competition between [123I]IBZM and endogenous dopamine has also been used to study baseline levels of endogenous dopamine, which have been found to be elevated in patients with schizophrenia (Abi-Dargham et al 2000). The first goal of this study was to evaluate D2 receptor availability in untreated patients with current major depression under sustained equilibrium conditions as achieved by constant infusion of [123I]IBZM (Laruelle et al 1995). This infusion technique enables measurement of [123I]IBZM specific binding that is not confounded by between-subject differences in cerebral blood flow or peripheral clearance. The second goal was to assess changes in [123I]IBZM binding following an acute amphetamine challenge, to test the hypothesis that amphetamine-induced dopamine release would be blunted in major depression, consistent with the general hypothesis of a deficiency in dopamine transmission in this condition.

of both genders and all ethnic origins; 2) age 18 45 years; 3) DSM-IV criteria for current major depressive disorder; 4) score of at least 16 on the first 17 items of the HAM-D scale at the time of the scan; 5) absence of any psychotropic medications for at least 2 weeks (6 weeks for fluoxetine, 4 weeks for neuroleptics), except benzodiazepines, which were discontinued three days before the scans; 6) no current or past history of major depressive episode with psychotic features; 7) absence of any other Axis I diagnosis, including bipolar disorder and current or lifetime history of alcohol or substance abuse or dependence; 8) absence of illicit or therapeutic lifetime exposure to psychostimulants; 9) absence of significant medical conditions; 10) absence of pregnancy; and 11) ability to provide informed consent. Study criteria for control subjects included the following: 1) subjects of both genders and all ethnic origins; 2) age 18 45 years; 3) absence of medical, neurologic, and psychiatric illness (including alcohol and substance abuse), as assessed by subject history, review of systems, physical examination, routine blood tests, urine toxicology, and EKG; 4) absence of any medications for at least 2 weeks; 5) absence of pregnancy; and 6) informed consent. The data of 2 of the 10 healthy subjects have been previously published in a study assessing the test/retest variability of the measurement of amphetamine-induced dopamine release in healthy subjects (Kegeles et al 1999). Administration of both [123I]IBZM and intravenous (IV) amphetamine were approved by the U.S. Food and Drug Administration under an Investigational New Drug protocol. The protocol was approved by the Institutional Review Boards of Columbia Presbyterian Hospital and of the New York State Psychiatric Institute. Subjects gave written informed consent after a detailed explanation of the study.

Radioligand Preparation
[123I]IBZM was prepared by direct electrophilic radioiodination of the phenolic precursor BZM([(S)(-)-N-[1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-6-methoxybenzamide] with high-purity sodium [123I]iodide solution, in the presence of a potassium biphthalate/sulfamic acid buffer, pH 2, and 3.2% aqueous peracetic acid, at 65C for 10 min (Zea-Ponce and Laruelle 1999). The reaction mixture was initially purified by passing it through a C-18 Light SepPak (120 mg; Waters; Milford, Mass.) followed by reverse phase high-performance liquid chromatography [PRP1-C-18 column; acetonitrile/ammonium phosphate (4 mmol/L, pH 7), 82:18]. The final product was formulated in sterile saline and sterilized by membrane filtration and confirmed to be pyrogen-free before its use. Under the described conditions, the labeling yield was 67 5%, the radiochemical yield 43 13% and the radiochemical purity 98 1% ( n 19).

Methods and Materials

Subjects
Nine patients who met DSM-IV criteria for unipolar major depression (seven female and two male) and ten healthy control subjects (seven female and three male) were included in this study. Inclusion and exclusion criteria were evaluated by history, chart review, Structured Clinical Interview for DSM-IV (SCID) (Spitzer et al 1992), Hamilton Depression Rating Scale for depression (HAM-D, Hamilton 1960), review of systems, physical examination, routine blood tests, pregnancy test, urine toxicology and electrocardiogram (EKG). All subjects had a SCID-II interview (Spitzer et al 1992). Study criteria for patients included the following: 1) patients

SPECT Scan Protocol

The previously described radiotracer constant infusion technique was used to perform the experiments under sustained equilibrium binding conditions (Laruelle et al 1995, 1996). To decrease radiation exposure to the thyroid gland, subjects received 0.6 g potassium iodide 60 min before [123I]IBZM injection. Four fiducial markers, each containing 3 Ci of [99mTc]NaTcO4, were

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glued on each side of the subjects head at the level of the cantho-meatal line. Two intravenous catheters were inserted (in the right and left arms, respectively), for drug administration and blood sampling, respectively. [123I]IBZM was administered as a priming bolus (3.92 0.57 mCi), immediately followed by a continuous infusion at a constant rate (0.77 0.06 mCi/hour) for the duration of the experiment (360 min), which corresponded to a total injected dose of 10.08 1.56 mCi, decay corrected to the beginning of the experiment. Patients and control subjects received similar doses of [123I]IBZM (patients: 9.9 1.0 mCi; control subjects: 10.1 1.9 mCi, p .75). Laruelle et al (1995) previously established that, using this administration schedule, stable activity levels in plasma and brain are observed from 150 min until the end of the infusion. During the first 180 min of the infusion, subjects were allowed to relax in a comfortable setting, in a room adjacent to the camera room. The first scanning session (preamphetamine) was initiated at 180 min, and lasted 60 min. Data from SPECT were obtained with the triple-head PRISM 3000 (Picker, Cleveland, OH), equipped with Low Energy Ultra High Resolution (LEUHR) fan beam collimators Full Width Half Max (FWHM 8 10 mm). Scanning was performed with the following acquisition parameters: continuous acquisition mode; matrix, 128 128 64; angular range, 120; angular steps, 3; seconds per step, 18; frame duration, 12 min; number of frames, 5; radius of rotation, 13.5 cm. At the end of the first scanning session (240 min), d-amphetamine (0.3 mg/kg) was administered intravenously over 30 sec. Electrocardiogram and vital signs were monitored continuously after the amphetamine injection. The second scanning session (postamphetamine) was obtained using similar parameters from 300 360 min.

Amphetamine Plasma Measurement

Amphetamine plasma concentration was measured (Analytical Psychopharmacology Laboratories, Nathan Kline Institute, Orangeburg, NY) at 10, 20, and 40 min post amphetamine injection with gas chromatography/mass spectroscopy as previously described (Kegeles et al 1999). Inter-assay variability is 5.2% (SD) at 5 ng/mL. Given the slow clearance of amphetamine, measurements at the three time points were averaged.

Image Analysis
Image analysis was performed as previously described (Kegeles et al 1999, 2000), blind to the diagnostic status. Briefly, frames were reconstructed using a Butterworth filter (cutoff 1 cm, power factor 10), transferred into the MEDx software system (Sensor Systems, Sterling, VA), and corrected for attenuation assuming uniform attenuation (attenuation coefficient 0.10 cm2/g) within an ellipse drawn around the skull as identified by the fiducial markers. Frames were realigned to each other, using a least-squares algorithm for within-modality coregistration (Automated Image Registration, AIR) (Woods et al 1992). Standard regions of interest of constant size (striatum 15,820 mm3; frontal 57,656 mm3; occipital 58,497 mm3) were used to analyze all studies. Right and left striatal regions were averaged. Specific binding was calculated as striatal minus nonspecific activity. Nonspecific activity was calculated as the average of the frontal and occipital regions because 1) the density of dopamine D2 receptors is negligible in these regions compared to the striatum (Lidow et al 1989); 2) these regions can be identified with greater reliability than the cerebellum; 3) in humans, [123I]IBZM activity in the neocortex is equal to the nonspecific activity in the striatum (Seibyl et al 1992); 4) the reproducibility of nonspecific binding measurement is improved when both occipital and frontal regions are used as opposed to using only one of these regions (Kegeles et al 1999). For each scanning session, D2 receptor availability was assessed by the specific to nonspecific equilibrium partition coefficient (V3), calculated as the ratio of striatal minus nonspecific to nonspecific activity (Laruelle et al 1994b). V3 is equal to the binding potential (BP, Bmax/KD) normalized by the nonspecific distribution volume (V2).

Clinical Response
Clinical response to the amphetamine administration was evaluated with two measures. First, four items (happiness, restlessness, energy, and anxiety) from the Amphetamine Interview Rating Scale (AIRS) (van Kammen and Murphy 1978) were self-rated during the 45 min following amphetamine injection. Each item was rated on a scale from 110, 1 being not at all and 10 being most ever. Self-ratings were obtained at baseline and at 5, 10, 20, 30, and 45 min post injection. These data were analyzed as previously described (Laruelle et al 1995). Following amphetamine, these ratings have been shown to peak at about 10 20 min, and to progressively decrease thereafter. As the peak response was shown to correlate with the area under the curve above baseline, peak response was used to assess response to amphetamine. Second, the Brief Psychiatric Rating Scale (BPRS; Overall and Gorham 1962) was administered just before and 30 min after amphetamine administration. Three outcomes were extracted from this scale: the total score, the sum of scores related to depressive symptomatology (somatization, anxiety, guilt, tension, depressive mood, blunted affect, and emotional withdrawal), and psychomotor retardation. Baseline anhedonia was evaluated with the Scale for the Assessment of Negative Symptoms (Andreasen 1982), as the sum of interest in recreational, sexual, and work-related activities.

V 3 B max/ K DV 2
Under steady-state conditions, and assuming that amphetamine does not affect [123I]IBZM nonspecific binding, the percent decrease in V3 (V3) is equal to that in BP (Laruelle et al 1997b). We previously reported that, using this protocol, the test/retest intraclass correlation coefficient of amphetamineinduced V3 is 0.89 (Kegeles et al 1999).

Statistical Analyses
All values are expressed as Mean SD. Statistical analyses were performed using factorial or repeated measures analysis of variance (ANOVA), as appropriate, and statistical significance was defined as p .05.

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Table 1. Demographics
Parameter n Age Gender (F/M) Race (C/H/AA) Smoking Status (NS/S/Hx) Subject SES Parental SES Control subjects 10 30 10 7/3 7/1/2 6/2/1 32 17 51 14 Depressed subjects 9 36 12 7/2 6/2/1 8/0/2 36 20 39 18 pa .23 .70 .70 .27 .65 .11

a p probability value by unpaired t test or 2, as appropriate. F, female; M, male; C, Caucasian; H, Hispanic; AA, African American; NS, nonsmoker; S, smoker; Hx, history of smoking; SES, socioeconomic status.

Results
Sample Composition
Subjects with major depression and control subjects were matched on age, gender, ethnicity, socioeconomic status, parental socioeconomic status, handedness, and smoking status (Table 1). Patients had HAM-D scores of 21 5 and BPRS depression scores of 23 3 at the time of the scan. Anhedonia scores were 7.2 2.1, ranging from 4 to 11. Four patients exhibited moderate levels of psychomotor retardation (scoring at least a 3 on the BPRS psychomotor retardation scale that ranges from 1 to 7), one patient scored 2 (very mild), and four patients scored 1 (none). Among the depressed subjects, three subjects also met DSM-IV criteria for borderline personality disorder, one subject met criteria for narcissistic personality disorder, and five did not meet criteria for an Axis II diagnosis. Four subjects had never received any antidepressant treatment, four had been treated once before with antidepressants, and one subject had two previously treated episodes. None had been treated with antidepressant medication in the last 6 months. None met criteria for melancholic depression. None had received electroconvulsive therapy. All subjects were free of benzodiazepines for at least 3 days before their scan (one subject received lorazepam up to 3 days before the scan; other subjects did not receive any psychotropic medication for at least 2 weeks before the scan).
Figure 1. Baseline [123I]IBZM V3 in striatum in ten control subjects (0.78 0.10) and nine depressed subjects (0.73 0.08). There is no significant difference in baseline D2 receptors availability between the two groups.

of psychomotor retardation (baseline [123I]IBZM V3 of 0.71 0.10) had similar baseline D2 receptor availability compared to the five patients with no psychomotor retardation (baseline [123I]IBZM V3 of 0.74 0.07, p .55).

Change in V3
Amphetamine-induced decrease in [123I]IBZM V3 (V3) was not statistically different between depressed patients

Baseline V3
No significant difference was observed in baseline [123I]IBZM V3 between patients (0.73 0.08) and control subjects (0.78 0.10, Figure 1, Table 2). Baseline V3 decreased with age (r2 .25, p .03), and there was no significant age by diagnosis interaction (p .81). In the depressed group, baseline V3 did not correlate with baseline HAM-D score (r2 .06, p .54), baseline BPRS depression score (r2 .02, p .72) or anhedonia scores (r2 .10, p .40). The four patients with moderate levels

Figure 2. Amphetamine-induced (0.3 mg/kg IV) changes in [123I]IBZM V3 in depressed subjects and control subjects. Values are expressed in percent decreases relative to preamphetamine values. No significant difference was observed between the two groups.

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Table 2. Baseline [123I]IBZM V3, Amphetamine-Induced Change in V3, and Plasma Amphetamine
Outcome Baseline [123I]IBZM V3 Amphetamine-induced V3 Amphetamine plasma levels (ng/mL) Control subjects 0.78 0.10 7.8 2.5% 38 14 Depressed subjects 0.73 0.08 9.8 5.5% 38 9 p .23 .32 .99

(9.8 5.5%) and control subjects (7.8 2.5%, Figure 2, Table 2). The variance of V3 was larger in patients compared to control subjects at a trend level (F test, F 0.28, p .08). No significant correlation was observed between V3 and age (r2 .09, p .21), and there was no significant age by diagnosis interaction (p .81). Plasma amphetamine levels did not differ between control subjects and depressed subjects (Table 2). No relationship was observed between plasma amphetamine levels and V3 (r2 .01, p .84).

Self-Ratings
Table 3 lists the four analog scores reported by the subjects at baseline and following amphetamine administration (peak scores, calculated as the difference between peak scores and baseline scores). At baseline, depressed subjects reported significantly lower scores on euphoria and energy, higher scores on anxiety, and similar scores on restlessness. Amphetamine administration resulted in a significant increase in euphoria and energy in both patients and healthy control subjects. Amphetamine induced a significant increase in restlessness in patients but not in control subjects. Amphetamine did not induce significant changes in anxiety ratings. Amphetamine-induce changes in self-ratings were not significantly different between the groups (no significant group by time interactions, Table 3). Thus, following amphetamine, ratings in euphoria and energy were still lower in depressed patients compared to control subjects, anxiety was still higher in depressed patients compared to control subjects, and restlessness did not differ between the groups. These changes are illustrated for euphoria in Figure 3. None of the self-rating
Table 3. Amphetamine Effect on Self-Reports
Control subjects Rating Euphoria Restlessness Energy Anxiety
a b c

Figure 3. Euphoria self-ratings in depressed subjects and control subjects, at baseline and following amphetamine injection (0.3 mg/kg IV). Both groups reported a transient increase in euphoria after administration of amphetamine. Ratings in depressed subjects remained consistently lower than in control subjects.

changes (increases in euphoria, energy, restlessness, and anxiety) were significantly correlated with [123I]IBZM V3, neither in the entire sample nor in each group considered separately.

Objective Clinical Ratings

Depressed subjects exhibited a moderate and transient improvement in symptomatology after amphetamine administration. Total BPRS scores decreased from 34 5 to 28 7 (repeated measures ANOVA, p .05). The depression items of the BPRS decreased from 23 3 to 15 6 (repeated measures ANOVA, p .004). [123I]IBZM V3 did not correlate with changes in total BPRS (r2 .04, p .87), or with changes in the sum of BPRS depression items (r2 .18, p .01). Following amphetamine, psychomotor retardation scores decreased

Depressed subjects Baseline 3.6 1.4 4.3 2.1 3.9 1.8 5.3 2.3 Peak 6.0 3.1c 7.1 2.5c 6.8 2.6c 6.1 3.4

Control subjects versus Depressed subjects (p) Baselinea 0.01 0.63 0.01 0.02 Peaka 0.02 0.14 0.08 0.02 Interactionb 0.73 0.07 0.29 0.49

Baseline 6.6 2.3 4.7 2.3 6.9 1.8 2.8 1.6

Peak 8.7 1.3c 5.1 2.9 8.6 1.9c 2.7 2.1

Unpaired t test. Repeated measures analysis of variance (ANOVA) group time interaction. Significantly different from baseline ratings (repeated measures ANOVA, p 0.05).

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to 1 (none) in all but one patient who scored 2 (very mild). Thus, psychomotor retardation decreased from 2.0 1.1 to 1.1 0.3 (repeated measures ANOVA, p .04). [123I]IBZM V3 did not correlate with changes in psychomotor retardation (r2 .24, p .17). No differences in V3 were observed between patients with moderate (V3 13 5%, n 4) and absent (V3 8 5%, n 4) psychomotor retardation at baseline (p .18). No relationship was observed between baseline anhedonia scores and [123I]IBZM V3 (r2 .02, p .71).

Discussion
This preliminary study in a small sample did not detect differences in baseline D2 receptor availability and in amphetamine-induced stimulation of D2 receptor transmission in untreated unipolar patients during a major depressive episode compared to matched healthy control subjects. The minimal effect size required to detect a between-group difference with adequate power (0.8) and confidence (p .05) with this sample size (n 9 per group) is 1 (using one-tailed test) or 1.5 (using two-tailed test). Thus, this study did not exclude the possibility of an association between major depression and moderate changes in dopamine D2 receptor availability or in amphetamine-induced stimulation of D2 receptor transmission. The involvement of the dopaminergic system in major depression could be conceptualized at several levels. First, alteration of dopamine function could be implicated in anhedonia, a core feature of major depression. Dopaminergic systems, and more specifically mesolimbic and mesocortical dopaminergic systems, play a key role in mediating reward and motivation (Mogenson et al 1980; Schultz et al 2000; Wise and Rompre ` 1989). As such, a deficiency in this system could be involved in anhedonia, viewed as a failure of the reward systems (Willner 1995; Willner et al 1992). Second, alterations in dopamine transmission could be involved in the pathophysiology of specific features of depressive symptomatology. Given the role of dopamine in locomotor activities, a deficiency of the nigrostriatal dopamine system might be involved in psychomotor retardation. Given the role of dopamine in the pathophysiology of psychotic symptoms, an excess of dopaminergic transmission in mesolimbic systems might be present in patients with psychotic depression. Finally, dopaminergic systems might be differentially involved in unipolar and bipolar disorder, or in depressed patients with or without personality disorders. Given these multiple sources of potential confounds, we studied a relatively homogenous group of unipolar depressed subjects. Patients with psychotic symptoms were excluded, and, although psychomotor retardation was not

an exclusion criteria, the group of patients recruited in this study did not present significant levels of psychomotor retardation. We also carefully excluded subjects with a lifetime history of alcoholism and substance abuse, given the evidence for alteration of dopamine systems in these conditions (Hietala et al 1994; Volkow et al 1997a). Recent evidence has emerged suggesting that low D2 receptor availability might be associated with personality traits such as detachment (Breier et al 1998; Farde et al 1997) or other conditions, such as social phobia (Schneier et al 2000), that might be associated with vulnerability to depression. Thus, any comorbid Axis I diagnoses were excluded, and all Axis II diagnoses were reported.

Baseline [123I]IBZM V3
Significant differences in baseline [123I]IBZM V3 between depressed patients and control subjects were not observed. This result confirms the results of Klimke et al (1999) and Ebert et al (1996). In the Klimke et al (1999) study, 15 DSM-IV depressed subjects were compared to 17 healthy control subjects. Patients were medication free for at least 1 week but permitted to remain on benzodiazepines. Ebert et al (1996) studied 20 DSM-III-R depressed subjects compared to 10 control subjects. The subjects were drug-free for 6 months. Our results contrast with results of Shah et al (1997), who studied 14 DSM-III-R depressed subjects and 15 control subjects. Eight patients were on antidepressants and three on benzodiazepines at the time of the scan. Thus, medication effects could have confounded their results. The depressed group included patients with psychotic features and bipolar patients, another potentially important difference. The striatal to frontal [123I]IBZM ratio was found to be greater in depressed subjects, but this difference reached significance in the right but not the left striata. Results for combined (right and left) striata are not reported. In our study, we did not find any statistically significant differences between patients and control subjects when right and left striatal [123I]IBZM V3 were analyzed separately (data not shown). Finally, our results vary from DHaenen and Bossuyt (1994), who studied 21 DSM-III-R depressed subjects compared to 11 control subjects. Patients were medication free for only 7 days, except for benzodiazepines. The differences between our study and the studies of Shah et al (1997) and DHaenen and Bossuyt (1994) may be related to differences in the clinical profiles of the investigated population, or to sampling errors due to the fact that all studies, including ours, had a limited sample size. The [123I]IBZM specific binding measured in this study was obtained under the condition of true binding equilibrium, as achieved by a constant infusion of the radiotracer.

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Table 4. Studies of D2 Receptor Availability in Major Depression

Control subjects Study DHaenen and Bossuyt 1994 Ebert et al 1996c Shah et al 1997c Klimke et al 1999 This studyd
a b

Depressed subjects
a

n 11 10 15 17 10

mean SD

n 21 20 14 15 9

mean SDa 1.94 0.27 1.75 0.26 1.88 0.16 1.88 0.15 1.72 0.08

Effect sizeb 0.91 0.66 0.72 0.06 0.67

1.74 0.17 1.61 0.19 1.79 0.11 1.89 0.21 1.78 0.10

Values are [123I]IBZM striatal uptake over region of reference uptake (in our study, equal to V3 1). Effect size was calculated as mean in depressed subjects minus mean in control subjects divided by average SD. c Values calculated by averaging mean of left and right striata. d Measures performed under equilibrium conditions.

This contrasts with the four previous studies, in which the [123I]IBZM striatal to nonspecific ratios were measured following a single bolus injection of the radiotracer. Because, under these conditions, [123I]IBZM binding is not at equilibrium, these ratios might be confounded by factors unrelated to receptor density, such as striatal blood flow and peripheral metabolism. These factors may also account for the differences in results. Given that three out of five studies failed to find significant differences between patients with major depression and control subjects in striatal [123I]IBZM specific binding, a parsimonious conclusion is that major depression is not consistently associated with changes in striatal D2 receptor availability. Another way to interpret these combined results is to calculate the weighted effect size. Table 4 lists the mean and SD of [123I]IBZM striatal to reference region ratios in the five studies, as well as the effect size of the difference between patients and control subjects. Weighting by the number of cases included in each study, the average effect size is 0.38. Given an average coefficient of variation of 10% (Table 4), this effect size corresponds to a 4% increase in D2 receptor availability in patients with depression compared to control subjects. A study with 99 subjects per group would be required to detect such a small effect with a power of 0.8 and a significance level of 0.05. Furthermore, it is unclear how such a small difference in receptor density might play a significant role in the pathophysiology of the condition.

Amphetamine-Induced Change in V3
In this study, we evaluated the magnitude of the increase in synaptic dopamine elicited by an amphetamine challenge in unipolar patients with major depression and found no differences between patients and control subjects. The amphetamine-induced decrease in [123I]IBZM V3 measured in this group of depressed subjects (9.8 5.5%, n 9) was similar to the effect seen in the matched comparison group of healthy subjects (7.8 2.5%, n 10), as well as in other cohorts of healthy subjects of comparable demographic characteristics: 7.6 8.0%,

n 15 (Laruelle et al 1996); 7.1 6.3%, n 15 (Abi-Dargham et al 1998); and 8.2 7.7% (Kegeles et al 1999). Keeping in mind the limitations due to the relatively small sample size, this result suggests that depression is neither associated with a significant alteration in the pool of dopamine available to amphetamine, nor in the feedback mechanisms involved in the regulation of dopaminergic cell activities following amphetamine exposure (Kegeles et al 2000). The mechanism by which amphetamine increases synaptic dopamine is complex, but is ultimately associated with dopamine transporter (DAT) function, since it is currently postulated that amphetamine-induced dopamine release is mediated by reverse transport of dopamine by DAT (Sulzer et al 1993). Thus, differences in DAT density between depressed subjects and control subjects might represent a possible confounding factor. While LaasonenBalk et al (1999) reported higher striatal DAT binding in 15 moderately depressed outpatients compared to 18 control subjects, Malison et al (1998) found no significant difference in DAT between 15 depressed subjects compared to 15 control subjects, and Allard and Norlen (1997) found no alteration in DAT density in striatal samples from depressed suicide victims. Interestingly, patients with major depression presented a significant (but transient) improvement in depressive symptomatology following amphetamine. Because this improvement was not correlated with the changes in striatal dopamine transmission induced by the challenge ([123I]IBZM V3), this study fails to support a key role of striatal dopamine in this symptomatic improvement. This negative finding suggests that other neurotransmitter systems activated by amphetamine (such as noradrenergic or serotonergic systems) are involved in this response. On the other hand, this improvement could be related to stimulation of dopaminergic transmission in discrete components of the dopaminergic system, such as the mesolimbic dopaminergic system. The resolution of the SPECT camera does not allow separate analysis of the contributions of the mesolimbic and nigrostriatal dopaminergic systems.

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Potential Role of Stress

Using the same scan protocol, we previously reported a significant increase in amphetamine-induced dopamine release in untreated patients with schizophrenia (Laruelle et al 1996), a finding that was confirmed in a second cohort (Abi-Dargham et al 1998), as well as in first-episode, neuroleptic-naive schizophrenic patients (Laruelle et al 1999). Similar results have been reported by Breier et al (1997) using [11C]raclopride and a lower dose of amphetamine (0.2 mg/kg). An important question raised by these studies is whether the stress associated with psychiatric hospitalization and/or the scanning procedure might account for the excess dopamine release measured in patients with schizophrenia, because stress activates dopamine release (Deutch et al 1990; Kalivas and Duffy 1995). In this regard, the group of unipolar depressed subjects studied here resembled the group of schizophrenic patients reported previously. Subjects from both groups were experiencing a severe psychiatric episode, had recently been admitted to the unfamiliar environment of a research ward, and were untreated at the time of the scan. Baseline self-reported anxiety, as assessed by the analog scale, was higher in patients with schizophrenia (4.2 1.9) compared to their control subjects (3.0 1.9, p .01) (Laruelle et al 1999). Similarly, in this study, baseline anxiety was higher in depressed subjects (5.3 2.3) compared to their control subjects (2.8 1.6, p .02). Despite elevated burden of stress, patients with depression did not show elevated activation of the dopamine system by amphetamine. This finding supports the hypothesis that the increased V3 observed in patients with schizophrenia was not a nonspecific consequence of stressful conditions (although it could represent a specific interaction between stress and schizophrenia). It is also possible that a blunted amphetamine response in the depressive patients was masked by an augmented response due to stress; however, if this was the case, we should observe a correlation between baseline levels of anxiety and amphetamine-induced reduction in [123I]IBZM V3 within the depressed group. Because we did not observe such a correlation, this interpretation is not supported by the data. It is currently unclear if the increase in amphetamineinduced dopamine release observed in patients with schizophrenia is specific to this illness or to general clinical conditions associated with propensity to psychosis. The study of psychotic patients with other Axis I conditions is needed to clarify this issue. The finding that depression per se is not associated with alterations in amphetamine-induced dopamine release is an important first step in the clarification of this question.

Conclusion
This study did not detect alterations in D2 receptor availability and amphetamine-induced dopamine release in a small sample of nonpsychotic patients hospitalized with major depression. These results do not support a major involvement of D2 receptor transmission in the pathophysiology of major depression. Further studies of depressed subjects with psychotic symptoms or marked psychomotor retardation are warranted to further explore the role of dopamine transmission in major depression.

The authors would like to acknowledge the financial support of the Stanley Foundation and the Public Heath Service (NIMH 2 P30 MH46745-10, NIMH 1-K02-MH01603-01, NIH M01RR00645) and the excellent technical support of Richard Weiss, Ted Posniakov, Analia Arevelo, and Monique Richards.

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