Relationship of the serotonin transporter with prolactin response to meta-chlorophenylpiperazine in cocaine dependence

Available online at www.sciencedirect.com

JOURNAL OF PSYCHIATRIC RESEARCH

Journal of Psychiatric Research 42 (2008) 1213–1219

www.elsevier.com/locate/jpsychires

Relationship of the serotonin transporter with prolactin response to meta-chlorophenylpiperazine in cocaine dependence Ashwin A. Patkar a,*, Paolo Mannelli a, Kathleen Peindl a, Kevin P. Hill b, Li-Tzy Wu a, Tong Lee a, Cynthia Kuhn c a

Department of Psychiatry and Behavioral Sciences, Duke University, Durham, NC, United States b Department of Medicine, Yale University, New Haven, CT, United States c Department of Pharmacology, Duke University, Durham, NC, United States Received 20 February 2007; received in revised form 4 January 2008; accepted 9 January 2008

Abstract Background: Preclinical evidence indicates that exposure to cocaine influences the activity of the serotonin transporter (5-HTT) as well as several 5-HT receptor subtypes. However, little is known about the relationship between the 5-HTT and 5-HT receptors following cocaine exposure in humans. Objective: We examined the relationship between platelet 5-HTT, a presynaptic 5-HT measure, and prolactin (PRL) response to meta-chlorophenylpiperazine (m-CPP), a postsynaptic 5-HT receptor agonist in cocaine dependent individuals. Methods: Platelet [3H] paroxetine binding sites were assayed and the m-CPP challenge test was performed in 35 African American cocaine dependent individuals and 33 controls. Clinical measures included assessments of drug use severity and depression. Results: Cocaine subjects showed reduced Bmax of [3H] paroxetine (t = 4.67, p < 0.01) and blunted PRL response to m-CPP (F = 21.86, p < 0.01) compared to controls. There was a positive correlation between Bmax and delta PRL [peak baseline PRL] in cocaine subjects (r = 0.50, p < 0.01) but not in controls (r = 0.19). ANCOVA analyses showed that the cocaine subgroup with moderate and severe reduction in Bmax showed a greater blunting in PRL response compared to the subgroup with mild Bmax reductions (F = 9.44, p < .005). Multivariate regression models showed that the main effects as well as the interaction of Bmax and severity of cocaine use significantly contributed to impaired PRL response (F = 17.90, p < .001). Conclusions: Disturbances in serotonin transporter binding and postsynaptic 5-HT receptor function seem to be associated in cocaine-dependent subjects. Severity of cocaine use appears to mediate this relationship. Whether there is a causal association between the two measures, or cocaine has separate and independent pre- and postsynaptic effects needs to be clarified. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Serotonin transporter; 5-HT; m-CPP; Cocaine; Substance abuse

1. Introduction The serotonin (5-HT) system in the brain regulates several processes including reward, mood, locomotor activity, sexual behavior, and feeding. The contribution of 5-HT system to the effects of cocaine is demonstrated by experimental modifications of the 5-HT system, lesion studies

*

Corresponding author. Tel.: +1 919 668 3626; fax: +1 919 668 5618. E-mail address: [email protected] (A.A. Patkar).

0022-3956/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2008.01.008

and knockout experiments in animals (Hall et al., 2004; Burmeister et al., 2003; Walsh and Cunningham, 1997; Przegalinski et al., 2003). An important target for cocaine is the serotonin transporter (5-HTT). Among the various components of the 5-HT system, the serotonin transporter (5-HTT) is critically important in regulating the reuptake of 5-HT into the presynaptic neuron and the platelet, and serves as a target site for selective serotonin uptake inhibitors (SSRIs) and cocaine (Graham and Langer, 1992; Ramamoorthy et al., 1993; Schloss and Williams, 1998). Cocaine binds to the 5-HTT and blocks 5-HT uptake, producing an

1214

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219

increase in extracellular 5-HT (Castanon et al., 2000; Cunningham et al., 1996). This increased synaptic 5-HT, secondary to administration of cocaine, has downstream effects on several subtypes of postsynaptic 5-HT receptors (Muller and Huston, 2006). Together, the data from animal experiments suggest that modifications in 5-HT activity may significantly contribute to drug seeking, self-administration, and reinforcement. Changes in 5-HTT have been reported in neuroimaging, peripheral, and post-mortem studies of cocaine abusing individuals (Jacobsen et al., 2000; Little et al., 1998; Patkar et al., 2003a). It also appears that alterations in 5-HTT may have clinical implications. Studies have found that 5-HTT abnormalities may be linked to impulsivity and aggression and also predict poor treatment-outcome in cocaine abusers (Patkar et al., 2002, 2003b). Despite the importance of 5HTT in mediating the effects of cocaine, SSRIs have not been shown to produce a consistent therapeutic benefit in the treatment of cocaine abuse (Vocci and Elkashef, 2005). A separate line of research has found abnormalities in neuroendocrine and behavioral responses to pharmacological challenges with 5-HT agents in cocaine abusers. Most of the challenge paradigms in cocaine abusers have employed a meta-chlorophenylpiperazine (m-CPP), a mixed postsynaptic 5-HT receptor agonist/antagonist or d,l-fenfluramine, a 5-HT releaser and uptake inhibitor. The results of the challenge protocols have mostly demonstrated disturbances in neuroendocrine responses in cocaine abusers and also found a relationship of the disturbed response to traits such as impulsivity and aggression (Buydens-Branchey et al., 1997; Coccaro et al., 1995; Handelsman et al., 1998; Patkar et al., 2006). However few studies have systematically examined whether cocaine use per se influences the challenge response. In a well-designed study, Buydens-Branchey et al. (1998) employed sequential challenges with d,l-fenfluramine to demonstrate that the blunted PRL response normalized with extended abstinence in cocaine abusers. To date there are no published data examining the relationship between alterations in 5-HTT alterations and neuroendocrine responses to 5-HT agents in cocaine dependent individuals. Such data could help to understand the relationships and interactions between pre- and postsynaptic effects of cocaine in humans and their clinical implications. The aims of the study were twofold: First to determine the relationship between 5-HTT density (Bmax) and prolactin (PRL) response to m-CPP in cocaine-dependent individuals; and second to examine whether measures of drug use influence changes in 5-HTT and m-CPP responses. Because an association between severity of cocaine use and biological measures have been previously reported (Patkar et al., 2006; Kampman et al., 2002), we hypothesized that (a) cocaine exposure will be associated with pre- and postsynaptic alterations in 5-HT measures, (b) a greater impairment in presynaptic measures (Bmax) will be associated with greater impairment in postsynaptic measures (PRL) and (c) severity of cocaine use will be positively associated with both pre- and postsynaptic 5-HT impairments.

2. Methods 2.1. Subjects Thirty five subjects were recruited from patients attending a publicly funded, intensive outpatient cocaine treatment program. The protocol was approved by the Institutional Review Board of the University and written informed consent was obtained from subjects. The Structured Clinical Interview (SCID) for DSM- IV Axis I Disorders (First et al., 1997) was then administered to volunteers. Inclusion Criteria were: Axis I diagnosis of cocaine dependence, and at least 2 weeks of abstinence to minimize the effects of recent drug use on biological parameters. Exclusion criteria were: a diagnosis of schizophrenia, bipolar disorder or major depression, unstable medical disorders, pregnancy, or treatment with psychotropic medication in the previous 4 weeks. Subjects who used or abused other substances (except nicotine) were included only if their primary drug was cocaine. Urine drug screens and breath alcohol levels were obtained for all subjects. Because over 90% of the patients in the treatment program were African–American (AA), the study sample was restricted to AA subjects to represent the clinical population. Thirty-three AA controls were recruited from those responding to local advertisements. Consent and screening procedures were similar to those followed for cocaine subjects. Control subjects were excluded if they had a history of substance abuse or dependence (except nicotine), a major psychiatric disorder, an unstable medical illness, a positive urine drug screen or who were taking psychotropic medications in the previous 4 weeks. 2.2. Clinical assessments Severity of drug use in cocaine subjects was assessed using the Addiction Severity Index (ASI) (McLellan et al., 1992). The Addiction Severity Index (ASI) is a 30– 40 min structured interview assessing seven problem areas in substance dependent persons. For each domain a composite score that ranges from 0 (minimum) to 1 (maximum) is provided to assess the adequacy of functioning in these areas during the previous 30 days. Cigarette smoking was assessed using the Fagerstrom Test for Nicotine Dependence (FTND), a widely used and validated 6-item questionnaire to assess severity of smoking (Heatherton et al., 1991). Depressive symptoms were assessed by the Beck Depression Inventory (BDI), a 21-item self report (Beck and Steer, 1987). 2.3. Paroxetine binding assay Details of the assay procedure have been published in our earlier studies (Patkar et al., 2003c). Subjects were instructed to fast overnight and women were studied in the initial follicular phase of the menstrual cycle. Twenty milliliters of venous blood was collected in ethylendiamine-

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219

tetraacetic acid (EDTA) containing tubes and processed within 4 hours to harvest platelets. The platelet pellets were immediately frozen at 80 °C until they were assayed. Paroxetine binding was performed using the technique described by Ozaki et al. (1994) with minor modifications. Specific binding of tritiated paroxetine (Life Science Products Inc., Boston, MA) was determined at 6 different concentrations in the presence and absence of fluoxetine (Sigma Labs, St Louis, MO). The data were analyzed by means of the EBDA software (McPherson, 1985) to determine maximum number of transporter sites (Bmax) and affinity constant (Kd). The Bmax of paroxetine binding was expressed as fentomoles/mg while the Kd was expressed as nanomoles/L.

1215

Variance (ANCOVA) with repeated measures to assess the main effects of group and time and their interaction. Unequally distributed baseline variables between the two groups were used as covariates. The maximum change in PRL after m-CPP (DPRL) was calculated by subtracting the baseline PRL values from the peak PRL response following m-CPP (120 min). Correlations between DPRL and Bmax and Kd values were performed using Pearson product moment correlations. To examine the relationship between [3H] paroxetine binding and m-CPP response, cocaine subjects were divided into 3 subgroups based on Bmax values of [3H] paroxetine binding and the PRL responses were compared across the 3 subgroups groups using ANCOVA with repeated measures. All data are reported as Mean ± Standard Deviation unless specified otherwise.

2.4. The m-CPP challenge test 3. Results 2.4.1. Procedure Women were studied in the initial follicular phase of the menstrual cycle. This phase was defined clinically as the 10day period following the end of menstrual phase. Subjects were instructed to have a low monoamine diet for 3 days prior to the procedure. On test day, subjects were asked to come in the morning after an overnight fast. Subjects were not allowed to eat, drink or smoke during the procedure. Two baseline blood samples were drawn at 30-min intervals and 0.5 mg/kg of oral m-CPP (Sigma-Aldrich, St Louis, MO) was administered. Six serial blood samples were obtained for PRL levels at 30-min intervals. The time schedule for blood draw included the period of increases in neuroendocrine parameters seen in previous studies (Kahn et al., 1992). The 60, 120 and 180 min samples were also examined for m-CPP levels. 2.4.2. PRL determination Blood samples were centrifuged within 2 h of collection, the sera were separated and frozen at 70 °C. PRL was assayed using the Active PRL DSL-4500 Immunoradiometric Assay Kit (Diagnostic Systems Laboratories, Texas). This assay employs a standardized two-site immunoradiometric assay technique (Miles et al., 1974) to provide quantitative estimates of PRL in the serum. The assay sensitivity was 0.1 ng/ml and specificity tests carried out by the manufacturer indicate that other hormones were unlikely to cross-react with the PRL-antibody. The interassay and intraassay coefficients of variation were 11% and 4.0% respectively. 2.5. Statistical analyses Cocaine dependent subjects and controls were compared on clinical and demographic variables using two-tailed ttests and chi square tests as appropriate. Two-tailed t tests were used to compare Bmax and Kd values of [3H] paroxetine binding between the subjects and controls. PRL response curves following m-CPP challenge were compared between subjects and controls employing Analysis of Co-

3.1. Sample characteristics Data are presented on 35 AA cocaine dependent subjects and 33 AA controls who had participated in the mCPP procedure. The subjects [22 (63%) male] and controls [22 (66%) male] did not differ significantly in gender or age (subjects = 34.3 ± 4.7 years, controls = 33.1 ± 4.15 years). Nearly 85% of subjects fulfilled all 7 DSM-IV criteria of cocaine dependence. The remaining 15% fulfilled 3–6 DSM-IV criteria for cocaine dependence (minimum of 3 criteria required for diagnosis). Subjects were abstinent for 15.2 ± 0.8 days prior to the m-CPP procedure. Reflecting the patient population in clinical settings, a significant proportion of the cocaine-dependent subjects had additional substance abuse diagnoses. Nicotine was the most common drug (80%) followed by alcohol (35%), marijuana (25%) and opioids (11%). About 30% of controls smoked and none used alcohol during the study period. The mean FTND scores were significantly higher among cocaine subjects (4.62 ± 2.17) compared to controls (2.11 ± 2.36) (t = 6.78, df = 66, p < .01) indicating that cocaine patients were more severely nicotine dependent compared to controls. Despite excluding individuals with major depression, cocaine subjects displayed higher scores on the BDI (14.94 ± 10.21) than controls (6.57 ± 3.75) (t = 6.82, df = 66, p < .001). 3.2. [3H] paroxetine binding between cocaine subjects and controls Confirming our previous published findings (Patkar et al., 2003c), cocaine subjects (626.9 ± 202.9) had significantly lower Bmax values as compared to controls (837.9 ± 211.2) fmol/mg (t = 4.65, df = 66, p < .001). However, there was no significant difference in Kd values between patients (.23 ± .21) and controls (.26 ± .18) (t = .79, df = 66, p = 52) and the correlation between Bmax and Kd values was not statistically significant (r = 0.09, p = .29). The platelet counts did not differ significantly

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219

between the cocaine patients (209 ± 31 B/L) and control subjects (233 ± 39 B/L). 3.3. PRL response to m-CPP: cocaine subjects versus controls Baseline PRL levels (average of the 2 baseline PRL values measured from blood draws 30-min apart) were higher in cocaine patients (9.28 ± 4.05) than controls (7.40 ± 2.88) (F = 8.98, df = 66, p < .01). We compared the PRL response curves between subjects and controls using baseline PRL, BDI and FTND scores as covariates. As shown in Fig. 1, repeated measures ANCOVA demonstrated a significant blunting in the PRL response in cocaine subjects compared to controls (F(1, 65) = 21.93, p < .001). Maximum change in PRL (DPRL) values were significantly lower in cocaine patients compared to controls (F(1, 67) = 22.8, p < .001). There were no significant differences in peak m-CPP concentrations between cocaine patients (27.3 ± 6.1 ng/ml) and controls (25.1 ± 4.6 ng/ml) (t = 1.09). 3.4. Correlation between Bmax values of [3H]-paroxetine binding and maximum change in PRL (DPRL) We explored the relationship between Bmax values and DPRL levels separately in cocaine subjects and controls. As shown in Fig. 2, there was a significant positive correlation between Bmax and DPRL in cocaine subjects (r = .50, p < .01). However Bmax and DPRL were not correlated in controls (r = 0.193, p = .41). We then performed subgroup analyses to test the hypothesis that there are interindividual differences among cocaine subjects based on 5-HT differences. Although we expected DPRL to differ across subgroups differing in Bmax, we felt this analysis will help to clarify the distribution of DPRL among cocaine subjects. We initially examined the distribution of Bmax between cocaine subjects and controls. There was over one standard deviation (SD) (203) difference in mean Bmax values between cocaine patients (627) and controls (828). We therefore subdivided cocaine subjects into 3 groups: mild

1200

[3H] ParoxetineBinding (fmol/mg)

1216

1000

800

600

400

200

0 0

1

2

4

3

5

6

7

8

9

Delta Prolactin(ng/ml) r=0.504, p=0.002

Fig. 2. Relationship between [H3] paroxetine binding and delta prolactin in cocaine dependent subjects.

(Bmax values <1 SD below the mean, N = 11), moderate (Bmax values between 1 and 2 SD below the mean, N = 14), and severe (Bmax values > 2 Standard Deviations (SD) below the mean, N = 10). The PRL response to mCPP was compared between the 3 subgroups. The results are summarized in Fig. 3. The ANCOVA model with basal PRL and FTND scores as covariates as well as no covariates in the equation showed that the PRL response to m-CPP differed between the 3 subgroup [ANCOVA with covariates: (F(2, 31) = 9.44, p < .005, ANCOVA without covariates: F(2, 31) = 10.26, p < .005)]. Pairwise comparisons showed that the mild subgroup showed a significant difference from the moderate (mean difference = 2.18, C.I. = .71–3.65, p < .01) and the severe (mean difference = 2.96, C.I. = 1.35–4.57, p < .001) subgroups; however the moderate and severe subgroups did not differ from each other (mean difference = .78, p = .223). We performed multivariate linear regressions to determine the contribution of Bmax toward predicting DPRL taking into account severity of drug use and Beck Depres-

20 >2SD below mean 2-1SD below mean <1SD below mean

18

mean prolactin (ng/ml)

Prolactin Levels (ng/ml)

control n=33 cocaine n=35

16

14

12

10

16 14 12 10 8 6 4

8

2 6 0

30

60

90

120

150

180

minutes ANCOVA with repeated measures F(1,65)= 21.93, p<0.001

Fig. 1. Prolactin response after a m-CPP challenge in cocaine users compared to controls.

0

30

60

90

120

150

180

minutes after challenge F(2,28)=19.851; p=0.001

Fig. 3. Paroxetine binding in cocaine users and prolactin response after a m-CPP challenge.

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219

sion Scores. Because ASI composite scores include multiple domains, some of which may not be directly related to drug use (e.g. employment, legal scores), we only included ASIdrug scores as a measure of severity of cocaine use. Bmax values, ASI-drug scores, basal PRL values and BDI scores were entered into the model. The main effects model was significant (F = 17.90, p < .001) and contributed 34.8% of the variance to the model. Both paroxetine binding (t = 3.12, p < .01) and severity of cocaine use (t = 3.61, p < .01) were significantly associated with a blunted prolactin response and the interaction between the two was also significant (t = 3.64, p < .01). Due to the possibility of gender differences in PRL response (New et al., 2004), we performed separate genderspecific analyses. DPRL values were not significantly different among men (n = 22) (5.81 ± 3.19) and women (n = 13) (6.85 ± 2.64) among cocaine patients (F(1, 34) = 2.42, p = .10) or controls (men (n = 22) = 8.97 ± 2.88, women (n = 11) = 9.28 ± 3.34, F(1,32) = .08, p = .89). 4. Discussion Although several human studies have reported disturbances in 5-HT function in cocaine abusers (BuydensBranchey et al., 1997; Jacobsen et al., 2000; Patkar et al., 2004), the findings have focused on a single measure of pre- or postsynaptic 5-HT function. This study combined assessments of presynaptic (platelet [3H]-paroxetine binding) and postsynaptic (m-CPP challenge response) functions along with clinical evaluations. There were three key findings: first, cocaine-dependent subjects showed significantly lower densities of platelet 5-HT transporter and greater blunting in prolactin response to m-CPP compared to controls. Second, there was a positive correlation between platelet 5-HT transporter densities and extent of blunted prolactin response in cocaine subjects, but not controls. Third, both 5-HT transporter densities and severity of cocaine use contributed to the blunted prolactin response in cocaine subjects. The reduction in 5-HTT densities in cocaine abusers is consistent with our previous findings from a separate data set (Patkar et al., 2003c); with reports of reduced 5-HTT in the brains of substance abusers (Heinz et al., 2002; Sekine et al., 2006) and also with investigations of various platelet 5-HT markers among psychiatric disorders characterized by poor impulse control (Brown et al., 1989; Marazziti and Conti, 1991; Coccaro et al., 1996). We found that the PRL response following m-CPP was significantly more blunted in cocaine patients than controls, even after a minimum of 2 weeks of abstinence, supporting previous findings using similar protocols (Buydens-Branchey et al., 1997). The PRL stimulating effects of m-CPP have been shown to be linked to its 5-HT effects, in particular its agonist actions at the postsynaptic 5-HT2C receptors (Murphy et al., 1991; Sleight et al., 1995; Thomas et al., 1996). Human studies have also indicated that doses of m-CPP used in challenge paradigms did not significantly alter

1217

dopamine or noradrenaline activity (Kahn et al., 1992; Silverstone et al., 1994). Therefore it may be reasonable to attribute the blunted PRL response observed in cocaine abusers to disturbances in 5-HT function, possibly postsynaptic 5-HT2C receptor subsensitivity. The principal finding from the study was the strong association between reduction in 5HTT densities and blunting in the PRL response that was only observed in cocaine dependent subjects. Essentially, the more severe the reduction in 5HTT, the greater was the impairment in PRL. This suggests that the alterations in m-CPP response may reflect downstream effects of changes in presynaptic serotonin transporter function following cocaine exposure in humans, consistent with preclinical data (Cunningham et al., 1992; Jacobsen et al., 2000). Because m-CPP has high affinities for the post synaptic 5-HT2C, 5-HT2A and 5-HT3 receptors, it is possible that the mechanism for the blunted neuroendocrine effects of m-CPP in cocaine abusers may also involve changes in post synaptic 5HT receptors. Our data also indicate that the influence of m-CPP on the serotonin transporter may be of importance for the functional effects of the drug. Confirming that the effects of m-CPP on the serotonin transporter may be of significance not only in rats but also in humans, m-CPP was recently reported to display affinity for the human serotonin transporter (Baumann and Rothman, 1995). Supporting our previous work (Patkar et al., 2006), we found that severity of cocaine use was also a strong contributor to DPRL and there was a significant interaction between clinical measures of drug severity and Bmax densities as determinants of DPRL. Our findings together with preclinical evidence that cocaine administration directs disrupts 5-HT mechanisms modulating neuroendocrine system (Baumann et al., 1995; Parsons et al., 1996), suggest that chronic cocaine use contributes to disturbances in PRL response to m-CPP in humans. It must be noted though that m-CPP has a modest dopaminergic activity. Preclinical studies have shown that the effect of m-CPP on extracellular dopamine levels is modest, as compared to the effect on serotonin. Eriksson et al. (1999) found that whereas the 5-HT concentrations after m-CPP administration were always >600% of baseline, dopamine concentrations increased to 120–170% of baseline only. The functional significance of such a weak increase in dopamine levels may be questioned; however, given the putative involvement of dopamine in reward and neuroendocrine mechanisms, the possibility that a dopamine-releasing effect of m-CPP may also play a role in the changes in PRL cannot be excluded. Investigations of the possible clinical correlates of 5-HT disturbances have provided some promising findings. Several studies have found impulsive and aggressive behaviors to be associated with deficits in central 5-HT function across a range of psychiatric disorders (Coccaro et al., 1996; Virkunnen et al., 1994). Alterations in the serotonin transporter have been related to behavioral characteristics such as impulsivity, aggression and self-harm among individuals with substance abuse and depression (Yudofsky

1218

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219

et al., 1993; Bligh-Glover et al., 2000). Along with these well- characterized clinical measures, the present study indicates that a more severe history of drug addiction could serve as a clinical marker for 5-HT disturbances in cocaine dependent subjects. Certain limitations of the study deserve comment. Ideally each subject should have undergone a placebo-controlled challenge on separate days. The single administration challenge paradigm we used was motivated by the logistical constraints of an outpatient sample and has been employed elsewhere (New et al., 2004). However, this design did not appear to compromise the data because the differences were robust, and personnel blinded to the clinical information performed the PRL estimations. Another limitation is that, reflecting the ‘real-world’ clinical setting, cocaine-dependent individuals had a history of using variety of other substances. While we controlled for the effects of tobacco smoking and monitored for abstinence from drug and alcohol for 2 weeks, we cannot exclude the possibility that use of other substances could have affected the findings. Finally, we did not systematically screen for antisocial personality disorder, a variable that has been associated with 5-HT disturbances. In conclusion, disturbances in serotonin transporter binding and post-synaptic 5-HT receptor function seem to be associated in cocaine-dependent subjects. Severity of cocaine use appears to mediate this relationship. Whether there is a causal association between the two measures, or cocaine has separate and independent pre- and post-synaptic effects needs to be clarified. Conflict of interest Drs. Patkar and Mannelli have received research support from GlaxoSmithKline, BristolMyers Squibb, Forest Pharmaceuticals and Pfizer. Dr. Patkar has served on Speakers Bureau for BristolMyersSquibb. Other authors disclose no conflicts of interest. Contributors Ashwin Patkar MD, designed the study, was the Principal Investigator and wrote the initial draft of the manuscript. Paolo Mannelli MD, and Kevin Hill MD, were co-investigators on the study and assisted with implementation of the study. Kathleen Peindl PhD, was the lead statistician on the study. Li-Tzy Wu PhD, helped with data interpretation. Cynthia Kuhn PhD, and Tong Lee MD, PhD, assisted with the laboratory assays, interpretation of results and revising the draft manuscript. All authors contributed substantially to the final version of the manuscript. Funding source This research was supported in part by grants DA00340 and DA015504 to AAP from the National Institute on

Drug Abuse (NIDA). NIDA had no further role in study design, in the collection, analysis and interpretation of data; in the writing of the report, and in the decision to submit the paper for publication. Acknowledgement This research was supported in part by grants DA00340 and DA015504 to AAP from the National Institute on Drug Abuse. References Baumann MH, Rothman RB. Repeated cocaine administration reduced 5HT1A-mediated prolactin secretion in rats. Neuroscience Letters 1995;193:9–12. Baumann MH, Mash DC, Staley JK. The serotonin agonist m-chlorophenylpiperazine (mCPP) binds to serotonin transporter sites in human brain. Neuroreport 1995;6:2150–2. Beck A, Steer R. Beck depression inventory. The Psychological Corporation. San Antonio, TX: Harcourt, Brace, and Jovanovich; 1987. Bligh-Glover W, Kolli TN, Shapiro-Kulnane L, Dilley GE, Friedman L, Balraj E, et al. The serotonin transporter in the midbrain of suicide victims with major depression. Biological Psychiatry 2000;47:1015–24. Brown CS, Kent TA, Bryant SG, Gevedon RM, Campbell JL, Felthous AR, et al. Blood platelet uptake of serotonin in episodic aggression. Psychiatry Research 1989;27:5–12. Burmeister J, Lungren E, Neiswander J. Effects of fluoxetine and dfenfluramine on cocaine-seeking behavior in rats. Psychopharmacology 2003;168:146–54. Buydens-Branchey L, Branchey M, Fergeson P, Hudson J, McKernin C. The meta-chlorophenylpiperazine challenge test in cocaine addicts: hormonal and psychological responses. Biological Psychiatry 1997;41: 1071–86. Buydens-Branchey L, Branchey M, Hudson J, Rothman M, Fergeson P, McKernin C. Effect of fenfluramine challenge on cocaine craving in addicted male users. The American Journal on Addictions 1998;7: 142–55. Castanon N, Scearce-Levie K, Lucas J, Rocha B, Hen R. Modulation of the effects of cocaine by 5-HT1B receptors: a comparison of knockouts and antagonists. Pharmacology, Biochemistry, and Behavior 2000;67: 559–66. Coccaro E, Kavoussi R, Hauger R. The meta-chlorophenylpiperazine challenge test in cocaine addicts: hormonal and psychological responses. International Clinical Psychopharmacology 1995;10:177–9. Coccaro E, Kavoussi R, Sheline Y, Berman M, Csernansky J. Impulsive aggression in personality disorder correlates with tritiated paroxetine binding in the platelet. Archives of General Psychiatry 1996;53:531–6. Cunningham KA, Paris JM, Goeders NE. Chronic cocaine enhances serotonin autoregulation and serotonin uptake binding. Synapse 1992;11:112–23. Cunningham K, Bradberry C, Chang A, Reith M. The role of serotonin in the actions of psychostimulants, molecular and pharmacological analyses. Behavioural Brain Research 1996;73:93–102. Eriksson E, Engberg G, Bing O, Nissbrandt H. Effects of mCPP on the extracellular concentrations of serotonin and dopamine in rat brain. Neuropharmacology 199;20:287–96. First M, Gibbon M, Spitzer R, Williams J. Structured clinical interview for DSM-IV Axis I disorders (SCID-IV): clinician version. Washington, DC: American Psychiatric Press; 1997. Graham D, Langer SZ. Advances in sodium-ion coupled biogenic amine transporters. Life Sciences 1992;51:631–45. Hall FS, Sora I, Drgonova J, Li XF, Goeb M, Uhl GR. Molecular mechanisms underlying the rewarding effects of cocaine. Annals of the New York Academy of Sciences 2004;1025:47–56.

A.A. Patkar et al. / Journal of Psychiatric Research 42 (2008) 1213–1219 Handelsman L, Kahn R, Sturiano C, Rinaldi P, Gabriel S, Schmeidler J, et al. Hostility is associated with a heightened prolactin response to meta-chlorophenylpiperazine in abstinent cocaine addicts. Psychiatry Research 1998;80:1–12. Heatherton T, Kozlowski L, Frecker R. The fagerstrom test for nicotine dependence: a revision of the Fagerstrom tolerance questionnaire. The British Journal of Addiction to Alcohol and Other Drugs 1991;86: 1119–27. Heinz A, Jones DW, Bissette G, Hommer D, Ragan P, Knable M, et al. Relationship between cortisol and serotonin metabolites and transporters in alcoholism. Pharmacopsychiatry 2002;35:127–34. Jacobsen L, Staley J, Malison R, Zoghbi S, Seibyl J, Kosten T, et al. Elevated central serotonin transporter binding availability in acutely abstinent cocaine-dependent patients. The American Journal of Psychiatry 2000;157:1134–40. Kahn R, Knott P, Gabriel S, DuMont K, Mastroianni L, Davidson M. Effect of m-chlorophenylpiperazine on plasma homovanillic acid concentrations in healthy subjects. Biological Psychiatry 1992;32: 1055–61. Kampman KM, Volpicelli JR, Mulvaney F, Rukstalis M, Alterman A, Pettinati H, et al. Cocaine withdrawal severity and urine toxicology results from treatment entry predict outcome in medication trials for cocaine dependence. Addictive Behaviors 2002;27:251–60. Little KY, McLaughlin DP, Zhang L, McFinton PR, Dalack GW, Cook Jr EH, et al. Brain dopamine transporter messenger RNA and binding sites in cocaine users: a postmortem study. Archives of General Psychiatry 1998;55:793–9. Marazziti D, Conti L. Aggression and suicide attempts: preliminary data. European Neuropsychopharmacoly 1991;1:169–72. McLellan A, Kushner H, Metzger D, Peters R, Smith I, Grissom G, et al. The fifth edition of the addiction severity index. Journal of Substance Abuse Treatment 1992;9:199–213. McPherson GA. Kinetic, EBDA, LIGAND. Lowry: a collection of radioligand binding analysis programs. Biosoft: Cambridge; 1985. Miles L, Lipschitz D, Bieber C, Cook J. Measurement of serum ferritin by a 2-site immunoradiometric assay. Analytical Biochemistry 1974;61: 209–24. Muller CP, Huston JP. Determining the region-specific contributions of 5HT receptors to the psychostimulant effects of cocaine. Trends in Pharmacological Sciences 2006;27:105–12. Murphy D, Lesch K, Aulakh C, Pigott T. Serotonin-selective arylpiperazines with neuroendocrine, behavioral, temperature and cardiovascular effects in humans. Pharmacological Reviews 1991;43:47–55. New A, Trestman R, Mitropoulou V, Goodman M, Koenigsberg H, Silverman J, et al. Low prolactin response to fenfluramine in impulsive aggression. Journal of Psychiatric Research 2004;38:223–30. Ozaki N, Rosenthal NE, Mazzola P, Chiveh CC, Hardin T, GarciaBorregeuro D, et al. Platelet [3H]paroxetine binding, 5-HT-stimulated Ca2+ response, and 5-HT content in winter seasonal affective disorder. Biological Psychiatry 1994;36:458–66. Parsons LH, Koob GF, Weiss F. Extracellular serotonin is decreased in the nucleus accumbens during withdrawal from cocaine self-administration. Behavioural Brain Research 1996;73:225–8. Patkar AA, Thornton CC, Berrettini WH, Gottheil E, Weinstein SP, Hill KP. Predicting treatment-outcome in cocaine dependence from admission urine drug screen and peripheral serotonergic measures. Journal of Substance Abuse Treatment 2002;23:33–40.

1219

Patkar AA, Gottheil E, Berrettini WH, Hill KP, Thornton CC, Weinstein SP. Relationship between platelet serotonin uptake sites and measures of impulsivity, aggression, and craving among African–American cocaine abusers. The American Journal on Addictions 2003a;12:432–7. Patkar AA, Gottheil E, Berrettini WH, Thornton CC, Hill KP, Weinstein SP. Relationship between platelet serotonin uptake sites and treatment outcome among African–American cocaine dependent individuals. The Journal of Addictive Diseases 2003b;22:79–92. Patkar AA, Berrettini WH, Lundy A, Murray H, Hill KP, Vergare MJ, et al. Seasonal variations in the binding of [3H] paroxetine to platelet serotonin transporter sites in African–American cocaine-dependent patients and healthy volunteers. Human Psychopharmacology Clinical and Experimental 2003c;18:103–11. Patkar AA, Berrettini WH, Mannelli P, Gopalakrishnan R, Hoehe MR, Bilal L, et al. Relationship between serotonin transporter gene polymorphisms and platelet serotonin transporter sites among African–American cocaine-dependent individuals and healthy volunteers. Psychiatr Genetics 2004;14:25–32. Patkar AA, Mannelli P, Peindl K, Hill KP, Gopalakrishnan R, Berrettini WH. Relationship of disinhibition and aggression to blunted prolactin response to meta-chlorophenylpiperazine in cocaine-dependent patients. Psychopharmacology (Berl) 2006;185:123–32. Przegalinski E, Czepiel K, Nowak E, Dlaboga D, Filip M. Withdrawal from chronic cocaine up-regulates 5-HT1B receptors in the rat brain. Neuroscience Letters 2003;351:169–72. Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T, Chang AS, et al. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proceedings of the National Academy of Sciences of the United States of America 1993;90:2542–6. Schloss P, Williams DC. The serotonin transporter: a primary target for antidepressant drugs. Journal of Psychopharmacology 1998;12:115–21. Sekine Y, Ouchi Y, Takei N, Yoshikawa E, Nakamura K, Futatsubashi M, et al. Brain serotonin transporter density and aggression in abstinent methamphetamine abusers. Archives of General Psychiatry 2006;63:90–100. Silverstone P, Rue J, Franklin M, Hallis K, Camplin G, Laver D, et al. The effects of administration of mCPP on psychological, cognitive, cardiovascular, hormonal and MHPG measurements in human volunteers. International Clinical Psychopharmacology 1994;9:173–8. Sleight A, Carolo C, Petit N, Zwingelstein C, Bourson A. Identification of 5-hydroxytryptamine7 receptor binding sites in rat hypothalamus: sensitivity to chronic antidepressant treatment. Molecular Pharmacology 1995;47:99–103. Thomas D, Gager T, Holland V, Brown A, Wood M. m-Chlorophenylpiperazine (mCPP) is an antagonist at the cloned human 5-HT2B receptor. Neuroreport 1996;17:1457–60. Virkunnen M, Kallio E, Rawlings R, Tokola R, Poland RE, Guidotti A, et al. Archives of General Psychiatry 1994;51:28–33. Vocci FJ, Elkashef A. Pharmacotherapy and other treatments for cocaine abuse and dependence. Current Opinion in Psychiatry 2005;18:265–70. Walsh S, Cunningham K. Serotonergic mechanisms involved in the discriminative stimulus, reinforcing and subjective effects of cocaine. Psychopharmacology 1997;130:41–58. Yudofsky SC, Silver JM, Hales RE. Cocaine and aggressive behavior: neurobiological and clinical perspectives. Bulletin of the Menninger Clinic 1993;57:218–26.