Antipsychotic Drug-Induced Weight Gain Mediated by Histamine H1 Receptor-Linked Activation of Hypothalamic AMP-Kinase

Antipsychotic drug-induced weight gain mediated by histamine H1 receptor-linked activation of hypothalamic AMP-kinase

Sangwon F. Kim*, Alex S. Huang*, Adele M. Snowman*, Cory Teuscher†, and Solomon H. Snyder*‡§¶

*The Solomon H. Snyder Department of Neuroscience, Departments of ‡Pharmacology and Molecular Sciences and §Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and †Departments of Medicine and Pathology, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405

Contributed by Solomon H. Snyder, December 21, 2006 (sent for review November 28, 2006)

The atypical antipsychotic drugs (AAPDs) have markedly enhanced after treatment with clozapine or olanzapine (Fig. 1 C and D). the treatment of schizophrenias but their use has been hindered by The drug actions are potent and substantial with EC50 values for the major weight gain elicited by some AAPDs. We report that both of Ϸ10 nM and with 6- and 3.5-fold maximal increases, orexigenic AAPDs potently and selectively activate hypothalamic respectively, with clozapine and olanzapine (Fig. 1 E–H). AMP-kinase, an action abolished in mice with deletion of histamine Clozapine also potently and selectively augments hypotha- H1 receptors. These ﬁndings may afford a means of developing lamic AMPK in intact animals. As little as 1 mg/kg of clozapine more effective therapeutic agents and provide insight into the markedly stimulates levels of phospho-AMPK (Fig. 2A) as well hypothalamic regulation of food intake. as AMPK catalytic activity, with 5 mg/kg producing a 3.5-fold augmentation of activity (Fig. 2B). The increase of phospho- atypical antipsychotic drugs ͉ obesity ͉ hypothalamus AMPK and AMPK catalytic activity is relatively selective for the hypothalamus, because clozapine (1 mg/kg) fails to increase he antipsychotic actions of classic neuroleptics revolution- phospho-AMPK levels in the cerebellum and liver [see support- Tized the therapy of schizophrenia, but their use has been ing information (SI) Fig. 5 A and B], and AMPK catalytic activity impeded by side effects such as extrapyramidal symptoms, is not affected in the cerebral cortex or cerebellum by clozapine tardive dyskinesia, a high incidence of nonresponders, and the (1 mg/kg) (Fig. 2C). At 5 mg/kg, clozapine elicits a 20% increase failure of negative symptoms such as apathy to respond. The in cortical AMPK activity, much less than the quadrupling of atypical antipsychotic drugs (AAPDs), pioneered by clozapine, hypothalamic AMPK activity, whereas no increase is apparent in represent an important advance in improving negative symp- the cerebellum (Fig. 2D). The effect of clozapine is maximal 3 h toms, benefiting patients who do not respond to the typical after drug administration and gradually decreases to basal levels drugs, and displaying fewer side effects (1–5). A major limitation in 24 h (SI Fig. 5F). of AAPDs is pronounced weight gain, predominantly mediated Kahn and colleagues (20) reported that the anorexigenic by increased food intake (6–10). Weight gain elicited by AAPDs peptide leptin reduces hypothalamic AMPK activity, which we is primarily related to increased food intake, although there may confirm. Clozapine reverses reductions in hypothalamic phos- also be metabolic alterations (11–13). pho-AMPK elicited by leptin (Fig. 3A) and insulin (20) (SI Fig. To directly address central systems that mediate appetite and 6). In intact mice, leptin (3 mg/kg) reduces hypothalamic phos- weight gain, we have explored hypothalamic AMPK phosphor- pho-AMPK (Fig. 3B) and catalytic activity (Fig. 3C), and ylation, which activates the enzyme (14, 15). In the periphery, clozapine reverses these actions. AMPK activation is associated with decreased lipid formation, The arcuate and paraventricular hypothalamic nuclei display because AMPK phosphorylates acetyl-CoA carboxylase (ACC) the greatest alterations of AMPK activity in response to feeding inhibiting the generation of malonyl-CoA. Malonyl-CoA is a stimuli (20). In immunohistochemical experiments phospho- substrate for fatty acid synthase so that inhibition of ACC AMPK is selectively augmented in these two nuclei with cloza- diminishes formation of fatty acids and lipid (14–16). In the pine (1 and 5 mg/kg), whereas much lesser effects are evident in hypothalamus, AMPK acts in a seemingly reciprocal fashion to the cerebral cortex (SI Fig. 7 A and B). By contrast, ziprasidone regulate food intake (15, 17–19). Kahn and collaborators (20) fails to alter phospho-AMPK in the paraventricular nucleus (SI showed that AMPK activity in the arcuate and paraventricular Fig. 8). hypothalamic nuclei is inhibited by anorexigenic agents such as We wondered whether the influence of AAPDs on hypotha- leptin and augmented by the orexigenic agouti-related protein lamic AMPK is secondary to actions of the drugs on specific (AGRP) (20). neuropeptide receptors that have been implicated in appetite We now show that orexigenic AAPDs selectively and potently regulation. Clozapine and olanzapine (10 and 100 nM) fail to stimulate hypothalamic AMPK, which has been linked to the influence ligand binding to receptors for leptin, ␣-MSH, and regulation of food intake (20), and reverse the actions of the neuropeptide Y (data not shown). anorexigenic hormone leptin. This action involves the histamine H1 receptor (H1R), because clozapine augmentation of AMPK is tm1Wat abolished in B6.129P-Hrh1 (H1RKO) mice, and orexigenic Author contributions: S.F.K. designed research; S.F.K., A.S.H., and A.M.S. performed re- potencies of neuroleptics correlate with their affinities for H1R. search; C.T. contributed new reagents/analytic tools; S.F.K., A.S.H., and S.H.S. analyzed data; and S.F.K. and S.H.S. wrote the paper. Results The authors declare no conﬂict of interest. In hypothalamic slices, clozapine and olanzapine markedly en- Abbreviations: AAPD, atypical antipsychotic drug; H1R, H1 receptor. hance levels of phospho-AMPK, and quetiapine, which is also See Commentary on page 3019. orexigenic, produces similar effects (Fig. 1A). However, risperi- ¶To whom correspondence should be addressed. E-mail: [email protected]. done, ziprasidone, haloperidol, and aripiprazole, which are much This article contains supporting information online at www.pnas.org/cgi/content/full/ less orexigenic (Table 1), fail to stimulate AMPK (Fig. 1B). 0611417104/DC1. Increased AMPK phosphorylation is observed as early as 5 min © 2007 by The National Academy of Sciences of the USA

3456–3459 ͉ PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611417104 Downloaded by guest on October 1, 2021 CTL 1mg/kg CLO A B idol A ine AMPK-P SEE COMMENTARY CTL Clozap HaloperAripipirazol CTL ClozapineOlanzapineQuetiapineRisperidoneZiprasidone AMPK AMPK-P AMPK-P CTL 5mg/kg CLO AMPK AMPK AMPK-P Clozapine 500nM Olanzapine 500nM C D AMPK 0 5 10 20 30 (min) 0 5 10 20 30 (min) * * P<0.001 AMPK-P AMPK-P B 10

AMPK AMPK 8

6 * P<0.01 Clozapine Olanzapine E F 50nM lo 0 5 50 500 5000 (nM) 0 5 50 500 5000 C (nM) 4

AMPK-P AMPK-P 2 0 AMPK AMPK (pmole/min/mg) -AMPK activity Saline 1mg/kg 5mg/kg α2 Clozapine G H C 7 5 Saline 7 6 1mg/kg CLO 6 5 4 5 4 4 3 * 3 3 2 2 2 1 1 -AMPK activity (pmole/min/mg) AMPK activity (pmole/min/mg) AMPK activity 0 0 1 α2− α2− 1 10 100 1000 10000 1 10 100 1000 10000

Clozapine (nM) Olanzapine (pmole/min/mg) -AMPK activity 0

α2 Hypothalamus Cortex Cerebellum Fig. 1. AAPDs activate AMPK in hypothalamic slices. (A and B) Hypothalamic slices were incubated in oxygenated artiﬁcial cerebrospinal ﬂuid buffer with D * Saline 500 nM drugs for 30 min. Phospho-AMPK (AMPK-P) and total AMPK were 8 CLO 5mg/Kg MEDICAL SCIENCES detected by Western blotting. (C and D) Hypothalamic slices were incubated ** with 500 nM clozapine or olanzapine for various times as indicated. Phospho- 6 AMPK and total AMPK were detected by Western blotting. (E and F) Hypo- thalamic slices were incubated with various concentrations of clozapine or 4 olanzapine for 30 min. (G and H) ␣2-APMK enzymatic activity was measured with SAMS peptides as a substrate. 2

0 -AMPK activity (pmole/min/mg) -AMPK activity Hypothalamus Cortex Cerebellum

Relative potencies of AAPDs in blocking H1R have been α2 reported to correlate with their orexigenic potencies (21, 22), Fig. 2. AAPDs activate AMPK in intact animals. (A and B) Mice received which we confirm (Table 1). Moreover, in hypothalamic slices, clozapine (1 or 5 mg/kg) and were killed at 3 h. Hypothalami were removed the H1R antagonist triprolidine stimulates phospho-AMPK to and tissue lysates analyzed for phospho-AMPK or ␣2-AMPK activity. (C and D) the same extent as clozapine both in hypothalamic slices (Fig. Mice received clozapine (1 or 5 mg/kg) and were killed at 3 h. Various parts of brain were isolated, and ␣2-AMPK activity was assayed. Bars represent the mean Ϯ SE of three independent lysates performed in triplicate. *, Student’s Table 1. Neuroleptic afﬁnities for the H1R correlate t test (n ϭ 5). with orexigenic actions

Drugs IC50, nM Orexigenic effects 4A) and in intact animals (SI Fig. 9). Conversely, histamine Clozapine 9 ϩϩϩϩ decreases phospho-AMPK with reversal of this effect by cloza- Olanzapine 13 ϩϩϩ pine (Fig. 4B). To explore whether augmentation of phospho- Quetiapine 40 ϩϩ AMPK by drugs stems from H1R blockade, we administered Risperidone 80 ϩ/Ϫ clozapine to H1R knockout mice. Whereas the drug elicits a Ziprasidone 150 Ϫ quadrupling of phospho-AMPK in wild-type mice, no effect is Haloperidol 2,000ϩϪevident in H1R knockout animals (Fig. 4 C and D). Aripipirazole 3,000ϩϪ Discussion Receptor binding was assayed by using rat brain membranes incubated Our findings indicate that the appetite stimulation–weight gain with [3H]mepyramine and 12 concentrations of drugs ranging from 30 pM to 10 ␮ M, in triplicate. Data are means of three independent determinations associated with AAPDs is mediated by activation of hypotha- that varied Ͻ10%. Orexigenic action of drugs was obtained from published lamic AMPK linked to blockade of the histamine H1R. AMPK literature, indicating reproducible differences among AAPDs in eliciting stimulation parallels the orexigenic actions of the drugs, with weight gain when administered at comparable therapeutic doses (5, 8, 14). clozapine and olanzapine producing the most marked effects.

Kim et al. PNAS ͉ February 27, 2007 ͉ vol. 104 ͉ no. 9 ͉ 3457 Downloaded by guest on October 1, 2021 A Clozapine 50nM 0 50 500 5,000 0 50 500 5,000 C 7 * AMPK-P 6 * 5 AMPK 4 3 Hypothalamus Cortex B 2 Leptin (3mg/kg) - - - + - + + + 1

Clozapine (5mg/Kg) ---+ + - + - + (pmole/min/mg) -AMPK activity

α2 0 AMPK-P Leptin (3mg/kg) - - ++ Clozapine (5mg/Kg) - + - + AMPK

Fig. 3. Clozapine reverses effects of leptin on phospho-AMPK in hypothalamic slices and in intact animals. (A) Hypothalamic slices were incubated with various concentrations of leptin in the absence or presence of 50 nM clozapine for 30 min. Phospho-AMPK and total AMPK were detected by Western blotting. (B and C) Mice received leptin (3 mg/kg), followed at1hbyclozapine (5 mg/kg) and were killed at 3 h. Phospho-AMPK and total AMPK were detected by Western blotting, and ␣2-AMPK activity was assayed. Bars represent the mean Ϯ SE of three independent lysates performed in triplicate. *, P Ͻ 0.001, Student’s t test (n ϭ 5).

The drug actions are very potent, with substantial effects evident between obesity and D2 receptor occupancy in humans (31). at 5 nM concentration. They are selective, with effects restricted Moreover, blocking D2 sites in the lateral hypothalamus in- largely to the arcuate and paraventricular nuclei of the hypo- creases feeding behavior in rodents (32). Some anorectic drugs thalamus. Orexigenic potencies of AAPDs parallel their affin- act by augmenting synaptic levels of norepinephrine in the ities for histamine H1Rs, and stimulation by AAPDs of AMPK hypothalamus (33), and affinity of drugs for noradrenergic ␣-2 is lost in H1R-deleted mice. These findings are in accord with receptors correlates with orexigenic potency (34). Although studies implicating central histamine (23, 24) and AMPK (20) in these correlations suggest some role for serotonin, norepineph- weight control as well as the orexigenic role of the paraventricu- rine, and dopamine in obesity, the present study establishes lar and arcuate nuclei (25, 26). Moreover, mice, like humans, definitively that orexigenic AAPDs act via histamine H1 recep- manifest weight gain in response to AAPDs, although inhibition tors and AMPK. Our findings predict that H1R-deleted mice of locomotor activity sometimes impairs characterization of should be resistant to the orexigenic actions of AAPDs and that orexigenic actions (27). H1 antihistamines should be orexigenic. In rats (35) and humans Numerous mechanisms have been advanced to explain the (36), H1 antihistamines have been reported to be orexigenic. orexigenic influences of AAPDs. Because therapeutic actions of Because these drugs typically are used sporadically and in the drugs have been linked to serotonin receptors, these have substantially lower doses than AAPDs, effects on weight are less also been hypothesized to mediate orexigenic effects. Thus, prominent for H1 antihistamines. agonists at 5HT2C receptors, such as fenfluramine and m- Weight gain elicited by AAPDs can be massive and associated chlorophenylpiperazine, are anorexigenic (28), whereas mice with the ‘‘metabolic syndrome’’ leading to diabetes (5, 7, 37, 38). with targeted deletion of 5HT2C receptors are obese (29). Thus, the orexigenic actions of AAPDs, especially olanzapine Orexigenic potencies of neuroleptics correlate significantly with and clozapine, have precluded their use in large numbers of affinity for 5HT2C receptors, although there are notable excep- patients. Ignorance of the mechanism of these orexigenic actions tions, such as ziprasidone, which is not orexigenic yet has high has hindered efforts to develop alternative therapeutic agents. receptor affinity (30). Neuroleptics elicit antipsychotic actions by Evaluation of candidate drugs for influences on H1R and blocking dopamine D2 receptors. Although blockade in vitro of hypothalamic AMPK provides a straightforward approach to these receptors does not correlate with orexigenic potencies, developing better drugs and may advance our understanding of positron emission tomographic studies reveal a relationship the hypothalamic regulation of food intake.

A B C 18 CLO 200 nM ARC PVC ARC PVC D 16 ** Tri 14 Saline 1 5 1 5 CLO 3 mg/kg Clozapine 12 (nM) Histamine (µg/ml) Saline 10 CTL 50 500 Clozapine * AMPK-P AMPK-P- 8 6 Arbitrary Units 4 AMPK AMPK CLO 2 3mg/kg 0 ARC PVN ARC PVN WT H1R -/- WT H1R -/-

Fig. 4. Clozapine activates AMPK through histamine H1 receptors. (A) Hypothalamic slices were incubated with triprolidine (50 or 500 nM) or clozapine (200 nM) for 30 min. Phospho-AMPK and total AMPK were detected by Western blotting. (B) Hypothalamic slices were incubated with various concentrations of histamine in the absence or presence of 200 nM clozapine for 30 min. Phospho-AMPK and total AMPK were detected by Western blotting. (C) Mice were administered saline or 3 mg/kg clozapine and perfused with 4% paraformaldehyde. Immunohistochemistry was performed with an antibody specific for phospho-AMPK. (D) Quantification of immunohistochemistry. Bars represent the mean Ϯ SE of five independent slides. *, P Ͻ 0.005; **, P Ͻ 0.001; Student’s t test (n ϭ 5).

3458 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611417104 Kim et al. Downloaded by guest on October 1, 2021 Methods temperature and incubated with a mouse anti-phospho-AMPK␣ Drug Preparation. All drugs were purchased from Toronto Re- antibody (Cell Signaling Technologies, Danvers, MA) diluted 1:200 SEE COMMENTARY search Chemicals (Toronto, ON, Canada). Clozapine was dis- into the blocking solution overnight at 4°C. Subsequent washes were solved in 0.1 M HCl (0.8 ml) and neutralized by 0.1 M NaOH (0.7 conducted in TBST, and labeling was visualized with the Vectastain ml). The drug was diluted with 8.5 ml of saline solution, and the Elite ABC kit (Vector Laboratories, Burlingame, CA). Images appropriate doses were administrated to mice. For control mice, were quantified with the AlphaEaseFC program. the same solution was injected without a drug. For in vitro assays, drugs were dissolved in DMSO. Effect of Neuroleptics on H1R Binding. The IC50 values of neuroleptics on H1R were determined as described (41). Briefly, rats were Hypothalamic Slices. Hypothalami from 8- to 10-week-old mice killed by decapitation and the forebrains removed. Brains were were cut at 0.4-mm intervals in sagittal and coronal planes by homogenized in 30 vol of Na-K phosphate buffer, pH 7.5, and ϫ using a Mcllwain tissue chopper. The slices were dispersed in centrifuged at 48,000 g for 10 min. The tissue was resuspended artificial cerebrospinal fluid buffer. in buffer and recentrifuged an additional three times. Tissue was resuspended in buffer at 15 mg/ml. ␮ Immunohistochemistry. Phospho-AMPK immunohistochemistry Tissue (0.2 ml) was added to tubes containing 25 lofdrug and 25 ␮lof[3H]mepyramine (30 nM). Nonspecific binding was was performed as described (39, 40), and all solutions before and ␮ including the primary antibody incubation contained 2 mM sodium determined in the presence of 1 M triprolidine. Tubes were tm1Wat incubated for1hat25°C, and the samples were filtered over fluoride. C57BL/6 mice or B6.129P-Hrh1 (H1R knockout) ϫ mice (8–10 weeks of age) were perfused with 4% paraformalde- 0.5% poly(ethyleneimine)-coated filters washed with 2 5mlof hyde maintained at 37°C. Organs were postfixed for2hatroom cold 50 mM NaCl. temperature and cryoprotected overnight at 4°C (30% sucrose in PBS). Free-floating sections (45 ␮m) were quenched with 3% H O This work was supported by U.S. Public Health Service Grants 2 2 DA000266 and MH18501 and Research Scientist Award DA00074 (to in water for 10 min at room temperature, washed in TBS-T (16 mM S.H.S.); Canadian Institute of Health Research Fellowship (to S.F.K.); Tris, pH 7.4/140 mM sodium chloride/0.1% Tween 20), and antigen and National Institutes of Health Grants NS36526, AI4515, AI41747, retrieved for 30 min in a 70°C water bath (10 mM sodium citrate in and AI45666 and National Multiple Sclerosis Society Grant RG-3129 TBS-T). Sections were blocked (5% NGS in TBS-T) for1hatroom (to C.T.).

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