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Dysregulation of the Mesolimbic Dopamine System and Reward in MCHⴚ/ⴚ Mice Pavlos Pissios, Lauren Frank, Adam R. Kennedy, Douglas R. Porter, Francis E. Marino, Fen-Fen Liu, Emmanuel N. Pothos, and Eleftheria Maratos-Flier Background: The hypothalamic neuropeptide melanin-concentrating hormone (MCH) plays a critical role in energy homeostasis. Abundant expression of the MCH receptor is observed outside the hypothalamus, especially in the dorsal and the ventral striatum, raising the possibility that MCH modulates the function of the midbrain dopamine neurons and associated circuitry. Methods: The MCH receptor 1 (MCHR1) expression was assessed by in situ hybridization. Expression of dopamine transporter (DAT) and the dopamine D1 and D2 receptor (D1R and D2R) subtypes in the caudate-putamen (CPu) and the nucleus accumbens (Acb) was evaluated by immunoblotting. Amperometry in ex vivo slices of the Acb was used to measure evoked-dopamine release in MCH⫺/ ⫺ mice. Catalepsy in MCH⫹/⫹ and MCH⫺/⫺ mice was assessed by the bar test after haloperidol injection. Locomotor activity was measured after acute and chronic treatment with amphetamine and after dopamine reuptake inhibitor GBR 12909 administration. Results: The psychostimulant amphetamine caused enhanced behavioral sensitization in MCH⫺/⫺ mice. We found significantly elevated expression of the DAT in the Acb of MCH⫺/⫺ mice. The DAT-mediated uptake of dopamine was also enhanced in MCH⫺/⫺ mice consistent with increased expression of DAT. We also found that evoked dopamine release is significantly increased in the Acb shell of MCH⫺/⫺ mice. The GBR 12909 administration increased the locomotor activity of MCH⫺/⫺ mice significantly above that of MCH⫹/⫹ mice. Conclusions: These results demonstrate that MCH, in addition to its known role in feeding and weight regulation, plays a critical role in regulating Acb dopamine signaling and related behavioral responses. Key Words: Amphetamine, appetite, dopamine, melanin-concentrating hormone, nucleus accumbens, obesity
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elanin-concentrating hormone (MCH) is expressed in a unique population of magnocellular neurons in the lateral hypothalamic area (LH) and the zona incerta, which make monosynaptic projections throughout the brain (1,2). Intracerebroventricular administration of MCH causes a significant increase in food intake (3,4), and long-term infusions of MCH produce obesity (5). In mice, genetic overexpression of MCH causes obesity (6), and deficiency of either MCH or the MCH receptor 1 (MCHR1) causes leanness and resistance to diet-induced obesity (DIO) and increases metabolic rate (7–13). Furthermore, pharmacological blockade of the MCHR1 results in hypophagia and resistance to DIO in rodents (14,15). The MCHR1 is robustly expressed in the ventral and dorsal striatum, the terminal fields of midbrain dopaminergic neurons. These findings suggest that MCH neurons might regulate motivated behaviors (16 –19). Consistent with this neuroanatomical pattern of MCH and MCHR1 expression, MCHR1⫺/⫺ mice display increased spontaneous motor activity while on chow diets (11,12,20), whereas MCH⫺/⫺ mice have increased activity when fed high-fat diet (13). Furthermore, deletion of the MCH gene in leptin-deficient ob/ob mice, which typically show little
From the Division of Endocrinology (PP, ARK, FEM, F-FL, EM-F), Department of Medicine, Beth Israel Deaconess Medical Center; Affiliated Neuroscience Program (EM-F), Harvard Medical School; Department of Pharmacology and Experimental Therapeutics and Program in Neuroscience (LF, DRP, ENP), Tufts University School of Medicine, Boston, Massachusetts. Address reprint requests to Pavlos Pissios, Ph.D., Division of Endocrinology, RN380G, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215; E-mail: [email protected]. Received July 20, 2007; revised December 17, 2007; accepted December 17, 2007.
0006-3223/08/$34.00 doi:10.1016/j.biopsych.2007.12.011
locomotor activity, leads to a threefold increase in spontaneous physical activity and substantial weight reduction (21). A recent study reported dysregulation of the mesolimbic system in MCHR1⫺/⫺ mice, providing direct evidence for MCH action in the striatum (22). Finally, injection of MCH directly into the medial nucleus accumbens shell (AcbSh) increases food intake, suggesting that this is an important site of MCH action outside of the hypothalamus (23). To directly assess the role of MCH in regulating the midbrain dopamine system, we evaluated the striatal-dependent functions in MCH⫺/⫺ mice with molecular, cellular, electrophysiological, and behavioral approaches. In the absence of MCH, we noted significant functional changes in the AcbSh, including increased electrically evoked dopamine release and increased reuptake of dopamine in the presynaptic terminals. These changes might explain the increased activity after injection of dopamine transporter (DAT) inhibitor and the enhanced behavioral sensitization to amphetamine that we observed in MCH⫺/⫺ mice. Our results suggest that MCH plays a critical role linking hypothalamic circuitry regulating homeostatic feeding drives with the mesolimbic dopamine system that influences hedonic responses to addictive stimuli, perhaps including those represented by palatable foods (24).
Methods and Materials Animals The MCH⫺/⫺ and MCH⫹/⫹ littermates were bred under our direction at Taconic (Hudson, New York). The MCH⫺/⫺ mice were originally generated by the Maratos-Flier group, and the physiology of these mice has been characterized (7,13). Mice used in this study had been backcrossed onto the C57BL6 background for at least 15 generations. Colonies are maintained as het ⫻ het breeders, progeny is genotyped, and MCH⫹/⫹ and MCH⫺/⫺ animals were shipped to us and individually housed in the Beth Israel Deaconess Medical Center (BIDMC) animal facility at 22°C ambient temperature. The MCHR1⫺/⫺ mice were BIOL PSYCHIATRY 2008;64:184 –191 © 2008 Society of Biological Psychiatry
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P. Pissios et al. obtained from Don Marsh (Merck, Rahway, New Jersey) and bred at BIDMC. Mice were fed standard laboratory chow, Harland-Teklad diet F6 (Madison, Wisconsin). All procedures were approved by the BIDMC Institutional Animal Care and Use Committee. Antibodies and Pharmacologic Agents Antibodies for dopamine D1 and D2 receptor (D1R and D2R) subtypes and DAT were from Chemicon (Temecula, California), and secondary antibodies were from Jackson Immunoresearch (West Grove, Pennsylvania). The D-amphetamine, SKF81297, quinpirole, haloperidol, and GBR 12909 were purchased from Sigma (St. Louis, Missouri). In Situ Hybridization Mice were deeply anesthetized and perfused transcardially with saline followed by 10% buffered formalin. Brains were postfixed overnight in formalin and cryoprotected in 20% sucrose solution before slicing on freezing-sliding microtome at 30-m thickness. Slices were stored in antifreeze at ⫺20°C. Hybridization was performed as previously described (25). Images were acquired on Zeiss Axioimager.Z1 with Axiovision 4.5 software (Oberkochen, Germany) at 20⫻ original magnification. Dopamine Radioimmunoassay Tissue punches from medial prefrontal cortex (mPFC), caudate putamen (CPu), and Acb were extracted with .1 N hydrochloric acid according to manufacturer’s protocol, and total dopamine levels in the extract were determined by radioimmunoassay (RIA) from IBL (Hamburg, Germany). Results were normalized and expressed as ng of dopamine/mg of total protein (Bradford, BioRad, Hercules, California). Immunoblotting Samples were obtained from 6-month-old males (6 – 8/group) not subjected to any pharmacologic treatment. Tissue punches from Acb and CPu were lysed in tissue lysis buffer (20 mmol/L Tris-Cl pH 7.5, 150 mmol/L sodium chloride (NaCl), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L -mercaptoethanol supplemented with protease inhibitors. Protein concentration was determined with the Bio-Rad protein assay (BioRad). Twenty microgram samples were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% mini-protean gel (BioRad) and transferred onto Protran nitrocellulose (Schleicher and Schuell, Keene, New Hampshire). Immunoblotting was performed according to the manufacturer’s recommendations (Chemicon). Blots were developed with the chemiluminescent reagent Super West Pico (Pierce, Rockford, Illinois) and quantitated with ImageQuant 5.1 (Amersham, Piscataway, New Jersey). Locomotor Activity The Comprehensive Laboratory Animal Monitoring System (CLAMS) apparatus (Columbus, Ohio) was used for monitoring locomotor activity. All mice used in the experiments were singly housed and between 12 and 24 weeks of age. Saline, SKF81297 (3 mg/kg), and quinpirole (2 mg/kg) were injected IP at a volume of 10 L/g of body weight (26). The GBR 12909 (20 mg/kg) was dissolved in water (because of its insolubility in saline). After overnight acclimatization MCH⫹/⫹ and MCH⫺/⫺ mice received vehicle (water) the first day and GBR 12909 the second day of the experiment. Ambulatory activity of MCH⫹/⫹
and MCH⫺/⫺ (10-min intervals) is shown for 4 hours after injections. Amphetamine sensitization: cohorts of mice received four separate injections at 10 L/g of body weight spaced 3 days apart. The order of the injections was: 1) saline, 2) amphetamine 1 mg/kg, 3) amphetamine 4 mg/kg, and 4) amphetamine 1 mg/kg. Ambulatory activity (10-min intervals) is shown for 2 hours after injections. All experiments were conducted between 12:00 PM and 4:00 PM (second half of the light cycle). Separate cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice received daily saline or amphetamine injections (1 mg/kg of body weight in a volume of 10 mL/kg of body weight) between 11:00 AM and 2:00 PM during the light cycle. Their ambulatory activity was monitored for 2 hours with the OptoM3 apparatus from Columbus Instruments (Columbus, Ohio). Haloperidol-Induced Catalepsy Catalepsy was measured by the bar test. A horizontal bar was positioned 3.5 cm above the cage floor. Six-month-old male mice (8/group) were injected with vehicle or haloperidol (total volume of 10 L/g of body weight) and positioned with front paws on the bar. Latency (sec) to removing front paws was evaluated 30 min and 60 min after IP injection. Cut-off time for the test was 90 sec. Slice Recordings Each animal was killed with a ketamine (200 mg/kg IP)/ xylazine (20 mg/kg IP) injection, and the brain was removed. It was immediately placed into ice-cold and carbogenated (95% oxygen, 5% carbon dioxide) dissection solution (Pelletier/ Carlen) containing 210 mmol/L sucrose, 3.5 mmol/L potassium chloride (KCl), 1 mmol/L calcium chloride (CaCl2) dihydrate, 4 mmol/L magnesium chloride hexahydrate, 1.25 mmol/L sodium phosphate monobasic hydrate, 10 mmol/L glucose, and 26 mmol/L sodium bicarbonate (NaHCO3). The brain was subsequently cut on a Leica VT1000S vibratome. Acute coronal slices were 300 m thick. After 1 hour recovery in carbogenated artificial cerebrospinal fluid solution (ACSF), the slice was transferred into the recording chamber with perfusion of ACSF set to 1 mL/min. Temperature was set to 37°C. The ACSF bath contained (in mmol/L): 124 NaCl, 2.0 KCl, 1.25 potassium phosphate monobasic, 2.0 magnesium sulfate, 25 NaHCO3, 1.0 CaCl2, 11 glucose, pH ⫽ 7.3. Details of the amperometric recordings have been described elsewhere (27,28). Local bath application of the dopamine reuptake blocker nomifensine (3 mol/L) and quinpirole (10 mol/L) for at least 30 min was also used to assess the contribution of reuptake and the D2R autoreceptor in the evoked dopamine signal. The five single pulses/slice are averaged into a grand mean, and the two groups (MCH⫹/⫹ vs. MCH⫺/⫺) are compared with one-way analysis of variance (ANOVA) of the means.
Results MCHR1 Is Highly Expressed in the Ventral Striatum But Not in the Ventral Tegmental Area Expression of MCHR1 in mouse brain was determined by in situ hybridization of antisense riboprobe according to published procedures (25). A strong signal was detected in the terminal regions of dopaminergic projections from the midbrain, especially in the ventral striatum, Acb, and the olfactory tubercle (Tu) (Figures 1A and 1B). The PFC and prelimbic (PrL) and infralimbic (IL) cortex showed intermediate levels of MCHR1 expression. More caudally, expression increased in the medial shell of www.sobp.org/journal
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P. Pissios et al. low dose of amphetamine (1 mg/kg) was chosen that does not produce a significant difference between saline- and amphetamine-treated MCH⫹/⫹ mice. The MCH⫺/⫺ mice developed a significant increase in activity compared with amphetaminetreated MCH⫹/⫹ mice on days 4 and 5 of the experiment (p ⬍ .05 by ANOVA for both days) (Figure S1 in Supplement 1). Seven days after the cessation of daily injections, all cohorts received amphetamine at 1 mg/kg of body weight. The MCH⫺/⫺ mice maintained significantly higher activity compared with the rest of the groups, demonstrating that chronic absence of MCH enhances the sensitizing effects of amphetamine in mice.
Figure 1. Melanin-concentrating hormone receptor 1 (MCHR1) is strongly expressed in the ventral striatum but not in the ventral tegmental area (VTA). (A–C) Coronal sections of mouse brain hybridized with antisense riboprobe to MCHR1. (D) No specific signal is detected in the striatum of the MCHR1 ⫺/⫺ mouse. Acb, nucleus accumbens; CPu, caudate-putamen; DTT, dorsal taenia tecta; IL, infralimbic cortex; Pir, Piriform cortex; PrL; prelimbic cortex; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; cc, corpus callosum; Tu, olfactory tubercle.
the Acb, but expression was also present throughout the CPu (Figure 1B). A minimal signal was seen in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) (Figure 1C). Slices from MCHR1⫺/⫺ mice were used as control for nonspecific hybridization, and no signal was detected (Figure 1D). MCHⴚ/ⴚ Mice Show Enhanced Sensitization to Repeated Amphetamine Administration One common assay for evaluating the mesolimbic system is behavioral sensitization to addictive drugs (29,30) wherein initial exposure to a psychostimulant such as amphetamine causes enhanced locomotor responses to subsequent administrations. Cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice were initially injected with saline, and no difference in locomotor activity between the two genotypes was observed (Figure 2A). Three days later, the same mice received a low dose of amphetamine (1 mg/kg of body weight), and the locomotor response was similar to that seen with saline, without any difference between the two genotypes (Figure 2B). Injection of a high dose of amphetamine (4 mg/kg) 3 days later produced a robust increase in locomotor activity in both MCH⫹/⫹ and MCH⫺/⫺ mice (Figure 2C). Although MCH⫺/⫺ mice tended to have more activity in the first hour, this difference did not reach statistical significance. Three days later, cohorts were re-challenged with the low dose of amphetamine (1 mg/kg). Both groups showed a robust increase in locomotor activity in response to this challenge, compared with their first exposure to the same dose, which confirmed behavioral sensitization (Figure 2B vs. 2D) (by paired t test within groups p ⬍ .0101 for MCH⫹/⫹ mice, p ⬍ .0003 for MCH⫺/⫺ mice for the first hour after injection). Importantly, MCH⫺/⫺ mice showed a significantly greater increase in locomotor activity compared with MCH⫹/⫹ mice [87% increase for the first hour after injection, F (1,15) ⫽ 7.49 p ⬍ .0153 by ANOVA]. Sensitization with a second paradigm of daily injections was also performed in separate cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice. To detect the increased sensitization of MCH⫺/⫺ mice, a www.sobp.org/journal
The Striatum of MCHⴚ/ⴚ Mice Shows Altered Expression of Proteins Involved in Dopamine Signaling and Turnover To understand the molecular basis for enhanced sensitization of MCH⫺/⫺ mice, we measured total dopamine levels in mPFC, Acb, and CPu of MCH⫹/⫹ and MCH⫺/⫺mice. We also determined the protein expression of striatal D1R, D2R, and DAT by immunoblotting extracts from CPu and Acb tissue punches. No difference was observed in total dopamine content (ng/mg of total protein) by RIA between the two groups in the mPFC (MCH⫹/⫹ 2.6 ⫾ .48 vs. MCH⫺/⫺ 3.36 ⫾ .28), CPu (MCH⫹/⫹ 296.24 ⫾ 6.33 vs. MCH⫺/⫺ 300.73 ⫾ 10.06), or Acb (MCH⫹/⫹ 150.37 ⫾ 10.22 vs. MCH⫺/⫺ 160.45 ⫾ 10.86). We also measured dopamine and DOPAC, a dopamine metabolite, by high-performance liquid chromatography (HPLC) in the Acb of MCH⫹/⫹ and MCH⫺/⫺ mice and again found no difference between the genotypes (dopamine [ng/mg of total protein]:
Figure 2. Enhanced sensitization of melanin-concentrating hormone (MCH)⫺/⫺ mice to systemic amphetamine injections. The MCH⫺/⫺ (black symbols) and MCH⫹/⫹ mice (open symbols) mice were subjected to a series of injections of (A) saline, (B) amphetamine (1 mg/kg of body weight), (C) amphetamine (4 mg/kg), and (D) amphetamine (1 mg/kg) separated by 72 hours. Ambulatory activity is represented as a number of beam breaks/10min interval for 2 hours. Statistical significant difference between groups (MCH⫹/⫹, MCH⫺/⫺) was detected only for the last amphetamine injection (D), [F(1,15) ⫽ 7.49 p ⬍ .0153 by analysis of variance, n ⫽ 9 –12 animals/ group]. Note the different scales of the y axes.
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BIOL PSYCHIATRY 2008;64:184 –191 187 completely blocked the increase in locomotor activity induced by SKF81297 in both groups (Figure 4). No differences in locomotor responses between the two genotypes tested were observed. We then tested the effects of the antipsychotic haloperidol on locomotor activity. Typical antipsychotic drugs produce side effects such as catalepsy, which is mediated through D2R (31). To evaluate the functional response of D2R, mice were injected with different doses of haloperidol (.2 mg/kg and 2 mg/kg/body weight), and the induction of catalepsy was assessed with the bar test 30 and 60 min after IP injection. Catalepsy was defined as the latency (sec) to removal of forepaws from the bar. The MCH⫺/⫺ mice exhibited significantly less catalepsy at the low-dose haloperidol (.2 mg/kg) (Figure S2A in Supplement 1; p ⬍ .05 by ANOVA) than MCH⫹/⫹ mice. No significant difference between genotypes was observed at the higher dose of haloperidol (2 mg/kg) (Figure S2B in Supplement 1). The decreased sensitivity of MCH⫺/⫺mice to haloperidol is consistent with decreased expression of D2R in the striatum of MCH⫺/⫺ mice, because D2R⫺/⫹ mice are less sensitive and D2R⫺/⫺mice are completely resistant to the cataleptic effects of haloperidol (32).
Figure 3. Altered expression of dopamine D2 receptor (D2R) and dopamine transporter (DAT) in the dorsal and the ventral striatum of MCH⫺/⫺mice. (A) Representative immunoblots and quantitation for dopamine D1 receptor (D1R), D2R, and DAT protein levels from CPu of MCH⫹/⫹ and MCH⫺/⫺ mice (p ⬍ .01, n ⫽ 6 – 8 animals/group for D2R by Student t test). (B) Representative immunoblots and quantitation for D1R, D2R, and DAT protein levels from Acb of MCH⫹/⫹ and MCH⫺/⫺ mice (p ⬍ .02, n ⫽ 6 – 8 animals/group for DAT by Student t test). (C) Quantitative polymerase chain reaction determination of messenger RNA (mRNA) levels for DAT from the VTA of MCH⫹/⫹ and MCH⫺/⫺ mice. Other abbreviations as in Figures 1 and 2.
MCH⫹/⫹ 91.62 ⫾ 9.06, MCH⫺/⫺ 96.52 ⫾4.48; DOPAC: MCH⫹/⫹ 13.86 ⫾ 1.21, MCH⫺/⫺ 12.20 ⫾ .54). Immunoblotting did not show any changes in the expression of D1R. In contrast, we found a 50% decrease in D2R expression in the CPu of MCH⫺/⫺ mice compared with MCH⫹/⫹ mice (p ⬍ .01) (Figure 3A). No difference in D2R expression was observed in the Acb. Immunoblotting also revealed an 80% increase of DAT in the Acb of MCH⫺/⫺ mice as compared with MCH⫹/⫹ mice (p ⬍ .02) (Figure 3B), whereas no difference was seen in the CPu. We also measured DAT messenger RNA in the VTA by quantitative polymerase chain reaction and found no difference between the genotypes (Figure 3C). These results indicate that the absence of MCH leads to region-specific compensatory adaptations in components of the dopamine system in the striatum. MCHⴚ/ⴚ Mice Show Normal Locomotor Responses to D1R and D2R Agonists But Are Less Sensitive to Haloperidol-Induced Catalepsy Altered expression of D2R and DAT suggest that mice lacking MCH might have abnormal responses to selective dopamine receptor agonists or antagonists. We examined the locomotor responses of MCH⫺/⫺ mice to the selective D1R agonist SKF81297 and the D2R agonist quinpirole. Injection of SKF81297 (3 mg/kg) produced a robust increase in locomotor activity in both MCH⫹/⫹ and MCH⫺/⫺ animals (Figure 4). Injection of quinpirole (2 mg/kg), as expected, significantly reduced the ambulatory activity of MCH⫹/⫹ and MCH⫺/⫺ mice (Figure 4). Co-injection of quiniprole with SKF81297
Acb Terminals of MCHⴚ/ⴚ Mice Exhibit Increased Evoked Dopamine Release and Reuptake Increased expression of DAT in the Acb of MCH⫺/⫺ mice should result in increased reuptake of dopamine into presynaptic terminals (33). To assess this possibility, we measured evoked release of dopamine from AcbSh terminals by amperometry in ex vivo slices. Under baseline conditions, no significant difference in dopamine signal (mean ⫾ SEM) was observed between the two genotypes (amplitude: MCH⫹/⫹ 16.9 ⫾ 1.7 pA vs. MCH⫺/⫺ 20.9 ⫾ 2.1 pA [n ⫽ 50 stimulations in 13 MCH⫹/⫹ slices; n ⫽ 75 stimulations in 16 MCH⫺/⫺ slices]) (Figure 5A). Application of a selective DAT inhibitor (3 mol/L nomifensine, for 30 min) significantly increased the dopamine signal in MCH–/– slices compared with slices from MCH⫹/⫹ mice (amplitude 20.0 ⫾ 2.0 pA in MCH⫹/⫹ vs. 48.0 ⫾ 9.0 pA in MCH⫺/⫺; n ⫽ 20 stimulations in four MCH⫹/⫹ slices, n ⫽ 20 stimulations in four MCH⫺/⫺ slices, *p ⬍ .01 by one-way ANOVA; Figure 5A). See also representative trace in Figure 5B. To assess the role of the inhibitory D2R autoreceptor, Acb slices were perfused with the D2R agonist quinpirole (10 mol/L
Figure 4. The MCH⫺/⫺ mice show normal locomotor responses to D1R and D2R agonists but are less sensitive to haloperidol-induced catalepsy. Ambulatory activity of MCH⫹/⫹ and MCH⫺/⫺ mice injected with either saline, SKF81297 (3 mg/kg), quinpirole (2 mg/kg), or SKF81297 ⫹ quinpirole. No difference was observed in the response of MCH⫺/⫺ (black symbols) and MCH⫹/⫹ mice (open symbols) to the D1R or D2R agonists. Abbreviations as in Figures 1–3.
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Figure 5. The MCH⫺/⫺mice exhibit increased electrically stimulated dopamine release in the presence of a DATspecific inhibitor. (A) The mean evoked dopamine signal amplitude shows no significant difference between MCH⫹/⫹ and MCH⫺/⫺ nucleus accumbens shell (AcbSh) slices in the absence of the DAT-specific inhibitor nomifensine. Signal amplitude increased significantly in AcbSh slices of MCH⫺/⫺ mice by application of nomifensine (3 mol/L) to the slice preparation for 30 min (*p ⬍ .01 by one-way analysis of variance). (B) Representative amperometric traces of electrical stimulationevoked dopamine release in acute coronal accumbens slices from MCH⫹/⫹ and MCH⫺/⫺ mice in the presence of the DAT-specific inhibitor nomifensine (3 mol/L). (C) Application of the D2R agonist quinpirole (10 mol/L, 30 min) causes significant reduction in nomifensinetreated slices of MCH⫹/⫹ and MCH⫺/⫺ mice. Increased evoked-dopamine signal in MCH⫺/⫺ slices persists even in the presence of quinpirole. (D) Representative amperometric traces of electrical stimulation-evoked dopamine release in acute coronal accumbens slices from MCH⫹/⫹ and MCH⫺/⫺ mice in the presence of nomifensine (3 mol/L for a total of 60 min) and quinpirole (10 mol/L for a total of 30 min). Other abbreviations as in Figures 1–3.
for 30 min) in the presence of 3 mol/L nomifensine, which caused a significant decrease in the evoked dopamine signal amplitude compared with vehicle in both genotypes: MCH⫹/⫹ with quinpirole 3.5 ⫾ .5 pA versus vehicle 20.4 ⫾ 2.2 pA (n ⫽ 28 stimulations in six slices from four mice; n ⫽ 20 stimulations in four slices in vehicle, p ⬍ .01 by ANOVA); MCH⫺/⫺ with quinpirole 7.0 ⫾ 1.5 pA versus vehicle 47.7 ⫾ 9.2 pA in vehicle (n ⫽ 20 stimulations in four slices from three MCH⫺/⫺ mice; n ⫽ 20 stimulations in four slices in vehicle, p ⬍ .01 by ANOVA; Figure 5C). Nevertheless, the evoked dopamine signal was significantly higher in MCH⫺/⫺ than MCH⫹/⫹ slices even after perfusion of saturating concentration of quinpirole (MCH⫹/⫹ 3.5 ⫾ .5 pA vs. MCH⫺/⫺ 7.0 ⫾ 1.5 pA, p ⬍ .01 by ANOVA), possibly suggesting an impaired D2R function in MCH⫺/⫺ mice (Figure 5C). See also representative trace in Figure 5D. These results suggest that increased DAT-mediated reuptake of dopamine likely acts to compensate for increased evoked
Figure 6. The activity of melanin-concentrating hormone (MCH)⫺/⫺ mice increases significantly above MCH⫹/⫹ mice by the dopamine reuptake inhibitor GBR 12909. The MCH⫺/⫺ and MCH⫹/⫹ mice were acclimatized overnight to the activity chambers and injected with vehicle IP the following day. The next day the same mice were injected with GBR 12909 (20 mg/kg) IP. Data are represented as beambreaks/10 min for 4 hours after injection. All runs were done between 12:00 PM and 4:00 PM; significant difference in activity between MCH⫹/⫹ and MCH⫺/⫺ mice was observed for the duration of the test (4 hours) [F(1,25) ⫽ 13.72, p ⬍ .001 by analysis of variance).
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dopamine release in Acb slices from MCH⫺/⫺ mice, an effect possibly enhanced by a defect in D2R autoreceptor function. Because of the compensation, increased dopamine release is not seen until application of DAT inhibitors blocks dopamine reuptake, unmasking increased dopamine release. MCHⴚ/ⴚ Mice Are More Sensitive to the LocomotorActivating Action of DAT Inhibitor GBR 12909 In Vivo The ex vivo results suggest that increased dopamine release might also occur in vivo and could be unmasked by the administration of dopamine reuptake blockers to mice. We used the highly selective dopamine reuptake blocker GBR 12909, which increases locomotor activity in mice. After injection with vehicle, no difference was noted in the locomotor activity of MCH⫹/⫹ and MCH⫺/⫺ mice over a 4-hour observation period (Figure 6). The next day, GBR 12909 was injected into the same mice at 20 mg/kg of body weight, and locomotor activity was
P. Pissios et al. measured as described in the preceding text. A significant increase in locomotor activity was observed within each genotype after GRB12909 injection compared with vehicle alone (p ⬍ .001 by paired t test for MCH⫹/⫹ and MCH⫺/⫺ mice). Importantly, MCH⫺/⫺ mice had substantially increased locomotor activity compared with MCH⫹/⫹ mice for the 4-hour duration of the test (Figure 6) [F (1,25) ⫽ 13.72, p ⬍ .001 by ANOVA]. Thus, compensatory adaptations in dopamine release and reuptake observed in ex vivo recordings seem to operate in MCH⫺/⫺ mice in vivo and, as is the case in vitro, can be unmasked by selective DAT blockers. MCHⴚ/ⴚ Mice Do Not Display Hyperphagia When Presented a High-Fat Palatable Diet We measured daily food consumption of standard chow compared with newly introduced high fat/sucrose palatable diet (HF) (Research Diets 12451) in singly housed MCH⫹/⫹ and MCH⫺/⫺ mice. No difference between the genotypes was observed in daily caloric intake of chow (days 1–3). When presented the HF diet, MCH⫹/⫹ mice increased their daily caloric intake above chow levels by 40% on Day 4 (first day of HF diet), slowly returning to chow levels by day 6. The MCH⫺/⫺ mice, in contrast, did not exhibit the hyperphagia on HF diet, and their daily caloric intake on HF was not different from chow (Figure 7).
Discussion Food intake is a complex behavior requiring integration of both homeostatic and hedonic inputs (34). Homeostatic signals provide information on energy status, whereas hedonic inputs mediate the rewarding aspects of feeding. Both inputs are essential, because animals must both perceive hunger and in consequence engage in activities that result in finding and consuming a meal. Furthermore, recent reports describe effects of peptides such as leptin (35,36) and ghrelin (37) on dopaminergic neurons, whose actions on feeding were thought to be limited to the hypothalamus. In this regard the relationship between the Acb and the LH is of interest, because these two areas have extensive reciprocal innervation. With multiple techniques we demonstrated that MCH⫺/⫺ mice have alterations in striatal-dependent behaviors. Total dopamine and dopamine metabolite content was not changed in MCH⫺/⫺ mice, indicating normal dopamine synthesis and degradation of MCH⫺/⫺ mice. However, we observed elevated DAT expression in the Acb, suggesting that clearance of extracellular dopamine might be increased in these animals. Amperometry recordings from AcbSh detected both increased dopamine release and increased dopamine reuptake in MCH⫺/⫺ mice. Furthermore, administration of selective DAT inhibitor increased locomotor activity significantly more in MCH⫺/⫺ mice than in MCH⫹/⫹ mice. We hypothesize that increased expression of DAT arises in compensation for increased release of dopamine from the VTA terminals in the Acb of MCH⫺/⫺ mice. However, the mechanism underlying increased dopamine release is not clear. It is possible that increased dopamine release results from impaired D2R negative feedback. This is consistent with the incomplete suppression of dopamine release by quinpirole in MCH⫺/⫺ mice. However, the effects of quinpirole are also subject to interpretation, because the incomplete suppression might also reflect the higher basal dopamine release seen MCH⫺/⫺ mice. Locomotor activity could in principle be affected as well by postsynaptic modifications of dopamine receptor signaling in
BIOL PSYCHIATRY 2008;64:184 –191 189 the medium spiny neurons. However, we were unable to demonstrate any such difference between MCH⫺/⫺ mice and MCH⫹/⫹ mice treated with D1R and D2R agonists. A similar response to D1R agonist is expected, because D1R expression is unchanged. In contrast D2R protein expression is 50% decreased in the CPu, and we expected an altered response to D2R agonists. Although a mechanism explaining the normal sensitivity of MCH⫺/⫺ mice to the D2R agonist quinpirole is lacking, it is possible that additional changes in the medium spiny neurons (possibly mediated by the glutamatergic input from the cortex to the Acb) might compensate for the decreased levels of D2R. Although decreased sensitivity to D2R agonist was not observed, MCH⫺/⫺ mice showed decreased sensitivity to the cataleptic actions of the D2R antagonist haloperidol, which has been reported in other models with decreased D2R expression (31,32). The striatum is the likely site of MCH action in the mesolimbic system. Although the VTA is important in mediating amphetamine sensitization, little MCH receptor is expressed in this area. Furthermore, a recent study failed to demonstrate acute effects of MCH on the firing pattern of dopaminergic and non-dopaminergic VTA neurons in brain slice recordings (38). Thus it is unlikely that our findings are mediated by direct effects of MCH on the VTA neurons. Although a direct action cannot formally be excluded, an indirect action through the mPFC and/or the Acb, which provide afferent connections to the VTA and also express significant levels of MCHR1, is more likely (Figure 1). Inhibition of D1R signaling by MCH has been demonstrated in the Acb (23). Another possibility is MCH acting presynaptically on afferents arriving from mPFC and Acb to the VTA (30,39). Although the precise contribution of these sites needs to be addressed in future experiments, the present data support a long-term inhibitory role of MCH within the mesolimbic system. Our results contrast with some of the findings in Smith et al. (22), who reported acute sensitivity of MCHR1⫺/⫺ mice to amphetamine and D1R agonists (although no sensitization experiments were conducted) and proposed a primarily postsynaptic mechanism for the amphetamine supersensitivity in MCHR1⫺/⫺ mice. Although the basis for a difference in acute amphetamine response between these models of ligand
Figure 7. Acute hyperphagia of high-fat palatable food (HF) is absent in melanin-concentrating hormone (MCH)⫺/⫺ mice. Daily consumption of standard low-fat chow was monitored for 3 days. Mice were then presented a HF diet for 3 more consecutive days. Acute overeating of HF diet is absent in MCH⫺/⫺ mice compared with wildtype mice on Day 4 (first day of HF diet) (p ⬍ .05 by analysis of variance).
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190 BIOL PSYCHIATRY 2008;64:184 –191 and receptor loss are not yet clear, it could be related to background strain differences—C57BL/6 in this report as opposed to C57BL/6X129SvEv hybrid background in Smith et al. (22). An intriguing alternative could be the activity of other peptides encoded by the MCH gene, such as neuropeptide-glutamic acid-isoleucine amide (NEI) (40). Despite the differences between the MCH⫺/⫺ models, the overall conclusion from MCHR1⫺/⫺ mice is consistent with our data in MCH⫺/⫺ mice in suggesting an inhibitory action of the MCH system in the striatum. A recent study also reported an atypical response of MCHR1⫺/⫺ mice to cocaine with absence of sensitization (41). The discrepancy between amphetamine and cocaine responses could simply be explained by the distinct mode of action of these drugs at different neural substrates and will need to be resolved by further experiments. Finally, we observed a potential consequence of the dysregulated mesolimbic system in MCH⫺/⫺ mice on eating behavior. The MCH⫺/⫺ mice did not exhibit a hyperphagic response when presented with a palatable diet. Because overeating of palatable foods is driven by hedonic factors, our results point to a disruption in food reward in MCH⫺/⫺ mice. A functional connection between the LH and reward was suggested almost 50 years ago when studies revealed that placement of electrodes in the LH resulted in robust electrical self-stimulation in rodents (42). Melanin-concentrating hormone neurons are located in the LH and are well positioned to integrate various inputs pertinent to homeostatic responses. Because the mesolimbic system plays a role in assigning an incentive salience to rewards such as food, it is tempting to speculate that MCH, which responds to nutritional status such as fasting, might modulate the adaptive behavioral responses of the animal through action on the mesolimbic circuitry. Energy homeostasis is a complex process that in addition to hunger and satiety involves devising strategies to seek, acquire, and consume food. The capacity of the MCH system to mediate integration among these diverse physiologic elements underscores its importance in the neural regulation of energy homeostasis.
This work was supported by National Institutes of Health DK56113 and DK56116 and a sponsored research agreement from Merck (EMF); DK069983 (EMF, ENP); and DK065872 and a Smith Family Investigator Award by the Medical Foundation (ENP). The CLAMS support was provided by the physiology core of PO1 DK 56116. Dr. Maratos-Flier is a recipient of a research award from the Yamanouchi Foundation. We would like to thank Drs. Joel Elmquist and Richard Palmiter for helpful discussions; Daniel Trombly and Xiaomei Wang for technical assistance; and Don Marsh (Merck) for providing us with the MCHR1-MCH⫺/⫺ mice. ARK is currently affiliated with the Jackson Laboratory at University of California at Davis-JAX West, West Sacramento, California. All authors reported no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. 1. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, et al. (1992): The melanin-concentrating hormone system of the rat brain: An immuno- and hybridization histochemical characterization. J Comp Neurol 319:218 –245.
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M
elanin-concentrating hormone (MCH) is expressed in a unique population of magnocellular neurons in the lateral hypothalamic area (LH) and the zona incerta, which make monosynaptic projections throughout the brain (1,2). Intracerebroventricular administration of MCH causes a significant increase in food intake (3,4), and long-term infusions of MCH produce obesity (5). In mice, genetic overexpression of MCH causes obesity (6), and deficiency of either MCH or the MCH receptor 1 (MCHR1) causes leanness and resistance to diet-induced obesity (DIO) and increases metabolic rate (7–13). Furthermore, pharmacological blockade of the MCHR1 results in hypophagia and resistance to DIO in rodents (14,15). The MCHR1 is robustly expressed in the ventral and dorsal striatum, the terminal fields of midbrain dopaminergic neurons. These findings suggest that MCH neurons might regulate motivated behaviors (16 –19). Consistent with this neuroanatomical pattern of MCH and MCHR1 expression, MCHR1⫺/⫺ mice display increased spontaneous motor activity while on chow diets (11,12,20), whereas MCH⫺/⫺ mice have increased activity when fed high-fat diet (13). Furthermore, deletion of the MCH gene in leptin-deficient ob/ob mice, which typically show little
From the Division of Endocrinology (PP, ARK, FEM, F-FL, EM-F), Department of Medicine, Beth Israel Deaconess Medical Center; Affiliated Neuroscience Program (EM-F), Harvard Medical School; Department of Pharmacology and Experimental Therapeutics and Program in Neuroscience (LF, DRP, ENP), Tufts University School of Medicine, Boston, Massachusetts. Address reprint requests to Pavlos Pissios, Ph.D., Division of Endocrinology, RN380G, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215; E-mail: [email protected]. Received July 20, 2007; revised December 17, 2007; accepted December 17, 2007.
0006-3223/08/$34.00 doi:10.1016/j.biopsych.2007.12.011
locomotor activity, leads to a threefold increase in spontaneous physical activity and substantial weight reduction (21). A recent study reported dysregulation of the mesolimbic system in MCHR1⫺/⫺ mice, providing direct evidence for MCH action in the striatum (22). Finally, injection of MCH directly into the medial nucleus accumbens shell (AcbSh) increases food intake, suggesting that this is an important site of MCH action outside of the hypothalamus (23). To directly assess the role of MCH in regulating the midbrain dopamine system, we evaluated the striatal-dependent functions in MCH⫺/⫺ mice with molecular, cellular, electrophysiological, and behavioral approaches. In the absence of MCH, we noted significant functional changes in the AcbSh, including increased electrically evoked dopamine release and increased reuptake of dopamine in the presynaptic terminals. These changes might explain the increased activity after injection of dopamine transporter (DAT) inhibitor and the enhanced behavioral sensitization to amphetamine that we observed in MCH⫺/⫺ mice. Our results suggest that MCH plays a critical role linking hypothalamic circuitry regulating homeostatic feeding drives with the mesolimbic dopamine system that influences hedonic responses to addictive stimuli, perhaps including those represented by palatable foods (24).
Methods and Materials Animals The MCH⫺/⫺ and MCH⫹/⫹ littermates were bred under our direction at Taconic (Hudson, New York). The MCH⫺/⫺ mice were originally generated by the Maratos-Flier group, and the physiology of these mice has been characterized (7,13). Mice used in this study had been backcrossed onto the C57BL6 background for at least 15 generations. Colonies are maintained as het ⫻ het breeders, progeny is genotyped, and MCH⫹/⫹ and MCH⫺/⫺ animals were shipped to us and individually housed in the Beth Israel Deaconess Medical Center (BIDMC) animal facility at 22°C ambient temperature. The MCHR1⫺/⫺ mice were BIOL PSYCHIATRY 2008;64:184 –191 © 2008 Society of Biological Psychiatry
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P. Pissios et al. obtained from Don Marsh (Merck, Rahway, New Jersey) and bred at BIDMC. Mice were fed standard laboratory chow, Harland-Teklad diet F6 (Madison, Wisconsin). All procedures were approved by the BIDMC Institutional Animal Care and Use Committee. Antibodies and Pharmacologic Agents Antibodies for dopamine D1 and D2 receptor (D1R and D2R) subtypes and DAT were from Chemicon (Temecula, California), and secondary antibodies were from Jackson Immunoresearch (West Grove, Pennsylvania). The D-amphetamine, SKF81297, quinpirole, haloperidol, and GBR 12909 were purchased from Sigma (St. Louis, Missouri). In Situ Hybridization Mice were deeply anesthetized and perfused transcardially with saline followed by 10% buffered formalin. Brains were postfixed overnight in formalin and cryoprotected in 20% sucrose solution before slicing on freezing-sliding microtome at 30-m thickness. Slices were stored in antifreeze at ⫺20°C. Hybridization was performed as previously described (25). Images were acquired on Zeiss Axioimager.Z1 with Axiovision 4.5 software (Oberkochen, Germany) at 20⫻ original magnification. Dopamine Radioimmunoassay Tissue punches from medial prefrontal cortex (mPFC), caudate putamen (CPu), and Acb were extracted with .1 N hydrochloric acid according to manufacturer’s protocol, and total dopamine levels in the extract were determined by radioimmunoassay (RIA) from IBL (Hamburg, Germany). Results were normalized and expressed as ng of dopamine/mg of total protein (Bradford, BioRad, Hercules, California). Immunoblotting Samples were obtained from 6-month-old males (6 – 8/group) not subjected to any pharmacologic treatment. Tissue punches from Acb and CPu were lysed in tissue lysis buffer (20 mmol/L Tris-Cl pH 7.5, 150 mmol/L sodium chloride (NaCl), 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L -mercaptoethanol supplemented with protease inhibitors. Protein concentration was determined with the Bio-Rad protein assay (BioRad). Twenty microgram samples were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% mini-protean gel (BioRad) and transferred onto Protran nitrocellulose (Schleicher and Schuell, Keene, New Hampshire). Immunoblotting was performed according to the manufacturer’s recommendations (Chemicon). Blots were developed with the chemiluminescent reagent Super West Pico (Pierce, Rockford, Illinois) and quantitated with ImageQuant 5.1 (Amersham, Piscataway, New Jersey). Locomotor Activity The Comprehensive Laboratory Animal Monitoring System (CLAMS) apparatus (Columbus, Ohio) was used for monitoring locomotor activity. All mice used in the experiments were singly housed and between 12 and 24 weeks of age. Saline, SKF81297 (3 mg/kg), and quinpirole (2 mg/kg) were injected IP at a volume of 10 L/g of body weight (26). The GBR 12909 (20 mg/kg) was dissolved in water (because of its insolubility in saline). After overnight acclimatization MCH⫹/⫹ and MCH⫺/⫺ mice received vehicle (water) the first day and GBR 12909 the second day of the experiment. Ambulatory activity of MCH⫹/⫹
and MCH⫺/⫺ (10-min intervals) is shown for 4 hours after injections. Amphetamine sensitization: cohorts of mice received four separate injections at 10 L/g of body weight spaced 3 days apart. The order of the injections was: 1) saline, 2) amphetamine 1 mg/kg, 3) amphetamine 4 mg/kg, and 4) amphetamine 1 mg/kg. Ambulatory activity (10-min intervals) is shown for 2 hours after injections. All experiments were conducted between 12:00 PM and 4:00 PM (second half of the light cycle). Separate cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice received daily saline or amphetamine injections (1 mg/kg of body weight in a volume of 10 mL/kg of body weight) between 11:00 AM and 2:00 PM during the light cycle. Their ambulatory activity was monitored for 2 hours with the OptoM3 apparatus from Columbus Instruments (Columbus, Ohio). Haloperidol-Induced Catalepsy Catalepsy was measured by the bar test. A horizontal bar was positioned 3.5 cm above the cage floor. Six-month-old male mice (8/group) were injected with vehicle or haloperidol (total volume of 10 L/g of body weight) and positioned with front paws on the bar. Latency (sec) to removing front paws was evaluated 30 min and 60 min after IP injection. Cut-off time for the test was 90 sec. Slice Recordings Each animal was killed with a ketamine (200 mg/kg IP)/ xylazine (20 mg/kg IP) injection, and the brain was removed. It was immediately placed into ice-cold and carbogenated (95% oxygen, 5% carbon dioxide) dissection solution (Pelletier/ Carlen) containing 210 mmol/L sucrose, 3.5 mmol/L potassium chloride (KCl), 1 mmol/L calcium chloride (CaCl2) dihydrate, 4 mmol/L magnesium chloride hexahydrate, 1.25 mmol/L sodium phosphate monobasic hydrate, 10 mmol/L glucose, and 26 mmol/L sodium bicarbonate (NaHCO3). The brain was subsequently cut on a Leica VT1000S vibratome. Acute coronal slices were 300 m thick. After 1 hour recovery in carbogenated artificial cerebrospinal fluid solution (ACSF), the slice was transferred into the recording chamber with perfusion of ACSF set to 1 mL/min. Temperature was set to 37°C. The ACSF bath contained (in mmol/L): 124 NaCl, 2.0 KCl, 1.25 potassium phosphate monobasic, 2.0 magnesium sulfate, 25 NaHCO3, 1.0 CaCl2, 11 glucose, pH ⫽ 7.3. Details of the amperometric recordings have been described elsewhere (27,28). Local bath application of the dopamine reuptake blocker nomifensine (3 mol/L) and quinpirole (10 mol/L) for at least 30 min was also used to assess the contribution of reuptake and the D2R autoreceptor in the evoked dopamine signal. The five single pulses/slice are averaged into a grand mean, and the two groups (MCH⫹/⫹ vs. MCH⫺/⫺) are compared with one-way analysis of variance (ANOVA) of the means.
Results MCHR1 Is Highly Expressed in the Ventral Striatum But Not in the Ventral Tegmental Area Expression of MCHR1 in mouse brain was determined by in situ hybridization of antisense riboprobe according to published procedures (25). A strong signal was detected in the terminal regions of dopaminergic projections from the midbrain, especially in the ventral striatum, Acb, and the olfactory tubercle (Tu) (Figures 1A and 1B). The PFC and prelimbic (PrL) and infralimbic (IL) cortex showed intermediate levels of MCHR1 expression. More caudally, expression increased in the medial shell of www.sobp.org/journal
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P. Pissios et al. low dose of amphetamine (1 mg/kg) was chosen that does not produce a significant difference between saline- and amphetamine-treated MCH⫹/⫹ mice. The MCH⫺/⫺ mice developed a significant increase in activity compared with amphetaminetreated MCH⫹/⫹ mice on days 4 and 5 of the experiment (p ⬍ .05 by ANOVA for both days) (Figure S1 in Supplement 1). Seven days after the cessation of daily injections, all cohorts received amphetamine at 1 mg/kg of body weight. The MCH⫺/⫺ mice maintained significantly higher activity compared with the rest of the groups, demonstrating that chronic absence of MCH enhances the sensitizing effects of amphetamine in mice.
Figure 1. Melanin-concentrating hormone receptor 1 (MCHR1) is strongly expressed in the ventral striatum but not in the ventral tegmental area (VTA). (A–C) Coronal sections of mouse brain hybridized with antisense riboprobe to MCHR1. (D) No specific signal is detected in the striatum of the MCHR1 ⫺/⫺ mouse. Acb, nucleus accumbens; CPu, caudate-putamen; DTT, dorsal taenia tecta; IL, infralimbic cortex; Pir, Piriform cortex; PrL; prelimbic cortex; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; cc, corpus callosum; Tu, olfactory tubercle.
the Acb, but expression was also present throughout the CPu (Figure 1B). A minimal signal was seen in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) (Figure 1C). Slices from MCHR1⫺/⫺ mice were used as control for nonspecific hybridization, and no signal was detected (Figure 1D). MCHⴚ/ⴚ Mice Show Enhanced Sensitization to Repeated Amphetamine Administration One common assay for evaluating the mesolimbic system is behavioral sensitization to addictive drugs (29,30) wherein initial exposure to a psychostimulant such as amphetamine causes enhanced locomotor responses to subsequent administrations. Cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice were initially injected with saline, and no difference in locomotor activity between the two genotypes was observed (Figure 2A). Three days later, the same mice received a low dose of amphetamine (1 mg/kg of body weight), and the locomotor response was similar to that seen with saline, without any difference between the two genotypes (Figure 2B). Injection of a high dose of amphetamine (4 mg/kg) 3 days later produced a robust increase in locomotor activity in both MCH⫹/⫹ and MCH⫺/⫺ mice (Figure 2C). Although MCH⫺/⫺ mice tended to have more activity in the first hour, this difference did not reach statistical significance. Three days later, cohorts were re-challenged with the low dose of amphetamine (1 mg/kg). Both groups showed a robust increase in locomotor activity in response to this challenge, compared with their first exposure to the same dose, which confirmed behavioral sensitization (Figure 2B vs. 2D) (by paired t test within groups p ⬍ .0101 for MCH⫹/⫹ mice, p ⬍ .0003 for MCH⫺/⫺ mice for the first hour after injection). Importantly, MCH⫺/⫺ mice showed a significantly greater increase in locomotor activity compared with MCH⫹/⫹ mice [87% increase for the first hour after injection, F (1,15) ⫽ 7.49 p ⬍ .0153 by ANOVA]. Sensitization with a second paradigm of daily injections was also performed in separate cohorts of MCH⫹/⫹ and MCH⫺/⫺ mice. To detect the increased sensitization of MCH⫺/⫺ mice, a www.sobp.org/journal
The Striatum of MCHⴚ/ⴚ Mice Shows Altered Expression of Proteins Involved in Dopamine Signaling and Turnover To understand the molecular basis for enhanced sensitization of MCH⫺/⫺ mice, we measured total dopamine levels in mPFC, Acb, and CPu of MCH⫹/⫹ and MCH⫺/⫺mice. We also determined the protein expression of striatal D1R, D2R, and DAT by immunoblotting extracts from CPu and Acb tissue punches. No difference was observed in total dopamine content (ng/mg of total protein) by RIA between the two groups in the mPFC (MCH⫹/⫹ 2.6 ⫾ .48 vs. MCH⫺/⫺ 3.36 ⫾ .28), CPu (MCH⫹/⫹ 296.24 ⫾ 6.33 vs. MCH⫺/⫺ 300.73 ⫾ 10.06), or Acb (MCH⫹/⫹ 150.37 ⫾ 10.22 vs. MCH⫺/⫺ 160.45 ⫾ 10.86). We also measured dopamine and DOPAC, a dopamine metabolite, by high-performance liquid chromatography (HPLC) in the Acb of MCH⫹/⫹ and MCH⫺/⫺ mice and again found no difference between the genotypes (dopamine [ng/mg of total protein]:
Figure 2. Enhanced sensitization of melanin-concentrating hormone (MCH)⫺/⫺ mice to systemic amphetamine injections. The MCH⫺/⫺ (black symbols) and MCH⫹/⫹ mice (open symbols) mice were subjected to a series of injections of (A) saline, (B) amphetamine (1 mg/kg of body weight), (C) amphetamine (4 mg/kg), and (D) amphetamine (1 mg/kg) separated by 72 hours. Ambulatory activity is represented as a number of beam breaks/10min interval for 2 hours. Statistical significant difference between groups (MCH⫹/⫹, MCH⫺/⫺) was detected only for the last amphetamine injection (D), [F(1,15) ⫽ 7.49 p ⬍ .0153 by analysis of variance, n ⫽ 9 –12 animals/ group]. Note the different scales of the y axes.
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BIOL PSYCHIATRY 2008;64:184 –191 187 completely blocked the increase in locomotor activity induced by SKF81297 in both groups (Figure 4). No differences in locomotor responses between the two genotypes tested were observed. We then tested the effects of the antipsychotic haloperidol on locomotor activity. Typical antipsychotic drugs produce side effects such as catalepsy, which is mediated through D2R (31). To evaluate the functional response of D2R, mice were injected with different doses of haloperidol (.2 mg/kg and 2 mg/kg/body weight), and the induction of catalepsy was assessed with the bar test 30 and 60 min after IP injection. Catalepsy was defined as the latency (sec) to removal of forepaws from the bar. The MCH⫺/⫺ mice exhibited significantly less catalepsy at the low-dose haloperidol (.2 mg/kg) (Figure S2A in Supplement 1; p ⬍ .05 by ANOVA) than MCH⫹/⫹ mice. No significant difference between genotypes was observed at the higher dose of haloperidol (2 mg/kg) (Figure S2B in Supplement 1). The decreased sensitivity of MCH⫺/⫺mice to haloperidol is consistent with decreased expression of D2R in the striatum of MCH⫺/⫺ mice, because D2R⫺/⫹ mice are less sensitive and D2R⫺/⫺mice are completely resistant to the cataleptic effects of haloperidol (32).
Figure 3. Altered expression of dopamine D2 receptor (D2R) and dopamine transporter (DAT) in the dorsal and the ventral striatum of MCH⫺/⫺mice. (A) Representative immunoblots and quantitation for dopamine D1 receptor (D1R), D2R, and DAT protein levels from CPu of MCH⫹/⫹ and MCH⫺/⫺ mice (p ⬍ .01, n ⫽ 6 – 8 animals/group for D2R by Student t test). (B) Representative immunoblots and quantitation for D1R, D2R, and DAT protein levels from Acb of MCH⫹/⫹ and MCH⫺/⫺ mice (p ⬍ .02, n ⫽ 6 – 8 animals/group for DAT by Student t test). (C) Quantitative polymerase chain reaction determination of messenger RNA (mRNA) levels for DAT from the VTA of MCH⫹/⫹ and MCH⫺/⫺ mice. Other abbreviations as in Figures 1 and 2.
MCH⫹/⫹ 91.62 ⫾ 9.06, MCH⫺/⫺ 96.52 ⫾4.48; DOPAC: MCH⫹/⫹ 13.86 ⫾ 1.21, MCH⫺/⫺ 12.20 ⫾ .54). Immunoblotting did not show any changes in the expression of D1R. In contrast, we found a 50% decrease in D2R expression in the CPu of MCH⫺/⫺ mice compared with MCH⫹/⫹ mice (p ⬍ .01) (Figure 3A). No difference in D2R expression was observed in the Acb. Immunoblotting also revealed an 80% increase of DAT in the Acb of MCH⫺/⫺ mice as compared with MCH⫹/⫹ mice (p ⬍ .02) (Figure 3B), whereas no difference was seen in the CPu. We also measured DAT messenger RNA in the VTA by quantitative polymerase chain reaction and found no difference between the genotypes (Figure 3C). These results indicate that the absence of MCH leads to region-specific compensatory adaptations in components of the dopamine system in the striatum. MCHⴚ/ⴚ Mice Show Normal Locomotor Responses to D1R and D2R Agonists But Are Less Sensitive to Haloperidol-Induced Catalepsy Altered expression of D2R and DAT suggest that mice lacking MCH might have abnormal responses to selective dopamine receptor agonists or antagonists. We examined the locomotor responses of MCH⫺/⫺ mice to the selective D1R agonist SKF81297 and the D2R agonist quinpirole. Injection of SKF81297 (3 mg/kg) produced a robust increase in locomotor activity in both MCH⫹/⫹ and MCH⫺/⫺ animals (Figure 4). Injection of quinpirole (2 mg/kg), as expected, significantly reduced the ambulatory activity of MCH⫹/⫹ and MCH⫺/⫺ mice (Figure 4). Co-injection of quiniprole with SKF81297
Acb Terminals of MCHⴚ/ⴚ Mice Exhibit Increased Evoked Dopamine Release and Reuptake Increased expression of DAT in the Acb of MCH⫺/⫺ mice should result in increased reuptake of dopamine into presynaptic terminals (33). To assess this possibility, we measured evoked release of dopamine from AcbSh terminals by amperometry in ex vivo slices. Under baseline conditions, no significant difference in dopamine signal (mean ⫾ SEM) was observed between the two genotypes (amplitude: MCH⫹/⫹ 16.9 ⫾ 1.7 pA vs. MCH⫺/⫺ 20.9 ⫾ 2.1 pA [n ⫽ 50 stimulations in 13 MCH⫹/⫹ slices; n ⫽ 75 stimulations in 16 MCH⫺/⫺ slices]) (Figure 5A). Application of a selective DAT inhibitor (3 mol/L nomifensine, for 30 min) significantly increased the dopamine signal in MCH–/– slices compared with slices from MCH⫹/⫹ mice (amplitude 20.0 ⫾ 2.0 pA in MCH⫹/⫹ vs. 48.0 ⫾ 9.0 pA in MCH⫺/⫺; n ⫽ 20 stimulations in four MCH⫹/⫹ slices, n ⫽ 20 stimulations in four MCH⫺/⫺ slices, *p ⬍ .01 by one-way ANOVA; Figure 5A). See also representative trace in Figure 5B. To assess the role of the inhibitory D2R autoreceptor, Acb slices were perfused with the D2R agonist quinpirole (10 mol/L
Figure 4. The MCH⫺/⫺ mice show normal locomotor responses to D1R and D2R agonists but are less sensitive to haloperidol-induced catalepsy. Ambulatory activity of MCH⫹/⫹ and MCH⫺/⫺ mice injected with either saline, SKF81297 (3 mg/kg), quinpirole (2 mg/kg), or SKF81297 ⫹ quinpirole. No difference was observed in the response of MCH⫺/⫺ (black symbols) and MCH⫹/⫹ mice (open symbols) to the D1R or D2R agonists. Abbreviations as in Figures 1–3.
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Figure 5. The MCH⫺/⫺mice exhibit increased electrically stimulated dopamine release in the presence of a DATspecific inhibitor. (A) The mean evoked dopamine signal amplitude shows no significant difference between MCH⫹/⫹ and MCH⫺/⫺ nucleus accumbens shell (AcbSh) slices in the absence of the DAT-specific inhibitor nomifensine. Signal amplitude increased significantly in AcbSh slices of MCH⫺/⫺ mice by application of nomifensine (3 mol/L) to the slice preparation for 30 min (*p ⬍ .01 by one-way analysis of variance). (B) Representative amperometric traces of electrical stimulationevoked dopamine release in acute coronal accumbens slices from MCH⫹/⫹ and MCH⫺/⫺ mice in the presence of the DAT-specific inhibitor nomifensine (3 mol/L). (C) Application of the D2R agonist quinpirole (10 mol/L, 30 min) causes significant reduction in nomifensinetreated slices of MCH⫹/⫹ and MCH⫺/⫺ mice. Increased evoked-dopamine signal in MCH⫺/⫺ slices persists even in the presence of quinpirole. (D) Representative amperometric traces of electrical stimulation-evoked dopamine release in acute coronal accumbens slices from MCH⫹/⫹ and MCH⫺/⫺ mice in the presence of nomifensine (3 mol/L for a total of 60 min) and quinpirole (10 mol/L for a total of 30 min). Other abbreviations as in Figures 1–3.
for 30 min) in the presence of 3 mol/L nomifensine, which caused a significant decrease in the evoked dopamine signal amplitude compared with vehicle in both genotypes: MCH⫹/⫹ with quinpirole 3.5 ⫾ .5 pA versus vehicle 20.4 ⫾ 2.2 pA (n ⫽ 28 stimulations in six slices from four mice; n ⫽ 20 stimulations in four slices in vehicle, p ⬍ .01 by ANOVA); MCH⫺/⫺ with quinpirole 7.0 ⫾ 1.5 pA versus vehicle 47.7 ⫾ 9.2 pA in vehicle (n ⫽ 20 stimulations in four slices from three MCH⫺/⫺ mice; n ⫽ 20 stimulations in four slices in vehicle, p ⬍ .01 by ANOVA; Figure 5C). Nevertheless, the evoked dopamine signal was significantly higher in MCH⫺/⫺ than MCH⫹/⫹ slices even after perfusion of saturating concentration of quinpirole (MCH⫹/⫹ 3.5 ⫾ .5 pA vs. MCH⫺/⫺ 7.0 ⫾ 1.5 pA, p ⬍ .01 by ANOVA), possibly suggesting an impaired D2R function in MCH⫺/⫺ mice (Figure 5C). See also representative trace in Figure 5D. These results suggest that increased DAT-mediated reuptake of dopamine likely acts to compensate for increased evoked
Figure 6. The activity of melanin-concentrating hormone (MCH)⫺/⫺ mice increases significantly above MCH⫹/⫹ mice by the dopamine reuptake inhibitor GBR 12909. The MCH⫺/⫺ and MCH⫹/⫹ mice were acclimatized overnight to the activity chambers and injected with vehicle IP the following day. The next day the same mice were injected with GBR 12909 (20 mg/kg) IP. Data are represented as beambreaks/10 min for 4 hours after injection. All runs were done between 12:00 PM and 4:00 PM; significant difference in activity between MCH⫹/⫹ and MCH⫺/⫺ mice was observed for the duration of the test (4 hours) [F(1,25) ⫽ 13.72, p ⬍ .001 by analysis of variance).
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dopamine release in Acb slices from MCH⫺/⫺ mice, an effect possibly enhanced by a defect in D2R autoreceptor function. Because of the compensation, increased dopamine release is not seen until application of DAT inhibitors blocks dopamine reuptake, unmasking increased dopamine release. MCHⴚ/ⴚ Mice Are More Sensitive to the LocomotorActivating Action of DAT Inhibitor GBR 12909 In Vivo The ex vivo results suggest that increased dopamine release might also occur in vivo and could be unmasked by the administration of dopamine reuptake blockers to mice. We used the highly selective dopamine reuptake blocker GBR 12909, which increases locomotor activity in mice. After injection with vehicle, no difference was noted in the locomotor activity of MCH⫹/⫹ and MCH⫺/⫺ mice over a 4-hour observation period (Figure 6). The next day, GBR 12909 was injected into the same mice at 20 mg/kg of body weight, and locomotor activity was
P. Pissios et al. measured as described in the preceding text. A significant increase in locomotor activity was observed within each genotype after GRB12909 injection compared with vehicle alone (p ⬍ .001 by paired t test for MCH⫹/⫹ and MCH⫺/⫺ mice). Importantly, MCH⫺/⫺ mice had substantially increased locomotor activity compared with MCH⫹/⫹ mice for the 4-hour duration of the test (Figure 6) [F (1,25) ⫽ 13.72, p ⬍ .001 by ANOVA]. Thus, compensatory adaptations in dopamine release and reuptake observed in ex vivo recordings seem to operate in MCH⫺/⫺ mice in vivo and, as is the case in vitro, can be unmasked by selective DAT blockers. MCHⴚ/ⴚ Mice Do Not Display Hyperphagia When Presented a High-Fat Palatable Diet We measured daily food consumption of standard chow compared with newly introduced high fat/sucrose palatable diet (HF) (Research Diets 12451) in singly housed MCH⫹/⫹ and MCH⫺/⫺ mice. No difference between the genotypes was observed in daily caloric intake of chow (days 1–3). When presented the HF diet, MCH⫹/⫹ mice increased their daily caloric intake above chow levels by 40% on Day 4 (first day of HF diet), slowly returning to chow levels by day 6. The MCH⫺/⫺ mice, in contrast, did not exhibit the hyperphagia on HF diet, and their daily caloric intake on HF was not different from chow (Figure 7).
Discussion Food intake is a complex behavior requiring integration of both homeostatic and hedonic inputs (34). Homeostatic signals provide information on energy status, whereas hedonic inputs mediate the rewarding aspects of feeding. Both inputs are essential, because animals must both perceive hunger and in consequence engage in activities that result in finding and consuming a meal. Furthermore, recent reports describe effects of peptides such as leptin (35,36) and ghrelin (37) on dopaminergic neurons, whose actions on feeding were thought to be limited to the hypothalamus. In this regard the relationship between the Acb and the LH is of interest, because these two areas have extensive reciprocal innervation. With multiple techniques we demonstrated that MCH⫺/⫺ mice have alterations in striatal-dependent behaviors. Total dopamine and dopamine metabolite content was not changed in MCH⫺/⫺ mice, indicating normal dopamine synthesis and degradation of MCH⫺/⫺ mice. However, we observed elevated DAT expression in the Acb, suggesting that clearance of extracellular dopamine might be increased in these animals. Amperometry recordings from AcbSh detected both increased dopamine release and increased dopamine reuptake in MCH⫺/⫺ mice. Furthermore, administration of selective DAT inhibitor increased locomotor activity significantly more in MCH⫺/⫺ mice than in MCH⫹/⫹ mice. We hypothesize that increased expression of DAT arises in compensation for increased release of dopamine from the VTA terminals in the Acb of MCH⫺/⫺ mice. However, the mechanism underlying increased dopamine release is not clear. It is possible that increased dopamine release results from impaired D2R negative feedback. This is consistent with the incomplete suppression of dopamine release by quinpirole in MCH⫺/⫺ mice. However, the effects of quinpirole are also subject to interpretation, because the incomplete suppression might also reflect the higher basal dopamine release seen MCH⫺/⫺ mice. Locomotor activity could in principle be affected as well by postsynaptic modifications of dopamine receptor signaling in
BIOL PSYCHIATRY 2008;64:184 –191 189 the medium spiny neurons. However, we were unable to demonstrate any such difference between MCH⫺/⫺ mice and MCH⫹/⫹ mice treated with D1R and D2R agonists. A similar response to D1R agonist is expected, because D1R expression is unchanged. In contrast D2R protein expression is 50% decreased in the CPu, and we expected an altered response to D2R agonists. Although a mechanism explaining the normal sensitivity of MCH⫺/⫺ mice to the D2R agonist quinpirole is lacking, it is possible that additional changes in the medium spiny neurons (possibly mediated by the glutamatergic input from the cortex to the Acb) might compensate for the decreased levels of D2R. Although decreased sensitivity to D2R agonist was not observed, MCH⫺/⫺ mice showed decreased sensitivity to the cataleptic actions of the D2R antagonist haloperidol, which has been reported in other models with decreased D2R expression (31,32). The striatum is the likely site of MCH action in the mesolimbic system. Although the VTA is important in mediating amphetamine sensitization, little MCH receptor is expressed in this area. Furthermore, a recent study failed to demonstrate acute effects of MCH on the firing pattern of dopaminergic and non-dopaminergic VTA neurons in brain slice recordings (38). Thus it is unlikely that our findings are mediated by direct effects of MCH on the VTA neurons. Although a direct action cannot formally be excluded, an indirect action through the mPFC and/or the Acb, which provide afferent connections to the VTA and also express significant levels of MCHR1, is more likely (Figure 1). Inhibition of D1R signaling by MCH has been demonstrated in the Acb (23). Another possibility is MCH acting presynaptically on afferents arriving from mPFC and Acb to the VTA (30,39). Although the precise contribution of these sites needs to be addressed in future experiments, the present data support a long-term inhibitory role of MCH within the mesolimbic system. Our results contrast with some of the findings in Smith et al. (22), who reported acute sensitivity of MCHR1⫺/⫺ mice to amphetamine and D1R agonists (although no sensitization experiments were conducted) and proposed a primarily postsynaptic mechanism for the amphetamine supersensitivity in MCHR1⫺/⫺ mice. Although the basis for a difference in acute amphetamine response between these models of ligand
Figure 7. Acute hyperphagia of high-fat palatable food (HF) is absent in melanin-concentrating hormone (MCH)⫺/⫺ mice. Daily consumption of standard low-fat chow was monitored for 3 days. Mice were then presented a HF diet for 3 more consecutive days. Acute overeating of HF diet is absent in MCH⫺/⫺ mice compared with wildtype mice on Day 4 (first day of HF diet) (p ⬍ .05 by analysis of variance).
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190 BIOL PSYCHIATRY 2008;64:184 –191 and receptor loss are not yet clear, it could be related to background strain differences—C57BL/6 in this report as opposed to C57BL/6X129SvEv hybrid background in Smith et al. (22). An intriguing alternative could be the activity of other peptides encoded by the MCH gene, such as neuropeptide-glutamic acid-isoleucine amide (NEI) (40). Despite the differences between the MCH⫺/⫺ models, the overall conclusion from MCHR1⫺/⫺ mice is consistent with our data in MCH⫺/⫺ mice in suggesting an inhibitory action of the MCH system in the striatum. A recent study also reported an atypical response of MCHR1⫺/⫺ mice to cocaine with absence of sensitization (41). The discrepancy between amphetamine and cocaine responses could simply be explained by the distinct mode of action of these drugs at different neural substrates and will need to be resolved by further experiments. Finally, we observed a potential consequence of the dysregulated mesolimbic system in MCH⫺/⫺ mice on eating behavior. The MCH⫺/⫺ mice did not exhibit a hyperphagic response when presented with a palatable diet. Because overeating of palatable foods is driven by hedonic factors, our results point to a disruption in food reward in MCH⫺/⫺ mice. A functional connection between the LH and reward was suggested almost 50 years ago when studies revealed that placement of electrodes in the LH resulted in robust electrical self-stimulation in rodents (42). Melanin-concentrating hormone neurons are located in the LH and are well positioned to integrate various inputs pertinent to homeostatic responses. Because the mesolimbic system plays a role in assigning an incentive salience to rewards such as food, it is tempting to speculate that MCH, which responds to nutritional status such as fasting, might modulate the adaptive behavioral responses of the animal through action on the mesolimbic circuitry. Energy homeostasis is a complex process that in addition to hunger and satiety involves devising strategies to seek, acquire, and consume food. The capacity of the MCH system to mediate integration among these diverse physiologic elements underscores its importance in the neural regulation of energy homeostasis.
This work was supported by National Institutes of Health DK56113 and DK56116 and a sponsored research agreement from Merck (EMF); DK069983 (EMF, ENP); and DK065872 and a Smith Family Investigator Award by the Medical Foundation (ENP). The CLAMS support was provided by the physiology core of PO1 DK 56116. Dr. Maratos-Flier is a recipient of a research award from the Yamanouchi Foundation. We would like to thank Drs. Joel Elmquist and Richard Palmiter for helpful discussions; Daniel Trombly and Xiaomei Wang for technical assistance; and Don Marsh (Merck) for providing us with the MCHR1-MCH⫺/⫺ mice. ARK is currently affiliated with the Jackson Laboratory at University of California at Davis-JAX West, West Sacramento, California. All authors reported no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. 1. Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, et al. (1992): The melanin-concentrating hormone system of the rat brain: An immuno- and hybridization histochemical characterization. J Comp Neurol 319:218 –245.
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