© 2001 Nature Publishing Group http://neurosci.nature.com
© 2001 Nature Publishing Group http://neurosci.nature.com
brief communications
Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice Christian Chiamulera1*, Mark P. Epping-Jordan2*, Alessandro Zocchi1,3, Clara Marcon1, Cécilia Cottiny2, Stefano Tacconi1, Mauro Corsi1, Francesco Orzi3,4 and François Conquet2 1 Department of Biology, GlaxoSmithKline Laboratories, via Fleming 4, 37100
Verona, Italy 2 GlaxoSmithKline R&D, Institut de Biologie Cellulaire et de Morphologie, rue
du Bugnon 9, 1005 Lausanne, Switzerland 3 Neuromed Institute, via Atinense 18, 86077 Pozzilli, Italy 4 Department of Neurological Sciences, University of Rome La Sapienza,
Piazzale Aldo Moro 5, 00185 Rome, Italy * The first two authors contributed equally to this work.
Correspondence should be addressed to F.C. (
[email protected])
Both ionotropic and metabotropic glutamate receptors (mGluRs) are involved in the behavioral effects of pyschostimulants1–3; however, the specific contributions of individual mGluR subtypes remain unknown. Here we show that mice lacking the mGluR5 gene do not self-administer cocaine, and show no increased locomotor activity following cocaine treatment, despite showing cocaine-induced increases in nucleus accumbens (NAcc) dopamine (DA) levels similar to wild-type (WT) mice. These results demonstrate a significant contribution of mGlu5 receptors to the behavioral effects of cocaine, and suggest that they may be involved in cocaine addiction. Both acute and repeated cocaine administration increase glutamate concentrations in the NAcc4,5, a brain region associated with the reinforcing and locomotor effects of cocaine6,7. Systemic and brain injections of non-selective mGluR agonists and antagonists mediate baseline and psychostimulant-induced locomotor activity 1–3 . mGluR5 is highly expressed in the NAcc 8 , and repeated systemic cocaine treatment increases mGluR5 mRNA levels in the NAcc shell and dorsolateral stria-
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nature neuroscience • volume 4 no 9 • september 2001
Fig. 1. Locomotor response to cocaine in mGluR5 WT (n = 14) and null mutant (n = 16) mice. Values represent mean percent activity counts ± s.e.m. *p < 0.05 versus saline; **p < 0.01 versus saline (Dunnett’s test after two-way repeated-measures analysis of variance; ANOVA). For detailed methods, see the supplementary information page of Nature Neuroscience online.
tum9; however, the functional role of mGluR5 in cocaine effects remains unknown. To investigate this role, we first examined the locomotor responses to cocaine in F5 generation mGluR5 WT and null mutant mice. Baseline locomotor activity did not differ between mutant and WT mice (mutant, 2710 ± 642; WT, 2393 ± 479, mean horizontal activity counts/45-min session ± s.e.m.). Cocaine induced a significant, dose-dependent increase in locomotor activity in WT mice, but did not alter locomotor activity in mutant mice at any time point or dose tested (Fig. 1). Although mice received repeated cocaine exposure that may have induced some behavioral sensitization in WT mice, locomotor activity was not increased in mutant mice. Our results indicate that mGluR5 is essential for cocaineinduced hyperactivity. To investigate whether the reinforcing properties of cocaine were affected by the mGluR5 mutation, we examined intravenous cocaine self-administration (SA) in WT and mutant mice. Acquisition of a discriminated two-lever food-reinforced task did not differ between WT and mutant mice (Fig. 2a). When intravenous cocaine was substituted for food, WT mice acquired stable cocaine SA across a typical dose range10, but mutant mice did not selfadminister cocaine at any dose tested (Fig. 2b). Active lever responding in mutant mice extinguished within three to five sessions at all cocaine doses, and no mutant mouse acquired stable SA, suggesting that the reinforcing properties of cocaine are absent in mice lacking mGluR5. Data from the food training suggest that the failure to acquire cocaine SA was not due to an inability to learn the lever-press task, and that the reinforcing properties of food are unchanged in mutant mice. The selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) 11 dose-dependently decreased cocaine SA (Fig. 2c) in C57Bl/6J mice. The effect of MPEP was specific to cocaine reinforcement, as MPEP had no effect on the rate of foodreinforced lever pressing (Fig. 2d) under the same schedule of reinforcement as during cocaine SA. These results suggest that Fig. 2. Food and cocaine reinforcement. (a) Acquisition to criterion of food-reinforced lever pressing did not differ in mGluR5 WT (n = 5) and null mutant (n = 6) mice. Values represent mean ± s.e.m. (Student’s t-test). (b) Cocaine SA in mice shown in (a). Values represent mean number of injections for 2 sessions at each dose ± s.e.m. *p < 0.05 versus saline within genotype; ***p < 0.05 WT at 0.4 versus WT at 3.2 mg/kg/injection (Bonferroni-corrected Student’s t-tests after two-way repeated-measures ANOVA). (c) MPEP dose-dependently decreased cocaine SA in C57Bl/6J mice (n = 5). Values represent mean percent of baseline number of injections per 1 h session at 0.8 mg/kg/injection, *p < 0.05 versus saline; ***p < 0.05 versus 3 mg/kg MPEP (means comparisons after one-way ANOVA). (d) MPEP (30 mg/kg i.v.) had no effect on the number of food-reinforced lever presses per minute in C57Bl/6J mice (n = 5) (repeated-measures t-test). For detailed methods, see the supplementary information page of Nature Neuroscience online. 873
brief communications
© 2001 Nature Publishing Group http://neurosci.nature.com
© 2001 Nature Publishing Group http://neurosci.nature.com
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the absence of cocaine SA in mutant mice was due to the loss of mGlu5 receptors and not to developmental alterations resulting from the genetic mutation. Numerous studies have reported that self-administered cocaine increases DA levels in the ventral striatum6, and psychostimulantinduced locomotor activity and increased mesoaccumbens DA levels in mice are closely correlated12. Because mutant mice showed neither a cocaine-induced locomotor response nor cocaine SA, we examined the effect of cocaine on NAcc DA levels using microdialysis in conscious, freely moving WT and mutant mice12. Wild-type and mutant mice had similar basal levels of extracellular DA. Cocaine-induced (10 mg/kg, i.p.) increases in extracellular DA levels did not differ between WT and mutant mice (Fig. 3b). These results suggest that the absence of mGluR5 affects neither baseline nor cocaine-induced increases in NAcc DA levels. To ensure that changes in responses to cocaine in mutant mice were not due to mGluR5 mutation-induced alterations in dopaminergic elements, we investigated the brain distribution and expression of DA receptors and the DA transporter (DAT) in mutant and WT mice. No differences were found between WT and mutant mice in binding of the selective radiolabeled compounds 3H-SCH23390 to D1-like and 3H-YMO91512 to D2-like DA receptors and 3-WIN35,428 to the DAT, or in expression of D1 or D2 DA receptor mRNA examined by in situ hybridization (data not shown). These findings indicated that the expression and distribution of DA receptors and of the DAT are not altered in the mutant mice. Several neurotransmitters and peptides contribute to cocaine dependence6,7. Although evidence supports a primary involvement for DA, the precise roles of specific DA receptor subtypes 874
Fig. 3. Extracellular NAcc DA levels in mGluR5 WT and null mutant mice. (a) Dialysis probe location. Solid and dashed boxes indicate the minimum and maximum extent of probe placements. ac, anterior commissure; CPu, caudate putamen; LV, lateral ventricle; NAccC, nucleus accumbens core; NAccSh, nucleus accumbens shell. (b) DA level analysis. 10 µl samples were collected every 20 min from WT and mutant mice (saline, n = 3/genotype; cocaine, n = 3/genotype) and were analyzed by HPLC. Values represent mean pg/sample DA ± s.e.m. *p < 0.05; **p < 0.01 cocaine versus saline at the same time point within genotype (Bonferroni-corrected Student’s t-test after separate two-way repeatedmeasures ANOVA). Extracellular DA levels were not significantly different between cocaine-treated WT and mutant groups (repeated-measures ANOVA, no significant main effects or interaction). For detailed methods, see the supplementary information page of Nature Neuroscience online.
remain unclear. D1- or D2-like DA receptor antagonists reduce the reinforcing effects of cocaine6,7; however, D1 receptor mutant mice acquire a cocaine-conditioned place preference13 and D2 receptor mutant mice self-administer cocaine (S.B. Caine et al., Soc. Neurosci. Abstr. 26, 681.8, 2000). Likewise, the exact mechanisms of the mGluR5 contribution to cocaine dependence are not known. It is possible that glutamate acts in synergy with mesolimbic DA afferents into the NAcc to mediate the effects of cocaine. Acute cocaine increases extracellular NAcc dopamine14 and glutamate4 levels, and these effects are enhanced after repeated cocaine administration5,14. Excitatory amino acid (most likely glutamatergic) and mesolimbic dopaminergic terminals form synapses on single NAcc neurons2,3. Nucleus accumbens output neurons express both DA2,6,7 and mGlu58 receptors. As has been suggested for locomotor activity2, mGluR subtypes expressed by NAcc projection neurons may interact with dopaminergic inputs through their respective intracellular signaling pathways to influence the reinforcing effects of cocaine. Regardless of the specific mechanisms involved, the present results suggest that mGluR5 is essential in cocaine SA and locomotor effects. Note: Supplementary methods are available on the Nature Neuroscience web site (http://neuroscience.nature.com/web_specials).
ACKNOWLEDGEMENTS We thank A. Morrison and C. Corti for their contribution in the establishment and detection of the mutation, F. Fornai and E. Grouzman for HPLC analyses, and M. Geyer, D. Lavery and E. Ratti for reviewing the manuscript. This work was supported by GlaxoSmithKline R&D.
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nature neuroscience • volume 4 no 9 • september 2001