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Cerebellar dopamine D2 receptors regulate social behaviors

Abstract

The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.

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Fig. 1: Distribution of D2R-expressing cells in the cerebellum of D2-RCE and D2-RiboTag mice.
Fig. 2: Molecular identity of cerebellar D2R cells.
Fig. 3: Activation of cerebellar D2R decreases GluA2 phosphorylation at S880 and modulates synaptic excitation onto Purkinje cells.
Fig. 4: Generation of mice lacking D2R in Purkinje cells by using the AAV8-CMV-Cre-eGFP.
Fig. 5: Enhanced social behaviors in mice lacking D2R in Purkinje cells.
Fig. 6: Purkinje cell D2R overexpression impairs social behavior.
Fig. 7: Enhanced social behaviors in mice lacking D2R in Purkinje cells Crus I/II lobules.
Fig. 8: Decreased sociability in mice overexpressing D2R in Purkinje cells Crus I/II lobules.

Data availability

All data are available in the main text, extended data figures and Supplementary Information. Data supporting the findings of this study are also available from the corresponding authors upon reasonable request. Source data are provided with this paper.

References

  1. Ito, M. Mechanisms of motor learning in the cerebellum. Brain Res. 886, 237–245 (2000).

    CAS  PubMed  Article  Google Scholar 

  2. Medina, J. F., Nores, W. L., Ohyama, T. & Mauk, M. D. Mechanisms of cerebellar learning suggested by eyelid conditioning. Curr. Opin. Neurobiol. 10, 717–724 (2000).

    CAS  PubMed  Article  Google Scholar 

  3. Carta, I., Chen, C. H., Schott, A. L., Dorizan, S. & Khodakhah, K. Cerebellar modulation of the reward circuitry and social behavior. Science https://doi.org/10.1126/science.aav0581 (2019).

  4. Locke, T. M. et al. Purkinje cell-specific knockout of tyrosine hydroxylase impairs cognitive behaviors. Front. Cell. Neurosci. 14, 228 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Locke, T. M. et al. Dopamine D1 receptor-positive neurons in the lateral nucleus of the cerebellum contribute to cognitive behavior. Biol. Psychiatry 84, 401–412 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Schmahmann, J. D. & Caplan, D. Cognition, emotion and the cerebellum. Brain 129, 290–292 (2006).

    PubMed  Article  Google Scholar 

  7. Ruigrok, T. J. Ins and outs of cerebellar modules. Cerebellum 10, 464–474 (2011).

    PubMed  Article  Google Scholar 

  8. Schweighofer, N., Doya, K. & Kuroda, S. Cerebellar aminergic neuromodulation: towards a functional understanding. Brain Res. Brain Res. Rev. 44, 103–116 (2004).

    PubMed  Article  Google Scholar 

  9. Nelson, T. E., King, J. S. & Bishop, G. A. Distribution of tyrosine hydroxylase-immunoreactive afferents to the cerebellum differs between species. J. Comp. Neurol. 379, 443–454 (1997).

    CAS  PubMed  Article  Google Scholar 

  10. Cutando, L. et al. Regulation of GluA1 phosphorylation by d-amphetamine and methylphenidate in the cerebellum. Addict. Biol. 26, e12995 (2021).

    CAS  PubMed  Article  Google Scholar 

  11. Ikai, Y., Takada, M. & Mizuno, N. Single neurons in the ventral tegmental area that project to both the cerebral and cerebellar cortical areas by way of axon collaterals. Neuroscience 61, 925–934 (1994).

    CAS  PubMed  Article  Google Scholar 

  12. Ikai, Y., Takada, M., Shinonaga, Y. & Mizuno, N. Dopaminergic and non-dopaminergic neurons in the ventral tegmental area of the rat project, respectively, to the cerebellar cortex and deep cerebellar nuclei. Neuroscience 51, 719–728 (1992).

    CAS  PubMed  Article  Google Scholar 

  13. Panagopoulos, N. T., Papadopoulos, G. C. & Matsokis, N. A. Dopaminergic innervation and binding in the rat cerebellum. Neurosci. Lett. 130, 208–212 (1991).

    CAS  PubMed  Article  Google Scholar 

  14. Barili, P., Bronzetti, E., Ricci, A., Zaccheo, D. & Amenta, F. Microanatomical localization of dopamine receptor protein immunoreactivity in the rat cerebellar cortex. Brain Res. 854, 130–138 (2000).

    CAS  PubMed  Article  Google Scholar 

  15. Bouthenet, M. L., Martres, M. P., Sales, N. & Schwartz, J. C. A detailed mapping of dopamine D2 receptors in rat central nervous system by autoradiography with [125I]iodosulpride. Neuroscience 20, 117–155 (1987).

    CAS  PubMed  Article  Google Scholar 

  16. Boyson, S. J., McGonigle, P. & Molinoff, P. B. Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain. J. Neurosci. 6, 3177–3188 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Camps, M., Kelly, P. H. & Palacios, J. M. Autoradiographic localization of dopamine D1 and D2 receptors in the brain of several mammalian species. J. Neural Transm. Gen. Sect. 80, 105–127 (1990).

    CAS  PubMed  Article  Google Scholar 

  18. Flace, P. et al. The cerebellar dopaminergic system. Front. Syst. Neurosci. 15, 650614 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Mengod, G., Martinez-Mir, M. I., Vilaro, M. T. & Palacios, J. M. Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry. Proc. Natl Acad. Sci. USA 86, 8560–8564 (1989).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Eisenstein, S. A. et al. Characterization of extrastriatal D2 in vivo specific binding of [18F](N-methyl)benperidol using PET. Synapse 66, 770–780 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Matuskey, D. et al. Age-related changes in binding of the D2/3 receptor radioligand [11C]+PHNO in healthy volunteers. Neuroimage 130, 241–247 (2016).

    CAS  PubMed  Article  Google Scholar 

  22. Andreasen, N. C. & Pierson, R. The role of the cerebellum in schizophrenia. Biol. Psychiatry 64, 81–88 (2008).

    PubMed  PubMed Central  Article  Google Scholar 

  23. Baldacara, L. et al. Is cerebellar volume related to bipolar disorder? J. Affect. Disord. 135, 305–309 (2011).

    CAS  PubMed  Article  Google Scholar 

  24. Courchesne, E., Yeung-Courchesne, R., Press, G. A., Hesselink, J. R. & Jernigan, T. L. Hypoplasia of cerebellar vermal lobules VI and VII in autism. N. Engl. J. Med. 318, 1349–1354 (1988).

    CAS  PubMed  Article  Google Scholar 

  25. D’Mello, A. M. & Stoodley, C. J. Cerebro-cerebellar circuits in autism spectrum disorder. Front Neurosci. 9, 408 (2015).

    PubMed  PubMed Central  Google Scholar 

  26. Murakami, J. W., Courchesne, E., Press, G. A., Yeung-Courchesne, R. & Hesselink, J. R. Reduced cerebellar hemisphere size and its relationship to vermal hypoplasia in autism. Arch. Neurol. 46, 689–694 (1989).

    CAS  PubMed  Article  Google Scholar 

  27. Webb, S. J. et al. Cerebellar vermal volumes and behavioral correlates in children with autism spectrum disorder. Psychiatry Res. 172, 61–67 (2009).

    PubMed  PubMed Central  Article  Google Scholar 

  28. Gangarossa, G. et al. Characterization of dopamine D1 and D2 receptor-expressing neurons in the mouse hippocampus. Hippocampus 22, 2199–2207 (2012).

    CAS  PubMed  Article  Google Scholar 

  29. Puighermanal, E. et al. drd2-cre:ribotag mouse line unravels the possible diversity of dopamine d2 receptor-expressing cells of the dorsal mouse hippocampus. Hippocampus 25, 858–875 (2015).

    CAS  PubMed  Article  Google Scholar 

  30. Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).

    CAS  PubMed  Article  Google Scholar 

  31. Puighermanal, E. et al. Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum. Nat. Commun. 11, 1957 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Gall, D. et al. Altered neuronal excitability in cerebellar granule cells of mice lacking calretinin. J. Neurosci. 23, 9320–9327 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. McDonough, A. et al. Unipolar (dendritic) brush cells are morphologically complex and require Tbr2 for differentiation and migration. Front Neurosci. 14, 598548 (2020).

    PubMed  Article  Google Scholar 

  34. Schilling, K. & Oberdick, J. The treasury of the commons: making use of public gene expression resources to better characterize the molecular diversity of inhibitory interneurons in the cerebellar cortex. Cerebellum 8, 477–489 (2009).

    CAS  PubMed  Article  Google Scholar 

  35. Dal Toso, R. et al. The dopamine D2 receptor: two molecular forms generated by alternative splicing. EMBO J. 8, 4025–4034 (1989).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. De Mei, C., Ramos, M., Iitaka, C. & Borrelli, E. Getting specialized: presynaptic and postsynaptic dopamine D2 receptors. Curr. Opin. Pharmacol. 9, 53–58 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. Montmayeur, J. P. et al. Differential expression of the mouse D2 dopamine receptor isoforms. FEBS Lett. 278, 239–243 (1991).

    CAS  PubMed  Article  Google Scholar 

  38. Surmeier, D. J., Ding, J., Day, M., Wang, Z. & Shen, W. D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons. Trends Neurosci. 30, 228–235 (2007).

    CAS  PubMed  Article  Google Scholar 

  39. Chung, H. J., Steinberg, J. P., Huganir, R. L. & Linden, D. J. Requirement of AMPA receptor GluR2 phosphorylation for cerebellar long-term depression. Science 300, 1751–1755 (2003).

    CAS  PubMed  Article  Google Scholar 

  40. Steinberg, J. P. et al. Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression. Neuron 49, 845–860 (2006).

    CAS  PubMed  Article  Google Scholar 

  41. de Leeuw, C. N. et al. rAAV-compatible MiniPromoters for restricted expression in the brain and eye. Mol. Brain 9, 52 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. Broekman, M. L., Comer, L. A., Hyman, B. T. & Sena-Esteves, M. Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain. Neuroscience 138, 501–510 (2006).

    CAS  PubMed  Article  Google Scholar 

  43. Yang, M., Silverman, J. L. & Crawley, J. N. Automated three-chambered social approach task for mice. Curr. Protoc. Neurosci. https://doi.org/10.1002/0471142301.ns0826s56 (2011).

  44. Stoodley, C. J. et al. Altered cerebellar connectivity in autism and cerebellar-mediated rescue of autism-related behaviors in mice. Nat. Neurosci. 20, 1744–1751 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Guell, X., Gabrieli, J. D. E. & Schmahmann, J. D. Triple representation of language, working memory, social and emotion processing in the cerebellum: convergent evidence from task and seed-based resting-state fMRI analyses in a single large cohort. Neuroimage 172, 437–449 (2018).

    PubMed  Article  Google Scholar 

  46. Guell, X., Schmahmann, J. D., Gabrieli, J. & Ghosh, S. S. Functional gradients of the cerebellum. Elife https://doi.org/10.7554/eLife.36652 (2018).

  47. Buckner, R. L., Krienen, F. M., Castellanos, A., Diaz, J. C. & Yeo, B. T. The organization of the human cerebellum estimated by intrinsic functional connectivity. J. Neurophysiol. 106, 2322–2345 (2011).

    PubMed  PubMed Central  Article  Google Scholar 

  48. Gehlert, D. R. & Wamsley, J. K. Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride. Neurochem. Int. 7, 717–723 (1985).

    CAS  PubMed  Article  Google Scholar 

  49. Martres, M. P., Sales, N., Bouthenet, M. L. & Schwartz, J. C. Localisation and pharmacological characterisation of D2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride. Eur. J. Pharmacol. 118, 211–219 (1985).

    CAS  PubMed  Article  Google Scholar 

  50. Bouthenet, M. L. et al. Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA. Brain Res. 564, 203–219 (1991).

    CAS  PubMed  Article  Google Scholar 

  51. Mansour, A. et al. Localization of dopamine D2 receptor mRNA and D1 and D2 receptor binding in the rat brain and pituitary: an in situ hybridization-receptor autoradiographic analysis. J. Neurosci. 10, 2587–2600 (1990).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Meador-Woodruff, J. H. et al. Distribution of D2 dopamine receptor mRNA in rat brain. Proc. Natl Acad. Sci. USA 86, 7625–7628 (1989).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Brock, J. W., Farooqui, S., Ross, K. & Prasad, C. Localization of dopamine D2 receptor protein in rat brain using polyclonal antibody. Brain Res. 578, 244–250 (1992).

    CAS  PubMed  Article  Google Scholar 

  54. Khan, Z. U. et al. Differential regional and cellular distribution of dopamine D2-like receptors: an immunocytochemical study of subtype-specific antibodies in rat and human brain. J. Comp. Neurol. 402, 353–371 (1998).

    CAS  PubMed  Article  Google Scholar 

  55. Levey, A. I. et al. Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies. Proc. Natl Acad. Sci. USA 90, 8861–8865 (1993).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Kim, Y. S., Shin, J. H., Hall, F. S. & Linden, D. J. Dopamine signaling is required for depolarization-induced slow current in cerebellar Purkinje cells. J. Neurosci. 29, 8530–8538 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Kelly, M. A. et al. Locomotor activity in D2 dopamine receptor-deficient mice is determined by gene dosage, genetic background, and developmental adaptations. J. Neurosci. 18, 3470–3479 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Radl, D. et al. Differential regulation of striatal motor behavior and related cellular responses by dopamine D2L and D2S isoforms. Proc. Natl Acad. Sci. USA 115, 198–203 (2018).

    CAS  PubMed  Article  Google Scholar 

  59. Neve, K. A. et al. Normalizing dopamine D2 receptor-mediated responses in D2 null mutant mice by virus-mediated receptor restoration: comparing D2L and D2S. Neuroscience 248, 479–487 (2013).

    CAS  PubMed  Article  Google Scholar 

  60. Bolbecker, A. R. et al. Eye-blink conditioning anomalies in bipolar disorder suggest cerebellar dysfunction. Bipolar Disord. 11, 19–32 (2009).

    PubMed  Article  Google Scholar 

  61. Bolbecker, A. R. et al. Eye-blink conditioning deficits indicate temporal processing abnormalities in schizophrenia. Schizophr. Res 111, 182–191 (2009).

    PubMed  PubMed Central  Article  Google Scholar 

  62. Millan, M. J. et al. Selective blockade of dopamine D3 versus D2 receptors enhances frontocortical cholinergic transmission and social memory in rats: a parallel neurochemical and behavioural analysis. J. Neurochem. 100, 1047–1061 (2007).

    CAS  PubMed  Article  Google Scholar 

  63. Watson, D. J. et al. Selective blockade of dopamine D3 receptors enhances while D2 receptor antagonism impairs social novelty discrimination and novel object recognition in rats: a key role for the prefrontal cortex. Neuropsychopharmacology 37, 770–786 (2012).

    CAS  PubMed  Article  Google Scholar 

  64. Loiseau, F. & Millan, M. J. Blockade of dopamine D3 receptors in frontal cortex, but not in sub-cortical structures, enhances social recognition in rats: similar actions of D1 receptor agonists, but not of D2 antagonists. Eur. Neuropsychopharmacol. 19, 23–33 (2009).

    CAS  PubMed  Article  Google Scholar 

  65. Bariselli, S. et al. Role of VTA dopamine neurons and neuroligin 3 in sociability traits related to nonfamiliar conspecific interaction. Nat. Commun. 9, 3173 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  66. Fields, H. L., Hjelmstad, G. O., Margolis, E. B. & Nicola, S. M. Ventral tegmental area neurons in learned appetitive behavior and positive reinforcement. Annu. Rev. Neurosci. 30, 289–316 (2007).

    CAS  PubMed  Article  Google Scholar 

  67. Gunaydin, L. A. & Deisseroth, K. Dopaminergic dynamics contributing to social behavior. Cold Spring Harb. Symp. Quant. Biol. 79, 221–227 (2014).

    PubMed  Article  Google Scholar 

  68. Prévost-Solié, C. et al. Superior colliculus to VTA pathway controls orienting response to conspecific stimuli. Nat. Commun. 13, 817 (2022).

    Article  CAS  Google Scholar 

  69. Wagner, M. J., Kim, T. H., Savall, J., Schnitzer, M. J. & Luo, L. Cerebellar granule cells encode the expectation of reward. Nature 544, 96–100 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. Hess, E. J. & Wilson, M. C. Tottering and leaner mutations perturb transient developmental expression of tyrosine hydroxylase in embryologically distinct Purkinje cells. Neuron 6, 123–132 (1991).

    CAS  PubMed  Article  Google Scholar 

  71. Jeong, Y. G., Kim, M. K. & Hawkes, R. Ectopic expression of tyrosine hydroxylase in zebrin II immunoreactive Purkinje cells in the cerebellum of the ataxic mutant mouse, pogo. Brain Res. Dev. Brain Res. 129, 201–209 (2001).

    CAS  PubMed  Article  Google Scholar 

  72. Kempadoo, K. A., Mosharov, E. V., Choi, S. J., Sulzer, D. & Kandel, E. R. Dopamine release from the locus coeruleus to the dorsal hippocampus promotes spatial learning and memory. Proc. Natl Acad. Sci. USA 113, 14835–14840 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Beas, B. S. et al. The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism. Nat. Neurosci. 21, 963–973 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. Candelas, M. et al. Cav3.2 T-type calcium channels shape electrical firing in mouse Lamina II neurons. Sci. Rep. 9, 3112 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  75. Biever, A. et al. PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons. J. Neurosci. 35, 4113–4130 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  76. Franklin, K. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates. 3rd edn, 256 (Academic Press, 2008).

  77. Ceolin, L. et al. Cell-type-specific mRNA dysregulation in hippocampal CA1 pyramidal neurons of the fragile X syndrome mouse model. Front. Mol. Neurosci. 10, 340 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  78. Saywell, V., Cioni, J. M. & Ango, F. Developmental gene expression profile of axon guidance cues in Purkinje cells during cerebellar circuit formation. Cerebellum 13, 307–317 (2014).

    PubMed  Article  Google Scholar 

  79. Belle, M. et al. Tridimensional visualization and analysis of early human development. Cell 169, 161–173 (2017).

    CAS  PubMed  Article  Google Scholar 

  80. Cutando, L. et al. Microglial activation underlies cerebellar deficits produced by repeated cannabis exposure. J. Clin. Invest. 123, 2816–2831 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. Bruinsma, C. F. et al. An essential role for UBE2A/HR6A in learning and memory and mGLUR-dependent long-term depression. Hum. Mol. Genet 25, 1–8 (2016).

    CAS  PubMed  Article  Google Scholar 

  82. Galliano, E. et al. Synaptic transmission and plasticity at inputs to murine cerebellar Purkinje cells are largely dispensable for standard nonmotor tasks. J. Neurosci. 33, 12599–12618 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  83. Martinez-Torres, S. et al. Monoacylglycerol lipase blockade impairs fine motor coordination and triggers cerebellar neuroinflammation through cyclooxygenase 2. Brain Behav. Immun. 81, 399–409 (2019).

    CAS  PubMed  Article  Google Scholar 

  84. Moy, S. S. et al. Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes Brain Behav. 3, 287–302 (2004).

    CAS  PubMed  Article  Google Scholar 

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Acknowledgements

We thank E. Shogo and P. Isope for providing antibodies against G-substrate and zebrin II and the iExplore, MRI and PVM Platforms of the IGF for their involvement in the maintenance and breeding of the colonies, imaging facilities and preparation of viruses. In addition, we thank G. Mel de Fontenay for essential and invaluable help in data analysis. This work was supported by Inserm, Fondation pour la Recherche Médicale (DEQ20160334919), La Marató de TV3 Fundació (113-2016), ANR EPITRACES (ANR-16-CE16-0018), ANR DOPAFEAR (ANR-16-CE16-0006), ANR Bergmann & Co (ANR-20-CE37-0024; to E.V.), the Swiss National Science Funds (31003A-175549; to M.M.) and a NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation (to E.P.). L. Cutando was supported by the postdoctoral Labex EpiGenMed fellowship (Investissements d’avenir, ANR-10-LABX-12-01) and Marie Curie Intra-European Fellowship (101028078). E.P. was a recipient of a Marie Curie Intra-European Fellowship (IEF327648), a recipient of a Beatriu de Pinós fellowship (2017BP00132) from the University and Research Grants Management Agency (Government of Catalonia, Spain) and is currently a recipient of a Ramon y Cajal contract (RYC2020-029596-I). L. Castell was supported by the predoctoral Labex EpiGenMed (Investissements d’avenir, ANR-10-LABX-12-01).

Author information

Authors and Affiliations

Authors

Contributions

L. Cutando, E.P. and E.V. conceived and led the project. L. Cutando, E.P. and E.V. designed the study. L. Cutando and E.P. performed brain dissections. L. Cutando and E.P. performed polysome immunoprecipitation and RT–qPCR experiments. L. Cutando performed western blot analyses. L. Cutando, E.P. and P.T. performed immunofluorescence assessments. L. Cutando and L. Castell performed in situ hybridization analysis. L. Cutando performed stereotaxic injections and behavioral experiments. L. Cutando, M.B. and A.C. performed iDISCO methodology. L. Cutando and F.B. performed in vivo cerebellar slices. L. Cutando and M.A.-L. designed AAVDJ-Pcp2-Cre. L. Cutando and F.A. performed PC sorting. M.R. provided D2R-floxed mice. A.Q. participated in data analysis. M.M. performed electrophysiological recordings and related analyses. E.V. supervised the project. L. Cutando and E.V. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Laura Cutando or Emmanuel Valjent.

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The authors declare no competing interests.

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Nature Neuroscience thanks Christoph Kellendonk and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Distribution of HA-positive cells throughout all cerebellar lobules.

Distribution of HA-positive cells in PC (green), molecular (orange) and granular (red) layers throughout all cerebellar lobules in four coronal levels (4–6 sections per animal; n = 4 animals). Total number of HA-positive cells counted in Supplementary Table 2. Sim: simple lobule; PM: paramedian lobule; Cop: copula pyramidis. PCl: Purkinje cell layer; gl: granular layer; ml: molecular layer.

Extended Data Fig. 2 Validation of the D2-RiboTag mice.

a, qRT-PCR analysis showing the enrichment of Drd2, the PCs marker Pcp2 and Cre after HA-immunoprecipitation on CC extracts. All genes were normalized to Tbp. The pellet fraction, containing all mRNAs from D2R-expressing cells, was compared to the input fraction (containing the mRNAs from all cell types). Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test, **p < 0.01. Complete statistical analysis provided in Source Data Extended Data Fig. 2. b, Single fluorescent in situ hybridization for Drd2 (green), Cre (red) and Pvalb (white) mRNA in the CC of D2-RiboTag (n = 2) mice. Slides were counterstained with DAPI (blue). PCl: Purkinje cell layer; gl: granular layer; ml: molecular layer.

Source data

Extended Data Fig. 3 Distribution of D2R-expressing cells in the molecular layer.

a, Single immunofluorescence for HA in the CC of D2-RiboTag mice. b, Distribution of D2R neurons in the molecular layer (ml) in the lateral cerebellum and vermis. Cells found in the inner third of the ml with their soma close to PCl were classified as Basket cells (BCs), while those localized in the middle and outer part of the ml were marked as Stellate cells (SCs). Note that BCs are the most abundant D2R-expressing ML-INs in all the lobules analyzed. Total number of HA-positive cells counted in the lateral cerebellum (Sim = 128; Crus I = 84; Crus II = 115; PM = 99 and Cop = 54). Total number of HA-positive cells counted in the vermis (lobule II = 114; III = 175; IV-V = 292; VI = 160; VII = 73; VIII = 119; IX = 88 and X = 24) (3-4 slices/mice, n = 5 mice). PCl: Purkinje cell layer; ml: molecular layer; Sim: simple lobule; PM: paramedian lobule; Cop: copula pyramidis.

Extended Data Fig. 4 Characterization of DCN D2R neurons.

a, Double immunofluorescence for HA (green) and G-Substrate (red) in the DCN of D2-RiboTag (n = 3) mice. (b) qRT-PCR performed after HA-immunoprecipitation in DCN extracts from D2-RiboTag mice. All genes were normalized with Tbp. Data presented as fold change comparing the pellet fraction (green bars) vs. input fraction (grey bars) (n = 5 pooled samples of 2 mice / pool). Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test, **p < 0.01, ***p < 0.001. Complete statistical analysis provided in Source Data Extended Data Fig. 4. (c) Single molecule fluorescent in situ hybridization for Drd2 (green), Slc6a5 (red, Glyt2) and Slc32a1 (cyan, VGAT) mRNA in the DCN (n = 2 mice). Slides counterstained with DAPI (blue) DCN: deep cerebellar nuclei.

Source data

Extended Data Fig. 5 Conditional D2R knockout mice generated by using the AAVDJ-Pcp2-Cre.

a, Schematic representation of the 9th injection sites for the AAVDJ-Pcp2-Cre injection in the CC of Drd2+/+-RiboTag (control) and Drd2LoxP/LoxP-RiboTag (PC-D2R-cKO) mice. b, Immunoblot for HA, Cre and β-actin in control (n = 9) and PC-D2R-cKO (n = 8) mice. Western blot quantification was calculated by HA / β-actin and Cre / β-actin and shown as the fold change compared with the control group. Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test. Complete statistical analysis provided in Source Data Extended Data Fig. 5. c, Drd2 mRNA expression analysis by qRT-PCR in the cerebellum of control and PC-D2R-cKO mice. A reduction in Drd2 mRNA was observed in PC-D2R-cKO mice compared with control animals, further validating the viral approach used. Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test, *p < 0.05. Complete statistical analysis provided in Source Data Extended Data Fig. 5, *p < 0.05. d, Double immunofluorescence performed in cerebellar slices of PC-D2R-cKO (n = 3) mice for HA (green), parvalbumin (PV), calbindin-D-28k (CB), G-substrate and Cre recombinase (red). e, Distribution of HA-transduced cells within PC (green), molecular (orange) and granular (red) layers. Numbers between parentheses indicate the total counted cells. Note that ~50% of HA-transduced cells were not in PCl indicating a lack of specificity of AAVDJ-Pcp2-Cre virus. PCl: Purkinje cell layer; gl: granular layer; ml: molecular layer.

Source data

Extended Data Fig. 6 Quantification of PCs transduced after injection of AAV8-CMV-Cre-eGFP in the CC of Drd2+/+ (control) and Drd2LoxP/LoxP (PC-D2R-cKO) mice.

a, b, Double immunofluorescence for GFP (green) and DARPP-32 (red) in the cerebellum of C57BL/6 mice injected with AAV8-CMV-Cre-eGFP to show the extension of the viral transduction throughout all cerebellar lobules at coronal (a) and sagittal (b) levels. c, d, Quantification of PCs transduced after AAV8-CMV-Cre-eGFP delivery in control mice (grey bars) and PC-D2R-cKO mice (green bars). Quantifications performed at sagittal (c) and coronal (d) cerebellar levels (sagittal: 4–6 sections analyzed per mouse, n = 4 control mice and n = 5 PC-D2R-cKO mice; coronal: 7–11 sections analyzed per mouse, n = 4 control mice and n = 5 PC-D2R-cKO mice). Data are presented as mean values ± s.e.m.. Sim: simple lobule; PM: paramedian lobule; Cop: copula pyramidis.

Extended Data Fig. 7 Ablation of cerebellar D2R cells enhances social behaviors.

a, Schematic representation of the 9th injection sites for the AAVDJ-Pcp2-Cre injection in the CC of Drd2+/+-RiboTag (control) and Drd2LoxP/LoxP-RiboTag (PC-D2R-cKO) mice. b, Locomotor activity assessment in the circular corridor (right panels). No differences were detected between control (n = 10) and PC-D2R-cKO (n = 9) mice neither in the horizontal nor in the vertical activity (rearings). c, Evaluation of the time spent grooming in control (n = 9) and PC-D2R-cKO (n = 9) mice. d, Motor coordination evaluation in the Beam walking test. Number of footslips displayed by control (n = 9) and PC-D2R-cKO (n = 9) mice on the wide and narrow beams. e, Motor coordination assessment using the coat-hanger test. No differences were observed between control mice (n = 9) and PC-D2R-cKO mice (n = 9) neither in the fall latency nor in the number of movements. f, Cerebellar motor adaptation evaluation using accelerating rotarod. The difference in the speed and the fall latency was compared between the 10th and the 1st trial during one-day session (control: n = 9; PC-D2R-cKO = 8). g, Accelerating Rotarod performance of control and PC-D2R-cKO mice. Speed and fall latency were evaluated across 5 consecutive days at 4–40 rpm and the 6th day at 4–80 rpm (control: n = 9; PC-D2R-cKO = 9). h, k, Diagram of the apparatus used to assess (h) sociability or (k) preference for social novelty. i, l, Representative heat maps of control and PC-D2R-cKO mice during (i) sociability and (l) preference for social novelty evaluation. j, m, Total sniffing time spent by control (n = 19) and PC-D2R-cKO (n = 15) mice toward (j) the object and stranger 1 or (m) toward stranger 1 (familiar mouse) and stranger 2 (novel mouse). n, o, Time spent and number of entries in each compartment during the evaluation of sociability. p, q, Time spent and number of entries in each compartment during the evaluation of preference for social novelty. Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test (b-f) or two-way ANOVA followed by Bonferroni’s post-hoc test (g, j and n-q), **p < 0.01, ***p < 0.001 (behavioral task effect); ##p < 0.01 (genotype effect). Complete statistical analysis provided in Source Data Extended Data Fig. 7.

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Extended Data Fig. 8 Quantification of PCs transduced after injection of AAV8-CMV-eGFP (control) or AAV8-CMV-mDrd2-IRES-eGFP (PC-D2R-OE) into the CC of C57BL/6 mice.

a-c, Distribution of GFP/DARPP-32-positive PCs throughout all cerebellar lobules in two sagittal (4-5 slices per mouse; n = 3 control mice and n = 3 PC-D2R-OE mice) (a and b) and coronal (c) (4-5 slices per mouse; n = 3 control mice and n = 3 PC-D2R-OE mice) levels of the cerebellum. Data are presented as mean values ± s.e.m.. Sim: simple lobule; PM: paramedian lobule; Cop: copula pyramidis.

Extended Data Fig. 9 Effect of quinpirole in PC-D2R-OE mice.

a, Plotted values of whole-cell voltage-clamp recordings of PF-PC EPSCs during 15 min in wild-type and PC-D2R-OE mice. A decrease in the EPSCs amplitude was observed when quinpirole was applied during the interval 5–10 in both groups, being slightly more pronounced although non-statistically significant in the PC-D2R-OE group (olive color bar, n = 7 neurons recorded from 3 mice) than in wild-type mice (green bar, n = 8 neurons recorded from 4 mice). Data analyzed by two-way ANOVA multiple comparisons followed by Bonferroni’s post-hoc test. Complete statistical analysis provided in Supplementary Table 8. b, Representation of the Area Under Curve (AUC) comparison between ‘Baseline’ (grey) and ‘After Quinpirole (10 µM) application’ (blue) in wild-type and PC-D2R-OE mice. A more robust reduction in the AUC was found in PC-D2R-OE mice than in wild-type animals. c, % of PF-PC EPSCs reduction between ‘Baseline’ (grey) and ‘After Quinpirole (10 µM) application’ in wild-type (green) and PC-D2R-OE (olive color) mice. In wild-type mice, 5 minutes of Quinpirole (10 µM) application triggered a reduction of ~13% in the PF-PC EPSCs amplitude (p = 0.048). Interestingly, Quinpirole (10 µM) application in PC-D2R-OE mice produced a slightly higher effect reaching a reduction around ~17% (p < 0.0001). However, when the effect of Quinpirole in both genotypes was compared (a), no differences were found. Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test, *p < 0.05, **p < 0.01, ***p < 0.001. Complete statistical analysis provided in Source Data Extended Data Fig. 9.

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Extended Data Fig. 10 Locomotor activity and motor coordination are not impaired in mice overexpressing D2R in PCs.

a, Schematic representation of the 9 injection sites for the AAV8-CMV-eGFP (control) or AAV8-CMV-mDrd2-IRES-eGFP (PC-D2R-OE) into the CC of C57BL/6 mice. b, Locomotor activity evaluation in the circular corridor. No differences were detected between control (n = 12) and PC-D2R-OE (n = 12) mice groups neither in the horizontal nor in the vertical activity (rearings). c, Time spent grooming was equivalent between control (n = 9) and PC-D2R-OE (n = 9) mice. d, Motor coordination evaluation in the Beam walking test. The number of footslips displayed by control (n = 12) and PC-D2R-OE (n = 12) mice in both wide and narrow beams was quantified. e, Motor coordination assessment using the Coat-hanger test. No differences were observed between control mice (n = 12) and PC-D2R-OE mice (n = 12) neither in the fall latency nor in the number of movements. f, Cerebellar motor adaptation evaluation using accelerating Rotarod. The difference in the speed and the fall latency were compared between the 10th and the 1st trial during one-day session (control: n = 12; PC-D2R-OE = 12). g, Accelerating Rotarod performance of control and PC-D2R-OE mice. Speed and fall latency were evaluated across 5 consecutive days at 4–40 rpm and the 6th day at 4–80 rpm (control: n = 9 mice; PC-D2R-OE: n = 9). Data are presented as mean values ± s.e.m. and analyzed by two-sided t-test (b-f) or two-way ANOVA followed by Bonferroni’s post-hoc test (g). Complete statistical analysis provided in Source Data Extended Data Fig. 10.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and Supplementary Information

Reporting Summary

Supplementary Video 1

Three-dimensional visualization of cerebellar D2R-expressing cells.

Supplementary Table

Supplementary Tables 1–7

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Cutando, L., Puighermanal, E., Castell, L. et al. Cerebellar dopamine D2 receptors regulate social behaviors. Nat Neurosci (2022). https://doi.org/10.1038/s41593-022-01092-8

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