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Cerebellar dopamine D2 receptors regulate social behaviors

Nature Neuroscience volume 25, pages 900–911 (2022)Cite this article

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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.

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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

Author notes

  1. Laura Cutando

    Present address: Institut de Neurociències and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain

  2. Emma Puighermanal

    Present address: Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain

  3. Fabrice Ango

    Present address: INM, Univ. Montpellier, CNRS, Inserm, Montpellier, France

Authors and Affiliations

  1. IGF, Univ. Montpellier, CNRS, Inserm, Montpellier, France

    Laura Cutando, Emma Puighermanal, Laia Castell, Pauline Tarot, Federica Bertaso, Margarita Arango-Lievano, Fabrice Ango & Emmanuel Valjent

  2. Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France

    Morgane Belle & Alain Chédotal

  3. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, CONICET; FCEN, Universidad de Buenos Aires, Buenos Aires, Argentina; and Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA

    Marcelo Rubinstein

  4. Institut de Neurociències and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain

    Albert Quintana

  5. Department of Fundamental Neuroscience, University of Lausanne, Lausanne, Switzerland

    Manuel Mameli

  6. Inserm UMR-S 1270, Paris, France

    Manuel Mameli

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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.

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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 25, 900–911 (2022). https://doi.org/10.1038/s41593-022-01092-8

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