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SHANK3 controls maturation of social reward circuits in the VTA

Nature Neuroscience volume 19, pages 926–934 (2016)Cite this article

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Abstract

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of autism spectrum disorder. How SHANK3 insufficiency affects specific neural circuits and how this is related to specific symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the ventral tegmental area of mice. We identified dopamine (DA) and GABA cell-type-specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate behavioral deficits, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors mGluR1 during the first postnatal week restored DA neuron excitatory synapse transmission and partially rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired ventral tegmental area function to social behaviors and identify mGluR1 modulation during postnatal development as a potential treatment strategy.

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Figure 1: Neonatal AAV-shShank3 infections target VTA DA and GABA neurons.
Figure 2: SHANK3 downregulation alters the postnatal development of AMPAR-mediated transmission.
Figure 3: SHANK3 downregulation affects excitatory transmission to VTA GABA neurons.
Figure 4: Stimulation of mGluR1 rescues synaptic deficits.
Figure 5: VTA SHANK3 insufficiency alters in vivo DA neuron activity.
Figure 6: VTA SHANK3 insufficiency induces social deficits that are reversed by PAM-mGluR1 treatment.
Figure 7: Synaptic and social deficits persist into adulthood and are reversed by treatment with PAM-mGluR1 during the critical period.
Figure 8: Optical stimulation of VTA DA neurons increases social preference.

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Acknowledgements

We thank M. Mameli, D. Jabaudon and F. Gardoni for the critical reading of the manuscript. C.B is supported by the Swiss National Science Foundation, Pierre Mercier Foundation and NCCR Synapsy. C.L. is supported by the Swiss National Science Foundation and by the European Research Council (MeSSI Advanced grant). This work was supported by a grant from the Simons Foundation (SFARI #239496 to C.L.).

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Author notes

  1. Sebastiano Bariselli and Stamatina Tzanoulinou: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland

    Sebastiano Bariselli, Stamatina Tzanoulinou, Christelle Glangetas, Clément Prévost-Solié, Luca Pucci, Paola Bezzi & Camilla Bellone

  2. Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, Bordeaux, France

    Christelle Glangetas & François Georges

  3. Université de Bordeaux, Bordeaux, France

    Christelle Glangetas & François Georges

  4. Department of Basic Neurosciences, Medical Faculty, University of Geneva, Geneva, Switzerland

    Joanna Viguié, Eoin C O'Connor & Christian Lüscher

  5. Centre National de la Recherche Scientifique, Neurodegenerative Diseases Institute, Bordeaux, France

    François Georges

  6. Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland

    Christian Lüscher

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Contributions

All VTA SHANK3 infections were performed by S.B. In vitro electrophysiology experiments were performed by S.B. Behavioral experiments were performed by S.T., with assistance from C.P.-S., S.B. and E.C.O'C. S.B. and S.T. performed the statistical analyses for the in vitro electrophysiology and the behavioral experiments, and contributed to the statistical analysis of the in vivo electrophysiology experiments. In vivo recordings were performed by C.G. and F.G. Immunohistochemistry was performed by S.B., J.V. and C.G. Western blots were performed by L.P. and P.B. The study was designed and the manuscript written by C.B., with assistance from S.B., E.C.O., C.L. and F.G.

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Correspondence to Camilla Bellone.

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Integrated supplementary information

Supplementary Figure 1 AAV-shShank3 targets DA and non-DA neurons within the VTA

(a) Top: schematic of the immunostaining experiment performed at P14 on VTA AAV-shShank3 infected mice. Representative confocal image shows ZsGreen expression in DA neurons (experiment repeated in 3 mice). (b) Top: schematic of cell counting experiments performed in adolescent WT animals injected with AAV-shShank3 at P5 (for details see Materials and Methods). Quantification of viral infection in TH+ (DA neurons; yellow, 61.1% ZsGreen+; red, 38.9% ZsGreen-) and TH- cells (non DA cells; pink, 39.5% ZsGreen+; blue, 60.5% ZsGreen-). (c) Left: representative images of immunostaining experiments performed at P28 on unilaterally injected mice at P5 shows TH and ZsGreen expression for the Uninfected (ZsGreen-) and the shShank3 infected (ZsGreen+) side (experiment repeated in 5 mice). Scale bar: 50 µm. Right: quantification of dopaminergic (TH+) neurons from infected (ZsGreen+) and Uninfected (ZsGreen-) side (U = 10, Mann-Whitney test). The numbers indicate the mice.

Supplementary Figure 2 Cell capacitance and input resistance in putative DA and GABA neurons

(a) Left: bar graph representing the mean value of cell capacitance of Uninfected Putative DA and GABA neurons (U < 0.001, Mann-Whitney test). Right: bar graph representing the mean value of input resistance of Uninfected Putative DA and GABA neurons (U = 13.0, Mann-Whitney test). (b) Left: bar graph representing the mean value of cell capacitance of shShank3 infected Putative DA and GABA neurons (U < 0.001, Mann-Whitney test). Right: bar graph representing the mean value of input resistance of shShank3 infected Putative DA and GABA neurons (U = 10, Mann-Whitney test). The numbers indicate cells and mice.

Supplementary Figure 3 Group I mGluR activation does not rescue synaptic deficits onto putative GABA neurons in the VTA

(a) Top: schematics of the experiment. Time course of AMPAR-EPSCs recorded at –60 mV before and after 5 minutes DHPG (20 µM) bath application from Uninfected and shShank3 infected VTA Putative GABA neurons. Inset: 1/2 example traces before and 25 minutes after DHPG application. (b) Top: schematics of the experiment. Bar graph representing the mean value of AMPA/NMDA recorded from shShank3 infected Putative GABA neurons from Vehicle and mGluR1-PAM Ro 677476 treated animals (t18 = 0.123, unpaired t-test). Right: example traces of AMPAR- and NMDAR-EPSCs recorded at +40 mV.

Supplementary Figure 4 Proportion of bursting and non-bursting DA neurons

(a) Experimental protocol and histological control of the injection site of the AAV-shShank3 virus in the VTA. Representative TH-immunostaining performed to delineate VTA dopaminergic area (red labeling). Right: co-localisation of TH positive neurons with shShank3 positive neurons in the VTA. (b) Graph representing the proportion of VTA DA neurons with a bursting pattern compared to VTA DA neurons without bursting activity in scrShank3 and in shShank3 mice treated with i.p. injection of Vehicle (U = 8, Mann-Whitney test). The numbers indicate the mice.

Supplementary Figure 5 Additional parameters of social behavior and locomotor activity in VTA SHANK3 mice

(a) Bar graph representing the duration of interaction with the social stimulus during the first half of the three-chamber social preference test (T1) (two-way ANOVA; no main effects and no interactions). (b) Bar graph representing the duration of interaction with the object during T1 (two-way ANOVA; Virus × drug interaction: F1,54 = 4.96, p = 0.030; main effect virus: F1,54 = 0.20, p = 0.655; main effect drug: F1,54 = 0.09, p = 0.763; followed by Tukey HSD post-hoc test). (c) Bar graph representing the duration of interaction with the object during the second half of the three-chamber social preference test (T2) (two-way ANOVA; Virus × drug interaction: F1,54 = 1.74, p = 0.192; main effect virus: F1,54 = 5.76, p = 0.020; main effect drug: F1,54 = 0.01, p = 0.931; followed by Tukey HSD post-hoc test). (d) Bar graph representing the number of entries in the virtual zone surrounding the social stimulus during T1. (two-way ANOVA; Virus × drug interaction: F1,54 = 0.67, p = 0.416; main effect virus: F1,54 = 4.33, p = 0.042; main effect drug: F1,54 = 0.45, p = 0.506; followed by Tukey HSD post-hoc test). (e) Bar graph representing the number of entries in the virtual zone surrounding the object during T1. (two-way ANOVA; Virus × drug interaction: F1,54 = 3.68, p = 0.060; main effect virus: F1,54 = 0.15, p = 0.703; main effect drug: F1,54 = 7.15, p = 0.010; followed by Tukey HSD post-hoc test). (f) Bar graph representing the number of entries in the virtual zone surrounding the object during T2. (two-way ANOVA; no main effects and no interactions). (g-h) Bar graphs reporting the change in social and object interaction from T2 to T1 between scrShank3 and shShank3 mice treated with Vehicle. (Social: two-way ANOVA; no main effects and no interactions; Object: two-way ANOVA; Virus × drug interaction: F1,54 = 4.88, p = 0.031; main effect virus: F1,54 = 0.90, p = 0.346; main effect drug: F1,54 = 0.06, p = 0.802; followed by Tukey HSD post-hoc test) (i) Bar graph representing the distance moved during the social preference test (two-way ANOVA; no main effects and no interactions). (j) Bar graph representing the velocity during the social preference test (two-way ANOVA; no main effects and no interactions). Error bars show SEM.

Supplementary Figure 6 Low volume of AAV-shShank3 alters social and sucrose preference

(a-c) Representative images of midbrain horizontal sections from three animals that underwent behavioral characterization injected with 50 nL of AAV-shShank3. (d) Schematic of the time course of the experiments and behavioral protocol of the social preference test. (e) Bar graph representing the normalized social preference at T2 (t32 = 2.27, unpaired t-test). (f) Bar graph representing the change in social interaction from T1 to T2 (t32 = 2.64, unpaired t-test). (g) Bar graph representing the change in object interaction from T1 to T2 (U = 122.00, Mann-Whitney test). (h) Schematic of the social memory test protocol in the three-chamber apparatus. (i) Bar graph representing the normalized social memory at T2 (t32 = 0.84, unpaired t-test). (j) Bar graph representing the change in novel mouse interaction from T1 to T2 (U = 133.00, Mann-Whitney test). (k) Bar graph representing the change in familiar mouse interaction from T1 to T2 (U = 132.00, Mann-Whitney test). (l) Bar graph representing the sucrose preference ratio at 1% sucrose concentration (t21 = 6.83, unpaired t-test). (m) Bar graphs representing water and sucrose consumption at 1% sucrose (water: t21 = 5.26; sucrose: t21 = -1.38, unpaired t-tests). (n) Bar graph representing the sucrose preference ratio at 8% sucrose concentration (U = 60.00, Mann-Whitney test). (o) Bar graphs representing water and sucrose consumption at 8% sucrose (water: U = 63.00, Mann-Whitney test; sucrose: t21 = 0.09, unpaired t-test). Error bars show SEM. The numbers indicate the mice.

Supplementary Figure 7 Further behavioral characterization of AAV-Shank3 infection in the VTA

(a) Bar graph of the distance moved during the open field test, which was performed with a separate cohort of scrShank3 and shShank3 mice (not treated with Vehicle or PAM i.p.) (U = 76.00, Mann-Whitney test). (b) Bar graph of the velocity during the open field test (U = 76.00, Mann-Whitney test). (c) Bar graphs representing the time in the virtual zones of the open field (wall: t28 = 0.37; intermediate: t28 = -0.63; center: t28 = 0.38; unpaired t-test) (d) Bar graph of self-grooming scored manually during the open field test (U = 38.50, Mann-Whitney test). (e) Bar graph with the weight of the animals at P28 (two-way ANOVA; Virus × drug interaction: F1,54 = 4.27, p = 0.044; main effect virus: F1,54 < 0.001, p = 0.995; main effect drug: F1,54 = 4.05, p = 0.049; followed by Tukey HSD post-hoc test). (f) Bar graph representing the weight of the animals at P31 (two-way ANOVA; Virus × drug interaction: F1,54 = 4.20, p = 0.045; main effect virus: F1,54 < 0.001, p = 0.971; main effect drug: F1,54 = 3.65, p = 0.061; followed by Tukey HSD post-hoc test). Error bars show SEM.

Supplementary Figure 8 Further behavioral characterization of AAV-Shank3 infection in the VTA during adulthood

(a) Left: Entries in the virtual zone around the social enclosure in T1 in adult shShank3 animals treated with Vehicle or Ro 677476 (t22 = 0.21, unpaired t-test). Middle: Bar graph representing the entries around the object enclosure during T1 (t22 = -1.30, unpaired t-test) Right: Bar graph representing the entries around the object enclosure in T2 (t22 = -1.24, unpaired t-test). (b) Left: Bar graph representing the time in social interaction in T1 in adult animals treated with Vehicle or Ro 677476 (t22 = 1.11, unpaired t-test). Middle: Time in object interaction in T1 (t22 = -0.05, unpaired t-test). Right: Time in object interaction in T2 (t22 = 0.69, unpaired t-test). Error bars show SEM. The numbers indicate the mice.

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Bariselli, S., Tzanoulinou, S., Glangetas, C. et al. SHANK3 controls maturation of social reward circuits in the VTA. Nat Neurosci 19, 926–934 (2016). https://doi.org/10.1038/nn.4319

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