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Regulation of autism-relevant behaviors by cerebellar–prefrontal cortical circuits

Nature Neuroscience volume 23, pages 1102–1110 (2020)Cite this article

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Abstract

Cerebellar dysfunction has been demonstrated in autism spectrum disorders (ASDs); however, the circuits underlying cerebellar contributions to ASD-relevant behaviors remain unknown. In this study, we demonstrated functional connectivity between the cerebellum and the medial prefrontal cortex (mPFC) in mice; showed that the mPFC mediates cerebellum-regulated social and repetitive/inflexible behaviors; and showed disruptions in connectivity between these regions in multiple mouse models of ASD-linked genes and in individuals with ASD. We delineated a circuit from cerebellar cortical areas Right crus 1 (Rcrus1) and posterior vermis through the cerebellar nuclei and ventromedial thalamus and culminating in the mPFC. Modulation of this circuit induced social deficits and repetitive behaviors, whereas activation of Purkinje cells (PCs) in Rcrus1 and posterior vermis improved social preference impairments and repetitive/inflexible behaviors, respectively, in male PC-Tsc1 mutant mice. These data raise the possibility that these circuits might provide neuromodulatory targets for the treatment of ASD.

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Fig. 1: Modulation of elevated PRL activity in PC-Tsc1 mutants rescues social deficits and repetitive/inflexible behaviors.
Fig. 2: Rcrus1 and PRL are functionally connected and play important roles in ASD behaviors.
Fig. 3: The cerebellar LN is functionally connected to the PRL cortex and regulates social behaviors.
Fig. 4: VM thalamus connects to the PRL cortex to regulate ASD behaviors.
Fig. 5: Posterior cerebellar vermis is structurally and functionally connected to PRL, and stimulation specifically rescues repetitive/inflexible behaviors.

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Data availability

The authors confirm that all relevant non-MRI data are included in the paper and/or its supplementary information files. Raw MRI data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

All code is publicly available on GitHub (https://github.com/Mouse-Imaging-Centre/RMINC) or is available upon reasonable request.

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Acknowledgements

P.T.T. acknowledges support from the National Institute of Neurologic Disorders and Stroke (NS083733), the National Institute of Mental Health (MH116882), the Tuberous Sclerosis Alliance and the Department of Defense. E.K. acknowledges support from National Institute of Neurologic Disorders and Stroke (NS107004) and Autism Speaks. H.F. and S.d.L. acknowledge support from the National Institute of Neurologic Disorders and Stroke (NS095232 and NS105039). F. Morgado, J.E. and J.P.L. acknowledge support from the Canadian Institute for Health Research and the Ontario Brain Institute. L.C.R and C.J.S. acknowledge support from the National Institute for Health (MH106957). M.A.B. acknowledges support from the Medical Research Council (MR/K022377/1). N.K. acknowledges support from the National Institute of Mental Health (MH094268) and declares that he is a paid consultant for Rescindo Therapeutics, although this does not provide a competing interest with this study. P.T.T. and E.K. acknowledge support from V. Jakkamsetti and J. Pascual for support with in vivo extracellular and acute slice recordings recordings and analysis; S. Birnbaum for assistance in behavioral studies; G. Konopka, C. Powell, L. Osburne, J. Foster, J. Lai, K. Rilett, E. Kim and A. Raznahan for generous provision of animal models; and J. Chadwick for graphics assistance.

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Authors and Affiliations

  1. Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Elyza Kelly, Fantao Meng, Yasaman Kazemi, Chongyu Ren, Christine Ochoa Escamilla, Jennifer M. Gibson, Sanaz Sajadi, Tommy Tan & Peter T. Tsai

  2. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Elyza Kelly, Jennifer M. Gibson, Robert J. Pendry, Brad E. Pfeiffer & Peter T. Tsai

  3. Departments of Otolaryngology-Head and Neck Surgery, Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Hirofumi Fujita & Sascha du Lac

  4. Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

    Felipe Morgado & Jason P. Lerch

  5. Mouse Imaging Centre, Toronto Hospital for Sick Children, Toronto, ON, Canada

    Felipe Morgado, Jacob Ellegood, Christopher Hammill & Jason P. Lerch

  6. Department of Neuroscience, Center for Behavioral Neuroscience, American University, Washington, DC, USA

    Laura C. Rice & Catherine J. Stoodley

  7. Centre for Craniofacial and Regenerative Biology and MRC Centre for Neurodevelopmental Disorders, Kingʼs College London, London, UK

    M. Albert Basson

  8. Department of Biomedical Science, Charles E. Schmidt College of Medicine and Brain Institute, Florida Atlantic University, Jupiter, Florida, USA

    Randy D. Blakely & Maureen K. Hahn

  9. Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA

    Scott V. Dindot

  10. Institut de Génétique et de Biologie Moléculaire et Cellulaire; Centre National de la Recherche Scientifique; Institut National de la Santé et de la Recherche Médicale; Université de Strasbourg, Illkirch, France

    Christelle Golzio

  11. ACT-GeM, Department of Human Genetics at Stanley Manne Children’s Research Institute; Department of Pediatrics and Cellular and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Nicholas Katsanis

  12. Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA

    Diane M. Robins

  13. MIND Institute and Department of Psychiatry and Behavioral Sciences, University of California, Davis, Davis, CA, USA

    Jill L. Silverman

  14. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada

    Karun K. Singh

  15. Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Rachel Wevrick

  16. Department of Medical Imaging and Psychology, University of Toronto; Diagnostic Imaging, Hospital for Sick Children, Toronto, ON, USA

    Margot J. Taylor

  17. Department of Pediatrics, University of Toronto, Holland Bloorview Kids Rehabilitation Hospital, Toronto, ON, USA

    Evdokia Anagnostou

  18. Wellcome Centre for Integrative Neuroimaging, FMRIB, Nuffield Department of Clinical Neuroscience, University of Oxford, Oxford, UK

    Jason P. Lerch

  19. Departments of Psychiatry and Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Peter T. Tsai

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  1. Elyza Kelly
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E.K., F. Meng, H. F., J.P.L., S.d.L. and P.T.T. formulated experiments and analysis. E.K., F. Meng, H.F., Y.K., C.O.E., J.M.G., S.S., C.R., D.J., R.P., T.T. and B.E.P. performed experiments and analysis. F. Morgado, J.E. and J.P.L. carried out the mouse structural imaging experiments and analysis. F. Morgado, J.E., M.J.T, C.H., E.A. and J.P.L. carried out the human structural imaging experiments and analysis. L.C.R. and C.J.S. performed functional imaging in humans and analysis of these studies. M.A.B., R.D.B., S.D., C.G., M.K.H., N.K., D.M.R., J.L.S, K.K.S. and R.W. provided critical reagents. E.K., H.F., J.P.L., S.d.L. and P.T.T. prepared the manuscript.

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Correspondence to Peter T. Tsai.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Ted Abel, Sarah Ferri 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 mPFC Gi DREADDs in PC-Tsc1 mutant mice.

a, Sample of injection site locations from PC-Tsc1 mutants injected with Gi (inhibitory) DREADDs/GFP into left prelimbic (PRL) medial prefrontal cortex (mPFC). b, Awake in vivo single unit recordings in the left PRL of control or PC- Tsc1 mutant mice. c, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO). d, Three chambered social novelty testing; time spent sniffing NA or familiar animal (FA). e, Time in the open arm and distance traveled in the elevated plus maze assay. f, Time in the center of the open field and g, distance traveled in the open field. h, Latency to fall in accelerating rotarod test. Box line denoted median/whiskers denoted 5–95%. n ≥ 10 for all experiments. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post test and single unit recordings were analyzed with Mann-Whitney test, shown as mean SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 2 Left Prelimbic Synaptic Properties in PC- Tsc1 Mutant Mice.

a, mini EPSC (mEPSC) representative traces with zoomed in views below initial traces as noted; b, mEPSC frequency; c, mEPSC amplitude; d, mEPSC rise time; e, mEPSC decay time; f, representative mIPSC traces with zoomed in views blow initial traces as noted; g, mIPSC frequency; h, mIPSC amplitude; i, mIPSC rise time; j, and mIPSC decay time in PC-Tsc1 mutant mice and control mice. Groups had 12–15 cells from 5 animals in each. Students t-test, ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

Extended Data Fig. 3 Impact of chemogenetic inhibition of mPFC on Rcrus1 PC inhibition-regulated behaviors.

a, Sample injection site locations in area Rcrus1. b, Awake in vivo single unit recordings in prelimbic cortex (PRL) in mice with Rcrus1 Gi DREADD inhibition (CNO) or vehicle treatment. c, In vivo single unit recordings in motor cortex of anesthetized mice with Rcrus1 Gi DREADDs inhibition compared to baseline. No significant change identified. d, In vivo single unit recordings in right PRL of anesthetized mice with Rcrus1 Gi DREADD inhibition compared to baseline. No significant change identified. e, In vivo single unit recordings in left PRL of anesthetized mice with Rcrus1 Gi DREADD inhibition and either GFP or Gi injection in the mPFC. f, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO). g, Three chambered social novelty testing; time spent sniffing NA, or familiar animal (FA). h, Time in the open arm and distance traveled in the elevated plus maze assay. i, Time in the center of the open field and j, distance traveled in the open field. k, Latency to fall in accelerating rotarod test. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post test. Single unit recordings were analyzed with Mann-Whitney test, shown as mean SEM. All raw values for frequency of spiking that are shown in figures as normalized values can be found in Supplementary Fig. 12. Box line denoted median/whiskers denoted 5–95%. n ≥ 10 for all behavioral experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 4 Inhibition of LN in PC-Tsc1 mutant mice.

a, Sample injection site locations and injection site pictures of Gi DREADDs injected into LN of PC-Tsc1 mutant mice. b, Single unit activity in the right lateral nucleus (LN) with chemogenetic inhibition (Gi) or control GFP injection. c, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO). d, Three chambered social novelty testing; time spent sniffing NA, or familiar animal (FA). e, Time in the open arm and distance traveled in the elevated plus maze assay. f, Time in the center of the open field and g, distance traveled in the open field. h, Latency to fall in accelerating rotarod test. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post hoc testing. Recordings were analyzed with Mann-Whitney test, shown as mean SEM. For frequency of spiking shown in figures as normalized values, raw values can be found in Supplementary Fig. 12. Box line denoted median/whiskers denoted 5-95%. n ≥ 10 for all behavioral experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 5 Thalamic and layer 1 cortical areas targeted by the medial and lateral cerebellar nuclei.

a, Thalamic nuclei with monosynaptic inputs from the right medial nucleus (MN) and lateral nucleus (LN) identified via AAV1-mediated anterograde transsynaptic tracing. The number of the trans-synaptically labeled thalamic neurons in each nucleus indicated as + + (high), + (low), and - (none). b, Disynaptic inputs from the right MN and LN to distinct cortical regions of the left cerebral cortex, identified via transsynaptic tracing. Labeled input density to layer 1 of each cortical area is indicated as + + (high), + (low), and - (none). c, Representative tracing results of disynaptic inputs from the right MN and LN to distinct areas in the left cerebral cortex. Axons and terminals of thalamic neurons trans-synaptically labeled from injections to MN and LN are indicated in black. Results from injections to MN and LN are in the left and right, respectively, of each panel. Top and bottom parts of each panel correspond to layer 1 and 6. Inputs to layer 1 originate predominantly from the VM thalamus. Arrowheads indicate layer 1. Scale bar applies to all panels. d, representative injection site in the LN and e, representative injection site in the MN. Scale bar applies to all panels. Abbreviations; Aud, auditory cortex; Cg, cingulate cortex; CL, centrolateral thalamic nucleus; FrA, frontal association cortex; IL, infralimbic cortex; LD, laterodorsal thalamic nucleus; LO, lateral orbital cortex; LP, lateroposterior thalamic nucleus; M1 and M2, primary and secondary motor cortex; MD, mediodorsal thalamic nucleus; MO, medial orbital cortex; PF, parafascicular thalamic nucleus; Po, posterior thalamic group; PrL, prelimbic cortex; Ptl, parietal association cortex; Rhi, ecto-/peri-/ento-entorhinal cortex; Rsp, retrosplenial cortex; S1 and S2, primary and secondary sensory cortex; Tem, temporal association cortex; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VO, ventral orbital cortex.

Extended Data Fig. 6 ChR2 activation of VM thalamus-PRL mPFC circuit.

a, Sample injection site locations for ChR2/Arch injection into the left VM thalamus. b, To test for possible antidromic activation, in vivo anesthetized single unit recordings in the VM thalamus-targeted parietal association cortex (PAC) were performed upon mPFC laser stimulation of ChR2 or GFP VM-thalamus terminals at 20 Hz or 4 Hz. Image of TdTomato positive terminals in PAC from LN AAV-1 tracing injection (top left). c, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO) with 20 Hz (left) or 4 Hz (right) stimulation. d, Three chambered social novelty testing; time spent sniffing NA, or familiar animal (FA) with 20 Hz (left) or 4 Hz (right) stimulation. e, Time in the open arm and distance traveled in the elevated plus maze assay. f, Distance traveled in the open field and g, time in the center of the open field. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post test and recordings were analyzed with two way ANOVA (unmarked = not significant). For frequency of spiking shown in figures as normalized values (mean SEM), raw values can be found in Supplementary Fig. 12. Box line denoted median/whiskers denoted 5-95%. n ≥ 10 for all behavioral experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 7 Arch inhibition of VM thalamus-PRL mPFC circuit on PC-Tsc1 mutant mice.

a, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO) with 20 Hz (left) or 4 Hz (right) stimulation. b, Three chambered social novelty testing; time spent sniffing NA, or familiar animal (FA) with 20 Hz (left) or 4 Hz (right) stimulation. c, Time in the open arm and distance traveled in the elevated plus maze assay. d, Time in the center of the open field and distance traveled in the open field. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post test. Box line denoted median/whiskers denoted 5-95%. N ≥ 10 for all behavioral experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 8 Structural covariance MRI of cerebellar vermis and mPFC.

Structural covariance MRI in mouse models of ASD vs. controls. Comparisons between vermis lobule VII (top), VIII (middle) or IX (bottom) and mPFC (left) or Prelimbic (PRL) mPFC (right). q values are stated. Each dot represents single imaged brain. Additional demographic information can be found in Supplementary Table 3.

Extended Data Fig. 9 Gi DREADDs inhibition of PCs in the posterior vermis and Gq DREADDs activation of posterior vermis in PC-Tsc1 mutant mice.

a, Injection sites from MN and LN tracing to VM thalamus (top) and sample locations of Gi (inhibitory) or Gq (excitatory) DREADD injections into posterior vermis of PC-Tsc1 mice (bottom). b, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO). c, Three chambered social novelty testing; time spent sniffing NA, or familiar animal (FA). d, Time in the open arm and e, distance traveled in the elevated plus maze assay. f, Time in the center of the open field and g, distance traveled in the open field. h, Latency to fall in accelerating rotarod test. i, Three chambered social approach assay; time spent sniffing novel animal (NA), or novel object (NO). j, Three chambered social novelty testing; time sniffing novel animal (NA), or familiar animal (FA). k, Time in the open arm and distance traveled in the elevated plus maze assay. l, Time in the center of the open field and distance traveled in the open field. m, Latency to fall in accelerating rotarod test. All behavioral tests were analyzed with two or three-way ANOVA and Sidak post test. Box line denoted median/whiskers denoted 5-95%. n ≥ 10 for all behavioral tests. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values and complete animal numbers can be found in Supplementary Table 2.

Extended Data Fig. 10 Raw frequency values for all normalized single unit graphs.

a, Frequency of firing of single units recorded in the left medial prefrontal cortex (mPFC) with control or inhibitory (Gi) DREADDs. b, Frequency of firing recorded in the left mPFC or motor cortex, with Rcrus1 Gi inhibition. c, Spike frequency in the mPFC with Rcrus1 Gi and mPFC Gi inhibition. d, Right mPFC spike frequency with Rcrus1 Gi. e, Spike Frequency in the Lateral Nucleus (LN) with LN Gi inhibition. f, mPFC spike frequency with LN Gi DREADDs. g, mPFC spike frequency with VM thalamic-mpfc Channel Rhodopsin (ChR2) activation of 4 Hz or h, 20 Hz. i, mPFC spike frequency with VM thalamus-mPFC Archaerhodopsin (Arch) inhibition at 4 Hz or j, 20 Hz. k, Parietal Association Cortex (PAC) firing with VM thalamic-mPFC ChR2 stimulation at 4 Hz and l, 20 Hz. m, mPFC spike frequency with posterior vermis Gi inhibition. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. P values complete animal numbers can be found in Supplementary Table 2. Statistical analysis was done with the Wilcoxon matched pairs signed ranks test.

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Kelly, E., Meng, F., Fujita, H. et al. Regulation of autism-relevant behaviors by cerebellar–prefrontal cortical circuits. Nat Neurosci 23, 1102–1110 (2020). https://doi.org/10.1038/s41593-020-0665-z

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