This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Strengthened inputs from secondary motor cortex to striatum in a mouse model of compulsive behavior Victoria L. Corbit1,2,3,4, Elizabeth E. Manning1,2,4, Aryn H. Gittis1,3 and Susanne E. Ahmari1,2,4 1Center for the Neural Basis of Cognition, Carnegie Mellon University, Pittsburgh, PA, USA 2Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA 3Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA 4Translational Neuroscience Program, Department of Psychiatry, University of Pittsburgh, PA, USA https://doi.org/10.1523/JNEUROSCI.1728-18.2018 Received: 2 July 2018 Revised: 19 November 2018 Accepted: 19 December 2018 Published: 8 February 2019 Author contributions: V.L.C., E.E.M., A.H.G., and S.E.A. designed research; V.L.C. and E.E.M. performed research; V.L.C., A.H.G., and S.E.A. analyzed data; V.L.C. and S.E.A. wrote the first draft of the paper; V.L.C., E.E.M., A.H.G., and S.E.A. edited the paper; V.L.C., A.H.G., and S.E.A. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by National Institute of Mental Health F31MH110125 (Victoria Corbit); National Science Foundation DMS 1516288 (Aryn Gittis) and National Institutes of Health R00 NS076524 (Aryn Gittis); BRAINS R01MH104255 (Susanne Ahmari), McKnight Neuroscience Scholar Award (Susanne Ahmari), MQ Fellows Award (Susanne Ahmari), Burroughs Wellcome Fund CAMS Award (Susanne Ahmari), and Klingenstein Fellowship in the Neurosciences (Susanne Ahmari). This data was previously presented in abstract/poster form. Correspondence should be addressed to To whom correspondence should be addressed: Bridgeside Point II, University of Pittsburgh, 450 Technology Drive, Suite 223, Pittsburgh, PA 15219, Tel: 412-624-3183 Email: [email protected] Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1728-18.2018 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 Strengthened inputs from secondary motor cortex to striatum in a mouse model of 2 compulsive behavior 3 4 Abbreviated title: Strengthened M2-striatal inputs in Sapap3-KOs 5 Victoria L. Corbit1,2,3,4, Elizabeth E. Manning1,2,4, Aryn H. Gittis1,3#, Susanne E. Ahmari1,2,4#* 6 7 1Center for the Neural Basis of Cognition, Carnegie Mellon University, Pittsburgh, PA, USA 8 2Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA 9 3Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA 10 4Translational Neuroscience Program, Department of Psychiatry, University of Pittsburgh, PA, 11 USA 12 #Co-senior author 13 14 15 * To whom correspondence should be addressed: 16 Bridgeside Point II, University of Pittsburgh 17 450 Technology Drive 18 Suite 223 19 Pittsburgh, PA 15219 20 Tel: 412-624-3183 21 Email: [email protected] 22 Pages 36 23 Figures 7 24 Tables 2 25 Abstract 241 words 26 Significance Statement 119 words 27 Introduction 662 words 28 Discussion 1430 words 29 1 30 31 Conflicts of Interest 32 The authors have no conflicts of interest. 33 34 Acknowledgements 35 This work was supported by National Institute of Mental Health F31MH110125 (Victoria 36 Corbit); National Science Foundation DMS 1516288 (Aryn Gittis) and National Institutes of 37 Health R00 NS076524 (Aryn Gittis); BRAINS R01MH104255 (Susanne Ahmari), McKnight 38 Neuroscience Scholar Award (Susanne Ahmari), MQ Fellows Award (Susanne Ahmari), 39 Burroughs Wellcome Fund CAMS Award (Susanne Ahmari), and Klingenstein Fellowship in the 40 Neurosciences (Susanne Ahmari). 41 This data was previously presented in abstract/poster form. 42 43 Abstract 44 Hyperactivity in striatum is associated with compulsive behaviors in Obsessive- 45 Compulsive Disorder (OCD) and related illnesses, but it is unclear if this hyperactivity is 46 due to intrinsic striatal dysfunction or abnormalities in corticostriatal inputs. 47 Understanding the cellular and circuit properties underlying striatal hyperactivity could 48 help inform the optimization of targeted stimulation treatments for compulsive behavior 49 disorders. To investigate the cellular and synaptic abnormalities that may underlie 50 corticostriatal dysfunction relevant to OCD, we utilized the Sapap3 knockout (Sapap3- 51 KO) mouse model of compulsive behaviors, which also exhibits hyperactivity in central 52 striatum. Ex vivo electrophysiology in double transgenic mice was used to assess 53 intrinsic excitability and functional synaptic input in spiny projection neurons (SPNs) and 54 fast-spiking interneurons (FSIs) in central striatum of Sapap3-KOs and wildtype (WT) 2 55 littermates. While we found no differences in intrinsic excitability of SPNs or FSIs 56 between Sapap3-KOs and WTs, excitatory drive to FSIs was significantly increased in 57 KOs. Contrary to predictions, lateral orbitofrontal cortex (LOFC)-striatal synapses were 58 not responsible for this increased drive; optogenetic stimulation revealed that LOFC 59 input to SPNs was reduced in KOs (~3-fold) and unchanged in FSIs. However, 60 secondary motor area (M2) post-synaptic responses in central striatum were 61 significantly increased (~6-fold) in strength and reliability in KOs relative to WTs. These 62 results suggest that increased M2-striatal drive may contribute to both in vivo striatal 63 hyperactivity and compulsive behaviors, and support a potential role for pre- 64 supplementary/ supplementary motor cortical regions in the pathology and treatment of 65 compulsive behavior disorders. 66 67 Significance Statement 68 These findings highlight an unexpected contribution of M2 projections to striatal 69 dysfunction in the Sapap3-KO OCD-relevant mouse model, with M2 inputs strengthened by at 70 least 6-fold onto both SPNs and FSIs in central striatum. Because M2 is thought to be 71 homologous to pre-supplementary/ supplementary motor areas (pre-SMA/SMA) in humans, 72 regions important for movement preparation and behavioral sequencing, these data are 73 consistent with a model in which increased drive from M2 leads to excessive selection of 74 sequenced motor patterns. Together with observations of hyperactivity in pre-SMA/SMA in both 75 OCD and Tourette Syndrome, and evidence that pre-SMA is a potential target for repetitive 76 transcranial magnetic stimulation treatment in OCD, these results support further dissection of 77 the role of M2 in compulsivity. 78 3 79 Introduction 80 Although stereotyped and compulsive behaviors are prominent, disabling, and 81 notoriously-treatment resistant symptoms in multiple severe neuropsychiatric disorders, 82 including Tourette Syndrome (TS) (Leckman et al., 2010), grooming disorders (e.g. skin-picking, 83 trichotillomania (Chamberlain et al., 2009; Flessner et al., 2012), and Obsessive Compulsive 84 Disorder (OCD) (Ayuso-Mateos, 2006; Karno et al., 1988; Menzies et al., 2008), little is known 85 about their underlying neural mechanisms. Imaging studies in patients with OCD and other 86 compulsivity-associated disorders have consistently identified both hyperactivity in the striatum 87 (caudate head) and increased corticostriatal functional connectivity at baseline and when 88 symptoms are expressed (Chamberlain et al., 2009; Del Casale et al., 2011; Denys et al., 2013; 89 Figee et al., 2013; Harrison et al., 2009; Leckman et al., 2010; Maia et al., 2008; Menzies et al., 90 2008; Rauch et al., 1994; Rauch et al., 1997; Saxena et al., 1998). However, the cellular and 91 synaptic abnormalities that underlie this hyperactivity are unclear. 92 Determining whether striatal hyperactivity originates in striatum or in upstream cortical 93 projections could help inform whether neuromodulatory treatments for OCD-related disorders 94 should target cortical or subcortical regions. Though striatal deep brain stimulation (DBS) has 95 reported efficacy in OCD (Figee et al., 2013; Greenberg et al., 2010), studies demonstrating 96 aberrant activity in corticostriatal circuits in OCD patients (Chamberlain et al., 2008; Del Casale 97 et al., 2011; Figee et al., 2013; Harrison et al., 2009; Menzies et al., 2008; Nakamae et al., 98 2014) suggest that cortical regions may be useful targets for non-invasive neurostimulation via 99 repetitive transcranial magnetic stimulation (rTMS), either through direct cortical effects or 100 modulation of connected subcortical structures. Consistent with this idea, orbitofrontal cortex 101 (OFC) and pre-supplementary motor area (pre-SMA), which are hyperactive in OCD patients 102 (de Wit et al., 2012b; Del Casale et al., 2011; Grützmann et al., 2016; Leckman et al., 2010; 103 Maltby et al., 2005; Yücel et al., 2007), have been identified as promising targets for rTMS 4 104 (Berlim et al., 2013). Determining how these hyperactive cortical regions interact with the 105 striatum to generate both dysfunctional striatal activity and compulsive behaviors could help 106 refine stimulation patterning for neuromodulatory treatments. 107 To begin to dissect the contributions of intrinsic striatal vs. extrinsic cortical factors to 108 abnormal OCD-relevant striatal activity, we used an animal model system that displays both 109 striatal hyperactivity and compulsive behavior: Sapap-3-knockout (KO) mice (Burguiere