Title Page Translational examination of the orbitofrontal cortex and striatum in obsessive compulsive disorder by Sean Christopher Piantadosi B.A., St. Mary’s College of Maryland, 2010 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2019 Committee Membership Page UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Sean Christopher Piantadosi It was defended on April 17, 2019 and approved by Anthony Grace, Professor, Department of Neuroscience, University of Pittsburgh Sandra Kuhlman, Associate Professor, Department of Biological Sciences, Carnegie Mellon University Byron Yu, Associate Professor, Departments of Electrical & Computer Engineering and Biomedical Engineering, Carnegie Mellon University Yanhua Huang, Associate Professor, Department of Psychiatry, University of Pittsburgh Bernardo Sabatini, Professor, Department of Neurobiology, Harvard Medical School Dissertation Director: Susanne Ahmari, Assistant Professor, Department of Psychiatry, University of Pittsburgh ii Copyright © by Sean Christopher Piantadosi 2019 iii Abstract Translational examination of the orbitofrontal cortex and striatum in obsessive compulsive disorder Sean Christopher Piantadosi, PhD University of Pittsburgh, 2019 For decades a causal role for orbitofrontal cortex (OFC) and striatal dysfunction in obsessive compulsive disorder (OCD) has been hypothesized. Structural as well as functional MRI studies have implicated these regions and their interconnections in OCD pathogenesis, though their precise roles in encoding obsessive or compulsive symptoms are still an area of active investigation. Several lingering questions therefore remain. First, what are the molecular adaptations in the OFC and striatum that give rise to these structural and functional deficits in OCD? Second, how do activity patterns in these regions give rise to compulsive behavior, and how do treatments that reduce compulsive behavior affect these activity patterns? Using a translational approach, we have investigated each of these questions for the first time. In Chapter 1, we have performed the first post-mortem analysis of OCD tissue looking at gene expression in the OFC and striatum. Targeting our analysis only to genes implicated in previous clinical and preclinical studies, we found that many genes critical for excitatory synapse function were reduced in the OFC of individuals with OCD. Several transcripts were also reduced across OFC and striatal brain regions, including DLGAP3 (also known as SAPAP3), which has been previously linked to OCD via preclinical studies showing that Sapap3-knockout mice display a compulsive behavioral phenotype and cortico-striatal alterations. In Chapter 2, we examine the functional consequences of reduced cortico-striatal Sapap3 expression on activity patterns of large groups of neurons in the OFC and central striatum using Sapap3-knockout (KO) mice and in vivo iv calcium imaging. Finally, in Chapter 3 we examine the role of genetically distinct striatal cell types in producing compulsive behavior. These data directly test several long-standing hypotheses regarding OCD pathogenesis and shed new light on how compulsive behavior may be generated in the brain. v Table of Contents Preface ........................................................................................................................................ xvii 1.0 Introduction ............................................................................................................................. 1 1.1 Obsessive Compulsive Disorder .................................................................................... 2 1.1.1 Burden of OCD .................................................................................................... 2 1.1.2 Heritability of OCD............................................................................................. 3 1.1.3 Genetics of OCD: Developing role for the glutamatergic system ................... 4 1.1.3.1 Candidate gene studies ............................................................................ 4 1.1.3.2 Genome-wide association studies ........................................................... 5 1.1.3.3 Post-mortem analysis of OCD associated genes .................................... 7 1.1.4 Neural basis of OCD ........................................................................................... 9 1.1.5 Functional CSTC disruption in OFC and striatum of OCD subjects .......... 11 1.1.6 Neurochemical findings in cortico-striatal circuits in OCD .......................... 13 1.2 Cortico-striatal dysfunction in OCD mouse models .................................................. 14 1.2.1 Transgenic mouse models of OCD-like behavior ........................................... 15 1.2.1.1 Hoxb8 mutant mice ................................................................................ 16 1.2.1.2 Slitrk5 mutant mice ................................................................................ 17 1.2.1.3 Sapap3/Dlgap3 mutant mice .................................................................. 18 1.2.2 Optogenetic manipulation of cortico-striatal circuits .................................... 19 1.2.3 Using in vivo single-photon calcium imaging to investigate neural circuits 21 1.3 Goals of the current dissertation ................................................................................. 23 vi 2.0 Decreases in excitatory synaptic gene expression in orbitofrontal cortex and striatum of subjects with obsessive compulsive disorder ........................................................ 25 2.1 Introduction .................................................................................................................. 26 2.2 Methods ......................................................................................................................... 30 2.2.1 Human post-mortem subjects .......................................................................... 30 2.2.2 Tissue collection and RNA extraction ............................................................. 30 2.2.3 Selection of transcripts for expression analysis .............................................. 31 2.2.4 Quantitative PCR (qPCR) ................................................................................ 31 2.2.5 Statistical Analysis ............................................................................................ 32 2.3 Results ............................................................................................................................ 34 2.3.1 Effect of OCD diagnosis on expression of excitatory synaptic structure transcripts in cortical and striatal regions ............................................................... 34 2.3.2 Effect of OCD diagnosis on expression of transcripts encoding excitatory synaptic receptors and transporters in cortical and striatal regions .................... 37 2.3.3 Effect of OCD diagnosis on expression of GABA synapse transcripts in cortical and striatal regions ....................................................................................... 39 2.3.4 Composite measures of transcript expression across cortical and striatal regions in OCD ........................................................................................................... 42 2.4 Discussion ...................................................................................................................... 44 2.4.1 Decreased levels of three key excitatory synaptic structure transcripts are observed in OFC and striatum.................................................................................. 44 2.4.2 Downregulation of multiple excitatory synaptic transcripts in OFC suggests an upstream causal event .......................................................................................... 46 vii 2.4.3 Synthesis of downregulation of glutamatergic transcripts with existing literature...................................................................................................................... 47 2.4.4 Potential caveats ................................................................................................ 48 2.4.5 Acknowledgments ............................................................................................. 49 3.0 Functional analysis of orbitofrontal cortex and central striatal activity during compulsive behavior and following treatment ......................................................................... 52 3.1 Introduction .................................................................................................................. 52 3.2 Methods ......................................................................................................................... 54 3.2.1 Animals .............................................................................................................. 54 3.2.2 Stereotactic surgery .......................................................................................... 55 3.2.2.1 Calcium imaging surgical methods ...................................................... 55 3.2.2.2 Optogenetic activation of central striatum surgical methods ............ 57 3.2.3 Drug preparation and administration ............................................................. 58 3.2.3.1 lOFC fluoxetine administration ............................................................ 58 3.2.3.2