Stephanie C. Dulawa - US grants

The symptoms of schizophrenia, obsessive-compulsive disorder (OCD), and Tourette's syndrome result in part from sensorimotor gating deficits. Patients diagnosed with these disorders have deficient prepulse inhibition (PPI) (a measure of sensorimotor gating). Additionally, schizophrenia patients have startle habitation deficits. The serotonin system has been implicated in both the pathophysiology and the mechanism of action of therapeutics for these disorders. A better understanding of the neural mechanisms that modulate PPI and habitation (two forms of startle plasticity) could permit the development of better treatments. This project proposes to investigate the role of pre- and postsynaptic serotonin-1B (5-HT1B) receptors and specific neural circuits in modulating PPI and habituation. 5-HT1B receptors regulate the activity of dopamine, serotonin, and GABA neurons that have been shown to modulate PPI.

DESCRIPTION (provided by applicant): This proposal aims to examine the role of 5-HT1A receptor in modulating anxiety. We aim to determine the brain region and developmental time point during which 5-HT1A receptors establish normal or "wild-type" (WT) anxiety levels for the lifetime of the animal. The proposed studies stem from our findings using inducible 5-HTIA rescue (1AR) mice, which conditionally express 5-HTIA receptors only in certain postsynaptic sites. Our findings suggest that the expression of postsynaptic 5-HT1A receptors is required sometime between postnatal days 5 and 21 for mice to exhibit WT rather than 5-HT1A knockout (1AKO) (increased) anxiety levels in adulthood. We will use a combination of molecular biological and behavioral tools to test our hypotheses. We propose to further behaviorally characterize the existing 1AR mice to more narrowly define the critical time for the 5-HT1A receptor to establish WT anxiety levels. We also propose to generate multiple novel 1AR lines using the bacterial artificial chromosome (BAC) strategy to dissect more precisely where and when the lasting effect on anxiety levels, established by the 5-HTIA receptor, takes place.

DESCRIPTION (provided by applicant): The proposed training award is designed to prepare the applicant for an independent research career studying the underlying neurobiology of depression and the antidepressant response. The training plan provides in-depth training in the transcriptional analysis of both micro-dissected brain regions and single-cells to investigate the molecular mechanisms underlying the antidepressant response. Trainning in transcriptional analysis and bioinformatics will be provided by collaborators at the Columbia Genome Center (CGC). Specific training in single-cell transcriptional analysis will occur in the laboratory of the co-sponsor, Dr. Dulac, at Harvard University. The applicant will also receive training in immunohistochemistry and transgenic technology in the laboratory of the sponsor, Dr. Hen. All individuals providing training are experts in their fields and have agreed to participate in the training program. The trainee will attend courses to prepare her to learn these techniques. The training environment at Columbia University is ideally suited to the goals of the training program, and fosters interactions among neurobiologists, geneticists, and statisticians to solve complex biological problems. The applicant has already generated mouse models of the behavioral response to chronic antidepressant treatment. The research plan proposes to use these models to examine the mechanisms underlying the antidepressant response. Using mice which inducibly express serotonin 1A (5-HT1A) receptors postsynaptically, but not presynaptically, the trainee will assess the role of pre vs. postsynaptic 5-HT1A receptors in the behavioral response to chronic fluoxetine. The role of these receptors on downstream targets of antidepressant treatment, including increases in brain-derived neurotrophic factor (BDNF) mRNA (exons 1-5), and neurogenesis, will also be assessed. Finally, using transcriptional analysis, the trainee will attempt to identify completely novel genes involved in the antidepressant response. Identifying such genes will greatly aid the applicant in beginning an independent research career studying the underlying neurobiology of depression and the antidepressant response. Thus, the proposed training program will prepare the candidate to successfully head a laboratory using molecular biology, behavior, and bioinformatics to elucidate the molecular mechanisms underlying mood regulation.

[unreadable] DESCRIPTION (provided by applicant): We propose to use behavioral and genetic mouse models to identify the neural mechanisms by which serotonin transporter (5-HTT) function modulates sensorimotor gating deficits and perseverative behaviors. Sensorimotor gating is a neural mechanism that filters excessive sensory, cognitive, and motor information, permitting mental and behavioral integration. Prepulse inhibition (PPI) is a form of startle plasticity that provides an operational measure of sensorimotor gating. Recent findings have implicated gain-of-function mutations in 5-HTT, and hypersensitivity of 5-HT1DP (homologous to mouse 5-HT1B) receptors, in two disorders characterized by deficient PPI and perseverative behaviors: obsessive-compulsive disorder (OCD) and autistic disorders. Only antidepressant drugs that potently block the reuptake of serotonin provide effective treatment for these disorders. Our recent findings in mice have identified a functional interaction between 5-HTT and 5-HT1B receptors in modulating PPI and perseverative behaviors. We found that the PPI deficits and perseverative behaviors induced by acute 5-HT1B agonist challenge are absent in 5-HTT knockout mice. We also found that the behavioral deficits induced by 5-HT1B agonists are absent in mice treated chronically, but not subchronically, with the selective serotonin reuptake inhibitor fluoxetine. Thus, we hypothesize that reducing 5-HTT function, genetically or pharmacologically, prevents the PPI deficits and perseverative behaviors induced by 5-HT1B receptor activation by desensitizing these receptors. We also hypothesize that increasing 5-HTT function, genetically or pharmacologically, will exacerbate the behavioral deficits induced by 5-HT1B receptor activation by sensitizing these receptors. Here, we propose to identify the mechanisms by which 5-HTT and 5-HT1B receptors interact to modulate these behaviors. First, we will test the hypothesis that potent blockade of 5-HTT is required to prevent 5-HT1B agonist-induced behavioral deficits by comparing the ability of different classes of antidepressants to reverse these effects. Second, we will assess 5-HT1B receptor expression and functional coupling to localize the brain regions in which 5-HT1B receptors are desensitized by antidepressant treatments. Third, we will generate two inducible transgenic mouse strains with increased 5-HTT function. One strain will overexpress mouse 5-HTT to evaluate the effects of increased 5-HTT availability, which may model the consequences of specific 5-HTT gene-linked polymorphic region (5-HTTLPR) alleles recently linked to OCD. The other strain will express human 5-HTT containing an uncommon mutation, Ne425Val, which renders 5-HTT constitutively active and was recently linked to OCD and autism. We hypothesize that both mouse strains will exhibit PPI deficits and perseverative behaviors, and increased behavioral responses to 5-HT1B agonists. Our unique approach could lead to novel animal models of the sensorimotor gating deficits and perseverative behaviors in OCD and autism. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Depression is identified among the leading global causes of disability by the World Health Organization, and affects 15-20% of individuals during their lifetime. All approved antidepressants require 2 - 4 weeks of treatment for their therapeutic effects to emerge, and 30-40% of depressed subjects do not respond adequately to current treatments. Fast-onset antidepressants with novel mechanisms of action are greatly needed. Few pharmacological agents have been identified which induce rapid antidepressant effects. None of these, including the noncompetitive NMDA antagonist ketamine, are approved for clinical use due to adverse side effects. We recently found that selective serotonin 2C (5-HT2C) receptor antagonists produce substantially faster-onset antidepressant effects than serotonin reuptake inhibitors using mouse models of chronic antidepressant action. This project proposes to identify the mechanisms underlying the rapid-onset antidepressant effects of 5-HT2C antagonists using mice. We already found that short-term treatment with 5- HT2C antagonists induces CREB phosphorylation, BDNF expression, and neuronal remodeling in the medial prefrontal cortex. We propose to use an integrated and multidisciplinary approach to further delineate these mechanisms which includes behavioral, molecular genetic, biochemical, morphological, and pharmacological approaches. Specific Aim 1 will identify the mechanisms by which 5-HT2C antagonist treatment induces BDNF expression. We will examine the role of 5-HT2C receptor blockade on pyramidal vs. GABAergic interneurons in the mPFC. We will also examine the role of the dopaminergic system, and will identify the specific dopamine receptor monomer and heteromers mediating this effect. Specific Aim 2 will determine which signaling molecules downstream of BDNF induce neuronal remodeling and the antidepressant behavioral response. We will assess whether 5-HT2C antagonists and ketamine utilize the same signaling cascade downstream of BDNF to induce rapid antidepressant effects. This work could provide evidence required to justify human studies examining selective 5-HT2C antagonists as potential fast-onset antidepressants. Importantly, this work could also identify novel targets for which new fast-onset antidepressants could be developed. Given the dearth of knowledge regarding the neural mechanisms underlying fast-onset antidepressant effects, these studies could provide critical insights into this phenomenon. In summary, the proposed work will provide valuable new insights into the mechanisms underlying fast-onset antidepressant action and lead to novel treatments.

DESCRIPTION (provided by applicant): Obsessive-compulsive disorder (OCD) is a severe anxiety disorder characterized by unwanted and intrusive thoughts, images, or impulses and/or repetitive behavior. OCD affects 1-3% of the world's population and is a leading cause of illness-related disability. Recently, the first genome-wide association study (GWAS) of OCD found a SNP, rs6131295, that achieved genome-wide significance in the trio portion of the sample. The gene closest to rs6131295 is BTBD3, and variation at this SNP regulates expression levels of BTBD3. BTBD3 is a member of a large family of transcription factors including BTBD9, a gene associated with Tourette's Syndrome (TS), a disorder frequently comorbid with OCD. We examined BTBD3 knockout (KO) mice for phenotypes relevant to OCD. Rather than considering BTBD3 KO mice a model for OCD as a categorical disorder, we focused on several behavioral domains including exploratory behavior, repetitive/compulsive behavior, and sensorimotor gating, that are altered in OCD and other related disorders including TS and autism. We found that BTBD3 KO mice exhibit increases in repetitive/compulsive behaviors, and reductions in exploratory behavior and sensorimotor gating. Our findings that BTBD3 KO mice have deficits across these domains increase the probability that BTBD3 was a true hit in the OCD GWAS. Far fewer risk genes have been identified for OCD than for other neuropsychiatric disorders such as schizophrenia and bipolar disorder, so increasing the number of genes that predispose to OCD would be highly significant. This proposal will use a novel F1 screening strategy in mice to identify epistatic modifiers of BTBD3. We will cross inbred C57BL/6J mice that are heterozygous for a KO allele of BTBD3 with 33 different inbred strains to produce large a F1 cohort. Half of the F1s will be wild type (WT), and half will be heterozygotes (HT) at the BTBD3 locus. F1 mice will be efficiently phenotyped for the behavioral traits we found to be altered in BTBD3 HT mice, including exploratory behavior, repetitive/compulsive behavior, and sensorimotor gating. These data will reveal which F1 genetic backgrounds modify the behavioral effects of carrying the BTBD3 KO allele. We will then perform a murine GWAS to identify genetic modifiers of BTBD3. Minimal genotyping will be required, since all of the genome besides the BTBD3 locus will be predicted from existing SNP databases. The genetic modifiers of murine BTBD3 that we identify can then be tested in the human GWAS datasets that were used to identify rs6131295 as a risk allele for OCD, and large GWAS datasets for TS and autism. This novel approach should greatly improve the power to detect epistatic modifiers in human genetic datasets by prioritizing specific comparisons and drastically reducing the number of statistical tests performed. Our approach could provide a novel strategy for identifying epistatic modifiers. Identifying genes that interact with BTBD3 will further our understanding of OCD susceptibility and pathophysiology, and other disorders involving similar behavioral domains including TS and autism.

PROJECT SUMMARY/ABSTRACT Compulsive behaviors are prominent, disabling, and often treatment-resistant symptoms of several neuropsychiatric disorders, including obsessive compulsive disorder (OCD). Dysfunction within fronto- subcortical brain structures is thought to underlie compulsive behaviors, but the precise circuits involved remain unknown. Currently, chronic (> 4 weeks) treatment with serotonin reuptake inhibitors (SRIs) provides the only effective pharmacological monotherapy for compulsive behaviors; yet, approximately 50% of OCD patients do not respond to SRIs. We have shown that serotonin 1B receptors (5-HT1BRs) regulate the expression of compulsive behaviors. 5-HT1BRs are located on axon terminals of serotonin-containing neurons (presynaptic), and neurons containing other neurotransmitters (postsynaptic)(3), where they inhibit neurotransmitter release when activated. Furthermore, 5-HT1BRs signal through both a canonical G protein- mediated pathway, and a noncanonical G protein-independent pathway. Canonical 5-HT1BR-mediated Gi- signaling requires direct interaction of 5-HT1BRs with glycogen synthase kinase-3 beta (GSK3?). On the other hand, the intracellular scaffolding protein beta arrestin-2 (?-arrestin2) mediates noncanonical 5-HT1BR signaling. We recently found that activation of 5-HT1BRs within the orbitofrontal cortex (OFC) is necessary and sufficient to induce compulsive behaviors in mice. The OFC contains both pre- and postsynaptic 5-HT1BRs, including those on dorsal raphe-OFC and basolateral amygdala (BLA)-OFC projections, respectively. We propose to develop and use a two virus, in vivo CRISPR-Cas9 system to dissect the role of 5-HT1BR density and canonical versus noncanonical signaling within these two circuits in modulating compulsive behaviors. In Specific Aim 1, we will either overexpress or knockout 5-HT1BR expression within these two projections. We will infuse 1) a Cre recombinase (Cre) dependent adeno-associated virus (AAV) which expresses either 5- HT1BR, or the S. aureus Cas9 (SaCas9) gene plus a guide sequence against 5-HT1BR, into the dorsal raphe or BLA, and 2) a Cre-expressing retrograde canine adenovirus (CAV2-cre) into the OFC. In Specific Aim 2, we will also use the two virus, in vivo CRISPR-Cas9 system, but will infuse a Cre-dependent AAV expressing SaCas9 and a guide sequence against either ?-arrestin2 or GSK3? into the dorsal raphe or BLA. Mice will be evaluated for 5-HT1BR agonist-induced compulsive behaviors in the open field, a delayed alternation task, and an operant paradigm assessing both acquisition and persistence of habitual lever pressing. The proposed work could establish a novel in vivo CRISPR-Cas9 system for manipulating gene expression within specific neural circuits, and could lead to innovative therapeutic strategies for treating compulsions.