ABSTRACT. Dr. Chesler will describe her work on the enhancement of integrative tools, resources and approaches for the use of model organism genetics for the understanding of human psychiatric and behavioral conditions.

Elissa Chesler Jackson Laboratory

ABSTRACT. Behavior is the primary output of the brain, so understanding its neural origins is key to advancing neuroscience and supporting brain health. Understanding behavior in its full complexity requires a detailed, multidimensional analysis of a broad range of behaviors in the context of the environment. Tools for quantifying neural activity with high temporal and spatial resolution already exist, but behavior is often measured at lower resolution, making discovery of causal linkages challenging. Tools for measuring the full richness of species-appropriate behaviors, and synchronizing these to neural activity, are presently lacking. Recognizing this gap, the Brain Research Through Advancing Innovative Neurotechnologies (BRAIN) Initiative 2.0 Report calls for more sophisticated methods of quantifying behavioral, environmental, and internal state influences on individuals. This talk will survey the current state of the art of brain-behavior quantification and synchronization, highlighting gaps where novel tool development could make a transformative impact. We envision opportunities to link brain and behavior at the same resolution in real time, to bring neural recording into real world settings with ambulatory systems, to infer internal states from quantified behaviors, and artificial intelligence tools to decode internal states from tagged neural activity. Realizing that vision will entail the development of novel sensors, data fusion platforms, and advanced computational approaches for high dimensionality and multi-modal data streams. New paradigms to establish causal relationships between neural activity and behavior across species may ultimately enable the development of closed-loop therapeutic interventions for patients with complex neurobehavioral disorders that currently lack effective treatments.

Sarah H Lisanby1,2

1Division of Translational Research, 2Noninvasive Neuromodulation Unit, National Institute of Health, Bethesda, Maryland, USA Funding Support: NIMH ZIA MH002955

ABSTRACT. Extracting actions and behaviors from video is a vital technique; however, supervised approaches are taxing and are prone to user bias. Recent computer vision advances (e.g. SLEAP, DeepLabCut, DeepLabStream) can provide pose estimation, but body-part position has low interpretability for segmenting behavior into complex movements, e.g. locomotion, grooming. B-SOiD (Hsu and Yttri, Nature Communications; www.bsoid.org) is an intuitive, open source platform that discovers spatiotemporal patterns in any pose data without user input. The algorithm can also be implemented in a semi-supervised fashion to extract specific experimenter-defined behaviors, but with far less algorithmic training than is usually required. Finally, B-SOiD can detect behaviors in real time, enabling close-loop experiments. We will demonstrate its use in individual and social behaviors in invertebrates, rodents, and humans. Then, we will provide examples of how the automated behavioral and kinematic outputs delivered real-world experimental insights.

EA Yttri 1 1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

ABSTRACT. In computational behavior analysis, computer vision tools are used to characterize the detailed posture and movement kinematics of human or animal subjects. The promise of these tools is enormous: detailed posture tracking could be used to diagnose and track progression ¬of movement disorders, run high-throughput screens of new drug treatments in animal models, track the sensory cues animals or people use when making decisions, and build models of the neural control of complex behavior. But computational behavior analysis is currently somewhat of a “wild west”, with no clear benchmark datasets or standards to evaluate the quality of newly proposed data analysis algorithms. This confusion makes it difficult for researchers to judge how well systems will work in their own labs. Unsupervised analyses— which are appealing for their more “unbiased” approach to behavior—are particularly difficult to judge, as there is little consensus in the field as to what constitutes a “good” unsupervised representation of animal pose and movement. Over the past two years, we have released new benchmark datasets of animal behavior and hosted online competitions to evaluate methods for supervised and unsupervised behavioral quantification, in hopes of establishing consensus measures of algorithm performance that will help researchers know what they can expect from these tools. In this talk, we will review potential applications of machine learning based behavior quantification, and discuss what we have learned from our competition tasks.

A Kennedy1, Jennifer J. Sun2 1 Department of Neuroscience, Northwestern University, 2 Computational and Mathematical Sciences, California Institute of Technology

ABSTRACT. Automated detection of complex animal behavior remains a challenging task in neurogenetics. We present an integrated rodent phenotyping platform, JAX Animal Behavior System (JABS) to the community for data acquisition, machine learning based behavior annotation, classifier sharing, and automated genetic analysis. Uniform data collection is carried out by the JABS-Data Acquisition Module, which includes 3D designs of hardware, software for real-time monitoring and video data collection. JABS-Classifier Module is an active learning system for labeling and validating behavior classifiers. JABS-Database Module allows users to share behavior classifiers, including many common single mouse behaviors. JABS-Genetics Module infers a deposited classifier on a library of 600 open field videos from 60 mouse trains, returns heritability, and GWAS results. In summary, we present an integrated platform for mouse behavior data collection, classification, and analysis with the core principles of standardized data collection and analytics pipeline, with a focus on genetics. This open-source tool is an ecosystem that allows the neuroscience community to build shared resources for behavior analysis.

Vivek Kumar The Jackson Laboratory Bar Harbor, ME 04609

ABSTRACT. There is a reciprocal interaction between diet and the chemical senses: sensations shape our dietary patterns, and diet, in turn, influences the way we sense food. Indeed, human and animal studies have shown that diet composition can regulate taste sensation and perception, but the causes and consequences of this chemosensory plasticity are still poorly understood. We have used the simple taste apparatus of the fly D. melanogaster to understand the molecular and neural mechanisms of diet-induced taste plasticity. We discovered that nutrients can directly regulate the responses of the taste cells and sensory neurons to sweetness via epigenetic mechanisms and that chemosensory alterations change meal size and intake by affecting the central processing of sensory information by dopaminergic neurons. We will summarize these findings and present new data showing how a high-sugar diet induces lasting changes in the sensory neurons to blunt their output and dull behavioral responses to food.

Anoumid Vaziri, Christina May, Thibaut Pardo, Hayeon Sung, Monica Dus The Department of Molecular, Cellular, and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109

ABSTRACT. Historically, it was assumed that any treatment targeting neurodevelopmental disorder risk genes would need to be administered early in development to be effective. However, a handful of studies have indicated that behavioural and neurophysiological phenotypes caused by impaired function of select neurodevelopmental disorder risk genes can be reversed by restoring gene function in adulthood. Despite wide interest, few of the large number of risk genes have been assessed for adult reversibility. Here, we developed a high-throughput strategy to assess the temporal requirements and phenotypic reversibility of neurodevelopmental disorder risk gene orthologs using a conditional protein degradation system and machine vision phenotypic profiling in Caenorhabditis elegans. Using this approach, we measured the effects of degrading and re-expressing four neurodevelopmental risk genes orthologs NLGN1/2/3/4x•nlg-1, EBF3•unc-3, BRN3A•unc-86, and DYNC1H1•dhc-1 across 26 morphological, locomotor, sensory, and learning phenotypes at multiple timepoints throughout development. We found that some degree of phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of phenotypic reversibility varied by gene and phenotype. For example, re-expression of some genes was able to reverse multiple phenotypic impairments across broad developmental time windows while others displayed temporally restricted reversibility windows. This work demonstrates the advantages of using high-throughput model systems to rapidly prioritize genes for future re-expression studies and therapeutic development. Further, our work offers novel insights into neural development and the temporal requirements of gene function for multiple behaviours implicated in neurodevelopmental disorders.

Lexis Kepler1*, Troy McDiarmid1,2*, Catharine Rankin1,3

1.Djavad Mowafaghian Centre for Brain Health, University of British Columbia, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada 2.Department of Genome Sciences, University of Washington School of Medicine, Foege Building S-250 3720 15th Ave NE, Seattle, Washington, United States of America 3.Department of Psychology, University of British Columbia, 2136 West Mall, Vancouver, British Columbia V6T 1Z4, Canada * Authors contributed equally

ABSTRACT. Animals modulate their social behaviors in response to both internal and environmental signals to increase fitness. The molecular and neural circuit-based mechanisms underlying behavioral regulation in response to sensory cues remains unclear. Emerging evidence in both mammals and insects indicates the intimate connection between experience-dependent changes in gene regulation and behavioral modifications. The Drosophila melanogaster is an excellent model to investigate this question, where links among stereotyped courtship behaviors, genes and circuits have been elucidated. In Drosophila melanogaster, FruitlessM (FruM) and DoublesexM (DsxM) are master transcription factors that control sex-specific innate and learned courtship behaviors, respectively. fru is expressed in 2000 interconnected neurons marking courtship circuits, whereas dsx is expressed in 700 neurons in the nervous system. Social experience has significant effects on Drosophila behaviors. For example, monosexual grouping suppresses wild type male courtship behaviors. Furthermore, socially isolated fru mutant males do not court, however if grouped, they use olfactory social cues to learn to court with flies around them. Socially isolated fru mutant males do not court. However, if grouped, they use olfaction to learn to court either males or females. This learning requires the gene dsx, which is co-expressed with fru in decision-making neurons of courtship circuits. How social experience regulates fru and dsx expression in the courtship circuits to modify circuit function remains unknown. We found that baseline activity of neurons within courtship circuits are increased in socially isolated males. To determine molecular changes that account for circuit activity and behavioral differences, we investigated transcriptional and chromatin profiles from the peripheral and central courtship circuits from males in different social conditions and mutants in pheromone receptors and fru. We found that in the olfactory receptor neurons (ORNs) chromatin around fru gene is modified with social experience and pheromone receptor mutants. These are associated with changes in fru splice patterns, which affects expression of downstream target genes with neuromodulatory functions, ultimately modifying neuronal sensitivity and courtship behaviors. Similar to the peripheral olfactory circuits, social isolation and pheromone receptor mutants regulate active chromatin marks around both fru and dsx in the central courtship circuits in the brain, which are associated with an increase in dsx expression and changes in fru splice patterns. Signaling from different pheromone receptors elicited differential effects on chromatin around both genes. In addition, dsx expression is also increased in grouped fru mutants, which might account for the learned courtship seen in grouped fru mutants. These data suggest that social experience and pheromone circuit activity can modify fru and dsx regulation in the courtship circuits to modulate behaviors with social experience. Our results provide insights into the fundamental mechanisms by which sensory experience drive behavioral modulation and learning, via chromatin-mediated changes in the expression of genes critical for neural circuit structure and function.

Chengcheng Du1, Qichen Duan1, Bryson Deanhardt2,3, Ali Carson1, Charlie Soeder3, Corbin Jones3, Pelin C. Volkan1,2 1 Duke University, Department of Biology, 2 Duke University, Department of Neurobiology, 3 University of North Carolina, Chapel Hill, Department of Biology

ABSTRACT. Humans and mice are social creatures, finding social interactions inherently rewarding. Social motivation, the processes that drive social interactions, is a keystone of development. Social motivation is also hypothesized to underlie social deficits in Autism Spectrum Condition (ASC). Social motivation encompasses multiple components, including social reward and social orienting. Current behavioral tasks focus on assaying general socialization, while social motivation assays are limited. Thus, we adapted standard operant conditioning into an automated social operant assay, rewarding nosepokes with opportunity for transient social interaction. We directly quantify social motivation by increasing the number of active nosepokes (work) required for a reward. Uniquely, we simultaneously assess social orienting with standard automated tracking. We established that C57 mice will work for access to a social interaction, increased relative to work for access to an empty chamber, and will simultaneously orient to a social stimulus. We found that individual motivation level is stable across testing days, and interestingly, that male mice display higher motivation than females. Then, we validated the assay with two test cases. Using the Shank3b knockout (KO) mouse ASC model, we demonstrate reduced social reward-seeking in Shank3B KOs and reduced social orienting in male Shank3B KOs. In line with oxytocin’s role in social reward circuitry, we show that administration of oxytocin antagonist reduces social motivation. Overall, this social operant assay provides a newly validated tool for future use in more thorough assessment of normal social development, social deficits in mouse models, and mapping social motivation brain circuits.

Simona Sarafinovska1,2, Susan E. Maloney1,3, Katherine B. McCullough1,2, Claire Weichselbaum1,2, Joseph D. Dougherty1,2,3.

1Dept of Psychiatry, 2Dept of Genetics, 3IDDRC, Washington University in St. Louis. Funding: Jakob Gene Fund; NIMH 1R01MH107515-01A1, R01MH12480, WashU IDDRC NIH/NICHD P50 HD103525.

ABSTRACT. We previously found that male rats exposed to chronic ethanol plus withdrawal had increased expression of genes involved in RNA splicing in the hippocampus as measured by RNA sequencing (RNA-Seq). In this study we examined the expression of splicing factors and alterations in RNA splicing and in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects with alcohol use disorder (AUD). We found that expression of Pcbp1 was elevated in the rat hippocampus during withdrawal from chronic ethanol exposure and PCBP1 was increased in the hippocampus of AUD subjects by qPCR. We next analyzed the rat RNA-Seq data for differentially expressed (DE) junctions during ethanol withdrawal and found 110 DE junctions which mapped to 53 annotated unique genes. One of these genes, Hapln2, had increased usage of a novel 3’ splice site during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD subjects. Use of this splice site is predicted to result in a frameshift and introduction of a premature stop codon. Putative PCBP1 binding motifs were found near the Hapln2 exon 3-4 splice sites. We performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat hippocampus to determine if PCBP1 was associated with the Hapln2 pre-RNA and observed enrichment of PCBP1 near the Hapln2 exon 3-4 3’ splice sites during ethanol withdrawal. These results implicate the splicing factor PCBP1 in altered RNA splicing in both rats and humans after chronic ethanol exposure.

L Martins de Carvalho1, M Maienschein-Cline1,2, H Chen1, SC Pandey1, AW Lasek1

1 Center for Alcohol Research in Epigenetics and Department of Psychiatry, University of Illinois at Chicago; 2 Research Informatics Core, University of Illinois at Chicago, Chicago, IL, USA Funding support: NIAAA P50 AA022538, R01 AA027231 and U01 AA020912.

ABSTRACT. The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability. Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene’s function in cognition, mice with targeted interference of the orthologous gene were used. Behavioural, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing. Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum–anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits. These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Mice with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centred on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.

Ahmed H. Al-Amri1,2, Paul Armstrong2, Mascia Amici3, Clemence Ligneul4, Jason P. Lerch4, Jack R. Mellor3, Manir Ali2, Chris F. Inglehearn2, Steven J. Clapcote2,*

1 National Genetic Centre, Royal Hospital, Muscat, Oman 2 University of Leeds, Leeds, United Kingdom 3 University of Bristol, Bristol, United Kingdom 4 University of Oxford, Oxford, United Kingdom

ABSTRACT. Complex motor behaviors are produced through the selection of different movement sequences. However, the organization of the neural circuits that drive these sequences remains unclear. To define the neural mechanisms responsible for complex behaviors, we study grooming in fruit flies which consists of a stereotyped sequence of leg movements elicited by mechanical stimuli to different body parts. We have found that each grooming movement is elicited by mechanosensory neurons receiving input coming from different body parts which relay information to postsynaptic interneurons, including a lineage of 21 morphologically distinct neurons that we have named “brain neurons 2” (BN2). We hypothesize that each BN2 neuron elicits grooming movement induced by input from mechanosensory neurons from a specific body part to which they are connected. We will test their individual function and define the extent to which they elicit site-directed movements given by their connectivity to mechanosensory neurons. Using GAL4 transgenic lines, we have identified the BN2 neurons individually by stochastic labeling and will further characterize grooming behavior elicited by each one. We used FlyWire, a platform based on a serial section EM-based dataset of a full adult Drosophila brain, to reconstruct mechanosensory neurons projecting from the fly’s head and have defined their postsynaptic connectivity to the BN2 neurons. We will test if grooming behavior is dependent on the BN2 neurons by blocking their neuronal activity while stimulating presynaptic mechanosensory neurons. The obtained results will help us elucidate the processes involved in sequential movement selection to produce complex behaviors.

AE Santana-Cruz1,2, K Eichler3, S Hampel2, S Calle2, L Kmecova2, A Seeds1,2

1Department of Anatomy and Neurobiology, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 2Institute of Neurobiology, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 3Department of Zoology, University of Cambridge, Cambridge, United Kingdom Funding support: NIGMS-RISE R25 GM061838, NINDS R01 NS121911, NIMHD-RCMI MD007600, NIH NIGMS-COBRE GM103642

ABSTRACT. The development of tools and techniques that allow for genetic characterization in a wide array of inbred mouse strains has driven the use of reduced complexity crosses (RCC) for identifying genes and genetic variants that contribute to behavioral variation. RCCs employ crosses between inbred mouse substrains to identify quantitative trait loci (QTL) that are associated with a phenotype of interest. RCCs result in the identification of large genomic regions, similar to standard genetic crosses between genetically divergent inbred strains, but the ability to identify the causal gene(s) and variant(s) is expedited due to the drastically reduced number of polymorphisms that exist between two closely related substrains. We tested 20 substrains from 6 inbred strain lineages to identify differences in cocaineinduced locomotor activation. Two substrains from the C3H/He lineage, C3H/HeJ (HeJ) and C3H/HeNTac (NTac), exhibited striking and replicable differences in locomotor response to cocaine and were selected for genetic mapping. 388 HeJ x NTac F2 mice were phenotyped for cocaine induced locomotor activation and genotyped using the Mouse Universal Genotyping Array. QTL analysis identified only a suggestive locus on Chr 19. We identified two candidate genes at this locus with intronic polymorphisms that differed between HeJ and NTac mice. Quantitative PCR experiments are underway to characterize substrain expression differences in these genes. We also characterized the gut microbiota in both C3H/He substrains and discovered significant differences in both abundance and diversity. However, cross-fostering experiments that shifted the microbiota of HeJ to NTac, and vice versa, had no effect on substrain behavioral differences in response to cocaine. Thus, we concluded that the composition of the gut microbiota at the time of behavioral testing does not contribute to observed phenotype differences. We also examined maternal care in the HeJ and NTac dams and observed no differences. Our results suggest that divergent phenotypes observed in some inbred mouse substrains may have more complex etiologies than previously appreciated. Additional tools and strategies that can be employed to identify mechanisms that drive behavioral differences will be discussed.

Gaines CH1,2, Schoenrock SA1, Farrington J1, Lee DF1,3, Aponte-Collazo LJ3,4, Shaw GD1,5, MillerDR1,5, Ferris MT1,5, de Villena FPM1 and Tarantino LM1,6

1Department of Genetics, School of Medicine, 2 Neuroscience Curriculum, 3 Pharmacology Curriculum, 4 Department of Pharmacology, 5Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill NC, United States and 6 Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill NC, United States.

ABSTRACT. Known for their incredible sociability, individuals with Williams Syndrome also deal with increased anxiety and altered motor coordination. Animal models investigating the effects of single genes within the Williams Syndrome Critical Region have highlighted Gtf2ird1 as a candidate gene linked to motor and behavioral differences, but lack the context of the full deletion in their interpretation, limiting the translational impact of their findings. By crossing heterozygous mice modeling a functionally complete deletion of the syntenic Williams Syndrome Critical Region with heterozygous mice from a novel transgenic line overexpressing the Gtf2ird1, we examine the effect of four distinct genotypes on motor and behavioral phenotypes using performance in a suite of behavioral tasks. By directly comparing these littermates (wildtype, complete deletion (CD), Gtf2ird1 overexpression, and Gtf2ird1 molecular rescue in the context of the complete deletion), we provide a comprehensive picture of the CD mouse line as a model for Williams Syndrome-relevant motor, social, and anxiety-like phenotypes and the role of Gtf2ird1, both overexpressed alone and on the CD background. The complete deletion of the Williams Syndrome region resulted in motor deficits in tasks utilizing strength and coordination, anxiety-like phenotypes in center avoidance behavior, and increased motivation in a social operant task. These differences were not significantly altered by Gtf2ird1 overexpression or rescue, revealing that Gtf2ird1 alone is not responsible for the changes in motor, social, and anxiety behaviors. Overall, the CD mouse line appears to be a suitable model to investigate the mechanisms underlying motor and social behaviors, though anxiety-related behaviors were not consistent across tasks.

KR Nygaard1,2, SE Maloney2,3, RG Swift1,2, KB McCullough1,2 JD Dougherty1,2.

1Department of Genetics, 2Department of Psychiatry, 3IDDRC, Washington University School of Medicine, St. Louis, MO, USA Funding Support: NSF DGE-1745038, 5R01MH107515-05, P50 HD103525. Mouse models were generously donated by V. Campuzano (CD) and J. Veenstra-Vanderweele (Gtf2ird1).

ABSTRACT. It is estimated that 3.9% of the global population uses cannabis annually and that those who use cannabis within their lifetime are at high risk to develop cannabis use disorder. Polymorphisms in Cell Adhesion Molecule 2 (CADM2), a gene associated with externalizing behaviours, were recently implicated in the risk for cannabis use initiation, though these associations are correlational. In the present study, we used a Cadm2 knockout mouse line to investigate the causal relationship of Cadm2 with voluntary cannabinoid intake, THC response, and cognitive phenotypes associated with substance use. During a two-choice edible preference test comparing drug and vehicle cookie dough consumption, Cadm2-/- mice showed lower consumption of and preference for THC- and cannabis oil-containing cookie dough compared to Cadm2+/+ and Cadm2+/- mice. Preliminary results also suggest that acute and repeated injection of THC (3 and 10mg/kg) to Cadm2 knockouts induces hyperlocomotion, and that the hypothermic effects of acute, high-dose THC seen in Cadm2+/+ mice are not observed in Cadm2-/- littermates. Finally, using the 5-choice serial reaction time task, we identified multiple genotype-dependent differences in executive functions in drug-naïve mice. Cadm2-/- mice acquired the task faster, committed fewer impulsive errors, and showed impaired attentional performance compared to their wildtype and/or heterozygous littermates. Together, these data implicate Cadm2 in the presentation of impulsive behaviour, physiological response to THC, and the rewarding potential of cannabinoid-containing edibles. These findings provide support for human correlational studies that propose CADM2 polymorphisms affect externalizing phenotypes, including drug initiation and regular use.

HHA Thorpe1, AM Talhat1, S Sanchez-Roige2, AA Palmer2,3, JY Khokhar1 1Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada Funding Support: Discovery Grant from the Natural Sciences and Engineering Research Council award (RGPIN-2019-05121) to JYK 2Department of Psychiatry, University of California San Diego, San Diego, CA, USA. Funding Support: California Tobacco-Related Disease Program (T29KT0526) and National Institute on Drug Abuse (DP1DA054394) to SSR 3Institute for Genomic Medicine, University of California San Diego, San Diego, CA, USA. Funding Support: California Tobacco-Related Disease Research Program (28IR-0070) to AAP

ABSTRACT. High intensity (alcohol) drinking (HID) is a relatively newly defined threshold whereby an individual achieves blood ethanol concentrations (BECs) at or exceeding 160 mg/dl (2X legal driving limit). Given the high propensity of HID in those with an alcohol use disorder (AUD), and the known role of corticostriatal function in AUD, there is a need to identify the neurobiological mechanisms regulating HID-induced plasticity in the corticostriatal circuit. To assess this, mice were given access to water or alcohol using drinking-in-the-dark (DID) procedures, and analyses were designed specifically to identify corticostriatal gene expression alterations unique to HID. Approximately 70% of subjects displayed HID after 2 weeks of daily DID (versus <20% in first time consumers). Gene co-expression networks were then identified from corticostriatal tissue that contained genes whose expression was correlated with BEC, and key hub genes in these networks as well as others discovered using alternative approaches were explored for HID-specific differences in expression. Many BEC co-expression network hub genes were identified, including the transcription factor gene E2f3, which was positively associated with BEC in cortex and the most biologically significant gene in the network. Additionally, 57/119 genes whose expression in cortex was correlated with BEC were also differentially expresses in HID subjects (including E2f3); no genes in striatal tissue differentiated HID subjects. These data indicate that DID can be used to model HID, and that HID may be associated with unique corticostriatal gene expression alterations, particularly in cortex.

D. N. Linsenbardt1, K. Patel1, R. Sena1, T. Huffman1, and E. Simpson2 1Department of Neurosciences, University of New Mexico, Albuquerque, NM 87131; 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis IN 46202

Acknowledgments: This work was supported in part by grant #s: AA025120 (DNL), the New Mexico Alcohol Research Center P50-AA022534 (Drs. Savage & Valenzuela), and the Substance Use Disorders Grand Challenge Initiative supported by the Center on Alcohol, Substance use, And Addictions (CASAA). RNA sequencing was carried out in the Center for Medical Genomics at Indiana University School of Medicine, which is partially supported by the Indiana Genomic Initiative at Indiana University (INGEN); INGEN is supported in part by the Lilly Endowment, Inc.

ABSTRACT. During adolescence, frequent and heavy cannabis use can lead to serious adverse health effects and cannabis use disorders (CUD). Rodent models of adolescent exposure to the main psychoactive component of cannabis, delta-9-tetrahydrocannabinol (THC), mimic the behavioral alterations observed in adolescent users. However, the underlying molecular mechanisms remain largely unknown. Here, we treated female and male mice with high doses of THC during early adolescence and assessed their memory and social behaviors in late adolescence. We then profiled the transcriptome of five brain regions involved in cognitive and addiction-related processes. We applied gene coexpression network analysis and identified five gene coexpression modules, termed cognitive modules, that simultaneously correlated with THC treatment and memory traits reduced by THC. The cognitive modules were related to endocannabinoid signaling in the female dorsal medial striatum, inflammation in the female ventral tegmental area, and synaptic transmission in the male nucleus accumbens. Moreover, cross-brain region module-module interaction networks uncovered intra- and inter-region molecular circuitries influenced by THC. Lastly, we identified key driver genes of gene networks associated with THC in mice and genetic susceptibility to CUD in humans. This analysis revealed a common mechanism regulating vulnerability to CUD in the nucleus accumbens of females and males, which shared four key drivers (Hapln4, Kcnc1, Elavl2, Zcchc12). These genes regulate transcriptional subnetworks implicated in addiction processes, synaptic transmission, brain development, and lipid metabolism. Our study provides novel insights into disease mechanisms regulated by adolescent exposure to THC in a sex- and brain region-specific manner.

Yanning Zuo1,2,3, Attilio Iemolo3, Patricia Montilla-Perez3, Hai-Ri Li3, Xia Yang1,4,5, Francesca Telese3

1Department of Integrative Biology and Physiology, University of California, Los Angeles, CA 2Neuroscience Interdepartmental Program, University of California Los Angeles, CA 3Department of Medicine, University of California, San Diego, CA 4Institute for Quantitative and Computational Biosciences, University of California, Los Angeles, CA 5Brain Research Institute, University of California, Los Angeles, CA

ABSTRACT. Socialization can both relieve and induce stress. This complex and reciprocal interaction can underlie onset and exacerbation of psychiatric disorders, such as depression, anxiety, and PTSD. The highly social nature of zebrafish, extraordinary sophistication in genetic tools available for zebrafish, and conservation of the HPA-axis across vertebrates makes zebrafish a valuable tool for studying stress biology & socialization. As social behaviour and responses to chronic stress can only be measured after 21-days of age, it is not known how mutations in several key HPA-axis signalling genes, particularly, glucocorticoid receptor (GR, nr3c1 gene), mineralocorticoid receptor (MR, nr3c2 gene), and adrenocortiocotropic hormone receptor (ACTH-R, mc2r gene) mutations affect social behaviour or chronic stress responses of adult fish. In the proposed project, I will examine if zebrafish lacking functional GR, MR, and ACTH-R have altered social behaviour & stress responses and if reverting the receptor mutation will normalize the HPA-axis function in adulthood. Groups of fish (males and females) will be tested in juvenile period (21-days) and adulthood (3-months) and duration of schooling and shoaling by mutant fish and wild-type sibling controls will be quantified. Response to acute and chronic stress will further characterize the HPA-axis mutant adult phenotype. This will be the first study of behaviour in adult conditional mutant zebrafish and pave way for targeting specific brain areas. This understanding will provide excellent basic insight into regulation of vertebrate socialization by stress and into onset and aggravation of a wide range of illnesses, including mood, anxiety- and stress- and trauma-related disorders.

S Shams1, Lee H1, & K Clark1

1Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America

ABSTRACT. Normal brain function and behavior depend on the proper assembly of complex and highly specialized neuronal circuits during brain development. Yet, how the remarkable neuroanatomical complexity of the adult brain arises from a comparatively simple, segmented embryonic nervous system remains poorly understood. Understanding the genetic mechanisms that regulate the emergence of segmental circuit specialization is thus important for uncovering the etiology of neurodevelopmental and psychiatric illnesses and remains a key aim of neuroscience research. Here, we take advantage of the highly conserved molecular logic that drives axon pathfinding and midline-crossing decisions to uncover some of the genetic mechanisms that enable the transformation of the relatively simple modular embryonic nervous system into the complex and segmentally specialized adult brain using the genetically tractable vinegar fly Drosophila melanogaster. In particular, we focus on pickpocket 23 (ppk23)-expressing gustatory receptor neurons, which play an important role in male courtship behavior. The ppk23-expressing (ppk23+) neurons that reside in the tarsal segments of the prothoracic (T1) legs project their axons across the midline of the T1 segment of the VNC in males but not females. In contrast, ppk23+ neurons in the mesothoracic (T2) and metathoracic (T3) legs do not cross the midline in either sex. We find that male-, T1-specific midline crossing of ppk23+ neurons is driven by an interaction between the non-cell autonomous activity of Sex combs reduced (Scr), a Hox gene that specifies the T1-segment, and the cell-autonomous activity of fruitless (fru), a male-specific sex determination gene. Ongoing studies aim to identify the signaling pathway by which these genes interact and how their interaction modulates the downstream activity of midline crossing genes.

Nicole Leitner, Yehuda Ben-Shahar Washington University in St. Louis

ABSTRACT. Although blood lead (Pb) levels have been declining in the US, Pb exposure remains a major public health issue. Pb poisoning negatively impacts health, including intelligence, behavior, and overall life achievement. There is no identified safety threshold for Pb since low-level Pb exposure can determine severe adverse health outcomes in susceptible individuals and late onset of neurological diseases from early life Pb exposure. Epidemiological studies have consistently reported positive associations between genetic factors with Pb sensitivity. However, few studies have disentangled their effects on onset of neurological diseases. To address the influence of genetic background on neurotoxicity from Pb exposure, we utilized Collaborative Cross Recombinant Inbred Intercross (CC-RIX) mice – a novel mouse epidemiological model – to better recapitulate genetic diversity found in humans with controlled Pb exposures to identify genetic polymorphisms that drive either susceptibility to Pb neurotoxicity. CC-RIX males and females received early-life Pb exposure through lactation and then through drinking water to mimic human exposure. Our results reveal significant variation in blood Pb levels between strains exposed to the same dose along with variation in long-term effects of Pb exposure on behavior and cognition in adult mice with early-life exposure. Furthermore, to predict the genetic basis for Pb-induced neurotoxicity, we are applying a haplotype-based computational genetic mapping (HBCGM) to provide an understanding of mechanisms by which genetics and toxic exposure contribute to neurological phenotypes. The data will provide new tools for exposure detection, risk assessment, and interventions to reduce lasting effects of early-life Pb exposure.

Danila Cuomo1 and David Threadgill1

1 Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, College Station, TX 77843, USA

ABSTRACT. Two independent cohorts of HS/Npt mice were bidirectionally selected for high and low nicotine preference using a 4-bottle choice procedure, resulting in four lines (High NIC-1 and -2, Low NIC-1 and -2). RNA-sequencing was performed on the striatum and frontal cortex of naive subjects from each line. We identified significant selection effects on gene expression in all four lines by performing a likelihood ratio test (LRT) using DESeq2. The cohort 1 striatum analysis revealed 383 genes with a sex-independent selection effect and 83 genes with a sex-dependent selection effect. The cohort 2 striatum analysis revealed 80 genes with a sex-independent selection effect and 17 genes with a sex-dependent selection effect. In addition, 12 genes overlapped between the striatum cohorts 1 and 2, including Slc35a5 and Uspl1, which were upregulated in both lines of High NIC mice. The cohort 1 frontal cortex analysis revealed 235 genes with a sex-independent selection effect and 45 genes with a sex-dependent selection effect. The cohort 2 frontal cortex analysis revealed 33 genes with a sex-independent selection effect and 5 genes with a sex-dependent selection effect. In addition, 5 genes overlapped between the frontal cortex cohorts 1 and 2, including Thrsp, which was downregulated in both lines of High NIC mice. Finally, preliminary analyses with Query CMap revealed the sex-independent striatum genes of both cohorts exhibit similar signatures to the reference perturbagen signatures associated with protein synthesis inhibitor, RNA binding motif, short chain dehydrogenase/reductase superfamily/extended SDR fold, and mitochondrial respiratory chain complex/complex IV.

WC Booher1,2, LA Vanderlinden3, HC O’Neill2, ZJ Werner2, E Meyers2, HL Mathews2, JA Stitzel2,4, RA Radcliffe1,2

University of Colorado Anschutz Medical Campus

ABSTRACT. Alcohol Use Disorder (AUD) is associated with repeated alcohol exposures and addiction behaviors. Annually, 5.3% of deaths worldwide are caused by alcohol abuse (Yerby, 2022). Despite the prevalence of AUD, we lack novel therapeutic approaches for treating the cellular mechanisms of the disease. Recently the conserved Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling pathway has been implicated in both mammalian and Drosophila models of AUD (Chen et al., 2021; Petruccelli et al., 2020). JAK/STAT signaling is involved in early development and innate immune signaling, but it is unclear how STAT activity influences specific ethanol induced behaviors, such as locomotion. To address these gaps in our knowledge, this proposed work will capitalize on the single Drosophila STAT homolog, Stat92E, and the genetic tools available in flies. Recent findings in our lab have shown that Stat92E may be important for reducing behavioral sensitization, which is a distinctive feature of addiction. To follow up on this work, we will use the Gal4/UAS expression system to knock-down Stat92E in neurons and knock-down or overexpress Stat92E in glia to provide insight as to how Stat92E functions in ethanol-induced locomotion. Second, we will conduct DNA or RNA extractions in STAT-reporter flies to investigate whether repeated ethanol exposures influence the preferential isoform usage of Stat92E transcripts and downstream transcriptional activity. Findings from this work will provide better resolution of STAT signaling in the context of AUD, informing the development of molecular strategies to manage the disorder.

Alexandria Wilson1, Erica Periandri1, Emily Petruccelli1 1Department of Biological Sciences, Southern Illinois University Edwardsville, Edwardsville, IL 62026

ABSTRACT. Alcohol Use Disorder (AUD) is a serious condition that heavily impairs the lives of those affected and is defined as the inability to stop or control alcohol use despite the consequences. Many environmental and genetics factors contribute to whether an individual will develop AUD, but the role of particular cellular pathways and neural circuitry are not yet fully understood. Recently, the conserved Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signaling pathway was implicated in both mammalian and Drosophila models of AUD (Chen et al., 2021; Petruccelli et al., 2020). The role of JAK/STAT signaling is well studied in the context of embryonic development, but it is unclear how repeated alcohol exposure influences adult STAT-mediated transcriptional activity. Using previously established STAT-GFP reporter (Bach et al., 2007) and overexpressing dominant-active UAS-STATNC (Ekas et al., 2010) fly stocks, I hypothesize that STAT signaling is altered in flies previously exposed to repeated intoxication. I will also determine if STAT activity is found in adult neurons, glia, or both and identify neural cells that are sensitive to overactive STAT. This work adds to our further understanding of the cellular events underlying AUD pathology.

Mackenzie Sievers1, Erica Periandri1, Emily Petruccelli1 1Department of Biological Sciences, Southern Illinois University Edwardsville, Edwardsville, IL 62026

ABSTRACT. Binge eating disorder (BED) is defined as chronic episodes of consuming large amounts of food in less than 2 hours. BED poses a serious public health problem, as eating disorders are associated with five to seven times higher mortality rates. Binge eating is a highly heritable trait; however, its genetic basis remains largely unexplored. Our lab established a mouse model for binge eating that focuses on heritable inbred strain differences in intake of sweetened palatable food versus control chow pellets following limited, intermittent access. In the present study, we examined two genetically similar substrains of BALB/c mice in our palatable food binge-eating paradigm in which we assessed escalation in food consumption, food consumption after a period of abstinence, and compulsive-like food intake in the light/dark conflict assay. BALB/cJ and BALB/cByJ mice showed comparable levels of acute and escalated consumption of palatable food across training trials. Surprisingly, BALB/cByJ mice also showed binge-like escalation in consumption of chow pellets that was indistinguishable from palatable food intake of both substrains. Despite comparable levels of binge-like eating between sexes, male mice showed a greater increase in body weight compared to female mice. Finally, we replicated the well-documented differences in anxiety-like behavior between BALB/cJ and BALB/cByJ mice, which was associated with substrain-dependent effects in compulsive-like intake. To summarize, BALB/cByJ mice show indiscriminate levels of binge-like eating, regardless of presumed palatability. The promiscuity in binge-like eating across diets could be mediated by genetic differences in motivational responses to novel food stimuli, impulsivity, or taste perception/processing.

Katherine D. Sena1,2,4, Jacob A. Beierle3,4, Kayla T. Richardson4,5, Camron D. Bryant4

1Undergraduate Program in Neuroscience, Boston University College of Arts and Sciences, Boston, MA 2Undergraduate Research Opportunity Program, Boston University 3PhD Program in Biomolecular Pharmacology, Boston University School of Medicine 4Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine 5Boston University Post-baccalaureate Research Education Program (BU PREP)

ABSTRACT. Irregular or excessive stress can dysregulate the negative feedback loop of the HPA axis. This overutilization of the stress response can aggravate the development of complex diseases including psychiatric disorders. A long-term goal of ours is to gain further insight into the connection between stress and disease to potentially develop novel therapeutics for stress aggravated disorders. Through a forward genetic screen to test numerous transposon-mediated insertional mutant alleles, we identified two candidate genes, pbx1a and diaph1, that impact locomotor responses to acute stress in larval zebrafish. Currently, we are generating conditional knock-in fish lines to disrupt and fluorescently tag endogenous pbx1a or diaph1 protein. Through our Gene Break Homology (GBH) vector series, we also introduce a fluorescent marker in the lens, heart, or pancreas for rapid genotyping as early as 3 dpf. These new integration alleles are Cre-reversible, and currently, we are developing a Cre-reporter line targeting the C-terminus of the zebrafish StAR gene. We will employ the StAR:Cre fish line to rescue pbx1a and diaph1 function specifically in interrenal cells (zebrafish adrenal cell population). Both pbx1a and diaph1 appear to be involved in cortisol production in human adrenal cell culture. However, both genes are expressed throughout much of the CNS, and their role could be more complex or take place outside of interrenal cells. None-the-less, we predict these genes are needed for normal ACTH stimulation of cortisol production in interrenal cells.

Rebecca L. Schnabel, Ryan P. Cotter, Tanya L. Schwab, Grace Boyum, Muriel Metko, Han B. Lee Peri Goldberg, and Karl J. Clark

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Funding Support: National Institutes of Health (USA) R01GM134732

ABSTRACT. There is evidence that excessive alcohol consumption can impact sleep, potentially via disruptions in circadian rhythms, but further research is needed in this area. The purpose of the present study was to assess the impact of daily binge drinking on circadian home cage behavior. Young adult male (n=20) and female (n=20) C57BL/6J mice (Postnatal day ≈ 60) were provided with access to either alcohol or water for 2 hours a day for 6 weeks while home cage activity was continuously monitored 24 hours a day, 7 days a week. For the first two weeks, mice were maintained on a reverse 12-hour light/dark cycle. Subjects were subsequently switched to constant low light conditions for two weeks to assess endogenous circadian behavior (i.e. free-running period; aka ‘tau’), and were then returned to their original 12-hour cycle for the remainder of the experiment. Female mice consumed more ethanol than males during the first 2 weeks, but this effect faded across the following 2 weeks during the free-running period when lights were constant, and then re-emerged when initial light cycle was reintroduced. Our evaluation of locomotion revealed bi-directional alterations in 24-hour home cage locomotion as a function of sex. Interestingly, only female 24-hour activity was altered following reinstatement of original light cycle. Together, these results suggest that alcohol consumption may lead to alterations in endogenous circadian rhythms, particularly in female mice.

Monserrat Orozco, Tameryn Huffman, Marissa Westenskow, and David N. Linsenbardt

Department of Neurosciences, University of New Mexico School of Medicine and Health Sciences Center, Albuquerque, NM

Acknowledgment Funding Support: This work was supported in part by grant #s: AA025120 (DNL), the New Mexico Alcohol Research Center P50-AA022534 (Drs. Savage & Valenzuela), and the Substance Use Disorders Grand Challenge Initiative supported by the Center on Alcohol, Substance use, And Addictions (CASAA).

ABSTRACT. Williams-Beuren Syndrome (WBS) is a multi-system disorder that results in characteristic social and cognitive phenotypes, including hypersociality and impaired learning. WBS is caused by a stereotypical ~1.8 Mb deletion in chromosome 7 which results in haploinsufficiency of 25-27 genes. While some genes in this “WBS critical region” have been functionally linked to certain behavioral traits, a complete understanding of how this deletion results in the social and cognitive defects observed in WBS is lacking. To address this, we recently performed a neuronal RNAi screen in Drosophila melanogaster and identified the gene eIF4H1, a fly ortholog of the human WBS gene eIF4H, as a regulator of social behavior. This gene encodes a component of the highly conserved eukaryotic translation initiation system, leading us to hypothesize that eIF4H1 impacts fly behavior by regulating the translation of a specific set of mRNAs in the fly nervous system. We will test this hypothesis by 1) establishing a clear role for eIF4H1 in Drosophila neuronal morphology, physiology, and/or development; and 2) identifying specific neuronal mRNA species that are bound and regulated by eIF4H1. This work will clarify the role of eIF4H1 in Drosophila behavior and neuronal translation, as well as provide a potential mechanistic model for the consequence of eIF4H haploinsufficiency in WBS patients.

Erik Nolan, Iris Chin, Cassondra Vernier, Yehuda Ben-Shahar Washington University in St. Louis, Department of Biology

ABSTRACT. Open-field activity is a commonly used measure of anxiety-related behavior in rodents. The inbred High and Low Activity strains of mice, selected for extreme differences in open-field activity, have been used as a genetic model of anxiety-related behaviors. The goal of this study was to determine if treatment of adult male and female High Activity (low anxiety) and Low Activity (high anxiety) mice with diazepam, an agonist at the benzodiazepine allosteric site on the GABAA receptor and a drug commonly prescribed to treat anxiety disorders in humans, leads to decreases in anxiety-like defensive behavioral responses as assessed in the open-field test (OFT) and elevated plus-maze (EPM). We have tested the effects of three doses of diazepam (0, 0.5, 1.0, 3.0 mg/kg, i.p.), given 30 min before behavioral testing. The only anxiolytic response observed was in the High Activity animals, with more entries into the open arms of the elevated plus-maze, similar to common mouse strains. Lack of response to diazepam suggests the Low Activity animals are not displaying classic conflict anxiety-like behavior, and instead may be displaying unconditioned fear-related behaviors, such as freezing behaviors, when exposed to novelty. Fear and anxiety are distinguishable traits, and both contribute to anxiety disorders in humans.

Erika A. Mehrhoff 1,2 , Winona C. Booher 1,2 , Julianna Hutchinson1 , Grace Schumacher 1 , Curtis Borski1,2 , Christopher A. Lowry 1 , Charles Hoeffer 1,2 , Marissa A. Ehringer 1,2

1 Department of Integrative Physiology, University of Colorado Boulder; 2 Institute for Behavioral Genetics, University of Colorado Boulder Supported by the University of Colorado internal funds

ABSTRACT. Impulsivity is defined as acting on the spur of the moment in response to immediate stimuli. Two forms of impulsivity are impulsive action involving deficiency in response inhibition and impulsive choice referring to impairment in delayed gratification. Both forms of impulsivity are associated with many psychiatric disorders including addiction, ADHD and bipolar disorder. Specifically, impulsivity is involved in many stages of addiction from experimentation to relapse. While impulse control disorders show moderate to high degrees of heritability, the genetics of impulsivity is not widely studied. We aimed to identify genes and pathways underlying impulsivity using a forward genetic screen of ENU-mutagenised zebrafish. We screened 102 families estimated to cover 7926 dominant and 3554 recessive alleles. We assessed impulsive action using a zebrafish version of 5-choice serial reaction time task. In this task, after the animal learns the association between a stimulus and a reward, a pre-stimulus interval is applied during which a premature response is recorded as a measure of impulsive action. We identified 7 impulsive families of which 3 has been further tested showing heritable impulsivity. The exome sequencing of the founders of 7 impulsive families identified 113 candidate mutations of which 10 are associated with impulse control disorders in GWASs. Site-specific PCR co-segregation analysis of 15 out of 113 mutations identified a mutation co-segregating with premature response. Confirmation and characterisation of ENU and CRISPR-Cas9 mutant lines are ongoing. We demonstrated for the first time the potential success of forward genetic screen of zebrafish for impulsivity.

Saeedeh Hosseinian, William Havelange, Adele Leggieri, Munise Merteroglu, Aleksandra Mech, Sofia Angianni, Ian Sealy, Elisabeth Busch-Nentwich, Caroline Brennan School of Biological and Behavioural Sciences, Queen Mary University of London, London E1 4NS

ABSTRACT. Misuse of psychostimulants including methamphetamine (MA) is a growing public health crisis with no current FDA-approved treatments, illustrating a critical need to understand the neurobiological mechanisms. Certain MA behaviors are heritable and thus identifying causal genetic factors can elucidate novel targets and mechanisms that inform new therapeutics. Our lab previously identified Hnrnph1, encoding the RNA-binding protein hnRNP H1 (H1) as a quantitative trait gene underlying MA stimulant sensitivity. However, the mechanisms by which Hnrnph1 regulates MA behaviors remain unclear. We identified Cacna2d2, encoding the voltage-gated calcium channel subunit 22, as a candidate downstream target by which H1 influences MA locomotor stimulant sensitivity. We observed reduced mRNA at the Cacna2d2 3’UTR (where H1 binds) in H1-mutant mice treated with MA compared to wildtypes, suggesting differential H1 binding affects 22 mRNA expression. We subsequently pretreated mice with the anti-epileptic drug pregabalin (PGB), which binds selectively to the 22/21 subunits to inhibit presynaptic calcium influx. PGB pretreatment (30 mg/kg, i.p.) reduced acute MA-induced locomotion in female wildtypes, but not female mutants or males of either genotype, without altering locomotion on its own. These results were complimented by 22 mRNA decreases at the 3’UTR in PGB (compared to saline)-pretreated female wildtypes in response to MA Furthermore, PGB pretreatment lowered acquisition of MA locomotor sensitization independent of genotype. Together, our results suggest the 22 subunit causally regulates MA locomotor sensitivity via differential hnRNP H1 binding, while also highlighting PGB as a potential therapeutic for modulating MA stimulant properties.

William B. Lynch1,2,3, Qiu T. Ruan1,3,4, Michael A. Rieger5, Jiayi W. Cox6, Jacob A. Beierle1,3,4, Emily J. Yao1, Amarpreet Kandola1, Melanie M. Chen1, Julia C. Kelliher1, Richard K. Babbs1, Peter E. A. Ash7, Benjamin Wolozin7, Karen K. Szumlinski8, W. Evan Johnson9, Joseph D. Dougherty5, and Camron D. Bryant1,2,3,4*

1. Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine. 2. Graduate Program for Neuroscience, Boston University. 3. Transformative Training Program in Addiction Science, Boston University School of Medicine. 4. Biomolecular Pharmacology Training Program, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine. 5. Department of Genetics, Department of Psychiatry, Washington University School of Medicine. 6. Programs in Biomedical Sciences, Boston University School of Medicine. 7. Department of Pharmacology and Experimental Therapeutics and Neurology, Boston University School of Medicine. 8. Department of Psychological and Brain Sciences, University of California, Santa Barbara. 9. Department of Medicine, Computational Biomedicine, Boston University School of Medicine

ABSTRACT. CHARGE syndrome is a rare congenital disorder characterized by a spectrum of physical manifestations including eye, heart, craniofacial, and ear defects. CHARGE patients frequently present with a range of behavioral difficulties such as autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (AD/HD), obsessive-compulsive disorder (OCD), anxiety, and sensory deficits. Most CHARGE cases arise from de novo, loss-of-function mutations in a master transcriptional regulator, chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is an ATP-dependent chromatin remodeling protein that regulates key neurodevelopmental factors such as SOX10, ALDH1A3, TWIST, and RELN and promotes neuronal differentiation, neural crest cell development, and is required for cerebellar organization and other neural processes. While it is clear that CHD7 is required for normal neural development, how it affects neural circuit formation and function to regulate behavior is unknown. To investigate the pathophysiology of behavioral symptoms associated with CHARGE syndrome, we established a mutant chd7 zebrafish line using CRISPR/Cas9. With a panel of unbiased and high-throughput behavioral assays, we have defined multiple sensorimotor behavioral phenotypes. Our data show chd7 mutants have specific auditory and visually-driven behavioral deficits that are independent of defects in sensory structures, implicating chd7 in the regulation of underlying brain circuits. Additionally, morphological, and behavioral phenotypes depend on the location of the mutation in the gene, providing a novel insight to phenotype penetrance. To identify brain regions impacted by chd7 loss of function, we are analyzing brain-wide activity and morphometry in vivo. Together, these studies will define mechanisms of chd7-dependent neurobehavioral phenotypes and empower future work to identify potential therapeutic targets.

Dana R. Hodorovich, Patrick Lindsley, Austen Berry, Derek Burton, Kurt C. Marsden drhodoro@ncsu.edu; pmlindsl@ncsu.edu; kcmarsde@ncsu.edu Department of Biological Sciences, NC State University, Raleigh NC 27695

ABSTRACT. Dysregulation in hypothalamic-pituitary-adrenal (HPA) axis signaling can increase risk for a variety of neuropsychiatric illnesses. While it is known that this pathway depends, in part, on glucocorticoid, mineralocorticoid, and ACTH receptor genes, exploring other genes that may be implicated in stress signaling offers further insight into the mechanisms by which this signaling operates. Genes that play a role in the stress response may serve as therapeutic targets for treating related disorders and could be investigated as predictors of susceptibility to HPA-axis dysregulation. Here, two genes, tgfbr1b and unkl, appear to be genetic modifiers of this pathway.

Zebrafish are suitable for stress-related work given that their stress signaling physiology is similar to humans. As larvae, wild-type zebrafish demonstrate a consistent locomotor response to an acute stressor. In this investigation, zebrafish mutant lines were generated for tgfbr1b and unkl using gene-break transposon mutagenesis. In preliminary acute stress assays, unkl+/- larvae exhibited increased locomotor activity following exposure to acute white light exposure, while tgfbr1b+/- larvae showed a decreased response when compared to wild-type siblings. Neither line showed significant differences in response to cinnamon oil, a noxious stimulant, compared with wild-type larvae, indicating that there is no inhibition or excitation of locomotion inherent within either mutation. Follow-up studies, including decoupling heterozygous and homozygous locomotor responses for each line, are discussed. While neither gene has traditionally been associated with stress signaling, these results warrant continued exploration to evaluate the interactions by which these genes may engage with the HPA-axis signaling or specific downstream responses.

Izzabella Green Mayo CLinic, USA

ABSTRACT. The opioid epidemic is an ongoing public health crisis in the United States, and little is currently known about genetic mechanisms that contribute to risk for opioid use disorder (OUD). We are using the Hybrid Rat Diversity Panel (HRDP) of 45 inbred rat strains to examine genetic contributions to OUD-related phenotypes. Here, we present initial findings from several HRDP strains using a longitudinal behavioral phenotyping protocol. Our protocol includes a self-administration paradigm to measure acquisition of oxycodone intake, motivation to obtain oxycodone, and progression to compulsive oxycodone use. Prior to and following the self-administration period, we are performing tests for allodynia and opioid analgesia to assess for opioid tolerance and withdrawal. Although preliminary, there appear to be emerging strain differences in several phenotypes. All strains acquired oxycodone self-administration similarly during the acquisition phase. While most strains appear to escalate oxycodone intake, HXB2/lpcv rats appear to escalate rapidly during long-access sessions. As indicated by the tail immersion test, all strains display initial opioid-induced analgesia. Following oxycodone self-administration, SHR/Ola rats appear to show a heightened tolerance to the analgesic effects of oxycodone. Upon conclusion of behavioral phenotypic characterization, we are collecting brain tissue from several addiction-related brain regions to examine potential changes in gene expression. RNA expression data will be integrated with existing genotypic data to identify genes and pathways involved with oxycodone-mediated behavioral phenotypes.

Eamonn Duffy2,4, Luanne Hale1, Lucy Hall2,4, Kyle Brown1, Andrew Kwilasz1, Laura Saba2,3, Marissa A. Ehringer2,4, Ryan Bachtell1,2

1 Department of Psychology and Neuroscience, University of Colorado Boulder; 2 Institute for Behavioral Genetics, University of Colorado Boulder; 3 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus; 4 Department of Integrative Physiology, University of Colorado Boulder

Funded by NIDA U01 DA051937.

ABSTRACT. Amidst a perpetual stream of sensory stimuli, animals must distinguish between salient and innocuous stimuli to survive. For instance, sudden loud sounds can trigger a highly conserved startle response that enables animals to evade danger. An appropriate threshold must be established such that only threatening stimuli elicit the response. Dysregulation of the startle threshold can produce hypersensitivity associated with autism and anxiety-related disorders, yet the genetic regulation of this threshold is poorly understood. A forward genetic screen in zebrafish identified a hypersensitive mutant harboring a causal nonsense mutation in the Cytoplasmic Fragile X Mental Retardation Protein Interacting Protein (FMRP) 2 (cyfip2). However, the molecular pathway(s) by which cyfip2 regulates the startle threshold remain unknown. Cyfip2 binds Rac1 to promote actin polymerization and regulates RNA translation through FMRP. To determine which pathway Cyfip2 uses to regulate the startle threshold, we created heatshock-inducible cyfip2 rescue constructs with specifically mutated residues that impair interaction with Rac1 or FMRP. While wild-type Cyfip2 restores normal sensitivity in cyfip2 mutants, mutants expressing altered Cyfip2 remain hypersensitive, indicating that Cyfip2 must interact with both Rac1 and FMRP to establish the startle threshold. To identify proteins downstream of Cyfip2-Rac1/FMRP interactions that may directly affect neuronal circuit function to regulate the startle threshold, we have performed a candidate-based drug screen as well as a discovery proteomics approach to define the molecular pathways impacted by loss of cyfip2. Together, my work will determine how cyfip2 regulates a clinically relevant behavior and identify molecular mechanisms that set the acoustic startle threshold.

Jacob Deslauriers, Lindsey Russ, Rohit Ghotkar, Jordan Jarman, Sanjana Pai, Derek Burton, Kurt Marsden

North Carolina State University, USA

ABSTRACT. Understanding the pharmacokinetic profile of an opioid drug is vital to therapeutic success, and mutations in human PK genes can alter the therapeutic efficacy of opioids. We observed that 30 min post-oxycodone administration (1.25 mg/kg, i.p.) BALB/cJ mice showed a higher whole brain concentration of oxycodone, and a female-specific increase in noroxycodone, and oxymorphone compared to BALB/cByJ. This observation could explain the sex-specific increase in oxycodone state-dependent conditioned place preference in BALB/cJ female mice. To potentially link behavioral differences with PK differences, we conducted quantitative trait locus (QTL) mapping of whole brain oxycodone and metabolite concentrations in a reduced complexity cross (RCC). QTL mapping in 133 F2 mice (68F, 65M) revealed a single QTL on chromosome 15 associated with brain oxymorphone concentration that explained 32% of the phenotypic variance in females. Oxymorphone is a full agonist at the mu opioid receptor, with 8x the potency of oxycodone, and likely contributes to oxycodone addictive properties. cis-eQTL analysis revealed genetically regulated expression of Zhx2, a gene coding for a transcriptional repressor that harbors a private BALB/cJ retroviral insertion that dramatically reduces protein expression and leads to sex specific dysregulation of CYP450 genes within the liver. Whole brain and liver mass spectroscopy proteomics in the parental strains corroborated the Zhx2 eQTL. We hypothesize that decreased Zhx2 expression leads to increased CYP450 expression, increased brain oxymorphone, and increased oxycodone-induced behaviors. Interestingly, human GWAS of nicotine consumption identified a nominal association (10^-7) with ZHX2, indicating this gene could influence metabolism of multiple drugs of abuse.

Jacob A. Beierle1,2,3, Emily J. Yao1, Stan I. Goldstein1, Julia L. Scotellaro1,Anyaa Shah1, Katherine D. Sena1, Olga Averin4, David E. Moody4, Christopher A. Reilly4, Andrew Emili5, Gary Peltz6, Martin T. Ferris7, Camron D. Bryant1

1Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston University School of Medicine; 2T32 Biomolecular Pharmacology Training Program, Boston University School of Medicine; 3Transformative Training Program in Addiction Science, Boston University School of Medicine; 4Department of Pharmacology and Toxicity, University of Utah, 5Department of Biology and Biochemistry, Boston University School of Medicine; 6Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University School of Medicine, 7Department of Genetics, University of North Carolina

ABSTRACT. Most behaviors are regulated by circadian circuits, including many types of learning and memory. The magnitude of the circadian clock effect on olfactory memory can be partially masked due to the strength of the training protocol used. The “short program” consists of a single pairing of the CS+ and the US and provides a modest amount of learning and memory formation, whereas the “long program” uses 12 shocks during the pairing and will provide a performance plateau (Beck et al., 2000; Tully & Quinn, 1985). We have shown the rate of olfactory learning in Drosophila melanogaster has circadian rhythms with a peak performance in the early to midevening (Lyons and Roman 2009). How the circadian circuit transmit time-ofday (TOD) information to downstream neurons, imposing rhythms on behavior, remains mostly unknown. The Serotonergic Dorsal Anterior Lateral (DAL) neurons are part of the circadian neural circuit and express the central molecular oscillator proteins (Chen, Wu et al. 2012). These DAL neurons innervate the a/b posterior neurons of the Mushroom Bodies (MB), sites where olfactory associative learning occurs within the Drosophila brain (Aso, Hattori et al. 2014). We are testing the hypothesis that DAL neurons carry TOD information to the mushroom bodies through the rhythmic release of 5-HT. The 5HT1A receptor is expressed in the MB a/b posterior neurons. We have found that mutations in this receptor result in loss of the circadian rhythm in the rate of learning, supporting a key prediction of our hypothesis.

MT Porter1, G Roman2 1Department of Biology, 2Department of BioMolecular Sciences, University of Mississippi, Oxford, MS 38677, United States of America Funding Support: NIH grant 1R15MH121859 (Roman, PI)

ABSTRACT. Drug use disorders are chronic, relapsing diseases that are partly under genetic control. The negative mood states that characterize drug withdrawal are associated with craving and relapse in humans, and some aspects of them can be modeled in mice. Here, we measured sucrose preference and immobility in the forced swim test in the 8 inbred Collaborative Cross (CC)/Diversity Outbred (DO) founder strains following repeated saline or 10 mg/kg cocaine treatment to evaluate evidence for interstrain variation in anhedonia and dysphoria. We observed a main effect of strain on sucrose preference, F(7, 115) = 4.3; p < 0.001, ƞ! " = .206. There was no effect of drug or a drug x strain interaction. In addition, we observed a significant effect of strain on immobility time in the forced swim test, F(7, 168) = 87.8, p < 0.001; ƞ! " = .79. There was no observed effect of drug (p = 0.73) or drug x strain interaction (p = 0.78). However, it may be possible that as we increase our sample size, we will be sufficiently powered to identify drug and strain x drug interactions for both assays. Given that we did observe significant strain differences on both tests, we have good evidence for heritability of the phenotypes. It is likely that we will need to investigate different cocaine administration paradigms in order to observe any main effects of drug on negative affective withdrawal. This information may ultimately be used to inform future QTL mapping studies in the CC or DO populations.

LW Gavette1, Riley Marchin1, Emma White1, Abed Abbas1, Miko Dai2, Jay-Ho Chung1, Sonya Farrell1, Pranav Kumar1, Connor Montgomery3, Samuel Pelletier3, JT Titmus1, Olivia Peterson1, Anna Spiro1, JTTitmus1, VM Philip4, EJ Chesler5, Abraham Palmer6, CC Parker1,2

1Middlebury College Department of Psychology, Program in Neuroscience, 2Middlebury College Department of Biochemistry, 3Middlebury College Department of Psychology, 4Center for Computational Sciences, The Jackson Laboratory, Bar Harbor, Maine,USA, 5Center for Mammalian Genetics, The Jackson Laboratory, Bar Harbor, Maine, USA, 6Institute for Genomic Medicine, University of California San Diego Funding Support: P50DA039841, Middlebury College Senior Research Project Supplement

ABSTRACT. Proper cAMP-dependent protein kinase (PKA) signaling is essential for processes ranging from glucose and lipid metabolism to nerve impulse transmission. We’ve previously shown that PKA regulatory subunit IIα (RIIα) plays a role in feeding behavior, with its deletion leading to moderated palatable high-fat diet (HFD) eating and sucrose drinking via decreased PKA enzymatic activity and altered interactions along the medial habenula (mHb)-interpeduncular nucleus (IPN) axis. We showed decreased basal Hb-IPN acetylcholine and Hb glutamate levels in RIIα-knockout mice; when challenged with HFD RIIα-knockout mice had decreased striatal DARPP-32T34 phosphorylation. Here, we’ve examined RNA and protein expression to better understand the mechanism behind the observed RIIα-knockout mouse phenotype. We hypothesized that differential dopamine and/or serotonin signaling in reward-pathway regions downstream of the mHb-IPN axis might underly the observed intake behaviors. We found increased protein expression of dopaminergic regulatory enzymes tyrosine hydroxylase, DOPA decarboxylase, and catechol-o-methyltransferase in the ventral tegmental area of RIIα-knockout mice compared to wildtype littermates. Targeted dopamine and serotonin mRNA array analyses showed expression differences of several important signaling molecules in ventral striatum in mice fed control diet (CD) and after 4-week HFD access. There was significant mRNA upregulation of the monoaminergic transport-related molecules Slc18a1, Slc18a2, Slc6a3, and Htr7 in CD-fed RIIα-knockout mice compared to wildtype littermates; differences were attenuated in HFD-fed mice. Combined, these data suggest differential basal signaling of dopamine and serotonin in RIIα-knockout mice and in response to HFD-feeding. Further studies will aim to understand how these changes may contribute to the RIIα-knockout mouse intake behavior.

M. Bloyd1, E. London1, C.A. Stratakis2, C.J. McBain1

1Section on Cellular & Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development 2Section on Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, USA; Human Genetics & Precision Medicine, IMBB, Foundation for Research & Technology Hellas, Greece; Research Institute, ELPEN, SA, Athens, Greece. Funding Support: Eunice Kennedy Shriver National Institute of Child Health and Human Development Intramural Research Program

ABSTRACT. Locomotor sensitization (LS) is an easily quantifiable behavior evident as an increased locomotor response that develops after repeated administration of psychostimulants. LS relates to intense craving in humans and identifying genes involved in LS in Drosophila has a big translational potential. Cellular mechanisms underlying LS are only partially elucidated and include neuroplastic processes which have lately been connected to redox modulation. Aim of our study was to identify genes that regulate LS, with emphasis on genes that regulate redox homeostasis. To validate our results, we combined two approaches: genetic screen and proteomic analysis. We performed genetic screen by silencing redox-related genes in a tissue-specific manner using UAS/GAL4 binary expression system and testing the ability of transgenic flies to develop LS to methamphetamine using FlyBong, a high-throughput method for the analysis of locomotor response to volatilized psychostimulants. We also used proteomic analysis to compare brains from sham treated flies with those that developed LS and those that did not. The ongoing genetic screen identified and confirmed 4 genes involved in the development of LS. Gene inactivation in the whole brain often resulted in lethality or other systemic effects. Inactivation of genes only in dopaminergic and serotonergic neurons, or dopaminergic neurons alone, affected LS. This combined approach resulted in several predicted candidate genes, such as malic enzyme, catalase, and superoxide dismutase. We show involvement of redox-related genes in LS which suggests a possibility of targeting redox pathways in prevention or for the therapeutic intervention of addiction.

Franka Rigo1, Ana Filošević Vujnović1, Rozi Andretić Waldowski1

1Department of Biotechnology, University of Rijeka, Rijeka, Croatia Funding Support: Croatian Science Foundation (#2794)

ABSTRACT. Dunc13, homologous to mammalian Munc13-1, is a presynaptic active zone protein. Diacyl glycerol binds to the C1 domain of Munc13-1/Dunc13 which decreases the energy barrier for vesicle fusion. Alcohol also binds to this C1 domain, which then inhibits DAG binding. Reduction of Dunc13 through mutation or transgenic Dunc13 RNAi expression leads to a decrease in alcohol sedation sensitivity. Moreover, targeted reduction of Dunc13 expression in the mushroom body leads to a decrease in alcohol sensitivity, whereas reduction of Dunc13 expression in the ellipsoid body seems to increase alcohol sensitivity. These data indicate a differential regulation of alcohol sensitivity by Dunc13 in different areas of the fly brain. To better understand how reducing Dunc13 activity changes alcohol sensitivity, we examined gene expression of presynaptic vesicle fusion proteins following an intoxicating dose of alcohol using RT-qPCR. Interesting RIM and Rab3 expression significantly increased, but Dunc13 and Synaptobrevin expression significantly decreased after 4 hours of alcohol exposure. Dunc-13P84200/+ heterozygotes have 50% wild type levels of Dunc-13 mRNA which closely approximated the levels found in ethanol exposed wild type flies. However the increase in expression of RIM and Rab3 were blunted in Dunc-13P84200/+ heterozygotes. Dunc13 functions along with RIM and Rab3 proteins as a heterotrimeric complex during presynaptic vesicle fusion. We further examined s RIMMB07541 insertional mutants and RIMEX73 deletion mutants for phenotypes in sedation sensitivity in. RIMMB07541 and RIMEX73 homozygotes and heterozygotes displayed a significant increase in alcohol sensitivity. Interestingly, the RIMMB07541/+; Dunc-13P84200/+ double heterozygotes were not significantly different from wild-type controls. Together these results suggest an interaction between these genes in regulating alcohol sedation sensitivity.

Gaurav Shrestha1, Gemunu Gunaratne2, Gregg Roman3 1Department of Biology, University of Mississippi, Oxford Mississippi, 2Department of Physics, University of Houston, Houston TX, 3Department of BioMolecular Sciences, University of Mississippi, Oxford Mississippi

ABSTRACT. Adolescence is sensitive to the effects of nicotine, as use increases likelihood of dependence to nicotine and drugs of abuse in adulthood. Genetics play a significant role in cognitive deficits and anxiety related adolescent nicotine use. To assess the role of genetic background on these responses, we utilized 4 mouse strains (C57BL/6J, A/J, NOD/ShiLtJ and 129S1). Male and female adolescent mice were exposed to nicotine via osmotic minipumps (PND 32- PND 44). To test the effects of adolescent nicotine exposure on learning in adulthood, we utilized a fear conditioning paradigm. We characterized anxiety-like phenotypes with the elevated plus maze (EPM). Lastly, we tested differences in adult nicotine-induced hypolocomotor activity. We found no evidence of treatment effect on learning (p>0.05), however, we found strain differences in freezing to the context, post hoc analyses revealed differences in context freezing as follows 129S1 = A/J >C57BL/6J>NOD/ShiLtJ. For cued learning, sex differences were seen with NOD/ShiLtJ females having higher freezing when compared to NOD/ShiLtJ males. Overall, for cued learning, we have the following results: 129S1>C57BL/6J>A/J>NOD/ShiLtJ. For EPM, a main effect of strain was found, with percent time spent in open arms listed as follows NOD/ShiLtJ>C57BL/6J>129S1> A/J. Lastly, nicotine sensitivity differed by sex and strain differences, with NOD/ShiLtJ mice being more sensitive to the effects of an acute nicotine dose. We concluded that genetic background plays an important role in learning, anxiety-like behavior and nicotine sensitivity. Current studies are assessing if differences in nicotine metabolism are related to behavioral differences.

Garcia-Trevizo, P.1, Novoa, C.1, Gould, T.J.1

1.The Pennsylvania State University, Biobehavioral Health, University Park, PA, US. Funding Support: NIDA R03DA048166, T32DA017629; Fulbright Colombia - Minciencias 20210798

ABSTRACT. Neurons in the arcuate nucleus of the hypothalamus (ARH) that co-express the neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) are essential for driving food intake. Consistent with their role in energy balance, their activity is tightly correlated with nutritional status: increased activity is associated with hunger while decreased activity is associated with satiety. Further, a rapid response to food cues suggests that this response does not require ingestion and supports evidence for “top-down” synaptic modulation of AgRP neurons. Our lab has previously demonstrated that AgRP neuronal activity is sensitive to diet thus diet-dependent plasticity may be a causal factor in obesity. However, up to 70% of the variability in BMI is attributable to genetic factors, most of which remains unexplained. Notably, genes within BMI-associated loci in human GWAS are highly enriched for the brain and CNS, suggesting the susceptibility to obesity may be strongly influenced by GxE effects on neuronal function. However, this research has primarily been conducted using inbred mouse models that lack genetic diversity, and do not capture the considerable influence that genetic factors play in controlling obesity and bodyweight. Here, we utilize mouse strains resilient and susceptible to diet-induced obesity to explore the impact of high-fat diet (HFD) feeding on AgRP neuronal function. Genetically resilient mice (WSB/EiJ) and susceptible (NZO/H1ltJ) mice of both sexes were assessed for feeding and anxiety-like behavior; we additionally assessed AgRP neuronal excitability and synaptic plasticity in identified NPY+ neurons using an F1 panel. Predictably, compared to B6 mice, genetically resilient mice consume less food while susceptible mice consume significantly more. This effect is sexually dimorphic as females in both the resilient and susceptible strains are less likely to gain weight on an obesogenic diet. AgRP neuronal activity mirrors diet-induced weight gain in most susceptible strains, with the exception of the particularly resilient WSB/EiJ. Synaptic plasticity onto AgRP neurons suggests that mechanisms that drive AgRP excitability in fasted B6 mice are similar to resilient and susceptible mice, while diet-induced excitability originates from a divergent etiology. Ongoing work will explore hypothalamic gene expression and the prevalence of inflammatory markers (GFAP and IBA1) in these mice. Overall, we hope these studies will identify genetic traits linked to weight gain and obesity, which might provide insight into the diversity of the human response to DIO.

Austin C. Korgan, Sophie L.A. Martin, Kristen M.S. O’Connell The Jackson Laboratory

ABSTRACT. Known for their incredible sociability, individuals with Williams Syndrome also deal with increased anxiety and altered motor coordination. Animal models investigating the effects of single genes within the Williams Syndrome Critical Region have highlighted Gtf2ird1 as a candidate gene linked to motor and behavioral differences, but lack the context of the full deletion in their interpretation, limiting the translational impact of their findings. By crossing heterozygous mice modeling a functionally complete deletion of the syntenic Williams Syndrome Critical Region with heterozygous mice from a novel transgenic line overexpressing the Gtf2ird1, we examine the effect of four distinct genotypes on motor and behavioral phenotypes using performance in a suite of behavioral tasks. By directly comparing these littermates (wildtype, complete deletion (CD), Gtf2ird1 overexpression, and Gtf2ird1 molecular rescue in the context of the complete deletion), we provide a comprehensive picture of the CD mouse line as a model for Williams Syndrome-relevant motor, social, and anxiety-like phenotypes and the role of Gtf2ird1, both overexpressed alone and on the CD background. The complete deletion of the Williams Syndrome region resulted in motor deficits in tasks utilizing strength and coordination, anxiety-like phenotypes in center avoidance behavior, and increased motivation in a social operant task. These differences were not significantly altered by Gtf2ird1 overexpression or rescue, revealing that Gtf2ird1 alone is not responsible for the changes in motor, social, and anxiety behaviors. Overall, the CD mouse line appears to be a suitable model to investigate the mechanisms underlying motor and social behaviors, though anxiety-related behaviors were not consistent across tasks.

KR Nygaard1,2, SE Maloney2,3, RG Swift1,2, KB McCullough1,2 JD Dougherty1,2.

1Department of Genetics, 2Department of Psychiatry, 3IDDRC, Washington University School of Medicine, St. Louis, MO, USA Funding Support: NSF DGE-1745038, 5R01MH107515-05, P50 HD103525. Mouse models were generously donated by V. Campuzano (CD) and J. Veenstra-Vanderweele (Gtf2ird1).

ABSTRACT. Humans and mice are social creatures, finding social interactions inherently rewarding. Social motivation, the processes that drive social interactions, is a keystone of development. Social motivation is also hypothesized to underlie social deficits in Autism Spectrum Condition (ASC). Social motivation encompasses multiple components, including social reward and social orienting. Current behavioral tasks focus on assaying general socialization, while social motivation assays are limited. Thus, we adapted standard operant conditioning into an automated social operant assay, rewarding nosepokes with opportunity for transient social interaction. We directly quantify social motivation by increasing the number of active nosepokes (work) required for a reward. Uniquely, we simultaneously assess social orienting with standard automated tracking. We established that C57 mice will work for access to a social interaction, increased relative to work for access to an empty chamber, and will simultaneously orient to a social stimulus. We found that individual motivation level is stable across testing days, and interestingly, that male mice display higher motivation than females. Then, we validated the assay with two test cases. Using the Shank3b knockout (KO) mouse ASC model, we demonstrate reduced social reward-seeking in Shank3B KOs and reduced social orienting in male Shank3B KOs. In line with oxytocin’s role in social reward circuitry, we show that administration of oxytocin antagonist reduces social motivation. Overall, this social operant assay provides a newly validated tool for future use in more thorough assessment of normal social development, social deficits in mouse models, and mapping social motivation brain circuits.

Simona Sarafinovska1,2, Susan E. Maloney1,3, Katherine B. McCullough1,2, Claire Weichselbaum1,2, Joseph D. Dougherty1,2,3.

1Dept of Psychiatry, 2Dept of Genetics, 3IDDRC, Washington University in St. Louis. Funding: Jakob Gene Fund; NIMH 1R01MH107515-01A1, R01MH12480, WashU IDDRC NIH/NICHD P50 HD103525.