ABSTRACT. We have developed imaging and computational workflows to accurately align multimodal 3D magnetic resonance histology (MRH) of the mouse brain with light sheet microscopy (LSM) and fully rendered 3D reference delineations. The method starts with geometrically accurate MRH of the brain in the skull by multi-gradient echo (MGRE) and diffusion tensor imaging (DTI) at an isotropic resolution of 15 m/voxel. Connectomes are generated using over 100 scan angles with a super-resolution of ~5 m. Brains are subsequently cleared with SHIELD, stained for selected proteins and imaged by LSM at 1.8 m/pixel. LSM data are registered into the reference space using a modified Allen Brain Atlas (ABA) Common Coordinate Framework. The result is a high-dimensional integrated volume with registration (HiDiver) that has a global alignment accuracy of ~ 50 µm. HiDiver now enables 3D quantitative and global analyses of cells, circuits, connectomes, and CNS regions of interest (ROIs). Throughput is sufficiently high that HiDiver is now being used in comprehensive quantitative studies of the impact of gene variants and aging on rodent brain cytoarchitecture.

G. Allan Johnson, Ph.D.1, Yuqi Tian, BS1, Gary P. Cofer, MS1, James J. Cook, BSCE1, James C. Gee, Ph.D.2, Adam Hall, Ph.D.3, Kathryn Hornburg, Ph.D.1, Yi Qi, MD1, Fang-Cheng Yeh, Ph.D.4, Nian Wang, Ph.D.5, Leonard E. White, Ph.D.6, Robert W. Williams, Ph.D.7

1 Duke University, Center for In Vivo Microscopy 2 University of Pennsylvania, Department of Radiology 3 LifeCanvas Technology 4 University of Pittsburgh, Department of Neurologic Surgery 5 Indiana University, Department of Radiology 6 Duke University, Department of Neurobiology 7 University of Tennessee Health Science Center, Department of Genetics, Genomics and Informatics

ABSTRACT. The ever-increasing use of mouse models in preclinical neuroscience research calls for an improvement in the methods used to translate findings between mouse and human brains. Using openly accessible brain-wide transcriptomic data sets, we evaluated the similarity of mouse and human brain regions on the basis of homologous gene expression. Our results suggest that mouse-human homologous genes capture broad patterns of neuroanatomical organization, but that the resolution of cross-species correspondences can be improved using a novel supervised machine learning approach. Using this method, we demonstrate that sensorimotor subdivisions of the neocortex exhibit greater similarity between species, compared with supramodal subdivisions, and that mouse isocortical regions separate into sensorimotor and supramodal clusters based on their similarity to human cortical regions. We also find that mouse and human striatal regions are strongly conserved, with the mouse caudoputamen exhibiting an equal degree of similarity to both the human caudate and putamen.

A. Beauchamp1,2,3, Y. Yee1,2,3, B. C. Darwin1,2, A. Raznahan4, R. B. Mars5,6, J. P. Lerch1,2,3,5

1Mouse Imaging Centre, Toronto, Ontario, Canada. 2The Hospital for Sick Children, Toronto, Ontario, Canada. 3Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 4Section on Developmental Neurogenomics, Human Genetics Branch, National Institute of Mental Health Intramural Research Program, Bethesda, MD, U.S.A. 5Wellcome Centre for Integrative Neuroimaging, Centre for Functional MRI of the Brain (FMRIB), Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom. 6Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, Netherlands.

ABSTRACT. Brain structure scaffolds intrinsic function, supporting cognition and, ultimately, behavioral flexibility. However, it remains unclear how a static, genetically controlled architecture may support such flexible cognition and behavior. In the current work, we evaluated genetic (twin-based heritability), phylogenetic (humans versus macaques) and cognitive analyses to understand how the macroscale organization of structure-function coupling across the cortex can inform its role in cognition. In humans, structure-function coupling was highest in regions of unimodal cortex and lowest in transmodal cortex, a pattern that was mirrored by a reduced alignment with heritable connectivity profiles. Structure-function uncoupling in non-human primates had a similar spatial distribution, but we observed an increased coupling between structure and function in association regions in macaques relative to humans. Meta-analysis suggested regions with the least genetic control (low heritable correspondence and different across primates) are linked to social cognition and autobiographical memory. Our findings establish that genetic and evolutionary uncoupling of structure and function in different transmodal systems may support complex forms of human cognition.

SL Valk1,2,3, T Xu4, C Paquola5,6, B Park5,7, RAI Bethlehem8, R Vos de Wael5, Jessica Royer5, S Kharabian Masouleh2,3, Ş Bayrak1, P Kochunov9, BTT Yeo10-14, D Margulies15, J Smallwood16, SB Eickhoff2,3*, BC Bernhardt5*

1. Otto Hahn Group Cognitive Neurogenetics, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany; 2. Institute of Neuroscience and Medicine, Brain & Behaviour (INM-7), Research Centre Jülich, Jülich, Germany; 3 Institute of Systems Neuroscience, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany;4. Center for the Developing Brain, New York City, USA; 5. Multimodal Imaging and Connectome Analysis Lab, McConnell Brain Imaging Centre, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada; 6. INM-1, FZ Jülich, Jülich, Germany; 7. Department of Data Science, Inha University, Incheon, South Korea; 8. Department of Psychiatry, Cambridge University, Cambridge UK; 9. Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, USA; 10. Department of Electrical and Computer Engineering, National University of Singapore, Singapore, Singapore; 11. Centre for Sleep and Cognition (CSC) & Centre for Translational Magnetic Resonance Research (TMR), National University of Singapore, Singapore, Singapore; 12. N.1 Institute for Health & Institute for Digital Medicine (WisDM), National University of Singapore, Singapore, Singapore; 13.Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America; 14 Integrative Sciences and Engineering Programme (ISEP), National University of Singapore, Singapore, Singapore; 15 Neuroanatomy and Connectivity Lab, Institut de Cerveau et de la Moelle epiniere, Paris, France; 16 Department of Psychology, Queen’s University, Kingston, Ontario, Canada

ABSTRACT. Human neuroimaging offers non-invasive insights into the structure and function of the living brain, allowing us to quantify variability in the brain that may confer risk for psychiatric and neurological disorders. However, statistical principles do not make exceptions for medical data, and the potential to use imaging to understand the variation in the brain that may confer risk for psychiatric and neurological disorders has been hampered by an overwhelming number of unreproducible findings. Studies with small samples, overly-lenient thresholds for statistical significance, and publication biases tend to seed hypothesis driven research, leading to a cyclic exaggeration of potentially incomplete or biased, unreliable findings. Recently, large-scale international consortia have been formed to address the reliability and reproducibility of biomedical findings. This includes neuroimaging genetic associations. I will be discussing the work and findings from the ENIGMA consortium, which began to perform unbiased genome-wide association scans of brain structure and has recently pooled together neuroimaging and genomic information from over 60 datasets around the world. We have identified genetic loci that help shape localized cortical and subcortical brain morphometry, their changes across the lifespan, and have identified genetic architectures that relate to genetic risk factors for disease. ENIGMA has expanded beyond studies of common genetics to incorporate over 30 clinical, methodological, and biologically focused working groups.This talk will highlight findings from a few of our ongoing initiatives in ENIGMA, touch upon new study directions currently underway, and aims to discuss open challenges in the field of collaborative neuroscience.

Neda Jahanshad(1) for the ENIGMA Consortium

1) Laboratory of Brain eScience, Imaging Genetics Center, Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, Marina dey Rey, CA 90292

ABSTRACT. White matter tracts form the structural basis of large-scale functional networks in the human brain. We applied brain-wide tractography to diffusion images from 30,810 adult participants (UK Biobank), and found significant heritability for 90 regional connectivity measures and 851 fiber tracts. Multivariate genome-wide association analyses identified 355 independently associated lead SNPs across the genome, of which 77% were not previously associated with human brain metrics. Enrichment analyses implicated neurodevelopmental processes including neurogenesis, neural differentiation, neural migration, neural projection guidance, and axon development, as well as prenatal brain expression especially in stem cells, astrocytes, microglia and neurons. We used the multivariate association profiles of lead SNPs to identify 26 genomic loci implicated in structural connectivity between core regions of the left-hemisphere language network, and also identified 6 loci associated with hemispheric left-right asymmetry of structural connectivity. Polygenic scores for schizophrenia, bipolar disorder, autism spectrum disorder, attention-deficit hyperactivity disorder, reading ability, left-handedness, Alzheimer’s disease, amyotrophic lateral sclerosis, and epilepsy showed significant multivariate associations with structural connectivity, each implicating specific sets of brain regions with trait-relevant functional profiles. This large-scale mapping study revealed common genetic contributions to the human brain's structural connectome in the general adult population, and its links with polygenic disposition to brain disorders and behavioral traits.

Zhiqiang Sha1, Dick Schijven1, Simon E. Fisher1,2, Clyde Francks1,2,3*

1 Language and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands 2 Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands 3Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

ABSTRACT. Although individual differences in animal behavior have been increasingly recognized in recent years, their biological and neural basis remains poorly understood. Here, we describe our progress in identifying the presence of individual differences in the exploratory behavior of adult zebrafish and mapping behavioral differences to patterns of whole brain activity. To identify the presence of individual differences, we tracked the three-dimensional trajectories from over four hundred fish from four inbred strains, and both sexes, during the exploration of a novel tank. Three-dimensional tracking was achieved by combining depth sensing cameras with a deep learning approach (DeepLabCut) for pose estimation. Using four exploratory behaviors (bottom distance, center distance, distance travelled, and percent tank explored) and community detection on a k-nearest neighbor network, we found that exploratory behavior stratified into four distinct clusters. These clusters included previously described bold and shy behaviors as well as two additional behavioral types. We also found that, upon repeated exposure to a novel tank, individual behaviors were consistent, and that the distribution of clusters differed based on the sex and strain of the fish. To identify the neural basis for these individual differences, we have incorporated our recently created adult zebrafish brain atlas (azba.wayne.edu) with the open source BrainGlobe (docs.brainglobe.info) suite of tools for brain mapping and cell counting. Whole-brain activity maps are generated by combining tissue clearing (iDISCO) with in situ hybridization chain reaction and light-sheet microscopy.

Justin W. Kenney, Neha Rajput, Kailyn Fields, Kush Parikh, Matheu Wong Wayne State University Department of Biological Sciences Detroit, MI 48202

ABSTRACT. Body temperature homeostasis is an essential function that relies upon the integration of the outputs from multiple classes of cooling- and warming-responsive cells. The computations that integrate these diverse outputs to control body temperature are not understood. Here we discover a new set of Warming Cells (WCs) and show that the outputs of these WCs and previously described Cooling Cells (CCs) are combined in a cross-inhibition computation to drive thermal homeostasis in larval Drosophila. We find that WCs and CCs are opponent sensors that operate in synchrony above, below, and near the homeostatic set-point, with WCs consistently activated by warming and inhibited by cooling, and CCs the converse. We show that molecularly, these opponent sensors rely on overlapping combinations of Ionotropic Receptors to detect temperature changes: Ir68a, Ir93a, and Ir25a for WCs; Ir21a, Ir93a, and Ir25a for CCs. Using a combination of optogenetics, sensory receptor mutants, and quantitative behavioral analysis, we find that the larva uses a flexible cross-inhibitory sensorimotor transformation of WC and CC outputs to locate and stay near the homeostatic set-point. Near the set-point, balanced cross inhibition suppresses avoidance to cooling and warming. Above the set-point, WCs mediate avoidance to warming while cross-inhibiting avoidance to cooling. Below the set-point, CCs mediate avoidance to cooling while cross-inhibiting avoidance to warming. We use electron microscopy to map all downstream synaptic partners of the WCs and CCs, and connectome based models to identify candidate circuits for the implementation of the cross-inhibitory sensorimotor transformation. Our results demonstrate how flexible integration between warming and cooling pathways can orchestrate homeostatic thermoregulation.

Luis Hernandez-Nunez Harvard University, USA

ABSTRACT. Ethanol disrupts learning and memory, and the severity of these effects may vary by genetic background, sex, and age. To understand how genetics and sex contribute to adolescent learning outcomes after ethanol, we surveyed fear conditioning after ethanol in a panel of inbred mice. Adolescent (PND 38 +/- 3) male and female mice from 9 inbred strains [C57BL/6J, C57BL/6NJ, DBA2/J, 129S1/SvImJ, A/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, and FVB/NJ (The Jackson Laboratory, Bar Harbor, ME; n=9/sex/strain/treatment)] were treated with ethanol (1.5 g/kg, i.p., 20% w/v in 0.9% saline) or saline 15 minutes prior to fear conditioning training. Contextual and cued learning were tested one day later. Contextual fear learning was most sensitive to disruption by pre-training ethanol, with heritable (31%) freezing outcomes dependent upon both strain (p<0.001) and sex (p=0.046; females more impaired than males). Cued fear learning was also impaired by ethanol, although to a lesser extent than contextual learning, with heritable (18%) freezing outcomes dependent upon strain (p<0.001) and not sex. Blood ethanol concentration (BEC) assessment suggested that strain differences in learning after ethanol were not related to ethanol metabolism. Next, we conducted RNA-sequencing of the dorsal hippocampus, a region uniquely involved in contextual learning, in C57BL/6J and DBA/2J strains to identify genetic mechanisms involved in ethanol-associated deficits. We found unique ethanol- and learning-associated transcripts and pathways across strains. Collectively, we demonstrated a genetic basis for learning outcomes after adolescent ethanol exposure in inbred mice and we have begun identifying neural pathways related to adolescent ethanol-related cognitive deficits.

Laurel R. Seemiller1, Lisa R. Goldberg1, Aswathy Sebastian2, Dana Zeid1, Istvan Albert3, and Thomas J. Gould1

1Department of Biobehavioral Health, Penn State University, University Park, PA, USA 2Huck Institutes of the Life Sciences, Penn State University, University Park, PA, USA 3Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA, USA

Funding: T32GM108563 (L.R.S.), 1U01DA041632 (T.J.G), the Jean Phillips Shibley Endowment (T.J.G.), and Penn State University (T.J.G.).

ABSTRACT. Over the last decade, there has been a steady rise in pregnant women diagnosed with opioid use disorder and increasing cases of infants born with neonatal opioid withdrawal syndrome (NOWS). The symptoms of NOWS include low body weight, impaired thermoregulation, irritability, and hyperalgesia. In our mouse model of NOWS, neonatal FVB/NJ, FVB/NCrl, and FVB/NHsd mice were administered morphine (10 mg/kg, s.c.) twice daily from postnatal day 1 (P1) to P15. Hot plate latency, tail-flick latency, and ultrasonic vocalization (USV) recordings were conducted on P7 and P14 during spontaneous morphine withdrawal, to determine the effects of repeated opioid exposure on pain sensitivity and irritability. We observed robust thermal hyperalgesia in all three substrains with no significant substrain x treatment interactions, Furthermore, we found main effect of morphine treatment on the number of USVs emitted in morphine- and saline-treated pups at P14. Using the deep learning software, DeepSqueak, we classified USV syllables to determine if certain syllables are associated with the effects of opioid exposure. Interestingly, some syllables were exclusive to one strain or one treatment group. We plan to conduct RNA sequencing on brain regions associated with withdrawal, such as the midbrain, brainstem, striatum, and hypothalamus, to identify brain mechanisms related to NOWS. Additionally, using quantitative trait locus (QTL) mapping, we aim to identify causal genetic variants responsible for differences in withdrawal-associated behaviors.

Kelly Wingfield1,2, Kayla Richardson1,3, Teodora Misic1,4, Emily Yao1, Jacob Beirle1,2, Camron D. Bryant1,4

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; 3Post-Baccalaureate Research Education Program, Boston University School of Medicine; 4Department of Neuroscience, Boston University.

ABSTRACT. The majority of people who try a given drug do not develop a corresponding substance use disorder (SUD). Part of the risk for developing an SUD is known to be mediated by genetic differences. Understanding the mechanism by which the genetic risk for SUDs is mediated has broad implications for prevention and treatment. Both SUDs and the differential genetic risk for SUD-relevant behavioral traits can be modeled in rodents. For some of these behavioral traits, rats offer unique advantages in comparison to mice. The NIDA Center for GWAS in Outbred Rats was initially funded in 2014 and has supported the use of HS rats in more than a dozen NIH-funded projects that are now using outbred heterogeneous stock (HS) rats. Currently more than 10,000 HS rats have been phenotyped and genotyped, with funding in place to support more than 5,000 additional HS rats. The majority of these studies examine behavioral traits relevant to SUDs such as self administration of nicotine, cocaine, oxycodone, and heroin, as well as measures of impulsivity and other behaviors that are associated with SUDs. In this talk I will describe the foundations of the center, which include the breeding and distribution of HS rats, the methods used for genotyping HS rats, and the standard analysis pipeline which includes GWAS, PheWAS, heritability estimates, genetic correlations and many other useful features. Specific results will be used to illustrate the importance of these key features of the center.

AA Palmer 1,2 1 Department of Psychiatry, University of California San Diego, La Jolla, USA, 2 Institute for Genomic Medicine, University of California San Diego, La Jolla, USA. Funding Support: NIDA P50 DA037844

ABSTRACT. Many personality traits are influenced by genetic factors. Rodents models provide an efficient system for analyzing genetic contribution to these traits. Using 1,246 adolescent heterogeneous stock (HS) male and female rats, we conducted a genome-wide association study (GWAS) of behaviors measured in an open field, including locomotion, novel object interaction, and social interaction. We identified 30 genome-wide significant quantitative trait loci (QTL). Using multiple criteria, including the presence of high impact genomic variants and co-localization of cis-eQTL, we identified 17 candidate genes (Adarb2, Ankrd26, Cacna1c, Cacng4, Clock, Ctu2, Cyp26b1, Dnah9, Gda, Grxcr1, Eva1a, Fam114a1, Kcnj9, Mlf2, Rab27b, Sec11a, and Ube2h) for these traits. Many of these genes have been implicated by human GWAS of various psychiatric or drug abuse related traits. In addition, there are other candidate genes that likely represent novel findings that can be the catalyst for future molecular and genetic insights into human psychiatric diseases. Together, these findings provide strong support for the use of the HS population to study psychiatric disorders.

Mustafa Hakan Gunturkun1, Tengfei Wang1, Apurva S. Chitre2, Angel Garcia Martinez1, Katie Holl3, Celine St. Pierre2, Hannah Bimschleger2, Jianjun Gao2, Riyan Cheng2, Oksana Polesskaya2, Leah C. Solberg Woods3, Abraham A. Palmer2,4 and Hao Chen1*

1Department of Pharmacology, Addiction Science and Toxicology, University of Tennessee Health Science Center, Memphis, TN, United States 2Department of Psychiatry, University of California, San Diego, La Jolla, CA, United States 3Department of Internal Medicine, Wake Forest School of Medicine, Winston Salem, NC, United States 4Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, United States

ABSTRACT. Addiction vulnerability is associated with non-drug traits such as the tendency to attribute incentive salience to reward cues, and is influenced by environmental and genetic factors. To characterize the genomic regions associated with these behaviors, we performed a genome-wide association study (GWAS) in two cohorts of phenotypically and genetically diverse N/NIH Heterogeneous Stock (HS) rats at two institutions, focusing on cue-responsivity measured during a Pavlovian conditioned approach paradigm. Many loci and candidate genes were identified using multiple bioinformatic approaches, including Dlg2, Tenm4, Mir708, and Taar1. Some of these genes have been previously associated with addiction-related behaviors, others some have not. Thus, we demonstrate that HS rats are useful for identifying previously unidentified genetic variants influencing addiction-related traits.

PJ Meyer1. CP King1, BM Thompson1, AS Chitre2, O Polesskaya2, SB Flagel3,4 TE Robinson5, LC Solberg Woods6, AA Palmer7,8,

1Department of Psychology, University at Buffalo, NY, USA, 2Department of Psychiatry, University of California San Diego, La Jolla, USA, Department of Psychiatry, University of Michigan, Ann Arbor, USA, 4Michigan Neuroscience Institute, University of Michigan, Ann Arbor, USA, 5Department of Psychology, University of Michigan, Ann Arbor, USA, 6Department of Internal Medicine, Molecular Medicine, Center on Diabetes, Obesity and Metabolism, Wake Forest School of Medicine, Winston-Salem, USA, 7Department of Psychiatry, University of California San Diego, La Jolla, USA, 8Institute for Genomic Medicine, University of California San Diego, La Jolla, USA. Funding Support: NIDA P50 DA037844

ABSTRACT. Substance use disorder is a complex trait influenced by genetic and environmental factors. Identifying the molecular mechanisms that give rise to addiction liability remains a challenge. One way to address this challenge is to use model organisms to determine the link between genetic variants, genes, signaling pathways, and relevant behaviors. In this study, we leverage the genetic diversity of the outbred population of HS rats combined with the extended access model of cocaine self-administration, a model that better reflects behavioral changes observed in humans with cocaine use disorder. We compared HS rats classified as having a low or high addiction index based on several behavioral measures of addiction severity. We used amygdala tissues collected after prolonged abstinence (5 weeks) from HS rats with low and high addiction indexes to profile gene expression and chromatin accessibility in individual cells by conducting single-nuclei RNA-seq and ATAC-seq, respectively. We identified cell type-specific differentially expressed genes and differentially accessible open chromatin regions associated with cocaine addiction-like behaviors. Pathway enrichment analysis revealed several signaling pathways perturbed by cocaine use in a cell type-specific manner, including changes in glycolysis pathways. By perturbing a step of the glycolysis pathway with a pharmacological agent, we rescued key cellular and behavioral measures associated with addiction-related phenotypes. Our work has identified new cell-type-specific transcriptional and regulatory mechanisms associated with addiction-related behaviors. These results advance our understanding of the molecular basis of the neuroadaptations induced by long-term use of addictive drugs, such as cocaine.

Telese Francesca1, Jessica Zhou2,3, Hairi Li1, Giordano de Guglielmo4, Marsida Kallupi4Lieselot Carrette4, Olivier George4, Abraham A. Palmer4, Graham McVicker3

1 Department of Medicine, University of California San Diego 2 Bioinformatics and Systems Biology Graduate Program, University of California San Diego 3 Integrative Biology Laboratory, Salk Institute for Biological Studies 4Department of Psychiatry, University of California San Diego

Funding: U01DA050239

ABSTRACT. While obesity is an increasingly prevalent disease, the genetics are poorly understood. Our lab previously identified Keratinocyte-associated protein 3 (Krtcap3) as a novel adiposity gene and sought to characterize its role using an in vivo whole-body knock-out (KO) rat model. We conducted two in vivo studies, where COVID-19 had a drastic change on the environment between the studies. In Study 1 (2019-2020), female KO rats had significantly increased food intake and fat mass compared to WT rats, as expected. In Study 2 (2020-2021), we were unable to replicate these differences. Study 2 WT rats ate more than Study 1 rats with a corresponding increase in adiposity. We hypothesized this was due to decreased stress in a quieter environment, which was supported by a decrease in serum corticosterone (CORT) in Study 2 WT rats. KO rats ate similar amounts between the two studies and had low CORT in both environments, suggesting a differential response to stress by genotype. We conducted a third in vivo study to examine how WT and KO rats respond to environmental stress. Surprisingly, the biggest effect was between KO control and stress rats, where KO stress rats had increased adiposity compared to controls, with no differences in adiposity between WT control and stress rats. KO stress rats also showed increased anxiety and passive coping relative to KO controls, with no changes in behavior between WT stress and controls. These data indicate that Krtcap3 plays a role in the stress response to impact both adiposity and behavior.

1Department of Molecular Medicine-Internal Medicine, Wake Forest University School of Medicine, Winston Salem, NC, USA, 2Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA, 3Department of Psychiatry & Behavioral Sciences, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Funding Support: R01 DK106386, T32 DA041349

ABSTRACT. Studies on the genetic basis of susceptibility to cocaine addiction in human populations are challenging due to limited sample sizes, heterogeneity of genetic backgrounds, and environmental variability. Drosophila present a powerful model system for investigation into the genetic underpinnings of cocaine addiction, using preference for cocaine as a proxy for addiction behavior. Utilizing the Microplate Feeder Assay (MFA), we quantified cocaine preference for 16,442 flies across 103 distinct genetic backgrounds of the Drosophila melanogaster Genetic Reference Panel (DGRP). We provided individual flies with a choice between 10µL of a control liquid food (4% sucrose, 1% yeast extract, and 0.004% FD&C Blue #1) and 10µL of the same liquid food supplemented with 0.02% cocaine. Solutions were delivered with the MFA, and consumption of each solution was quantified using a plate reader following a 22-hour exposure. Normalized cocaine preference was calculated for each fly as the difference in consumption between the two solutions divided by their total consumption. We found significant, naturally-occurring genetic variation for cocaine preference across these DGRP lines with significant sexual dimorphism, where male flies on average exhibit higher cocaine preference than female flies of the same line. Overall, males of 18.4% of tested DGRP lines preferred cocaine-supplemented food over control food, while females of only 3.9% of lines preferred the cocaine solution. Estimates of broad sense heritability of consumption were calculated using individual level data, as well as using DGRP line means, and were found to be H ̂2 = 0.19 and H ̂2 = 0.97, respectively. These data will facilitate future genome-wide association analyses, as well as identify D. melanogaster genetic backgrounds from the DGRP that can better model cocaine addiction. Our observations that innate cocaine preference is dependent on genetic background and sex will likely also apply to genetic risk susceptibility for cocaine addiction in human populations.

Jeffrey S. Hatfield1, Trudy F. C. Mackay1, Robert R. H. Anholt1 1Department of Genetics and Biochemistry and Center for Human Genetics, Clemson University, Greenwood, S.C., U.S.A.

ABSTRACT. Interactions between genetics and environment (GxE) have effects on brain development, behavior, and risk for disease, such as obesity and type II diabetes. Further, epigenetic modifiers, such as imprinted genes, drive food intake and body composition throughout development and into adulthood. Parent-of-origin specific effects have been identified in F1 mouse panels, with significant differences in behavior and differential allele-specific gene expression in various brain regions. The arcuate nucleus of the hypothalamus (ARH) was a particular ‘hot-spot’ in these studies. One of the primary neuronal subtypes, agouti-related peptide (AgRP) and neuropeptide Y (NPY) neurons, is essential for driving food intake. These neurons play a key role in integrating peripheral and central signals to modulate food intake, body weight (fat mass), and locomotor (food seeking) behavior. 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. Our lab and others have previously demonstrated that AgRP neuronal activity is sensitive to diet thus diet-dependent plasticity may be a causal factor in obesity. However, this research has primarily been conducted using inbred mouse models and single ‘parent-of-origin’ designs that lack genetic and epigenetic diversity, and do not capture the considerable influence that genetic factors play in controlling obesity and bodyweight. Here, we utilize an F1 panel of mice that are resilient and susceptible to diet-induced obesity (DIO). We selected the wild-derived PWK/PhJ mouse as the resilient strain because this mouse consumes fewer calories and gains less weight on HFD, compared to C57BL/6J mice. Resilient PWK/PhJ mice bred with susceptible C57BL/6J mice (expressing a NPYGFP reporter) created F1 lines from both paternal PWK/PhJ (PWKxB6) and paternal B6 (B6xPWK) mice. We found that fat-content, locomotor activity, feeding behavior, and AgRP neuronal activity was influenced by parent-of-origin in a sex-specific manner. B6xPWK male mice had increased body fat (%), an effect that was attenuated by eight weeks HFD feeding and were less active in the elevated plus maze, compared to PWKxB6 male mice. Further, only PWKxB6 male offspring were susceptible to diet-induced increases in AgRP neuronal activity, an effect that is driven by an elevated baseline (chow) firing rate in B6xPWK offspring. HFD fed B6xPWK female mice had increased body fat (%), compared to B6xPWK chow as well as both chow and HFD PWKxB6 mice. However, both chow and HFD fed B6xPWK female offspring were less active in the open field and elevated plus maze. B6xPWK females had decreased AgRP neuronal activity but we did not identify diet-induced changes in AgRP neuronal activity in female mice. This effect aligns with previous research, which has demonstrated more resistance to diet-induced plasticity in AgRP neurons from female mice. Ongoing work will explore parent-of-origin dependent changes in food preference, hypothalamic gene expression, and will explore the impact of maternal care (based on maternal genotype; PWK/PhJ vs. C57BL/6J) on offspring development. 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, Zoey J.D. Bridges, Kristen M.S. O’Connell Jackson Laboratory, USA

ABSTRACT. Putative loss-of-function variants in the gene Myelin Transcription Factor 1-like (MYT1L) located on chromosome 2p25.3 have been associated with a variety of neuropsychiatric and behavioral features including intellectual disability (ID), autism spectrum condition (ASC), attention-deficit hyperactivity disorder (ADHD), hypotonia, motor and speech delays, and obesity. However, little is known about the function of MYT1L or how it participates in behavioral circuits. To better understand MYT1L function in vivo and the impact of its loss on behavioral outcomes, we generated a novel mouse model of Myt1l haploinsufficiency by mimicking a frameshift mutation observed in an index patient with ID, ASC, ADHD, obesity, and developmental delays. Previously, we showed our model recapitulates several clinical features of MYT1L Syndrome including obesity, strength impairments, social deficits, and hyperactivity. In the current study, we extended our investigation to understand motor function and sensory responsivity in the presence of Myt1l haploinsufficiency by examining gait development, motor coordination, visual acuity, tactile sensation, olfactory function, and thermal sensation. We observed alterations in motor coordination, tactile discrimination, and sex-specific cold and heat sensitivities. Together, our current data suggest that MYT1L plays a role in motor and sensory functions, which may be sex-dependent. The recapitulation of clinical features in this model will allow us to next dive deeper into examining how MYT1L participates in behavioral circuits (both spatially and temporally), and in what manner, genetically and pharmacologically, we can rescue or ameliorate these behavioral difficulties.

Susan E. Maloney1,2, Katherine B. McCullough2,3, Manish Madasu4,5, Ream Al-Hasani4,5, Joseph D. Dougherty1,2,3

1IDDRC, Depts. of 2Psychiatry, 3Genetics, 4Anesthesiology at Washington University School of Medicine, 5Center for Clinical Pharmacology, University of Health Science and Pharmacy in St. Louis, MO, USA.

Funding Support: The Jakob Gene Fund (JDD,SEM), NIMH (R01MH124808, JDD,SEM), and NICHD (P50HD103525, IDDRC@WUSTL).

ABSTRACT. Behavior is fundamental to life and every individual has their own way of behaving and responding to their environment. For instance, some people like taking risks while others do not. Examples of individual behavioral differences are abundant, but how they arise remains elusive. Biological factors, such as sex and genetics, are known to play a role in these behavioral differences. To study the effect of these factors on individual differences in behavior and to determine the presence of distinct behavioral clusters, we examined exploratory behavior from over 400 zebrafish from four strains, and both sexes. When exposed to a novel environment, zebrafish exhibit a variety of exploratory behaviors that we captured in three-dimensions using depth-sensing cameras. Markerless tracking of the fish was done using a deep learning approach (DeepLabCut). We found that fish behavior stratifies into four distinct clusters. Behavior represented by these clusters are bold and shy, along with the two novel behavior types: wall huggers and active explorers. Further, we observed that cluster membership is influenced by sex and genetics, and we found that individual differences in behavior are consistent over multiple days.

Neha Rajput 1Department of Biological Sciences, Wayne State University, Detroit, MI 48202 Funding Support: NIH grant from NIGMS: R35GM142566

ABSTRACT. RNA binding proteins are master regulators of protein synthesis that control the translation, localization, polyadenylation, and splicing of their target mRNAs. RNA binding proteins are critical for behaviors that require protein synthesis, such as memory formation and consolidation of long-term memories. RNA binding protein dysfunction is associated with a growing number of neurological disorders including Autism and Alzheimer’s disease, so it is critical to understand how RNA binding proteins contribute to cognition. Recent studies show that mRNAs encoding RNA binding proteins are locally translated in synapses during memory training, suggesting that local translation of RNA binding proteins in synapses is a critical part of plasticity and behavior. Supporting this idea, our lab has previously utilized subcellular sequencing of neuronal somas and synapses in the nematode worm Caenorhabditis elegans to show that synaptic areas are significantly enriched for mRNA transcripts encoding RNA binding proteins. We hypothesize that locally translated RNA binding proteins may regulate protein synthesis in synapses to regulate learning and memory. Few RNA binding proteins have been tested for direct functional roles in learning and memory, and even fewer have been characterized as molecular learning and memory regulators. To address this gap, our lab is testing whether evolutionarily conserved RNA binding proteins are required for learning and memory using RNA interference targeting mRNAs encoding RNA binding proteins as well as assays measuring molecularly conserved positive olfactory associative memory in C. elegans. We have identified several RNA binding proteins that regulate associative memory, including novel memory regulators. To determine the precise molecular mechanisms by which these RNA binding proteins regulate memory, we are using eCLIP, or enhanced cross-linking immunoprecipitation. Our current focus is on the translational suppressor PUF-8, an ortholog of mammalian Pumilio 2 for which we have identified a novel memory suppressing role. Developing eCLIP will allow us to identify memory-regulating pathways that are downstream of not just PUF-8, but several RNA binding proteins to characterize the memory-regulating RNA binding protein network in synapses and provide novel insight into the role of synaptic transcripts in learning and memory. More specifically, this research addresses the mechanisms of RNA binding protein control of downstream mRNAs during learning and memory.

Ashley Hayden Baylor College of Medicine, USA

ABSTRACT. Introduction: Lifetime incidence of epilepsy is 1 in 26 individuals, with many diagnoses occurring during childhood. Pediatric epilepsy strongly correlates with behavioral, cognitive, and psychiatric comorbidities which can be more detrimental to quality of life than the actual seizures. Early life seizures (ELS) are associated with learning and memory deficits due to lasting modification of neuronal activation patterns in a manner that is brain-region specific. One emerging hallmark of ELS in rodent models is impairment of fear extinction learning; however, it is unclear how neural ensembles engaged by seizures are implicated in subsequent cognitive impairment to produce fear-extinction deficits. We hypothesize that exposure to ELS may drive epigenomic changes to produce persistent dysregulation of neuronal activation patterns in specific regions of the epileptic brain. In this study, we sought to first characterize brainregion specific neuronal activation in an early life seizure mouse model. Additionally, we compared regional patterns of neuronal engagement in ELS and later activation during fear extinction.

Methods: This study utilized a transgenic mouse model to visualize neuronal activation in a flurothyl-induced generalized seizure. Dual immunofluorescent staining of both ELS-tagged neurons (GFP) and NeuN, a pan-neuronal marker, was performed to localize regions of neuronal activation and compare activation amongst anatomic regions implicated in prior seizure models. The immediate early gene c-Fos served as an independent marker of neuronal activation following fear extinction. Dual immunofluorescent staining of GFP and cFos was used to evaluate the degree of co-activation between seizure and fear extinction states.

Results: In dual immunostaining of GFP with NeuN of the ELS model, only a modest proportion of the total neuron population were found to be activated in ELS. NeuN-positive neurons of the periaqueductal gray demonstrated greatest seizure activation as measured by GFP co-localization, compared to forebrain regions (e.g., piriform cortex) or midbrain regions (e.g., dentate gyrus). In dual immunostaining of ELSGFP with cFos after fear extinction, cFos-labelling of fear-activated cells was most frequent in forebrain regions, including the infralimbic area and piriform cortex. GFP-labelling of seizure-activated cells was most abundant in the periaqueductal gray. Co-localization of GFP and cFos cell populations was strongest in the infralimbic area and periaqueductal gray.

Conclusions: This study establishes a reliable immunofluorescent method for visualization of neuronal activation in seizure and extinction learning models. Extinction learning appears to engage ELS-activated neurons in a brain-region specific manner, with strongest incidence of overlapping activation in the infralimbic area of prefrontal cortex and the midbrain periaqueductal gray. Future work will aim to correlate seizure- induced neuronal activation and fear extinction with epigenetic modifiers hypothesized to participate in brain region-specific regulation, and ultimately will seek to determine if targeted intervention can ameliorate cognitive deficits associated with early life seizures.

Christina Hansen University of Vermont, USA

ABSTRACT. Drugs of abuse such as alcohol disrupt dopaminergic reward pathways, leading to maladaptive goal-directed behaviors. In both mammals and Drosophila, evidence suggests that dopamine also mediates ethanol-induced locomotor activity, however, the underlying neural circuitry remains poorly understood. Using flyGrAM, an automated group activity monitor, we investigated whether distinct populations of DANS are differentially recruited to support ethanol-induced activity in a dose-dependent manner. We then performed high-content behavior analysis using FlyTracker and C-trax. We found that both the PAM and PPL1 subsets of DANs are involved in the ethanol response and have ethanol-dependent modulatory activity. Additionally, we discovered neuronal subsets with dynamic roles in modulating ethanol-induced activity. Specifically, increased DAN involvement occurred at higher doses of ethanol, suggesting that more DANs are recruited at higher doses to counter the sedating effects of ethanol. Overall, this study clarifies our understanding of the dose-dependent activity of specific subsets of DANs and serves as a starting point for more detailed circuit analyses.

Caldarone KD1,2, Song SL3, Savory NT3, Nyugen A1,2, Pollack S1,2, Kaun KR3, Scaplen KM1,2,3.

1 Department of Psychology 2 Center for Health and Behavioral Sciences, Bryant University, Smithfield, RI, USA 3 Department of Neuroscience Brown University, Providence, RI, USA

Funding Support: This work was supported by the Rhode Island Institutional Development Award (IdeA) Network of Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430, the Rhode Island Foundation Medical Research Fund 20210957, and the generous support of the Center for Health and Behavioral Sciences at Bryant University.

ABSTRACT. Substance use disorder (SUD) is a chronic disease characterized by compulsive drug seeking and use, despite negative consequences. SUD is highly heritable, and many genes have been identified as associated with addiction. However, these only accounts for a small proportion of the observed variation, suggesting a role for many other biological and environmental factors. Accumulating evidence proposes that the gut microbiome plays a significant role in the behavioral responses to cocaine, with the abundance and variety of the microbial metabolites altering host behavior. Our objective is to understand the interaction of the microbiome and host genetics in SUD. Host genetics influences behavior directly or can influence behavior by altering the composition of the gut microbiome. Leveraging the data collected by the P50 Center for Systems Neurogenetics of Addiction, we used fecal samples and phenotype data from Diversity Outbred mice to test our hypothesis. We have identified microbial abundance QTLs at each level of microbial classification (e.g., family, genus) in both the fecal boli and the cecal contents and made them accessible through an online QTL viewer. In addition, we have utilized Collaborative Cross mice and gene knock-out mutants to test the haplotype and gene effects of the loci on microbial abundance. Using multiple statistical approaches, we have identified numerous microbe and behavior associations. Together these results support the role of host genetics controlling the microbial abundance and microbial community composition being associated with behavior.

Jason A. Bubier1, Dong-Binh Tran2, Asaf Peer2, Hoan Nguyen2, Robyn Ball1, Belinda Cornes2, Elissa J. Chesler1, George Weinstock2

1The Jackson Laboratory 600 Main St. Bar Harbor ME 04609 2 The Jackson Laboratory for Genomic Medicine, 10 Discovery Dr. Farmington CT 06032

U01DA043809 to GMW/JAB and P50DA039841to EJC

ABSTRACT. Model organism research is essential for discovering the mechanisms of gene to disease relationships. At RGD (https://rgd.mcw.edu) model organism research is enhanced by the integration of 9 other comparative research species’ data. Alzheimer’s Disease is a human disease for which rat and mouse models have been studied. Alzheimer’s Disease at RGD can be found by navigating the disease ontology from the Neurological or Age-Related Disease portals. The gene list for Alzheimer’s is given for rat by default, but other species are available for selection. The inbred and mutant rat strains listed are links to strain report pages. The Annotations button opens a disease report page that details genes, references and data sources. Embedded within the Disease Portal page is the ability to perform ontology term enrichment using the Multi Ontology Enrichment Tool (MOET). After launching an analysis, more focused analyses are possible by selecting a subcategory term and choosing to explore that more restricted gene list. For example, the Alzheimer’s Disease gene list enriched for mouse Mammalian Phenotypes ontology annotations, produces a powerful analysis utilizing MP annotations imported from MGI. Further granularity can be achieved by interrogating the enriched subsets, e.g. the 286 gene list annotated specifically for abnormal behavior phenotype. Resultant gene lists can be downloaded for use in RGD’s suite of bioinformatic tools. For example, Variant Visualizer can be used to find damaging variants for human, rat, or dog. We illustrate how data can be interrogated using multiple species and bioinformatic tools, empowering genes research in human afflictions.

Mary L Kaldunski1, Jennifer R Smith1, G Thomas Hayman1, Stanley JF Laulederkind1, Shur-Jen Wang1, Mahima Vedi1, Morgan Hill1, Wendy Demos1, Monika Tutaj1, Adam Gibson1, Logan Lamers1, Harika S Nalabolu1, Ketaki Thorat1, Jyothi Thota1, Marek A Tutaj1, Jeffrey L De Pons1, Melinda R Dwinell1,2, Anne E Kwitek1,2. 1Rat Genome Database, Joint Department of Biomedical Engineering, Medical College of Wisconsin and Marquette University 2Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

ABSTRACT. Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disorder caused by a complete loss of the lysosomal enzyme alpha-N-acetylglucosaminidase, encoded by the gene NAGLU, which results in the inhibition of the breakdown of large sugars leading to the accumulation of heparin sulfate and serious neurological issues. Patients experience behavioral impairments beginning in early childhood with developmental delay, somatic issues like hearing loss, and progressing into cognitive deterioration, sleep disturbances, aggression, hyperactivity, social and emotional irregularities, and finally a lack of fear, motor decline, and dementia with early death. The MPS IIIB mouse model recapitulates many clinical features including altered circadian rhythms, juvenile-onset hearing loss, later-age onset of balance issues and phenotypes suggestive of reduced fear. To understand the onset and progression of unexplored clinically-relevant areas in this model, we evaluated various domains of social, sensory, and conditioning behaviors with validated tasks. Behavior and health were monitored between 5-10 weeks of age. Both male and female MPS IIIB homozygous knockout mice weighed consistently more than their heterozygous controls littermates. Visual sensory thresholds performed using the Virtual Optometry System (VOS) in all mice at 6 weeks of age revealed deficits in acuity or contrast in KO mice. Social aspects such as preference, novelty and dominance were assessed using the three-chamber social approach assay and tube test. Territorial aggression toward a conspecific was evaluated in males with a resident intruder paradigm and processed using a deep learning pose estimation and behavioral predictive classifier software. Sensorigating ability was assessed using auditory startle/pre-pulse inhibition and associative learning in the fear conditioning task. With life expectancy in the teens, early identification and intervention is vital. Our study using a mouse model of MPS IIIB provides much needed phenotypic information that will be useful in future evaluations of potential treatments.

Katherine B. McCullough1,2,6, Mark Sands3, Patricia Dickson2,4, Joseph D. Dougherty1,2,6, Carla M. Yuede1,5,6, Susan E. Maloney1,6. Departments of 1Psychiatry, 2Genetics, 3Medicine, 4Pediatrics, 5Neurology, 6IDDRC at Washington University School of Medicine. Funding Support: NICHD (P50HD103525, IDDRC@WUSTL).

ABSTRACT. The HS-CC (heterogeneous stock- collaborative cross) mouse population encompasses 89% of the genetic diversity available in Mus musculus and is approximately 10 times more genetically diverse than Homo sapiens. The HS-CC was formed from an 8-way cross of inbred strains, including 3 wild-derived strains, and importantly contains no rare alleles. The Portland Alcohol Research Center has used the HS-CC to develop a genetic mouse model of high vs. low alcohol preference using two-bottle choice short-term selective breeding. We present gene expression profiles for the 8 founder strains, known to have wide variation in alcohol preference, across sex and three brain regions known to be involved in excessive alcohol drinking: (1) the nucleus accumbens core, (2) the central nucleus of the amygdala and (3) the prelimbic cortex. Results across and within groups (sex, strain and brain region) are presented for the 240 samples sequenced (5 per strain/region/sex). Differential expression analysis is additionally investigated for the three wild derived founder strains (WKB, CAST and PWK) and the C57BL/6J genetic reference strain. This selection includes the two founder strains known to have the highest preference for drinking alcohol (PWK and C57BL/6J). Overall, we observe that the differences among strains are lower than the differences across regions.

JQ Anderson1, P Darakjian1, TJ Phillips1, RJ Hitzemann1, AR Ozburn1

1Portland Alcohol Research Center, Department of Behavioral Neuroscience, Oregon Health & Science University, and VA Portland Health Care System, Portland, OR, 97239, USA Funding Support: Supported by the NIH grants (AA013519, AA010760, AA07468, AA13484); US Department of Veterans Affairs Grant (BX004699), and a gift from the John R. Andrews Family.

ABSTRACT. Negative mood states characterize drug withdrawal and are associated with craving and relapse in humans. Mice can model aspects of negative mood states associated with amphetamine (AMP) withdrawal, yet conventional crosses lack resolution needed for fine mapping. We investigated negative mood states associated with acute AMP withdrawal using highly recombinant, commercially available, outbred CFW mice to determine their feasibility for future QTL mapping studies. We tested ~1,000 CFW mice on the Elevated Zero Maze (EZM), Porsolt Forced Swim Test (FST), and Sucrose Preference Test to assess changes in anxiety-like behavior, dysphoria, and anhedonia respectively, before and after 14 consecutive days of 2.5 mg/kg d-AMP administration. While we observed variation in sucrose preference in CFW mice, a paired-samples t-test failed to identify significant differences. For the EZM, CFW mice exhibited significant decreases in time in open quadrants during withdrawal, indicating increased anxiety-like behavior, t(656) = 4.2; p < 0.001. For the FST, we observed significant increases in immobility time from before AMP administration (M = 146.4 s, SD = 47.3 s) to after (M = 178.6 s, SD = 38.5 s), suggesting the mice exhibited dysphoric behavior due to amphetamine withdrawal t(780) = -24.5; p < 0.0001. By exploiting the higher number of recombinations in CFW mice, we hope to map behavioral and gene expression QTLs with high precision. This will allow for the identification of plausible biological explanations for how alleles influence behavior and thereby implicate specific genes.

Riley Marchin1, Emma White1, Abed Abbas1, Miko Dai3, Jay-Ho Chung1, Sonya Farrell1,Levi Gavette1, Pranav Kumar1, Connor Montgomery2, Samuel Pelletier2, JT Titmus1, Olivia Peterson1, Anna Spiro1, JT Titmus1, Abraham Palmer4, Clarissa C Parker1, 2

1 Program in Neuroscience, Middlebury College; 2 Department of Psychology, Middlebury College; 3 Program in Molecular Biology and Biochemistry, Middlebury College; 4 Institute for Genomic Medicine, University of California San Diego

ABSTRACT. Animals must weigh the potential risks and benefits of novelty in deciding how to react to new stimuli in their environment. Neophilia, or novelty seeking, may drive an animal to discover a new food source or shelter, while neophobia, or novelty avoidance, may protect an animal from predator exposure. Generalists such as Drosophila simulans and polyphagous species such as D. mojavensis baja, tend to respond with more neophilia than the closely related specialists / monophagous species of D. sechellia and D. mojavensis wrigleyi. It is possible that specialists receive less benefit from novelty seeking behaviors, since fewer novel stimuli are suitable for them. Using the behavioral assays of open field exploration and food choice, we hope to explore the link between specialization and evolved neophobia. In order to examine the genetic basis of these traits, the Roman Lab has generated a collection of interspecific Recombinant Inbred (RI) Lines between the parental species of D. simuans and sechellia. These RI lines segregate in their response to novelty, as indicated by their varied level of exploration when placed in an open field arena, and are a valuable tool that can be used to map major effect loci for a variety of quantitative traits in the future. Identification of major effect loci in this project will not just further our understanding of specialization-related neophobia, but may also shed light on the genetic underpinnings of neophilia and neophobia more generally.

E. McMullen1, A. Kalluchi1, G. Roman1

1 Department of BioMolecular Sciences, University of Mississippi, Oxford, MS, USA Funding Support: NSF Grant 2135305 (Roman, PI), NIH Grant 1R15MH121859 (Roman, PI)

ABSTRACT. Rat is an important model organism for the study of mammalian genetics. Rats have a large behavioral repertoire, especially for addiction-related traits, where novel paradigms have been recently explored and established. The P50 Center for GWAS in Outbred Rats focuses on N/NIH heterogeneous stock (HS) rats, which were created by intercrossing 8 inbred strains and maintained as an outbred population for almost 100 generations. This population enables precise mapping of alleles that affect a given phenotype. The Center serves as the foundation for more than 15 studies that use HS rats, collectively funded to test more than 16,000 rats. This data has been accumulated and organized in relational database, it includes extensive addiction-related behavioral and physiological data, DNA sequencing; subsets of rats have RNASeq, gut microbiome and metabolomics, single cell RNASeq, and ATACSeq data. We have created a public-facing portal (CGORD.ORG) that organizes these data according to FAIR (Findable, Accessible, Interoperable, Reusable) standards and is optimized for machine learning (ML) applications. The datasets associated with publications resolve to DOIs and will be permanently hosted by the UCSD library, SRA, NCBI and similar resources. The unpublished datasets have detailed descriptions and a data dictionary that maps to existing ontologies and data elements registry and can be requested. The landing pages for individual datasets have additional fields designed to be machine-readable, but not visible to human users. The size and scope of this data is unique and can be used for a variety of unforeseen analyses in the future.

O Polesskaya1, GJ Barrero,1, AS Chitre,1, J Grethe2, A Bandrowski2, M Martone2, AA Palmer1,3

1Department of Psychiatry, 2Center for Research in Biological Structure, 3Institute of Genomic Medicine, University of California San Diego, San Diego, CA, USA Funding Support: NIH/NIDA P50DA037844

ABSTRACT. Patients diagnosed with Major Depressive Disorder (MDD) have elevated expression of pro-inflammatory genes, and patients diagnosed with chronic inflammatory disorders often develop symptoms of depression. The hippocampus is important in mood and memory and is also affected by both depression and neuroinflammation. Currently, the molecular mechanisms underlying the relationship between depression and inflammation are poorly understood. We hypothesize that both chronic stress and systemic inflammation can induce inflammation in the hippocampus. In this study, we examine changes in the activation and concentration of microglia and astrocytes by systemic inflammation or chronic stress in mice. We utilized the Unpredictable Chronic Mild Stress (UCMS) model to induce chronic stress, and lipopolysaccharide (LPS) to induce systemic inflammation. We hypothesized that UCMS and LPS will induce increased inflammation in the hippocampus, and ultimately result in pro-inflammatory gene expression changes. Our preliminary data suggests that inflammation induced by chronic stress or LPS activates glia in the hippocampus. Understanding the relationship between depression is important in expanding our knowledge of the etiology of depression and identifying new targets for treatment. The result of this study will offer insights on the effect of inflammation on MDD pathophysiology.

Ariel Y Zhang, Elias Elias, Andrew Labeeb, Rachel Prutzman, Melissa T Manners, PhD Univerisity of the Sciences, USA

ABSTRACT. Cocaine use disorder (CUD) affects over 1 million people in the US. However, there are no FDA approved treatments for CUD. A recent study (Silverman et. al, 2016) showed that administration of a GABA-A agonist, Gaboxadol, potentiated the locomotor activation caused by cocaine, which has been previously associated with abuse liability. Extending on our prior work on Glyoxalase 1, which metabolizes methylglyoxal, a GABA-A agonist, we investigated whether administration of the GLO1 inhibitor S-bromobenzylglutathione cyclopentyl diester (pBBG) influenced the locomotor response to cocaine in male and female C57BL/B6 mice. Next, we explored the ability of the pBBG to inhibit the rewarding effects of cocaine using the conditioned place preference (CPP). We also conducted preliminary studies to measure if these results could be replicated by using genetically modified animals with a knockdown of the Glo1 gene using the same paradigm. We found that co-administering pBBG with cocaine potentiated the locomotor response to cocaine; however, pBBG did not alter CPP for either drug. Similarly, we saw that GLO1 knockdown mice showed increase cocaine activation compared to wildtype littermates. Although, inhibiting GLO1 pharmacologically with pBBG and genetically with knockdown animals influenced the locomotor response to cocaine, which is sometimes used as a proxy for the rewarding effects of drugs, we did not observe any effect on CPP. These data provide further evidence that the gene Glo1 and more broadly GABA-A signaling, is related to CUD, however the role of Glo1 in the rewarding effects of cocaine was less clear.

E Alcantara 1, C Ortez 1, A Ilustrisimo 1, A.M. Barkley-Levenson 1, AA Palmer 1

1Department of Psychiatry, University of California San Diego, La Jolla, California, U.S. Funding Support: NIAAA R01AA026281

ABSTRACT. Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become longlasting and inflexible remain unclear. We recently demonstrated that formation of these memories results in expression of alternative transcript isoforms in memory-encoding neurons in Drosophila melanogaster. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Decreasing expression of genes that play a role in splicing in adult MB neurons reduces formation of these memories, demonstrating the necessity of RNA processing in ethanol memory formation. Moreover, decreasing expression of genes that are alternatively spliced, like Dop2R in adult MB neurons reduces ethanol memory formation. This suggests that the splicing changes in these genes has functional implications for future memory formation. To test this, we generated mutant Drosophila that have forced expression of the naïve or trained isoform of Dop2R. Using a novel three-choice assay we assessed mutant preference for an odor previously associated with ethanol following Pavlovian conditioning. We hypothesized that mutants expressing the naïve isoform will have reduced preference for an odor associated with alcohol, and mutants expressing the trained isoform exhibited greater preference. Our results suggest that dynamic splicing within memory-encoding neurons has functional consequences. These findings highlight the functional role of dynamic splicing in the nervous system, and demonstrate a role for alternative splicing in the development of AUD.

T. M. BROWN1, E. PETRUCCELLI2, A. G. WATERMAN3, K. NUÑEZ4, K. O’CONNOR-GILES3 K.R.KAUN3 1Neuroscience Graduate Program, Brown University, Providence, RI 2Dept. Bio. Sci., Southern Illinois University, Edwardsville, Edwardsville, Il 3Dept. Neurosci., Brown Univ. Providence, RI; 4Mol. Pharm. Phys. Graduate Program, Brown Univ. Providence, RI; Disclosures T. Brown: None. E. Petruccelli: None. A. G. Waterman: None. K. Nuñez: None. K. O’ConnorGiles: None. K. R. Kaun: None. Corresponding author / contact: tariq_brown@brown.edu Funding Robert J. and Nancy D. Carney Institute for Brain Science Innovation Award NIAAA R01AA024434 IMSD: 5R25GM083270-11 (MPI)

ABSTRACT. Substance use disorders are characterized by a shift in voluntary drug-taking from recreational to compulsive, which can be characterized by drug-seeking behaviors that persists despite negative consequences and periods of abstinence. The fragile x mental retardation protein (FMRP), an RNA-binding protein that regulates synapse plasticity, is required for activity-dependent excitatory synapse elimination, including that induced in dopamine-receiving striatal cells by cocaine exposure. Total absence of FMRP (Fmr1 KO) significantly dampens multiple cocaine-induced behaviors. For example, in intravenous cocaine self-administration, Fmr1 KO mice fail to make normal upward shifts in responding when lower doses are introduced and earn fewer infusions than wildtype (WT) mice under increased schedules of reinforcement. Using a cortical-striatal co-culture model to examine how loss of FMRP affects synapse plasticity in striatal medium spiny neurons (MSNs), we find deficits in the basal spontaneous firing rate of KO, compared to WT, dopamine D1 receptor (D1R)-expressing MSNs, as well as impaired synaptic responses to D1R signaling. In adult mice, we find that loss of FMRP in D1R-expressing cells of the nucleus accumbens (part of the ventral striatum) increases both cocaine conditioned place preference and propensity to self-administer low dose cocaine, suggesting that FMRP functions in this region and cell type to suppress early drug-taking. Alternatively, its absence in D1R-, but not D2R-, expressing cells may alter the balance of signaling in the nucleus accumbens during early drug experience. Overall, our findings suggest that FMRP is critical to the responses of D1R-expressing MSNs to dopaminergic signaling and influences cocaine-induced behaviors.

J.L. Huebschman1,2, E. Chow1, E. Harrison1, H. Gangal1,2, Y. Guo1, J. Wang1,2, L.N. Smith1,2

1Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, Bryan, TX 77807, USA, 2Texas A&M Institute for Neuroscience, Texas A&M University, College Station, TX 77843, USA Funding Support: NIH NIDA (DA051727) and Texas A&M University internal funding

ABSTRACT. Mammalian pair bonding is a distinct phenotype in which mating pairs of individuals form a strong bond. Sometimes also referred to as "social monogamy", pair bonds are characterized by two individuals' enduring mutual preference for each other. While neurobiology models have demonstrated the importance of oxytocin for social recognition and dopamine activating reward centers in the brain to establish the pair bond, relatively less work has addressed the genomic underpinnings of the phenotype. One strategy to investigate the genomics of pair bonding is to identify shared genetic changes in species that have independently acquired the pair bonding phenotype in a process known as convergent evolution. Modern advances in sequencing technology make it possible to perform such comparisons across large numbers of species to identify shared genomic changes underlying pair bonding. In this work, I used a computational method called RERconverge to identify genes evolving convergently in association with pair bonding in 173 mammal species (24 pair bonding species). I identified brain-related genomic functionalities involved in sensory processing and memory, as well as non-brain-related functionalities that may be important to group living like immune function, that show convergent evolution in association with pair bonding. These findings provide valuable information about shared mechanisms in the evolution of pair bonding behavior across mammals.

A Kowalczyk1,2, A Pfenning1,2

1Department of Computational and Systems Biology, 2Neuroscience Institute, Carnegie Mellon University, Pittsburgh, PA, USA Funding Support: Carnegie Mellon University Neuroscience Institute Distinguished Postdoctoral Fellowship Program

ABSTRACT. Genetics has a huge impact on all traits, influencing susceptibility to disease and response to treatment. Many studies in animals are carried out on a single, inbred strain, in which all members are genetically identical to each other. By not including genetic variation, these studies can produce both false positive and false negative findings. The AD-BXD are a mouse model of Alzheimer’s disease (AD), produced by crossing the 5XFAD transgenic AD model to the BXD family of mice. This family of mice includes over 6 million variations, and has been used for over 50 years to collects tens of thousands of phenotypes. By adding the BXD genetic variation into the 5XFAD AD model, we can identify different aspects of disease which are under different genetic control, and more accurately model the human population. By using an animal model, we can carefully control or manipulate the environment, and cross every level of biology, from genetic variants, to protein expression, to neuroanatomy, to behavior. Preliminary results of our current study show different genetic effects on aspects of cognitive aging and longevity, and a link to mitochondrial DNA copy number.

David Ashbrook University of Tennessee Health Science Center, USA

ABSTRACT. Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is an autosomal recessive lysosomal storage disease caused by a deficiency in the lysosomal enzyme galactosylceramidase, resulting in progressive accumulation of toxic metabolites in all cells. GLD is characterized by limb stiffness, weakness, blindness, and development delay/regression, and there is currently no cure. The mouse model of infantile GLD (Twitcher, Twi) exhibits motor deficits that are largely refractory to treatment. Twitcher mice display a persistent tremor and gait abnormalities near disease end stage, but little is known about the trajectory of these traits. We tracked the progression of such behaviors using quantitative tremor assessment and gait analysis. At all timepoints (p21, 28, and 35) Twi mice run slower than WT mice. Across time, Twi mice develop a narrower front paw stance and a wider hind paw stance. Notable kyphosis means Twi mice take longer strides relative to body length compared to WT mice at p28 and p35. Quantitative measurement via force-plate actometer showed that Twi mice have a significant tremor at p28 and p35, but not at p21. Our data reveal how these reliable Twi behavioral markers develop and change from weaning, when mice largely appear normal, through late in disease.

JT Dearborn1, CR Mikulka2, MS Sands1,3

1Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA 2Casma Therapeutics, Cambridge, MA, USA 3Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA Funding Support: NIH R01NS100779-04

ABSTRACT. Besides significant benefits to physical health, exercise can reduce symptoms of mental illness, enhance psychological development, and improve the pathophysiology of numerous diseases. The effects of exercise on the symptoms of chronic stress have been previously studied, but data are contradictory due to the limited number of studies incorporating sex as a variable. To further examine the effects of exercise on chronic stress, male and female mice were subjected to an Unpredictable Chronic Mild Stress (UCMS) paradigm with daily accessibility to running wheels for 2 hours per day. Physiological and behavioral tests were conducted throughout the stress paradigm to determine if exercise promotes resilience to the expected symptoms of UCMS. Voluntary wheel running (VWR) was increased by chronic stress, with females choosing to run more compared to males. Females also displayed resilience to the weight loss effects of chronic stress compared to males. Results of the behavioral tests reflected a sex-specific exercise-induced resilience. Interestingly, exercise reduced the latency to enter the light side in the light dark test, regardless of stress. These results indicate that exercise concurrent with chronic stress can lead to resilience to the behavioral effects of chronic stress, with sex-specific differences.

Elias Elias University of the Sciences, USA

ABSTRACT. Virtual Poster

The bidirectional selection of mice for the successful elementary logic task solution and for the lack of such success (using the shortened version of “puzzle-box” test) proceeded for 5 generations and demonstrated the significant differences both in the duration of solution latencies and in the proportion of animals which were able to solve the most complicated stages of this test. The puzzle-box test requires that an animal is eager to penetrate (enter) the comfortable dark part of the experimental box from the brightly lit one. The test contains four stages – (1) with no obstacle for such avoidance reaction, when the underpass, leading to the dark was masked (2) by wood shavings and when this underpass was blocked (3, 4) by the plug (carton + plastic), which animal can remove either by teeth or by muzzle. The data obtained show the significant differences in “plug” stages solutions in mice of selected lines (“plus” and “minus” lines respectively) as well as the differences of “plus” line scores from those of animals from the initial unselected genetically heterogeneous population. The technique used permitted not only to evaluate animals’ capacity to operate the “object permanence” rule (the object which had been perceived recently still exists and could be found), but also the short term and long-term memories. It should be noted as well that “plus” selected mice ate significantly more new food in new environment (hyponeophagia test) that “minus” selected animals. The general conclusion from data obtained demonstrates that the more broad issue - that of “executive functions” expression in laboratory mouse population - was affected by selection process. The investigation was performed with the financial support of RFBR, grant № 20-015-00287, Project of State Assignment of Moscow State University No.121032500080-8 and Interdisciplinary Scientific and Educational School of Moscow University

Perepelkina O.V., Poletaeva I.I. Moscow State University, Biology department

ABSTRACT. Over the last decade, there has been a steady rise in pregnant women diagnosed with opioid use disorder and increasing cases of infants born with neonatal opioid withdrawal syndrome (NOWS). The symptoms of NOWS include low body weight, impaired thermoregulation, irritability, and hyperalgesia. In our mouse model of NOWS, neonatal FVB/NJ, FVB/NCrl, and FVB/NHsd mice were administered morphine (10 mg/kg, s.c.) twice daily from postnatal day 1 (P1) to P15. Hot plate latency, tail-flick latency, and ultrasonic vocalization (USV) recordings were conducted on P7 and P14 during spontaneous morphine withdrawal, to determine the effects of repeated opioid exposure on pain sensitivity and irritability. We observed robust thermal hyperalgesia in all three substrains with no significant substrain x treatment interactions, Furthermore, we found main effect of morphine treatment on the number of USVs emitted in morphine- and saline-treated pups at P14. Using the deep learning software, DeepSqueak, we classified USV syllables to determine if certain syllables are associated with the effects of opioid exposure. Interestingly, some syllables were exclusive to one strain or one treatment group. We plan to conduct RNA sequencing on brain regions associated with withdrawal, such as the midbrain, brainstem, striatum, and hypothalamus, to identify brain mechanisms related to NOWS. Additionally, using quantitative trait locus (QTL) mapping, we aim to identify causal genetic variants responsible for differences in withdrawal-associated behaviors.

Kelly Wingfield1,2, Kayla Richardson1,3, Teodora Misic1,4, Emily Yao1, Jacob Beirle1,2, Camron D. Bryant1,4

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; 3Post-Baccalaureate Research Education Program, Boston University School of Medicine; 4Department of Neuroscience, Boston University.

ABSTRACT. Many studies in neuroscience and translational analysis of disease models use quantitative analysis of locomotive behavior from different animal genotypes to decipher and comprehend neurological mechanisms or test the efficiency of new therapeutic techniques. However, there has been no study that attempts to predict the genotype of an animal entirely from their locomotive trajectories. Such studies have tremendous application in the area of integrated pest management (IPM), which requires monitoring and identification of different species of pests. To overcome current techniques in IPM which rely on an optimized and high imagery system, we propose an alternative method for the identification of the genotype of animals from their locomotive trajectories. We provide a proof of concept of this approach by using open-field arena trajectories of four different genotypes of fruit flies. We demonstrate that features of trajectories such as turn angles and distance traveled can be used by supervised machine learning models to predict fruit flies’ genotype with an accuracy of 83%. Using an interpretable machine learning model, we show that turn angles are better predictors for fruit fly genotype compared to the distance traveled. Our approach only relies on trajectory (x,y) positions of the flies and doesn’t need body structures such as wings, legs, etc.

Minh Nguyen1, Gregg Roman2, Benjamin Soibam1, * 1 Department of computer science and engineering technology, University of HoustonDowntown 2 Department of Biomolecular Sciences, School of Pharmacy, University of Mississippi 3 Department of Natural Sciences, University of Houston-Downtown