Ashley E. Marquardt¹, Aidan L. Pham¹, Christi V. Dionisis¹, Gabrielle E. Voithofer¹, Lucy A. Sirrs¹, Jonathan W. Van Ryzin² and Margaret M. McCarthy¹

Physical play among adolescents is a unique social behavior notable for its transient expression and independence from reproductive drive and aggression. Both males and females engage in social play but on average play by males is more intense in frequency and duration, a feature common to every species that exhibits adolescent play. Work in the laboratory rat establishes that the neural network of play is overlapping with circuits controlling adult reproductive and aggressive behaviors as well as the reward circuitry. Interestingly, the sex bias in play is not a distributed property of these circuits but instead localized to the medial amygdala (MeA) and programmed by androgens in developing males, although play is expressed at a time of life free of gonadal steroids. This led us to ask the following questions; 1) what are the cellular mechanism by which androgens developmentally masculinize play behavior, 2) is there a sex difference in the transcriptomics of play, 3) does engaging in play impart value to animals and, if so, does it differ by sex, and 4) are the lower play rates in females a default due to lack of androgen or the result of active feminization? This talk will provide answers to each and highlight new avenues for exploration of this often over looked, under appreciated yet vital social behavior.

1- Department of Pharmacology and Physiology, Program in Neuroscience, UM-MIND, University of Maryland School of Medicine.

2- Department of Psychology and Neuroscience, University of North Carolina

TJovanovic1, K Prendergast2, AC Korgan2, SA George1, TL Bale2

Trauma exposure during development significantly increases risk for psychopathology, including posttraumatic stress disorder (PTSD), which is twice as common in women as men. One of the hallmark symptoms of PTSD is threat reactivity, which can be measured in the laboratory using fear conditioning methods. In our prior work we have found that fear-potentiated startle to threat signals was heightened in women who experienced sexual trauma during adolescence. This same sensitive period of development was associated with increased blood levels of keratinocytes and keratin-associated proteins. Our current work is examining the long-term impact of adolescent exposure to interpersonal violence in men and women using fear-potentiated startle and proteomic analyses related to the 17q21 gene cluster. Preliminary results of this ongoing study replicate prior findings showing increased fear responses, altered fear extinction, and changes associated with keratinocytes. In addition, we have observed significant sex differences in the above measures. These data suggest robust long-term effects of interpersonal trauma on skin cells in women, which may be associated with Merkel cell signaling, and could lead to novel interventions for PTSD. Such interventions could be optimized and personalized based on an individual’s proteomic and psychophysiological biomarkers.

1Department of Psychiatry, Wayne State University, Detroit, MI

2Department of Psychiatry, University of Colorado Anschutz Medical Campus, Aurora, CO

TL Bale1, AC Korgan1, IJ Sibley1, T Jovanovic2

Childhood trauma experience increases risk for neuropsychiatric and neurodevelopmental disorders, including posttraumatic stress disorder (PTSD), autism spectrum disorders (ASDs), and attention deficit/hyperactivity disorder (ADHD). We recently utilized a Gene x Environment x Development (GxExD) interactions approach in a study where we found that women exposed to interpersonal violence trauma (the E) uniquely during adolescence (the D), but not childhood or adulthood, had novel protein biomarkers (the G) associated with a sensory cell system in the skin, Merkel cells (MC). Merkel cell mechanosensory signaling is important in gentle and social touch, inflammation-induced pain, and the skin’s neuroendocrine stress response. Interestingly, many of the genes identified in our study belong to a 17q21 gene cluster, suggesting an epigenetically regulated site altered by adolescent trauma. We developed a preclinical mouse model using chemogenetics in which studies can examine biological mechanisms connecting developmental timing of stress with sensory alterations in MC responses. To validate this model using a KRT14^Cre x DREADD-Gq transgenic cross to activate MCs, we identified expected somatosensory brain regions activated by MC stimulation following CNO treatment. Further, we identified the behavioral changes and valence of acute and chronic MC stimulation in male and female mice. Finally, we determined a significant effect of MC activation prior to conditioned fear extinction that did not impact fear conditioning. Extinction effects were also context dependent but did not show an effect of sex. Combined, these experiments provide validation of a preclinical mouse model in which further studies can now examine a potential functional link between mechanosensory Merkel cells and the pathology and sensory symptomatology in PTSD.

1Department of Psychiatry, University of Colorado Anschutz Medical Campus, Aurora, CO

2Department of Psychiatry, Wayne State University, Detroit, MI

Diego Luiz Rovaris, Ph.D.

In this talk, I will present findings from a collaborative effort within the Latin American Genomics Consortium (LAGC) that aims to deepen our understanding of the genetic architecture of Attention-Deficit/Hyperactivity Disorder (ADHD) in ancestrally and environmentally diverse populations. I will explore how Latin America's contrasting environments — shaped by regional socioeconomic, cultural, and healthcare disparities — influence genetic signals and add complexity to psychiatric genomic interpretation. I will also share recent findings on the high prevalence of childhood trauma among adults with ADHD, and discuss how early life stress is linked to increased comorbidity and greater symptom severity. Beyond advocating for equitable representation, I will highlight how including underrepresented populations can uncover novel insights into gene–environment interactions and the biological mechanisms underlying mental illness.

Department of Physiology and Biophysics,
Institute of Biomedical Sciences, University of São Paulo, Brazil

Rodrigo Grassi-Oliveira, MD, PhD

Cocaine use disorder (CUD) is a severe psychiatric condition shaped by cumulative exposure to environmental adversities across the lifespan. In this talk, I will present human data highlighting how both prenatal cocaine exposure and lifetime exposure to potentially traumatic events (PTEs) influence the epigenetic landscape in individuals with CUD. In newborns with prenatal cocaine exposure (PCE), we observed significant alterations in DNA methylation profiles at birth, including reduced gestational epigenetic age and lower BDNF levels, alongside elevated epigenetic scores for psychiatric and metabolic phenotypes. These findings indicate that the biological imprint of prenatal adversity is detectable at birth. In adulthood, analysis from a large Brazilian cohort (N = 1,250) revealed that 84.9% of individuals with CUD had a history of PTEs, compared to 33.3% in non-psychiatric controls. Traumatic exposures included life-threatening events, physical violence, sexual assault, and severe childhood adversities. Our epigenome-wide association study identified distinct DNA methylation profiles associated with both CUD and PTE exposure, particularly at loci such as AHRR, SOCS3, and FKBP5, which are involved in immune regulation and stress response. Importantly, we detected independent effects of trauma, as well as group × trauma interactions, at multiple CpG sites, underscoring the complex biological interplay between addiction and early adversity. These human data emphasize that trauma across the lifespan—beginning in utero—modifies key epigenetic pathways, contributing to vulnerability for cocaine use disorder. Such insights advance our understanding of trauma-informed addiction science and may inform future prevention and treatment strategies.

Aarhus University, Denmark

C. Reed1, E. Walker-Ziegler1, S. Aldrich1, J. Erk1, S.H. Mitchell1, T.J. Phillips1,2

A relationship between low initial cognitive flexibility and subsequent high ethanol intake has been demonstrated in non-human primates (NHPs) by members of the Portland Alcohol Research Center (PARC). We designed an operant set-shifting task for mice that parallels that used in NHPs to examine this relationship in mouse lines selectively bred for high and low ethanol preference (HP, LP). This task incorporates a series of fixed rule sets beginning with simple discrimination (SD), followed by intra- and extra-dimensional shifts (ID and ED respectively), and reversal of each ruleset. In two studies, we manipulated mode of responding (lever or nosepoke), method for obtaining the reward (dipper or sipper), and reward type (saccharin or sucrose solution). The number of days to complete training criteria of 30 operant responses in a 30-minute session for saccharin/nosepoke and sucrose/lever testing conditions did not differ between the HP and LP lines. There were no line differences in the number of days to complete the SD rule when the signal light location was fixed. In the final testing week of the sucrose/lever study, there was a significant line difference, only in females, with HP mice reaching a higher rule set than LP mice. However, a majority (97%) of mice were unable to move beyond following the signal light when it was variably presented above the nosepoke/lever (rule 3 out of 7). NHPs are able to complete complex operant set-shifting tasks that are challenging to model in mice.

1Department of Behavioral Neuroscience and the Portland Alcohol Research Center, Oregon Health & Science University, 2VA Portland Health Care System, Portland, OR 97239, USA

Funding Support: NIH/NIAAA P60AA010760 (USA) and Department of Veterans Affairs Senior Research Career Scientist Award 15F-RCS-010 (USA).

Erika Cortez ^1,2 and Kristin M Scaplen ^1,2,3

Alcohol use disorder (AUD) is a chronic relapsing disorder that manifests as problematic patterns of alcohol use. At the core of AUD’s behavioral manifestations is alcohol’s profound effect on the brain. Alcohol engages and ultimately subordinates memory circuits resulting in enduring preferences, habitual behaviors, and persistent cravings. Insight to the circuit mechanisms that underlie how alcohol-associated memories are encoded, maintained, and expressed is critical to understanding why these memories are remarkably resistant to change. However, understanding the circuit basis of
reward-related learning and addiction has been challenging. Efforts are often hindered by the neuronal heterogeneity that exists within dopamine (DA) sub-regions, as well as the lack of spatial and temporal resolution to distinguish neuronal subpopulations or isolate individual neurons. Drosophila melanogaster is a powerful model organism to address these challenges because they offer a rich genetic toolkit that permits dissection of heterogenous circuits with precise temporal and spatial resolution. Importantly, Drosophila form persistent alcohol reward memories, which impel them to
overcome aversive stimuli in pursuit of alcohol-associated cues. Previous work identified discrete circuits that underlie alcohol reward memories and their temporal requirements. In Drosophila alcohol reward memory requires the mushroom body (MB), a region essential for learning and memory as well DA modulation of the MB which shifts from an entire population during acquisition to two discrete DA subsets known to process memories with opposing valences. However, it remains unclear the mechanisms by which these memories are consolidated. The Dorsal Paired Medial neurons (DPM), which innervate the MB have an established role in sleep regulation and memory consolidation. Using neurogenetic tools available in Drosophila we investigated the role of DPM neurons in alcohol reward memories and other alcohol associated behaviors. Further, we report postsynaptic targets of DPM using the genetic anterograde transsynaptic tracing tool, trans-Tango. Preliminary data suggest that DPM is required for the consolidation of alcohol reward memories but not acquisition or retrieval and is synaptically connected with neurons previously identified to be important for the consolidation of alcohol reward memories. These findings provide insight to the circuit mechanisms underlying the consolidation of alcohol associated memories and why they are so resistant to change.

1 Department of Psychology, Bryant University, Smithfield, RI, 02917

2 Center for Health and Behavioral Sciences, Bryant University, Smithfield, RI 02917

3 Department of Neuroscience, Brown University, Providence, RI, 02912

Barry Dickson

How are instinctive behaviours wired into an animal’s nervous system? Mating is one of the strongest instincts in most animal species, and we’re trying to figure out what is going on in the brain of the fruit fly Drosophila as it seeks to fulfil this primal urge. We’ve identified sex-specific neurons in male and female brains that encode their respective states of sexual arousal and traced out the sensory pathways that induce these states. In the aroused state, specific sensorimotor pathways are activated to choreograph the song-and-dance routine that the pair perform as a prelude to copulation. Flies, like most animals, are also excellent navigators, capable of traversing variable terrain in search of mates or food. We have recently elucidated the central steering circuit in Drosophila that couples the navigation and locomotor systems in both goal-directed and exploratory turning. I will present our recent findings on the mating and steering circuits in Drosophila and outline future research directions that aim to use both mate choice and spatial navigation as models to reverse engineer animal cognition.

Queensland Brain Institute

Cook, MN*, Hartless, CB and Lester, DB.

Stress may contribute to the development and/or emergence of neuropsychiatric disorders, including those related to changes in reward and motivation. Anhedonia is a common outcome of chronic unpredictable stress (CUS) paradigms and may be indicator of resilience or vulnerability to the effects of chronic stress. Here, we exposed male and female C57BL/6J (B6) and DBA2/J (D2) mice to four weeks of chronic unpredictable stress, then used fixed potential amperometry to examine stimulated dopamine (DA) release, half-life, and auto receptor functioning. Overall, D2 mice exposed to CUS tended to have increased dopamine release compared to their control counterparts or B6 mice. D2 mice exposed to stress also had a shorter DA half-life than D2 control mice. CUS tended to decrease DA autoreceptor functioning in B6 but not D2 mice. Finally, CUS increased DA half-life in D2 mice following administration of nomifensine, a DA agonist. These strain-related differences in the effects of CUS on autoreceptor functioning and DA half-life may help in understanding reward- and motivation-related changes following exposure to chronic unpredictable stress.

Department of Psychology, The University of Memphis, Memphis, TN USA.

Funding support: Dunavant Professorship

Renee Papaluca1,2, Eva Guerrero-Hreins1,2, Aneta Stefanidis4, Claire Foldi4, Brian Oldfield4, Priya Sumithran*3, Robyn Brown*1

Bariatric surgery is the most effective long-term obesity treatment, reducing appetite and improving glycaemic control. These benefits are due to profound changes to the gut-brain axis. However, a small patient subset experience adverse mental health outcomes post-surgery, such as depression. While longitudinal studies begin to explore this, research exploring how gut-brain axis alterations could mechanistically contribute to these adverse mental health outcomes post-surgery is lacking. Therefore, this study investigates individual differences in depressive-like behaviour after vertical sleeve gastrectomy (VSG) in mice. Methods: Male and female C57BL/6 mice (n=57) were fed a high-fat, high-sugar diet (11 weeks) before VSG or sham surgery. Depression-like behaviour was assessed pre- and post-surgery. Based on test performance, VSG mice were then separated into tertiles and the upper and lower tertiles were used as ‘depression-susceptible and ‘depressionprone’ mice respectively. From these subgroups, RNA was extracted from tissue punches of the dorsal striatum and nucleus accumbens, key regions implicated in major depressive disorder and subjected to bulk RNA-sequencing. Results: VSG led to significant, sustained weight loss and reduced food intake. Differential gene expression analysis revealed downregulated neuroinflammation and tight junction-related genes in VSG depression-susceptible mice. Further gene set enrichment analysis displays an upregulation of TNF-α-NF-κB and PI3K-AKT/mTOR pathways. Additional chow-diet cohort data is pending, with preliminary findings to be presented at the meeting. Conclusion: This study provides insight into the possible neurobiological mechanisms underlying post-bariatric surgery mental health outcomes, shedding light on potential new biomarkers for depression risk associated with this common weight-loss procedure.

1University of Melbourne, School of Biomedical Sciences, Department of Biochemistry and Pharmacology, 2Florey Institute of Neuroscience and Mental Health, 3Department of Surgery, School of Translational Medicine, Monash University, 4Department of Physiology, Monash University.

*equal contribution Introduction:

CA Moul, 1

While there is considerable evidence for a role of the serotonin system in the aetiology of antisocial and aggressive behaviour, parsing out the mechanisms responsible for this relationship have remained elusive. The body of work presented here focusses on evidence to link the serotonin 1B receptor gene with callous-unemotional (psychopathic) traits and proposes a cognitive mechanism that may explain the relationship.

First, evidence is provided from genetic and epigenetic studies of clinical samples of children and adults with antisocial behaviour problems to demonstrate an association between HTR1B and callous-unemotional (psychopathic) traits. Next, a theory of amygdala function in psychopathy is used to provide the rationale for animal studies that tested the role of serotonin 1B in basic aspects of associative learning (response-reversal). Finally, the translation of this to a human study shows how callous-unemotional (psychopathic) traits in healthy, non-criminal young adults are associated with a deficit in the allocation of attention to, and concurrent encoding of, outcome stimuli.

Data from these three lines of work will be presented and entwined with a narrative that seeks to emphasise the importance of cognitive endophenotypes in genetic association studies of psychopathology. These data will illustrate how deviations in simple cognitive mechanics (e.g. attention and associative learning) can cause potentially dramatic variations in social behaviour. Missing pieces of the puzzle and future directions will be discussed.

1 School of Psychology, University of Sydney

Funding Support: Australian Research Council DP200102371

AR McEwan 1, G Hutchings 1, A Rattray 1, C Murgatroyd 2, A MacKenzie 1

Human development and health depend on the precise expression of genes in specific cells, at the right times, and in response to appropriate stimuli, known as context-dependent gene regulation. This regulation involves a variety of DNA elements called context-dependent cis-regulatory elements (cdCREs), which may constitute up to 10% of the genome. These include enhancers, silencers, and promoters that are crucial for complex multicellularity. Vertebrates share many phenotypic similarities, cell types, and gene expression patterns, often regulated by conserved cdCREs. The regulation of these genes has been implicated in the progression of disorders in humans. Obesity and anxiety have significantly impacted society during the COVID-19 pandemic. Understanding the role of cdCREs behind these conditions can improve our quality of life and resilience. We identified the BE5.1 region within the BDNF gene as of interest, being associated with obesity (rs10767664; p = 4.69 × 10^–26). The major T-allele of BE5.1 acted as an enhancer, while the obesity-linked A-allele did not. Deleting BE5.1 in mice showed no significant effect on BDNF expression in the hypothalamus or weight gain but increased anxiety-like behaviour in female mice, reversible by diazepam. Human studies also linked the rs10767664 polymorphism to anxiety. The findings suggest a novel mechanism regulating BDNF expression, affecting mood and anxiety in both mice and humans.

1. Institute of Medical Sciences, University of Aberdeen, Ashgrove Road West, Aberdeen, Scotland. AB25 2ZD, United Kingdom
2. Manchester Metropolitan University, Ormond Building, Lower Ormond Street, Manchester, M15 6BX, United Kingdom

J. Almeida¹, H. Chen¹, E. Legge¹, M. Sutter², and A.W. Lasek¹.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that primarily surround parvalbumin (PV)-expressing GABAergic interneurons in cortical regions. We previously showed that binge-like ethanol consumption resulted in increased accumulation of PNNs in the insular cortex (insula) and that PNNs in the insula regulate aversion-resistant ethanol consumption, as measured by adulteration of ethanol with quinine, in male mice. To investigate mechanisms contributing to increased brevican accumulation following chronic ethanol exposure, C57BL/6J mice underwent 6 weeks of binge-like drinking in the drinking in the dark (DID) procedure. Western blots of insula protein lysates showed reduced brevican cleavage product following chronic DID, suggesting reduced proteolytic activity of matrix metalloproteinases (MMPs). We performed qPCR to measure transcript levels of MMPs and their endogenous inhibitors (TIMPs). Timp3 transcript levels were increased in the insula after chronic DID, consistent with reduced activity of MMPs. We next investigated whether brevican is involved in ethanol consumption. Since brevican cell-autonomously regulates the excitability of PV neurons, we generated mice with knockout of the brevican gene (Bcan) in PV neurons (Bcan^PVKO) to test the hypothesis that brevican may regulate aversion-resistant ethanol consumption. Male Bcan^PVKO exhibited decreased aversion-resistant ethanol consumption when compared to their wild-type counterparts. We also found that knockout of Bcan in anterior insula neurons (Bcan^insnKO) by injecting AAV-hSyn-EGFP-Cre into the insula of male homozygous Bcan floxed mice resulted in reduced aversion-resistant ethanol consumption. Interestingly, female Bcan^PVKO and Bcan^insnKO mice did not exhibit this phenotype, suggesting a sex-specific role for brevican in insular cortex neurons in aversion-resistant ethanol consumption.

¹Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298, USA.

²Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, 60612, USA.

Funding support: NIAAA U01AA020912 and R01AA027231.

CL Barr 1,2,3, Y Feng 1, KG Wigg 1G

Genome wide association studies (GWAS) have implicated the OTX2 (Orthodenticle homeobox 2) gene locus in major depressive disorders (MDD) as well as genetically correlated traits (alcohol consumption, smoking initiation, chronotype). Of the genes identified by MDD GWAS, the gene for the transcription factor OTX2 stands out as it is responsible for both opening and closing of critical and sensitive brain periods. These are developmental periods where the brain is more sensitive to environmental input and are critical for normal brain development. Evidence suggests that the brain may also be more sensitive to negative environmental impact during sensitive periods. Critically, human and animal models both specifically implicate OTX2 gene expression in the response to stress and risk for depression. Based on the genetic findings, and the potential role of OTX2 as a mediator of environmental risk for depression, we identified genes regulated by OTX2 in human neural precursor cells (NPCs) using CRISPR activation (CRISPRa) to increase expression. We identified 17 significantly differentially expressed genes, including OTX2 which was increased 4-fold. In addition to OTX2, 4 genes of the 17 have been directly implicated in depression/depressive behaviours from human and animal studies (GPER1, VGF, TAFA5, P3H2). GPER1 has been indicated as serum biomarkers for depression. Additional differentially expressed genes are involved in processes implicated in depression (e.g. neurogenesis, neuroplasticity, response to stress). These novel findings link OTX2 expression with genes previously implicated in depression from human and animal studies, suggesting OTX2 as a master regulator of depression risk.

1 Division of Experimental and Translational Neuroscience, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada

2 Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada

3 Departments of Psychiatry and Physiology, University of Toronto, Toronto, Ontario, Canada

Funding Support: Labatt Family Chair in Depression Biology in Children

Youssef S¹, Johal K¹, Stefanelli G¹.

Neurogenesis and neurodevelopment are complex processes that heavily rely on dynamic epigenetic mechanisms and their ability to regulate gene expression. Recently, a role for histone variants and their dynamics has been shown to be crucial for neurodevelopment. Our lab is focused on Anp32e, a histone chaperone specific for H2A.Z variant. Notably, Anp32e has been shown to regulate H2A.Z levels in the genome and contribute to higher brain functions like memory formation. In addition, the lack of Anp32e has been associated with impaired dendritic morphology and downregulation of genes involved in neuronal development, highlighting its critical role in development. Yet, Anp32e role in neurodevelopment remains unexplored. Here, we use a knock-out mouse model to investigate how Anp32e regulates H2A.Z distribution and gene expression during critical stages of embryonic cortical development. We show that knock-out of Anp32e result in accumulation of H2A.Z throughout cortical development, severely impacting gene expression. In addition, we will perform behavioral assays to investigate if the epigenomic and transcriptomic alterations result in altered adult behaviors. Overall, our studies investigate for the first time the contribution of a new epigenetic mechanism involving H2A.Z and Anp32e to neurodevelopment and the regulation of adult behaviors.

¹ University of Ottawa, Department of Biology, Ottawa, ON, Canada

WC Booher^1,2, K Lanier¹, K Doenges¹, J Manke¹, M Armstrong¹, L Saba^1, N Reisdorph¹, RA Radcliffe^1,2

Oral consumption of Cannabis carries many of the same well-documented negative consequences associated with smoking Cannabis, along with additional health concerns specific to oral ingestion. It is essential to gain a deeper understanding of the factors contributing to the health risks of oral consumption, including individual differences in how people metabolize delta-9-tetrahydrocannabinol (∆9-THC), which is likely influenced by genetic factors. We hypothesized that genetically distinct mouse strains would exhibit variations in metabolism of ∆9-THC. To test this hypothesis, we administered 10 or 15 mg/kg ∆9-THC via oral gavage to four mouse strains (A/J, C57BL/6J, CAST/EiJ, and NZO/HILtJ) that are among the founders of two well-characterized experimental genetic mouse populations, the Collaborative Cross recombinant inbred strains and the Diversity Outbred heterogenous stock mice. Blood was collected at 60-, 90-, and 240-minutes post administration and plasma levels of ∆9-THC and its primary metabolites were measured using mass spectrometry. There were significant differences between the A/J and C57BL/6J ∆9-THC plasma levels and some of the metabolites, with few notable sex differences. The overall pattern of these differences suggest that C57BL/6J mice metabolize ∆9-THC more rapidly than A/J mice. CAST/EiJ and NZO/HILtJ mice exhibit significant differences in ∆9-THC plasma concentrations, but not other metabolites. To our knowledge, this is the first study to include A/J, CAST/EiJ, and NZO/HILtJ mice in a ∆9-THC oral administration mouse model. These findings underscore the need for further research to explore genetic differences in the pharmacokinetics of ∆9-THC in preclinical models of oral Cannabis consumption.

¹Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045

²Institute for Behavioral Genetics, University of Colorado, Boulder, CO

Funding support: Skaggs School of Pharmacy and Pharmaceutical Science ADR Seed Grant Program, Institute of Cannabis Research, and NIH-NCRR grant 1S10OD010366-01A1

Emmanuel S. Onaivi1 , Anna Bukiya2 , Venkat Sharma1 , Shilpa Buch3

There is an explosion of new knowledge that the effect of cannabis is encoded in our genome on chromosomes 1 and 6, and the biological effects is mediated by action on human endocannabinoid system (ECS) that are involved in reproduction and implantation of fertilized egg in the uterus. The identification of additional lipid mediators, enzymes and receptors is expanding the ECS, to endocannabinoidome (eCBome). Together with the ECS, eCBome are composed of genes coding for CB1 and CB2 cannabinoid receptors (CB1Rs, CB2Rs), endocannabinoids (eCBs), and their metabolic enzyme machinery for their biosynthesis and catabolism. The remarkable progress and new understanding are unravelling the role of the eCBome genetics, influence on behavior and providing deeper insights on the biological actions of cannabinoids in many disorders. CB2R conditional knockout (cKO) mice with deletion of CB2Rs from dopamine neurons, DAT-Cnr2 and those with deletion from microglia Cx3Cr1- Cnr2 were used and they displayed differential phenotypes. CB2Rs in dopamine neurons and microglia upregulated the expression of NLRP3 inflammasome pathway including NLRP3, and increased expression of proinflammatory cytokines, TNF-α, IL-6, IL-1α and IL-1β in the frontal cortices of the cKO mice following subacute treatment with 8% alcohol. The molecular pathways of fetal damage by alcohol, from cerebral artery to developmental delay highlights the functional presence and interaction of the ECS in baboons at different stages of development, including the effects of simultaneous alcohol and tetrahydrocannabinol on cerebral artery. An insight into the emerging eCBome genetics and neuroimmune crosstalk offers opportunities for continued preclinical and clinical studies.

1Biology department, College of Science and Health, William Paterson University, Wayne NJ, 2University of Tennessee Health Science Center, Memphis, TN, 3University of Nebraska Medical Center, Omaha, Nebraska.

RNA modifications have emerged as key regulators of gene expression, affecting translation, RNA stability and subcellular localization. In recent years, one of the most common RNA modifications, N6-methyladenosine (m6A), has been shown to influence brain development, cognitive functions, and behavior. We will discuss work using Drosophila as a model organism to explore the role of RNA modifications, specifically m6A, in brain development and behavior. We will focus on sex-specific roles for mRNA modifications in regulating developmental and behavioral phenotypes, including stress response and food choice.

MN Flamand1,2

RNA modifications have emerged as a pervasive feature of cellular mRNAs with diverse impacts on gene expression. N⁶-methyladenosine (m⁶A), the most abundant internal mRNA modification, is involved in the control of mRNA processing, stability, and translation and has important roles within the nervous system. Studies in animal models have revealed severe neurodevelopmental and cognitive deficits linked to disruptions in mRNA modification machinery. However, the specific molecular mechanisms through which m⁶A mediates these effects remain poorly defined. We previously found that m⁶A contributes to the localization of select mRNAs to dendrites and axons in mouse hippocampal neurons. We propose that m⁶A readers interact with distinct proteins in both the soma and synapses, thereby locally regulating gene networks crucial for synaptic plasticity. We hypothesize that disruption in these networks cause defects in synaptic function and contribute to the progression of neurodegenerative disorders associated with cognitive impairment. Advancements in this line of research critically rely on a better understanding of the local interactions and functions of RBPs in the brain. However, the tools to identify interactions in neurites harboring functional synapses are lacking. To overcome this obstacle, we are developing proximity labeling methods to map protein-protein and protein-RNA interactions in neuronal projections in vitro and at synapses in vivo. By integrating biochemical assays, transcriptomic analyses, and advanced imaging techniques, we aim to elucidate the complex roles of m⁶A readers at synapses and understand how their functions are altered during neurodegeneration.

1Neuroscience Unit, CHU de Québec Research Centre – Université Laval, 2Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval

Circular RNAs (circRNAs) are a class of regulatory RNAs that are enriched in synapses and recently implicated in cognitive processes. Emerging studies have also shown that circRNAs can encode micropeptides, which suggests that there are novel proteoforms in the adult brain awaiting discovery. Here, using a high-throughput IRES reporter screen and ribosome sequencing, together with mass spectrometry on synaptosomes isolated from the medial prefrontal cortex (mPFC) of behaviorally trained male C57/Bl6 mice, we have found widespread local circRNA translation in response to fear extinction learning. More than 1500 synapse-enriched circRNAs contain active IRES elements, with 324 circRNAs interacting with the ribosome, and 241 ribosome-bound circRNAs exhibiting direct evidence of activity-induced translation.Furthermore, we have identified a novel micropeptide (P1) that is derived from a single exon circRNA, the linear mRNA host of which encodes an enzyme involved in protein repair. Although P1 is only a third of the size of the full-length protein, it is locally expressed, enzymatically active, and interacts with plasticity-related synaptic proteins, including CamKIIα. In addition, targeted P1 knockdown impairs, whereas its overexpression enhances fear extinction memory. The discovery of an experience-dependent circRNA-derived micropeptide that is biologically active in the adult brain sheds new light on the ‘dark’ proteome and expands the notion that synapse-enriched circRNAs are key drivers of localized plasticity and memory

Professor Timothy Bredy, Queensland Brain Institute, University of Queensland

Yanhong Shi, Ph.D.

RNA modifications occur in all classes of cellular RNAs and play important roles in important biological processes and pathological events. Increasing studies demonstrate that RNA modifications could have key regulatory roles parallel to those of DNA and protein modifications. The biological relevance of RNA modifications in development and human diseases has been explored extensively in recent years. Our study aims to decipher the functional roles and underlying mechanisms of RNA modification in brain disorders. We will explore how RNA modification impacts the nervous system and dysregulated RNA modification links to brain disorders.

Beckman Research Institute of City of Hope

Renee Papaluca1,2, Eva Guerrero-Hreins1,2, Aneta Stefanidis4, Claire Foldi4, Brian Oldfield4, Priya Sumithran*3, Robyn Brown*1

Bariatric surgery is the most effective long-term obesity treatment, reducing appetite and improving glycaemic control. These benefits are due to profound changes to the gut-brain axis. However, a small patient subset experience adverse mental health outcomes post-surgery, such as depression. While longitudinal studies begin to explore this, research exploring how gut-brain axis alterations could mechanistically contribute to these adverse mental health outcomes post-surgery is lacking. Therefore, this study investigates individual differences in depressive-like behaviour after vertical sleeve gastrectomy (VSG) in mice. Methods: Male and female C57BL/6 mice (n=57) were fed a high-fat, high-sugar diet (11 weeks) before VSG or sham surgery. Depression-like behaviour was assessed pre- and post-surgery. Based on test performance, VSG mice were then separated into tertiles and the upper and lower tertiles were used as ‘depression-susceptible and ‘depressionprone’ mice respectively. From these subgroups, RNA was extracted from tissue punches of the dorsal striatum and nucleus accumbens, key regions implicated in major depressive disorder and subjected to bulk RNA-sequencing. Results: VSG led to significant, sustained weight loss and reduced food intake. Differential gene expression analysis revealed downregulated neuroinflammation and tight junction-related genes in VSG depression-susceptible mice. Further gene set enrichment analysis displays an upregulation of TNF-α-NF-κB and PI3K-AKT/mTOR pathways. Additional chow-diet cohort data is pending, with preliminary findings to be presented at the meeting. Conclusion: This study provides insight into the possible neurobiological mechanisms underlying post-bariatric surgery mental health outcomes, shedding light on potential new biomarkers for depression risk associated with this common weight-loss procedure.

1University of Melbourne, School of Biomedical Sciences, Department of Biochemistry and Pharmacology, 2Florey Institute of Neuroscience and Mental Health, 3Department of Surgery, School of Translational Medicine, Monash University, 4Department of Physiology, Monash University.

*equal contribution Introduction:

mmanuel S. Onaivi1 , Anna Bukiya2 , Venkat Sharma1 , Shilpa Buch3

There is an explosion of new knowledge that the effect of cannabis is encoded in our genome on chromosomes 1 and 6, and the biological effects is mediated by action on human endocannabinoid system (ECS) that are involved in reproduction and implantation of fertilized egg in the uterus. The identification of additional lipid mediators, enzymes and receptors is expanding the ECS, to endocannabinoidome (eCBome). Together with the ECS, eCBome are composed of genes coding for CB1 and CB2 cannabinoid receptors (CB1Rs, CB2Rs), endocannabinoids (eCBs), and their metabolic enzyme machinery for their biosynthesis and catabolism. The remarkable progress and new understanding are unravelling the role of the eCBome genetics, influence on behavior and providing deeper insights on the biological actions of cannabinoids in many disorders. CB2R conditional knockout (cKO) mice with deletion of CB2Rs from dopamine neurons, DAT-Cnr2 and those with deletion from microglia Cx3Cr1- Cnr2 were used and they displayed differential phenotypes. CB2Rs in dopamine neurons and microglia upregulated the expression of NLRP3 inflammasome pathway including NLRP3, and increased expression of proinflammatory cytokines, TNF-α, IL-6, IL-1α and IL-1β in the frontal cortices of the cKO mice following subacute treatment with 8% alcohol. The molecular pathways of fetal damage by alcohol, from cerebral artery to developmental delay highlights the functional presence and interaction of the ECS in baboons at different stages of development, including the effects of simultaneous alcohol and tetrahydrocannabinol on cerebral artery. An insight into the emerging eCBome genetics and neuroimmune crosstalk offers opportunities for continued preclinical and clinical studies.

1Biology department, College of Science and Health, William Paterson University, Wayne NJ, 2University of Tennessee Health Science Center, Memphis, TN, 3University of Nebraska Medical Center, Omaha, Nebraska.

Alexander S. Hatoum¹, Alex Miller², Aaron J. Gorelik³, David A.A. Baranger¹, Emma C. Johnson¹, Arpana Agrawal¹, Ryan Bogdan³

Background: Alcohol use disorder (AUD) presents with substantial heterogeneity.

Rational: Attempts to distinguish SUDs such as AUD by clinical features alone have had limited translational success.

Hypothesis: Objective biomarkers, like those derived from neuroimaging, will help parse AUD heterogeneity based on biology.

Methods. We developed neuroimaging derived cortical thickness biotypes of AUD in the European subset of the UK biobank (N=22,321) and conducted GWAS of biotype membership vs. controls (i.e., AUD biotype 1 vs. controls from biotype 2 and non-affected individuals). We also conducted GWAS contrasting biotypes in all individuals with neuroimaging data (N = 32,287). Results: Differences in regional cortical thickness within the salience network (e.g., regions) differentiated two AUD biotypes from controls. AUD biotype 1 was characterized by greater cortical thickness and biotype 2 had less cortical thickness, within the salience network. GWAS of biotype 1 (vs controls) identified one suggestive hit (rs60657889, p = 5.93e-08). GWAS contrasting the two biotypes revealed two significant hits including lead variant rs12355217 on FAM107B, (P=1.77e-09), a gene known to influence neurological pathways.

Discussion. Using a data-forward approach, we can parse heterogeneity in AUD and discover potential genetic pathways contributing to heterogeneity. Future analyses will examine loci from a joint analysis of biotypes with the largest GWAS of problematic alcohol use using a multivariate GWAS (N=1,079,947) and conduct multi-ancestry fine-mapping to determine likely causal variants. Finally, genetic correlations and drug repurposing analyses will determine the clinical and pharmacotherapeutic separability of brain-imaging derived biotypes.

1. Washington University School of Medicine, Department of Psychiatry, 2. Indiana University School of Medicine, Department of Psychiatry, 3. Washington University in St. Louis, Department of Psychological & Brain Sciences. Funding: NIAAA K01030083

William B. Lynch1,2,3, Jacob A. Beierle1,3,4, Bryan McKiver5, Arthur J. Vanvalkenburg6, Jared Mann5, Emily J. Yao1, Milad Mortazavi7, Yu-Yu Ren7, Binh-Minh Nguyen1, Kayla T. Richardson1,8, Abraham A. Palmer7, W. Evan Johnson6, M. Imad Damaj5, Camron D. Bryant1,3,4

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of cancer treatment with an unclear genetic etiology. Paclitaxel (PAC) is an anti-mitotic agent that causes CIPN in 60-70% of patients. Quantitative genetics in near-isogenic mouse substrains [C57BL/6J (J) and C57BL/6NCrl (N)] can facilitate gene, biological, and therapeutic discovery. J, but not N, showed robust PAC-induced mechanical hypersensitivity (von Frey) and cold hypersensitivity (acetone) that emerged, peaked, and recovered at 7, 14, and 28 days post-first PAC treatment (4X 2 mg/kg, i.p.). We identified a genome-wide significant quantitative trait locus (QTL) for mechanical hypersensitivity on chromosome 1 (LOD=7.7; p=0.001; 40-78 Mb; 11% variance explained) and a suggestive QTL for cold hypersensitivity on chromosome 14 (LOD=4.4; p=0.095; 100-124 Mb; 8% variance explained). Polymorphic, positional, differentially expressed genes (effect of Substrain, Substrain x Treatment) across DRG, PAG, and spinal cord (SC) at 7, 14, and 28 days post-first PAC revealed Cryba2, Aox3, Utp14b, Il1r2, and Wdfy1 as candidate genes for mechanical hypersensitivity and Slitrk1, Slitrk6, Lmo7, Cldn10, Hs6st3, Mbnl2, and Ndfip2 for cold hypersensitivity. Threshold-free analysis of gene expression signatures across substrains, tissues, treatments and time using rank-rank hypergeometric overlap indicated earlier, post-PAC changes in DRG and SC and later changes in PAG. Dynamic, biological pathways associated with peak substrain behavioral differences and recovery included mitochondrial function (NADH:ubiquinone oxidoreductase and Cytochrome c Oxidase genes) and ribosomal function. To summarize, we identified genetic and biological sources of variation following the emergence and recovery of CIPN that have implications for understanding risk and time-dependent therapeutics.

1Laboratory of Addiction Genetics. Current Location: Center for Drug Discovery, Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, 2Graduate Program for Neuroscience, Boston University, Boston, MA USA, 3Transformative Training Program in Addiction Science, Boston University, Boston, MA USA, 4T32 Biomolecular Pharmacology PhD Training Program, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., L-603, Boston, MA 02118 USA, 5Department of Pharmacology and Toxicology, Molecular Medicine Research Building, Room 3044, Virginia Commonwealth University, 6Division of Infectious Disease, Department of Medicine and Center for Data Science, Rutgers University - New Jersey Medical School, Newark, NJ USA, 7Department of Psychiatry, University of California, San Diego, Biomedical Research Facility II (BRF2), 3A24, 9500 Gilman Drive, La Jolla, CA 92093-0667 USA, 8BU Post—Post-baccalaureate Research Education Program (PREP), Graduate Medical Sciences, Boston University, 72 E. Concord St., L-317, L-309, Boston, MA 02118

Joseph Liang1, Kristen Tsoi1, Anushka Sood1, Christopher Mok1, Ben Westmore1, Catharine Rankin1,2

Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder that affects up to 1% of all people older than 65 years old. Our current understanding of the genetic contributions of PD has been expanded by genome-wide association studies in recent years. However, two major challenges plague researchers studying the genetic underpinnings of the disease today: There is a need to functionally characterize newly identified risk loci, and there remains a signification portion of loci for which a best gene candidate has yet to be assigned. We established a pipeline for in vivo characterization of C. elegans orthologs of newly identified PD risk loci. C. elegans have orthologs to many PD-associated and biologically relevant genes, there are strains with loss-of-function mutations available for almost every gene in the C. elegans genome. Notably, our lab developed the Multi-Worm Tracker for high-throughput characterization of behavioural and morphological phenotypes in populations of freely behaving animals in real time. Studying more than 150 mutant strains harbouring loss-of-function mutations in orthologs of PD-linked have yielded unique phenotypic profiles spanning up to 30 phenotypes ranging from morphology, baseline behaviours, non-associative learning and a dopamine-dependent behaviour for 83 PD-linked gene orthologs. From the data generated, we built a machine learning model to identify the best candidate gene for yet-to-be assigned loci. This research will establish high-throughput genotype-to-phenotype characterization of newly identified risk genes for PD to inform future disease modelling efforts and further our understanding of the biological processes underlying PD.

1Graduate Program of Neuroscience, University of British Columbia, Vancouver, B.C., Canada. 2Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, B.C., Canada. 3Department of Psychology, University of British Columbia, Vancouver, B.C., Canada.

Funding Support: Canadian Institutes of Health Research, Project Grant (CIHR MOP PJT-165947).

R-K Thériault¹, JD Manduca¹, Nolan CJ¹, J Lalonde², B Labonté³, IC Tobias¹, ML Perreault¹

Eph receptors are the largest family of tyrosine receptor kinases and are involved in neuronal growth and differentiation during development. The EphA2 receptor (EphA2R) has unique cell signalling characteristics and has been additionally implicated in processes causally linked to depression including vascular permeability, inflammation, and synaptic plasticity. In this work we explored the role of the EphA2R in regulating neuronal systems function in adults and its potential involvement in depression. Using rats, we first showed that chronic unpredictable stress (CUS) suppressed hippocampal (HIP) theta power and elevated HIP EphA2R signalling selectively in female rats that were stress-susceptible. Conversely, stress-susceptible males had lower high gamma power in the HIP and no alterations in EphA2R signalling. To further explore the functional role of prefrontal cortical EphA2Rs, we used a selective peptide to activate the receptor (5 nmoles/side). In female, but not male rats, acute prefrontal cortical EphA2R activation induced a depression-like phenotype and suppressed HIP theta power. HIP gene expression analysis using RNA-seq, analyzed 75 minutes post-injection, showed a rapid female-specific elevation in the gene expression of markers of inflammation, as well as microglial and immune activation. In human studies, postmortem analysis of frontal cortical gene expression in those that had depression showed significant elevations in the gene expression of several EphA2R downstream effectors in women, but not men. Together these findings suggest that heightened EphA2R activity may be associated with enhanced depression risk in women. A role for the EphA2R as a potential female-specific therapeutic target in depression warrants further investigation.

¹Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada

²Department of Molecular and Cellular Biology, University of Guelph, University of Guelph, Guelph, ON, Canada

³Department of Psychiatry and Neurosciences, Faculty of Medicine, Université Laval, Quebec City, QC, Canada.

Funding Support: University of Guelph Research Leadership Chairs Program, Canadian Institutes for Health Research, 450277

J Minerva1, JA Krawitz1, N Rashkover1, N Ma1, RA Jain1

When experiencing sudden acoustic stimuli, animals must integrate stimulus characteristics, context, and history to select if and how they respond. One aspect of this decision-making is habituation learning, where individuals diminish their responses to repeated innocuous stimuli, allowing them to attend to more relevant stimuli. A key gene regulating this habituation learning is ap2s1, a subunit of the AP2 complex which regulates clathrin-mediated endocytosis. Human AP2S1 alleles are associated with autism spectrum disorder and learning deficits, though the mechanisms of how this gene regulates learning are unknown.

To identify neural mechanisms through which ap2s1 regulates learning, we used a fluorescent calcium indicator (GCaMP5G) to quantify neuronal activity and pre- and postsynaptic calcium transients in critical circuit components for acoustically-evoked behavior in zebrafish larvae. We specifically examined the reticulospinal Mauthner command neurons initiating rapid escape responses, and their major sources of excitatory drive: the auditory nerve exciting the Mauthner lateral dendrites, and spiral fiber neurons synapsing on the Mauthner axon initial segments (AIS). ap2s1 mutants bias behavioral selection toward Mauthner-driven responses, and we observe increased Mauthner firing underlying their impaired habituation behavior. While auditory nerve driven dendritic activation is unaffected in ap2s1 mutants, we find elevated calcium transients at spiral fiber axon termini presynaptic to Mauthner AIS in naïve ap2s1 mutants. Presynaptic AIS activation attenuates with repeated stimuli, but remains elevated in mutants. These data suggest ap2s1 regulates excitatory synaptic strength to modulate habituation learning and behavioral choice through the spiral fiber pathway, suggesting AP2S1 similarly regulates excitatory drive in humans.

1Department of Biology, Haverford College, Haverford, PA, USA

Funding Support: NIH R15EY031539

BK Leung1, S Merlin5, AK Walker2,6, V Eapen2,7, R Clarke2,3, BW Balleine1, TM Furlong4,6

1Decision Neuroscience Laboratory, School of Psychology, 2Discipline of Psychiatry and Mental Health, 3Ingham Institute, 4School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia. 5Medical Science, School of Science, Western Sydney University, Campbelltown, Sydney, NSW, Australia. 6Neuroscience Research Australia, Randwick, NSW, Australia. 7Mental Health Research Unit, South Western Sydney Local Health District, Liverpool, Australia.

Symptoms of autism spectrum disorder (ASD) and Gilles de la Tourette syndrome (GTS) may emerge because of imbalances in cortico-striatal neurocircuits. We have recently developed a mouse-model of ASD and GTS by knocking down Immp2l, a mitochondrial gene associated with these disorders. Immp2l knock-out (KD) mice and wild type-like mice (WT) were trained on Pavlovian and instrumental learning procedures where auditory cues predicted food delivery and lever-press responses earned a food outcome. It was demonstrated that goal-directed learning was not changed for Immp2l KD mice, as lever-press responses were sensitive to changes in outcome value, and to contingency reversal and degradation. However, Immp2l KD mice were more responsive to auditory stimuli paired with food as indicated by a non-specific increase in lever response rates during Pavlovian-to-instrumental transfer. Finally, there were no alterations to neuron density in striatum or any prefrontal cortex or limbic brain structures examined. The current study suggests that the Immp2l gene is not necessary for learned maladaptive goal driven behaviours in ASD or GTS, but that it may contribute to increased capacity for external stimuli to drive behaviour. Such alterations could potentially influence the expression of tics and repetitive behaviours. Given that this is the first application of this battery of instrumental learning procedures to a mouse model of ASD or GTS, it is an important initial step in determining the contribution of known risk-genes to goal-directed behaviours, which should be more broadly applied to other rodent models of ASD and GTS.

Funding Support: Strategic Initiative Project from the Australian Research Council (CE140100007), Sydney Partnership for Health Education Research and Enterprise (SPHERE) Grant, and Ingham Institute Mental Health Research Award.

AR Dearman¹, P Vrtička², J Moore¹, M Kumari¹, L Schalkwyk³

Commonly-used genetic predictors of poor mental health (polygenic indices; PGIs) are based on genome-wide association studies (GWAS) of neuropsychiatric conditions (NCs). Intuitively, such PGIs should capture the direct effects of a person's genetic make-up on their risk of poor mental health. However, typical GWAS do not account for within-family effects, resulting in PGIs which capture risk factors from the rearing environment. Furthermore, endophenotypes (such as brain structure and function) may be better suited to genetic characterisation than NC diagnoses, which are considered behavioural phenotypes. We use sex-stratified path models to estimate the direct and environmentally-mediated effects of PGIs on mental health in four UK population datasets. We include eleven PGIs based on NCs and 30 PGIs based on endophenotypes. Each model simultaneously regresses a PGI onto four outcomes: psychological distress, and three measures of the rearing environment (parental separation, father's employment and mother's education), along with paths between environments and psychological distress. We also run regressions to examine bivariate associations. Bivariate and path model coefficients are compared to estimate the extent to which PGI effects are environmentally mediated. Eleven PGIs are significantly associated with psychological distress. The only PGI consistently associated with psychological distress is depression – all other significant associations are sex- and dataset-specific, including six other NCs and four endophenotypes. The only PGIs without environmental mediation are two endophenotypes. In conclusion, the depression PGI is the most consistent predictor of poor mental health, however its effects may be partly mediated by the rearing environment.

¹Institute for Social and Economic Research, ²Department of Psychology, ³School of Life Sciences;University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SQ, UK

Funding Support: Economic and Social Research Council & Biotechnology & Biological Sciences Research Council (UK), ES/T00200X/1

Jamie Peters^1,2, Giuseppe Giannotti*^1,3, Jasper A. Heinsbroek^1,2, Robin Vareed^1,4, Nathaniel P. Kregar^1,2, Francesca Mottarlini⁵, Christopher M. Driskill², Kasey L. Brida², and Jeremy J. Day²

Addiction to opioids is characterized by pervasive opioid seeking that results from dysfunction in brain circuits after chronic opioid use. The prefrontal cortex is central to these dysfunctional circuits, but little is known about the microcircuitry and cell types within the prefrontal cortex that regulate opioid addiction and relapse. Using a preclinical model of opioid addiction, we trained rats to self-administer intravenous heroin over a period of weeks. We implemented in vivo fiber photometry and optogenetics combined with a neuroanatomical viral approach to examine the neuronal activity and function of two distinct prefrontal cortex ensembles. One ensemble projects to the nucleus accumbens shell and the other to the lateral hypothalamus, with only a small percentage of collateralization between these targets observed (~5%). These anatomically distinct neuronal ensembles were found to be activated during heroin-associated cues (tone+light paired with heroin infusions) in a similar fashion based on calcium transients recorded at the cell bodies. However, distinct functions for each ensemble were revealed by optogenetic manipulation during the cue-reactivity window, with the prefrontal ensemble projecting to the nucleus accumbens shell functioning as a limiter of heroin seeking, and the lateral hypothalamus-projecting ensemble functioning as a driver of heroin seeking. These prefrontal ensembles also arise from distinct layers of the cortex, and single nucleus RNA sequencing is currently underway to determine whether they also exhibit unique transcriptomes. Importantly, targeting both cue-reactive ensembles bidirectionally was necessary to reduce heroin relapse, underscoring the importance of cell-type and circuit-specific intervention strategies.

¹Department of Anesthesiology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA

²Present affiliation: Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

³Present affiliation: Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA 99164, USA

⁴Biomedical Sciences Graduate Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA

⁵Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy

Funding support: NIDA/NIH R01 DA045836 (J.P.), F31 DA059203 (R.V.), DP5 OD026407 (J.A.H.), and K99 DA048974 (G.G).

KJ Clemens1, S Hesam-Shariati1, K Zimmermann1, R Ceccin2, PD Waters3, NA Habib4, KV Morris5

Recent research highlights dysregulation in Brain-Derived Neurotrophic Factor (BDNF) expression may be linked to a range of neuropsychiatric and neurodegenerative disorders. Targeting BDNF-related pathways has emerged as a potential treatment strategy, specifically through manipulation of long non-coding RNA (BDNF-antisense), although safe therapeutic delivery remains a problem. Here, we targeted Bdnf-AS using short hairpin RNA (shRNA) encapsulated in Integrating Lipid Nanoparticles (iLNPs) injected intravenously into rats. The iLNPs deliver and integrate Bdnf-AS shRNA and exosome packaging machinery to liver cells, which are converted into stable producers expressing Extracellular Vesicle (EV) loaded with the shRNAs. The EVs are hypothesised to cross the blood-brain barrier, regulating Bdnf-AS expression in the brain. Rats receiving shRNA against Bdnf-AS exhibited influenced working memory retention compared to controls. Preliminary molecular analyses confirmed the presence of the vector in the liver gDNA in all treated rats and of the shRNA in the brain. Further analysis of brain, blood, spleen and liver is ongoing. The use of iLNPs for shRNA delivery represents a promising innovative strategy, although further characterization of molecular mechanisms and efficacy of this approach is needed.

1School of Psychology, University of New South Wales, Sydney, NSW, Australia, 2Menzies Health Institute Queensland and School of Pharmacy and Medical Science, Griffith University, Gold Coast, QLD, Australia, 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia, 4Imperial College London, London, United Kingdom, 5Centre for Genomics and Personalised Health, School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD, Australia

Funding Support: National Health and Medical Research Council Ideas Grant APP2027730

S Gottlieb1,3 , SM Scarboro1,2,3, MF Lopez,1,3 , and HC Becker,1,2,3,4

Oxytocin (OT) has gained interest in the field of alcohol use disorder (AUD), having been shown to reduce alcohol consumption in multiple drinking models when given exogenously. Here we chemogenetically activated OT neurons in the paraventricular nucleus (PVN) of the hypothalamus to investigate the neural mechanism of OT effects on alcohol consumption. Oxt-IRES-Cre mice underwent viral stereotaxic injections to express an excitatory DREADD or control virus in the PVN (n=4-7/sex/virus). After recovery, mice were trained to respond (FR4) for 12% (v/v) alcohol in daily 20- min sessions. After stable baseline responding/intake was established, testing was conducted over several sessions. Mice were tested 30-min following injection (IP) of vehicle, the DREADD-activator CNO (3mg/kg), or OT (0.5mg/kg) in a within-subjects. Lever responses and alcohol intake (g/kg) were measured. Systemic administration of OT significantly reduced lever responses and alcohol intake in both sexes regardless of which virus was injected (p<0.05). Animals who received control virus showed no effect of CNO administration on either behavioral outcome. In animals who received experimental virus, males significantly reduced lever pressing and alcohol intake (p<0.05), and females trended toward decrease (p=0.08). Systemic oxytocin shows promise as a potential therapeutic in AUD. Here we show targeted PVN neural activation of OT decreases alcohol self-administration in a way similar to systemic, elucidating a key brain region in this pathway. Larger sample sizes plan to be incorporated, and further studies aim to investigate the contribution of vasopressin in this oxytocin-induced reduction of alcohol self-administration.

1Department of Psychiatry and Behavioral Sciences, 2Department of Neuroscience, 3Charleston Alcohol Research Center, Medical University of South Carolina, 4RHJ VA Medical Center, Charleston, SC 29401-USA. Funding Support: NIAAA grants R01 AA026536, P50 AA10761, U01 AA014095, U24 AA29968, T32 AA007474, F31 AA026483 & VA Medical Research (BX000813)

Brad Balderson1 , Narayan Pokhrel2 , Yanning Zuo2 , Benjamin Johnson2 , Olivier George2, Abraham A. Palmer2,3, Graham McVicker1 , Francesca Telese2

The nucleus accumbens (NAc) is a critical brain region for reward processing and addiction. However, the impact that genetic variants associated with addiction have on the gene regulatory networks of specific NAc cell types remains unclear. To investigate this, we utilized an outbred population of rats, which were genotyped and assessed for drug-taking behavior, using an oxycodone intravenous self-administration model. We profiled gene expression and chromatin accessibility in ~450,000 single nuclei from NAc tissue collected from 85 rats following five weeks of oxycodone abstinence. Our analysis identified 15 major cell types within the NAc, including a rare population of D3 medium spiny neurons (MSNs), which express the dopamine receptor Drd3 and comprise ~2% of NAc cells. Differential abundance analysis revealed that higher D3 MSN abundance was associated with increased oxycodone intake. Additionally, we conducted a latent factor analysis to uncover cell type-specific gene expression and chromatin accessibility patterns across individuals. This analysis identified a distinct molecular signature linked to increased drug intake, composed of genes and open chromatin regions enriched in specific cell types, including D3 MSNs and opioid receptor Oprm1-expressing MSNs. Finally, we leveraged the rat genotypes to identify genetic variants associated with the gene expression and chromatin accessibility patterns linked to oxycodone consumption. This analysis yielded significant associations, suggesting a genetic basis for variability in gene expression and chromatin accessibility associated with drugtaking behavior in HS rats. Overall, our single-cell multiomic approach identified genetically encoded regulatory networks that are linked to oxycodone addiction-related behaviors in rats.

1 Salk Institute for Biological Studies, Integrative Biology Laboratory, La Jolla, CA 2 Department of Psychiatry, University of California San Diego, La Jolla, CA, USA 3 Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA Funding Support: NIDA U01DA050239

Lewis M Sherer¹, Vivian M Chan¹, R Steven Stowers², Sarah J Certel³, Karla R Kaun¹

Co-transmission, or the ability of a neuron to release more than one neurotransmitter, neuromodulator, or neuropeptide, has been increasingly recognized as a critical mechanism by which individual neurons can more precisely control their output to downstream circuits. Despite its importance to the nervous system, however, the functional effects of co-transmission are not fully understood. We use the well-characterized neural circuitry and powerful genetic toolkit of Drosophila melanogaster to examine properties of two octopamine/glutamate co-transmitting neurons with known roles in feeding and social behavior. VPM1 is a sexually dimorphic neuron that responds to male pheromones, and VPM4 is not dimorphic and responds to food. We characterize the stimulus-evoked release dynamics of octopamine and glutamate from these neurons using volumetric two-photon imaging of calcium and neurotransmitter-specific fluorescent sensors. Finally, to examine how housing experience and associated internal state impacts co-transmission, we examine changes in activity and transmitter release from VPM1 and VPM4 that occur as a consequence of socialization. These results are critical for a complete understanding of how neurons modulate their outputs to downstream circuitry and lay the foundation for the development of novel therapeutic strategies that target specific neurotransmitter systems.

1 - Department of Neuroscience, Brown University, Providence, RI, USA

2 - Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA

3 - Division of Biological Sciences, Center for Structural and Functional Neuroscience, The University of Montana, Missoula, MT, USA

Funding Support: Brown University Training Program in the Molecular Biology of Aging (5T32AG041688)

B. Martin1, E. Zeitoun1, G. Dieuset1, N. Costet2, V. Latapie3, S. Jamain3, A. Biraben1
The audiogenic seizure (AGS) is a widely used model for preclinical studies of death in epilepsy. Immediately after exposure to the sound, the mouse manifests a stereotyped behavior, which can be successively characterized by a wild running, clonic seizures, a tonic-clonic seizure in which the mouse falls onto its flanks, and a tonic seizure with an extension of the limbs towards the tail, followed or not by death. Our laboratory has recently developed two genetic mouse lines, LAGS+ and LAGS-, based on a bidirectional genetic selection on phenotypic criteria, initiated by a four-way cross derived from DBA/1J, DBA/2J, BALB/cJ and 129/SvTer. Now, both LAGS+ and LAGS- present 100% of tonic seizures when stimulated with a 110 dB white noise. However, they diverge on pronostic. Effectively, after 15 generations of selection, LAGS+ (for lethal audiogenic seizure), presents &gt;99% of lethal AGS while LAGS- (for non-lethal audiogenic seizure) presents &lt;2% of lethal AGS. With the aim of identifying chromosomal regions contributing to AGS-related mortality, we have genotyped LAGS+ and LAGS- mice and compared their allelic frequencies. Directional selection was. At each generation, 10 couples were constituted. At generation 11 for LAGS+ and generation 10 for LAGS-, we genotyped the 10 couples of each line using Illumina GGP GIGA-MUGA Arrays allowing the genotyping of more than 143,000 SNPs. We then performed a homozygosity mapping to identify shared genomic regions between animals with the same phenotype. The analyses identified a multitude of chromosomal regions as potentially involved in mortality in this model of epilepsy.

1Univ Rennes, CHU Rennes, INSERM, Laboratoire de Traitement de Signal et d’Image - UMR 1099, 35000 Rennes, France
2Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, F-35000 Rennes, France
3Univ Paris Est Créteil, INSERM, IMRB, Neuropsychiatrie Translationnelle, 94010 Créteil, France

K Huang^1,2, K Conn^1,2, E Greaves^1,2, LK Milton^1,2& CJ Foldi^1,2

Excessive exercise has been reported in up to 80% of patients with anorexia nervosa (AN) and is an essential component of the activity-based anorexia (ABA) paradigm. Exercise in the ABA model is often called “compulsive” and may share similar underlying biological mechanisms with obsessive-compulsive disorder (OCD), including disrupted dorsal striatum (DStr) activity. However, specific effects on compulsive behaviours or differentiated actions of D2R in dorsomedial striatum (DMS) from those in dorsolateral striatum (DLS) remain unknown. In order to determine the involvement of DStr D2R neurons in compulsive exercise and operant behaviours in ABA rats, we injected D2R-Cre driven inhibitory (Gi) DREADDs into the DMS, and operant-paired fibre photometry (D2-Cre driven GCaMP). Female Sprague-Dawley rats were either exposed to ABA conditions (n=15) or underwent the outcome-specific devaluation task (ODT; n=15). CNO was injected during ABA exposure or the instrumental training phase of ODT. We also monitored D2 dynamics in DMS (n=2) or DLS (n=6) during ODT. We showed that the suppression of D2R in DMS specifically increased running wheel activity in ABA (p=0.012) but did not affect compulsive operant responding in ODT. Moreover, changes in calcium release that correlated with operant actions during instrumental learning showed distinct patterns in DMS and DLS that changed throughout training. The results demonstrate the specific involvement of D2R in DStr in compulsive exercise in ABA. This knowledge will aid in the development of targeted treatments for patients with AN that exercise excessively and potentially a reframing of AN definition based on clinical subtypes.

¹Department of Physiology, Monash University, Clayton VIC, Australia; ²Monash Biomedicine Discovery Institute, Clayton VIC, Australia

Pozeilov H¹, Levi M¹, Dayan E¹, Forkosh O² & Shohat-Ophir G ¹

Individual differences, also referred to as personality, are a fundamental characteristic of living organisms and play a crucial role in our understanding of behavior. In humans, the most widely accepted model of personality is based on five continuous dimensions, with each individual scoring differently across these factors. Recent developments in machine vision and learning algorithms have made it possible to detect and analyze a continuous range of inter-individual differences in non-human organisms. To explore this in Drosophila, we employed linear discriminant analysis (LDA) to identify new behavioral dimensions, which we refer to as identity domains. These domains reflect maximal behavioral variation between individuals while maintaining consistency within individuals over time. Using this computational framework, we investigated the complex relationships between social interaction, personality, and motivational states in flies. Our experimental paradigm involved male and female flies subjected to different social and sexual experiences for four days, after which their social group interactions were recorded. The findings offer compelling evidence of distinct inter-individual differences in Drosophila, pointing toward the presence of personality-like traits in flies. We identified four novel identity domains that differentiate between males and females and distinguish between different social and sexual experiences. In addition, we examined how the microbiome's absence affects flies' personality. Our data suggest that the lack of a microbiome leads to a significant shift in the distribution of individual flies across identity space. We developed a single-fly two-choice, operant learning paradigm, in which flies learn to self-stimulate their Neuropeptide F expressing (NPF) neurons (presumably due to its rewarding value), by touching an electrode that induces optogenetic activation of a channel rhodopsin expressed in NPF neurons. We find inter-individual differences in learning kinetics associated with motivational state and NPF levels.

¹ The Mina & Everad Goodman Faculty of Life Sciences, The Multidisciplinary Brain Research Center and the Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Israel

² Department of Cognitive and brain Science, Department of Animal Sciences, Israel

Jean-Richard-dit-Bressel, Philip¹

Individuals differ in their responsivity to the adverse consequences of their actions, with many persisting in highly detrimental behaviour. Despite its relevance to everyday decision-making and clinical conditions, experimental tasks used to study this phenomenon often fail to reveal psychological causes for these inter-individual differences. We addressed this by developing rat, human, and mouse versions of a conditioned punishment task that efficiently disentangles key psychological underpinnings for these differences. Across species, we find avoidance of negative consequences is bimodally distributed across individuals. We show these differences in avoidance are not primarily driven by differences in appetitive or aversive motivation, nor differences in behavioural control. Rather, behavioural differences were primarily due to failures in Action-Punisher learning, indicating an overlooked source of punishment insensitivity. Preliminary data showing how this task is being leveraged for deeper insight into neural mechanisms will also be discussed.

1. School of Psychology, University of New South Wales, Sydney, Australia.

J.D. Jentsch¹

Operant responding for natural or pharmacological reinforcers exhibits substantial individual variation, with a sizeable fraction of that variability being explained by heritable genetic factors. Consistent with our observation that strains from the hybrid mouse diversity panel (HMDP) exhibit extreme differences in operant intravenous cocaine self-administration, we evaluated the acquisition and performance of an action that was reinforced with sucrose solution, using Fixed/variable ratio schedules of reinforcement across 3 long-access sessions in 81 HMDP strains. Across the panel, total lever presses made during the 36 hours of testing ranged from ~0 to almost 7,000 (~195 presses/hr), with a heritability estimate of ~28%. We next studied 8 inbred strains that spanned the range of non-zero operant responding (MRL/MpJ>C57BL/10J>=BALB/cJ>C57BL/6J>DBA/2J>A/J); mice were trained to lever press for a chocolate-flavored BOOST reinforcer. Mice were evaluated using a progressive ratio schedule of reinforcement, in which the response requirements doubled after each schedule completion. Strain differences in responding on the progressive ratio test were observed, with a heritability estimate of ~20%. When different mice from the same strains were evaluated for nucleus accumbens core dopamine release using fast scan cyclic voltammetry in acute brain slices, we found a positive genetic correlation between lever responding in the progressive ratio task and uM dopamine release (r[4]=.831, p=.041). These results suggest that genetic variation present in common inbred strains alters the excitability of dopamine release into the nucleus accumbens and creates substantial differences in motivation to obtain a salient positive reinforcer.

¹Department of Psychology, Binghamton University, Binghamton NY U.S.A.

Funding Support: US Public Health Service Grants P50-DA039841 and T32-AA025606

Hernandez, John S¹., Le, Nelson.², Azanchi, Reza.¹, Glenn, Eve.¹, and Kaun, Karla .R.¹

The escalation of alcohol self-administration is a critical factor in the transition from alcohol use to compulsive drinking, a significant global health concern. While much research has focused on the neural mechanisms driving excessive alcohol intake, there is a pressing need to understand the neural substrates that underlie individual differences in alcohol preference and seeking. This understanding is essential for unraveling why escalation occurs in some individuals but not others, which is key to addressing variability in motivated response. Using Drosophila melanogaster, an ideal model due to its well-characterized neural circuits and genetic tools, we developed a 3-day operant paradigm to assess behaviors related to self-administration of volatilized ethanol. Our findings reveal a simple 2-neuron cholinergic and dopaminergic circuit within the mushroom body that modulates ethanol preference, with implications for how experience shapes this preference over time. We identified dopamine receptors within this circuit that influence ethanol preference, highlighting how individual variation in motivated behavior may emerge from specific neural pathways. Finally, we identified transcriptional changes that are epigenetically regulated within our genetically homogenous population. These insights are critical for understanding the mechanisms that drive alcohol preference, which can predict the likelihood of developing dependence.

1 - Brown University, RI. Department of Neuroscience

2 - Post-baccalaureate Research Education Program, Brown University, Providence, RI

Funding support was provided by the NIAAA R01AA024434 (supporting N.J.M, R.A., K.R.K.), NIAAA F32AA29595 (supporting J.S.H.) and NIGMS R25GM125500 (supporting N.L.).

WC Booher^1,2, K Lanier¹, K Doenges¹, J Manke¹, M Armstrong¹, L Saba^1, N Reisdorph¹, RA Radcliffe^1,2

Oral consumption of Cannabis carries many of the same well-documented negative consequences associated with smoking Cannabis, along with additional health concerns specific to oral ingestion. It is essential to gain a deeper understanding of the factors contributing to the health risks of oral consumption, including individual differences in how people metabolize delta-9-tetrahydrocannabinol (∆9-THC), which is likely influenced by genetic factors. We hypothesized that genetically distinct mouse strains would exhibit variations in metabolism of ∆9-THC. To test this hypothesis, we administered 10 or 15 mg/kg ∆9-THC via oral gavage to four mouse strains (A/J, C57BL/6J, CAST/EiJ, and NZO/HILtJ) that are among the founders of two well-characterized experimental genetic mouse populations, the Collaborative Cross recombinant inbred strains and the Diversity Outbred heterogenous stock mice. Blood was collected at 60-, 90-, and 240-minutes post administration and plasma levels of ∆9-THC and its primary metabolites were measured using mass spectrometry. There were significant differences between the A/J and C57BL/6J ∆9-THC plasma levels and some of the metabolites, with few notable sex differences. The overall pattern of these differences suggest that C57BL/6J mice metabolize ∆9-THC more rapidly than A/J mice. CAST/EiJ and NZO/HILtJ mice exhibit significant differences in ∆9-THC plasma concentrations, but not other metabolites. To our knowledge, this is the first study to include A/J, CAST/EiJ, and NZO/HILtJ mice in a ∆9-THC oral administration mouse model. These findings underscore the need for further research to explore genetic differences in the pharmacokinetics of ∆9-THC in preclinical models of oral Cannabis consumption.

¹Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045

²Institute for Behavioral Genetics, University of Colorado, Boulder, CO

Funding support: Skaggs School of Pharmacy and Pharmaceutical Science ADR Seed Grant Program, Institute of Cannabis Research, and NIH-NCRR grant 1S10OD010366-01A1

Thibault Renoir^1,2, James J Gattuso¹, Bilgenur Bezcioglu¹, Geraldine Kong³, Millicent Ekwudo¹, Carey Wilson¹, Nina Kleditzsch¹, Ahmed Kamal¹, Jenna Hendey¹, Carolina Gubert¹, Anthony J Hannan^1,2

The evident limitations of current treatments for depression, anxiety and obsessive-compulsive disorder (OCD), underscore the need for a paradigm shift in how we understand and treat these disorders. There is enormous public and research enthusiasm for the potential that psychedelic drugs like psilocybin, the psychoactive compound produced by “magic mushrooms” will drive a new paradigm of care for mental ill health. Although promising results are emerging for the treatment of depression, anxiety and OCD, questions remain about the biological mechanisms underlying therapeutic outcomes. Also, the recent approval for the use of psilocybin for treatment-resistant depression in Australia has been criticized as premature considering (i) the difficulty separating therapeutic effects from expectancy (placebo effects), (ii) remaining questions on the biological mechanisms underpinning efficacy and (iii) a lack of well-designed long-term studies assessing potential deleterious effects.

In this talk, Dr Renoir will present behavioral and molecular data relevant to both acute and chronic psilocybin in transgenic mouse models relevant to depression, anxiety and obsessive/compulsive-like disorders. Dr Renoir will also discuss recent evidence suggesting the potential involvement of the serotonin transporter (5-HTT) in mediating some effects of psilocybin as well as limitations/questions around the psychedelic actions in the context of schizophrenia.

This is especially timely as the acceptance and approval of psychedelic-assisted psychotherapy becomes adopted worldwide and there are concerns about the increasing use of repeated low doses of psychedelics (i.e. at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy - often referred to as ‘microdosing’).

¹Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, Australia.

²Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia.

³Peter Doherty Institute of Infection and Immunity, University of Melbourne, Parkville, Victoria 3000, Australia

CJ Foldi1,2,3

Psilocybin, the psychoactive compound produced by so-called “magic” mushrooms has shown promise in alleviating symptoms of a range of psychiatric conditions including depression, anxiety and substance use disorder. Recently, a small Phase 1 trial showed psilocybin to be safe and tolerable for individuals with anorexia nervosa (AN), with 4/10 participants experiencing long-lasting improvements in eating disorder symptoms. A major challenge in understanding the mechanisms through which psilocybin acts to cause enduring changes in brain function and behaviour is that it is impossible to effectively blind participants to treatment, making clinical studies particularly susceptible to placebo effects. Animal models are necessary for this mechanistic investigation, and this presentation will focus on data accumulated in the lab over recent years using an animal model known as activity-based anorexia in combination with operant learning paradigms, behavioural pharmacology, RNAscope and in-vivo fiber photometry. We demonstrate that psilocybin elicits a specific increase in cognitive flexibility to improve body weight outcomes in activity-based anorexia. Moreover, we show effects of psilocybin on learning are mediated by actions at specific serotonin receptor subtypes, the transcription of which is transiently altered in the prefrontal cortex across the 24 hours after psilocybin administration. Finally, we applied computational modelling to understand how performance strategies are altered by psilocybin over time and reveal that enhanced cognitive flexibility is underpinned by augmented dopamine signalling in the ventral striatum. Our results are translationally relevant for the clinical application of psilocybin for AN, considering these individuals display both impaired cognitive flexibility and diminished reward processing.

1Monash University Department of Physiology, 2Monash Biomedicine Discovery Institute 3Australian Eating Disorders Research & Translation Centre Melbourne, VIC, Australia. Funding Support: National Health & Medical Research Council (NHMRC) of Australia (GNT: 2011334)

Hayk Davtyan^1,2, Jean Paul Chadarevian^1,2, Jonathan Hasselmann^1,2, Ghazaleh Eskandari-Sedighi¹, Sherry Lin-Koch⁴, Alina L Chadarevian^2,3, Joia Kai Capocchi¹, Duc Duong⁵, Jasmine Nguyen¹, Christina Tu^1,2, Sepideh Kiani Shabestari³, Fang Wu⁵, Anantharaman Shantaraman⁵, Kimiya Mansour², William Carlen-Jones¹, Michael Mgerian³, Katia Deynega¹, Tau En Lim¹, Alan Khoi Mai¹, Lauren Le¹, Ani Agababian³, David A Hume⁶, Clare Pridans⁷, Elizabeth Head¹, Sandeep Robert Datta⁴, Nicholas T Seyfried⁵, Mathew Blurton-Jones^1,2,3

Hematopoietic stem cell transplantation (HSCT) in combination with microglia depletion is increasingly being examined as a potential therapy for neurological disorders. The premise of this approach is that HSCT-derived monocytes (MN) may infiltrate the brain and differentiate into “microglia-like” cells. However, induced pluripotent stem cells derived microglia (iMG) provides a potential alternative source of therapeutic cells. As many questions remain regarding the similarities and differences between microglia and monocytes, we utilized a xenotransplantation-compatible model that lacks endogenous microglia (hFIRE mice) to examine the transcriptional and functional properties of human iMG and MN. iMG and MN from four patients were transplanted into adult hFIRE brains and four months later behavioral testing was performed. Brains were then examined via spatial RNA sequencing, proteomics, histological, and biochemical approaches. Despite four months of brain residence and near complete chimerism, human iMG and MN continued to exhibit many important differences. In particular, we found that MN, but not iMG, induced a chronic proinflammatory state associated with significant levels of astrogliosis, demyelination, synaptic loss, and behavioral impairment. Taken together, these results demonstrate the critical role of ontogeny on myeloid cell function within the brain and provide important implications for the development of CNS-wide microglial replacement therapies.

¹Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA

²Stem Cell Research Center, University of California, Irvine, CA, USA

³Department of Neurobiology & Behavior, University of California, Irvine, CA, USA

⁴Harvard Medical School, Boston, MA, USA

⁵Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

⁶Mater Research Institute, University of Queensland, Brisbane, Australia

⁷Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

L Perez Mederos¹, JMHM Reul¹, KR Mifsud^1,2

Hyaluronan (HA) is an important extracellular matrix (ECM) component of the brain. Disruption of HA has been linked to both neurodevelopmental and neurodegenerative disorders, potentially due to the emerging role it has in cognition. To investigate how HA contributes to hippocampal learning, rats were subjected to the Morris water maze (MWM). This paradigm includes a swim control (SC) group to control for the ‘stress’ component of this task. This is important because our previous work has indicated the acute stress can increase binding of the glucocorticoid receptor transcription factor to hyaluronidase 2 (HYAL2), a gene directly involved in HA turnover in the hippocampus.

Rats were trained to find a submerged platform using distal cues to form an internal spatial map (MWM-t). SC rats were exposed to the maze for the same duration but with no platform. Baseline control rats were killed direct from home cage. HA concentration [HA] was measured by ELISA in six brain regions. RNA expression was analysed by qPCR.

MWM-t rats showed a learning-specific increase in hippocampal [HA] compared with SC/BL rats. This was accompanied by a learning-specific increase in expression of hyaluronan synthase mRNA indicating a transcription-dependent process. In the amygdala, [HA] was significantly increased above BL in both MWM and SC groups.

Early results indicate that both the stress and spatial learning aspects of the MWM paradigm are affecting HA concentration in a transcription-dependant and region specific manner. Further validation work is required to ascertain the behavioural consequences of these changes for long-term health.

¹Translational Health Sciences, Bristol Medical School, University of Bristol ²Animal Welfare and Behavioural Group, Bristol Veterinary School, University of Bristol

Funding Support: University of Bristol Internal funding, The Physiological Society RKEA TPSRKE08

Charles Hoeffer^1,2, Andrew M. Lombardi¹, Mina Griffioen^1,2, Helen Wong², Ryan Milstead^1,2,3, Curtis Borski², Erin Shiely^1,2, Myra E. Bower ^1,2, Emily Schmitt¹, Lauren LaPlante², Marissa A. Ehringer^1,2, Jerry Stitzel^1,2.

Improved understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate detrimental nicotine withdrawal effects, and improve cessation rates. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4β2 and α7. Protein kinase B-β (Pkb-β or Akt2) expression is restricted to astrocytes in mice and humans and is activated by nicotine. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice. For in/ex vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 μg/mL) or following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed enhanced acute responses and reduced area following chronic exposure. In culture, we found that 100μM nicotine was sufficient for morphological changes and blocking α7 or α4β2 nAChRs prevented observed morphologic changes. We performed conditioned place preference (CPP) in Akt2 cKO mice which revealed reduced nicotine preference in cKO mice compared to controls. Finally, we performed RNASeq experiments comparing nicotine- and LPS-mediated gene expression identifying robust differences between these two astrocytic stimuli. These findings show the importance of nAChRs and AKT2 signaling in the astrocytic response to nicotine.

¹Department of Integrative Physiology, University of Colorado at Boulder; ²Institute for Behavioral Genetics, University of Colorado at Boulder. Boulder, CO, 80303 USA.

MK Mulligan1, A Zhang1, EA Duecker1, S Saxena1, S Khanam1, X Wang2, BM Moore II3

C57BL/6J and DBA/2J mice exhibit differential responses to acute high-dose (10 mg/kg, i.p.) THC. At 1h and 4h post-injection, C57BL/6J show greater sensitivity (p<0.01) to THC’s inhibitory effects on motor activity. Females of both strains are more sensitive to THC-induced hypothermia at 1h and temperature in C57BL/6J females remains below baseline at 4h. To identify pathways mediating these differences we conducted bulk RNA-sequencing and proteomics/phosphoproteomics of matched cortical tissue. 96 samples (six replicates across treatment, strain, sex, and time) and 16 samples (two replicates across treatment, strain, and sex) were used for RNA-sequencing and proteomics, respectively. Consistent with behavioral differences, the transcriptional response to THC (p<0.05) was more pronounced in C57BL/6, and at 4h vs. 1h. The number of differentially expressed (DE) genes was: 4,250 vs.1,872 (C57BL/6J females); 2,911 vs. 768 (C57BL/6J males); 2,364 vs. 801 (DBA/2J females); 2,202 vs. 785 (DBA2/J males). At 1h, 11,384 unique proteins were detected across all samples. Our JUMP pipeline identified 46,108 modified peptides and 34,961 modified sites, including 26,951 serine, 6,644 threonine, and 1,366 tyrosine phosphorylation sites. 1,033 phosphopeptides and 95 proteins were DE between THC and controls (p<0.01). Enriched pathways included neuron projection morphogenesis, dendrite development, and synapse signaling. Several DE phosphorylation sites were detected on the Cannabinoid 1 Receptor that were significantly (p<0.01) elevated by THC, particularly in DBA/2J, possibly indicating earlier termination of THC activation and diminished sensitivity relative to C57BL/6J. This multiomic dataset provides insight into the molecular mechanisms underlying genetic differences in behavioral response to THC.

1Department of Genetics, Genomics and Informatics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, 2Department of Neurology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA, 3Deparment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA

Funding Support: NIH/NIDA R01DA056523

Priscila A. Costa^1,2, Brendan Navaneethan^1,3, Sophie Debs^1,2,3, Illya Conn^1,4, Andriane Penklis^1,3,4, Cynthia Shannon Weickert^2,4,5, and Tertia D. Purves-Tyson^1,2

Maternal immune activation (MIA) induces striatal hyperdopaminergia in rat offspring comparable to psychosis in schizophrenia. Raloxifene, a selective estrogen receptor modulator, can alleviate psychosis in schizophrenia and reduces psychosis-like behaviour in male MIA offspring, but potentiates it in healthy rats. The molecular mechanisms underlying raloxifene's behavioural effects are unknown. We examined whether raloxifene alters dopaminergic-, GABAergic-, and glutamatergic-related gene expression in the dorsal striatum (dSTR) of male and female MIA offspring. Offspring of Wistar dams injected with saline or poly(I:C) on GD19 received daily raloxifene (oral,5mg/kg) or placebo between postnatal days 58-84. Dopaminergic, GABAergic, and glutamatergic transcripts were measured in the dSTR using RT-qPCR. Although neither MIA nor raloxifene altered GABAergic or glutamatergic transcripts, MIA reduced dopamine breakdown enzyme transcripts, Comt and Maob, in females and Maob in males, however this was not prevented by raloxifene. Raloxifene reduced Comt mRNA in MIA offspring compared to controls, and reduced Maoa and Maob mRNAs in all male offspring. Dopamine receptor transcripts were unchanged by MIA or raloxifene. These data suggest reduced dopamine breakdown may contribute to striatal hyperdopaminergia in male and female MIA offspring. Raloxifene and MIA do not appear to alter behaviour by changing striatal GABAergic or glutamatergic transcripts. Raloxifene reduced Comt mRNA in MIA-exposed offspring, but not in controls, indicating distinct effects of treatment in offspring with MIA-induced dopamine dysregulation. This suggests raloxifene may potentiate striatal hyperdopaminergia in some contexts, with further research required to distinguish molecular mechanisms underlying its variable effects to leverage benefits and avoid potential detrimental effects.

1- Preclinical Neuropsychiatry Laboratory, Neuroscience Research Australia, Randwick, NSW, 2031, Australia

2- Discipline of Psychiatry and Mental Health, Faculty of Medicine, University of New South Wales (UNSW), Sydney NSW 2052, Australia,

3- School of Biomedical Sciences, UNSW, Sydney NSW 2052, Australia

4- Schizophrenia Research Laboratory, Neuroscience Research Australia, Randwick, NSW 2031, Australia,

5- Department of Neuroscience & Physiology, Upstate Medical University, Syracuse, NY 13210, USA.

E Kinstler 1, A Aggarwal 1, SE Paul 1, A J Gorelik 1, 23andMe Research Team 2, EC Johnson 3, A Agrawal 3, R Bogdan 1, AP Miller 4

Background. We examined the extent to which polygenic liability to distinct impulsivity domains are associated with substance use initiation in early adolescence in the Adolescent Brain Cognitive Development Study^SM (ABCD Study®). Methods. Data were obtained from participants (n=5,556; ages 9-13) of European ancestry in the ABCD Study from baseline to year 3 follow-ups. Initiation included endorsement of any alcohol, nicotine, cannabis, or other undirected/recreational use of any other substances in non-religious ceremonies. Five domains of impulsivity were assessed using the UPPS-P Impulsive Behavior Scale short form: 1) sensation-seeking, 2) lack of perseverance, 3) lack of premeditation, 4) positive urgency, and 5) negative urgency. UPPS-P polygenic scores (PGSs) for these 5 domains were generated using summary statistics from 23andMe, Inc. (n>132,000) and PRS-CS. Associations between PGSs and substance initiation (0/1) were tested using logistic mixed-effects models, and mediation models evaluated indirect effects via baseline impulsivity. Results. Higher sensation-seeking PGSs predicted any substance use (OR=1.12) and alcohol use initiation (OR=1.13), while higher lack of perseverance PGSs predicted alcohol initiation (OR=1.08). Higher negative urgency PGSs and positive urgency PGSs predicted nicotine use initiation (OR=1.21 and 1.23). Premeditation PGS were not associated with substance use initiation and the initiation of cannabis use was not associated with any UPPS PGS. Reported baseline impulsivity mediated 9-17% of observed PGS associations with substance initiation (β≥0.002). Conclusions. These findings underscore the importance of trait-specific genetic and behavioral pathways to substance use initiation, supporting the role of impulsivity-related traits in early substance use risk.

1. Psychological & Brain Sciences, Washington University in Saint Louis, 2. 23andMe, Inc., Sunnyvale, CA, USA, 3. Department of Psychiatry, Washington University School of Medicine, 4. Department of Psychiatry, Indiana University School of Medicine

Funding Support: Data for this study were provided by the Adolescent Brain Cognitive Development (ABCD) Study℠, which was funded by awards U01DA041022, U01DA041025, U01DA041028, U01DA041048, U01DA041089, U01DA041093, U01DA041106, U01DA041117, U01DA041120, U01DA041134, U01DA041148, U01DA041156, U01DA041174, U24DA041123, and U24DA041147 from the NIH and additional federal partners (https://abcdstudy.org/federal-partners.html). This study was supported by R01 DA054750. Authors received funding support from NIH: SEP was supported by F31AA029934. AJG was supported by NSF DGE-213989. ECJ was supported by K01DA051759. ASH was supported by K01AA030083. DAAB was supported by K99AA030808. R01DA054750 (RB & AA), K01AA031724 (APM)

Tariq Brown¹, Emily Petruccelli², John Hernandez³, Reza Azanchi³, Kristin Scaplen⁴, Erica Larschan⁵, Kate O’Connor-Giles² and Karla R. Kaun²

Tight regulation of transcription and RNA processing is required for appropriate cellular homeostasis, neurogenesis, and synaptic plasticity. This regulation can be dynamically and persistently perturbed by addiction-associated substances. Alcohol use has been associated with disrupted alternative splicing in humans, non-human primates, rats, mice, chickens, and flies, suggesting a fundamental mechanism through which alcohol affects the molecular processing and function of cells. However, the functional consequences of transcript switching, and how it can affect subsequent substance use are almost entirely unknown. We found that lasting memory for an odor associated with the rewarding properties of alcohol causes differential transcript usage in memory-encoding neurons. These switches included alternative start sites, exon skipping, and alternative use of splice donor/acceptor sites, and occurred in a variety of different genes including the Dopamine-2-like Receptor (Dop2R) and the Drosophila signal transducer and activator of transcription (Stat92E) genes. We hypothesized that alcohol-induced transcript switching causes functional changes to the plasticity of memory circuits and behavioral response to alcohol and other psychoactive drugs like nicotine and methamphetamine. Using a combination of functional in vivo imaging and high resolution behavior assays to test the rewarding properties of volatile substances, we demonstrate that alternative splicing contributes to memory circuit plasticity and behavioral decision-making. The conservation of drug-induced alternative splicing from flies to humans suggests it is likely a molecular mechanism through which addictive substances affect memory and consequent behavioral responses.

¹Neuroscience Graduate Program, Brown University; ²Department of Biological Sciences, Southern Illinois University; ³Department of Neuroscience, Brown University; ⁴Department of Psychology, Bryant University; ⁵Department of Molecular Biology, Cell Biology and Biochemistry, Brown University

K Czarnecki 1, K Krick 1, EA Heller 1

Alternative splicing differentiates identity and function of neuronal subtypes and is activity dependent. Defects in neuronal splicing are associated with neurological and psychiatric disorders. The hPTM, H3K36me3, is implicated in splicing and interacts with a variety of splicing-related proteins. The H3K36me3 writer, SETD2, directly interacts with splicing factors. We find that H3K36me3 is causally linked to alternative splicing in the context of cocaine reward behavior in mice. However, we still lack a mechanistic understanding of the interplay between hPTMs and alternative splicing. This project aims to define the regulation of alternative splicing by SETD2 and/or H3K36me3 by decoupling these putative splicing regulators. To accomplish this, we acutely depleted either H3K36me3 and SETD2 and then measured global splicing changes using RNA-sequencing in ex-vivo mouse neurons. We achieved depletion of H3K36me3 using a newly SETD2 inhibitor, EZM0414, and of SETD2 through a dTAG degron at the endogenous locus. In addition, we apply a CRISPR epigenetic editing tool to specifically deposit H3K36me3 and assess the sufficiency of H3K36me3 to drive splicing at target exons. This project will be the first to determine the relative contributions of SETD2 and H3K36me3 to alternative splicing to uncover how these factors might regulate diverse neuronal functions.

1 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania USA

Funding Support: NIH/NIDA R01-DA052465

Luana Carvalho¹, Jonathas Almeida¹ and Amy W. Lasek¹

¹Department of Pharmacology and Toxicology, Virginia Commonwealth University

Alternative splicing is highly prevalent in the brain and tightly regulated by RNA-binding proteins (RBPs). Disruptions in this regulation have been implicated in psychiatric disorders, including alcohol use disorder (AUD). Poly(rC) binding protein 1 (PCBP1) is a multifunctional RBP that regulates exon retention and exclusion, influencing RNA splicing and protein function. Using the Lieber-DeCarli liquid diet model, we previously found that Pcbp1 expression is upregulated in the male rat hippocampus during alcohol withdrawal (AW), a period characterized by anxiety- and depressive-like behaviors. We demonstrated that PCBP1 modulates alternative splicing of Hapln2, a gene encoding an extracellular matrix (ECM) protein critical for nerve conduction velocity. PCBP1-driven splicing of Hapln2 leads to either nonsense-mediated decay or the production of a non-functional protein, potentially impairing neurotransmission and contributing to withdrawal-induced emotional and cognitive dysfunction. Beyond Hapln2, we hypothesized that PCBP1 regulates a broader network of genes involved in withdrawal-related behavioral deficits. To investigate this, we performed RNA Immunoprecipitation Sequencing (RIP-Seq) on hippocampal tissue from 12 males (6 control, 6 withdrawal) and 12 females (6 control, 6 withdrawal). Both IP and input samples were sequenced, and PCBP1-bound peaks were identified using MACS3. Differential enrichment analysis was performed with edgeR (FDR < 0.1). In males, we identified 73 highly enriched and 474 lowly enriched PCBP1 peaks, corresponding to 70 and 368 unique genes, respectively, compared to controls. These findings suggest a reduction in PCBP1 interaction with its RNA targets during AW. In contrast, no significant differences in PCBP1-enriched peaks were observed in females during withdrawal compared to female controls, suggesting potential sex-specific regulatory mechanisms. Notably, genes with high PCBP1 enrichment in males are involved in glycosaminoglycan metabolism, which plays a key role in ECM composition and cellular signaling. In contrast, genes with low PCBP1 enrichment are associated with synaptogenesis, glutamatergic receptor function, and myelination signaling, highlighting potential disruptions in neuronal connectivity and oligodendrocyte function during AW.

These findings provide new insights into PCBP1-mediated splicing regulation during alcohol withdrawal, linking altered RNA processing to ECM remodeling, synaptic plasticity, and myelination, while also revealing potential sex differences in PCBP1-RNA interactions. Understanding these mechanisms may reveal novel therapeutic targets for AUD-related neuroadaptations and withdrawal-induced behavioral deficits.

Rudong Li ^1,2, Jill L. Reiter ^1,2, Season K. Wyatt-Johnson ^3,4, Chuanpeng Dong ^1,2, Caine S. Smith ^5,6, Sheketha R. Hauser ^4,7, Manav Kapoor ^8,9, Julia Stevens ^5,6, R. Dayne Mayfield ^10,11, Alison M. Goate ^8,9, Yue Wang ², Howard J. Edenberg ^2,12, Richard L. Bell ⁷, Greg T. Sutherland ^5,6, Randy R. Brutkiewicz ^3,4_, Yunlong Liu ^1,2

Intron retention (IR) can be either a type of regulated alternative RNA splicing event or a consequence of dysregulated RNA splicing machinery. IR has been studied in neurological disease and cancer, particularly in the context of antiviral immune response, immune checkpoints, and spliceosome-targeted therapies. However, its involvement in complex neuropsychiatric disorders, such as alcohol use disorder (AUD), remains largely unexplored.

Here, we systematically identified IR events in post-mortem brain tissue of individuals with and without AUD. We analyzed the transcriptome of 320 samples from the superior frontal cortex, nucleus accumbens, central nucleus, and basolateral amygdala of 142 independent subjects (66 AUD and 76 controls). We observed an increase of total IR in the AUD samples, after adjusting for age, sex, sequencing cohort, brain regions and individuals. The same observation was confirmed in a well-characterized animal model of alcoholism. Significantly higher levels of total IR were found in the brains of selectively bred alcohol-preferring P rats consuming alcohol compared to water. Additionally, we found more IR events in the brains of individuals with AUD compared to controls. These AUD-associated introns exhibited lower splicing strength at the acceptor site compared to constitutively excised introns, suggesting that splicing of these introns were more susceptible to disease or environmental factors. Expression of the host genes of these IR events was enriched in several neuronal and glial cell types, including Purkinje neurons, visual cortex neurons, and oligodendrocytes. We also found a significantly higher proportion of long introns (>1 kilobase) among these IR events. Because longer introns could potentiate the formation of double-stranded RNA (dsRNA), we performed immunostaining of dsRNA in brain tissue sections from P rats. The dsRNA signal was co-localized with neurons and was significantly higher in multiple brain regions from P rats consuming alcohol compared to water. Moreover, in the human AUD samples, dsRNA-related response pathways were activated and neuronal counts were significantly decreased compared to non-AUD controls.

In conclusion, our findings indicate that chronic alcohol consumption is associated with elevated IR in the brain, particularly in key cell types such as GABAergic regulatory cells, excitatory neurons and oligodendrocytes. Our study provides evidence that IR may lead to the formation of dsRNA, which in turn can trigger immune signaling and apoptosis that contribute to neuroimmune activity and the loss of neurons observed in AUD. Thus, these findings support a potential dsRNA-mediated mechanism for the involvement of IR in AUD.

Rudong Li ^1,2, Jill L. Reiter ^1,2, Season K. Wyatt-Johnson ^3,4, Chuanpeng Dong ^1,2, Caine S. Smith ^5,6, Sheketha R. Hauser ^4,7, Manav Kapoor ^8,9, Julia Stevens ^5,6, R. Dayne Mayfield ^10,11, Alison M. Goate ^8,9, Yue Wang ², Howard J. Edenberg ^2,12, Richard L. Bell ⁷, Greg T. Sutherland ^5,6, Randy R. Brutkiewicz ^3,4_, Yunlong Liu ^1,2

¹ Center for Computational Biology and Bioinformatics (CCBB), Indiana University (IU) School of Medicine, IN, USA

² Department of Medical and Molecular Genetics (MMGE), IU School of Medicine, IN, USA

³ Department of Microbiology and Immunology, IU School of Medicine, IN, USA

⁴ Stark Neurosciences Research Institute, IU School of Medicine, IN, USA

⁵ New South Wales Brain Tissue Research Centre, University of Sydney, NSW, Australia

⁶ Charles Perkins Centre and School of Medical Sciences, University of Sydney, NSW, Australia

⁷ Department of Psychiatry, IU School of Medicine, IN, USA

⁸ Ronald M. Loeb Center for Alzheimer’s Disease, Department of Neuroscience, Icahn School of Medicine at Mount Sinai, NY, USA

⁹ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, NY, USA

¹⁰ Waggoner Center for Alcohol and Addiction Research (WCAAR), University of Texas at Austin, TX, USA

¹¹ Department of Neuroscience, University of Texas at Austin, TX, USA

¹² Department of Biochemistry and Molecular Biology, IU School of Medicine, IN

Hayk Davtyan^1,2, Jean Paul Chadarevian^1,2, Jonathan Hasselmann^1,2, Ghazaleh Eskandari-Sedighi¹, Sherry Lin-Koch⁴, Alina L Chadarevian^2,3, Joia Kai Capocchi¹, Duc Duong⁵, Jasmine Nguyen¹, Christina Tu^1,2, Sepideh Kiani Shabestari³, Fang Wu⁵, Anantharaman Shantaraman⁵, Kimiya Mansour², William Carlen-Jones¹, Michael Mgerian³, Katia Deynega¹, Tau En Lim¹, Alan Khoi Mai¹, Lauren Le¹, Ani Agababian³, David A Hume⁶, Clare Pridans⁷, Elizabeth Head¹, Sandeep Robert Datta⁴, Nicholas T Seyfried⁵, Mathew Blurton-Jones^1,2,3

Hematopoietic stem cell transplantation (HSCT) in combination with microglia depletion is increasingly being examined as a potential therapy for neurological disorders. The premise of this approach is that HSCT-derived monocytes (MN) may infiltrate the brain and differentiate into “microglia-like” cells. However, induced pluripotent stem cells derived microglia (iMG) provides a potential alternative source of therapeutic cells. As many questions remain regarding the similarities and differences between microglia and monocytes, we utilized a xenotransplantation-compatible model that lacks endogenous microglia (hFIRE mice) to examine the transcriptional and functional properties of human iMG and MN. iMG and MN from four patients were transplanted into adult hFIRE brains and four months later behavioral testing was performed. Brains were then examined via spatial RNA sequencing, proteomics, histological, and biochemical approaches. Despite four months of brain residence and near complete chimerism, human iMG and MN continued to exhibit many important differences. In particular, we found that MN, but not iMG, induced a chronic proinflammatory state associated with significant levels of astrogliosis, demyelination, synaptic loss, and behavioral impairment. Taken together, these results demonstrate the critical role of ontogeny on myeloid cell function within the brain and provide important implications for the development of CNS-wide microglial replacement therapies.

¹Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA

²Stem Cell Research Center, University of California, Irvine, CA, USA

³Department of Neurobiology & Behavior, University of California, Irvine, CA, USA

⁴Harvard Medical School, Boston, MA, USA

⁵Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

⁶Mater Research Institute, University of Queensland, Brisbane, Australia

⁷Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

Charles Hoeffer^1,2, Andrew M. Lombardi¹, Mina Griffioen^1,2, Helen Wong², Ryan Milstead^1,2,3, Curtis Borski², Erin Shiely^1,2, Myra E. Bower ^1,2, Emily Schmitt¹, Lauren LaPlante², Marissa A. Ehringer^1,2, Jerry Stitzel^1,2.

Improved understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate detrimental nicotine withdrawal effects, and improve cessation rates. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4β2 and α7. Protein kinase B-β (Pkb-β or Akt2) expression is restricted to astrocytes in mice and humans and is activated by nicotine. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice. For in/ex vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 μg/mL) or following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed enhanced acute responses and reduced area following chronic exposure. In culture, we found that 100μM nicotine was sufficient for morphological changes and blocking α7 or α4β2 nAChRs prevented observed morphologic changes. We performed conditioned place preference (CPP) in Akt2 cKO mice which revealed reduced nicotine preference in cKO mice compared to controls. Finally, we performed RNASeq experiments comparing nicotine- and LPS-mediated gene expression identifying robust differences between these two astrocytic stimuli. These findings show the importance of nAChRs and AKT2 signaling in the astrocytic response to nicotine.

¹Department of Integrative Physiology, University of Colorado at Boulder; ²Institute for Behavioral Genetics, University of Colorado at Boulder. Boulder, CO, 80303 USA.

Bhim B. Biswa 1,2, Bharathi Venkatachalam 1,2, Hiroshi Mori 2,3, Atsushi Toyoda 4, Ken Kurokawa 2,5, Tsuyoshi Koide 1,2

Domestication, a longstanding process of selective breeding for human benefits, fundamentally alters the behaviour of animals, with tameness being a key trait. In order to study factors associated with domestication, we have applied selective breeding for higher motivation to approach humans (active tameness) using genetically diverse wild-derived heterogeneous stock (WHS) mice from eight wild strains. As a result, the selected groups exhibit higher active tameness than the non-selected groups. Furthermore, the selected groups exhibit higher sociability as well as higher levels of blood oxytocin, a hormone associate with social behavior, than the non-selected groups. We examined gut microbiota in mice selectively bred for increased human interaction (active tameness) compared to control groups. We analysed faecal samples from 80 mice, comprising 40 from the selected groups and 40 controls, through shotgun metagenomic analysis. Leveraging our shotgun metagenomic data, we compiled a collection of 374 high-quality metagenome-assembled genomes (MAGs) of bacteria across 11 phyla. The results indicate that selection for tameness does not significantly alter the taxonomic and functional diversity of the gut microbiota, but there is a notable increase in the abundance of Limosilactobacillus reuteri in the selected groups. We isolated multiple lines of L. reuteri from the faeces obtained from the selected mice. When one of these lines was administered to control mice via drinking water, an increase in tameness behaviour was observed with higher blood oxytocin levels. Our findings suggest that gut microbiota, particularly species like L. reuteri, can influence active tameness or social behavior in mice.

1 Mouse Genomics Resource Laboratory, National Institute of Genetics (NIG), Japan. 2 Graduate Institute for Advanced Studies (SOKENDAI), Japan. 3 Genome Diversity Laboratory, NIG, Japan. 4 Comparative Genomics Laboratory, NIG, Japan. 5 Genome Evolution Laboratory, NIG, Japan

SM Heyworth1, F Cortesi1,2, M Lührmann1,2, R Carew1, KL Cheney1

Seahorses hunt using pivot feeding, a highly effective strategy relying on advanced visual capabilities to identify planktonic prey against the background, maintain focus despite movement, and crucially, perceive depth allowing accurate strike. However, the retinal structure of the seahorse visual system remains largely unstudied. Here, we present our findings of how the seahorse retina supports targeted prey capture.

In all five species studied, we identified multiple retinal regions characterised by differences in morphology and gene expression. Histological analysis and MRI scans revealed the presence of multiple foveae, while slicing and wholemount techniques confirmed changes in cell density throughout the retina, extending beyond the foveal regions. Transcriptome sequencing revealed the expression of three opsin genes in the retina: sws2 (blue), rh2a (green) and lws (red). Notably, fluorescent in situ hybridisation showed distinct differences in opsin gene expression and co-expression within the same cone photoreceptor cells across the dorsal-ventral and nasal-temporal axes, including expression of only one opsin per cone within the fovea and a ventronasal absence of lws. These differences demonstrate support for dichromatic, trichromatic, and potentially tetrachromatic retinal regions. To further investigate the role of retinal regionalisation in prey strike, we conducted feeding trials on Hippocampus whitei to determine the effect on foraging when ambient light matched the sensitivity of the foveal single cones. Our study reveals that seahorses have one of the most complex fish visual systems to date, highlighting the benefit of multi-technique, multi-species comparisons in sensory ecology.

1School of the Environment, The University of Queensland, 2The Queensland Brain Institute, The University of Queensland

Funding Support: Australian Research Council Future Fellowship (FT190100313; awarded to KLC); Holsworth Wildlife Research Endowment, The Ecological Society of Australia (awarded to SMH).

Jingjing Yan, Kelsey Mainard, Fumihiro Ito, Yangyuan Li, Andrew Nguyen, Aabha Vora, Lijuan Feng and Wanhe Li

The molecular clock drives the circadian rhythm and its behavioral and physiological outputs through tightly regulated gene expression. Histone post-translational modifications (PTMs) alter chromatin structure and recruitments of transcriptional factors, thereby epigenetically regulating gene expression. Many types of PTMs, including acetylation and methylation, exhibit rhythms at promoters, enhancers, and gene bodies, hence modifying the accessibility of genes to transcriptional machinery. Histone monoaminylation is a recently recognized kind of PTM, where neurotransmitters, such as excessive dopamine or serotonin resulting from pathological conditions, are covalently bonded to the histone tail. This process regulates neuronal transcription. Most recently, a previously unreported histone monoaminylation called histaminylation was identified. Characterized as a PTM, histaminylation cycles in mice’s hypothalamic tuberomammillary nucleus (TMN) throughout their sleep/wake cycle. The functions of these emerging types of PTMs in epigenetic regulation in the nervous system, both in healthy and diseased brain states, are being uncovered, making them highly intriguing.

In this study, we built a neurogenetics toolbox to study histone modifications using the model organism Drosophila melanogaster. We found that perturbing histone monoaminylation resulted in a unique sleep phenotype. This phenotype reflects a defect in sleep maintenance during the nighttime but not sleep initiation, because sleep deprivation can induce normal sleep rebound. Subsequently, we employed a comprehensive set of molecular, genetics, and genomics approaches to further explore histone monoaminylation-dependent sleep regulation. We identified the neural circuit in which the dynamics of gene expression are regulated through epigenetic mechanisms. Since the monoamine biochemistry and histone proteins are remarkably conserved between humans and flies, the discovery of histone monoaminylation-dependent sleep regulation may reveal a conserved sleep regulatory mechanism in an epigenetic setting.

Center for Biological Clocks Research, Department of Biology, Texas A&M University

AM Barkley-Levenson1 , ALS Borges1,2, R Sultana1 , D-J Paredes1 , V Cordova3

Recent genome wide association studies (GWAS) have identified numerous novel hits for problematic alcohol use and alcohol consumption. However, follow-up studies are still needed to demonstrate a causal relationship between implicated genes and alcohol-related traits. Here, we describe the functional validation of a novel genetic association for problematic alcohol use and alcohol drinking (Fut2). In humans, common nonfunctional FUT2 variants are associated with increased risk of inflammatory bowel disease and may worsen alcohol-related liver disease, but these variants have not been investigated in relation to alcohol intake. We found that male Fut2 homozygous knockout mice have greater binge-like ethanol consumption in a drinking in the dark (DID) test than wild type littermates, and knockout mice of both sexes show greater ethanol conditioned place preference. To begin to investigate a potential mechanism underlying the role of Fut2 in binge-like drinking, we also examined gut microbiota composition before and after three cycles of ethanol or water DID in the homozygous knockout and wild type mice. We found reduced alpha diversity in the Fut2 knockout mice compared to wild type animals in both drinking groups. Additionally, the ethanol drinking knockout mice had increased relative abundance of Prevotella compared to wild types, which has been associated with increased inflammatory responses. Taken together, these findings provide evidence for a causal role of Fut2 in ethanol consumption and reward sensitivity. The identification of a gene x alcohol interaction effect on gut microbiota composition suggests a possible mechanism by which Fut2 genotype can impact alcohol-related traits. Funding: NIH-NIAAA grant R00AA027835, NIH-NIGMS grant P20GM103451, University of New Mexico College of Pharmacy Pilot Project Research Award, 1Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, NM, USA. 2Biomedical Sciences Graduate Program, University of New Mexico Health Sciences Center, Albuquerque, NM, USA. 3New Mexico IMBRE Student Experience, New Mexico State University, Las Cruses, NM, USA.

Hernandez, John S¹., Moges, S¹., Le, Nelson²., Azanchi, Reza.¹, and Kaun, Karla R¹.

Animals are strongly motivated to seek high-quality food while avoiding low-quality options and are adept at adjusting their foraging strategies to maximize food rewards. Drosophila melanogaster is no exception; they actively seek out food-associated odors and tastes while avoiding aversive ones. Despite its importance for understanding the mechanisms of motivation, how experience shapes the way flies engage with odors and tastes remains poorly understood. Here, we describe how smell and taste shape operant engagement and strategies in Drosophila, by analyzing the nuanced behaviors that emerge when flies self-administer odors and activate taste receptors. We demonstrate that the valence of odor and taste generated different behavioral repertoires, and flies increased engagement with appetitive outcomes and reduced engagement with aversive outcomes. We inquired whether behaviors that emerged were a consequence of engagement with odor or taste, and noted that behaviors were odor-driven, but shaped by taste such that combined odor and taste caused a restriction of operant strategies compared to odor alone. Our approach provides the behavioral framework to examine which neural circuits uniquely contribute to operant strategies for pursuing positive outcomes and avoiding negative outcomes.

1 - Brown University, RI. Department of Neuroscience

2 - Post-baccalaureate Research Education Program, Brown University, Providence, RI

Funding support was provided by the NIAAA R01AA024434 (supporting N.J.M, R.A., K.R.K.), NIAAA F32AA29595 (supporting J.S.H.) and NIGMS R25GM125500 (supporting N.L.).

David N. Linsenbardt, Rebecca M. Sena, Nora Perrone-Bizzozero

The nucleus accumbens (Acb) is a key brain area for processing the rewarding properties of alcohol, and as such has become a target for interventions aimed at decreasing excessive alcohol consumption. However, we still know relatively little about the molecular genetic mechanism by which excessive alcohol consumption alters the function of the Acb. To address this gap in knowledge, mice were given daily access to water or alcohol for several weeks using drinking-in-the-dark (DID) procedures, and Acb brain tissue was collected, sequenced, and evaluated for alternative splicing events using Multivariate Analysis of Transcript Splicing (rMATs-turbo) using a cut-off of FDR q<0.05. Repeated binge alcohol drinking using 6-10 mice per group led to significant splicing differences in 112 transcripts. There were 73 exon skipping events (ES), 14 mutually exclusive exon events (MXE), 13 alternative 5' splice sites (A5SS), 11 alternative 3' splice sites (A3SS), and a single intron retention event (IR). Among these alterations, we identified several that occurred in key genes known to regulate neural function. Key ES events were detected in Kif21a, Caprin2 and other genes involved in the establishment of cell polarity, Rho protein signal transduction, endocytosis and dendritic spine morphology. MXE events were in the vesicle uptake proteins (AtpV6v1d and Atp6v0b) and proteins regulating neurotransmission (e.g., Stau2, Vapa, Pvr, and Kidins220). A3SS events included the A-kinase anchor Akap8 and the membrane transport protein Hook2, while A5SS events included Camk1d, Stat1 and the voltagegated potassium channel Kcnq2. The only significant IR event was observed in Hdac10. Collectively these findings identify alcohol-induced splicing alterations in the Acb that may be involved in functional alterations associated with repeated binge alcohol exposure. Acknowledgments:

Dept. of Neurosciences, University of New Mexico, School of Medicine and Health Sciences Center, Albuquerque, NM 87131

This work was supported in part by grant #s: AA025120, AA015614, AA014127, and the New Mexico Alcohol Research Center P50-AA022534.

J. SINGH¹, A.H.M. WONG², J. PRESSEY^3, K. SHAHABI⁴, M. A. WOODIN³, C. DOCKSTADER⁴;

Undergraduate research allows faculty to teach, conduct research, and serve, while building a community of student scholars ready to lead future research. Although undergraduate thesis projects offer an early research experience, most students don't get this opportunity.

Over the past five years, the Human Biology Program at the University of Toronto has run a free, two-week 'Lab Bootcamp' for over 250 life science undergraduates. The program bridges pedagogical theory with research practice, fostering technical skills and community building. Bootcamp simulates an 80-hour independent neuroscience research project. Small groups design and execute the molecular cloning and insertion of the KCC2 protein, crucial for synaptic activity regulation. They develop strategies to upregulate KCC2 in a mouse model of Huntington’s Disease, considering sensitivity, time, and limitations. By the end, students have designed and executed a research problem, and developed technical, communication, and collaborative skills.

Across 250+ students, qualitative analyses indicate substantial increases in students' technical and critical-thinking skills, as well as their communicative and collaborative abilities. Increased confidence in conducting research was rated highest by students, followed by the importance of community and collaboration. A four-month follow-up revealed that many participants continue in research through work-study positions, research assistant roles, and independent projects.

HMB Lab Bootcamp offers an accessible, transformative opportunity in neuroscience research enabling skill acquisition, and fostering confidence and community-building in an inquiry-based, experiential setting.

¹ Department of Immunology, University of Toronto, Toronto, Canada; ² Hong Kong Baptist University, Kowloon Tong, Hong Kong; ³Department of Cell & Systems Biology, University of Toronto, Toronto, Canada; ³Human Biology Program, University of Toronto, Toronto, ON, Canada

Britahny M. Baskin^1,2, Grace L. Whitney¹, Emma J. Sandago¹, Kelly K. Wingfield¹, Caroline Topping¹, Camily Hidalgo-Goncalves^1,3, Nina Garbarino^1,4, Mengyuan Liu^1,5, Camron D. Bryant^1,2

Accompanying the opioid epidemic is an increase in neonatal opioid withdrawal syndrome (NOWS), defined by a set of withdrawal symptoms in infants born to mothers who are dependent on opioids, including weight loss, agitation, excessive crying, and hyperalgesia. Despite having a heritable component, the genetic basis of NOWS remains largely unknown. We used a third trimester-approximate model for NOWS in mice comprising twice daily morphine (10 mg/kg, s.c.) from P1-P14 and assessment of opioid withdrawal signs on P7 and P14 (hypothermia, weight loss, locomotor agitation, ultrasonic vocalizations (USVs), and hyperalgesia and developmental milestones (self-righting, negative geotaxis, forlimb grasp, eye opening, pinnae attachment, fur cover, pivot extinction, cliff aversion, and vertical screen test). Ongoing studies implicate strain differences depending on morphine treatment, including blunted developmental weight gain and hypothermia in DBA/2J. There is also a trending Strain x Treatment interaction in thermal hyperalgesia on in the tail withdrawal on P7 (p=0.07) and a Strain x Sex interaction on P14 in the tail withdrawal (p=0.02). For USVs, spectrotemporal analysis of syllable types using supervised and unsupervised machine learning is ongoing. There is a trending Strain x Treatment x Sex interaction on P7 for total USVs (p=0.055) and a significant interaction for percentage of emitted “downward” syllables (p<0.01). Regarding milestones, we observed strain effects on self-righting and pivot extinction and treatment effects on pivot extinction, negative geotaxis, cliff aversion, and forelimb grasp. The Strain x Treatment interactions across NOWS model traits indicate that the BXD-RI panel will be effective for quantitative genetic analysis.

1. Laboratory of Addiction Genetics, Center for Drug Discovery, Department of Pharmaceutical Sciences, Northeastern University
2. NIH/NIDA T32 Training Program on Development and Medications for Substance Use Disorder
3. Williams College Alumni Sponsored Internship Program (ASIP)
4. Peak Awardee, Northeastern University
5. Master’s Program in Biomedical Sciences, Department of Pharmaceutical Sciences, Northeastern University

Sonali N. Reisinger^a, Nicholas van de Garde^a,b, Asim Muhammad^a,b, Pranav Adithya^a,b, Riki Dingwall^a,b, Carolina Gubert^a, Gabriel Dabscheck^d,e, Jonathan M. Payne^d,e,f, Anthony J. Hannan^a,b,g

Neurofibromatosis type I (NF1) is caused by a mutation in the neurofibromin 1 gene. Patients exhibit diverse symptoms, including tumour formation, hyperpigmentation, and vision disorders. Most also experience learning difficulties and psychiatric conditions like ADHD, autism, anxiety, or depression, which standard therapies do not address.
Here we employed a mouse model of NF1, the heterozygous Nf1 +/- mouse line. The initial approach involved the phenotypic characterisation of this mouse model using a broad battery of behavioural tests to examine specific aspects of cognition (including attention, working memory, short-term memory, spatial memory, associative memory, cognitive flexibility), social function, and psychiatric traits (including anxiety and depression-like behaviours). Male and female Nf1 +/- mice showed globally reduced cognitive performance and an increase in traits relevant to autism, but no changes in emotionality or locomotion, according to integrative behavioural analyses. Brain weights of Nf1 +/- mice were significantly higher compared to controls, and several behavioural measures correlated with brain weights, providing supportive evidence for a mechanistic link. In light of human evidence showing higher brain volumes in NF1 patients, this should be urgently investigated.
Touchscreen testing was then employed to evaluate sustained attention using the rodent continuous performance test (rCPT). Initial evaluation of performance in this task revealed specific attentional deficits in Nf1 +/- mice. This was not confounded by differential reward motivation, as +/- performed similarly to controls in the touchscreen progressive ratio task. Future experiments will use touchscreen testing to evaluate promising drug candidates in a preclinical study using Nf1 +/- mice.

a Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia

b Florey Department of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia

c Peter Doherty Institute of Infection and Immunity, University of Melbourne, Parkville, VIC, Australia

d Royal Children's Hospital Melbourne, Parkville, VIC, Australia

e Murdoch Children’s Research Institute, Parkville, VIC, Australia

f Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia

g Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC, Australia

Funding support: Flicker of Hope Foundation

Paul J. Meyer¹, Christopher P. King^1,2, Thiago M. Sanches³, Apurva S. Chitre³, Nana K. Amissah¹, Karissa T. Reyes¹, Connor Martin², Oksana Polesskaya³, Keita Ishiwari², Hao Chen⁵, Leah C. Solberg Woods⁶, Craig C. Colder¹, David M. Dietz⁴ Abraham A. Palmer^3,7

Individual differences in behavioral regulation, including sensation seeking, attentional control, impulsivity, and cue reactivity, are linked to psychological disorders including substance use disorder (SUD). To examine the phenotypic and genetic relationships in animal models of these traits, we analyzed behavioral and genomic data from a large sample of heterogeneous stock (HS) rats (n ≈ 1600). Seven behavioral tasks assessed key measures, including locomotor response to novelty, social and light reinforcement, reaction time, delay discounting, Pavlovian cue-reactivity, and cocaine-conditioned cue preference. Significant individual variation and sex differences were seen, with females generally displaying higher locomotion, social and light reinforcement, reaction time, cue-reactivity, and cocaine cue preference, while males exhibited greater delay discounting and goal-directed approach. Factor analysis showed most measures clustered within individual tasks, though cocaine cue preference was linked to impulsivity-related traits, including delay discounting and false alarms in the reaction time task. Genome-wide association (GWA) analysis revealed heritabilities ranging from (0.12–0.30) for these traits, with several traits being genetically correlated to drug self-administration traits measured in other HS cohorts. Despite power limitations, preliminary sex-specific GWA for cue-reactivity revealed loci not identified in the pooled analysis. GWA using principal components derived from this test’s measures did not identify additional loci. Across all tasks, several candidate genes were identified; examples include Tenm4 on chromosome 1 (cue-reactivity), Abcfl on chromosome 20 (reaction time) and Otx2 on chromosome 15 (delay discounting). Many loci overlap with human GWA studies for SUD, underscoring the translational relevance of these results.

¹Department of Psychology, University at Buffalo, Buffalo, USA.

²Clinical and Research Institute on Addictions, Buffalo, USA.

³Department of Psychiatry, University of California San Diego, La Jolla, USA.

⁴Department of Pharmacology and Toxicology, University at Buffalo, Buffalo USA.

⁵Department of Pharmacology, Addiction Science and Toxicology, University of Tennessee Health Science Center, Memphis, USA.

⁶Department of Internal Medicine, Molecular Medicine, Center on Diabetes, Obesity and Metabolism, Wake Forest School of Medicine, Winston-Salem, USA.

⁷Institute for Genomic Medicine, University of California San Diego, La Jolla, USA.

Funding Support: DA037844, DA060810

André Lucas Silva Borges1,2 , Donna-Jael G Paredes1,3, Rebeka Sultana1,3, Amanda M Barkley-Levenson1,3

Anxiety and depression are some of the most prevalent psychiatric disorders, affecting approximately 4 and 5% of the worldwide population, respectively. Low efficacy of existing treatments for depression has directed research efforts toward the investigation of novel therapies such as micronutrients. Zinc is one such micronutrient, and zinc dysregulation has been repeatedly linked to the pathophysiology of depression. Interestingly, genome-wide association studies (GWAS) have found that a zinc transporter gene (Slc39a8) is associated with depression and bipolar disorder, as well as other psychiatric disorders such as alcohol use disorder (AUD) and schizophrenia. Therefore, this study aimed to assess how Slc39a8 genotype contributes to different anxiety and depression-like phenotypes in mice that are relevant to these psychiatric disorders. We tested naive Slc39a8 heterozygous knockout mice (HET) and their wild-type littermates (WT) of both sexes on an open field test (OFT) and forced-swim test (FST) to investigate anxiety and depression-like behaviors, respectively. Our results showed no differences between the genotypes in either test. Considering the correlation of Slc39a8 with AUD, we tested a separate cohort of mice on the OFT and FST following 3 weeks of intermittent access to ethanol to determine whether there were genotypic differences in these behaviors during withdrawal. Again, we saw no significant differences between the genotypes on either test. These results suggest that heterozygous Slc39a8 knockout does not alter baseline depression- and anxiety-like behavior or withdrawalassociated negative affective changes. Further investigation is necessary to unravel the mechanisms underlying the association between Slc39a8 and depression and anxiety phenotypes.

1 Department of Pharmaceutical Sciences, 2 School of Medicine, 3 University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA Funding Support: NIH-NIAAA grant #R00 AA027835

Minjeong Shin, Isabella Hajdu, Lillian Wright, Olga Akay, Peter S. Johnstone, Deniz Top

Circadian rhythms are behavioural and physiological responses to rhythmic environmental changes, such as photoperiod and temperature. Such behavioural rhythms are regulated by a transcription/translation negative feedback loop called the circadian clock, which regulates the daily expression of several hundred genes. In Drosophila, the circadian clocks are found in ~240 neurons that are arranged into distinct clusters across the brain, suggesting a need for communication across the clusters to maintain coherent behavioural rhythms. Neurotransmitters connect of these clusters, often acting through G-protein coupled receptors (GPCRs), forming a neuronal network. Among the functions of GPCRs is regulation of cytosolic cAMP levels that serve as a secondary signal to relay extracellular instructions to internal cellular machinery. PKA is a kinase that responds to cAMP and activates a wide variety of proteins through phosphorylation. We have found that PKA phosphorylates CLK protein at a single residue to repress function of the transcriptional activator complex of the circadian clock. This site is conserved across various species of insects, fish and mammals. When PKA is knocked down in distinct circadian neuronal clusters, both the local circadian clocks and overall behavioural rhythms change differently. Thus, PKA is a signaling molecule that responds to various instructions in distinct circadian neurons to regulate the circadian clock, maintaining both circadian clock synchrony and ensuring coherent behavioural rhythms.

Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.

Funding Support: CIHR (Canada) PJT 178220

Olivia O.F. Williams1, Joshua D. Manduca1, and Melissa L. Perreault1

Autism Spectrum Disorders (ASD) exhibit sex differences in age of onset, prevalence, etiology, and presentation, however the molecular underpinnings have yet to be understood. Using one of the most widely used models to study attributes of ASD, the valproic acid (VPA) rat model, sex differences in cortical gene expression at postnatal days 0 and 35 were assessed. Sex-specific differences in neuronal oscillatory activity and behaviour were also evaluated. At birth, the sex-specific regulation of genes involved in growth factor signaling (egfr, fgf15, and hgf), neuropeptides (tac1, cartpt, pdyn, and penk) and receptors (drd1a, drd2, adora2a, mc4r, and htr3a) were observed. In adolescence, genes involved in vascular permeability (gpr116, cldn5, flt1, rgs5, angptl4, esam, and decorin) were downregulated in female VPA rats whereas in males, genes involved in neurotransmitter or neuropeptide signalling (drd2, cartpt, adora2a, and pdyn) were upregulated. Female VPA rats displayed greater anxiety, reduced recognition memory, and socialization. Male VPA rats exhibited difficulties in location memory and sociality. Sex- and frequency-specific changes in cortical spectral power were observed, and male VPA animals selectively displayed lower cortical-hippocampal communication. These findings identify key sex differences in gene expression, oscillatory function, and behaviour in rats with VPA exposure that may have relevance to the sex-specific symptoms observed in some types of ASD.

1University of Guelph, Department of Biomedical Sciences, Guelph, Ontario, Canada

M Hernández¹, AM Barkley-Levenson¹

Alcohol use disorder (AUD) is among one of the leading causes of preventable death in the United States. Genome wide association studies (GWAS) have identified specific genes that may influence AUD and related phenotypes. Recent GWAS have found the gene dipeptidyl peptidase like 6 (DPP6) to be associated with problematic alcohol use. To determine whether this gene alters sensitivity to alcohol’s rewarding properties, we conducted an experiment assessing conditioned place preference (CPP) in Dpp6 knockout mice. We tested male heterozygous (HET; n=10) and homozygous (HOM; n=5) knockout mice and wild type littermates (WT; n=10) on a three-week alcohol CPP protocol. A biased conditioning approach was used wherein animals received alcohol paired with the chamber that was least preferred at baseline. Mice received 12 conditioning sessions in total (6 alcohol and 6 saline), with a preference test given after each set of 4 conditioning sessions. We found that HOMs displayed significant CPP, while WTs and HETs did not. On the third CPP test, HOMs had a significantly stronger preference for the alcohol-paired chamber than WTs (p < 0.05). Two days after the final CPP test, alcohol intake was assessed using a 2-hr preference drinking test during the light cycle. No significant genotypic differences were observed in alcohol or water intake, or alcohol preference. Collectively, these data suggest that Dpp6 may alter sensitivity to the rewarding effects of alcohol while not directly altering alcohol intake. Further research is warranted to clarify its contribution to AUD phenotypes in males and females.

¹Department of Pharmaceutical Sciences, University of New Mexico Health Science Center, Albuquerque, NM, Funding Support: NIH-NIAAA grant R00 AA027835

Jaegeon Lee1,2, Seung Ha Kim1,2, Yong-Seok Lee1,2,3, Yu Kyeong Kim3,4, Sang Jeong Kim1,2,3

Bergmann glia (BG) are radial glial cells in the cerebellar cortex that have multiple functions. They have been shown to interact with Purkinje cells in several ways: they modulate synaptic inputs to Purkinje cells, regulate excitability through external potassium concentration, and directly affect Purkinje cells through neurotransmitter release. In this study, we genetically expressed hM3Dq in BG through GFAP-promoter-contained AAV virus. We showed that this BG-PC regulation is mediated by the NMDA receptor and GABA_A receptor. Furthermore, using calcium imaging, we confirmed that BG activation induces MLI activation. Taken together, we concluded that glutamate released from BG induces GABA release from MLI, which in turn inhibits PC activity. Our findings provide a cellular mechanism for understanding the role of BG in the modulation of neuronal activity in the cerebellum.

1Department of Physiology, 2Department of Biomedical Sciences, 3Memory Network Medical Research Center, Neuroscience Research Institute, Wide River Institute of Immunology, Seoul National University College of Medicine, Seoul, Korea, 4 Department of Nuclear Medicine, Seoul National University Boramae Medical Center.

Britahny M. Baskin^1,2, Grace L. Whitney¹, Emma J. Sandago¹, Kelly K. Wingfield¹, Caroline Topping¹, Camily Hidalgo-Goncalves^1,3, Nina Garbarino^1,4, Mengyuan Liu^1,5, Camron D. Bryant^1,2

Accompanying the opioid epidemic is an increase in neonatal opioid withdrawal syndrome (NOWS), defined by a set of withdrawal symptoms in infants born to mothers who are dependent on opioids, including weight loss, agitation, excessive crying, and hyperalgesia. Despite having a heritable component, the genetic basis of NOWS remains largely unknown. We used a third trimester-approximate model for NOWS in mice comprising twice daily morphine (10 mg/kg, s.c.) from P1-P14 and assessment of opioid withdrawal signs on P7 and P14 (hypothermia, weight loss, locomotor agitation, ultrasonic vocalizations (USVs), and hyperalgesia and developmental milestones (self-righting, negative geotaxis, forlimb grasp, eye opening, pinnae attachment, fur cover, pivot extinction, cliff aversion, and vertical screen test). Ongoing studies implicate strain differences depending on morphine treatment, including blunted developmental weight gain and hypothermia in DBA/2J. There is also a trending Strain x Treatment interaction in thermal hyperalgesia on in the tail withdrawal on P7 (p=0.07) and a Strain x Sex interaction on P14 in the tail withdrawal (p=0.02). For USVs, spectrotemporal analysis of syllable types using supervised and unsupervised machine learning is ongoing. There is a trending Strain x Treatment x Sex interaction on P7 for total USVs (p=0.055) and a significant interaction for percentage of emitted “downward” syllables (p<0.01). Regarding milestones, we observed strain effects on self-righting and pivot extinction and treatment effects on pivot extinction, negative geotaxis, cliff aversion, and forelimb grasp. The Strain x Treatment interactions across NOWS model traits indicate that the BXD-RI panel will be effective for quantitative genetic analysis.

1. Laboratory of Addiction Genetics, Center for Drug Discovery, Department of Pharmaceutical Sciences, Northeastern University
2. NIH/NIDA T32 Training Program on Development and Medications for Substance Use Disorder
3. Williams College Alumni Sponsored Internship Program (ASIP)
4. Peak Awardee, Northeastern University
5. Master’s Program in Biomedical Sciences, Department of Pharmaceutical Sciences, Northeastern University

Paul J. Meyer¹, Christopher P. King^1,2, Thiago M. Sanches³, Apurva S. Chitre³, Nana K. Amissah¹, Karissa T. Reyes¹, Connor Martin², Oksana Polesskaya³, Keita Ishiwari², Hao Chen⁵, Leah C. Solberg Woods⁶, Craig C. Colder¹, David M. Dietz⁴ Abraham A. Palmer^3,7

Individual differences in behavioral regulation, including sensation seeking, attentional control, impulsivity, and cue reactivity, are linked to psychological disorders including substance use disorder (SUD). To examine the phenotypic and genetic relationships in animal models of these traits, we analyzed behavioral and genomic data from a large sample of heterogeneous stock (HS) rats (n ≈ 1600). Seven behavioral tasks assessed key measures, including locomotor response to novelty, social and light reinforcement, reaction time, delay discounting, Pavlovian cue-reactivity, and cocaine-conditioned cue preference. Significant individual variation and sex differences were seen, with females generally displaying higher locomotion, social and light reinforcement, reaction time, cue-reactivity, and cocaine cue preference, while males exhibited greater delay discounting and goal-directed approach. Factor analysis showed most measures clustered within individual tasks, though cocaine cue preference was linked to impulsivity-related traits, including delay discounting and false alarms in the reaction time task. Genome-wide association (GWA) analysis revealed heritabilities ranging from (0.12–0.30) for these traits, with several traits being genetically correlated to drug self-administration traits measured in other HS cohorts. Despite power limitations, preliminary sex-specific GWA for cue-reactivity revealed loci not identified in the pooled analysis. GWA using principal components derived from this test’s measures did not identify additional loci. Across all tasks, several candidate genes were identified; examples include Tenm4 on chromosome 1 (cue-reactivity), Abcfl on chromosome 20 (reaction time) and Otx2 on chromosome 15 (delay discounting). Many loci overlap with human GWA studies for SUD, underscoring the translational relevance of these results.

¹Department of Psychology, University at Buffalo, Buffalo, USA.

²Clinical and Research Institute on Addictions, Buffalo, USA.

³Department of Psychiatry, University of California San Diego, La Jolla, USA.

⁴Department of Pharmacology and Toxicology, University at Buffalo, Buffalo USA.

⁵Department of Pharmacology, Addiction Science and Toxicology, University of Tennessee Health Science Center, Memphis, USA.

⁶Department of Internal Medicine, Molecular Medicine, Center on Diabetes, Obesity and Metabolism, Wake Forest School of Medicine, Winston-Salem, USA.

⁷Institute for Genomic Medicine, University of California San Diego, La Jolla, USA.

Funding Support: DA037844, DA060810

Adele Leggieri1 , Judit García-González2 , Saeedeh Hosseinian1 , Peter Ashdown1 , Sofia Anagianni1 , Xian Wang1 , William Havelange1 , Noèlia Fernàndez-Castillo3,4,5,6 , Bru Cormand3,4,5,6 and Caroline H. Brennan1

RBFOX1 regulates transcriptional networks linked to synaptic transmission and neurodevelopment. Mutations in the RBFOX1 gene are associated with psychiatric disorders but how RBFOX1 influences psychiatric disorder vulnerability remains unclear. Recent studies showed that RBFOX1 mediates the alternative splicing of PAC1, a critical HPA axis activator. Further, RBFOX1 dysfunction is linked to dysregulation of BDNF/TrkB, a pathway promoting neuroplasticity, neuronal survival, and stress resilience. Hence, RBFOX1 dysfunction may increase psychiatric disorder vulnerability via HPA axis dysregulation, leading to disrupted development and allostatic overload. To test this hypothesis, we generated a zebrafish rbfox1 loss of function (LoF) line and examined behavioural and molecular effects during development. In larvae and adults, rbfox1 LoF resulted in hyperactivity, impulsivity and hyperarousal, and alterations in proliferation, fertility and survival, traits associated with allostatic overload. In larvae, rbfox1 LoF disrupted expression of pac1a, bdnf, trkb2, and HPI axis genes. These latter were restored after chronic TrkB agonist/antagonist treatment. In adults, bdnf/trkb2 and HPI axes dysregulation was only seen following acute stress. Our findings revealed a strict interplay between RBFOX1 and BDNF/TrkB in stress resilience and suggest that RBFOX1 LoF predisposes to psychiatric diseases through HPA axis hyperactivation during development, impairing adaptation and heightening vulnerability to allostatic overload.

1 School of Biological and Behavioural Sciences, Queen Mary University of London, Mile End Rd, London, E1 4NS, United Kingdom 2 Department of Genetics and Genomic Sciences, Icahn School of Medicine, Mount Sinai, New York City, NY 10029, USA 3 Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Universitat de Barcelona, Barcelona, Catalunya, 08028, Spain 4 Centro de Investigación Biomédica en Red de Enfermedades raras (CIBERER), Spain 5 Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Catalunya, 08028, Spain 6 Institut de recerca Sant Joan de Déu, Espluges de Llobregat, Catalunya, 08950, Spain

Alanna E. Carey, Halley L. Dante, Kevin M. Delgado, Rhea Singh, Jerry L. Chen

Individuals exhibit significant variation in their ability to learn complex tasks. This variation could reflect intrinsic or environmental factors. This project aims to understand the neurobiology of individual heritable differences using rodent models and examine how genetic variation alters gene expression and cellular function that influences individual learning and behavior. Utilizing an automated homecage, goal-directed learning paradigm, we assayed learning in Diversity Outbred (DO) mice (n=244) and groups consisting of the eight founder inbred lines (129S1, NZO, CAST, A/J, WSB, C57, NOD, PWK, n=6 per strain). Learning performance highly varied for DO mice, and differences were observed between inbred lines that grouped strains into three learning variance groups. The low-variance learner strain (NZO) learned the task to expert levels compared to the total inbred founder lines, while low-variance non-learner strains (PWK, NOD) failed to advance through procedural learning. Genetic contributions to learning performance were identified in DO animals through Quantitative Trait Loci analysis (QTL), expression QTL (eQTL), and differential expression of genes (DEG) across the neocortex, hippocampus, and striatum. DEG revealed genes influenced by multiple learning phenotypes, and eQTL analysis aligned with QTL and DEG results. We performed spatial transcriptomics utilizing 10X Xenium, exploring candidate genes’ cellular representation in low-variance founder strains. We found cell-type-specific differences between learner and non-learner strains in oligodendrocytes and cholinergic striatal neurons. These findings offer valuable insights into how genetic variation influences goal-directed learning at the cellular and molecular levels. Moreover, these experiments will lay the foundational work toward understanding heterogeneity in cognitive abilities.

Jacob A Beierle¹, Gautam Sabnis¹, Brian Geuther¹, Elaina Cote¹, Stephanie Puig², and Vivek Kumar¹

Migraine is a chronic neurovascular disorder that affects ~15% of the global population. There is a high need for novel migraine therapeutics, as a large percentage of migraineurs do not respond to existing treatment options. Mouse models provide an opportunity to study the biology of migraine to improve treatment, but migraine intensity is typically quantified through mechanical allodynia, which reduces the complex symptomology of migraine to a single feature and suffers from low face validity. Using ethology to infer migraine intensity is an enticing option but determining behaviors that are associated with migraine and manually scoring them has previously been too laborious to be feasible. In this work I leverage machine vision to empower the quantification of a diverse index of voluntary behaviors in the open field arena and from these features construct a score predicting nitroglycerin evoked migraine in male and female C57BL/6J mice. Using this score, I observe sex differences in the male and female response to nitroglycerin, in agreement with previous work in this model and the human migraine literature. I then use Trpa1 knock out mice to validate the specificity of this model to predict migraine, deconvoluting it from other effects of nitroglycerin administration. In the future the throughput of this approach will provide an efficient way to validate human GWAS candidate genes in single gene knockout mice, to assess the impact of genetic background on migraine intensity, and to screen putative therapeutics.

1. The Jackson Laboratory; 2, UMass Chan Medical School

MS Totten1 , AL Peterson1 , DM Pierce2 and KM Erikson2

High fat diets (HFD) have been linked to gene expression alterations, negative behavior changes, and brain disease. Genes such as alpha-synuclein (SNCA) and amyloid precursor protein (APP) are expressed in the hippocampus and are associated with many behaviors. The aims of this study were to evaluate the impact of a HFD on various behaviors and hippocampal SNCA and APP expression in male and female mice from different strains. We hypothesized that behavior and gene expression would be impacted the most in male mice fed a HFD based on our previous diet studies. Mice from strains C57BL/6J and DBA/2J (n=36 per strain; n=36 per sex) were randomly assigned either a control diet (10% kcal fat) or a HFD (60% kcal fat) for 16 weeks. Behavior was measured using the open field test for anxiety, nestlet shredding for general welfare, and novel object recognition for memory. Male C57BL/6J mice fed a HFD had a 7-fold upregulation of hippocampal SNCA expression (p<0.0001) and a 10-fold upregulation of APP expression (p<0.0001) compared to the control diet group. Furthermore, HFD-fed male C57BL/6J mice showed higher anxiety-like behavior assessed by fecal boli (p=0.021) and 183% less nestlet shredding (p=0.017). We found no significant impact of diet on memory. Overall, the HFD treatment impacted male C57BL/6J mice the most in terms of mRNA expression, anxiety, and general welfare. This study demonstrates important biological sex and genetic factors that should be considered when examining the impact of diet on behavior and the brain.

1 Chemistry, Biochemistry, and Nutrition Program, Salem College, Winston-Salem, NC, USA 2 Department of Nutrition, University of North Carolina Greensboro, Greensboro, NC, USA Funding Support: UNC Greensboro Health and Human Sciences Research Grant and Faculty First Award

Utsav Gyawali, Jacqueline B Mehr, Abnanoub Armanious, Nicholas T Bello, Morgan H James

Background: It has been proposed that binge eating can serve as a form of ‘self-medication’ against dysphoric states in persons of higher weight. Despite this, the neurobiological system(s) underlying this phenomenon remain uncharacterized. Here, using rats, we developed a model of diet-induced obesity model that promotes a depression-like phenotype. We tested the hypothesis that binge eating would restore hedonic deficits associated with higher weight, in part via recruitment of a hypothalamic-midbrain circuit. Methods: Female Long Evans rats were maintained on a regular chow or high fat diet (HFD; 45% fat) for 8w; binge-like eating was then promoted via intermittent access to sweetened fat for 4w. Hedonic tone was measured using intracranial self-stimulation (n=6-7/group). We determined brain levels of orexin, a hypothalamic peptide involved in reward, using immunohistochemistry (n=8-10/group), qPCR (n=6-9/group), and fiber photometry recordings of the OxLight1 sensor (n=5-6/group) in ventral tegmental area (VTA). We also assessed ‘food addiction’ behaviors by measuring sucrose seeking (n=12-15/group). Results: HFD rats had higher ICSS thresholds vs. controls, indicating reward system deficit. These changes were associated with blunted orexin levels and lower orexin release in VTA. Binge eating partially restored reward thresholds and orexin release in VTA, but also promoted the development of several 'food addiction' behaviors, which were normalized by inhibition of the orexin-VTA circuit. Conclusions: Binge-like eating is 'self-medicating' against dysphoric states associated with higher weight, however this pattern of food intake promotes 'food addiction' behaviors. The orexin-VTA circuit is a target to normalize mood and eating outcomes in higher weight states.

Renee Papaluca¹, Eva Guerrero-Hreins¹, Nikeisha J Caruana¹, David A Stroud^1,2,3, Michael P Mendon^1,4, Aneta Stefanidis^5,6, Brian J Oldfield^5,6, Claire J Foldi^5,6, Priya Sumithran^7,8, Robyn M Brown¹*

People seeking obesity treatment often display compulsive disordered eating behaviours towards calorie-dense food high in fat and sugar which parallels behaviour observed in substance use disorder towards drugs. Indeed, chronic overconsumption of high-fat/high-sugar (HFHS) foods is associated with dysregulation of striatal brain reward areas. Evidence suggests bariatric surgery reduces motivation for HFHS foods post-surgery, however the gut-brain mechanisms underlying this change are unknown. Interestingly, a small subset of patients receiving bariatric surgery report adverse mental health outcomes following surgery such as depression, yet the determinants of this phenomenon are also unknown. The striatum is a key brain region at the centre of both hedonic processing of food reward and mood. This presentation will report on differentially expressed genes and proteins in the striatum underlying changes in food motivated behaviour and propensity for negative changes in mood following bariatric surgery in a mouse model.

1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.

1. Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia.

1. Victorian Clinical Genetics Services, Royal Children’s Hospital, Melbourne, Victoria, Australia.

1. Institute of Computational Biology, Computational Health Center, Helmholtz Munich, Munich, Germany.

1. Department of Physiology, Monash University, Clayton, Victoria, Australia.
2. Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.
3. Department of Surgery, School of Translational Medicine, Monash University, Victoria, Australia
4. Department of Endocrinology and Diabetes, Alfred Health, Victoria, Australia

K Huang^1,2, MA Magateshvaren Saras^3,4, K Conn^1,2, H Munguba⁵, E Greaves^1,2, F Reed^1,2, S Tyagi^4,6 & CJ Foldi^1,2

Activity-based anorexia (ABA) is a biobehavioural rodent model recapitulating key phenotypes of anorexia nervosa (AN), and we have previously shown that suppressing neural activity in the medial prefrontal cortex (mPFC)-nucleus accumbens shell (AcbSh) circuit could prevent pathological weight loss. Here, we used the translating ribosome affinity purification (TRAP) technique within this neural pathway to identify risk genes associated with susceptibility and resistance to pathological weight loss in female ABA rats. Female Sprague-Dawley rats (n=12) with viral TRAP construct expression were exposed to ABA conditions, which consisted of 90-minute food access and voluntary running wheel access, until they lost >20% body weight (Susceptible) or for a maximum of 10 days (Resistant). Following weight recovery, pathway-specific RNA was extracted and differentially expressed genes (DEGs) were identified. Gene ontology (GO) and Gene-set enrichment analysis (GSEA) were used for biological interpretation of DEGs. 1424 DEGs between Resistant and Susceptible were identified, including five risk genes previously reported to be associated with AN. The DEGs were involved in pathways associated with metabolic functions and neurodegenerative diseases, including Parkinson’s, Alzheimer’s and Huntington’s disease. Our conservative GO analysis revealed 2 upregulated and 24 downregulated genes in Resistant rats, with upregulated genes related to metabolism and downregulated genes associated with postsynaptic, cytoskeletal and axonal functions. This study revealed transcriptional changes in a specific neural circuit related to vulnerability to pathological weight loss that were not influenced by current calorie deficit. It highlights the utility of this model for examining the causal role these genes play in anorectic behaviours.

¹Department of Physiology, Monash University, Clayton VIC, Australia; ²Monash Biomedicine Discovery Institute, Clayton VIC, Australia; ³IITB-Monash Research Academy, Mumbai, Maharashtra, India; ⁴Central Clinical School, Monash University, Melbourne VIC, Australia; ⁵Department of Biochemistry, Weill Cornell Medicine, New York, USA; ⁶School of Computing Technologies, RMIT, Melbourne VIC, Australia

Maia Choi¹, Holly Poore¹, Nathaniel Thomas¹, Travis Mallard², Fazil Aliev¹, Jonathan Coleman³, Cynthia Bulik⁴, Danielle Dick¹

Binge eating disorder is the most common eating disorder, yet its etiology remains poorly understood. In epidemiological data, binge eating is correlated with both internalizing (e.g., depression, anxiety) and externalizing (e.g., substance use problems, ADHD) disorders. Further, preliminary analyses from the first genome wide association study (GWAS) of binge eating suggested that binge eating showed moderate genetic correlations with both externalizing and internalizing outcomes. In our project, we used data from the first GWAS of binge eating, a multivariate GWAS of externalizing, and a multivariate GWAS of internalizing, to assess the extent to which genetic influences on binge eating are shared with internalizing and/or externalizing, and/or specific to binge eating. By combining data from several million individuals, we find evidence that binge eating is genetically correlated with both externalizing and internalizing behaviors, indicating that genes underlying both behavioral and emotional regulation are involved in binge eating disorder. Additional analyses parsed out the effect of comorbid anorexia to better understand the nature of these associations. Although we find that binge eating is significantly genetically associated with both internalizing and externalizing psychopathology, the associations with externalizing are weaker and seem to be driven by the shared variance with internalizing. Further, associations between binge eating and internalizing psychopathology are in part unique from the comorbidity between binge eating and anorexia. These analyses provide insight into the genetic nature of influences on human eating disorders.

¹Rutgers Addiction Research Center, Rutgers University

²Massachusetts General Hospital, Harvard University

³King’s College, London

⁴UNC Center of Excellence for Eating Disorders

Rachel C. Rice, Remy R. Frawley, Daniela V. Gil, Annalisa M. Baratta, Shirley Y. Hill, Gregg E. Homanics, Sean P. Farris

Alcohol Use Disorder (AUD) is a serious neuropsychiatric disorder caused by genetic and environmental factors impacting widespread changes in molecular pathways that may be inherited across generations. Our current study investigated unbiased system-wide changes in gene expression in the blood and brain (i.e., medial prefrontal cortex and central nucleus of amygdala) of alcohol naïve male and female F1 C57BL/6J offspring from paternal preconception ethanol exposure (PPE), maternal preconception ethanol exposure (MPE), and biparental preconception alcohol exposure (BPE). In parallel, we conducted an unbiased RNA-Seq analysis of human blood lymphocytes from a 30+ year longitudinal multigenerational study of families with and without a history of AUD to identify evolutionary conserved changes in gene expression related to disease. Differential gene expression analyses revealed sex- and alcohol-dependent changes in gene expression that were evolutionary conserved across species. Only approximately 40% of differentially expressed genes were protein-coding, suggesting a potential epigenetic role for non-coding RNAs in the risk of developing AUD. A systematic meta-analysis identified shared enriched biological pathways for protein-coding genes across PPE, MPE, and BPE groups related to proinflammatory-, metabolic-, growth-, and cancer-related processes, with differing directionalities pathways depending on sex of the offspring. Overall, this work encompasses the first comprehensive cross-generational transcriptomic study on preconception ethanol exposure and may identify potential biomarkers of cross-generational alcohol-related disease risk. We gratefully acknowledge the support of NIH/NIAAA grants AA020889, AA030257, and AA031168 as well as internal support from Bridging Connections in Addiction Research at the University of Pittsburgh.

University Of Pittsburgh Center For Neuroscience, Pittsburgh, Pa 15261; University Of Pittsburgh Department Of Anesthesiogy And Perioperative Medicince, Pittsburgh, Pa 152161; University Of Pittsburgh Department Of Psychiatry, Pittsburgh, Pa 15261; University Of Pittsburgh Department Of Biomedical Informatics, Pittsburgh, Pa 15261

A Takahashi1, N Mimura1, B Hu1, K Okamoto1, K Ohashi2, T Kawase2, A Toyoda3, T Tsukahara2, K Mitsui1

There are a large individual differences in aggression within a species, influenced by genetic and environmental factors, including gut microbiota. In this study, we found significant differences in aggression between ICR mouse strains obtained from CLEA Japan (ICR^CLEA) and Charles River Laboratories Japan (ICR^CR; currently the Jackson Laboratory Japan). In a 3-day resident-intruder test, 90% of ICR^CR males showed aggressive behavior, compared to only 37% of ICR^CLEA males. Analysis of fecal samples using 16S rRNA sequencing showed distinct differences in gut microbiota composition between breeders. Fecal microbiota transplantation (FMT) between ICR^CLEA and ICR^CR males demonstrated bidirectional changes in aggression: FMT from ICR^CLEA donors to ICR^CR males significantly reduced aggressive behavior, whereas FMT from ICR^CR donor to ICR^CLEA males increased aggression. These results indicate that the gut microbiota composition influences aggressive behavior. Further, 16S rRNA sequencing of fecal samples identified five bacterial genera significantly enriched in high-aggression groups related to ICR^CR and one bacterial genus characteristic of low-aggression groups related to ICR^CLEA. When low-aggressive ICR^CLEA was subjected to post-weaning social isolation stress, which is known to escalate aggressive behavior of male rodents, the bacterial genus characteristic of ICR^CLEA–associated low aggression showed significant reduction in the socially isolation group. These results suggest that gut microbiome associated with aggression are sensitive to social isolation stress.

1 Laboratory of Behavioral Neurobiology, University of Tsukuba, Tsukuba, Ibaraki, Japan, 2 Kyoto Institute of Nutrition & Patholohy, Tsuzuki, Kyoto, Japan, 3 College of Agriculture, Ibaraki University, Ami, Ibaraki, Japan

Funding Support: JST FOREST Program JPMJFR214A, JSPS KAKENHI 22K19744, KAKENHI 22H02660, Astellas Foundation for Research on Metabolic Disorders, and Project for University-Industry Cooperation Strengthening in Tsukuba (Ibaraki Prefecture), JAPAN

Tatsuhiko Goto¹, Prudence Nyirimana¹, Riku Sasaki¹, Dipson Gyawali¹, Atsuhi J. Nagano², Akira Ishikawa³

Animal tameness is one of key behavioral characteristics in domestic animals. Several experimental models including foxes, rats, and mice have been investigated in behavior genetics of tameness. In the livestock industry, tame behavior is desirable for animal production. Given that the genetic basis of tame behavior is revealed using Japanese indigenous chicken resources, tamed chickens will be created efficiently. In this study, we aim to find quantitative trait loci (QTLs) for tame behavior in chickens. From 491 segregating individuals based on a cross between Darumachabo and Tosa-jidori breeds, phenotypes and genotypes were collected. Tame behaviors (number of “steps”, “avoiding”, and “heading”) at 30 weeks of age were evaluated by a 1-min handling test. RAD-seq was used to collect genotypic data. Fully informative 425 SNPs on Chrs. 1-27 were used for QTL analysis of tame behavior using R/qtl. Genome-wide thresholds were calculated by permutation tests. This study revealed two significant QTLs on Chr. 2 (LOD = 4.2) and Chr. 5 (LOD = 3.8) in activity (“step”) during handling test. In addition, two suggestive QTLs were found for “heading” on Chr. 2 (LOD = 3.4) and “avoiding” on Chr. 3 (LOD = 3.3). In these four QTLs, homozygotes for the Tosa-jidori allele indicated high avoidance activity. These QTLs explained a small part (around 8%) of phenotypic variance in tame behavior. Therefore, further genetic mapping is needed for a better understanding of the genetic basis underlying animal tameness.

¹Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan, ²Ryukoku University, Otsu, Japan, ³Nagoya University, Nagoya, Japan