Conference Agenda

HHA Thorpe1, P Fontanillas2, JJ Meredith3, MV Jennings3, RB Cupertino3, S Pakala3, 23andMe Research Team2, SL Elson2, JY Khokhar1, LK Davis4,5,6, EC Johnson7, AA Palmer3,8, S Sanchez-Roige3,4

While genome-wide association studies (GWASs) associate over 100 genetic variants with cannabis use disorder, these GWASs focus on the addiction extreme and neglect pre-addiction traits. Collaborating with 23andMe, Inc., we present GWASs of cannabis initiation (N=131,895) and use frequency (N=73,374). Cannabis initiation GWAS identified two loci near Glutamate Metabotropic Receptor 3 (rs12673181, p=6.90E-09) and near Cell Adhesion Molecule 2 (CADM2; rs11922956, p=2.40E-11), a gene implicated in cannabis and other substance use, impulsivity, and risk-taking. Consistently, cannabis use frequency GWAS identified one locus near CADM2 (rs4856591, p=8.10E-09). Both traits were heritable (h₂SNP: initiation=0.13, frequency=0.04) and genetically correlated with cannabis use disorder (r_g: initiation=0.62, frequency=0.45). Cannabis initiation and use frequency genetically correlated with 115 and 40 other traits, respectively, notably with other substance use but also with physical (e.g., pain) and psychiatric (e.g., depression) conditions. We therefore examined polygenic score (PGS) associations with various phenotypes in medical and population cohorts. Phenome-wide association study in BioVU participants identified 22 cannabis initiation PGS associations, replicating positive associations with cannabis and other substance use disorders and mood disorders, and uncovering novel associations with anxiety, celiac disease, diabetes, HIV, and viral hepatitis. In All of Us participants, cannabis initiation PGS positively associated with lifetime, daily, and problematic cannabis use, whereas cannabis use frequency PGS positively associated with lifetime and problematic use. The identification of novel genetic/phenotypic associations with cannabis initiation and use frequency endorses future, better-powered population cohort studies of cannabis pre-addiction genetics, which will identify cannabis use disorder transition risk factors and enable translational research.

1 Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
2 23andMe, Inc., Sunnyvale, California, United States
3 Department of Psychiatry, 8 Institute for Genomic Medicine, University of California San Diego, La Jolla, California, United States
4 Department of Medicine, Division of Genetic Medicine, 5 Department of Psychiatry and Behavioral Sciences, 6 Department of Biomedical Informatics, Vanderbilt University, Nashville, TN, USA
7 Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA

Funding Sources: CIHR Postdoctoral Fellowship, California Tobacco-Related Disease Research Program (T32IR5226 and 28IR-0070), NIDA (DP1DA054394), CIHR Canada Research Chair in Translational Neuropsychopharmacology

M Courchesne1, RA Muhammad1, RC Cumming1

The cellular mechanisms that underly learning and memory consolidation in the brain are bioenergetically demanding. With the decline in neuronal glucose utilization that occurs during advanced ageing, the brain relies on alternate metabolites such as lactate and lipids as fuel sources to meet energy demands. The improper processing of these metabolites may underlie memory deficits typically observed in later life. Lactate production in neurons is primarily governed by lactate dehydrogenase-A (LDHA) activity, the rate-limiting enzyme that catalyses pyruvate to lactate conversion. In the present study, the effects of neuronal-specific ldha induction and knockout on learning and memory was examined in aged mice. The Morris water maze (MWM) was used to test spatial learning and 24-hour spatial memory in aged neuronal ldha induction mice. Additionally, the object location task (OLT) was used to test pattern separation ability and assess spatial contextual learning and memory. Neuronal ldha induction mice exhibited reduced spatial memory in males compared to age-matched controls. In contrast, memory was improved in old ldha knockout mice compared to age-matched controls. Brain sections of neuronal-specific ldha induction and knockout aged mice were collected to assess changes in lipid dynamics using immunofluorescence microscopy. Interestingly, levels of perilipin2, which plays an important role in maintaining droplet integrity to prevent free-fatty acid toxicity, was reduced in certain hippocampal subregions of ldha induction mice. Overall, the present study suggests that excess neuronal-specific LDHA accumulation is cognitively detrimental in aged mice, and these findings could be attributed to aberrant lipid processing and metabolism.

1Department of Biology, Western University, London, Ontario, Canada Funding Support: Government of Ontario, Western University, Children’s Health Research Institute

EY Choi¹, HHA Thorpe¹, A Hassan², M Pusparajah³, KK Yeung³, JY Khokhar¹

CADM2, located on chromosome 3, is a synaptic adhesion molecule associated with cannabis use and cognition. It is primarily expressed in the brain, especially in addiction-related regions. Our lab has previously used a Cadm2 knockout (KO) mouse line to investigate the association between CADM2 and voluntary cannabinoid intake, THC response and cognitive phenotypes. Prepulse Inhibition (PPI) of startle is one form of sensorimotor gating that has been widely studied in mice and THC has been shown to impair sensorimotor gaining in rodents. Here we investigated the effects of CADM2 genotype and THC exposure on PPI. PPI trials were run on WT, HET and KO mice both before and after THC treatment. Our findings suggested that baseline PPI was attenuated in the KO mice. Additionally, KO mice were observed to be hyperactive compared to the WT and HET, even without the startle stimulus. Furthermore, after 2mg/kg dose of THC, the differences previously seen between the KO and HET mice disappeared, showing that this dose may attenuate PPI in HET, but not in KO and WT mice.We are conducting Matrix-assisted laser desorption/ionization (MALDI) on THC-treated brains in WT, HET, and KO mice to evaluate differences in glutamate, GABA, dopamine, serotonin, and choline levels in the PFC and NAc. Since the same mice were used for PPI and MALDI, we will also be able to correlate neurotransmitter levels to their PPI measures. These studies aim to clarify THC’s effect on behavioural phenotypes and neurotransmitter levels in relation to variation in Cadm2 expression.

¹Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada ²Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada

³Department of Chemistry, University of Western Ontario, London, ON, Canada

Funding Support: EYC is funded by a CIHR CGS-M grant. JYK is funded by a Canada Research Chair in Translational Neuropsychopharmacology from CIHR. HT is funded by a post-doctoral fellowship from CIHR.

¹Troy D. Wilcox, ¹Heidi S. Fisher,¹ Robyn L. Ball, ¹Hao He, ¹Vivek M. Philip, ¹ Ashley A. Olson, ¹Lacey Kent-Webber, ¹ Leona H. Gagnon, ¹ Erik B. Bloss, ¹ Elissa J. Chesler

Here we used advanced transcriptomic, neuroanatomical, and behavioral techniques in divergent inbred mouse strains that display a wide range of addiction-like phenotypes, vulnerable or resistant, to understand how changes in neurocircuitry and neural gene expression differ across the behavioral phases of drug self-administration in genetically distinct populations. We compared neuroanatomy and transcriptional profiles of three mouse stains with differing genetic backgrounds and disparate behavioral patterns of cocaine intravenous self-administration. Using a multifaceted design, we find that all strains share some transcriptional response patterns during acquisition and extinction of drug seeking behavior, as compared to mice with no cocaine exposure and those that were exposed but failed to acquire drug seeking behavior, while other changes in gene regulation are strain specific. In addition, we contrast these transcriptional changes with preliminary data on dendritic morphology and density confirming acquisition of cocaine self-administration is associated with neuronal morphology change. We found that the spine head diameter distribution changes during acquisition and extinction of drug seeking behavior in a manner that differs across strains. This functional data reveals that the behavioral differences in the strains correspond to strain differences in structural plasticity. Together, these findings suggest that both previously discovered and unknown transcriptional and neuroanatomical correlates of drug self-administration differ among strains with different vulnerability to acquisition and maintenance of drug seeking behavior. Identifying the causal genes, their regulatory mechanisms, and the role they play in neurobiological mechanisms of addiction is critical to unraveling and addressing their effects on substance use disorder in people.

¹ The Jackson Laboratory, Bar Harbor ME

Funding Support: The Jackson Laboratory, R01 DA37937, and P50 DA039841.

Nuñez KM*¹, Sherer LM*², Walley A², Salamon S², Selcho M³, Barnea G², Kaun KR²

Internal states such hunger shape how an organism responds to external cues from its environment. The internal state of an organism can affect sensory processing, decision-making, and behavior from flies to humans. Ethanol exhibits particularly strong effects on internal state and decision-making; however, we have little understanding of the complex molecular mechanisms through which this occurs. We identified a parallel circuit through which hunger facilitates cue-induced ethanol seeking, and describe the mechanisms used within this circuit that influence ethanol preference. We first determined that the norepinephrine analogue octopamine (OA) is required for ethanol preference in hungry and not sated flies. We narrowed this effect to a single asymmetrical OA neuron that innervates mushroom body circuitry. Using in vivo calcium imaging, we determined that hunger increases activity of this neuron and identified a postsynaptic mushroom body output neuron that drives changes in ethanol preference. Together, these results demonstrate a parallel ethanol memory circuit that activates upon food deprivation to motivate ethanol seeking, and pave the way for future experiments identifying the neurotransmitters, neuromodulators, and receptors/autoreceptors that impact stress-induced ethanol seeking.

¹ New York University School of Medicine, New York City, United States

² Department of Neuroscience, Brown University, Providence, United States

³ Department of Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, University of Würzburg, Würzburg, Germany.

* Co-first author

Michael Leonardo¹, Alexis Kastigar¹, Josiah Comstock², Shelby Towers², Benjamin Gourley¹, Jonathan Lipovich¹, Amber Walden¹; Sarah Brunty¹, Jessica Huffman^1; Jun Fan¹,Travis Salisbury^1, Donald Primerano1, James Denvir¹, Alejandro Q. Nato^1, Deranda B. Lester^{2 ,}Price E. Dickson¹

Environmental impoverishment may interact with the genome to cause addiction vulnerability through effects on the transcriptome and, ultimately, neurophysiology within reward-circuitry. In a series of experiments, we tested these hypotheses using mice from the genetically diverse Collaborative Cross (CC) recombinant inbred panel, CC founder strains, and BXD founder strains. Mice from a subset of these strains were housed in one of two conditions (environmental enrichment or impoverishment) beginning at 3 weeks of age and continuing throughout the experiment. Starting at 13 weeks of age, mice were tested on behavioral assays to quantify sensation seeking, risk taking, and intravenous cocaine self-administration. In a separate group of drug-naïve mice, we quantified striatal and hippocampal gene expression using bulk RNA-seq; snRNA-seq was used to quantify cell-type specific gene expression in differentially housed C57BL/6J mice. In a separate set of experiments using the BXD founder strains (C57BL/6J and DBA/2J), we compared the relative effects of environmental enrichment, social enrichment, and environmental impoverishment on NAc dopamine dynamics, striatal gene expression, sensation seeking, and systemic corticosterone. Following analysis of these data, we reached two primary conclusions. First, we observed robust effects of housing condition on dependent variables across biological scale including behavior, neurophysiology, and gene expression. Second, many of these effects were strongly dependent on mouse strain. Collectively, these data reveal previously unknown gene-by-environment interactions influencing addiction-relevant endpoints across biological scale and indicate that the underlying genetic mechanisms driving these phenomena can be dissected using systems genetics and recombinant inbred mouse panels.

1. Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV

2. Department of Psychology, University of Memphis, Memphis, TN

Funding Support: NIDA R00DA043573, NIDA R21DA054929, NIGMS P20GM103434, NIDA Drug Supply Program

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. In the past decade, a novel epigenetic mechanism involving histone variants and their chaperones has been found to be fundamental to regulate gene expression in post-mitotic cells like neurons. Histone variants’ dynamics have been implicated in the regulation of higher brain functions like learning and memory and are involved in numerous neurological disorders. However, little is known about histone variants’ dynamics and their role in neurogenesis and neurodevelopment.

In our lab, we focus on histone variant H2A.Z and its specific chaperones Anp32e, which removes the variant from chromatin, and Srcap, with the opposite function. We show that H2A.Z is relocated at different genes during neuronal development, with genes losing the variant over time and others gaining it. H2A.Z changes precede gene expression changes, suggesting that H2A.Z’s relocation in the genome is fundamental to regulate gene expression. Consistently with this hypothesis, viral-mediated knock-down of Anp32e at early stages of in vitro neuronal development impairs the natural loss of Anp32e from candidate genes and alters their expression. Notably, bulk RNA-seq showed that knock-down of Anp32e causes specific downregulation of genes involved in neurogenesis and neurodevelopment and significantly impacts dendritic arborization. Overall, these data suggest that H2A.Z’s dynamics, orchestrated by its chaperones, during development play a fundamental role in the regulation of neurodevelopment, opening the road for the study of histone variants’ genomic relocation as a novel epigenetic mechanism implicated in nervous system development.

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

WC Booher^1,2, KE Lanier¹, RA Radcliffe^1,2

There is a growing interest in the use of Cannabis for therapeutic purposes; however, effective drug therapy requires an understanding of a drug’s pharmacokinetic (PK), pharmacodynamic (PD), and pharmacogenomic (PG) interrelationships. By dosing male and female C57Bl/6J and A/J mice with 15 mg/kg ∆-9-tetrahydrocannabinol (∆9 -THC), we measured ∆9 -THC, blood plasma levels, as well as the major ∆9-THC metabolites 11-OH-THC, THC-COOH, and THC-COOH glucuronide, at 60-, 90-, and 240-minutes. A two-way repeated measures ANOVA revealed a significant effect of strain across time, where compared to A/J mice, C57Bl/6J mice had significantly less ∆9 -THC in their plasma across all three time points. Additionally, a two-way repeated measures ANOVA demonstrated a significant effect of strain across time, where compared to A/J mice, C57Bl/6J mice had significantly more 11-OH-THC and 11-COOH-THC glucuronide in their plasma across all three time points. To summarize, C57Bl/6J mice had significantly less ∆9 -THC, but significantly more 11-OH-THC and 11-COOH-THC glucuronide in their plasma, suggesting that C57Bl/6J mice are metabolizing ∆9-THC significantly faster than the A/J mice. Thus, we propose that variation in the PK of cannabinoids is due in part to genetics and, in turn, this effect contributes to individual differences in responses to Cannabis.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
Institute for Behavioral Genetics, University of Colorado Boulder, 447 UCB, Boulder, CO, USA.

Funding support: Departmental funds

Dana Guhle 1, Devang Mehta 1, Nika Farivar 1, Glen Uhrig 1, Ronald Davis 2, and Jacob Berry 1.

A critical function of the brain is to elicit optimal and adaptive behavioural responses needed to survive. An animal’s response adapts based on past events (i.e. memory) or internal state (i.e. starvation-altered innate behaviour). Interestingly, in Drosophila melanogaster, dopaminergic (DA) signalling through a receptor, Dop1R1, regulates both learned and state driven behavioural plasticity. Dop1R1is highly expressed in the memory center and is critical for encoding memories through adenylyl cyclase (ADCY1 ortholog) driven synaptic depression. In contrast, signaling through a separate adenylyl cyclase (ADCY2 ortholog) strengthens synapses and drives state dependent changes to innate odour preference. It’s unclear how these Dop1R1 pathways bi-directionally regulate memory synapses. To understand Dop1R1 signaling environments, we utilized TurboID proximity labelling proteomics and RNAi screening to identify interactors that help regulate learned and innate behaviour. In vivo Proximity labelling using Turbo-V5 fused to endogenous Dop1R1 identified candidate proteins significantly enriched around the Dop1R1 receptor compared to another memory regulator, Dop1R2. Disruption of candidate proteins in memory circuits lead to significantly altered behavioural plasticity in starved odour preference and associative learning contexts. Interestingly, many candidate interactors affect either internal state or learned behavoural plasticity but not both, indicating distinct pathways. In vivo functional imaging experiments will be conducted to reveal if these candidates alter Dop1R1 receptor mediated effects on synaptic strength, and downstream cAMP signalling. Altogether, our study will identify and characterize novel pathways regulating dopaminergic signalling to illicit two distinct behaviours and illuminate how the brain fine-tunes these behaviours through one receptor.

1University of Alberta, 2The Scripps Research Institute

AT Bechard¹ and AF Simon¹

Social interactions between animals depend upon where they settle in relation to each other; a behaviour called social spacing. This behaviour is defined as the distance between individuals in a stable group and has been shown to be affected by life experience, genetics, and the environment. Simple interactions like social spacing that mediate and precede more complex behaviours serve as a practical means to elucidate the basic neurogenetics involved in a variety of social behaviours. Here I used the fruit fly, Drosophila melanogaster, as a model to investigate the neural circuitry underlying social spacing at the level of brain structures.

I used genetic tools available in the fruit fly to precisely hyperactivate or inhibit neurons in brain structures of interest while assaying social space. Two structures, the mushroom bodies and the protocerebral bridge, are of interest because they show enrichment of Neuroligin 3 (Nlg3); a postsynaptic cell adhesion protein that we previously found to have a strong association with social spacing behaviour. Additionally, I manipulated nlg3-expressing neurons to test their involvement in social spacing. I determined that both brain structures, as well as nlg3-expressing neurons, are involved in social spacing neural circuitry, and likely in a sexually dimorphic manner. This research contributes to understanding the role Nlg3 plays in social spacing and reveals more routes of neural connectivity to be investigated.

¹Department of Biology, Western University, London, ON, Canada

Funding Support: Ontario Graduate Scholarship to ATB and NSERC Discovery grants 05054-2022 to AFS

J. Wesley Robinson, Ryley Yost, Abbie Bechard, Waliu Alaka, Avneet Sahota, Khadijat O. Mosuro, Jade de Belle, Anne F. Simon

Social behaviour can be defined as a response to individuals in a group and is required for organisms to survive and reproduce. As such, social behaviour relies on the perception, integration, and response to social stimuli, from the interaction or communication with other individuals. The integration of, and response to, these cues require signalling throughout neural circuitry.

The focus of our lab is to better understand the neurogenetic underpinnings of Drosophila melanogaster social space determination. We and others have shown that social spacing in Drosophila can be influenced by a variety of intrinsic and extrinsic factors, such as mating status, social enrichment, genes, and environmental conditions. We have shown that dopaminergic signalling is an important signalling molecule to modulate social space. Others have also implicated acetylcholine in the mushroom bodies as well, a region of the fly brain associated with sensory integration. Recent research has also begun to connect neuropils or specific neural circuits to their associated social behaviours. Here, we show that genetically controlling the activation of individual circuits, and reducing specific neurotransmitters, modifies fly behaviour and provides insight into the neural circuitry controlling social space.

Our findings identify specific neuropil involvement in social space and the causal neural circuits for the sex-specific determination of social spacing.

Department of Biology, Western University, London, ON

A Sghaier1, JS Dason1

Numerous studies have focused on how various proteins regulate synaptic development and plasticity. However, the roles of lipids, such as phosphatidylserine (PS), have been less studied. PS is a phospholipid synthesized by Phosphatidylserine synthase (Pss) and upon localization to the cell membrane is transported from the outer to inner leaflet by dATP8B (ATP8B), a phospholipid flippase. Drosophila Pss loss-of-function mutants have previously been shown to have reduced synaptogenesis/axonal growth. The effects of loss of dATP8B on synaptic growth have not been characterized. The primary objective of this study is to determine if PS is required for normal levels of synaptic growth and plasticity. We hypothesize that PS is required for proper synaptic growth as well as activity-dependent synaptic growth. We found that synaptic growth in ATP8B mutants were generally not significantly different in the number of boutons or active zones when compared to their controls. Drosophila larvae raised at high temperatures (30°C) have previously been shown to have increased locomotion, resulting in activity-dependent synaptic growth. We found that this activity-dependent synaptic growth is largely absent in ATP8B mutants. Current experiments are examining whether presynaptic or glial Pss and ATP8B are required for activity-dependent growth. Future experiments will use Lactadherin-C2-mCherry, a fluorescent probe specific to PS, to determine if PS localization changes in response to increased synaptic activity and whether these changes are important for activity-dependent synaptic growth.

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

Funding Support: NSERC

S Saxena1, EA Duecker1, S Khanam1, BM Moore II2, MK Mulligan1

Levels of Δ9- tetrahydrocannabinol (THC) in cannabis and consumables are rising. THC is the major psychoactive component of cannabis and has a profound effect on brain function and behavior. However, many of the specific effectors mediating these responses have not yet been identified and it is not known how these signaling pathways differ across genotypes to influence behavioral and physiological responses to THC. To address these issues, we developed a pharmacogenomic screen leveraging the BXD recombinant inbred mouse population to identify gene variants in effector proteins regulating cannabinoid 1 receptor (CB1) levels, signal transduction, termination of signaling, and/or trafficking following acute and repeated exposure to high-dose (10 mg/kg, i.p.) THC. The screen consists of a battery of tests directly related to THC-mediated CB1 activation—hypothermia, antinociception (tail flick response to a thermal stimulus), and motor responses (activity in an open field). Our long-term goal is to screen 80 BXD strains of both sexes. Using only a subset of ~20 BXDs we found sex differences and correlations between some motor traits (e.g., time mobile) and hypothermia following an acute dose of THC. We identified a quantitative trait locus (QTL) on Chromosome 11 containing Rps6kb1 as a potential candidate gene modulating both response traits. Here we report progress following the screening of 10 additional BXD strains. We address whether additional strains improve QTL detection, identify additional associations between response traits, and identify traits and QTLs related to rapid tolerance following repeated exposure to THC.

1Department of Genetics, Genomics and Informatics, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy, The University of Tennessee Health Science Center, Memphis, Tennessee, USA

Funding Support: NIH R01 DA056523

NE Bulthuis¹, LI Quintana², M Stackmann¹, CA Denny^3,4

The encoding or retrieval of a contextual fear memory recruits the activity of thousands of neurons across the mammalian brain. This ensemble of cells is often identified by immediate-early genes (IEGs), which are expressed following neuronal activity, but the extent to which the expression pattern of one IEG reflects another is unclear. Here, we examined the IEGs Arc/Arg3.1 and c-Fos across several brain regions during fear memory encoding or retrieval. Mice were administered a 3-shock contextual fear conditioning (CFC) paradigm and sacrificed 1h later (encoding), 1 h after context re-exposure 1 day later (1-day retrieval), or 1 h after context re-exposure 5 days later (5-day retrieval) (n = 8 per group). Brain tissue was immunostained for Arc and c-Fos protein. We found that the two IEGs showed stark differences in expression, with fewer than 50% of Arc⁺ or c-Fos⁺ cells in any brain region expressing both markers. While c-Fos⁺ cell density remained constant across memory states in both CA3 and the dentate gyrus (DG) of the hippocampus, Arc⁺ cell density increased from encoding to retrieval in CA3 yet remained constant in the DG. In subcortical areas like the basolateral amygdala complex (BLA) and the paraventricular nucleus of the thalamus (PVT), expression of Arc and c-Fos did not vary with memory state, but c-Fos expression exceeded that of Arc across all states. Our results warrant further study of the conditions that induce expression of these markers, and they suggest that ensembles identified by either IEG may be anatomically or functionally distinct.

¹Doctoral Program in Neurobiology & Behavior, Columbia University; ²Barnard College, Columbia University; ³Department of Psychiatry, Columbia University Irving Medical Center (CUIMC); ⁴Division of Systems Neuroscience, Research Foundation for Mental Hygiene, Inc. (RFMH) / New York State Psychiatric Institute (NYSPI), New York, NY, USA

Funding Support: NSF GRFP DGE2036197, NINDS F99NS129178 & R21NS114870, NIMH F31MH125656, NICHD T32HD007430 & R01HD101402, NIA R21AG064774, the Barnard Summer Science Research Institute, NYSPI, and a gift from For the Love of Travis, Inc., USA.

Ella Doornaert¹, Dorit Moehrle¹, Alaa El-Cheikh Mohamad¹, Gurwinder Johal¹, Brian Allman¹, Susanne Schmid¹²

Autism spectrum disorder (ASD) is a neurodevelopmental condition affecting one in 160 children worldwide. Homozygous loss-of-function mutation in the CNTNAP2 gene has been linked to a syndromic form of ASD and multiple studies have identified other CNTNAP2 mutations as risk factors for ASD. The homozygous Cntnap2 knockout (KO) rat has been proven to be a valid genetic rat model for studying ASD, including associated sensory disruptions. The Cntnap2-KO rat has consistently shown increased startle and reduced prepulse inhibition (PPI). Here, we introduced a comprehensive method to assess startle and PPI in the Cntnap2-KO rat that distinguishes between startle and sound scaling components. These pertain to a reduction in response amplitude and sound sensitivity, respectively. Furthermore, we implemented a new statistical tool, ARTool, enabling an in-depth examination of sex effects. Through this, we discovered that sex, prepulse intensity, and startle stimulus intensity profoundly impact whether PPI deficits are evident in the Cntnap2-KO rat or not. Our results highlight that the Cntnap2-KO rat does not universally exhibit a PPI deficit, but rather that impaired PPI is contingent on specific testing conditions. This finding could explain the notoriously inconsistent findings on PPI deficits across animal models and different labs, emphasizing the importance of considering testing conditions in PPI assessments. This research was funded by a CIHR and NSERC-USRA grant.

¹Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario; ²Psychology, University of Western Ontario

André Lucas S. Borges¹, Donna-Jael G Paredes¹, Rebeka Sultana¹, Amanda M Barkley-Levenson, Ph.D.¹

Recent genome-wide association studies (GWAS) have identified multiple genes that are significantly associated with alcohol consumption and problematic use. One of these genes, fucosyltransferase 2 (Fut2), encodes the galactoside 2-L-fucosyltransferase enzyme. Fut2 genotype has been shown to affect the presence of gut-epithelium structures that are important for bacterial anchoring, and there is evidence for a bidirectional relationship between Fut2 expression and gut microbiome composition. Therefore, microbiome-driven changes in the gut-brain axis are one possible mechanism by which Fut2 might influence alcohol consumption. We have seen previously that Fut2 homozygous knockout male mice have enhanced binge-like drinking compared to wild-type (WT) littermates. Here, we aimed to assess Fut2 genotype effects on gut microbiota after binge-like alcohol drinking and in water drinking controls. For this study, adult male and female mice homozygous for the Fut2 knockout and WT littermates were used. Mice received three repeated 4-day drinking in the dark (DID) exposures with either 20% ethanol or water. Fecal microbiome analysis after the final DID test showed reduced alpha diversity, increased abundance of Prevotella and decreased abundance of Oscillibacter in homozygous mice compared to WT. We also saw general trends towards greater genotypic differences in the alcohol DID group vs. the water DID group, suggesting that Fut2 knockout mice may be more susceptible than WT mice to the effects of alcohol on the gut microbiome. Our results demonstrate the potential role of the Fut2 gene in binge drinking and may provide possible microbiome targets to modulate this behavior.

1 Department of Pharmaceutical Sciences, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.

Funding Support: NIH grant R00AA027835, UNM College of Pharmacy Pilot Project Award

Esmin Unaran, Olamide Adebiyi, Amr Eed, Medha Krishnan, Tim Bussey, Lisa Saksida and Ravi S. Menon

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by distinct pathologies, including extracellular amyloid beta (AB) deposition and intracellular tau burden. Functional connectivity (FC), the correlation of neuronal signalling between different brain regions, is often impaired in neurological diseases, including AD. Our research focused on investigating the impact of AB pathology on FC using a murine model. We utilized the APP-NLGF (APPKI3) mice as our AD model and compared them with APP-NL (APPKI1) mice as controls. We examined FC changes at two time points: 3 months old (session 1), representing initial plaque deposition, and 7 months old (session 2), representing plaque density saturation.To investigate the alterations between two time points within groups, we conducted resting state functional magnetic imaging (rsfMRI) at 9.4 T and performed paired t-tests on correlation values of resting state timeseries between different brain regions. This comparison revealed no change in APPKI1 group between time points as expected. However, in the APPKI3 group, we observed an increase in interhemispheric FC in the CA1 region of the hippocampus in session 2. To further investigate the alterations between groups within sessions, we performed unpaired t-tests on the correlation values. This investigation revealed that FC between CA1, dentate gyrus (DG) and CA2+CA3 (grouped together) was impaired at 3-months in KI3 relative to KI1, suggesting a loss in FC between hippocampal subfields at this earlier time point. These results align with previous human literature, suggesting that APP-NLGF is a suitable AD model to study FC.

Western University

Fariya Zaheer¹, Gabriel Levine¹, Ana Leticia Simal¹, Paige O. Reid¹, Tami Martino^1,2, Giannina Descalzi¹

Chronic pain is a debilitative disease affecting 1 in 5 adults globally and is a major risk factor for developing anxiety and depression. Our current understanding of chronic pain remains inadequate, resulting in few efficacious treatment options, and few therapeutics for the treatment of associated mental health disorders. Cellular homeostasis is crucial for normal bodily functions and investigation at the cellular levels may reveal a better understanding of the processes that occur leading to the development of chronic pain. Using the spared nerve injury (SNI) model of neuropathic pain, we found that adult male mice with impaired BECLIN-1 function show enhanced mechanical and thermal hypersensitivity compared to wildtype controls. Remarkably, we found that while SNI induced increases in anxiety-like behaviours in wildtype mice, this was not observed in mice with impaired BECLIN-1 protein function. We also found that BECLIN-1 protein influence is sex-specific and that its activity reduction only seems to impact male rodents as female transgenic mice and the wildtype cohort showed no significant differences in SNI induced changes in pain thresholds or anxiety-like behaviours. Our data thus indicates that BECLIN-1 activity is differentially involved in the nociceptive and emotion related effects of chronic neuropathic pain in a sex-specific manner.

¹Department of Biomedical Sciences, ²Center for Cardiovascular Investigations, University of Guelph

Justine Anne Guevarra¹, Isabella Salguero¹, Hanna Jin¹, and Fernando Vonhoff¹

Amyloid precursor protein (APP) is a single-pass transmembrane protein encoded by the human APP gene. It is expressed in several tissues and organs, but it is primarily found in the brain. Its physiological functions remain elusive, but APP is thought to be involved in various biological processes including neuronal development and survival, learning and memory, synaptic plasticity and transmission. It is highly conserved across species with related proteins such as APLP1 and APLP2 in mammals, and APPL in fruit flies. Studies in mice and flies show that lack of APP proteins results in developmental and locomotor deficits, which can be rescued by the expression of the human APP, confirming its conserved properties. APP is also a metalloprotein, which can bind to copper (Cu) and zinc (Zn) ions and influence homeostasis regulation. Moreover, APP mRNA contains iron-response elements (IRE) in its structure that can bind to iron-regulatory proteins (IRP), allowing intracellular iron (Fe) to regulate APP translation. Interestingly, Fe shares similar chemical characteristics with aluminum (Al), allowing Al to bind to iron-binding proteins involved in transporting Fe and disrupt Fe homoeostasis. Moreover, recent studies display increasing evidence on the possible link between metals and neurodegenerative disorders. This project aims to investigate the effects of metals associated with APPL in aging-dependent locomotor decline in Drosophila melanogaster. Overall, the knowledge gained will serve as a reference for future studies in mammals and broaden the understanding of the fundamental mechanisms involving APP, metals, and aging in flies.

¹Department of Biological Sciences, University of Maryland, Baltimore County, USA

Heba Mahmood, James Thompson-Dick, Anna Phan

Loneliness and social isolation negatively impact mental and physical health. The health consequences of loneliness and isolation are estimated to be greater than for obesity and smoking combined. The public health measures enacted during the COVID-19 pandemic also had the unintended side effect of increasing social isolation. Fruit flies (Drosophila melanogaster) are a social species that demonstrate cooperative behavior and a preference for social stimuli over non-social stimuli. We assessed the effects of chronic social isolation on social interactions and locomotion within an arena, and on sleep. Flies were isolated throughout development and for 5 days as adult flies, and behavioral tests were conducted on 5do adults. We found isolated males have altered social interactions, decreased locomotion, and fragmented sleep at night compared to grouped housed males. We are currently analyzing the female social interaction data. Isolated females also show reduced locomotion, but no nighttime sleep disturbances compared to grouped housed females. We are also testing whether flies isolated during development versus flies isolated in adulthood have differing sleep behavior. We hypothesize developmental and adult isolation would both result in sleep alterations, but perhaps they may differ in the specific aspects or magnitude of sleep disturbances. This research is significant because determining the behavioral effects of social isolation lays the foundation for future research that will examine how isolation alters the brain to cause these behaviors. Investigating these cellular mechanisms is critical for attenuating negative consequences of social isolation.

AP St. Louis1, J Yu1, JS Dason1

Chronic pain can develop with the chemotherapy medication Taxol, in which prolonged use can result in nociceptive hypersensitivity. Neuronal Calcium Sensor 1, the human orthologue for Drosophila calcium binding proteins Frequenin1 (Frq1) and Frequenin2 (Frq2), has been shown in vitro to be reduced upon Taxol treatment through cleavage by the calcium-dependent protease Calpain (Calp). We hypothesize that Frq1 and Frq2 are required for nociception and that changes in Frq1 and Frq2 expression is an underlying mechanism of Taxol induced-nociceptive hypersensitivity. To determine if Frq1 and Frq2 are required for nociception, we examined frq1 and frq2 mutants and found that these mutant larvae displayed nociceptive hypersensitivity to thermal stimuli in comparison to their genetic controls. Next, we used CRISPR-Cas9 to tag the endogenous frq1 and frq2 genes with a flag epitope. We found that Frq1 and Frq2 are widely expressed in the Drosophila central nervous system, and this localization overlaps with the nociceptive circuit. We additionally found that Taxol-treated control larvae phenocopied frq1 and frq2 loss-of-function mutants, and overexpression of Frq prevented nociceptive hypersensitivity. Lastly, we used CRISPR to mutate the Calp cleavage site on Frq1 and Frq2 from Isoleucine-35 to Alanine-35, which was shown in vitro to prevent Calp cleavage, and are currently testing to determine if this protects against Taxol-induced nociceptive hypersensitivity. Collectively, our data demonstrates a novel role for Frq1 and Frq2 in nociception and Taxol induced-nociceptive hypersensitivity.

1 Department of Biomedical Sciences, University of Windsor, Windsor, Ontario, Canada.

Funding Support: CIHR

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

When presented with acoustic stimuli, zebrafish larvae integrate stimulus characteristics, context, and their recent history to select if and how to respond. One aspect of this decision-making is habituation learning, where individuals diminish their response to repeated innocuous stimuli, allowing them to focus on more relevant stimuli. We have identified that 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 the neural mechanisms through which ap2s1 regulates learning, we are using live calcium imaging to compare neuronal activity patterns during habituation between ap2s1 mutant and sibling zebrafish. We are focusing on several candidate neural populations that regulate acoustically-evoked responses: the pair of bilateral command-like Mauthner cells (M-cells) which directly initiate rapid escape responses, and hindbrain glycinergic inhibitory neurons including a cluster of feedforward inhibitory neurons directly synapsing on the M-cells. This work will test the roles of these populations in acoustically-evoked habituation learning, and parse the neuronal and functional impacts of ap2s1 on this circuitry. By investigating how zebrafish ap2s1 regulates habituation, we may further understand the impact of AP2S1 on human learning and cognitive function.

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

Funding Support: NIH R15EY031539

AL Simal 1, J Tuling 1, G Descalzi 1

Chronic pain impacts 25% of Canadians aged fifteen and above, especially marginalized groups, with women making up 67% of those affected. Despite this, pre-clinical research has predominantly prioritized male rodent models, leaving a knowledge gap regarding female chronic pain mechanisms.

Mounting evidence indicates neuroplastic changes within the anterior cingulate cortex (ACC) as pivotal in chronic pain development. Responding to neuronal activity, the astrocyte-neuronal lactate shuttling (ANLS) can rapidly provide lactate to neurons, to meet large metabolic demands required for neuroplasticity. However, its role in chronic pain-induced neuroplasticity remains unknown.

This study investigates ANLS in the ACC of female and male mice, exploring its involvement in chronic neuropathic pain development. Using the spared nerve injury (SNI) model in adult (8wks) female and male C57BL/6 mice, we assessed gene expression in the ACC of ANLS pathways at 5, 14, 30, and 60 days post-surgery using RT-qPCR. We also confirmed mechanical allodynia for each timepoint using the Von Frey Test prior to sample collection.

Despite similar patterns of SNI-induced pain hypersensitivity in both sexes, we found that long-term SNI increased ANLS-related gene expression in the ACC of male but not female mice. We thus conclude that neuropathic pain affects ANLS in the mouse ACC in a sexually dimorphic manner. Furthermore, these observed sex differences highlight the need to include both females and males when investigating novel molecular targets for chronic pain treatment, deepening our knowledge of pain chronification.

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

Funding Support: Funds provided by the Canadian Pain Society Early Career Investigator Pain Research Grant and an NSERC DG.

Brian P. Wang1 , Nailah Fatima2 , Munich Villanueva2 , and Fred W. Wolf1,2 1 QSB Graduate Program, UC Merced, CA 95343 2 Department of Molecular and Cell Biology, UC Merced, CA 95343

Drosophila continually sense the external environment and internal signals to drive behavioral choices that optimally fulfill current needs, like finding and drinking water when thirsty. Water seeking, unlike water taste and ingestion, can be effortful, and so it is weighed against water availability, temperature, and competing needs. The circuity controlling water seeking requires AstA-expressing neurons (Janu-AstA) to promote thirsty water seeking with positive valence, and to inhibit feeding behavior. Janu-AstA project from the sensory processing subesophageal zone to the higher order processing region, the superior medial protocerebrum (SMP), which contains significant volume sexual dimorphism. Other behavioral circuits recruit the SMP, but it is unclear how these circuits are integrated in choice determination for complex situations. We uncovered water seeking properties in Janu-AstA output neurons that also exhibit sex-specific behaviors. Janu-AstA synapse onto the NPF-Ms (male-specific Neuropeptide F neurons that suppress male-male courtship) and the OviINs (female-relevant GABAergic oviposition inhibitory neurons). The NPF-Ms and the OviINs promote water seeking when inhibited in males. Surprisingly, the water seeking function of Janu-AstA is also male-specific, mirroring the NPF-Ms. Females use a distinct circuit for thirsty water seeking. Our results demonstrate the intersection between behavioral circuits where Janu-AstA relays thirsty water seeking drive to courtship and oviposition circuits that may coordinate task- and sex-appropriate responses. The male-specificity of water seeking in Janu-AstA is likely transmitted by the NPF-Ms or other sexspecific SMP circuitry. Our results suggest that neural circuits innervating the SMP may be endowed with sexually dimorphic functions, thus it is important to look closer at the SMP to understand how complex behaviors can be modulated by the sex of the animal.

Akhila Eswaran¹, Anna Phan¹

Over the past several decades, the focus of research has mainly been on the identification of genes required for normal memory processes. Recent studies, however, have shown that genes functioning to suppress memory also exist. sec22 was identified to constrain memory as a novel memory suppressor, through a targeted small scale RNAi memory screen conducted using Drosophila melanogaster. The ubiquitously expressed sec22 plays a pivotal role in the transport of vesicles between the endoplasmic reticulum and Golgi apparatus. However, the role of sec22 in learning and memory is unclear. Using the aversive olfactory behavioral assay, we found that knockdown (KD) of sec22 in all neurons, dopamine neurons and mushroom body neurons (MBNs), significantly improved memory due to a specific enhancement of learning. We also found that this effect is due to sec22’s function during development as well as in adulthood, using a temporal gene expression system (GAL80^ts). sec22 is part of the Synaptobrevin family of genes consisting of the vesicle fusion and secretion associated genes, ykt6, vamp7, syb and nsyb. Intriguingly, we found that ykt6 KD in MBNs enhanced learning while KD of vamp7, syb and nsyb, impaired it. To unveil sec22’s mechanism of memory suppression, we will test its KD effects on live neuronal function, neuronal anatomy and neurotransmission. Characterizing sec22 as a memory suppressor may reveal key insights into cellular mechanisms of learning and memory which may help reveal therapeutic targets for memory loss associated neurological disorders.

¹Department of Biological Sciences, University of Alberta, Edmonton, Canada.

Funding Support: Natural Sciences and Engineering Research Council grant RGPIN-2020-04009

FD Rogers 1,2, R Mallarino 2, CJ Peña 1

The neural mechanisms that underly naturally occurring paternal care remain largely undiscovered. In this work, we have leveraged the natural behavior of African striped mice (Rhabdomys pumilio) in conjunction with histology and single nucleus RNA-sequencing to uncover putative neural mechanisms of paternal care. African striped mice are murine rodents in which both paternal and male alloparental care is naturally occurring and common. Using brain-wide cFOS quantification, we have identified a significant positive correlation (p < .005) between immediate early gene activity (i.e., cFOS+ cells by IHC) in the medial preoptic area (MPOA) and paternal phenotypes (vs. infanticidal and ambivalent phenotypes) in sexually naïve male striped mice. To further determine whether distinct cell types are activated or show variable molecular profiles corresponding to behavioral phenotype, we applied single-nucleus RNA-sequencing to the MPOA of 20 individuals across phenotypes: infanticidal (in Mus musculus and African striped mice), allopaternal, paternal (i.e., genetic fathers), maternal (i.e., genetic mothers), and behaviorally naïve controls. Within African striped mice, we find that the general cell-type composition of the MPOA is comparable across the five contrasting phenotypes. However, at variable levels (i.e., all neurons, subtypes of neurons, and activated neurons), we have identified divergent patterns of gene expression in contrasts between caring and non-caring phenotypes. Progress in understanding the mechanisms of paternal care has long been hampered by inaccessibility of modern molecular tools in novel, naturally paternal species. This combination of approaches gives us unparalleled access to finally defining molecular and cellular mechanisms driving individual differences in paternal care.

1 Princeton Neuroscience Institute, Princeton University, Princeton, NJ; 2 Department of Molecular Biology, Princeton University, Princeton, NJ

Vangel Matic_­­­­­­^1,2, Klaus-Peter Ossenkopp^1,2, Martin Kavaliers^1,2

The mesolimbic dopamine pathway is known to be involved in mediating pathological motivational processes in drug addiction. In rodents, common drugs of abuse induce a sensitization of locomotor activity that is thought to depend on drug-induced neuroadaptations in the mesolimbic pathway. Sucrose, a highly palatable food, is known to increase mesolimbic dopamine signalling and enhance locomotor sensitization induced by drugs of abuse, however the effect of sucrose alone in facilitating locomotor sensitization has not been evaluated. Compared to male rats, females are known to have a greater preference and motivation for sucrose, suggesting that a sex difference may exist in sucrose-induced locomotor sensitization. The goal of this study was to identify if sucrose induces a sensitization of locomotor activity and evaluate if sex differences exist in this effect. Long-Evans adult male (n = 16) and female rats (n = 16) were given nine-day access to sucrose (0.3 M) or water for 30-min daily with activity assessment on the 1^st, 5^th and 9^th day immediately following fluid access. Female + sucrose rats showed an increase in activity that was not observed in other groups. Further, female + sucrose rats displayed greater activity than both male + sucrose and female + water groups while male + sucrose and male + water rats did not significantly differ. These results suggest that sucrose induces a sensitization of locomotor activity in female but not male rats, perhaps reflecting sex differences in the neuroanatomical organization of the mesolimbic pathway in development.

¹Graduate Program in Neuroscience and ²Department of Psychology, Western University, London, Ontario, Canada

Funding Support: Natural Sciences and Engineering Research Council of Canada (NSERC).

Perepelkina O.V., Poletaeva I.I.

The mouse strains, selected for “plus” and “minus” puzzle-box solution, are now at F9 “stage”. The puzzle box test @evaluates the ability of an animal to use the “object permanence” rule (the object perceived recently could be found even if not seen anymore). The statistically significant interstrain differences are still present between “plus” and “minus” strain mice in test presentations when the underpass (leading to safe dark box compartment) was blocked by a (removable) plug. In F6, F7 and later the differences were found also in the neophagophobia test (new food in new environment) as well as in the test on attention. During the latter test an animal is presented with an object, which moves along the circular experimental box. The approaches to this object as well as exploration behavior were registered during the object presentation. There were 5 different object (plastic toys, small plastic spoon and discs) shown to animals in the chance sequence. The expression of active attention in mice of “plus” strain was higher (indexed by different variables), than in “minus” mice. The comparison of recent memory in mice of F5-F8 also demonstrated that in “plus” strain this character had higher scores, than in “minus” mice. The increased attention scores reported here are another evidence of the prevalence in cognitive traits expression in selected strain “plus”. Earlier we described that while trying to penetrate into the dark compartment of the puzzle-box “plus” mice removed the plug successfully, while “minus” mice failed to do so in significantly higher proportions of cases. Although “minus” mice tried to do so - they actively manipulated the plug, but were unable to remove it. We may conclude that that both – plus and minus mice “understand” the object permanence rule, as they try to use the hidden underpass. But only “plus” strain animals were able to realize such solution. Together with increased attention abilities this could mean that “plus” mice are superior in “executive” functions expression.

Lomonossov Moscow State University, Moscow, Russia

Supported by Russian Scientific Foundation N 23-25-00042

Christian Del Valle-Colón¹, Miguel J. Álvarez-Cortés¹, Sebastián I. Morales-Cancio¹, Airined Montes-Mercado¹, Nicolás L. Fuenzalida-Uribe^1,2, José L. Agosto¹, Alfredo Ghezzi¹

Alcoholism is a condition characterized by behavioral and physiological changes, which can significantly impact interpersonal relationships. Through disrupts circadian rhythms, sleep patterns and genetic expression, after a prolonged alcohol consumption induces neuroadaptations, leading to tolerance and dependence. Drosophila melanogaster has emerged as an effective model for studying alcohol addiction due to its behavioral responses to alcohol that closely resemble those observed in humans. We explored the impact of Tip60 knockdown on the ventrolateral neurons (LNv) that regulate the sleep/wake cycle in Drosophila. We hypothesized that Tip60 would have a major role to acquire ethanol tolerance. Using the UAS-Gal4 system to knockdown Tip60 expression into LNv neurons (pdf-Gal4/UAS-Tip60-RNAi flies) we record the group flies activity during alcohol exposure and measure alcohol sensitivity and tolerance in age-matched adult female flies. For other hand, we assess to brain expression of the neuropeptide PDF to study Tip60 regulation, analyzing his branching pattern. We found that Tip60 knockdown flies display significant alcohol sensitivity and reduced alcohol tolerance. Moreover we found that Tip60 knockdown in LNv leads to a significant reduction in PDF branching and the ethanol exposure exacerbates this reduction in pdf-Gal4/UAS-Tip60-RNAi flies, decreasing PDF brain branching. These results suggest that Tip60 in LNv is involved in regulation of alcohol tolerance acquisition and brain branching patterns of PDF neuropeptide. Understanding the molecular and cellular mechanisms underlying ethanol neuroadaptations, potentially leading to the identification of new therapeutic targets for alcohol-induced disorders.

¹ Department of Biology at University of Puerto Rico, Río Piedras Campus, PR

² Institute of Neurobiology, San Juan, PR

Johal K.S1, Youssef S.A.1, Stefanelli G.1*

Snf-2-related CREB-binding protein activator protein (SRCAP) is a histone chaperone specific for the histone variant H2A.Z that is mutated in numerous neurodevelopmental disorders. Across development, H2A can be removed and deposited at different genes to regulate their expression and assist in developmental transitions. We hypothesize that SRCAP regulates neurodevelopment by incorporating H2A.Z at different genes. We show that the knock-down of Srcap in N2A cells leads to a reduction of H2A.Z at candidate genes and altered gene expression. In addition, we show that depletion of Srcap impairs retinoic acid (RA) induced N2A differentiation and impairs gene expression in N2A cells. Overall, our data strongly support a role for Srcap in neuronal differentiation and neurodevelopment by regulating H2A.Z genomic levels and location.

1 Department of Biology, University of Ottawa (ON), Canada * To whom correspondence should be addressed: gilda.stefanelli@uottawa.ca

Samina Panjwani¹, Oren Princz-Lebel¹, Miguel Skirzewski¹, Lisa Saksida^1,2, Marco Prado^1,2, Tim Bussey^1,2

Introduction:Synucleinopathies are a group of neurodegenerative disorders characterized by the abnormal misfolding and aggregation of the protein alpha-synuclein (α-syn) throughout the nervous system. Evidence suggests that α-syn aggregation disrupts critical neural networks, which may underlie many motor and non-motor deficits. Recently, there has been a shift towards understanding cognitive, as they have the potential to serve as early biomarkers for these disorders. A key cognitive domain of interest is attention, as patients with synucleinopathies often present with impaired sustained and selective attention, yet the mechanisms and molecular basis underlying these deficits are not well understood.

Methods:Human-derived α-syn pre-formed fibrils (PFFs) will be injected unilaterally into the striatum of a transgenic M83 line of mice. The PFFs will mimic alpha-synuclein pathology, creating a mouse model for synucleinopathies. The mice will then be tested on the Continuous Performance Task (CPT), a widely used test to assess attentional processing in human patients. Using cutting-edge rodent touchscreen cognition devices, the mice will perform a rodent CPT paradigm analogous to those used on humans to investigate selective and sustained attentional deficits.

Expected Results:We expect to see impairments in sustained and selective attention in the synuclein mice on the CPT touchscreen task.

Discussion:Synucleinopathies are currently classified as an uncurable group of disorders, and the development of cognitive impairments can be debilitating. A stronger understanding of how α-syn pathology can impair attention can guide the development of novel disease-modifying treatments and reduce the burden on the healthcare system.

1Program of Neuroscience, 2| Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, London, Ontario, Canada.

Funding Support: Initiative for Translational Neuroscience (ITN)

D. N. Linsenbardt, N. M. Maphis, M. D. Morningstar

Regularly drinking alcohol to intoxication, or binge drinking (BD), has very recently emerged as a modifiable risk factor for the development and progression of Alzheimer’s disease (AD). To evaluate potential neurobehavioral mechanisms mediating this increased AD risk, we recorded neural activity in alcohol consuming mice that develop pathological Tau (pTau; ‘P301S’). We were surprised to observe extremely large and rhythmic depolarization events known as spike and wave discharges (SWDs) commonly associated with absence seizures, and quickly confirmed the existence of many previous reports of this phenomenon in other mouse models of AD, but not P301S. Importantly, we did not observe SWDs in non-transgenic (nTG) littermates. We discovered from the literature that this transgenic mouse was derived on a B6C3H/F1 background strain, that the C3H/HeJ substrain exhibits SWDs, and that this is due to the presence of a hypomorphic retroviral-like insertion mutation in the Gria4 gene that results in an 8-fold reduction in its expression. To test the possibility that this mutation is present in our P301S mice and somehow not in nTG littermates, we re-evaluated sequenced brain tissue from two separate brain regions (hippocampus and hindbrain) of experimental subjects from a larger study. We found two-fold differences in expression between brain regions, but no differences between P301S and nTG littermates. Thus, although we have no evidence to date supporting Gria4 mutation as the cause of our observed SWDs in P301S mice, it seems likely that there is at the very least an interaction with this AMPA receptor mutation that is masked at the level of its brain expression.

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

Acknowledgments: This work was supported in part by grant #s: AA025120, AA015614, The New Mexico Alcohol Research Center (P50-AA022534), and the Substance Use Disorders Grand Challenge Initiative supported by the Center on Alcohol, Substance use, And Addictions (CASAA).

O El-Deeb1

The alternatively-spliced fruitless gene contributes to species-isolation in Drosophila. Our lab has shown that P2 transcripts, specifically, influence female receptivity to male courtship. Neurons that express P2 transcripts were visualized in the central nervous system, and hyperactivation and silencing of those neurons led to rejection of male courters. Consequently, the objectives of my work are to further specify the neurons that regulate female receptivity and explore the genetic mechanisms behind their functions.

I used a Gal4-driver and a set of UAS-linked responder genes to replicate the hyperactivation and silencing of the neurons that express P2. I then added the GAL4 repressor, Gal80, to repress GAL4 activity specifically in the thorax and abdomen. The results were D. melanogaster females who only had the P2 neurons in their heads hyperactivated or silenced. In courtship assays, these females mated at control levels, indicating that the critical P2 neurons for regulating receptivity are in the ventral nerve cord. The same concepts are now being used to explore whether these neurons act through distinct mechanisms of fruitless or in conjunction with other genes such as doublesex, pickpocket, and Sex-peptide (SP).

Sex-peptide, a gene that produces a seminal protein transferred to females after copulation, has emerged as a candidate. Sex-Peptide receptors in the uterus bind Sex Peptide and trigger a neural cascade that results in rejection of subsequent courters. Fluorescent imaging and genetic modification of neurons that co-express P2 fruitless and Sex-peptide-receptor provide promising evidence of the neurological niche of fruitless in female mating behaviours.

Department of Biology, Western University, London, Ontario, Canada

Mírian Velten Mendes^1*, Renato Elias Moreira Júnior¹, Mariana Siqueira Amormino¹, Guilherme Henrique de Asevede¹, Isabela Nascimento¹, Tatiani Uceli Maioli², Ana Maria Caetano Faria², Ana Lúcia Brunialti Godard¹

Alcohol Use Disorder (AUD) is a complex condition with many different aspects and effects in the body, and one that has come to light recently is how the changes it causes in gut microbiota could affect addictive behaviors. Disruption of intestinal barrier (leaky gut), and dysbiosis, leads to translocation of bacterial DNA into the bloodstream inducing the chronic inflamed state common in AUD. Moreover, there is a reduction in butyrate producing bacteria and changes in concentration of other Short-Chain Fatty Acids (SCFA). Those factors can trigger epigenetic changes and weaken the gut, causing leaky gut. Still there a lot of microbiome changes yet to be documented in AUD. Said that, our research group aims to elucidate these changes using a mice model of alcohol free-choice model to simulate AUD. Our finding reveals that, (1) cecum and colon microbiome from mice that consumed EtOH 10% did significantly chance, (2) microbiome changes directly affect metabolic pathways (3) genes associated with bacterial population abundance changes are corelated with human genes known to cause genetic metabolic diseases. We hypothesize that these findings could lead to elucidating how changes in metabolites concentration affect behavior, and try to propose new targets for treatment using probiotics,

1 Laboratório de genética Animal e Humana. Departamento de genética, ecologia e evolução; Universidade Federal de Minas Gerais – Brazil.

2 Laboratório de Imunobiologia. Departamento de Bioquímica e Imunologia; Universidade Federal de Minas Gerais - Brazil.

Funding Support: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG APQ-04517-22), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Pró-Reitoria de Pesquisa - UFMG (PRPq-UFMG), and Pós-Graduação em Genética – UFMG, Brazil.

EA Mehrhoff1,2, L Bunch1, M Bower1,2, N Fowler1, E Yang1, L Hendricks3, G Verner1, H Lee4, C Aki1, M Branney1, A Funke1, MA Ehringer1,2

Multiple large Genome Wide Association Studies (GWAS) of alcohol behaviors identified a single nucleotide polymorphism (SNP) in the human gene for the glucokinase regulatory protein (GCKR). SNP rs1260326 corresponds to an amino acid change at position 446 from Proline to Leucine (P446L), where the P allele is associated with increased levels of consumption (“risk” allele). Gckr 446P and 446L mice have been tested for voluntary alcohol consumption, early measures of alcohol response, and alcohol metabolism. Female PP and PL mice voluntarily consume and prefer alcohol compared to the female LL mice (p=.003), consistent with the direction of association in human genetic studies. However, there are no differences in voluntary alcohol consumption due to genotype among the males. The alcohol intake differences in females do not appear to be due to taste preference, because there are no differences in saccharin or quinine intake in either sex. No genotypic differences are found in the drinking-in-the-dark “binge” model of alcohol consumption or in the rate of alcohol metabolism. These animals were also tested for early measures of alcohol response using loss of righting reflex (LORR), balance beam, and stationary dowel test. However, there are no genotypic differences in response for any of these behaviors, so our focus is now on assessing chronic tolerance in these mice. Characterization of this Gckr P446L mouse model will aid in understanding the mechanism of GCKR’s effect on alcohol behaviors in humans, where we hypothesize a liver-brain axis is at play.

1Department of Integrative Physiology, University of Colorado Boulder, 2Institute for Behavioral Genetics, University of Colorado Boulder, 3Department of Asian Languages and Civilizations, University of Colorado Boulder, 4Department of Molecular, Cellular & Developmental Biology, University of Colorado Boulder

Funding support: R03 AA026733.

Dibyadeep Datta, Ph.D.1,2*, Shengtao Yang, Ph.D.1*, Mary Kate P. Joyce, Ph.D.1*, Elizabeth Woo, M.A.1, Steven McCarroll, Ph.D.3, Guillermo Gonzalez-Burgos, Ph.D.4, Isabella Perone, M.A.1, Stacy Uchendu, B.S.1, Emi Ling, Ph.D.5, Melissa Goldman, Ph.D.5, Sabina Berretta, Ph.D.6,7, John Murray, Ph.D.2, Yury Morozov, Ph.D.1, Jon Arellano, Ph.D.1, Alvaro Duque, Ph.D.1, Pasko Rakic, M.D.1, Ryan O’Dell, M.D.2, Christopher H. van Dyck, M.D.1,2, David A. Lewis, M.D.4, Min Wang, Ph.D.1,§, Fenna Krienen, Ph.D.8,§, Amy F.T. Arnsten, Ph.D.1,§

*Equal contributions

Background: The risk of mental disorders is consistently linked to mutations in CACNA1C (encoding L-type-calcium-channel Cav1.2) but it is not known why these channels are so critical to cognition, and whether they affect the layer III pyramidal cells in dorsolateral prefrontal cortex (DLPFC) that are especially vulnerable in cognitive disorders.
Methods: The design included transcriptomic analyses from human and macaque DLPFC, and connectivity, subcellular localization, cell-type specific protein expression, physiology and cognitive behavior in macaques.

Results: Layer III pyramidal cells in DLPFC express a calcium-related functional interactome, with elevated CACNA1C, GRIN2B, KCNN3 and CALB1, and protein co-expression, concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type-calcium channels powerfully influenced neuronal firing needed for working memory, where either blockade or increased drive by β1-adrenoceptors, reduced neuronal firing, the latter via SK potassium channel opening. An L-type-calcium channel blocker or β1-adrenoceptor antagonist protected working memory from stress.

Conclusions: The L-type calcium channel Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B) and calbindin (CALB1) are enriched in the layer III pyramidal cells in DLPFC most vulnerable in cognitive disorders, where these channels powerfully influence neuronal firing needed for working memory. The finding that either inadequate or excessive L-type-calcium-channel activation reduced neuronal firing explains why either loss- or gain-of-function mutations in CACNA1C increase risk for cognitive disorders. The selective expression of calbindin in these DLPFC pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer’s disease tau pathology emerging when calbindin is lost with age or inflammation.

Department of Neuroscience1 and Psychiatry2, Yale University School of Medicine, New Haven, CT USA; Department of Genetics3, Harvard Medical School, Boston, MA USA;
Departments of Psychiatry and Neuroscience4, University of Pittsburgh, 3811 O'Hara Street, Pittsburgh, PA USA; Stanley Center for Psychiatric Research5, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Basic Neuroscience Division6, McLean Hospital, Belmont MA USA and Department of Psychiatry7, Harvard Medical School, Boston, MA USA; Princeton Neuroscience Institute8, Princeton University, Princeton, NJ, USA

Josianne Clavel^1,2, Karine Doiron², Serge McGraw^1,2,3

During embryonic development, DNA methyltransferase 3A (DNMT3A) plays a crucial role in DNA methylation, an epigenetic mechanism regulating gene expression. Pathogenic alterations in DNMT3A are implicated in Tatton-Brown-Rahman syndrome (TBRS), a condition characterized by abnormal cognitive impairments, intellectual disability, facial dysmorphia, and overgrowth. To better understand the effects of DNMT3A deficiency on brain development, we generated a mouse model with a heterozygous Dnmt3a loss-of-function mutation (R684H) recreating a mutation observed in a TBRS patient (R688H). Our hypothesis suggests that this mutation disrupts the establishment of DNA methylation profiles, which leads to alterations in cell function during brain development. To uncover the impact on brain function, we performed a variety of behavioral tests (open field, elevated plus maze (EPM), marbles burying task, rotarod, gait analysis, four-limb suspension, and balance beam test) on 40-day-old Dnmt3a^R684H/+ mice, as the homozygous mutation is lethal postnatally. Our assessments reveal traits consistent with those observed in individuals with TBRS, including behavioral abnormalities, obesity, signs of neurodegeneration, and ataxia-like behavior. The Dnmt3a^R684H/+ mice show motor deficits, including reduced performance on the rotarod test and difficulties with suspension strength and balance tasks. These impairments suggest compromised motor function and neuromuscular coordination in the mice. These results will now enable us to narrow our focus on specific brain regions (e.g., cerebellum, cortex) likely to be influenced by these assessments. Thus, these results pave the way for a deeper understanding of the underlying mechanisms governing disorders of brain development and uncover how DNMT3A pathogenetic variants lead to the neurological complications associated with TBRS.

1 Department of Biochemistry, Faculty of Medicine, Université de Montréal, Canada

2 CHU Sainte-Justine Research Center, Montreal, Canada

3 Department of Obstetrics and Gynecology, Faculty of Medicine, Université de Montréal, Canada

Karine Doiron¹, Diego A. Camacho-Hernandez^1,2, Anthony Lemieux¹, and Serge McGraw^1,3

DNMT3A is crucial for DNA methylation, controlling gene regulation in development. In brain development, DNMT3A suppresses germline- and pluripotency-specific genes by adding DNA methylation marks to their promoters in progenitor cells. Conversely, removal of DNA methylation from promoters of neuron-specific genes promotes their expression. Alterations in these processes are tied to neurological diseases. DNMT3A loss-of-function causes overgrowth and abnormal brain development, as in Tatton-Brown-Rahman Syndrome (TBRS). Our understanding of how DNMT3A mutations affect neural progenitor cell differentiation and contribute to neurological disorders is limited due to a lack of targeted studies, especially in patient-derived brain cells.

We hypothesize that DNMT3A loss-of-function disrupts DNA methylation marks and affects the programming of progenitors into terminally differentiated neurons, leading to neurodevelopmental disorders. We generated patient-derived induced pluripotent stem cells (iPSC) with a heterozygous DNMT3A mutation, that we differentiated into neuronal progenitor cells (NPC), cortical neurons and 3D organoids.

We show that DNMT3A loss-of-function in iPSC initiates alterations in DNA methylation and gene expression in NPC and cortical neurons. Notably, we observe alterations in the expression of genes pivotal for neurogenesis and cell fate commitment (e.g., WNT7A, NEUROD1), reminiscent of characteristic phenotypes observed in various neurodevelopmental disorders. Interestingly, DNMT3A loss-of-function leads to larger expansion of derived 3D cortical organoids, which show cortical disorganization due to dysregulated fate specification.

We show that DNMT3A loss-of-function in iPSC disrupts DNA methylation profiles during neural lineage specification. This interference triggers alterations in gene expression, impacting cell fate commitment and potentially resulting in aberrant functioning of TBRS brain cells.

¹Centre de Recherche du CHU Ste-Justine, Montréal, Québec, Canada

²Département de Biochimie et Médecine Moléculaire, Université de Montréal, Québec, Canada

³Département d’Obstétrique et Gynécologie, Université de Montréal, Québec, Canada

R Gibson1, K Simon2, M Vekariya3, A Sahibzada1, M Arai1, T Yildirim1, J Yue4, R Mistlberger1, B Kent1

Deprivation or continuous fragmentation of sleep suppresses the proliferation and maturation of new neurons in the hippocampus of adult rodents. A common type of sleep disruption occurs in humans working nightshifts, when sleep is displaced from night to day. We evaluated hippocampal neurogenesis in mice (C57BL6/j, ~3 months age) subject to sleep disruption procedures. A sleep deprivation group (n=6, 3 females) was deprived of sleep for 12 hours each day during the typical rest phase (12h lights-on) and left undisturbed during the active phase (lights off) for 7 consecutive days. A sleep fragmentation group (n=8, 4 females) were disturbed every 2 minutes during their rest phase for 7 days. The control group (n=14, 7 females) remained undisturbed in their home cage. Activity during the active phase was recorded via infrared motion sensors. Mice were injected with 200mg/kg of BrdU 2-hours prior to brain and blood sample collection at the end of the light period on day 7. Preliminary analysis suggests the week-long procedures induced an accumulating sleep deficit, as indicated by a progressive increase in rest bout duration and total rest time at night. Plasma corticosterone quantified by ELISA assay did not differ across conditions or between sexes; effects of the procedures on neurogenesis were independent of this stress hormone. To assess neurogenesis, brain sections spanning the range of the dentate gyrus were stained using immunohistochemistry. All sections have been imaged using a Nikon A1R Laser Scanning Confocal system and cell counting is ongoing.

1Department of Psychology, 3Department of Biological Sciences, 4Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, B.C., Canada

2Department of Molecular Medicine, Georg-August-Universität Göttingen, Göttingen, Lower Saxony, Germany

Funding Support: NSERC Discovery Grant (BA Kent) RGPIN/03909-2021

*G. Modara1,2, I. Schwein3, J. S. Snyder3, M. S. Madhav2.

Neurogenesis in the dentate gyrus continuously generates new granule cells (GCs) that gradually integrate into the hippocampal network and are required for functions such as learning, memory, and pattern separation. In mice, GC generation peaks at birth and declines with age. It’s been shown that adult-born neurons pass through immature critical periods of experience-dependent plasticity, which underlie their important role in hippocampal-dependent learning. However, the functional development of neurons born soon after birth remains unclear, even though they make up a large portion of the GC population and are likely in their critical period for plasticity in adolescence, when hippocampal memory functions develop. It is proposed that these developmentally born GCs become attuned to environmental features present during their adolescence, enabling enhanced learning of situations containing those features later in adulthood. We aim to test this proposal by exposing adolescent mice to textured visual stimuli and assessing their ability to discriminate these textures from other stimuli in adulthood. Our method includes a custom-built exposure setup and a touchscreen testing chamber that will be available as open-source material. Furthermore, considering the role of the hippocampus in the development of Alzheimer’s disease and depression, our work will establish a framework for how early-life experiences shape circuits in mental health disorders. Additionally, this project highlights the significance of open-source innovations in expanding the scope of scientific research, allowing researchers to bypass traditional financial constraints and promote a more inclusive and collaborative scientific community.

1Univ. of British Columbia, Vancouver, BC, Canada, 2Sch. Of Biomed. Eng., 3Dept. of Psychology,

Habibe K. Üçpunar¹, Haoze Li¹, Qian Lin¹

Memories serve as a toolbox for adjusting experiences to be used in favor of the survival and adaptation of the organism. Therefore, the duration of how long an experience is kept in memory varies. Although both classical and operant conditioning have been extensively studied in zebrafish, most of those studies require training with multiple sessions lasting for hours to generate a memory trace. Moreover, those memories usually do not last longer than 24 hours.

In this study, we investigated long-term memory formation by using an associative learning paradigm with a single conditioning event. We have built an Arduino-controlled behavioral setup where we established fear conditioning in juvenile fish by pairing a color (CS) with an electric shock stimulus (US). Fish were tested for fear memories in the form of freezing responses after 24h, 1 week, and 2 weeks. TLWT fish were used and tracked by using DeepLabCut. Although the control fish and most of the test fish did not show a freezing response to the stimulus during the two-week assessment, a portion of the test fish displayed freezing behavior in response to the conditioned color during this test period. The next task is to perform whole-brain imaging and observe the whole-brain dynamics for long-term fear memory retention by leveraging the small and transparent skull of juvenile fish. The findings of the study may shed light on differential memory retention mechanisms.

¹Department of Cell and Systems Biology, University of Toronto, Ontario, Canada

Funding Support: TUBİTAK 2219 postdoctoral fellowship to HKÜ

Richard E. Brown¹, Wyatt Ortibus¹, Kyle Roddick^1, Hong Lu² and Ann Marie Craig²

The incidence of epilepsy increases in people over 50 years of age and more people over 70 years of age develop epilepsy than young children. (Sen et al., 2020. Lancet 395:735-748). We discovered a novel mouse model that (1) spontaneously develops seizures with no external induction and (2) develops these seizures in old age. We tested mouse lines with mutations in the neurexin-1 synaptic organizing protein: newly generated Nrxn1+/- mice with one allele disrupted, Nrxn1 ΔS5/ΔS5 mice with altered splicing at splice site 5 (Hong et al., 2023. Cell Reports 42:112714), Nrxn1 ΔS5/- mice, and their WT (C57BL/J) control mice. We video-recorded spontaneous seizures and scored their severity on a 10-point revised Racine scale (Van Erum, et al., 2019. Epilepsy & Behavior 95:51-55). Mice began to show seizures at 11 months of age. We recorded seizures from 3 of 37 male and none of 19 female Nrxn1+/- and WT mice. However, 15 of 46 male and 9 of 47 female Nrxn1 ΔS5/- and Nrxn1 ΔS5/ΔS5 mice showed seizures. The seizures were scored from 1 to 9 on the revised Racine scale. Electrophysiological data from young mice showed elevated excitatory but not inhibitory synaptic activity in the Nrxn1 ΔS5/ΔS5 mice (Hong et al., 2023. Cell Reports 42:112714) but no elevation of excitatory synaptic activity in the Nrxn1 ΔS5/- mice. This suggests that the elevated glutamergic transmission associated with Nrxn1 ΔS5 altered the E/I balance and predisposed them to have seizures and suggests that drugs reducing excitatory transmission will reduce seizure activity.

¹Department of Psychology & Neuroscience, Dalhousie University, Halifax, Nova Scotia Canada B3H 4R2

²Djavad Mowafaghian Centre for Brain Health and Department of Psychiatry, University of British Columbia, Vancouver, BC V6T 2B5, Canada