Conference Agenda

Aya Arrar^1,2, Madison R. Longmuir^1,2, Kate M. Onuska^1-3, Bryan Lung^2,4,6,7, Scheila Schmidt², Arash Salahinejad², Lisa M. Saksida^2,4, Timothy J. Bussey^2,4, Tallulah S. Andrews^6,7, Taylor W. Schmitz^2-4, Vania F. Prado^2,4,5, Marco A.M Prado^{2,4, 5}

Apolipoprotein E (ApoE) exhibits isoform-specific interactions with Alzheimer’s disease (AD)-related pathology, promoting amyloid-β (Aβ) deposition and tau-mediated neurodegeneration. However, lack of appropriate preclinical models limits abilities to evaluate the synergistic effect of Aβ, tau and ApoE on cognition and disease progression. To overcome this, we generated mice which enable systematic combination of humanized variants of the ApoE genotypes (hApoE3 or hApoE4), amyloid (hApp^NL or hApp^NL-F) and tau (hMAPT). We then evaluated whether the interaction among these AD-related factors influences cognition and pathological load.

Attentional demand was assessed, using the cross-species Continuous Performance Task (CPT), an automated touchscreen test. Biochemical and imaging techniques including single-nuclei (sn)RNA sequencing, structural magnetic resonance imaging, immunofluorescence microscopy, and ELISA.

Attentional deficits were observed at 6,9 and 12 months of age in App^NL-F ApoE4 mice. Increased levels of insoluble Aβ, plaque burden, and size were observed in the cortex of App^NL-F ApoE4 mice at 9 and 12 months. snRNA sequencing indicated a reduction of excitatory neurons in the cortex and upregulation of gliosis related genes at 9 months in App^NL-F ApoE4 mice. ApoE4 mice also showed increased levels of cortical atrophy compared to Apoe3 mice.

Our results provide new insight into how amyloid, ApoE, and tau conspire to accelerate disease progression and affect cognition. We show that amyloid interacts with both ApoE3 and ApoE4 to drive cell type specific reduction of cortical excitatory neurons, with a faster progression rate in the ApoE4 background. Our findings also demonstrate cortical neurodegeneration and cognitive deficits prior and parallel to amyloid plaque formation.

1 Schulich Medicine and Dentistry, Western University, London, ON, Canada

2 Robarts Research Institute, Western University, London, ON, Canada

3 Lawson Health Research Institute, London, ON, Canada

4 Department of Physiology and Pharmacology, Western University, London, ON, Canada

5 Department of Anatomy and Cell Biology, Western University, London, ON. Canada

6 Department of Biochemistry, Schulich School of Medicine & Dentistry

7 Department of Computer Science, University of Western Ontario

WB Lynch1,2, SI Goldstein1,3, SA Miracle1,2, KK Wingfield1,3, R Bhandari1, ET Gerhardt1, A Farnan1, B Nguyen1, I Kazerani1, GA Saavedra1, O Averin4, BM Baskin1, JA Beierle1, MT Ferris4, CA Reilly5, A Emili6, CD Bryant1

Opioid Use Disorder (OUD) maintains epidemic proportions in the U.S. with limited pharmacological treatments. Sensitivity to the opioids’ rewarding properties has a genetic component and can predict addiction liability. We identified Zhx2 as a candidate gene underlying increased oxycodone (OXY) metabolite brain concentration in BALB/cJ (J) vs. BALB/cByJ (By) females. The metabolite, oxymorphone (OMOR), is more potent than OXY and could explain the enhancement of state-dependent learning of OXY conditioned place preference (CPP) in J vs. By females. A structural intronic variant significantly reduces in Zhx2 expression in J vs. By mice, which we hypothesize could enhance OMOR levels and OXY addiction-model behaviors. We are currently testing this hypothesis in Zhx2 knockout mice and measuring OXY metabolite levels and addiction model behaviors. Consistent with our hypothesis, Zhx2 KO females showed an increase in brain OMOR levels and OXY-induced locomotor activity compared to WT females. However, in contrast to our hypothesis, we found that state-dependent expression of OXY-CPP was actually decreased in KO females and increased in males. Brain proteomic analysis of Zhx2 KO mice identified multiple proteins implicated in small-molecule metabolism and inflammatory processes that could contribute to behavioral differences. We are currently conducting brain CUT&RUN-seq and bulk RNA-seq to complement the proteomic analyses and identify functional DNA-binding targets of Zhx2. Our work supports validation of Zhx2 as a quantitative trait gene underlying brain OMOR concentration and behavior and candidate quantitative trait mechanisms, which could increase our understanding of Zhx2 brain function and OXY addiction liability in humans.

1Laboratory of Addiction Genetics, Department of Pharmaceutical Sciences, Center for Drug Discovery, Northeastern University; 2Graduate Program for Neuroscience, Boston University; 3Biomolecular Pharmacology PhD Program, Department of Pharmacology, Physiology & Biophysics, Boston University Chobanian & Avedisian School of Medicine; 4Department of Genetics, UNC Chapel Hill; 5Center for Human Toxicology, University of Utah Health; 6Knight Cancer Institute, Oregon Health & Science University

Funding Support: NIDA U01DA050243-04, NIDA F31DA056217-01, Boston University Transformative Training Program in Addiction Science, NIDA Summer Research Internship Program, Boston University Research in Science and Engineering Program

DY Assaf1, JS Dason1

The Drosophila melanogaster foraging gene (for) encodes a cyclic GMP-dependent protein kinase (PKG) that regulates nociception. Furthermore, PKG expression is increased in nerve injury-induced nociceptive hypersensitivity mouse models. It is unknown if for expression changes in response to injury in Drosophila. Additionally, the mechanisms by which PKG expression are regulated in response to injury remains unknown. The histone methyltransferase G9a has previously been shown to regulate for mRNA expression, and we previously found higher for levels resulted in nociceptive hypersensitivity, whereas loss of for reduced nociceptive sensitivity. However, a direct link between for and G9a with respect to nociception has not been established. We used a thermal nociception assay and found that G9a null mutants displayed nociceptive hypersensitivity in comparison to their genetic control. Next, we examined FOR protein levels using Western blots and an antibody that recognizes all FOR isoforms. We found that G9a null mutants had a higher amount of FOR protein in comparison to the control. We then used an antibody specific for the FOR P1 isoform, which was previously shown to be important for nociception, and found that FOR P1 expression was upregulated in G9a null mutants. Current experiments involve examining the G9a for null double mutants to determine if this increase in FOR expression is required for the nociceptive hypersensitivity seen in G9a null mutants. Collectively, our data demonstrates that G9a negatively regulates nociception and FOR P1 protein expression.

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

Funding Support: CIHR

Catharine Rankin, Lexis D. Kepler, DM Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada

Glia play essential roles in nervous system development and regulating neuron function throughout the lifespan. The nervous system of the nematode, C. elegans, includes 56 glia cells which have recently been shown to directly modulate complex behaviours through multiple mechanisms such as controlled phagocytosis, neurotransmitter release, and clearance of neurotransmitters from the synapse. In this work, we assessed whether glia play a role in non-localized touch-induced reversals and habituation, a form of non-associated learning where one learns to stop responding to repetitive stimuli. We assessed knockout strains for genes documented to be important for different glia functions in C. elegans. Using our lab’s high through-put machine tracking system, we captured the effects on phenotypes spanning animal locomotion, morphology, sensory sensitivity, and habituation. We found genes involved in different glia functions altered response components of the non-localized harsh touch-induced reversals. In addition, we found that multiple genes involved in amphid sensory organ function share strikingly similar phenotypic impairments. Interestingly, the amphid sensory organ is not a component of the non-localized touch-induced reversal circuit. Taking advantage of the temporal and spatial specificity of the Auxin-Inducible Degradation system, we are currently working to create glia-specific degradation lines to confirm the involvement of glia in habituation and assess whether the impairments are caused by glia dysfunction during development or acutely during the behaviour.

A Bahi^1,2, JL Dreyer³

Patients with stress-triggered major depression disorders (MDD) can often seek comfort or temporary relief through alcohol consumption, as they may turn to it as a means of self-medication or coping with overwhelming emotions. The use of alcohol as a coping mechanism for stressful events can escalate, fostering a cycle where the temporary relief it provides from depression can deepen into alcohol dependence, exacerbating both conditions. Although, the specific mechanisms involved in stress-triggered alcohol dependence and MDD comorbidities are not well understood, a large body of literature suggests that the serotonin transporter (SERT) plays a critical role in these abnormalities. To further investigate this hypothesis, we used a lentiviral-mediated loss-off-function approach to examine the role of hippocampal SERT knockdown in social defeat stress-elicited depression like behavior and ethanol-induced place preference (CPP). The results showed that social stress prodepressant effects were reversed following SERT knockdown demonstrated by increased sucrose preference, shorter latency to feed in the novelty suppressed feeding test, and decreased immobility time in the tail suspension and forced swim tests. Moreover, and most importantly, social stress-induced ethanol-CPP acquisition and reinstatement were significantly reduced following hippocampal SERT knockdown using shRNA-expressing lentiviral vectors. Finally, we confirmed that SERT hippocampal mRNA expression correlated with measures of depression- and ethanol-related behaviors by Pearson’s correlation analysis. Taken together, our data suggest that hippocampal serotoninergic system is involved in social stress-triggered mood disorders as well as in the acquisition and retrieval of ethanol contextual memory and that blockade of this transporter can decrease ethanol rewarding properties.

¹College of Medicine, ²Center of Medical and Bio-Allied Health Sciences Research, Ajman University, Ajman, United Arab Emirates

³Division of Biochemistry, Department of Medicine, University of Fribourg, CH-1700, Fribourg, Switzerland

Funding Support: AB was supported by grants from Ajman University (2019-IRG-MED-1) and the National Research foundation (31M082). JLD received grants from the Swiss National Science Foundation 3100-059350 and 3100AO-100686

Neha Rajput¹, Matheu Wong¹, Dea Kanani¹, Ada Squires¹, Kush Parikh¹, Kailyn Fields¹, and Justin W. Kenney¹

Individual differences in behavior have been observed across wide range of taxa, including humans, rodents, and fish. However, the biological mechanisms that underlie these differences are not fully understood. To explore the neural mechanisms that underpin behavioral differences, we use adult zebrafish as a model. Previously, we have identified four distinct behavioral types in adult zebrafish when exploring a novel environment: bold, shy, wall-huggers and active explorers. To gain a better understanding of the neural basis of these differences, we developed tools for whole-brain activity mapping. We used situ hybridization chain reaction (HCR) to detect the expression of c-fos, an immediate early gene, as a means of labeling active neurons. To visualize brain-wide c-fos expression, we utilize a tissue clearing technique and light sheet microscopy to generate high resolution images. For automatic detection of c-fos positive cells, we have utilized CellFinder, a deep learning-based cell identification approach integrated into the BrainGlobe computational environment. Subsequently, the images were registered to our recently developed adult zebrafish brain atlas (AZBA) using advanced normalization tools (ANTs). We have successfully trained CellFinder to identify c-fos positive cells with an accuracy of 96% and found that c-fos expression peaks at 15-30 minutes following exposure to a novel tank. With this approach, we identified brain regions activation associated with bold and shy behavior.

¹Department of Biological Sciences, Wayne State University, Detroit, MI 48202

Funding Support: NIH grant from NIGMS: R35GM142566

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.

Burt M Sharp¹, Shuangying Leng², Caroline Jones¹, Hao Chen²

Most individuals affected in the national epidemic of oxycodone abuse began taking oral oxycodone by prescription. We studied vulnerability to oxycodone intake in a rat model of oral drug self-administration (SA), since pharmacokinetics affect abuse potential. Females (33 inbred strains) and males (26 strains) obtained oxycodone at increasing concentrations in operant sessions (FR5; 1-16-h) followed by extinction and reinstatement. Active spout licks were greater in females than males during 4-h and 16-h sessions (p<0.001 for all). Across all stages of oxycodone SA, intake/session was greater in females (p<0.001). Both sexes escalated intake during 16-h extended access vs 4-h sessions (p<2e-16). Intake and active licks varied greatly by strain. The heritability (h2) of active licks/4-h at increasing oxycodone dose was larger in males (h2 females: 0.30-0.39 vs. males: 0.41-0.53). Under a progressive ratio schedule, breakpoints differed by strain (p<2e-16) and by sex in some strains (p=0.018). For cue-induced reinstatement, active licks were greater in females than males (p<0.001). Behavior in naive rats was assessed using elevated plus maze (EPM), open field (OF) and novel object interaction. (NOI) tests. EPM-defining traits were most commonly associated with SA in both sexes, whereas more OF and NOI traits were SA-associated in males. Overall, sex and heredity are major determinants of the motivation to take and seek oxycodone, which escalates during extended access. The correlation of EPM, a measure of anxiety, with multiple SA parameters indicates the influence of pleiotropic genes. Funding provided by NIH/NIDA U01DA053672 and U01DA047638.

1. Department of Genetics, Genomics and Informatics, 2. Department of Pharmacology, Addiction Science And Toxicology. University of Tennessee Health Science Center, Memphis, TN.

Madison Longmuir^1,2, Aya Arrar^1,2, Kate M Onuska^1-3, Arash Salahinejad,² Tim J. Bussey^1,4, Lisa M. Saksida^1,4, Taylor W. Schmitz^2,3,4 Marco A.M. Prado^2,4,5, Vania F. Prado^2,4,5

Apolipoprotein (ApoE4) is the strongest genetic risk factor for late onset Alzheimer’s Disease (AD). In comparison with the most common isoform ApoE3, ApoE4 accelerates hippocampal amyloid-β deposition, increases the formation of neurofibrillary tangles, and increase hippocampal degeneration. Here we explore the interplay of AD related pathologies on cognition using knock-in mouse models that express humanized versions of App (hApp^NL or hApp^NL-F), hMAPT, and hApoE3 or E4 genes. We performed biochemical techniques including immunofluorescence microscopy, ELISAs (Aβ), and Western blots (tau) at different ages. Hippocampal dependent cognitive function was evaluated with the cross-species touchscreen-based Location Discrimination task that tests for pattern separation ability. Our analysis reveals that plaque pathology and insoluble Aβ are only present in App^NL-F (ApoE3 or ApoE4) mice. Levels of insoluble Aβ, plaque burden and neuroinflammation were increased in the hippocampus of App^NL-F ApoE4 mice in comparison to App^NL-F ApoE3 mice at 9, 12, and 16 months of age and at 6-7 months ApoE4 mice presented hippocampal atrophy. Behavioral analysis demonstrated that mice expressing Apoe4, both App^NL and App^NL-F, exhibit hippocampal dependent deficits at 6, 9, 12 months of age compared to Apoe3 mice. However, deficits were still exasperated in Apoe4 mice expressing the App^NL-F mutation with accumulation of amyloid plaques.

These new mouse models, along with translatable touchscreen cognition tests, can be used to explore mechanisms by which ApoE4 impairs adult hippocampal neurogenesis and plasticity, and consequently, patten separation. The mechanistic insights from these models will help to guide future work in patients at risk for AD.

1 Schulich Medicine and Dentistry, Western University, London, ON, Canada
2 Robarts Research Institute, Western University, London, ON, Canada
3 Lawson Health Research Institute, London, ON, Canada
4 Department of Physiology and Pharmacology, Western University, London, ON, Canada
5 Department of Anatomy and Cell Biology, Western University, London, ON. Canada

RM Jolicoeur Alfaro1, I Anreiter1,2, H Rodd1

Transgenerational epigenetic inheritance is the transmission of epigenetic modifications and altered phenotypes beyond the generations exposed directly or indirectly to a stressor. Stimulant drugs, such as cocaine and nicotine, can induce transgenerational changes in behavior through alterations to DNA methylation. Our group previously showed that chronic exposure to the commonly prescribed stimulant Ritalin (methylphenidate hydrochloride) in the first generation (G1) of Trinidadian guppies (Poecilia reticulata) led to alterations in exploratory behaviour that were present in the G1, as well as the untreated G2 and G4. Our aim was to investigate the details of this transgenerational transmission, both with regards to the specific behaviours transmitted and with the molecular mechanisms associated with this transmission. To identify potential mechanisms, we investigated the DNA methylation and transcriptomic patterns of several generations. Brains of G4 individuals were sequenced using reduced representation bisulfite sequencing and we identified several genes that were differentially methylated between the untreated G4 descendants of G1 control fish and G1 Ritalin-treated fish. In addition, the transcriptomes of G1 brains were investigated to identify genes that were differentially expressed after chronic Ritalin treatment. To uncover the specific behaviours that were inherited transgenerationally, we analyzed the individual behaviors that had previously been analyzed in combination. Our study provides insights into the molecular mechanisms underlying Ritalin-induced transgenerational effects on guppy brain function and into the specific behavioral alterations that were inherited. As the epigenetic effects of Ritalin exposure have not been previously studied, our results may have important implications for understanding the long-term effects of Ritalin exposure in humans.

1 Department of Ecology and Evolutionary Biology, University of Toronto, Ontario, Canada

2 Department of Biological Sciences, University of Toronto Scarborough, Toronto, Canada

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

Renato Elias Moreira Júnior¹ , Mariana Siqueira Amormino¹ , Luana Martins de Carvalho^1,2, Mírian Velten Mendes¹ , Guilherme Henrique de Asevede¹ , Isabela Nascimento¹, Tatiani Uceli Maioli³ , Ana Maria Caetano Faria³ , Ana Lúcia Brunialti Godard¹

Alcohol Use Disorder is characterized as a complex condition that may activate innate immune receptors in the striatum, a key region of the Mesolimbic Dopaminergic System. This potential activation could result in alterations in the transcriptional regulation of cytokines, inflammatory markers, and regulatory proteins (notably LRRK2). These changes are believed to induce neuroinflammation, which in turn could contribute to the behavioral alterations observed post-alcohol consumption. In addition, it appears that other factors also influence these behavioral outcomes, including the gut microbiota, neuropeptides and neurotransmitters genes, and epigenetic mechanisms. Given this background, our research group used a mouse model characterized by high alcohol consumption and preference to explore the connections between behavior, neuroinflammation, gut microbiota, transcriptional regulation, and alcohol. Our observations revealed that high alcohol consumption and preference led to (1) an increase in brain inflammatory cells; (2) upregulation of cytokines genes (Il6 and Il1β) and pro-inflammatory signals (Inos and Nfat), downregulation of the anti-inflammatory cytokine (Il10) and the Lrrk2 gene in the striatum; (3) alterations in the transcriptional and epigenetic regulation of genes linked to neuropeptides and neurotransmitters; (4) changes in the composition and structure of the gut microbiota, and (5) behavioral alterations marked by decreased anxiety and depression like behaviors along with an increase in compulsive-like behavior. These findings indicate that the interaction among the immune system, behavior, gut microbiota, and the brain epigenetic and transcriptional profile plays a critical role in high alcohol consumption and preference. We hypothesize that these findings are intricately linked via the gut-brain axis.

¹ Laboratório de genética Animal e Humana. Departamento de genética, ecologia e evolução; Universidade Federal de Minas Gerais – Brazil. ² Molecular Medicine Research Building. Department of Pharmacology and Toxicology; Virginio Commonwealth University – USA. ³ 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 (PRPqUFMG), and Pós-Graduação em Genética – UFMG, Brazil.

MR Evans, RT Yost, AF Simon

Social spacing is the distance organisms choose to settle from their closest neighbours. Many species display this form of social behaviour, including Drosophila melanogaster, and the underlying neural circuitry of social spacing is under investigation.A region of the fly brain considered the functional equivalent of the mammalian hippocampus, the mushroom bodies (MBs), is known to be important for social space and is neuromodulated by extensive dopaminergic signalling. Dopamine (DA) is known to play a role in controlling social space, but the specific involvement of the four DA receptors to which DA binds is currently unexplored (Dop1R1, Dop1R2, Dop2R, DopEcR).I generated fly lines that use the Gal4/UAS system to drive RNA interference against each DA receptor in the MBs of the fly brain, silencing receptor expression in this region. Flies were then tested in the social space assay to assess the impact of DA receptor knockdown on social spacing.Silencing any of the four DA receptors in the MBs significantly increased social space in males, causing flies to settle farther apart. In contrast, females exhibited no disruption to social spacing when any of the DA receptors in the MBs were silenced. These findings imply that sexually dimorphic circuitry controls social spacing. This work furthers understanding of social spacing in D. melanogaster and may provide insights into social space regulation in other organisms, as pathways underlying social behaviour are likely conserved in many species.

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

Funding support: NSERC USRA to MRE, NSERC Fellowship to RTY and NSERC Discovery grants 05054-2022 to AFS

Wyatt Ortibus¹ Kyle Roddick¹ Ian Weaver¹ Richard Brown¹

Individual differences in learning and memory performance of mice introduces within-group variability to experimental data and masks genotype, age and sex differences in measures of cognition. These individual differences provide insight into the different learning strategies of mice in olfactory discrimination and reversal learning. Using an operant olfactometer we tested the cognitive flexibility in wildtype (C57BL/6J) and Nrxn1+/- mice which were developed as a model for human Autism. Mice were trained to discriminate an initial odor pair (A vs B) to a criterion of 85% accuracy, then to a criterion of 85% accuracy on a second odor pair (C vs D) and then to a criterion of 85% accuracy on reversal learning of the second odor pair where the rewarded outcomes are switched (CS+ becomes CS- and vice-versa). We used signal detection theory to examine the reversal-response patterns in the olfactometer based on four possible outcomes: hits, misses, false alarms (FA), and correct rejections (CR). Using these responses we coded reversal learning performance into four different learning strategies which we define as Perseverance (Misses & FA), Ignore Both Stimuli (Misses & CR), Respond to Both Stimuli (FA & Hits) and Respond Only to CS+ (Hits & CR). These phases of reversal learning allow us to analyze individual differences in performance and to determine genotype effects in decision making strategies that are missed when only percent correct is analyzed.

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

A Attaran 1, M Skirzewski 1, O Princz-Lebel 1,2, V Novikov 1,2, R Sandoval 1, D Palmer1, A Panchbhaya 3, V Mendez Diaz 3, JA McPhail 4, A Louwrier 4, LM Saksida 1,2,3, TF Busy 1,2,3, VF Prado1,2,3,5, MAM Prado1,2,3,5.

Synucleinopathies are neurodegenerative disorders characterized by abnormal accumulation of misfolded alpha-synuclein (a-Syn) protein in aggregates known as Lewy bodies in different brain areas. The dorsal striatum is associated with motor and cognitive impairment in synucleinopathies like Parkinson’s disease and dementia with Lewy bodies and dysregulation of neurochemical signals is likely implicated in these diseases. Dopamine is one of the key neurotransmitters regulating striatal function and its optinal activity is required for control of cognition, learning and memory, and movement. In this study, we investigated how toxic a-Syn compromises neurochemical circuits involved in cognitive function in a mouse model of synucleinopathy. M83 hemizygous mice were injected with pre-formed fibrils (PFFs) into the dorsal striatum, and behavioral and dopamine dynamic changes were assessed through the visuomotor conditional learning (VMCl) task on a touchscreen, coupled with dopamine dynamic recordings using fiber photometry and a genetically-encoded dopamine sensor (GRABDA). Mice injected with PFFs presented a significant cognitive deficit compared to controls, with a signifficant larger number of correct choices. Additionally, we detected a difference in dopamine dynamics recorded from PFF-injected mice during VMCL, including when mice were making correct choices, incorrect choices, and collecting rewards. These changes in dopamine dynamics were associated with elevated phosphorylated a-Syn in PFF-injected brains, as confirmed by immunofluorescence and Western blotting. These results provide insights into the early neurochemical changes during synucleinopathy progression associated with cognitive dysfunction.

1. Robarts Research Institute, The University of Western Ontario, London, ON, N6A 5B7, Canada.
2. Neuroscience Program, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON N6A5K8, Canada.
3. Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON N6A5K8, Canada.
4. StressMarq Biosciences Inc., Vancuver, BC, V8T 4Y2, Canada.
5. Department of Anatomy & Cell Biology, The University of Western Ontario, London, ON N6A5K8, Canada.

Emily A. Mehle¹, Alber Aqil¹, Erik C. Andersen², Omer Gokcumen¹ and Denise M. Ferkey¹

Chemosensation (the combined senses of smell and taste) allows animals to detect and navigate towards a potential food source or mate, while avoiding harmful/toxic environments and predation. However, much of what we know about these senses comes from study of laboratory strains of animals. In the case of C. elegans, the isogenic laboratory strain named N2 was first isolated in 1951 from mushroom compost in Bristol, England and has been used for chemosensory experiments for over 60 years. Little is known about how naturally occurring genetic diversity among C. elegans wild isolates affects chemosensory responses, or how adaptive forces may shape the response profiles of C. elegans populations throughout the world. A collection of ~400 wild isolate strains of C. elegans has become available through the Caenorhabditis Natural Diversity Resource (CaeNDR, https://caendr.org), along with full genome sequence data and habitat isolation information for each strain, making genome-wide association (GWA) studies now possible. Utilizing the wild strains and resources from CaeNDR, we are investigating natural variation in aversive chemosensory response to quinine, copper and SDS and have found a considerable degree of natural variation in behavioral sensitivity to each stimulus, with responses between stimuli not strongly correlated. Using GWA analysis, we are working to identify genomic regions that are associated with differential behavioral sensitivity to each aversive stimulus.

¹Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260 USA

²Department of Biology, Johns Hopkins University, Baltimore, MD 21218 USA

SM Miracle^1,2, WB Lynch^1,2, SI Goldstein^1,3, KK Wingfield^1,3, I Kazerani¹, GA Saavedra¹, R Bhandari¹, A Farnan¹, B Nguyen¹, ET Gerhardt¹, BM Baskin¹, JA Beierle¹, A Emili⁴, CD Bryant¹

Oxycodone (OXY) misuse is integral to the opioid addiction epidemic. OXY is metabolized by CYP2D enzymes into oxymorphone (OMOR), a much more potent and efficacious mu opioid receptor agonist that could enhance OXY behaviors. We mapped Zhx2 (zinc-finger homeobox 2) as a candidate gene underlying brain [OMOR] in a BALB/c reduced complexity cross. Zhx2 is a transcriptional repressor of CYP450 enzymes. Here, we addressed whether Zhx2 overexpression in Zhx2-deficient BALB/cJ (J) mice would decrease CYP2D expression, reduce brain [OMOR], and reduce OXY-induced behaviors. For liver, J mice received retro-orbital AAV (AAV8-TBG-mZhx2-P2A-eGFP) injections. For brain, J females received AAV (AAV/F-CMV-mZhx2-P2A-eGFP.miR122) intracerebroventricular (ICV) injections. After 3 weeks, mice underwent OXY-conditioned place preference (OXY-CPP). Following initial preference on Day (D) 1, on D2-D5, mice received alternating OXY injections (1.25 mg/kg, IP) and saline (IP). Mice were assessed for drug-free and state-dependent OXY-CPP on D8 and D9. For female Zhx2 liver overexpression, there was increased Cyp2d22 transcript, no effect on brain [OXY] or metabolite levels, and increased time spent on the OXY-paired side on Day 1,8, and 9. For males, there was no effect on Cyp2d transcripts, a decrease in brain [OXY], and no effect on preference. Consistent with our hypothesis, female Zhx2 brain overexpression decreased OXY-induced locomotion during training and state-dependent OXY-CPP. Immunohistochemical analysis showed focal viral spread to brain reward regions, including septal nuclei. Overall, our results support Zhx2 in OXY behaviors. Current data show increased brain CYP2D in Zhx2 loss-of-function mice. We will soon conduct functional analysis of CYP2D6 overexpression in wild-types.

¹Laboratory of Addiction Genetics, Department of Pharmaceutical Sciences, Center for Drug Discovery, Northeastern University; ²Graduate Program for Neuroscience, Boston University; ³Biomolecular Pharmacology PhD Program, Department of Pharmacology, Physiology & Biophysics, Boston University Chobanian & Avedisian School of Medicine; ⁴Knight Cancer Institute, Oregon Health & Science University

Sissi Yuli1, Devang Mehta1, Dana Guhle1, Glen Uhrig1, Ronald Davis2, Jacob Berry1

Sleep is an essential behaviour that restores energy and resources to maintain physical and mental health. The sleep/wake balance is dynamically regulated by distributed neural circuits in the brain in both mammals and Drosophila, and the neurotransmitter dopamine (DA) is critical for this. Recent Drosophila studies indicate that sleep is regulated in part by DA signaling through the Dop1R2 receptor. Interestingly, Dop1R2 loss in either sleep or wake promoting circuits increases sleep, suggesting this receptor might have differential synaptic and molecular functions depending on the circuit. While we confirmed that pan-neuronal knockdown of Dop1R2 increases sleep, we found that RNAi disruption of Dop1R2 in different mushroom body compartments have varying effects on sleep. We hypothesize that Dop1R2 inhibits sleep through differential cellular effects on sleep/wake circuits potentially through differential interaction partners. To this end, we have used TurboID-catalyzed proximity labeling to uncover Dop1R2’s interactome in vivo and have identified new sleep-regulating genes through RNAi-based screening of these candidates for sleep phenotypes. Ongoing in vivo imaging of DA mediated synaptic physiology across these circuits will advance our understanding of how Dop1R2 and its interactor genes mediate sleep regulation at the molecular, circuit, and synaptic levels. This work will provide important insights into how spatially distinct receptor signaling regulates sleep and what protein interactions might differentiate synaptic effects. This knowledge will aid future development of targeted interventions for various sleep disorders such as insomnia and narcolepsy.

1Department of Biological Sciences, University of Alberta, 2Department of Neuroscience, Wertheim UF Scripps Institute

Funding Support: Berry Lab Start-Up, NSERC Discovery Grant (RES0052937)

De Oliveira, Cleusa; Tassin, Nusaibah and Schmid, Susanne.

The extracellular matrix (ECM) makes approximately 20% of the brain volume, and it is crucial for improving communication between neurons and glia. One specific type of ECM, the perineuronal nets (PNNs) is characterized by its distinct morphology wrapping around neurons in the brain and in the spinal cord. PNN development is advanced by neural activity, therefore coinciding with the period where neuronal plasticity is critical. It has been shown that alterations in PNNs may lead to some neurodevelopmental disorders including autism spectrum disorder (ASD). The CNTNAP2 rat model of ASD is characterized, among other things, by increased neuronal activity in some auditory areas, strongly increased startle responses and altered auditory processing. Although the PNNs have been studied in different mice models of autism, it has not been investigated in the rats. We evaluated the expression of the PNNs in brain areas relevant for auditory processing and auditory evoked behaviors in the CNTNAP2 rats comparing KO and WT, males, and females. We found no sex differences in the PNN expression in all the brain areas studied, including the pedunculopontine tegmental nucleus (PPT), the pontine caudal reticular nucleus (PnC), the central inferior colliculus (CIC), the auditory cortex (AC) and the prefrontal cortex (PFC). Interestingly, we found a very strong PNN expression surrounding startle mediating (glutamatergic) PnC giant neurons in the brainstem. We also found an increased expression of PNN in the CIC of CNTNAP2 KO rats in comparison with the WT rats, suggesting that the altered PNN in the CIC might be involved in the altered auditory processing in KO rats.

University of Western Ontario

Quintin A. Whitman1,2* , Julia S. Krimberg1,2 , Samantha C. Lauby1,2,3,4 , Patrick O. McGowan1,2,5,6

Maternal care in rats is a crucial component in establishing normative neurodevelopment in offspring. However, it is known that maternal care can be variable across litters, even in rats that share close genetic similarities. Previous data in our lab indicate that in control conditions, individual dam-pup interactions and subsequent offspring behaviour show significant variability within a given litter. Alongside known inter-litter differences, these intra-litter differences may be an important source of variation within maternal care and general offspring behaviour. Here, we analyze a collection of behavioural variables with respect to maternal care, specifically licking measures, and general offspring behaviour to identify the impact of intra-litter differences on total litter variation. To do this, we calculate a coefficient of variation (CV) for individual offspring scores and inter-litter means for each variable. We find that the CVs for individual offspring scores nested within litters show significantly higher variance than inter-litter scores. Additionally, we assess a subset of normalized maternal care and general behaviour variables using linear-mixed effects modelling, accounting for litter membership and offspring identification as random effects. We report that over half of the variables tested in this model return greater intra-litter standard deviations, indicating a strong impact of intra-litter over interlitter variance. Overall, alongside inter-litter variance we find that intra-litter variance is an important consideration when examining maternal and offspring behaviour in the rat model.

1Center for Environmental Epigenetics and Development, Department of Biological Sciences, University of Toronto Scarborough, Toronto, 2Department of Cell and Systems Biology, University of Toronto, Toronto, 3Department of Psychology, College of Liberal Arts, University of Texas at Austin, Austin, 4Center for Molecular Carcinogenesis and Toxicology, University of Texas at Austin, Austin, 5Department of Physiology, University of Toronto, Toronto, 6Department of Psychology, University of Toronto, Toronto

EA Duecker1, S Saxena1, S Khanam1, KM Hamre2, BM Moore II3, GA Johnson4, MK Mulligan1

It is unclear how prenatal exposure to THC influences brain development and later life outcomes. Human studies are often correlative, meaning that causal associations cannot be made between exposure and outcomes later in life. Causal associations are possible in rodents but may suffer from a lack of translatability. We introduce a novel mouse model of developmental THC exposure that incorporates features designed to increase translational relevance: exposure to high-dose THC prior to pregnancy and analysis of brain structural outcomes using high-resolution MRI. 16 adult C57BL/6J female mice received daily injections of vehicle (VEH, n=8) or high-dose THC (n=8, 10 mg/kg, i.p.) for 7 days before pairing with a male for 5 days. Daily injections continued during mating and gestation and were discontinued at birth. Pups were weighed weekly starting at postnatal day (PND)14. Habituation to a novel environment, anxiety, and activity were measured on PND27. Brains were collected for MRI and spatial transcriptomics on PND28. Four litters were conceived for each condition. Pregnancy success rates were 50% in both conditions, indicating that THC treatment prior to pregnancy does not impact fertilization/implantation. THC had a small effect on litter size (p<0.05) but no effect on weight. There was no effect on activity or habituation, but there was a trend (p=0.09) for higher anxiety associated with THC. Brains from each sex and litter (n=16) have been scanned on a 7-tesla scanner at 35μm resolution. Brains from one litter have been processed for spatial expression. Analysis of expression and MRI-derived phenotypes is ongoing.

1Department of Genetics, Genomics and Informatics, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA; 2Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA; 3Department of Pharmaceutical Sciences, College of Pharmacy, The University of Tennessee Health Science Center, Memphis, Tennessee, USA;4Center for In Vivo Microscopy, Duke University Medical Center, Department of Radiology, Duke University Medical Center, Durham, North Carolina, USA

Funding Support: NIH R01 DA056523

IA Sanchez-Sanchez1, DL Figueroa-Vargas1, M Vazquez-Berrios2, B Marie2, A Ghezzi1

Alcohol Use Disorder is a global public health concern. The addictive nature of alcohol is rooted, in part, in the homeostatic adaptation of the nervous system to the effects of the drug. These adaptations manifest as an increase in alcohol tolerance and the emergence of physiological dependence. Interestingly, upon sustained exposure, the same adaptations that lead to tolerance can lead to cognitive alcohol adaptations, where the presence of alcohol is now a requirement for proper cognitive function. Despite advancements in understanding the neuromolecular targets of alcohol, the molecular and cellular mechanisms underlying neuroadaptations and cognitive impairments remain unresolved. Here, we use Drosophila melanogaster larvae as a genetic model system to study the mechanism underlying cognitive neuroadaptation to alcohol. Using an olfactory conditioning assay, where an odorant is paired with a heat shock, previous studies have shown that acute alcohol exposure impairs learning in Drosophila larvae. However, upon chronic alcohol exposure, larvae exhibit successful avoidance of the conditioned stimulus, indicative of alcohol-induced neuroadaptations. Here, we show that these behavioral changes are not only accompanied by transcriptional alterations in the brain that affect different synaptic processes but are also paired with significant changes in synapse morphology at the larval NMJ during cognitive neuroadaptation to alcohol. We are currently testing candidate genes implicated in this process through targeted RNAi knocked down. The findings may contribute to the development of targeted interventions for AUD and illuminate potential therapeutic strategies for mitigating alcohol-related cognitive deficits.

1 Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, PR,

USA, 2 Institute of Neurobiology, University of Puerto Rico, Medical Science Campus, San

Juan, PR, USA

Funding Support: Puerto Rico Louis Stokes Alliance for Minority Participation (PR-LSAMP)

NSF-HRD 2008186; NIH COBRE: Puerto Rico Center for Neuroplasticity P20 GM103642; NIH PR-INBRE: Advancing Competitive Biomedical Research in Puerto Rico P20 GM103475.

Ryan Oliver, Srujan Yamali, Sabina Knox, Tarandeep Dadyala, and Lisha Shao

Courtship in Drosophila has traditionally been attributed to male behavior, with females often perceived as passive participants. Recent studies have challenged this notion, revealing a more active role for females in shaping courtship dynamics. However, quantifying female behaviors during courtship remains challenging due to their subtle nature. In this study, we developed a novel high-throughput assay that aims to quantify the courtship index of female fruit flies within a controlled environment that allows for visual and olfactory cues without physical contact between males and females. The expansive arena allocated for the females ensures behavior tracking over time. Preliminary results revealed a diverse array of female courtship behaviors. Our ongoing work aims to translate female locomotor features during courtship into a measurable courtship index. We further seek to identify specific neurons and genes associated with these behaviors, unveiling the neural and genetic basis of female courtship. Our research will enrich the current understanding of Drosophila mating behaviors and have broader implications for the study of evolutionary biology in courtship.

University of Delaware

D Halasz¹, AL Simal¹, P Reid¹, J Anderson¹, F Zaheer¹, G Descalzi¹.

Chronic pain is a major risk factor for depression and anxiety, and rodent models of chronic pain correspond with altered activity in the Nucleus Accumbens (NAc). Accordingly, activity in the NAc is involved in the maintenance of pain hypersensitivity and emotion-related changes associated with chronic pain. However, the underlying molecular mechanisms driving this process have yet to be fully elucidated. Neuroplasticity is a mechanism that is highly involved in chronic pain development. In this work, differences between neuroplasticity-related markers in the NAc in a chronic pain state were compared using two injury models in C57 male and female mice. A complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain model as well as a spared-nerve injury (SNI)-induced neuropathic pain model was used to assess changes in relative levels of NR2B, pNR2B, CREB, pCREB, and PSD-95. Specific attention was also paid to the temporal aspects of pain development, with assessment of these molecular markers post-injury occurring at time points of 3 hours, 24 hours, 3 days, and 7 days in the CFA model, and time points of 5, 14, 30, and 60 days in the SNI model. Significant sex differences concerning the injury condition were identified, with elevated levels of specific markers observed in the SNI group. This work not only further reveals the underlying molecular mechanisms occurring in the NAc, but also suggests that chronic-pain models that allow sufficient time for depression-like and anxiety-like changes to develop are more appropriate for assessing changes in the NAc.

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

Funding Support: Funds provided by CIHR, PG 44370.

Britahny M Baskin¹, Emma J Sandago¹, Hong S Choi², Carissa J Stots², Isabel Bojorquez-Emmons³, Reilly N Thompson¹, Megan L Quinn¹, Olivia F Barclay², Alexandra G Panepinto², Daniel Schmidlin², Soumyak Mattagajasingh³, Sophia A Miracle¹, Yahia Adla¹, Kathleen M Kantak², Camron D Bryant¹

Psychostimulant use disorders have ~40-50% heritability. Quantitative trait locus (QTL) mapping in nearly identical rodent substrains facilitates identification of quantitative trait genes/variants underlying behavior. We previously found enhanced cocaine locomotor sensitivity and self-administration in SHR/NCrl vs. SHR/NHsd substrains purchased from their respective vendors (Charles River Laboratories, Harlan Envigio Laboratories). Following in-house breeding of parental substrains and F2 crosses, adult rats underwent locomotor activity assessments following saline, and cocaine (5, 20mg/kg; i.p), and a sucrose preference task. Additionally, rats from parental substrains were tested on a Differential Reinforcement of Low-Rate Responding (DRL) operant task which required response inhibition to assess impulsivity and intravenous operant cocaine (0.25 mg/kg) self-administration sessions under an FR1 reinforcement schedule. SHR/NCrl rats exhibited greater locomotor activity when first injected with saline (novelty response) and greater conditioned hyperactivity compared to SHR/NHsd following repeated injections of cocaine. Regardless of substrain, females displayed a greater novelty response and cocaine-induced locomotion than males. SHR/NHsd showed a stronger sucrose preference, indicating reward-type sensitivity. Consistent with a more addiction-prone phenotype, on the DRL task, SHR/NCrl rats exhibited lower response efficiency and sex-dependent increases in burst responding, indicative of impulsivity. Females from both substrains self-administered more cocaine than males. Reciprocal F2 offspring (one-half had SHR/NCrl grandsire, one-half had SHR/NHsd grandsire) revealed higher cocaine-induced locomotion and sensitization in females regardless of paternal lineage of origin, however, the F2s with SHR/NHsd grandsires exhibited higher sucrose preference scores than those with SHR/NCrl grandsires, indicating a parent of origin effect. These results and the coming QTL analysis demonstrate heritable differences in pro-addiction phenotypes amongst near-isogenic substrains and their reciprocal F2 offspring.

¹Laboratory of Addiction Genetics, Department of Pharmaceutical Sciences, Center for Drug Discovery, Northeastern University; ²Department of Psychological and Brain Sciences, Boston University, Boston, MA, 02215; ³Undergraduate Program in Neuroscience, Boston University, Boston, MA 02215

A Zheng1,2, T Rahman3, P Patel3, B Allman1, S Schmid1

Mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are associated various neurodevelopmental disorders in humans, most notably autism spectrum disorder. Rats with a loss-of-function mutation in the Cntnap2 gene (Cntnap2^-/- rats) show an increased acoustic startle response, which parallels acoustic hyperreactivity in autistic individuals. The acoustic startle response is mediated by the caudal pontine reticular nucleus (PnC) in the brainstem, specifically giant neurons in the PnC. Previous studies from our lab have found that, in response to startle sounds, PnC firing rates are increased in female Cntnap2^-/- rats and PnC giant neuron recruitment is increased in both female and male Cntnap2^-/- rats compared with wildtype littermates. This raises the question of why PnC giant neurons are more excitable in Cntnap2^-/- rats. Considering that Cntnap2 has been implicated in AMPA receptor (AMPAR) expression levels and since glutamate receptors mediate the acoustic startle response, we hypothesized that Cntnap2^-/- rats have increased expression of AMPARs. Using RNAscope in situ hybridization, we fluorescently labelled mRNA for Cntnap2, glutamate AMPAR subunit 1 (GluA1), and GluA4 in PnC giant neurons. Cntnap2^-/- rats showed decreased mean signal intensity for Cntnap2 mRNA compared with wildtype littermates, indicating that transcription of the Cntnap2 gene is altered. However, transcription of the genes for GluA1 and GluA4 does not seem to be altered by Cntnap2^-/- as mean signal intensities were similar between the genotypes. Overall, our results show that altered glutamate receptor expression in PnC giant neurons likely does not contribute to increased excitability of these neurons in Cntnap2^-/- rats.

1Department of Anatomy and Cell Biology, 2Neuroscience Graduate Program, 3Department of Physiology and Pharmacology, Western University, London, Ontario, Canada

Funding Support: Natural Sciences and Engineering Research Council of Canada (NSERC) 04472-2018RGPIN

Mariana Siqueira Amormino¹, Fernanda Alvarenga Lima Barroso², Mírian Velten Mendes¹, Rafaella Antunes Vasconcelos ¹, Renato Elias Moreira Júnior ¹, Vasco Ariston de Azevedo², Ana Lúcia Brunialti Godard ¹.

The bidirectional relationship between gut microbiota and the brain in the genesis and maintenance of alcohol use disorder guides our understanding of how restoring the gut ecosystem impacts the modulation of neuroregulatory pathways involved in the drug's positive reinforcement. This study investigates the capacity of probiotic strains to mitigate ethanol preference behavior by correcting the imbalance of bacterial communities and the potential loss of their substrates. We employed an experimental design with male C57/BL6 mice to validate the involvement of brain systems in ethanol-seeking behavior, developed on previous studies from our laboratory. The selection of lactic acid bacteria, Lacticaseibacillus rhamnosus L156.4 and Lactococcus lactis NCDO 2118, was based on their high synthesis capacity when combined and their effectiveness in controlling Gram-negative bacteria competition for gut regulation post-ethanol ingestion. Preliminary results are promising, showing a significant reduction in beverage consumption by the animals after two weeks of administering a blend of these lactic bacteria, across various dietary protocols, and alleviation of behaviors associated with anxiety and long-term memory. Additionally, histological analyses of the colon revealed a notable reduction in inflammatory infiltrates and differential regulation of goblet cells post-probiotic blend administration, maintaining intestinal barrier integrity. We infer neuronal modulation through the reestablishment of the gut microbiota previously altered by ethanol.

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

²Laboratório de Genética Molecular e Celular. Departamento de genética, ecologia e evolução, 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.

¹Rush D., ¹Blackmore P., ¹LeFevre L., ¹Wudyka W., ¹Britton J., ¹Holly J., ^2,3Phillips T.J., and ¹Shabani S.

The mu-opioid receptor systems mediate powerful rewarding effects, and also contribute to methamphetamine (MA) consumption-related behaviors. Selective breeding based on a MA consumption trait discovered two genes that influence several MA-related traits. These two genes are: trace-amine associated receptor 1 (Taar1) and mu-opioid receptor 1 (Oprm1). A Taar1 variant that codes for a functional receptor is associated with low MA consumption and high sensitivity to aversive and hypothermic effects of MA. Some Taar1-mediated effects are modified by Oprm1 variants. In the first study we used a conditioned place preference (CPP) procedure to determine whether the selective TAAR1 agonist, RO5256390, blocks rewarding effects of morphine, a mu-opioid receptor agonist. Mice were first injected with 0 or 0.1 mg/kg RO5256390, and 15 minutes later with 0 or 10 mg/kg morphine, and then were placed in a CPP apparatus for several conditioning trials. One day after preference testing, after the same injections, animals had rectal temperature measured immediately before morphine injection (time 0 min) and then at 60, 120 and 180 min after injection. In a second study, we used multiple doses of each drug to determine thermal effects after single injections. RO5256390 plus morphine induced rewarding effects only when drugs were given prior to the preference test. In the first study, RO5256390 alone induced hypothermia; however, RO5256390 plus morphine induced significantly greater hypothermia. Similar results were obtained in the second study. These studies indicate that the interaction from activation of these two receptors determine drug-related motivational and physiological outcomes.

¹ Dept of Biomedical Science, Grand Valley State University, Allendale, MI USA. ² Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR. ³ Veterans Affairs Portland Health Care System, Portland, OR USA.

Hana Terada, Kyle M. Roddick and Richard E. Brown

Neurexins are a group of presynaptic cell adhesion molecules that form complexes with postsynaptic neuroligins, which influence the development and function of excitatory and inhibitory synapses. Individuals with autism exhibit inhibitory deficits likely due to issues in sensorimotor gating. Recent findings show that deletion of splice site 5 may recover ASD-related symptoms, which we tested by investigating prepulse inhibition of startle and acoustic startle, comparing male and female Nrxn1 knockouts (Neurexin1+/- mice) with dS5 knock-ins (ds5/- and ds5/ds5 mice) and wildtype (C57BL6/J ) mice from 6 to 24 months of age. We found that acoustic startle declined with age for all genotypes, and percent prepulse inhibition increased with decibel intensity for age groups 0-6 and 7-12. Surprisingly, +/- mouse models had higher %PPI than WT controls.

Department of Psychology & Neuroscience, Dalhousie University,Halifax NS Canada B3H 4R2

Amoolya Sai Dwijesha1, Celina Phan2,5, and Anna Phan1,3,4

Cohesinopathies are a group of multisystem developmental disorders that arise from mutations in the cohesin complex (CC) subunits and accessory proteins and are characterized by neurological and biological impairments. As an evolutionarily conserved structure, the CC comprises of 4 subunits: Stromalin (STAG1/2), Rad21, Smc1 and Smc3, and accessory proteins that assist in chromosome segregation and gene regulation. Recently, Stromalin was identified as a memory suppressor that produced its neurological effects by repressing synaptic vesicle (SV) pool sizes in Drosophila dopamine neurons. Previous studies on animal and cellular models of cohesinopathies have shown that perturbed gene regulation rather than imperfect chromosome segregation may underlie these disorders. We hypothesized that Stromalin’s SV pool size effects might be due to CC’s role in transcriptional regulation; these effects may produce certain neurological symptoms seen in cohesinopathies like higher seizures incidences. We found that decreasing Stromalin in the whole brain caused an increase in seizure frequencies. We then tested Stromalin’s 5 potential downstream targets previously identified by our lab through RNA-sequencing: nep1, CG17698, cox7c, ttm2 and su(z)12, for seizure behavior. Besides cox7c, the mammalian homologues of these genes were found to be dysregulated or mutated in some human disorders characterized by seizures, and in rat models of status epilepticus. We identified CG17698 and cox7c as potential candidates for causing the seizure phenotypes in our cohesinopathy model. Presently, we are testing if a reduction in SV numbers at the presynaptic terminals can prevent the seizure phenotype in our cohesinopathy model.

1Department of Biological Sciences, 2Department of Laboratory Medicine & Pathology,

3Neuroscience and Mental Health Institute, 4Women and Children’s Health Research Institute, University of Alberta, Edmonton, Alberta, Canada, 5Innovation and Portfolio Management, Canadian Blood Services, Edmonton, Alberta, Canada

Funding support: This research is supported by the Natural Sciences and Engineering Research Council (NSERC) grant (RGPIN-2020-04009).

Gurlaz Kaur¹, James Thompson-Dick¹, Anna Phan¹

Social behaviors and interactions are essential for physiological development, learning, and survival of many organisms. Social isolation has been shown to be detrimental both physiologically and mentally in humans, leading to higher levels of cardiovascular disease, mortality, anxiety, and depression. When subjected to social isolation, humans, rodents, and Drosophila melanogaster exhibit increased aggression, feeding, disrupted sleep, and in the case of humans and rodents, impaired cognition. Despite the generally accepted assertion that social isolation leads to profound behavioral effects, very little is known about the mechanisms by which isolation alters neural physiology to affect behavior. In this study, we demonstrate for the first time that chronic social isolation in Drosophila also impairs learning, as evidenced by the olfactory aversive conditioning assay. Drosophila were isolated during development until 5 days old adults and compared to group-housed controls. Both males and females were found to have impaired learning. Isolated individuals did not differ in their responses to the conditioned and unconditioned stimuli in the learning paradigm, suggesting this deficit is specific to learning and does not result from stimulus-response changes. We are currently conducting single-cell RNA-sequencing experiments to identify (1) the gene expression changes that occur upon social isolation in neurons and (2) the neuronal subtypes that respond most strongly to isolation. We anticipate this information will help reveal the mechanisms by which isolation alters neural physiology and behavior.

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

Funding Support: This research is supported by the Natural Sciences and Engineering Research Council (NSERC) grant (RGPIN-2020-04009).

Helen M. Kamens, Emma K. Anziano, William J. Horton, and Sonia A. Cavigelli

Adolescence is a critical developmental period when the brain is plastic, and stress exposure can have lasting physiological and behavioral consequences. One mechanism by which adolescent stress may have lasting effects is through altering microRNAs (miRNAs), which lead to broad-scale changes in gene expression. In three independent studies of male rodents that utilized unbiased approaches (RNA sequencing or microarray) aimed at identifying miRNA altered by stress, miRNA-200a was altered in areas of the brain associated with emotion regulation. The current study extended this research to investigate the effect of chronic adolescent restraint stress on miRNA-200a expression in 2 strains (BALB/cJ and C57BL/6J) of male and female mice. We utilized a 14-day (2-hours/day) restraint stress protocol and verified stress effects on body weight gain and circulating corticosterone concentrations relative to non-restraint controls. After adolescent stress or control conditions, mice were undisturbed until sacrifice in adulthood. At sacrifice 3 weeks after completion of adolescent stress, there were no longer significant differences in body weight between groups; however, animals exposed to stress had less miRNA-200a expression in the ventral hippocampus than control animals. These data extend prior results implicating miRNA-200a expression as stress-dependent in both male and female mice. Future work should evaluate if miRNA-200a is a causal mechanism by which stress leads to lasting physiological and behavioral consequences.

Department of Biobehavioral Health, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States of America.

Funding: The Pennsylvania State University Consortium on Substance Use and Addiction and the Social Science Research Institute supported this work.

S Heine¹, BS Charles^1,2, S Savona-Jofre¹, and AJ Moehring^1,2

Aggression can be a critical behaviour necessary for the procurement of mates or resources. While the neural basis of aggression has been extensively studied in males, it remains poorly understood in females. Here, we present our results on the underlying basis of female aggression in Drosophila. We find that the hyperactivation of a small subset of doublesex neurons can induce high levels of aggression in females, but not males. We then identify the key visual and chemical cues that trigger aggressive behaviours in females. Lastly, we determine whether the winner-loser effect observed in males, whereby a male who wins an aggressive encounter is more likely to initiate subsequent aggressive encounters, also holds true in females.

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

²Neuroscience Program, Western University, London, ON, Canada.

EM Norman, P Rajashekar, KM Hamre

Fetal alcohol spectrum disorders (FASDs) are neurodevelopmental disorders that result in many behavioral phenotypes including hyperactivity and increased anxiety. In the present study, we exposed C57BL/6J mice to ethanol during midgestation in a binge paradigm and subsequently behaviorally tested the offspring as adults. In brief, dams were given 5.8 g/kg ethanol via gavage on embryonic day 9 with controls given isocaloric maltose/dextrin. Behavioral phenotypes were examined in a battery of tests including an activity chamber and several mazes. Activity was tested on 2 consecutive days to be able to assess habituation to a novel environment. Comparisons between day 1 and day 2 demonstrated treatment effects on habituation such that both male and female ethanol-treated mice showed less habituation than controls. Because habituation is a well-studied phenotype, we used bioinformatic analyses to identify potential genes that could contribute to the ethanol-mediated effect. To identify potential candidates, we first correlated open field phenotypes with striatum gene expression from adult BXD mice using the GeneNetwork repository of behavioral and expression data. From these analyses, over 40 genes were correlated with the phenotypes of interest. In order to further evaluate these candidates, we used PubMed to assess whether any of these genes have previously been shown to be affected by prenatal ethanol exposure. However, we found no evidence for any of these genes being altered by prenatal ethanol exposure. Further analyses will be conducted with gene expression from other brain regions to further identify potential candidates.

Department of Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN USA.

Support: R01AA023508, NIH MSRF, Rhodes/UT Neuroscience Fellowship

Jude Frie

Methods: The device uses RFID to identify rats, a lickometer to activate fluid delivery, a custom low-profile PCB that sits on top of an Arduino-based microcontroller, fluid delivery via custom peristaltic pump for accurate measurement of consumption volume, OLED display, and continuous data logging to an SD card. A validation of the design was conducted with two devices (one water and one 10% ethanol) using four female rats. Data was collected for nine days. Pump error was evaluated by simple linear regression and descriptive statistics. Changes in daily alcohol consumption and preference were analyzed via one-way ANOVA with day as a repeated measure. Individual hourly water and alcohol consumption were evaluated via two-way ANOVA with time as a repeated measure. Effect of light cycle and bout range on each subject’s water and alcohol consumption was evaluated via two-way ANOVA. Max and mean bout sizes, volume per lick, and bout frequency between fluid types were analyzed via two-tailed paired t-test. Bouts were defined as a drinking event containing at least 20 licks, similar to previous literature.

Results: Pump accuracy following 20 measurements of 1.00 mL was high, with an average error of 0.5% (95% CI 0.3-0.7) based on weight and remained stable across tests. Rats consumed a much greater volume of alcohol during the dark cycle (F(1,64)=494.7, P<0.001). Alcohol resulted in greater average bout size (t(3)=3.659, P=0.0353), max bout size (t(3)=7.088, P=0.0058), and volume per lick (t(3)=5.255, P=0.0134)). Importantly, the device was also able to delineate individual variation in fluid consumption.

Conclusions: Having a robust, affordable method for measuring drinking microstructure in socially housed animals will be of considerable use in preclinical addiction research and a step toward more translationally relevant animal models of fluid consumption. The added dimension of time allows for the analysis of circadian-linked consumption and the discrimination of continuous or binge-like drinking behaviours. Additionally, being open-source enables researchers to customize the device for more advanced applications such as sending signals to additional peripherals (e.g., optogenetic stimulation) or software on drinking initiation for time-locked or closed-loop interventions, manipulations, and measurements.

Western University

Naira Rashid and Sayeed ur Rehman

HSPB8 also known as the Alpha-crystallin C chain , Protein kinase H11 or Small stress protein-like protein HSP22 is a small heat shock protein. This protein functions as a chaperone in association with Bag3, a stimulator of macroautophagy. It intervenes in the governance of cell proliferation, apoptosis, and carcinogenesis. Mutations in this gene have been linked with various neurodegeneration. HSPB8 manoeuvres its role by expediting autophagy by inhibiting the deposition of misfolded proteins in afflicted cells. Ameliorating the discerning attempt of misfolded proteins by HspB8-BAG3-Hsp70 to autophagy might also result in reduced delivery to the proteasome by the BAG1-Hsp70 complex so that the probable proteasome overwhelming is reduced.

With the help of bioinformatics, we have predicted novel exons of the human HSPB8 gene. The addition of novel exons in the mature RNA transcript resulted in the removal of the essential residues of the HSPB8 protein. These novel isoforms can be analysed to understand their role in neuroprotection and several other processes in which HSPB8 plays a cardinal role. These novel isoforms can also be targeted for therapeutic purposes by developing splice-switching antisense oligonucleotides (SSOs) in order to regulate their functioning.

Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, India.

Kisby, Brent; Bustamante, Christian; Shanmugam, Sambantham; Castro-Piedras, Isabel; Ponomarev, Igor

Toll-like receptor 3 (Tlr3) – dependent innate immune activation in rodents contributes to escalated ethanol consumption in a sex and genotype-dependent manner. Our preliminary data suggests these effects may be mediated by Tlr3 activation in microvasculature cell types. Micro-vessels consist of cell populations that form the blood brain barrier (BBB), including endothelial cells (ECs), smooth muscle cells, and pericytes. We hypothesize that Tlr3 activation-induced changes in gene expression in these cell types contribute to BBB dysfunction and escalated alcohol intake. The goal of this study was to determine the effects of Tlr3 activation and/or chronic alcohol drinking on gene expression in enriched brain microvasculature in high-drinking FVB/NJ X C57BL/6J F1 hybrid mice. Male mice were randomly assigned to receive repeated injections of Poly(I:C) (PIC), a Tlr3 agonist, or saline (9 injections total). Mice were allowed to choose between alcohol or water every other day (17 dinking sessions) and were assigned to one of four groups: saline/water (SW), saline/ethanol (SE), PIC/water (PW), and PIC/ethanol (PE). Brains were harvested 24 hours after the final alcohol session, micro-vessels from the frontal cortex were purified using mechanical homogenization and density-gradient centrifugation, and RNA sequencing and bioinformatics was performed to compare gene expression between groups. We identified 1,588 genes differentially expressed (DEGs) between PE and SE groups at nominal p-value of less than 0.05 and 74 DEGs at 5% FDR. The top DEGs were Rsad2, H2-K1, Slc16a1, and Ifi44, which are implicated in the immune response and are cell type-specific to the brain microvasculature. Taken together, these data suggest that vascular cell types are responsive to repeated Tlr3 activation and could contribute to excessive ethanol consumption.

1Texas Tech University Health Sciences Center, Lubbock, TX 79430 T

Aboubacrine Mahamane Touré^1,2,3, Lisa-Marie Legault¹, Michelle Robb^1,3, Serge McGraw^1,3,4 and Elsa Rossignol ^1,5,6

Exposure to alcohol at critical periods of prenatal life can alter DNA methylation profiles of brain cells and contribute to the molecular mechanisms by which Fetal Alcohol Spectrum Disorder (FASD) emerges. However, the effects of alcohol exposure at the very beginning of gestation on brain development remain largely unexplored. Using a translational mouse model, we show that pre-implantation alcohol exposure (PAE) induces a neurobehavioral phenotype with autistic-like behaviors in a sex-dependent fashion in adult mice. Males display reduced anxiety with no hyperactivity while females are anxious and hypoactive. Regarding social interactions, males display altered sociability with an intact preference for social novelty while the opposite scenario is seen in females. Analysis of PAE embryonic brains shows no overt effect on cortical thickness but reveals a strong reduction in GABAergic interneuron (IN) density in the dorsal migratory streams. This reflects a reduction of IN precursor proliferation in the medial ganglionic eminence, together with a delay in tangential migration of IN towards the dorsal pallium. Disorders of IN migration or proliferation have been previously associated with autistic-like phenotype, suggesting a contribution of this cellular deficit to the overall phenotype. Mechanistically, we find that PAE induces lasting stage- and sex-specific changes in DNA methylation of multiple brain-expressed genes, including regulators of IN development. Interesting candidate genes are currently being further investigated and validated. Altogether, our results provide new insights on the impact of PAE on IN development and functions before and after birth and on the disease mechanisms of alcohol-related neurodevelopmental disorders.

1 CHU Sainte-Justine Research Center

2 Department of Biological Sciences, Université des Sciences des Techniques et des Technologies de Bamako, Bamako, Mali

3 Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada

4 Department of Obstetrics and Gynecology, Université de Montréal, Montreal, Quebec, Canada

5 Department of Neurosciences, Université de Montréal, Montreal, Quebec, Canada

6 Department of Pediatrics, Université de Montréal, Montreal, Quebec, Canada

Funding Support: CIHR project fund (S. McGraw and E. Rossignol)

Pedro Marinho, Jaiden Smith, Hakan Kayir, Patrick McCunn, Jibran Khokhar

Cannabis use is common in adolescence and there is evidence for sex differences regarding the long-term effect of cannabis use. We aimed to investigate how exposure to 3 types of cannabis vapour in adolescent rats impacts brain development through behavioural tests and magnetic resonance imaging. Male and female Sprague Dawley rats were divided into four groups and exposed to high-CBD, high-THC, balanced CBD + THC, or air at post-natal days 28-42 using a vaporizer. On days 0 and 14 of exposure, rats underwent cannabinoid tetrad test. In adulthood, rats underwent Pavlovian autoshaping, active avoidance, prepulse inhibition and diffusion and functional MRI. Results indicated sex-dependent differences in the physiological effects of cannabis exposure in the cannabinoid tetrad test. Pavlovian autoshaping showed that male rats exposed to high-THC and high-CBD had less lever-directed behaviour compared to controls and balanced groups, while all female rats showed no significant differences between the exposure groups. All three male cannabis-exposed groups showed impaired avoidance learning. The female active avoidance data showed significant differences between the THC and CBD-exposed groups only. Both males and females showed no group differences in PPI. Regarding MRI data in male rats, we found a single network with altered functional connectivity amongst the four groups and two networks with altered structural connectivity amongst the four groups. Females showed no altered networks. Adolescent cannabis vapour exposure can lead to long-lasting effects in adulthood, with males possibly being more vulnerable.

Schulich School of Medicine and Dentistry, University of Western Ontario

Robb M^1,2, Legault LM^2,3, Dupas T^2,4, McGraw S^1,2,3,4

Prenatal alcohol exposure (PAE) impairs embryonic development and can cause Fetal Alcohol Spectrum Disorder (FASD). Phenotypes include developmental delays, intellectual deficits and morphological abnormalities. Various studies have shown that FASD is also associated with changes in epigenetic profiles and that maternal diet can have a large impact on embryonic development. For instance, methyl donors such as folate, choline and vitamin B12 are particularly crucial for the establishment of an appropriate embryonic epigenetic profile. Our previous data have shown that a methyl donor enriched diet before and during gestation attenuates the effects of PAE, notably by reducing morphological defects and growth retardation in mouse embryos. We hypothesize that, conversely, a maternal diet deficient in methyl donors would amplify the phenotype of embryos exposed to alcohol.

Our mouse model recreates a single binge-like alcohol exposure during the preimplantation period. Diets with varying levels of depletion are currently being tested to find one that is severe enough without being detrimental to reproductive health. Samples will be collected at E13.5 and E18.5 to analyze gross morphology and identify anomalies. Also, neuronal development, migration and function will be evaluated through immunofluorescence and epigenetic health will be measured through transcriptomic analyses.

In this study, we aim to demonstrate that methyl donor deficiencies deregulate the epigenetic landscape of developing embryos, making them more vulnerable to alcohol exposure and amplifying their brain abnormalities. This information will be crucial to guide pregnant and conceiving women in making appropriate lifestyle and nutrition choices to ensure the health of their child.

¹Department of Neuroscience, Université de Montréal, Canada, ² CHU Sainte-Justine Research Institute, Montréal, Canada, ³Department of Biochemistry, Université de Montréal, Canada, ⁴Department of Obstetrics and Gynecology, Université de Montréal, Canada