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

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

1 - Brown University, RI. Department of Neuroscience

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Funding support: Flicker of Hope Foundation

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

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

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

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

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

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

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

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

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

Funding Support: DA037844, DA060810

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

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

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

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

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

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

Funding Support: CIHR (Canada) PJT 178220

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

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

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

M Hernández¹, AM Barkley-Levenson¹

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

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