Kevin Christie^1#, Tarandeep Dadyala^1#, Irina Sinakevitch¹, Nicholas Collins¹, Phuong Chung², Masayoshi Ito^2,3, Lisha Shao^1,*

Assigning valence—appeal or aversion—to gustatory stimuli and relaying it to higher‑order brain regions to guide flexible behaviors is crucial to survival. Yet the neural circuits that transform taste into motivationally relevant signals remain poorly defined in any model system. In Drosophila melanogaster, substantial progress has been made in mapping the sensorimotor pathways encoding intrinsic valence for feeding and the architecture of the dopaminergic reinforcement system. However, where and how "effective" (i.e., real-time) valence is first imposed on a taste has long been a mystery. Here, we identified a pair of subesophageal zone interneurons in Drosophila, termed Fox, that impart reinforcing positive valence to sweet taste and convey this signal to the mushroom body, the fly’s associative learning center. We show that Fox neuron activity is necessary and sufficient to drive appetitive behaviors and can override a tastant’s intrinsic neutral or aversive valence without impairing taste quality discrimination. Furthermore, Fox neurons relay the positive valence to specific dopaminergic neurons that mediate appetitive memory formation. Our findings reveal a circuit mechanism through which effective valence is bestowed upon sweet sensation and transformed into a reinforcing signal that supports learned sugar responses. Preliminary data further suggest that Fox function may extend beyond sweetness: Fox may amplify real-time valence for the tastant most valuable to the animal’s current physiological state, including during sugar–protein choice. Fox neurons thus form a convergent–divergent “hourglass” circuit motif, acting as a bottleneck for valence assignment and distributing motivational signals to higher-order centers. This architecture confers both robustness and flexibility in reward processing—an organizational principle that may generalize across species.

¹ Department of Biological Sciences, University of Delaware, Newark, DE 19711

² Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147

³ Current address: Lib-Gate Co., Ltd.

^# These authors contributed equally to this work

^* Correspondence: shaol@udel.edu

Rajee Ganesan 1 , Andrew Wang 2 , BaDoi N. Phan 2,3 , Michael J. Leone 2,3 , Heather Sestili Harper 2 , Andreas Pfenning 2,4

Vocal production learning is the ability to imitate sounds through social exposure. The rare trait is believed to have independently evolved three times in birds and five times in mammals, a prime example of convergent evolution. All studied vocal learning species have evolved a specialized forebrain sensorimotor learning circuit that is either absent or rudimentary in their closer, vocal non-learning relatives. Previous work within the lab has found that the “regulatory code” linking genome sequence to cell-type-specific function is highly conserved across mammals. In this study, we leverage this principle to identify candidate enhancers from publicly available Zoonomia datasets, and trained machine learning models predicting open and closed chromatin across all 240 mammalian motor cortex regions. We screened for differences in regulation between vocal learning and non-learning species by applying the Tissue-Aware Conservation Inference Toolkit, a machine learning approach to study how enhancer activity conservation relates to phenotype evolution. These machine learning models can learn sequence patterns of enhancers that robustly predict conserved activity across evolutionary distances, allowing us to test whether enhancer conservation patterns are associated with the evolution of vocal learning. Our results revealed lineage-specific gains and losses of regulatory elements, and we show that L6 corticothalamic neurons and oligodendrocytes had the strongest enrichment with the vocal learning phenotype, suggesting key roles in vocalization-related traits through motor learning and synaptic plasticity. Future studies will include single cell integration across species to identify orthologous cell populations and to better understand molecular mechanisms associated with the evolution of vocal learning.

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA 2 Computational Biology Department, Carnegie Mellon University, Pittsburgh, PA, USA 3 Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 4 Neuroscience Institute, Carnegie Mellon University, Pittsburgh, PA, USA

Sophia A. Miracle1,2, Morgan L. Hofmeyer1,3, Ava B. Glavine1,4, Isabella C. Conti1,5, Sophia V. Pavlidis1,6 , Hala Ajjawi1 , Aleksandra G. Gorelik1,6 , Bryce Axe7,8 , Priyanka Thareja1,9 , Angelique Buton10, Kaylie R. Kaneshiro1,11 , William B. Lynch1,2 , Kelly Wingfield1,12 , Praveen P. Kulkarni8,13 , Joseph Rower14, Lili Sun14 , Stephanie G. Puig10 , Ralph Loring15 , Christopher A. Reilly14, Craig F. Ferris8,13,15, Camron D. Bryant1,2

Oxycodone (OXY; active ingredient of OxyContin®) is a major contributor to the opioid epidemic. We genetically mapped and validated loss-of-function in zinc-fingers and homeobox 2 (Zhx2) underlying increased brain oxymorphone (OMOR) in female mice. OMOR, an OXY metabolite, is a much more potent and efficacious mu opioid receptor agonist that could increase OXY addiction risk. Transcriptome analysis of Zhx2 knockout (KO) brains via bulk RNA-seq identified enrichment of extracellular matrix, endothelial cells, and cell-to-cell adhesion, suggesting Zhx2 KO compromises blood brain barrier (BBB) integrity. In support, there was a significant reduction in transcript levels of the BBB marker Claudin5 in KO females. Furthermore ex vivo analysis indicated increased permeability of sodium fluorescein but not Evans blue, specifically in hippocampus of KO females, suggesting brain region-dependent disruption of BBB. In vivo structural imaging revealed reduced water diffusion throughout the brain of KO females and enlarged ventricles. In response to OXY in awake mice, KO females showed increased OXYinduced negative bold signal in midbrain and increased OXY-induced positive bold signal in brainstem. Functional connectivity analysis identified decreased brain-wide connectivity in Zhx2 KOs. In addition to a BBB mechanism, KO females also showed increased plasma [OMOR] following systemic OXY, suggesting increased liver metabolism of OXY also contributes to increased brain [OMOR]. To summarize, multiple lines of evidence support a BBB mechanism underlying increased brain [OMOR] in Zhx2 KO females. We are currently conducting functional enzymatic assays of liver microsomes to determine whether increased liver OMOR production also contributes to the phenotype.

1Laboratory of Addiction Genetics, Department of Pharmaceutical Sciences and Center for Drug Discovery, Northeastern University, Boston, MA USA; 2Graduate Program for Neuroscience, Graduate Medical Sciences, Boston University Chobanian and Avedisian School of Medicine, Boston, MA USA; 3Undergraduate Program of Neuroscience, College of Arts and Sciences, Boston University, Boston, MA USA; 4Undergraduate program of Health Science, College of Health Sciences, Northeastern University, Boston, MA USA; 5Undergraduate Program of Behavioral Neuroscience, College of Science, Northeastern University, Boston, MA USA; 6Undergraduate Program of Biology, College of Science, Northeastern University, Boston, MA USA; 7Masters Program of Bioengineering, College of Engineering, Northeastern University, Boston, MA USA; 8Center for Translational Neuroimaging, Northeastern University, Boston, MA USA; 9Masters Program of Bioinformatics, College of Science, Northeastern University, Boston, MA USA 10Department of Psychiatry, University of Massachusetts Chan Medical School, Worcester, MA, USA; 11Undergraduate Program of Biology, College of Arts and Science, Tufts University, Boston, MA USA 12Graduate Program of Pharmacology, Boston University, Boston, MA, USA; 13Department of Psychology, Northeastern University, Boston, MA USA; 14Center for Human Toxicology, University of Utah Health, Salt Lake City, UT USA; 15Department of Pharmaceutical Sciences, Northeastern University, Boston, MA USA

Nana K. Amissah¹, Christopher P. King¹, Sydney David¹, Destiny Brakey², Luke T Hannan¹, Oksana Polesskaya³, Quinn Carroll², Thiago Missfeldt Sanches³, K. Linnea Volcko²; Apurva Chitre³; Denghui Chen³, Maggie Postolache², Hannah Bimschleger³, Jianjun Gao³, Khai -Ming Nguyen³, Beverly Peng³, Riyan Cheng³, Leah C. Solberg Woods⁴, Abraham A. Palmer^{3, 5}, Derek Daniels^1,2, Paul J. Meyer¹

Maintaining fluid homeostasis is critical for life. Although there are individual variations in the behavioral regulation of fluid homeostasis in rats, the source of these variations is poorly understood. To address this, we conducted a genome-wide association study (GWAS) in 826 male and female heterogenous stock (HS) rats examining multiple phenotypes related to 24-hour food and water intake. Rats were housed in hanging wire cages for 24 hours. Total food intake over the 24-hour test was measured, and drinking was measured via a contact lickometer with millisecond resolution and were subjected to GWAS analyses. Total water intake had moderate genetic correlation with total food intake (r_g = .533), and licks (r_g = .565). There was moderate heritability for traits such as mean licks per burst (h² = .261), total water intake (h² = .224), and burst number (h² = .241). Eight unique loci on chromosomes 1, 2, 7, 12, 14 and 20 were associated with several measures of food and water intake. The locus on chromosome 1 was linked with burst number and mean licks per burst. This locus contained the candidate gene Stx11 which mediates lipid metabolism (Zhang et al., 2022). The locus on chromosome 2 was associated with water intake, and contained the candidate gene Syt6, which is involved in synaptic modulation thorough the brain derived neurotrophic factor (Wong et al., 2015). Food intake linked to a locus on chromosome 14, and contained Paqr3 a gene that regulates glucose and lipid metabolism disorders caused by insulin resistance. These candidate genes were identified by examining eQTL and coding variants. These results demonstrate that the individual differences in food and fluid intake have genetic components. Further studies will examine causal links between the identified candidate genes and ingestive behaviors by directly manipulating these genes using CRISPR-mediated approaches.

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

² Department of Biological Sciences, University at Buffalo, Buffalo, USA.

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

⁴ Department of Internal Medicine, Molecular Medicine, Center on Diabetes, Obesity and Metabolism, Wake Forest School of Medicine, Winston-Salem, USA.

⁵ Institute for Genomic Medicine, University of California San Diego, La Jolla, USA.

Supported by P50DA037844, U01DA060669, P30DA060810, and R01DK133818

Helen M. Kamens¹, Tanzil M. Arefin^2,3,4,5,6, Hayreddin Said Unsal^3,7, Thomas Neuberger^2,3, Nanyin Zhang^2,3,4

Mouse models are an essential tool for understanding behavior and disease states in neuroscience research. While genetic and sex-specific effects have been reported in many neurodegenerative and psychiatric illnesses, these factors may also alter baseline neuroanatomical features of mice. This raises the question of whether the observed changes are related to the disease being studied (i.e., pathological differences) or if there are baseline strain or sex differences that may potentially predispose animals to different responses. Over the past decade, tremendous effort has been made in mapping neural architecture at various scales; however, the complex relationships including identifying genetic and sex-specific differences in brain structure and function remain understudied. To bridge this gap, we used C57BL/6J and DBA/2J mice, two of the most widely used inbred mouse strains in neuroscience research, to investigate strain and sex-specific features of the brain connectome in awake animals using magnetic resonance imaging (MRI). By combining resting-state fMRI and diffusion MRI, we found that the motor, sensory, limbic, and salience networks exhibit significant differences in both functional and structural domains between C57BL/6J and DBA/2J mice. Further, functional and structural properties of the brain were significantly correlated in both strains. Our results underscore the importance of considering these baseline differences when interpreting the brain-behavior interactions in mouse models of human disorders.

¹Department of Biobehavioral Health, The Pennsylvania State University, University Park, USA

²Huck Institutes of Life Science, The Pennsylvania State University, University Park, PA, USA.

³Department of Biomedical Engineering, The Pennsylvania State University, University Park, USA.

⁴Center for Neurotechnology in Mental Health Research, The Pennsylvania State University, University Park, USA.

⁵Department of Neuroscience, University of Rochester Medical Center, Rochester, New York, USA

⁶Center for Advanced Brain Imaging and Neurophysiology, University of Rochester Medical Center, Rochester, New York, USA

⁷Department of Electrical and Electronics Engineering, Abdullah Gul University, Kayseri, Türkiye

Acknowledgments

This work was supported by the National Institute on Drug Abuse (DA060335, H.M.K.), Penn State’s Department of Biobehavioral Health, Social Science Research Institute, and Consortium on Substance Use and Addiction. The authors would like to acknowledge the Huck Institutes High Field Magnetic Resonance Imaging Core Facility (RRID:SCR_024461) for use of their Bruker Biospec 70/30.

Perham Black, Kelcey Stapleton, Geanette Lam, Aylin Rodan, *Adrian Rothenfluh

Animal life is full of daily decision making. These are easy when rewards are available at no cost or peril. However, many situations require a weighing of the cost vs. benefits of specific decisions. To model decisions that require a cost/benefit analysis, we have established a novel assay where Drosophila choose between a small reward (eg. 10mM sucrose) at little cost (liquid solution) and a larger reward (30mM sucrose) at a higher cost (food embedded in agarose, which requires work to get the sucrose out). We find that the internal state of a fly (eg. food deprivation) will motivate them to prefer the high-reward/high-cost option over the low/low one. Silencing ~half of the flies’ brain dopamine neurons causes them to show the same motivation for sucrose, but reduced motivation to ‘work for’ amino acids when amino acid-deprived. In lieu of a classical anatomical screen, we have simulated this situation in the connectome-derived virtual brain and screened for dopaminergic neurons that affect this cost/benefit calculation. We focus on 2 sets of in silico-identified DA neurons and predicted that one set will be involved in signaling satiety but will not alter the hi reward+cost vs. low reward+cost calculation. For the other set of DA neurons, we predicted the opposite result. Manipulating these two sets of DA neurons in vivo confirmed our prediction. We thus identify: 1] a set of DA neurons mediating satiety (anti-motivation). 2] a second set that is specifically involved in motivating flies to consume amino acids at the cost of work (hard food), but not of bitterness (aversive due to the potential cost of toxicity). 3] the value of using a virtual brain simulation to find motivation-relevant neurons in vivo. We next seek to understand the cellular and molecular correlates of the motivated state in these neurons.

Huntsman Mental Health Institute, Dept. Psychiatry, University of Utah, SLC.

Hao Chen1, Shuangying Leng1, Jun Huang1, Caroline Jones2, Robert W Williams2, and Burt M Sharp2

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) under a fixed ratio 5 schedule, where licking was used as the operant behavior. Rats were not water or food deprived. Training started with 0.025 mg/ml oxycodone, gradually increased to 0.1 mg/ml, and session length was extended from 1-h to 16-h, followed by extinction and reinstatement sessions. Females (49 strains) and males (45 strains) licked significantly more on the active spout compared to the inactive spout (p<0.001). The number of active licks were greater in females than males during 4-h and 16-h sessions (p<0.001 for all). Both sexes escalated intake during 16-h extended access vs 4-h sessions (p<2e-16). The heritability of active licks has a range from h2 of 0.22 to 0.59, while that for inactive licks ranged from 0.08, 0.34 at different stages of self-administration. Initial QTL mapping using GEMMA with LOCO identified several significant loci, among them, a region in Chr 1 between 159-172 Mb was associated with oxycodone intake at 0.025, 0.05 and and 0.1 mg/ml, 4h sessions, with max – log10(p) values of 6.1, 5.1 and 5.6, respectively. Potential candidate genes within this range include Cyp2r1 and Pde3b, both have strong cis-eQTL in the brain and are involved in vitamin D metabolism.

1 Department of Pharmacology, Addiction Science And Toxicology

2 Department of Genetics, Genomics and Informatics University of Tennessee Health Science Center, Memphis, TN

Funding provided by NIH/NIDA U01DA053672.

BN Kuhn1 (presenting author underlined)

The rise of opioid use disorder (OUD) worldwide makes it imperative to disentangle the behavioral, genetic and neurobiological correlates associated with both OUD vulnerability and resiliency. Using a novel preclinical rat model of OUD that captures the multi-symptomatic diagnosis and complex multidimensional interactions between symptoms conferring OUD propensity, we have shown distinct behavioral and neurobiological profiles associated with each phenotype (n>1000). Additionally, genome-wide association study (GWAS; n=874) analysis indicates both resiliency and vulnerability to OUD are heritable states. GWAS identified genetic variants for nociception, heroin consumption and motivation to obtain heroin, with OUD vulnerability associated with the latter two. Several of the identified genes are known regulators of neuroplasticity, thereby prompting further investigation into neuroplastic mechanisms contributing to OUD propensity. Guided by findings from GWAS, we are assessing OUD phenotypic differences in components of the extracellular matrix (ECM), microglia and dendritic spine morphology within a canonical circuit necessary for OUD-like behaviors (prelimbic cortex, PrL; nucleus accumbens core, NAc; ventral pallidum, VP). Opposing phenotypic differences in PrL and VP ECM and microglia plasticity are evident, suggesting a mechanistic role for these neuroplastic components in mediating OUD vulnerability and resiliency. Furthermore, cell-specific alterations in NAc dendritic spine morphology are currently underway. Together these data identify novel genetic loci associated with OUD behaviors and vulnerability which further guided the assessment into neuroplastic measures that are likely contributing to OUD vulnerability and resiliency.

¹Department of Psychology and Neuroscience, Baylor University, Waco, TX, USA

Tolulope J Ajanaku^1,2, Eamonn P. Duffy^1,2 , Jonathon O. Ward³, Luanne H. Hale³, Caleb I. Hodges³, Laura M. Saba⁴, Marissa A. Ehringer^1,2, Ryan K. Bachtell^2,3

Prescription opioid use is limited by the development of analgesic tolerance and opioid‑induced hyperalgesia (OIH), yet the extent to which these adaptations are shaped by genetic background versus drug exposure remains unclear. Here, we used 20 inbred Hybrid Rat Diversity Panel (HRDP) strains to quantify strain differences in baseline thermal sensitivity, oxycodone analgesia, the development of tolerance, and OIH following voluntary oxycodone self‑administration. Rats completed a tail immersion test before (Pre‑SA, before self‑administration) and after (Post‑SA, after self‑administration) intravenous oxycodone or saline self‑administration. Analgesia was summarized as the area under the withdrawal‑latency curve, with tolerance defined as the change in area under the curve between trials. Heritability was estimated from the mixed‑effects models of various phenotypes. Baseline and post‑exposure analgesia and thermal sensitivity were moderately heritable (H² ≈ 0.24–0.30), whereas tolerance and change in thermal sensitivity showed much lower heritability (H² ≤ 0.10), indicating a larger contribution of non‑genetic factors to these adaptations. Most strains exhibited classic tolerance to oxycodone, but a few showed sensitization or resistance. Most strains also displayed increased thermal sensitivity after oxycodone self‑administration, indicative of OIH. Surprisingly, total oxycodone intake was only weakly related to tolerance at both individual‑ and strain‑mean levels, suggesting that the mechanisms regulating oxycodone consumption and those governing analgesic tolerance are at least partly dissociable. Together, these findings indicate that opioid analgesia and baseline pain sensitivity are strongly shaped by genetic background, whereas tolerance and OIH that emerge following volitional oxycodone intake are less heritable and loosely related to total drug exposure.

1Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, USA

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

3Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO,

USA

4Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical

Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA

Micah A. Shelton¹, Nicole Horan¹, Xiangning Xue², Lisa Maturin³, Darrell Eacret⁴, Julie Michaud⁵, Navsharan Singh⁶, Benjamin R. Williams⁷, Mackenzie C. Gamble^6,7, Joseph A. Seggio⁵, Madeline Kuppe-Fish⁷, BaDoi N. Phan⁸, George C. Tseng², Julie A. Blendy⁴, Leah C Solberg Woods⁹, Abraham A. Palmer³, Olivier George³, Marianne L. Seney^1*, Ryan W. Logan^7,10*

Opioid use disorder (OUD) has emerged as a severe, ongoing public health emergency. Current, frontline addiction treatment strategies fail to produce lasting abstinence in most users. This underscores the lasting effects of chronic opioid exposure and emphasizes the need to understand the molecular mechanisms of drug seeking and taking, but also how those alterations persist through acute and protracted withdrawal. Here, we used RNA sequencing in post-mortem human tissue from males (n=10) and females (n=10) with OUD and age and sex-matched comparison subjects. We compared molecular alterations in the nucleus accumbens (NAc) and dorsolateral prefrontal cortex (DLPFC) between humans with OUD and rodent models across distinct stages of opioid use and withdrawal (acute and prolonged) using differential gene expression and network-based approaches. We found that the molecular signature in the NAc of females with OUD mirrored effects seen in the NAc of female mice at all stages of exposure. Conversely, males with OUD showed strong overlap in expression profile with rats in acute withdrawal. Co-expression networks involved in post-transcriptional modification of RNA and epigenetic modification of chromatin state. This study provides fundamental insight into the converging molecular pathways altered by opioids across species. Further, this work helps to disentangle which alterations observed in humans with OUD are driven by acute drug exposure and which alterations are consequences of chronic exposure.

¹Department of Psychiatry, University of Pittsburgh School of Medicine
²Department of Biostatistics, University of Pittsburgh
³Department of Psychiatry, University of California, San Diego
⁴Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania
⁵Department of Biology, Bridgewater State University
⁶Department of Pharmacology, Physiology & Biophysics, Boston University School of Medicine
⁷Department of Psychiatry, University of Massachusetts Chan Medical School
⁸Medical Scientist Training Program, University of Pittsburgh School of Medicine
⁹Department of Internal Medicine, Section on Molecular Medicine, Wake Forest University School of Medicine
¹⁰Department of Neurobiology, University of Massachusetts Chan Medical School
*Corresponding authors

Rebecca Voss¹, Rebecca Osbaldeston², Matt Curran², Kevin Villafañe², Cole Roland², Roshan A. Jain^1,2

Serotonin (5-HT) regulates many aspects of behavior including mood, sleep, appetite, social interactions, and decision-making. A major challenge in untangling serotonin’s many distinct functions in humans is determining which of its 14 receptors are responsible for these diverse processes. We are modeling serotonin’s decision-making role through a simple response selection of zebrafish larvae, where serotonin modulates how zebrafish larvae bias their selection between two different acoustically evoked escape behaviors: an explosive short-latency response (SLC) and a kinematically and neuronally distinct long-latency response (LLC). Through a pharmacological screen, we found that 5-HT2B/2C receptor agonists shift behavioral bias towards SLC responses and antagonists shift bias towards LLC responses. To distinguish which specific receptors drive this modulation of decision-making, we used CRISPR-directed mutagenesis to disrupt each of the three zebrafish genes encoding 5-HTR2B/C subtypes, and then assessed the behavioral impacts. Mosaic larvae in which htr2cl1 was disrupted showed a behavioral shift towards LLC response, consistent with the drug data. In contrast, disrupting htr2b and htr2cl2 produced the opposite shift in escape behavior selection bias phenotype, towards SLC responses. Because mosaic G0 individuals may vary in the degree and type of molecular disruption, we have generated a set of novel germline-transmitted htr2b, htr2cl1, and htr2cl2 mutations. We are now using this set of mutations to clarify the individual and combined roles of 5-HTR2 receptors in simple acoustic decision-making. Together, our pharmacological and genetic results support a model in which 5-HT2B and 5-HT2C receptors bidirectionally modulate vertebrate decision-making following acoustic threat.

1 Bi-College Interdisciplinary Neuroscience Program, Haverford College, Haverford, PA 19041

2 Department of Biology, Haverford College, Haverford, PA 19041

H Gifford1, S Lin1, Y Ding1

From the vibrant dances of Birds of Paradise to the smelly nuptial gifts of dung beetles, mating signals have incredible diversity across the animal kingdom. The evolution of mating signals is a question with major implications for speciation and sexual isolation. Drosophila courtship song is an innate behavior where males vibrate their wings to produce context-dependent and species-specific acoustic signals for potential female mates. We found that the hybrid offspring of D. teissieri and D. santomea, two closely related species that sing distinct song types, produce “chimeric” courtship songs. These chimeric song types consist of simultaneous production of different parental song elements used for similar social contexts, suggesting that the species divergence of song types lie in the motor patterning circuits downstream of courtship song decision-making nodes. The genetic differences underlying this change are unknown. To explore the genetic differences, we generated a backcross F2 population of 570 individuals, scored song phenotypes, and used multiplexed shotgun genotyping to genotype the population. Quantitative trait locus mapping reveals a highly polygenic basis underlying the divergence in courtship song traits. Intriguingly, we also uncover pervasive inter-chromosomal epistasis, with any song phenotype effect being highly contingent on the extent of D. teissieri chromosomal introgression into the D. santomea genetic background. Our results indicate that species divergence in courtship song types involves changes at many gene loci that work together in a highly non-additive manner to shape the evolution of motor patterning circuits, highlighting the complexity of the genetic basis for neuronal and behavioral evolution.

1Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

Funding Support: NIGMS Grant GM142678

Antonio Marini-Davis and Fernando J. Vonhoff.

Datasets in the field of neurobiology are becoming increasingly complex, and common statistical methods such as T-test and ANOVA are struggling to capture this complexity. Researchers often discard informative details of their data through use of improperly specified models or non-parametric tests to conclude treatment effects. I detail here the increasingly used method of generalized linear (mixed) models to make more precise statistical conclusions when data doesn’t fit the normal assumptions for traditionally used statistical tests. Using a real behavioral dataset generated using a two choice preference based assay using Drosophila Melanogaster, I delineate the process of recognizing when more complex modeling is necessary, and show the diagnostic tools to ensure proper conclusions are drawn when using model-based statistical inference. Proper model specification allows future researchers to retain valuable information in their data and make better informed conclusions about their experimental outcomes.

University of Maryland, Baltimore County. US

Takeshi Murai¹, Lauren Mongeau¹, Lauren Bailey¹, Abbey Setlik¹, Stacey J. Sukoff Rizzo^1,2

Episodic memory is one of the long-term memories that involves the recollection of personal experiences or events (“What”), linked directly with the time (“When”) and place (“Where”). This is also one of the earliest cognitive domains that are impaired in age-related neurodegenerative disorders such as Alzheimer’s disease. We have been establishing a comprehensive battery of touchscreen-based tasks in marmosets that captures a spectrum of cognitive domains sensitive to detect aging-related cognitive decline. The present study describes the establishment of a Paired Associates Learning (PAL) test, which has been successfully used to evaluate episodic-like memory in humans. Initially, marmosets are trained on an FR-1 schedule of positive reinforcement using touchscreens mounted to their home cages. For the PAL task, subjects were trained to associate touch with a specific stimulus (“What”) presented in a specific location (“Where”) to receive a reward. Baseline testing presented two unique stimulus-location pairs. Once the subject learned the stimulus-location combinations, they were assessed in probe trials at 1 day, 2 weeks, 4 weeks, 3 months and 6 months of post-training. Our preliminary data demonstrate that marmosets have the ability to retain intact episodic-like memory over time. Interestingly, we observed the earliest decay in episodic-like memory in aged marmosets within 3 months, while younger marmosets retain episodic-like memory beyond 6 months. Ongoing studies continue to evaluate natural decay of episodic-like memory in this task in aged versus young subjects with longitudinal studies planned as annual assessments throughout the subject's lifespan.

¹Aging Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

²Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Funding Support: National Institute on Aging NIA R24AG073190, NIAU19AG074866

Robert van de Weerd

Neural circuit interventions hold promise for treating nervous system disorders but are limited by lack of specificity, inadvertently affecting nearby neurons and causing side effects. Cis-regulatory elements (CREs) offer a promising strategy to restrict optogenetic or chemogenetic tool expression to specific neuron subtypes. However, CRE discovery faces significant challenges including low in vivo success, species-specific activity, difficulties with multiplexed AAV screening, and limited spatial resolution. Here, we developed ESCargoT (Engineered Specificity of Cargo Transcription), a platform combining machine learning-guided CRE prioritization, modular AAV assembly, and multiplexed in situ spatial screening focused on the spinal cord, a critical region for pain and itch. First, we trained CRE prioritizing ML models on cross-species chromatin accessibility data from 15 dorsal horn neural subtypes and identified Excit-1, enabling chemogenetic inhibition that reversed inflammatory mechanical allodynia. Subsequently, we accelerated CRE discovery by developing SPRA (spatial parallel reporter assay) for multiplexed screening of a 27-candidate library, identifying new candidates that drive cargo expression in oligodendrocytes and several neuronal subtypes. We then validated two CREs targeting Exc-LMO3 and Exc-SKOR2 neurons and demonstrate that the Exc-SKOR2 enhancer, unlike Excit-1, suppressed chemical itch in mice. Together, our platform enables multiplexed in vivo enhancer profiling that accelerates CRE discovery and gene therapy development.

Carnegie Melon University

DV Gil¹, C Ferguson², M Miskanic², SS Mrozowski², GE Homanics^2,3, SP Farris^2,4

Alcohol activates the neuroimmune system, triggering inflammatory signaling thought to promote alcohol consumption and contribute to alcohol use disorder (AUD). However, the molecular mechanisms linking neuroinflammation to drinking behavior remain unclear. Malat1 is a widely expressed, evolutionarily conserved long non-coding RNA that promotes pro-inflammatory signaling, including in the central nervous system. Malat1 is upregulated in post-mortem brains from AUD subjects and rodents chronically exposed to alcohol, yet its causal role in regulating alcohol consumption remains unexplored. We hypothesized that Malat1 promotes immune-induced increases in alcohol consumption. To assess whether modulation of Malat1-associated neuroinflammatory signaling alters alcohol intake, tamoxifen-inducible Malat1 homozygous floxed, hemizygous CreERT2 (Malat1 global KO) mice and Cre negative littermate controls (WT) underwent ten days of baseline two-bottle choice (2BC; 10% v/v), followed by ten additional days of drinking post-treatment. One cohort received a single lipopolysaccharide (LPS; 1 mg/kg, i.p.) injection to induce inflammation before this period; another received daily quercetin (30 mg/kg, i.p.), an anti-inflammatory compound reported to regulate Malat1 function and attenuate alcohol reward, prior to each post-treatment session. A subset of mice from each group was monitored with sipper devices to characterize temporal drinking patterns. LPS increased alcohol intake in females regardless of genotype. Quercetin produced a treatment-by-genotype interaction in females, suggesting Malat1 differentially modulates quercetin’s effects on drinking. Neither treatment altered male drinking, though Malat1 global KO males consistently consumed more alcohol than WT controls. Together, these findings suggest greater female sensitivity to inflammation-associated drinking, and a sex-specific role for Malat1 in mediating alcohol intake.

¹Center for Neuroscience at the University of Pittsburgh ²Department of Anesthesiology & Perioperative Medicine, ³Department of Pharmacology and Chemical Biology, ⁴Department of Biomedical Informatics, University of Pittsburgh, Pittsburgh, PA, USA

Funding Support: NIAAA F31 AA032172, NIAAA U01 AA020889, NIAAA R01 AA030257

Alp M. Ummet ¹, Trudy F. C. Mackay¹, Robert R. H. Anholt¹

Previous studies revealed natural genetic variation in cocaine consumption and preference among the wild-derived, inbred lines of the Drosophila Genetic Reference Panel and implicated the mushroom bodies, brain structures that mediate experience-dependent behavior (Highfill et al., PLoS Genet. (2019) 15, e1007834). Previous studies also showed correlations between variation in mushroom body structure and behavior such as aggression and sleep. (Zwarts et al., Nat. Commun. (2015) 6, 10115). To assess whether variation in mushroom body morphology is correlated with variation in cocaine preference, we conducted an initial study by selecting six DGRP lines, three cocaine-preferring lines in which at least one sex showed preference for a cocaine-supplemented sucrose solution over control solution and three lines with mean aversion scores for cocaine preference. We dissected brains and stained mushroom bodies from males and females separately with an anti-fasciclin-II antibody. We quantified three-dimensional morphometrics of the alpha and beta lobes by confocal microscopy. We also observed the absence of lobes, bilateral asymmetry, and anatomical abnormalities. Our initial studies showed variation in mushroom body morphology and suggested a correlation of cocaine preference with alpha lobe structure and asymmetry. To consolidate these observations with statistical significance, we will expand this initial study to a larger sample of 48 lines. Based on evolutionary conservation of fundamental biological processes, correlations between variation in mushroom body morphology and cocaine preference in the fly brain raise the possibility that subtle variations in neural circuitry in the human brain could contribute to risk for cocaine use disorder.

1 Institute for Human Genetics, Department of Genetics and Biochemistry, Clemson University, Greenwood SC, USA.

Funding Support: Supported by grant DA041613 from the National Institute on Drug Abuse (NIDA) to TFC Mackay and RRH Anholt.

Andras Szabados, Kateryna M. Dukh, Ivanna Ihnatovych, Kinga Szigeti

α7nAChR is a key element of cholinergic anti-inflammatory pathway (CAIP); its activation leads to the inhibition of NF-κB complex. CHRFAM7A, a human-specific gene detected in 99% of the human population is present in different copy numbers and orientation (direct, inverted). The direct allele of CHRFAM7A is translated and gets incorporated into the α7 nAChR. The inverted CHRFAM7A allele is not translated and is a functional null from the α7 nAChR perspective. We have previously shown that the direct allele prolongs NFKB presence in the nucleus in iPSC derived microglia-like cells. We performed a human macrophage ex vivo study (N=70) to characterize NFKB translocation and Il-6 expression on the CHRFAM7A genetic background.

Primary human macrophages were treated with TLR agonists LPS, imiquimod, PAM2CSK4. NF-κB translocation dynamics was quantified over time using Manders’ coefficient. IL-6 expression was measured by ELISA.

In the iPSC model, the direct isogenic MGL cells demonstrated prolonged NF-κB nuclear translocation compared to null in response to LPS. In the human ex vivo model TLR agonists induced NF-κB translocation was prolonged compared to the null MGL on all 3 genetic background: translocation to LPS 4h (direct), 6h (heterozygous), up to 24 h (inverted); response to PAM2CSK4 - 45 min (direct), 2h (heterozygous), 4h (inverted). No NF-κB translocation was detected in response to imiquimod. The level of IL-6 was significantly elevated in response to all treatments, including imiquimod. The presence of both the direct and inverted CHRFAM7A alters the immune response to TLR agonists likely via distinct mechanism.

Department of Neurology, State University of New York at Buffalo, 875 Ellicott St., Buffalo, NY, 14203, USA

Funding Support: Community Foundation for Greater Buffalo (Kinga Szigeti).

Nicolás Rosas¹, Ivanna Ihnatovych¹, Kinga Szigeti¹

Human‑specific genes contribute to the unique structural and functional features of the human brain. CHRFAM7A is one such gene, created by the fusion of the α7 nicotinic acetylcholine receptor (α7nAChR) and ULK4, a member of the ULK kinase family. CHRFAM7A has been associated with neuropsychiatric diseases.

Three CHRFAM7A alleles exist in the human population: the ancestral allele (0‑copy), allele harboring the direct‑orientation fusion gene (CHRFAM7A), and the allele with inverted fusion gene carrying a 2‑bp deletion in exon 6 (CHRFAM7AΔ2bp). Because animal models lack CHRFAM7A and its variants, studying its role in brain development and disease has been challenging. We developed a human isogenic iPSC model to elucidate the function of CHRFAM7A. We have previously shown that the direct allele modulates α7nAChR activity.

Here, we use iPSC‑derived medial ganglionic eminence (MGE) neuronal progenitors and primary human monocytes to investigate how the inverted CHRFAM7AΔ2bp allele influence neuronal development and decipher the underlying genetic mechanism. Through quantitative live-cell imaging, we assessed cytoskeletal debundling, growth cone dynamics, cell motility, and neurite arborization to characterize cellular processes affected by this human-specific variant.

We found that the inverted CHRFAM7A allele modulates ULK4 expression through a distinct genetic epistasis, driven by the emergence of an alternatively spliced ULK4 exon 12-18sh variant. The splice variant shifts ULK4 isoform balance toward the long isoform, leading to a microtubule cytoskeleton gain‑of‑function that enhances neuronal complexity and cell motility, ultimately strengthening functional connectivity in the human brain.

¹Department of Neurology, State University of New York at Buffalo, 875 Ellicott St., Buffalo, NY, 14203, USA

This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti).

Nour Selim 1, Romeo Aiyabei 1, Ivan Soto 1, Gauri Paul 1, and Cynthia T. Hsu 1

Parkinson’s pathology, in which the progressive loss of dopamine neurons contributes to debilitating loss of motor control, is an important and active area of biomedical research. While oxidative stress is known to contribute to neurodegeneration, the potential for environmental influences to exacerbate the condition are not well understood. Blue light is a prevalent environmental factor that is attributed to the habitual use of electronics; as such, exploring its adverse effects on humankind is a necessity. Previous literature has shown that blue light induces oxidative stress in D. melanogaster, which may indicate the risk for increased damage to DA neurons and perpetuate the loss of motor functions. We have found that blue light exposure not only increases oxidative stress but also sleep in a cryptochrome dependent manner. This raises two questions that we are currently investigating: (1) if blue light is sufficient to increase oxidative stress specifically in the dopaminergic neurons, and (2) if dopaminergic neurons increase activity in response to blue light or if they are downregulated to promote sleep and thus recovery from oxidative damage. This will help determine whether the oxidative stress caused by blue light is sufficient to affect dopaminergic neuronal function and thus lead to neurodegenerative ailments.

1 California State University, Fresno, CA, USA

N Fairbanks1, C Forbes1,2, S Stringfield1,2, A Sved1,2, J Zeak1, M Seney1,2, Y Huang1,2, M Torregrossa1,2, C McClung1,2

Adolescence is a period of substantial neurodevelopment and behavioral changes, increasing their vulnerability to drug use and developing substance use disorders. Developmental changes in sleep and circadian rhythms, primarily represented by a shift towards a more evening chronotype, are increasingly recognized as relevant to understanding adolescent addiction vulnerability. Thus, this natural variation in sleep and circadian traits seen in adolescence may be associated with indicators of risk for substance use, such as altered cognitive function, and impulsivity. To investigate this interaction, male and female adolescent heterogenous stock rats were screened on measures of sleep duration and circadian chronotype prior to being either tested on measures of impulsivity, attention and cognition using the 5-choice serial reaction time task (5-CSRTT), and for propensity to self-administer nicotine, or underwent electrophysiology recordings of Nucleus Accumbens (NAc) action potential firing. Our results showed that circadian period had the strongest relationship with impulsivity. Specifically, rats with a short circadian period were more impulsive and made more premature responses. We also found that circadian period was strongly associated with measures of nicotine self-administration, where a longer circadian period was associated with more total nicotine intake and greater lever pressing. These results suggest that natural variation in sleep and circadian traits, like having a short or long circadian period, may be associated with different risk factors of substance use disorders.

1Department of Psychiatry, 2Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA

Funding Support: National Institute on Drug Abuse (USA), P50DA046346

VD Knickerbocker1, T Laster1, J Osgood1, K Perron1, N Stroud1, J Suckovic1, C Wise2, JM Wotton2, and Z Bichler1

C57BL/6J mice represent the predominant inbred background in preclinical research, with extensive phenotypic characterization available through resources such as the Mouse Phenome Database (https://phenome.jax.org/). Despite this breadth, inter‑laboratory variability in experimental design, protocol implementation, and under‑powered designs frequently limit the reliability and reproducibility of reported phenotypes. At the same time, many additional mouse strains are routinely used, further highlighting the need for harmonized reference data.

To address this, we leveraged the large, continuously expanding dataset generated at The Jackson Laboratory’s Neurobehavioral Phenotyping Core since 2019, comprising mice tested under harmonized protocols, controlled environmental parameters, and validated operator procedures. Using aggregated multi‑assay datasets, we derived strain‑specific and age‑stratified reference ranges for key motor and activity‑related phenotypes. Analytical efforts included composite score generation, inter‑assay correlation matrices, and cross‑modal concordance analyses to evaluate redundancy and discriminative sensitivity across widely used motor assays.

Our initial objective was to develop recommendations for aging studies using C57BL/6J as a benchmark strain. We identified assays most sensitive to age- and sex-related differences, estimated empirically supported group‑size thresholds, and examined correlations across assays to highlight complementary or redundant motor measures. These analyses provide a framework for selecting reliable phenotypes and designing efficient behavioral pipelines.

Ultimately, we hope this effort will contribute to improved reproducibility, more strategic and ethical study design, and broader alignment with the 3Rs (Replacement, Reduction, Refinement) principles in in vivo behavioral research.

1Neurobehavioral Phenotyping Core at the Center for Biometric Analysis, 2Center for Biometric Analysis, The Jackson Laboratory, Bar Harbor, Maine, USA.

Acknowledgement: The authors would like to extend the co-authorship to all former staff

members of the Neurobehavioral Phenotyping core at the Center for Biometric Analysis at The Jackson Laboratory as they have generated or help generate essential data needed for this work.

JW DeMarsh^1,6, AJ Brandner^1,6, GE Homanics^3,4, BK Taylor^2,3,6, SP Farris^2,5,6

Alcohol Use Disorder (AUD) is a chronic, relapsing condition in which pain during withdrawal- collectively termed chronic alcohol withdrawal-induced pain (CAWIP)- contributes to continued alcohol use and relapse. Despite its clinical relevance, the behavioral and neurophysiological mechanisms underlying CAWIP remain poorly understood. This study broadly characterizes CAWIP using complementary behavioral and electrophysiological measures across two mouse models of chronic alcohol exposure. Adult C57BL/6J mice underwent either chronic intermittent ethanol vapor (CIEV) exposure to model dependence or a novel one-bottle access (1BA) voluntary drinking paradigm as a comparison model. Pain-related behaviors were assessed via von Frey testing for cold hypersensitivity, and conditioned place pereference/aversion (CPP/CPA) for spontaneous pain-like behaviors. To investigate underlying neurophysiological changes, excitatory neurons in the parabrachial nucleus were examined for markers of hyperexcitability, spontaneous activity, and membrane resistance. Withdrawal from both CIEV and 1BA exposure produced cold hypersensitivity and mechanical hypersensitivity alongside electrophysiological recordings that confirmed increased neuronal hyperexcitability, spontaneous activity, and altered membrane resistance. Together, these findings suggest that CAWIP involves intrinsic central neuronal changes that may drive sensory hypersensitivity during withdrawal. These results advance our understanding of the multidimensional nature of CAWIP and set the stage for future studies to uncover the central neuronal mechanisms driving this phenomenon and discover more clinically-relevant therapeutic targets.

¹University of Pittsburgh, Center for Neuroscience, Pittsburgh, PA, 15261, USA ²University of Pittsburgh, Department of Anesthesiology & Perioperative Medicine, Pittsburgh, PA, 15261, USA

³University of Pittsburgh, Department of Pharmacology & Chemical Biology, Pittsburgh, PA, 15261, USA

⁴University of Pittsburgh, Department of Neurobiology, Pittsburgh, PA, 15261, USA

⁵University of Pittsburgh, Department of Biomedical Informatics, Pittsburgh, PA, 15261, USA

⁶University of Pittsburgh, Center for Pain Research, Pittsburgh, PA, 15261, USA

Mandi Luellen, Melody S. Kirby, and Cynthia T. Hsu

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors that regulate neurotransmission, synaptic plasticity, and neuronal excitation. Disruptions to mGluR function have been implicated in psychiatric disorders including schizophrenia and mood disorders. Here, we present preliminary data characterizing knockdown of two metabotropic glutamate receptor homologs in Drosophila, mangetout (mtt) and metabotropic glutamate receptor (mGluR), in sleep, locomotor, and feeding assays. We find that constitutive pan-neuronal knockdown of mangetout is lethal while adult-specific knockdown leads to drastic impairments in locomotion. In contrast, while previous studies have reported sleep deficits in constitutive pan-neuronal knockdown of mGluR, we find that adult-specific knockdown does not compromise sleep or locomotion. This suggests that the role of mGluR in sleep is developmental, in contrast to mtt, which has significant effects in adult behavior. Supplementing mtt knockdown flies with octopamine and serotonin precursors does not rescue locomotor phenotypes. Our findings may indicate an overlooked role of mtt as a homolog for metabotropic glutamate receptors and a potential target for pharmacological screens.

California State University, Fresno

Abigail Setlik1, Rishabh Choudhary1, Lauren Bailey1, Takeshi Murai1, Lauren Mongeau1, Emily Rothwell2, Afonso Silva2, Stacey J. Sukoff Rizzo1,2

Emerging data indicate that impairments in motor function and alterations in sleep may precede Alzheimer’s disease (AD) pathology and related cognitive decline by several years. The present studies investigated early changes in activity and circadian patterns in common marmosets (Callithrix jacchus) with genetic mutations in the PSEN1 gene which confers early onset AD in humans. Nanowatches (Camntech LTD) were attached to the collars of freely moving male (n=16) and female (n=22) marmosets (aged 1-6 yrs). Individuals were evaluated longitudinally over up to a 3-year period which revealed behavioral changes with aging and disease progression measure by AD related biomarkers. Ongoing studies continue to investigate the earliest behavioral changes associated with genetic risk for AD as a functional prodromal biomarker to reveal the earliest activity changes that precede disease and cognitive decline.

1Aging Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 2Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Adore D. Ferguson-Richards, Justin Minerva, Jacob A. Krawitz, Nicky Rashkover, Roshan A. Jain

To adapt to our environment, our nervous system is constantly interpreting sensory information allowing us to react to relevant stimuli while ignoring the irrelevant ones. Which allows for the appropriate response to the stimuli based on the current environmental constraints. One of the main contributors to this is a form of non-associative learning called habituation, characterized by a decline in responsiveness to a stimulus after repeated exposure. Habituation learning is evolutionarily conserved across all animals from invertebrates up to humans. Habituation learning can vary in a number of neuropsychiatric conditions in humans including Attention-Deficit/Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD). There are multiple genes associated with these conditions; the one we chose to focus on is AP2S1. This gene encodes a subunit of the AP2 complex which assists in clathrin mediated endocytosis however its role in behavioral regulation is not well understood. When ap2s1 is mutated in zebrafish, larvae show deficits in habituation of the acoustically-evoked escape response, making this a useful model to understand the mechanism of this learning. The fast escape response of five day old larval zebrafish is controlled by a pair of command neurons called the Mauthner cells. When a Mauthner cell fires, larvae perform a fast escape, so we reason that habituation requires regulating these critical neurons. The Spiral Fiber neurons are excitatory neurons that are presynaptic to the Mauthner cells and are thought to regulate Mauthner firing. We investigated the activity of the Spiral Fiber termini, which bundle together around the start of the Mauthner axon as well as the Spiral Fiber cell bodies. We used a fluorescent calcium indicator (GCaMP5G) to visualize Spiral fiber structure and activity to determine if there is an association between the development of these neuronal structures and the altered habituation of the mutants. For the wild type siblings there is activity at the Spiral Fiber termini which reduces as the fish habituate. In the mutants there is higher activity observed at the Spiral Fiber termini compared to the wild type. To understand why I am measuring the dimensions of the bundle of Spiral Fiber termini as well as counting the cell bodies to determine if there’s a developmental difference between the mutants and the siblings. By characterizing the circuit development of Spiral Fiber neurons we hope to better understand how ap2s1 regulates habituation learning.

Dakota F. Brockway, Samuel L. Boehm, Nilah D. Jordan, Max E. Joffe

Alcohol use disorder is characterized by maladaptive decision making and persistent alcohol seeking, yet the circuit mechanisms within the prefrontal cortex that drive alcohol motivation remain poorly understood. Vasoactive intestinal peptide (VIP) interneurons are powerful regulators of cortical network activity through disinhibitory circuit motifs and VIP neuromodulatory signaling. However, the role of VIP interneurons in alcohol-related behaviors has not been established. We investigated how VIP signaling in the prelimbic prefrontal cortex regulates alcohol motivation and prefrontal circuit activity. Whole-cell patch clamp electrophysiology revealed that VIP directly depolarizes VIP interneurons, suggesting that VIP signaling can amplify VIP interneuron activity within cortical circuits. Consistent with this mechanism, intoxicating doses of systemic alcohol administration increased calcium activity in VIP neurons measured using fiber photometry in vivo. To determine whether VIP neuron activity contributes to alcohol seeking, we manipulated VIP neurons during operant alcohol self-administration. Chemogenetic activation of VIP neurons significantly increased motivation for alcohol under progressive ratio schedules, indicating that VIP neuron activity promotes alcohol-seeking behavior. In addition to regulating interneuron activity, VIP signaling differentially modulated layer 5 pyramidal neurons depending on projection subtype. Electrophysiological recordings revealed that VIP excitation enhanced excitability in extratelencephalic neurons while suppressing action potential firing in intratelencephalic neurons, suggesting that VIP signaling may bias prefrontal output toward specific downstream circuits. Together, these findings identify VIP interneurons as a previously unrecognized regulator of alcohol motivation and suggest that alcohol recruits VIP signaling to reshape prefrontal cortical output.

Justine Anne A. Guevarra¹ and Fernando Vonhoff¹

Alzheimer’s Disease (AD) is the most common form of dementia and is associated with mutations in certain genes including APP. The human APP gene encodes for amyloid precursor protein (App), which is a transmembrane protein expressed in several tissues and organs, including the brain. Although its physiological functions remain elusive, studies in mice and fruit flies show that lack of App proteins results in developmental and locomotor deficits, which can be rescued by the expression of the human App, confirming its conserved properties. App is also a metalloprotein, which can bind to metals such as copper and zinc. Interestingly, recent studies display increasing evidence on the possible link between metals and neurodegenerative disorders. Post-mortem AD brains reveal accumulation of metals such as aluminum, copper, zinc, etc. To our knowledge, there is no known study investigating the possible protective role of the fly ortholog, appl, on metal exposure in flies. This project aims to investigate the effects of metal exposure in flight behavior in Drosophila melanogaster. Current observations showed a decline in flight performance in appl null flies compared to wild-type flies following aluminum treatment, suggesting a protective role for appl. Surprisingly, copper treatment did not influence the appl null flies but resulted in lower survival rate and flight performance in wild-type flies. Overall, the knowledge gained from the experiments will serve as a reference for future studies in mammals and broaden the understanding of the mechanisms involving App / Appl and metal toxicity in flies.

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

Lauren R. Mongeau¹, Lauren Bailey¹, Takeshi Murai¹, Abbey Setlik¹, Andrew DeSana¹, Afonso Silva², Stacey J. Sukoff Rizzo^1,2

¹Aging Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

²Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Introduction: Alzheimer's disease (AD) is a neurodegenerative disorder and the most common form of dementia. While a primary symptom is cognitive decline, sensory deficits including impairments in taste and smell have emerged as early indicators of amyloid and tau pathology that precede cognitive impairment. The ability to discriminate the functional changes related to variation in normal healthy aging from those that lead to pathological aging, and the onset of AD may help predict the earliest accumulation of AD pathology and enable better detection, earlier diagnosis, and interventions for prevention and treatment. For these studies we leveraged a large population of marmosets including marmosets seeded with tau to evaluate the trajectory and progression of AD pathology and sensory and cognitive impairments.

Methods: Male and female marmosets across an age span ranging from 18 months to 16 years utilized for this task. Marmosets were initially habituated to the presentation of a panel fixed with two drinking bottles placed on the front of their homecages that both contained a 20% solution of marshmallow juice (MJ) formulated from dehydrated marshmallows into drinking water for a 10-minute session. Following the habituation session in which we verified that marmosets sampled from both bottles, we conducted a two-choice preference test in subsequent sessions with varying solutions of MJ (0.5%, 1%, 5%, 10%) or water, randomized for testing order and counterbalanced for side using a modified latin-square design. All 4 sessions were conducted on consecutive days of the week with a second week of testing presenting the same testing order but the opposite presentation for side from the previous week. The bottles were weighed before and after the session to determine the amount consumed and the preference ratio of the water to MJ. A dummy bottle was placed on the opposite side of the panel to account for potential leakage.

Results: Preference ratio was evaluated of animals that have tau pathology. All Marmosets could discriminate against the higher concentrations of 10% and 5% MJ relative to water. We saw differential responses at the 1% and 5%,

Conclusion: With this, we can establish typical marmoset taste discrimination longitudinally in wild-type marmosets, and marmosets with higher risk of tau pathology. Analysis of these studies provide characterization of gustation discrimination and decline in healthy marmosets and marmosets with risk of tau pathology across lifespan, which are being correlated with aging and AD related biomarkers to inform and predict cognitive decline associated with non-specific dementia.[LM1]

*add funding information here

• [LM1]Dementia progression (non-specific) impairs taste and smell (Waldton, 1974; Lang et al., 2006 Steinbach et al., 2010)

Shani Beicher (Raphael) and Sigal Ben-Zaken, PhD

Creativity—the capacity to generate novel and valuable solutions—represents a complex cognitive phenotype with substantial inter-individual variation. While heritability estimates from twin studies suggest 40-70% genetic contribution to creative abilities, the molecular genetic architecture underlying creativity remains poorly understood. The dopaminergic system, particularly genes regulating dopamine metabolism and signaling in the prefrontal cortex, has emerged as a promising candidate pathway given dopamine's established role in cognitive flexibility and executive function.

Motor creativity, defined as the ability to generate diverse and novel motor solutions to environmental challenges, represents a specialized form of creative cognition critical for performance in dynamic, unpredictable contexts such as combat sports. Elite Judo—a combat sport requiring rapid tactical decision-making and execution of complex motor sequences under pressure—provides an ideal model system for investigating the genetic basis of motor creativity. Furthermore, Judo offers unique advantages for studying sex differences, as it is one of few combat sports with gender parity in competitive participation and achievement.

Despite extensive research on cognitive creativity, motor creativity has received minimal attention, and potential sex-specific genetic influences have been virtually unexplored. Understanding these mechanisms has broad implications for behavioral genetics, extending from talent identification in specialized domains to personalized cognitive enhancement strategies.

Methods

We conducted a genetic association study in 77 elite Israeli Judo athletes (32 females, 45 males; aged 16-25 years) representing national and international competitive levels. Participants completed validated assessments of divergent thinking (verbal and figural creativity measuring fluency, flexibility, and originality) and sport-specific motor creativity tasks. Buccal epithelial samples were genotyped for three functionally relevant polymorphisms in the dopaminergic system: COMT A/G rs4680 (catechol-O-methyltransferase, regulating prefrontal dopamine catabolism), BDNF C/T rs6265 (brain-derived neurotrophic factor, modulating dopamine neuron survival), and DRD2 C/T rs6277 (dopamine receptor D2, affecting receptor expression and signaling).

Results

Female athletes exhibited significantly higher scores across all creativity dimensions compared to males (fluency, flexibility, and originality; all p < 0.05). Moderate to high correlations emerged between divergent thinking measures and motor creativity scores (r = 0.45-0.68), particularly in males, suggesting partially shared cognitive mechanisms.

Critically, we identified a significant sex-by-genotype interaction for COMT rs4680 across all figural divergent thinking dimensions (p < 0.05). Female athletes carrying the GG genotype (low COMT activity, higher prefrontal dopamine) demonstrated the highest creativity scores, while male athletes with the AA genotype (high COMT activity, lower prefrontal dopamine) showed superior performance. This interaction remained significant after controlling for training history and competitive achievement level. No significant main effects of genotype or interactions were observed for BDNF rs6265 or DRD2 rs6277 polymorphisms.

Discussion

Our findings reveal sex-specific genetic influences on creative cognition, with opposite effects of the COMT rs4680 polymorphism in males versus females. This pattern suggests that sex hormones modulate the relationship between prefrontal dopamine tone and creative thinking. Estrogen and testosterone receptors are abundantly expressed in prefrontal cortex and interact with dopaminergic signaling pathways. The observed interaction may reflect sex-differential optimal dopamine levels for creative cognition, consistent with inverted-U models of prefrontal dopamine function.

These results extend beyond athletic performance, providing insight into fundamental mechanisms of sex differences in cognition and the complex interplay between genetic variation, sex hormones, and neurotransmitter systems in shaping behavioral phenotypes. Future research should examine additional dopaminergic polymorphisms, investigate the neural mechanisms mediating these sex-specific effects using neuroimaging approaches, and determine whether similar gene-by-sex interactions influence creativity in non-athletic populations and other cognitive domains.

F. Paliarin, E. Doré, P. Panthagani, S. Mirza, L. Finlay, T. Nguyen, E. Weiser, C. Duplantis, and R. Maiya

Social stress is a critical driver of escalated alcohol use and relapse; however, the associated molecular mechanisms are poorly understood, limiting the identification and evaluation of therapeutic targets. We have demonstrated using Social Defeat Stress (SDS) model that alcohol consumption was escalated in both male and female C57BL/6J mice. Stress related behaviors have been shown to be mediated via Dynorphin/Kappa opioid receptor (Dyn/KOR) system. When administered systemically with a long-acting KOR antagonist like Norbinaltorphimine (NorBNI), both male and female stress animals reduced alcohol consumption, with minimal effect on unstressed controls. Stress escalated alcohol consumption was ameliorated in Oprk1-Cre male mice after chemogenetic activation of KOR expressing neurons in Basolateral Amygdala (BLA^KOR). Both KOR antagonism and chemogenetic activation of BLA^KOR terminals in Bed Nucleus of Stria Terminalis (BNST) attenuated escalation of alcohol consumption in both males and females, implicating the BLA^KOR-BNST pathway in SDS induced drinking. In line with these findings, we found that, KOR deletion in the BLA diminished SDS-escalated alcohol consumption in female mice. We further observed increased prodynorphine (pDyn) expression in Dorsal raphe nucleus (DRN) and enhanced activation of BNST-projecting DRN^Dyn neurons following social stress, suggesting these neurons as a key source of Dyn recruited by SDS. Future experiments will examine the causal role of DRN^Dyn neurons in SDS-induced alcohol escalation following deletion of KOR in BLA-BNST pathway.

Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, 70112, USA

Funding: R01AA031733, R01AA027293 and LSUHSC startup funds.

A.T. Bechard¹, R. Ataei¹, & A.F. Simon¹

Sleep disturbance is strongly connected to increases in disordered behaviour such as inattention, social avoidance, and hyperactivity across species. There is increasing evidence that the bidirectional relationship between disturbed sleep and disordered behaviour may be modulated by changes in gene expression. Mutations in genetic regulators of dopamine availability and circadian rhythm have been associated to this relationship, but how expression of such genes may mediate these effects has yet to be explored. To investigate this connection, flies (Drosophila melanogaster) are a great model due to their genetic tractability and thoroughly characterized behavioural assays. In this study, the effect of sleep disturbance on daily locomotor activity and sleep are measured in flies with altered expression of primary circadian regulator period (per) or dopamine transporter (dat). Preliminary results indicate that dat mutants of both sexes display an exaggerated locomotor response to the mechanical stimulation used to disrupt sleep. Also, dat may play a more prominent role in female sleep compared to males, and per a stronger role in males. In addition to gene expression analysis via RT-qPCR, next steps include behavioural assays on dat and per mutants to measure the effect of sleep disruption on hyperactive, inattentive, and social behaviours. The results of this study will help to establish the symptomatological relationship between sleep disturbance and the expression of genes regulating circadian rhythm and dopamine availability.

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

Funding support: internal grants to AFS and ATB; NSERC Fellowships to ATB; NSERC Discovery grants 05054-2022 to AFS.