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