Danielle Posthuma Genome-wide association studies (GWAS) have successfully identified many novel loci for neuropsychiatric traits. At the same time the results of GWAS showed that these traits are highly polygenic, mostly influenced by large numbers of weakly associated variants. Interpreting such polygenic results is challenging. Recent large-scale initiatives, such as those from the Allen Brain Institute and the PyschEncode consortium provide fine-scaled atlases of functional genetic elements at cellular level. This novel information can be used to interpret results from GWAS studies and facilitate biological understanding of complex traits. In this session, I will discuss how we can leverage both GWAS results and novel functional genomic resources to formulate hypotheses that can be tested in functional experiments. I will discuss current and just finished work on gene finding for brain related traits, including strategies to link genetic findings to biology or drug targets. This includes the latest results - hot off the press - from a large GWAS on Alzheimer’s disease for the PGC-ALZ working group, and several new findings for psychiatric disorders, including schizophrenia. Vrije Universiteit, Amsterdam, NL

M Hernández¹, R Sultana¹, D-J Paredes¹, AM Barkley-Levenson¹

Recent genome wide association studies (GWAS) have identified numerous novel hits for problematic alcohol use and alcohol consumption. However, follow-up studies are still needed to demonstrate a causal relationship between implicated genes and alcohol-related traits. Here, we describe the functional validation of Dpp6, which is a novel genetic association for problematic alcohol use and alcohol drinking. Dpp6 encodes an auxiliary subunit of A-type voltage-gated potassium channels and is involved in modulating dendritic excitability and synaptic plasticity. We have found that global knockout of Dpp6 does not alter ethanol binge-like drinking or total consumption in a chronic intermittent two-bottle choice procedure, but does produce an escalation in ethanol preference over time in this procedure compared to wild type (WT) littermates. Knockout mice also show greater binge-like sucrose intake in a single bottle procedure, but do not differ from WT in sucrose preference in a two-bottle choice test. However, we do see a significant increase in ethanol sensitivity in the knockout mice compared to WTs across multiple behaviors (ethanol conditioned place preference, locomotor sedation, and ethanol-induced anxiolysis) following ethanol injections, suggesting that route of administration may be relevant for the genotypic differences observed in this model. Taken together, these findings confirm that loss of Dpp6 does impact multiple ethanol-associated behavioral phenotypes, even without significantly altering voluntary ethanol consumption.

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

Funding support: NIH-NIAAA grant R00AA027835, NIH-NIGMS grant K12GM088021

Karina Piotrowska1 , Annie Park1 , Scott Waddell1 1

Nicotinic acetylcholine receptors (nAChRs) are implicated in the reinforcing properties of addictive substances. However, their heterogeneous composition makes it difficult to resolve how cell-type specific subunit combinations contribute towards addictive behaviour in vivo. Neonicotinoid insecticides are known nAChR agonists, and have been implicated in pollinator decline due to their addictive properties. We are studying these addictive behaviours in Drosophila, which permits an analysis of the roles of specific nAChR subunits within the dopaminergic system. Quantitative analysis of feeding revealed that flies form a robust dietary preference for sucrose laced with neonicotinoids. Moreover, chronic neonicotinoid exposure results in further elevated consumption of pesticide-laced sucrose, reflecting an experience-dependent adaptation in reward valuation. Optogenetic inhibition revealed an unexpected role for aversively reinforcing dopaminergic neurons in promoting neonicotinoid preference. Single-cell sequencing data of subtypes of dopaminergic neurons reveals differential cell-type specific expression of nAChR subunits. We are currently investigating whether this potential heterogeneity of nAChR composition drives functionally distinct cellular responses to neonicotinoids in vivo. Our studies will generate a cell-type resolved model of neonicotinoid function within dopaminergic circuits, which should uncover conserved neural mechanisms underlying neonicotinoid, and nicotine, addiction.

Centre for Neural Circuits and Behaviour, Department of Physiology, Anatomy and Genetics, University of Oxford

Myra Bower^1,2, Andrew Lombardi¹, Cate Hensley ², Curtis Borski^1,2, Kora Kastengren^1,2, Erika Mehrhoff^1,2, Charles Hoeffer^1,2 Marissa Ehringer^1,2, Jerry Stitzel^1,2

Tobacco use remains the world’s leading cause of preventable death and disease. Nicotine use disorder is characterized by immediate neuronal adaptations that may promote use. While studies of neurons are informative, a critical perspective is missing for other cell types. Astrocytes are dynamic regulators of brain homeostasis and active participants of neurocircuitry underlying substance use disorders. Clusterin (CLU), a gene identified by large scale human genome wide association studies (GWAS) of smoking, is a gene also primarily expressed by astrocytes. Using a mouse model, we investigated the role of Clusterin in nicotine induced astrocyte morphology and reward behavior. Immunohistochemical staining for area and Sholl analysis of tissue collected 24 hours after nicotine or saline control identified differences due to Clusterin knockout. Mouse astrocytes were assessed in both primary cell culture exposed to nicotine (100 um) and in adult hippocampus after subcutaneous nicotine injection (0.35 mg/kg). Clusterin loss reduced the morphological response of astrocytes to nicotine in vitro and in vivo. Clusterin knock-out and wild-type mice were also tested for nicotine reward by conditioned place preference at the same dose. Additionally, we also see a trend (p = 0.07) for Clusterin knock-out towards a place aversion to nicotine, suggesting loss of the gene induces a more unpleasant experience at this dose. These data support Clusterin as a genetic modulator of nicotine-conditioned responses and glial plasticity that may prevent nicotine use in mice.

¹Department of Integrative Physiology, University of Colorado at Boulder;

²Institute for Behavioral Genetics, University of Colorado at Boulder.

Zachary Tatom1 , Maya Eid2 , Thiago Missfeldt Sanches1 , Apurva S. Chitre1 , Denghui Chen1 , Benjamin Johnson1 , Elaine Keung1 , Oksana Polesskaya1 , Tom Jhou2 , Abraham A. Palmer1,3

Many substances of abuse produce initial rewarding eHects (euphoria) followed by a period of aversive eHects including anxiety, craving, anhedonia, and withdrawal. Dysregulation of these aversive eHects has been suggested to contribute to the etiology of substance use disorders through aberrant avoidance-based learning. We hypothesize that GWAS of avoidance-based learning using both food-based and cocaine-based behavioral assays in Heterogenous Stock (HS) rats will enable estimation of the heritability of these behaviors and identify new loci associated with them in rats. We exposed 1,074 HS rats (379 male, 695 female) to behavioral testing including runway operant cocaine-seeking, food-based progressive ratio and punishment testing, and locomotion assays. Our results demonstrate that behavioral phenotypes derived from these assays are significantly heritable (with h2 estimates ranging from 0-0.307) and identify significant (p < 0.05) genetic loci related to avoidance-based learning including from the punishment task on Chromosomes 2, 3, 5, and 6, and the cocaine-operant runway latency task on Chromosome X. 172 positional candidate genes were identified from significant and suggestive loci, including genes from the cadherin family and genes associated with primary neuronal cilia. Lead GWAS SNPs were also associated with novelty-related and social interaction phenotypes in PheWAS from independent samples of HS Rats. Our results suggest that these avoidance-based learning traits are themselves complex heritable traits which may pleiotropically aHect other aspects of addiction biology.

1 Department of Psychiatry, University of California San Diego, La Jolla, CA, USA; 2 Department of Neurobiology, School of Medicine, University of Maryland, Baltimore, MD, USA; 3 Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA Funding Support: R37DA054370, P30DA060810, T32AA013525

Rose Marie Akiki, Sierra Simmerson, Caila Worley, Rebecca G Cornbrooks, Kosuke Magami, Alain Greige, Kristen K Snyder, Daniel J. Wood, Mary Claire Herrington, Philip Mace, Kyle Blidy, Nobuya Koike, Stefao Berto, Christopher W. Cowan, Makoto Taniguchi

Emotional experiences often drive behavioral responses that enable organisms adapt to their environment. These experiences trigger the expression of specific gene, initiating transcriptional programs that underlie cellular, synaptic, and behavioral plasticity. This gene expression is tightly regulated and plays critical role in adaptive responses to emotional stimuli. Conversely, dysregulation of these transcriptional program is implicated in maladaptive behaviors associated with neuropsychiatric disorders, including mood and substance use disorder (SUD). We recently demonstrated that long non-coding RNAs (lncRNAs) form DNA:RNA hybrid structure, R-loops, across the genome. Genes associated with these R-loops are identified in the glutamatergic and dopaminergic signaling pathways, calcium signaling pathways, and synaptic plasticity-related genes. We discovered that neuronal activity induces the formation of R-loop at the enhancer region of immediate early gene, Npas4, a gene that we previously identified as a key regulator of stress-induced anhedonia-like behavior and drug reward-associated contextual memory. This R-loop is essential for linking distal enhancer to the proximal promoter, facilitating Npas4 gene expression, and regulating cocaine reward-associated contextual memory. Together, these data identify R-loops as novel regulatory mechanism that translates emotional experience into gene expression and behavioral plasticity. In this presentation, we will discuss the functional mechanisms of R-loops in gene expression and behavior in response to emotional experiences.

Department of Neuroscience, Medical University of South Carolina

Funding support: NIH_T32 DA07288, R01 DA032708, R01 MH129521, P20 GM140964, P20 GM148302, P50 DA046373, and UL1 TR001450.

Brad Balderson1 , Narayan Pokhrel2 , Arnav Gurha2 , Yanning Zuo2 , Benjamin Johnson2 , Olivier George2 , Abraham A. Palmer2,3 , Graham McVicker1 , Francesca Telese2

The nucleus accumbens (NAc) is a key subcortical brain structure that regulates reward and is involved in addiction. However, the molecular basis of individual differences in addiction phenotypes are not well characterized. To dissect the molecular basis of oxycodone addiction, we used an intravenous self administration assay (IVSA) to measure oxycodone addiction-like behaviours in outbred Heterogeneous Stock (HS) rats. NAc tissues were collected from 85 HS rats after 5 weeks of abstinence from oxycodone. We obtained whole genome sequencing, and also 10X multi-ome from 500,000 single-cell nuclei. Latent factor analysis on gene expression and chromatin accessibility across 14 NAc cell types identified a molecular pattern of changes significantly associated with oxycodone in-take (factor of oxycodone, FOXY). A major driver of FOXY was the abundance of Drd3-expressing medium spiny neurons (MSNs), with higher abundance associated with increased oxycodone in-take. We also identified gene expression and chromatin accessibility changes in Grm8-expressing MSNs and Chat expressing interneurons as a major component of FOXY. These cell types exclusively express the opioid receptor Oprm1 in the NAc. To evaluate if FOXY was genetically driven, we called cis-acting eQTLs and caQTLs across all cell types, and trained Predixscan models to predict gene expression and chromatin accessibility for identified eGenes and caPeaks in each cell type. These cis-variant predicted expressions indicated 18% of the variance in FOXY could be explained by genetic differences between individuals, and this variance was marginally predictive of oxycodone-intake (R=0.18, p=0.14). Overall, we identified a pattern of molecular and cell abundance changes in the NAc associated with oxycodone in-take, and at least 18% of the variance in this pattern was explained by genetic differences between individuals.

1 Salk Institute for Biological Studies, Integrative Biology Laboratory, La Jolla, CA 2 Department of Psychiatry, University of California San Diego, La Jolla, CA, USA 3 Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA

Ana Pombo

Midbrain dopamine neurons (DNs) are the first responders to the acute exposure of cocaine and are central to the development of drug addiction. Following a single cocaine injection, DNs undergo synaptic and transcriptional changes that are resolved within days but preserve a long-term cellular memory of the exposure by a mechanism that remains unknown. To investigate whether persistent alterations in 3D genome organization are involved in the cellular memory of an acute drug exposure, we applied Genome Architecture Mapping (GAM), single nucleus transcriptomics (snRNA-seq) and chromatin accessibility mapping (snATAC-seq) in the mouse midbrain 24 hours or 14 days after a single injection of cocaine or saline control. We found that DNs retain extensive chromatin reorganization 14 days after a single exposure across multiple genomic scales, including topologically associating domains (TADs), chromatin condensation and looping interactions, which affect genes previously associated with chronic cocaine addiction, synaptic plasticity and metabolic adaptation.To assess whether altered 3D genome structure 14 days post exposure has consequences for the transcriptional responses to cocaine, we re-exposed animals to a second injection of cocaine after 14 days of abstinence. Transcriptional responses were more extensive and often did not match the direction or intensity of changes seen upon the first exposure. Importantly, transcriptional responses following re-injection occurred predominantly at genes with reorganized chromatin architecture. Together, these findings identify 3D genome remodelling as a key mechanism underlying cellular memory of cocaine exposure, providing new insight into the earliest molecular events in addiction and the plasticity of the genome.

Max Delbrück Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Epigenetic Regulation and Chromatin Architecture Group, Berlin, Germany.

Department of Biology and Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD, USA.

Gabriella M. Silva¹, Joseph A. Picone¹, Annalise Hassan¹, Natalie L. Truby¹, R. Kijoon Kim¹, Corinne Smith¹, Shelbey S. Strandberg¹, Jessica L. Bell¹, Xiaohong Cui¹, Theingi Aung¹, Peter J. Hamilton¹

Zfp189, which encodes a Krüppel-associated box zinc finger protein (KZFP) transcription factor (TF), differentially accumulates in rodent cortical and limbic brain regions in response to stress- or drug-use experience. Here, we aimed to illuminate the brain cell-type specific molecular mechanisms through which this and other KZFP TFs produce cocaine-related brain changes, with emphasis on investigating transposable elements (TEs), since KZFPs like ZFP189 are known regulators of TEs. To investigate this, we quantified TE transcripts in existing single nuclei and bulk RNA-sequencing datasets of rodents exposed to cocaine. TE transcripts often go un-analyzed in transcriptomic data due to multi-mapping reads. We identified dynamic TE transcript expression across cocaine exposure, especially in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes. To directly regulate brain TEs, we created novel synthetic ZFP189 TFs and synthetic KZFP-interacting TRIM28, each capable of exerting distinct forms of transcriptional control at KZFP-regulated genes, including TEs. These KZFP TFs were virally delivered to the NAc of mice, including conditional delivery to MSNs, and the consequences on cocaine-related behaviors, including intravenous self-administration, and transcriptional response, by bulk and snRNAseq, were characterized. We discover that normal KZFP function in brain is critical to produce cocaine-induced NAc transcription and an escalation of cocaine-taking behaviors, and this can be impeded with synthetic KZFP TFs. Our synthetic KZFPs release TEs that form cis-regulatory contacts with down-regulated, predominantly immune-related genes. Collectively this work points to the KZFP-mediated transcriptional repression of brain TEs as an important mechanistic step in cocaine-induced gene expression and behavioral changes.

¹Department of Neuroscience and Anatomy; Virginia Commonwealth University School of Medicine

Funding from NIH grants: R01DA058089, R01DA058958

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

KR Robbins1, A Bredbenner1, RA Osbaldeston2, KS Villafañe2, EE Shin2, E Merkulova1, A Clevenger1, PB Delean2, C Campos2, GC Peet2, RA Jain1,2

Visualizing active neurons and circuits in vivo is critical for investigating the neural activity that underlies behavior. While several established methodologies are available to achieve this end in larval zebrafish, they are limited by the scale of tissue visualization, temporal resolution, need to restrain larvae, and/or accessibility of necessary instruments. Here, we establish a pipeline for the visualization and quantification of spatiotemporally precise whole-brain neural activity in larval zebrafish using CaMPARI2, a genetically encoded photoconvertible calcium indicator. Using temporally specific photoconverting UV light exposures, we capture whole-brain “snapshots” of neural activity time-locked to stimuli during unrestrained larval behavior. We optimized experimental conditions for establishing sub-second neuronal activity changes across acoustically-evoked behavioral paradigms spanning minutes to hours. We then leveraged this system to pinpoint brain-wide neural activity changes during nonassociative habituation learning, observing distinct activity signatures in the subpallium, preoptic area, and habenulae that are altered through pharmacological and/or genetic disruption of habituation learning. This approach effectively complements the temporal precision achievable through post hoc activity detection methods and expands the accessibility of large-scale behavioral circuit dissection beyond highly specialized real-time volumetric imaging equipment.

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

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

Funding Support: NIH R15EY031539

Peyton Presto¹, Julian Cardenas¹, Christian Bustamante¹, Brent Kisby^1,2, Guangchen Ji^1,2, Olga Ponomareva¹, Volker Neugebauer^1,2,3*, Igor Ponomarev^1,2*

Neuropathic pain is a chronic pain condition that results from damage or dysfunction in the nervous system. While mechanisms of neuropathic pain at the peripheral and spinal cord level have been extensively studied, pain mechanisms in the brain remain underexplored. The amygdala, a limbic brain region, has emerged as a critical brain area for the emotional-affective dimension of pain and pain modulation. Amygdala neuroplasticity has been associated with pain states, but exact molecular and cellular mechanisms underlying these states and the transition from acute to chronic pain are not well understood. Here, we used the spinal nerve ligation model of neuropathic pain in male rats to investigate changes in gene expression in two amygdala nuclei, basolateral (BLA) and central (CeA) at the chronic pain stage using RNA sequencing. We used an integrative approach that focuses on functional significance and cell type specificity of differentially expressed genes to nominate mechanistic targets for central regulation of chronic pain. Our integrative transcriptomic and bioinformatic analyses identified individual genes (e.g., Cxcl10, Cxcl12, Mbp, Plp1, Mag, Mog, Slc17a6, Gad1, Sst), molecular pathways (e.g., cytokine-mediated signaling pathway), biological processes (e.g., myelination, synaptic transmission), and specific cell types (e.g., oligodendrocytes, glutamatergic and GABAergic neurons) affected by chronic pain. Our results also provide evidence for hemispheric lateralization of pain processing in the amygdala. Overall, our study proposes oligodendrocyte dysfunction in the amygdala, neuroimmune signaling in the CeA, and glutamatergic neurotransmission in the BLA as mechanistic determinants of and potential therapeutic targets for the management of chronic neuropathic pain.

1. Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, 3601 4^th Street, Lubbock, Texas 79430, U.S.A. 2. Center of Excellence for Translational Neuroscience and Therapeutics, Texas Tech University Health Sciences Center, 3601 4^th Street, Lubbock, Texas 79430, U.S.A. 3. Garrison Institute on Aging, Texas Tech University Health Sciences Center, 3601 4^th Street, Lubbock, Texas 79430, U.S.A.

Funding Support: National Institutes of Health grants R01 NS038261 to V.N. and I.P. and R01 AA027096 to I.P.

Emma Gnatowski^1,2, Jessikah Buys^1,2, Jensen Goulette^2,3, Andrew A. George¹ and Michael F. Miles^1,2

Acute ethanol reduces anxiety in humans and animal models. Anxiety disorders increase risk for Alcohol Use Disorder (AUD) and human subjects report that stress and anxiety increase ethanol consumption. The Miles laboratory previously identified the microtubule binding protein Ninein (Nin) as a candidate gene underlying ethanol’s acute anxiolytic-like properties in BXD recombinant inbred mice. Here we report on behavioral, gene expression and GABAergic function consequences of Nin deletion in central amygdala (CeA). Deletion of Nin in CeA was done using stereotactic injections of AAV8-hSyn-GFP (control) or AAV8-hSyn-CRE-GFP (deletion) virus in Ninfl/fl mice. CeA Nin deletion increased acute ethanol anxiolysis in the light-dark box assay in male and female mice and reduced intermittent access 2-bottle choice ethanol consumption and preference x 5 weeks in female but not male mice. There were no changes in ethanol sedation (loss-of-righting reflex) or pharmacokinetics. Taste preference for quinine or saccharin were also unaffected. Bulk RNAseq analysis of stereotactic injection sites in CeA revealed striking evidence of neuroinflammatory and GABAergic gene expression alterations in Nin deletion mice. Preliminary electrophysiological studies on CeA IPSP activity measured by voltage clamp analysis showed Nin deletion altered IPSC duration, suggesting a post-synaptic site of action. Conclusions: These studies document that Nin function in CeA modulates the acute anxiolytic and consumption properties of ethanol, with the latter showing a striking sex preference. Initial mechanistic studies suggest that disruption of Nin expression in CeA produces changes in post-synaptic GABA receptor function, with coincident gene expression changes consistent with altered GABAergic neuron homeostasis and possible synaptic remodeling.

¹Dept. of Pharmacology and Toxicology, ²VCU Alcohol Research Center, and ³Dept. of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA USA

Funding Support: NIAAA grants F31AA030727, P50AA022537, and R01AA027581.

RC Rice,¹ MN Wauhop,² GE Homanics,^3,4 SP Farris^2,5

The long noncoding RNA growth arrest specific 5 (Gas5) is differentially methylated in blood and brain of individuals with alcohol use disorder and has multiple proposed functions, including immune- and glucocorticoid signaling modulation. In mouse, we previously observed a male-specific increase in nondependent voluntary ethanol consumption following Gas5 knockdown (KD) in medial prefrontal cortex (mPFC) and persistent downregulation of mPFC Gas5 following chronic intermittent ethanol vapor (CIEV). We hypothesize mPFC Gas5 modulates CIEV-induced escalation of ethanol consumption and stress-related behaviors during ethanol abstinence. To test this, we performed mPFC-specific Gas5 KD in adult male mice (C57BL/6J background), exposed them to CIEV-2BC, then tested stress-related behaviors and peak serum CORT levels during abstinence. mPFC Gas5 KD did not alter voluntary ethanol consumption, but modulated CIEV effects on stress-related behaviors during abstinence. In the open field test, Gas5 KD in air controls mimicked the stress-like phenotype of CIEV, measured by decreased entries and time spent in the inner zone, whereas Gas5 KD attenuated this phenotype in CIEV mice. In the elevated plus maze, CIEV resulted in decreased stretch-attend postures, which was ablated in Gas5 KD CIEV mice, suggesting Gas5 KD reversed CIEV impairment on risk assessment behavior. Peak serum CORT increased in CIEV mice seven days into abstinence, with no effect of Gas5 KD. However, 21 days into abstinence, CIEV mice with Gas5 KD displayed a persistent increase in serum CORT, while their CIEV control counterparts displayed levels resembling air controls. These findings suggest Gas5 modulates stress-related phenotypes of ethanol abstinence.

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 Pharma cology & 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

Funding Support: NIAAA AA031168 (PI: Rice), U01 AA020889 (PI: Farris, MPI: Homanics), and R01 AA024836 (PI: Farris).

Markos Michail Chatzigiannis1,2, Hirotaka Shoji2 , Daiki Sato2,3,4 , Keizo Takao, Tsuyoshi Miyakawa2

Behavioral phenotyping across genetically modified mouse strains is extensive but lacks a coherent framework for cross strain comparison. We assembled a large scale dataset comprising more than 10,000 mice from 167 strains across 15 behavioral assays. Multifactor analysis identified two principal dimensions, locomotor activity and learning/memory, that captured the dominant components of cross strain covariance. Clustering along these axes defined six behavioral phenotypes reflecting systematic variation in activity and cognitive performance. To assess clinical relevance, each strain was assigned a disorder association score derived independently of mouse behavioral data from publicly available human gene–disease association resources. Scores were calculated for intellectual disability (ID), autism spectrum disorder (ASD), schizophrenia, and major depressive disorder. Disorder association differed across endotypes, with the strongest and most consistent enrichment observed for ID and ASD. Strains with high ID or ASD burden were concentrated in the same two profiles characterized by comparable learning impairments but opposite locomotor patterns: one predominantly hypoactive and the other hyperactive. Across disorders, specific behavioral indices showed selective correlation with disorder burden, identifying the most informative measures for distinguishing disorder relevant models. These results indicate that clinically distinct diagnostic categories share underlying behavioral structure in mouse models that is not captured by disorder titles alone. This framework enables the interpretation of large scale behavioral data and the evaluation of disorder relevance for genetically modified mice.

1. Department of Systems Medical Science, Fujita Health University Graduate School of Medicine, Kutsukake-cho, Toyoake, Japan 2. Division of Systems Medical Science, Center for Medical Science, Fujita Health University, Kutsukake-cho, Toyoake, Japan 3. Institute for Advanced Academic Research, Chiba University, Chiba, Japan 4. Graduate School of Science, Chiba University, Chiba, Japan

Naureen Hameed¹, Sergio L. Crespo Flores¹, Evan Cirone¹, Chenyue Zhao¹, Annika F. Barber^1,2,*

Central pacemaker neurons use a combination of external stimuli and neuropeptide signaling to synchronize molecular oscillations leading to circadian behaviors. The clock network structure and signaling between these pacemaker neuron groups have been well described, but how these pacemakers communicate with specific brain output regions remains poorly understood. Here, we identified how “core” clock neurons in Drosophila, the ventrolateral neurons (LNvs), signal to the proto-hypothalamic region, the pars intercerebralis (PI). Previously thought to communicate with the PI only indirectly, we provide evidence to show that LNvs functionally modulate, the PI’s insulin-producing cells (IPCs) in a time-of-day-dependent manner. This functional connectivity relies on neuropeptidergic signaling of two canonical clock neuropeptides: pigment dispersing factor (PDF) and short Neuropeptide F (sNPF). Loss of either receptor alone in PI subpopulations does not alter feeding or locomotor rhythmicity. Further, we provide insight into how these two neuropeptides may be acting together via their receptors to signal to IPCs. We identify sexually dimorphic responses of IPC response to LNv stimulation, which may be partially explained by sex differences in proximity of clock neurons to the PI. Our findings indicate that LNvs form both direct peptidergic signaling but also form indirect multisynaptic circuits with IPCs, which may model more broadly how they communicate with various other clock output regions.

¹ Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.

² Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.

* annika.barber@waksman.rutgers.edu

GE Homanics¹, SJ Sukoff Rizzo², AC Silva², PL Strick², GW Carter³, JE Park²

Mutations in the presenilin 1 (PSEN1) gene are the most common cause of familial, early-onset Alzheimer's disease (AD), yet rodent models fail to fully recapitulate human AD pathology because they do not naturally develop amyloid plaques or tau aggregates. The common marmoset (Callithrix jacchus) offers a compelling alternative. It is a small non-human primate whose brain closely resembles the human brain, and it develops spontaneous age-related amyloid and tau pathology. We used CRISPR/Cas9 gene editing to independently introduce two PSEN1 point mutations (C410Y and A426P) — the same single-nucleotide changes found in human patients — into marmoset embryos. Several genetically engineered founders were produced; however, most died prematurely. One C410Y founder survived to adulthood and sired germline offspring. Phenotypic outcomes observed in these animals will be the subject of companion talks. In addition, experiments are underway to develop marmoset models of sporadic, late-onset AD. These nonhuman primate models are unprecedented for studying the earliest molecular events that initiate AD, evaluating preventive interventions, and bridging the rodent-to-human translational gap.

Departments of ¹Anesthesiology and ²Neurobiology, University of Pittsburgh, Pittsburgh PA, 15261. ³The Jackson Laboratory, Bar Harbor, ME 04609.

Funding Support: NIA U19 AG074866 and UPMC-ITTC grant IPA 2019 No. 16.

Lauren Bailey, PhD¹, Takeshi Murai, PhD¹, Lauren Mongeau¹, Abbey Setlik¹, Tingting Zhang, PhD¹, Seung-Kwon Ha, PhD, DVM¹, Gregory W. Carter², Afonso C Silva, PhD¹ and Stacey J Sukoff Rizzo, PhD¹,

Fundamental questions remain regarding the mechanisms that initiate Alzheimer’s disease (AD), drive its progression, and link pathology to cognitive impairment. As part of our MARMO-AD consortium, we established a comprehensive testing battery sensitive to detecting age-dependent cognitive decline across the lifespan in our colony of aging marmosets, and marmosets genetically engineered with mutations in the PSEN1 gene which confers early onset AD in humans. Beginning in adolescence, marmosets are trained using touchscreens through a battery of tests that captures a spectrum of cognitive domains including spatial working memory (delayed match to position), behavioral flexibility and reversal learning (delayed non-match to position), recognition memory (trial unique delayed match to sample), attention (serial reaction time task), and episodic-like memory (paired associative learning task). Behavioral and cognitive function are aligned with longitudinal PET neuroimaging and blood-based biomarkers to track AD progression. Similar to human PSEN1 mutation carriers, plasma Aβ42:40 is significantly elevated relative to non-carrier controls Despite robust biomarker and pathological changes, PSEN1 marmosets show no significant deficits in task acquisition or performance across cognitive domains up to 4 years of age. These data are not surprising and recapitulate the disease trajectory of increased amyloid in plasma and brain years before cognitive decline. These ongoing longitudinal studies are enabling the identification of the molecular and cellular mechanisms other than amyloid that contribute to and precede cognitive decline associated with Alzheimer’s disease progression.

(1)University of Pittsburgh School of Medicine, Pittsburgh, PA, USA (2) The Jackson Laboratory, Bar Harbor, Maine, USA

This work is supported by U19AG074866 and 5T32AG021885-19.

Thais Rafael Guimarães¹, Jung Eun Park¹, Catrina Spruce², Stephanie Hachem¹, Swati Banerjee¹, Lauren K Hayrynen Schaeffer¹, Gregg E Homanics¹, Stacey J Sukoff Rizzo¹, Gregory W Carter,², Afonso C Silva¹, and Amantha Thathiah¹

Progress in preclinical Alzheimer’s disease (AD) research has been constrained by models that fail to faithfully recapitulate human aging and overt AD neuropathology. The common marmoset (Callithrix jacchus), a New World non-human primate, exhibits aging trajectories, genetic heterogeneity, and complex social behaviors closely resembling those of humans, providing a highly translational platform for age-related neurodegenerative research. Importantly, marmoset studies uniquely enable longitudinal correlation of in vitro cellular models with in vivo assessments across the marmoset lifespan. We performed an integrated ex vivo and in vitro characterization of marmoset AD and tauopathy models. Immunohistochemical analyses of postmortem brains revealed robust amyloid-β (Aβ) and tau pathology, along with the associated cellular pathology. To establish an in vitro cellular system, we adapted a well-established human direct reprogramming protocol to generate age-conserved induced neurons (iNs) from marmoset fibroblasts. Comparative, unbiased RNA-seq analyses of marmoset and human iN conversion trajectories revealed significant species-specific differences, guiding targeted optimization of the reprogramming strategy. The refined protocol achieved high-efficiency neuronal conversion, improved cell survival and maturation, and preserved AD-relevant protein expression, including amyloid precursor protein (APP)/Aβ and tau. Together, this integrated framework establishes the marmoset as a powerful translational model for AD research. This platform enables minimally invasive mechanistic studies, longitudinal analyses, high-throughput drug screening, and therapeutic discovery aimed at accelerating disease-modifying strategies for AD.

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

²The Jackson Laboratory, Bar Harbor, ME, USA

Gregory W. Carter

Laboratory mice and marmosets provide potential models of aging and Alzheimer’s disease with the capacity to track the initiation and progress of pathology across compressed lifespans. We have performed genetic, genomics, and proteomic analyses of plasma and postmortem tissues to map multi-modal aspects of disease development and progression in these model species. We have drawn from human genetic studies to engineer multiple mouse strains carrying candidate genetic factors for late-onset Alzheimer's disease. In the marmoset, we are combining genetic engineering of select loci with outbred genetic variability to analyze biomarkers of aging and dementia. We assembled a new telomere-to-telomere reference genome for the common marmoset (Callithrix jacchus) and performed whole-genome Illumina sequencing on over 230 marmosets. For both model systems, we have used high-coverage proteomics, Alamar NULISA, and transcriptomics to assess the disease-relevant consequences of genetic factors. We have identified a broad range of disease-associated signatures in knock-in mouse and marmoset models, including immune, metabolic, and synaptic alterations. These outcomes frequently correlate with clinically relevant biomarkers and behavioral outcomes. We have also used multi-omic signatures to understand the molecular pharmacodynamics of candidate drugs. Our findings constitute a data and model resource for identifying the appropriate animal model for understanding genetic liability in Alzheimer's and preclinical testing of targeted therapeutics.

The Jackson Laboratory, Bar Harbor, ME, USA

Leslie C. Griffith MD PhD

Neurons are among the most structurally and functionally specialized cells in the body, capable of processing, integrating, and transmitting information. Unlike many other cell types, neurons exhibit extreme morphological polarity, with distinct compartments—dendrites, soma, axon, and synaptic terminals—each requiring specifically tailored protein populations to support their localized functions. The recognition that cells are capable of locally synthesizing proteins marked a major shift in our thinking about cellular organization. Local translation enables neurons to establish functionally distinct subdomains and it allows rapid, site-specific responses to activity, supporting structural changes that are required for long-term plasticity. Localized protein synthesis and compartment-specific protein turnover allow neurons to dynamically respond to activity and environmental changes.

I will talk today about the first plasticity-related protein to be shown to be locally synthesized in neurons: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Activity-dependent CaMKII synthesis is conserved across phyla and occurs in both pre- and postsynaptic compartments. Activity also has an additional effect on CaMKII that is equally conserved: it causes a subcellular redistribution of the protein. How the dynamic regulation of CaMKII levels and subcellular localization are related is not understood, and I will discuss recent results from my lab that address these questions. My talk will highlight how the unique biology of neurons depends on highly specialized protein landscapes—proving that in neuroscience, just like real estate, success ultimately comes down to location, location, location.

Nancy Lurie Marks Professor of Neuroscience, Brandeis University