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