Sex, Drugs, and the Microbiome: Goal/Sign-Tracking Phenotype Reveals Associations Between Behavior and Microbiome in a Sex-Dependent Manner in the Rat

Introduction: Multiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. For example, individuals that are predisposed to assign incentive salience to food stimuli ("sign-trackers", ST), are not only more impulsive compared to those that do not ("goal-trackers", GT), but are also more sensitive to drugs and drug stimuli as well. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors. Methods: Outbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype. Results: Microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. In the female STs, attention, as measured by omissions in the reaction time test revealed notable microbiome correlations. In both males and females, many measures were correlated with bacterial family Ruminocococcaceae and Lachnospiraceae. Conclusions: These data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex. Funding Statement: Funding sources: P50DA037844, 12/RC/2273. AB was supported by a fellowship from the Wellcome Trust (105941/Z/14/Z). Declaration of Interests: JFC & TGD are in receipt of research funding from 4D‐Pharma, Mead Johnson, Suntory Wellness, Nutricia, and Cremo. Timothy Dinan has been an invited speaker at meetings organized by Servier, Lundbeck, Janssen, and AstraZeneca. John Cryan has been an invited speaker at meetings organized by Mead Johnsen, Alkermes, and Janssen. Ethics Approval Statement: Animals were treated in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the experiments were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University at Buffalo, The State University of New York.