Environmental enrichment augments behavioral and epigenetic response to heroin self‐administration
Authors: McFalls Ashley Jane , Imperio Ceasar G. , Masser Dustin R. , Bixler Georgina V. , Wronowski Benjamin C. , Vrana Kent E. , Grigson Patricia Sue , Freeman Willard M. 
Published: November 2023

From the investigation of heroin use amongst American soldiers in Vietnam (Robins et al. (2010) Am J Addict 19:203) to environmental enrichment studies in rodents (Solinas et al. (2010) Prog Neurobiol 92:572), it is well documented that the quality of one's environment profoundly affects the addictive potential of a drug. Further, environment (e.g., nurture, stress, cues, exercise) and drugs of abuse have the potential to create persistent changes in brain chemistry that leave an addicted individual vulnerable to relapse behavior. This study sought to explore the molecular phenomena that lie at the intersection of environment and drug abuse. Thirty‐one male Sprague‐Dawley rats were randomly assigned to 2 housing conditions: standard (1 subject/cage) and enriched (2–3 subjects/cage + novel objects and a running wheel). Rats that had been housed under different conditions were given the opportunity to self‐administer heroin or saline within a 3 hour session (within identical (non‐enriched) self‐administration chambers). We found that environmental enrichment (n=10) attenuated motivation to work for heroin and drug‐seeking after 14 days abstinence following two‐weeks of self‐administration. We then assessed mRNA expression and epigenetic status for a panel of genes found to be associated with drug‐seeking, taking and abstinence. We have previously found increased expression of the early growth response genes (EGR1 and EGR2) following abstinence from heroin (Kuntz‐Melcavage et al. (2008) Subst Abuse 2:1). In the present study, EGR1 and EGR2 mRNA expression in medial prefrontal cortex (mPFC) was decreased in enriched subjects compared to standard heroin (n=10), standard saline (n=5) and enriched saline (n=6). Similarly, enriched subjects displayed decreased EGR2 mRNA in the nucleus accumbens (NAcc) and hippocampus compared to standard heroin, standard saline and enriched saline. Whole genome ELISA, as well as LINE‐1 and ID element‐selective analysis revealed no changes in global 5‐methylcytosine (5‐mC) between groups in all three brain regions. We examined methylation status of promoter regions and 1st and 2nd exons of EGR1 and EGR2 using Bisulfite amplicon sequencing (BSAS). Counter to what we predicted from the gene expression profiles, standard saline animals were found to have hyper‐methylation of specific CpG sites in the proximal promoter region of EGR2. mPFC and NAcc showed similar profiles of CpG and non CpG methylation of both the EGR1 and EGR2 gene, although no other differences between groups was found. This may indicate that gene expression of EGR1 and EGR2 are mediated through other mechanisms. Experiments are currently being conducted to assess gene expression and epigenetic status of EGR1 and EGR2 in the Ventral Tegmental Area.Support or Funding InformationThis work was funded, in part, by a grant from the Pennsylvania Department of Health using Tobacco CURE Funds to P.S.G, K.E.V and W.M.F., the Donald W. Reynolds Foundation and the National Institute on Aging (P30AG050911) to W.M.F, the National Institute on Drug Abuse F31DA036322 to C.G.I.