An RNA-seq study of the mPFC of rats with different addiction phenotypes.

Addiction is a disease of brain-reward circuitry whereby attention, motivation, memory and emotional systems become enslaved to the goal of seeking and acquiring drug, instead of responding to the natural rewards for which these systems evolved. At the intersection of reward/limbic structures, the medial prefrontal cortex (mPFC) receives and consolidates signals regarding environment and orchestrates the most appropriate response (i.e., decision-making and attention). As such, mPFC function plays a critical role in the vulnerability or resilience to drug addiction. In our model of drug-induced reward devaluation, an outbred group of Sprague-Dawley rats parsed into two distinct drug-taking phenotypes: those, referred to as small suppressors (SS) that readily ingest a heroin-paired sweet cue and then take little drug, and those, referred to large suppressors (LS), that avoid the heroin-paired cue, but then respond greatly for the drug of abuse. In the present study, we analyzed the mPFC transcriptome of rats from these divergent groups to discover differences in gene expression that underlie these distinct phenotypes. Genes found to be differentially expressed were those associated with schizophrenia and dopamine signaling, signal transduction, development and synaptic plasticity. These genes may underlie the circumstance whereby some individuals succumb to addiction, while others do not, and may reveal new pharmacological targets for the treatment of drug addiction.

Greater avoidance of a saccharin cue paired with passive delivery of heroin is associated with a select increase in expression of CRFR2 and CRFbp in the hippocampus in rats.

As a drug of abuse tightens its hold on addicted individuals, aspects of life that once brought pleasure lose their appeal while attention and motivation are turned toward acquiring drug. In a rodent model of self-administration and reward devaluation, we previously showed that animals that suppress intake of a drug-paired saccharin cue show greater addiction-like behaviors, as well as increased gene-expression of elements of the corticotropin releasing factor (CRF) pathway in the prefrontal cortex (mPFC), hippocampus (Hipp), and ventral tegmental area (VTA). In the present study, we explored whether the observed differences in components of the CRF signaling pathway were a function of self-administration or devaluation of the cue. Moreover, as an increasing body of work illustrates, functional and molecular hemispheric differences in reward pathway components, we examined whether these CRF pathway components exhibited hemispheric differences in response to heroin administration. Over a period of 7 trials, 30 male rats received brief access to saccharin followed by passive (IP) injection of heroin (n = 20) or saline (n = 10). Saccharin intakes between large saccharin suppressors (LS; 12 animals) and small suppressors (SS; 8 animals) were statistically different after trial 1 and separated further with ensuing trials. We then assessed gene expression for components of the CRF pathway in the mPFC, Hipp, VTA, Amygdala, and nucleus accumbens (NAc). Within the Hipp, LS showed greater expression of CRF binding protein (CRFbp). No differences were observed in the mPFC, VTA, NAc or Amygdala. Several hemisphere differences in CRF signaling pathway genes were detected. These findings indicate that avoidance of the experimenter delivered heroin-paired saccharin cue, do not recapitulate findings observed for avoidance of the iv self-administered heroin-paired saccharin cue, at least in terms of the expression of genes within the CRF pathway, and provide further evidence that consideration should be given to hemisphere differences when exploring molecular phenomena.

Exposure to environmental enrichment attenuates addiction-like behavior and alters molecular effects of heroin self-administration in rats.

Environmental factors profoundly affect the addictive potential of drugs of abuse and may also modulate the neuro-anatomical/neuro-chemical impacts of uncontrolled drug use and relapse propensity. This study examined the impact of environmental enrichment on heroin self-administration, addiction-related behaviors, and molecular processes proposed to underlie these behaviors. Male Sprague-Dawley rats in standard and enriched housing conditions intravenously self-administered similar amounts of heroin over 14 days. However, environmental enrichment attenuated progressive ratio, extinction, and reinstatement session responding after 14 days of enforced abstinence. Molecular mechanisms, namely DNA methylation and gene expression, are proposed to underlie abstinence-persistent behaviors. A global reduction in methylation is reported to coincide with addiction, but no differences in total genomic methylation or repeat element methylation were observed in CpG or non-CpG (CH) contexts across the mesolimbic circuitry as assessed by multiple methods including whole genome bisulfite sequencing. Immediate early gene expression associated with drug seeking, taking, and abstinence also were examined. EGR1 and EGR2 were suppressed in mesolimbic regions with heroin-taking and environmental enrichment. Site-specific methylation analysis of EGR1 and EGR2 promoter regions using bisulfite amplicon sequencing (BSAS) revealed hypo-methylation in the EGR2 promoter region and EGR1 intragenic CpG sites with heroin-taking and environmental enrichment that was associated with decreased mRNA expression. Taken together, these findings illuminate the impact of drug taking and environment on the epigenome in a locus and gene-specific manner and highlight the need for positive, alternative rewards in the treatment and prevention of drug addiction.

Assessment of individual differences in the rat nucleus accumbens transcriptome following taste-heroin extended access.

Heroin addiction is a disease of chronic relapse that harms the individual through devaluation of personal responsibilities in favor of finding and using drugs. Only some recreational heroin users devolve into addiction but the basis of these individual differences is not known. We have shown in rats that avoidance of a heroin-paired taste cue reliably identifies individual animals with greater addiction-like behavior for heroin. Here rats received 5min access to a 0.15% saccharin solution followed by the opportunity to self-administer either saline or heroin for 6h. Large Suppressors of the heroin-paired taste cue displayed increased drug escalation, motivation for drug, and drug loading behavior compared with Small Suppressors. Little is known about the molecular mechanisms of these individual differences in addiction-like behavior. We examined the individual differences in mRNA expression in the nucleus accumbens (NAc) of rats that were behaviorally stratified by addiction-like behavior using next-generation sequencing. We hypothesized that based on the avoidance of the drug-paired cue there will be a unique mRNA profile in the NAc. Analysis of strand-specific whole genome RNA-Seq data revealed a number of genes differentially regulated in NAc based on the suppression of the natural saccharine reward. Large Suppressors exhibited a unique mRNA prolife compared to Saline controls and Small Suppressors. Genes related to immunity, neuronal activity, and behavior were differentially expressed among the 3 groups. In total, individual differences in avoidance of a heroin-paired taste cue are associated with addiction-like behavior along with differential NAc gene expression.

Reward devaluation and heroin escalation is associated with differential expression of CRF signaling genes.

One of the most damaging aspects of drug addiction is the degree to which natural rewards (family, friends, employment) are devalued in favor of seeking, obtaining and taking drugs. We have utilized an animal model of reward devaluation and heroin self-administration to explore the role of the coricotropin releasing factor (CRF) pathway. Given access to a saccharin cue followed by the opportunity to self-administer heroin, animals will parse into distinct phenotypes that suppress their saccharin intake (in favor of escalating heroin self-administration) or vice versa. We find that large saccharin suppressors (large heroin takers) demonstrate increased mRNA expression for elements of the CRF signaling pathway (CRF, CRF receptors and CRF binding protein) within the hippocampus, medial prefrontal cortex and the ventral tegmental area. Moreover, there were no gene expression changes of these components in the nucleus accumbens. Use of bisulfite conversion sequencing suggests that changes in CRF binding protein and CRF receptor gene expression may be mediated by differential promoter methylation.