Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

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.

Once is too much: Early development of the opponent process in taste reactivity behavior is associated with later escalation of cocaine self-administration in rats.

Evidence suggests that the addiction process may begin immediately in some vulnerable subjects. Specifically, some rats have been shown to exhibit aversive taste reactivity (gapes) following the intraoral delivery of a cocaine-predictive taste cue after as few as 1-2 taste-drug pairings. After only 3-4 trials, the number of gapes becomes a reliable predictor of later cocaine self-administration. Given that escalation of drug-taking behavior over time is recognized as a key feature of substance use disorder (SUD) and addiction, the present study examined the relationship between early aversion to the cocaine-predictive flavor cue and later escalation of cocaine self-administration in an extended-access paradigm. The data show that rats who exhibit the greatest conditioned aversion early in training to the intraorally delivered cocaine-paired cue exhibit the greatest escalation of cocaine self-administration over 15 extended-access trials. This finding suggests that early onset of the conditioned opponent process (i.e., the near immediate shift from ingestion to rejection of the drug-paired cue) is a reliable predictor of future vulnerability and resilience to cocaine addiction-like behavior. Future studies must determine the underlying neural mechanisms associated with this early transition and, hence, with early vulnerability to the later development of SUD and addiction. In so doing, we shall be in position to discover novel diagnostics and novel avenues of prevention and treatment.

Heroin-induced suppression of saccharin intake in OPRM1 A118G mice.

The single nucleotide polymorphism of the µ-opioid receptor, OPRM1 A118G, has been associated with greater drug and alcohol use, increased sensitivity to pain, and reduced sensitivity to the antinociceptive effects of opiates. In the present studies, we employed a 'humanized' mouse model containing the wild-type (118AA) or variant (118GG) allele to examine behavior in a model of heroin-induced devaluation of an otherwise palatable saccharin cue when repeated saccharin-heroin pairings occurred every 24h (Experiment 1) or every 48h (Experiment 2). The results showed that, while both the 118AA and 118GG mice demonstrated robust avoidance of the heroin-paired saccharin cue following daily taste-drug pairings, only the 118AA mice suppressed intake of the heroin-paired saccharin cue when 48h elapsed between each taste-drug pairing. Humanized 118GG mice, then, defend their intake of the sweet cue despite saccharin-heroin pairings and this effect is illuminated by the use of spaced, rather than massed, trials. Given that this pattern of strain difference is not evident with saccharin-cocaine pairings (Freet et al., 2015), reduced avoidance of the heroin-paired saccharin cue by the 118GG mice may be due to an interaction between the opiate and the subjects' drive for the sweet or, alternatively, to differential downstream sensitivity to the aversive kappa mediated properties of the drug. These alternative hypotheses are addressed.

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.

Greater avoidance of a heroin-paired taste cue is associated with greater escalation of heroin self-administration in rats.

Heroin addiction is a disease of chronic relapse affecting over half of its users. Therefore, modeling individual differences in addiction-like behavior is needed to better reflect the human condition. In a rodent model, avoidance of a cocaine-paired saccharin cue is associated with greater cocaine seeking and taking. Here, we tested whether rats would avoid a saccharin cue when paired with the opportunity to self-administer heroin and whether the rats that most greatly avoid the heroin-paired taste cue would exhibit the greatest drug escalation over time, the greatest willingness to work for drug, and the greatest heroin-induced relapse. Adult male Sprague-Dawley rats received 5 min access to a 0.15% saccharin solution followed by the opportunity to self-administer either saline or heroin for 3 hr (short access) or 6 hr (extended access). Following 16 to 18 pairings, terminal saccharin intake was used to categorize the rats into small (>200 licks/5min) or large (<200 licks/5min) suppressors and responding for drug was examined accordingly. Only 5% of the short access rats reached the criteria for large suppressors. This large suppressor did not differ from the small suppressors in drug-taking behavior. On the other hand, 50% of the extended access saccharin-heroin rats were large suppressors and showed the largest escalation of drug intake, drug-loading behavior, and the greatest relapse-like behaviors. Extended access small suppressors displayed drug-taking behaviors that were similar to rats in the short access heroin condition. Avoidance of a heroin-paired taste cue reliably identifies individual differences in addiction-like behavior for heroin using extended drug access.

Cocaine-induced suppression of saccharin intake and morphine modulation of Ca²âº channel currents in sensory neurons of OPRM1 A118G mice.

Several studies have shown that human carriers of the single nucleotide polymorphism of the µ-opioid receptor, OPRM1 A118G, exhibit greater drug and alcohol use, increased sensitivity to pain, and reduced sensitivity to the antinociceptive effects of opiates. In the present study, we employed a 'humanized' mouse model containing the wild-type (118AA) or variant (118GG) allele to examine behavior in our model of drug-induced suppression of a natural reward cue and to compare the morphine pharmacological profile in acutely isolated sensory neurons. Compared with 118AA mice, our results demonstrate that homozygous 118GG mice exhibit greater avoidance of the cocaine-paired saccharin cue, a behavior linked to an aversive withdrawal-like state. Electrophysiological recordings confirmed the reduced modulation of Ca(2+) channels by morphine in trigeminal ganglion (TG) neurons from 118GG mice compared to the 118AA control cells. However, repeated cocaine exposure in 118GG mice led to a leftward shift of the morphine concentration-response relationship when compared with 118GG control mice, while a rightward shift was observed in 118AA mice. These results suggest that cocaine exposure of mice carrying the 118G allele leads to a heightened sensitivity of the reward system and a blunted modulation of Ca(2+) channels by morphine in sensory neurons.

Once is too much: conditioned aversion develops immediately and predicts future cocaine self-administration behavior in rats.

Rats emit aversive taste reactivity (TR) behavior (i.e., gapes) following intraoral delivery of a cocaine-paired taste cue and greater conditioned aversive TR at the end of training predicts greater drug-seeking and taking. Here, we examined the development of this conditioned aversive TR behavior on a trial-by-trial basis in an effort to determine when the change in behavior occurs and whether early changes in this behavior can be used to predict later drug taking. The results show that conditioned aversive TR to a cocaine-paired cue occurs very early in training (i.e., following as few as 1-2 taste-drug pairings) and, importantly, that it can be used to predict later drug seeking and drug taking in rats.

Neuroglial expression of the MHCI pathway and PirB receptor is upregulated in the hippocampus with advanced aging.

The hippocampus undergoes changes with aging that impact neuronal function, such as synapse loss and altered neurotransmitter release. Nearly half of the aged population also develops deficits in spatial learning and memory. To identify age-related hippocampal changes that may contribute to cognitive decline, transcriptomic analysis of synaptosome preparations from adult (12 months) and aged (28 months) Fischer 344-Brown Norway rats assessed for spatial learning and memory was performed. Bioinformatic analysis identified the MHCI pathway as significantly upregulated with aging. Age-related increases in mRNAs encoding the MHCI genes RT1-A1, RT1-A2, and RT1-A3 were confirmed by qPCR in synaptosomes and in CA1 and CA3 dissections. Elevated levels of the MHCI cofactor (B2m), antigen-loading components (Tap1, Tap2, Tapbp), and two known MHCI receptors (PirB, Klra2) were also confirmed. Protein expression of MHCI was elevated with aging in synaptosomes, CA1, and DG, while PirB protein expression was induced in both CA1 and DG. MHCI expression was localized to microglia and neuronal excitatory postsynaptic densities, and PirB was localized to neuronal somata, axons, and dendrites. Induction of the MHCI antigen processing and presentation pathway in hippocampal neurons and glia may contribute to age-related hippocampal dysfunction by increasing neuroimmune signaling or altering synaptic homeostasis.