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Several studies demonstrated the role of the endogenous opioid system in the development of susceptibility to alcohol dependence. Recently, we reported that binge intragastric administration of ethanol induces selective alterations of pronociceptin and prodynorphin gene expression in the rat amygdala complex depending on the days of exposures and on the development of tolerance and dependence. The aim of the present study was to investigate the potential epigenetic mechanisms leading to these alcohol-induced changes in gene expression. Specific histone modifications and DNA methylation at opioid peptide precursor promoters were analyzed by chromatin immunoprecipitation and real-time methylation-specific PCR, respectively. We found a linkage between gene expression alterations and epigenetic modulation at pronociceptin and prodynorphin promoters following alcohol treatment. In animals treated for 1 day, we observed a reversed correlation, with a decrease of histone 3 lysine 27 trimethylation (repressive mark) and an increase of histone 3 lysine 9 acetylation (activating mark), associated with both gene expression up-regulation. In rats treated with alcohol for up to 5 days, we found an increase in histone 3 lysine 9 acetylation in the pronociceptin promoter providing further evidence of the already proposed possible role for histone deacetylases for addiction treatment. No significant alterations in DNA methylation and histone 3 lysine 4 trimethylation following different alcohol exposures were present, suggesting the selectivity of epigenetic effects induced by alcohol. These data demonstrate that ethanol induces selective epigenetic changes, thus better defining the role of opioid peptides in the ethanol-induced effects in the amygdala complex.

The interaction of opioids and stimulants is well established; however, the mechanisms that underlie the role that opioid receptors play in psychostimulant action are not. Nociceptin/orphaninFQ (N/OFQ), the endogenous agonist at NOP receptors, attenuates the behavioral effects of cocaine. The effects of cocaine on N/OFQ were examined in rats using immunoautoradiographic and RIA techniques. Chronic administration of cocaine decreased N/OFQ in medial regions of the caudate putamen, the nucleus accumbens shell, and the substantia nigra. These studies show that N/OFQ levels are altered by treatment with cocaine. Furthermore, the changes in N/OFQ parallel those seen for kappa-opioid receptors, suggesting that the interactions between cocaine and these systems might be similar.

The effects of kappa-opioid agonist treatment on kappa-opioid receptor density and on dynorphin A levels in the rat brain were studied. Rats were treated with the selective kappa-opioid agonist U-69593 or vehicle for 5 d. Dynorphin A levels and kappa-opioid receptor binding were measured on day 8 (3 d after the last injection) or 22 (17 d after the last injection). On day 8, kappa-opioid receptor density was increased in the hypothalamus of rats treated with U-69593; there were no changes in the frontal cortex or caudate putamen. In contrast, there was an increase in dynorphin A levels in the frontal cortex and no changes in hypothalamus and caudate putamen in response to U-69593. On day 22, Bmax was decreased in frontal cortex and caudate putamen of U-69593-treated rats, whereas dynorphin A levels were increased in the caudate putamen and in the frontal cortex. These findings suggest that kappa-opioid receptor agonist treatment has long-term, continually changing effects on the kappa-opioid system.

The effect of the selective serotonin uptake inhibitor fluoxetine was examined on prodynorphin gene expression. Fluoxetine or vehicle was infused continuously for 7 d via osmotic minipumps into male rats. Northern blot analysis showed significant increases in prodynorphin gene expression in the hypothalamus (171% of controls) and significant decreases in the caudate putamen and nucleus accumbens (62% and 70% of controls, respectively). There were no significant changes in the hippocampus. Thus, chronic inhibition of serotonin uptake can regulate prodynorphin gene expression in the hypothalamus, caudate putamen, and nucleus accumbens. Fluoxetine effects were also evaluated in rats treated with p-chloroamphetamine (PCA), a neurotoxin that depletes serotonin. Because we previously reported that continuous infusion of cocaine for 7 d (which inhibits dopamine, serotonin, and norepinephrine uptake), or GBR 12909 (a selective dopamine uptake inhibitor), produced significant decreases in the hypothalamus and cocaine also produced a significant increase in prodynorphin gene expression in caudate putamen, regulation of prodynorphin gene expression by fluoxetine is suggested to be different from that by cocaine. Because neither a selective dopamine uptake inhibitor nor a selective serotonin uptake inhibitor produced the same effect as cocaine in the caudate putamen, this effect is likely regulated by the inhibition of norepinephrine uptake, by a combination of effects on two or three neurotransmitter transporters, or by a mechanism unrelated to transporter inhibition.