The escalation of cocaine SA under LgA conditions is dose-dependent and is associated with heightened susceptibility to drug-induced relapse. The characterization of neurobiological alterations that accompany escalated SA should facilitate the identification of mechanisms underlying the onset of human addiction.
The inhibitory effect of salvinorin A on striatal dopamine levels may contribute to its induction of conditioned place aversion and decreases in locomotion in mice. These findings are consistent with the in vitro characterization of salvinorin A as a kappa opioid receptor agonist. It is of interest that a compound such as salvinorin A, that lowers striatal dopamine levels and leads to conditioned place aversion in rodents, is self-administered by humans under certain conditions.
The most common single nucleotide polymorphism in the coding region of the human mu opioid receptor gene is the A118G variant, an adenine to guanine transition at nucleotide position 118 of the coding sequence of the gene. This polymorphism codes for an asparagine to aspartic acid substitution at amino acid 40 in the amino-terminus, thereby removing a potential extracellular glycosylation site. Using in vitro cellular expression assays, this variant has been reported to change binding of the endogenous agonist beta-endorphin and signaling of the receptor following binding of beta-endorphin. Three clinical studies report that A118G genotype affects opioid antagonist-mediated increases in cortisol levels. These studies demonstrate a functional role of this variant in responses to endogenous and exogenous opioids. To further characterize function, we expressed the prototype and variant receptors in two types of cells (human 293 embryonic kidney cells and Syrian hamster adenovirus-12-induced tumor cells). Stable expression of variant and prototype receptors was characterized by differences in levels of cell surface binding capacity (B max ), forskolin-induced cAMP accumulation, as well as agonist-induced accumulation of cAMP (EC 50 ) for several agonists, but not for beta-endorphin. In contrast, transiently expressed variant receptors showed only a minor difference in cell surface binding capacity compared to the prototype, and no differences in cAMP EC 50 values. Opioid receptors modulate many endogenous physiological and neurobiological systems. They are also essential drug targets in the treatment of pain and pivotally involved in addiction to drugs of abuse, including opiates, cocaine and alcohol. Mu, kappa, and delta opioid receptors (encoded by the OPRM1, OPRK1, and OPRD1 genes, respectively), are G-protein coupled receptors (GPCRs), which couple to inhibitory (G i /G o ) heterotrimeric G-proteins. Several polymorphic variants of the human OPRM1 gene have been described (e.g. Bergen et al. 1997;Berrettini et al. 1997;Bond et al. 1998;Hoehe et al. 2000), including variants that alter amino acid sequence of the receptor. Some of these naturally occurring sequence alterations have also been reported to alter properties of receptor function, studied using in vitro expression systems (Bond et al. 1998;Koch et al. 2000;Befort et al. 2001;Margas et al. 2007).The A118G variant of the human mu opioid receptor gene (OPRM1) is in the coding region of the first exon and is the single nucleotide polymorphism (SNP) with the highest overall allelic frequency of any OPRM1 coding region variant reported, although its heterozygosity varies widely across populations, from 1% to 2% frequencies of the minor (118G) allele reported in African Americans up to 50% in Japanese (e.g. Bond et al. 1998;Gelernter et al. 1999;Szeto et al. 2001;Tan et al. 2003;Bart et al. 2004;Kim et al. 2004). This SNP encodes an amino acid substitution of asparagine to
The present findings indicate that predictable individual differences in cocaine SA under extended access conditions are relevant only at low doses and are surmountable by increasing the available dose of cocaine.
The m-opioid receptor is the site of action of opiates and opioids. We examined whether there are differences in cytosine : guanine (CpG) dinucleotide methylation in the OPRM1 promoter between former heroin addicts and controls. We analyzed methylation at 16 CpG dinucleotides in DNA obtained from lymphocytes of 194 Caucasian former severe heroin addicts stabilized in methadone maintenance treatment and 135 Caucasian control subjects. Direct sequencing of bisulfite-treated DNA showed that the percent methylation at two CpG sites was significantly associated with heroin addiction. The level of methylation at the À18 CpG site was 25.4% in the stabilized methadonemaintained former heroin addicts and 21.4% in controls (p ¼ 0.0035, generalized estimating equations (GEE); p ¼ 0.0077, t-test; false discovery rate (FDR) ¼ 0.048), and the level of methylation at the + 84 CpG dinucleotide site was 7.4% in cases and 5.6% in controls (p ¼ 0.0095, GEE; p ¼ 0.0067, t-test; FDR ¼ 0.080). Both the À18 and the + 84 CpG sites are located in potential Sp1 transcription factor-binding sites. Methylation of these CpG sites may lead to reduced OPRM1 expression in the lymphocytes of these former heroin addicts.
In this study, we investigated the effects of acute morphine administration, chronic intermittent escalating-dose morphine administration and spontaneous withdrawal from chronic morphine on mRNA levels of mu opioid receptor (MOP-r), and the opioid peptides pro-opiomelanocortin (POMC) and preprodynorphin (ppDyn) in several key brain regions of the rat, associated with drug reward and motivated behaviors: lateral hypothalamus (lat.hyp), nucleus accumbens (NAc) core, amygdala, and caudate-putamen (CPu). There was no effect on MOP-r mRNA levels in these brain regions 30 min after either a single injection of morphine (10 mg/kg, i.p.) or chronic intermittent escalating-dose morphine (from 7$5 mg/kg per day on day 1 up to 120 mg/kg per day on day 10). Activation of the stress-responsive hypothalamic-pituitary-adrenal axis by 12 h withdrawal from chronic morphine was confirmed; both POMC mRNA levels in the anterior pituitary and plasma adrenocorticotropic hormone levels were significantly elevated. Under this withdrawal-related stress condition, there was an increase in MOP-r mRNA levels in the lat.hyp, NAc core, and CPu. Recent studies have demonstrated a novel role for the lat.hyp orexin (or hypocretin) activation in both drug-related positive rewarding, and withdrawal effects. Around 50% of lat.hyp orexin neurons express MOP-r. Therefore, we also examined the levels of lat.hyp orexin mRNA, and found them increased in morphine withdrawal, whereas there was no change in levels of the lat.hyp ppDyn mRNA, a gene coexpressed with the lat.hyp orexin. Our results show that there is an increase in MOP-r gene expression in a region-specific manner during morphine withdrawal, and support the hypothesis that increased lat.hyp orexin activity plays a role in morphine-withdrawalrelated behaviors.
Rat genome U34A (Affymetrix) oligonucleotide microarrays were used to analyze changes in gene expression in the caudate putamen (CPu) of Fischer rats induced by 1 and 3 days of "binge" cocaine (or saline) administration. A triplicate array assay of pooled RNA of each treatment group was used to evaluate the technical variability and sensitivity of microarrays. Cocaine-regulated genes were identified using the Affymetrix MAS 5.0 and Data Mining Tool v. 3. Eighty-nine upregulated and eight downregulated genes/ESTs were found after 1 day of "binge" cocaine. Following 3 days of cocaine treatment we identified 21 upregulated and 17 downregulated genes/ESTs. RNase protection assays of selected genes confirmed reliability of changes identified by the microarrays at the level of > or =1.40-fold increase. Many genes upregulated in the CPu by cocaine were immediate early genes for transcription factors and for "effector" proteins (e.g., vesl/Homer1a, Arc, synaptotagmin IV). Acute "binge" cocaine also increased mRNA levels for glutamate receptor GluR2, dopamine receptor D1, and a number of phosphatases. Genes downregulated by cocaine include several genes associated with energy metabolism in mitochondria, as well as the phosphatydylinositol-4 kinase and the regulator of G-protein signaling protein 4 (RGS4). A differential expression of somatostatin receptor SSTR2, not known to be a cocaine-responsive gene, as well as the clock gene Per2, were found by microarrays and confirmed by RNase protection assay. These results demonstrate the potential of microarrays in profiling gene expression with > or =40% increase or > or =14% decrease in mRNA levels for discovery of novel cocaine-responsive genes.
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