Astrocytes are a neural target of morphine action via glucocorticoid receptor-dependent signaling.

Chronic opioid use leads to the structural reorganization of neuronal networks, involving genetic reprogramming in neurons and glial cells. Our previous in vivo studies have revealed that a significant fraction of the morphine-induced alterations to the striatal transcriptome included glucocorticoid (GC) receptor (GR)-dependent genes. Additional analyses suggested glial cells to be the locus of these changes. In the current study, we aimed to differentiate the direct transcriptional effects of morphine and a GR agonist on primary striatal neurons and astrocytes. Whole-genome transcriptional profiling revealed that while morphine had no significant effect on gene expression in both cell types, dexamethasone significantly altered the transcriptional profile in astrocytes but not neurons. We obtained a complete dataset of genes undergoing the regulation, which includes genes related to glucose metabolism (Pdk4), circadian activity (Per1) and cell differentiation (Sox2). There was also an overlap between morphine-induced transcripts in striatum and GR-dependent transcripts in cultured astrocytes. We further analyzed the regulation of expression of one gene belonging to both groups, serum and GC regulated kinase 1 (Sgk1). We identified two transcriptional variants of Sgk1 that displayed selective GR-dependent upregulation in cultured astrocytes but not neurons. Moreover, these variants were the only two that were found to be upregulated in vivo by morphine in a GR-dependent fashion. Our data suggest that the morphine-induced, GR-dependent component of transcriptome alterations in the striatum is confined to astrocytes. Identification of this mechanism opens new directions for research on the role of astrocytes in the central effects of opioids.

Regulation of the immediate-early genes arc and zif268 in a mouse operant model of cocaine seeking reinstatement.

Reinstatement of extinguished operant responding for drug is an appropriate model of relapse to drug abuse. Due to the difficulty of implementing in mice the procedure of instrumental intravenous self-administration, mechanisms of reinstatement have so far been studied almost exclusively in rats. A mouse model of reinstatement of cocaine seeking has recently been characterized (Soria et al. 2008). The aim of the present study was to assess regional brain activation, as measured by induction of the immediate early genes (IEG) arc and zif268, during priming- or cue-elicited reinstatement of cocaine seeking using this new mouse model and the in situ hybridization technique. We have demonstrated that cue-elicited reinstatement of cocaine seeking was associated with induction of the IEG in the medial prefrontal cortex (prelimbic and infralimbic) and basolateral amygdala. Priming-induced reinstatement produced a more widespread up-regulation of those genes in forebrain regions including medial prefrontal, orbitofrontal and motor cortex, dorsal striatum and basolateral amygdala. These patterns of IEG expression are in agreement with previous results obtained in rats and thus indicate that the new mouse model of reinstatement is functionally equivalent to rat models. That comparability adds to the usefulness of the mouse model as a tool for addressing neurobiological mechanisms of addiction.

alpha-Synuclein expression in the brain and blood during abstinence from chronic alcohol drinking in mice.

alpha-Synuclein is a presynaptic protein proposed to serve as a negative regulator of dopaminergic neurotransmission. Recent research has implicated alpha-synuclein in chronic neuroadaptations produced by psychostimulant and opiate use, as well as in genetically determined susceptibility to alcoholism in humans. The aim of our study was to characterize the changes in alpha-synuclein expression after short-term abstinence from chronic alcohol drinking in mice. Male C57BL/6J mice were allowed to drink increasing concentrations of alcohol in the two-bottle choice procedure. Then the mice were given constant access to an 8% alcohol solution and water for 32 days, and were sacrificed 2 h, 24 h or 48 h after alcohol withdrawal. RT-PCR, in situ hybridization and Western blotting techniques were used to measure alpha-synuclein mRNA and protein levels in the brain and blood. alpha-Synuclein protein levels were elevated by up to 80% in the amygdala of mice withdrawn from alcohol for 24 h or 48 h. No changes in alpha-synuclein levels were found in the mesencephalon or striatum/accumbens. The levels of alpha-synuclein mRNA remained unchanged in all brain regions examined (the striatum, nucleus accumbens, amygdala, substantia nigra, ventral tegmental area). alpha-Synuclein mRNA was up-regulated in the whole blood 48 h after alcohol withdrawal. The accumulation of alpha-synuclein in the amygdala, observed in this study, seems to be a common feature of alcohol and opiate abstinence. This finding suggests a role of alpha-synuclein in common neuroadaptations produced by long-term alcohol and drug use. Although alpha-synuclein expression in the blood seems unrelated to that in the brain, it may serve as a peripheral biomarker of chronic alcohol consumption.

Prenatal exposure to valproic acid disturbs the enkephalinergic system functioning, basal hedonic tone, and emotional responses in an animal model of autism.

RATIONALE: It has been suggested that behavioral aberrations observed in autism could be the result of dysfunction of the neuroregulatory role performed by the endogenous opioid peptides. Many of those aberrations have been recently modeled in rats exposed to valproic acid (VPA) on the 12th day of gestation (VPA rats). OBJECTIVES: The aim of the present study was to elucidate functioning of the enkephalinergic system, one of the endogenous opioid peptide systems strongly involved in emotional responses, in VPA rats using both biochemical and behavioral methods. MATERIALS AND METHODS: In situ hybridization was used to measure proenkephalin mRNA expression in adult VPA rats' central nucleus of the amygdala, the dorsal striatum, and the nucleus accumbens. Additional groups of animals were examined in a conditioned place aversion to naloxone, the elevated plus maze, and object recognition tests to assess their basal hedonic tone, anxiety, learning and memory, respectively. RESULTS: Prenatal exposure to VPA decreased proenkephalin mRNA expression in the dorsal striatum and the nucleus accumbens but not in the central nucleus of the amygdala. It also increased anxiety and attenuated conditioned place aversion to naloxone but had no impact on learning and memory. CONCLUSIONS: The present results suggest that prenatal exposure to VPA may lead to the decreased activity of the striatal enkephalinergic system and in consequence to increased anxiety and disregulated basal hedonic tone observed in VPA rats. Presented results are discussed in light of interactions between enkephalinergic, GABAergic, and dopaminergic systems in the striatum and mesolimbic areas of the brain.

Regulation of alpha-synuclein expression in limbic and motor brain regions of morphine-treated mice.

Chronic exposure to opiates produces dependence and addiction, which may result from neuroadaptations in the dopaminergic reward pathway and its target brain regions. The neuronal protein alpha-synuclein has been implicated in neuronal plasticity and proposed to serve as a negative regulator of dopamine neurotransmission. Thus, alpha-synuclein could mediate some effects of opiates in the brain. The present study investigated the influence of acute and chronic morphine administration on alpha-synuclein mRNA and protein expression in the brains of mice. Downregulation of alpha-synuclein mRNA was observed in the basolateral amygdala, dorsal striatum, nucleus accumbens, and ventral tegmental area of mice withdrawn from chronic morphine treatment. The changes were the most pronounced after longer periods of withdrawal (48 h). In contrast, levels of alpha-synuclein protein, as assessed by Western blotting, were significantly increased in the amygdala and striatum/accumbens (but not in the mesencephalon) of morphine-withdrawn mice. In both brain regions, levels of alpha-synuclein were elevated for as long as 2 weeks after treatment cessation. Because alpha-synuclein is a presynaptic protein, the detected opposite changes in its mRNA and protein levels are likely to take place in different populations of projection neurons whose somata are in different brain areas. Axonal localization of alpha-synuclein was confirmed by immunofluorescent labeling. An attempt to identify postsynaptic neurons innervated by alpha-synuclein-containing axon terminals revealed their selective apposition to calbindin D28K-negative projection neurons in the basolateral amygdala. The observed changes in alpha-synuclein levels are discussed in connection with their putative role in mediating suppression of dopaminergic neurotransmission during opiate withdrawal.

New operant model of reinstatement of food-seeking behavior in mice.

RATIONALE: A major problem in treating obesity is the high rate of relapse to abnormal food-taking behavior when maintaining diet. OBJECTIVES: The present study evaluates the reinstatement of extinguished palatable food-seeking behavior induced by cues previously associated with the palatable food, re-exposure to this food, or stress. The participation of the opioid and dopamine mechanisms in the acquisition, extinction, and cue-induced reinstatement was also investigated. MATERIALS AND METHODS: C57BL/6 mice were first trained on a fixed-ratio-1 schedule of reinforcement to obtain chocolate-flavored pellets during 20 days, which was associated to a stimulus light. Operant behavior was then extinguished during 20 daily sessions. mRNA levels of opioid peptide precursors and dopamine receptors were evaluated in the brain by in situ hybridization and RT-PCR techniques. RESULTS: A reinstatement of food-seeking behavior was only obtained after exposure to the food-associated cue. A down-regulation of prodynorphin mRNA was found in the dorsal striatum and nucleus accumbens after the acquisition, extinction, and reinstatement of the operant behavior. Extinction and reinstatement of this operant response enhanced proenkephalin mRNA in the dorsal striatum and/or the nucleus accumbens core. Down-regulation of D2 receptor expression was observed in the dorsal striatum and nucleus accumbens after reinstatement. An up-regulation of PDYN mRNA expression was found in the hypothalamus after extinction and reinstatement. CONCLUSIONS: This study provides a new operant model in mice for the evaluation of food-taking behavior and reveals specific changes in the dopamine and opioid system associated to the behavioral responses directed to obtain a natural reward.

Forebrain PENK and PDYN gene expression levels in three inbred strains of mice and their relationship to genotype-dependent morphine reward sensitivity.

RATIONALE: Vulnerability to drug abuse disorders is determined not only by environmental but also by genetic factors. A body of evidence suggests that endogenous opioid peptide systems may influence rewarding effects of addictive substances, and thus, their individual expression levels may contribute to drug abuse liability. OBJECTIVES: The aim of our study was to assess whether basal genotype-dependent brain expression of opioid propeptides genes can influence sensitivity to morphine reward. METHODS: Experiments were performed on inbred mouse strains C57BL/6J, DBA/2J, and SWR/J, which differ markedly in responses to morphine administration: DBA/2J and SWR/J show low and C57BL/6J high sensitivity to opioid reward. Proenkephalin (PENK) and prodynorphin (PDYN) gene expression was measured by in situ hybridization in brain regions implicated in addiction. The influence of the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI), which attenuates effects of endogenous PDYN-derived peptides, on rewarding actions of morphine was studied using the conditioned place preference (CPP) paradigm. RESULTS: DBA/2J and SWR/J mice showed higher levels of PDYN and lower levels of PENK messenger RNA in the nucleus accumbens than the C57BL/6J strain. Pretreatment with nor-BNI enhanced morphine-induced CPP in the opioid-insensitive DBA/2J and SWR/J strains. CONCLUSIONS: Our results demonstrate that inter-strain differences in PENK and PDYN genes expression in the nucleus accumbens parallel sensitivity of the selected mouse strains to rewarding effects of morphine. They suggest that high expression of PDYN may protect against drug abuse by limiting drug-produced reward, which may be due to dynorphin-mediated modulation of dopamine release in the nucleus accumbens.

The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum.

BACKGROUND: Various drugs of abuse activate intracellular pathways in the brain reward system. These pathways regulate the expression of genes that are essential to the development of addiction. To reveal genes common and distinct for different classes of drugs of abuse, we compared the effects of nicotine, ethanol, cocaine, morphine, heroin and methamphetamine on gene expression profiles in the mouse striatum. RESULTS: We applied whole-genome microarray profiling to evaluate detailed time-courses (1, 2, 4 and 8 hours) of transcriptome alterations following acute drug administration in mice. We identified 42 drug-responsive genes that were segregated into two main transcriptional modules. The first module consisted of activity-dependent transcripts (including Fos and Npas4), which are induced by psychostimulants and opioids. The second group of genes (including Fkbp5 and S3-12), which are controlled, in part, by the release of steroid hormones, was strongly activated by ethanol and opioids. Using pharmacological tools, we were able to inhibit the induction of particular modules of drug-related genomic profiles. We selected a subset of genes for validation by in situ hybridization and quantitative PCR. We also showed that knockdown of the drug-responsive genes Sgk1 and Tsc22d3 resulted in alterations to dendritic spines in mice, possibly reflecting an altered potential for plastic changes. CONCLUSIONS: Our study identified modules of drug-induced genes that share functional relationships. These genes may play a critical role in the early stages of addiction.

Alterations of prodynorphin gene expression in the rat mesocorticolimbic system during heroin self-administration.

Opiate-induced alterations in the gene expression of the opioid propeptides prodynorphin (PDYN) and proenkephalin (PENK) in the brain have previously been described. However, there are no studies examining opioid propeptide system alterations due to long-lasting heroin self-administration. In our study, using in situ hybridization, we measured PDYN and PENK mRNA levels in the dorsal striatum, central nucleus of amygdala (CEA), and nucleus accumbens (NAcc) shell and core in rats after 6 weeks of heroin self-administration (fixed ratio 5, 0.02 mg/kg/infusion of heroin i.v.) and their respective saline or heroin "yoked" control. Our results show an increase in the PDYN mRNA level in the CEA and NAcc shell and no changes of PENK gene expression after heroin self-administration. In addition, to dissociate pharmacological effects of heroin from those produced by motivational processes driving active heroin intake on the PDYN and PENK gene expression, we compared effects of response-dependent (contingent) and response-independent (noncontingent--"yoked" heroin control) heroin administration. We found no differences between contingent and noncontingent groups of rats. In conclusion, our results indicate neuroadaptations in the PDYN but not PENK gene expression in rat limbic forebrain during heroin self-administration. We show that such changes depend on direct pharmacological actions of heroin rather than contingent heroin intake. These neuroalterations probably occur as an adaptation to long-lasting heroin exposure and may underlie changes leading to compulsive drug use and addiction.