Vulnerability to opiate intake in maternally deprived rats: implication of MeCP2 and of histone acetylation.

We previously showed that maternal deprivation predisposes male rats to anxiety, accompanied with an increase in their opiate consumption. In the present report, we searched for brain epigenetic mechanisms that possibly underlie this increase. For that, we examined the expression of the methyl-CpG-binding protein MeCP2 and of the histone deacetylases HDAC2 and HDAC3, as well as the acetylation status of histone H3 and H4 in mesolimbic structures of adult maternally deprived rats, using immunohistochemistry and Western blot analysis. A long-lasting increase in MeCP2 expression was found throughout the striatum of deprived rats. Enhanced HDAC2 expression and increased nuclear HDAC activity in the nucleus accumbens of deprived rats were associated with lower acetylation levels of histone H3 and H4. Treatment for 3 weeks with the HDAC inhibitor sodium valproate abolished HDAC activation together with the decrease in the acetylation levels of histone H4, and was accompanied with normalized oral morphine consumption. The data indicate that epigenetic mechanisms induced by early adverse environment memorize life experience to trigger greater opiate vulnerability during adult life. They suggest that sodium valproate may lessen vulnerability to opiate intake, particularly in subgroups of individuals subjected to adverse postnatal environments.

[Epigenetics and drug addiction: a focus on MeCP2 and on histone acetylation]. / Addiction et régulations épigénétiques - Implications de MeCP2 et de l'acétylation des histones.

Chronic drug exposure alters gene expression in the brain, which is believed to underlie compulsive drug seeking and drug taking behavior. Recent evidence shows that drug-induced long-term neuroadaptations in the brain are mediated in part by epigenetic mechanisms. By remodeling chromatin, this type of regulation contributes to drug-induced synaptic plasticity that translates into behavioral modifications. How drug-induced alterations in DNA methylation regulate gene expression is reviewed here, with a focus on MeCP2, a protein binding methylated DNA. The importance of histone modifications, especially acetylation is also discussed, with an emphasis on the effects of inhibitors of histone deacetylases on drug-induced behavioral changes. The precise identification of the epigenetic mechanisms that are under the control of drugs of abuse may help to uncover novel targets for the treatment of drug seeking and relapse.

Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food.

Cocaine exposure induces changes in the expression of numerous genes, in part through epigenetic modifications. We have initially shown that cocaine increases the expression of the chromatin remodeling protein methyl-CpG binding protein 2 (MeCP2) and characterized the protein phosphatase-1Cß (PP1Cß) gene, as repressed by passive i.p. cocaine injections through a Mecp2-mediated mechanism involving de novo DNA methylation. Both proteins being involved in learning and memory processes, we investigated whether voluntary cocaine administration would similarly affect their expression using an operant self-administration paradigm. Passive and voluntary i.v. cocaine intake was found to induce Mecp2 and to repress PP1Cß in the prefrontal cortex and the caudate putamen. This observation is consistent with the role of Mecp2 acting as a transcriptional repressor of PP1Cß and shows that passive intake was sufficient to alter their expression. Surprisingly, striking differences were observed under the same conditions in food-restricted rats tested for food pellet delivery. In the prefrontal cortex and throughout the striatum, both proteins were induced by food operant conditioning, but remained unaffected by passive food delivery. Although cocaine and food activate a common reward circuit, changes observed in the expression of other genes such as reelin and GAD67 provide new insights into molecular mechanisms differentiating neuroadaptations triggered by each reinforcer. The identification of hitherto unknown genes differentially regulated by drugs of abuse and a natural reinforcer should improve our understanding of how two rewarding stimuli differ in their ability to drive behavior.

Cocaine self-administration by rats is inhibited by cyclic GMP-elevating agents: involvement of epigenetic markers.

The C-type natriuretic peptide (CNP) exerts its action via stimulation of the cyclic GMP (cGMP) signalling pathway, which includes the activation of cGMP-dependent protein kinases. The pathway can also be activated by inhibitors of phosphodiesterases (PDE) that hydrolyse cGMP. The present report shows that activation of the cGMP pathway by CNP, by bromo-cGMP, a cell-permeant cGMP analogue, or by the PDE inhibitor zaprinast dose dependently reduces intravenous cocaine self-administration by rats. The effect was found when the compounds were injected in situ into the prefrontal cortex, but not when they were injected into the nucleus accumbens. A decrease in the number of cocaine infusions performed by rats was obtained under the fixed ratio-1 schedule of reinforcement as well as under a progressive ratio schedule, which evaluates the motivation of the animals for the drug. Decrease in cocaine self-administration was accompanied with reduced expression of the epigenetic markers methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in dopaminergic projection areas. An increase in the acetylation level of histone H3, but not of histone H4, was also noticed. Since MeCP2 and HDAC2 are known to modulate dynamic functions in the adult brain, such as synaptic plasticity, our results showing that activation of the cGMP signal transduction pathway decreased both cocaine intake and expression of the epigenetic markers strongly suggest that the MeCP2/HDAC2 complex is involved in the analysis of the reinforcing properties of cocaine in the prefrontal cortex.

Cocaine induces the expression of MEF2C transcription factor in rat striatum through activation of SIK1 and phosphorylation of the histone deacetylase HDAC5.

Distinct forms of MEF2 transcription factor act as positive or negative regulators of dendritic spine formation, with MEF2C playing a key regulator role in synapse plasticity. We report here that acute cocaine treatment of rats induced the expression of MEF2C in the striatum through a recently discovered transduction pathway. Repeated injections were found to induce MEF2C to a lesser extent. The mechanism by which MEF2C was induced involves the subsequent activation of the salt-inducible kinase SIK1 and the phosphorylation of HDAC5, a member of the class IIa of HDACs. Cocaine activated SIK1 by phosphorylation on Thr-182 residue, which was accompanied by the nuclear import of the kinase. In the nuclear compartment, SIK1 then phosphorylated HDAC5 causing the shuttling of its phospho-form from the nucleus to the cytoplasm of striatal cells. Activation of SIK1 by cocaine was further validated by the phosphorylation of TORC1/3, which was followed by the shuttling of TORC proteins from the nucleus to the cytoplasm. Activation of MEF2C was assessed by measuring the expression of the MEF2C gene itself, since the gene is known to be under the control of its own product. Since MEF2C plays a key role in memory/learning processes, activation of this pathway by cocaine is probably involved in plasticity mechanisms whereby the drug establishes its long-term effects such as drug dependence.

Hippocampal Cannabinoid 1 Receptors Are Modulated Following Cocaine Self-administration in Male Rats.

Cocaine addiction is a complex pathology inducing long-term neuroplastic changes that, in turn, contribute to maladaptive behaviors. This behavioral dysregulation is associated with transcriptional reprogramming in brain reward circuitry, although the mechanisms underlying this modulation remain poorly understood. The endogenous cannabinoid system may play a role in this process in that cannabinoid mechanisms modulate drug reward and contribute to cocaine-induced neural adaptations. In this study, we investigated whether cocaine self-administration induces long-term adaptations, including transcriptional modifications and associated epigenetic processes. We first examined endocannabinoid gene expression in reward-related brain regions of the rat following self-administered (0.33 mg/kg intravenous, FR1, 10 days) cocaine injections. Interestingly, we found increased Cnr1 expression in several structures, including prefrontal cortex, nucleus accumbens, dorsal striatum, hippocampus, habenula, amygdala, lateral hypothalamus, ventral tegmental area, and rostromedial tegmental nucleus, with most pronounced effects in the hippocampus. Endocannabinoid levels, measured by mass spectrometry, were also altered in this structure. Chromatin immunoprecipitation followed by qPCR in the hippocampus revealed that two activating histone marks, H3K4Me3 and H3K27Ac, were enriched at specific endocannabinoid genes following cocaine intake. Targeting CB1 receptors using chromosome conformation capture, we highlighted spatial chromatin re-organization in the hippocampus, as well as in the nucleus accumbens, suggesting that destabilization of the chromatin may contribute to neuronal responses to cocaine. Overall, our results highlight a key role for the hippocampus in cocaine-induced plasticity and broaden the understanding of neuronal alterations associated with endocannabinoid signaling. The latter suggests that epigenetic modifications contribute to maladaptive behaviors associated with chronic drug use.

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine.

Epigenetic mechanisms have recently been shown to be involved in the long-term effects of drugs of abuse. A well described epigenetic mechanism modulating transcriptional activity consists in the binding to DNA of methyl-CpG binding proteins, such as MeCP2, recruiting histone deacetylases (HDACs). Nicotine causes long-term changes in the brain, but little is known concerning the mechanisms involved in nicotine-preference. Using a nicotine-conditioned place preference protocol, we demonstrate here that the histone deacetylase inhibitor phenylbutyrate was able to dramatically reduce the preference for nicotine, without altering the aversive properties of the drug. We measured immunohistochemically the acetylation of lysine-9 of histone H3, and the expression of phosphorylated cAMP-response element-binding protein, HDAC2 and methyl-CpG-binding protein 2 in the striatum and prefrontal cortex of rats displaying nicotine-preference or aversion and treated with phenylbutyrate. We show that, at the dose administered, the inhibitor was effective in inhibiting HDAC activity. The data suggest that phosphorylated cAMP-response element-binding protein participates in the establishment of conditioned place preference, but not in the reduction of nicotine-preference in response to phenylbutyrate. Moreover, striatal expression of HDAC2 in response to phenylbutyrate mirrored the behavioral effects of the inhibitor, suggesting that HDAC2 is involved in promoting synaptic plasticity underlying the preference for nicotine.

Rhythmic Regulation of DNA Methylation Factors and Core-Clock Genes in Brain Structures Activated by Cocaine or Sucrose: Potential Role of Chromatin Remodeling.

The circadian system interacts with the mesocorticolimbic reward system to modulate reward and memory in a time-of-day dependent manner. The circadian discrimination of reward, however, remains difficult to address between natural reinforcers and drugs of abuse. Circadian rhythms control cocaine sensitization and conversely cocaine causes long-term alteration in circadian periodicity in part through the serotonergic neurotransmission. Since neural circuits activated by cocaine and natural reinforcers do not completely overlap, we compared the effect of cocaine with that of sucrose, a strong reinforcer in rodents, by using passive chronic administration. The expression of fifteen genes playing a major role in DNA methylation (Dnmts, Tets), circadian rhythms (Clock, Bmal1, Per1/2, Cry1/2, Rev-Erbß, Dbp1), appetite, and satiety (Orexin, Npy) was analyzed in dopamine projection areas like the prefrontal cortex, the caudate putamen, and the hypothalamus interconnected with the reward system. The corresponding proteins of two genes (Orexin, Per2) were examined by IHC. For many factors controlling biological and cognitive functions, striking opposite responses were found between the two reinforcers, notably for genes controlling DNA methylation/demethylation processes and in global DNA methylation involved in chromatin remodeling. The data are consistent with a repression of critical core-clock genes by cocaine, suggesting that, consequently, both agents differentially modulate day/night cycles. Whether observed cocaine and sucrose-induced changes in DNA methylation in a time dependent manner are long lasting or contribute to the establishment of addiction requires further neuroepigenetic investigation. Understanding the mechanisms dissociating drugs of abuse from natural reinforcers remains a prerequisite for the design of selective therapeutic tools for compulsive behaviors.

Epigenetic Regulation of Circadian Clocks and Its Involvement in Drug Addiction.

Based on studies describing an increased prevalence of addictive behaviours in several rare sleep disorders and shift workers, a relationship between circadian rhythms and addiction has been hinted for more than a decade. Although circadian rhythm alterations and molecular mechanisms associated with neuropsychiatric conditions are an area of active investigation, success is limited so far, and further investigations are required. Thus, even though compelling evidence connects the circadian clock to addictive behaviour and vice-versa, yet the functional mechanism behind this interaction remains largely unknown. At the molecular level, multiple mechanisms have been proposed to link the circadian timing system to addiction. The molecular mechanism of the circadian clock consists of a transcriptional/translational feedback system, with several regulatory loops, that are also intricately regulated at the epigenetic level. Interestingly, the epigenetic landscape shows profound changes in the addictive brain, with significant alterations in histone modification, DNA methylation, and small regulatory RNAs. The combination of these two observations raises the possibility that epigenetic regulation is a common plot linking the circadian clocks with addiction, though very little evidence has been reported to date. This review provides an elaborate overview of the circadian system and its involvement in addiction, and we hypothesise a possible connection at the epigenetic level that could further link them. Therefore, we think this review may further improve our understanding of the etiology or/and pathology of psychiatric disorders related to drug addiction.

Methionine Supplementation Abolishes Nicotine-Induced Place Preference in Zebrafish: a Behavioral and Molecular Analysis.

In zebrafish, nicotine is known to regulate sensitivity to psychostimulants via epigenetic mechanisms. Little however is known about the regulation of addictive-like behavior by DNA methylation processes. To evaluate the influence of DNA methylation on nicotine-induced conditioned place preference (CPP), zebrafish were exposed to methyl supplementation through oral L-methionine (Met) administration. Met was found to reduce dramatically nicotine-induced CPP as well as behaviors associated with drug reward. The reduction was associated with the upregulation of DNA methyltransferases (DNMT1 and 3) as well as with the downregulation of methyl-cytosine dioxygenase-1 (TET1) and of nicotinic receptor subunits. Met also increased the expression of histone methyltransferases in nicotine-induced CPP groups. It reversed the nicotine-induced reduction in the methylation at α7 and NMDAR1 gene promoters. Treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine (AZA) was found to reverse the effects of Met in structures of the reward pathway. Interestingly, Met did not modify the amount of the phospho-form of CREB (pCREB), a key factor establishing nicotine conditioning, whereas AZA increased pCREB levels. Our data suggest that nicotine-seeking behavior is partially dependent on DNA methylation occurring probably at specific gene loci, such as α7 and NMDAR1 receptor gene promoters. Overall, they suggest that Met should be considered as a potential therapeutic drug to treat nicotine addiction.

LSP2-9166, an orthosteric mGlu4 and mGlu7 receptor agonist, reduces cocaine self-administration under a progressive ratio schedule in rats.

Cocaine addiction is a serious health issue in Western countries. Despite the regular increase in cocaine consumption across the population, there is no specific treatment for cocaine addiction. Critical roles for glutamate neurotransmission in the rewarding effects of psychostimulants as well as relapse have been suggested and accumulating evidence indicates that targeting mGlu group III receptors could represent a promising strategy to develop therapeutic compounds to treat addiction. In this context, the aim of our study was to examine the effect of LSP2-9166, a mGlu4/mGlu7 receptor orthosteric agonist, on the motivation for cocaine intake. We used an intravenous self-administration paradigm in male Wistar rats as a reliable model of voluntary drug intake. We first evaluated the direct impact of cocaine on Grm4 and Grm7 gene expression. Voluntary cocaine intake under a fixed ratio schedule of injections induced an increase of both mGlu4 and mGlu7 receptor transcripts in nucleus accumbens and hippocampus. We then evaluated the ability of LSP2-9166 to affect cocaine self-administration under a progressive ratio schedule of reinforcement. We found that this compound inhibits the motivation to obtain the drug, although it induced a hypolocomotor effect which could biais motivation index. Our findings demonstrate that mGlu group III receptors represent new targets for decreasing motivation to self-administer cocaine.

Inhibition of histone deacetylases in rats self-administering cocaine regulates lissencephaly gene-1 and reelin gene expression, as revealed by microarray technique.

Injection of the histone deacetylase inhibitor trichostatin A (TsA) to rats has been shown to decrease their motivation to self-administer cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex and nucleus accumbens of rats self-administering cocaine and treated with TsA. Using oligonucleotide microarrays, we identified 722 probe sets in the cortex and 136 probe sets in the nucleus accumbens that were differentially expressed between vehicle and TsA-treated rats that self-administered cocaine. Microarray data were validated by real-time PCR for seven genes. Using immunohistochemistry, we further investigated the expression of Lis1 and reelin genes, because (i) they were similarly regulated by TsA at the mRNA level; (ii) they belong to the same signal transduction pathway; (iii) mutations within both genes cause lissencephaly. Cocaine self-injection was sufficient to activate the two genes at both the mRNA and protein levels. TsA treatment was found to up-regulate both Lis1 and reelin protein expression in the cortex and to down-regulate it in the nucleus accumbens of rats self-administering cocaine. The data suggest that the two proteins contribute to establish neurobiological mechanisms underlying brain plasticity whereby TsA lowers the motivation for cocaine.

CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats.

Regulation of gene expression is known to contribute to the long-term adaptations taking place in response to drugs of abuse. Recent studies highlighted the regulation of gene transcription in neurons by chromatin remodeling, a process in which posttranslational modifications of histones play a major role. To test the involvement of epigenetic regulation on drug-reinforcing properties, we submitted rats to the cocaine operant self-administration paradigm. Using the fixed ratio 1 schedule, we found that the histone deacetylase (HDAC) inhibitors trichostatin A and phenylbutyrate dose-dependently reduced cocaine self-administration. Under the progressive ratio schedule, both trichostatin A and depudecin significantly reduced the breaking point, indicating that HDAC inhibition attenuated the motivation of rats for cocaine. Conversely, HDAC inhibition did not decrease self-administration for the natural reinforcer sucrose. This observation was correlated with measurements of HDAC activity in the frontal cortex, which was inhibited in response to cocaine, but not to sucrose self-administration. Control experiments showed that the decrease in the motivation for the drug was not attributable to a general motivational dysfunction because trichostatin A had no adverse effect on locomotion during the habituation session or on cocaine-induced hyperlocomotion. It was not attributable to anhedonia because the inhibitor had no effect on the sucrose preference test. In contrast, trichostatin A completely blocked the cocaine-induced behavioral sensitization. Together, the data show that epigenetic regulation of gene transcription in adult brain is able to influence a motivated behavior and suggest that HDAC inhibition may counteract the neural sensitization leading to drug dependence.

Regulation of Brain DNA Methylation Factors and of the Orexinergic System by Cocaine and Food Self-Administration.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

Induction by cocaine of the serotonergic 5-HT3 receptor in rat cerebellum.

The expression of the 5-HT(3) receptor, a member of the serotonin receptor family, was examined in rat cerebellum of saline- or cocaine-treated rats. Both immunohistochemistry and Western blot analysis were used. We found that the expression of this serotonin receptor subtype was increased in the cerebellum of rats injected either acutely or repeatedly (1 injection/day for 10 days) with cocaine. The stimulation was more pronounced after a single injection than after a series of 10 injections. Surprisingly, the expression of the 5-HT(3) receptor was mainly localized in Bergmann glial cells, as assessed from the co-localization of the receptor with the glial cell marker glial fibrillary acidic protein (GFAP). Our data emphasize the importance of the 5-HT(3) receptor induction in the cerebellum as part of the neuroadaptations taking place in rat brain in response to the psychostimulant cocaine.

Odor increases [3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion.

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium.

Alpha-adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-mu2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, mu2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.

Activation of the cGMP pathway in dopaminergic structures reduces cocaine-induced EGR-1 expression and locomotor activity.

Nitric oxide (NO) and the C-type natriuretic peptide (CNP) exert their action on brain via the cGMP signaling pathway. NO, by activating soluble guanylyl cyclase, and CNP, by stimulating membrane-bound guanylyl cyclase, cause intracellular increases of cGMP, activating cGMP-dependent protein kinases (PKGs). We show here that injection of CNP into the rat ventral tegmental area strongly reduced cocaine-induced egr-1 expression in the nucleus accumbens in a dose-dependent manner. The effect of CNP was reversed by the previous injection of a selective PKG inhibitor, KT5823. Activation of PKG by 8-bromo-cGMP reduced, like CNP, cocaine-induced gene transcription in dopaminergic structures. To confirm the involvement of PKG, this was overexpressed in either the mesencephalon or the caudate-putamen. Using the polyethyleneimine delivery system, an active protein was expressed by injecting a plasmid vector containing the human PKG-Ialpha cDNA. PKG was overexpressed in dopaminergic and GABAergic neurons when the plasmid was injected in the ventral tegmental area, whereas overexpression was observed in medium spiny GABAergic neurons and in both cholinergic and GABAergic interneurons when the PKG vector was injected into the caudate-putamen. Activation of the overexpressed PKG reduced cocaine-induced egr-1 expression in dopaminergic structures and affected behavior (i.e., locomotor activity). These effects were again reversed by previous injection of the selective PKG inhibitor. The current data suggest that NO and the neuropeptide CNP are potential regulators of cocaine-related effects on behavior.

Acute or repeated cocaine administration generates reactive oxygen species and induces antioxidant enzyme activity in dopaminergic rat brain structures.

Either a single (acute) or repeated daily (chronic) injections (1 injection/day) of 20 mg/kg cocaine for 10 days to rats was found to increase reactive oxygen species production in two dopaminergic brain structures, the frontal cortex and the striatum. We found that the mitochondrial genome was down-regulated after acute cocaine injection. Hydroperoxide and lipid peroxide generation was correlated with an increase in mitochondrial hydrogen peroxide generation and with a reduced functioning of mitochondrial complex I in response to cocaine. As judged from the measurement of caspase-3 activity and TUNEL labeling, neither acute nor chronic cocaine treatment has been found to induce apoptosis in any of the structures examined. This differs dramatically from what has been described for methamphetamine. Cocaine-induced radical formation was accompanied by the induction of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, after both acute and chronic cocaine treatment. In addition, proteasome chymotrypsin-like activity was enhanced following a single cocaine injection in both cortex and striatum. It is proposed that the compensatory mechanisms to oxidative stress occurring in response to cocaine were effective in scavenging reactive oxygen species and in preventing subsequent cellular damage, thus explaining why no significant cell death was found in these brain structures.