Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study.

DNA methylation is a major epigenetic process which regulates the accessibility of genes to the transcriptional machinery. In the present study, we investigated whether modifying the global DNA methylation pattern in the brain would alter cocaine intake by rats, using the cocaine self-administration test. The data indicate that treatment of rats with the DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (dAZA) and zebularine enhanced the reinforcing properties of cocaine. To obtain some insights about the underlying neurobiological mechanisms, a genome-wide methylation analysis was undertaken in the prefrontal cortex of rats self-administering cocaine and treated with or without dAZA. The study identified nearly 189 000 differentially methylated regions (DMRs), about half of them were located inside gene bodies, while only 9% of DMRs were found in the promoter regions of genes. About 99% of methylation changes occurred outside CpG islands. Gene expression studies confirmed the inverse correlation usually observed between increased methylation and transcriptional activation when methylation occurs in the gene promoter. This inverse correlation was not observed when methylation took place inside gene bodies. Using the literature-based Ingenuity Pathway Analysis, we explored how the differentially methylated genes were related. The analysis showed that increase in cocaine intake by rats in response to DNA methyltransferase inhibitors underlies plasticity mechanisms which mainly concern axonal growth and synaptogenesis as well as spine remodeling. Together with the Akt/PI3K pathway, the Rho-GTPase family was found to be involved in the plasticity underlying the effect of dAZA on the observed behavioral changes.

Effects of combined nicotine and fluoxetine treatment on adult hippocampal neurogenesis and conditioned place preference.

Adult neurogenesis occurs in mammals within the dentate gyrus, a hippocampal subarea. It is known to be induced by antidepressant treatment and reduced in response to nicotine administration. We checked here whether the antidepressant fluoxetine would inverse the decrease in hippocampal neurogenesis caused by nicotine. It is shown that repeated, but not a single injection of rats with fluoxetine was able to abolish the decrease in adult dentate cell proliferation produced by nicotine treatment. We measured the expression of several biochemical parameters known to be associated with neurogenesis in the dentate gyrus. Both drugs increased the expression of p75 neurotrophin receptor, which promotes proliferation and early maturation of dentate gyrus cells. Using the conditioned place preference (CPP) paradigm, we also gave both drugs in a context in which their rewarding properties could be measured. Fluoxetine produced a significant but less robust CPP than nicotine. A single injection of fluoxetine was found to reduce nicotine-induced CPP. Moreover, the rewarding properties of nicotine were completely abolished in response to repeated fluoxetine injections. Expression of nicotine-induced CPP was accompanied by an increase of phospho-CREB (cyclic AMP-responsive element-binding protein) and HDAC2 (histone deacetylase 2) expression in the nucleus accumbens. The data suggest that fluoxetine reward, as opposed to nicotine reward, depends on dentate gyrus neurogenesis. Since fluoxetine was able to disrupt the association between nicotine and the environment, this antidepressant may be tested as a treatment for nicotine addiction using cue exposure therapy.

Bilateral lesions of the entorhinal cortex differentially modify haloperidol- and olanzapine-induced c-fos mRNA expression in the rat forebrain.

Lesions of the entorhinal cortex are now an accepted model for mimicking some of the neuropathological aspects of schizophrenia, since evidence has accumulated for the presence of cytoarchitectonic abnormalities within this cortex in schizophrenic patients. The present study was undertaken to address the functional consequences of bilateral entorhinal cortex lesions on antipsychotic-induced c-fos expression. After a 15-day recovery period, the effect of a typical antipsychotic, haloperidol (1 mg/kg), on c-fos mRNA expression was compared with that of an atypical one, olanzapine (10 mg/kg), in both sham-lesioned and entorhinal cortex-lesioned rats. In sham-lesioned rats, both haloperidol and olanzapine induced c-fos expression in the caudal cingulate cortex, dorsomedial and dorsolateral caudate-putamen, nucleus accumbens core and shell and lateral septum. In addition, olanzapine, but not haloperidol, increased c-fos expression within the central amygdala. In entorhinal cortex-lesioned rats, haloperidol-induced c-fos expression was markedly reduced in most areas. In contrast, the olanzapine-induced c-fos expression was not altered in the nucleus accumbens shell and lateral septum of the lesioned rats. These findings reveal that entorhinal cortex lesions affect c-fos expression in a compound- and regional-dependent manner. Our results further emphasize the importance of the exploration of the mechanisms of action of antipsychotic drugs in the context of an associated cortical pathology.

Induction of the immediate early genes egr-1 and c-fos by methamphetamine in mouse brain.

Methamphetamine (METH) is one of the most commonly abused psychostimulant, and is known to induce dopaminergic neurotoxicity by generating oxidative stress and free radicals. In the present study we investigated the effects of METH on egr-1 and c-fos immediate early gene induction in different regions of mouse brain, at different doses and different time courses. We also measured the tissue levels of monoamines in order to correlate their changes with gene expression. A single injection of METH (40 mg/kg) significantly increased egr-1 and c-fos mRNA expression within 30 min in frontal cortex, nucleus accumbens, caudate putamen, septum and CA1 region of hippocampus. Time course studies showed that in most cases, both genes were expressed within 30 min and decreased after 60 min. METH produced a significant decrease in striatal dopamine level, reaching a very low level after 24 h. Striatal serotonin level significantly increased and returned to control levels after 2 h. These data show that METH induced egr-1 and c-fos mRNA expression in selective brain areas, which correlated with an alteration in monoamines.

Cyclic GMP-dependent protein kinase potentiates serotonin-induced Egr-1 binding activity in PC12 cells.

The NO/cyclic GMP (cGMP) signal transduction pathway, which involves the cGMP-dependent protein kinase (PKG), regulates transcription of several genes, including immediate early genes. Using transfection experiments with the PKG-Ialpha cDNA cloned from human aorta, we show here that addition of membrane-permeable cGMP analogues to PC12 cells slightly upregulated ERK MAP (mitogen-activated protein) kinase. Likewise, PKG-Ialpha was found to activate weakly DNA binding activity of the Egr-1 transcription factor. On the other hand, PKG-Ialpha overexpression was shown to tremendously amplify the Egr-1 binding activity induced by the neurotransmitter serotonin, which activates egr-1 gene expression also via the stimulation of the ERK MAP kinase pathway. Since this potentiation occurred neither at the level of ERK nor at the egr-1 transcriptional level, the mechanism of amplification probably results from the convergence of ERK and PKG pathways at the level of the transcription factor Egr-1.

Mechanisms underlying neuronal death induced by chromogranin A-activated microglia.

The neurotoxic effects of activated microglia in neurodegenerative diseases are well established. We recently provided evidence that chromogranin A (CGA), a multifunctional protein localized in dystrophic neurites and in senile plaques, induces an activated phenotype and secretion of neurotoxins by rat microglia in culture. In the present study, we focused on the mechanisms underlying neuronal degeneration triggered by CGA-activated microglia. We found that neuronal death exhibits apoptotic features, characterized by the externalization of phosphatidylserine and the fragmentation of DNA. Microglial neurotoxins markedly stimulate the phosphorylation and activity of neuronal p38 mitogen-activated protein kinase and provoke the release of mitochondrial cytochrome c, which precedes apoptosis. Inhibition of p38 kinase with SB 203580 partially protects neurons from death induced by CGA-activated microglia. Furthermore, neurons are also protected by Fas-Fc, which antagonizes the interactions between the death receptor Fas and its ligand FasL and by cell-permeable peptides that inhibit caspases 8 and 3. Thus, CGA triggers the release of microglial neurotoxins that mobilize several death-signaling pathways in neurons. Our results further support the idea that CGA, which is up-regulated in many neuropathologies, represents a potent endogeneous inflammatory factor possibly responsible for neuronal degeneration.

C-type natriuretic peptide (CNP) regulates cocaine-induced dopamine increase and immediate early gene expression in rat brain.

The neuropeptide C-type natriuretic peptide (CNP) is the primary biologically active natriuretic peptide in brain. Using in situ hybridization, the present report demonstrates that CNP regulates egr-1, c-fos and junB immediate early gene expression in rat brain. In the frontal cortex, CNP induced immediate early gene expression whereas it inhibited dose-dependently the cocaine-induced early gene expression in the dopaminergic projection fields nucleus accumbens and caudate-putamen. CNP may produce its effect directly on dopaminergic neurons because we found that its receptor, guanylyl cyclase GC-B, was expressed in the mesencephalon where dopaminergic neurons originate, as well as in their projection fields. The inhibition by CNP of the early gene expression elicited by cocaine in the caudate-putamen is correlated with a CNP-evoked decrease in cocaine-induced rise in extracellular dopamine, measured by in vivo microdialysis experiments. The significance of the inhibition of cocaine-induced dopamine release and early gene induction by the endogenous peptide CNP is demonstrated by data indicating that CNP reduced the cocaine-induced spontaneous locomotor activation. By inhibiting dopaminergic neuronal activity, CNP represents a potential negative regulator of related behavioural effects of cocaine.

Cyclic AMP-elevating agents induce the expression of MAP kinase phosphatase-1 in PC12 cells.

Stimulation of pheochromocytoma PC12 cells by cAMP-elevating agents caused the induction of the immediate early gene 3CH134, which encodes MAP kinase phosphatase-1 (MKP-1). Forskolin was as potent as serum in stimulating MKP-1 gene expression, whereas dibutyryl-cAMP and neuropeptide PACAP were less effective. Induction of the MKP-1 gene was accompanied by neo-synthesis of MKP-1 protein. MAP kinase activation was not involved in the cAMP-induced MKP-1 gene expression. The MAP kinase inactivation, that would result from MKP-1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.

C-fos and egr-1 immediate-early gene induction by cocaine and cocaethylene in rat brain: a comparative study.

The induction of immediate-early genes can now be considered as a tool to study neuronal activation in different brain structures. These genes, which are rapidly and transiently induced in response to diverse extracellular stimulation, coordinate alterations in gene expression underlying neuronal plasticity. Using in situ hybridization, we found that acute i.p. cocaine (20 mg/kg) injection produced a strong expression of egr-1 and c-fos genes in the nucleus accumbens, caudate-putamen, and frontal cortex in the rat. Cocaethylene is an active metabolite of cocaine that is formed when cocaine is consumed together with ethyl alcohol. Injection of cocaethylene at a dose equivalent to cocaine induced the expression of the two immediate-early genes in the same brain structures, but to a lesser extent. A high dose of ethanol increased egr-1 and c-fos expression in the frontal cortex and in the lateral part of the caudate-putamen. Since cocaine is known to potently inhibit both dopamine and serotonin transporters, whereas cocaethylene only inhibits the dopamine transporter, our results strongly suggest that the serotonergic system participates in the mode of action of cocaine in its ability to trigger immediate-early gene transcription.

Odor regulates the expression of the mitogen-activated protein kinase phosphatase gene hVH-5 in bilateral entorhinal cortex-lesioned rats.

Since it is known that several immediate early genes are induced by olfactory stimuli, we determined whether an olfactory stimulus also induces the expression of the mitogen-activated protein kinase (MAPK) phosphatase gene hVH-5 (homologue of vaccinia virus H1 phosphatase gene, clone 5), a member of a novel class of immediate early genes encoding dual-specificity protein phosphatases. The expression was studied by in situ hybridization in different brain structures involved in odor processing, in control and bilateral entorhinal cortex (EC) lesioned rats. EC-lesion did not significantly affect hVH-5 gene expression in the glomerular cell layer of the olfactory bulb (OB), while odor stimulation induced it in both control and EC-lesioned groups. In contrast, odor-induced expression of hVH-5 gene in mitral/granular cell layers was only evident after lesion of the EC. Similar results were obtained in the piriform cortex (PCx), a structure intimately connected to the mitral cell layer. In the CA1 hippocampal subfield, odor stimulation induced hVH-5 gene expression in both control and EC-lesioned animals, the increase being potentiated in lesioned rats. CA3 and dentate gyrus exhibited a similar pattern of gene expression, the odor stimulating gene expression in both control and lesioned groups. The amygdala (Am) displayed no significant change. It appears that through the induction of a MAPK phosphatase, the EC controls MAPK activities differently after odor stimulation in OB, PCx and hippocampus (Hip). The results illustrate the notion that odor representation in the brain requires plastic modifications at both anatomical and functional levels.

Gamma-hydroxybutyrate and cocaine administration increases mRNA expression of dopamine D1 and D2 receptors in rat brain.

The effects of acute and repeated gamma-hydroxybutyrate (GHB) and cocaine administration on D1 and D2 dopamine receptor mRNA expression were examined using in situ hybridization histochemistry in different rat brain structures rich in GHB receptors. Six hours after a single GHB administration (500 mg/kg i.p.), an increase in D1 and D2 mRNA expression was observed in almost all regions examined; whereas, acute cocaine injection (20 mg/kg i.p.) had no effect. Repeated exposure to GHB (500 mg/kg i.p. twice daily) for 10 days, followed by a 14-h withdrawal period, induced increasing effects on D1 and D2 dopamine receptor mRNA expression, similar to those caused by chronic treatment with cocaine (20 mg/kg i.p. once a day). These effects of GHB and cocaine on dopamine receptor mRNA expression could be a consequence, for both compounds, of the modulation of dopaminergic activity; thus, supporting the benefit of GHB in cocaine substitution therapy.

Acute ibogaine injection induces expression of the immediate early genes, egr-1 and c-fos, in mouse brain.

The aim of the present study was to evaluate if an acute injection of ibogaine (IBO) induces immediate early gene expression in different regions of mouse brain. Adult male C57 mice received a single injection of IBO and were perfused transcardially with 1% paraformaldehyde 30 min after the drug administration. A single injection of IBO produced a significant increase of egr-1 messenger RNA induction in nucleus accumbens (NAc), caudate-putamen (CPu), frontal cortex (FCx), septum, dentate gyrus (DG) and CA3 region of hippocampus, whereas c-fos gene was induced in CPu, FCx, DG, septum and CA1 region of hippocampus. This gene expression may be due, in part, to the stimulant properties of IBO, as we found with other psychostimulants.

Cocaine and fluoxetine induce the expression of the hVH-5 gene encoding a MAP kinase phosphatase.

A novel class of immediate early genes that encode enzymes of the MAP kinase phosphatase family has recently been described. These enzymes are dual-specificity protein phosphatases and some show tissue-specific distribution, like the hVH-5 gene (homologue of vaccinia virus H1 phosphatase gene clone 5), which is expressed predominantly in the adult brain. In this paper, we investigated whether the hVH-5 gene is induced by psychostimulants in rat brain, as has been demonstrated for immediate early genes encoding transcription factors. Using in situ hybridization, we found that i.p. injection of cocaine, amphetamine and caffeine induced hVH-5 mRNA expression within 40 min in the nucleus accumbens (NAc), caudate putamen, frontal cortex and hippocampus, with a maximal effect in the NAc. The cocaine-induced hVH-5 gene induction involves the serotonergic system, since it was abolished in the NAc by lesioning serotonergic raphé projections with 5,7-dihydroxytryptamine. Moreover, the effect of cocaine was fully mimicked by the selective serotonin uptake inhibitor fluoxetine. In contrast to what has been described for c-fos and egr-1 immediate early genes, we found that hVH-5 mRNA expression in the NAc and hippocampus was as significant after repeated cocaine injections for 10 days as after a single injection. The considerable and prolonged induction of the MAP kinase phosphatase hVH-5 gene by psychostimulant drugs in postmitotic brain cells, particularly in the NAc, could indicate that MAP kinase substrates are involved in the reinforcing properties of drugs of abuse.

The serotonergic system modulates the cocaine-induced expression of the immediate early genes egr-1 and c-fos in rat brain.

Transcription regulatory factors are rapidly induced in brain by a wide variety of stimuli and may be important in coordinating changes in gene expression under-lying neuronal plasticity. Using in situ hybridization, we found that acute cocaine administration (20 mg/kg, intraperitoneally (i.p.)) produced a robust induction of both c-fos and egr-1 immediate early genes. Egr-1 messenger RNA induction was highest in the caudate putamen and in the shell of the nucleus accumbens. No significant induction was noticed after injection of fluoxetine, a selective inhibitor of serotonin uptake. Cocaine-induced egr-1 and c-fos expression was substantially reduced in the brain areas from rats in which the serotonergic projections were lesioned by injection of the neurotoxin 5,7-dihydroxytryptamine and in rats that have been injected with tropisetron, an antagonist of the 5-hydroxytryptamine (5-HT3) receptor. Conversely, the 5-HT3 receptor agonist 2-methylserotonin induced the expression of these early genes in structures including the caudate putamen and nucleus accumbens.

The Serotonergic System Modulates the Cocaine-Induced Expression of the Immediate Early Genes egr-1 and c-fos in Rat Brain.

Transcription regulatory factors are rapidly induced in brain by a wide variety of stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. Using in situ hybridization, we found that acute cocaine administration (20 mg/kg, intraperitoneally (i.p.)) produced a robust induction of both c-fos and egr-1 immediate early genes. Egr-1 messenger RNA induction was highest in the caudate putamen and in the shell of the nucleus accumbens. No significant induction was noticed after injection of fluoxetine, a selective inhibitor of serotonin uptake. Cocaine-induced egr-1 and c-fos expression was substantially reduced in the brain areas from rats in which the serotonergic projections were lesioned by injection of the neurotoxin 5,7-dihydroxytryptamine, and in rats that have been injected with tropisetron, an antagonist of the 5-hydroxytryptamine (5-HT3) receptor. Conversely, the 5-HT3 receptor agonist 2-methyl-serotonin induced the expression of these early genes in structures including the caudate putamen and nucleus accumbens.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

5-Hydroxytryptamine induces TIS8/egr-1 and c-fos expression in PC12 cells. Involvement of tyrosine protein phosphorylation.

The TIS8/egr-1 gene is a member of the class of immediate early genes. Originally discovered as a mitogen-induced gene, it can also be induced by synaptic activity. We report here the induction of the TIS8/egr-1 gene by the neurotransmitter 5-hydroxytryptamine (5-HT) in cultured rat phaeochromocytoma PC12 cells. Induction was maximal 40 min after addition of 5-HT to the cells, and declined very rapidly to reach the basal level after 90 min. The electrophoretic mobility-shift assay showed that induction of the TIS8/egr-1 gene by 5-HT was accompained by increased Egr-1 protein binding to its DNA consensus sequence. We found an overall correlation of 5-HT-induced egr-1 expression with that of c-fos expression. The kinetics of the ability of both gene products to bind to their respective DNA consensus sequence was also similar. The 5-HT-induced activation in Egr-1 binding was inhibited by ketanserin and mesulergine, indicating that 5-HT exerted its action via a 5-HT2 receptor subtype. The tyrosine protein kinase inhibitor genistein abolished induction of the TIS8/egr-1 gene, suggesting that tyrosine kinase activity is required for the induction of early genes by 5-HT. Genistein also inhibited 5-HT-induced Egr-1 binding activity. The increase in phosphotyrosine content of focal adhesion kinase we noticed upon addition of 5-HT to cells suggests that this cytoplasmic tyrosine protein kinase mediates the effect of 5-HT in eliciting early gene induction.

Immediate early gene induction by natriuretic peptides in PC12 phaeochromocytoma and C6 glioma cells.

The effect of the natriuretic peptides ANP, BNP and CNP on cGMP formation and immediate early gene expression was investigated in PC12 phaeochromocytoma and C6 glioma cell lines. The three natriuretic peptides were shown to rapidly induce c-fos, TIS8/egr-1 and junB mRNA expression in both cell lines, via stimulation of the cGMP pathway. CNP stimulated cGMP formation and gene induction more potently than the other peptides in C6 cells, and this was statistically significant. In contrast, the three peptides produced similar gene induction in PC12 cells, despite the higher cGMP accumulation evoked by ANP or BNP. CNP was also found to increase DNA binding activity of the transcription factor AP1 in both cell types, demonstrating that natriuretic peptides potentially regulate key cellular gene expression.

Induction of c-fos, jun B and egr-1 expression by haloperidol in PC12 cells: involvement of calcium.

Acute injection of haloperidol, a dopamine D2 receptor antagonist, is known to increase immediate early gene expression of the fos and jun families in rodent striatal neurons. A set of gene induction, including c-fos, jun B and TIS8/egr-1, was found when haloperidol was added to PC12 cells in culture. Electrophoretic mobility-shift assays show that haloperidol-evoked gene induction was accompanied by a transient and dose-dependent increase in AP1 and EGR-1 binding activities in these cells. Gene expression is tentatively explained by the rapid and transient increase in cytosolic free Ca2+ concentration observed upon haloperidol addition. The cytosolic calcium rise and AP1 binding activation elicited by haloperidol were dependent on extracellular Ca2+, suggesting that haloperidol exerted its effects by promoting Ca2+ entry into PC12 cells. The haloperidol-induced increase in AP1 binding activity and intracellular Ca2+ was not reproduced by two other dopamine D2 receptor antagonists, sulpiride and (+)-butaclamol.

5'-(N-ethylcarboxamido)adenosine inhibits Ca2+ influx and activates a protein phosphatase in bovine adrenal chromaffin cells.

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.