Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R-or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R-and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.addiction | psychostimulant drugs | chromatin | nucleus | fluorescence-activated cell sorting I n both humans and rodents, repeated self-administration of addictive substances can lead to a state of addiction characterized by a loss of control over drug use, with compulsive selfadministration despite its severe negative consequences (1). The addictive state is persistent, with drug craving and relapse occurring even after decades of abstinence. All addictive drugs share the ability to increase extracellular dopamine (DA) levels in the ventral striatum (2) and are thought to exert their longlasting effects by "hijacking" the normal reward systems, including the mesolimbic-striatal DA pathway and the nucleus accumbens (NAc) (3-5).A single exposure to psychostimulants triggers long-lasting alterations in behavior and synaptic physiology (6), revealing their powerful control of neuronal plasticity. Repeated exposures induce further changes that contribute to the occurrence of addiction (7-10). Growing evidence suggests that long-lasting changes in the properties of striatal neurons involve epigenetic processes (11). In particular, histone posttranslational modifications (PTMs), including phosphorylation, acetylation, and methylation play a role in promoting transcriptional alterations following cocaine administration ...
Neuronal DNA modifications differ from those in other cells, including methylation outside CpG context and abundant 5-hydroxymethylation whose relevance for neuronal identities are unclear. Striatal projection neurons expressing D1 or D2 dopamine receptors allow addressing this question, as they share many characteristics but differ in their gene expression profiles, connections, and functional roles. We compare translating mRNAs and DNA modifications in these two populations. DNA methylation differences occur predominantly in large genomic clusters including differentially expressed genes, potentially important for D1 and D2 neurons. Decreased gene body methylation is associated with higher gene expression. Hydroxymethylation differences are more scattered and affect transcription factor binding sites, which can influence gene expression. We also find a strong genome-wide hydroxymethylation asymmetry between the two DNA strands, particularly pronounced at expressed genes and retrotransposons. These results identify novel properties of neuronal DNA modifications and unveil epigenetic characteristics of striatal projection neurons heterogeneity.
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