Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons

Abstract

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 ...

Fluorescence‐activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post‐translational modifications

“…NeuN labelling (Fig. B) revealed that non‐neuronal (presumably mostly glial) cells were not more numerous than neurons in the mouse striatum, contrary to a common belief but in accordance with previous studies (Herculano‐Houzel & Lent, ; Matamales & Girault, ; Jordi et al ., ). Actually, in our experiments the proportion of neuronal to non‐neuronal nuclei in the mouse striatum was close to a 1 : 1 ratio with slightly more neuronal nuclei (Fig.…”

Epigenetic Signaling in Psychiatric Disorders

“…The next important challenge will be to understand cell-type specific regulation. Recent findings suggest that cocaine regulation of H3K9me2 and H3K9me3 follows a cell-type specific time course 151 . Interestingly, although cocaine represses G9a expression in both D1 and D2 medium spiny neurons (MSNs) in NAc, cell-type specific manipulation suggest that G9a exerts opposing effects 152 .…”

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

“…An added benefit of the TRAP approach due to the labelling of the ribosomal protein is the presence of strong immunoreactive signal in the nucleolus of target cell types. This enables FANS purification of target nuclei for assessment of DNA, nascent RNA transcription in the specific cell-type of interest, as well as proteomic analysis of histone modifications or transcription factor binding 120,121 .…”