pCREB-dependent transcriptional activation is a mediator of molecular neuroadaptations induced by methamphetamine self-administration in the rat dorsal striatum

Neuroplastic changes in the dorsal striatum participate in the transition from casual drug use to habitual and compulsive drug taking. These alterations might also play a critical role in the development of methamphetamine (METH) addiction. Nevertheless, the molecular substrates that underlie habitual METH consumption have yet to be elucidated. Therefore, in the present study, we examined the influence of METH self-administration on the expression of genes and proteins of interest, as potential substrates of METH-induced neuronal plasticity in the dorsal striatum. Rats self-administered METH (0.1 mg/kg/injection, i.v.) during 15 h sessions for 8 d and were euthanized after 2 h, 24 h, or 1 month of abstinence. Compared to yoked saline control, METH self-administration induced increases in the mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and of the synaptic protein, synaptophysin (Syp) at 2 h after cessation of drug exposure. METH self-administration also caused changes in FosB, BDNF and TrkB protein levels, with increases at 2 and 24 h but decreases observed after 1 month of drug abstinence. Importantly, METH exposure caused increases in the levels of H3K4me3 and pCREB after 2 and 24 h of abstinence. Chromatin immunoprecipitation followed by qPCR was used to clarify the role of these proteins in the regulation of gene expression. We found that METH self-administration caused enrichment of pCREB, but not of H3K4me3, on the promoters of c-fos, fosb, Bdnf and Syp at 2 h after drug cessation. These data indicate that METH-induced activation of their transcription is mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that caused alterations in the expression of genes and proteins serving as substrates for addiction-related synaptic plasticity. Animals were trained to self-administer METH for 8 d as described in the paper, dorsal striata were isolated 2 h, 24 h and 1 month after the final self-administration session. RNA extraction, RNA labeling and microarray hybridization were performed. In brief, total RNA was isolated from the samples using RNeasy Mini Kit (QIAGEN, Valencia, CA). RNA concentration and integrity was determined using Agilent BioAnalyzer (Agilent, Santa Clara, CA). Samples were stored at -80ºC. Microarray hybridization was done using RatRef-12 Expression BeadChips arrays (22 523 probes) (Illumina Inc., San Diego, CA). A 600 ng of total RNA from each sample was amplified using Illumina RNA Amplification kit (Ambion, Austin, TX). Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics, Indianapolis, IN). 750 ng of each cRNA sample were hybridized to Illumina arrays at 55°C overnight according to the Gene Expression Protocol for BeadStation (Illumina Inc.). Hybridized cRNA was detected with cyanine3-streptavidin (GE Healthcare, Piscataway, NJ) and quantified using Illumina's BeadStation 500GX scanner.