Dataset Information

Cocaine self-administration induces divergent protein expression in the nucleus accumbens of male and female mice to eliminate basal sex differences

ABSTRACT: Substance use disorder (SUD) is a chronic neuropsychiatric condition characterized by long-lasting alterations in the neural circuitry regulating reward and motivation. Substantial work has focused on characterizing the molecular substrates which underlie these persistent changes in neural function and behavior; however, this work has overwhelmingly focused on male subjects, despite mounting clinical and preclinical evidence that females demonstrate dissimilar progression to SUD and responsivity to drugs of abuse, such as cocaine. Here, we show that sex is a critical biological variable that defines drug-induced plasticity in the NAc. Using quantitative mass spectrometry, we assessed the protein expression patterns altered by cocaine self-administration and demonstrate unique molecular profiles between males and females. We show that 1. Cocaine self-administration induces non-overlapping protein expression patterns in males and females and 2. Cocaine specifically acts on baseline sexual dimorphisms to exert these effects. Critically, we find that cocaine administration blunts not only basal sex-differences in the accumbens proteome, but also the pre-existing sex differences in behavior for natural rewards. Together, these data suggest that chronic cocaine is capable of rewriting baseline proteomic function to maintain cocaine-specific behaviors.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Striatum, Brain

SUBMITTER: Erin Calipari

LAB HEAD: Erin S. Calipari

PROVIDER: PXD022832 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Cocaine self-administration induces sex-dependent protein expression in the nucleus accumbens.

López Alberto J AJ Johnson Amy R AR Euston Tanner J TJ Wilson Rashaun R Nolan Suzanne O SO Brady Lillian J LJ Thibeault Kimberly C KC Kelly Shannon J SJ Kondev Veronika V Melugin Patrick P Kutlu M Gunes MG Chuang Emily E Lam TuKiet T TT Kiraly Drew D DD Calipari Erin S ES

Communications biology 20210716 1

Substance use disorder (SUD) is a chronic neuropsychiatric condition characterized by long-lasting alterations in the neural circuitry regulating reward and motivation. Substantial work has focused on characterizing the molecular substrates that underlie these persistent changes in neural function and behavior. However, this work has overwhelmingly focused on male subjects, despite mounting clinical and preclinical evidence that females demonstrate dissimilar progression to SUD and responsivity ...[more]

PMID: 34272455

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Project description:Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1-important for synaptic connections during development-as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine. c57bl/6 mice were given IP injections of chronic cocaine 20mg/kg once per day for 7 days and sacrificed 30 minutes after the final dose of cocaine. Control animals were given saline for 7 days and sacrificed 30 minutes after the final dose of saline. Nucleus accumbens (NAc) tissue was collected and then PARP-1 ChIP-seq was performed. Three sequencing replicates were performed on each group.

Project description:Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a novel mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine, and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine. Mice were treated for 7 days with daily injections of cocaine (20 mg/kg) and sacrificed 24 hrs later. Chromatin from bilateral punches of NAc was immunoprecipitated with an antibody against MEF2A as described previously with minor modifications (Renthal et al., 2007). Chromatin was sonicated to an average of ~500 bp and immunoprepitated with antibody against MEF2A (Santa Cruz, sc-313) or an IgG control (Upstate/Millipore). Antibody-bound chromatin was precipitated using Protein A beads from Upstate (06-157), which were washed with low salt, high salt, and LiCl buffers to remove non-specific DNA binding. Eluted chromatin was reverse-crosslinked at 65oC in the presence of proteinase K and EDTA. DNA was purified by chloroform extraction/ethanol precipitation and the enrichment of specific promoters was amplified by ligation-mediated PCR for genome-wide analysis (Sikder et al., 2006). Amplified DNA was then labeled with Cy3 (input-enriched) or Cy5 (MEF2-enriched) and hybridized to Nimblegen (Madison, WI) MM8 mouse promoter arrays. Bilateral nucleus accumbens from eight mice were pooled for microarray analysis.

Project description:Repeated administration of the psychostimulant cocaine has been shown to produce persistent alterations in genome-wide transcriptional regulatory networks, chromatin remodeling activity and, ultimately, gene expression profiles in the brain’s reward circuitry. Virtually all previous investigations have centered on drug-mediated effects occurring throughout active euchromatic regions of the genome, such that very little is known concerning the impact of cocaine exposure on the regulation and maintenance of heterochromatin in adult brain. Here, we report that cocaine administration dramatically and dynamically alters heterochromatic histone H3 lysine 9 trimethylation (H3K9me3) in the nucleus accumbens (NAc), a key brain reward region. Furthermore, we demonstrate that repeated cocaine exposure induces persistent decreases in heterochromatization in this brain region, suggesting a potential role for heterochromatic regulation in the long-term actions of cocaine. To identify precise genomic loci affected by these alterations, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) was performed on NAc. ChIP-Seq analyses confirmed the existence of the H3K9me3 mark mainly within inter-genic regions of the genome and identified specific patterns of cocaine-induced H3K9me3 regulation at repetitive genomic sequences. Cocaine-mediated decreases in H3K9me3 enrichment at specific genomic repeats (e.g., LI repeats) were further confirmed by the increased expression of LINE-1 and IAP retrotransposons in NAc. Such increases likely reflect global patterns of genomic de-stabilization in this brain region following repeated cocaine administration and open the door for future investigations into the epigenetic and genetic basis of drug addiction. Animals received daily i.p. injections of either 'saline' (7 treatments of saline) or 'repeated' cocaine (7 treatments of 20 mg/kg cocaine). 24 hours after the last dose, chromatin immunoprecipitation was performed utilizing previously validated methods. Each experimental condition was performed in either duplicate or triplicate (2 cocaine, 3 saline). Briefly, for each ChIP, bilateral 14-gauge NAc punches (anterior and posterior) were pooled from 5 mice (20 punches). Tissue was lightly fixed to cross-link DNA with associated proteins and the material was further sheared and immunoprecipitated using sheep anti-rabbit magnetic beads conjugated to an antibody that specifically recognizes H3K9me3. Resulting immunoprecipitated DNA and total (input) genomic DNA were prepared for ChIP-sequencing using an Illumina kit according to manufacturer’s instructions. 20 ng of starting material, as determined by PicoGreen concentrations, was used in each case. Briefly, each sample underwent end repair followed by addition of an A base to the 3’ end. Proprietary adapters were then ligated to the ends, followed by size selection on a 3% agarose gel. The range of excision was 175-225 bp. Following DNA clean up, samples were amplified with 21 cycles of PCR. Amplification and size selection were confirmed with a BioAnalyzer. 5 pM concentrations were used to generate clusters for sequencing analysis.

Dataset Information

Cocaine self-administration induces divergent protein expression in the nucleus accumbens of male and female mice to eliminate basal sex differences

Publications

Cocaine self-administration induces sex-dependent protein expression in the nucleus accumbens.

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