Project description:Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological, and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we find that G9a repression by cocaine occurs in both Drd1 (striatonigral)- and Drd2 (striatopallidal)-expressing medium spiny neurons (MSNs). Conditional knockout and overexpression of G9a within these distinct cell types, however, reveals divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicate that such developmental deletion of G9a selectively in Drd2 neurons results in the unsilencing of transcriptional programs normally specific to striatonigral neurons, and the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral ‘switching’ phenotype in mice indicates a novel role for G9a in contributing to neuronal subtype identity, and suggests a critical function for cell-type specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli. Polyribosome associated mRNAs from 2-5 month old, age and sex matched Drd1-Cre; Drd1-TRAP; G9afl/fl and Drd1-TRAP; G9afl/fl, Drd2-Cre; Drd2-TRAP; G9afl/fl and Drd2-TRAP; G9afl/fl mice (n = 2-4 mice/genotype/drug treatment, 2 hours after the last of eight repeated cocaine injections of 20 mg/kg/day) were obtained as previously described. EGFP labeled ribosomes and associated mRNAs were immunoprecipitated using a mix of two monoclonal anti-GFP antibodies (50 μg of clones #19C8 and #19F7 for each IP, available at Sloan-Kettering Monoclonal Antibody Facility). Purified mRNA was amplified and processed for microarray and qPCR analysis using the Affymetrix two-cycle cDNA Synthesis kit (Affymetrix) as previously described. Affymetrix Mouse Genome 430 2.0 arrays were used in all experiments. Information regarding the array design and features can be found at www.affymetrix.com. Mouse Genome 430 2.0 arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and globally scaled to 150 using the Affymetrix GeneChip Operating Software (GCOS v1.4).
Project description:RNAseq of FACS Drd1 and Drd2 receptor expressing medium spiny neurons (direct and indirect medium spiny neurons) from 5-6 month Q175 Huntington's disease model mice, compared to WT littermates.
Project description:RNAseq of FACS Drd1 and Drd2 receptor expressing medium spiny neurons (direct and indirect medium spiny neurons) from 5-6 month BACHD Huntington's disease model mice, compared to WT littermates
Project description:Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological, and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we find that G9a repression by cocaine occurs in both Drd1 (striatonigral)- and Drd2 (striatopallidal)-expressing medium spiny neurons (MSNs). Conditional knockout and overexpression of G9a within these distinct cell types, however, reveals divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicate that such developmental deletion of G9a selectively in Drd2 neurons results in the unsilencing of transcriptional programs normally specific to striatonigral neurons, and the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral ‘switching’ phenotype in mice indicates a novel role for G9a in contributing to neuronal subtype identity, and suggests a critical function for cell-type specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli.
Project description:Goal of the experiment: Analysis of gene expression changes in the cortex, striatum, hippocampus, hypothalamus, Drd2-MSNs and Drd1-MSNs of mice with a postnatal, neuron-specific ablation of GLP or G9a as compared to control mice. For microarray analysis, hippocampus, hypothalamus, cortex and striatum of Camk2a-Cre; GLPfl/fl, Camk2a-Cre; G9afl/fl and age (10-14 week old) and sex matched littermate controls were used for total RNA purification. Four biological replicates were performed for each experiment. Polyribosome associated mRNAs from five, age (10-14 week old) and sex matched Drd1-Cre; Drd1-bacTRAP; G9afl/fl, or Drd2-Cre; Drd2-bacTRAP; G9afl/fl and Drd1-bacTRAP; G9afl/fl or Drd2-bacTRAP; G9afl/fl control mice were used. Three biological replicates were performed for each experiment.
Project description:TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in medium spiny neurons in the whole striatum of adult Drd1-EGFP/Rpl10a (CP73) mice that were administered either saline or cocaine.
Project description:TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in medium spiny neurons in the accumbens nucleus of adult Drd1-EGFP/Rpl10a (CP73) mice that were administered either saline or cocaine under long-access conditions