RESUMO
Drugs of abuse activate defined neuronal ensembles in brain reward structures such as the nucleus accumbens (NAc), which are thought to promote the enduring synaptic, circuit, and behavioral consequences of drug exposure. While the molecular and cellular effects arising from experience with drugs like cocaine are increasingly well understood, the mechanisms that sculpt NAc ensemble participation are largely unknown. Here, we leveraged unbiased single-nucleus transcriptional profiling to identify expression of the secreted glycoprotein Reelin (encoded by the Reln gene) as a marker of cocaine-activated neuronal ensembles within the rat NAc. Multiplexed in situ detection confirmed selective expression of the immediate early gene Fos in Reln+ neurons after cocaine experience, and also revealed enrichment of Reln mRNA in Drd1 + medium spiny neurons (MSNs) in both the rat and human brain. Using a novel CRISPR interference strategy enabling selective Reln knockdown in the adult NAc, we observed altered expression of genes linked to calcium signaling, emergence of a transcriptional trajectory consistent with loss of cocaine sensitivity, and a striking decrease in MSN intrinsic excitability. At the behavioral level, loss of Reln prevented cocaine locomotor sensitization, abolished cocaine place preference memory, and decreased cocaine self-administration behavior. Together, these results identify Reelin as a critical mechanistic link between ensemble participation and cocaine-induced behavioral adaptations.
RESUMO
Drugs of abuse increase extracellular concentrations of dopamine in the nucleus accumbens (NAc), resulting in transcriptional alterations that drive long-lasting cellular and behavioral adaptations. While decades of research have focused on the transcriptional mechanisms by which drugs of abuse influence neuronal physiology and function, few studies have comprehensively defined NAc cell type heterogeneity in transcriptional responses to drugs of abuse. Here, we used single nucleus RNA-seq (snRNA-seq) to characterize the transcriptome of over 39,000 NAc cells from male and female adult Sprague-Dawley rats following acute or repeated cocaine experience. This dataset identified 16 transcriptionally distinct cell populations, including two populations of medium spiny neurons (MSNs) that express the Drd1 dopamine receptor (D1-MSNs). Critically, while both populations expressed classic marker genes of D1-MSNs, only one population exhibited a robust transcriptional response to cocaine. Validation of population-selective transcripts using RNA in situ hybridization revealed distinct spatial compartmentalization of these D1-MSN populations within the NAc. Finally, analysis of published NAc snRNA-seq datasets from non-human primates and humans demonstrated conservation of MSN subtypes across rat and higher order mammals, and further highlighted cell type-specific transcriptional differences across the NAc and broader striatum. These results highlight the utility in using snRNA-seq to characterize both cell type heterogeneity and cell type-specific responses to cocaine and provides a useful resource for cross-species comparisons of NAc cell composition.
Assuntos
Cocaína , Masculino , Feminino , Ratos , Animais , Camundongos , Cocaína/farmacologia , Neurônios Espinhosos Médios , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Ratos Sprague-Dawley , Neurônios/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Núcleo Accumbens/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MamíferosRESUMO
Drugs of abuse increase extracellular concentrations of dopamine in the nucleus accumbens (NAc), resulting in transcriptional alterations that drive long-lasting cellular and behavioral adaptations. While decades of research have focused on the transcriptional mechanisms by which drugs of abuse influence neuronal physiology and function, few studies have comprehensively defined NAc cell type heterogeneity in transcriptional responses to drugs of abuse. Here, we used single nucleus RNA-seq (snRNA-seq) to characterize the transcriptome of over 39,000 NAc cells from male and female adult Sprague-Dawley rats following acute or repeated cocaine experience. This dataset identified 16 transcriptionally distinct cell populations, including two populations of medium spiny neurons (MSNs) that express the Drd1 dopamine receptor (D1-MSNs). Critically, while both populations expressed classic marker genes of D1-MSNs, only one population exhibited a robust transcriptional response to cocaine. Validation of population-selective transcripts using RNA in situ hybridization revealed distinct spatial compartmentalization of these D1-MSN populations within the NAc. Finally, analysis of published NAc snRNA-seq datasets from non-human primates and humans demonstrated conservation of MSN subtypes across rat and higher order mammals, and further highlighted cell type-specific transcriptional differences across the NAc and broader striatum. These results highlight the utility in using snRNA-seq to characterize both cell type heterogeneity and cell type-specific responses to cocaine and provides a useful resource for cross-species comparisons of NAc cell composition.
RESUMO
Exposure to drugs of abuse produces robust transcriptional and epigenetic reorganization within brain reward circuits that outlives the direct effects of the drug and may contribute to addiction. DNA methylation is a covalent epigenetic modification that is altered following stimulant exposure and is critical for behavioral and physiological adaptations to drugs of abuse. Although activity-related loss of DNA methylation requires the Gadd45 (Growth arrest and DNA-damage-inducible) gene family, very little is known about how this family regulates activity within the nucleus accumbens or behavioral responses to drugs of abuse. Here, we combined genome-wide transcriptional profiling, pharmacological manipulations, electrophysiological measurements, and CRISPR tools with traditional knockout and behavioral approaches in rodent model systems to dissect the role of Gadd45b in dopamine-dependent epigenetic regulation and cocaine reward. We show that acute cocaine administration induces rapid upregulation of Gadd45b mRNA in the rat nucleus accumbens, and that knockout or site-specific CRISPR/Cas9 gene knockdown of Gadd45b blocks cocaine conditioned place preference. In vitro, dopamine treatment in primary striatal neurons increases Gadd45b mRNA expression through a dopamine receptor type 1 (DRD1)-dependent mechanism. Moreover, shRNA-induced Gadd45b knockdown decreases expression of genes involved in psychostimulant addiction, blocks induction of immediate early genes by DRD1 stimulation, and prevents DRD1-mediated changes in DNA methylation. Finally, we demonstrate that Gadd45b knockdown decreases striatal neuron action potential burst duration in vitro, without altering other electrophysiological characteristics. These results suggest that striatal Gadd45b functions as a dopamine-induced gene that is necessary for cocaine reward memory and DRD1-mediated transcriptional activity.
Assuntos
Cocaína , Animais , Antígenos de Diferenciação , Cocaína/farmacologia , Dopamina , Epigênese Genética , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens , RatosRESUMO
Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. Here, we leveraged single-nucleus RNA-sequencing to generate a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified an immediate early gene expression program that is up-regulated following cocaine experience in vivo and dopamine receptor activation in vitro. Multiplexed induction of this gene program with a large-scale CRISPR-dCas9 activation strategy initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Together, these results define the transcriptional response to cocaine with cellular precision and demonstrate that drug-responsive gene programs can potentiate both physiological and behavioral adaptations to drugs of abuse.
Assuntos
Cocaína , Animais , Cocaína/farmacologia , Dopamina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/metabolismo , Ratos , TranscriptomaRESUMO
CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.