Biology subjects

Tuscher, J. J.

Publications and source records attributed to Tuscher, J. J..

9 recordsLinked to original sources

bioRxiv2025

Morphine regulates astrocyte transcriptional dynamics in the ventral tegmental area by stimulation of glucocorticoid signaling

Opioids are potent analgesics often prescribed for the treatment of chronic pain, a condition affecting millions worldwide. Although pain states increase vulnerability to opioid use disorders, the neural mechanisms underlying this interaction remain incompletely understood. The ventral tegmental area (VTA) is a key site for opioid actions, and emerging evidence suggests that pain states and opioid experience both induce transcriptional, molecular, and circuit adaptations in the VTA that contribute to motivated behaviors. However, the transcriptional responses of distinct VTA cell types to each of these factors (alone or in combination) have not been identified. Here, we employed single-nucleus RNA sequencing to comprehensively define transcriptional alterations in the rat VTA to acute morphine administration in a chronic inflammatory pain model. We report that morphine induces gene expression changes primarily in glial cells and dopamine neurons, with minimal effects in other neuronal cell types. Surprisingly, VTA astrocytes and oligodendrocytes exhibited the most robust transcriptional responses to opioid exposure, despite lacking detectable opioid receptor expression. Among the most highly regulated glial genes was Fkbp5, which encodes a co-chaperone protein that acts in concert with heat shock proteins to modulate stress responses. Using pharmacological and CRISPR-based approaches in rat glial cells and human astrocytes, we demonstrate that regulation of Fkbp5 is mediated indirectly through glucocorticoid signaling rather than direct opioid receptor activation. These findings reveal that glial cells within reward circuits undergo profound transcriptional reprogramming in response to opioids through indirect, stress-hormone mediated mechanisms, highlighting a previously unappreciated non-neuronal contribution to opioid-induced neural adaptations.

neuroscience↗

bioRxiv2024

Reelin marks cocaine-activated striatal ensembles, promotes neuronal excitability, and regulates cocaine reward

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.

neuroscience↗

bioRxiv2023

Temporally Specific Gene Expression and Chromatin Remodeling Programs Regulate a Conserved Pdyn Enhancer

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 h) enriched for inducible transcription factors and later changes (4 h) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 h, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 h after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuro-psychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

neuroscience↗

bioRxiv2023

Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation (CRISPRa) system in which catalytically-dead Cas9 (dCas9) fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs (sgRNAs), resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

neuroscience↗

bioRxiv2023

Distinct subpopulations of D1 medium spiny neurons exhibit unique transcriptional responsiveness to cocaine

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.

neuroscience↗

bioRxiv2021

An atlas of transcriptionally defined cell populations in the rat ventral tegmental area

The ventral tegmental area (VTA) is a complex brain region that is essential for reward function but is also implicated in neuropsychiatric diseases including substance abuse. While decades of research on VTA function have focused on the role of dopaminergic neurons, recent evidence has identified critical roles for VTA GABAergic and glutamatergic neurons in reward processes as well. Interestingly, molecular characterization has revealed that subsets of these neurons express genes involved in the transport, synthesis, and vesicular packaging of multiple neurotransmitters, providing evidence for co-release neurons. However, these studies have largely relied on low-throughput methods, and the molecular architecture of the VTA has not been comprehensively examined. Here, we performed single nucleus RNA-sequencing (snRNA-seq) on 21,600 VTA cells from male and female Sprague-Dawley rats to generate a transcriptional atlas of the rat VTA. We identified 16 transcriptionally distinct cell types within the VTA, including 7 neuronal populations. Further subclustering revealed several VTA neuronal populations expressing markers for more than one neurotransmitter system, with one cluster exhibiting high expression levels of genes involved in the synthesis and transport of GABA, glutamate, and dopamine. Finally, snRNA-seq enabled the de novo identification of thousands of marker genes for each transcriptionally distinct population, revealing cluster-specific enrichment of gene sets implicated in neuropsychiatric and neurodevelopmental disorders, as well as specific phenotypes associated with alcohol and tobacco use. Together, these results highlight the heterogeneity of cellular populations in the VTA and identify novel markers and disease-linked genes enriched in distinct neuronal subtypes.

neuroscience↗

bioRxiv2020

A Cre-dependent CRISPR/dCas9 activation system for gene expression regulation in neurons

Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customizations for different applications. The simplicity and modularity of CRISPR-based systems have transformed this aspect of genome editing, providing a variety of possible applications and targets. However, there are currently few available tools for cell-selective CRISPR regulation in neurons. Here, we designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction in the absence of Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in both HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate highly inducible genes (GRM2, Tent5b, and Fos) as well as moderately inducible genes (Sstr2 and Gadd45b) in a Cre-specific manner. Furthermore, to illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from of a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and will enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.

neuroscience↗

bioRxiv2019

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged high-throughput single-nucleus RNA-sequencing to create 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 specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.

neuroscience↗

bioRxiv2018

A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation

Recent developments in CRISPR-based gene editing have provided new avenues to interrogate gene function. However, application of these tools in the central nervous system has been delayed due to difficulties in transgene expression in post-mitotic neurons. Here, we present a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system to drive gene expression in primary neuronal cultures and the adult brain of rodent model systems. We demonstrate robust, modular, and tunable induction of endogenous target genes as well as multiplexed gene regulation necessary for investigation of complex transcriptional programs. 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.

neuroscience↗