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.

Transcriptomic characterization of human lateral septum neurons reveals conserved and divergent marker genes across species.

The lateral septum (LS) is a midline, subcortical structure, which regulates social behaviors that are frequently impaired in neurodevelopmental disorders including schizophrenia and autism spectrum disorder. Mouse studies have identified neuronal populations within the LS that express a variety of molecular markers, including vasopressin receptor, oxytocin receptor, and corticotropin releasing hormone receptor, that control specific facets of social behavior. Despite its critical role in the regulation of social behavior and notable gene expression patterns, comprehensive molecular profiling of the human LS has not been performed. Here, we conducted single nucleus RNA-sequencing (snRNA-seq) to generate the first transcriptomic profiles of the human LS using postmortem human brain tissue samples from 3 neurotypical donors. Our analysis identified 4 transcriptionally distinct neuronal cell types within the human LS that are enriched for TRPC4, the gene encoding Trp-related protein 4. Differential expression analysis revealed a distinct LS neuronal cell type that is enriched for OPRM1, the gene encoding the µ-opioid receptor. Leveraging recently collected mouse LS snRNA-seq datasets, we also conducted a cross-species analysis. Our results demonstrate that TRPC4 enrichment in the LS is highly conserved between human and mouse, while FREM2, which encodes FRAS1 related extracellular matrix protein 2, is enriched only in the human LS. Together, these results highlight transcriptional heterogeneity of the human LS, and identify robust marker genes for the human LS.

TrkB-dependent regulation of molecular signaling across septal cell types.

The lateral septum (LS), a GABAergic structure located in the basal forebrain, is implicated in social behavior, learning, and memory. We previously demonstrated that expression of tropomyosin kinase receptor B (TrkB) in LS neurons is required for social novelty recognition. To better understand molecular mechanisms by which TrkB signaling controls behavior, we locally knocked down TrkB in LS and used bulk RNA-sequencing to identify changes in gene expression downstream of TrkB. TrkB knockdown induces upregulation of genes associated with inflammation and immune responses, and downregulation of genes associated with synaptic signaling and plasticity. Next, we generated one of the first atlases of molecular profiles for LS cell types using single nucleus RNA-sequencing (snRNA-seq). We identified markers for the septum broadly, and the LS specifically, as well as for all neuronal cell types. We then investigated whether the differentially expressed genes (DEGs) induced by TrkB knockdown map to specific LS cell types. Enrichment testing identified that downregulated DEGs are broadly expressed across neuronal clusters. Enrichment analyses of these DEGs demonstrated that downregulated genes are uniquely expressed in the LS, and associated with either synaptic plasticity or neurodevelopmental disorders. Upregulated genes are enriched in LS microglia, associated with immune response and inflammation, and linked to both neurodegenerative disease and neuropsychiatric disorders. In addition, many of these genes are implicated in regulating social behaviors. In summary, the findings implicate TrkB signaling in the LS as a critical regulator of gene networks associated with psychiatric disorders that display social deficits, including schizophrenia and autism, and with neurodegenerative diseases, including Alzheimer's.

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 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr 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 neuropsychiatric 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.

Chst9 Marks a Spatially and Transcriptionally Unique Population of Oprm1 -Expressing Neurons in the Nucleus Accumbens.

Opioids produce addictive, analgesic, and euphoric effects via actions at mu opioid receptors (µORs). The µOR is encoded by the Oprm1 gene and is expressed in multiple brain regions that regulate reward and motivation, such as the nucleus accumbens (NAc). Oprm1 expression in NAc medium spiny neurons (MSNs) mediates opioid place preference, seeking, and consumption. However, recent single nucleus RNA sequencing (snRNA-seq) studies in rodent, primate, and human NAc have revealed that multiple subpopulations of NAc neurons express Oprm1 mRNA, making it unclear which populations mediate diverse behaviors resulting from µOR activation. Using published snRNA-seq datasets from the rat NAc, we identified a novel population of MSNs that express the highest levels of Oprm1 of any NAc cell type. Here, we show that this population is selectively marked by expression of Chst9 , a gene encoding a carbohydrate sulfotransferase. To validate this observation and characterize spatial localization of this population in the rat NAc, we performed multiplexed RNAscope fluorescence in situ hybridization studies to detect expression of Oprm1 and Chst9 mRNA along with well-validated markers of MSNs. Notably, Chst9 + neurons exhibited more abundant expression of Oprm1 as compared to other cell types, and formed discrete cellular clusters along the medial and ventral borders of the NAc shell subregion. Moreover, CHST9 mRNA was also found to mark specific MSN populations in published human and primate snRNA-seq studies, indicating that this unique population may be conserved across species. Together, these results identify a spatially and transcriptionally distinct NAc neuron population characterized by the expression of Chst9 . The abundant expression of Oprm1 in this population and the conservation of these cells across species suggests that they may play a key functional role in opioid response and identify this subpopulation as a target for further investigation.

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 neuropsychiatric 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.

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.

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.

Morphological and Molecular Identification of Culicoides (Diptera: Ceratopogonidae) Species of the Southern California Desert.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are vectors of important animal pathogens including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). While some Culicoides species present in the southern California desert are implicated in the transmission of these viruses to ruminant animals, these species have not been extensively studied due in part to the challenge of identifying Culicoides to species and to the lack of published gene sequences for these species to support their molecular identification. In this study, Culicoides were captured using suction traps baited with either carbon dioxide or UV light from transitional habitat between the southern California peninsular mountain ranges and the Colorado desert of southeastern California. Captured midges were initially identified using traditional morphological methods, with species identification subsequently confirmed by sequence analysis of COI and 28S rDNA genes. Phylogenetic analyses support that some Culicoides subgenera are not monophyletic. Two recognized species (C. sitiens Wirth and Hubert and C. bakeri Vargas) shared the same COI and 28S sequences. An additional cryptic species may be present within C. sitiens. Two additional recognized species (C. cacticola Wirth and Hubert and C. torridus Wirth and Hubert) may be conspecific or cryptic to each other. In total, 19 Culicoides species (or species aggregate) were collected in this study, with genetic sequences published for the first time for 16 of them. Published genetic sequences will support future research on these species, including studies on the ecology and habits of their immature stages which are often tedious to identify using morphology.

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 and frequently implicated in neuropsychiatric disease. While decades of research on VTA function have focused on dopamine neurons, recent evidence has identified critical roles for GABAergic and glutamatergic neurons in reward processes. Additionally, although subsets of VTA neurons express genes involved in the synthesis and transport of multiple neurotransmitters, characterization of these combinatorial populations has largely relied on low-throughput methods. To comprehensively define the molecular architecture of the VTA, we performed single-nucleus RNA sequencing on 21,600 cells from the rat VTA. Analysis of neuronal subclusters identifies selective markers for dopamine and combinatorial neurons, reveals expression profiles for receptors targeted by drugs of abuse, and demonstrates population-specific enrichment of gene sets linked to brain disorders. These results highlight the heterogeneity of the VTA and provide a resource for further exploration of VTA gene expression.

Comparison of LACE and HOSPITAL Readmission Risk Scores for CMS Target and Nontarget Conditions.

This study evaluated the utility and performance of the LACE index and HOSPITAL score with consideration of the type of diagnoses and assessed the accuracy of these models for predicting readmission risks in patient cohorts from 2 large academic medical centers. Admissions to 2 hospitals from 2011 to 2015, derived from the Vizient Clinical Data Base and regional health information exchange, were included in this study (291 886 encounters). Models were assessed using Bayesian information criterion and area under the receiver operating characteristic curve. They were compared in CMS diagnosis-based cohorts and in 2 non-CMS cancer diagnosis-based cohorts. Overall, both models for readmission risk performed well, with LACE performing slightly better (area under the receiver operating characteristic curve 0.73 versus 0.69; P ≤ 0.001). HOSPITAL consistently outperformed LACE among 4 CMS target diagnoses, lung cancer, and colon cancer. Both LACE and HOSPITAL predict readmission risks well in the overall population, but performance varies by salient, diagnosis-based risk factors.

Assessing Patient Needs During Natural Disasters: Mixed Methods Analysis of Portal Messages Sent During Hurricane Harvey.

BACKGROUND: Patient portals play an important role in connecting patients with their medical care team, which improves patient engagement in treatment plans, decreases unnecessary visits, and reduces costs. During natural disasters, patients' needs increase, whereas available resources, specifically access to care, become limited. OBJECTIVE: This study aims to examine patients' health needs during a natural crisis by analyzing the electronic messages sent during Hurricane Harvey to guide future disaster planning efforts. METHODS: We explored patient portal use data from a large Greater Houston area health care system focusing on the initial week of the Hurricane Harvey disaster, beginning with the date of landfall, August 25, 2017, to August 31, 2017. A mixed methods approach was used to assess patients' immediate health needs and concerns during the disruption of access to routine and emergent medical care. Quantitative analysis used logistic regression models to assess the predictive characteristics of patients using the portal during Hurricane Harvey. This study also included encounters by type (emergency, inpatient, observation, outpatient, and outpatient surgery) and time (before, during, and after Hurricane Harvey). For qualitative analysis, the content of these messages was examined using the constant comparative method to identify emerging themes found within the message texts. RESULTS: Out of a total of 557,024 patients, 4079 (0.73%) sent a message during Hurricane Harvey, whereas 31,737 (5.69%) used the portal. Age, sex, race, and ethnicity were predictive factors for using the portal and sending a message during the natural disaster. We found that prior use of the patient portal increased the likelihood of portal use during Hurricane Harvey (odds ratio 13.688, 95% CI 12.929-14.491) and of sending a portal message during the disaster (odds ratio 14.172, 95% CI 11.879-16.907). Having an encounter 4 weeks before or after Hurricane Harvey was positively associated with increased use of the portal and sending a portal message. Patients with encounters during the main Hurricane Harvey week had a higher increased likelihood of portal use across all five encounter types. Qualitative themes included: access, prescription requests, medical advice (chronic conditions, acute care, urgent needs, and Hurricane Harvey-related injuries), mental health, technical difficulties, and provider constraints. CONCLUSIONS: The patient portal can be a useful tool for communication between patients and providers to address the urgent needs and concerns of patients as a natural disaster unfolds. This was the first known study to include encounter data to understand portal use compared with care provisioning. Prior use was predictive of both portal use and message sending during Hurricane Harvey. These findings could inform the types of demands that may arise in future disaster situations and can serve as the first step in intentionally optimizing patient portal usability for emergency health care management during natural disasters.

Disparities in COVID-19 hospitalizations and mortality among black and Hispanic patients: cross-sectional analysis from the greater Houston metropolitan area.

BACKGROUND: Disparate racial/ethnic burdens of the Coronavirus Disease 2019 (COVID-19) pandemic may be attributable to higher susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) or to factors such as differences in hospitalization and care provision. METHODS: In our cross-sectional analysis of lab-confirmed COVID-19 cases from a tertiary, eight-hospital healthcare system across greater Houston, multivariable logistic regression models were fitted to evaluate hospitalization and mortality odds for non-Hispanic Blacks (NHBs) vs. non-Hispanic Whites (NHWs) and Hispanics vs. non-Hispanics. RESULTS: Between March 3rd and July 18th, 2020, 70,496 individuals were tested for SARS-CoV-2; 12,084 (17.1%) tested positive, of whom 3536 (29.3%) were hospitalized. Among positive cases, NHBs and Hispanics were significantly younger than NHWs and Hispanics, respectively (mean age NHBs vs. NHWs: 46.0 vs. 51.7 years; p < 0.001 and Hispanic vs. non-Hispanic: 44.0 vs. 48.7 years; p < 0.001). Despite younger age, NHBs (vs. NHWs) had a higher prevalence of diabetes (25.2% vs. 17.6%; p < 0.001), hypertension (47.7% vs. 43.1%; p < 0.001), and chronic kidney disease (5.0% vs. 3.3%; p = 0.001). Both minority groups resided in lower median income (median income [USD]; NHBs vs. NHWs: 63,489 vs. 75,793; p < 0.001, Hispanic vs. non-Hispanic: 59,104 vs. 68,318; p < 0.001) and higher population density areas (median population density [per square mile]; NHBs vs. NHWs: 3257 vs. 2742; p < 0.001, Hispanic vs. non-Hispanic: 3381 vs. 2884; p < 0.001). In fully adjusted models, NHBs (vs. NHWs) and Hispanics (vs. non-Hispanic) had higher likelihoods of hospitalization, aOR (95% CI): 1.42 (1.24-1.63) and 1.61 (1.46-1.78), respectively. No differences were observed in intensive care unit (ICU) utilization or treatment parameters. Models adjusted for demographics, vital signs, laboratory parameters, hospital complications, and ICU admission vital signs demonstrated non-significantly lower likelihoods of in-hospital mortality among NHBs and Hispanic patients, aOR (95% CI): 0.65 (0.40-1.03) and 0.89 (0.59-1.31), respectively. CONCLUSIONS: Our data did not demonstrate racial and ethnic differences in care provision and hospital outcomes. Higher susceptibility of racial and ethnic minorities to SARS-CoV-2 and subsequent hospitalization may be driven primarily by social determinants.

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Analysis of the Implementation of Telehealth Visits for Care of Patients With Cancer in Houston During the COVID-19 Pandemic.

PURPOSE: The purpose of this study was to evaluate the use of telemedicine amid the SARS-CoV-2 pandemic in patients with cancer and assess barriers to its implementation. PATIENTS AND METHODS: Telehealth video visits, using the Houston Methodist MyChart platform, were offered to patients with cancer as an alternative to in-person visits. Reasons given by patients who declined to use video visits were documented, and demographic information was collected from all patients. Surveys were used to assess the levels of satisfaction of treating physicians and patients who agreed to video visits. RESULTS: Of 1,762 patients with cancer who were offered telehealth video visits, 1,477 (83.8%) participated. The patients who declined participation were older (67.7 v 60.2 years; P < .0001), lived in significantly lower-income areas (P = .0021), and were less likely to have commercial insurance (P < .0001) than patients who participated. Most participating patients (92.6%) were satisfied with telehealth video visits. A majority of physicians (65.2%) were also satisfied with its use, and 74% indicated that they would likely use telemedicine in the future. Primary concerns that physicians had in using this technology were inadequate patient interactions and acquisition of medical data, increased potential for missing significant clinical findings, decreased quality of care, and potential medical liability. CONCLUSION: Oncology/hematology patients and their physicians expressed high levels of satisfaction with the use of telehealth video visits. Despite recent advances in technology, there are still opportunities to improve the equal implementation of telemedicine for the medical care of vulnerable older, low-income, and underinsured patient populations.

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

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.

An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.