The lateral septum (LS) is a midline, subcortical structure that is a critical regulator of social behaviors. Mouse studies have identified molecularly distinct neuronal populations within the LS, which control specific facets of social behavior. Despite its known molecular heterogeneity in the mouse and critical role in regulating social behavior, comprehensive molecular profiling of the human LS has not been performed. Here, we conducted single-nucleus RNA sequencing (snRNA-seq) to generate transcriptomic profiles of the human LS and compared human LS profiles to recently collected mouse LS snRNA-seq datasets. Our analyses identified TRPC4 as a conserved molecular marker of the mouse and human LS, while FREM2 is enriched only in the human LS. We also identify a distinct neuronal cell type marked by OPRM1, the gene encoding the μ-opioid receptor. Together, these results highlight transcriptional heterogeneity of the human LS and identify robust marker genes for the human LS.

Drugs of abuse activate defined neuronal populations in reward structures such as the nucleus accumbens (NAc), which 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, mechanisms that dictate NAc neuronal recruitment remain unknown. Here, we leveraged unbiased single-nucleus transcriptional profiling and targeted in situ detection to identify Reln (encoding the secreted glycoprotein, Reelin) as a marker of cocaine-activated neuronal populations within the rat NAc. A CRISPR interference approach enabling selective Reln knockdown in the adult NAc altered expression of calcium signaling genes, promoted a transcriptional trajectory consistent with loss of cocaine sensitivity, and decreased MSN excitability. Behaviorally, Reln knockdown prevented cocaine locomotor sensitization, abolished cocaine place preference memory, and decreased cocaine self-administration behavior. These results identify Reelin as a critical mechanistic link between neuronal activation and cocaine-induced behavioral adaptations.

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.

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.

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.

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.

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.

Abstract 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.

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.

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.

Abstract Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer–gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer–gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

Blue wavelength light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue wavelength light in cultured primary rat cortical cells. Exposure to blue light (470 nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1 , Cry1 , Cry2, Clock, or Per2. IEG expression was increased following 4 h of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Induction of IEGs by blue light was maintained in cortical cultures treated with AraC to block glial proliferation, indicating that induction occurred selectively in postmitotic neurons. Importantly, changes in gene expression induced by blue wavelength light were prevented when cultures were maintained in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.

BACKGROUND:Gene annotations, such as those in GENCODE, are derived primarily from alignments of spliced cDNA sequences and protein sequences. The impact of RNA-seq data on annotation has been confined to major projects like ENCODE and Illumina Body Map 2.0.RESULTS:We aligned 21,504 Illumina-sequenced human RNA-seq samples from the Sequence Read Archive (SRA) to the human genome and compared detected exon-exon junctions with junctions in several recent gene annotations. We found 56,861 junctions (18.6%) in at least 1000 samples that were not annotated, and their expression associated with tissue type. Junctions well expressed in individual samples tended to be annotated. Newer samples contributed few novel well-supported junctions, with the vast majority of detected junctions present in samples before 2013. We compiled junction data into a resource called intropolis available at http://intropolis.rail.bio . We used this resource to search for a recently validated isoform of the ALK gene and characterized the potential functional implications of unannotated junctions with publicly available TRAP-seq data.CONCLUSIONS:Considering only the variation contained in annotation may suffice if an investigator is interested only in well-expressed transcript isoforms. However, genes that are not generally well expressed and nonetheless present in a small but significant number of samples in the SRA are likelier to be incompletely annotated. The rate at which evidence for novel junctions has been added to the SRA has tapered dramatically, even to the point of an asymptote. Now is perhaps an appropriate time to update incomplete annotations to include splicing present in the now-stable snapshot provided by the SRA.

We aligned 21,504 publicly available Illumina-sequenced human RNA-seq samples from the Sequence Read Archive (SRA) to the human genome and compared detected exon-exon junctions with junctions in several recent gene annotations. 56,865 junctions (18.6%) found in at least 1,000 samples were not annotated, and their expression associated with tissue type. Newer samples contributed few novel well-supported junctions, with 96.1% of junctions detected in at least 20 reads across samples present in samples before 2013. Junction data is compiled into a resource called intropolis available at http://intropolis.rail.bio. We discuss an application of this resource to cancer involving a recently validated isoform of the ALK gene.

All exon-exon junctions detected by Rail-RNA across 21,504 human RNA-seq samples from the Sequence Read Archive, as studied in the paper "Human splicing diversity across the Sequence Read Archive" by Abhinav Nellore, Andrew E. Jaffe, Jean-Philippe Fortin, José Alquicira-Hernández, Leonardo Collado-Torres, Siruo Wang, Robert A. Phillips III, Nishika Karbhari, Kasper D. Hansen, Ben Langmead, and Jeffrey T. Leek. File formats are described at http://intropolis.rail.bio .