Cross-Platform Synaptic Network Analysis of Human Entorhinal Cortex Identifies TWF2 as a Modulator of Dendritic Spine Length.

Proteomic studies using postmortem human brain tissue samples have yielded robust assessments of the aging and neurodegenerative disease(s) proteomes. While these analyses provide lists of molecular alterations in human conditions, like Alzheimer's disease (AD), identifying individual proteins that affect biological processes remains a challenge. To complicate matters, protein targets may be highly understudied and have limited information on their function. To address these hurdles, we sought to establish a blueprint to aid selection and functional validation of targets from proteomic datasets. A cross-platform pipeline was engineered to focus on synaptic processes in the entorhinal cortex (EC) of human patients, including controls, preclinical AD, and AD cases. Label-free quantification mass spectrometry (MS) data (n = 2260 proteins) was generated on synaptosome fractionated tissue from Brodmann area 28 (BA28; n = 58 samples). In parallel, dendritic spine density and morphology was measured in the same individuals. Weighted gene co-expression network analysis was used to construct a network of protein co-expression modules that were correlated with dendritic spine metrics. Module-trait correlations were used to guide unbiased selection of Twinfilin-2 (TWF2), which was the top hub protein of a module that positively correlated with thin spine length. Using CRISPR-dCas9 activation strategies, we demonstrated that boosting endogenous TWF2 protein levels in primary hippocampal neurons increased thin spine length, thus providing experimental validation for the human network analysis. Collectively, this study describes alterations in dendritic spine density and morphology as well as synaptic proteins and phosphorylated tau from the entorhinal cortex of preclinical and advanced stage AD patients.SIGNIFICANCE STATEMENT Proteomic studies can yield vast lists of molecules that are altered under various experimental or disease conditions. Here, we provide a blueprint to facilitate mechanistic validation of protein targets from human brain proteomic datasets. We conducted a proteomic analysis of human entorhinal cortex (EC) samples spanning cognitively normal and Alzheimer's disease (AD) cases with a comparison of dendritic spine morphology in the same samples. Network integration of proteomics with dendritic spine measurements allowed for unbiased discovery of Twinfilin-2 (TWF2) as a regulator of dendritic spine length. A proof-of-concept experiment in cultured neurons demonstrated that altering Twinfilin-2 protein level induced corresponding changes in dendritic spine length, thus providing experimental validation for the computational framework.

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 system in which catalytically dead Cas9 fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs, 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.

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.

Multigenerational epigenetic inheritance: One step forward, two generations back.

Modifications to DNA and histone proteins serve a critical regulatory role in the developing and adult brain, and over a decade of research has established the importance of these "epigenetic" modifications in a wide variety of brain functions across the lifespan. Epigenetic patterns orchestrate gene expression programs that establish the phenotypic diversity of various cellular classes in the central nervous system, play a key role in experience-dependent gene regulation in the adult brain, and are commonly implicated in neurodevelopmental, psychiatric, and neurodegenerative disease states. In addition to these established roles, emerging evidence indicates that epigenetic information can potentially be transmitted to offspring, giving rise to inter- and trans-generational epigenetic inheritance phenotypes. However, our understanding of the cellular events that participate in this information transfer is incomplete, and the ability of this transfer to overcome complete epigenetic reprogramming during embryonic development is highly controversial. This review explores the existing literature on multigenerational epigenetic mechanisms in the central nervous system. First, we focus on the cellular mechanisms that may perpetuate or counteract this type of information transfer, and consider how epigenetic modification in germline and somatic cells regulate important aspects of cellular and organismal development. Next, we review the potential phenotypes resulting from ancestral experiences that impact gene regulatory modifications, including how these changes may give rise to unique metabolic phenotypes. Finally, we discuss several caveats and technical limitations that influence multigenerational epigenetic effects. We argue that studies reporting multigenerational epigenetic changes impacting the central nervous system must be interpreted with caution, and provide suggestions for how epigenetic information transfer can be mechanistically disentangled from genetic and environmental influences on brain function.

The genome of the vervet (Chlorocebus aethiops sabaeus).

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

Chemogenetic inactivation of the dorsal hippocampus and medial prefrontal cortex, individually and concurrently, impairs object recognition and spatial memory consolidation in female mice.

The dorsal hippocampus (DH) and medial prefrontal cortex (mPFC) are brain regions essential for processing and storing episodic memory. In rodents, the DH has a well-established role in supporting the consolidation of episodic-like memory in tasks such as object recognition and object placement. However, the role of the mPFC in the consolidation of episodic-like memory tasks remains controversial. Therefore, the present study examined involvement of the DH and mPFC, alone and in combination, in object and spatial recognition memory consolidation in ovariectomized female mice. To this end, we utilized two types of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to inactivate the DH alone, the mPFC alone, or both brain regions concurrently immediately after object training to assess the role of each region in the consolidation of object recognition and spatial memories. Our results using single and multiplexed DREADDS suggest that excitatory activity in the DH and mPFC, alone or in combination, is required for the successful consolidation of object recognition and spatial memories. Together, these studies provide critical insight into how the DH and mPFC work in concert to facilitate memory consolidation in female mice.

Estradiol-Mediated Spine Changes in the Dorsal Hippocampus and Medial Prefrontal Cortex of Ovariectomized Female Mice Depend on ERK and mTOR Activation in the Dorsal Hippocampus.

Dendritic spine plasticity underlies the formation and maintenance of memories. Both natural fluctuations and systemic administration of 17ß-estradiol (E2) alter spine density in the dorsal hippocampus (DH) of rodents. DH E2 infusion enhances hippocampal-dependent memory by rapidly activating extracellular signal-regulated kinase (ERK)-dependent signaling of mammalian target of rapamycin (mTOR), a key protein synthesis pathway involved in spine remodeling. Here, we investigated whether infusion of E2 directly into the DH drives spine changes in the DH and other brain regions, and identified cell-signaling pathways that mediate these effects. E2 significantly increased basal and apical spine density on CA1 pyramidal neurons 30 min and 2 h after infusion. DH E2 infusion also significantly increased basal spine density on pyramidal neurons in the medial prefrontal cortex (mPFC) 2 h later, suggesting that E2-mediated activity in the DH drives mPFC spinogenesis. The increase in CA1 and mPFC spine density observed 2 h after intracerebroventricular infusion of E2 was blocked by DH infusion of an ERK or mTOR inhibitor. DH E2 infusion did not affect spine density in the dentate gyrus or ventromedial hypothalamus, suggesting specific effects of E2 on the DH and mPFC. Collectively, these data demonstrate that DH E2 treatment elicits ERK- and mTOR-dependent spinogenesis on CA1 and mPFC pyramidal neurons, effects that may support the memory-enhancing effects of E2. SIGNIFICANCE STATEMENT: Although systemically injected 17ß-estradiol (E2) increases CA1 dendritic spine density, the molecular mechanisms regulating E2-induced spinogenesis in vivo are largely unknown. We found that E2 infused directly into the dorsal hippocampus (DH) increased CA1 spine density 30 min and 2 h later. Surprisingly, DH E2 infusion also increased spine density in the medial prefrontal cortex (mPFC), suggesting that estrogenic regulation of the DH influences mPFC spinogenesis. Moreover, inhibition of ERK and mTOR activation in the DH prevented E2 from increasing DH and mPFC spines, demonstrating that DH ERK and mTOR activation is necessary for E2-induced spinogenesis in the DH and mPFC. These findings provide novel insights into the molecular mechanisms through which E2 mediates dendritic spine density in CA1 and mPFC.

Inhibition of local estrogen synthesis in the hippocampus impairs hippocampal memory consolidation in ovariectomized female mice.

The potent estrogen 17ß-Estradiol (E2) plays a critical role in mediating hippocampal function, yet the precise mechanisms through which E2 enhances hippocampal memory remain unclear. In young adult female rodents, the beneficial effects of E2 on memory are generally attributed to ovarian-synthesized E2. However, E2 is also synthesized in the adult brain in numerous species, where it regulates synaptic plasticity and is synthesized in response to experiences such as exposure to females or conspecific song. Although de novo E2 synthesis has been demonstrated in rodent hippocampal cultures, little is known about the functional role of local E2 synthesis in mediating hippocampal memory function. Therefore, the present study examined the role of hippocampal E2 synthesis in hippocampal memory consolidation. Using bilateral dorsal hippocampal infusions of the aromatase inhibitor letrozole, we first found that blockade of dorsal hippocampal E2 synthesis impaired hippocampal memory consolidation. We next found that elevated levels of E2 in the dorsal hippocampus observed 30min after object training were blocked by dorsal hippocampal infusion of letrozole, suggesting that behavioral experience increases acute and local E2 synthesis. Finally, aromatase inhibition did not prevent exogenous E2 from enhancing hippocampal memory consolidation, indicating that hippocampal E2 synthesis is not necessary for exogenous E2 to enhance hippocampal memory. Combined, these data are consistent with the hypothesis that hippocampally-synthesized E2 is necessary for hippocampus-dependent memory consolidation in rodents.

Sex steroid hormones matter for learning and memory: estrogenic regulation of hippocampal function in male and female rodents.

Ample evidence has demonstrated that sex steroid hormones, such as the potent estrogen 17ß-estradiol (E2), affect hippocampal morphology, plasticity, and memory in male and female rodents. Yet relatively few investigators who work with male subjects consider the effects of these hormones on learning and memory. This review describes the effects of E2 on hippocampal spinogenesis, neurogenesis, physiology, and memory, with particular attention paid to the effects of E2 in male rodents. The estrogen receptors, cell-signaling pathways, and epigenetic processes necessary for E2 to enhance memory in female rodents are also discussed in detail. Finally, practical considerations for working with female rodents are described for those investigators thinking of adding females to their experimental designs.

Canonical Wnt signaling is necessary for object recognition memory consolidation.

Wnt signaling has emerged as a potent regulator of hippocampal synaptic function, although no evidence yet supports a critical role for Wnt signaling in hippocampal memory. Here, we sought to determine whether canonical ß-catenin-dependent Wnt signaling is necessary for hippocampal memory consolidation. Immediately after training in a hippocampal-dependent object recognition task, mice received a dorsal hippocampal (DH) infusion of vehicle or the canonical Wnt antagonist Dickkopf-1 (Dkk-1; 50, 100, or 200 ng/hemisphere). Twenty-four hours later, mice receiving vehicle remembered the familiar object explored during training. However, mice receiving Dkk-1 exhibited no memory for the training object, indicating that object recognition memory consolidation is dependent on canonical Wnt signaling. To determine how Dkk-1 affects canonical Wnt signaling, mice were infused with vehicle or 50 ng/hemisphere Dkk-1 and protein levels of Wnt-related proteins (Dkk-1, GSK3ß, ß-catenin, TCF1, LEF1, Cyclin D1, c-myc, Wnt7a, Wnt1, and PSD95) were measured in the dorsal hippocampus 5 min or 4 h later. Dkk-1 produced a rapid increase in Dkk-1 protein levels and a decrease in phosphorylated GSK3ß levels, followed by a decrease in ß-catenin, TCF1, LEF1, Cyclin D1, c-myc, Wnt7a, and PSD95 protein levels 4 h later. These data suggest that alterations in Wnt/GSK3ß/ß-catenin signaling may underlie the memory impairments induced by Dkk-1. In a subsequent experiment, object training alone rapidly increased DH GSK3ß phosphorylation and levels of ß-catenin and Cyclin D1. These data suggest that canonical Wnt signaling is regulated by object learning and is necessary for hippocampal memory consolidation.

17ß-estradiol is necessary for extinction of cocaine seeking in female rats.

Human and preclinical models of addiction demonstrate that gonadal hormones modulate acquisition of drug seeking. Little is known, however, about the effects of these hormones on extinction of drug-seeking behavior. Here, we investigated how 17ß-estradiol (E2) affects expression and extinction of cocaine seeking in female rats. Using a conditioned place preference (CPP) paradigm, ovariectomized rats were maintained throughout conditioning with 2 d of E2 treatment followed by 2 d of vehicle treatment, or were injected with E2 daily. Hormone injections were paired or explicitly unpaired with place conditioning sessions. Expression of a cocaine CPP was of equal magnitude regardless of conditioning protocol, suggesting that E2 levels during conditioning did not affect subsequent CPP expression. During extinction, daily E2 administration initially enhanced expression of the cocaine CPP, but resulted in significantly faster extinction compared to controls. Whereas E2-treated rats were extinguished within 8 d, vehicle-treated rats maintained CPP expression for more than a month, indicative of perseveration. To determine whether E2 could rescue extinction in these rats, half were given daily E2 treatment and half were given vehicle. E2-treated rats showed rapid extinction, whereas vehicle-treated rats continued to perseverate. These data demonstrate for the first time that E2 is necessary for extinction of cocaine seeking in female rats, and that it promotes rapid extinction when administered daily. Clinically, these findings suggest that monitoring and maintaining optimal E2 levels during exposure therapy would facilitate therapeutic interventions for female cocaine addicts.

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.

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.

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.

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.

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.

Trafficking, persistence, and activation state of adoptively transferred allogeneic and autologous Simian Immunodeficiency Virus-specific CD8(+) T cell clones during acute and chronic infection of rhesus macaques.

Despite multiple lines of evidence suggesting their involvement, the precise role of CD8(+) T cells in controlling HIV replication remains unclear. To determine whether CD8(+) T cells can limit retroviral replication in the absence of other immune responses, we transferred 1-13 x 10(9) allogeneic in vitro expanded SIV-specific CD8(+) T cell clones matched for the relevant restricting MHC-I allele into rhesus macaques near the time of i.v. SIV challenge. Additionally, in vitro expanded autologous SIV-specific CD8(+) T cell clones were infused 4-9 mo postinfection. Infused cells did not appreciably impact acute or chronic viral replication. The partially MHC-matched allogeneic cells were not detected in the blood or most tissues after 3 d but persisted longer in the lungs as assessed by bronchoalveolar lavage (BAL). Autologous cells transferred i.v. or i.p. were found in BAL and blood samples for up to 8 wk postinfusion. Interestingly, despite having a nominally activated phenotype (CD69(+)HLA-DR(+)), many of these cells persisted in the BAL without dividing. This suggests that expression of such markers by T cells at mucosal sites may not reflect recent activation, but may instead identify stable resident memory T cells. The lack of impact following transfer of such a large number of functional Ag-specific CD8(+) T cells on SIV replication may reflect the magnitude of the immune response required to contain the virus.

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.

A Cre-Dependent CRISPR/dCas9 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 customization for different applications. The simplicity and modularity of clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have transformed genome editing and expanded the gene regulatory toolbox. However, there are few available tools for cell-selective CRISPR regulation in neurons. 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 even without 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 HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate numerous rat or human genes (GRM2, Tent5b, Fos, Sstr2, and Gadd45b) in a Cre-specific manner. To illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from 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 enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.

Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media.

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.