Biology subjects

Phillips, R. A.

Publications and source records attributed to Phillips, R. A..

17 recordsLinked to original sources

bioRxiv2026

Three dimensional reconstruction of the human nucleus accumbens reveals topographic organization and molecular heterogeneity of D1-islands across the anterior posterior axis

Spatial transcriptomics has transformed molecular characterization of the human brain, but most studies profile individual two-dimensional tissue sections and therefore cannot capture molecular organization across 3D neuroanatomical axes. Here, we developed a spatial genomics framework to reconstruct the 3D cellular architecture of the human nucleus accumbens (NAc) by densely sampling and aligning serial sections across its anterior-posterior (AP) extent. Combining single-cell Xenium profiling with near-transcriptome-wide VisiumHD, we mapped cell type composition and topography across AP, mediolateral and dorsoventral axes. Neuronal populations varied in relative abundance across the AP axis while maintaining characteristic spatial topographies. These differences were particularly pronounced among D1-islands, a topographically organized structure within the NAc in which transcriptionally distinct subpopulations of DRD1-expressing medium spiny neurons (MSNs) aggregate into discrete cellular formations. D1-islands comprised two molecularly distinct spatial domains with conserved counterparts across species. Both domains varied in relative abundance across the AP axis, but demonstrated distinct 3D distribution; one was present throughout the NAc, whereas the other, corresponding to islands of Calleja (ICj), was more restricted to intermediate-to-posterior levels. Local spatial analyses revealed region- and AP-dependent cellular relationships, including segregation of the two island populations despite their anatomical proximity. Together, these findings establish a framework for three dimensional spatial genomics of human brain tissue and reveal molecular and cellular organization not captured by individual tissue sections.

neuroscience↗

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↗

bioRxiv2025

Spatiomolecular mapping reveals anatomical organization of heterogeneous cell types in the human nucleus accumbens

The nucleus accumbens (NAc) is a key component of the mesolimbic dopamine system that critically regulates many behaviors related to reward and motivation. The NAc is implicated in several neuropsychiatric disorders, including major depressive disorder, schizophrenia, and substance use disorders. Rodent studies have identified spatial organization of heterogeneous medium spiny neuron (MSN) subtypes across the NAc core and shell, but the extent to which this cellular diversity and spatial organization is conserved in the human brain remains unclear. Here, we generated a spatiomolecular atlas of NAc cell types and spatial domains by integrating spatial transcriptomics and single-nucleus RNA sequencing data from postmortem NAc tissue from 10 neurotypical adult donors. We identified 20 transcriptionally unique cell populations and 8 spatial domains, including specialized D1 islands composed of distinct dopamine receptor 1 (DRD1) MSN subtypes, which were enriched for OPRM1. In contrast to a discrete core vs. shell division, we observed continuous spatial gradients of gene expression across MSN domains, suggesting a more complex organization of DRD1 and DRD2 MSNs. Cross-species comparisons demonstrated conservation of MSN subtypes and spatial features between human, rodent, and nonhuman primate NAc. Genetic enrichment analysis with stratified linkage disequilibrium score regression revealed specific spatial domains associated with risk for psychiatric and addiction-related traits. To investigate this further, we spatially mapped ligand-receptor interactions involving neuropsychiatric risk genes. Finally, we leveraged existing rodent NAc data to identify drug-responsive transcriptional programs and predict their spatial distribution in the human NAc. Collectively, we provide a spatiomolecular framework for understanding the human NAc and its relevance to neuropsychiatric disease.

neuroscience↗

bioRxiv2025

Variability in foraging ranges of snow petrels and implications for breeding distribution and use of stomach-oil deposits as proxies for paleoclimate

BackgroundBreeding pelagic seabirds feed over wide areas. Their diet and space use can therefore provide proxies for otherwise be difficult to observe environmental conditions but interpretation of these proxies requires knowledge of foraging range. Antarctic sea ice regulates global climate, but a paucity of data on its past extent causes uncertainty in climate reconstruction and prediction. Deposits of stomach oils, produced defensively by snow petrels Pagodroma nivea (near-obligate sea-ice foragers), reflect sea ice conditions, sometimes over tens of millennia, but spatial interpretation of these biological archives is hampered by lack of data on foraging range, and how this varies seasonally, among colonies and with sex. MethodsWe tracked 34 and 60 snow petrels using light-based geolocators and GPS loggers, respectively to estimate foraging ranges at three colonies located 180-200 km inland in Dronning Maud Land. We tested whether foraging latitude is associated with ice edge latitude, estimated via satellite remote sensing. We then projected potential foraging ranges for all known colonies on the study area to reexamine assumptions made in paleoclimate studies. ResultsDuring most breeding stages, and across breeding seasons, core home ranges were centred approximately 2{degrees} south of the outer sea-ice edge and tracked this habitat as it receded during the spring melt. Females were approximately 7% lighter than males but foraged at similar distances and in similar areas. Foraging ranges differed little between colonies but substantially between breeding stages. For example, median range was [~]1,400 km during the pre-laying exodus vs. [~]550 km during brood-guard. ConclusionsSnow petrel stomach-oil deposits potentially integrate environmental conditions over greater and more seasonally variable areas than previously assumed, probably with a bias towards conditions in the outer Marginal Ice Zone during the early summer when stomach oil deposition due to nest competition is likely greatest. Projected potential home ranges of snow petrel colonies in the western Weddell Sea suggest that breeding is limited by access to foraging habitat, such as coastal polynyas. We hypothesise that as sea ice fluctuated over previous glacial-interglacial cycles, this also regulated breeding distribution across the region.

ecology↗

bioRxiv2024

Accurate sample deconvolution of pooled snRNA-seq using sex-dependent gene expression patterns

Single-nucleus RNA sequencing (snRNA-seq) technology offers unprecedented resolution for studying cell type-specific gene expression patterns. However, snRNA-seq poses high costs and technical limitations, often requiring the pooling of independent biological samples and the loss of individual sample-level data. Deconvolution of sample identity using inherent features would enable the incorporation of pooled barcoding and sequencing protocols, thereby increasing data throughput and analytical sample size without requiring increases in experimental sample size and sequencing costs. In this study, we demonstrate a proof of concept that sex-dependent gene expression patterns can be leveraged for the deconvolution of pooled snRNA-seq data. Using previously published snRNA-seq data from the rat ventral tegmental area, we trained a range of machine learning models to classify cell sex using genes differentially expressed in cells from male and female rats. Models that used sex-dependent gene expression predicted cell sex with high accuracy (93-95%) and outperformed simple classification models using only sex chromosome gene expression (88-90%). The generalizability of these models to other brain regions was assessed using an additional published data set from the rat nucleus accumbens. Within this data set, model performance remained highly accurate in cell sex classification (90-92% accuracy) with no additional training. This work provides a model for future snRNA-seq studies to perform sample deconvolution using a two-sex pooled sample sequencing design and benchmarks the performance of various machine learning approaches to deconvolve sample identification from inherent sample features.

genomics↗

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↗

bioRxiv2024

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, which control specific facets of social behavior. Despite its critical role in regulating 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 5 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 {micro}-opioid receptor. Leveraging recently generated mouse LS snRNA-seq datasets, we 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.

neuroscience↗

bioRxiv2023

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 (ORs). The OR 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 OR 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.

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

Ocean-scale variation in migration schedules of a long-distance migratory seabird is fully compensated upon return to the breeding site

BackgroundMigratory birds generally have tightly scheduled annual cycles, in which delays can have carry-over effects on the timing of later events, ultimately impacting reproductive output. Whether temporal carry-over effects are more pronounced among migrations over larger distances, with tighter schedules, is a largely unexplored question. MethodsWe tracked individual Arctic Skuas Stercorarius parasiticus, a long-distance migratory seabird, from eight breeding populations between Greenland and Siberia using light-level geolocators. We tested whether migration schedules among breeding populations differ as a function of their use of seven widely divergent wintering areas across the Atlantic Ocean, Mediterranean Sea and Indian Ocean. ResultsBreeding at higher latitudes led not only to later reproduction and migration, but also faster spring migration and shorter time between return to the breeding area and clutch initiation. Wintering area was consistent within individuals among years; and more distant areas were associated with more time spent on migration and less time in the wintering areas. Skuas adjusted the period spent in the wintering area, regardless of migration distance, which buffered the variation in timing of autumn migration. Choice of wintering area had only minor effects on timing of return at the breeding area and timing of breeding and these effects were not consistent between breeding populations. ConclusionThe lack of a consistent effect of wintering area on timing of return between breeding areas indicates that individuals synchronize their arrival with others in their population despite extensive individual differences in migration strategies.

ecology↗

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

Rapid radiation of Southern Ocean shags in response to receding sea ice

AimUnderstanding how wild populations respond to climatic shifts is a fundamental goal of biological research in a fast-changing world. The Southern Ocean represents a fascinating system for assessing large-scale climate-driven biological change, as it contains extremely isolated island groups within a predominantly westerly, circumpolar wind and current system. The blue-eyed shags (Leucocarbo spp.) represent a paradoxical Southern Ocean seabird radiation; a circumpolar distribution implies strong dispersal capacity yet their speciose nature suggests local adaptation and isolation. Here we use genetic tools in an attempt to resolve this paradox. LocationSouthern Ocean. Taxa17 species and subspecies of blue-eyed shags (Leucocarbo spp.) across the geographical distribution of the genus. MethodsHere we use mitochondrial and nuclear sequence data to conduct the first global genetic analysis of this group using a temporal phylogenetic framework to test for rapid speciation. ResultsOur analysis reveals remarkably shallow evolutionary histories among island-endemic lineages, consistent with a recent high-latitude circumpolar radiation. This rapid sub-Antarctic expansion contrasts with significantly deeper lineages detected in more temperate regions such as South America and New Zealand that may have acted as glacial refugia. The dynamic history of high-latitude expansions is further supported by ancestral demographic and biogeographic reconstructions. Main conclusionsThe circumpolar distribution of blue-eyed shags, and their highly dynamic evolutionary history, potentially make Leucocarbo a strong sentinel of past and ongoing Southern Ocean ecosystem change given their sensitivity to climatic impacts.

evolutionary biology↗

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

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 promoter human synapsin 1 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.

neuroscience↗

bioRxiv2020

Identification of genes required for enzalutamide resistance in castration-resistant prostate cancer cells in vitro

Castration-resistant prostate cancer can be treated with the anti-androgen 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 eleven 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, while 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. Financial supportThe authors would like to acknowledge funding from the Joe C. Davis Foundation (to RJM), the Vanderbilt Institute for Clinical and Translational Research (VICTR, to YY, PEC, and RJM). The Vanderbilt Institute for Clinical and Translational Research (VICTR) is funded by the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, Award Number 5UL1TR002243. The content of this manuscript solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would also like to acknowledge the Case Research Institute, a joint venture between University Hospitals and Case Western Reserve University, start-up funds (to MMG), and the Cell and Molecular Biology Training Program (T32 GM 008056 to SEK).

cancer biology↗

bioRxiv2019

Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

Blue waveform light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue waveform 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 waveform light in cultured primary rat cortical neurons. Exposure to blue light (470nm) 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 hours 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. Changes in gene expression induced by blue waveform light were prevented when neurons were cultured 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.

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↗