Transcriptional signatures of fentanyl use in the mouse ventral tegmental area.

Synthetic opioids such as fentanyl contribute to the vast majority of opioid-related overdose deaths, but fentanyl use remains broadly understudied. Like other substances with misuse potential, opioids cause lasting molecular adaptations to brain reward circuits, including neurons in the ventral tegmental area (VTA). The VTA contains numerous cell types that play diverse roles in opioid use and relapse; however, it is unknown how fentanyl experience alters the transcriptional landscape in specific subtypes. Here, we performed single nuclei RNA sequencing to study transcriptional programs in fentanyl-experienced mice. Male and female C57/BL6 mice self-administered intravenous fentanyl (1.5 µg/kg/infusion) or saline for 10 days. After 24 h abstinence, VTA nuclei were isolated and prepared for sequencing on the 10× platform. We identified different patterns of gene expression across cell types. In dopamine neurons, we found enrichment of genes involved in growth hormone signalling. In dopamine-glutamate-GABA combinatorial neurons, and some GABA neurons, we found enrichment of genes involved in Pi3k-Akt signalling. In glutamate neurons, we found enrichment of genes involved in cholinergic signalling. We identified transcriptional regulators for the differentially expressed genes in each neuron cluster, including downregulated transcriptional repressor Bcl6, and upregulated transcription factor Tcf4. We also compared the fentanyl-induced gene expression changes identified in mouse VTA with a published rat dataset in bulk VTA, and found overlap in genes related to GABAergic signalling and extracellular matrix interaction. Together, we provide a comprehensive picture of how fentanyl self-administration alters the transcriptional landscape of the mouse VTA that serves as the foundation for future mechanistic studies.

Multimodal Interrogation of Ventral Pallidum Projections Reveals Projection-Specific Signatures and Effects on Cocaine Reward.

The ventral pallidum (VP) is a central hub in the reward circuitry with diverse projections that have different behavioral roles attributed mostly to the connectivity with the downstream target. However, different VP projections may represent, as in the striatum, separate neuronal populations that differ in more than just connectivity. In this study, we performed in mice of both sexes a multimodal dissection of four major projections of the VP-to the lateral hypothalamus (VP→LH), ventral tegmental area (VP→VTA), lateral habenula (VP→LHb), and mediodorsal thalamus (VP→MDT)-with physiological, anatomical, genetic, and behavioral tools. We also tested for physiological differences between VP neurons receiving input from nucleus accumbens medium spiny neurons (MSNs) that express either the D1 (D1-MSNs) or the D2 (D2-MSNs) dopamine receptor. We show that each VP projection (1) when inhibited during a cocaine conditioned place preference (CPP) test affects performance differently, (2) receives a different pattern of inputs using rabies retrograde labeling, (3) shows differentially expressed genes using RNA sequencing, and (4) has projection-specific characteristics in excitability and synaptic input characteristics using whole-cell patch clamp. VP→LH and VP→VTA projections have different effects on CPP and show low overlap in circuit tracing experiments, as VP→VTA neurons receive more striatal input, while VP→LH neurons receive more olfactory input. Additionally, VP→VTA neurons are less excitable, while VP→LH neurons are more excitable than the average VP neuron, a difference driven mainly by D2-MSN-responding neurons. Thus, VP→VTA and VP→LH neurons may represent largely distinct populations of VP neurons.

in silico transcriptome dissection of neocortical excitatory neurogenesis via joint matrix decomposition and transfer learning.

The rising quality and amount of multi-omic data across biomedical science demands that we build innovative solutions to harness their collective discovery potential. From publicly available repositories, we have assembled and curated a compendium of gene-level transcriptomic data focused on mammalian excitatory neurogenesis in the neocortex. This collection is open for exploration by both computational and cell biologists at nemoanalytics.org, and this report forms a demonstration of its utility. Applying our novel structured joint decomposition approach to mouse, macaque and human data from the collection, we define transcriptome dynamics that are conserved across mammalian excitatory neurogenesis and which map onto the genetics of human brain structure and disease. Leveraging additional data within NeMO Analytics via projection methods, we chart the dynamics of these fundamental molecular elements of neurogenesis across developmental time and space and into postnatal life. Reversing the direction of our investigation, we use transcriptomic data from laminar-specific dissection of adult human neocortex to define molecular signatures specific to excitatory neuronal cell types resident in individual layers of the mature neocortex, and trace their emergence across development. We show that while many lineage defining transcription factors are most highly expressed at early fetal ages, the laminar neuronal identities which they drive take years to decades to reach full maturity. Finally, we interrogated data from stem-cell derived cerebral organoid systems demonstrating that many fundamental elements of in vivo development are recapitulated with high-fidelity in vitro, while specific transcriptomic programs in neuronal maturation are absent. We propose these analyses as specific applications of the general approach of combining joint decomposition with large curated collections of analysis-ready multi-omics data matrices focused on particular cell and disease contexts. Importantly, these open environments are accessible to, and must be fueled with emerging data by, cell biologists with and without coding expertise.

Cochlear organoids reveal transcriptional programs of postnatal hair cell differentiation from supporting cells.

We explore the changes in chromatin accessibility and transcriptional programs for cochlear hair cell differentiation from postmitotic supporting cells using organoids from postnatal cochlea. The organoids contain cells with transcriptional signatures of differentiating vestibular and cochlear hair cells. Construction of trajectories identifies Lgr5+ cells as progenitors for hair cells, and the genomic data reveal gene regulatory networks leading to hair cells. We validate these networks, demonstrating dynamic changes both in expression and predicted binding sites of transcription factors (TFs) during organoid differentiation. We identify known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and the analysis predicts the regulatory factors Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt-signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for hair cell (HC) regeneration, which is limited in the adult.

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus.

The development and diversity of neuronal subtypes in the human hypothalamus has been insufficiently characterized. To address this, we integrated transcriptomic data from 241,096 cells (126,840 newly generated) in the prenatal and adult human hypothalamus to reveal a temporal trajectory from proliferative stem cell populations to mature hypothalamic cell types. Iterative clustering of the adult neurons identified 108 robust transcriptionally distinct neuronal subtypes representing 10 hypothalamic nuclei. Pseudotime trajectories provided insights into the genes driving formation of these nuclei. Comparisons to single-cell transcriptomic data from the mouse hypothalamus suggested extensive conservation of neuronal subtypes despite certain differences in species-enriched gene expression. The uniqueness of hypothalamic neuronal lineages was examined developmentally by comparing excitatory lineages present in cortex and inhibitory lineages in ganglionic eminence, revealing both distinct and shared drivers of neuronal maturation across the human forebrain. These results provide a comprehensive transcriptomic view of human hypothalamus development through gestation and adulthood at cellular resolution.

Genome wide association joint analysis reveals 99 risk loci for pain susceptibility and pleiotropic relationships with psychiatric, metabolic, and immunological traits.

Chronic pain is at epidemic proportions in the United States, represents a significant burden on our public health system, and is coincident with a growing opioid crisis. While numerous genome-wide association studies have been reported for specific pain-related traits, many of these studies were underpowered, and the genetic relationship among these traits remains poorly understood. Here, we conducted a joint analysis of genome-wide association study summary statistics from seventeen pain susceptibility traits in the UK Biobank. This analysis revealed 99 genome-wide significant risk loci, 65 of which overlap loci identified in earlier studies. The remaining 34 loci are novel. We applied leave-one-trait-out meta-analyses to evaluate the influence of each trait on the joint analysis, which suggested that loci fall into four categories: loci associated with nearly all pain-related traits; loci primarily associated with a single trait; loci associated with multiple forms of skeletomuscular pain; and loci associated with headache-related pain. Overall, 664 genes were mapped to the 99 loci by genomic proximity, eQTLs, and chromatin interaction and ~15% of these genes showed differential expression in individuals with acute or chronic pain compared to healthy controls. Risk loci were enriched for genes involved in neurological and inflammatory pathways. Genetic correlation and two-sample Mendelian randomization indicated that psychiatric, metabolic, and immunological traits mediate some of these effects.

A single-cell genomic atlas for maturation of the human cerebellum during early childhood.

Inflammation early in life is a clinically established risk factor for autism spectrum disorders and schizophrenia, yet the impact of inflammation on human brain development is poorly understood. The cerebellum undergoes protracted postnatal maturation, making it especially susceptible to perturbations contributing to the risk of developing neurodevelopmental disorders. Here, using single-cell genomics of postmortem cerebellar brain samples, we characterized the postnatal development of cerebellar neurons and glia in 1- to 5-year-old children, comparing individuals who had died while experiencing inflammation with those who had died as a result of an accident. Our analyses revealed that inflammation and postnatal cerebellar maturation are associated with extensive, overlapping transcriptional changes primarily in two subtypes of inhibitory neurons: Purkinje neurons and Golgi neurons. Immunohistochemical analysis of a subset of these postmortem cerebellar samples revealed no change to Purkinje neuron soma size but evidence for increased activation of microglia in those children who had experienced inflammation. Maturation-associated and inflammation-associated gene expression changes included genes implicated in neurodevelopmental disorders. A gene regulatory network model integrating cell type-specific gene expression and chromatin accessibility identified seven temporally specific gene networks in Purkinje neurons and suggested that inflammation may be associated with the premature down-regulation of developmental gene expression programs.

Transcriptomic profiling of reward and sensory brain areas in perinatal fentanyl exposed juvenile mice.

Use of the synthetic opioid fentanyl increased ~300% in the last decade, including among women of reproductive ages. Adverse neonatal outcomes and long-term behavioral disruptions are associated with perinatal opioid exposure. Our previous work demonstrated that perinatal fentanyl exposed mice displayed enhanced negative affect and somatosensory circuit and behavioral disruptions during adolescence. However, little is known about molecular adaptations across brain regions that underlie these outcomes. We performed RNA sequencing across three reward and two sensory brain areas to study transcriptional programs in perinatal fentanyl exposed juvenile mice. Pregnant dams received 10 µg/ml fentanyl in the drinking water from embryonic day 0 (E0) through gestational periods until weaning at postnatal day 21 (P21). RNA was extracted from nucleus accumbens (NAc), prelimbic cortex (PrL), ventral tegmental area (VTA), somatosensory cortex (S1) and ventrobasal thalamus (VBT) from perinatal fentanyl exposed mice of both sexes at P35. RNA sequencing was performed, followed by analysis of differentially expressed genes (DEGs) and gene co-expression networks. Transcriptome analysis revealed DEGs and gene modules significantly associated with exposure to perinatal fentanyl in a sex-wise manner. The VTA had the most DEGs, while robust gene enrichment occurred in NAc. Genes enriched in mitochondrial respiration were pronounced in NAc and VTA of perinatal fentanyl exposed males, extracellular matrix (ECM) and neuronal migration enrichment were pronounced in NAc and VTA of perinatal fentanyl exposed males, while genes associated with vesicular cycling and synaptic signaling were markedly altered in NAc of perinatal fentanyl exposed female mice. In sensory areas from perinatal fentanyl exposed females, we found alterations in mitochondrial respiration, synaptic and ciliary organization processes. Our findings demonstrate distinct transcriptomes across reward and sensory brain regions, with some showing discordance between sexes. These transcriptome adaptations may underlie structural, functional, and behavioral changes observed in perinatal fentanyl exposed mice.

Predicting Heterogeneity in Patient Response to Morphine Treatment for Neonatal Opioid Withdrawal Syndrome.

Infants with neonatal opioid withdrawal syndrome commonly receive morphine treatment to manage their withdrawal signs. However, the effectiveness of this pharmacotherapy in managing the infants' withdrawal signs vary widely. We sought to understand how information available early in infant monitoring can anticipate this treatment response, focusing on early modified Finnegan Neonatal Abstinence Scoring System (FNASS) scores, polygenic risk for opioid dependence (polygenic risk score (PRS)), and drug exposure. Using k-means clustering, we divided the 213 infants in our cohort into 3 groups based on their FNASS scores in the 12 hours before and after the initiation of pharmacotherapy. We found that these groups were pairwise significantly different for risk factors, including methadone exposure, and for in-hospital outcomes, including total morphine received, length of stay, and highest FNASS score. Whereas PRS was not predictive of receipt of treatment, PRS was pairwise significantly different between a subset of the groups. Using tree-based machine learning methods, we then constructed network graphs of the relationships among these groups, FNASS scores, PRS, drug exposures, and in-hospital outcomes. The resulting networks also showed meaningful connection between early FNASS scores and PRS, as well as between both of those and later in-hospital outcomes. These analyses present clinicians with the opportunity to better anticipate infant withdrawal progression and prepare accordingly, whether with expedited morphine treatment or non-pharmacotherapeutic alternative treatments.

A guide to the BRAIN Initiative Cell Census Network data ecosystem.

Characterizing cellular diversity at different levels of biological organization and across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also essential to manipulate cell types in controlled ways and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data-generating centers, data archives, and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain with demonstration of prototype feasibility for human and nonhuman primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed by the BICCN, and to accessing and using these data and extensive resources, including the BRAIN Cell Data Center (BCDC), which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted toward Findable, Accessible, Interoperable, and Reusable (FAIR) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain.

The brain's dark transcriptome: Sequencing RNA in distal compartments of neurons and glia.

Transcriptomic approaches are powerful strategies to map the molecular diversity of cells in the brain. Single-cell genomic atlases have now been compiled for entire mammalian brains. However, complementary techniques are only just beginning to map the subcellular transcriptomes from distal cellular compartments. We review single-cell datasets alongside subtranscriptome data from the mammalian brain to explore the development of cellular and subcellular diversity. We discuss how single-cell RNA-seq misses transcripts localized away from cell bodies, which form the 'dark transcriptome' of the brain: a collection of subtranscriptomes in dendrites, axons, growth cones, synapses, and endfeet with important roles in brain development and function. Recent advances in subcellular transcriptome sequencing are beginning to reveal these elusive pools of RNA. We outline the success stories to date in uncovering the constituent subtranscriptomes of neurons and glia, as well as present the emerging toolkit that is accelerating the pace of subtranscriptome discovery.

Genome-wide significant risk loci for mood disorders in the Old Order Amish founder population.

Genome-wide association studies (GWAS) of mood disorders in large case-control cohorts have identified numerous risk loci, yet pathophysiological mechanisms remain elusive, primarily due to the very small effects of common variants. We sought to discover risk variants with larger effects by conducting a genome-wide association study of mood disorders in a founder population, the Old Order Amish (OOA, n = 1,672). Our analysis revealed four genome-wide significant risk loci, all of which were associated with >2-fold relative risk. Quantitative behavioral and neurocognitive assessments (n = 314) revealed effects of risk variants on sub-clinical depressive symptoms and information processing speed. Network analysis suggested that OOA-specific risk loci harbor novel risk-associated genes that interact with known neuropsychiatry-associated genes via gene interaction networks. Annotation of the variants at these risk loci revealed population-enriched, non-synonymous variants in two genes encoding neurodevelopmental transcription factors, CUX1 and CNOT1. Our findings provide insight into the genetic architecture of mood disorders and a substrate for mechanistic and clinical studies.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Depression, stress and regional cerebral blood flow.

Decreased cerebral blood flow (CBF) may be an important mechanism associated with depression. In this study we aimed to determine if the association of CBF and depression is dependent on current level of depression or the tendency to experience depression over time (trait depression), and if CBF is influenced by depression-related factors such as stressful life experiences and antidepressant medication use. CBF was measured in 254 participants from the Amish Connectome Project (age 18-76, 99 men and 154 women) using arterial spin labeling. All participants underwent assessment of symptoms of depression measured with the Beck Depression Inventory and Maryland Trait and State Depression scales. Individuals diagnosed with a unipolar depressive disorder had significantly lower average gray matter CBF compared to individuals with no history of depression or to individuals with a history of depression that was in remission at time of study. Trait depression was significantly associated with lower CBF, with the associations strongest in cingulate gyrus and frontal white matter. Use of antidepressant medication and more stressful life experiences were also associated with significantly lower CBF. Resting CBF in specific brain regions is associated with trait depression, experience of stressful life events, and current antidepressant use, and may provide a valuable biomarker for further studies.

Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons.

Altered activity of the ventral pallidum (VP) underlies disrupted motivation in stress and drug exposure. The VP is a very heterogeneous structure composed of many neuron types with distinct physiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for motivational behavior. While Npas1+ neurons have been characterized in the globus pallidus external, there is limited information on these neurons in the VP. To address this limitation, we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-sequencing on ribosome-associated mRNA from VP Npas1+ neurons to determine their molecular identity. Finally, we used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We used a similar approach in females using the chronic witness defeat stress (CWDS). We identified VP Npas1+ projections to the nucleus accumbens, ventral tegmental area, medial and lateral habenula, lateral hypothalamus, thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct translatome representing distinct biological processes. Chemogenetic activation of hM3D(Gq) receptors in VP Npas1+ neurons increased susceptibility to a subthreshold SDS and anxiety-like behavior in the elevated plus maze and open field while the activation of hM4D(Gi) receptors in VP Npas1+ neurons enhanced resilience to chronic SDS and CWDS. Thus, the activity of VP Npas1+ neurons modulates susceptibility to social stressors and anxiety-like behavior. Our studies provide new information on VP Npas1+ neuron circuitry, molecular identity, and their role in stress response.SIGNIFICANCE STATEMENT The ventral pallidum (VP) is a structure connected to both reward-related and aversive brain centers. It is a key brain area that signals the hedonic value of natural rewards. Disruption in the VP underlies altered motivation in stress and substance use disorder. However, VP is a very heterogeneous area with multiple neuron subtypes. This study characterized the projection pattern and molecular signatures of VP Neuronal PAS 1-positive (Npas1+) neurons. We further used tools to alter receptor signaling in VP Npas1+ neurons in stress to demonstrate a role for these neurons in stress behavioral outcomes. Our studies have implications for understanding brain cell type identities and their role in brain disorders, such as depression, a serious disorder that is precipitated by stressful events.

Adaptations in Nucleus Accumbens Neuron Subtypes Mediate Negative Affective Behaviors in Fentanyl Abstinence.

BACKGROUND: Opioid discontinuation generates a withdrawal syndrome marked by increased negative affect. Increased symptoms of anxiety and dysphoria during opioid discontinuation are significant barriers to achieving long-term abstinence in opioid-dependent individuals. While adaptations in the nucleus accumbens are implicated in opioid abstinence syndrome, the precise neural mechanisms are poorly understood. Additionally, our current knowledge is limited to changes following natural and semisynthetic opioids, despite recent increases in synthetic opioid use and overdose. METHODS: We used a combination of cell subtype-specific viral labeling and electrophysiology in male and female mice to investigate structural and functional plasticity in nucleus accumbens medium spiny neuron (MSN) subtypes after fentanyl abstinence. We characterized molecular adaptations after fentanyl abstinence with subtype-specific RNA sequencing and weighted gene co-expression network analysis. We used viral-mediated gene transfer to manipulate the molecular signature of fentanyl abstinence in D1-MSNs. RESULTS: Here, we show that fentanyl abstinence increases anxiety-like behavior, decreases social interaction, and engenders MSN subtype-specific plasticity in both sexes. D1-MSNs, but not D2-MSNs, exhibit dendritic atrophy and an increase in excitatory drive. We identified a cluster of coexpressed dendritic morphology genes downregulated selectively in D1-MSNs that are transcriptionally coregulated by E2F1. E2f1 expression in D1-MSNs protects against loss of dendritic complexity, altered physiology, and negative affect-like behaviors caused by fentanyl abstinence. CONCLUSIONS: Our findings indicate that fentanyl abstinence causes unique structural, functional, and molecular changes in nucleus accumbens D1-MSNs that can be targeted to alleviate negative affective symptoms during abstinence.

The Neuroscience Multi-Omic Archive: a BRAIN Initiative resource for single-cell transcriptomic and epigenomic data from the mammalian brain.

Scalable technologies to sequence the transcriptomes and epigenomes of single cells are transforming our understanding of cell types and cell states. The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network (BICCN) is applying these technologies at unprecedented scale to map the cell types in the mammalian brain. In an effort to increase data FAIRness (Findable, Accessible, Interoperable, Reusable), the NIH has established repositories to make data generated by the BICCN and related BRAIN Initiative projects accessible to the broader research community. Here, we describe the Neuroscience Multi-Omic Archive (NeMO Archive; nemoarchive.org), which serves as the primary repository for genomics data from the BRAIN Initiative. Working closely with other BRAIN Initiative researchers, we have organized these data into a continually expanding, curated repository, which contains transcriptomic and epigenomic data from over 50 million brain cells, including single-cell genomic data from all of the major regions of the adult and prenatal human and mouse brains, as well as substantial single-cell genomic data from non-human primates. We make available several tools for accessing these data, including a searchable web portal, a cloud-computing interface for large-scale data processing (implemented on Terra, terra.bio), and a visualization and analysis platform, NeMO Analytics (nemoanalytics.org).

Transcriptional signatures of fentanyl use in the mouse ventral tegmental area.

Synthetic opioids such as fentanyl contribute to the vast majority of opioid-related overdose deaths, but fentanyl use remains broadly understudied. Like other substances with misuse potential, opioids cause lasting molecular adaptations to brain reward circuits, including neurons in the ventral tegmental area (VTA). The VTA contains numerous cell types that play diverse roles in opioid use and relapse, however it is unknown how fentanyl experience alters the transcriptional landscape in specific subtypes. Here, we performed single nuclei RNA sequencing to study transcriptional programs in fentanyl experienced mice. Male and female C57/BL6 mice self-administered intravenous fentanyl (1.5 µg/kg/infusion) or saline for 10 days. After 24 hr abstinence, VTA nuclei were isolated and prepared for sequencing on the 10X platform. We identified different patterns of gene expression across cell types. In dopamine neurons, we found enrichment of genes involved in growth hormone signaling. In dopamine-glutamate-GABA combinatorial neurons, and some GABA neurons, we found enrichment of genes involved in Pi3k-Akt signaling. In glutamate neurons, we found enrichment of genes involved in cholinergic signaling. We identified transcriptional regulators for the differentially expressed genes in each neuron cluster, including downregulation of transcriptional repressor Bcl6, and upregulation of Wnt signaling partner Tcf4. We also compared the fentanyl-induced gene expression changes identified in mouse VTA with a published rat dataset in bulk VTA, and found overlap in genes related to GABAergic signaling and extracellular matrix interaction. Together, we provide a comprehensive picture of how fentanyl self-administration alters the transcriptional landscape of the mouse VTA, that serves for the foundation for future mechanistic studies.

Identifying enhancer properties associated with genetic risk for complex traits using regulome-wide association studies.

Genetic risk for complex traits is strongly enriched in non-coding genomic regions involved in gene regulation, especially enhancers. However, we lack adequate tools to connect the characteristics of these disruptions to genetic risk. Here, we propose RWAS (Regulome Wide Association Study), a new application of the MAGMA software package to identify the characteristics of enhancers that contribute to genetic risk for disease. RWAS involves three steps: (i) assign genotyped SNPs to cell type- or tissue-specific regulatory features (e.g., enhancers); (ii) test associations of each regulatory feature with a trait of interest for which genome-wide association study (GWAS) summary statistics are available; (iii) perform enhancer-set enrichment analyses to identify quantitative or categorical features of regulatory elements that are associated with the trait. These steps are implemented as a novel application of MAGMA, a tool originally developed for gene-based GWAS analyses. Applying RWAS to interrogate genetic risk for schizophrenia, we discovered a class of risk-associated AT-rich enhancers that are active in the developing brain and harbor binding sites for multiple transcription factors with neurodevelopmental functions. RWAS utilizes open-source software, and we provide a comprehensive collection of annotations for tissue-specific enhancer locations and features, including their evolutionary conservation, AT content, and co-localization with binding sites for hundreds of TFs. RWAS will enable researchers to characterize properties of regulatory elements associated with any trait of interest for which GWAS summary statistics are available.

Early and Late Transcriptional Changes in Blood, Neural, and Colon Tissues in Rat Models of Stress-Induced and Comorbid Pain Hypersensitivity Reveal Regulatory Roles in Neurological Disease.

Background: Irritable bowel syndrome (IBS) and temporomandibular disorder (TMD) are two chronic pain conditions that frequently overlap in the same individual, more commonly in women. Stress is a significant risk factor, exacerbating or triggering one or both conditions. However, the mechanisms underlying IBS-TMD co-morbidity are mostly unknown. Aim: To detect both specific and common stress-induced visceral hypersensitivity (SIH) and comorbid TMD-IBS pain hypersensitivity (CPH) genetic signatures over time. Method: Twenty-four female rats were randomly assigned to one of three experimental groups: naïve, SIH, and CPH (orofacial pain plus stress). RNA was extracted from blood, colon, spinal cord, and dorsal root ganglion 1 or 7 weeks after the stress paradigm. We combined differential gene expression and co-expression network analyses to define both SIH and CPH expression profiles across tissues and time. Results: The transcriptomic profile in blood and colon showed increased expression of genes enriched in inflammatory and neurological biological processes in CPH compared to SIH rats, both at 1 and 7 weeks after stress. In lumbosacral spinal tissue, both SIH and CPH rats compared to naïve revealed decreased expression of genes related to synaptic activity and increased expression of genes enriched in "angiogenesis," "Neurotrophin," and "PI3K-Akt" pathways. Compared to SIH, CPH rats showed increased expression of angiogenesis-related genes 1 week after exposure to stress, while 7 weeks post-stress the expression of these genes was higher in SIH rats. In dorsal root ganglia (DRG), CPH rats showed decreased expression of immune response genes at week 1 and inhibition of nerve myelination genes at 7 weeks compared to naïve. For all tissues, we observed higher expression of genes involved in ATP production in SIH compared to CPH at 1 week and this was reversed 7 weeks after the induction of stress. Conclusion: Our study highlights an increased inflammatory response in CPH compared to SIH rats in the blood and colon. DRG and spinal transcriptomic profiles of both CPH and SIH rats showed inhibition of synaptic activity along with activation of angiogenesis. Targeting these biological processes may lead to a more profound understanding of the mechanisms underlying IBS-TMD comorbidities and new diagnostic and therapeutic strategies.