RESUMEN
Microglia, the innate immune cells in the central nervous system, exhibit distinct transcriptional profiles across brain regions that are important for facilitating their specialized function. There has been recent interest in identifying the epigenetic modifications associated with these distinct transcriptional profiles, as these may improve our understanding of the underlying mechanisms governing the functional specialization of microglia. One obstacle to achieving this goal is the large number of microglia required to obtain a genome-wide profile for a single histone modification. Given the cellular and regional heterogeneity of the brain, this would require pooling many samples which would impede biological applications that are limited by numbers of available animals. To overcome this obstacle, we have adapted a method of chromatin profiling known as Cleavage Under Targets and Tagmentation (CUT&Tag-Direct) to profile histone modifications associated with regional differences in gene expression throughout the brain reward system. Consistent with previous studies, we find that transcriptional profiles of microglia vary by brain region. However, here we report that these regional differences also exhibit transcriptional network signatures specific to each region. Additionally, we find that these region-dependent network signatures are associated with differential deposition of H3K27ac and H3K7me3, and while the H3K27me3 landscape is remarkably stable across brain regions, the H3K27ac landscape is most consistent with the anatomical location of microglia which explain their distinct transcriptional profiles. Altogether, these findings underscore the established role of H3K27me3 in cell fate determination and support the active role of H3K27ac in the dynamic regulation of microglial gene expression. In this study, we report a molecular and computational framework that can be applied to improve our understanding of the role of epigenetic regulation in microglia in both health and disease, using as few as 2,500 cells per histone mark.
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Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are currently no FDA-approved cessation therapeutics. Repeated methamphetamine (METH) use induces long-term gene expression changes in brain regions associated with reward processing and drug-seeking behavior, and recent evidence suggests that methamphetamine-induced neuroinflammation may also shape behavioral and molecular responses to the drug. Microglia, the resident immune cells in the brain, are principal drivers of neuroinflammatory responses and contribute to the pathophysiology of substance use disorders. Here, we investigated transcriptional and morphological changes in dorsal striatal microglia in response to methamphetamine-taking and during methamphetamine abstinence, as well as their functional contribution to drug-taking behavior. We show that methamphetamine self-administration induces transcriptional changes associated with protein folding, mRNA processing, immune signaling, and neurotransmission in dorsal striatal microglia. Importantly, many of these transcriptional changes persist through abstinence, a finding supported by morphological analyses. Functionally, we report that microglial ablation increases methamphetamine-taking, possibly involving neuroimmune and neurotransmitter regulation. In contrast, microglial depletion during abstinence does not alter methamphetamine-seeking. Taken together, these results suggest that methamphetamine induces both short and long-term changes in dorsal striatal microglia that contribute to altered drug-taking behavior and may provide valuable insights into the pathophysiology of MUD.
Asunto(s)
Trastornos Relacionados con Anfetaminas , Comportamiento de Búsqueda de Drogas , Metanfetamina , Microglía , Autoadministración , Metanfetamina/farmacología , Microglía/metabolismo , Microglía/efectos de los fármacos , Animales , Masculino , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Comportamiento de Búsqueda de Drogas/fisiología , Ratones , Trastornos Relacionados con Anfetaminas/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Ratones Endogámicos C57BL , Refuerzo en Psicología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacosRESUMEN
Aversive stimuli activate corticotropin-releasing factor (CRF)-expressing neurons in the paraventricular nucleus of hypothalamus (PVNCRF neurons) and other brain stress systems to facilitate avoidance behaviors. Appetitive stimuli also engage the brain stress systems, but their contributions to reward-related behaviors are less well understood. Here, we show that mice work vigorously to optically activate PVNCRF neurons in an operant chamber, indicating a reinforcing nature of these neurons. The reinforcing property of these neurons is not mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis. We found that PVNCRF neurons send direct projections to the ventral tegmental area (VTA), and selective activation of these projections induced robust self-stimulation behaviors, without activation of the HPA axis. Similar to the PVNCRF cell bodies, self-stimulation of PVNCRF-VTA projection was dramatically attenuated by systemic pretreatment of CRF receptor 1 or dopamine D1 receptor (D1R) antagonist and augmented by corticosterone synthesis inhibitor metyrapone, but not altered by dopamine D2 receptor (D2R) antagonist. Furthermore, we found that activation of PVNCRF-VTA projections increased c-Fos expression in the VTA dopamine neurons and rapidly triggered dopamine release in the nucleus accumbens (NAc), and microinfusion of D1R or D2R antagonist into the NAc decreased the self-stimulation of these projections. Together, our findings reveal an unappreciated role of PVNCRF neurons and their VTA projections in driving reward-related behaviors, independent of their core neuroendocrine functions. As activation of PVNCRF neurons is the final common path for many stress systems, our study suggests a novel mechanism underlying the positive reinforcing effect of stressful stimuli.
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Hormona Liberadora de Corticotropina , Hormonas Liberadoras de Hormona Hipofisaria , Ratones , Animales , Hormona Liberadora de Corticotropina/metabolismo , Hormonas Liberadoras de Hormona Hipofisaria/metabolismo , Hormonas Liberadoras de Hormona Hipofisaria/farmacología , Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , Hipotálamo/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Neuronas Dopaminérgicas/metabolismoRESUMEN
Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are currently no FDA-approved cessation therapeutics. Repeated methamphetamine (METH) use induces long-term gene expression changes in brain regions associated with reward processing and drug-seeking behavior, and recent evidence suggests that methamphetamine-induced neuroinflammation may also shape behavioral and molecular responses to the drug. Microglia, the resident immune cells in the brain, are principal drivers of neuroinflammatory responses and contribute to the pathophysiology of substance use disorders. Here, we investigated transcriptional and morphological changes in dorsal striatal microglia in response to methamphetamine-taking and during methamphetamine abstinence, as well as their functional contribution to drug-taking behavior. We show that methamphetamine self-administration induces transcriptional changes associated with protein folding, mRNA processing, immune signaling, and neurotransmission in dorsal striatal microglia. Importantly, many of these transcriptional changes persist through abstinence, a finding supported by morphological analyses. Functionally, we report that microglial ablation increases methamphetamine-taking, possibly involving neuroimmune and neurotransmitter regulation, and that post-methamphetamine microglial repopulation attenuates drug-seeking following a 21-day period of abstinence. In contrast, microglial depletion during abstinence did not alter methamphetamine-seeking. Taken together, these results suggest that methamphetamine induces both short and long-term changes in dorsal striatal microglia that contribute to altered drug-taking behavior and may provide valuable insights into the pathophysiology of MUD.
RESUMEN
Microglia are widely known for their role in immune surveillance and for their ability to refine neurocircuitry during development, but a growing body of evidence suggests that microglia may also play a complementary role to neurons in regulating the behavioral aspects of substance use disorders. While many of these efforts have focused on changes in microglial gene expression associated with drug-taking, epigenetic regulation of these changes has yet to be fully understood. This review provides recent evidence supporting the role of microglia in various aspects of substance use disorder, with particular focus on changes to the microglial transcriptome and the potential epigenetic mechanisms driving these changes. Further, this review discusses the latest technical advances in low-input chromatin profiling and highlights the current challenges for studying these novel molecular mechanisms in microglia.
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Microglía , Trastornos Relacionados con Sustancias , Humanos , Microglía/metabolismo , Epigénesis Genética , Cromatina/metabolismo , Trastornos Relacionados con Sustancias/genética , Trastornos Relacionados con Sustancias/metabolismo , TranscriptomaRESUMEN
The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc's anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.
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Neuronas , Núcleo Accumbens , Animales , Encéfalo , Hibridación Fluorescente in Situ , Ratones , Núcleo Accumbens/fisiologíaRESUMEN
Coordinated activity-induced transcriptional changes across multiple neuron subtypes of the prefrontal cortex (PFC) play a pivotal role in encoding and regulating major cognitive behaviors. Yet, the specific transcriptional programs in each neuron subtype remain unknown. Using single-cell RNA sequencing (scRNA-seq), here we comprehensively classify all unique cell subtypes in the PFC. We analyze transcriptional dynamics of each cell subtype under a naturally adaptive and an induced condition. Adaptive changes during adolescence (between P21 and P60), a highly dynamic phase of postnatal neuroplasticity, profoundly impacted transcription in each neuron subtype, including cell type-specific regulation of genes implicated in major neuropsychiatric disorders. On the other hand, an induced plasticity evoked by chronic cocaine addiction resulted in progressive transcriptional changes in multiple neuron subtypes and became most pronounced upon prolonged drug withdrawal. Our findings lay a foundation for understanding cell type-specific postnatal transcriptional dynamics under normal PFC function and in neuropsychiatric disease states.
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Conducta Adictiva/fisiopatología , Corteza Prefrontal/fisiología , Animales , Conducta Adictiva/genética , Masculino , Ratones , Neurociencias , Corteza Prefrontal/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Neuronas Dopaminérgicas/metabolismo , Porción Compacta de la Sustancia Negra/metabolismo , Factores de Transcripción/genética , Tirosina 3-Monooxigenasa/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Mesencéfalo/citología , RatonesRESUMEN
Tobacco smokers titrate their nicotine intake to avoid its noxious effects, sensitivity to which may influence vulnerability to tobacco dependence, yet mechanisms of nicotine avoidance are poorly understood. Here we show that nicotine activates glucagon-like peptide-1 (GLP-1) neurons in the nucleus tractus solitarius (NTS). The antidiabetic drugs sitagliptin and exenatide, which inhibit GLP-1 breakdown and stimulate GLP-1 receptors, respectively, decreased nicotine intake in mice. Chemogenetic activation of GLP-1 neurons in NTS similarly decreased nicotine intake. Conversely, Glp1r knockout mice consumed greater quantities of nicotine than wild-type mice. Using optogenetic stimulation, we show that GLP-1 excites medial habenular (MHb) projections to the interpeduncular nucleus (IPN). Activation of GLP-1 receptors in the MHb-IPN circuit abolished nicotine reward and decreased nicotine intake, whereas their knockdown or pharmacological blockade increased intake. GLP-1 neurons may therefore serve as 'satiety sensors' for nicotine that stimulate habenular systems to promote nicotine avoidance before its aversive effects are encountered.
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Reacción de Prevención/fisiología , Péptido 1 Similar al Glucagón/fisiología , Habénula/fisiología , Nicotina/farmacología , Animales , Exenatida , Femenino , Técnicas de Silenciamiento del Gen , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/fisiología , Núcleo Interpeduncular/fisiología , Masculino , Ratones , Ratones Noqueados , Vías Nerviosas/fisiología , Nicotina/antagonistas & inhibidores , Péptidos/farmacología , Ratas , Recompensa , Autoestimulación , Fosfato de Sitagliptina/farmacología , Núcleo Solitario/fisiología , Ponzoñas/farmacologíaRESUMEN
Inducible nitric oxide synthase (iNOS) is a potent mediator of oxidative stress during neuroinflammation triggered by neurotrauma or neurodegeneration. We previously demonstrated that acute iNOS inhibition attenuated iNOS levels and promoted neuroprotection and functional recovery after spinal cord injury (SCI). The present study investigated the effects of chronic iNOS ablation after SCI using inos-null mice. iNOS-/- knockout and wild-type (WT) control mice underwent a moderate thoracic (T8) contusive SCI. Locomotor function was assessed weekly, using the Basso Mouse Scale (BMS), and at the endpoint (six weeks), by footprint analysis. At the endpoint, the volume of preserved white and gray matter, as well as the number of dorsal column axons and perilesional blood vessels rostral to the injury, were quantified. At weeks two and three after SCI, iNOS-/- mice exhibited a significant locomotor improvement compared to WT controls, although a sustained improvement was not observed during later weeks. At the endpoint, iNOS-/- mice showed significantly less preserved white and gray matter, as well as fewer dorsal column axons and perilesional blood vessels, compared to WT controls. While short-term antagonism of iNOS provides histological and functional benefits, its long-term ablation after SCI may be deleterious, blocking protective or reparative processes important for angiogenesis and tissue preservation.
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Óxido Nítrico Sintasa de Tipo II/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Axones/metabolismo , Modelos Animales de Enfermedad , Femenino , Sustancia Gris/metabolismo , Filamentos Intermedios/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Estrés Oxidativo , Células del Asta Posterior/metabolismo , Recuperación de la Función , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/rehabilitación , Sustancia Blanca/metabolismoRESUMEN
Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.
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Epigénesis Genética/genética , Aprendizaje/fisiología , Memoria/fisiología , Trastornos Relacionados con Sustancias/genética , Metilación de ADN/genética , Histonas/metabolismo , HumanosRESUMEN
Schwann cell (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair, and functional recovery. Reparative efficacy, however, may be limited because of the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by overexpressing polysialic acid (PSA) has been shown to promote SC migration. In this study, a SCI contusion model was used to evaluate the migration, supraspinal axon growth support, and functional recovery associated with polysialyltransferase (PST)-overexpressing SCs [PST-green fluorescent protein (GFP) SCs] or controls (GFP SCs). Compared with GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, beyond the modest improvements provided by GFP SC controls. This study for the first time demonstrates that a lack of migration by SCs may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA-modified SCs will be a potent reparative approach for SCI. © 2012 Wiley Periodicals, Inc.
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Movimiento Celular/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Células de Schwann , Ácidos Siálicos/farmacología , Traumatismos de la Médula Espinal , Animales , Proteínas Bacterianas/genética , Biotina/análogos & derivados , Recuento de Células , Dextranos , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Lineales , Proteínas Luminiscentes/genética , Regeneración Nerviosa/fisiología , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Endogámicas F344 , Recuperación de la Función/fisiología , Células de Schwann/efectos de los fármacos , Células de Schwann/fisiología , Células de Schwann/trasplante , Nervio Ciático/citología , Serotonina/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Factores de TiempoRESUMEN
Tobacco smoking is one of the leading causes of disease and premature death in the United States. Nicotine is considered the major reinforcing component in tobacco smoke responsible for tobacco addiction. Nicotine acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The predominant nAChR subtypes in mammalian brain are those containing α4 and ß2 subunits. The α4ß2 nAChRs, particularly those located in the mesoaccumbens dopamine pathway, play a key role in regulating the reinforcing properties of nicotine. Considering that twelve mammalian nAChR subunits have been cloned, it is likely that nAChRs containing subunits in addition to, or other than, α4 and ß2 also play a role in the tobacco smoking habit. Consistent with this possibility, human genome-wide association studies have shown that genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster located in chromosome region 15q25, which encode the α5, α3 and ß4 nAChR subunits, respectively, increases vulnerability to tobacco addiction and smoking-related diseases. Most recently, α5-containing nAChRs located in the habenulo-interpeduncular tract were shown to limit intravenous nicotine self-administration behavior in rats and mice, suggesting that deficits in α5-containing nAChR signaling in the habenulo-interpeduncular tract increases vulnerability to the motivational properties of nicotine. Finally, evidence suggests that nAChRs may also play a prominent role in controlling consumption of addictive drugs other than nicotine, including cocaine, alcohol, opiates and cannabinoids. The aim of the present review is to discuss recent preclinical findings concerning the identity of the nAChR subtypes that regulate self-administration of nicotine and other drugs of abuse.
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Etanol/farmacología , Drogas Ilícitas/farmacología , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Etanol/administración & dosificación , Humanos , Nicotina/administración & dosificación , Subunidades de Proteína , Receptores Nicotínicos/genética , Refuerzo en Psicología , AutoadministraciónRESUMEN
Trauma to the spinal cord produces endogenously irreversible tissue and functional loss, requiring the application of therapeutic approaches to achieve meaningful restoration. Cellular strategies, in particular Schwann-cell implantation, have shown promise in overcoming many of the obstacles facing successful repair of the injured spinal cord. Here, we show that the implantation of Schwann cells as cell suspensions with in-situ gelling laminin:collagen matrices after spinal-cord contusion significantly enhances long-term cell survival but not proliferation, as well as improves graft vascularization and the degree of axonal in-growth over the standard implantation vehicle, minimal media. The use of a matrix to suspend cells prior to implantation should be an important consideration for achieving improved survival and effectiveness of cellular therapies for future clinical application.