RESUMO
Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic ß cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total ß-cell mass, and the role of Gi-GPCRs in establishing ß-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates ß-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic ß cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal ß cells decreased ß-cell proliferation, reduced adult ß-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal ß-cell proliferation, increased adult ß-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult ß cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of ß-cell replication. These studies link Gi-GPCR signaling to ß-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase ß-cell mass in patients with diabetes.
Assuntos
Proliferação de Células , Diabetes Mellitus Tipo 2/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Glucose/genética , Glucose/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Transgênicos , Receptores Adrenérgicos alfa 2/genéticaRESUMO
UNLABELLED: Serotonin (5-HT) is a crucial neuromodulator linked to many psychiatric disorders. However, after more than 60 years of study, its role in behavior remains poorly understood, in part because of a lack of methods to target 5-HT synthesis specifically in the adult brain. Here, we have developed a genetic approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system by stereotaxic injection of an adeno-associated virus expressing Cre recombinase (AAV-Cre) into the midbrain/pons of mice carrying a loxP-conditional tryptophan hydroxylase 2 (Tph2) allele. We investigated the behavioral effects of deficient brain 5-HT synthesis and discovered a unique composite phenotype. Surprisingly, adult 5-HT deficiency did not affect anxiety-like behavior, but resulted in a robust hyperactivity phenotype in novel and home cage environments. Moreover, loss of 5-HT led to an altered pattern of circadian behavior characterized by an advance in the onset and a delay in the offset of daily activity, thus revealing a requirement for adult 5-HT in the control of daily activity patterns. Notably, after normalizing for hyperactivity, we found that the normal prolonged break in nocturnal activity (siesta), a period of rapid eye movement (REM) and non-REM sleep, was absent in all animals in which 5-HT deficiency was verified. Our findings identify adult 5-HT as a requirement for siestas, implicate adult 5-HT in sleep-wake homeostasis, and highlight the importance of our adult-specific 5-HT-synthesis-targeting approach in understanding 5-HT's role in controlling behavior. SIGNIFICANCE STATEMENT: Serotonin (5-HT) is a crucial neuromodulator, yet its role in behavior remains poorly understood, in part because of a lack of methods to target specifically adult brain 5-HT synthesis. We developed an approach that reproducibly achieves near-complete elimination of 5-HT synthesis from the adult ascending 5-HT system. Using this technique, we discovered that adult 5-HT deficiency led to a novel compound phenotype consisting of hyperactivity, disrupted circadian behavior patterns, and elimination of siestas, a period of increased sleep during the active phase. These findings highlight the importance of our approach in understanding 5-HT's role in behavior, especially in controlling activity levels, circadian behavior, and sleep-wake homeostasis, behaviors that are disrupted in many psychiatric disorders such as attention deficit hyperactivity disorder.
Assuntos
Encéfalo/metabolismo , Transtornos Cronobiológicos/genética , Proteínas de Fluorescência Verde/deficiência , Hipercinese/genética , Parassonias/genética , Serotonina/deficiência , Análise de Variância , Animais , Cromatografia Líquida de Alta Pressão , Transtornos Cronobiológicos/patologia , Comportamento Exploratório , Feminino , Proteínas de Fluorescência Verde/genética , Hipercinese/patologia , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Transdução Genética , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
Serotonin (5-hydroxytryptamine or 5-HT) is thought to regulate neurodevelopmental processes through maternal-fetal interactions that have long-term mental health implications. It is thought that beyond fetal 5-HT neurons there are significant maternal contributions to fetal 5-HT during pregnancy but this has not been tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used Pet1(-/-) (also called Fev) mice in which most dorsal raphe neurons lack 5-HT. We detected previously unknown differences in accumulation of 5-HT between the forebrain and hindbrain during early and late fetal stages, through an exogenous source of 5-HT which is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source. We uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor in both mice and humans. This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and indicates that maternal-placental-fetal interactions could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.
Assuntos
Feto/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Serotonina/biossíntese , Animais , Embrião de Mamíferos/metabolismo , Feminino , Feto/embriologia , Humanos , Técnicas In Vitro , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Núcleos da Rafe/citologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Serotonina/análise , Serotonina/metabolismo , Fatores de Tempo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Serotonin 2c receptors (5-HT2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.
Assuntos
Ansiedade/fisiopatologia , Retroalimentação Fisiológica , Neurônios GABAérgicos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Inibição Neural , Receptor 5-HT2C de Serotonina/metabolismo , Serotonina/metabolismo , Potenciais de Ação/efeitos da radiação , Animais , Ansiedade/metabolismo , Ansiedade/patologia , Cálcio/metabolismo , Regulação para Baixo/efeitos da radiação , Retroalimentação Fisiológica/efeitos da radiação , Neurônios GABAérgicos/patologia , Neurônios GABAérgicos/efeitos da radiação , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Espaço Intracelular/efeitos da radiação , Luz , Camundongos , Inibição Neural/efeitos da radiação , Optogenética , Estrutura Terciária de Proteína , Núcleos da Rafe/metabolismo , Núcleos da Rafe/efeitos da radiação , Opsinas de Bastonetes/química , Opsinas de Bastonetes/metabolismo , Transdução de Sinais/efeitos da radiaçãoRESUMO
Analysis of constitutive Engrailed (En) null mice previously implicated the two En homeobox paralogs in the development of serotonin (5-HT) neurons. An unresolved question is whether En plays intrinsic roles in these neurons. Here, we show that En1 and En2 are expressed in maturing 5-HT neurons that will form the dorsal raphe nucleus (DRN) and part of the median raphe nucleus. Although En1 expression in 5-HT neurons persists postnatally, En2 expression is extinguished by embryonic day 17.5. To investigate intrinsic serotonergic functions for En1/2, we generated compound conditional En mutants with floxed alleles and a cre recombinase line that becomes active in postmitotic fetal 5-HT neurons. We present evidence in support of a requirement for En1/2 in the maturation of DRN cytoarchitecture. The disruption of DRN cytoarchitecture appears to result from a defect in secondary migration of serotonergic cell bodies toward the midline rather than disruption of their primary ventral migration away from the ventricular zone. Furthermore, En1/2 are required for perinatal maintenance of serotonergic identity and postnatal forebrain 5-HT levels. Increased numbers of caspase-3-expressing cells and loss of significant numbers of 5-HT neuron cell bodies, indicative of apoptosis, occurred after loss of serotonergic identity. Analysis of an allelic series of conditional mutants showed that En1 is the predominant functional En paralog in maturing 5-HT neurons, although a small contribution from En2 was reproducibly detected. Together, our findings reveal complex intrinsic functions for En in maturing 5-HT neurons, hence necessitating a reinterpretation of their roles in 5-HT system development.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Núcleos da Rafe/fisiologia , Serotonina/fisiologia , Alelos , Animais , Animais Recém-Nascidos , Contagem de Células , Morte Celular/genética , Morte Celular/fisiologia , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mitose/genética , Mitose/fisiologia , RNA/genética , Núcleos da Rafe/citologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or ß1 integrin than cortical neurons. Blocking ß1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.
Assuntos
Axônios/fisiologia , Sistema Nervoso Central/lesões , Neuroglia/fisiologia , Neurônios/fisiologia , Serotonina/fisiologia , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Condroitina ABC Liase/farmacologia , Cicatriz/patologia , Corpo Caloso/citologia , Feminino , Imunofluorescência , Proteína GAP-43/metabolismo , Cones de Crescimento/fisiologia , Imuno-Histoquímica , Integrina beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/fisiologia , Neuritos/fisiologia , Neuritos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores de Laminina/biossíntese , Receptores de Laminina/genéticaRESUMO
Central serotonin-producing neurons are heterogeneous-differing in location, morphology, neurotoxin sensitivity and associated clinical disorders-but the underpinnings of this heterogeneity are largely unknown, as are the markers that distinguish physiological subtypes of serotonergic neurons. Here we redefined serotonergic subtypes on the basis of genetic programs that are differentially enacted in progenitor cells. We uncovered a molecular framework for the serotonergic system that, having genetic lineages as its basis, is likely to have physiological relevance and will permit access to genetically defined subtypes for manipulation.
Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/citologia , Serotonina/genética , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Rombencéfalo/citologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Serotonina/metabolismo , Células-Tronco/metabolismo , Transgenes/genética , Transgenes/fisiologiaRESUMO
Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. Here, we show that unique distal enhancers define the Pet1 neuron lineage that generates serotonin (5-HT) neurons in mice. Heterogeneous single-cell chromatin landscapes are established early in postmitotic Pet1 neurons and reveal the putative regulatory programs driving Pet1 neuron subtype identities. Distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. We find that Pet1 and Lmx1b control chromatin accessibility to select Pet1-lineage-specific enhancers for 5-HT neurotransmission. Additionally, these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. Together, our findings reveal postmitotic transcription factors that reorganize accessible chromatin for neuron specialization.
Assuntos
Cromatina , Serotonina , Animais , Camundongos , Neurônios/fisiologia , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
Neurons must function for decades of life, but how these non-dividing cells are preserved is poorly understood. Using mouse serotonin (5-HT) neurons as a model, we report an adult-stage transcriptional program specialized to ensure the preservation of neuronal connectivity. We uncover a switch in Lmx1b and Pet1 transcription factor function from controlling embryonic axonal growth to sustaining a transcriptomic signature of 5-HT connectivity comprising functionally diverse synaptic and axonal genes. Adult-stage deficiency of Lmx1b and Pet1 causes slowly progressing degeneration of 5-HT synapses and axons, increased susceptibility of 5-HT axons to neurotoxic injury, and abnormal stress responses. Axon degeneration occurs in a die back pattern and is accompanied by accumulation of α-synuclein and amyloid precursor protein in spheroids and mitochondrial fragmentation without cell body loss. Our findings suggest that neuronal connectivity is transcriptionally protected by maintenance of connectivity transcriptomes; progressive decay of such transcriptomes may contribute to age-related diseases of brain circuitry.
Assuntos
Serotonina , Fatores de Transcrição , Animais , Axônios/metabolismo , Camundongos , Neurônios/metabolismo , Serotonina/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Embryonic CNS neurons can migrate from the ventricular zone to their final destination by radial glial-guided locomotion. Another less appreciated mechanism is somal translocation, where the young neuron maintains its primitive ventricular and pial processes, through which the cell body moves. A major problem in studying translocation has been the identification of neuronal-specific markers that appear in primitive, radially shaped cells. We used enhanced yellow fluorescent protein under control of the Pet-1 enhancer/promoter region (ePet-EYFP), a specific marker of early differentiated serotonergic neurons, to study their migration via immunohistology and time-lapse imaging of living slice cultures. As early as E10.0, ePet-EYFP-expressing neurons were axonless, radially oriented, and spanned the entire neuroepithelium. The soma translocated within the pial process toward the pial surface and could also translocate through its neurites, which sprouted from the pial process. The dynamin inhibitor dynasore significantly reduced translocation velocity, while the nonmuscle myosin II inhibitor blebbistatin and the kinesin inhibitor AMP-PNP had no significant effect. Here we show for the first time that serotonergic neurons migrate by somal translocation mediated, in part, by dynamin.
Assuntos
Movimento Celular/fisiologia , Dinaminas/metabolismo , Epitélio/fisiologia , Matriz Extracelular/fisiologia , Neurônios/fisiologia , Serotonina/metabolismo , Fatores Etários , Animais , Proteínas de Bactérias/genética , Encéfalo/citologia , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Hidrazonas/farmacologia , Técnicas In Vitro , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Gravidez , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/genéticaRESUMO
The molecular architecture of developing serotonin (5HT) neurons is poorly understood, yet its determination is likely to be essential for elucidating functional heterogeneity of these cells and the contribution of serotonergic dysfunction to disease pathogenesis. Here, we describe the purification of postmitotic embryonic 5HT neurons by flow cytometry for whole-genome microarray expression profiling of this unitary monoaminergic neuron type. Our studies identified significantly enriched expression of hundreds of unique genes in 5HT neurons, thus providing an abundance of new serotonergic markers. Furthermore, we identified several hundred transcripts encoding homeodomain, axon guidance, cell adhesion, intracellular signaling, ion transport, and imprinted genes associated with various neurodevelopmental disorders that were differentially enriched in developing rostral and caudal 5HT neurons. These findings suggested a homeodomain code that distinguishes rostral and caudal 5HT neurons. Indeed, verification studies demonstrated that Hmx homeodomain and Hox gene expression defined an Hmx(+) rostral subtype and Hox(+) caudal subtype. Expression of engrailed genes in a subset of 5HT neurons in the rostral domain further distinguished two subtypes defined as Hmx(+)En(+) and Hmx(+)En(-). The differential enrichment of gene sets for different canonical pathways and gene ontology categories provided additional evidence for heterogeneity between rostral and caudal 5HT neurons. These findings demonstrate a deep transcriptome and biological pathway duality for neurons that give rise to the ascending and descending serotonergic subsystems. Our databases provide a rich, clinically relevant resource for definition of 5HT neuron subtypes and elucidation of the genetic networks required for serotonergic function.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/classificação , Neurônios/metabolismo , Serotonina/metabolismo , Fatores Etários , Análise de Variância , Animais , Proteínas de Bactérias/genética , Análise por Conglomerados , Biologia Computacional/métodos , Embrião de Mamíferos , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Rombencéfalo/citologiaRESUMO
Neonatal rodents deficient in medullary serotonin neurons have respiratory instability and enhanced spontaneous bradycardias. This study asks if, in Pet-1(-/-) mice over development: 1) the respiratory instability leads to hypoxia; 2) greater bradycardia is related to the degree of hypoxia or concomitant hypopnea; and 3) hyperthermia exacerbates bradycardias. Pet-1(+/+), Pet-1(+/-), and Pet-1(-/-) mice [postnatal days (P) 4-5, P11-12, P14-15] were held at normal body temperature (T(b)) and were then made 2 degrees C hypo- and hyperthermic. Using a pneumotach-mask system with ECG, we measured heart rate, metabolic rate (Vo(2)), and ventilation. We also calculated indexes for apnea-induced hypoxia (total hypoxia: apnea incidence x O(2) consumed during apnea = microl.g(-1).min(-1)) and bradycardia (total bradycardia: bradycardia incidence x magnitude = beats missed/min). Resting heart rate was significantly lower in all Pet-1(-/-) animals, irrespective of T(b). At P4-5, Pet-1(-/-) animals had approximately four- to eightfold greater total bradycardia (P < 0.001), owing to an approximately two- to threefold increase in bradycardia magnitude and a near doubling in bradycardia incidence. Pet-1(-/-) animals had a significantly reduced Vo(2) at all T(b); thus there was no genotype effect on total hypoxia. At P11-12, total bradycardia was nearly threefold greater in hyperthermic Pet-1(-/-) animals compared with controls (P < 0.01). In both genotypes, bradycardia magnitude was positively related to the degree of hypopnea (P = 0.02), but there was no genotype effect on degree of hypopnea or total hypoxia. At P14-15, genotype had no effect on total bradycardia, but Pet-1(-/-) animals had up to seven times more total hypoxia (P < 0.001), owing to longer and more frequent apneas and a normalized Vo(2). We infer from these data that 1) Pet-1(-/-) neonates are probably not hypoxic from respiratory dysfunction until P14-15; 2) neither apnea-related hypoxia nor greater hypopnea contribute to the enhanced bradycardias of Pet-1(-/-) neonates from approximately P4 to approximately P12; and 3) an enhancement of a temperature-sensitive reflex may contribute to the greater bradycardia in hyperthermic Pet-1(-/-) animals at approximately P12.
Assuntos
Apneia/fisiopatologia , Bradicardia/fisiopatologia , Febre/fisiopatologia , Insuficiência Respiratória/fisiopatologia , Serotonina/deficiência , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Apneia/genética , Apneia/patologia , Tamanho Corporal/fisiologia , Bradicardia/genética , Bradicardia/patologia , Tronco Encefálico/anormalidades , Modelos Animais de Doenças , Feminino , Febre/genética , Febre/patologia , Genótipo , Frequência Cardíaca/fisiologia , Humanos , Lactente , Masculino , Mesencéfalo/anormalidades , Camundongos , Camundongos Mutantes , Insuficiência Respiratória/genética , Insuficiência Respiratória/patologia , Morte Súbita do Lactente , Fatores de Transcrição/metabolismoRESUMO
Altered expression of the human FEV (fifth Ewing variant) ETS transcription factor gene impacts the level of CNS serotonin (5-HT) neuron gene expression and maternal nurturing. However, the regulatory mechanisms that determine FEV expression are poorly understood. Here, we investigated the cis-regulatory control of FEV to begin to identify the upstream transcription factors that restrict FEV expression to 5-HT neurons. We find that sequences extending only 275 bp upstream of the FEV 5' untranslated region are sufficient to direct FEV transgene expression to embryonic 5-HT neurons, although sequences farther upstream are required for maintenance in adult 5-HT neurons. Two highly conserved consensus GATA factor binding sites within the 275 bp region interact with GATA factors in vitro. Chromatin immunoprecipitations with embryonic hindbrain demonstrated Gata-2 interactions with the orthologous mouse Pet-1 ETS cis-regulatory region. Mutagenesis of GATA sites revealed that one or the other site is required for serotonergic FEV transgene expression. Unexpectedly, FEV-LacZ transgenes enabled determination of 5-HT neuron precursor fate in the adult Pet-1(-/-) dorsal and median raphe nuclei and thus provided additional insight into FEV/Pet-1 function. Comparable numbers of FEV-LacZ-positive cells were detected in Pet-1(+/-) and Pet-1(-/-) adult dorsal raphe nuclei, indicating that the majority of mutant serotonergic precursors are not fated to apoptosis. However, B7 dorsal raphe cells were aberrantly distributed, suggesting a role for FEV/Pet-1 in their midline organization. Our findings identify a direct transcriptional interaction between Gata-2 and FEV and a unique marker for new insight into FEV/Pet-1 function in 5-HT neuron development.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Transcrição GATA/genética , Imunoglobulinas/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Serotonina/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Regiões 5' não Traduzidas/genética , Animais , Apoptose/genética , Tronco Encefálico/citologia , Tronco Encefálico/embriologia , Tronco Encefálico/metabolismo , Moléculas de Adesão Celular , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reporter/genética , Humanos , Imunoglobulinas/genética , Óperon Lac/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neurogênese/genética , Núcleos da Rafe/citologia , Núcleos da Rafe/embriologia , Núcleos da Rafe/metabolismo , Elementos Reguladores de Transcrição/genética , Células-Tronco/citologia , Transcrição Gênica/genéticaRESUMO
Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1b(f/f/p)), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1b(f/f/p) mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b(f/f/p) mice rapidly became hypothermic when exposed to an ambient temperature of 4 degrees C, decreasing core temperature to 30 degrees C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO(2) chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO(2). We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.
Assuntos
Regulação da Temperatura Corporal/genética , Neurônios/fisiologia , Respiração/genética , Serotonina/deficiência , Serotonina/genética , Animais , Sistema Nervoso Central/fisiologia , Células Quimiorreceptoras/fisiologia , Camundongos , Camundongos Knockout , Serotonina/biossínteseRESUMO
The nicotinic acetylcholine receptor (nAChR) beta4/alpha3/alpha5 gene cluster encodes several heteromeric transmitter receptor subtypes that are essential for cholinergic synaptic transmission in adrenal gland, autonomic ganglia, pineal gland, and several nuclei in the central nervous system. However, the transcriptional mechanisms coordinating expression of these subunit genes in different cell populations are unknown. Here, we used transgenic methods to investigate long-range transcriptional control of the cluster. A 132-kb P1-derived artificial chromosome (PAC) encoding the rat cluster recapitulated the neurally- and endocrine-restricted expression patterns of the endogenous beta4/alpha3/alpha5 genes. Mutation of ETS factor binding sites in an enhancer, beta43', embedded in the beta4 3'-untranslated exon resulted in greatly diminished beta4, alpha3, and alpha5 expression in adrenal gland and to a lesser extent in the superior cervical ganglion (SCG) but not in other tissues. Phylogenetic sequence analyses revealed several conserved noncoding regions (CNRs) upstream of beta4 and alpha5. Deletion of one of them (CNR4) located 20 kb upstream of beta4 resulted in a dramatic decrease in beta4 and alpha3 expression in the pineal gland and SCG. CNR4 was sufficient to direct LacZ transgene expression to SCG neurons, which express the endogenous beta4alpha3alpha5 subunits, and pineal cells, which express the endogenous beta4alpha3 combination. Finally, CNR4 was able to direct transgene expression to major sites of expression of the endogenous cluster in the brain. Together, our findings support a model in which cell type-specific shared long-range regulatory elements are required for coordinate expression of clustered nAChR genes.
Assuntos
Família Multigênica , Isoformas de Proteínas , Receptores Nicotínicos , Sequências Reguladoras de Ácido Nucleico , Animais , Animais Geneticamente Modificados , Sequência de Bases , Cromossomos Artificiais de Mamíferos , Éxons , Genes Reporter , Humanos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/fisiologia , Glândula Pineal/citologia , Glândula Pineal/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Alinhamento de Sequência , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismo , Distribuição Tecidual , TransgenesRESUMO
Central serotonin (5-HT) orchestrates myriad cognitive processes and lies at the core of many stress-related psychiatric illnesses. However, the basic relationship between its brain-wide axonal projections and functional dynamics is not known. Here we combine optogenetics and fMRI to produce a brain-wide 5-HT evoked functional map. We find that DRN photostimulation leads to an increase in the hemodynamic response in the DRN itself, while projection areas predominately exhibit a reduction of cerebral blood volume mirrored by suppression of cortical delta oscillations. We find that the regional distribution of post-synaptically expressed 5-HT receptors better correlates with DRN 5-HT functional connectivity than anatomical projections. Our work suggests that neuroarchitecture is not the primary determinant of function for the DRN 5-HT. With respect to two 5-HT elevating stimuli, we find that acute stress leads to circuit-wide blunting of the DRN output, while the SSRI fluoxetine noticeably enhances DRN functional connectivity. These data provide fundamental insight into the brain-wide functional dynamics of the 5-HT projection system.
Assuntos
Córtex Cerebral/diagnóstico por imagem , Núcleo Dorsal da Rafe/diagnóstico por imagem , Fluoxetina/farmacologia , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Estresse Psicológico/metabolismo , Animais , Mapeamento Encefálico/métodos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Núcleo Dorsal da Rafe/efeitos dos fármacos , Núcleo Dorsal da Rafe/metabolismo , Núcleo Dorsal da Rafe/fisiopatologia , Potenciais Evocados Visuais/efeitos dos fármacos , Feminino , Imobilização , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Optogenética , Estimulação Luminosa , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Estresse Psicológico/fisiopatologiaRESUMO
Formation of long-range axons occurs over multiple stages of morphological maturation. However, the intrinsic transcriptional mechanisms that temporally control different stages of axon projection development are unknown. Here, we addressed this question by studying the formation of mouse serotonin (5-HT) axons, the exemplar of long-range profusely arborized axon architectures. We report that LIM homeodomain factor 1b (Lmx1b)-deficient 5-HT neurons fail to generate axonal projections to the forebrain and spinal cord. Stage-specific targeting demonstrates that Lmx1b is required at successive stages to control 5-HT axon primary outgrowth, selective routing, and terminal arborization. We show a Lmx1bâPet1 regulatory cascade is temporally required for 5-HT arborization and upregulation of the 5-HT axon arborization gene, Protocadherin-alphac2, during postnatal development of forebrain 5-HT axons. Our findings identify a temporal regulatory mechanism in which a single continuously expressed transcription factor functions at successive stages to orchestrate the progressive development of long-range axon architectures enabling expansive neuromodulation.
Assuntos
Axônios/fisiologia , Proteínas com Homeodomínio LIM/metabolismo , Neurônios Serotoninérgicos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Perfilação da Expressão Gênica , Proteínas com Homeodomínio LIM/deficiência , Camundongos , Prosencéfalo/citologia , Medula Espinal/citologia , Fatores de Transcrição/deficiênciaRESUMO
Aggressive behavior in our modern, civilized society is often counterproductive and destructive. Identifying specific proteins involved in the disease can serve as therapeutic targets for treating aggression. Here, we found that overexpression of RGS2 in explicitly serotonergic neurons augments male aggression in control mice and rescues male aggression in Rgs2-/- mice, while anxiety is not affected. The aggressive behavior is directly correlated to the immediate early gene c-fos induction in the dorsal raphe nuclei and ventrolateral part of the ventromedial nucleus hypothalamus, to an increase in spontaneous firing in serotonergic neurons and to a reduction in the modulatory action of Gi/o and Gq/11 coupled 5HT and adrenergic receptors in serotonergic neurons of Rgs2-expressing mice. Collectively, these findings specifically identify that RGS2 expression in serotonergic neurons is sufficient to drive male aggression in mice and as a potential therapeutic target for treating aggression.
Assuntos
Agressão/fisiologia , Proteínas RGS/metabolismo , Neurônios Serotoninérgicos/metabolismo , Potenciais de Ação , Animais , Ansiedade/metabolismo , Cálcio/metabolismo , Células Cultivadas , Depressão/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas RGS/genética , RNA Mensageiro/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serotonina/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismoRESUMO
The central serotonin (5-HT) neurotransmitter system is an important modulator of diverse physiological processes and behaviors; however, the transcriptional mechanisms controlling its development are largely unknown. The Pet-1 ETS factor is a precise marker of developing and adult 5-HT neurons and is expressed shortly before 5-HT appears in the hindbrain. Here we show that in mice lacking Pet-1, the majority of 5-HT neurons fail to differentiate. Remaining ones show deficient expression of genes required for 5-HT synthesis, uptake, and storage. Significantly, defective development of the 5-HT system is followed by heightened anxiety-like and aggressive behavior in adults. These findings indicate that Pet-1 is a critical determinant of 5-HT neuron identity and implicate a Pet-1-dependent program in serotonergic modulation of behavior.
Assuntos
Agressão/fisiologia , Ansiedade/genética , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neurônios/fisiologia , Serotonina/fisiologia , Fatores de Transcrição/genética , Alelos , Animais , Comportamento Animal/fisiologia , Monoaminas Biogênicas/biossíntese , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Cromatografia Líquida de Alta Pressão , Imuno-Histoquímica , Hibridização In Situ , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Sistema Nervoso/embriologia , Equilíbrio Postural/fisiologia , Rombencéfalo/fisiologia , Serotonina/biossíntese , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
A large body of literature has implicated serotonin [5-hydroxytryptamine (5-HT)] in descending modulation of nociceptive transmission. Here, we have studied the pain behavior of Lmx1b conditional knock-out mice (Lmx1b(f/f/p)), which lack 5-HT neurons in the CNS. Lmx1b(f/f/p) mutant mice showed normal thermal and visceral pain responses but were less sensitive to mechanical stimuli and exhibited enhanced inflammatory pain compared with their littermate control mice. Importantly, the analgesic effect of several antidepressant drugs, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and tricyclic antidepressants, was either abolished or greatly attenuated in Lmx1b(f/f/p) mice. Moreover, in the acute versus persistent pain settings, the analgesic actions of the SNRI duloxetine and the SSRI fluoxetine were differentially affected. Together, our results provide in vivo genetic evidence demonstrating that although the predominant role of the central 5-HT system in inflammatory pain is inhibitory, its role in acute mechanical pain is facilitatory. The findings that the analgesic effects of various antidepressant drugs are differentially dependent on the central 5-HT system should help us to understand the mechanism of the analgesic action of different classes of antidepressants in the management of persistent pain.