RESUMEN
Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via ß3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.
Asunto(s)
Movimiento Celular , Colinérgicos/farmacología , Ritmo Circadiano , Células Madre Hematopoyéticas/fisiología , Leucocitos/fisiología , Sistema Nervioso Parasimpático/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Adhesión Celular , Células Cultivadas , Quimiotaxis , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Femenino , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 3/fisiología , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an â¼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.
Asunto(s)
Membrana Celular/metabolismo , Hipocampo/metabolismo , Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Receptores de Ácido Kaínico/metabolismo , Simportadores/metabolismo , Animales , Membrana Celular/genética , Células Cultivadas , Hipocampo/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Neuronas/citología , Receptores de Ácido Kaínico/genética , Simportadores/genética , Cotransportadores de K Cl , Receptor de Ácido Kaínico GluK2RESUMEN
KCC2 is the neuron-specific member of the of K(+)-Cl(-) cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl(-) from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl(-), which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K(+)-Cl(-) cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl(-) through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl(-) regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K(+)-Cl(-) cotransport in neurons.
Asunto(s)
Simportadores/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Transporte Biológico Activo/fisiología , Tamaño de la Célula , Cloruros/metabolismo , ADN/genética , ADN/aislamiento & purificación , Femenino , Hipocampo/citología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Transporte Iónico , Microscopía Confocal , Oocitos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Xenopus , Cotransportadores de K ClRESUMEN
Efferent signals from the vagus nerve are thought to mediate both basal and meal-induced gastric acid secretion, and provide trophic support of the mucosa. However, the underlying mechanisms are incompletely understood. Neurturin, signalling via glial cell line-derived neurotrophic factor (GDNF)-family receptor α2 (GFRα2), is essential for parasympathetic innervation of many target tissues but its role in gastric innervation is unknown. Here we show that most nerve fibres in wild-type mouse gastric mucosa, including all positive for gastrin-releasing peptide, are cholinergic. GFRα2-deficient (KO) mice lacked virtually all cholinergic nerve fibres and associated glial cells in the gastric (oxyntic and pyloric) mucosa but not in the smooth muscle, consistent with the selective expression of neurturin mRNA in the gastric mucosa. 2-Deoxyglucose and hexamethonium failed to affect acid secretion in the GFRα2-KO mice indicating the lack of functional innervation in gastric mucosa. Interestingly, basal and maximal histamine-induced acid secretion did not differ between wild-type and GFRα2-KO mice. Moreover, circulating gastrin levels in both fasted and fed animals, thickness of gastric mucosa, and density of parietal and different endocrine cells were similar. Carbachol-stimulated acid secretion was higher in GFRα2-KO mice, while atropine reduced basal secretion similarly in both genotypes. We conclude that cholinergic innervation of gastric mucosa depends on neurturin-GFRα2 signalling but is dispensable for gastrin secretion and for basal and maximal acid output. Basal acid secretion in the KO mice appears to be, at least partly, facilitated by constitutive activity of muscarinic receptors.
Asunto(s)
Ácido Gástrico/metabolismo , Mucosa Gástrica/inervación , Gastrinas/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Animales , Neuronas Colinérgicas/fisiología , Femenino , Gastrinas/sangre , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Noqueados , Neuroglía/fisiología , Neurturina/fisiologíaRESUMEN
A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA(A)-mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factores de Transcripción de la Respuesta de Crecimiento Precoz/fisiología , Simportadores/biosíntesis , Animales , Sitios de Unión , Células Cultivadas , Hipocampo/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Neuronas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transducción de Señal , Simportadores/genética , Transcripción Genética , Cotransportadores de K ClRESUMEN
The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.
Asunto(s)
Citoesqueleto/fisiología , Dendritas/ultraestructura , Espinas Dendríticas/fisiología , Neuronas/citología , Simportadores/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Proteínas del Citoesqueleto , Dendritas/metabolismo , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Técnicas In Vitro , Lisina/análogos & derivados , Lisina/metabolismo , Proteínas de la Membrana , Ratones , Ratones Noqueados , Mutación/fisiología , Proteínas del Tejido Nervioso , Neuropéptidos , Técnicas de Placa-Clamp/métodos , Simportadores/deficiencia , Transmisión Sináptica/fisiología , Transfección/métodos , Cotransportadores de K ClRESUMEN
The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.
Asunto(s)
Factores de Transcripción de la Respuesta de Crecimiento Precoz/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/metabolismo , Neurturina/fisiología , Simportadores/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Factores de Transcripción de la Respuesta de Crecimiento Precoz/fisiología , Hipocampo/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/fisiología , Técnicas de Cultivo de Órganos , Simportadores/fisiología , Cotransportadores de K ClRESUMEN
Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Cloruros/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Receptor trkB/metabolismo , Simportadores/metabolismo , Animales , Regulación hacia Abajo/efectos de los fármacos , Electrofisiología , Hipocampo/citología , Transporte Iónico , Excitación Neurológica/efectos de los fármacos , Excitación Neurológica/fisiología , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor trkB/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Cotransportadores de K ClRESUMEN
The neuron-specific K-Cl cotransporter (KCC2) maintains a low intracellular Cl(-) concentration in neurons and is necessary for fast hyperpolarizing responses to GABA and glycine. The mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. Expression of the major isoform, KCC2b, is strongly upregulated during neuronal maturation, and is modulated by neuronal activity, trauma, and neurotrophic factors. In the present study, we have focused on the regulatory influence of the upstream stimulating factors USF1 and USF2 via an E-box control element in the KCC2b promoter (E-boxKCC2b). Electrophoretic mobility shift assay in cell lines and chromatin immunoprecipitation in neurons demonstrated binding of endogenous USF1 and USF2 to the E-box(KCC2b) element. Mutation of the E-boxKCC2b site resulted in reduced KCC2b promoter activity in cell lines and cortical neurons. Overexpression of a dominant-negative form of USF confirmed the involvement of endogenous USF proteins in the regulation of the KCC2b gene. The results suggest that binding of USF proteins to the E-boxKCC2b may contribute to the upregulation of KCC2b gene expression in developing brain.
Asunto(s)
Elementos E-Box/fisiología , Neuronas/metabolismo , Simportadores/metabolismo , Regulación hacia Arriba/fisiología , Factores Estimuladores hacia 5'/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratas , Simportadores/genética , Transfección , Cotransportadores de K ClRESUMEN
In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Cl- concentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/- mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.
Asunto(s)
Neuronas/metabolismo , Simportadores/metabolismo , Animales , Tronco Encefálico/metabolismo , Bulbo Raquídeo/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Parto/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Frecuencia Respiratoria , Simportadores/genética , Cotransportadores de K ClRESUMEN
To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19+ progenitor compartment.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Compartimento Celular , Linfopoyesis , Células Madre/citología , ADP-Ribosil Ciclasa/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos Ly/metabolismo , Médula Ósea/metabolismo , Linaje de la Célula , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Ratones , Modelos BiológicosRESUMEN
Most unmyelinated nociceptive neurons that mediate pain and temperature sensation from the skin bind isolectin B4 (IB4)-lectin and express Ret, the common signaling component of glial cell line-derived neurotrophic factor (GDNF) family. One of these factors, neurturin, is expressed in the epidermis, whereas its GDNF family receptor alpha2 (GFRalpha2) is expressed in the majority of unmyelinated Ret-positive sensory neurons. However, the physiological roles of endogenous neurturin signaling in primary sensory neurons are poorly understood. Here, we show that the vast majority (approximately 85%) of IB4 binding and P2X3 purinoreceptor-positive neurons, but virtually none of the calcitonin gene-related peptide (CGRP) or vanilloid receptor transient receptor potential vanilloid 1-positive neurons in mouse dorsal root ganglion (DRG) express GFRalpha2. In GFRalpha2 knock-out (KO) mice, the IB4-binding and P2X3-positive DRG neurons were present but reduced in size, consistent with normal number but reduced caliber of unmyelinated axons in a cutaneous nerve. Strikingly, nonpeptidergic (CGRP-negative) free nerve endings in footpad epidermis were >70% fewer in GFRalpha2-KO mice than in their wild-type littermates. In contrast, the density of CGRP-positive epidermal innervation remained unaffected. In the formalin test, the KO mice showed a normal acute response but a markedly attenuated persistent phase, indicating a deficit in inflammatory pain response. Behavioral responses of GFRalpha2-KO mice to innocuous warm and noxious heat were not blunted; the mice were actually markedly hypersensitive to noxious cold in tail immersion test. Overall, our results indicate a critical role for endogenous GFRalpha2 signaling in maintaining the size and terminal innervation of the nonpeptidergic class of cutaneous nociceptors in vivo.
Asunto(s)
Epidermis/inervación , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Inflamación/fisiopatología , Neuroglía/fisiología , Animales , Recuento de Células , Línea Celular , Ganglios Espinales/fisiología , Ganglios Espinales/fisiopatología , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/deficiencia , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Calor , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/fisiología , Dolor/fisiopatologíaRESUMEN
The expression of the neuron-specific K+/Cl- cotransporter (KCC2) is restricted to the CNS and is strongly upregulated during neuronal maturation, yielding a low intracellular chloride concentration that is required for fast synaptic inhibition in adult neurons. To elucidate the mechanisms of KCC2 gene regulation, we analyzed the KCC2 (alias Slc12a5) promoter and proximal intron-1 regions and revealed 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. Here we focus on one of these factors, early growth response 4 (Egr4), which shows a similar developmental upregulation in CNS neurons as KCC2. KCC2 luciferase reporter constructs containing the Egr4 site (Egr4(KCC2)) were strongly induced by Egr4 overexpression in neuro-2a neuroblastoma cells and in cultured neurons. Egr4-mediated induction was decreased significantly by point-mutating the Egr4(KCC2). Insertion of Egr4(KCC2) into the KCC2 basal promoter in the endogenous reverse, but not in the opposite, orientation reestablished Egr4-mediated induction. Electrophoretic mobility shift assay confirmed specific Egr4 binding to Egr4(KCC2). Interference RNA-mediated knock-down of Egr4 and a dominant-negative isoform of Egr4 significantly inhibited KCC2 reporter induction and endogenous KCC2 expression in cultured neurons. Together, the results indicate an important role for Egr4 in the developmental upregulation of KCC2 gene expression.
Asunto(s)
Cloruros/fisiología , Factores de Transcripción de la Respuesta de Crecimiento Precoz/biosíntesis , Neuronas/metabolismo , Potasio/fisiología , Simportadores/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Secuencia de Bases , Sitios de Unión/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Línea Celular Tumoral , Cloruros/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Pan troglodytes , Mutación Puntual , Potasio/metabolismo , Ratas , Simportadores/antagonistas & inhibidores , Simportadores/genética , Simportadores/metabolismo , Cotransportadores de K ClRESUMEN
Subsets of parasympathetic and enteric neurons require neurturin signaling via glial cell line-derived neurotrophic factor family receptor alpha2 (GFRalpha2) for development and target innervation. Why GFRalpha2-deficient (Gfra2-/-) mice grow poorly has remained unclear. Here, we analyzed several factors that could contribute to the growth retardation. Neurturin mRNA was localized in the gut circular muscle. GFRalpha2 protein was expressed in most substance P-containing myenteric neurons, in most intrapancreatic neurons, and in surrounding glial cells. In the Gfra2-/- mice, density of substance P-containing myenteric ganglion cells and nerve bundles in the myenteric ganglion cell layer was significantly reduced, and transit of test material through small intestine was 25% slower compared to wild-type mice. Importantly, the knockout mice had approximately 80% fewer intrapancreatic neurons, severely impaired cholinergic innervation of the exocrine but not the endocrine pancreas, and increased fecal fat content. Vagally mediated stimulation of pancreatic secretion by 2-deoxy-glucose in vivo was virtually abolished. Retarded growth of the Gfra2-/- mice was accompanied by reduced fat mass and elevated basal metabolic rate. Moreover, the knockout mice drank more water than wild-type controls, and wet-mash feeding resulted in partial growth rescue. Taken together, the results suggest that the growth retardation in mice lacking GFRalpha2 is largely due to impaired salivary and pancreatic secretion and intestinal dysmotility.
Asunto(s)
Motilidad Gastrointestinal , Trastornos del Crecimiento/etiología , Intestinos/inervación , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Femenino , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/genética , Neurturina , Trastornos Nutricionales/etiología , Páncreas/inervación , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/análisis , Saliva/metabolismo , Sustancia P/análisis , Sustancia P/fisiología , Nervio Vago/fisiologíaRESUMEN
GABA(A) receptors mediate both fast phasic inhibitory postsynaptic potentials and slower tonic extrasynaptic inhibition. Hyperpolarizing phasic GABAergic inhibition requires the activity of neuron-specific chloride-extruding potassium-chloride cotransporter KCC2 in adult CNS. However, the possible role of KCC2 in tonic GABAergic inhibition and the associated behaviors is unknown. Here, we have examined the role of KCC2 in phasic vs tonic GABA inhibition by measuring the behavioral effects of pharmacological agents that presumably enhance phasic vs tonic inhibition in mice that retain 15-20% of normal KCC2 protein levels. These KCC2-deficient mice show decreased sensitivity to diazepam-induced sedation and motor impairment consistent with the reported role for KCC2 in fast hyperpolarizing inhibition. In contrast, the mice exhibit normal responses to low-dose alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis; behaviors thought to be associated with tonic GABAergic inhibition. Electrophysiological recordings show that the tonic conductance is not affected. The results suggest that KCC2 activity is more critical for behaviors dependent on phasic than tonic GABAergic inhibition.
Asunto(s)
Anticonvulsivantes/farmacología , Depresores del Sistema Nervioso Central/farmacología , Diazepam/farmacología , Etanol/farmacología , Pérdida de Tono Postural/fisiología , Isoxazoles/farmacología , Simportadores/deficiencia , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Conducta Animal/efectos de los fármacos , Conducta Exploratoria/efectos de los fármacos , Hipocampo/citología , Hipocampo/fisiología , Hipocampo/efectos de la radiación , Pérdida de Tono Postural/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos de la Destreza Motora/inducido químicamente , Trastornos de la Destreza Motora/tratamiento farmacológico , Neuronas/efectos de los fármacos , Cotransportadores de K ClRESUMEN
The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2 pan antibody but not by the N-terminal KCC2a antibody in KCC2b-deficient neurons. One possible cause for the epitope masking is the binding site of Ste20-related proline-alanine-rich kinase (SPAK) in the KCC2a N-terminus. SPAK, a known regulator of K-Cl cotransporters, was co-immunoprecipitated in a complex with KCC2a but not KCC2b isoform. Moreover, SPAK overexpression decreased the transport activity of KCC2a but not that of KCC2b, as revealed by rubidium flux assay in HEK293 cells. Thus, our data indicate that both KCC2 isoforms perform as chloride cotransporters in neuronal cells, while their N-terminal heterogeneity could play an important role in fine-tuning of the K-Cl transport activity.
Asunto(s)
Neuronas/fisiología , Simportadores/fisiología , Secuencia de Aminoácidos , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/fisiología , Ratas , Cotransportadores de K ClRESUMEN
Glial cell line-derived neurotrophic factor family receptor (GFRalpha) 4, the binding receptor for persephin, is coexpressed with the signaling Ret receptor tyrosine kinase predominantly in thyroid calcitonin-producing C cells. We show by in situ hybridization and immunohistochemistry that the functional, glycolipid-anchored form of GFRalpha4 is produced in mouse only in the C cells but not in parathyroid gland or in the brain. C cells expressed functional GFRalpha4 throughout postnatal development, whereas Ret expression in these cells decreased postnatally and was undetectable in adults. To understand the physiological role of GFRalpha4, we produced GFRalpha4-deficient [knockout (KO)] mice. No differences were observed between wild-type and GFRalpha4-KO littermate animals in growth, gross behavior, or viability. The number and morphology of the thyroid C cells were indistinguishable between the genotypes in both newborn and adult age. However, thyroid tissue calcitonin content was reduced by 60% in newborn and by 45% in 3-wk-old GFRalpha4-KO mice compared with wild-type controls. In contrast, thyroid calcitonin levels were similar in adult animals. Consistent with the reduced calcitonin levels, bone formation rate in juvenile GFRalpha4-KO mice was increased. In conclusion, this study indicates a novel role for endogenous GFRalpha4 signaling in regulating calcitonin production in thyroid C cells of young mice.
Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Receptores de Calcitonina/metabolismo , Glándula Tiroides/metabolismo , Animales , Huesos/metabolismo , Encéfalo/metabolismo , Calcitonina/metabolismo , Línea Celular , Cromosomas Artificiales Bacterianos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos , Genotipo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Modelos Estadísticos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/citología , Factores de Tiempo , Distribución TisularRESUMEN
Vagal parasympathetic input to the islets of Langerhans is a regulator of islet hormone secretion, but factors promoting parasympathetic islet innervation are unknown. Neurturin signaling via glial cell line-derived neurotrophic factor family receptor alpha2 (GFRalpha2) has been demonstrated to be essential for the development of subsets of parasympathetic and enteric neurons. Here, we show that the parasympathetic nerve fibers and glial cells within and around the islets express GFRalpha2 and that islet parasympathetic innervation in GFRalpha2 knockout (KO) mice is reduced profoundly. In wild-type mice, neuroglucopenic stress produced a robust increase in plasma levels of islet hormones. In the GFRalpha2-KO mice, however, pancreatic polypeptide and insulin responses were completely lost and glucagon response was markedly impaired. Islet morphology and sympathetic innervation, as well as basal secretions of the islet hormones, were unaffected. Moreover, a glucose tolerance test failed to reveal differences between the genotypes, indicating that direct glucose-stimulated insulin secretion was not affected by GFRalpha2 deficiency. These results show that GFRalpha2 signaling is needed for development of the parasympathetic islet innervation that is critical for vagally induced hormone secretion. The GFRalpha2-KO mouse represents a useful model to study the role of parasympathetic innervation of the endocrine pancreas in glucose homeostasis.
Asunto(s)
Islotes Pancreáticos/fisiología , Neuroglía/fisiología , Sistema Nervioso Parasimpático/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Glucemia/metabolismo , Línea Celular , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Prueba de Tolerancia a la Glucosa , Islotes Pancreáticos/inervación , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Polipéptido Pancreático/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Nervio Vago/fisiologíaRESUMEN
Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.
Asunto(s)
Neuronas Motoras/fisiología , Músculo Liso/fisiología , Neuronas/fisiología , Pezones/fisiología , Piloerección/fisiología , Animales , Diferenciación Celular/fisiología , Femenino , Ganglios Simpáticos/fisiología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.