RESUMO
The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.
Assuntos
Sistema Nervoso Entérico/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Tretinoína/farmacologia , Animais , Linhagem Celular , Humanos , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Nervo Vago/citologiaRESUMO
The jugular ganglion (JG) contains sensory neurons of the vagus nerve which innervate somatic and visceral structures in cranial and cervical regions. In this study, the number of sensory neurons in the human JG was investigated. And, the morphology of sensory neurons in the human JG and nodose ganglion (NG) was compared. The estimated number of JG neurons was 2721.8-9301.1 (average number of sensory neurons⯱â¯S.D.â¯=â¯7975.1⯱â¯3312.8). There was no significant difference in sizes of the neuronal cell body and nucleus within the JG (cell body, 1128.8⯱â¯99.7 µâ¯m2; nucleus, 127.7⯱â¯20.8 µâ¯m2) and NG (cell body, 963.8⯱â¯225.7 µâ¯m2; nucleus, 123.2⯱â¯32.3 µâ¯m2). These findings indicate that most of sensory neurons show the similar morphology in the JG and NG. Our immunohistochemical method also demonstrated the distribution of ion channels, neurotransmitter agents and calcium-binding proteins in the human JG. Numerous JG neurons were immunoreactive for transient receptor potential cation channel subfamily V member 1 (TRPV1, mean⯱â¯SDâ¯=â¯19.9⯱â¯11.5 %) and calcitonin gene-related peptide (CGRP, 28.4⯱â¯6.7 %). A moderate number of JG neurons contained TRPV2 (12.0⯱â¯4.7 %), substance P (SP, 15.7⯱â¯6.9 %) and secreted protein, acidic and rich in cysteine-like 1 (SPARCL1, 14.6⯱â¯7.4 %). A few JG neurons had vesicular glutamate transporter 2 (VGLUT2, 5.6⯱â¯2.9 %) and parvalbumin (PV, 2.3⯱â¯1.4 %). SP- and TRPV2-containing JG neurons had mainly small and medium-sized cell bodies, respectively. TRPV1- and VGLUT2- containing JG neurons were small to medium-sized. CGRP- and SPARCL1-containing JG neurons were of various cell body sizes. Sensory neurons in the human JG were mostly free of vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY). In the external auditory canal skin, subepithelial nerve fibers contained TRPV1, TRPV2, SP, CGRP and VGLUT2. Perivascular nerve fibers also had TRPV1, TRPV2, SP, CGRP, VIP, NPY and TH. However, PV- and SPARCL1-containing nerve endings could not be seen in the external auditory canal. It is likely that sensory neurons in the human JG can transduce nociceptive and mechanoreceptive information from the external auditory canal. Theses neurons may be also associated with neurogenic inflammation in the external auditory canal and ear-cough reflex through the vagus nerve.
Assuntos
Gânglios , Neuropeptídeos/metabolismo , Canais de Cátion TRPV/metabolismo , Idoso , Autopsia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Meato Acústico Externo/citologia , Meato Acústico Externo/metabolismo , Feminino , Gânglios/citologia , Gânglios/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neurotransmissores/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Substância P/metabolismo , Nervo Vago/citologia , Nervo Vago/metabolismo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Vagal afferents innervating the small intestinal mucosa regulate feeding, gastrointestinal (GI) digestive, and immune functions. Their anatomical-functional characterization has been impeded by the inability to selectively label and manipulate them. Nav 1.8-Cre-tdTomato mice label 80% of nodose and dorsal root ganglia neurons. Here, the origin of these neuron's terminals and their distribution in the small intestinal mucosa were examined by quantitatively comparing tdTomato-labeled innervation in nonoperated (control), subdiaphragmatic vagotomy (VAGX), and sham-operated mice. Control mice exhibited a large proximal-to-distal decrease and a moderate mesentery-to-antimesentery decrease in villus innervation. VAGX reduced this innervation to a greater degree proximally (91-93%) than distally (65-72%), resulting in flat proximal-distal distributions. Therefore, estimates of vagal villus afferent distributions (control minus VAGX) paralleled control distributions, but were slightly reduced in magnitude. Compared with villus afferents, crypt innervation exhibited a muted proximal-to-distal decrease in control mice and a smaller loss after VAGX (45-48%). Sham-operated mice exhibited similar distributions of villus and crypt afferents as control mice, suggesting surgery did not contribute to the effects of VAGX. Most crypt and villus afferent terminals along the entire proximal-distal small intestinal axis had similar morphology to those previously reported in the proximal duodenum, but the density of terminal branches varied. Our findings suggest the majority of small intestinal mucosal innervation labeled in Nav 1.8-Cre-tdTomato mice is vagal in origin. Therefore, these mice will be valuable for studying vagal mucosal afferent morphology, interactions with other GI elements, plasticity, and function.
Assuntos
Mucosa Intestinal/inervação , Intestino Delgado/inervação , Neurônios Aferentes/citologia , Nervo Vago/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , VagotomiaRESUMO
Deletion of the leptin receptor from vagal afferent neurons (VAN) using a conditional deletion (Nav1.8/LepRfl/fl) results in an obese phenotype with increased food intake and lack of exogenous cholecystokinin (CCK)-induced satiation in male mice. Female mice are partially protected from weight gain and increased food intake in response to ingestion of high-fat (HF) diets. However, whether the lack of leptin signaling in VAN leads to an obese phenotype or disruption of hypothalamic-pituitary-gonadal axis function in female mice is unclear. Here, we tested the hypothesis that leptin signaling in VAN is essential to maintain estrogen signaling and control of food intake, energy expenditure, and adiposity in female mice. Female Nav1.8/LepRfl/fl mice gained more weight, had increased gonadal fat mass, increased meal number in the dark phase, and increased total food intake compared with wild-type controls. Resting energy expenditure was unaffected. The decrease in food intake produced by intraperitoneal injection of CCK (3 µg/kg body wt) was attenuated in female Nav1.8/LepRfl/fl mice compared with wild-type controls. Intraperitoneal injection of ghrelin (100 µg/kg body wt) increased food intake in Nav1.8/LepRfl/fl mice but not in wild-type controls. Ovarian steroidogenesis was suppressed, resulting in decreased plasma estradiol, which was accompanied by decreased expression of estrogen receptor-1 (Esr1) in VAN but not in the hypothalamic arcuate nucleus. These data suggest that the absence of leptin signaling in VAN is accompanied by disruption of estrogen signaling in female mice, leading to an obese phenotype possibly via altered control of feeding behavior.
Assuntos
Ingestão de Alimentos/genética , Comportamento Alimentar/fisiologia , Neurônios Aferentes/metabolismo , Obesidade/genética , Receptores para Leptina/genética , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/genética , Colecistocinina/farmacologia , Dieta Hiperlipídica , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Feminino , Grelina/farmacologia , Camundongos , Obesidade/metabolismo , Saciação , Nervo Vago/citologia , Aumento de Peso/genéticaRESUMO
The nervous system maintains physiological homeostasis through reflex pathways that modulate organ function. This process begins when changes in the internal milieu (e.g., blood pressure, temperature, or pH) activate visceral sensory neurons that transmit action potentials along the vagus nerve to the brainstem. IL-1ß and TNF, inflammatory cytokines produced by immune cells during infection and injury, and other inflammatory mediators have been implicated in activating sensory action potentials in the vagus nerve. However, it remains unclear whether neural responses encode cytokine-specific information. Here we develop methods to isolate and decode specific neural signals to discriminate between two different cytokines. Nerve impulses recorded from the vagus nerve of mice exposed to IL-1ß and TNF were sorted into groups based on their shape and amplitude, and their respective firing rates were computed. This revealed sensory neural groups responding specifically to TNF and IL-1ß in a dose-dependent manner. These cytokine-mediated responses were subsequently decoded using a Naive Bayes algorithm that discriminated between no exposure and exposures to IL-1ß and TNF (mean successful identification rate 82.9 ± 17.8%, chance level 33%). Recordings obtained in IL-1 receptor-KO mice were devoid of IL-1ß-related signals but retained their responses to TNF. Genetic ablation of TRPV1 neurons attenuated the vagus neural signals mediated by IL-1ß, and distal lidocaine nerve block attenuated all vagus neural signals recorded. The results obtained in this study using the methodological framework suggest that cytokine-specific information is present in sensory neural signals within the vagus nerve.
Assuntos
Interleucina-1beta/farmacologia , Receptores Tipo I de Interleucina-1/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Nervo Vago/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Teorema de Bayes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacosRESUMO
The non-neuronal, immunological effects of the cholinergic signaling are exerted on the system's scale of observation via the vagus nerve and on the cellular scale via α7 nicotinic acetylcholine receptor (nAChR) signaling in myeloid cells of the periphery or brain's microglia and astrocytes. The developmental effects of such multi-scale signaling can be conceived of as an example of psychoneuroimmunological (PNI) homeokinesis and, while reported in the literature, are not yet systematically well studied. To be better understood, the intricacy of the multi-scale interactions requires relevant preclinical animal models. Chronically instrumented non-anesthetized fetal sheep model comes with a strong track record of bench-to-bed translation and a large body of evidence for its strong resemblance to and relevance for human physiology on various scales of organization. Recently, there has been growing interest in pleiotropic effects of vagus nerve stimulation (VNS) on various organ systems such as innate immunity, metabolism, and emotion with implications for programming of PNI phenotype. Here we describe the procedures required to record and manipulate the vagus nerve activity in this large pregnant mammalian organism. Extending this in vivo model to in vitro, on the cellular scale, we present the method to manipulate the cholinergic signaling in ovine fetal microglia and astrocytes and analyze their responses on protein and RNA levels. Together these models can provide multi-scale-level mechanistic insights into the effects of cholinergic signaling on PNI phenotype.
Assuntos
Acetilcolina/metabolismo , Feto/metabolismo , Microglia/metabolismo , Psiconeuroimunologia/métodos , Estimulação do Nervo Vago/métodos , Nervo Vago/metabolismo , Animais , Colinérgicos/farmacologia , Feminino , Feto/citologia , Feto/efeitos dos fármacos , Microglia/citologia , Microglia/efeitos dos fármacos , Ovinos , Transdução de Sinais , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.
Assuntos
Sistema Nervoso Entérico/citologia , Gânglios Simpáticos/citologia , Trato Gastrointestinal/embriologia , Crista Neural/citologia , Neuregulina-1/genética , Receptor ErbB-3/genética , Células de Schwann/citologia , Animais , Embrião de Galinha , Trato Gastrointestinal/inervação , Doença de Hirschsprung/genética , Camundongos , Neuregulina-1/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Receptor ErbB-3/metabolismo , Nervo Vago/citologiaRESUMO
The pituitary adenylyl cyclase-activating polypeptide (PACAP) and its G protein-coupled receptors, PAC1, VPAC1 and VPAC2 form a system involved in a variety of biological processes. Although some sympathetic stimulatory effects of this system have been reported, its central cardiovascular regulatory properties are poorly characterized. VPAC1 receptors are expressed in the nucleus ambiguus (nAmb), a key center controlling cardiac parasympathetic tone. In this study, we report that selective VPAC1 activation in rhodamine-labeled cardiac vagal preganglionic neurons of the rat nAmb produces inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization, membrane depolarization and activation of P/Q-type Ca2+ channels. In vivo, this pathway converges onto transient reduction in heart rate of conscious rats. Therefore we demonstrate a VPAC1-dependent mechanism in the central parasympathetic regulation of the heart rate, adding to the complexity of PACAP-mediated cardiovascular modulation.
Assuntos
Bulbo/metabolismo , Neurônios/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bradicardia/induzido quimicamente , Bradicardia/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Masculino , Bulbo/citologia , Bulbo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Rastreamento Neuroanatômico , Neurônios/citologia , Neurônios/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Ratos Sprague-Dawley , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/metabolismoRESUMO
KEY POINTS: Heart Failure (HF) is accompanied by reduced ventricular function, activation of compensatory neurohormonal mechanisms and marked autonomic dysfunction characterized by exaggerated sympathoexcitation and reduced parasympathetic activity. With 6 weeks of exercise training, HF-related loss of choline acetyltransferase (ChAT)-positive vagal preganglionic neurones is avoided, restoring the parasympathetic tonus to the heart, and the immunoreactivity of dopamine ß-hydroxylase-positive premotor neurones that drive sympathetic outflow to the heart is reduced. Training-induced correction of autonomic dysfunction occurs even with the persistence of abnormal ventricular function. Strong positive correlation between improved parasympathetic tonus to the heart and increased ChAT immunoreactivity in vagal preganglionic neurones after training indicates this is a crucial mechanism to restore autonomic function in heart failure. ABSTRACT: Exercise training is an efficient tool to attenuate sympathoexcitation, a hallmark of heart failure (HF). Although sympathetic modulation in HF is widely studied, information regarding parasympathetic control is lacking. We examined the combined effects of sympathetic and vagal tonus to the heart in sedentary (Sed) and exercise trained (ET) HF rats and the contribution of respective premotor and preganglionic neurones. Wistar rats submitted to coronary artery ligation or sham surgery were assigned to training or sedentary protocols for 6 weeks. After haemodynamic, autonomic tonus (atropine and atenolol i.v.) and ventricular function determinations, brains were collected for immunoreactivity assays (choline acetyltransferase, ChATir; dopamine ß-hydroxylase, DBHir) and neuronal counting in the dorsal motor nucleus of vagus (DMV), nucleus ambiguus (NA) and rostroventrolateral medulla (RVLM). HF-Sed vs. SHAM-Sed exhibited decreased exercise capacity, reduced ejection fraction, increased left ventricle end diastolic pressure, smaller positive and negative dP/dt, decreased intrinsic heart rate (IHR), lower parasympathetic and higher sympathetic tonus, reduced preganglionic vagal neurones and ChATir in the DMV/NA, and increased RVLM DBHir. Training increased treadmill performance, normalized autonomic tonus and IHR, restored the number of DMV and NA neurones and corrected ChATir without affecting ventricular function. There were strong positive correlations between parasympathetic tonus and ChATir in NA and DMV. RVLM DBHir was also normalized by training, but there was no change in neurone number and no correlation with sympathetic tonus. Training-induced preservation of preganglionic vagal neurones is crucial to normalize parasympathetic activity and restore autonomic balance to the heart even in the persistence of cardiac dysfunction.
Assuntos
Fibras Autônomas Pré-Ganglionares/fisiologia , Insuficiência Cardíaca/fisiopatologia , Neurônios/fisiologia , Condicionamento Físico Animal , Nervo Vago/fisiologia , Animais , Pressão Sanguínea , Coração/inervação , Frequência Cardíaca , Masculino , Ratos , Ratos Wistar , Nervo Vago/citologiaRESUMO
AIM: To investigate the expression of CD56 in dog odontogenesis in order to elucidate the expression found in ameloblastomas. MATERIALS AND METHODS: Immunohistochemical analysis of CD56 expression of developing dog teeth in the bud, cap and bell stages including the remnants of the dental lamina. RESULTS: Weak CD56 expression was observed in the dental epithelium during the bud stage with intense staining of certain peripheral epithelial cells. Positive staining of epithelial cells was also observed in the cap stage with intense staining of the inner enamel epithelium at this stage. During the bell stage the staining was concentrated on the cervical loop areas. The dental papilla revealed positive staining throughout the cap and bell stages while the dental follicle stained intensely positive throughout all the phases examined. The dental lamina and Serres rests also stained positive for CD56. CONCLUSIONS: The expression of CD56 in dog odontogenic tissue varies according to the stage of tooth development. There is a positive correlation between the positive staining observed in ameloblastomas and their odontogenic cells of origin.
Assuntos
Antígeno CD56/biossíntese , Cães/crescimento & desenvolvimento , Cães/metabolismo , Odontogênese/fisiologia , Ameloblastoma/metabolismo , Ameloblastoma/veterinária , Animais , Diferenciação Celular/fisiologia , Papila Dentária/citologia , Papila Dentária/metabolismo , Órgão do Esmalte/citologia , Órgão do Esmalte/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Imuno-Histoquímica/veterinária , Germe de Dente/citologia , Germe de Dente/metabolismo , Nervo Vago/citologia , Nervo Vago/metabolismoRESUMO
Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios Motores/metabolismo , Nervo Vago/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Nervo Vago/citologia , Nervo Vago/fisiologiaRESUMO
The main goal of our research was to study the possible alterations of the chemical coding of the dorsal motor vagal nucleus (DMX) neurons projecting to the porcine stomach prepyloric region following prolonged acetylsalicylic acid supplementation. Fast Blue (FB) was injected into the studied area of the stomach. Since the seventh day following the FB injection, acetylsalicylic acid (ASA) was given orally to the experimental gilts. All animals were euthanized on the 28th day after FB injection. Medulla oblongata sections were then processed for double-labeling immunofluorescence for choline acetyltransferase (ChAT), pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the control DMX, only PACAP was observed in 30.08 ± 1.97 % of the FB-positive neurons, while VIP, NOS, GAL, SP, LENK, and CART were found exclusively in neuronal processes running between FB-labeled perikarya. In the ASA DMX, PACAP was revealed in 49.53 ± 5.73 % of traced vagal perikarya. Moreover, we found de novo expression of VIP in 40.32 ± 7.84 %, NOS in 25.02 ± 6.08 %, and GAL in 3.37 ± 0.85 % of the FB-labeled neurons. Our results suggest that neuronal PACAP, VIP, NOS, and GAL are mediators of neural response to aspirin-induced stomach inflammatory state.
Assuntos
Gastrite/metabolismo , Bulbo/metabolismo , Neurônios Eferentes/metabolismo , Nervo Vago/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Aspirina/efeitos adversos , Encefalina Leucina/genética , Encefalina Leucina/metabolismo , Galanina/genética , Galanina/metabolismo , Mucosa Gástrica/metabolismo , Gastrite/induzido quimicamente , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Estômago/inervação , Estômago/patologia , Substância P/genética , Substância P/metabolismo , Suínos , Nervo Vago/citologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
The aim of our study was to localize and define immunocytochemical characteristic of the dorsal motor nucleus of the vagus (DMX) neurons projecting to the porcine stomach prepyloric region in the physiological state and after gastric partial resection. To identify the stomach-projecting perikarya, the neuronal retrograde tracer--Fast Blue (FB) was injected into the studied region of control and resection group (RES). In the RES group, on 22nd day after FB injection, the partial resection of the stomach region previously injected with FB was performed. Sections were immunostained with ChAT, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu-enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the DMX of control and RES group, the stomach-projecting perikarya were found in the entire extent of the nucleus bilaterally. Within control animals, 30.08 ± 1.97 % of the gastric DMX perikarya expressed PACAP, while other substances were found only in the neuronal fibers. In the RES group DMX, PACAP was found in 45.58 ± 2.2 %, VIP in 28.83 ± 3.63 %, NOS in 21.22 ± 3.32 %, and GAL in 5.67 ± 1.49 % of the FB-labeled gastric perikarya. Our data implicate PACAP, VIP, NOS, and GAL as neuronal survival promoting substances and the CART-, LENK-, SP- NOS-, and GAL-immunoreactive processes in control of the gastric vagal neurons in the pig.
Assuntos
Neurônios Motores/metabolismo , Antro Pilórico/inervação , Nervo Vago/citologia , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Encefalina Leucina/genética , Encefalina Leucina/metabolismo , Galanina/genética , Galanina/metabolismo , Gastrectomia/veterinária , Neurônios Motores/classificação , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Especificidade de Órgãos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Antro Pilórico/cirurgia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Substância P/genética , Substância P/metabolismo , Suínos , Nervo Vago/metabolismo , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
BACKGROUND: The involvement of vagal parasympathetic efferents in esophageal myenteric neurons in vagal inhibitory pathways to the lower esophageal sphincter (LES) is not clear. Thus, this study was performed to demonstrate morphologically the presence of vagal inhibitory pathways to the LES via esophageal neurons. METHODS: Fast Blue (FB) was injected into the LES of Wistar rats, and 3 days after injection, the animals were subjected to electrical stimulation of the vagus nerve. The esophagus was processed for immunohistochemistry for Fos that was an immediate-early gene as a marker of neuronal activity, nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The immunoreactivities were then compared with the FB labeling in esophageal neurons. KEY RESULTS: Fast Blue-labeled neurons were observed within an esophageal area of 30 mm oral to the LES, with the highest frequency in the esophagus just above the LES. Most of the FB-labeled neurons were positive for NOS and VIP, but a few for ChAT. Following vagal-electrical stimulation, one fourth of the FB-labeled neurons presented nuclei expressing Fos and most of these Fos/FB neurons were NOS-positive. CONCLUSIONS & INFERENCES: A majority of the FB-labeled esophageal neurons appeared to be descending motor neurons innervating the LES. Moreover, the colocalization of VIP and NOS in most of the LES-projecting neurons suggests that VIP and NO released from these neurons induce LES relaxation, and the innervation of the vagal efferents to the LES-projecting esophageal neurons in the distal esophagus implies a vagal inhibitory pathway responsible for LES relaxation.
Assuntos
Esfíncter Esofágico Inferior/inervação , Esôfago/inervação , Neurônios Nitrérgicos/citologia , Nervo Vago/citologia , Amidinas/farmacologia , Animais , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/biossíntese , Estimulação Elétrica , Corantes Fluorescentes/farmacologia , Imuno-Histoquímica , Masculino , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Vias Neurais/citologia , Vias Neurais/metabolismo , Neurônios Eferentes/citologia , Neurônios Eferentes/metabolismo , Neurônios Nitrérgicos/metabolismo , Ratos , Ratos Wistar , Nervo Vago/metabolismo , Peptídeo Intestinal Vasoativo/análise , Peptídeo Intestinal Vasoativo/biossínteseRESUMO
BACKGROUND: Recently, vagus nerve preservation or reconstruction of vagus has received increasing attention. The present study aimed to investigate the feasibility of reconstructing the severed vagal trunk using an autologous sural nerve graft. METHODS: Ten adult Beagle dogs were randomly assigned to two groups of five, the nerve grafting group (TG) and the vagal resection group (VG). The gastric secretion and emptying functions in both groups were assessed using Hollander insulin and acetaminophen tests before surgery and three months after surgery. All dogs underwent laparotomy under general anesthesia. In TG group, latency and conduction velocity of the action potential in a vagal trunk were measured, and then nerves of 4 cm long were cut from the abdominal anterior and posterior vagal trunks. Two segments of autologous sural nerve were collected for performing end-to-end anastomoses with the cut ends of vagal trunk (8-0 nylon suture, 3 sutures for each anastomosis). Dogs in VG group only underwent partial resections of the anterior and posterior vagal trunks. Laparotomy was performed in dogs of TG group, and latency and conduction velocity of the action potential in their vagal trunks were measured. The grafted nerve segment was removed, and stained with anti-neurofilament protein and toluidine blue. RESULTS: Latency of the action potential in the vagal trunk was longer after surgery than before surgery in TG group, while the conduction velocity was lower after surgery. The gastric secretion and emptying functions were weaker after surgery in dogs of both groups, but in TG group they were significantly better than in VG group. Anti-neurofilament protein staining and toluidine blue staining showed there were nerve fibers crossing the anastomosis of the vagus and sural nerves in dogs of TG group. CONCLUSION: Reconstruction of the vagus nerve using the sural nerve is technically feasible.
Assuntos
Abdome/inervação , Nervo Sural/cirurgia , Nervo Vago/cirurgia , Abdome/fisiologia , Potenciais de Ação , Animais , Cães , Esvaziamento Gástrico/fisiologia , Nervo Sural/citologia , Nervo Sural/fisiologia , Transplantes , Nervo Vago/citologia , Nervo Vago/fisiologiaRESUMO
The mechanism that initiates regeneration of pancreatic ß-cells is not clear at present. The vagal nerve is implicated in the regulation of gastrointestinal functions, glucose metabolism and proliferation of pancreatic ß-cells under physiological conditions. To elucidate the triggering mechanism of the regeneration of pancreatic ß-cells, we examined the involvement of the vagal nerve. To this end, we employed a rat pancreatic duct ligation (DL) model, in which profound ß-cell neogenesis and ß-cell proliferation were observed within a week. We administered atropine to block the vagal nerve. Administration of atropine inhibited proliferation of ß-cells in both islets and islet-like cell clusters (ICC), without affecting ductal cell proliferation in the ligated pancreas. The numbers of PDX-1 and MafB-positive cells in or attaching to the ducts were significantly reduced by atropine. MafB/glucagon and MafB/insulin double-positive cells were also decreased by atropine. Finally, atropine reduced the number of MafA-positive ductal cells, all of which were positive for insulin, by 50% on day 5. These results strongly suggest that the vagal nerve is involved in ß-cell proliferation, induction of endocrine progenitors and neogenesis of α- and ß-cells.
Assuntos
Células Secretoras de Insulina/fisiologia , Pâncreas/inervação , Sistema Nervoso Parassimpático/fisiologia , Regeneração , Células-Tronco/citologia , Animais , Atropina/farmacologia , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Glucagon/metabolismo , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ligadura , Fator de Transcrição MafB/metabolismo , Masculino , Proteínas Oncogênicas/metabolismo , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/cirurgia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/metabolismo , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiologiaRESUMO
The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent adeno-associated virus (AAV2) DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-ß-hydroxylase Cre transgenic mice (DßH(Cre/0)), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive(ir) axonal varicosities were tyrosine hydroxylase (TH)-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry colocalization was virtually absent when AAV2 was injected into the RVLM of DßH(Cre/0);VGLUT2(flox/flox) mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DßH(Cre/0) mice or into the raphe of ePet(Cre/0) mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase and VGLUT2 immunoreactivities were highly colocalized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DßH(Cre/0) mice made predominantly asymmetric synapses with choline acetyl-transferase-ir DMV neurons. Photostimulation of ChR2-positive axons in DßH(Cre/0) mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/s (current-clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine, and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic PGNs monosynaptically and this connection uses glutamate as an ionotropic transmitter.
Assuntos
Glutamina/metabolismo , Bulbo/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Imuno-Histoquímica , Bulbo/citologia , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Técnicas de Patch-Clamp , Nervo Vago/citologia , Nervo Vago/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
Peripheral injection of pancreatic polypeptide (PP) and peptide YY(3-36) (PYY(3-36)), the hormones released in response to meals, reduce food intake, in which the rank order of the potency is PP>PYY(3-36). These anorectic effects are abolished in abdominal vagotomized rats, suggesting that PP and PYY(3-36) induce anorexia via vagal afferent nerves. However, it is not clear whether PP and PYY(3-36) directly act on vagal afferent neurons. In this study, we examined the effects of PP and PYY(3-36) on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in isolated nodose ganglion neurons of the mouse vagal afferent nerves. At 10(-11)M, PP but not PYY(3-36) recruited a significant population of nodose ganglion neurons into [Ca(2+)](i) increases. PP at 10(-11) to 10(-7) and PYY(3-36) at 10(-10) to 10(-7)M increased [Ca(2+)](i) in a concentration-dependent manner. At submaximal to maximal concentrations of 10(-10) and 10(-8)M, PP increased [Ca(2+)](i) in approximately twice greater population of nodose ganglion neurons than PYY(3-36). Furthermore, the majority of PP-responsive neurons also exhibited [Ca(2+)](i) responses to cholecystokinin-8, a hormone known to induce satiety through activating nodose ganglion neurons. The results demonstrate that PP and PYY(3-36) directly activate nodose ganglion neurons and suggest that the marked effect of PP on cholecystokinin-8-responsive nodose ganglion neurons could be linked to the regulation of feeding.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Gânglio Nodoso/efeitos dos fármacos , Polipeptídeo Pancreático/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeo YY/farmacologia , Animais , Capsaicina/farmacologia , Ingestão de Alimentos/fisiologia , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios Aferentes/efeitos dos fármacos , Gânglio Nodoso/citologia , Cloreto de Potássio/farmacologia , Ratos , Sincalida/metabolismo , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacosRESUMO
BACKGROUND/PURPOSE: In Hirschsprung's disease (HD), thick extrinsic nerve fibers can be associated with the aganglionic segment in the anorectum. We surgically disrupted the migration of vagal neural crest cell-derived cells (vagal NCC) in embryos from transgenic mice we created previously (SOX10-VENUS Tg) which have the SOX10 gene labeled with Venus (V), a green fluorescent protein, to observe sacral NCC activity in the anorectum. METHOD: Proximal colon harvested from SOX10-VENUS Tg embryos on day 10.5 (n = 10) was transected at the ascending colon. V-positive sacral NCC in the anorectum were observed during organ culture under fluorescence stereoscopic microscopy, and compared with non-transected control specimens (n = 10). RESULTS: In transected specimens, no V-positive sacral NCC were identified initially in the anorectum. By day 2, there were thick beaded sacral NCC in the anorectum in 6/10 (60 %) that migrated steadily to the transected end over 3-4 days. In controls, thinner and shorter V-positive sacral NCC began migrating cranially on day 2, and were met by distally migrating vagal NCC. CONCLUSION: Disruption of vagal NCC migration appears to induce sacral NCC activity in the anorectum, suggesting that thick extrinsic nerve fibers seen in HD may be a secondary phenomenon.
Assuntos
Movimento Celular , Doença de Hirschsprung/etiologia , Crista Neural/citologia , Nervo Vago/citologia , Animais , Camundongos , Fibras NervosasRESUMO
Obstructive sleep apnea, manifested by intermittent hypoxia and excess production of reactive oxygen species (ROS) in airways, is associated with hyperreactive airway diseases, but the mechanism remains unclear. Sensitization of lung vagal C fibers (LVCFs) contributes to the airway hypersensitivity. We investigated the mechanisms underlying the sensitization of LVCFs with acute intermittent hypoxia (AIH), by 10 episodes of exposure to 30 s of hypoxic air (0%, 5%, or 10% O(2)) followed by 30 s of room air in anesthetized, open-chest, and artificially ventilated rats. Reflex apneic response to intravenous capsaicin (an LVCF stimulant), as measured by phrenic nerve activity, was concentration dependently augmented by AIH. Similarly, reflex apneic response to intravenous α,ß-methylene-ATP (another LVCF stimulant) was augmented by AIH (0% O(2)). The reflex apnea evoked by these two stimulants was abolished by bilateral vagotomy, which suggests the involvement of lung vagal afferents. The AIH-augmented apneic response to these two stimulants was prevented by pretreatment with dimethylthiourea (a hydroxyl radical scavenger), N-acetyl-l-cysteine (an antioxidant) and HC-030031 [a transient receptor potential ankyrin 1 (TRPA1) receptor antagonist]. Consistently, electrophysiological study revealed the afferent responses of LVCFs to capsaicin or α,ß-methylene-ATP were augmented by AIH, and this sensitization of LVCFs was prevented by dimethylthiourea, N-acetyl-l-cysteine, and HC-030031. In contrast, AIH did not alter the afferent response of LVCFs to mechanical stimulation by lung hyperinflation. We concluded that AIH sensitizes LVCFs in rats, thus resulting in exaggerated airway reflexogenic responses to chemical stimulants, possibly by ROS action and activation of TRPA1 receptors.