RESUMO
Only a few papers exist dealing with the development and aging of the autonomic nervous system - and even rarer are studies that investigated the otic ganglion. Using a special trepan, we removed and investigated 172 samples from 86 corpses, ranging from 20 weeks of gestational age (GA) to 95 years of age. The aim of the study was to measure different morphometric parameters of the ganglionic neurons in order to study age-related changes from early development until old age. Fetuses show the highest numerical density of neurons. Then, in the first years of life, a rapid growth of the cytoplasm takes place, which is the main reason for the neuronal growth and the increase of the general size of the otic ganglion at this age. Also, the number of satellite cells increases till puberty. In adults, the parameters are relatively stable over decades and decrease slowly, in contrast to the steep increase in the first years of life. Moreover, neuronal degeneration, storage of pigments, neuro-axonal dystrophy, and lymphocytic infiltrates increase with age.
Assuntos
Desenvolvimento Embrionário/fisiologia , Gânglios Parassimpáticos/crescimento & desenvolvimento , Gânglios Parassimpáticos/ultraestrutura , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Structural alterations in the central autonomic nuclei (dorsal vagal complex and intermediolateral nucleus) of the centrifuged random-bred male rats subjected to +Gz loads were examined. Acute exposure to gravitational loads predominantly produced the reactive changes in these nuclei, while persistently repeated regular loads resulted in cumulation of the destructive alterations. The structural perturbations in the central autonomic nuclei can disturb the autonomic regulation of physiological functions. The character of such disturbances is partially determined by the peculiarities in structural organization of these nuclei.
Assuntos
Área Postrema/ultraestrutura , Gânglios Parassimpáticos/ultraestrutura , Hipergravidade , Neurônios/ultraestrutura , Corno Lateral da Medula Espinal/ultraestrutura , Animais , Área Postrema/irrigação sanguínea , Área Postrema/patologia , Centrifugação , Gânglios Parassimpáticos/irrigação sanguínea , Gânglios Parassimpáticos/patologia , Masculino , Microscopia Eletrônica de Transmissão , Microtomia , Neurônios/patologia , Ratos , Ratos Wistar , Corno Lateral da Medula Espinal/irrigação sanguínea , Corno Lateral da Medula Espinal/patologiaRESUMO
PURPOSE: The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprung's disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest-derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest-derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS(+)/EDNRB(sl/sl) to investigate intestinal innervation in HD. METHODS: SOX10-VENUS(+)/EDNRB(sl/sl) (n = 30) were compared with wild-type littermates as controls (EDNRB(s/s), n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining. RESULTS: In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS(+)/EDNRB(sl/sl) mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS(+)/EDNRB(sl/sl) mice appeared the same. Innervation patterns did not change over time. CONCLUSION: This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.
Assuntos
Colo/inervação , Modelos Animais de Doenças , Sistema Nervoso Entérico/anormalidades , Gânglios Parassimpáticos/ultraestrutura , Doença de Hirschsprung/patologia , Camundongos Transgênicos , Fibras Nervosas/ultraestrutura , Reto/inervação , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Linhagem da Célula , Colo/patologia , Sistema Nervoso Entérico/patologia , Imunofluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/efeitos da radiação , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Luminescentes/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Confocal , Microscopia de Fluorescência , Crista Neural/patologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor de Endotelina B/biossíntese , Receptor de Endotelina B/deficiência , Receptor de Endotelina B/genética , Reto/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXE/genética , Raios UltravioletaRESUMO
BACKGROUND AND PURPOSE: Prostaglandin E(2) (PGE(2)) modulates autonomic transmission in the peripheral circulation. We investigated the role of endogenous PGE(2) and its presynaptic EP(1) receptor subtype in modulating the autonomic neurotransmission in cerebral vasculature. METHODS: The standard in vitro tissue-bath technique was used for measuring changes in isolated porcine basilar arterial tone. Calcium imaging and nitric oxide estimation along with immunohistochemical analysis for cyclo-oxygenase-1, cyclo-oxygenase-2, EP(1) receptor, PGE synthase, and neuronal nitric oxide synthase were done in cultured sphenopalatine ganglia and basilar artery. RESULTS: Selective EP(1) receptor antagonists (SC-19220 and SC-51322) inhibited relaxation of endothelium-denuded basilar arterial rings elicited by transmural nerve stimulation (2 and 8 Hz) without affecting that induced by nicotine or sodium nitroprusside (a nitric oxide donor). The SC-19220 inhibition of transmural nerve stimulation-elicited relaxation was blocked by cyclo-oxygenase inhibitors (salicylic acid and naproxen) but was not affected by guanethidine (a sympathetic neuronal blocker) or atropine. Perivascular cyclo-oxygenase-1- and cyclo-oxygenase-2-immunoreactive fibers were observed in basilar arteries. PGE synthase and EP(1) receptor immunoreactivities were coincident with neuronal nitric oxide synthase immunoreactivities in perivascular nerves of the basilar arteries and the sphenopalatine ganglia. omega-conotoxin (an N-type calcium channel blocker) significantly blocked transmural nerve stimulation-induced relaxation, which was further attenuated by SC-19220. In cultured sphenopalatine ganglia neurons, exogenous PGE(2) significantly increased calcium influx and diaminofluorescein fluorescence indicative of nitric oxide synthesis. Both responses were blocked by SC-19220. CONCLUSIONS: These results suggest that neuronal PGE(2) facilitates nitric oxide release from the cerebral perivascular parasympathetic nitrergic nerve terminals by increasing neuronal calcium influx through activation of presynaptic EP(1) receptors. PGE(2) may play an important role in regulating the nitrergic neurovascular transmission in the cerebral circulation.
Assuntos
Vias Autônomas/metabolismo , Artérias Cerebrais/inervação , Dinoprostona/metabolismo , Neurônios Nitrérgicos/metabolismo , Receptores de Prostaglandina E/metabolismo , Vasodilatação/fisiologia , Animais , Vias Autônomas/efeitos dos fármacos , Vias Autônomas/ultraestrutura , Artéria Basilar/inervação , Artéria Basilar/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Artérias Cerebrais/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilidrazida/farmacologia , Gânglios Parassimpáticos/efeitos dos fármacos , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Imuno-Histoquímica , Oxirredutases Intramoleculares/metabolismo , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/ultraestrutura , Óxido Nítrico Sintase Tipo I/metabolismo , Antagonistas de Prostaglandina/farmacologia , Prostaglandina-E Sintases , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP1 , Sus scrofa , Vasodilatação/efeitos dos fármacosRESUMO
We have examined the ultrastructure of the myenteric ganglion of the subdiaphragmatic esophagus and determined whether the ganglion neurons receive direct projections from the dorsal motor nucleus of the vagus (DMV) using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) as an anterograde tracer. The neurons (22.2 microm x 13.3 microm) of myenteric ganglion in the esophagus contained dark cytoplasm having many free ribosomes, mitochondria, and an oval nucleus, and received only a few axon terminals contacting somata. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Approximately 85% of the axon terminals contacting dendrites and about 50% of the axon terminals contacting somata contained pleomorphic vesicles, while the rest contained round synaptic vesicles. When WGA-HRP was injected into the DMV, anterogradely labeled fibers and terminals were found in the myenteric ganglia. The WGA-HRP labeled terminals were large (1.97 microm) and contained round clear vesicles and small granular vesicles. These labeled terminals contacted exclusively the small dendrites, but not the somata. These results suggest that the DMV neurons project directly to the myenteric ganglion neurons and regulate the esophageal muscles via the ganglion neurons.
Assuntos
Vias Eferentes/fisiologia , Gânglios Parassimpáticos/fisiologia , Bulbo/fisiologia , Sinapses/fisiologia , Nervo Vago/fisiologia , Animais , Vias Eferentes/ultraestrutura , Gânglios Parassimpáticos/ultraestrutura , Masculino , Bulbo/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Neurônios/fisiologia , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestrutura , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre/metabolismoRESUMO
CaV2.2 channels play a key role in the gating of transmitter release sites (TRS) at presynaptic terminals. Physiological studies predict that the channels are linked directly to the TRS but the molecular composition of this complex remains poorly understood. We have used a high-affinity anti-CaV2.2 antibody, Ab571, to test a range of proteins known to contribute to TRS function for both an association in situ and a link in vitro. CaV2.2 clusters were isolated intact on immunoprecipitation beads and coprecipitated with a number of these proteins. Quantitative staining covariance analysis (ICA/ICQ method) was applied to the transmitter release face of the giant calyx terminal in the chick ciliary ganglion to test for TRS proteins with staining intensities that covary in situ with CaV2.2, resulting in a covariance sequence of NSF>RIM>spectrin>Munc18>VAMP>alpha-catenin, CASK>SV2>Na+-K+ approximately 0. A high-NaCl dissociation challenge applied to the immunoprecipitated complex, using the fractional recovery (FR) method [Khanna, R., Li, Q. & Stanley, E.F. (2006) PLoS.ONE., 1, e67], was used to test which proteins were most intimately associated with the channel, generating an FR sequence for CaV2.2 of: VAMP>or=actin>tubulin, NSF, Munc18, syntaxin 1>spectrin>CASK, SNAP25>RIM, Na+-K+ pump, v-ATPase, beta-catenin approximately 0. Proteins associated with endocytosis are considered in a companion paper [Khanna et al. (2007)Eur. J. Neurosci., 26, 560-574]. With the exception of VAMP and RIM, the ICQ and FR sequences were consistent, suggesting that proteins that covary the most strongly with CaV2.2 in situ are also the most intimately attached. Our findings suggest that the CaV2.2 cluster is an integral element of a multimolecular vesicle-fusion module that forms the core of a multifunctional TRS.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Membranas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação/fisiologia , Canais de Cálcio Tipo N/química , Sinalização do Cálcio/fisiologia , Galinhas , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Fusão de Membrana/fisiologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/química , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007)Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (approximately 1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Membranas Sinápticas/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Canais de Cálcio Tipo N/química , Galinhas , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Substâncias Macromoleculares/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/química , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/química , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestruturaRESUMO
The myenteric ganglia regulate not only gastric motility but also secretion, because a submucous plexus is sparsely developed in the rodent stomach. We have examined whether the neurons of the dorsal motor nucleus of the vagus (DMV) have direct synaptic contacts on the myenteric ganglia and the ultrastructure of the vagal efferent terminals by using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The myenteric ganglia of the rat were composed of four types of neurons, i.e., small, medium-sized, large, and elongated neurons. The average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. More than half of the axosomatic terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized, and large neurons. About 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. When WGA-HRP was injected into the DMV, many anterogradely labeled terminals were found around the myenteric neurons. The labeled terminals were large (3.16 +/- 0.10 microm) and contacted exclusively the somata. Most of them (about 90%) contained round vesicles and formed asymmetric synaptic contacts. Serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the DMV. The vagal axon terminals generally contacted the medium-sized or the elongated neurons, whereas a few labeled terminals contacted the small and the large neurons. The present results indicate that the DMV projects to all types of neurons and that their axon terminals contain mostly round synaptic vesicles and form asymmetric synaptic contacts.
Assuntos
Vias Eferentes/ultraestrutura , Gânglios Parassimpáticos/ultraestrutura , Bulbo/ultraestrutura , Plexo Mientérico/ultraestrutura , Sinapses/ultraestrutura , Nervo Vago/ultraestrutura , Animais , Vias Eferentes/fisiologia , Gânglios Parassimpáticos/fisiologia , Masculino , Bulbo/fisiologia , Microscopia Eletrônica de Transmissão , Músculo Liso/inervação , Músculo Liso/ultraestrutura , Plexo Mientérico/fisiologia , Peristaltismo/fisiologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Estômago/inervação , Estômago/ultraestrutura , Sinapses/fisiologia , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Nervo Vago/fisiologia , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano SilvestreRESUMO
The otic ganglion is a cranial component of the parasympathetic division of the autonomic nervous system (ANS). Similar to other parasympathetic ganglia, otic ganglion presents multipolar neurons that are completely surrounded by satellite cells and intercellular substance as well, which allow us to use this ganglion as a good experimental model for studying the relationship neuron / extracellular matrix. We have studied rat otic ganglion in 10 animals through light microscopy. After routine histological methods, 5 µm sections were obtained and coloured by Gomori`s trichrome, periodic acid-Schiff (PAS), Alcian Blue pH 2.5 and pH1.0, acetylation + PAS, acetylation + deacetylation + PAS, acid hydrolysis + PAS, phenylhydrazine + PAS and thiosemicarbazide + PAS. The presence of neutral glycoproteins was demonstrated by PAS reactivity. PAS inhibition following Alcian Blue staining in pH 2.5 and 1.0 showed the presence of a small quantity of acid glycoprotein. The extracellular matrix analysis showed the presence of neutral and acid glycoconjugates. These findings suggests a mutual interaction and a complex role in ganglionic physiology.
El ganglio ótico es un componente craneal de la división parasimpática del sistema nervioso autónomo (SNA). Similar al otro ganglio parasimpático, el ganglio ótico presenta neuronas multipolares que están rodeadas totalmente por las células satélites y la sustancia intercelular, lo cual permite la utilización de este ganglio como un buen modelo experimental para estudiar las relaciones matriz extracelular/neurona. Examinamos, con microscopio de luz, el ganglio ótico 10 ratones. Con los métodos histológicos rutinarios fueron obtenidas 5 secciones y coloreadas con tricrómico de Gomori, PAS, Azul de Alcián pH 2.5 y pH1.0, acetilación + PAS, acetilación + desacetilación + PAS, hidrólisis de ácido + PAS, fenilhidrazina + PAS y tiosemicarbacida + PAS. La presencia de glicoproteínas neutras fue demostrada por la reactividad de PAS. La inhibición de PAS y la posterior tinción con Azul de Alcian en pH 2.5 y 1.0, demostró la presencia de una cantidad pequeña de glicoproteínas ácidas. El análisis extracelular de la matriz demostró la presencia de glicoconjugados neutros y ácidos. Estos resultados sugieren una interacción mutua y un papel complejo en la fisiología ganglionar.
Assuntos
Animais , Masculino , Ratos , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Reação do Ácido Periódico de Schiff , Sistema Nervoso Autônomo , Ratos Wistar , Gânglios Parassimpáticos/anatomia & histologiaRESUMO
Neurotransmitter release is well known to occur at specialized synaptic regions that include presynaptic active zones and postsynaptic densities. At cholinergic synapses in the chick ciliary ganglion, however, membrane formations and physiological measurements suggest that release distant from postsynaptic densities can activate the predominantly extrasynaptic alpha7 nicotinic receptor subtype. We explored such ectopic neurotransmission with a novel model synapse that combines Monte Carlo simulations with high-resolution serial electron microscopic tomography. Simulated synaptic activity is consistent with experimental recordings of miniature excitatory postsynaptic currents only when ectopic transmission is included in the model, broadening the possibilities for mechanisms of neuronal communication.
Assuntos
Simulação por Computador , Gânglios Parassimpáticos/fisiologia , Modelos Neurológicos , Método de Monte Carlo , Sinapses/fisiologia , Transmissão Sináptica , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Algoritmos , Animais , Embrião de Galinha , Difusão , Potenciais Pós-Sinápticos Excitadores , Gânglios Parassimpáticos/ultraestrutura , Processamento de Imagem Assistida por Computador , Matemática , Microscopia Eletrônica , Técnicas de Patch-Clamp , Probabilidade , Receptores Nicotínicos/metabolismo , Sensibilidade e Especificidade , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
The neuropeptide tachykinins and their receptors have been implicated in the pathogenesis of lung disease, although the role of the tachykinin neurokinin-3 receptor has not been elucidated. Using confocal microscopy, we identified tachykinin neurokinin-3 receptors on human bronchial parasympathetic ganglion neurons. Electrophysiologic recordings demonstrated that activation of sensory nerve fibers, either by antidromic stimulation or capsaicin, depolarized these neurons. This response was mimicked by exogenously applied tachykinin neurokinin-3 receptor-selective agonist, senktide analogue, but not significantly by tachykinin neurokinin-1 or neurokinin-2 receptor-selective agonists. Responses to endogenous tachykinins or exogenous selective tachykinin neurokinin-3 receptor activation with senktide analogue were inhibited by the selective tachykinin neurokinin-3 receptor antagonists, SB 223412 or SB 235375. We provide the first evidence that tachykinin neurokinin-3 receptors regulate human bronchial parasympathetic ganglion neurotransmission by activation of a peripheral reflex. This pathway may play a significant role in controlling bronchomotor tone and air flow to the lung.
Assuntos
Brônquios/inervação , Gânglios Parassimpáticos/fisiologia , Neurônios Aferentes/fisiologia , Receptores da Neurocinina-3/fisiologia , Substância P/análogos & derivados , Acetatos/farmacologia , Adulto , Capsaicina/farmacologia , Estimulação Elétrica , Potenciais Evocados/fisiologia , Feminino , Gânglios Parassimpáticos/efeitos dos fármacos , Gânglios Parassimpáticos/ultraestrutura , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/ultraestrutura , Fragmentos de Peptídeos/farmacologia , Quinolinas/farmacologia , Receptores da Neurocinina-3/agonistas , Receptores da Neurocinina-3/antagonistas & inibidores , Substância P/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
A unique costorage of neuropeptides was recently found in the abdominal perisympathetic organs (PSOs) of the American cockroach, Periplaneta americana. Having specific antisera directed against all peptides belonging to this neurosecretory system, we examined the sorting of PSO-peptides in the soma of the median neurosecretory cells of abdominal ganglia by using immunoelectron microscopic double stainings. The data indicate that all six abundant neuropeptides of this neurohormonal system, which includes three capa-gene related products, are primarily incorporated into separate vesicles. These vesicles fuse with each other in the cytoplasm and become translucent on their way to the axon hillock. By means of light microscopy and MALDI-TOF mass spectrometry, an identical population of neuropeptides was found in interneurons of the brain. As revealed by subsequent immunoelectron microscopic analysis, the peptides of these cells are separately packed into dense core vesicles but do not fuse with each other. Thus, hitherto unknown cell-type-specific sorting mechanisms occur in neurosecretory cells and interneurons, respectively.
Assuntos
Proteínas de Insetos/metabolismo , Interneurônios/metabolismo , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Periplaneta/metabolismo , Abdome/inervação , Animais , Feminino , Imunofluorescência , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Interneurônios/ultraestrutura , Masculino , Sistemas Neurossecretores/ultraestrutura , Periplaneta/ultraestrutura , Transporte Proteico/fisiologia , Vesículas Secretórias/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
1. The 5-HT receptor subtype that mediates bronchocontraction and the involvement of neuronal and non-neuronal acetylcholine was assessed in murine isolated tracheae. 2. Atropine (1-10 nM) caused a rightward shift of the methacholine concentration-effect curves (pA(2)=9.0) but reduced the maximum response to 5-HT, suggesting that 5-HT acts as an indirect agonist. The potency of 5-HT receptor agonists (alpha-methyl-5-HT approximately 5-HT>5-carboxamidotryptamine), together with the competitive antagonism of 5-HT by ketanserin (pA(2)=9.4), suggests the involvement of the 5-HT(2A) receptor. 3. While cholinergic twitch responses to electrical field stimulation were abolished by the fast sodium channel inhibitor tetrodotoxin (300 nM), as well as by combined blockade of N-, P- and Q-type voltage-operated calcium channels by omega-conotoxin GVIA (30 nM) and agatoxin IVA (100 nM), responses to 5-HT were unaffected. Similarly, botulinum toxin A (50 nM) inhibited EFS twitch responses, but not contractions to 5-HT. 4. Choline acetyltransferase immunoreactivity was localised to ganglia and nerve fibres as well as approximately half the epithelial cells in the preparation. Removal of the epithelial layer markedly attenuated the contractile response to 5-HT, but had no effect on contractions to either methacholine or EFS. 5. These findings suggest that 5-HT, acting at 5-HT(2A) receptors on mouse tracheal epithelial cells, stimulates these cells to release acetylcholine, which then causes contraction of airway smooth muscle. This phenomenon should be borne in mind in when interpreting studies of murine models of airway disease.
Assuntos
Acetilcolina/fisiologia , Epitélio/fisiologia , Contração Muscular/efeitos dos fármacos , Serotonina/análogos & derivados , Serotonina/farmacologia , Traqueia/efeitos dos fármacos , 8-Hidroxi-2-(di-n-propilamino)tetralina/administração & dosagem , Animais , Atropina/farmacologia , Compostos de Boro/farmacologia , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Cartilagem/efeitos dos fármacos , Cartilagem/fisiologia , Estimulação Elétrica/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/efeitos dos fármacos , Feminino , Gânglios Parassimpáticos/efeitos dos fármacos , Gânglios Parassimpáticos/ultraestrutura , Receptores de Inositol 1,4,5-Trifosfato , Ketanserina/farmacologia , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Tensoativos/efeitos adversos , Tensoativos/química , Tetrodotoxina/farmacologia , Traqueia/inervação , ômega-Conotoxina GVIA/farmacologiaRESUMO
1. We tested the hypothesis that arterial baroreceptor reflexes modulate cerebrovascular tone through a pathway that connects the cardiovascular nucleus tractus solitarii with parasympathetic preganglionic neurons in the pons. 2. Anesthetized rats were used in all studies. Laser flowmetry was used to measure cerebral blood flow. We assessed cerebrovascular responses to increases in arterial blood pressure in animals with lesions of baroreceptor nerves, the nucleus tractus solitarii itself, the pontine preganglionic parasympathetic neurons, or the parasympathetic ganglionic nerves to the cerebral vessels. Similar assessments were made in animals after blockade of synthesis of nitric oxide, which is released by the parasympathetic nerves from the pterygopalatine ganglia. Finally the effects on cerebral blood flow of glutamate stimulation of pontine preganglionic parasympathetic neurons were evaluated. 3. We found that lesions at any one of the sites in the putative pathway or interruption of nitric oxide synthesis led to prolongation of autoregulation as mean arterial pressure was increased to levels as high as 200 mmHg. Conversely, stimulation of pontine parasympathetic preganglionic neurons led to cerebral vasodilatation. The second series of studies utilized classic anatomical tracing methods to determine at the light and electron microscopic level whether neurons in the cardiovascular nucleus tractus solitarii, the site of termination of baroreceptor afferents, projected to the pontine preganglionic neurons. Fibers were traced with anterograde tracer from the nucleus tractus solitarii to the pons and with retrograde tracer from the pons to the nucleus tractus solitarii. Using double labeling techniques we further studied synapses made between labeled projections from the nucleus tractus solitarii and preganglionic neurons that were themselves labeled with retrograde tracer placed into the pterygopalatine ganglion. 4. These anatomical studies showed that the nucleus tractus solitarii directly projects to pontine preganglionic neurons and makes asymmetric, seemingly excitatory, synapses with those neurons. These studies provide strong evidence that arterial baroreceptors may modulate cerebral blood flow through direct connections with pontine parasympathetic neurons. Further study is needed to clarify the role this pathway plays in integrative physiology.
Assuntos
Vias Autônomas/fisiologia , Circulação Cerebrovascular/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Ponte/fisiologia , Pressorreceptores/fisiologia , Núcleo Solitário/fisiologia , Animais , Vias Autônomas/efeitos dos fármacos , Vias Autônomas/ultraestrutura , Barorreflexo/efeitos dos fármacos , Barorreflexo/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Artérias Cerebrais/inervação , Artérias Cerebrais/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Denervação , Gânglios Parassimpáticos/efeitos dos fármacos , Gânglios Parassimpáticos/fisiologia , Gânglios Parassimpáticos/ultraestrutura , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Microscopia Eletrônica , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/ultraestrutura , Ponte/efeitos dos fármacos , Ponte/ultraestrutura , Pressorreceptores/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/ultraestruturaRESUMO
Peripheral or central interruption of the baroreflex or the parasympathetic innervation of cerebral vessels leads to similar changes in regulation of cerebral blood flow. Therefore, we sought to test the hypothesis that the cardiovascular nucleus tractus solitarii, the site of termination of arterial baroreceptor nerves, projects to pontine preganglionic neurons whose stimulation elicits cerebral vasodilatation. The current study utilized both light and electron microscopic techniques to analyze anterograde tracing from the cardiovascular nucleus tractus solitarii to preganglionic parasympathetic neurons in the pons. We further used retrograde tracing from that same pontine region to the cardiovascular nucleus tractus solitarii and evaluated the confluence of tracing from the cardiovascular nucleus tractus solitarii to pontine preganglionic neurons labeled retrogradely from the pterygopalatine ganglia. The cardiovascular nucleus tractus solitarii projected to pontine preganglionic parasympathetic neurons, but more rostral and caudal regions of nucleus tractus solitarii did not. In contrast, all three regions of nucleus tractus solitarii projected to the nucleus ambiguus and dorsal motor nucleus of the vagus. Although not projecting to pontine preganglionic parasympathetic neurons, regions lateral, rostral, and caudal to cardiovascular nucleus tractus solitarii sent projections through the pons medial to the preganglionics. The study establishes the presence of a direct monosynaptic pathway from neurons in the cardiovascular nucleus tractus solitarii to pontine preganglionic parasympathetic neurons that project to the pterygopalatine ganglia, the source of nitroxidergic vasodilatory innervation of cerebral blood vessels. It provides evidence that activation of those preganglionic neurons can cause cerebral vasodilatation and increased cerebral blood flow. Finally, it demonstrates differential innervation of medullary and pontine preganglionic parasympathetic neurons by different regions of the nucleus tractus solitarii.
Assuntos
Lisina/análogos & derivados , Ponte/irrigação sanguínea , Núcleo Solitário/irrigação sanguínea , Animais , Transporte Axonal , Barorreflexo/fisiologia , Circulação Cerebrovascular/fisiologia , Gânglios Autônomos/fisiologia , Gânglios Parassimpáticos/fisiologia , Gânglios Parassimpáticos/ultraestrutura , Masculino , Microscopia Eletrônica , Ponte/fisiologia , Ponte/ultraestrutura , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/fisiologia , Núcleo Solitário/ultraestruturaRESUMO
Rapid synaptic transmission in the embryonic chicken ciliary ganglion occurs through the activation of two distinct classes of nicotinic acetylcholine receptors (AChRs): those containing alpha3 subunits (alpha 3*-AChRs) and those containing alpha7 subunits (alpha 7*-AChRs). alpha3*-AChRs are found on ciliary neurons in clusters at synaptic sites on the cell body, whereas alpha7* -AChRs are found on somatic spines, which historically were thought not to have release sites in the embryo. However, Shoop et al. (Shoop et al. [1999] J. Neurosci. 19:692-704) recently described release sites having pre- and postsynaptic densities on somatic spines. We used transmission electron microscopy to compare the structure of synaptic sites on spines with those on the smooth surfaced part of the cell. We find that the two populations of sites are similar in active zone length, number of vesicles, and distance between vesicles and active zone. To study the functional properties of these sites, we examined their stimulation-dependent uptake and release of the extracellular tracer horseradish peroxidase (HRP). We found that each class of release sites both took up and released HRP in a stimulation- and calcium-dependent manner. The mean fraction of synaptic vesicles labeled with tracer was similar for the two populations, both after loading ( approximately 45%) and after unloading ( approximately 7%). Thus we detect no differences between these two anatomically distinct classes of release sites, other than their incidence: sites on spines occurred only 12% as often as those on the cell body. The release sites on somatic spines presumably underlie synaptic responses attributable to alpha7*-AChRs.
Assuntos
Gânglios Parassimpáticos/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico/fisiologia , Receptores Nicotínicos/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/ultraestrutura , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Embrião de Galinha , Estimulação Elétrica , Endocitose/fisiologia , Exocitose/fisiologia , Gânglios Parassimpáticos/embriologia , Gânglios Parassimpáticos/metabolismo , Peroxidase do Rábano Silvestre , Microscopia Eletrônica , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/metabolismo , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Selective sympathetic nerve dysfunction occurs during aging and in certain disease states. Here, we review findings concerning the effects of chronic sympathetic denervation on parasympathetic innervation to orbital target tissues in the adult rat. Long-term sympathetic denervation was induced by excising the ipsilateral superior cervical ganglion for 5-6 weeks prior to analyses. Following sympathectomy, pterygopalatine ganglion parasympathetic neurons show reduced nitric oxide synthase protein in their somata and projections to vascular targets. Laser Doppler measurements of ocular blood flow indicate that sympathectomy is also accompanied by reduced nitrergic vasodilatation. In the superior tarsal muscle of the eyelid, parasympathetic varicosities, normally, are distant to smooth muscle cells but make axo-axonal contacts with sympathetic nerves, consistent with physiological evidence showing only prejunctional inhibitory effects on sympathetically mediated smooth muscle contraction. Following sympathectomy, parasympathetic varicosities proliferate and closely appose smooth muscle cells, and this is accompanied by establishment of parasympathetic-smooth muscle excitatory neurotransmission. Many pterygopalatine parasympathetic neurons normally contain nerve growth factor (NGF) protein and express NGF mRNA. However, following chronic sympathectomy or elimination of sympathetic impulse activity, NGF mRNA and protein are markedly reduced, indicating that sympathetic neurotransmission enhances NGF expression in parasympathetic neurons. Together, these findings portray a striking dependency of parasympathetic neurons on sympathetic nerves to maintain normal phenotype and function. Sympathetic influences on parasympathetic neurons may be mediated, in part, through axo-axonal synapses. NGF synthesis and release by parasympathetic neurons may represent a molecular basis underlying the formation of these synapses, and up-regulation of NGF synthesis by sympathetic nerve activity may act to reinforce these associations.
Assuntos
Comunicação Celular/fisiologia , Regulação para Baixo/fisiologia , Gânglios Parassimpáticos/crescimento & desenvolvimento , Órbita/inervação , Terminações Pré-Sinápticas/metabolismo , Simpatectomia/efeitos adversos , Fibras Simpáticas Pós-Ganglionares/fisiopatologia , Animais , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/ultraestrutura , Humanos , Fator de Crescimento Neural/metabolismo , Órbita/fisiologia , Fenótipo , Terminações Pré-Sinápticas/ultraestrutura , Fibras Simpáticas Pós-Ganglionares/lesões , Fibras Simpáticas Pós-Ganglionares/metabolismoRESUMO
Chick ciliary neurons have somatic spines grouped in discrete clumps or mats tightly folded against the soma and enriched in nicotinic receptors containing alpha7 subunits. An embryonic ciliary neuron has one to two dozen such spine mats, all overlaid by a large presynaptic calyx engulfing the cell. Three-dimensional tomographic reconstruction from serial thick sections revealed 13 somatic spines in one complete spine mat on a ciliary neuron late in embryogenesis. The spines varied in morphology and usually were branched but had numerous similarities to dendritic spines, including mean length, volume, surface area, presence of endoplasmic reticulum, and occasional multivesicular bodies. The spines invariably were connected to the soma via a narrow neck of approximately 0.2 micrometer in diameter as found for dendritic spines, suggesting restricted access from spine lumen to soma. A prominent difference between dendritic and somatic spines is the absence of postsynaptic densities from most somatic spines both on embryonic and adult ciliary neurons. Transmitter access to receptors on the spines may occur either by lateral diffusion from release sites over nearby postsynaptic densities or by release directly onto spines from the overlying calyx lined with vesicles. The latter is less likely in the adult, where some spines are adjacent to but not overlaid by vesicle-enriched presynaptic structures. The anatomical configuration of spine mats suggests coordinate spine activation by transmitter release into a confined volume while spine morphology is used to control the chemical consequences of synaptic signaling.
Assuntos
Extensões da Superfície Celular/ultraestrutura , Microscopia Eletrônica , Neurônios/ultraestrutura , Receptores Nicotínicos , Transmissão Sináptica/fisiologia , Envelhecimento/fisiologia , Animais , Embrião de Galinha , Galinhas , Gânglios Parassimpáticos/embriologia , Gânglios Parassimpáticos/ultraestrutura , Processamento de Imagem Assistida por Computador , Neurônios/metabolismo , Receptores Nicotínicos/biossíntese , Vesículas Sinápticas/ultraestruturaRESUMO
The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), or the desensitising agonist, alpha,beta-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 microM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 microM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gamma S, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATP gamma S on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 microM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 microM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.
Assuntos
Trifosfato de Adenosina/fisiologia , Gânglios Parassimpáticos/metabolismo , Glândula Submandibular/inervação , Transmissão Sináptica/fisiologia , Acetilcolina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Fibras Autônomas Pré-Ganglionares/metabolismo , Potenciais Pós-Sinápticos Excitadores , Gânglios Parassimpáticos/ultraestrutura , Imuno-Histoquímica , Potenciais da Membrana , Antagonistas Nicotínicos/farmacologia , Fibras Parassimpáticas Pós-Ganglionares/metabolismo , Terminações Pré-Sinápticas/metabolismo , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Purinérgicos P2/metabolismoRESUMO
Parasympathetic ganglia neurons in the lower airway of laboratory animals have membrane properties associated with integration of signals from the central nervous system. In this study, intracellular recordings were made from parasympathetic ganglia located on bronchi from human lungs in order to determine the level of integration provided by human neurons. Ganglion neurons were characterized as either tonic or phasic: tonic neurons responded with repetitive action potentials sustained throughout a depolarizing current step whereas phasic neurons generated one action potential and accommodated. Phasic neurons could be further differentiated as having either short or long duration after hyperpolarizing potentials following single action potentials. In phasic neurons, stimulation of preganglionic nerves elicited one or two populations of nicotinic fast excitatory postsynaptic potentials (fEPSPs) that were graded in amplitude, subthreshold for action potential generation, and decreased in amplitude during higher frequency stimulation. In tonic neurons, single preganglionic stimuli evoked two to five populations of fEPSPs, one to three of which were at threshold for action potential generation. Dye injection into the neurons revealed multiple, branching dendrites. These results provide evidence that human bronchial ganglion neurons have unique membrane properties and anatomical characteristics associated with integrating presynaptic stimuli. Changes in these properties may thus affect output from these ganglia and, consequently, autonomic tone in the lower airways.