RESUMO
The active zone (AZ) of chemical synapses is a specialized area of the presynaptic bouton in which vesicles fuse with the plasma membrane and release neurotransmitters. Efficient signaling requires synaptic vesicles (SVs) to be recruited, primed, and retained at the AZ, in close proximity to voltage-dependent calcium channels that are activated during presynaptic depolarization. The electron-dense specializations at the AZ might provide a molecular platform for the spatial coordination of these different processes. To investigate this hypothesis, we examined high-resolution three-dimensional models of Caenorhabditis elegans cholinergic neuromuscular junctions generated by electron tomography. First, we found that SVs are interconnected within the bouton by filaments similar to those described in vertebrates. Second, we resolved the three-dimensional structure of the dense projection centered in the AZ. The dense projection is a more complex structure than previously anticipated, with filaments radiating from a core structure that directly contact SVs in the interior of the bouton as well as SVs docked at the plasma membrane. Third, we investigated the functional correlate of these contacts by analyzing mutants disrupting two key AZ proteins: UNC-10/RIM and SYD-2/liprin. In both mutants, the number of contacts between SVs and the dense projection was significantly reduced. Similar to unc-10 mutants, the dependence of SV fusion on extracellular calcium concentration was exacerbated in syd-2 mutants when compared with the wild type. Hence, we propose that the dense projection ensures proper coupling of primed vesicles with calcium signaling by retaining them at the AZ via UNC-10/RIM and SYD-2/liprin-dependent mechanisms.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Fosfoproteínas/fisiologia , Terminações Pré-Sinápticas/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteínas de Transporte/genética , Tomografia com Microscopia Eletrônica , Fenômenos Eletrofisiológicos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intercelular , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação/fisiologia , Vias Neurais/fisiologia , Vias Neurais/ultraestrutura , Sistema Nervoso Parassimpático/ultraestrutura , Fosfoproteínas/genética , Terminações Pré-Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Fixação de TecidosRESUMO
Narcine brasiliensis electric organ was stimulated to fatigue in vivo. Electrical display of organ output and biochemical assay of bound acetylcholine (ACh) and ATP in isolated vesicles were used to assess the state of fatigue relative to denervated control organs of the same fish. A morphometric analysis of the fate of the synaptic vesicle populations in the nerve terminals was carried out. Statistically significant morphological changes in vesicle populations and plasma membranes were observed between control and fatigued electroplaque stacks from individual fish. Pooled data from several fish were used to evaluate the possible role of the different vesicle types in neurotransmission. Fatigue resulted in the loss of 49% of the total vesicle population and a 76% loss of vesicles with bound calcium (Ca). An approximately equivalent increase in the nerve-terminal plasma membrane area was measured. This was predominantly in the form of fingerlike protrusions and/or invaginations of the terminals which were present in the control organs but which were significantly increased by stimulation. Vesicle attachments to the nerve terminal membrane were reduced by 90%. This suggests that the failure in transmission may be due to reduction in the number of vesicles which are loaded with transmitter and can attach to the terminal membrane. The Ca-binding capacity of the lost vesicles was not transferred to the plasma membranes. This result was interpreted as support for the hypothesis that vesicle-bound ATP provides the Ca-binding site.
Assuntos
Órgão Elétrico/fisiologia , Sistema Nervoso Parassimpático/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Acetilcolina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Órgão Elétrico/ultraestrutura , Estimulação Elétrica , Peixes/anatomia & histologia , Mitocôndrias/ultraestrutura , Vesículas Sinápticas/metabolismoRESUMO
Modulation of evoked quantal transmitter release by protein kinase C (PKC) was investigated at an identified cholinergic neuro-neuronal synapse of the Aplysia buccal ganglion. Evoked acetylcholine release was increased by a diacylglycerol analog that activates PKC and was decreased by H-7, a blocker of PKC. FLRFamide facilitated evoked quantal release by increasing presynaptic Ca2+ influx. The inhibition of PKC by H-7 prevented both the increase of presynaptic Ca2+ influx and the facilitation of evoked acetylcholine release induced by the activation of presynaptic FLRFamide receptors. These results provide evidence that the activation of PKC could be a step in the intracellular pathway by which FLRFamide receptors increase evoked quantal acetylcholine release.
Assuntos
Neurotransmissores/metabolismo , Oligopeptídeos/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Proteína Quinase C/metabolismo , Receptores de Neurotransmissores/metabolismo , Sinapses/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Acetilcolina/metabolismo , Animais , Aplysia , Cálcio/fisiologia , Eletrofisiologia , Ativação Enzimática , Isoquinolinas/farmacologia , Oligopeptídeos/farmacologia , Sistema Nervoso Parassimpático/ultraestrutura , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Receptores Nicotínicos/metabolismoRESUMO
The mammalian mesopontine tegmentum (MPT) contains two cholinergic nuclei, the pedunculopontine tegmental nucleus (PPTg) and the laterodorsal tegmental nucleus (LDTg). These provide the cholinergic innervation of, among other brain areas, the dopaminergic A9 and A10 cell groups. Their axons are thus the source of endogenous acetylcholine (ACh) acting on somato-dendritic acetylcholine receptors in the substantia nigra (SN) and ventral tegmental area (VTA). The anatomy, physiology, functional and pathological implications of these interactions with the nicotinic subtype of acetylcholine receptors (nAChRs) are discussed with a view of the important role of the MPT as a master regulator of nicotinic dopaminergic signalling in the brain, including for nicotine addiction.
Assuntos
Dopamina/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Receptores Nicotínicos/fisiologia , Transtornos Relacionados ao Uso de Substâncias/fisiopatologia , Tegmento Mesencefálico/fisiologia , Animais , Comportamento/fisiologia , Humanos , Vias Neurais/fisiologia , Sistema Nervoso Parassimpático/patologia , Sistema Nervoso Parassimpático/ultraestrutura , Transtornos Relacionados ao Uso de Substâncias/patologia , Tegmento Mesencefálico/patologia , Tegmento Mesencefálico/ultraestruturaRESUMO
Structural transformations in the dorsal vagal complex and intermediolateral nucleus due to +G, loads were studied in white outbred male rats centrifuged according to the standard procedure (P.S.Paschenko, 1995). Methods of investigation included light and electron microscopy, morphometric analysis and statistical analysis. Acute exposure to +Gz loads resulted in essentially reactive changes in the centers under study. At the same time, regular exposure to this extreme factor led to cumulation of destructive changes. The peculiar structure of the centers governs uniqueness of disorders which may unbalance the autonomous regulation of organism functions.
Assuntos
Encéfalo/fisiopatologia , Hipergravidade , Exposição Ocupacional/efeitos adversos , Sistema Nervoso Parassimpático/fisiopatologia , Medula Espinal/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Animais , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Masculino , Microscopia Eletrônica , Sistema Nervoso Parassimpático/ultraestrutura , Ratos , Voo Espacial , Medula Espinal/ultraestrutura , Sistema Nervoso Simpático/ultraestruturaRESUMO
In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting.
Assuntos
Vias Eferentes/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Glândula Submandibular/fisiologia , Sinapses/metabolismo , Células Acinares/fisiologia , Células Acinares/ultraestrutura , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Vias Eferentes/crescimento & desenvolvimento , Vias Eferentes/ultraestrutura , Fluoresceína-5-Isotiocianato , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/ultraestrutura , Imagem Óptica , Sistema Nervoso Parassimpático/crescimento & desenvolvimento , Sistema Nervoso Parassimpático/ultraestrutura , Glândula Submandibular/crescimento & desenvolvimento , Glândula Submandibular/ultraestrutura , Sinapses/ultraestrutura , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismoRESUMO
Trauma to the conus medullaris and cauda equina may result in autonomic, sensory, and motor dysfunctions. We have previously developed a rat model of cauda equina injury, where a lumbosacral ventral root avulsion resulted in a progressive and parallel death of motoneurons and preganglionic parasympathetic neurons, which are important for i.e. bladder control. Here, we report that an acute implantation of an avulsed ventral root into the rat conus medullaris protects preganglionic parasympathetic neurons and motoneurons from cell death as well as promotes axonal regeneration into the implanted root at 6 weeks post-implantation. Implantation resulted in survival of 44+/-4% of preganglionic parasympathetic neurons and 44+/-4% of motoneurons compared with 22% of preganglionic parasympathetic neurons and 16% of motoneurons after avulsion alone. Retrograde labeling from the implanted root at 6 weeks showed that 53+/-13% of surviving preganglionic parasympathetic neurons and 64+/-14% of surviving motoneurons reinnervated the graft. Implantation prevented injury-induced atrophy of preganglionic parasympathetic neurons and reduced atrophy of motoneurons. Light and electron microscopic studies of the implanted ventral roots demonstrated a large number of both myelinated axons (79+/-13% of the number of myelinated axons in corresponding control ventral roots) and unmyelinated axons. Although the diameter of myelinated axons in the implanted roots was significantly smaller than that of control roots, the degree of myelination was appropriate for the axonal size, suggesting normal conduction properties. Our results show that preganglionic parasympathetic neurons have the same ability as motoneurons to survive and reinnervate implanted roots, a prerequisite for successful therapeutic strategies for autonomic control in selected patients with acute conus medullaris and cauda equina injuries.
Assuntos
Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Radiculopatia/cirurgia , Compressão da Medula Espinal/cirurgia , Raízes Nervosas Espinhais/fisiologia , Raízes Nervosas Espinhais/cirurgia , Transplante de Tecidos/métodos , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Sobrevivência Celular/fisiologia , Citoproteção/fisiologia , Modelos Animais de Doenças , Sobrevivência de Enxerto/fisiologia , Masculino , Microscopia Eletrônica de Transmissão , Neurônios Motores/ultraestrutura , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Paralisia/fisiopatologia , Paralisia/cirurgia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Parassimpático/ultraestrutura , Radiculopatia/fisiopatologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Compressão da Medula Espinal/fisiopatologiaRESUMO
Longitudinal electron-microscopic and morphometric studies of autonomic nerves containing predominantly parasympathetic fibers were undertaken in the spontaneously diabetic BB rat. Unmyelinated fibers of the diabetic vagus nerve and myelinated fibers of the penile nerve showed increased numbers of axonal glycogenosomes and axonal sequestration. Morphometric examination of myelinated and unmyelinated fibers of the vagus nerve revealed diminished fiber size compared with age-matched control animals. The distal myenteric nerve showed marked degenerative changes, whereas no structural changes could be demonstrated in intra-myenteric ganglion cells. These changes are similar to those described previously in somatic nerves of this model but different from those seen in sympathetic nerves of the diabetic BB rat.
Assuntos
Doenças do Sistema Nervoso Autônomo/patologia , Neuropatias Diabéticas/patologia , Sistema Nervoso Parassimpático/ultraestrutura , Animais , Axônios/ultraestrutura , Colo/inervação , Masculino , Plexo Mientérico/ultraestrutura , Fibras Nervosas/patologia , Fibras Nervosas/ultraestrutura , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/ultraestrutura , Sistema Nervoso Parassimpático/patologia , Pênis/inervação , Ratos , Ratos Endogâmicos BB , Nervo Vago/ultraestruturaRESUMO
We examined Fisher 344 female rats aged 6, 27, and 33 months old. Prior to sacrifice and morphometric analyses of forebrain cholinergic neurons all rats underwent behavioral characterization in a spatial learning task using the Morris water maze. Performance on the spatial task permitted subsequent grouping of the 27- and 33-month-old animals into impaired or nonimpaired groups. Importantly, the percentage of animals that displayed spatial impairments increased sharply with advancing age. Quantitative assessment of the size and density of choline acetyltransferase (ChAT)-positive neurons throughout the basal forebrain revealed a significant enlargement of forebrain cholinergic neurons within 27-month-old nonimpaired rats compared to 6-month-old rats and 27- and 33-month-old impaired animals. This increase in size was most noted in the medial septum and nucleus of the diagonal band. Significant decreases in the density of ChAT-positive neurons was observed only in the nucleus of the diagonal band of 27-month-old impaired rats compared to 6-month-old controls. Although the significance of enlarged forebrain cholinergic neurons is unclear, we discuss the possibility that within aged rodents neuronal swelling is an active event and represents an early manifestation of the aging process and may constitute a restorative and/or compensatory event in that these rats are relatively asymptomatic with respect to their behavioral deficits. In addition, we discuss in some detail various technical and life effect issues which may vary the outcome of investigations of aged rodents.
Assuntos
Envelhecimento/patologia , Comportamento Animal/fisiologia , Colina O-Acetiltransferase/metabolismo , Neurônios/ultraestrutura , Prosencéfalo/ultraestrutura , Envelhecimento/metabolismo , Animais , Feminino , Imuno-Histoquímica , Aprendizagem/fisiologia , Neurônios/enzimologia , Sistema Nervoso Parassimpático/enzimologia , Sistema Nervoso Parassimpático/ultraestrutura , Prosencéfalo/enzimologia , Ratos , Ratos Endogâmicos F344 , Percepção Espacial/fisiologiaRESUMO
Nerve growth factor receptor immunoreactivity in the septohippocampal pathway of adult Fischer 344 rats was assessed at the light and electron microscope level. The medial septum possesses immunoreactive somata, dendrites, axons, and terminals. Immunostained somata are either bipolar or multipolar in appearance. Dendritic processes of immunoreactive septal neurons are categorized into two groups: proximal dendrites with smooth plasma membranes and distal dendrites with numerous swellings. Immunoreactive axons within the septum are long and slender and do not possess varicosities. At the electron microscope level, immunoreactivity is confined predominantly to the plasma membrane of cell bodies and dendrites of septal neurons, as well as to the plasma membrane of axons and terminals. Both immunoreactive and nonimmunoreactive terminals that contain clear, spherical vesicles are observed contacting immunoreactive dendrites and somata. Although accumulations of vesicles are evident within these terminals at sites of contact, distinct synaptic specializations are difficult to distinguish due to the localization of reaction product on the apposing plasma membranes. Axons possessing immunoreactivity are also observed in the fimbria-fornix pathway, a major source of afferent inputs to the hippocampus. Immunoreactive axons and terminals are topographically organized in the hippocampal dentate gyrus. The density of immunostained axons and terminals is highest immediately adjacent to the granular layer. In comparison, a moderate density of immunoreactive axons is found in the outer molecular layer and a weak density in the inner molecular, granular, and polymorphic layers. Immunoreactivity is found on the plasma membrane of small unmyelinated axons and terminals aggregated into clusters throughout the dentate gyrus. Definitive examples of axosomatic and axodendritic synapses possessing immunoreactivity presynaptically are not observed. Immunoreactive profiles within the medial septum and hippocampus also circumfuse a small number of intracerebral vessels. Ultrastructural examination reveals that immunoreactivity is present within a narrowed extension of the subarachnoid space and appears to be closely associated with the plasma membrane of leptomeningeal cell processes. The present study provides direct evidence for the cellular distribution of nerve growth factor receptor immunoreactivity in the medial septum and dentate gyrus in the adult rat and offers new insight into the ultrastructural localization of nerve growth factor receptor among septal cholinergic neurons and their efferent projections to the hippocampus.
Assuntos
Hipocampo/citologia , Receptores de Superfície Celular/análise , Animais , Anticorpos Monoclonais , Axônios/fisiologia , Vasos Sanguíneos/ultraestrutura , Circulação Cerebrovascular , Dendritos/ultraestrutura , Feminino , Hipocampo/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica , Vias Neurais/citologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/ultraestrutura , Gravidez , Ratos , Ratos Endogâmicos F344 , Receptores de Fator de Crescimento NeuralRESUMO
The cat superior colliculus (SC) receives a dense cholinergic input from three brainstem nuclei, the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus, and the parabigeminal nucleus (PBG). The tegmental inputs project densely to the intermediate gray layer (IGL) and sparsely to the superficial layers. The PBG input probably projects only to the superficial layers. In the present study, the morphology of choline acetyltransferase (ChAT)-immunoreactive axons and synaptic endings in the superficial and deep layers of the SC was examined by light and electron microscopy to determine whether these cholinergic afferents form different types of synapses in the superficial and deep layers. Two types of fibers were found within the zonal (ZL) and upper superficial gray layers (SGL): small diameter fibers with few varicosities and larger diameter fibers with numerous varicosities. Quantitative analysis demonstrated a bimodal distribution of axon diameters, with one peak at approximately 0.3-0.5 micron and the other at 0.9-1.0 micron. On the other hand, ChAT-immunoreactive fibers in the IGL were almost all small and formed discrete patches within the IGL. Two types of ChAT-immunoreactive synaptic profiles were observed within the ZL and upper SGL using the electron microscope. The first type consisted of small terminals containing predominantly round synaptic vesicles and forming asymmetric synaptic contacts, mostly on dendrites. The second type was comprised of varicose profiles that also contained round synaptic vesicles. Their synaptic contacts were always symmetric in profile. ChAT-immunoreactive terminals in the IGL patches contained round or pleomorphic synaptic vesicles, and the postsynaptic densities varied from symmetric to asymmetric, including intermediate forms. However, no large varicose profiles were observed. This study suggests that cholinergic fibers include at least two different synaptic morphologies: small terminals with asymmetric thickenings and large varicose profiles with symmetric terminals. The large varicose profile in the superficial layers is absent in the IGL. This result suggests that the cholinergic inputs that innervate the superficial layers and the patches in the IGL of the cat SC differ in their synaptic organization and possibly also in their physiological actions.
Assuntos
Gatos/anatomia & histologia , Sistema Nervoso Parassimpático/ultraestrutura , Colículos Superiores/ultraestrutura , Sinapses/ultraestrutura , Animais , Gatos/fisiologia , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Microscopia Eletrônica , Fibras Nervosas/enzimologia , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Vias Neurais/anatomia & histologia , Vias Neurais/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Colículos Superiores/fisiologia , Sinapses/fisiologiaRESUMO
The ultrastructural organization of cholinergic afferents to the rat olfactory bulb (OB) was studied with the aid of choline acetyltransferase (ChAT) immunocytochemistry in electron microscopy. Particular attention has been paid to a subset of glomeruli characterized by a remarkably high density of cholinergic afferents. Numerous cholinergic terminals making symmetric or asymmetric synaptic contacts were observed in the periglomerular area. ChAT-labelled terminals have a diameter ranging from 0.3 to 1.5 micron and contain numerous clear agranular vesicles. Axo-somatic and axo-dendritic contacts were both observed in contact with several types of target neurons. Three types of cholinoceptive, noncholinergic neurons could be identified: periglomerular cells, superficial short-axon cells, and external tufted cells. Our results provide an anatomical substrate for the hypotheses concerning the complex effects of acetylcholine in the processing of sensory information in the olfactory bulb.
Assuntos
Bulbo Olfatório/ultraestrutura , Sistema Nervoso Parassimpático/ultraestrutura , Animais , Axônios/ultraestrutura , Colina O-Acetiltransferase/imunologia , Colina O-Acetiltransferase/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Bulbo Olfatório/enzimologia , Bulbo Olfatório/fisiologia , Sistema Nervoso Parassimpático/enzimologia , Sistema Nervoso Parassimpático/fisiologia , Ratos , Ratos Wistar , Sinapses/ultraestruturaRESUMO
Immunocytochemical double-labeling techniques were used at the light and electron microscopic levels to investigate whether dopamine-beta-hydroxylase and tyrosine hydroxylase-containing axons contact basal forebrain cholinergic neurons. Dopamine-beta-hydroxylase- and tyrosine hydroxylase-positive fibers and terminals were found in close proximity to cholinergic neurons throughout extensive basal forebrain areas, including the vertical and horizontal limb of the diagonal band nuclei, the sublenticular substantia innominata, bed nucleus of the stria terminalis, ventral pallidum, and ventrolateral globus pallidus. Cholinergic cells in some aspects of the globus pallidus appeared to be contacted by tyrosine hydroxylase-positive but not dopamine-beta-hydroxylase-positive fibers, suggesting dopaminergic input to cholinergic neurons in these regions. Direct evidence for the termination of dopamine-beta-hydroxylase and tyrosine hydroxylase-positive fibers on cholinergic neurons was obtained in electron microscopic double-immunolabeling studies. Using high magnification light microscopic screening, both qualitative and quantitative differences were noted in the catecholaminergic innervation of forebrain cholinergic neurons. For example, while many cholinergic neurons were in close proximity to single dopamine-beta-hydroxylase-positive varicosities, others, particularly those located in the substantia innominatabed nucleus of the stria terminalis continuum, were apparently contacted by labeled fibers in repetitive fashion. The findings of the present study, together with our preliminary biochemical experiments (Zaborszky et al. [1993] Prog. Brain Res. 98:31-49) suggest that catecholaminergic afferents can differentially modulate forebrain cholinergic neurons. Such interactions may be important in learning and memory processes, and their perturbations may contribute to the cognitive decline seen in aging and in disorders such as Alzheimer's and Parkinson's diseases.
Assuntos
Catecolaminas/fisiologia , Dopamina beta-Hidroxilase/metabolismo , Neurônios/enzimologia , Sistema Nervoso Parassimpático/fisiologia , Prosencéfalo/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Axônios/enzimologia , Axônios/fisiologia , Axônios/ultraestrutura , Mapeamento Encefálico , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Sistema Nervoso Parassimpático/enzimologia , Sistema Nervoso Parassimpático/ultraestrutura , Prosencéfalo/enzimologia , Prosencéfalo/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies have indicated that galanin is one of the most abundant peptides in the basal forebrain and that it has a significant modulatory influence on cholinergic transmission. The aim of the present study was to use a light electron microscopic correlation technique to determine whether galanin-immunoreactive terminals form synaptic contacts with basal forebrain cholinergic cells of the rat. Sections from fixed-perfused brains were stained at the light and electron microscopic levels for galanin and choline acetyltransferase immunoreactivity in the same section by using a dual-colour immunohistochemical method. The results showed that galanin-immunoreactive axonal terminals are unevenly distributed in the medial septal nucleus, the diagonal band, and the nucleus basalis. Galanin-positive synapses were most prominent on choline acetyltransferase-positive neurons in the lateral parts of the nucleus of the diagonal band and in the posterior half of the nucleus basalis, which is where there was the greatest overlap between the distribution of galanin-immunoreactive terminals and choline acetyltransferase-positive neurons. The origins of these galanin-positive terminals are not known, but the results confirm that the basal forebrain galaninergic system has a synaptic influence on basal forebrain cholinergic neurons in the rat.
Assuntos
Galanina/metabolismo , Neurônios/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Terminações Pré-Sinápticas/metabolismo , Prosencéfalo/metabolismo , Animais , Gânglios da Base/metabolismo , Gânglios da Base/ultraestrutura , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Prosencéfalo/citologia , Prosencéfalo/ultraestrutura , Ratos , Ratos Wistar , Sinapses/ultraestruturaRESUMO
Previous observations indicate that the basal forebrain receives dopaminergic input from the ventral midbrain. The present study aimed at determining the topographic organization of these projections in the rat, and whether this input directly terminates on cholinergic neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into discrete parts of the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNC) labeled axons and terminals in distinct parts of the basal forebrain, including medial and lateral septum, diagnoal band nuclei, ventral pallidum, globus pallidus, substantia innominata, globus pallidus, and internal capsule, where PHA-L-labeled terminals abutted cholinergic (choline acetyltransferase = ChAT-containing) profiles. Three-dimensional (3-D) computerized reconstruction of immunostained sections clearly revealed distinct, albeit overlapping, subpopulations of ChAT-immunoreactive neurons apposed by PHA-L-labeled input from medial VTA (mainly in vertical and horizontal diagonal band nuclei), lateral VTA and medial SNC (ventral pallidum and anterior half of substantia innominata), and lateral SNC (caudal half of the substantia innominata and globus pallidus). At the ultrastructural level, about 40% of the selected PHA-L-labeled presynaptic terminals in the ventral pallidum and substantia innominata were found to establish synaptic specializations with ChAT-containing profiles, most of which on the cell body and proximal dendritic shafts. Convergent synaptic input of unlabeled terminals that formed asymmetric synapses with the ChAT-immunoreactive profiles were often found in close proximity to the PHA-L-labeled terminals. These observations show that the cholinergic neurons in the basal forebrain are targets of presumably dopaminergic SNC/VTA neurons, and suggest a direct modulatory role of dopamine in acetylcholine release in the cerebral cortical mantle.
Assuntos
Catecolaminas/fisiologia , Neurônios/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Prosencéfalo/fisiologia , Substância Negra/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Vias Autônomas/citologia , Vias Autônomas/fisiologia , Vias Autônomas/ultraestrutura , Axônios/fisiologia , Axônios/ultraestrutura , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Neurônios/enzimologia , Neurônios/ultraestrutura , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/ultraestrutura , Fito-Hemaglutininas , Prosencéfalo/citologia , Prosencéfalo/ultraestrutura , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia , Substância Negra/ultraestrutura , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/ultraestruturaRESUMO
The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles.
Assuntos
Dendritos/fisiologia , Glicina/metabolismo , Sistema Nervoso Parassimpático/citologia , Retina/citologia , Sarcosina/farmacologia , Animais , Autorradiografia , Colina/metabolismo , Dendritos/efeitos dos fármacos , Imunofluorescência , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Confocal , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/ultraestrutura , Coelhos , Ratos , Retina/efeitos dos fármacos , Retina/ultraestruturaRESUMO
A combination of choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry was used to characterize the ultrastructural distribution of acetylcholinesterase in identified cholinergic and non-cholinergic neurons in the ferret brain. Previous studies have shown that most of the cholinergic input to the brain arises from choline acetyltransferase-positive neurons found in the neostriatum, basal forebrain and dorsolateral pontine tegmentum. In all these cells intense staining for acetylcholinesterase was localized within the cisternae of the rough endoplasmic reticulum, in the nuclear envelope and Golgi apparatus, and along the plasma membranes of the soma and dendrites. In contrast, the distribution of acetylcholinesterase in non-cholinergic neurons was restricted mainly to the cisternae of the endoplasmic reticulum and the nuclear envelope. Since previous studies have associated high levels of acetylcholinesterase staining with non-cholinergic neurons in the locus coeruleus and substantia nigra zona compacta, these areas were examined as well. The ultrastructural localization of acetylcholinesterase in the principal locus coeruleus neurons was as observed in typical non-cholinergic neurons. On the other hand, the distribution of acetylcholinesterase in the principal cells of the zona compacta of the substantia nigra was more like that found in cholinergic neurons. In conclusion, the subcellular distribution of acetylcholinesterase in the principal cholinergic neurons of the brain follows a characteristic pattern which, with one exception, is different from that of acetylcholinesterase-positive non-cholinergic neurons.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/enzimologia , Dendritos/enzimologia , Neurônios/enzimologia , Sistema Nervoso Parassimpático/enzimologia , Animais , Encéfalo/ultraestrutura , Colina O-Acetiltransferase/metabolismo , Feminino , Furões , Histocitoquímica , Imuno-Histoquímica , Locus Cerúleo/enzimologia , Masculino , Microscopia Eletrônica , Neurônios/ultraestrutura , Sistema Nervoso Parassimpático/ultraestrutura , Coloração e Rotulagem , Substância Negra/enzimologia , Distribuição TecidualRESUMO
The distribution of specifically-labeled neurotensin binding sites was examined in relation to that of cholinergic neurons in the rat nucleus basalis magnocellularis at both light and electron microscopic levels. Lightly prefixed forebrain slices were either labeled with [125I](Tyr3) neurotensin alone or processed for combined [125I]neurotensin radioautography and acetylcholinesterase histochemistry. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of single-labeled slices, silver grains were found to be preferentially localized over perikarya and proximal processes of nucleus basalis cells. The label was distributed both throughout the cytoplasm and along the plasma membrane of magnocellular neurons all of which were found to be cholinesterase-positive in a double-labeled material. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that the major fraction (80-89%) of specifically-labeled binding sites associated with cholinesterase-reactive cell bodies and dendrites was intraneuronal. These intraneuronal sites were mainly dispersed throughout the cytoplasm and are thus likely to represent receptors undergoing synthesis, transport and/or recycling. A proportion of the specific label was also localized over the nucleus, suggesting that neurotensin could modulate the expression of acetylcholine-related enzymes in the nucleus basalis. The remainder of the grains (11-20%) were classified as shared, i.e. overlied the plasma membrane of acetylcholinesterase-positive neuronal perikarya and dendrites. Extrapolation from light microscopic data, combined with the observation that shared grains were detected at several contact points along the plasma membrane of cells which also exhibited exclusive grains, made it possible to ascribe these membrane-associated receptors to the cholinergic neurons themselves rather than to abutting cellular profiles. Comparison of grain distribution with the frequency of occurrence of elements directly abutting the plasma membrane of neurotensin-labeled/cholinesterase-positive perikarya indicated that labeled cell surface receptors were more or less evenly distributed along the membrane as opposed to being concentrated opposite abutting axon terminals endowed or not with a visible junctional specialization. The low incidence of labeled binding sites found in close association with abutting axons makes it unlikely that only this sub-population of sites corresponds to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of cholinergic neurons suggests that neurotensin acts primarily in a paracrine mode to influence the magnocellular cholinergic system in the nucleus basalis.
Assuntos
Gânglios da Base/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Receptores de Neurotransmissores/análise , Acetilcolinesterase/química , Animais , Autorradiografia , Gânglios da Base/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Radioisótopos do Iodo , Masculino , Microscopia Eletrônica , Sistema Nervoso Parassimpático/ultraestrutura , Ratos , Ratos Endogâmicos , Receptores de Neurotensina , Coloração e Rotulagem , Cloreto de TolônioRESUMO
Nerve growth factor selectively prevents the degeneration of cholinergic neurons following intrastriatal infusion but rescues both cholinergic and noncholinergic striatal neurons if the nerve growth factor is secreted from grafts of genetically modified fibroblasts. The present study evaluated whether grafted fibroblasts genetically modified to secrete human nerve growth factor could provide trophic influences upon intact cholinergic and noncholinergic striatal neurons. Unilateral striatal grafts of polymer-encapsulated cells genetically modified to secrete human nerve growth factor induced hypertrophy and significantly increased the optical density of choline acetyltransferase-immunoreactive striatal neurons one, two, and four weeks post-transplantation relative to rats receiving identical grafts missing only the human nerve growth factor construct. Nerve growth factor secreting grafts also induced a hypertrophy of noncholinergic neuropeptide Y-immunoreactive striatal neurons one, two, and four weeks post-transplantation. Glutamic acid decarboxylase-immunoreactive neurons were unaffected by the human nerve growth factors secreting grafts. The effects upon choline acetyltransferase-immunoreactive and neuropeptide Y-immunoreactive striatal neurons dissipated following retrieval of the implants. Immunocytochemistry for nerve growth factor revealed intense graft-derived immunoreactivity for up to 1000 microns from the capsule extending along the dorsoventral axis of the striatum. Nerve growth factor-immunoreactivity was also observed within a subpopulation of striatal neurons and may represent nerve growth factor consumer neurons which retrogradely transported graft-derived nerve growth factor. When explanted, grafts produced 2-4 ng human nerve growth factor/24 h over the time course of this study indicating that this level of continuous human nerve growth factor secretion was sufficient to mediate the effects presently observed.
Assuntos
Transplante de Células/métodos , Neostriado/fisiologia , Fatores de Crescimento Neural/biossíntese , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Linhagem Celular , Colina O-Acetiltransferase/metabolismo , Cricetinae , Ensaio de Imunoadsorção Enzimática , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Humanos , Imuno-Histoquímica , Masculino , Neostriado/citologia , Neostriado/ultraestrutura , Fatores de Crescimento Neural/genética , Neuropeptídeo Y/metabolismo , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/ultraestrutura , Polímeros , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/ultraestruturaRESUMO
The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.