RESUMO
Functional properties of the neural cell adhesion molecule (NCAM) are strongly influenced by polysialylation. We used gene-targeting to generate mice lacking ST8SiaIV/PST-1, one of the polysialyltransferases responsible for addition of polysialic acid (PSA) to NCAM. Mice homozygous for the null mutation reveal normal development of gross anatomical features. In contrast to NCAM-deficient mice, olfactory precursor cells in the rostral migratory stream express PSA and follow their normal pathway. Furthermore, delamination of mossy fibers in the hippocampal CA3 region, as found in NCAM-deficient mice, does not occur in ST8SiaIV mutants. However, during postnatal development these animals show a decrease of PSA in most brain regions compared to wild-type animals. Loss of PSA in the presence of NCAM protein but in the absence of obvious histological changes allowed us to directly address the role of PSA in synaptic plasticity. Schaffer collateral-CA1 synapses, which express PSA in wild types, showed impaired long-term potentiation (LTP) and long-term depression (LTD) in adult mutants. This impairment was age-dependent, following the time course of developmental disappearance of PSA. Contrary to NCAM mutant mice, LTP in ST8SiaIV mutants was undisturbed at mossy fiber-CA3 synapses, which do not express PSA in wild-type mice. The results demonstrate an essential role for ST8SiaIV in synaptic plasticity in hippocampal CA1 synapses, whereas PSA produced by different polysialyltransferase or polysialyltransferases at early stages of differentiation regulates migration of neural precursor cells and correct lamination of mossy fibers. We suggest that NCAM but not PSA is likely to be important for LTP in the hippocampal CA3 region.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/deficiência , Envelhecimento , Animais , Encéfalo/metabolismo , Potenciais Pós-Sinápticos Excitadores , Hipocampo/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Inibição Neural/genética , Neurônios/citologia , Sialiltransferases/genética , Sinapses/metabolismoRESUMO
The Neural Cell Adhesion Molecule (NCAM) serves as a temporally and spatially regulated modulator of a variety of cell-cell interactions. This review summarizes recent results of studies aimed at understanding its regulation of expression and biological function, thereby focussing on its polysialylated isoforms (PSA-NCAM). The detailed analysis of the expression of PSA and NCAM in the hippocampal mossy fiber system and the morphological consequences of PSA-NCAM deficiency in mice support the notion that the levels of expression of NCAM are important not only for the regulation and maintenance of structural changes, such as migration, axonal growth and fasciculation, but also for activity-induced plasticity. There is evidence that PSA-NCAM can specifically contribute to a presynaptic form of plasticity, namely long-term potentiation at hippocampal mossy fiber synapses. This is consistent with previous observations that NCAM-deficient mice show deficits in spatial learning and exploratory behavior. Furthermore, our data points to an important role of the hypothalamic-pituitary-adrenal axis, which is the principle adaptive response of the organism to environmental challenges, in the control of PSA-NCAM expression in the hippocampal formation. In particular, we evidence an inhibitory influence of corticosterone on PSA-NCAM expression.
Assuntos
Hipocampo/fisiologia , Molécula L1 de Adesão de Célula Nervosa , Moléculas de Adesão de Célula Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Ácidos Siálicos/fisiologia , Animais , Hipocampo/químicaRESUMO
In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively gamma-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.
Assuntos
Moléculas de Adesão de Célula Nervosa/genética , Neurônios Aferentes/metabolismo , Bulbo Olfatório/citologia , Olfato/genética , Animais , Bromodesoxiuridina , Divisão Celular/genética , Imuno-Histoquímica , Masculino , Memória de Curto Prazo , Camundongos , Camundongos Transgênicos , Mutação , Prosencéfalo/metabolismo , Desempenho PsicomotorRESUMO
In vertebrates, interneurons of the olfactory bulb (OB) are generated postnatally and throughout life at the subventricular zone of the forebrain. The neuronal precursors migrate tangentially through the forebrain using a well defined pathway, the rostral migratory stream (RMS), and a particular mode of migration in a chain-like organization. A severe size reduction of the OB represents the most striking morphological phenotype in neural cell adhesion molecule (NCAM)-deficient mice. This defect has been traced back to a migration deficit of the precursors in the RMS and linked to the lack of the polysialylated form of NCAM. In this study we investigate the morphological alterations and functional properties of the RMS in mice totally devoid of all isoforms of NCAM and polysialic acid (PSA). We show that a morphologically altered, but defined and continuous pathway exists in mutants, and we present in vivo and in vitro evidence that PSA-NCAM in the RMS is not essential for the formation and migration of chains. Instead, we find a massive gliosis associated with the formation of membrane specializations in a heterotypic manner, linking precursors to astrocytes. This finding and the over-representation and defasciculation of axons in the pathway suggest that important interactions between migrating cells and their stationary environment are perturbed in the mutants. Finally, we used transplantation experiments to demonstrate that lack of PSA-NCAM leads to a decrease but not a total blockade of migration and demonstrate that the mutant RMS is functional in transporting normal neuronal precursors to the OB.
Assuntos
Movimento Celular/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Prosencéfalo/fisiologia , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/fisiologia , Cruzamentos Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/genética , Neuroglia/citologia , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Neurônios/citologia , Neurônios/ultraestrutura , Bulbo Olfatório/anormalidades , Bulbo Olfatório/ultraestrutura , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Técnicas de Cultura de Órgãos , Prosencéfalo/citologia , Prosencéfalo/ultraestrutura , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Ácidos Siálicos/genética , Células-Tronco/fisiologiaRESUMO
Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.
Assuntos
Caspases/fisiologia , Proteínas Fetais/fisiologia , Membro Posterior/embriologia , Necrose , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/fisiologia , Inibidores de Caspase , Cromatina/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Desenvolvimento Embrionário e Fetal , Proteínas Fetais/antagonistas & inibidores , Membro Posterior/anormalidades , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Camundongos Mutantes , Morfogênese/fisiologia , Organelas/ultraestrutura , Proteínas/genética , Receptores de Fatores de Crescimento/agonistas , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
Cell adhesion molecules (CAMs) are known to be involved in a variety of developmental processes that play key roles in the establishment of synaptic connectivity during embryonic development, but recent evidence implicates the same molecules in synaptic plasticity of the adult. In the present study, we have used neural CAM (NCAM)-deficient mice, which have learning and behavioral deficits, to evaluate NCAM function in the hippocampal mossy fiber system. Morphological studies demonstrated that fasciculation and laminar growth of mossy fibers were strongly affected, leading to innervation of CA3 pyramidal cells at ectopic sites, whereas individual mossy fiber boutons appeared normal. Electrophysiological recordings performed in hippocampal slice preparations revealed that both basal synaptic transmission and two forms of short-term plasticity, i.e., paired-pulse facilitation and frequency facilitation, were normal in mice lacking all forms of NCAM. However, long-term potentiation of glutamatergic excitatory synapses after brief trains of repetitive stimulation was abolished. Taken together, these results strongly suggest that in the hippocampal mossy fiber system, NCAM is essential both for correct axonal growth and synaptogenesis and for long-term changes in synaptic strength.
Assuntos
Hipocampo/fisiopatologia , Deficiências da Aprendizagem/genética , Transtornos Mentais/genética , Fibras Nervosas/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Axônios/patologia , Axônios/fisiologia , Axônios/ultraestrutura , Hipocampo/citologia , Hipocampo/patologia , Técnicas In Vitro , Deficiências da Aprendizagem/patologia , Deficiências da Aprendizagem/fisiopatologia , Potenciação de Longa Duração , Transtornos Mentais/patologia , Transtornos Mentais/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas/patologia , Fibras Nervosas/ultraestrutura , Moléculas de Adesão de Célula Nervosa/análise , Moléculas de Adesão de Célula Nervosa/genética , Plasticidade Neuronal/genética , Sinapses/ultraestrutura , Transmissão Sináptica , Sinaptofisina/análise , Sinaptofisina/biossínteseRESUMO
The aim of the present study was to provide a quantitative analysis of the synapses made onto the axon hillock and initial segment of rat abducens motoneurons retrogradely or intracellularly stained with HRP. GABA-immunoreactive terminals contacting these axons were visualized using a postembedding procedure. The presynaptic terminals contained either spherical or pleomorphic vesicles. gamma-Aminobutyric acid (GABA)-immunoreactive axon terminals, which belonged to this last category, were distributed both onto axon hillocks and the proximal part of initial segments. The percentage of axonal membrane covered by synapses ranged from 44.1 to 68.2%. A quantitative analysis performed on a series of ultrathin sectioned terminals contacting the axon of an intracellularly labeled motoneuron revealed a significant correlation between the length of membrane apposition of the terminals and their perimeter or surface area, and also between the area of membrane apposition and terminal volume. GABA-immunoreactive terminals had a mean perimeter and volume that were larger than those of unlabeled axon terminals. The number of active zones was correlated with the area of apposition. Some hypotheses concerning the functional role of the GABAergic innervation of this particular part of the neuron are discussed.
Assuntos
Nervo Abducente/fisiologia , Axônios/fisiologia , Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Ácido gama-Aminobutírico/fisiologia , Nervo Abducente/citologia , Nervo Abducente/ultraestrutura , Animais , Axônios/ultraestrutura , Peroxidase do Rábano Silvestre , Imuno-Histoquímica , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
The neural cell adhesion molecule (NCAM), probably the best characterized and most abundant cell adhesion molecule on neurons, is thought to be a major regulator of axonal growth and pathfinding. Here we present a detailed analysis of these processes in mice deficient for all NCAM isoforms, generated by gene targeting. The hippocampal mossy fiber tract shows prominent expression of polysialylated NCAM and the generation of new axonal projections throughout life. Focusing on this important intrahippocampal connection, we demonstrate that in the absence of NCAM, fasciculation and pathfinding of these axons are strongly affected. In addition we show alterations in the distribution of mossy fiber terminals. The phenotype is more severe in adult than in young animals, suggesting an essential role for NCAM in the maintenance of plasticity in the mature nervous system.
Assuntos
Axônios/fisiologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Envelhecimento/fisiologia , Animais , Complexo de Golgi/ultraestrutura , Hipocampo/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout/genética , Terminações Nervosas/ultraestrutura , Fibras Nervosas/ultraestrutura , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/ultraestrutura , Coloração e RotulagemRESUMO
Interactions mediated by cell surface glycoproteins are considered to be crucial during the formation of the nervous system. Using a monoclonal antibody directed to mCD24, a glycosylphos-phatidylinositol-anchored membrane glycoprotein, we have mapped its distribution throughout the mouse cerebral cortex during development and in young adult. Before birth, mCD24 immunoreactivity was observed in the intermediate zone, the cortical plate and the marginal zone, whereas the ventricular zones were immunonegative. After birth, mCD24 expression declined rapidly in the cortex, except in the corpus callosum (and other commissures in the brain) where immunoreactivity was still found until P20. Furthermore, mCD24 expression was maintained in young adults (until P60, at least) in zones of secondary neurogenesis, such as the granule cells of the dentate gyrus, the subventricular zone lining the anterior part of the lateral ventricles and a zone of cells extending between the striatum and the corpus callosum to the centre of the olfactory bulb. In this area mCD24 and polysialic acid neural cell adhesion molecule stainings were superimposed, and this corresponded to the pathway of migration of the olfactory immature neurons (subependymal layer). A layer of ciliated ependymal cells, lining all the ventricular walls, was also immunoreactive for mCD24. Thus, except for these epithelial-like cells, mCD24 was essentially found associated with differentiating postmitotic neurons. Its spatiotemporal expression, both during development and in the adult, is compatible with a role for this glycoprotein in cell surface recognition and in signalling events occurring during neuronal migration and axonal growth.
Assuntos
Envelhecimento/metabolismo , Antígenos CD/biossíntese , Encéfalo/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Animais , Antígenos CD/análise , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Antígeno CD24 , Feminino , Idade Gestacional , Glicosilfosfatidilinositóis , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Moléculas de Adesão de Célula Nervosa/análise , Especificidade de Órgãos , GravidezRESUMO
The coexistence of gamma-aminobutyric acid (GABA) and glycine in axon terminals impinging on rat abducens motoneurones was investigated using a double staining procedure combining retrograde labelling of the motoneurones with HRP and post-embedding immunocytochemical staining of axon terminals. Adjacent ultrathin sections of cell bodies of identified motoneurones were individually treated with GABA or glycine antibodies. The terminals single labelled for GABA represented 11.4% of the terminals analyzed, while 8% of them were glycine immunoreactive and 9% were both GABA and glycine immunoreactive. All the labelled terminals contained pleomorphic vesicles. The mean length of apposition of the double labelled terminals was statistically larger (2.20 +/- 0.97 microns) than the GABA (1.65 +/- 0.57 microns) or glycine immunoreactive ones (1.37 +/- 0.35 microns).
Assuntos
Nervo Abducente/química , Glicina/análise , Neurônios Motores/química , Ácido gama-Aminobutírico/análise , Nervo Abducente/citologia , Vias Aferentes/química , Animais , Axônios/química , Imuno-Histoquímica , Masculino , Neurônios Motores/ultraestrutura , Terminações Nervosas/química , Ratos , Ratos WistarRESUMO
In this quantitative electron microscopic study we investigated the distribution of GABA axon terminals on rat abducens motoneurons by combining retrograde labelling of montoneurons with post-embedding immunodetection of GABA. We analysed the synapses on 13 cell bodies and 60 proximal dendritic profiles distributed along the entire rostro-caudal extent of the nucleus. For each of these two compartments, we analysed 1754 and 1176 axon terminals in contact with 6042 and 3299 microns of postsynaptic membrane. The axon terminals were classified as Sv-type (containing spherical vesicles) or Pv-type (containing pleomorphic vesicles). The GABAergic terminals contained pleomorphic vesicles and established mainly symmetrical synaptic contacts. Their apposition lengths were greater than those of unlabelled terminals. On cell bodies, the percentage of GABAergic synaptic covering varied from 2.5% to 14.1% and the synaptic frequency of GABAergic axon terminals varied from 0.6% to 8.9%. These two parameters were significantly correlated with the diameter of the motoneurons. The percentage of synaptic covering and synaptic frequency were smaller on dendrites of small motoneurons than on those of large ones. The proximal dendrites of small motoneurons had a lesser GABAergic innervation than large ones. The total synaptic covering and frequency were smaller on somata than on dendrites. However, the percentage of synaptic covering by GABA terminals was higher on cell bodies than on proximal dendrites.
Assuntos
Nervo Abducente/ultraestrutura , Dendritos/ultraestrutura , Neurônios Motores/ultraestrutura , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/análise , Nervo Abducente/química , Animais , Tamanho Celular , Peroxidase do Rábano Silvestre , Masculino , Microscopia Eletrônica , Terminações Nervosas/ultraestrutura , Ratos , Ratos Wistar , Sinapses/químicaRESUMO
This electron microscopic study, based on serial section analysis, describes the synaptic organization of the interstitial subnucleus of the nucleus of the solitary tract and identifies the terminals of the vagal primary afferents utilizing degeneration and HRP transport. The interstitial subnucleus contains sparsely scattered cell bodies, numerous dendrites and axon terminals, and bundles of unmyelinated and myelinated axons. The cell bodies which are small in diameter have an organelle poor cytoplasm and a large invaginated nucleus. Axon terminals can be classified into two main types according to their vesicular shape. The first type contains clear, round vesicles and can be further subdivided into two subgroups on the basis of their morphology and the size of their vesicles. In the first subgroup the terminals are small, contain a few mitochondria and their vesicles are densely packed with an homogeneous size. In the second subgroup the terminals which vary from small to large, contain many mitochondria and contain round vesicles which are heterogeneous in size. The second main terminal type consists of axon terminals containing pleomorphic vesicles which are associated with asymmetrical or symmetrical synaptic contacts on dendrites. Axo-axonic contacts are present in the interstitial subnucleus. In general, the presynaptic axon terminals contain pleomorphic vesicles and the postsynaptic elements contain round vesicles of varying size. In some dendrites, identified by the presence of ribosomes, groups of round and/or pleomorphic vesicles are found associated with synaptic contacts. These dendrites are presynaptic to conventional dendrites and postsynaptic to axon terminals. After removal of the nodose ganglion, degenerative alterations are seen only at the caudal and middle levels of the interstitial subnucleus. Degeneration occurs in a few myelinated axons and in axon terminals which usually contain a mixture of small and larger round, clear vesicles. After HRP injection into the vagus nerve, the HRP reaction product is visible in axon terminals filled with clear, round vesicles which are heterogeneous in size. The labelled axon terminals establish single or multiple synaptic contacts. This study demonstrates that terminals of vagal primary afferents consist principally of terminals of the second subgroup. The morphology of these terminals are compared to primary afferents in the brainstem and spinal cord.
Assuntos
Bulbo/ultraestrutura , Nervo Vago/ultraestrutura , Vias Aferentes/ultraestrutura , Animais , Axônios/ultraestrutura , Gatos , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Dendritos/ultraestrutura , Feminino , Peroxidase do Rábano Silvestre , Masculino , Microscopia Eletrônica , Degeneração NeuralRESUMO
Serotonin (5-HT) immunocytochemistry was used at the electron microscopic level to examine 5-HT neurons reinnervating and hyperinnervating the hippocampus of adult rat, three to four months after a total 5-HT denervation and subsequent graft of embryonic raphe cells. The study focused on immunostained nerve cell bodies, dendrites and axon terminals (varicosities) in the core of grafts, and on a large single section sampling of axon terminals from a CA3 and a dentate gyrus sector of the outgrowth, which were systematically compared to the endogenous 5-HT innervation of the same regions described in a companion paper. The shape, size and synaptic investment of the grafted 5-HT somata and their dendrites resembled those of in situ 5-HT neurons. Clusters of small, clear vesicles were sometimes seen along these 5-HT dendrites. 5-HT axonal varicosities were fairly numerous in the core. A few were directly apposed to, or made asymmetrical synaptic contact with the immunostained dendrites and perikarya, but the vast majority showed no indication of junctional specialization (synaptic incidence of 19%, as stereologically extrapolated for whole varicosities). Occasional myelinated 5-HT axons were also present in the core of grafts. In the two outgrowth sectors, the graft-borne 5-HT varicosities were similar in size, content, frequency of synaptic contact and identity of junctional and appositional elements, irrespective of their laminar location. Moreover, none of these parameters were significantly different from those of the endogenous innervation. Notably, in spite of their excessive number, the synaptic incidence of the outgrowth 5-HT varicosities remained inferior to 20%. The similarity between the respective microenvironments of the supernumerary, graft-borne 5-HT terminals and of their normal counterparts could only be explained by a random intratissular distribution of these varicosities in both the normal and the grafted hippocampus. Thus, in spite of their transplantation and growth into an abnormal milieu, and the fact that they hyperinnervated the host tissue, the grafted embryonic 5-HT neurons appeared committed to express a particular set of intrinsic and relational morphological features corresponding to their normal adult characteristics.
Assuntos
Transplante de Tecido Encefálico , Transplante de Tecido Fetal , Hipocampo , Neurônios/transplante , Serotonina/análise , 5,7-Di-Hidroxitriptamina/toxicidade , Animais , Axônios/ultraestrutura , Transplante de Tecido Encefálico/fisiologia , Dendritos/química , Dendritos/ultraestrutura , Denervação , Feminino , Transplante de Tecido Fetal/fisiologia , Sobrevivência de Enxerto , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Microscopia Eletrônica , Neurônios/química , Neurônios/fisiologia , Neurônios/ultraestrutura , Núcleos da Rafe/citologia , Núcleos da Rafe/embriologia , Ratos , Ratos Endogâmicos , Sinapses/química , Sinapses/ultraestruturaRESUMO
This study describes the ventrobasal complex of the primate by using GABA immunocytochemistry at the electron microscopic level. The primate ventrobasal complex has a similar synaptic organization to sensory thalamic nuclei in other species. Two synaptic profiles within the ventrobasal complex contain flattened or pleomorphic synaptic vesicles and are GABA-immunoreactive. F-boutons (= F1 type, Guillery's classification; Guillery: Z. Zellforsch. 96:1-38, '69) are located principally in the extraglomerular neuropil and contain densely packed flattened synaptic vesicles and several elongate mitochondria and establish symmetric (Gray's type II) synaptic contacts. These boutons are not found postsynaptic to any other element and are presynaptic principally to nonimmunoreactive elements that are thought to be thalamocortical relay cell dendrites. PSD-boutons (= F2 type, Guillery's classification) contain a moderate number of flattened or pleomorphic synaptic vesicles and fewer mitochondria than F-boutons. PSD-boutons are found in glomerular and extraglomerular areas of neuropil and establish symmetric synaptic contacts. These boutons are considered to be appendages of interneuron dendrites and are postsynaptic to RL-, RS (Guillery's classification)-, F-, and other PSD-boutons. PSD-boutons are presynaptic to thalamocortical relay neurons and interneuron dendrites including PSD-boutons. Problems in distinguishing F- from PSD-boutons are addressed by comparing immunostained and nonimmunostained material and by the use of serial sections. The majority of synaptic contacts between pleomorphic vesicle-containing profiles appear to be between PSD-boutons and other components of interneurons. Few contacts between F-boutons and local circuit neurons are seen. These data suggest the principal GABAergic input to interneurons in the primate ventrobasal complex is derived from other interneurons.
Assuntos
Macaca/anatomia & histologia , Núcleos Talâmicos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Imuno-Histoquímica , Macaca/metabolismo , Microscopia Eletrônica , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Núcleos Talâmicos/ultraestruturaRESUMO
The synaptic organization of serotonin elements in the nucleus raphe magnus of adult rat was investigated by electron microscopic immunocytochemistry with an antiserum against serotonin itself. Immunoreactive somata in the nucleus raphe magnus were usually of the same size (7-15 microns) and showed similar cytological features as their unlabeled congeners. The serotonin perikarya were contacted by a few unlabeled axon terminals containing round synaptic vesicles, and gave rise to dendrites which often ran perpendicularly to the midline. These dendrites received many contacts from axon terminals containing round or pleomorphic synaptic vesicles, but themselves failed to show vesicular aggregates or membrane differentiations suggestive of synaptic specialization. Immunoreactive axon terminals were numerous and mostly contained round or pleomorphic vesicles. Several exhibited synaptic contacts on dendrites, but there were also others which did not show any synaptic membranous differentiation, even when followed in serial sections. Immunostained myelinated as well as unmyelinated axons could be observed. These results provide a first description of the morphology and synaptic organization of the serotonin neurons in adult rat nucleus raphe magnus.
Assuntos
Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Animais , Dendritos/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Núcleos da Rafe/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
This electron microscopic study describes the different types of synaptic terminals found in the nucleus raphe dorsalis of the adult cat. Serial section analysis was used extensively to confirm the nature of the synaptic contact established by the various classes of terminals. Five different classes of terminals are identified according to the shape and packing density of the synaptic vesicles and type of contact they establish. The most common class (RDI-type) contains densely packed, round, agranular synaptic vesicles and establishes asymmetrical synaptic contacts. A second class (RDII-type) also contains spherical synaptic vesicles, but establishes symmetrical synaptic contacts with dendrites of all sizes. Most of the terminals in these two classes contain a few dense-cored synaptic vesicles, but a small sub-group contains many dense-cored vesicles. A third, less frequent, class (RSI-type) contains sparsely packed spherical synaptic vesicles and the majority of these terminals have asymmetrical contacts. A fourth terminal class contains pleomorphic synaptic vesicles (P-type), contacts dendrites of all sizes, and usually establishes symmetrical synaptic contacts. Finally, boutons thought to be the vesicle-filled excrescences of dendrites (postsynaptic dendrites) are found and in some cases the dendritic origin of these profiles was confirmed by serial sectioning. Such boutons containing pleomorphic vesicles are presynaptic to other such dendrites as well as conventional dendrites, and are postsynaptic to the other terminal types described. Somata within the nucleus exhibit somatic spines but receive few synaptic contacts. Most axo-somatic terminals contain either round or pleomorphic vesicles and have postsynaptic thickenings intermediate to the symmetric and asymmetric types.
Assuntos
Núcleos da Rafe/ultraestrutura , Sinapses/ultraestrutura , Animais , Axônios/ultraestrutura , Gatos , Dendritos/ultraestrutura , Microscopia Eletrônica , Vesículas Sinápticas/ultraestruturaRESUMO
In the nucleus raphe dorsalis of the cat, an electron microscopic immunocytochemistry method was used to identify the fine structure of serotoninergic dendritic profiles and axon terminals analyzed in serial sections. Two classes of serotoninergic dendrites were distinguished in the nucleus. The first class was constituted by conventional serotonin (5-HT) dendrites that were contacted by unlabeled axon terminals containing differing populations of synaptic vesicles. The second class consisted of serotoninergic dendrites that contained vesicles in their dendritic shafts. Such 5-HT dendrites were further subdivided into two groups according to their synaptic contacts. In some 5-HT vesicle-containing dendrites, the vesicles were densely packed in small clusters and were associated with a well-defined synaptic specialization. These dendrites were classified as serotoninergic presynaptic dendrites and established synaptic contacts with unlabeled and labeled dendrites and were contacted by unlabeled axon terminals. In other 5-HT vesicle-containing dendrites, extensive serial section examination showed that the vesicles could be observed near the membrane but were never found to be associated with any synaptic membrane specialization. Serotoninergic axon terminals that were presumed to be recurrent collaterals of 5-HT neurons were present in the nucleus. Some of them were observed in synaptic contact with dendrites or dendritic protrusions whereas others did not exhibit synaptic specializations. The existence of serotoninergic dendrodendritic synaptic contacts and axon terminals suggests direct local interactions between serotoninergic neurons within the nucleus raphe dorsalis.
Assuntos
Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Animais , Axônios/metabolismo , Gatos , Dendritos/metabolismo , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Núcleos da Rafe/ultraestrutura , Sinapses/metabolismoRESUMO
The nucleus raphe dorsalis of the cat was examined by serial section electron microscopy and the presence of vesicle-containing dendrites is reported. Such dendrites were divided into two classes according to their synaptic contact. Dendrites containing round and/or pleomorphic vesicles associated with a clear synaptic specialization which was generally intermediate between a Gray's type I or II were classified as presynaptic dendrites. These presynaptic dendrites were presynaptic to conventional dendrites and dendritic spines. In addition, some profiles containing a sparse population of vesicles which may be dendritic in nature were observed involved in serial synaptic arrangements. A second class of dendrites were characterized by the presence of vesicles which were never found associated with any synaptic membrane specialization. Commonly, the vesicles were densely packed and associated with unusual densities. Serial section analysis of these densities showed that they were not presynaptic dense projections. We suggest that the existence of vesicle-containing dendrites in the nucleus raphe dorsalis of the cat constitute the morphological support for the dendritic release of neurotransmitters.
Assuntos
Dendritos/ultraestrutura , Núcleos da Rafe/ultraestrutura , Animais , Gatos , Técnicas Histológicas , Microscopia Eletrônica , Sinapses/ultraestruturaRESUMO
Antibodies to 5-hydroxytryptamine (5-HT) were obtained from 4 rabbits after injections of 5-HT coupled to bovine serum albumin by means of paraformaldehyde (PF). Two methods were used to monitor the development of antibodies (AB): the one based on the "in vitro" competitive binding properties of the antibodies with 3(H)5-HT, the other, on their "in situ" binding properties to endogenous 5-HT, using the peroxidase-anti-peroxidase immunohistochemical technique, applied to paraffin embedded sections of cat brainstem. No pharmacological processing, detergents or proteolytic enzymes were used. The specificity of the antiserum was tested by competitive procedures with 20 analogs using the "in vitro" and "in situ" techniques. "In vitro" studies were performed with 5-HT free analogs and with analogs previously coupled with PF to lysine. Radioimmunological tests showed that the antibodies recognize mainly the ethylamine (CH2-CH2-NH2)-chain of the free analogs and that the best specificity was obtained with the 5-HT conjugate (5-HT-lysine-PF). The results suggest that the hapten is coupled through the phenolic positions C4 or C5. The "in situ" immunohistochemical extinction assays also revealed a distinct specificity for 5-HT. Possible optical and ultrastructural applications are illustrated in the raphé nuclei of the cat. These results confirm the reliability of radioimmunological tests for studying the specificity of AB directed against haptens, provided that haptens and analogs tested were first chemically transformed to resemble the immunogen (herewith lysine-PF coupling) with regard to its antigenic structure.
Assuntos
Anticorpos , Núcleos da Rafe/metabolismo , Serotonina/imunologia , Animais , Especificidade de Anticorpos , Gatos , Técnicas Imunoenzimáticas , Serotonina/metabolismoRESUMO
The distributions of ganglionar cell bodies, specifically labeled with 3H-5-HT, were studied in light microscope autoradiographs of the cat nodose ganglion. The size, number and localization of labeled cells were examined under different experimental conditions: after in vitro incubation of the ganglion with 3H-5-HT and after retrograde transport of 3H-5-HT injected into the nucleus of the solitary tract (NST) which receives primary visceral sensory projections from nodose ganglia cells of the vagal nerve. Following incubation of the nodose ganglia with a low concentration of 3H-5-HT (10(-6) M), some ganglionar cell bodies took up and retained the tracer. In both the right and left ganglia, they were significantly smaller in size than the unreactive neurones. The mean diameter of their perikaryon was 36.97 +/- 0.52 microns, compared with 45.76 +/- 0.87 microns in unreactive neurones. About 600 labeled cell bodies were counted in each ganglion, corresponding to 2-3% of the total nodose ganglion cell population. These reactive neurones were not localized in one particular area of the ganglia, but scattered throughout both of them. Following bilateral or unilateral microinjections of 3H-5-HT in the NST, retrogradely labeled cell bodies were observed, 24 h later, in the nodose ganglia. Their mean diameter was estimated to be 36.14 +/- 0.69 microns and they represented approximately 2% of the total ganglion cell population. As in in vitro experiments, the labeled cells were not grouped in any particular region of the ganglion. These experiments show that the distribution of both populations of labeled cells, observed under these conditions, are comparable. On an anatomical and quantitative basis one may reasonably suppose that the perikaryal or terminal uptakes concern the same neuronal population.