RESUMO
The experimental model of cortical dysplasia (CD) obtained by administering carmustine (1-3-bis-chloroethyl-nitrosurea [BCNU]) in pregnant rat uterus mimics the histopathological abnormalities observed in human CD patients: altered cortical layering, and presence of heterotopia and dysmorphic/heterotopic neurons. To investigate further the cortical layering disruption and the neuronal composition of heterotopia in BCNU-exposed cortex, we analyzed the expression pattern of the transcription factors Nurr1, Er81, Ror-beta, and Cux2 (respectively specific markers of layers VI, V, IV and superficial layers) in the cortical areas of BCNU-treated rats by means of in situ hybridization, and compared the findings with those observed in adult control rats. Combining in situ hybridization and immunohistochemistry we also investigated the origin of dysmorphic or heterotopic neurons. The main results of the present study are (i) the analysis of cortical layer thickness revealed that the cortical thinning in the BCNU model was prevalently restricted to the superficial layers; (ii) in cortical and periventricular heterotopia, the prevalent presence of superficial layer neurons in the internal areas, and deeper layer neurons in a more peripheral region, demonstrated a rudimentary pattern of laminar organization in nodule formation; and (iii) the Er81 signal in the dysmorphic and heterotopic pyramidal neurons located in layers I/II showed that they belong to layer V. These results shed light on the disorganization of the laminar architecture of the BCNU model by providing correlations with normal cortical layering and revealing the ontogenesis of heterotopia and heterotopic/dysmorphic neurons. They also provide strong evidence of the usefulness of layer-specific markers in investigating the neuropathology of CD, thus opening up the possibility of expanding their application to human neuropathology.
Assuntos
Carmustina , Malformações do Desenvolvimento Cortical/induzido quimicamente , Malformações do Desenvolvimento Cortical/patologia , Neocórtex/metabolismo , Neocórtex/patologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Malformações do Desenvolvimento Cortical/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Joubert syndrome (JS) is characterized by hypotonia, ataxia, developmental delay, and a typical neuroimaging finding, the so-called "molar tooth sign" (MTS). The association of MTS and polymicrogyria (PMG) has been reported as a distinct JS-related disorder (JSRD). So far, five patients have been reported with this phenotype, only two of them being siblings. We report on one additional family, describing a living child with JS and PMG, and the corresponding neuropathological picture in the aborted brother. No mutations were detected in the AHI1 gene, the only so far associated with the JS + PMG phenotype. Moreover, linkage analysis allowed excluding all known gene loci, suggesting further genetic heterogeneity.
Assuntos
Anormalidades Múltiplas/diagnóstico , Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/patologia , Irmãos , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Aborto Eugênico , Criança , Análise Mutacional de DNA , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/genética , Doenças Fetais/patologia , Humanos , Masculino , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/genética , Gravidez , SíndromeRESUMO
We investigated the expression of gamma-aminobutyric acid (GABA) in the developing rat thalamus by immunohistochemistry, using light, confocal and electron microscopy. We also examined the relationship between the expression of the homeoprotein Otx2, a transcription factor implicated in brain regionalization, and the radial and non-radial migration of early generated thalamic neurons, identified by the neuronal markers calretinin (CR) and GABA. The earliest thalamic neurons generated between embryonic days (E) 13 and 15 include those of the reticular nucleus, entirely composed by GABAergic neurons. GABA immunoreactivity appeared at E14 in immature neurons and processes laterally to the neuroepithelium of the diencephalic vesicle. The embryonic and perinatal periods were characterized by the presence of abundant GABA-immunoreactive fibers, mostly tangentially oriented, and of growth cones. At E15 and E16, GABA was expressed in radially and non-radially oriented neurons in the region of the reticular thalamic migration, between the dorsal and ventral thalamic primordia, and within the dorsal thalamus. At these embryonic stages, some CR- and GABA-immunoreactive migrating-like neurons, located in the migratory stream and in the dorsal thalamus, expressed the homeoprotein Otx2. In the perinatal period, the preponderance of GABAergic neurons was restricted to the reticular nucleus and several GABAergic fibers were still detectable throughout the thalamus. The immunolabeling of fibers progressively decreased and was no longer visible by postnatal day 10, when the adult configuration of GABA immunostaining was achieved. These results reveal the spatio-temporal features of GABA expression in the developing thalamus and suggest a novel role of Otx2 in thalamic cell migration.
Assuntos
Movimento Celular/fisiologia , Neurônios/metabolismo , Fatores de Transcrição Otx/biossíntese , Tálamo/crescimento & desenvolvimento , Ácido gama-Aminobutírico/fisiologia , Animais , Calbindina 2 , Contagem de Células , Interpretação Estatística de Dados , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica , Neurônios/ultraestrutura , Fatores de Transcrição Otx/genética , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Tálamo/citologia , Tálamo/fisiologia , Fixação de TecidosRESUMO
During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.
Assuntos
Proteínas de Membrana/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Glutamato/metabolismo , Sinaptofisina/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Hipocampo , Substâncias Macromoleculares , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Proteínas R-SNARE , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Tetrodotoxina/farmacologiaRESUMO
Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.
Assuntos
Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Biomarcadores , Toxinas Botulínicas Tipo A/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Hipocampo/citologia , Imuno-Histoquímica , Interneurônios/metabolismo , Masculino , Proteínas de Membrana/imunologia , Camundongos , Proteínas do Tecido Nervoso/imunologia , Fármacos Neuromusculares/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma , Ácido gama-Aminobutírico/fisiologiaRESUMO
A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex. The phenotype of cell subpopulations within the heterotopic structures was analyzed by means of antibodies raised against glial and neuronal markers, calcium binding proteins, GABA, and AMPA glutamate receptors. Neurons within the subcortical heterotopic nodules were characterized by abnormal firing properties, with sustained repetitive bursts of action potentials. The subcortical nodules were surrounded by cell clusters with ultrastructural features of young migrating neurons. The immunocytochemical data suggested, moreover, that the subcortical heterotopia were formed by neurons originally committed to the neocortex and characterized by morphological features similar to those found in human periventricular nodular heterotopia. The present study demonstrates that double MAM treatment at gestational day 15 induces in rats developmental brain abnormalities whose anatomical and physiological features bear resemblance to those observed in human brain dysgeneses associated with intractable epilepsy. Therefore, MAM treated rats could be considered as useful tools in investigating the pathogenic mechanisms involved in human developmental brain dysgeneses.
Assuntos
Anormalidades Induzidas por Medicamentos , Encéfalo/anormalidades , Acetato de Metilazoximetanol/análogos & derivados , Neurotoxinas/toxicidade , Animais , Encéfalo/patologia , Ventrículos Cerebrais , Coristoma , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Hipocampo , Humanos , Imuno-Histoquímica , Injeções Intraperitoneais , Acetato de Metilazoximetanol/toxicidade , Microscopia Eletrônica , Neurônios/patologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Experiments have been performed on adult albino rats in order to study the cellular organization of the thalamic reticular nucleus. For this purpose four approaches have been used: Nissl stain, Golgi impregnation, retrograde transport of horseradish peroxidase after injection in different thalamic nuclei, and immunocytochemistry with antibodies against GABA and glutamic acid decarboxylase. In sections through the horizontal plane, three morphologically different neurons have been observed. Cells with round perikarya and with multipolar dendrites were found predominantly in the rostral pole of the nucleus. Neurons with large fusiform cell body and with dendrites arborizing mainly on the horizontal plane were detected through the whole extent of the nucleus. Small fusiform neurons were observed almost exclusively in the medial third of the dorso-ventral extent of the nucleus. The Golgi impregnation method demonstrated that dendrites of small fusiform neurons develop in the vertical plane perpendicular to the dendritic arborization of large fusiform neurons. In coronal sections neurons with round perikarya and with large fusiform cell bodies are detectable while small fusiform neurons are only rarely visible. These data have been confirmed by statistical form factor analysis. Moreover, by means of the horseradish peroxidase and the immunocytochemical study, it has been confirmed that all three groups of neurons project within the thalamus and that they are GABAergic. The data concerning the distribution within the nucleus of the three morphologically different neurons are discussed in relation to the topographic distribution of cortical sensory afferents and to the topographic maps within different sectors of the reticular nucleus.
Assuntos
Ratos/anatomia & histologia , Núcleos Talâmicos/ultraestrutura , Animais , Glutamato Descarboxilase/análise , Peroxidase do Rábano Silvestre , Imuno-Histoquímica , Coloração e Rotulagem , Núcleos Talâmicos/química , Ácido gama-Aminobutírico/análiseRESUMO
The organization of glia and its relationship with migrating neurons were studied in the rat developing thalamus with immunocytochemistry by using light, confocal, and electron microscopy. Carbocyanine labeling in cultured slice of the embryonic diencephalon was also used. At embryonic day (E) 14, vimentin immunoreactivity was observed in radial fascicles spanning the neuroepithelium and extending from the ventricular zone to the lateral surface of the diencephalic vesicle. Vimentin-immunopositive fibers orthogonal to the radial ones were also detected at subsequent developmental stages. At E16, radial and non-radial processes were clearly associated with migrating neurons identified by the neuronal markers calretinin and gamma-aminobutyric acid. Non-radial glial fibers were no longer evident by E19. Radial fibers were gradually replaced by immature astrocytes at the end of embryonic development. In the perinatal period, vimentin immunoreactivity labeled immature astrocytes and then gradually decreased; vimentin-immunopositive cells were only found in the internal capsule by the second postnatal week. Glial fibrillary acidic protein immunoreactivity appeared at birth in astrocytes of the internal capsule, but was not evident in most of the adult thalamic nuclei. Confocal and immunoelectron microscopy allowed direct examination of the relationships between neurons and glial processes in the embryonic thalamus, showing the coupling of neuronal membranes with both radial and non-radial glia during migration. Peculiar ultrastructural features of radial glia processes were observed. The occurrence of non-radial migration was confirmed by carbocyanine-labeled neuroblasts in E15 cultured slices. The data provide evidence that migrating thalamic cells follow both radial and non-radial glial pathways toward their destination.
Assuntos
Neuroglia/classificação , Neuroglia/ultraestrutura , Ratos/embriologia , Ratos/crescimento & desenvolvimento , Tálamo/embriologia , Tálamo/crescimento & desenvolvimento , Fatores Etários , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Movimento Celular/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ratos/anatomia & histologia , Ratos Wistar , Tálamo/citologia , Vimentina/metabolismoRESUMO
Apoptosis is a form of naturally occurring cell death that plays a fundamental role during development and is characterized by internucleosomal DNA fragmentation. In this study we used specific in situ labeling of DNA breaks (Gavrieli et al. [1992] J. Cell. Biol. 119:493-501) to analyze the distribution of apoptotic cells in rat cerebral cortex and thalamus at different developmental stages from embryonic day 16 to adulthood. Control experiments and electron microscopy confirmed that the reaction product was confined to the nucleus of selected cells. Plotting and counting of labeled nuclei in counterstained paraffin sections showed that apoptosis occurred mainly during the first postnatal week and was absent in embryonic and adult samples. In the cortex, the number of apoptotic cells progressively increased from birth to the first postnatal week, with a peak between postnatal (P) day 5 and P8, and subsequently decreased. At the time of maximal expression of apoptosis, labeled nuclei were present mainly in layer VIb and underlying white matter and at the border between cortical plate and layer I. Only a few apoptotic cells were found scattered in the thalamus, without a particular concentration in selected areas, but with a peak at P5. Differences in the number of apoptotic cells between cortex and thalamus suggest that apoptotic cell death may have a different functional significance in the two brain areas.
Assuntos
Apoptose/fisiologia , Córtex Cerebral/citologia , Dano ao DNA , Técnicas Genéticas , Tálamo/citologia , Animais , Biotina , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Nucleotídeos de Desoxiuracil/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Microscopia Eletrônica , Ratos , Ratos Wistar , Tálamo/embriologia , Tálamo/crescimento & desenvolvimentoRESUMO
The perireticular thalamic nucleus (PRT) consists of scattered neurons that are located in the internal capsule adjacent to the gamma aminobutyric acid (GABA)-immunoreactive (ir) reticular thalamic nucleus (RT) and whose number decreases during development. The common feature of PRT neurons in different species is the immunoreactivity for the calcium binding protein parvalbumin (PV), which is also expressed by RT cells. In this study, we analyzed, at the light and electron microscopic level, the distribution and morphology of PV-ir neurons and their relationship with GABA in adult and developing rats. We found that the rostrocaudal distribution and the morphology of PV-ir neurons of the PRT were different at each stage of postnatal development examined. The adult configuration of the PV-ir population in the PRT was achieved at postnatal day 21. With electron microscopy, the developing PRT was observed to contain PV-ir neuronal cell bodies and dendrites contacted by several PV-negative synaptic terminals, some of which were GABA-ir, whereas the adult PRT contained also large PV-ir boutons, generally GABA-ir. Very few GABA-ir neurons were found in the PRT region and only during the first postnatal week, thus indicating that the PV-ir neurons of PRT represent a distinct population from those of RT. Our results demonstrate a morphological, neurochemical, and ultrastructural complexity of the PRT not only during development, but also in adulthood. These findings provide new data supporting the suggested roles of the PRT during postnatal development, and may indicate that in adult life it can play other so far unknown functions.
Assuntos
Neurônios/química , Parvalbuminas/análise , Núcleos Talâmicos/química , Núcleos Talâmicos/ultraestrutura , Animais , Imuno-Histoquímica , Microscopia Eletrônica , Parvalbuminas/imunologia , Ratos , Ratos Wistar , Núcleos Talâmicos/crescimento & desenvolvimento , Ácido gama-Aminobutírico/análiseRESUMO
The development of layer I was studied in the human frontal cortex from 21 weeks of gestation (GW) to 2.5 postnatal months in series of adjacent sections processed for thionin staining, Bodian silver staining, and immunocytochemical labeling of neurons and glia. In addition, the terminal dUTP nick-end labeling (TUNEL) method was used to label in situ DNA fragmentation. A progressive decrease of cell density and the disappearance of the subpial granular layer (SGL) appeared as distinctive developmental features of human layer I, consistently with previous investigations. The neuronal antigen microtubule-associated protein2 was found to label preferentially Cajal-Retzius cells and dendritic processes extending from the cortical plate. At midgestation, the calcium binding protein calretinin stained in the marginal zone numerous neurons, including the Cajal-Retzius cells and their processes. Calretinin-immunoreactive neurons decreased during the subsequent maturation: such decline was abrupt in the SGL, whereas bipolar calretinin-immunopositive cells accumulated in the inner marginal zone to be presumably incorporated into the cortical plate. Cajal-Retzius cells expressed calretinin throughout the examined developmental stages. The glial antigen vimentin was already expressed at midgestation, and vimentin immunopositivity decreased progressively in cell bodies and fibers of layer I during development. Glial fibrillary acidic protein-positive elements gradually matured, and the positive cell bodies displayed the features of mature astrocytes at the end of gestation. Moreover, a decrease of free glial cells was observed in layer I, suggesting their progressive incorporation into the cortical plate. TUNEL-positive cells were detected at midgestation in the marginal zone, and they were concentrated in the SGL until its disappearance; their number decreased dramatically throughout layer I after 30 gestational weeks. TUNEL-positive nuclei or regressive changes were not detected in Cajal-Retzius cells throughout the examined developmental stages. Thus, our data point out that naturally occurring cell death is an active mechanism contributing to the disappearance of the SGL but not to the subsequent developmental reshaping of human layer I, in which, instead, migratory phenomena should play a major role. In addition, our findings argue against a disappearance of Cajal-Retzius cells due to regressive processes.
Assuntos
Apoptose/fisiologia , Lobo Frontal/embriologia , Neuroglia/citologia , Neurônios/citologia , Calbindina 2 , Contagem de Células , Fragmentação do DNA/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Lobo Frontal/citologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lactente , Recém-Nascido , Masculino , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
The authors report the immunocytochemical localization in rat brain of affinity-purified anti-GluR3 (glutamate receptor) antibodies from two patients with Rasmussen encephalitis (RE) and from immunized rabbits. The distribution of immunolabeling was similar using antibodies from rabbits and patients with RE. No electrophysiologic responses were elicited from acutely dissociated kainate-responsive neurons isolated from rat brain when these antibodies were applied. These findings show that anti-GluR3 antibodies purified from patients with RE bind to specific regions of the CNS but do not act through an excitotoxic mechanism.
Assuntos
Encefalite/imunologia , Neurônios/imunologia , Receptores de AMPA/análise , Receptores de AMPA/imunologia , Animais , Encefalite/patologia , Encefalite/fisiopatologia , Humanos , Imuno-Histoquímica , Neurônios/patologia , Neurônios/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
The present work was aimed to study the immunocytochemical localization of the calcium-binding protein, calretinin, in the rat thalamus from embryonic day 14 to the third postnatal week. In the adult rat thalamus, calretinin immunoreactivity is intensely expressed in some intralaminar and midline nuclei, as well as in selected regions of the reticular nucleus. At embryonic day 14, calretinin was expressed by immature and migrating neurons and fibres laterally to the neuroepithelium of the diencephalic vesicle in the region identified as reticular neuroepithelium. At embryonic day 16, immunoreactive neurons were present in the primordium of the reticular nucleus and in the region of the reticular thalamic migration, where neurons showed the morphology of migratory cells. At the end of embryonic development and in the first postnatal week, calretinin-positive neurons were observed in selected region of the reticular nucleus and it was intensely expressed in some intralaminar and midline nuclei. Bands of immunopositive fibres were also observed crossing the thalamus. During the second postnatal week, the immunolabelling in the reuniens, rhomboid, paraventricular and central medial thalamic nuclei remains very intense while a decrease of immunoreactivity in mediodorsal, centrolateral and laterodorsal nuclei was observed. The immunostaining of fibres, particularly evident in the perinatal period, progressively decreased and it was no longer visible by the end of the second postnatal week when the distribution and intensity of calretinin immunostaining was similar to that observed in the adult rat thalamus. The present findings indicate that the immunolocalization of calretinin can be used to identify subsets of thalamic neuronal population during pre- and postnatal maturation allowing also the detection of the migratory pattern of early generated reticular thalamic neurons.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/biossíntese , Tálamo/metabolismo , Envelhecimento/metabolismo , Animais , Encéfalo/embriologia , Calbindina 2 , Desenvolvimento Embrionário e Fetal , Imuno-Histoquímica , Fibras Nervosas/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Especificidade de Órgãos , Ratos , Ratos Wistar , Proteína G de Ligação ao Cálcio S100/análise , Tálamo/embriologia , Tálamo/crescimento & desenvolvimentoRESUMO
This study is aimed at the investigation of the morphological and electrophysiological characteristics of neurons from the nucleus reticularis thalami in rat thalamic slices incubated in vitro. Ten neurons were recorded in the ventrobasal complex, four of which were successfully injected following horseradish peroxidase injection. Two main types of reticular thalamic neurons were morphologically identified: (1) the small fusiform 'f' cells characterized by a very elongated perikaryon, dendritic arborization prevalent in the rostrocaudal and dorsoventral planes, and an axon without any collaterals branching within the nucleus reticularis thalami; and (2) the large fusiform 'F' neurons with dendrites arborizing mainly in the horizontal plane and with axonal branches within the nucleus reticularis thalami. The electrophysiological properties of the neurons were similar in F and f cells. The reticular neurons showed, in resting conditions, a single spike response followed by a postexcitatory hyperpolarizing potential. The hyperpolarization of these neurons transformed the single spike response into a burst discharge similar to that observed in thalamic relay neurons at resting membrane potential. The same phenomenon was observed when bicuculline was administered by perfusion to the slices and, in this case, a recovery to a single spike response was obtained by a depolarizing d.c. current injection. By contrast, the local administration of GABA induced a depolarization with a pronounced decrease in input resistance. The present data demonstrate the presence of at least two neuronal subtypes within the nucleus reticularis thalami, suggesting that only one is responsible for the phenomenon of auto-inhibition by means of intrinsic axon collaterals. Moreover, it is hypothesized that intranuclear GABAergic collaterals could control neuronal excitability of reticular thalamic cells by both shunting the membrane and shifting the burst firing to a single spike firing mode.
Assuntos
Neurônios/fisiologia , Núcleos Talâmicos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Bicuculina/farmacologia , Dendritos/análise , Estimulação Elétrica , Peroxidase do Rábano Silvestre , Técnicas In Vitro , Neurônios/classificação , Neurônios/efeitos dos fármacos , Ratos , Núcleos Talâmicos/citologia , Núcleos Talâmicos/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
This work was performed to confirm previous data reporting the presence of GABAergic interneurons in the ventrobasal complex of guinea-pig, and to investigate the intrinsic organization of this nucleus compared to that of thalamic nuclei lacking interneurons. Immunocytochemical experiments were performed on the thalamus of adult guinea-pigs perfused with mixed aldehydes using an anti-GABA serum. At light microscopy, the immunoreaction on floating Vibratome sections showed that GABAergic neurons are present only in the reticular and lateral geniculate nuclei and in the ventrobasal complex. Quantitative evaluation of their number indicated that they are 20 and 15% of the total neuronal population in lateral geniculate nucleus and ventrobasal complex, respectively, while they are less than 1% in ventrolateral nucleus. At the ultrastructural level, the postembedding immunogold procedure showed the presence, in the ventrobasal complex, of GABA-labeled profiles involved in complex synaptic arrangements similar to those found in carnivores and primates. Conversely, GABA-labeled terminals in thalamic nuclei devoid of interneurons formed exclusively axo-dendritic or axo-somatic contacts, like in rats and mice. The present data suggest that GABAergic neurons in the ventrobasal complex of guinea-pigs give rise to functionally important rearrangements of its intrinsic synaptic organization and that they represent the morphological basis for an intrinsic modulatory mechanism that is absent in other thalamic nuclei lacking inhibitory interneurons. The phylogenetic implications of these findings are also discussed in comparison to other animal species.
Assuntos
Cobaias/anatomia & histologia , Interneurônios/fisiologia , Núcleos Talâmicos/citologia , Ácido gama-Aminobutírico/fisiologia , Animais , Mapeamento Encefálico , Contagem de Células , Corpos Geniculados/química , Corpos Geniculados/citologia , Imuno-Histoquímica , Masculino , Camundongos , Neurônios/química , Neurônios/ultraestrutura , Filogenia , Especificidade da Espécie , Sinapses/ultraestrutura , Transmissão Sináptica , Núcleos Talâmicos/química , Ácido gama-Aminobutírico/análiseRESUMO
The postnatal development of inhibitory GABAergic circuits in the thalamic reticular and ventrobasal nuclei was studied in rats ranging from the day of birth to the end of the third postnatal week by means of a postembedding immunogold staining procedure to visualize GABA. In the reticular nucleus, GABA labeling was present from birth in cell bodies, dendrites, growth cones and a few synaptic terminals, whereas in the ventrobasal nucleus it was exclusively in axonal processes identifiable as growth cones, vesicle-rich profiles and synaptic terminals. In both nuclei, GABA-labeled synaptic terminals were, however, very scarce and immature in neonatal animals and they became numerous and morphologically mature only after the end of the second postnatal week. These findings suggest that inhibitory synaptic responses in the somatosensory thalamus are not yet fully mature throughout the first two postnatal weeks and support the hypothesis that GABA may initially play trophic roles. The relatively late maturation of the thalamic GABAergic system may have important functional consequences, as the reticulothalamic circuits are responsible for the generation of spindle wave oscillations whose cellular mechanisms are also involved in the generation of spike-and-wave (absence) seizures in humans and in animal models.
Assuntos
Gânglios da Base/crescimento & desenvolvimento , Gânglios da Base/fisiologia , Terminações Pré-Sinápticas/fisiologia , Formação Reticular/crescimento & desenvolvimento , Formação Reticular/fisiologia , Núcleos Talâmicos/crescimento & desenvolvimento , Núcleos Talâmicos/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Animais Recém-Nascidos , Gânglios da Base/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/fisiologia , Vias Neurais/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Formação Reticular/ultraestrutura , Núcleos Talâmicos/ultraestruturaRESUMO
Selective immunostaining for calcium-binding proteins identifies subpopulations of neurons with hypothetical distinct functional roles. The neuronal localization of calcium-binding proteins calretinin, parvalbumin and calbindin is here correlated to GABA and glutamate immunoreactivity in the guinea-pig piriform cortex. In the external border of the molecular layer, neurons positive for calretinin with morphological features of Cajal-Retzius cells were found. Rare GABA immunoreactive cells were observed in the same subpial region, whereas neurons containing GABA were abundant within layers Ia and Ib. Aspartate- and glutamate-immunoreactive cells were also found in the outer Ia layer. A distinct band of calretinin-immunoreactive fibres and terminals localized in layer Ia, where the afferent fibres originating from the olfactory bulb are segregated. In layer II the number of cells containing calretinin exceeded the number of neurons positive for the anti-GABA antibody. Part of the layer II calretinin-positive neurons with pyramidal shape and large apical dendrites directed toward the surface were found to be immunoreactive for the anti-glutamate antibody on adjacent sections. Neurons in layer II immunoreactive for either parvalbumin or calbindin showed morphological features of interneurons, and their number matched the count of GABA containing cells. Calretinin-positive neurons with the general morphological features of interneurons were scarcely represented in the deep piriform cortical layers, where large multipolar and small bipolar calbindin-positive cells prevailed. The present data show that in the piriform cortex of the guinea-pig calretinin, although expressed in Cajal-Retzius-like cells as in other cortical areas, also marks a subpopulation of glutamate containing pyramidal-like neurons in layer II.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Proteína G de Ligação ao Cálcio S100 , Animais , Ácido Aspártico/metabolismo , Calbindina 2 , Córtex Cerebral/citologia , Corantes , Ácido Glutâmico/metabolismo , Cobaias , Imuno-Histoquímica , Neurônios/ultraestruturaAssuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Knock-out Otx1 mice show brain hypoplasia, spontaneous epileptic seizures and abnormalities of the dorsal region of the neocortex. We investigated structural alterations in excitatory and inhibitory circuits in somatosensory cortex of Otx1(-/-) mice by immunocytochemistry using light, confocal and electron microscopy. Immunostaining for non-phosphorylated neurofilament SMI311 and subunit 1 of the NMDA receptor - used as markers of pyramidal neurons - showed reduced layer V pyramidal cells and ectopic pyramidal cells in layers II and III of the mutant cortex. Immunostaining for calcium-binding proteins calbindin, calretinin and parvalbumin - markers of non-overlapping types of GABAergic interneurons - showed no differences between wild-type and knock-out cortex for calbindin and calretinin neurons, while parvalbumin neurons were only patchily distributed in Otx1(-/-) cortex. The pattern of positivity of the GABAergic marker glutamic acid decarboxylase in Otx1(-/-) cortex was also altered and similar to that of parvalbumin. GABA transporter 1 immunoreactivity was greater in Otx1(-/-) than wild-type; quantitation of structures immunoreactive for this transporter in layer V showed that they were increased overall in Otx1(-/-) but the density of inhibitory terminals on pyramidal neurons in the same layer labeled with this transporter was similar to that in wild-type mice. No differences in the distribution or intensity of the glial markers GABA transporter 3 or glial fibrillary acidic protein were found. The defects found in the cortical GABAergic system of the Otx1(-/-) mouse can plausibly explain the cortical hyperexcitability that produces seizures in these animals.
Assuntos
Epilepsia/genética , Malformações do Sistema Nervoso/genética , Vias Neurais/anormalidades , Vias Neurais/metabolismo , Neurônios/metabolismo , Transportadores de Ânions Orgânicos , Córtex Somatossensorial/anormalidades , Córtex Somatossensorial/metabolismo , Fatores de Transcrição/deficiência , Animais , Biomarcadores , Proteínas de Transporte/metabolismo , Epilepsia/metabolismo , Epilepsia/patologia , Proteínas da Membrana Plasmática de Transporte de GABA , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Descarboxilase/metabolismo , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Inibição Neural/fisiologia , Vias Neurais/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Neurônios/ultraestrutura , Fatores de Transcrição Otx , Parvalbuminas/metabolismo , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Somatossensorial/ultraestrutura , Fatores de Transcrição/genéticaRESUMO
Cajal-Retzius cells, identified using calretinin antiserum, were studied in layer I (LI) of adult human temporal cortex from epileptic patients with Taylor's focal cortical dysplasia and architectural dysplasia, in comparison with normal cortex. Both types of dysplasia showed LI hypercellularity, but only in architectural dysplasia was the density of Cajal-Retzius cells significantly increased. A subset of Cajal-Retzius cells were reelin immunoreactive, but none were GABA positive. These findings suggest that differences in the persistence of Cajal-Retzius cells, which probably reflect different types of alteration during brain development, can assist in characterizing different forms of cortical dysplasia.