RESUMO
An antiserum to mGluR1 alpha labeled a 160 kd protein in immunoblots of membranes derived from rat brain or cells transfected with mGluR1 alpha. Immunoreactivity for mGluR1 alpha was present in discrete subpopulations of neurons. The GABAergic neurons of the cerebellar cortex were strongly immunoreactive; only some Golgi cells were immunonegative. Somatostatin/GABA-immunopositive cells in the neocortex and hippocampus were enriched in mGluR1 alpha. The hippocampal cells had spiny dendrites that were precisely codistributed with the local axon collaterals of pyramidal and granule cells. Electron microscopic immunometal detection of mGluR1 alpha showed a preferential localization at the periphery of the extensive postsynaptic densities of type 1 synapses in both the cerebellum and the hippocampus. The receptor was also present at sites in the dendritic and somatic membrane where synapses were not located.
Assuntos
Encéfalo/citologia , Neurônios/citologia , Receptores de Glutamato/análise , Membranas Sinápticas/ultraestrutura , Sequência de Aminoácidos , Animais , Linhagem Celular , Cerebelo/citologia , Cerebelo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Hipocampo/citologia , Hipocampo/ultraestrutura , Soros Imunes , Imuno-Histoquímica/métodos , Proteínas de Membrana/análise , Proteínas de Membrana/isolamento & purificação , Microscopia Imunoeletrônica/métodos , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/isolamento & purificação , Neurônios/ultraestrutura , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Especificidade de Órgãos , Ratos , Proteínas Recombinantes/análise , Somatostatina/análise , Transfecção , Ácido gama-Aminobutírico/análiseRESUMO
It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.
Assuntos
Hipocampo/ultraestrutura , Interneurônios/ultraestrutura , Fibras Nervosas/ultraestrutura , Células Piramidais/ultraestrutura , Receptores de AMPA/análise , Sinapses/ultraestrutura , Envelhecimento/fisiologia , Animais , Células COS , Calibragem , Feminino , Hipocampo/crescimento & desenvolvimento , Interneurônios/fisiologia , Masculino , Microscopia Imunoeletrônica/métodos , Fibras Nervosas/fisiologia , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Sinapses/fisiologia , TransfecçãoRESUMO
Networks of GABAergic interneurons are implicated in synchronizing cortical activity at gamma frequencies (30-70 Hz). Here we demonstrate that the combined electrical and GABAergic synaptic coupling of basket cells instantaneously entrained gamma-frequency postsynaptic firing in layers 2/3 of rat somatosensory cortex. This entrainment was mediated by rapid curtailment of gap junctional coupling potentials by GABAA receptor-mediated IPSPs. Electron microscopy revealed spatial proximity of gap junctions and GABAergic synapses on somata and dendrites. Electrical coupling alone entrained postsynaptic firing with a phase lag, whereas unitary GABAergic connections were ineffective in gamma-frequency phasing. These observations demonstrate precise spatiotemporal mechanisms underlying action potential timing in oscillating interneuronal networks.
Assuntos
Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Interneurônios/fisiologia , Sinapses/fisiologia , Ácido gama-Aminobutírico/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Fatores Etários , Animais , Bicuculina/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Junções Comunicantes/ultraestrutura , Interneurônios/citologia , Microscopia Eletrônica , Ratos , Ratos Wistar , Sinapses/ultraestruturaRESUMO
In neocortical circuits, repetitively active neurons evoke unitary postsynaptic potentials (PSPs) whose peak amplitudes either increase (facilitate) or decrease (depress) progressively. To examine the basis for these different synaptic responses, we made simultaneous recordings from three classes of neurons in cortical layer 2/3. We induced repetitive action potentials in pyramidal cells and recorded the evoked unitary excitatory (E)PSPs in two classes of GABAergic neurons. We observed facilitation of EPSPs in bitufted GABAergic interneurons, many of which expressed somatostatin immunoreactivity. EPSPs recorded from multipolar interneurons, however, showed depression. Some of these neurons were immunopositive for parvalbumin. Unitary inhibitory (I)PSPs evoked by repetitive stimulation of a bitufted neuron also showed a less pronounced but significant difference between the two target neurons. Facilitation and depression involve presynaptic mechanisms, and because a single neuron can express both behaviors simultaneously, we infer that local differences in the molecular structure of presynaptic nerve terminals are induced by retrograde signals from different classes of target neurons. Because bitufted and multipolar neurons both formed reciprocal inhibitory connections with pyramidal cells, the results imply that the balance of activation between two recurrent inhibitory pathways in the neocortex depends on the frequency of action potentials in pyramidal cells.
Assuntos
Neocórtex/fisiologia , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas In Vitro , Interneurônios/metabolismo , Interneurônios/fisiologia , Neocórtex/citologia , Inibição Neural/fisiologia , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Células Piramidais/fisiologia , Ratos , Somatostatina/metabolismo , Sinapses/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Animais , Composição de Bases , Sequência de Bases , Primers do DNA , Mutação da Fase de Leitura , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Plasmídeos , Coelhos , Mapeamento por Restrição , Reticulócitos/metabolismo , Transcrição GênicaRESUMO
Glutamate receptors activated by NMDA (NMDARs) or AMPA (AMPARs) are clustered on dendritic spines of pyramidal cells. Both the AMPAR-mediated postsynaptic responses and the synaptic AMPAR immunoreactivity show a large intersynapse variability. Postsynaptic responses mediated by NMDARs show less variability. To assess the variability in NMDAR content and the extent of their coexistence with AMPARs in Schaffer collateral-commissural synapses of adult rat CA1 pyramidal cells, electron microscopic immunogold localization of receptors has been used. Immunoreactivity of NMDARs was detected in virtually all synapses on spines, but AMPARs were undetectable, on average, in 12% of synapses. A proportion of synapses had a very high AMPAR content relative to the mean content, resulting in a distribution more skewed toward larger values than that of NMDARs. The variability of synaptic NMDAR content [coefficient of variation (CV), 0.64-0.70] was much lower than that of the AMPAR content (CV, 1.17-1.45). Unlike the AMPAR content, the NMDAR content showed only a weak correlation with synapse size. As reported previously for AMPARs, the immunoreactivity of NMDARs was also associated with the spine apparatus within spines. The results demonstrate that the majority of the synapses made by CA3 pyramidal cells onto spines of CA1 pyramids express both NMDARs and AMPARs, but with variable ratios. A less-variable NMDAR content is accompanied by a wide variability of AMPAR content, indicating that the regulation of expression of the two receptors is not closely linked. These findings support reports that fast excitatory transmission at some of these synapses is mediated by activation mainly of NMDARs.
Assuntos
Células Piramidais/química , Receptores de AMPA/análise , Receptores de N-Metil-D-Aspartato/análise , Sinapses/química , Animais , Linhagem Celular , Dendritos/química , Humanos , Imuno-Histoquímica , Ratos , Ratos WistarRESUMO
The morphology and postsynaptic targets of GABA-containing boutons were determined in the striate cortex of cat, using a postembedding immunocytochemical technique at the electron microscopic level. Two types of terminals, both making symmetrical synaptic contacts, were GABA-positive. The first type (95% of all GABA-positive boutons) contained small pleomorphic vesicles, the second type (5%) contained larger ovoid vesicles. Furthermore, 99% of all cortical boutons containing pleomorphic vesicles were GABA positive, and all boutons with pleomorphic vesicles made symmetrical synaptic contacts. These results together with previously published stereological data (Beaulieu and Colonnier, 1985, 1987) were used to estimate the density of GABA-containing synapses, which is about 48 million/mm3 in the striate cortex. The postsynaptic targets of GABA positive boutons were also identified and the distribution was calculated to be as follows: 58% dendritic shafts, 26.4% dendritic spines, 13.1% somata and 2.5% axon initial segments. A total of 11% of the postsynaptic targets were GABA immunoreactive and therefore originated from GABAergic neurons. The results demonstrate that the majority of GABAergic synapses exert their action on the membrane of dendrites and spines rather than on the somata and axons of neurons.
RESUMO
The cerebral cortex is an area rich in taurine (2-aminoethanesulphonic acid), but only limited information exists regarding its cellular distribution. We therefore examined taurine-like immunoreactivity in the cerebral cortex of the rat, cat and macaque monkey using antiserum directed against glutaraldehyde-conjugated taurine. Immunostaining was assessed at the light and electron microscopic level, and patterns obtained in light microscopic studies were compared to those produced with antiserum to gamma-aminobutyric acid (GABA) and homocysteic acid (HCA). In all three species, strong taurine-like immunoreactive perivascular endothelial cells, pericytes and oligodendrocytes were found. These cells were located throughout the neuropil, which itself showed a low level of immunoreactivity. In rats and cats, a small number of weakly taurine-enriched neurons were observed, particularly in superficial layers. In all cortical areas of the macaque, however, glial staining was matched by strong, selective staining of subpopulations of cortical neurons which were distributed in a bilaminar pattern involving layers II/III and VI. In addition, in primary visual cortex, area 17, immunopositive neurons were also present in sublayer IVCbeta, while in the hippocampus strongly taurine-positive neurons were most conspicuous in the granule cell layer of the dentate gyrus. In all regions, strongly taurine-positive neurons constituted only a subpopulation of the neurons occupying a given layer. Examination of adjacent sections for GABA immunoreactivity showed that the most strongly taurine-positive neurons in layers II/III were immunoreactive for GABA. The cells located in layers IVCbeta and VI, and the granule cells of the dentate gyrus, however, were GABA-negative. The morphological features of these latter groups suggested that the antiserum to taurine identifies subsets of spiny stellate, small pyramidal and dentate granule cells. None of these neurons showed immunoreactivity with antiserum to HCA in the primate; HCA-positive glia were found along the pial and white matter boundaries of the cortex, and showed no overlap with strongly taurine-positive glial elements. Although a transmitter role for taurine may be unlikely, particularly in view of its enrichment in subpopulations of both inhibitory and excitatory cells, the capacity of taurine to influence membrane-associated functions in excitable tissues, and its selective distribution demonstrated here, provides the potential for a contribution to communication between cortical cells.
RESUMO
The synaptic circuits underlying cholinergic activation of the cortex were studied by establishing the quantitative distribution of cholinergic terminals on GABAergic inhibitory interneurons and on non-GABAergic neurons in the striate cortex of the cat. Antibodies to choline acetyltransferase and GABA were used in combined electron microscopic immunocytochemical experiments. Most of the cholinergic boutons formed synapses with dendritic shafts (87.3%), much fewer with dendritic spines (11.5%), and only occasional synapses were made on neuronal somata (1.2%). Overall, 27.5% of the postsynaptic elements, all of them dendritic shafts, were immunoreactive for GABA, thus demonstrating that they originate from inhibitory neurons. This is the highest value for the proportion of GABAergic postsynaptic targets obtained so far for any intra- or subcortical afferents in cortex. There were marked variations in the laminar distribution of targets. Spines received synapses most frequently in layer IV (23%) and least frequently in layers V-VI (3%); most of these spines also received an additional synapse from a choline acetyltransferase-negative bouton. The proportion of GABA-positive postsynaptic elements was highest in layer IV (49%, two-thirds of all postsynaptic dendritic shafts), and lowest in layers V-VI (14%). The supragranular layers showed a distribution similar to that of the average of all layers. The quantitative distribution of targets postsynaptic to choline acetyltransferase-positive terminals is very different from the postsynaptic targets of GABAergic boutons, or from the targets of all boutons in layer IV reported previously. In both cases the proportion of GABA-positive dendrites was only 8-9% of the postsynaptic elements. At least 8% of the total population of choline acetyltransferase-positive boutons, presumably originating from the basal forebrain, were also immunoreactive for GABA. This raises the possibility of cotransmission at a significant proportion of cholinergic synapses in the cortex. The present results demonstrate that cortical GABAergic neurons receive a richer cholinergic synaptic input than non-GABAergic cells. The activation of GABAergic neurons by cholinergic afferents may increase the response specificity of cortical cells during cortical arousal thought to be mediated by the basal forebrain. The laminar differences indicate that in layer IV, at the first stage of the processing of thalamic input, the cholinergic afferents exert substantial inhibitory influence in order to raise the threshold and specificity of cortical neuronal responses. Once the correct level of activity has been set at the level of layer IV, the influence can be mainly facilitatory in the other layers.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Sinapses/metabolismo , Córtex Visual/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Gatos , Neurônios/metabolismo , Neurônios Aferentes/metabolismo , Córtex Visual/enzimologia , Córtex Visual/ultraestruturaRESUMO
The morphology of certain Golgi-stained cells was examined in the striate and peristriate cortex of the cat and in the striate cortex of the rhesus monkey. Neurons in layer III were selected on the basis of their characteristic vertical axon bundles, which are 20-150 microns in diameter and traverse layers II-V. Selected neurons were examined under the electron microscope to characterize their synapses and to establish their postsynaptic targets. It was found that double bouquet cells form symmetrical or type II synapses. In the cat the postsynaptic membrane specialization was more extensive than in the monkey. After removing the Golgi precipitate from boutons of two cells in the cat, small pleomorphic and flattened vesicles were found in the boutons. Earlier suggestions that double bouquet cells make synapses preferentially with spines of apical dendrites could not be confirmed. Out of 66 boutons in area 17 of the cat, 86.4% formed synapses with dendritic shafts, many of them belonging to nonpyramidal cells, 9% with perikarya of nonpyramidal cells, and only 4.6% with spines. Out of 19 synapses examined in area 18, 74% were contacting dendritic shafts and the rest contacted spines. In the monkey 60% of a total of 35 double bouquet cell synapses made synapses with dendritic shafts. A different type of double bouquet cell with densely spiny dendrites is also described in layer IV of the monkey striate cortex. This neuron formed asymmetrical synapses. It is suggested that layer III double bouquet cells with vertical axon bundles are probably inhibitory and act on other nonpyramidal cells and certain parts of pyramidal cells.
Assuntos
Córtex Visual/citologia , Animais , Axônios/ultraestrutura , Gatos , Dendritos/ultraestrutura , Interneurônios/citologia , Interneurônios/ultraestrutura , Macaca mulatta , Microscopia Eletrônica , Especificidade da Espécie , Sinapses/ultraestrutura , Córtex Visual/ultraestruturaRESUMO
The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.
Assuntos
Córtex Cerebral/fisiologia , Sinapses/fisiologia , Córtex Visual/fisiologia , Animais , Gatos , Córtex Cerebral/citologia , Dendritos/imunologia , Dendritos/metabolismo , Imuno-Histoquímica , Interneurônios/fisiologia , Iontoforese , Terminações Nervosas/metabolismo , Perfusão , Fito-Hemaglutininas , Córtex Visual/citologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The neuropeptide substance P is a transmitter candidate for certain primary afferent fibers which terminate in the substantia gelatinosa. In this study the light and electron microscopic localization of substance P in the substantia gelatinosa of the spinal trigeminal nucleus of the rat has been studied using immunocytochemical procedures. Substance P immunoreactive fibers were observed mainly in lamina I and outer lamina II. Ultrastructural analysis revealed immunoreactivity in unmyelinated fibers and in axon terminals which contained agranular spherical vesicles and large dense-cored vesicles and which made predominantly simple asymmetric axodendritic synaptic contacts. Immunoreactive terminals only rarely formed the central terminal of synaptic glomeruli and in only one example was a stained terminal possibly postsynaptic to an unstained terminal. The majority of synapses were onto small dendrites in outer lamina II and in some cases these dendrites were themselves presynaptic to other dendrites. Immunoreactive terminals also synapsed with the soma and proximal dendrites of large neurons on the border of laminae I and II. The results show that there are at least two distinct targets for substance P immunoreactive terminals in the substantia gelatinosa, namely the large lamina I neurons and lamina II probable interneurons. Some of the former may be projection neurons while some of the latter may correspond to the inhibitory islet cells described by Gobel and colleagues in the cat. In addition the results indicate that few substance P immunoreactive terminals receive axoaxonic synapses and emphasize instead the role of postsynaptic interactions. In particular the results suggest several sites at which substance P might interact postsynaptically with the neuropeptide enkephalin.
Assuntos
Substância P/metabolismo , Núcleo Espinal do Trigêmeo/metabolismo , Vias Aferentes/metabolismo , Animais , Transporte Axonal , Dendritos/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Interneurônios/metabolismo , Bulbo/metabolismo , Microscopia Eletrônica , Fibras Nervosas/metabolismo , Neurônios/metabolismo , Ratos , Medula Espinal/metabolismo , Substância Gelatinosa/metabolismo , Sinapses/metabolismoRESUMO
Following the injection of horseradish peroxidase into the ipsilateral substantia nigra, 36 retrogradely labelled neurons in the striatum were characterized (in three rats) by Golgi staining and gold toning: each neuron was of the medium-size, densely spinous type. Prior to the injection of horseradish peroxidase, two of the rats had had lesions placed in the ipsilateral motor cortex, the third rat had had a lesion placed in the ipsilateral frontal and prefrontal cortex. In the electron microscope, degenerating boutons of cortical neurons were found in asymmetrical synaptic contact with the spines of proximal and distal dendrites of all six of the identified striatonigral neurons that were studied. Some of the degenerating boutons were small (diameter 0.1-0.3 micron), while others were larger (1-2 microns). An individual dendrite of a striatonigral neuron was in symaptic contact with very few degenerating boutons. Local axon collaterals in the striatum could be traced from two of the identified striatonigral neurons that received degenerating cortical boutons. These were studied in the electron microscope; their boutons formed symmetrical synapses with spines or dendritic shafts of other striatal neurons. The synaptic boutons contained large, clear, round and pleomorphic vesicles. The postsynaptic targets of these boutons morphologically resemble the dendrites of medium-size spiny neurons. It is concluded that afferents from the cortex make monosynaptic contact with the dendritic spines of medium-size spiny striatonigral neurons and that such neurons have local axon collaterals in the striatum that form synapses with other spiny neurons.
Assuntos
Córtex Cerebral/anatomia & histologia , Corpo Estriado/anatomia & histologia , Substância Negra/anatomia & histologia , Animais , Axônios/ultraestrutura , Mapeamento Encefálico , Corpo Estriado/citologia , Corpo Estriado/ultraestrutura , Feminino , Peroxidase do Rábano Silvestre , Microscopia Eletrônica , Vias Neurais/anatomia & histologia , Ratos , Coloração e Rotulagem , Sinapses/ultraestruturaRESUMO
Light and electron microscopic quantitative analysis was carried out on a type of neuron intracellularly filled with horseradish peroxidase. Two cells were studied in area 17, one of which was injected intra-axonally, and its soma was not recovered. One cell was studied in area 18. The two somata were on the border of layers IVa/b; they were radially elongated and received synapses from numerous large boutons with round synaptic vesicles. The dendrites were smooth and remained largely in layer IV. The cells can be recognised on the basis of their axonal arbor, which was restricted to layer IV (90-95% of boutons) with minor projections to layers III, V, and VI. Many of the large, bulbous boutons contacted neuronal somata, short collaterals often forming "claw"-like configurations around cells. The name "clutch cell" is suggested to delineate this type of neuron from other aspiny multipolar cells. Computer-assisted reconstruction of the axon showed that in layer IV the axons occupied a rectangular area about 300 X 500 microns, elongated anteroposteriorly in area 17 and mediolaterally in area 18. The distributions of synaptic boutons and postsynaptic cells were patchy within this area. A total of 321 boutons were serially sectioned in area 17. The boutons formed type II synaptic contacts. The postsynaptic targets were somata (20-30%), dendritic shafts (35-50%), spines (30%), and rarely axon initial segments. Most of the postsynaptic somata tested were not immunoreactive for GABA and their fine structural features suggest that they are spiny stellate, star pyramidal, and pyramidal neurons. The characteristics of most of the postsynaptic dendrites and spines also suggest that they belong to these spiny neurons. A few of the postsynaptic dendrites and somata exhibited characteristics of cells with smooth dendrites and these somata were immunoreactive for GABA. It is suggested that clutch cells are inhibitory interneurons exerting their effect mainly on layer IV spiny neurons in an area localised perhaps to a single ocular dominance column. The specific laminar location of the axons of clutch cell also suggests that they may be associated with the afferent terminals of lateral geniculate nucleus cells, and could thus be responsible for generating some of the selective properties of neurons of the first stage of cortical processing.
Assuntos
Córtex Visual/citologia , Animais , Gatos , Dendritos/ultraestrutura , Corpos Geniculados/anatomia & histologia , Interneurônios/citologia , Microscopia Eletrônica , Inibição Neural , Sinapses/ultraestrutura , Córtex Visual/metabolismo , Córtex Visual/ultraestrutura , Ácido gama-Aminobutírico/metabolismoRESUMO
An interlaminar, ascending, and GABAergic projection is demonstrated in the striate cortex of the cat. We have examined a basket cell, with soma and smooth dendrites in layers V and VI, that was injected intracellularly with HRP in the kitten. Three-dimensional reconstruction of its axon revealed a horizontal plexus in layer V and upper VI, extending about 1.8 mm anteroposteriorly and 0.8 mm mediolaterally; a dense termination in the vicinity of the soma in layers V and VI; and an ascending tuft terminating in layers II and III in register above the soma and about 250 microns in diameter. Many boutons of this cell contacted neuronal somata and apical dendrites of pyramidal cells and subsequent electron microscopy showed that these boutons formed type II synaptic contacts with these structures. A random sample of postsynaptic targets (n = 199) in layers III, V, and VI showed that somata (20.1%), dendritic shafts (38.2%), and dendritic spines (41.2%) were contacted. The fine structural characteristics of postsynaptic elements indicated that the majority originated from pyramidal cells. Direct identification of postsynaptic neurons was achieved by Golgi impregnation of four large pyramidal cells in layer V, which were contacted on their somata and apical dendrites by between three and 34 boutons of the HRP-filled basket cell. Layer IV neurons were not contacted. Golgi-impregnated neurons similar to the HRP-filled basket cell were also found in the deep layers. The axonal boutons of one of them were studied; it also formed type II synapses with somata and apical dendrites of pyramidal cells. Boutons of the HRP-filled neuron were shown to be GABA-immunoreactive by the immunogold method. This is direct evidence in favour of the GABAergic nature of deep layer basket cells with ascending projections. The existence of an ascending GABAergic pathway was also demonstrated by injecting [3H]GABA into layers II and III. The labelled amino acid was transported retrogradely by a subpopulation of GABA-immunoreactive cells in layers V and VI, in addition to cells around the injection site. The axonal pattern and mode of termination of deep basket cells make them a candidate for producing or enhancing directional selectivity, a characteristic of layer V cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Gatos/anatomia & histologia , Córtex Visual/anatomia & histologia , Ácido gama-Aminobutírico/metabolismo , Animais , Autorradiografia , Microscopia Eletrônica , Inibição Neural , Sinapses/fisiologia , Córtex Visual/fisiologia , Córtex Visual/ultraestruturaRESUMO
The intrahippocampal distribution of axon collaterals of individual CA3 pyramidal cells was investigated in the rat. Pyramidal cells in the CA3 region of the hippocampus were physiologically characterized and filled with biocytin in anesthetized animals. Their axonal trees were reconstructed with the aid of a drawing tube. Single CA3 pyramidal cells arborized most extensively in the CA1 region, covering approximately two-thirds of the longitudinal axis of the hippocampus. The total length of axon collaterals in the CA3 region was less than in CA1 and the axon branches tended to cluster in narrow bands (200-800 microns), usually several hundred microns anterior or posterior to the cell body. The majority of the recurrent collaterals of a given neuron remained in the same subfield (CA3a, b, or c) as the parent cell. CA3a neurons innervated predominantly the basal dendrites, whereas neurons located proximal to the hilus (CA3c) terminated predominantly on the apical dendrites of both CA1 and CA3 cells. Two cells, with horizontal dendrites and numerous thorny excrescences at the CA3c-hilus transitional zone, were also labeled and projected to both CA3 and CA1 regions. All CA3 neurons projected some collaterals to the hilar region. Proximal (CA3c) neurons had numerous collaterals in the hilus proper. One CA3c pyramidal cell in the dorsal hippocampus sent an axon collaterals to the inner third of the molecular layer. CA3c pyramidal cells in the ventral hippocampus had extensive projections to the inner third of the dentate molecular layer, as well as numerous collaterals in the hilus, CA3, and CA1 areas, and several axon collaterals penetrated the subiculum. The total projected axon length of a single neuron ranged from 150 to 300 mm. On the basis of the projected axon length and bouton density (mean interbouton distance: 4.7 microns), we estimate that a single CA3 pyramidal cell can make synapses with 30,000-60,000 neurons in the ipsilateral hippocampus. The concentrated distribution of the axon collaterals ("patches") indicates that subpopulations of neurons may receive disproportionately denser innervation, whereas innervation in the rest of the target zones is rather sparse. These observations offer new insights into the physiological organization of the CA3 pyramidal cell network.
Assuntos
Hipocampo/citologia , Animais , Feminino , Hipocampo/ultraestrutura , Histocitoquímica , Lisina/análogos & derivados , Masculino , Fibras Nervosas/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Células Piramidais/fisiologia , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestrutura , Terminologia como AssuntoRESUMO
The precise location of physiologically identified specific afferent input on the different types of cell in the visual cortex and the identification of the neurotransmitters of these cells are essential to a better understanding of the first stage of cortical processing. A combination of anatomical, neurochemical, and physiological methods was used to identify the cortical neurones that receive synaptic input from X- and Y-type afferents, which are thought to originate from cells of the lateral geniculate nucleus. One method relied on chance contacts made between single physiologically characterised axons, which had been injected with horseradish peroxidase (HRP), and the processes of cells impregnated by the Golgi method. These experiments revealed that both X and Y axons formed synapses on the dendrites of spiny stellate cells in layer 4. Y axons in both areas 17 and 18 established multiple synaptic contacts on basal dendrites of layer 3 pyramidal cells. One X axon contacted the apical dendrite of a layer 5 pyramidal cell and one Y axon contacted the dendrite of a large cell with smooth dendrites in layer 3. The maximum number of synapses made between one axon and a single postsynaptic cell was eight, although in most cases it was only one. It was concluded that one axon only provides a small fraction of the geniculate afferent input to an individual cell. A second method revealed that the somata in layer 4 in synaptic contact with the HRP-filled axon terminals were GABA-immunoreactive, and therefore might be involved in inhibitory processes. From light microscopic data it was found that somata receiving contacts from X axons in area 17 were significantly smaller (average diameter 15 microns) than those contacted by the Y axons in areas 17 and 18 (average diameter 24 microns). Somatic contacts were extremely rare in layer 6. These data show that the X and Y afferents may activate separate subsets of inhibitory neurones.
Assuntos
Corpos Geniculados/análise , Sinapses/análise , Córtex Visual/análise , Vias Visuais/análise , Ácido gama-Aminobutírico/análise , Animais , Gatos , Comunicação Celular , Corpos Geniculados/ultraestrutura , Peroxidase do Rábano Silvestre , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Terminações Nervosas/análise , Terminações Nervosas/fisiologia , Terminações Nervosas/ultraestrutura , Neurônios/análise , Neurônios/fisiologia , Neurônios/ultraestrutura , Prata , Coloração e Rotulagem , Sinapses/fisiologia , Córtex Visual/fisiologia , Vias Visuais/ultraestruturaRESUMO
Somatostatin-immunoreactive neurons in the rat neostriatum were studied by correlated light and electron microscopy using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactivity was localized in neuronal perikarya and processes. The perikarya were of spindle or fusiform shape (average length 16.9 microns) and were found in all parts of the neostriatum. From each neuron there arose two to four straight immunoreactive dendritelike processes, which could frequently be traced as far as about 130 microns from their perikaryon. Immunoreactive varicose axonlike processes were occasionally found, some of which were proximal axons of identified immunoreactive cells. Nine of the light microscopically identified neurons showing somatostatin-immunoreactivity were studied in the electron microscope; two of them had proximal axons with varicosities. Each neuron had an oval or elongated nucleus, which was always indented. These morphological features correspond well to those of certain "medium-size aspiny" neurons classified by Golgi studies. Although the immunoreactive endproduct was diffusely located throughout the neuron, it was characteristically located in the saccules and large granules (diameter 133 nm) of the Golgi apparatus, and large immunoreactive vesicles of similar size to those in the Golgi apparatus frequently occurred in all parts of axon. Very little synaptic input was found on the perikarya and dendrites of somatostatin-immunoreactive neurons. The perikarya and proximal dendrites received both symmetrical and asymmetrical synaptic input, while the distal dendrites usually received boutons that formed asymmetrical contacts. The somatostatin-immunoreactive boutons contained pleomorphic electron-lucent vesicles (diameter 39.3 nm) and a few large immunoreactive granular vesicles; these boutons always formed symmetrical synapses. Their postsynaptic targets were dendritic shafts, spines, and unclassified dendritic profiles. On the other hand, the varicosities of identified proximal axons of somatostatin-positive neurons did not form typical synapses, since they lacked clusters of small vesicles, but some of them were in direct apposition (via membrane specializations) to unlabelled perikarya or dendrites. It is concluded that somatostatin is a useful marker for a particular type of neuron in the neostriatum. The presence of somatostatin immunoreactivity in synaptic boutons is consistent with the view that somatostatin could be a neurotransmitter in the neostriatum.
Assuntos
Corpo Estriado/metabolismo , Somatostatina/metabolismo , Animais , Axônios/metabolismo , Corpo Estriado/citologia , Corpo Estriado/ultraestrutura , Dendritos/metabolismo , Complexo de Golgi/metabolismo , Técnicas Imunoenzimáticas , Masculino , Microscopia Eletrônica , Ratos , Sinapses/metabolismoRESUMO
Light microscopic autoradiography was used to identify cells in the neostriatum that became labelled after the local injection of [3H]gamma-aminobutyrate (GABA). The GABA-accumulating cells comprised up to 15% of the total population of neurons. Thirty-seven of these cells were examined in the electron microscope and it was found that they all had similar cytological characteristics, i.e., prominent nuclear indentations, a moderate volume of cytoplasm, rich in organelles, and sparse synaptic input to the perikaryon. Nine of the cells that had accumulated GABA were also impregnated following Golgi staining. These Golgi-impregnated neurons were of medium size and all had dendrites that were aspiny, often varicose, and that occasionally followed a recurving path. After gold toning, the Golgi-impregnated, GABA-accumulating neurons were examined in the electron microscope and were found to receive boutons forming symmetrical or asymmetrical synaptic contacts on their somata and dendrites; the symmetrical synapses were most common on the cell body and proximal dendrites, while the distal dendrites mainly received boutons forming asymmetrical contacts. We conclude that one type of GABAergic neuron in the neostriatum is a type of medium-sized aspiny neuron and that this neuron is likely to receive synaptic input both from neurons within the striatum and from neurons in distant brain regions. We suggest that this neuron is a local circuit neuron in the neostriatum since its morphological features are quite distinct from those of identified projecting neurons.
Assuntos
Corpo Estriado/citologia , Ácido gama-Aminobutírico/metabolismo , Animais , Autorradiografia , Axônios/ultraestrutura , Dendritos/ultraestrutura , Feminino , Microscopia Eletrônica , Vias Neurais/citologia , Neurônios/classificação , Ratos , Ratos Endogâmicos , Substância Negra/citologia , Sinapses/ultraestruturaRESUMO
Glutamate decarboxylase (GAD)-immunoreactive varicosities were found around cell bodies of nonimmunoreactive and immunoreactive neurons in the cat's visual cortex; they also occurred along apical dendrites and axon initial segments of pyramidal neurons. By examination in the electron microscope of structures first identified in the light microscope, it was established that the GAD-immunoreactive varicosities were boutons in symmetrical synaptic contact with pyramidal cells in layers II-IV. More than 90% of 142 boutons surrounding the cell bodies of 20 pyramidal neurons were immunoreactive for GAD. Since such a high proportion of the axosomatic boutons are GAD-immunoreactive, it is likely that the terminals of basket cells are included in this population and so the basket cell probably uses gamma-aminobutyrate as a transmitter, as suggested by previous authors. Almost all the 68 boutons in symmetrical contact with the axon initial segments of six pyramidal neurons could be shown to be GAD-immunoreactive, which makes it very likely that the boutons of axoaxonic cells contain GAD-immunoreactivity. This was established unequivocally for an individual Golgi-impregnated axoaxonic cell by combining Golgi impregnation and immunocytochemistry in the same sections: A Golgi-impregnated axoaxonic cell whose cell body was in layer II gave rise to numerous terminal segments, some of which were examined in the electron microscope after gold-toning. These boutons were in synaptic contact with axon initial segments and not only contained the Golgi precipitate but were also immunoreactive for GAD. It is concluded that the axoaxonic cell in the visual cortex uses gamma-aminobutyrate as a transmitter. An individual axoaxonic cell in layer II/III was filled with horseradish peroxidase by intracellular iontophoresis. The very extensive local axonal field was composed of 330 terminal bouton rows in layer II/III and a sparse descending collateral projection to infragranular layers. A computer-assisted reconstruction of the axonal field in three dimensions revealed the following: The main output of the cell is to pyramidal neurons that lie deeper than the soma; the axonal arborization occupies an area of 400 micron in the anteroposterior axis and extends 200 micron along the mediolateral axis; the terminal bouton rows in layer II/III form clusters about 50 micron wide running approximately at right angles to the border between areas 17 and 18, with an intercluster interval of about 100 micron. These findings suggest that the terminals of an individual axoaxonic cell could be contained within one ocular dominance column but that there may be inhomogeneities in the weighting of the axoaxonic input to pyramidal cells in the supragranular layers.