RESUMO
Substance P (SP) is known to be a peptide that facilitates epileptic activity of principal cells in the hippocampus. Paradoxically, in other models, it was found to be protective against seizures by activating substance P receptor (SPR)-expressing interneurons. Thus, these cells appear to play an important role in the generation and regulation of epileptic seizures. The number, distribution, morphological features and input characteristics of SPR-immunoreactive cells were analyzed in surgically removed hippocampi of 28 temporal lobe epileptic patients and eight control hippocampi in order to examine their changes in epileptic tissues. SPR is expressed in a subset of inhibitory cells in the control human hippocampus, they are multipolar interneurons with smooth dendrites, present in all hippocampal subfields. This cell population is considerably different from SPR-positive cells of the rat hippocampus. The CA1 (cornu Ammonis subfield 1) region was chosen for the detailed morphological analysis of the SPR-immunoreactive cells because of its extreme vulnerability in epilepsy. The presence of various neurochemical markers identifies functionally distinct interneuron types, such as those responsible for perisomatic, dendritic or interneuron-selective inhibition. We found considerable colocalization of SPR with calbindin but not with parvalbumin, calretinin, cholecystokinin and somatostatin, therefore we suppose that SPR-positive cells participate mainly in dendritic inhibition. In the non-sclerotic CA1 region they are mainly preserved, whereas their number is decreased in the sclerotic cases. In the epileptic samples their morphology is considerably altered, they possessed more dendritic branches, which often became beaded. Analyses of synaptic coverage revealed that the ratio of symmetric synaptic input of SPR-immunoreactive cells has increased in epileptic samples. Our results suggest that SPR-positive cells are preserved while principal cells are present in the CA1 region, but show reactive changes in epilepsy including intense branching and growth of their dendritic arborization.
Assuntos
Epilepsia/patologia , Hipocampo/patologia , Interneurônios/metabolismo , Interneurônios/patologia , Substância P/metabolismo , Sinapses/patologia , Adulto , Idoso , Contagem de Células/métodos , Dendritos/metabolismo , Dendritos/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica/métodos , Interneurônios/classificação , Interneurônios/ultraestrutura , Masculino , Microscopia Imunoeletrônica/métodos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Mudanças Depois da Morte , Sinapses/classificação , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
A model of epileptic cell death has been developed employing unilateral injections of kainic acid, a glutamate agonist, into the CA3 subfield of the hippocampus. The contralateral hippocampus, where neuronal damage is induced by hyperactivity in afferent pathways, served as the model structure. The pattern of cell death in this model was shown earlier to correspond to the vulnerable regions in human temporal lobe epilepsy. In the present time-course study we demonstrated that the different subpopulations of vulnerable cells in the contralateral hippocampus of the rat degenerate at different times following kainate injection. Spiny calretinin-containing cells in the hilus and CA3 stratum lucidum disappear at 12-24 h, other types of hilar neurons and CA3c pyramidal cells show shrinkage and argyrophilia at two days, whereas CA1 pyramidal cells degenerate at three days postinjection. The majority of cells destined to die showed a transient expression of the heatshock protein 72, approximately one day (for hilar-CA3c) or two days (for CA1) before degeneration. Parvalbumin-immunoreactivity transiently disappeared from the soma and dendrites of interneurons between the first and the fourth day. The results suggest that seizure-induced cell death is delayed, therefore acute oedema, even if it occurs, is insufficient to kill neurons. The only exception is the population of calretinin-containing interneurons degenerating at 12-24 h. The further one day delay between hilar-CA3c and CA1 cell death is likely to be due to differences in the relative density of glutamate receptor types (kainate versus NMDA) and the source of afferent input of these subfields. Thus, simple pharmacotherapy targeting only one of the excitotoxic mechanisms (i.e. acute oedema of calretinin cells versus delayed death of hilar-CA3c and CA1 cells at different time points) is likely to fail.
Assuntos
Morte Celular/efeitos dos fármacos , Epilepsia do Lobo Temporal/induzido quimicamente , Lateralidade Funcional/fisiologia , Hipocampo/efeitos dos fármacos , Ácido Caínico/farmacologia , Degeneração Neural/efeitos dos fármacos , Animais , Calbindina 2 , Morte Celular/fisiologia , Modelos Animais de Doenças , Hipocampo/citologia , Hipocampo/fisiologia , Imuno-Histoquímica , Masculino , Degeneração Neural/fisiologia , Células Piramidais/citologia , Células Piramidais/imunologia , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Proteína G de Ligação ao Cálcio S100/imunologia , Coloração pela Prata , Fatores de TempoRESUMO
The mechanism of delayed death of pyramidal cells in the hippocampal CA1 region and the acute death of various types of hilar neurons after ischemia is still unknown. Excitotoxicity may play a role in ischemic cell death, a prerequisite of which is the development of increased excitability or an enhanced excitatory transmission in the selectively vulnerable subfields of the hippocampus. Such changes may take place upon the loss or malfunction of local inhibitory neurons in the early postischemic period. In the present study we examined the vulnerability of non-pyramidal neurons containing a recently discovered calcium binding protein, calretinin, in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for calretinin enabled us to visualize a new type of spiny non-pyramidal cell in the hippocampus specifically associated with the mossy fiber system. This cell type is present exclusively in regions where mossy fiber terminals occur, i.e. in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. A selective loss of immunoreactivity in these neurons was already observed at 12-24 h after ischemia, when the pyramidal cells in the CA1 region showed no signs of damage. At a survival time of two to three days, most if not all spiny calretinin-immunoreactive cells had disappeared from the hippocampus. Other types of calretinin-containing GABAergic neurons were also reduced in number, but only at a time when CA1 pyramidal cells also started to degenerate, i.e. two to three days after ischemia. We speculate that the early loss of spiny calretinin-containing cells, together with other non-pyramidal cells associated with the mossy fiber system (somatostatin-containing neurons and mossy cells of the hilus), may result in pathological network activity in the hippocampus, which may ultimately lead to an increased excitatory transmission and delayed pyramidal cell death in the CA1 region.
Assuntos
Isquemia Encefálica/patologia , Hipocampo/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Animais , Isquemia Encefálica/metabolismo , Calbindina 2 , Morte Celular , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Degeneração Neural , Proteínas do Tecido Nervoso/imunologia , Neurônios/metabolismo , Células Piramidais/metabolismo , Células Piramidais/patologia , Ratos , Ratos Wistar , Proteína G de Ligação ao Cálcio S100/imunologia , Coloração pela PrataRESUMO
Intracerebral or intraperitoneal injections of kainic acid, an agonist at a class of glutamate receptors, have been extensively used to model temporal lobe epilepsy. In the present study we compared the types and distributions of selectively vulnerable neurons in the ipsi- and contralateral hippocampi following unilateral kainate injections into the CA3 subfield in order to examine whether "proximal" or "distant" neuronal damage resembled the pathology, and possibly also the mechanism, of human temporal lobe epilepsy. The degeneration of principal cells in the different hippocampal subfields was visualized by silver impregnation, and the loss of various types of non-principal cells was studied by immunostaining for the calcium binding proteins parvalbumin, calbindin-D28k and calretinin, as well as for somatostatin. In the first series of experiments various concentrations (ranging from 0.1 to 1 mg/ml) and volumes (0.5-2 microliters) of kainate were tested to induce reproducible damage in the contralateral hippocampus. The optimal dose, employed in the subsequent vulnerability studies, was found to be 3 x 0.5-microliter injections (over a period of 10 min) of a concentration of 0.33 mg/ml under ether anaesthesia, which was discontinued immediately after injection. Anaesthesia with equithesin was found to prevent contralateral cell death. Most if not all pyramidal cells in the CA3 region degenerated on the ipsilateral side, whereas the dentate granule cells, and the majority of CA1 pyramidal cells were resistant. A strikingly different pattern was found on the contralateral side, where CA1 pyramidal cells were almost completely lost, but the CA3 region (with the exception of CA3c) and the dentate gyrus remained intact. Three subpopulations of non-principal cells were found to be vulnerable in both hemispheres, the hilar somatostatin cells, spiny calretinin cells and mossy cells, as well as the spiny calretinin cells in stratum lucidum of CA3. The other subpopulations were resistant, except for those within the effective injection site. We propose that the "distant" (contralateral) damage resembles the pattern, and probably also the mechanism, of cell death in human temporal lobe epilepsy, whereas the ipsilateral damage does not.
Assuntos
Modelos Animais de Doenças , Dominância Cerebral , Epilepsia do Lobo Temporal , Hipocampo/patologia , Ácido Caínico/toxicidade , Neurônios/patologia , Convulsões/induzido quimicamente , Anestésicos/farmacologia , Animais , Biomarcadores , Calbindina 2 , Morte Celular/efeitos dos fármacos , Hidrato de Cloral/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Éter/farmacologia , Hipocampo/efeitos dos fármacos , Injeções , Injeções Intraperitoneais , Ácido Caínico/administração & dosagem , Sulfato de Magnésio/farmacologia , Masculino , Necrose , Degeneração Neural/efeitos dos fármacos , Proteínas do Tecido Nervoso/análise , Vias Neurais , Pentobarbital/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Ratos , Ratos Wistar , Proteína G de Ligação ao Cálcio S100/análise , Convulsões/patologiaRESUMO
A subpopulation of gamma-aminobutyric acid (GABA) containing neurons was reported to contain cholecystokinin-immunoreactive material in the visual cortex of cat [Somogyi et al., J. Neurosci. (1984) 4, 2590-2603]. In the present study pre-embedding immunocytochemistry was used to identify which of the several types of presumed GABAergic nonpyramidal cells in areas 17 and 18 contain cholecystokinin immunoreactivity. Most of the cholecystokinin-immunoreactive somata were found in layers II-III, they were less frequent in layers I and VI, and relatively rare in layers IV and V. The distribution and density of the axon terminals resembled that of the cell bodies. Two well defined types of cholecystokinin-immunoreactive neuron were distinguished: (1) double bouquet cells in layers II-III with vertically projecting axons, and (2) small basket cells with local axons either restricted to layers II-III, or descending to layer V. Additional cholecystokinin-positive cells showed features of bitufted or multipolar neurons in layers II-VI and horizontal cells in layer I, but these cells could be defined less well due to partial staining. Cholecystokinin-immunoreactive dendrites were found to run horizontally in layer I for several hundred micrometers. Some of the cholecystokinin-immunoreactive cells in layer VI had very long dendrites ascending radially up to layer III, as did their axons. A few cholecystokinin-immunoreactive cells appeared to have two axons and this was confirmed by electron microscopy. All cholecystokinin-immunoreactive neurons and terminals were separated from the basal lamina of blood vessels by glial endfeet. Random samples of boutons from each layer as well as identified terminals traced to their origin from local neurons were examined in the electron microscope. All of the boutons established symmetrical (type II) synaptic contacts with dendritic shafts, spines or somata. Quantitative electron microscopy of the postsynaptic targets of double bouquet cells and small basket cells demonstrated clear differences between these two types of neuron; basket cells having a higher proportion of their terminals in synaptic contact with somata. The findings that several distinct types of cortical neurons, as defined by their synaptic connections, contain cholecystokinin-immunoreactive material and that identified axons of all examined neurons form type II synaptic contacts suggests that the majority, if not all cholecystokinin-positive boutons forming type II contacts originate from local cortical cells. The distribution of targets postsynaptic to cholecystokinin-positive neurons is compared to those of cells labelled by other methods.
Assuntos
Colecistocinina/metabolismo , Córtex Visual/metabolismo , Animais , Gatos , Colecistocinina/fisiologia , Técnicas Imunoenzimáticas , Microscopia Eletrônica , Vias Neurais/anatomia & histologia , Sinapses/ultraestrutura , Transmissão Sináptica , Córtex Visual/citologia , Córtex Visual/fisiologiaRESUMO
In the present study, the connectivity between two subcortical nuclei involved in hippocampal theta activity, the supramammillary nucleus and the medial septum-diagonal band of Broca complex, was examined. Targets of the supramammillary afferents in the medial septum-diagonal band of Broca complex were identified by combining anterograde transport of Phaseolus vulgaris leucoagglutinin with immunostaining for putative postsynaptic neurons, i.e. for parvalbumin and choline acetyltransferase that are known to label the GABAergic and cholinergic neurons, respectively, of the medial septum-diagonal band of Broca complex. Double retrograde transport experiments using the tracers horseradish peroxidase and wheat germ agglutinin-conjugated colloidal gold were employed to identify supramammillary neurons that project both to the hippocampus and the medial septum-diagonal band of Broca complex. Phaseolus vulgaris leucoagglutinin injections into the supramammillary nucleus of the rat resulted in dense fibre and terminal labelling in the medial septum-diagonal band of Broca complex. Labelled terminals formed asymmetric synapses mainly on distal dendrites of medial septal neurons. Proximal dendrites and somata were rarely contacted. The supramammillary afferents showed no target selectivity for a particular cell type; they innervated both cholinergic and GABAergic cells. Occasionally, perisomatic, basket-like terminals of supramammillary origin were found around parvalbumin-containing neurons. Double-retrograde experiments revealed that at least 25% of the supramammillo-hippocampal cells also projected to the medial septum-diagonal band of Broca. These data suggest that the nucleus, known to modulate the hippocampal electrical activity directly by the supramammillo-hippocampal pathway, also has the potential for an indirect action via the innervation of both the GABAergic and cholinergic septohippocampal neurons. This dual modulation may originate, at least in part, from the same population of supramammillary neurons.
Assuntos
Lobo Frontal/fisiologia , Corpos Mamilares/fisiologia , Neurônios/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Colina O-Acetiltransferase/metabolismo , Lobo Frontal/citologia , Lobo Frontal/ultraestrutura , Imuno-Histoquímica , Masculino , Corpos Mamilares/citologia , Corpos Mamilares/ultraestrutura , Microscopia Eletrônica , Fibras Nervosas/diagnóstico por imagem , Fibras Nervosas/fisiologia , Neurônios/ultraestrutura , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/ultraestrutura , Parvalbuminas/metabolismo , Fito-Hemaglutininas , Ratos , Ratos Wistar , UltrassonografiaRESUMO
The role of ATP as a fast neurotransmitter is emerging from several lines of physiological and pharmacological studies. The bulk of experimental data on release properties and purinergic receptor-mediated postsynaptic potentials derives from studies in the habenula, but the source of the stimulation-evoked ATP release in this region is still unknown. In the present study, retrograde and anterograde tracing techniques were used to establish that both calretinin-containing and calretinin-negative neurons in the triangular septal and septofimbrial nuclei send a massive projection to the medial habenula, where they form asymmetrical synapses with their target neurons. The cells of origin, their axon terminals, as well as their synaptic targets remained unstained in sections immunostained for GABA. Electrolytic lesions of this anatomically circumscribed pathway resulted in an over 80% decrease in ATP release from habenula slices evoked by electric field stimulation. The possibility of transneuronal effects and release from local collaterals of habenular projection neurons accounting for the decreased ATP release has been excluded, since (i) there were no signs of neuronal degeneration, chromatolysis or atrophy in the habenula, (ii) the projection neurons have extremely sparse local collaterals and (iii) there are apparently no interneurons in the habenula. We conclude that the projection from the triangular septal and septofimbrial nucleus to the habenula uses ATP as a fast neurotransmitter, and its co-transmitter, if any, is likely to be glutamate.
Assuntos
Trifosfato de Adenosina/metabolismo , Habenula/metabolismo , Núcleos Talâmicos/metabolismo , Animais , Estimulação Elétrica , Habenula/anatomia & histologia , Habenula/citologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Núcleos Talâmicos/anatomia & histologia , Núcleos Talâmicos/citologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The topographic distribution of calretinin-immunoreactive neurons was studied in the medial septum diagonal band of Broca complex of the rat, in relation to the localization of other neurochemically identified cell groups containing choline acetyltransferase, parvalbumin or calbindin D28k. Double-labelling experiments revealed that these four antigen-containing cells formed distinct dorsoventrally running lamellae overlayed on top of each other similar to onion leaves. There was only a slight overlapping of the various cell groups. None of the four antigens were co-localized in the same cells. The lamella occupied by calretinin-positive neurons is situated at the border of the medial septum and the intermediolateral septal nucleus, and shows some overlap with the area occupied by cholinergic neurons. Retrograde transport of horseradish peroxidase from the hippocampus combined with immunostaining for calretinin revealed that calretinin-containing neurons do not participate in the septohippocampal projection. The lack of projection to the amygdala was also confirmed. Thus, calretinin-containing neurons represent a distinct cell group in the medial septal region, which either projects to subcortical areas, or may function as interneurons relaying hippocampal feedback to the medial septal projection neurons.
Assuntos
Química Encefálica/fisiologia , Encéfalo/anatomia & histologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Animais , Encéfalo/citologia , Calbindina 2 , Colina O-Acetiltransferase/metabolismo , Lobo Frontal/citologia , Lobo Frontal/fisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Parvalbuminas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The loss of the calcium binding protein, Calbindin-D28k, from dentate granule cells has been observed in different animal models of epilepsy and in ischaemia. This decrease is accompanied by alterations of calcium and N-methyl-D-aspartate currents, which may explain the hyperexcitability of the dentate gyrus. In the present study, we found a loss of calbindin immunoreactivity from over 90% of the dentate granule cells in lobectomy samples from four of 10 temporal lobe epilepsy patients. In another four patients, over 50%, of dentate granule cells were devoid of calbindin immunoreactivity, whereas the remaining two cases showed a 20-30% decrease. Electron microscopy revealed a normal ultrastructure both in calbindin-containing and calbindin-negative granule cells. Both calbindin-positive and -negative mossy fibre collaterals participated in supragranular sprouting. As inferred from data in animal models, the lack of calbindin in dentate granule cells of human epileptic subjects is likely to result in hyperexcitability of the dentate gyrus, which may then function as a "motor" for seizures.
Assuntos
Giro Denteado/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Adolescente , Adulto , Calbindina 1 , Calbindinas , Criança , Giro Denteado/patologia , Epilepsia do Lobo Temporal/patologia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Fibras Nervosas/metabolismoRESUMO
The distribution, size, dendritic morphology and synaptic connections of calbindin-, calretinin- and substance P receptor-positive interneurons and pathways have been examined in control and epileptic human dentate gyrus. In the epileptic dentate gyrus, calbindin-containing interneurons are preserved, but their dendrites become elongated and spiny, and several cell bodies appear hypertrophic. The relative laminar distribution of calretinin-containing cells did not change, but their number was considerably reduced. The calretinin-positive axonal bundle at the top of the granule cell layer originating from the supramammillary nucleus expanded, forming a dense network in the entire width of the stratum moleculare. Substance P receptor-immunopositive cells were partially lost in epileptic samples, and in addition, the laminar distribution and dendritic morphology of the surviving cells differed considerably from the controls. In the control human dentate gyrus, the majority of substance P receptor-positive cells can be seen in the hilus, while most are present in the stratum moleculare in the epileptic tissue. Their synaptic input is also changed. The extent of individual pathological abnormalities correlates with each other in most cases. Our data suggest, that although a large proportion of inhibitory interneurons are preserved in the epileptic human dentate gyrus, their distribution, morphology and synaptic connections differ from controls. These functional alterations of inhibitory circuits in the dentate gyrus are likely to be compensatory changes with a role to balance the enhanced excitatory input in the region.
Assuntos
Giro Denteado/patologia , Epilepsia do Lobo Temporal/patologia , Interneurônios/patologia , Adolescente , Adulto , Calbindina 2 , Calbindinas , Criança , Giro Denteado/metabolismo , Giro Denteado/ultraestrutura , Epilepsia do Lobo Temporal/metabolismo , Feminino , Humanos , Interneurônios/metabolismo , Interneurônios/ultraestrutura , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Receptores da Neurocinina-1/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
The distribution, morphology, synaptic coverage and postsynaptic targets of calbindin-containing interneurons and afferent pathways have been analyzed in the control and epileptic CA1 region of the human hippocampus. Numerous calbindin-positive interneurons are preserved even in the strongly sclerotic CA1 region. The morphology of individual cells is altered: the cell body and dendrites become spiny, the radially oriented dendrites disappear, and are replaced by a large number of curved, distorted dendrites. Even in the non-sclerotic epileptic samples, where pyramidal cells are present and calbindin-immunoreactive interneurons seem to be unchanged, some modifications could be observed at the electron microscopic level: they received more inhibitory synaptic input, and the calbindin-positive excitatory afferents - presumably derived from the CA1, the CA2 and/or the dentate gyrus - are sprouted. In the strongly sclerotic tissue, with the death of pyramidal cells, calbindin-positive terminals (belonging to interneurons and the remaining excitatory afferents) change their targets. Our data suggest that an intense synaptic reorganization takes place in the epileptic CA1 region, even in the non-sclerotic tissue, before the death of considerable numbers of pyramidal cells. Calbindin-positive interneurons participate in this reorganization: they show plastic changes in response to epilepsy. The enhanced inhibition of inhibitory interneurons may result in the disinhibition of pyramidal cells or in an abnormal synchrony in the output region of the hippocampus.
Assuntos
Vias Aferentes/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Interneurônios/metabolismo , Plasticidade Neuronal/fisiologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Sinapses/metabolismo , Adulto , Vias Aferentes/patologia , Vias Aferentes/ultraestrutura , Calbindinas , Dendritos/metabolismo , Dendritos/patologia , Dendritos/ultraestrutura , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Hipocampo/patologia , Hipocampo/ultraestrutura , Humanos , Imuno-Histoquímica , Interneurônios/patologia , Interneurônios/ultraestrutura , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/metabolismo , Células Piramidais/patologia , Células Piramidais/ultraestrutura , Sinapses/patologia , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/patologia , Membranas Sinápticas/ultraestruturaRESUMO
Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus. We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the axon loses parvalbumin or immunoreactivity for parvalbumin. The hyperinnervation of axon initial segments might be a compensatory change in the inhibitory network, but at the same time may lead to a more effective synchronization of granule cell firing that could contribute to the generation or amplification of epileptic seizures.
Assuntos
Giro Denteado/patologia , Epilepsia do Lobo Temporal/patologia , Interneurônios/química , Inibição Neural , Adolescente , Adulto , Axônios/química , Axônios/ultraestrutura , Sincronização Cortical , Dendritos/química , Dendritos/ultraestrutura , Epilepsia do Lobo Temporal/cirurgia , Feminino , Humanos , Interneurônios/ultraestrutura , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Vias Neurais , Parvalbuminas/análise , Sinapses/química , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/análiseRESUMO
Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.
Assuntos
Canabinoides/metabolismo , Hipocampo/metabolismo , Interneurônios/metabolismo , Receptores de Droga/metabolismo , Ácido gama-Aminobutírico/metabolismo , Idoso , Membrana Celular/metabolismo , Hipocampo/citologia , Hipocampo/ultraestrutura , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Terminações Pré-Sinápticas/metabolismo , Receptores de Canabinoides , Receptores de Droga/agonistasRESUMO
Day-old domestic chicks (Gallus domesticus) were trained on a one-trial passive avoidance task in which the aversive stimulus was a bitter tasting substance, methylanthranilate. Thirty minutes later, localization of binding of highly specific ligands (([D-Ala2, Gly-ol]-enkephalin ([3H]DAGO) for mu (mu) receptor sites, [D-Pen2,D-Pen5]-enkephalin ([3H]-DPDPE) for delta (delta) sites, and [3H]-U- 69593 for kappa (kappa 1) sites) to opioid receptors in various regions of the forebrain of methyl-anthranilate trained (M-) and control (water trained (W-)) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to delta ([3H]-DPDPE), but not mu or kappa receptors, were found in several regions of the forebrain, of trained compared to control chicks. There were decreases in binding in the hyperstriatum dorsale of the left hemisphere (14%) and a decrease in binding in the lateral hyperstriatum ventrale of the right hemisphere (14%). However, significant increases were observed in delta binding in the paleostriatum augmentatum of the right hemisphere (16%) and the lobus parolfactorius of both hemispheres (left, 20%; right, 21%). In a control experiment designed to determine whether the taste of methylanthranilate contributed to the increase in 3H-DPDPE binding, there was no significant difference in the level of binding between blindfolded birds in which methylanthranilate was placed in the beak, and blindfolded birds in which water was placed on the bead and inserted into the beak. These findings demonstrate that changes occur in an opioid receptor sub-type in specific regions of forebrain of the chick following passive avoidance training which may be related to events concerned with the process of memory formation.
Assuntos
Aprendizagem da Esquiva/fisiologia , Prosencéfalo/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Animais , Autorradiografia , Galinhas , D-Penicilina (2,5)-Encefalina , Encefalinas/metabolismo , Filtração , Ensaio Radioligante , ortoaminobenzoatosRESUMO
Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.
Assuntos
Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Endocanabinoides , Glicerídeos/metabolismo , Hipocampo/metabolismo , Lipase Lipoproteica/metabolismo , Sinapses/enzimologia , Animais , Espinhas Dendríticas/enzimologia , Hipocampo/ultraestrutura , Humanos , Imuno-Histoquímica , Lipase Lipoproteica/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Terminações Pré-Sinápticas/enzimologia , Transdução de Sinais , Especificidade da EspécieRESUMO
In the Golgi preparation of visual Wulst of chicken four types of projection neurons were found: type 1a, 1b, 1c, and 1d neurons, which are distinguished on the basis of their dendritic ramification pattern and especially on the density of the dendritic spines. Type 1a has large dendritic tree with moderately dense spiny dendrites. Type 1b has very dense spiny dendrites. Type 1c is a small neuron with moderately dense spiny dendrites. Type 1d is a star-pyramid-like neuron with a short, bifurcating apical dendrite. The final dendritic pattern and spine density of projection neurons was found to develop at the end of the first month. In the Wulst short axon cells (interneurons-INs) of different types occur: large, medium-sized and small INs can be observed. Their axon-arborizations are significantly different from each other. Some of them are GABA-immuno-positive neurons, which are very probably inhibitory interneurons. Stellate-like neurons also occur in IHA and HIS. These neurons have sparsely spiny dendrites and locally arborizing axons.
Assuntos
Complexo de Golgi/ultraestrutura , Neurônios/citologia , Telencéfalo/citologia , Animais , Axônios/ultraestrutura , Diferenciação Celular , Galinhas , Imuno-Histoquímica , Neurônios/ultraestrutura , Telencéfalo/irrigação sanguínea , Telencéfalo/ultraestruturaRESUMO
Our investigations try to reveal the morphological changes found postnatally at different levels and centres of central nervous system of kittens of cats chronically treated with ethanol during their pregnancy and lactation. The changes found in neurons of lateral geniculate nucleus (LGN) were analyzed in Golgi-stained preparations from brains removed on the first or on the ninth postnatal days. The data were computerized. The changes measured in neurons refer to their maturation process: a significant delay of differentiation of the neurons occurs in LGN. Also a delayed gyrification of brains was observed.
Assuntos
Encéfalo/patologia , Etanol/toxicidade , Corpos Geniculados/patologia , Neurônios/ultraestrutura , Efeitos Tardios da Exposição Pré-Natal , Animais , Peso Corporal/efeitos dos fármacos , Gatos , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Feminino , Corpos Geniculados/efeitos dos fármacos , Corpos Geniculados/ultraestrutura , Histocitoquímica , Neurônios/efeitos dos fármacos , Gravidez , Coloração e RotulagemRESUMO
Neurons of the supramammillary nucleus are known to fire phase-locked to hippocampal theta rhythm. Stimulation of this area induces theta activity in the hippocampus via the medial septum and facilitates perforant pathway stimulation-evoked population spikes in the dentate gyrus even if the medial septum is inactivated. This latter effect was suggested to be due to a direct inhibitory input from the supramammilary nucleus to hippocampal nonpyramidal cells resulting in disinhibition. In the present study, using anterograde tracing with Phaseolus vulgaris leucoagglutinin, we aimed to identify the types of neurons innervated by the supramammillary projection in the dentate gyrus and Ammons horn, with particular attention to the presumed postsynaptic inhibitory neurons, which may mediate the proposed disinhibitory action. Double-immunostaining for the tracer and different neuropeptides (somatostatin, cholecystokinin, neuropeptide Y) or calcium binding proteins (calretinin, parvalbumin, calbindin D28K) present in different subpopulations of interneurons revealed no multiple contacts between supramammillary afferents and labeled inhibitory cells at the light microscopic level. Furthermore, postembedding immunostaining of electron microscopic sections for GABA demonstrated that none of the 68 PHAL-labeled supramammillary boutons examined and none of their postsynaptic targets were immunoreactive for the inhibitory neurotransmitter. We conclude, therefore, that most if not all postsynaptic targets of the supramammillary projection are principal cells both in the dentate gyrus and in the CA2-CA3a subfields. This suggests that a mechanism other than disinhibition is responsible for the facilitatory effect of this pathway on hippocampal evoked activity.
Assuntos
Vias Aferentes , Hipocampo/fisiologia , Corpos Mamilares/fisiologia , Sinapses/fisiologia , Vias Aferentes/citologia , Animais , Transporte Axonal , Hipocampo/citologia , Peroxidase do Rábano Silvestre , Imuno-Histoquímica , Masculino , Corpos Mamilares/citologia , Microscopia Eletrônica , Neurônios/fisiologia , Fito-Hemaglutininas , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/análiseRESUMO
Chicken ectostriatum was studied in Golgi preparations. In the two parts of the area (ectostriatum centrale and ectostriatum periphericum) the cell types were analysed. Projection neurons with long axon, and interneurons with short axon, could be distinguished in both parts of the region. The projection neurons are different in the two parts of the area, whilst the interneurons, with regard to their morphological features, are completely similar. The stellate-like projection neurons in ectostriatum centrale have 4-6 main dendrites, which ramify only slightly, and are densely covered by characteristic spines. The projection neurons in ectostriatum periphericum emit several (7-10) main dendrites, which have numerous bifurcations establishing a dense dendritic tree. The dendrites are densely covered by regular spines. The interneurons--in both parts of ectostriatum--have three subtypes. All have characteristic local axonal arborizations with varicose terminal sections. Some quantitative parameters of Golgi impregnated neurons were also measured and analysed by computer. Preliminary results of this study are included in the present publication.
Assuntos
Galinhas/anatomia & histologia , Telencéfalo/anatomia & histologia , Vias Visuais/anatomia & histologia , Animais , Dendritos/análise , Processamento Eletrônico de Dados , Neurônios/classificação , Neurônios/citologia , Telencéfalo/citologiaRESUMO
There are numerous hypotheses for the role of the axon collaterals of pyramidal cells. Most hypotheses predict that pyramidal cells activate specific classes of postsynaptic cells. We have studied the postsynaptic targets of two layer III pyramidal cells, that were of special interest because of their clumped axon arborization near, and also 0.4-1.0 mm from the cell body, in register in both layers III and V. 191 terminations from four sites (layers III and V, both in the column of the cell and in distant clumps) were analysed by electron microscopy. Only one bouton contacted a cell body and that was immunoreactive for GABA. The major targets were dendritic spines (84 and 87%), and the remainder were dendritic shafts. Of these 13 were classed as pyramidal-like (P), 8 smooth cell-like (S) and three could not be classified. Four of five S types, but none of the seven P types tested were immunoreactive for GABA, supporting the fine structural classification. The putative inhibitory cells therefore formed not more than 5% of the postsynaptic targets, and their activation could only take place through the convergence of pyramidal cells onto a select population of GABA cells. The results show that the type of pyramidal cells with clumped axons studied here make contacts predominantly with other pyramidal cells. Thus the primary role of both the intra and intercolumnar collateral systems is the activation of other excitatory cells.