Biomedical subjects

T F Freund

Publications and source records attributed to T F Freund.

At least 109 records · Page 6Linked to original sources

PubMed1993

Hippocampal pyramidal cells excite inhibitory neurons through a single release site.

Morphologically a synapse consists of a presynaptic release site containing vesicles, a postsynaptic element with membrane specialization, and a synaptic cleft between them. The number of release sites shapes the properties of synaptic transmission between neurons. Although excitatory interactions between cortical neurons have been examined, the number of release sites remains unknown. We have now recorded excitatory postsynaptic potentials evoked by single pyramidal cells in hippocampal interneurons and visualized both cells using biocytin injections. Light and electron microscopy showed that excitatory postsynaptic potentials were mediated by a single synapse. We also reconstructed the entire axon arborization of single pyramidal cells, filled in vivo, in sections counterstained for parvalbumin, which selectively marks basket and axo-axonic cells. Single synaptic contacts between pyramidal cells and parvalbumin-containing neurons were dominant (> 80%), providing evidence for high convergence and divergence in hippocampal networks.

Animals↗

PubMed1993

Complete axon arborization of a single CA3 pyramidal cell in the rat hippocampus, and its relationship with postsynaptic parvalbumin-containing interneurons.

The complete axon arborization of a single CA3 pyramidal cell has been reconstructed from 32 (60 microns thick) sections from the rat hippocampus following in vivo intracellular injection of neurobiotin. The same sections were double-immunostained for parvalbumin--a calcium-binding protein selectively present in two types of GABAergic interneurons, the basket and chandelier cells--in order to map boutons of the pyramidal cell in contact with dendrites and somata of these specific subsets of interneurons visualized in a Golgi-like manner. The axon of the pyramidal cell formed 15,295 boutons, 63.8% of which were in stratum oriens, 15.4% in stratum pyramidale and 20.8% in stratum radiatum. Only 2.1% of the axon terminals contacted parvalbumin-positive neurons. Most of these were single contacts (84.7%), but double or triple contacts (15.3%) were also found. The majority of the boutons terminated on dendrites (84.1%) of parvalbumin-positive cells, less frequently on cell bodies (15.9%). In order to estimate the proportion of contacts representing synapses, 16 light microscopically identified contacts between boutons of the filled pyramidal cell axon and the parvalbumin-positive targets were examined by correlated electron microscopy. Thirteen of them were found to be asymmetrical synapses, and in the remaining three cases synapses between the labelled profiles could not be confirmed. We conclude that the physiologically effective excitatory connections between single pyramidal cells and postsynaptic inhibitory neurons are mediated by a small number of contacts, mostly by a single synapse. This results in a high degree of convergence and divergence in hippocampal networks.

Animals↗

PubMed1993

Precision and variability in postsynaptic target selection of inhibitory cells in the hippocampal CA3 region.

Non-pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea-pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo-axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritic region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritic segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum-moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo-axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo-axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramidale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons--often in conjunction with particular excitatory afferents (Han et al., Eur. J. Neurosci., 5, 395-410, 1993)--underlies functional differences in inhibitory synaptic actions.

Animals↗

PubMed1993

Distribution, morphological features, and synaptic connections of parvalbumin- and calbindin D28k-immunoreactive neurons in the human hippocampal formation.

Calcium binding proteins calbindin D28k (CaBP) and parvalbumin (PV) are known to form distinct subpopulations of gamma-aminobutyric acid (GABA)ergic neurons in the rodent hippocampal formation. Light and electron microscopic morphology and connections of these protein-containing neurons are only partly known in the primate hippocampus. In this study, CaBP and PV were localized in neurons of the human hippocampal formation including the subicular complex (prosubiculum, subiculum, and presubiculum) in order to explore to what extent these subpopulations of hippocampal neurons differ in phylogenetically distant species. CaBP immunoreactivity was present in virtually all granule cells of the dentate gyrus and population of in a proportion of pyramidal neurons in the CA1 and CA2 regions. A distinct population of CaBP-positive local circuit neurons was found in all layers of the dentate gyrus and Ammon's horn. Most frequently they were located in the molecular layer of the dentate gyrus and the pyramidal layer of Ammon's horn. In the subicular complex pyramidal neurons were not immunoreactive for CaBP. In the prosubiculum and subiculum immunoreactive nonpyramidal neurons were equally distributed in all layers, whereas in the presubiculum they occurred mainly in the superficial layers. Electron microscopy showed typical somatic and dendritic features of the granule, pyramidal, and local circuit neurons. CaBP-positive mossy fiber terminals in the hilus of the dentate gyrus and terminals of presumed pyramidal neurons of Ammon's horn formed asymmetric synapses with dendrites and spines. CaBP-positive terminals of nonprincipal neurons formed symmetric synapses with dendrites and dendritic spines, but never with somata or axon initial segments. PV was exclusively present in local circuit neurons in both the hippocampal formation and subicular complex. Most of the PV-positive cell bodies were located among or close to the principal cell layers. However, large numbers of immunoreactive neurons were also found in the molecular layer of the dentate gyrus and in strata oriens of Ammon's horn. PV-positive cells were equally distributed in all layers of the subicular complex. Electron microscopy showed the characteristic somatic and dendritic features of local circuit neurons. PV-positive axon terminals formed exclusively symmetric synapses with somata, axon initial segments and dendritic shafts, and in a few cases with dendritic spines. The CaBP- and PV-containing neurons formed similar subpopulations in rodents, monkeys, and humans, although the human hippocampus displayed the largest variability of these immunoreactive neurons in their morphology and location. Calcium binding protein-containing neurons frequently occurred in the molecular layer of the human dentate gyrus and in the stratum lacunosum-moleculare of Ammon's horn.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗

PubMed1993

Selective neuronal death in the contralateral hippocampus following unilateral kainate injections into the CA3 subfield.

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.

Anesthetics↗

PubMed1993

Early degeneration of calretinin-containing neurons in the rat hippocampus after ischemia.

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.

Animals↗

PubMed1993

Calretinin is present in non-pyramidal cells of the rat hippocampus--III. Their inputs from the median raphe and medial septal nuclei.

The subcortical innervation of a recently described subpopulation of non-pyramidal neurons, containing the calcium binding protein, calretinin, was investigated in the rat hippocampus using the anterograde tracer Phaseolus vulgaris-leucoagglutinin and double immunocytochemistry for calretinin and serotonin at the light and electron microscopic levels. Our results show that the GABAergic component of the septohippocampal pathway and the serotonergic raphe afferents establish multiple synaptic contacts with the calretinin-immunoreactive interneurons. The majority of the targets of both pathways were spine-free calretinin neurons known to innervate the dendritic region of the principal cells, but the GABAergic septal pathway was found to terminate also on the spiny neurons of stratum lucidum of the CA3 region and in the dentate hilus. The present results demonstrate that the serotonergic raphe-hippocampal and the GABAergic septohippocampal pathways are able to modulate dendritic inhibition of principal cells via calretinin-containing GABAergic interneurons.

Animals↗

PubMed1993

Postsynaptic targets of GABAergic hippocampal neurons in the medial septum-diagonal band of broca complex.

The termination pattern of hippocamposeptal nonpyramidal cells was investigated by injecting Phaseolus vulgaris leucoagglutinin (PHAL) into stratum oriens of the CA1 region. Electron microscopic analysis showed that the majority of the anterogradely labeled boutons formed symmetric synapses with dendrites and occasionally with cell bodies located in the medial septum-diagonal band of Broca complex. We have demonstrated with postembedding GABA immunocytochemistry that the majority of PHAL-labeled axon terminals were GABAergic. The neurochemical character of the postsynaptic target cells was also investigated using immunocytochemical double staining. Our data showed that the majority of the labeled hippocamposeptal axons innervated parvalbumin-immunoreactive cells representing GABAergic projection neurons, and a smaller number of contacts were found on ChAT-positive neurons. Septohippocampal neurons identified by retrograde HRP transport were also shown to reactive direct hippocamposeptal input. According to recent results, the lateral septum is unlikely to relay the hippocampal feedback to the medial septum; therefore, the direct hippocampal projection to the medial septum, arising from GABAergic nonpyramidal cells, seems to be the only feedback pathway to the area containing septohippocampal neurons. A novel circuit diagram, based on our recent morphological-immunocytochemical findings, is shown for the synaptic organization of the septo-hippocampo-septal loop. We suggest that the GABAergic hippocamposeptal feedback controls the activity of septal (mostly GABAergic) projection neurons as a function of hippocampal synchrony. The newly discovered reciprocal interactions may give a better insight into septohippocampal physiology.

Afferent Pathways↗

PubMed1992

Convergence and segregation of septal and median raphe inputs onto different subsets of hippocampal inhibitory interneurons.

The convergence and segregation of medial septal and median raphe afferents in the innervation of different subpopulations of GABAergic interneurons was investigated in the rat hippocampal formation. Following local injections of 5,7-dihydroxytryptamine into the median raphe nucleus destroying all serotonergic neurons, iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHAL) into the medial septum resulted in anterograde labelling of axons in the hippocampus. The labelled varicose fibres made multiple contacts with calbindin D28K-, parvalbumin-, and cholecystokinin-immunoreactive interneurons. These results disproved the possibility that PHAL-labelled afferents innervating hippocampal interneurons following septal PHAL injections would have been raphe axons passing through the injection site. In the second set of experiments a double anterograde tracing technique (PHAL from the septum and biotinylated-PHAL from the median raphe) and a triple or double immunostaining procedure was used to determine the types of interneurons (calbindin D28K-, parvalbumin-, or cholecystokinin-immunoreactive) innervated by one, or the other, or both pathways. The results showed that parvalbumin-containing neurons were innervated by septal afferents but avoided by raphe axons, whereas calbindin D28K-containing cells, and to a smaller extent cholecystokinin-containing cells served as targets for both pathways. In some cases the same individual calbindin D28K- or cholecystokinin-containing neurons received multiple contacts from afferents of both septal and raphe origin. Thus, our results indicate that different subcortical nuclei modulate largely different inhibitory circuits in the hippocampal formation. However, considering the occasional convergence of the two subcortical nuclei not only onto the same type, but even onto the same individual calbindin D28K-containing interneurons, we propose that a particular inhibitory function, most probably feed-forward inhibition in the distal dendritic region, is under the control of both pathways.

Afferent Pathways↗

PubMed1992

Distribution of GABAergic interneurons immunoreactive for calretinin, calbindin D28K, and parvalbumin in the cerebral cortex of the lizard Podarcis hispanica.

The types and distribution of cells containing three calcium-binding proteins, calretinin, calbindin D28K, and parvalbumin, have been studied by immunocytochemistry in different areas of the cerebral cortex of lizards. Cross-reactivity of the antisera has been excluded by demonstrating the existence of several cell groups immunoreactive for one but not the other two calcium-binding proteins. In the dorsal and dorsomedial cortices all three proteins coexist in a single subpopulation of gamma-aminobutyric acid (GABA)ergic neurons, the terminals of which form pericellular baskets around cell bodies of bipyramidal neurons. The somata of these neurons are largely restricted to the cellular and inner plexiform layers, but the dendrites usually penetrate all layers, allowing the neurons to sample input from all possible sources. A small number of parvalbumin-containing neurons in the outer plexiform layer do not contain the other two proteins. The medial cortex, which is likely to be homologous to the mammalian dentate gyrus, only contains parvalbumin-immunoreactive neurons. The dendritic trees of these cells appear to avoid the Timm-positive fields receiving input from zinc-rich fiber collaterals, originating from principal cells. The lateral cortex contains calbindin D28K-immunoreactive GABAergic neurons, which lack the other two calcium-binding proteins. These neurons have horizontally running dendrites in the outer plexiform layer, but their axon terminals could not be visualized. The present study uncovered important similarities and differences between the lizard and the mammalian archicortex in the types of neurons containing calcium-binding proteins. As in mammals, different cell types evolved in the lizard to inhibit the perisomatic versus the distal dendritic region of principal cells, the calcium-binding protein-containing neurons being responsible for the former, and neuropeptide-containing neurons for the latter. The results also suggest that further neurochemical diversion of GABAergic interneurons coupled to a functional specialization took place during phylogenetic development from reptiles to mammals.

Animals↗

PubMed1992

Pyramidal neurons are immunoreactive for calbindin D28k in the CA1 subfield of the human hippocampus.

The calcium binding protein calbindin D28k (CaBP) is localized in the granule cells of the dentate gyrus, in pyramidal neurons of the CA1 and CA2 subfields of the Ammon's horn as well as in distinct groups of local circuit neurons in both parts of the human hippocampal formation. Immunostaining was performed on human brain perfused 2 h after death. The localization of CaBP in the human was found to be similar to that in the monkey hippocampus suggesting that there is no species difference in the cellular localization of CaBP among different primates.

Animals↗

PubMed1992

gamma-Aminobutyric acid-containing basal forebrain neurons innervate inhibitory interneurons in the neocortex.

The basal forebrain-neocortex pathway--involved in higher cognitive processing, selective attention, and arousal--is considered one of the functionally most important ascending subcortical projections. The mechanism by which this relatively sparse subcortical pathway can control neuronal activity patterns in the entire cortical mantle is still unknown. The present study in the cat provides evidence that gamma-aminobutyric acid-containing basal forebrain neurons participate in the neocortical projection and establish multiple synaptic connections with gamma-aminobutyric acid-releasing interneurons containing somatostatin or parvalbumin. We propose that a mechanism by which the numerically small ascending pathways can exert a powerful global effect in the neocortex is by the selective innervation of gamma-aminobutyric acid-releasing interneurons, which, in turn, control the activity of large populations of pyramidal cells through their extensive axon arborizations. Finally, these results demonstrate a direct anatomical link between two cell populations implicated in Alzheimer disease pathology: basal forebrain neurons and cortical somatostatin cells.

Animals↗

PubMed1992

Neuropeptide Y-containing interneurons in the hippocampus receive synaptic input from median raphe and GABAergic septal afferents.

Neuropeptide Y has been extensively studied in the central nervous system due to a possible involvement of neuropeptide Y-containing neurons in cognitive functions. In the hippocampus neuropeptide Y is present in a subpopulation of nonpyramidal cells, which control the firing of hippocampal output neurons. In the present study we examined whether septohippocampal and raphe-hippocampal afferents--known to have a powerful effect on hippocampal electrical activity patterns--innervate neuropeptide Y-containing neurons in the hippocampal formation of the rat. Using a combination of pre- and postembedding immunostaining and tracing with Phaseolus vulgaris leucoagglutinin (PHAL) we showed that GABAergic afferents arising from the medial septal area extensively innervate neuropeptide Y-containing neurons. Afferents of median raphe origin, most of which are thought to be serotonergic, were also found to make multiple synaptic contacts with these cells. Thus, the neuropeptide Y-containing subpopulation of interneurons--which innervate distal dendrites of principal cells--are also among those through which different subcortical nuclei modulate information processing in the hippocampal formation.

Afferent Pathways↗

PubMed1992

GABA-immunoreactive basal forebrain afferents innervate GABA-immunoreactive non-pyramidal cells in the cerebral cortex of the lizard Podarcis hispanica.

The basal forebrain projection to the cerebral cortex was studied in the lizard Podarcis hispanica by anterograde transport of Phaseolus vulgaris leucoagglutinin. After injections of the lectin into the septal-basal forebrain area, Phaseolus vulgaris leucoagglutinin-labelled fibres were mainly detected in the outer plexiform layer of the medial cortex and in the inner plexiform layer of the dorsal and dorsomedial cortices. Ultrathin sections from these areas were obtained and processed for postembedding immunogold staining for GABA. Most of the Phaseolus vulgaris leucoagglutinin-labelled boutons in the dorsal and dorsomedial cortical areas were GABA immunoreactive and all the double-labelled boutons established symmetric synaptic contacts on cell bodies and dendrites that were also found to be GABA immunoreactive in all cases. In contrast, Phaseolus vulgaris leucoagglutinin-labelled varicosities in the outer plexiform layer of the medial cortex made asymmetric synaptic contacts on GABA-immunonegative profiles and they were themselves negative for GABA. In double-labelled sections, GABA-, calbindin D28k- and neuropeptide Y-immunoreactive neurons were found to be innervated by multiple Phaseolus vulgaris leucoagglutinin-labelled varicosities in the dorsal and dorsomedial cortical areas, whereas in the medial cortex Phaseolus vulgaris leucoagglutinin-labelled fibres were not observed in contact with any subpopulation of GABAergic cells. The results demonstrate that in lizards the septal-basal forebrain projection to the cortex has a GABAergic component, which selectively terminates on GABAergic non-pyramidal cells including the neuropeptide Y- and the calbindin D28k-containing subpopulations. This synaptic organization is remarkably similar to that in mammals, and suggests that the mechanisms of control of the cortical activity by the basal forebrain have been highly preserved during phylogeny.

Afferent Pathways↗

PubMed1992

Calretinin is present in non-pyramidal cells of the rat hippocampus--I. A new type of neuron specifically associated with the mossy fibre system.

Calretinin-containing cells were visualized with immunocytochemistry in the rat dorsal hippocampal formation. Calretinin immunoreactivity was present exclusively in non-pyramidal cells in all layers of the dentate gyrus and the CA1-3 areas. Calretinin-positive neurons and processes were most abundant in the hilus of the dentate gyrus and in the stratum lucidum of the CA3 region. Several calretinin-immunoreactive cells were located within the hippocampal fissure. A distinct band of calretinin-immunoreactive fibres occupied the superficial part of the granule cell layer and the lowest part of the molecular layer. Closer examination of the calretinin-positive cells revealed that they formed two distinct cell groups. One group of cells, found exclusively in the stratum lucidum of the CA3 area and in the hilus of the dentate gyrus, was covered with numerous spines. Their somata and dendrites were restricted to stratum lucidum and to the hilus. Cells of the other group had smooth, often varicose, radially running dendrites, and were present in all areas and layers of the hippocampal formation. Two to three thick primary dendrites arose from the irregularly shaped cell body of spiny cells and emitted fine secondary branches only distally (70-100 microns) from the soma, where they formed a profuse network. The extensive dendritic tree of the cells spread horizontally within stratum lucidum and span a distance of 400-600 microns both in the septotemporal and in the transverse directions. The layer-specific location of these cells and their processes suggested that the majority of their input may derive from mossy fibres. This presumption has been confirmed by electron microscopic examination. A large number of asymmetrical synapses were found to cover the soma, the dendritic shafts and the spines (four to six synapses/spine) of the cells. A large proportion of the synapses were formed by boutons, which showed the distinctive features of mossy fibre terminals. Three to six primary dendrites arose from the multipolar, bipolar or pyramidal-shaped somata of spine-free cells, which were smaller than the somata of spiny cells. The smooth and frequently varicose dendrites branched proximally and ran primarily radially. Dendrites ascended or descended through several layers and received both asymmetrical and symmetrical synapses. In the CA1 subfield, the vertically running dendrites frequently contacted other calretinin-immunoreactive spine-free dendrites or cell bodies. Two or three calretinin-immunoreactive dendrites were often seen to be attached for over 100 or, occasionally, 200 microns and several puncta adherentia were observed between them using the electron microscope.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Calretinin is present in non-pyramidal cells of the rat hippocampus--II. Co-existence with other calcium binding proteins and GABA.

The possible co-existence of calretinin with other calcium binding proteins, parvalbumin and calbindin D28k, and with GABA, was studied in non-pyramidal cells of the rat dorsal hippocampal formation, using the mirror technique. The majority of the calretinin-containing neurons (83%) were found to be immunoreactive for GABA (79% in the dentate gyrus, 84% in the CA2-3, and 88% in the CA1 subfield). Most of the GABA-negative calretinin-immunoreactive neurons were located in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. Detailed analysis of the calretinin-immunoreactive cells of these subfields revealed that the two morphologically distinct types of calretinin neurons, i.e. the spiny and the spine-free cells, differ in their immunoreactivity for GABA. The overwhelming majority (92%) of the spine-free neurons were GABA-positive, whereas the immunoreactivity of spiny cells was ambiguous. At the sensitivity threshold of the immunocytochemical techniques used in the present study, most of the spiny cells (89%) had to be considered as GABA-negative, although the staining intensity in their cell bodies was somewhat above background level. Colchicine treatment resulted in a degeneration of calretinin-immunoreactive neurons; therefore, its effect on the GABA content of spiny neurons could not be evaluated. Nevertheless, the observations suggest that calretinin-containing neurons are heterogeneous both morphologically and neurochemically. Examination of the co-existence of calcium binding proteins revealed that none of the hippocampal cells contained both calretinin and parvalbumin in any regions of the hippocampal formation. Some overlap was detected between the calretinin- and the calbindin D28k-containing cell populations, 5.1% of the former and 6.2% of the latter were immunoreactive for both calcium binding proteins. This may be due to a small degree of cross-reactivity of the calbindin D28k antiserum with calretinin. Thus, our results demonstrate that the majority of calretinin-immunoreactive neurons are GABAergic and represent a subpopulation of non-pyramidal cells with no or only a negligible overlap with the subpopulations containing the other calcium binding proteins, parvalbumin and calbindin.

Animals↗

PubMed1992

Calbindin D28k-containing nonpyramidal cells in the rat hippocampus: their immunoreactivity for GABA and projection to the medial septum.

Calbindin D28k-containing non-pyramidal cells were found in all layers and subfields of the hippocampus, with the highest frequency in stratum radiatum of the CA1-CA3 subfields. A large number of these neurons had a vertically oriented dendritic tree, often restricted to to stratum radiatum. In stratum oriens and near to the border of strata radiatum and lacunosum moleculare cells with horizontally running dendrites were also found. Multipolar cells were most common in stratum radiatum of the CA3 region. The GABAergic nature of the calbindin D28k-containing non-pyramidal cells was studied using the "mirror" technique. Adjacent thick sections were immunostained for calbindin D28k and GABA, and halved neurons were identified on the common surfaces. The majority of calbindin D28k-containing non-pyramidal cells were shown to be GABAergic. The GABA-negative calbindin cells were found in relatively large numbers in stratum oriens of the CA1-CA3 region, and occasionally in strata radiatum and pyramidale of CA2, and in stratum radiatum of the CA3c region near to the border of the dentate hilus. However, even in these cells a weak immunostaining, only slightly but consistently above background level, was always observed. Earlier studies have demonstrated that the somata of GABAergic neurons with distant projections may contain a level of GABA that is below the detection threshold of immunocytochemistry. Here we provide direct evidence that the calbindin-containing non-pyramidal cells were among those projecting to the medial septum. Following horseradish peroxidase injections into the medial septum 80% of the retrogradely labelled non-pyramidal cells were found to be immunoreactive for calbindin D28k, and 20% contained neuropeptide Y. These results suggest that the calbindin D28k-containing and apparently GABA-immunonegative non-pyramidal cells in stratum oriens of the CA1-CA3 regions may also be GABAergic, but have a distant projection, that is, to the medial septum.

Animals↗

PubMed1992

Pattern of neuronal death in the rat hippocampus after status epilepticus. Relationship to calcium binding protein content and ischemic vulnerability.

The pattern of hippocampal cell death has been studied following hippocampal seizure activity and status epilepticus induced by 110-min stimulation of the perforant pathway in awake rats. The order of vulnerability of principal cells in the different hippocampal subfields--as determined by silver impregnation--was found to be very similar to the pattern found in ischemia; i.e., dentate hilus greater than CA1, subiculum greater than CA3c greater than CA3a,b greater than dentate granule cells. The hilar somatostatin-containing cells were the most vulnerable cell type, whereas all other subpopulations of nonprincipal neurons--visualized by immunocytochemistry for the calcium binding proteins parvalbumin and calbindin--were remarkably resistant. Pyramidal cells in the CA3 region containing neither of the examined calcium binding proteins were more resistant to overexcitation than CA1 pyramidal cells, most of which do contain calbindin. This indicates that no simple relationship exists between vulnerability in status epilepticus and neuronal calcium binding protein content, and that local and/or systemic hypoxia during status epilepticus may be responsible for the ischemic pattern of cell death.

Animals↗