Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material

The coexistence of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and cholecystokinin (CCK)- or somatostatin-immunoreactive material in the same neurons was studied in the hippocampus and visual cortex of the cat. One-micrometer-thick serial sections of the same neuron were reacted to reveal different antigens by the unlabeled antibody enzyme method. All CCK- and somatostatin-immunoreactive neurons in the cortex and all CCK-immunoreactive and the majority of somatostatin-immunoreactive neurons in the hippocampus that could be examined in serial sections were also immunoreactive for GABA. In neurons that were immunoreactive for GAD it was often possible to demonstrate immunoreactivity for one of the peptides as well as for GABA. GABA-immunoreactive neurons, as revealed by an antiserum to GABA, were present in all layers of the cortex and hippocampus, and their shape, size, and distribution were similar to GAD-immunoreactive neurons. All GAD-immunoreactive neurons were also positive for GABA, but the latter staining revealed additional neurons. CCK/GABA- and somatostatin/GABA-immunoreactive neurons were present mainly in layers II and upper III and in layers V and VI in the visual cortex. CCK/GABA-immunoreactive neurons were most frequently present in the strata oriens, pyramidale, and moleculare of the hippocampus and in the polymorph cell layer of the dentate gyrus. Somatostatin/GABA-immunoreactive neurons were localized mainly in the stratum oriens and in the hilus of the fascia dentata. The two peptides could not be found in the same neuron. The majority of neurons that were GABA immunoreactive did not stain for either peptide. The presence of CCK- and somatostatin-immunoreactive material in GABAergic cortical neurons raises the possibility that neuroactive peptides affect GABAergic neurotransmission.     

Visualization of taurine, GABA and glutamate in developing feline cerebellum by immunohistochemistry

The localization of taurine, GABA and glutamate in developing feline cerebellum was performed using antibodies raised against the amino acids conjugated to bovine serum albumin with glutaraldehyde. Distinct patterns of immunostaining were observed for each of the amino acids. Taurine-like immunoreactivity reached a peak at 4 weeks after birth, as did GABA-like immunoreactivity, whereas glutamate-like immunoreactivity was greatest in the mature cerebellum. Purkinje cells are all taurine-positive in cerebellum from neonatal animals, whereas in the mature cerebellum they appear to contain only GABA and glutamate, with virtually no taurine, in contrast to observations reported with rodent cerebellum. Ultrastructural studies and immunogold labelling visualized by electron microscopy show that the band of taurine-like immunoreactivity observed in newborn feline cerebellum is localized within dendrites, axons and glial processes. Granule cells migrating through this region also show prominent taurine-like immunoreactivity.     

GABA plasma membrane transporters,GAT-1 and GAT-3, display different distributions in the rat hippocampus

This study evaluates the distribution of two high affinity gamma-aminobutyric acid (GABA) transporters (GAT-1 and GAT-3) in the rat hippocampus using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the dentate gyrus. In Ammon's horn, immunoreactive processes were concentrated around the somata of pyramidal cells, particularly at their basal regions. The apical and basal dendritic fields of pyramidal cells also displayed numerous GAT-1 immunoreactive punctate structures and axons. The zone of termination of the mossy fibers that includes both the hilus of the dentate gyrus and stratum lucidum of the CA3 area was the lightest immunolabeled region of the hippocampal complex. Electron microscopic preparations demonstrated that GAT-1 immunoreactive axon terminals form symmetric synapses with somata, axon initial segments, and dendrites of granule and pyramidal cells in the dentate gyrus and Ammon's horn, respectively. Immunoreactivity was localized to the plasma membrane and the cytoplasm of axon terminals. The somata of previously described local circuit neurons in the dentate gyrus and Ammon's horn contained GAT-1 immunoreactivity associated with the Golgi complex. Light, diffuse GAT-3 immunoreactivity was present throughout the hippocampal formation. Thin, astrocytic glial processes displayed GAT-1 and GAT-3 immunoreactivity. This localization of GAT-1 and GAT-3 indicates that they are involved in the uptake of GABA from the extracellular space into GABAergic axon terminals and astrocytes. © 1996 Wiley-Liss, Inc.     

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

Calcium binding proteins calbindin D_28k (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 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. The corresponding areas of the rat or monkey hippocampus were devoid of such neurons. In both rodents and primates similar populations of principal neurons contained CaBP. In addition, CaBP and PV were localized in distinct and nonoverlapping populations of nonprincipal cells. Their target selectivity did not change during phylogeny (e.g., PV-positive cells mainly innervate the perisomatic region and CaBP-positive cells the distal dendritic region of principal cells). © 1993 Wiley-Liss,Inc.     

GABABR1 receptor protein expression in human mesial temporal cortex: changes in temporal lobe epilepsy

Immunocytochemistry was used to examine gamma-aminobutyric acid beta (GABA)(B)R1a-b protein expression in the human hippocampal formation (including dentate gyrus, hippocampus proper, subicular complex, and entorhinal cortex) and perirhinal cortex. Overall, GABA(B)R1a-b immunostaining was intense and widespread but showed differential areal and laminar distributions of labeled cells. GABA(B)R1a-b-immunoreactive (-ir) neurons were found in the three main layers of the dentate gyrus, the most intense labeling being present in the polymorphic layer, whereas the granule cells were moderately immunoreactive. Except for slight variations, similar distribution patterns of GABA(B)R1a-b immunostaining were found along the different subfields of the Ammon's horn (CA1-CA4). The highest density of GABA(B)R1a-b-ir neurons was localized in the stratum pyramidale, where virtually every pyramidal cell was intensely immunoreactive, including the proximal part of the apical dendrites. Within the subicular complex, a more intense GABA(B)R1a-b immunostaining was found in the subiculum than in the presubiculum or parasubiculum, especially in the pyramidal and polymorphic cell layers. In the entorhinal cortex, distribution of GABA(B)R1a-b immunoreactivity was localized mainly in both pyramidal and nonpyramidal cells of layers II, III, and VI and in the superficial part of layer V, with layers I, IV, and deep layer V being less intensely stained. In the perirhinal cortex, the most intense GABA(B)R1a-b immunoreactivity was located in the deep part of layer III and in layer V and was mainly confined to medium-sized and large pyramidal cells. Thus, the differential expression, but widespread distribution, of GABA(B)R1a-b protein found in the present study suggests the involvement of GABA(B) receptors in many circuits of the human hippocampal formation and adjacent cortical structures. Interestingly, the hippocampal formation of epileptic patients (n = 8) with hippocampal sclerosis showed similar intensity of GABA(B)R1a-b immunostaining in the surviving neurons located within or adjacent to those regions presenting neuronal loss than in the controls. However, surviving neurons in the granule cell layer of the dentate gyrus displayed a significant reduction in immunostaining in 7 of 8 patients. Therefore, alterations in inhibitory synaptic transmission through GABA(B) receptors appears to affect differentially certain hippocampal circuits in a population of epileptic patients. This reduction in GABA(B)R1a-b expression could contribute to the pathophysiology of temporal lobe epilepsy.     

Colocalization of taurine- and cysteine sulfinic acid decarboxylase-like immunoreactivity in the hippocampus of the rat

It is proposed that taurine is an inhibitory neurotransmitter/neuromodulator in the CNS. The present study localized taurine-containing neurons within the rat hippocampus with the use of a monoclonal antibody against conjugated taurine (Tau2) in conjunction with an antiserum against cysteine sulfinic acid decarboxylase (CSADC), a synthesizing enzyme for taurine. Taurine-like immunoreactivity Tau-LI) and CSADC-LI were colocalized in neurons of the dentate gyrus, CA1(/CA2), CA3, and CA4. Of all the cells examined, pyramidal basket cells within the granule cell layer of the dentate gyrus were most intensely stained with both Tau2 and CSADC. Granule cells were also double-labeled with Tau-LI and CSADC-LI. Cell nuclei and dendrites in the CA1 region stained more intensely with Tau2 than somata. CSADC-LI was colocalized with Tau-LI within these neurons. Light staining with both Tau2 and the CSADC antiserum was inconsistently present in CA3 and CA4 neurons and was found to be highly dependent on the type of fixation and delay to fixation. Tau-LI was more consistently present in increased numbers of neurons in CA3 when glutaraldehyde was added to the paraformaldehyde fixative solution. Hippocampi which were immersion-fixed in paraformaldehyde following a 0-, 6-, or 24-hour postmortem delay exhibited a lack of Tau2 staining in the CA3 region in the majority of animals studied, similar to some paraformaldehyde perfusion-fixed rats. These studies suggest that taurine was present in the majority of neurons within the major cell layers of the rat hippocampus, but Tau-LI was more easily lost from neurons in the CA3 region following delay to fixation. The localization of Tau-LI in excitatory neurons such as granule cells and pyramidal cells is not consistent with its proposed inhibitory transmitter role. However, the prominent Tau2 staining in dendrites of the CA1 region provides anatomical support for the hypothesis that taurine may be released from dendrites in the CA1 region and may function as a neuromodulator of calcium flux in these pyramidal neurons.     

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e, dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentale granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral coritcal hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.     

Localization of the ras-like rab3A protein in the adult rat brain

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e, dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentale granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral coritcal hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.     

Precocious development of parvalbumin-like immunoreactive interneurons in the hippocampal formation and entorhinal cortex of the fetal cynomolgus monkey

The calcium-binding protein parvalbumin (PV), a reliable marker of the hippocampal basket and chandelier cells, is first expressed on embryonic day 83 (E83), corresponding to midgestation of the macaque monkey, in restricted hippocampal groups of immature neurons (Berger and Alvarez [1996] J. Comp. Neurol. 366:674–699). In the present study, PV-like immunoreactivity (LIR) was used to follow the further development of this subclass of interneurons. Asynchronous area-specific developmental sequences were observed, predominating initially in the caudal half of the hippocampal formation and the laterocaudal division of the entorhinal cortex and occurring relatively simultaneously in the interconnected hippocampal and entorhinal subfields. Dendritic elongation of PV-like immunoreactive interneurons and perisomatic distribution of PV-like immunoreactive terminal boutons on their cellular targets were first observed in the subiculum around E127; then from E127 to E142 in CA3/CA2 and layers III–V of the entorhinal cortex and, to a lesser extent in CA1, the dentate hilus and deep granule cell layer; and finally from E156 to postnatal day 12 in the rest of the dentate gyrus, the presubiculum and parasubiculum, and layers III-II-I of the entorhinal cortex. These data provide the first indication that a population of basket cells, a major γ-aminobutyric acid (GABA)ergic component of the hippocampal intrinsic inhibitory circuitry, reaches its cellular targets several weeks before birth in primates in contrast to rodents. The role of the prenatal PV expression in the hippocampal formation of nonhuman primates and whether it coincides with the onset of postsynaptic inhibitory potentials or is accompanied or preceded by a period of γ-aminobutyric acid-–mediated excitatory effects as in rat pups, are crucial questions. They underline the need to pursue direct investigations on primates to be able to legitimately extrapolate the data obtained in rodents. J. Comp. Neurol. 403:309–331, 1999. © 1999 Wiley-Liss, Inc.     

Localization and induction of c-fos protein-like immunoreactive material in the nuclei of adult mammalian neurons

The distribution of the proto-oncogene product, c-fos protein, has been studied in tissue from adult rat brain using immunocytochemical techniques. Sections of rat brain were incubated with a polyclonal antibody to a synthetic fragment of the c-fos protein (M-peptide) and visualized for immunoreactivity using the avidin-biotin technique. Low levels of c-fos protein-like immunoreactivity were concentrated in the nucleus. Dark nuclear staining of cells was observed in the cerebral cortex, in hippocampal pyramidal neurons, in granule cells of the dentate gyrus and other regions. Neurons in the cat visual cortex also showed low levels of c-fos protein-like immunoreactivity. Pre-absorbing the antibody with the M-peptide abolished the immunostaining. Generalized seizures evoked by injection of pentylenetetrazol produced a massive induction of fos protein(s) in the piriform and cingulate cortices as well as the dentate gyrus of rats. These results demonstrate that c-fos protein-like immunoreactive materials is found within the nuclei of fully differentiated adult mammalian neurons at low basal levels and that activation of nerve cells leads to an induction of c-fos proteins.     

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design‐based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5–10‐year‐old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5–10‐year‐old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.     

Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two “fast-acting” amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects. © 1996 Wiley-Liss, Inc.     

Evidence for commissurally projecting parvalbumin-immunoreactive basket cells in the dentate gyrus of the rat.

The fluorescent retrograde tracer, fluorogold, was used to identify commissurally projecting neurons in the hippocampus and dentate gyrus. After injection of fluorogold into the hippocampus, the contralateral hippocampus was evaluated for fluorogold-immunoreactive or fluorescent neurons. In addition to observing labeled hilar neurons and CA3 pyramidal cells that previously have been reported to send commissurally projecting axons to the contralateral hippocampus, the authors unexpectedly found a population of fluorogold-labeled cells in the granule cell layer with the morphology and location of GABA-immunoreactive basket cells. Immunocytochemical staining revealed that all fluorogold-labeled cells of the granule cell layer were immunoreactive for parvalbumin. However, not all parvalbumin cells, shown previously to be a subset of GABA neurons, were fluorogold-labeled. The association between fluorogold transport and parvalbumin immunoreactivity was unique for these cells of the granule cell layer. In the adjacent hilus, relatively few of the many fluorogold-labeled cells were parvalbumin- or GABA-immunoreactive. These results (1) identify a population of presumed inhibitory neurons that apparently form commissural projections; (2) document that all of these cells contain the calcium-binding protein parvalbumin; and (3) indicate that the vast majority of commissurally projecting hilar neurons are neither parvalbumin- nor GABA-immunoreactive.     

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus

There is considerable evidence that pathways of the hippocampus use an excitatory amino acids as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.     

Parvalbumin and calbindin D-28K in the human entorhinal cortex. An immunohistochemical study.

Research is here reported on the distribution of immunoreactivities of the calcium-binding proteins parvalbumin and calbindin D-28K in the entorhinal cortex of normal human brains. Topographically, parvalbumin immunoreactive neurons were only seen in the lateral portion of the rostral entorhinal cortex, in continuity with the adjacent perirhinal cortex. The intermediate and caudal portions gave positive results along the mediolateral extension of the entorhinal cortex. The laminar distribution of parvalbumin immunoreactive neurons was similar throughout the entorhinal cortex. Heavy immunostaining, largely coincident with cell islands, was observed in cells and fibers in layer II, being densest in the deep half of layer III and more sparsely distributed in layers V and VI. Calbindin D-28K immunoreactivity was found throughout the entorhinal cortex. In contrast to parvalbumin immunoreactivity, calbindin D-28K was present from layer I up to upper layer III, the neurons being most numerous in the cell islands of layer II. These results show that rostromedial portions of the human entorhinal cortex contain calbindin immunoreactivity, but not parvalbumin, while the lateral, intermediate and caudal portions of the entorhinal cortex contain both calcium-binding proteins. As it is known that these two proteins belong to a subset of GABAergic neurons, we suggest that a topographical diversity in some of the cells may be responsible for inhibitory effects in the human entorhinal cortex. This proposed diversity might be relevant to the processing of information that the entorhinal cortex conveys to the dentate gyrus and receives from various components of the hippocampus, the subicular complex and other cortical and subcortical sources.     

Expression of the mu-opioid receptor is induced in dentate gyrus granule cells after focal cerebrocortical ischaemia and stimulation of entorhinal afferents

Focal ischaemia in the cerebral cortex affects the inducibility of long-term potentiation (LTP) in the hippocampus. This impairment of hippocampal function may result from excessive activation of cortico-hippocampal afferents and subsequent perturbation of hippocampal LTP-relevant transmitter systems, which include opioids. Here, we tested if permanent focal ischaemia and electrical afferent stimulation influence the expression of the mu-opioid receptor (MOR) in the rat hippocampus. In the applied ischaemia model, the entire ipsilateral cortical hemisphere and hippocampus experienced sustained excitation as indicated by a long-lasting increase in the expression of arg 3.1/arc (ARG) mRNA, a marker for neuronal activity. Expression of MOR mRNA and protein was strongly increased in granule cells, which contain very low MOR levels under normal conditions, but not in gamma-aminobutyric acid (GABA)ergic neurons, which express the MOR constitutively. In the molecular layer, which contains the dendrites of granule cells, focal ischaemia caused a redistribution of MOR-like immunoreactivity. In contrast to the dentate gyrus, MOR expression was unaltered in the hippocampus proper and in non-infarcted cortical areas. Repetitive high-frequency stimulation of cortico-hippocampal perforant path afferents induced strong MOR mRNA expression throughout the granular layer. However, weak tetanization sufficient to induce LTP and ARG expression did not influence MOR mRNA levels. Taken together, we provide direct evidence for the induction of MOR expression in granule cells experiencing sustained excitation by cortical afferents. In activated, MOR-expressing granule cells, inhibitory opioids may counter-regulate glutamatergic excitation by the perforant path.     

Calretinin/PSA-NCAM immunoreactive granule cells after hippocampal damage produced by kainic acid and DEDTC treatment in mouse

There is a dramatic increase in the number of lightly immunoreactive calretinin cells in the granular layer of the dentate gyrus of the mouse hippocampus 1 day after excitotoxic injury using kainic acid combined with the zinc chelator diethyldithiocarbamate. At 7 days after treatment, these cells are strongly immunoreactive for calretinin and for the polysialated form of the glycoprotein neural cell adhesion molecule (PSA-NCAM). The reexpression of calretinin and PSA-NCAM after treatment corresponds well with the loss of input from the damaged hilar mossy cells. These cells could be considered immature granule cells since they are immunoreactive to markers for immature cells such as PSA-NCAM, and are not immunoreactive to calbindin D28k and neuronal nuclear specific protein NeuN (present in mature granule cells), or GABA (present in interneurons). Ultrastructural analysis of these cells indicates that they are immature. Labelling of cell proliferation with 5-bromo-2'-deoxyuridine (BrdU) shows that by day 1 no calretinin immunoreactive cell of the dentate gyrus corresponds to newly generated cells. By day 7 only 6% of the calretinin immunoreactive cells in the dentate gyrus are marked for BrdU. Our data indicate that the CR/PSA-NCAM immunoreactive cells of the dentate gyrus, in spite of their immature characteristics, are not the products of reactive neurogenesis. These cells could represent a reservoir of pre-existing not completely differentiated granule cells that react to damage.