Toshio Kosaka

GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus

The distribution of Ca2+-binding protein, parvalbumin (PV), containing neurons and their colocalization with glutamic acid decarboxylase (GAD) were studied in the rat hippocampus and dentate gyrus using immunohistochemistry. PV immunoreactive (PV-I) perikarya were concentrated in the granule cell layer and hilus in the dentate gyrus and in the stratum pyramidale and stratum oriens in the CA3 and CA1 regions of the hippocampus. They were rare in the molecular layer of the dentate gyrus, in the stratum radiatum and in the stratum lacunosum-moleculare of the hippocampus. PV-I axon terminals were restricted to the granule cell layer, the stratum pyramidale and the immediately adjoining zones of these layers. Almost all PV-I neurons were also GAD immunoreactive (GAD-I), whereas only about 20% of GAD-I neurons also contained PV. The percentages of GAD-I neurons which were also immunoreactive for PV were dependent on the layer in which they were found; i.e. 40-50% in the stratum pyramidale, 20-30% in the dentate granule cell layer and in the stratum oriens of the CA3 and CA1 regions, 15-20% in the hilus and in the stratum lucidum of CA3 region and only 1-4% in the dentate molecular layer and in the stratum radiatum and the stratum lacunosum-moleculare of the CA3 and CA1 regions. PV-I neurons are a particular subpopulation of GABAergic neurons in the hippocampal formation. Based on their morphology and laminar distribution, they probably include basket cells and axo-axonic cells.

Fast-spiking cells in rat hippocampus (CA1-region) contain the calcium-binding protein parvalbumin

Fast spiking cells in the CA1 region of the rat hippocampus were revealed as gamma-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with postembedding parvalbumin immunohistochemistry.

Structural and quantitative analysis of astrocytes in the mouse hippocampus

We revealed the structural features of astrocytes by means of light microscopy, confocal laser scanning microscopy and high voltage electron microscopy, and estimated their numerical densities in the mouse hippocampus. The high voltage electron microscope examinations of Golgi-impregnated astrocytes clearly disclosed their fine leaflet-like processes in the masses occupied by individual astrocytes. The intracellular injection of two different fluorescent tracers into two neighboring astrocytes revealed that each astrocyte occupied a discrete area with a limited overlap only at its peripheral portion. In a quantitative analysis using an optical dissector, the numerical densities of astrocytes identified as S100-immunoreactive cells were only slightly different in their areal and laminar distributions. The numerical densities were higher in the stratum lacunosum-moleculare and dentate hilus, while they were slightly lower in the principal cell layers than the average (24.2 x 10(3) mm(-3)) in whole hippocampal regions. As for the dorsoventral difference, the numerical densities were significantly larger at the ventral level in the dentate gyrus, whereas such tendency was not apparent in the hippocampus proper. The projection area of the astrocytes estimated from Golgi-impregnated samples was roughly in inverse relation to the numerical densities; the areas in the stratum lacunosum-moleculare were somewhat smaller than the other layers, where the numerical densities were high. The present study indicates that astrocytes are distributed rather evenly without any prominent areal or laminar differences and that the individual astrocytes have their own domains; the periphery of the domain of a given astrocyte is interdigitated intricately with the processes of adjacent astrocytes whereas its inner core portion is not penetrated by them.

Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus.

SummaryThe structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic cells.

Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter-synthesizing enzymes in the central nervous system

Aldehyde fixatives containing high concentrations of glutaraldehyde, usually used for conventional electron microscope studies, were successfully used for immunocytochemistry of transmitter synthesizing enzymes, glutamate decarboxylase and tyrosine hydroxylase, in the rat central nervous system. Although a high concentration of glutaraldehyde could cause tremendous non-specific staining, this was almost completely absent after treating sections with 1% sodium borohydride for 30 min. Furthermore, it was shown that a high concentration of glutaraldehyde might cause no appreciable reduction of the antigenicities of glutamate decarboxylase and tyrosine hydroxylase when compared with fixatives containing a low concentration of glutaraldehyde. It is suggested that fixatives containing high concentrations of glutaraldehyde are very useful, not only for conventional electron microscope studies, but also for light and electron microscope immunocytochemistry of some antigens, including glutamate decarboxylase and tyrosine hydroxylase.

Coexistence of immunoreactivities for glutamate decar☐ylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: possible coexistence of gamma-aminobutyric acid (GABA) and dopamine

The coexistence of immunoreactivities for glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH) was revealed in some neurons in the periglomerular region and in the superficial part of the external plexiform layer of the rat main olfactory bulb. In neurons showing the immunoreactivity for either GAD or TH, about 10-55% showed both immunoreactivities.

Gap junctions among dendrites of cortical GABAergic neurons establish a dense and widespread intercolumnar network

Gap junctions are common between cortical GABAergic interneurons but little is known about their quantitative distribution along dendritic profiles. Here, we provide direct morphological evidence that parvalbumin-containing GABAergic neurons in layer 2/3 of the cat visual cortex form dense and far-ranging networks through dendritic gap junctions. Gap junction-coupled networks of parvalbumin neurons were visualized using connexin36 immunohistochemistry and confocal laser-scanning microscopy (CLSM). The direct correspondence of connexin36-immunopositve puncta and gap junctions was confirmed by examining the same structures in both CLSM and electron microscopy. Single parvalbumin neurons with large somata (≥200 μm2) formed 60.3 ± 12.2 (mean ± SD) gap junctions with other cells whereby these contacts were not restricted to proximal dendrites but occurred at distances of up to 380 μm from the soma. In a Sholl analysis of large-type parvalbumin neurons, 21.9 ± 7.9 gap junctions were within 50 μm of the soma, 21.7 ± 7.6 gap junctions in a segment between 50 and 100 μm, 11.2 ± 4.7 junctions between 100 and 150 μm, and 5.6 ± 3.6 junctions were in more distal segments. Serially interconnected neurons could be traced laterally in a boundless manner through multiple gap junctions. Comparison to the orientation-preference columns revealed that parvalbumin-immunoreactive cells distribute randomly whereby their large dendritic fields overlap considerably and cover different orientation columns. It is proposed that this dense and homogeneous electrical coupling of interneurons supports the precise synchronization of neuronal populations with differing feature preferences thereby providing a temporal frame for the generation of distributed representations.

Selective staining of a population of parvalbumin-containing GABAergic neurons in the rat cerebral cortex by lectins with specific affinity for terminal N-acetylgalactosamine.

Lectins with specific affinity for terminal N-acetylgalactosamine, Vicia villosa agglutinin (VVA) and Glycine max agglutinin (soybean agglutinin; SBA), are shown to stain selectively a subpopulation of GABAergic neurons in the rat cerebral cortex. About 90% of VAA- and/or SBA-stained cells are also parvalbumin-immunoreactive, but no VVA-stained cells showed somatostatin-28-like or cholecystokinin-8-like immunoreactivities.

Distribution of calretinin immunoreactivity in the mouse dentate gyrus

Calretinin containing elements were visualized with immunocytochemistry in the adult mouse dentate gyrus (DG). In the ventral DG calretinin immunoreactive (CR-IR) large multipolar cells were clustered; they extended between two and four thick cylindrical dendrites which further branched into several thinner processes. Characteristic grape like spiny appendages were occasionally observed on these thick and thinner dendritic processes. On the basis of these structural features these large CR-IR cells were identified as hilar mossy cells. At the supragranular zone a dense CR-IR band was seen, where numerous CR-IR punctae and fibers were packed tightly among putative granule cell dendrites. In the granule cell layer, especially at the dorsal DG, numerous faintly CR-IR cells were located at the interface with the hilus. They were triangular in shape and neither calbindin D28k nor GABA positive, but were immunoreactive for highly polysialylated neural cell adhesion molecule (NCAM-H) and thus considered as newly generated neurons. In the molecular layer CR-IR cells were also scattered; they were mainly located near the pial surface and the hippocampal fissure, small in size, ovoid in shape and usually gave rise to one very thin axon like and one thin cylindrical dendritic process. These cells were assumed to be Cajal-Retzius cells. Throughout the layers, that is, the molecular layer, the granule cell layer and the hilus, CR-IR multipolar and/or fusiform cells were encountered. They resembled those reported in the rat DG in their structural features and usually extended smooth or varicose or sparsely spiny dendritic processes; some of them were confirmed to be GABA-like immunoreactive and/or glutamic acid decarboxylase immunoreactive. The present study showed that CR immunoreactivity in the mouse DG differed significantly from that in the rat and monkey dentate gyri reported previously.

Gap junctions on GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus (CA1 region)

SummaryGap junctions were identified for the first time on chemically defined neurons in the central nervous system. Gap junctions were thus demonstrated on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 μm thick) sections were cut alternately and consecutively from osmium-fixed tissue which was embedded in epoxy resin and usable for conventional electron microscopic studies. The semithin sections were processed for postembedding immunocytochemistry using an anti-PV serum. Structures corresponding to the PV-immunoreactive (PV-I) profiles on the semithin sections were easily identified on electron micrographs from the adjacent thin sections. Using this technique gap junctions were found (1) between PV-I dendrites, (2) between PV-I dendrites and PV-I somata and (3) between PV-I dendrites and small processes whose origin could not be identified. Despite a systematic search, we did not find gap junction between PV-negative processes.