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The laminar development of the external plexiform layer (EPL) in the rat main olfactory bulb and the postnatal development of parvalbumin-immunoreactive [PV(+)] neurons mainly located in this layer were studied in animals at postnatal week 1-4 at a light microscopic level. The EPL in the adult olfactory bulb consists of two sublayers, the inner sublayer (ISL) and the outer sublayer (OSL). The ISL was already developed well even at postnatal day 7 (P7), whereas the OSL was first recognized at P10 as a thin zone consisting of more or less loosely packed large-sized and small-to-medium-sized somata subjacent to the glomerular layer (GL). The OSL increased in thickness and came to occupy nearly one-third to -half of the EPL at P14. PV(+) neurons first appeared at P10 mainly in the inner border of EPL. Only a few PV(+) neurons were scattered in the EPL at P10, but they increased remarkably in number during P14-21. Some of these PV(+) neurons at P10 had an intensely immunoreactive soma, extending relatively long processes with varicosities and/or spines. At P14, PV(+) neurons were located not only in the ISL but also at the border between the ISL and OSL, but in the OSL proper they were rarely observed. These PV(+) neurons showed branched and complicated processes with numerous varicosities and spines, displaying more mature features than those in previous stages. Even at P14 many of these PV(+) neurons appeared to exhibit some characteristic structural features of those in the adult stage.(ABSTRACT TRUNCATED AT 250 WORDS)

The laminar distribution and morphological features of parvalbumin-immunoreactive [PV(+)] neurons, one of the subpopulations of GABAergic neurons, were studied in the rat olfactory bulb at a light microscopic level. In the main olfactory bulb of adult rats, PV(+) neurons were mainly located in the external plexiform layer (EPL), and a few were scattered in the glomerular layer (GL), mitral cell layer (ML), and granule cell layer (GRL); whereas PV(+) neurons were rarely seen in the accessory olfactory bulb. The inner and outer sublayers of the EPL (ISL and OSL) appeared to be somewhat different in the distribution of PV(+) somata and features of PV(+) processes. PV(+) somata were located throughout the OSL, and PV(+) processes intermingled with one another, making a dense meshwork in the OSL; whereas, in the ISL, PV(+) somata were mainly located near the inner border of the EPL, and PV(+) processes made a sparser meshwork than that in the OSL. PV(+) neurons in the EPL were apparently heterogeneous in their structural features and appeared to be classifiable into several groups. Among them there appeared five distinctive types of PV(+) neurons. The most prominent group of PV(+) neurons in the OSL were superficial short-axon cells, located in the superficial portion of this sublayer and giving rise to relatively thick processes, in horizontal or oblique directions, which usually bore spines and varicosities. Another prominent group of PV(+) neurons extended several short, branched dendrites with spines and varicosities, which appeared to intermingle with one another, making a relatively small, spherical or ovoid dendritic field around the cell bodies; most of them resembled Van Gehuchten cells reported in previous Golgi studies. A third distinctive and most numerous group of PV(+) neurons were of the multipolar type; their somata and processes were located throughout the EPL. Their relatively smooth processes with frequent varicosities and a few spines were extended horizontally or diagonally throughout the EPL. A fourth group, which could be a subtype of the multipolar type, were located in or just above th ML and extended several thin, smooth dendrites in the EPL, some of which appeared to reach the border between the GL and EPL. Occasionally, axon-like processes arose from their cell bodies and extended into the ML. This fourth type of PV(+) neuron was named inner short-axon cells. A fifth group of neuron was located in the ML; processes of these neurons were extended horizontally, so they were named inner horizontal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The immunointensities of calcium-binding proteins parvalbumin (PV) and calbindin D28K were quantified in different parts of Purkinje cells and interneurons (basket cells and stellate cells) of the rat cerebellum. An electron microscopic, postembedding immunogold procedure on Lowicryl K4M-embedded thin sections was applied. Neuronal profiles were identified by double-labeling immunocytochemistry using the combination of the two primary antibodies, mouse monoclonal anti-rat calbindin D28K and rabbit polyclonal anti-rat PV. The secondary antibodies were conjugated with colloidal gold of different sizes (10 and 15 nm diameter). In the cerebellar cortex, double-labeled profiles were identified as Purkinje cells and profiles labeled only with anti-PV were identified as inteneurons. The densities of gold particles were used for statistical comparison of the relative levels of PV and calbindin D28K in somata, dendrites, dendritic spines, axons and axon terminals of Purkinje cells, and interneurons. The axons and axon terminals of Purkinje cells and basket cells had significantly higher levels of PV immunoreactivity than Purkinje cell somata, primary, secondary, and tertiary dendrites, and dendritic spines, as well as interneuron somata. On the other hand, the present study could not determine conclusively whether calbindin D28K was distributed homogeneously throughout soma, dendrites, and axons of Purkinje cells or was also concentrated in Purkinje cell axons. To estimate absolute PV concentrations, we made a series of artificial standard samples which were aldehyde-fixed 10% bovine serum albumin containing given concentrations of PV (0, 12.5, 25, 50, 100, 200, and 400 microM, 1 and 2 mM), and calibration curves were deduced from quantitative immunogold analyses of these standard samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody (MAb) 473 is shown to outline selectively a subpopulation of GABAergic neurons containing a specific calcium-binding protein parvalbumin (PV) in the adult rat parietal cortex, using preembedding immunocytochemistry at the light microscopic level. About 90% of MAb 473 stained cells in the rat parietal cortex were PV immunoreactive. Thus we compared MAb 473 staining with that of three chemical probes, previously shown to stain selectively a subpopulation of PV-containing GABAergic neurons in this brain region, namely, a lectin, Vicia villosa agglutinin (VVA), with a specific affinity for terminal N-acetyl-galactosamine, MAb 3B3 which is specific for chondroitin sulfate proteoglycan and MAb HNK-1 which is specific for some types of carbohydrate epitope containing a sulfated derivative of glucuronic acid. About 85% of MAb 473 immunoreactive cells were shown to be stained with VVA. Furthermore about 90% of MAb 473 immunoreactive cells were also stained with MAb 3B3. Thus MAb 473 positive cells were almost included into VVA and/or MAb 3B3 positive cells. On the other hand only about 34% of MAb 473 positive cells were HNK-1 positive, whereas about 44% of HNK-1 positive cells were MAb 473 positive. Thus these two MAbs defined different, though partially overlapping, subsets of PV-containing GABAergic neurons in the rat parietal cortex.

In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects of olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) - 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) - 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) - 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70-80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) - 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) - 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.

The number of neuronal and glial cells in the rat somatosensory cortex (barrel area) has been estimated by a stereological method, the disector, using pairs of toluidine blue-stained, plastic-embedded 0.5-microns-thick sections, 1.5 microns distant from each other. Chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent semithin sections. Thus we were able to analyze quantitatively number, distribution, and proportion of five cell types: (1) gamma-aminobutyric acid-(GABA)-negative neurons; (2) GABA-like immunoreactive (GABA-LIR) neurons; (3) a specific calcium-binding protein parvalbumin-immunoreactive (PV-IR) neurons, a subpopulation of GABA-LIR neurons; (4) S-100 beta-LIR glial cells (astrocytes); and (5) S-100 beta-negative glial cells (oligodendrocytes and microglia). The densities of total cells, glial cells, and neurons in the rat somatosensory cortex were 85.4 +/- 10(3)/mm3, 30.5 x 10(3)/mm3, and 54.9 x 10(3)/mm3, respectively. Of all neurons 25% and 14% were GABA-LIR and PV-IR, respectively; all PV-IR neurons are GABA-LIR, and thus about 54% of GABA-LIR neurons are PV-positive. The number of total cells under a unit surface area of 1 mm2 through the thickness of the somatosensory cortex was 171.6 x 10(3); the number of neurons and glial cells were 110.2 x 10(3) and 61.4 x 10(3), respectively. There were 27.7 x 10(3) GABA-LIR neurons and 15.0 x 10(3) and 12.7 x 10(3) PV-IR neurons and PV-negative GABA-LIR neurons, respectively. The laminar distribution of each group of cells shows prominent differences, indicating that the cellular composition was different from layer to layer. The density of GABA-LIR neurons was highest in layer IV. The numerical density of PV-IR neurons was 2-4 times higher in layer IV than in layers II/III, V, and VI, whereas that of PV-negative GABA-LIR neurons was almost constant throughout the layers.

Immunocytochemistry revealed that in the cat dorsal lateral geniculate nucleus (dLGN) almost all parvalbumin-positive cells are GABAergic and about 56% of the calbindin D-28K (calbindin-immunoreactive neurons are also GABA-positive. On the other hand, in the same nucleus, almost all GABAergic neurons contain parvalbumin, and about 89% of the GABA-immunoreactive neurons contain calbindin. Double-labeling with calbindin and parvalbumin revealed that approximately 50% of the immunoreactive neurons are double-stained. In the PGN, virtually all neurons are GABA and parvalbumin-positive. Only a few scattered cells were also calbindin-immunoreactive. These results show that GABAergic geniculate cells can be differentiated on the basis of their calcium-binding protein immunoreactivity. Four types of immunoreactive cells are described here: (1) cells positive for GABA, parvalbumin and calbindin, (2) cells positive for GABA and parvalbumin, but negative for calbindin, (3) cells negative for GABA and parvalbumin, but positive for calbindin, (4) cells negative for GABA, parvalbumin and calbindin.

In the cat visual cortex, almost all parvalbumin-positive cells are GABAergic, and about 80% of the calbindin D-28K-positive neurons are also GABA-immunoreactive. About 37% of the GABAergic neurons contain parvalbumin and a smaller fraction (about 18%) contains calbindin. Furthermore, parvalbumin and calbindin are localized in two separate neuronal populations in the cat visual cortex, suggesting that two GABAergic populations can be distinguished, one containing parvalbumin and one containing calbindin. Double staining for parvalbumin and neuropeptides (CCK, SRIF and NPY), revealed no double-labeled cells, with the exception of a few SRIF- and parvalbumin-positive neurons. These results show that cortical GABAergic cells can be differentiated on basis of their calcium binding protein and neuropeptide immunoreactivity.

Monoclonal antibody HNK-1 is shown to outline selectively a subpopulation of GABAergic neurons containing a specific calcium-binding protein parvalbumin (PV) in the adult rat parietal cortex, using pre- and postembedding immunocytochemistry at light microscopic level. About 98% of HNK-1 stained cells in the rat parietal cortex were PV immunoreactive. About 95% of HNK-1 immunoreactive cells were also shown to be stained with a lectin, Vicia villosa agglutinin (VVA), with a specific affinity for terminal N-acetylgalactosamine, which has been previously shown to stain selectively a subpopulation of PV-containing GABAergic neurons in this region. Furthermore almost all HNK-1 immunoreactive cells were also stained with a monoclonal antibody, 3B3, which is specific for chondroitin sulfate proteoglycan. 3B3 was shown in the present study to stain selectively a subpopulation of PV-immunoreactive neurons in the adult rat parietal cortex. In addition, a direct comparison of two monoclonal antibodies HNK-1 and VC1.1 revealed that these two were identical in their staining properties and that they defined the same subset of PV-containing GABAergic neurons in the rat parietal cortex.

Parvalbumin mRNA was localized in rat brain by in situ hybridization using a 35S labelled rat parvalbumin cDNA and a synthetic oligodeoxyribonucleotide (corresponding to base sequences 140 to 183 of rat parvalbumin cDNA). Strongest hybridization signals were detected in the Purkinje cells of the cerebellum and in neurones of the reticular nucleus of the thalamus. Signal was also detected in the cerebral cortex, hippocampus, basal ganglia and brain stem in agreement with the distribution of parvalbumin immunoreactivity.

Monoclonal antibody VC1.1 is shown to stain selectively a subpopulation of GABAergic neurons in the rat cerebral cortex. Almost all VC1.1 immunoreactive cells were also GABA-like immunoreactive (GABA-LI) and parvalbumin (PV) immunoreactive, whereas they were about 30% and 65% of GABA-LI and PV-positive cells in the parietal cortex and about 13% and 32% in the occipital cortex, respectively. Although a few VC1.1 positive cells showed somatostatin-like and/or cholecystokinin-like immunoreactivities, they were exceptional (less than 1% of VC1.1 positive cells). Furthermore about 90% of VC1.1 positive cells were also stained with a lectin, Vicia villosa agglutinin, with a specific affinity for terminal N-acetylgalactosamine.

Calcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurons show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Gap 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 on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 micron 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.

The 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.