T. Kosaka

Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons.

SummaryThe 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-μm-thick sections, 1.5 μm 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-100β-LIR glial cells (astrocytes); and (5) S-100β-negative glial cells (oligodendrocytes and microglia). The densities of total cells, glial cells, and neurons in the rat somatosensory cortex were 85.4 × 103/mm3, 30.5 × 103/mm3, and 54.9 × 103/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 × 103; the number of neurons and glial cells were 110.2 × 103 and 61.4 × 103, respectively. There were 27.7 × 103 GABA-LIR neurons and 15.0 × 103 and 12.7 × 103 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.

Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method, the disector

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-μm-thick sections, 3 μm distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 × 103/mm3; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 × 103/mm3, 272.4 × 103/mm3, and 1.9 × 103/mm3, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 × 103/mm3 and 1.1 × 103/mm3, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV + neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA + and/or PV + neurons was 2.5 × 103/mm3 and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 × 103/mm3, 6.2 × 103/mm3, and 5.4 × 103/mm3, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.

Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling

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 μM, 1 and 2 mM), and calibration curves were deduced from quantitative immunogold analyses of these standard samples. We also analyzed a fast twitch muscle, the superficial part of the gastrocnemius muscle (GCM), whose PV content was previously reported in a biochemical study; the comparison between gold particle densities of GCM and standard samples indicated that these artificial standard samples could be used to estimate the approximate intracellular concentrations of PV. Based on these analyses PV concentrations were estimated as 50-100 μM in Purkinje cell somata and dendrites as well as interneuron somata, and as 1 mM or more in axons and axon terminals of Purkinje cells and basket cells.

Calcium-binding protein parvalbumin-immunoreactive neurons in the rat olfactory bulb

The laminar distribution and morphological features of parvalbumin-immunoreactive [PV(+l)] 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, axonlike 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. PV(+) processes from the fourth and the fifth group of cells appeared to make contacts on mitral cell somata. In the GL some presumably periglomerular cells were also PV(+). In the GRL, PV(+) neurons were small in number, but they were also heterogeneous in their structural features; Some were identified as Golgi cells. This study shows a tremendous heterogeneity in morphological features of a chemically defined subpopulation of GABAergic interneurons in the olfactory bulb.

Synaptic contacts between mitral/tufted cells and GABAergic neurons containing calcium-binding protein parvalbumin in the rat olfactory bulb, with special reference to reciprocal synapses between them

It has been believed so far that in the mammalian olfactory bulb, only granule cells form reciprocal synapses with mitral/tufted cells in the external plexiform layer (EPL). However, the present electron microscopic study demonstrated for the first time that another group of immunocytochemically defined interneurons different from granule cells, viz., neurons containing a specific calcium-binding protein parvalbumin, also made reciprocal synapses with mitral/tufted cells in the EPL.

Distribution of the calcium binding proteins, calbindin D-28K and parvalbumin, in the subicular complex of the adult mouse

The immunohistochemical localizations of two specific calcium binding proteins, calbindin D-28K (calbindin) and parvalbumin (PV) were examined in the subicular complex, that is, the subiculum, presubiculum, and parasubiculum, of the adult mouse and were compared in detail with staining pattern of the acetylcholinesterase (AChE) histochemistry. The calbindin immunoreactivity exhibited a conspicuous regional and laminar pattern of distribution, which somewhat resembled the AChE staining pattern but was apparently different from the latter in various points. The PV immunoreactivity also exhibited a characteristic regional difference, although less prominent. The subiculum could be divided into two subregions, intensely calbindin-immunoreactive (calbindin-IR) and AChE stained proximal subiculum and only faintly calbindin-IR and AChE stained distal subiculum. In the subiculum most of calbindin-IR neurons were pyramidal cells which were clustered in the superficial half of the cell layer in the proximal subiculum and appeared to be segregated from calbindin negative pyramidal cells located in the distal subiculum and in the basal part of the proximal subiculum. In the presubiculum calbindin-IR neurons were clustered in layer 2, most of which were supposed to be presubicular pyramidal cells. In the parasubiculum, the overall immunostaining pattern of PV and calbindin were somewhat complementary. In the transition area calbindin-IR neurons were clustered but few PV-IR neurons were located, and thus the distribution of immunoreactive neuronal somata was apparently different from the adjacent parts of the parasubiculum, indicating that the transition area might be a separate entity. In addition to calbindin-IR presumable principal neurons, calbindin-IR and PV-IR nonpyramidal cells were scattered throughout the subicular complex. Furthermore, these two calcium binding proteins were colocalized in some nonpyramidal cells in the subicular complex. The present study revealed some new aspects of the areal and laminar organization of the subicular complex, which had not been shown by previous classical purely morphological approaches.

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis

SummaryIn 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 on 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.

Neurobiological applications of high voltage electron microscopy

Abstract The high penetration power of electrons at a very high accelerating voltage enables the examines of thick biological specimens. This property of the high voltage electron microscope is especially useful for the morphological study of neurons and glial cells with complicated terminal arborizations. The present paper deals with the basic procedure and some applications of high voltage electron microscopy of Golgi impregnated materials. High voltage electron microscopy can also be effectively used in autoradiographic studies with remarkable reduction in exposure time.

Regional difference in the distribution of parvalbumin-containing neurons immunoreactive for monoclonal antibody HNK-1 in the mouse cerebral cortex

Using preembedding immunocytochemistry at light microscopic level, we found that, although monoclonal antibody HNK-1 selectively outlined a subpopulation of GABAergic neurons containing a specific calcium-binding protein parvalbumin (PV) in the adult mouse cerebral cortex, the proportion of HNK-1-positive cells to PV-containing cells showed prominent regional difference. In the parietal cortex, approximately 50% of PV-positive cells were HNK-1-positive whereas only approximately 10% of PV-positive cells were HNK-1-immunoreactive in the occipital and temporal cortices. There were also prominent differences in this proportion among allocortical areas. These observations indicate that the cellular composition of chemically defined subpopulations of GABAergic neurons are different from area to area in mouse cerebral cortex.