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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.

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-microns-thick sections, 3 microns 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 x 10(3)/mm3; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 x 10(3)/mm3, 272.4 x 10(3)/mm3, and 1.9 x 10(3)/mm3, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 x 10(3)/mm3 and 1.1 x 10(3)/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 x 10(3)/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 x 10(3)/mm3, 6.2 x 10(3)/mm3, and 5.4 x 10(3)/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.

Intraglomerular dendritic tufts of Golgi-impregnated and biotinylated dextran amine (BDA)-labeled mitral cells in the rat main olfactory bulb were analyzed in detail. In particular, the relationships of BDA-labeled tufts with olfactory nerve (ON) terminals and processes of calbindin D-28K-immunoreactive (CB-IR) cells were investigated with confocal laser-scanning light microscopic (CLSM) and electron microscopic (EM) analyses. CB-IR cells were type 2 periglomerular cells that restricted their processes in the ON-free (non-ON) zone of the glomerulus and received few synapses from ON terminals. The mitral tufts varied in complexity, but individual branches were rather simple, smooth processes that bore some branchlets and spines and extended more or less in a straight line or a gentle curve rather than winding tortuously within glomeruli as though they did not consider the compartmental organization, which consisted of ON and non-ON zones that interdigitated in a complex manner with one another. Conventional EM analysis revealed that both thin and thick, presumed proximal branches of mitral/tufted cell dendritic tufts received asymmetrical synapses from ON terminals. Correlated CLSM-EM analysis confirmed direct contacts between the BDA- and CB-labeled processes detected in the CLSM examinations, and synapses were recognized at some of those sites. Furthermore, ON terminals and CB-IR processes were distributed on both proximal and distal dendritic branches in a more or less mosaic pattern. These findings revealed that, on the mitral dendritic tufts, ON terminals and processes of type 2 periglomerular neurons were not clearly segregated proximodistally but, rather, were arranged in a mosaic pattern, which may be important in fine tuning the output from individual glomeruli.

Glutamic acid decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthetic enzyme, consists of two isoforms, GAD67 and GAD65. Although distributions of the two GAD isoforms at the somatic level are known to be heterogeneous among different subpopulations of GABAergic neurons, those at the synaptic level have not been investigated. In order to analyze quantitatively the two GAD-isoform immunoreactivities in axon terminals, we combined confocal laser scanning microscopy with digitized image analysis to measure the gray levels of immunofluorescent signals for the two GAD isoforms in a large number of individual boutons in each hippocampal and dentate layer of the mouse. Synaptic boutons exhibited lamina-specific immunoreactivities against the GAD isoforms. Boutons in the principal cell layers (stratum pyramidale of the hippocampus proper and the granule cell layer of the dentate gyrus) showed more intense immunoreactivity against GAD67 than those in the dendritic layers (strata lacunosum-moleculare, radiatum, and oriens of the hippocampus proper and the molecular layer of the dentate gyrus). By contrast, boutons in the dendritic layers showed more intense immunoreactivity against GAD65 than those in the principal cell layers. Such differential distributions could be correlated to the GAD-isoform immunoreactivities in the axon terminals originating from parvalbumin-containing neurons, a particular subpopulation of hippocampal GABAergic neurons mainly innervating the perisomatic domain of principal neurons. In addition to previously reported physiological and pharmacological differences between the GABAergic synapses on perisomatic domain and those on distal dendrites, the present results suggest a functional differentiation of GABAergic synapses between these two inhibitory sites.