Yusuke Aika

HOW SIMPLE IS THE ORGANIZATION OF THE OLFACTORY GLOMERULUS? : THE HETEROGENEITY OF SO-CALLED PERIGLOMERULAR CELLS

Recent progress in the studies of the olfactory system, especially in the molecular biological studies, makes it one of the useful sensory model systems for understanding neural mechanisms for the information processing. In the olfactory bulb, the primary center of the olfactory system, glomeruli are regarded as important functional units in the transmission of odorant signals and in processing the olfactory information, but have been believed to be composed by only a small number of neuronal types and thus to be simple in their neuronal and synaptic organization. However, accumulating morphological data reveal that each type of neurons might further consist of several different subpopulations, indicating that the organization of glomeruli might not be so simple as it was believed. Here we describe an aspect of the structural organization of glomeruli, focusing on the heterogeneities of periglomerular neurons in mammalian main olfactory bulb.

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.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb—IV. Intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons

Synapses of intraglomerular processes of tyrosine hydroxylase-immunoreactive neurons in the rat main olfactory bulb were examined by electron microscopic immunocytochemistry. Prominent characteristics of intraglomerular synapses of tyrosine hydroxylase-immunoreactive elements were that the vast majority (about 80%) of their synaptic inputs were asymmetrical synapses from olfactory nerve terminals and, though far smaller in proportion, one half of the remaining were asymmetrical synapses from mitral/tufted cell dendrites and the other half were symmetrical synapses from gamma-aminobutyric acid-like immunoreactive elements. So far, we have observed no typical reciprocal synapses between tyrosine hydroxylase-immunoreactive processes and mitral/tufted dendrites; however, we have often identified serial synapses; that is, asymmetrical synapses from olfactory nerve terminals or mitral/tufted cell dendrites to tyrosine hydroxylase-immunoreactive processes, and then symmetrical synapses from the latter to different mitral/tufted cell dendrites. These synaptic connections of tyrosine hydroxylase-immunoreactive neurons were very different from those of Calbindin-D(28k)-immunoreactive neurons, which received no synaptic contact directly from olfactory nerve terminals but formed reciprocal synapses with mitral/tufted cells as we analysed previously.Thus, our present and previous electron microscopic studies combined with confocal laser scanning light microscopy clearly indicated for the first time the heterogeneity of periglomerular neurons, not only in their chemical and morphological features, but also in their synaptic organization in the olfactory glomerulus.

Quantitative analysis of GABAergic neurons in the mouse hippocampus, with optical disector using confocal laser scanning microscope

The numerical densities (NDs) of glutamic acid decarboxylase (GAD) 67 immunoreactive (IR) neurons in the mouse hippocampus were estimated according to the optical disector method using a confocal laser scanning microscope (CLSM), and the cell sizes of disector-counted neurons were measured. Particularly, we focused on the dorsoventral differences of the NDs and cell sizes in individual subdivisions and layers. The NDs of GAD67-IR neurons were larger at the ventral level than at the dorsal level in most subdivisions and layers, except in the stratum pyramidale (SP) of the CA1 region and stratum radiatum (SR) of the CA3 region. In the whole hippocampus, the ND of GAD67-IR neurons was 5.7+/-0.2x103/mm3 at the dorsal level, and 7.3+/-0.3x103/mm3 at the ventral level. The laminar differences showed that the NDs of GAD67-IR neurons in the principal cell layers were generally larger than those in the dendritic layers in each subdivision. The ND of GAD67-IR neurons was largest in the SP of the CA1 region at the dorsal level (13.5+/-0.9x103/mm3), and smallest in the molecular layer (ML) of the dentate gyrus (DG) at the dorsal level (1.7+/-0.2x103/mm3). The mean cell sizes of GAD67-IR neurons also showed prominent dorsoventral and laminar differences. In the CA3 region, the mean cell size of GAD67-IR neurons was smaller at the dorsal level than at the ventral level, while in the DG, it was larger at the dorsal level than at the ventral level. On the other hand, the mean cell size of GAD67-IR neurons in the CA1 region showed no significant dorsoventral difference. In the whole hippocampus, the mean cell size of GAD67-IR neurons was slightly smaller at the dorsal level (somatic profile area 149.2+/-2.5 microm2) than at the ventral level (154.2+/-2.9 microm2). The laminar differences showed that the mean cell sizes of GAD67-IR neurons in the principal cell layers were generally larger than those in the dendritic layers in each subdivision. The mean cell size of GAD67-IR neurons was largest in the SP of the CA3 region at the ventral level (180.7+/-8.7 microm2), and smallest in the stratum lacunosum-moleculare (SLM) of the CA3 region at the dorsal level (115.9+/-7.9 microm2). The cell size distributions in individual layers revealed that GAD67-IR neurons were roughly classified into two subgroups. The composition of these subgroups suggested the heterogeneity of GAD67-IR neurons in the mouse hippocampus in view of cell size

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.

Structure of intraglomerular dendritic tufts of mitral cells and their contacts with olfactory nerve terminals and calbindin-immunoreactive type 2 periglomerular neurons

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. J. Comp. Neurol. 440:219–235, 2001.

Dense GABAergic input on somata of parvalbumin-immunoreactive GABAergic neurons in the hippocampus of the mouse.

GABAergic neurons in the hippocampus proper are greatly diverse in their morphological and physiological features. In the present study we examined whether or not they are also diverse regarding the density of GABAergic input on their somata. GABAergic neurons were immunocytochemically identified with antibodies against glutamic acid decarboxylase (GAD), and the densities of GAD-immunoreactive (GAD-IR) boutons that abutted on GAD-IR somata were estimated by conventional light microscopic, combined light and electron microscopic, and confocal laser scanning microscopic analyses. GAD-IR somata were apparently diverse regarding the density of GABAergic input on them, and those surrounded by higher densities of GAD-IR boutons were distributed mainly in the strata pyramidale and oriens of the CA3 and CA1 regions and could be correlated to a parvalbumin (PV)-IR subpopulation of GABAergic neurons. Quantitative analysis clearly revealed the statistically significant difference between PV-positive and PV-negative GAD-IR neurons in the densities of their somatic GAD-IR boutons. Particularly, most of PV-IR neurons in the CA3 stratum pyramidale as well as some in other layers are characterized by an exceedingly high density of perisomatic GAD-IR boutons. Furthermore, the majority of GAD-IR boutons on PV-IR somata in the stratum pyramidale were also PV-IR. Bilateral transection of the fimbria-fornix, which was supposed to remove GABAergic afferents from the septum, had only partial effects on the densities of PV-IR boutons on PV-IR somata, indicating these PV-IR boutons mainly originated from intrinsic PV-IR neurons. These observations indicate the dense mutual connection between PV-IR GABAergic neurons through perisomatic synaptic contacts, particularly in the stratum pyramidale.

Quantitative analysis of neuronal nitric oxide synthase-immunoreactive neurons in the mouse hippocampus with optical disector

A detailed quantitative analysis of immunocytochemically identified nonprincipal neurons containing neuronal nitric oxide synthase (nNOS) was performed on the mouse hippocampus, with particular reference to the dorsoventral gradient. The present study applied two variations of a stereologic technique, the optical disector—one that used confocal laser‐scanning microscope optical sections to examine colocalization of nNOS and glutamic acid decarboxylase 67 (GAD67), and the other that used conventional thick sections to examine numerical densities (NDs) and cell sizes of nNOS‐immunoreactive (IR) neurons. Colocalization analysis indicated that practically all nNOS‐IR neurons (97.6%) were GAD67‐IR, whereas a part of the GAD67‐IR neurons (about 30%) were nNOS‐IR in the whole hippocampus at both dorsal and ventral levels. The percentages of GAD67‐IR neurons containing nNOS were higher in the dentate gyrus (DG, about 50%), and lower in the Ammons horn (about 20%). Laminar analysis revealed that the majority of GAD67‐IR neurons contained nNOS in the stratum lacunosum‐moleculare of the CA3 region (about 60%) and in the molecular layer of the DG (about 80%). The NDs of nNOS‐IR neurons in the whole hippocampus showed a dorsoventral gradient, which increased from dorsal (1.6 × 103/mm3) to ventral (2.2 × 103/mm3) levels. The NDs were relatively higher in the principal cell layers, where about 40% of nNOS‐IR neurons were situated both in the Ammons horn and DG. The mean cell sizes of nNOS‐IR neurons showed no remarkable laminar differences or dorsoventral gradient in the Ammons horn, but they were extensively larger in the hilus of the DG than in other layers. These results indicate that nNOS‐IR neurons in the mouse hippocampus represent a subpopulation of γ‐aminobutyric acid (GABA)ergic neurons and suggest that the laminar distributions of nNOS‐IR neurons related to possible functional heterogeneity of GABAergic neurons in each hippocampal layer. J. Comp. Neurol. 410:398–412, 1999.

Cathecolaminergic Neurons in the Olfactory Bulb

The olfactory bulb (OB) is an expedient region for analyzing neuronal organization in the central nervous system, as it displays a simple and distinctly laminar cytoarchitecture consisting of a relatively small number of neuron types, but is also so rich in chemical and neuroactive substances located in a variety of neuron types in distinct layers.1

Corrigendum: GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms, GAD67 and GAD65, in the mouse hippocampus: A digitized quantitative analysis. J. Comp. Neurol. 395:177–194.

Fukuda, T., Y. Aika, C.W. Heizmann, and T. Kosaka (1998) GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms, GAD67 and GAD65, in the mouse hippocampus: A digitized quantitative analysis. J. Comp. Neurol. 395:177–194.