Heterogeneity of tyrosine hydroxylase expressing neurons in the main olfactory bulb of the mouse.

The structural features of dopamine (DA)-GABAergic neurons in the mouse main olfactory bulbs were examined, using both wild type and transgenic TH-GFP mice, with the combination of several methods; the immunocytochemistry, biotinylated dextran amine labeling, lucifer yellow injection in fixed slices, biocytin injection in live slice and the functional olfactory deprivation. DA-GABAergic neurons were clustered in the glomerular layer (GL) but they also scattered in other layers. DA-GABAergic juxtaglomerular neurons, were classified into 5 groups based on their structural features and named as follows: 1) Large periglomerular (LPG) cells with tuft-like glomerular dendritic branches and apparent axons extending to the distant glomeruli, which correspond to the " inhibitory juxtaglomerular association (IJGA) neurons" participating in the interglomerular association system. 2) Small periglomerular (SPG) cells including both axonic and anaxonic ones; the axonic SPG cells might correspond to the classical periglomerular cells. 3) Transglomerular cells extending dendritic processes spanning 2 or more glomeruli. 4) Incrusting cells extending their dendritic branches mainly in the periphery of the glomeruli. 5) Other various neurons not-yet classified. In the layers other than the GL various types of TH expressing neurons were scattered; some of them extended dendritic processes into the GL.

Calcium-binding protein, secretagogin, specifies the microcellular tegmental nucleus and intermediate and ventral parts of the cuneiform nucleus of the mouse and rat.

Secretagogin (SCGN) is a recently discovered calcium binding protein of the EF hand family, cloned from ß cells of pancreatic island of Langerhans and endocrine cells of the gastrointestinal gland. SCGN characterizes some particular neuron groups in various regions of the nervous system and is considered as one of the useful neuron subpopulation markers. In the present study we reported that SCGN specifically labelled a particular neuronal cluster in the brainstem of the mice and rats. The comparison of the SCGN immunostaining with the choline acetyltransferase immunostaining and acetylcholinesterase staining clearly indicated that the particular cluster of SCGN positive neurons corresponded to the microcellular tegmental nucleus (MiTg) and the ventral portion of the cuneiform nucleus (CnF), both of which are components of the isthmus. The analyses in mice indicated that SCGN positive neurons in the MiTg and CnF were homogeneous in size and shape, appearing to compose a single complex: their somata were small comparing with the adjacent cholinergic neurons in the pedunculotegmantal nucleus, 10.5 vs 16.0 µm in diameter, and extended 2-3 slender smooth processes. SCGN might be one of significant markers to reconsider the delineations of the structures of the mouse, and presumably rat, brainstem.

Calcium-binding protein, secretagogin, characterizes novel groups of interneurons in the rat striatum.

In the rat striatum numerous secretagogin (SCGN) positive neurons were scattered. They were heterogeneous in their morphological and chemical properties. We examined the colocalization of SCGN with known four interneuron markers, parvalbumin (PV), calretinin (CR), nitric oxide synthase (NOS) and choline acetyl transferase (ChAT). 60-70% of SCGN positive striatal neurons contained either PV or CR or ChAT, but none contained NOS. On the other hand the remaining 30-40% expressed none of these markers, most of which were GAD positive. The present study indicates that there are hitherto unknown groups of striatal interneurons in the rat striatum.

Neuronal organization of the main olfactory bulb revisited.

The main olfactory bulb is now one of the most interesting parts of the brain; firstly as an excellent model for understanding the neural mechanisms of sensory information processing, and secondly as one of the most prominent sites whose interneurons are generated continuously in the postnatal and adult periods. The neuronal organization of the main olfactory bulb is fundamentally important as the basis of ongoing and future studies. In this review we focus on four issues, some of which appear not to have been recognized previously: (1) axons of periglomerular cells, (2) the heterogeneity and peculiarity of dopamine-GABAergic juxtaglomerular cells, (3) neurons participating in the interglomerular connections, and (4) newly found transglomerular cells.

Secretagogin-containing neurons in the mouse main olfactory bulb.

Secretagogin (SCGN) is a recently discovered calcium binding protein of the EF hand family. We studied the structural features of SCGN-positive neurons in the mouse main olfactory bulb (MOB). SCGN-positive neurons were localized throughout layers but clustered in the glomerular layer (GL), mitral cell layer (MCL) and granule cell layer (GCL). They were heterogeneous, including numerous juxtaglomerular neurons, granule cells, small to medium-sized neurons in the external plexiform layer (EPL), and a few small cells in the ependymal/subependymal layer. Calretinin and/or tyrosine hydroxylase occasionally colocalized in SCGN-positive juxtaglomerular neurons. Calretinin also frequently colocalized in SCGN-positive EPL and GCL neurons. Morphologically some of juxtaglomerular SCGN-positive neurons were classical periglomerular cells, whereas others were apparently different from those periglomerular cells, although they were further heterogeneous. Some extended one slender process into a glomerulus which passed the glomerulus and further penetrated into another nearby glomeruli, and thus their dendritic processes spanned two or three or more glomeruli. We named this type of juxtaglomerular neurons "transglomerular cells." With the stereological analysis we estimated total number of juxtaglomerular SCGN-positive neurons at about 80,000/single MOB. The present study revealed the diversity of SCGN-positive neurons in the mouse MOB and their particular structural properties hitherto unknown.

Further characterization of the juxtaglomerular neurons in the mouse main olfactory bulb by transcription factors, Sp8 and Tbx21.

Juxtaglomerular neurons in the mouse main olfactory bulb consist of various types of neurons, especially classified by their chemical properties such as transmitter-related molecules and calcium binding proteins. In addition several transcription factors have been revealed to characterize neuronal subpopulations. In this study we examined the immunoreactivities of two transcription factors, Sp8 and Tbx21, in the juxtaglomerular neuronal subpopulations containing calretinin, calbindin, secretagogin, tyrosine hydroxylase (TH) and nitric oxide synthase (NOS). Both Sp8 and Tbx21 immunoreactivities were so diverse in their staining intensities. Almost all calretinin and secretagogin positive neurons were relatively strongly Sp8 positive, whereas none of calbindin positive neurons were Sp8 positive. TH positive neurons were also usually Sp8 positive, although some were faintly positive. These four types of interneurons were Tbx21 negative. On the other hand large faintly NOS positive external tufted cells were occasionally Tbx21 positive but always Sp8 negative, whereas small NOS positive periglomerular cells without distinctly stained dendrites were usually Sp8 positive and Tbx21 negative. Strangely, most of strongly NOS positive periglomerular cells with distinctly stained dendritic processes were Sp8 negative and Tbx21 negative. Thus Sp8 and Tbx21 immunoreactivities further characterized juxtaglomerular neurons and, especially confirmed the heterogeneity of NOS positive juxtaglomerular neurons.

"Interneurons" in the olfactory bulb revisited.

The main olfactory bulbs (MOBs) are now one of the most interesting parts of the brain in at least two points; the first station of the olfaction as an excellent model for understanding the neural mechanisms of sensory information processing and one of the most prominent sites whose interneurons are generated continuously in the postnatal and adult periods. Here we point out some new aspects of the MOB organization focusing on the following 4 issues: (1) there might be both axon-bearing and anaxonic periglomerular cells (PG cells), (2) most parvalbumin positive medium-sized neurons in the external plexiform layer as well as a few nitric oxide synthase positive PG cells and calretinin positive granule cells are anaxonic but display dendritic hot spots with characteristics of axon initial segments, (3) some of so-called "short-axon cells" project to the higher olfactory related regions and thus should be regarded as "nonprincipal projection neurons" and (4) tyrosine hydroxylase positive GABAergic (DA-GABAergic) juxtaglomerular neurons (JG neurons) are a particular type of JG neurons as a main source of the interglomerular connection, forming an intrabulbar association system.

Heterogeneity of calbindin-containing neurons in the mouse main olfactory bulb: I. General description.

The structural features of calbindin-positive neurons were studied in the mouse main olfactory bulb (MOB). Calbindin-positive neurons were heterogeneous, including numerous periglomerular cells, a few granule cells, small to medium-sized interneurons in the external plexiform layer, and large short-axon cells located in the external plexiform layer, internal plexiform layer, granule cell layer and ependymal/subependymal layer. These large short-axon cells were also heterogeneous; some corresponded to classically identified short-axon cells such as Blanes cells, Golgi cells, horizontal cells and vertical cells, but some others appeared to be previously unidentified. A few faintly calbindin-positive presumed tufted cells were also encountered. Near the ependymal/subependymal layer of the MOB some calbindin-positive short-axon cells extended their dendritic processes more or less parallel to the sagittal plane, presumably corresponding to medullary cells named recently. In addition we encountered a few calbindin-positive horizontal cells in the internal plexiform layer extending their axons toward the lateral olfactory tract, one of which was confirmed to extend its axon into the lateral olfactory tract, indicating that they were presumed to be one of projection neurons. The present study revealed the diversity of calbindin-positive neurons in the mouse MOB and their particular structural properties hitherto unknown.

Distinct domanial and lamellar distribution of clustered lipofuscin granules in microglia in the main olfactory bulb of young mice.

Lipofuscin granules are generally considered as age-pigment. However, we encountered numerous large irregular clusters of lipofuscin granules in the olfactory nerve layer and glomerular layer of the main olfactory bulb (MOB) of young adult and even juvenile mice of C57BL/6J strain. Those numerous autofluorescent irregular lipofuscin granules were contained in the cytoplasm of microglial cells. Importantly they showed a prominent pattern of distribution; that is, they were rather restricted to the OCAM positive ventro-lateral domain (V-domain) of the MOB but few in the OCAM negative dorso-medial domain (D-domain), even when microglia distributed rather homogeneously in both OCAM positive V-domain and OCAM negative D-domain. Those lipofuscin granules were not seen in MOBs of 10 days and 2w old C57BL mice, but usually encountered in the MOBs of 3w old mice. Similar clusters of lipofuscin granules in the olfactory nerve layer and glomerular layer were also encountered in BALB/c strain, and, although less prominent, in ICR and ddY strains. However, they were not encountered in young adult rats of three strains, Wistar, Sprague-Dawley and Long-Evans, indicating one of prominent species differences between mice and rats.

Two types of tyrosine hydroxylase positive GABAergic juxtaglomerular neurons in the mouse main olfactory bulb are different in their time of origin.

Tyrosine hydroxylase (TH) positive juxtaglomerular neurons in the olfactory bulb consist of at least two groups with different soma sizes. On the other hand TH positive neurons are known to be generated continuously in the postnatal and even adult periods. We investigated the birth dates of small- and large-sized TH positive juxtaglomerular neurons. 5-Bromo-2'-deoxyuridine (BrdU) was injected at various developmental and juvenile stages and BrdU-labeled neurons were examined in adult mice. We revealed that both small- and large-sized TH positive neurons were generated at embryonic and perinatal periods and that TH positive neurons generated at the juvenile period were small-sized but not large-sized juxtaglomerular cells.

Sodium channel cluster, betaIV-spectrin and ankyrinG positive "hot spots" on dendritic segments of parvalbumin-containing neurons and some other neurons in the mouse and rat main olfactory bulbs.

Axon initial segments (AISs) and nodes of Ranvier are considered as the sites for spike generation, which are highly enriched in sodium channels and some cytoskeletal molecules such as ankyrinG, betaIV-spectrin. Previously, we showed that most parvalbumin positive cells in the external plexiform layer (EPL) of the mouse main olfactory bulb (MOB) were anaxonic but displayed some patch-like betaIV-spectrin and sodium channel cluster positive segments on their dendrites. In this study we further characterized those particular dendritic segments. AnkyrinG was also located there, whereas phospho-IkappaBalpha was not. Electron-microscopically those dendritic segments displayed the membrane undercoating characteristic to the AISs and nodes of Ranvier, further confirming their resemblance to the spike generation sites, "hot spots". Three-dimensional analysis revealed that each parvalbumin positive EPL neuron had 2-7 hot spots, 3-28 microm in length and located 7-50 microm from the somata. Similar "hot spots" were also encountered on a few calretinin positive granule cells and nitric oxide synthase positive periglomerular cells in the mouse MOB. In addition parvalbumin positive EPL cells in the rat MOB displayed similar multiple dendritic "hot spots". Our study suggested that these morphologically identified dendritic "hot spots" might correspond to dendritic spike generation sites of those neurons.

Tyrosine hydroxylase-positive GABAergic juxtaglomerular neurons are the main source of the interglomerular connections in the mouse main olfactory bulb.

The interglomerular connections in the mouse olfactory bulb were examined with the retrograde-tracer experiments using Fluorogold. When the injections were restricted to the glomerular layer, we encountered tracer-labeled cells in the glomerular layer and the superficial part of the external plexiform layer, not only near the injection sites but also more than 500 microm distant from the injection sites. Almost of those tracer-labeled neurons distant from the injection sites were large tyrosine hydroxylase-positive juxtaglomerular neurons, some of which were confirmed to have intraglomerular dendrites. Thus, the long interglomerular connections were mainly made by a particular type of dopaminergic-GABAergic juxtaglomerular neurons.

Heterogeneity of parvalbumin-containing neurons in the mouse main olfactory bulb, with special reference to short-axon cells and betaIV-spectrin positive dendritic segments.

The structural features of parvalbumin-positive neurons were studied in the mouse main olfactory bulb (MOB). Parvalbumin-positive neurons were heterogeneous, including numerous medium-sized interneurons in the external plexiform layer (EPL), some few large short-axon cells and a few periglomerular cells. Their overall distribution pattern and structural features resembled those of the rat MOB. However, large short-axon cells were frequently encountered in the internal plexiform and granule cell layers, which were rare in the rat MOB. In addition a few large short-axon cells were also encountered throughout the EPL. These short-axon cells extended their axons mainly in the EPL, usually making columnar axonal fields. Most parvalbumin-positive cells except periglomerular cells were confirmed to be glutamic acid decarboxylase positive. We examined the immuno-localization of the markers for the axon initial segments (AISs), betaIV-spectrin and sodium channels, to determine whether or not heterogeneous parvalbumin-positive neurons have axons. We confirmed their localization on the AISs of the large short-axon cells and periglomerular cells. However, these markers were encountered on some patch-like segments on the dendritic processes instead of the thin axon-like processes of the medium-sized EPL interneurons. The present study revealed the diversity of parvalbumin-positive neurons in the mouse MOB and their particular structural properties hitherto unknown.

Chemical properties of type 1 and type 2 periglomerular cells in the mouse olfactory bulb are different from those in the rat olfactory bulb.

We analyzed the cellular composition of the juxtaglomerular region in the main olfactory bulb of C57B/6J strain mice, focusing on 1) the compartmental organization of the glomerulus and the presence of type 1 and 2 periglomerular cells, 2) the colocalization relationships among the 4 major chemically identified groups of periglomerular cells, glutamic acid decarboxylase (GAD)/gamma-aminobutyric acid (GABA), tyrosine hydroxylase, calretinin and calbindin D28k positive periglomerular cells, and 3) the chemical properties of the nitric oxide synthase (NOS)-positive juxtaglomerular cells. We confirmed the compartmental organization of the glomerulus and the presence of both type 1 and 2 periglomerular cells in the mice. Similar to rat periglomerular cells, the tyrosine hydroxylase-positive cells were type 1 and GAD/GABA-positive. On the other hand, both the calbindin D28k-positive and calretinin-positive cells were type 2 periglomerular cells, but in contrast to those in rats, which are GAD/GABA-negative, all of the calbindin D28k-positive periglomerular cells and 65% of the calretinin-positive periglomerular cells were GAD/GABA-positive. The GAD/GABA-positive cells thus included both type 1 and type 2 periglomerular cells. Juxtaglomerular NOS-positive cells have been proposed as a subgroup of type 1 periglomerular cells that are separate from the calretinin-positive and calbindin D28k-positive cells in rats. However, in the mice, about 70% of the NOS-positive cells were calretinin-positive, and 50% of the calretinin-positive cells were NOS-positive. We herein reveal the significant species differences in the chemical properties of periglomerular cells and suggest that the cellular organization of the mouse main olfactory bulb cannot be extrapolated from that of rats.

Heterogeneity of nitric oxide synthase-containing neurons in the mouse main olfactory bulb.

The distribution and structural features of nitric oxide [corrected] synthase (NOS) containing intrinsic neurons were studied in the mouse main olfactory bulb (MOB). NOS positive neurons were heterogeneous, including some subpopulations of periglomerular cells, granule cells, interneurons in the external plexiform layer, superficial and deep short-axon cells and stellate cells. NOS positive periglomerular cells were frequently calretinin immunoreactive and, although rarely, calbindin positive. Importantly, some middle and external tufted cells were also confirmed to be NOS positive, some of which were also cholecystokinin (CCK) positive. Retrograde tracer experiments showed that some NOS positive tufted cells, which were also CCK positive, constitute the intrabulbar association system and the projection system to the olfactory tubercle. In addition, another particular subpopulation of NOS positive neurons with no or little CCK immunoreactivity appeared to project to areas covering the dorsal endopiriform nucleus, claustrum and insular cortex. Furthermore, diverse types of neurons other than mitral/tufted cells were also suggested to be projection neurons of the MOB. The present study revealed the diversity of NOS positive neurons in the mouse MOB and further revealed that they were different from those reported previously in the rat MOB in structural and chemical properties.

Organization of the main olfactory bulb of lesser hedgehog tenrecs.

Using a confocal laser scanning microscope (CLSM) and an electron microscope, we investigated the organization of the main olfactory bulb (MOB) of tenrecs, which were previously included into insectivores but now considered to be in a new order "Afrosoricida" in the superclade 'Afrotheria'. We confirmed that the overall structural organization of the tenrec MOB was similar to that of rodents: (1) the compartmental organization of glomeruli and two types of periglomerular cells we proposed as the common organizational principles were present; (2) there were characteristic dendrodendritic and axo-dendritic synapses in the glomerulus and external plexiform layer (EPL) and gap junctions in glomeruli; and (3) no nidi, particular synaptic regions reported only in laboratory musk shrew and mole MOBs, were encountered. However, instead of nidi, we often observed a few tangled olfactory nerves (ONs) with large irregular boutons in the glomerular-external plexiform layer border zone, with which dendrites of various displaced periglomerular cells were usually found to be intermingled. Electron microscopic (EM) examinations confirmed characteristic large mossy terminal-like ON terminals making asymmetrical synapses to presumed mitral/tufted cell and displaced periglomerular cell dendrites. In addition, gap junctions were also encountered between dendritic processes in these tiny particular regions, further showing their resemblance to glomeruli.

synaptic organization of the glomerulus in the main olfactory bulb: compartments of the glomerulus and heterogeneity of the periglomerular cells.

According to the combinatorial receptor and glomerular codes for odors, the fine tuning of the output level from each glomerulus is assumed to be important for information processing in the olfactory system, which may be regulated by numerous elements, such as olfactory nerves (ONs), periglomerular (PG) cells, centrifugal nerves and even various interneurons, such as granule cells, making synapses outside the glomeruli. Recently, structural and physiological analyses at the cellular level started to reveal that the neuronal organization of the olfactory bulb may be more complex than previously thought. In the present paper, we describe the following six points of the structural organization of the glomerulus, revealed by confocal laser scanning microscopy and electron microscopy analyses of rats, mice and other mammals: (i) the chemical heterogeneity of PG cells; (ii) compartmental organization of the glomerulus, with each glomerulus consisting of two compartments, the ON zone and the non-ON zone; (iii) the heterogeneity of PG cells in terms of their structural and synaptic features, whereby type 1 PG cells send their intraglomerular dendrites into both the ON and non-ON zones and type 2 PG cells send their intraglomerular dendrites only into the non-ON zone, thus receiving either few synapses from the ON terminals, if present, or none at all; (iv) the spatial relationship of mitral/tufted cell dendritic processes with ON terminals and PG cell dendrites; (v) complex neuronal interactions via chemical synapses and gap junctions in the glomerulus; and (vi) comparative aspects of the organization of the main olfactory bulb.

Neuronal gap junctions between intraglomerular mitral/tufted cell dendrites in the mouse main olfactory bulb.

In the mouse main olfactory bulb (MOB) gap junction-forming processes in glomeruli were analyzed by means of the serial electron microscopical reconstruction. Gap junctions were encountered between diverse types of dendritic processes and thus confirming our previous study on gap junctions in the rat MOB. Importantly, among more than 30 gap junctions examined in serial sections, we encountered 3 gap junctions made between mitral/tufted cell dendrites in the glomerulus. Then we must consider both direct coupling between mitral/tufted cells via gap junctions and indirect coupling between mitral/tufted cells via intervening interneuronal processes as suggested previously.