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.     

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.     

"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.     

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.     

Vasoactive intestinal polypeptide-containing elements in the olfactory bulb of the hedgehog (Erinaceus europaeus)

The distribution of vasoactive intestinal polypeptide (VIP)-immunopositive elements was analyzed in the olfactory bulb (OB) of the Western European hedgehog (Erinaceus europaeus) under light and electron microscopy. The immunoreactivity appeared in an abundant population of periglomerular cells of the glomerular layer, in interneurons of the external plexiform layer, and in a restricted group of deep short-axon cells of the internal plexiform layer, the granule cell layer and the white matter. In the glomerular layer, VIP-containing periglomerular cells constituted a population of non-GABAergic neurons and did not receive synapses from olfactory axons. In the EPL, VIP-immunoreactivity appeared in a morphologically heterogeneous population of GABAergic interneurons, most of them identified as satellite cells and Van Gehuchten cells. These interneurons exerted an abundant and selective innervation of the somata, primary and secondary dendrites of the principal mitral and tufted cells, but did not contact granule cells. Perisomatic innervation of the principal cells followed two different patterns. The first included 'normal' basket-like arrangements of VIP-containing varicosities surrounding the somata of mitral and tufted cells. In the second, a set of satellite cells gave rise to short dendritic shafts that embraced the somata of principal cells in an 'exuberant' basket-like arrangement. These two morphological patterns of perisomatic innervation of principal cells were correlated with a neurochemical specificity of the target. In this sense, the 'exuberant' basket-like structures were always found surrounding a subpopulation of principal cells that did not contain the calcium-binding protein parvalbumin (PV). By contrast, they were never found surrounding the subpopulation of PV-containing principal cells, which only showed 'normal' basket-like structures. This study provides new data on the connectivity and neurochemical features of the hedgehog olfactory bulb and suggests that the olfactory circuits in this species are more complex than those described in other mammals.     

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.     

Tyrosine hydroxylase-like immunoreactive neurons in the olfactory bulb of the snake,Elaphe quadrivirgata,with special reference to the colocalization of tyrosine hydroxylase- and GABA-like immunoreactivities

Summary The distribution and structural features of tyrosine hydroxylase-like immunoreactive (TH-LI) neurons were studied in the olfactory bulb of a snake, Elaphe quadrivirgata, by using pre-and post-embedding immunocytochemistry at the light microscopic level. In contrast to rodent olfactory bulbs previously reported, many TH-LI neurons were seen not only in the main olfactory bulb (MOB) but also in the accessory olfactory bulb (AOB). With regard to the TH-like immunoreactivity, there appeared no appreciable differences between MOB and AOB. As in mammalian MOB, the majority of TH-LI neurons were clustered in the periglomerular region and appeared to send their dendritic branches into glomeruli, which as a whole make an intense TH-LI band in the glomerular layer (GML). In the external plexiform/mitral cell layer (EPL/ML) of MOB and AOB as well as in the outer sublamina of the internal plexiform layer (OSL) of AOB, an appreciable number of TH-LI neurons were scattered, extending dendritic processes which appeared to make a loose meshwork. TH-LI neurons in EPL/ML (including OSL) appeared to consist of at least two morphologically different types. The first had a small perikaryon and one or two smooth dendrites which usually extended to GML and were frequently confirmed to enter into glomeruli. The second had a larger perikaryon and 2–3 dendrites which branched into several varicose processes extending in EPL/ML/OSL but appeared not to enter into glomeruli. The TH-like immunoreactivity was rarely seen in the internal plexiform layer and internal granule cell layer. The colocalization of GABA-like and TH-like immunoreactivities was further studied. Almost all TH-LI neurons in both EPL/ ML/OSL and GML contained GABA-like immunoreactivity irrespectively of the type of TH-LI cells.Abbreviations in Figures AOB accessory olfactory bulb - MOB main olfactory bulb - Hem hemisphere - ON olfactory nerve layer - VN vomeronasal nerve layer - GM glomerular layer - EP/M external plexiform layer/Mitral cell layer - IP internal plexiform layer - IG internal granular layer - OS outer sublamina of the IPL of AOB - MS middle sublamina of the IPL of AOB - IS inner sublamina of the IPL of AOB     

Postnatal changes in expression of vesicular glutamate transporters in the main olfactory bulb of the rat

Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization–immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events.     

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation

After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvO1, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.     

Properties of external plexiform layer interneurons in mouse olfactory bulb slices

In the external plexiform layer (EPL) of the main olfactory bulb, apical dendrites of inhibitory granule cells form large numbers of synapses with mitral and tufted (M/T) cells, which regulate the spread of activity along the M/T cell dendrites. The EPL also contains intrinsic interneurons, the functions of which are unknown. In the present study, recordings were obtained from cell bodies in the EPL of mouse olfactory bulb slices. Biocytin-filling confirmed that the recorded cells included interneurons, tufted cells, and astrocytes. The interneurons had fine, varicose dendrites, and those located superficially bridged the EPL space below several adjacent glomeruli. Interneuron activity was characterized by high frequency spontaneous excitatory postsynaptic potential/currents that were blocked by the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and largely eliminated by the voltage-sensitive Na+ channel blocker, tetrodotoxin. Interneuron activity differed markedly from that of tufted cells, which usually exhibited spontaneous action potential bursts. The interneurons produced few action potentials spontaneously, but often produced them in response to depolarization and/or olfactory nerve (ON) stimulation. The responses to depolarization resembled responses of late- and fast-spiking interneurons found in other cortical regions. The latency and variability of the ON-evoked responses were indicative of polysynaptic input. Interneurons expressing green fluorescent protein under control of the mouse glutamic acid decarboxylase 65 promoter exhibited identical properties, providing evidence that the EPL interneurons are GABAergic. Together, these results suggest that EPL interneurons are excited by M/T cells via AMPA/kainate receptors and may in turn inhibit M/T cells within spatial domains that are topographically related to several adjacent glomeruli.     

Cholinergic innervation of the main and the accessory olfactory bulbs of the rat as revealed by a monoclonal antibody against choline acetyltransferase

Summary The main and accessory olfactory bulbs (MOB and AOB) of the rat were immunohistochemically stained with a monoclonal antibody against choline acetyltransferase (ChAT) in order to know the difference in the distribution patterns of cholinergic fibers between these two structures. A few ChAT-immunoreactive cell bodies were found in the superficial and middle parts of the external plexiform layer (EPL) of the MOB, in the granule cell layer (GCL) of the MOB, and in the GCL of the AOB. The frequency in appearance of these cells was 0.9 cells/section in the MOB and 0.3 cells/section in the AOB. While the glomerular layer (GL) and the superficial part of the EPL were most densely innervated in the MOB, the internal plexiform layer received the richest innervation in the AOB. There were no immunoreactive structures in the olfactory nerve layer of the MOB and in the vomeronasal nerve layer and glomerular layer of the AOB. In addition to a relatively homogenous distribution of cholinergic fibers in the MOB and AOB, there were several foci of very dense network of immunoreactive fibers at the posterior level of the OB. These foci formed a part of the modified glomerular complex that was recently identified using 2-deoxyglucose method and was presumed to be related to suckling behaviour in the neonatal rat.     

Complementary postsynaptic activity patterns elicited in olfactory bulb by stimulation of mitral/tufted and centrifugal fiber inputs to granule cells

Main olfactory bulb (MOB) granule cells receive spatially segregated glutamatergic synaptic inputs from the dendrites of mitral/tufted cells as well as from the axons of centrifugal fibers (CFFs) originating in olfactory cortical areas. Dendrodendritic synapses from mitral/tufted cells occur on granule cell distal dendrites in the external plexiform layer (EPL), whereas CFFs preferentially target the somata/proximal dendrites of granule cells in the granule cell layer (GCL). In the present study, tract tracing, and recordings of field potentials and voltage-sensitive dye optical signals were used to map activity patterns elicited by activation of these two inputs to granule cells in mouse olfactory bulb slices. Stimulation of the lateral olfactory tract (LOT) produced a negative field potential in the EPL and a positivity in the GCL. CFF stimulation produced field potentials of opposite polarity in the EPL and GCL to those elicited by LOT. LOT-evoked optical signals appeared in the EPL and spread subsequently to deeper layers, whereas CFF-evoked responses appeared in the GCL and then spread superficially. Evoked responses were reduced by N-methyl-d-aspartate (NMDA) receptor antagonists and completely suppressed by AMPA receptor antagonists. Reduction of extracellular Mg(2+) enhanced the strength and spatiotemporal extent of the evoked responses. These and additional findings indicate that LOT- and CFF-evoked field potentials and optical signals reflect postsynaptic activity in granule cells, with moderate NMDA and dominant AMPA receptor components. Taken together, these results demonstrate that LOT and CFF stimulation in MOB slices selectively activate glutamatergic inputs to the distal dendrites versus somata/proximal dendrites of granule cells.     

Tufted cell dendrodendritic inhibition in the olfactory bulb is dependent on NMDA receptor activity

Mitral and tufted cells constitute the primary output cells of the olfactory bulb. While tufted cells are often considered as "displaced" mitral cells, their actual role in olfactory bulb processing has been little explored. We examined dendrodendritic inhibition between tufted cells and interneurons using whole cell voltage-clamp recording. Dendrodendritic inhibitory postsynaptic currents (IPSCs) generated by depolarizing voltage steps in tufted cells were completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2amino-5-phosphonopentanoic acid (D,L-AP5), whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2-3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) had no effect. Tufted cells in the external plexiform layer (EPL) and in the periglomerular region (PGR) showed similar behavior. These results indicate that NMDA receptor-mediated excitation of interneurons drives inhibition of tufted cells at dendrodendritic synapses as it does in mitral cells. However, the spatial extent of lateral inhibition in tufted cells was much more limited than in mitral cells. We suggest that the sphere of influence of tufted cells, while qualitatively similar to mitral cells, is centered on only one or a few glomeruli.     

Intraglomerular dendritic link connected by gap junctions and chemical synapses in the mouse main olfactory bulb: electron microscopic serial section analyses

Glomeruli of the main olfactory bulb are considered to serve as functional units in processing the olfactory information. Thus the fine tuning of the output level from each glomerulus is important to the information processing in the olfactory system. The interactions among neuronal elements in glomeruli might be one of main mechanisms regulating this output level. In the mouse main olfactory bulb neuronal connections via chemical synapses and gap junction in glomeruli were analyzed by the serial electron microscopical reconstruction. Gap junctions were encountered between diverse types of dendritic processes, between mitral/tufted cell dendrites, between mitral/tufted cell dendrites and periglomerular cell dendrites and between mitral/tufted cell dendrites and dendrites of some interneurons different from periglomerular cells. Then these morphological observations indicate that we must consider both direct coupling between mitral/tufted cells via gap junctions and indirect coupling between mitral/tufted cells via intervening interneuronal processes. One of gap junction-forming processes presynaptic in asymmetrical synapses was traced back to the soma of its origin located in the glomerular layer, which was thus identified as an external tufted cell. However, interestingly, it showed apparently different ultrastructural features from other external tufted cells located at the border between the glomerular and external plexiform layers; the latter resemble so-called mitral/tufted cells located in the external plexiform and mitral cell layers. Then external tufted cells were assumed to be heterogeneous in their ultrastructural features. We occasionally encountered several dendrites connected by gap junctions, which furthermore made chemical synapses with each other and with other surrounding processes. Thus both chemical synapses and gap junctions interconnect complexly various processes in the glomerulus, where the local circuit among intermingled olfactory nerves, mitral/tufted cell dendrites and interneuron dendrites is far more complex than previously schematized.     

Neuronal gap junctions in the mouse main olfactory bulb: morphological analyses on transgenic mice

In the present study we analyzed the structural features of extraglomerular gap junction-forming processes in mouse olfactory bulb electron microscopically. This work complements a previous study in which we analyzed the structural features of neuronal gap junction-forming processes within the glomerulus itself. Furthermore we examined connexin 36 expressing cells in the mouse olfactory bulb by analyzing transgenic mice in which the connexin 36 coding sequence was replaced with histological reporters. In extraglomerular regions, the mitral/tufted cell somata, dendrites and axon hillocks made gap junctions and mixed synapses with interneuronal processes. These gap junctions and synapses were associated with various types of interneuronal processes, including a particular type of sheet-like or calyx-like process contacting the somata or large dendrites of mitral/tufted cells. In the olfactory bulbs of the transgenic mice, connexin 36 was expressed in mitral cells, tufted cells, presumed granule cells and periglomerular cells. Multiple immunofluorescent labelings further revealed that presumed interneurons expressing connexin 36 in the periglomerular region rarely expressed calbindin, calretinin or tyrosine hydroxylase and are likely to comprise a chemically uncharacterized class of neurons. Similarly, interneurons expressing connexin 36 in the granule cell layer were rarely positive for calretinin, which was expressed in numerous presumed granule cells in the mouse main olfactory bulb. In summary, these findings revealed that mitral/tufted cells make gap junctions with diverse types of neurons; in the glomeruli gap junction-forming interneuronal processes originated from some types of periglomerular cells but others from a hitherto uncharacterized neuron type(s), and in the extraglomerular region gap-junction forming processes originate mainly from a subset of cells within the granule cell layer.     

Distribution of enkephalin-like immunoreactivity in the rat main olfactory bulb

Enkephalin-like immunoreactivity was localized within the main olfactory bulb of the rat using immunohistochemical techniques. These studies utilized well characterized antisera directed to either leu⁵- or met⁵-enkephalin. Specificity was established by absorption of the antisera with either 10 μM synthetic leu⁵- or met⁵-enkephalin.Specific enkephalin-like immunoreactivity was observed within several different cell populations including (1) periglomerular cells, (2) granule cells and their processes within the external plexiform layer and (3) occasional short-axon (horizontal) cells within the granule and external plaxiform layers. The granule cell layer contained the greatest number of immunoreactive cells. Only a limited number of immunoreactive cells were found in both the periglomerular and granule cell layers, suggesting the enkephalin-containing neurons represent a sub-population within each layer.The absence of immunoreactive processes in the periventribular white matter, as well as the morphologies of immunoreactive bulbar neurons, indicates that enkephalin is found exclusively within intrinsic olfactory bulb neurons.     

Parvalbumin-containing neurons in the rat basolateral amygdala: morphology and co-localization of Calbindin-D(28k)

Parvalbumin is a calcium-binding protein that is contained in certain neuronal populations in the brain. Although the exact function of parvalbumin is not clear, it has been found to be a useful marker for studying the connections of specific cell types in immunohistochemical studies. In the present investigation immunohistochemical techniques were used to study the morphology of parvalbumin-containing neurons in the rat basolateral amygdala. These neurons were found to be a morphologically heterogeneous subpopulation of non-pyramidal interneurons. Parvalbumin-positive axons in the basolateral amygdala were observed to form "pericellular baskets" that enveloped the perikarya of pyramidal neurons. In addition, some parvalbumin-immunoreactive axons formed "cartridges" that appeared to surround non-immunoreactive processes. The morphology of parvalbumin-positive neurons closely resembled that of neurons containing calbindin, a related calcium-binding protein. Analysis of adjacent sections stained for each protein using the mirror technique revealed that approximately 80% of parvalbumin neurons also contained calbindin, and that approximately 60% of calbindin neurons also contained parvalbumin.This study demonstrates that parvalbumin-containing neurons constitute an important subpopulation of non-pyramidal interneurons in the rat basolateral amygdala. The axonal configurations of these cells indicate that they may exert a potent inhibitory influence over pyramidal projection neurons. We suggest that parvalbumin-containing neurons can control emotional responses mediated by the basolateral amygdala by controlling the output from this important brain region.     

Neuronal gap junctions in the rat main olfactory bulb,with special reference to intraglomerular gap junctions

The structural features of neuronal gap junction-forming processes in the rat olfactory bulb were analyzed electron microscopically. Gap junctions were present in glomeruli and extraglomerular regions. In extraglomerular regions, mitral/tufted cell somata, dendrites and axon hillock-initial segments made gap junctions and mixed synapses with interneuronal processes, some of which were confirmed to be GABA positive. In glomeruli gap junctions were encountered mainly between mitral/tufted cell dendrites and diverse types of processes; a small population of them were conclusively identified as periglomerular cell dendrites. Gap junction-forming processes frequently received synapses from olfactory nerve terminals, suggesting that they could be type 1 periglomerular cells. However, the majority were GABA negative or only faintly positive and none were tyrosine hydroxylase positive, indicating that they were different from previously reported type 1 periglomerular cells. Furthermore serial sectioning analyses revealed that the majority of those processes forming gap junctions with mitral/tufted dendrites were smooth cylindrical and had few presynaptic sites, indicating that they were different from previously described periglomerular cells. These findings revealed that mitral/tufted cells make gap junctions with diverse types of neurons; and some of these gap junction-forming processes originated from some types of periglomerular cells but others from hitherto uncharacterized neuron type(s).     

Localization of the CB1 type cannabinoid receptor in the rat basolateral amygdala: high concentrations in a subpopulation of cholecystokinin-containing interneurons.

The neuronal localization of the CB1 cannabinoid receptor in the rat basolateral amygdala was studied using peroxidase and fluorescence immunohistochemical techniques. All nuclei of the basolateral amygdala contained a large number of lightly stained pyramidal neurons and a small number of more intensely stained non-pyramidal neurons. Most of the latter cells had medium-sized to large multipolar somata and three to four aspiny dendrites, but some exhibited smaller oval somata. The axon initial segments of some of these non-pyramidal neurons exhibited large swollen varicosities in colchicine-injected animals, suggesting that much of the CB1 receptor protein is transported down the axons of these cells. Double-labeling studies using immunofluorescence histochemistry combined with confocal laser scanning microscopy revealed that the great majority of non-pyramidal neurons with CB1 receptor immunoreactivity belonged to a cholecystokinin-containing subpopulation. Whereas none of the other subpopulations of non-pyramidal neurons (exhibiting immunoreactivity for calretinin, parvalbumin, or somatostatin) expressed high levels of CB1 receptor immunoreactivity, a small percentage of these cells exhibited low levels of immunoreactivity.The results indicate that cannabinoids may modulate the activity of pyramidal projection neurons as well as a subpopulation of cholecystokinin-containing non-pyramidal neurons in the basolateral amygdala. Previous studies indicate that most of the latter are inhibitory interneurons that utilize GABA as a neurotransmitter. The intense staining of the cholecystokinin-containing interneurons and the evidence that large amounts of CB1 receptor protein are transported down the axons of these cells suggests that, as in the hippocampus, cannabinoids may inhibit the release of GABA from the axon terminals of these neurons.     

Immunocytochemical localization of the GABAB2 receptor subunit in the glomeruli of the mouse main olfactory bulb

The olfactory input to the brain is carried out by olfactory nerve axons that terminate in the olfactory bulb glomeruli and make synapses onto dendrites of glutamatergic projection neurons, mitral and tufted cells, and GABAergic interneurons, periglomerular cells. The dendrites are reciprocally connected through asymmetric synapses of mitral/tufted cells with periglomerular cells and symmetric synapses of the opposite direction. Transmission at the first synapse in the olfactory pathway is regulated presynaptically, and this regulation is mediated, in part, by metabotropic GABAB receptors that, when activated, inhibit transmitter release from the olfactory nerve. Functional GABAB receptors are heterodimers composed of the GABAB1 and GABAB2 subunits. Studies using double immunofluorescence have shown colocalization of both subunits in the glomerular neuropil, and ultrastructural studies have localized GABAB1 to extrasynaptic, synaptic, and perisynaptic sites on the plasma membrane of olfactory nerve terminals. We studied the subcellular localization of GABAB2 in the mouse olfactory glomeruli using a subunit-specific antibody and preembedding immunogold labeling. Immunoreactivity for GABAB2 was associated with symmetric dendrodendritic synapses of periglomerular cells with mitral/tufted cells and was localized to the extrasynaptic plasma membrane of presynaptic dendrites, and extrasynaptic, synaptic, and perisynaptic sites on the plasma membrane of postsynaptic dendrites. The results suggest that postsynaptic, and perhaps presynaptic, GABAB receptors may be expressed at GABAergic synapses between dendrites of periglomerular interneurons and projection neurons. Immunolabeling was observed at junctions of the olfactory nerve with mitral/tufted cell dendrites, providing ultrastructural evidence for the expression of the GABAB2 subunit at the primary olfactory synapse.