Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb

To determine the dendritic fields, mitral, displaced mitral, middle tufted, and granule cells in the rabbit olfactory bulb were stained by intracellular injection of HRP. The secondary dendrites of mitral cells were distributed mostly in the inner half of the external plexiform layer (EPL). Those of displaced mitral cells extended mainly into the middle and superficial sublayers in the EPL. The secondary dendrites of middle tufted cells were distributed mostly in the superficial portion of the EPL. Mitral cells extended their secondary dendrites in virtually all directions within a plane tangential to the mitral cell layer (MCL) and thus had a disklike projection field with a radius of about 850 microns. Displaced mitral cells had similar dendritic projection fields in the tangential plane but with somewhat distorted shapes. The secondary dendrites of middle tufted cells had a tendency to extend in particular directions. From the projection pattern of the gemmules on the peripheral processes, granule cells were classified into three types. Type I granule cells had gemmules both in the superficial and in the deep sublayers of the EPL. The peripheral processes of Type II granule cells were confined to the deep half of the EPL. The gemmules of Type III granule cells ere distributed in the superficial half of the EPL. The differing dendritic ramification among mitral, displaced mitral, and middle tufted cells suggests the separation of the dendrodendritic synaptic interactions with granule cells in different sublayers in the EPL. It also suggests a functional separation of the sublayers of the EPL.     

Nectin-1 spots regulate the branching of olfactory mitral cell dendrites

Olfactory mitral cells extend lateral secondary dendrites that contact the lateral secondary and apical primary dendrites of other mitral cells in the external plexiform layer (EPL) of the olfactory bulb. The lateral dendrites further contact granule cell dendrites, forming dendrodendritic reciprocal synapses in the EPL. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. We recently showed that the immunoglobulin-like cell adhesion molecule nectin-1 constitutes a novel adhesion apparatus at the contacts between mitral cell lateral dendrites, between mitral cell lateral and apical dendrites, and between mitral cell lateral dendrites and granule cell dendritic spine necks in the deep sub-lamina of the EPL of the developing mouse olfactory bulb and named them nectin-1 spots. We investigated here the role of the nectin-1 spots in the formation of dendritic structures in the EPL of the mouse olfactory bulb. We showed that in cultured nectin-1-knockout mitral cells, the number of branching points of mitral cell dendrites was reduced compared to that in the control cells. In the deep sub-lamina of the EPL in the nectin-1-knockout olfactory bulb, the number of branching points of mitral cell lateral dendrites and the number of dendrodendritic reciprocal synapses were reduced compared to those in the control olfactory bulb. These results indicate that the nectin-1 spots regulate the branching of mitral cell dendrites in the deep sub-lamina of the EPL and suggest that the nectin-1 spots are required for odor information processing in the olfactory bulb.     

Segregated labeling of olfactory bulb projection neurons based on their birthdates

Mitral and tufted cells are the projection neurons of the olfactory bulb (OB). We previously reported that somata location and innervation patterns were different between early‐ and late‐born mitral cells (Imamura et al., 2011). Here, we introduced a plasmid that drives the expression of a GFP gene into the mouse OB using in utero electroporation, and demonstrated that we can deliver the plasmid vectors into distinct subsets of OB projection neurons by changing the timing of electroporation after fertilisation. The electroporation performed at embryonic day (E)10 preferentially labeled mitral cells in the accessory OB and main OB mitral cells in dorsomedial mitral cell layer (MCL). In contrast, the E12 electroporation introduced the plasmid vectors preferentially into main OB mitral cells in the ventrolateral MCL and tufted cells. Combining these data with BrdU injections, we confirmed that E10 and E12 electroporation preferentially labeled early‐ and late‐born projection neurons, respectively. This work introduces a novel method for segregated labeling of mouse olfactory bulb projection neurons based on their birthdates. With this technique we found that early‐ and late‐born projection neurons extend their secondary dendrites in the deep and superficial external plexiform layer (EPL), respectively. Although a similar segregation has been suggested for mitral vs. tufted cell dendrites, we found mitral cells projecting secondary dendrites into the superficial EPL in E12‐electroporated main OB. Our observations indicate that timing of neurogenesis regulates not only somata location and innervation patterns but also the laminar organisation of projection neuron dendrites in the EPL.     

Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb

Studies on the morphological organization of the main olfactory bulb have indicated that there are subpopulations of granule cells with different dendritic patterns in the external plexiform layer (EPL). Small, extracellular injections of horseradish peroxidase (HRP) were made iontophoretically into superficial and deep parts of the EPL and the granule cell layer (GCL) in adult rats. Superficial EPL injections principally labeled superficial granule cell somata, whereas deep EPL injections labeled both superficial and deep granule cell somata. Injections in the superficial GCL labeled granule cell dendritic processes extending across the entire EPL. However, deep GCL injections labeled few granule cell dendrites in the superficial EPL, but labeled many such processes in the deep EPL. These results were the same in material processed with the Hanker-Yates procedure, where the morphology of individual neurons could be studied, and in the more sensitive tetramethyl benzidineprocedure. Serial reconstructions of individual granule cells were made from both HRP and Golgi-Kopsch material. The distal dendrites of deep granule cells reached only as far as the deep EPL, where they branched extensively and had many dendritic spines. The dendrites of superficial granule cells, however, reached the most superficial part of the EPL where they ramified most extensively. The superficial granule cells typically had a higher spine density in the superficial part of the EPL than in the deep part. On the basis of these results, we conclude that the superficial granule cells predominantly innervate the superficial EPL and that the deep granule cells exclusively innervate the deep EPL. Granule cells are believed to exert inhibitory influences on the bulbar output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses. Since the secondary dendrites of the tufted cells ramify in the superficial EPL and the dendrites of most mitral cells ramify in deep EPL, the superficial and deep granule cells may preferentially modulate the responses of tufted and mitral cells, respectively.     

Immunolocalization of metabotropic glutamate receptor 7 in the rat olfactory bulb

Metabotropic glutamate receptors (mGluRs) constitute a large family of G-protein-coupled receptors that are subdivided into three groups based on sequence similarity, pharmacological profiles, and coupling to second messengers. Although mRNAs for seven of the eight mGluRs are expressed in the olfactory system, the localization and function of specific subtypes have not been fully characterized. Mitral cells of the olfactory bulb express mRNA for several mGluRs, including mGluR7, which has been suggested as a presynaptic glutamate autoreceptor. To investigate the immunolocalization of mGluR7 in the olfactory system, we used a polyclonal antiserum specific for the carboxy terminus of the receptor. Mitral cell somata and proximal dendrites were strongly labeled by the mGluR7 antibody. Electron microscopic analysis revealed that most of the mitral cell somatic staining was cytoplasmic. In olfactory bulb glomeruli, immunoreactivity was present in axons and dendrites. In the piriform cortex, diffuse staining was present in layer Ia that was markedly reduced following bulbectomy, consistent with expression of mGluR7 in mitral cell axon terminals. Electron microscopic analysis of this region confirmed the presence of mGluR7 in multiple axon terminals. Distinct labeled fibers in all levels of layer I appeared to originate from labeled piriform cortex pyramidal cells in layers II and III. Our results indicate that mGluR7 is primarily presynaptic at olfactory bulb synapses. However, the postsynaptic localization of mGluR7 at selected synapses indicates that mGluR7 is not targeted exclusively to axonal compartments. J. Comp. Neurol. 385:372–384, 1997. © 1997 Wiley-Liss, Inc.     

Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb

The output cells of the main olfactory bulb, the mitral and tufted cells, can be categorized into subclasses on the basis of their intrabulbar dendritic and axonal characteristics. Their form was studied in rats following labeling by iontophoretic injection of horseradish peroxidase into the external plexiform layer (EPL). The fact that these extracellular injections labeled small numbers of neurons permitted reconstruction of individual cells. The injection depth within the EPL determined the type of cells labeled. Secondary dendrites of each cell type lay in one of three partially overlapping zones in the EPL. The deepest zone contained the secondary dendrites of one group of mitral cells (Type I), which had the deepest and longest dendrites of the output cells. An intermediate zone of the EPL contained the secondary dendrites of middle tufted and a second class of mitral cells (Type II). The superficial zone, adjacent to the glomerular layer, contained the relatively short, asymmetric dendritic fields of external tufted cells. The few labeled internal tufted cells had secondary dendrites in either the intermediate or deep zones. Every cell type, except the Type I mitral cells, had axon collaterals in the internal plexiform and upper granule cell layers. No cell types had axons re-entering the EPL. These results for output cells combined with our previous observations on granule cells point to a functional sublaminar organization of the EPL that has not previously been proposed.     

Nectin‐1 spots as a novel adhesion apparatus that tethers mitral cell lateral dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb

Mitral cells project lateral dendrites that contact the lateral and primary dendrites of other mitral cells and granule cell dendrites in the external plexiform layer (EPL) of the olfactory bulb. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. In immunofluorescence microscopy, the immunofluorescence signal for the cell adhesion molecule nectin‐1 was concentrated on mitral cell lateral dendrites in the EPL of the developing mouse olfactory bulb. In electron microscopy, the immunogold particles for nectin‐1 were symmetrically localized on the plasma membranes at the contacts between mitral cell lateral dendrites, which showed bilateral darkening without dense cytoskeletal undercoats characteristic of puncta adherentia junctions. We named the contacts where the immunogold particles for nectin‐1 were symmetrically accumulated "nectin‐1 spots." The nectin‐1 spots were 0.21 μm in length on average and the distance between the plasma membranes was 20.8 nm on average. In 3D reconstruction of serial sections, clusters of the nectin‐1 spots formed a disc‐like structure. In the mitral cell lateral dendrites of nectin‐1‐knockout mice, the immunogold particles for nectin‐1 were undetectable and the plasma membrane darkening was electron‐microscopically normalized, but the plasma membranes were partly separated from each other. The nectin‐1 spots were further identified between mitral cell lateral and primary dendrites and between mitral cell lateral dendrites and granule cell dendritic spine necks. These results indicate that the nectin‐1 spots constitute a novel adhesion apparatus that tethers mitral cell dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb. J. Comp. Neurol. 523:1824–1839, 2015. © 2015 Wiley Periodicals, Inc.     

Characterization of CGRP protein expression in “satellite‐like” cells and dendritic arbours of the mouse olfactory bulb

The main olfactory bulb (OB) is made up of several concentric layers, forming circuitries often involving dendro‐dendritic synapses. Important interactions between OB neurons occur in the external plexiform layer (EPL), where dendrites of tufted and Van Gehuchten cells form synapses with dendrites of deeper lying mitral, tufted, and granule cells. OB neurons display a variety of neurotransmitters. Here, the focus is on calcitonin gene‐related peptide (CGRP), a 37‐amino acid neuropeptide transmitter that is widely distributed in the central and peripheral nervous system. In the OB, CGRP‐immunoreactive (ir) cell bodies were mostly observed in the mitral cell layer (MCL) of normal mice, and their number increased following colchicine treatment. Sparsely distributed CGRP‐ir cell bodies were also found in the EPL and granular cell layer. Double‐immunofluorescence experiments revealed a lack of co‐localization between CGRP‐like immunoreactivity (LI) and corticotropin‐releasing factor‐ or galanin‐LI, two markers for mitral cells, and no CGRP‐LI was found in cholecystokinin‐, parvalbumin‐, or vasoactive intestinal polypeptide‐ir tufted/Van Gehuchten cells. CGRP‐ir cell bodies were not found to co‐localize glutamic acid decarboxylase 67 (GAD67)‐green fluorescence protein, γ‐aminobutyric acid (GABA)‐, or calretinin‐LI, although the possibility remains that CGRP‐ir cells may contain low levels of GABA and/or GAD67 not detected by our methodology. Dendrites of CGRP‐ir cells extensively ramified deep in the EPL and double‐immunofluorescence revealed them to be adjacent with, often apparently contacting, dendrites of granule, mitral, tufted, and Van Gehuchten cells. We propose that these CGRP‐ir cell bodies in the mouse OB are “satellite‐like” cells within and, occasionally, close to the MCL. J. Comp. Neurol. 518:770–784, 2010. © 2009 Wiley‐Liss, Inc.     

Cytochrome oxidase staining marks dendritic zones of the rat olfactory bulb external plexiform layer

Cytochrome oxidase staining of the rat olfactory bulb external plexiform layer (EPL) produces a darkly stained intermediate zone bordered by lightly stained superficial and deep zones. Similar zonal staining was seen in cats, rabbits, and hamsters. These zones vary in relative thickness around the circumference of the olfactory bulb; the deep zone is proportionally thicker in the most dorsal and ventral parts of the bulb. Tufted cell somata are unevenly distributed within the EPL; the outer part of the EPL has more somata. The distribution of the cytochrome oxidase reaction product shows that the darkly stained intermediate zone is not produced by staining of tufted cell somata. Zones of cytochrome oxidase staining correspond to the sublaminar distribution of mitral and tufted cell basal dendrites. This was demonstrated by labeling mitral and tufted cells with small extracellular horseradish peroxidase injections and processing alternate sections for horseradish peroxidase and for cytochrome oxidase. Because there was cross-reaction of the cytochrome oxidase procedure with horseradish peroxidase, it was possible to trace many neurons through both series of sections. Middle tufted cells of the superficial EPL have basal dendrites confined to the superficial zone of light cytochrome oxidase staining. Internal tufted cells and middle tufted cells of the intermediate zone send their basal dendrites into the intermediate zone. One group of mitral cells (type I) has basal dendrites confined to the deep zone of lighter cytochrome oxidase staining. A second group of mitral cells (type II) and tufted cells of the intermediate EPL has basal dendrites primarily confined to the intermediate zone of dark cytochrome oxidase staining. The correlation of the enzyme staining with the dendritic laminar patterns supports the existence of three relatively distinct sublaminae in the EPL and supports the designation of two types of mitral cells. The staining pattern also provides an independent method for evaluating the sublaminae of the EPL without the necessity of labeling individual groups of cells. Finally, the staining pattern suggests that the intermediate zone of the EPL may be subjected to more tonic synaptic input, causing it to have an increased level of metabolic activity.     

Golgi analyses of dendritic organization among denervated olfactory bulb granule cells

In the vertebrate olfactory bulb, the primary projection neurons, mitral and tufted cells, have reciprocal dendrodendritic synapses with respective subpopulations of anaxonic interneurons called granule cells. In the neurological murine mutant Purkinje Cell Degeneration (PCD), all mitral cells are lost during early adulthood. As a consequence, a subpopulation of granule cells is deprived of both afferent input and efferent targets. The effect of this event on the morphology and sublaminar distribution of granule cells was studied with light microscopic Golgi procedures in affected homozygous recessive PCD mutants and normal heterozygous littermate controls. In the control mice, a minimum of three subpopulations were identified predominantly on the basis of the topology of apical dendrites and their spinous processes within the external plexiform layer (EPL) of the olfactory bulb: type I had dendrites extending across the full width of the EPL and a homogeneous distribution of spines; type II had dendritic arbors confined to the deeper EPL; type III had apical dendrites that arborized extensively within the superficial EPL with no arbors or spines present in the deeper EPL. Prior studies suggest that type II cells form connections with mitral cells; type III cells form connections with tufted cells; and type I cells may integrate information from both populations of projection neurons. In the mutant PCD mice, the classification of subpopulations of granule cells proved difficult due to a compression of dendritic arbors within the EPL. Dendritic processes followed a more horizontal tangent relative to the radial orientation seen in control mice. The length of dendritic branches was reduced by approximately 20% with a corresponding decrease in the number of spines. The density of spines (#/1 micron of dendrite) was constant in both controls and mutants at approximately 0.21. Truncation of the dendrites in the PCD mutants appeared to occur at terminal portions because the number of dendritic bifurcations was equal in both groups of mice. The data are discussed in terms of subpopulations of granule cells in the mouse olfactory bulb, the sublaminar organization of olfactory bulb circuits, and the capacity for survival and plasticity in the reciprocal dendrodendritic circuits mediated by the granule cell spines.     

Molecular determinants of metabotropic glutamate receptor 1B trafficking

The metabotropic glutamate receptor mGluR1 undergoes alternative splicing to generate isoforms differing in C-terminal sequence. The mechanism by which these isoforms give different functional responses to agonists in vitro is so far unclear. Using the native mGluR1 and CD2-mGluR1 chimeric molecules, as well as their C-terminal truncations and mutants, we identified an endoplasmic reticulum (ER) retention signal Arg-Arg-Lys-Lys within the C-terminal sequence of mGluR1b. Its presence results in a much reduced cell surface expression of the receptor and chimeric molecules in cell lines and their restricted trafficking in neurones. This motif is also present in the C-terminus of mGluR1a, but its effect is overcome by a region of the mGluR1a-specific C-terminal sequence (amino acids 975-1098). Our results indicate that these splice variants of mGluR1 utilize different targeting pathways and suggest that this may be a general phenomenon in the metabotropic glutamate receptor gene family.     

A leucine-rich repeat membrane protein, 5T4, is expressed by a subtype of granule cells with dendritic arbors in specific strata of the mouse olfactory bulb

Segregation of neuron-type-specific synaptic connections in different strata is a characteristic feature shared by the olfactory bulb (OB) and retina. In the mammalian OB, mitral cells form dendrodendritic synapses with granule cells (GCs) in the deep stratum of the external plexiform layer (EPL), whereas tufted cells form dendrodendritic synapses in the superficial stratum. In the search for membrane proteins with strata-specific expression patterns, we found that a leucine-rich repeat membrane protein (5T4 oncofetal trophoblast glycoprotein) was expressed selectively by a subset of superficial GCs. The somata of 5T4-positive GCs were localized in or near the mitral cell layer, and their apical dendrites ramified preferentially in the superficial stratum of the EPL, where tufted cell dendrites ramified. Strata-specific expression of 5T4 was found also in the retina: 5T4 was expressed selectively by rod-bipolar cells and a subset of amacrine cells whose dendrites ramified in a specific sublamina of the inner plexiform layer. During the perinatal and postnatal development of the OB, 5T4 expression paralleled in time the formation of dendrodendritic synapses in the EPL. Odor deprivation during the first postnatal month selectively reduced the thickness of the superficial stratum of the EPL and the number of 5T4-positive GCs. Because 5T4 is known to interact with actin cytoskeleton, these observations suggest that 5T4 is involved in the formation or maintenance of strata-specific dendritic ramification or synaptic connection of subsets of local interneurons.     

Transient ischemia-induced changes of neurofilament 200 kDa immunoreactivity and protein content in the main olfactory bulb in gerbils

This study was carried out to investigate alterations of neurofilament 200 kDa (NF-200) and its polyphosphorylation form (RT97) immunoreactivity and protein content in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, weak NF-200 immunoreactivity was detectable in a few somata of mitral cells, which projected weak NF-200-immunoreactive processes to the external plexiform layer (EPL). At 1-5 days after ischemia, strong NF-200 and RT97 immunoreactivity was shown by the mitral cell processes; however, somata of mitral cells did not show NF-200 immunoreactivity. At this time point, strong NF-200-immunoreactive mitral cell processes ran to the EPL and glomerular layer (GL). Thereafter, NF-200 and RT97 immunoreactivity was decreased up to 30 days after ischemia. In the 15 days post-ischemic group, the distribution pattern of NF-200 and RT97 immunoreactivity was slightly lower than that in the 1-5 days post-ischemic groups. In the 30 days post-ischemic group, moderate NF-200 and RT97 immunoreactivity was found in the mitral cells processes, but the immunoreactivity in the EPL and GL nearly disappeared. A Western blot study showed a pattern of NF-200 and RT97 expression at all post-ischemic time points similar to that of immunohistochemistry after ischemia. This result indicates that NF-200 and RT97 accumulates in injured mitral cell processes a few days after transient ischemia, which suggests that the axonal transport in the MOB may be disturbed during this period after transient ischemia.     

Glomerular formation in the developing rat olfactory bulb.

Using the confocal microscope together with markers for the cellular components of glomeruli, we examined the spatiotemporal cellular interactions that occur between the axons of olfactory receptor cells, their dendritic targets, and glial cells during the critical period of glomerular formation. We have employed markers of immature and mature olfactory receptor cell axons, mitral/tufted cell dendrites, and glial cells as well as a synapse-associated protein for double- and triple-label immunocytochemistry. Axons of olfactory receptor cells grew into a dense dendritic zone of the olfactory bulb (comprising the dendrites of both mitral and tufted cells) between E17 and E18. At E19, these axons coalesced into protoglomeruli, which continued to develop until birth, when the basic anatomical structure of adult glomeruli emerged. Neither mitral/tufted cell dendrites nor olfactory bulb astrocytes became specifically associated with these protoglomeruli until E21. Ensheathing cells remained restricted to the outer nerve fiber layer and did not appear to contribute to glomerular formation. Finally, the synaptophysin staining has shown that synaptic constituents are expressed as early as E17, prior to the appearance of mature olfactory receptor cell axons. Based on these data, we have established a time line detailing the temporal and spatial interactions that occur between cell types during late embryonic rat olfactory bulb development. We conclude that the initial event in the formation of glomeruli is the penetration of the mitral/tufted cell dendritic zone by olfactory receptor cell axons. The coalescence of dendritic and glial processes into glomerular structures appears secondary to the arrival of the olfactory receptor cell axons.     

Laminar distributions of interneurons in the main olfactory bulb of the adult hamster

The laminar distributions of intemeurons in the hamster olfactory bulb were studied with rapid Golgi techniques. Eight morphologically distinct classes of intemeurons were characterized according to their somal locations and their dendritic and axonal properties. Two of these classes, Blanes and Golgi cells, had somata restricted to the deeper layers of the bulb and were not observed more superficially than the mitral body layer (MBL). Their dendrites and axons were predominantly situated within the granule cell layer (GRL) and internal plexiform layer (IPL) but occassionally could be traced as superficially as the deeper portion of the external plexiform layer (EPL). Neither their dendrites nor their axons were oriented consistently with respect to the layers of the bulb. Blanes cells had numerous dendritic spines whereas Golgi cells were relatively spine-poor. Two other classes had somata that were restricted to the IPL and MBL, and had dendrites that exhibited clear orientations with respect to these layers. One class, horizontal cells, had dendrites that ran tangentially within the IPL and MBL. The other class, Cajal cells, had radially oriented dendrites that extended peripherally into the superficial region of the EPL and centrally for greater distances into the GRL. Both classes had axons that projected superficially into the EPL. The granule cells in our material were similar to those described in other species. The sixth class of intemeurons was designated as Van Gehuchten cells. The somata of Van Gehuchten cells were restricted to the EPL and MBL. Their processes branched elaborately within the EPL and MBL but were not oriented consistently with respect to these layers. It is unclear from our material whether these cells bear axons or whether they are amacrine intemeurons. Another class, superficial short axon cells, had somata that were predominantly located at the boundary between the EPL and the glomerular layer (GL) but could also be found throughout the periglomerular region and the superficial half of the EPL. These cells had dendritic and axonal processes that extended predominantly into the GL and appeared to branch around and between individual glomeruli. The eighth class, periglomerular cells, had somata located throughout the GL. Most of these cells gave rise to one dendritic trunk that arborized within a single glomerulus. Occassionally a second, less elaborate dendritic process emerged from the main trunk or from the soma and extended into the periglomerular space or encroached upon an adjacent glomerulus. However, we did not find any periglomerular cells with extensive dendritic arbors in more than one glomerulus. These distributions are discussed in relation to the notion that olfactory bulb interneurons may be assigned to two functional groups associated with two distinct levels of integration.     

Distribution of VIP- and NPY-like immunoreactivities in rat main olfactory bulb

The distribution of vasoactive intestinal peptide (VIP)- and neuropeptide Y (NPY)-like immunoreactivities in the Sprague-Dawley rat main olfactory bulb was analyzed using the peroxidase-antiperoxidase light microscopic immunocytochemical technique. VIP-like immunoreactivity was most prominently localized within a large number of intermediate-sized neurons whose perikarya and extensively branched varicose processes remained confined to the external plexiform layer (EPL). A few small short-axon type neurons in the mitral cell layer and granule cell layer (GRL) and even fewer large neurons in the glomerular layer (GL)/EPL border region contained immunoreactivity for VIP as well. Neuropeptide Y-like immunoreactivity (NPY-I) was principally localized within sparsely distributed large multipolar neurons of the deep GRL and within axons distributed with diminishing density from deep to superficial GRL. In addition, dense NPY-I was localized within very few large superficial short-axon type neurons of the GL/EPL border region. The restricted laminar and cellular distribution of NPY-I and VIP-I suggests that both peptides may act to modulate granule cell activity, and therefore, indirectly, olfactory bulb output.     

Heterogeneity of gamma-aminobutyric acid type A receptors in mitral and tufted cells of the rat main olfactory bulb

Mitral and tufted cells of the olfactory bulb receive strong gamma-aminobutyric acid (GABA)-ergic input and express GABA(A) receptors containing the alpha1 or alpha3 subunit. The distribution of these subunits was investigated in rats via multiple immunofluorescence and confocal microscopy, by using gephyrin as a marker of GABAergic synapses. A prominent immunoreactivity was detected throughout the external plexiform layer (EPL) and glomerular layer (GL). However, although staining for the alpha1 subunit was uniform throughout the EPL, that of the alpha3 subunit was most intense in the outer one-third of this layer. All mitral cells were positive for the alpha1 subunit. In contrast, the alpha3 subunit was restricted to a subpopulation of mitral cells, many of which also expressed calretinin. Likewise, external tufted cells could be subdivided into distinct groups, either singly labeled for the alpha1 or alpha3 subunit or doubly labeled. At the subcellular level, staining for the alpha1 and alpha3 subunits was punctate, forming clusters partially colocalized with gephyrin. However, many alpha1- and alpha3-positive clusters lacked gephyrin, suggesting the existence of either nonsynaptic GABA(A) receptor clusters or synaptic receptors not associated with gephyrin. Quantitative analysis of colocalization among the three markers in the inner EPL, outer EPL, and GL revealed considerable heterogeneity, suggestive of a differential organization of GABA(A) receptor subtypes in the apical and basal dendrites of mitral and tufted cells. Together these results reveal a complex subunit organization of GABA(A) receptors in the olfactory bulb and suggest that mitral and tufted cells participate in different synaptic circuits controlled by distinct GABA(A) receptor subtypes.     

Bidirectional Regulation of Neurite Elaboration by Alternatively Spliced Metabotropic Glutamate Receptor 5 (mGluR5) Isoforms

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP.     

Laminar distributions of internuerons in the main olfactory bulb of the adult hamster

The laminar distributions of intemeurons in the hamster olfactory bulb were studied with rapid Golgi techniques. Eight morphologically distinct classes of intemeurons were characterized according to their somal locations and their dendritic and axonal properties. Two of these classes, Blanes and Golgi cells, had somata restricted to the deeper layers of the bulb and were not observed more superficially than the mitral body layer (MBL). Their dendrites and axons were predominantly situated within the granule cell layer (GRL) and internal plexiform layer (IPL) but occassionally could be traced as superficially as the deeper portion of the external plexiform layer (EPL). Neither their dendrites nor their axons were oriented consistently with respect to the layers of the bulb. Blanes cells had numerous dendritic spines whereas Golgi cells were relatively spine-poor. Two other classes had somata that were restricted to the IPL and MBL, and had dendrites that exhibited clear orientations with respect to these layers. One class, horizontal cells, had dendrites that ran tangentially within the IPL and MBL. The other class, Cajal cells, had radially oriented dendrites that extended peripherally into the superficial region of the EPL and centrally for greater distances into the GRL. Both classes had axons that projected superficially into the EPL. The granule cells in our material were similar to those described in other species. The sixth class of intemeurons was designated as Van Gehuchten cells. The somata of Van Gehuchten cells were restricted to the EPL and MBL. Their processes branched elaborately within the EPL and MBL but were not oriented consistently with respect to these layers. It is unclear from our material whether these cells bear axons or whether they are amacrine intemeurons. Another class, superficial short axon cells, had somata that were predominantly located at the boundary between the EPL and the glomerular layer (GL) but could also be found throughout the periglomerular region and the superficial half of the EPL. These cells had dendritic and axonal processes that extended predominantly into the GL and appeared to branch around and between individual glomeruli. The eighth class, periglomerular cells, had somata located throughout the GL. Most of these cells gave rise to one dendritic trunk that arborized within a single glomerulus. Occassionally a second, less elaborate dendritic process emerged from the main trunk or from the soma and extended into the periglomerular space or encroached upon an adjacent glomerulus. However, we did not find any periglomerular cells with extensive dendritic arbors in more than one glomerulus. These distributions are discussed in relation to the notion that olfactory bulb interneurons may be assigned to two functional groups associated with two distinct levels of integration.