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     

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.     

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.     

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis

Summary In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects on olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) – 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) – 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) – 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70–80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) – 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) – 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.     

1-甲基-4-苯基-1,2,3,6-四氢吡啶对食蟹猴嗅球多巴胺能神经元的影响

目的 建立食蟹猴1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)帕金森病系统性模型,探讨嗅球中多巴胺（DA）及多巴胺/cAMP调节磷蛋白（DARPP32）的表达情况。
方法 3只成年健康食蟹猴,静脉注射MPTP,建立帕金森病系统性模型,取出嗅球,切片,免疫组织化学染色DA和DARPP32,摄片并观察DA和DARPP32在食蟹猴嗅球中的分布及表达情况,采用Image Pro-Plus软件,半定量分析模型组和正常组之间DA和DARPP32的表达差异。 结果 食蟹猴嗅球中DA和DARPP32神经元集中分布于突触小球层,DA神经纤维分布于突触小球层,而DARPP32神经纤维分布于嗅球各层,以突触小球层（GL）和外网状层（EPL）最为密集。MPTP损伤后,与正常对照组比较,DA和DARPP32神经元及神经纤维均减少,以DA神经元及神经纤维减少明显。 结论 食蟹猴嗅球中DA神经元和神经纤维分布于突触小球层。食蟹猴MPTP帕金森病系统性模型的嗅球DA能神经元和纤维明显减少,可能与帕金森病嗅觉障碍有关。     

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.     

The bilateral bulbar projections of the primary olfactory neurons in the frog

Summary Whether or not the frog olfactory neuroreceptor cells project bilaterally to the olfactory bulb is still a debated question. We therefore decided to ascertain whether bilateral projections of the primary olfactory input exist and if so to investigate their extent. Reproducible extracellular bilateral bulbar potentials were recorded in the frog following electrical stimulation of dorsal or ventral olfactory nerve bundles. The general features of the contralateral evoked responses were very similar to those of the ipsilateral response. The contralateral response disappeared after transection of the rostral part of the olfactory interbulbar adhesion but not following transection of the habenular or anterior commissures. Horseradish peroxidase labelling showed that the fiber terminations of the olfactory nerve bundle was not restricted to the ipsilateral olfactory bulb but included the medial aspects of the contralateral bulb. The intertelencephalic sections increased the magnitude of the ipsilateral evoked responses. Olfactory bulb isopotential maps revealed a rough topographical correspondence between the olfactory neuroepithelium and bulb along the medio-lateral axis as well as along the dorso-ventral axis. In addition, a projection of the medial and central part of the olfactory sac to the medial part of the contralateral olfactory bulb through the interbulbar adhesion was confirmed. These findings suggest first, that the fibers from the neuro-receptors located in either the ventral or the dorsal olfactory mucosae project to both olfactory bulbs, and second, that the left and right bulbs exert a constant inhibition on each other via the habenular commissure.Abbreviations AON anterior olfactory nucleus - ax olfactory neuroreceptor axon - BA bulbar adhesion - D_I latero-dorsal olfactory nerve bundle - D_II centro-dorsal olfactory nerve bundle - D_III mediodorsal olfactory nerve bundle - EPL external plexiform layer - GL glomerular layer - gl glomerulus - GRL granular cell layer - MOB main olfactory bulb - m mitral cell - MBL mitral cell body layer - ON olfactory nerve - V lateral ventricule - V_I latero-ventral ol-factory nerve bundle - V_II centro-ventral olfactory nerve bundle - V_III medio-ventral olfactory nerve bundle - VN vomero-nasal nerve     

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.     

帕金森病MPTP食蟹猴模型嗅球细胞凋亡

目的 建立1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶（MPTP）帕金森病（PD）模型,探讨模型组嗅球细胞凋亡和胶质细胞增生情况。方法 3只成年健康食蟹猴,静脉注射MPTP建立PD模型,另3只静脉注射生理盐水作为对照。取出嗅球,免疫组织化学染色检测Caspase-3、Bcl-2、离子钙接头蛋白分子1（Iba-1）和胶质纤维酸性蛋白（GFAP）在食蟹猴嗅球中的表达情况,采用Image J v1.8.0软件分析模型组和对照组之间的差异。 结果 MPTP损伤后,与对照组相比,模型组嗅球突触小球层Caspase-3阳性细胞数明显增加,而Bcl-2表达减少;与对照组相比,模型组嗅球突触小球层和外网状层的GFAP和Iba-1阳性细胞数增加。 结论 MPTP可诱导食蟹猴嗅球突触小球层细胞凋亡,并伴有星形胶质细胞增生和小胶质细胞激活,这可能与帕金森病的功能障碍有关。     

Localization of C-PON immunoreactivity in the rat main olfactory bulb. Demonstration that the population of neurons containing endogenous C-PON display NADPH-diaphorase activity

The presence of the neuropeptide C-terminal flanking peptide of neuropeptide-Y, C-PON, has been investigated in the main olfactory bulb of the rat using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. The distribution of immunoreactive structures to C-PON was examined in both horizontal and coronal sections. Endogenous C-PON was localized within two types of short-axon cells including (1) superficial short-axon cells in the glomerular layer and (2) deep short-axon cells lying in the deepest portion of the granule cell layer and in the adjacent white matter. In addition, varicose immunoreactive processes were detected in all layers, although they were more numerous in the deepest portion of the granule cell layer. Immunoreactive cell bodies and processes were also observed in the nucleus olfactorius anterior and in the intrabulbar portion of the anterior commissure. Nevertheless, immunoreactive structures were not localized in the lateral olfactory tract. The indirect immunofluorescence technique to detect endogenous C-PON in combination with the enzyme histochemical demonstration of NADPH-diaphorase activity, in single sections, showed that the NADPH-diaphorase procedure is a reliable marker for these C-PON positive cells. Also, indirectly, that, in the rat main olfactory bulb, C-PON and neuropeptide-Y are contained in the same cell types. Many glomeruli were stained following the NADPH-diaphorase procedure, but they were not C-PON immunoreactives. Results of this study provide evidence suggesting that C-PON may influence polysynaptically the function of mitral cells and, therefore, the olfactory bulb output.     

Zincergic innervation from the anterior olfactory nucleus to the olfactory bulb displays plastic responses after mitral cell loss

Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.     

Zincergic innervation from the anterior olfactory nucleus to the olfactory bulb displays plastic responses after mitral cell loss

Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.     

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.     

Morphological characteristics of cultured olfactory bulb cells

Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types in the intact adult mouse olfactory bulb. Identified cultured cells included mitral/tufted cells, granule cells, short-axon cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters consisted of a loose array of neurons that appeared to be densely interconnected by neuntes. However, few neurites or fascicles emanated from clusters to adjoining areas. Aggregates consisted of many small, usually rounded, neurons piled on top of one larger neuron, or on more than one, with typically many neuntes and fascicles projecting to adjacent aggregates, clusters or individual neurons. Neurites of cultured olfactory bulb cells were well developed, and some were several millimeters long. Synapses were very prominent in these cultures, especially in aggregates, clusters, and fascicles. Electron-lucent, dense-core, and coated vesicles were present. Polarity, shape, and length of the long axis (size) of 815 cultured neurons, identified by positive anti-microtubule-associated protein 2 staining, were documented. Cultured neurons varied in size from 9 to 27 μm, with an average size of 16 μm. Elliptical bipolar (35%), triangular multipolar (21%), and round unipolar (15%) were the most common polarity/shape combinations found in culture. Multipolar, triangular, triangular multipolar, and elliptical bipolar cells increased in size with increasing age of culture. The relative proportions of triangular, multipolar, elliptical multipolar, and triangular multipolar cells decreased, whereas the relative proportions of round, unipolar, and round unipolar cells increased with increasing age of culture. These changes in population subtypes and cell size may indicate continued differentiation and maturation of cultured neurons.     

Serial reconstruction of Lucifer yellow-labeled supraoptic nucleus neurons in perfused rat hypothalamic explants

Intracellular recordings from supraoptic nucleus neurons in perfused explants of rat hypothalamus were followed by intracellular injections of the fluorescent dye, Lucifer yellow. Following fixation, 40 microns sections were processed for whole cell light-microscopic reconstruction in the horizontal or coronal plane. The somata of most supraoptic neurons were elongated (mean 25 X 13 microns) with 1-3 sparsely branched dendrites (length 30-725 microns) that displayed numerous spines. Most (95%) dendrites turned in the ventral direction to end in the glial lamina along the base of the nucleus. Each neuron had one axon: in 60% of cells, the axon arose from a dendritic profile and immediately assumed a varicose appearance; in the other 40% of cells, the axon appeared to arise directly from the soma and demonstrated in its initial 80-200 microns numerous spines and few varicosities, i.e. the morphological features of a dendrite. All axons coursed in a dorsomedial direction over the optic tract. At this point, most axons revealed smaller secondary processes 2-15 microns in length. Axons then turned ventrally towards the basal hypothalamus; some could be followed for up to 2100 microns from the cell somata. This approach to the light microscope morphology of supraoptic neurons provides a surprising array of detail on soma, dendrite and axon characteristics, while retaining the overall relationship between individual neurons and neighboring structures, including the boundaries of the nucleus itself.     

A light and electron microscopic study of taurine-like immunoreactivity in the main olfactory bulb of frogs

The distribution of taurine in the frog olfactory bulb was studied using light and electron microscopic immunohistochemical techniques. At the light microscopic level, taurine-like immunoreactivity (taurine-LI) was found in (i) fibers coursing from the olfactory nerve layer to the glomerular layer, (ii) cell bodies and processes primarily located in the caudal part of the granule cell layer (GCL), and (iii) puncta outlining unstained somata of mitral cells and cells in the GCL. In consecutive sections processed for taurine or GABA, numerous cells of the caudal GCL displayed taurine-LI and GABA-like immunoreactivity (GABA-LI). A bimodal distribution of the cross-sectional cell area for GABA-LI cells implied their morphological diversity, and the peak for larger GABA-LI cells coincided with the maximum for taurine-LI cells. At the electron microscopic level, single immunogold labeling showed that GABA-LI, but not taurine-LI, is present in granule cells, whereas both taurine-LI and GABA-LI were localized in a ‘non-granule’ type of cell. The double labeling procedure demonstrated coexistence of taurine-LI and GABA-LI in neurons of a ‘non-granule’ type. These cells had some ultrastructural features typical of short axon cells in the GCL of the mammalian olfactory bulb and were tentatively considered as short axon-like cells. Results suggest that, in the frog olfactory bulb, taurine is contained in primary olfactory afferents and short axon-like cells of the GCL co-localizing GABA and taurine.     

Supraoptic nucleus afferents from the main olfactory bulb--I. Anatomical evidence from anterograde and retrograde tracers in rat

The morphological features of a putative connection between the main olfactory bulb and the supraoptic nucleus of the rat was studied using a combination of anatomical techniques. Immunocytochemistry of neurophysin-containing processes were employed to delineate morphological features of supraoptic dendrites. Main olfactory bulb efferents to the supraoptic nucleus were studied by injection of the anterogradely transported substances, wheatgerm agglutinin conjugated horseradish peroxidase or Phaseolus vulgaris leucoagglutinin, into the main olfactory bulb. To confirm the results of these studies, the distribution of retrogradely labeled cells within the main olfactory bulb was determined after injection of rhodamine-labeled latex microspheres or Fluoro-Gold into the supraoptic nucleus. Neurophysin immunocytochemistry revealed the supraoptic nucleus dendritic plexus which coursed anteroposteriorly beneath supraoptic somata. Additionally, a portion of this plexus also projected ventrolaterally into periamygdaloid areas, a feature of supraoptic architecture which is not generally appreciated. The anterograde tracers labeled main olfactory bulb efferents including a dense plexus of terminals and fibers ventrolateral to the ipsilateral supraoptic nucleus. The pattern of anterogradely labeled fibers and terminals appeared to overlap with the distribution of ventrolaterally projecting neurophysin-containing processes. Since the latter consists of dendritic processes of supraoptic origin, this suggests that the main olfactory bulb projects to the supraoptic nucleus. Injections of rhodamine-labeled latex microspheres or Fluoro-Gold resulted in retrogradely labeled mitral cells throughout the ipsilateral main olfactory bulb. Taken together, these anatomical studies demonstrate a direct projection from the main olfactory bulb to the supraoptic nucleus of the rat. A comparison electrophysiological study confirmed these results.     

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.     

Ultrastructural identification of substance P immunoreactive neurons in the main olfactory bulb of the hamster

The neurons containing substance P immunoreactivity in the main olfactory bulb of the hamster are located in the glomerular layer. Their cell bodies lie in the periglomerular region and contain spherical or ovoid nuclei which lack invaginations of the nuclear membrane and tend to be positioned eccentrically in the cell body. Dendrites of these neurons extend throughout the periglomerular region and project into the glomerular neuropil. Within the glomerular neuropil, processes with substance P immunoreactivity contain agranular, spherical synaptic vesicles. Primary olfactory axons, and processes of uncertain origin which contain pleomorphic synaptic vesicles, form synaptic contacts with substance P immunoreactive processes.These ultrastructural findings confirm that the substance P immunoreactive neurons are external tufted cells. Their likely physiological properties are considered in relation to the synaptic organization in the glomerular layer of the main olfactory bulb and to the other putative neurotransmitters or neuromodulators located in this layer.