Expression of the small conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channel,SK3, in the olfactory ensheathing glial cells of rat brain

Olfactory sensory neurons are wrapped by ensheathing glial cells in the olfactory nerve layer (ONL). Neither functional roles nor electrical properties of ensheathing glial cells have been, as yet, fully clarified. Four subunits (SK1–4) of small conductance Ca²⁺-activated K⁺ (SK) channels have been cloned. In the present study, immunohistochemical analyses showed that SK3 channels are expressed in ensheathing glial cells in the rat olfactory bulb, in addition to neuronal cells in other regions. Western blotting analysis demonstrated that SK3 was predominantly expressed in the olfactory bulb, thalamus, moderately in the hippocampus and cerebellum and modestly in the cerebral cortex of the rat brain. SK3 immunoreactivity was detected in the ONL of the olfactory bulb, neural cell body and fibers of the substantia nigra and hypothalamus. SK3 immunoreactivity was quite intense in the outer (superficial) part of the ONL. SK3-immunoreactive structures were overlapped with glial fibrillary acidic protein (GFAP), but not with vimentin, markers for glial cells and olfactory sensory axons, respectively. Immunoelectron microscopy showed that SK3 immunoreactivity was localized in thin processes that enfolded fascicles of immunonegative olfactory nerve axons. These results indicate that SK3 is expressed specifically in the olfactory ensheathing glial cells in olfactory regions.This work was supported in part by a Grant-in-Aid to A.F. for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by scholarship from Ono Pharmaceutical Company, and by Narishige Neuroscience Research Foundation.     

Expression of cell adhesion molecules in the olfactory system of the adult mouse: presence of the embryonic form of N-CAM

The expression of the neural cell adhesion molecules N-CAM and L1 was investigated in the olfactory system of the mouse using immunocytochemical and immunochemical techniques. In the olfactory epithelium, globose basal cells and olfactory neurons were stained by the polyclonal N-CAM antibody reacting with all three components of N-CAM (N-CAM total) in their adult and embryonic states. Dark basal cells and supporting cells were not found positive for N-CAM total. The embryonic form of N-CAM (E-N-CAM) was only observed on the majority of globose basal cells, the precursor cells of olfactory neurons, and some neuronal elements, probably immature neurons, since they were localized adjacent to the basal cell layer. Differentiated neurons in the olfactory epithelium did not express E-N-CAM. In contrast to N-CAM total, the 180-kDa component of N-CAM (N-CAM180) and E-N-CAM, L1 was not detectable on cell bodies in the olfactory epithelium. L1 and N-CAM180 were strongly expressed on axons leaving the olfactory epithelium. Olfactory axons were also labeled by antibodies to N-CAM180 and L1 in the lamina propria and the nerve fiber and glomerular layers of the olfactory bulb, but only some axons showed a positive immunoreaction for E-N-CAM. Ensheathing cells in the olfactory nerve were observed to bear some labeling for N-CAM total, L1, and N-CAM180, but not E-N-CAM. In the olfactory bulb, L1 was not present on glial cells. In contrast, N-CAM180 was detectable on some glia and N-CAM total on virtually all glia. Glia in the nerve fiber layer were labeled by E-N-CAM antibody only at the external glial limiting membrane. In the glomerular layer, E-N-CAM expression was particularly pronounced at contacts between olfactory axons and target cells. The presence of E-N-CAM in the adult olfactory epithelium and bulb was confirmed by Western blot analysis. The continued presence of E-N-CAM in adulthood on neuronal precursor cells, a subpopulation of olfactory axons, glial cells at the glia limitans, and contacts between olfactory axons and their target cells indicates the retention of embryonic features in the mammalian olfactory system, which may underlie its remarkable regenerative capacity.     

Responses of the astroglia in sensory deprived olfactory bulb of developing rats.

The effects of unilateral olfactory deprivation on the glial population during the olfactory bulb development have been studied. The lack of sensory stimulation has been found to be related to an increase in gliofibrillary acid protein (GFAP) in the three layers of the deprived bulbs. This increase is due to the higher number of astrocytes in the deprived bulb, which is much more noticeable in the plexiform layer than in the other two, together with a hypertrophy of the reactive astrocytes resulting in an increase in the number and thickness of their prolongations. Our results demonstrate that sensory olfactory deprivation acts as other noxius agents on the CNS, causing gliosis in the olfactory bulb. This gliosis is revealed by astrocytic hyperplasia and hypertrophy.     

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.     

The glucose transporter GLUT1 and the tight junction protein occludin in nasal olfactory mucosa.

The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.     

猫嗅球外丛层和僧帽细胞层年龄相关形态学变化

分别用Nissl法及免疫组织化学ABC法标记青、老年猫嗅球中嗅觉二级神经元和外丛层胶质细胞,显微镜下观察其分布并计数,对嗅觉二级神经元胞体直径和外丛层厚度进行测量,比较其年龄相关性变化,研究神经元与胶质细胞之间的关系,探讨老年性嗅觉功能衰退的相关神经机理。结果显示,老年猫嗅觉二级神经元胞体直径和分布密度均有不同程度的显著性下降(P<0.05);外丛层厚度变化不明显(P>0.05);外丛层胶质细胞特别是星形胶质细胞显著性增生(P<0.05)。表明在衰老过程中嗅觉二级神经元有丢失,并呈现功能下降,可能是老年性嗅觉功能衰退的原因之一。同时外丛层胶质细胞增生以进一步保护神经元,延缓其衰老。     

Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

Novel subdomains of the mouse olfactory bulb defined by molecular heterogeneity in the nascent external plexiform and glomerular layers

Background  

In the mouse olfactory system, the role of the olfactory bulb in guiding olfactory sensory neuron (OSN) axons to their targets is poorly understood. What cell types within the bulb are necessary for targeting is unknown. What genes are important for this process is also unknown. Although projection neurons are not required, other cell-types within the external plexiform and glomerular layers also form synapses with OSNs. We hypothesized that these cells are important for targeting, and express spatially differentially expressed guidance cues that act to guide OSN axons within the bulb.     

Association of substance-P-immunoreactive nerves with the murine olfactory mucosa

Location and distribution of nerve fibers immunoreactive to substance P were studied in the mouse olfactory mucosa. A moderately dense plexus of fibers is present at the interface of the olfactory epithelium and the connective tissue of the lamina propria. In addition, many immunoreactive nerve fibers are noted in close association with Bowman's glands and blood vessels in the lamina propria. However, such fibers were not observed in olfactory epithelium proper nor in the fila olfactoria. Substance-P-immunoreactivity is almost totally abolished by treatment of animals with capsaicin, an agent known to deplete substance P from primary sensory neurons. It is suggested that the substance-P-immunoreactive fibers are of sensory origin, with their perikarya most likely located in the trigeminal ganglia. Functionally, they might influence local blood flow and/or the secretion of Bowman's glands.     

An ultrastructural and immunohistological study of the rat olfactory epithelium: Unique properties of olfactory sensory cells

The olfactory epithelium contains three cell types: basal cells, supporting cells and sensory neurons. Electron microscopy as well as immunofluorescence microscopy with intermediate-filament antibodies were used to study the rat olfactory epithelium in order to obtain more information about these different cell types and to try to investigate their histogenetic origins. We found mitoses in the basal-cell layer, as well as multiple centrioles and tonofilaments in some basal cells. As revealed by electron microscopy, the supporting cells contained tonofilaments and reacted strongly with antibodies to keratin, in line with their known epithelial nature. When antibodies to other intermediate-filament types were used, i.e. glial fibrillary acidic protein, vimentin, desmin and neurofilaments, no reaction was seen in the cells of the olfactory epithelium, with the exception of occasional staining of a few axons in the subepithelial layer by neurofilament antibodies. In particular, the cell bodies, dendrites and most axons of the sensory neurons were negative for a variety of antibodies against neurofilaments. Olfactory sensory neurons therefore belong to the very few cells in adult animals which seem to lack intermediate filaments. We discuss whether this finding is related to the fact that these cells are also unique among neurons in that they are not permanent cells but constantly turn over.     

Volumetric and horseradish peroxidase tracing analysis of rat olfactory bulb following reversible olfactory nerve lesions

Olfactory receptor neurons can regenerate from basal stem cells. Receptor neuron lesion causes degenerative changes in the olfactory bulb followed by regeneration as new olfactory receptor axons innervate the olfactory bulb. To our knowledge, parametric analyses of morphometric changes in the olfactory bulb during degeneration and regeneration do not exist except in abstract form. To better characterize olfactory bulb response, we performed morphometric analysis in rats following reversible olfactory nerve lesion with diethyldithiocarbamate. We also performed anterograde tracing of the olfactory nerve with wheatgerm agglutinin linked to horseradish peroxidase. Results of morphometry and tracing were complementary. The glomerular layer and external plexiform layer showed shrinkage of 45 and 26%, respectively, at 9 days. No significant shrinkage occurred in any other layer. Individual glomeruli shrank by 40-50% at 3 and 9 days following lesion. These data show that degenerative changes occur both in the glomeruli and transneuronally in the external plexiform layer. Olfactory nerve regeneration (identified by WGA-HRP transport) paralleled volumetric recovery. Recovery occurred first in ventral and lateral glomeruli between 9 and 16 days followed by recovery in medial and dorsal glomeruli. These data indicate substantial transynaptic degeneration in the olfactory bulb and a heretofore unrecognized gradient in olfactory nerve regeneration that can be used to systematically study recovery of a cortical structure.     

Structural and immunohistological modifications in olfactory bulb of the staggerer mutant mouse.

In the present study, we describe the structural and cytological changes observed in staggerer mutant olfactory bulbs, as compared to normal mice. On the basis of photonic and ultrastructural observations we tried to define the alterations induced by the mutation: i.e. a reduction of bulb size, a reduction in the volume of three out of the six architectonic layers (glomerular, external and internal plexiform), a reduction of glomeruli size, a loss of half the mitral cells and a slight decrease in juxtaglomerular interneuron number. In staggerer, an hypertrophy of glial ensheathing cell processes was especially evident at the level of each glomerulus, whereas the density of the astrocyte network was weaker in the granular layer and the nerve layer not apparently impaired. An immunofluorescent labelling study combined with confocal scanning microscopy was performed in order to identify the cellular type and the differentiation degree of the various elements. Antibodies anti-GFAP, a protein present in both ensheathing cells and astrocytes, and anti-OMP, the specific maturation protein of the nerve layer, were used for that purpose. Data confirmed the reality of the gliosis and the persistence of the sensory component in the mutant. All the structural alterations described in staggerer olfactory bulb were in close agreement with the functional troubles previously recorded. Our results are discussed in connection with the present knowledge on embryonal origin, fetal development and adult cellular renewal of the olfactory bulb.     

Neuron-specific enolase,neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

Summary Immunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.     

Immunohistochemical Studies of the Cellular Changes in the Peripheral Olfactory System After Zinc Sulfate Nasal Irrigation

The peripheral olfactory system has a remarkable capacity for repair. We have performed an immunohistochemical study of the cellular changes that occur after zinc sulfate irrigation of the nasal cavity. The rapid loss of epithelial cells was followed by the proliferation of basal cells and the restoration of the epithelium with olfactory tissue. Horizontal basal cell markers, anti-cytokeratin 5/6 (CK5/6), and the Bandeiraea simplicifolia (BS-1) lectin initially co-localized on day 1 after treatment but rapidly displayed a disparity in their staining profile, with CK5/6 immunoreactive cells having a profile more akin to cells expressing the sustentacular marker cytokeratin 18 (CK18). This suggests CK5/6 and BS-1 label a different subset of horizontal basal cells. Axonal degeneration and regeneration was studied with a panel of markers to olfactory receptor neurons, their terminals, and olfactory bulb dendrites. The glial cells of the peripheral olfactory system, olfactory ensheathing cells, remained in position, with little change in immunoreactivity to laminin, although an increase in the expression of glial fibrillary acidic protein was observed. The events and the extent of reconstitution of the olfactory system after degeneration serves as a foundation for future studies designed to understand the unique regenerative capacity of the olfactory system.