Primary Culture of Adult Mouse Olfactory Receptor Neurons

Olfactory receptor neurons (ORNs) are unique because they can be replaced by stem cells throughout life. Previous studies have demonstrated that adult mouse olfactory epithelium (OE) injured by exposure to ZnSO₄through nasal irrigation can stimulate stem cell mitotic activityin situ, which continues when placed in culture. We report on an improved ZnSO₄delivery method, mist inhalation, which produces more consistent and greater yields of OE cells. Cultures established following this method contained bipolar, nest, fusiform, and giant cells. The bipolar cells usually underwent asymmetric process development. Some bipolar cells reacted positively to neuron-specific antibodies and were immunonegative for keratin and glia-specific proteins, suggesting that they were ORNs. Those that were negative for the neuron-specific proteins may represent either neuron progenitors or olfactory ensheathing cells. The fusiform cells were relatively small and undifferentiated, exposure to brain-derived neurotrophic factor resulted in their decrease and an increase in bipolar cells. Therefore, they might be the stem cells. The nest cells had morphological characteristics of epithelia and bound keratin antibodies. The giant cells had the morphology of epithelial cells but were negative for keratin; they may represent a unique cell population induced by the ZnSO₄. These results indicate that the major cell types of intact OE are present in our cultures, and each retains characteristics foundin situ. The mist inhalation method provides anin vitropopulation of adult mitotically active neurons for study.     

Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures

In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes. Central to this approach is a rigorous characterization of the cells that reside in culture. Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from both the epithelium and lamina propria). To identify olfactory receptor neurons in the cultures, staining with antibodies against neuron-specific tubulin was compared to staining with antibodies to neuron-specific enolase, the neural cell adhesion molecule, N-CAM, and the adhesion molecule, Ll. Staining of mature olfactory neurons in culture, with an antibody against the olfactory marker protein, was compared to staining with antibodies to carnosine. In contrast to tissue section staining, the overlap between carnosine and olfactory marker protein staining was not complete. Olfactory nerve glial cells were immunoreactive for the S100β protein and nestin, an intermediate filament found in early neuronal progenitor cells and Schwann cells. Antibodies to nestin did not label olfactory neurons or progenitor cells. An antibody to an oligodendrocyte-Schwann cell enzyme, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, did not label olfactory glia, but did label oligodendrocyte-like cells that appeared to be derived from the CNS glial feeder layer. An antibody against the heavy (200 kDa) neurofilament protein stained a minor subset of cells. The cultures also contained muscle cells, cartilage cells and macrophages (and/or microglia). These results demonstrate that multiple cell types either maintain or re-establish differentiated, cell type-specific phenotypes in dissociated olfactory cell cultures.     

Rat olfactory neurons express a 200 kDa neurofilament

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.     

Expression of stathmin and SCG10 proteins in the olfactory neurogenesis during development and after lesion in the adulthood

Stathmin and SCG10 belong to a family of phosphoproteins associated to cell proliferation and differentiation. In the present study, we have analyzed immunocytochemically the distribution of these proteins during neurogenesis in the mouse olfactory system, from midgestation to adulthood. Data show that already at embryonic day 12, stathmin and SCG10 immunoreactivities were present in the olfactory and vomeronasal neurons, and their number increased greatly, colocalizing with neuronal specific tubulin, a marker of immature neurons. Later on up to adulthood, the distribution of stathmin and SCG10 became progressively restricted to a few immature receptor and chemosensory neurons. Significantly, in the olfactory epithelium, stathmin was seen in immature neurons and also in basal cells representing precursors of neuronal elements. Interestingly, before birth stathmin and SCG10 immunopositive cells were seen outside the olfactory epithelium, seemingly migrating toward the olfactory bulb. After regeneration in the adult following peripheral lesion of the olfactory epithelium, stathmin and SCG10 were again strongly expressed and generally colocalized with neuronal specific tubulin immunoreactivity. Overall these results indicate that stathmin and SCG10 are expressed in immature olfactory neurons as well as in the migrating cells generated from the olfactory epithelium, supporting the role of these proteins in neurogenesis and cell migration.     

Phosphorylated neurofilament expression and resistance to kainate toxicity

Antibodies directed against phosphorylated neurofilaments, which are major proteins of the neuronal cytoskeleton, usually do not label neuronal cell bodies except in some neurological diseases. In the present study, we show that in rat cortical cell cultures exposed to kainate there is an inverse relation between neuronal survival and the proportion of neuronal cell bodies stained by a monoclonal antibody (clone SMI₃₁) that recognizes extensively phosphorylated neurofilament proteins (150 kDa and 200 kDa). The immunoblot analysis also revealed an increase in 150-kDa phosphorylated neurofilament expression in kainate-treated cell cultures. Furthermore, the direct quantification of viable neurons SMI₃₁-immunopositive or immunonegative in perikarya showed that the majority of neurons resistant to kainate toxicity expressed phosphorylated neurofilaments in their cell bodies. The percentage of viable neurons displaying SMI₃₁-immunoreactivity in their cell bodies increased from 14.7% in control cultures to 30.0% in cultures treated with 10 μM kainate. These data suggest that phosphorylated neurofilament expression is associated with a reduced cell vulnerability to excitotoxicity induced by kainate.     

FGF2 promotes neuronal differentiation in explant cultures of adult and embryonic mouse olfactory epithelium

Neurogenesis in the adult olfactory epithelium is highly regulated in vivo. Little is known of the molecular signals which control this process, although contact with the olfactory bulb or with astrocytes has been implicated. Explants of mouse olfactory epithelium were grown in the presence or absence of several peptide growth factors. Basic fibroblast growth factor (FGF2) stimulated differentiation of sensory neurons in adult and embryonic olfactory epithelium. Other growth factors tested were ineffective. FGF2-stimulated neurons were born in vitro and expressed neurofilament, neural cell adhesion molecule, and β-tubulin. The cells also expressed olfactory marker protein, a marker for mature olfactory sensory neurons in vivo. These bipolar neurons did not express glial fibrillary acidic protein or low-affinity nerve growth factor receptor. These results indicate that neither astrocytes nor olfactory bulb are necessary for differentiation of olfactory sensory neurons in vitro. © 1996 Wiley-Liss, Inc.     

Olfactory epithelial organotypic slice cultures: A useful tool for investigating olfactory neural development

Anin vitro slice culture was established for investigating olfactory neural development. The olfactory epithelium was dissected from embryonic day 13 rats; 400μm slices were cultured for 5 days in serum-free medium on Millicell-CM membranes coated with different substrates. The slices were grown in the absence of their appropriate target, the olfactory bulb, or CNS derived glia. The cultures mimic many features ofin vivo development. Cells in the olfactory epithelium slices differentiate into neurons that express olfactory marker protein (OMP). OMP-positive cells have the characteristic morphology of olfactory receptor neurons: a short dendrite and a single thin axon. The slices support robust axon outgrowth. In single-label experiments, many axons expressed neural specific tubulin, growth-associated protein 43 and OMP. Axons appeared to grow equally well on membranes coated with type I rat tail collagen, laminin or fibronectin. The cultures exhibit organotypic polarity with an apical side rich in olfactory neurons and a basal side supporting axon outgrowth. Numerous cells migrate out of the slices, of which a small minority was identified as neurons based on the expression of neural specific tubulin and HuD, a nuclear antigen, expressed exclusively in differentiated neurons. Most of the migrating cells, however, were positive for glial fibrilary acidic protein and S-100, indicating that they are differentiated glia. A subpopulation of these glial cells also expressed low-affinity nerve growth factor receptors, indicating that they are olfactory Schwann cells. Both migrating neurons and glia were frequently associated with axons growing out of the slice. In some cases, axons extended in advance of migrating cells. This suggests that olfactory receptor neurons in organotypic cultures require neither a pre-established glial/neuronal cellular terrain nor any target tissue for successful axon outgrowth. Organotypic olfactory epithelial slice cultures may be useful for investigating cellular and molecular mechanisms that regulate early olfactory development and function.     

Transection of the rat olfactory nerve increases glial fibrillary acidic protein immunoreactivity from the olfactory bulb to the piriform cortex

Astrocytic glial fibrillary acidic protein (GFAP) immunoreactivity in response to retrograde changes of motoneurons after axotomy has been the subject of a number of reports. In contrast, this study examined the astrocytic GFAP immunoreactivity in response to axotomy in a sensory system, the adult rat olfactory system. The purpose of this study was to determine, by immunolabeling GFAP, the extent and transience of astrocytic reactivity in the olfactory system. Unilateral transection of the olfactory nerve fascicles was performed intracranially at the level of the cribriform plate. Rats were allowed to survive from 24 hours to 1 month after axotomy. GFAP immunolabeling was examined throughout the rat olfactory system using the peroxidase-anti-peroxidase method. After axotomy, a transient increase occurred in the astrocytic GFAP immunoreactivity in the ipsilateral olfactory system. The greatest enhancement of GFAP immunoreactivity in the olfactory system occurred at 48 hours post-axotomy. Increased GFAP immunoreactivity occurred not only along the axons and synaptic endings of the injured primary olfactory neurons, but also along the dendrites, cell bodies, axons, and synaptic endings of the secondary sensory neurons. The increased GFAP immunoreactivity was specifically associated with the anatomical distribution pathways of the primary and secondary olfactory neurons. Increased GFAP immunoreactivity was not altered until 14 days post-axotomy. At 1 month post-axotomy, GFAP immunoreactivity returned to control levels. The time course and transience of increased GFAP immunoreactivity closely correlates with the time course of rat primary olfactory neuronal degeneration and regeneration after axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione levels in olfactory and non-olfactory neural structures of rats

Olfactory receptor neurons are a CNS entry point for a wide variety of airborne substances. Therefore, it is probable that detoxification mechanisms are present in these neurons to neutralize such agents. Glutathione (GSH) is an essential component of several detoxification schemes, and in this study we examined the distribution and levels of GSH in the olfactory epithelium, olfactory bulb, cortex, hippocampus and cerebellum in neonatal, weanling, adult and aged rats. We report that GSH is primarily localized to the olfactory receptor neurons and their oxons within the olfactory epithelium. It is also localized within the glomerular neuropil and granule cells of the olfactory bulb. Levels of GSH in the olfactory epithelium and hippocampus do not change as a function of age, although GSH levels decrease in several brain regions, including the olfactory bulb, cerebellum and cortex.     

Structure and function of long-lived olfactory organotypic cultures from postnatal mice

The first synapse in the olfactory pathway mediates a significant transfer of information given the restricted association of specific olfactory receptor neurons with specific glomeruli in the olfactory bulb. To understand better how this connection is made and what the functional capacities of the participating cells are, we created a long-lived culture system composed of olfactory epithelium and olfactory bulb tissues. Using the roller tube method of culturing, we grew epithelium-bulb cocultures, explanted from 1-4-day-old Swiss Webster mice, on Aclar for periods ranging from 18 hr to 68 days. The explants flattened so that in some areas the culture was only a few cells thick, making individual cells distinguishable. From 107 cultures studied, we identified the following cell types by expression of specific markers (oldest culture expressing marker, days in vitro, DIV): olfactory receptor neurons (neural cell adhesion molecule, 42 DIV); mature receptor neurons (olfactory marker protein, 28 DIV); postmitotic olfactory receptor neurons and olfactory bulb neurons (beta-tubulin, 68 DIV); astrocytes (glial fibrillary acidic protein, glutamate/aspartate transporter, 68 DIV); olfactory horizontal basal cells (cytokeratin, 22 DIV). Neuronal processes formed glomeruli in 2-4-week-old cultures. We also recorded electro-olfactography responses to puffs of vapor collected over an odorant mixture containing ethyl butyrate, eugenol, (+) carvone, and (-) carvone from cultures as old as 21 DIV. These features of our olfactory culture system make this model useful for studying properties of immature and mature olfactory receptor neurons, pathfinding strategies of receptor axons, and mechanisms of information transfer in the olfactory glomerulus.     

Esthesioneuroepithelioma: a tumor of true olfactory epithelium origin

Summary A case of esthesioneuroepithelioma was investigated ultrastructurally and immunohistochemically, using antibodies against neurofilament protein (NFP), glial fibrillary acidic protein (GFAP), keratin, neuron-specific enolase (NSE), S-100 protein (S-100), and tyrosine hydroxylase (TH). The tumor initially manifested as an epidural mass in the anterior cranial fossa in a 64-year-old man, and about 31/2 years later, autopsy further revealed extensive metastases to the lymph nodes of the neck and thoracic cavity. In the cranial and nasal cavities, the tumor was composed of fairly uniform, ill-defined cells arranged in nests which were surrounded by a fibrovascular stroma. These histological features were reproduced in the metastatic tumor nodules with frequent occurrence of tubular arrangements of the tumor cells. Ultrastructurally, two different cell types were well recognized by their characteristic morphological features, which were reminiscent of sensory neurons and sustentacular cells of the olfactory epithelium. No dense-cored secretory granules were observed in the tumor cells. Immunohistochemically, the tumor showed a variable number of cells positive for NFP, keratin, NSE and S-100. NFP was present in a relatively small number of cells, which were found diffusely in the nests. Keratin was observed in the cells mainly located at the periphery. NSE-positive cells tended to form irregular clusters in the center. A few S-100-positive cells were found, without any particular arrangement. These findings indicated that the present tumor, which actually arose in the superior nasal cavity, consisted of cells differentiating in at least two distinct directions, neuronal and epithelial, and strongly suggested that the tumor was of true olfactory epithelium origin, or more precisely, derived from the bipotential, undifferentiated basal cells of this epithelium.     

Continuous culture of neuronal cells from adult human olfactory epithelium

Cells from the olfactory epithelium of adult human cadavers have been propagated in primary culture and subsequently cloned. These cells exhibit neuronal properties including: neuron-specific enolase, olfactory marker protein, neurofilaments, and growth-associated protein 43. Simultaneously, the cells exhibit nonneuronal properties such as glial fibrillary acidic protein and keratin, the latter suggesting properties of neuroblasts or stem cells. These clonal cultures contain 5-10% of cells sufficiently differentiated to show odorant-dependent cyclic adenosine 3',5'-monophosphate (cAMP) or calcium-release responses when challenged with submicromolar concentrations of odorants. The potential of culturing neuronal cells from patients with neuropsychiatric disorders, such as Alzheimer's disease or schizophrenia, could enable the study of the pathophysiology of these neurons in the culture dish and allow new approaches to the study of mental illness.     

Characteristics of odorant elicited calcium changes in cultured human olfactory neurons

An important step in establishing and utilizing a cell culture system for the in vitro study of olfaction is assessing whether the cultured cells possess physiological properties similar to those of mature olfactory neurons. Various investigators have successfully established proliferating cell lines from olfactory tissue, but few have demonstrated the characteristics of odor sensitivity of these cells. We successfully established cultured cell lines from adult human olfactory tissue obtained using an olfactory biopsy procedure and measured their ability to respond to odor stimulation using calcium imaging techniques. A subset of the human olfactory cells in culture displayed a distinct morphology and specifically expressed immunocytochemical markers characteristic of mature human olfactory neurons such as OMP, G(olf), NCAM and NST. Under defined growth conditions, these cultured cells responded to odorant mixes that have been previously shown to elicit intracellular calcium changes in acutely-isolated human olfactory neurons. These odorant-elicited calcium responses displayed characteristics similar to those found in mature human olfactory neurons. First, cultured cells responded with either increases or decreases in intracellular calcium. Second, increases in calcium were abolished by removal of extracellular calcium. Third, inhibitors of the olfactory signal transduction cascades reversibly blocked these odorant-elicited intracellular calcium changes. Our results demonstrate that cultures of adult human olfactory cells established from olfactory biopsies retain some of the in vivo odorant response characteristics of acutely isolated cells from the adult olfactory epithelium. This work has important ramifications for investigation of olfactory function and dysfunction using biopsy procedures and in vitro assays of odor sensitivity.     

Neurons of the olfactory epithelium in adult rats contain vimentin

In the developing nervous system, the intermediate filament protein vimentin is found in the proliferating neuroepithelium and neural crest. As development proceeds, postmitotic neurons cease vimentin expression and neurofilament proteins begin to accumulate. We have shown that olfactory receptor neurons deviate from the general pattern of neuronal intermediate filament expression, in that they continue to express vimentin or a highly vimentin-like protein rather than neurofilament proteins in the adult rat. With light-microscopic immunohistochemistry, three independently derived antibodies to vimentin label all portions of the primary olfactory projection, including the sensory neuron cell bodies in the olfactory epithelium, the fascicles of the olfactory nerve, and their axonal arbors in the glomeruli of the olfactory bulb. In contrast, anti-neurofilament antisera stain only rare scattered receptor cells and a small number of axons in the olfactory nerve. Electron-microscopic immunohistochemistry shows dense staining of olfactory axons with anti-vimentin. The vimentin-like immunoreactive material in the olfactory nerve layer was characterized by SDS-PAGE and by immunoblotting. On immunoblots of homogenates of the olfactory nerve, the anti-vimentin monoclonal antibody SBV-21 (Blose et al., 1984) stains only a single protein of Mr = 55 kDa. This band comigrates with vimentin in crude cytoskeletal material from the neonatal rat brain prepared according to the method of Dahl et al. (1981). SBV-21 does not stain neurofilament triplet proteins or glial fibrillary acidic protein, which are also present in these blots. These results demonstrate that the vast majority of olfactory receptor neurons and their axons contain vimentin or a protein of similar immunological character and electrophoretic mobility, while identifiable expression of neurofilament proteins is confined to a very small subpopulation. Hence, the switch in intermediate filament proteins that normally accompanies neuronal maturation is arrested in most olfactory neurons, and a "juvenile" biochemical marker is retained. This population of neurons is also unique among mammalian neurons in several other respects, including that olfactory neurons die during normal adult life or following injury and then are replaced from a proliferating pool of stem cells.     

Amphibian olfactory receptor neurons express olfactory marker protein

Expression of olfactory marker protein (OMP) in olfactory receptor neurons (ORNs) in two amphibians was investigated by immunohistochemical methods. The OMP immunoreactivity was observed in the cilia, apical dendritic knobs, dendrites and somas of ORNs; the axons of ORNs also showed intense immunoreactivity for OMP throughout their course from the olfactory epithelium to the glomerular layer of the olfactory bulb. Seven days after olfactory nerve transection in salamander, the number of OMP-positive ORNs was markedly reduced in the ipsilateral epithelium. The results demonstrate that amphibian ORNs express OMP and confirm its phylogenetic conservation across diverse species.     

The generation of olfactory epithelial neurospheres in vitro predicts engraftment capacity following transplantation in vivo

The stem and progenitor cells of the olfactory epithelium maintain the tissue throughout life and effectuate epithelial reconstitution after injury. We have utilized free-floating olfactory neurosphere cultures to study factors influencing proliferation, differentiation, and transplantation potency of sphere-grown cells as a first step toward using them for therapeutic purposes. Olfactory neurospheres form best and expand most when grown from neonatal epithelium, although methyl bromide-injured or normal adult material is weakly spherogenic. The spheres contain the full range of epithelial cell types as marked by cytokeratins, neuron-specific antigens, E-cadherin, Sox2, and Sox9. Globose basal cells are also prominent constituents. Medium conditioned by growth of phorbol ester-stimulated, immortalized lamina propria-derived cells (LP_Imm) significantly increases the percentage of Neurog1eGFP(+) progenitors and immature neurons in spheres. Sphere-forming capacity resides within selected populations; FACS-purified, Neurog1eGFP(+) cells were poorly spherogenic, while preparations from ΔSox2eGFP transgenic mice that are enriched for Sox2(+) basal cells formed spheres very efficiently. Finally, we compared the potency following transplantation of cells grown in spheres vs. cells derived from adherent cultures. The sphere-derived cells engrafted and produced colonies with multiple cell types that incorporated into and resembled host epithelium; cells from adherent cultures did not. Furthermore, cells from spheres grown in conditioned media from the phorbol ester-activated LP_Imm line gave rise to significantly more neurons after transplantation as compared with control. The current findings demonstrate that sphere formation serves as a biomarker for engraftment capacity and multipotency of olfactory progenitors, which are requirements for their eventual translational use.     

New structure,the “olfactory pit,” in human olfactory mucosa

A whole-mount immunocytochemical method was devised to study the olfactory receptor neurons on the surface of the human olfactory mucosal sheet. Antibodies to neuron-specific tubulin and/or microtubule-associated protein 5 and phosphorylated neurofilament protein were used. Specimens taken at autopsy from 56 patients ranging in age from 2 days to 92 years revealed a structure not previously described, an olfactory pit. Round or oval openings with a diameter of 50 to 500 μm were observed on the surface of the olfactory epithelium in the whole-mount specimen. The morphology, number, and distribution of these openings varied among the different individuals. A detailed analysis of these structures was carried out by rehydrating and sectioning the whole-mount specimens. The olfactory pit (OP) is a blind pouch lined with olfactory epithelium (OE), which appears as an invagination of OE into the connective tissue, with a depth varying between 150 and 200 μm. In some sections through an OP, a thick axon bundle emerging from the bottom of the pouch was visible. The extension and termination of this axon bundle in the central nervous system has not been explored. We have found OPs in monkey olfactory mucosa, but none in rodents. The function of the pit specialization is unclear, but it appears to be a feature of normal, young epithelium. The configuration of the blind pouch may prolong odorant association with the olfactory receptor neurons, or the OP may contain specialized neurons that have not yet been recognized by morphological, biochemical, or functional techniques. J. Comp. Neurol. 378:443–453, 1997. © 1997 Wiley-Liss, Inc.     

Oxidative damage in the olfactory system in Alzheimer's disease

Increased oxidative damage is a prominent and early feature of vulnerable neurons in Alzheimer's disease (AD). However, while damage to proteins, sugars, lipids, nucleic acids and organelles such as lysosomes, mitochondria, and endoplasmic reticulum are evident, the source of increased reactive oxygen species has not been determined. Furthermore, a major limitation in further determining the source, as well as finding a means to arrest damage, is the paucity of cellular models directly homologous to AD since the vulnerable neurons of the brain in AD cannot be studied in vitro. Here, we examined the olfactory epithelium in situ to see if neurons there exhibit a similar pathological oxidative balance to vulnerable neurons in AD. In biopsy specimens, (eight AD and three controls) we found that neurons, and also the surrounding epithelial cells, show an increase in oxidative damage for a subset of the markers increased in the brain of cases of AD. Lipid peroxidation and heme oxygenase-1, a stress response protein, were increased, while nucleic acid or protein oxidation, demonstrated in vulnerable neurons in AD, were not increased. These findings highlight the systemic nature of oxidative abnormalities in AD, but that different cell types may express this abnormality by a different array of oxidative stress markers, supporting the potential for using olfactory neurons or other cells derived from AD patients in culture to understand the mechanistic basis for increased oxidative damage in AD and as a model to screen compounds for therapeutic intervention.