ISSN:0736-5748    

DOI:10.1016/S0736-5748(97)87096-7

出版商:Wiley    

年代:2005

数据来源: WILEY

摘要:

AbstractThe growth and differentiation of olfactory sensory neurons are regulated tightly. We had shown previously, by immunohistochemistry, that transforming growth factor‐α (TGF‐α) and epidermal growth factor (EGF) receptor are present in the olfactory epithelium of untreated adult rats and that TGF‐α is a potent mitogen of olfactory epitheliumin vitro. Expression of EGF receptor and TGF‐α was detected primarily in horizontal basal cells and supporting cells but rarely in globose basal cells, which suggested that EGF receptor is not a likely candidate for the mitotic regulator of sensory neurons. In order to expand the search for candidate regulators, we have now examined other members of the EGF family of receptors and ligands. By utilizing reverse transcriptase‐polymerase chain reaction (RT‐PCR) methodology, we have detected the messenger RNA encoding the protein of theneugene (p185neu) and Neu differentiation factor (NDF) isoforms in the olfactory mucosa. Immunohistochemical localization of p185neuand NDF indicates expression of these proteins in the olfactory epithelium of adult rats in regions where globose basal cells and immature sensory neurons are found, as well as in the ensheathing cells of the olfactory nerve. The presence ofneuand NDF transcripts in the olfactory tissue and the localization of their encoded polypeptides to proliferative regions of the epithelium suggest involvement of these gene products in the regulated proliferation/differentiation of the sensory neurons.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00039-1

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractThe ontogeny and cellular specificity of expression of β‐galactosidase activity and olfactory marker protein (OMP) are compared in olfactory tissue of the H‐OMP‐lacZ‐3 line of transgenic mice. In this line the expression oflacZis driven by a 0.3 kb fragment of the rat OMP promoter. During fetal development,lacZexpression is detectable in olfactory receptor neurons (ORNs) shortly after the initial appearance of endogenous OMP. The β‐galactosidase marker was observed only in mature olfactory receptor neurons where it co‐localized with endogenous OMP. It was absent from immature neurons that express the growth associated phosphoprotein B50/GAP43. Lesion of the peripheral olfactory pathway by intranasal irrigation with Triton X‐100 eliminated expression of both OMP andIacZin the olfactory neuroepithelium. Subsequent regeneration of the full complement of olfactory receptor neurons was associated with co‐expression of both OMP and β‐galactosidase activity. Neither OMP nor β‐galactosidase activity was induced in any other cell type of the regenerating olfactory mucosa. Thus, as little as 0.3 kb of the OMP promoter has the ability to targetlacZexpression to olfactory receptor neurons in a temporally and spatially defined manner. We discuss the potential utility of this transgenic line for future studies of the olfactory system.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00063-9

出版商:Wiley    

年代:2005

数据来源: WILEY

摘要:

AbstractIn 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.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00057-3

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractAnin vitroslice 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 vivodevelopment. 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.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00056-1

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractThe formation and development of primary olfactory axons was studied in the rat embryo using acetylcholinesterase histochemistry, immunocytochemistry for neuron‐specific β‐tubulin (TuJ1) and growth associated protein 43 (GAP43), and a fluorescent tracer DiI. Olfactory axons extend from the olfactory receptor neurons localized in the olfactory epithelium. These fibers grow to reach and enter the olfactory bulbs, where they form the first relay and integrative synaptic station in the olfactory system: the olfactory glomerulus. In this communication we address the development of primary olfactory fibers: first from the olfactory placode and later from the olfactory epithelium. Olfactory fibers enter the olfactory bulbs apparently in a disordered manner but soon arrange themselves in hook shaped aggregates of fibers, with many boutons (inmature synaptic terminals), to form the glomeruli. We detected this kind of structure for the first time at embryonic day 16. The olfactory receptor cells are usually anchored in the basal lamina of the olfactory epithelium but some of them, after reaching their targets, lose their epithelial attachment, leave the olfactory epithelium and migrate to and enter the olfactory bulbs. The traffic of cells between the olfactory epithelium and the brain lasts late into embryonic development. We describe four types of migratory mechanism used by different populations of cells to reach their targets in the telencephalic vesicle and propose the existence of migrating cells that enter the telencephalon. These data were corroborated by injections into the olfactory epithelium a of murine retrovirus carrying theEscherichia coli lac‐Zgene.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00055-X

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractNeurogenesis and proliferation of olfactory receptor neurons (ORNs) in the olfactory epithelium (OE) are reduced in postnatal hypothyroid rats and upregulated following restoration of thyroid function, leading to compensatory growth and restitution of these deficits [Paternostro M. A. and Meisami E. (1993).Dev. Brain Res.76, 151–161; Paternostro M. A. and Meisami E. (1994).Dev. Brain Res.83, 151–162]. To investigate thyroid hormonal role on maturation of ORNs, serial sections of the septal OE from normal newborn, 25‐ and 90‐day‐old rats were immunostained for olfactory marker protein (OMP), a marker for mature ORNs, and compared with the same from age‐matched hypothyroid rats and those allowed to recover from thyroid deficiency at the time of weaning (day 25). The parameters studied were the localization and distribution of the OMP(+) cells within the OE and their density and total number. Hypothyroidism was induced by adding the reversible goitrogen propylthiouracil (PTU) to the rats' drinking water (1 g/l) from birth to days 25 or 90. Recovery from hypothyroidism was induced by withdrawal of PTU at day 25. The OMP(+) cells occupied a distinct, broad band in the normal rat OE, while in hypothyroid animal, this band was narrow and restricted to OE's apical zones. Recovery resulted in broadening of the OMP(+) cell band and normalized distribution of OMP(+) cells as evident in the 90‐day‐old recovery animals. In normal control rats, density of OMP(+) cells increased by 2.5‐ and 1.3‐fold during the suckling and post‐weaning period (days 25–90), while total numbers of these cells increased by 12‐ and 3‐fold, respectively, during the same age periods. Hypothyroidism decreased the growth in density by 25 and 30%, while total number of OMP(+) neurons were reduced by 40 and 70% in the 25‐ and 90‐day‐old animals, respectively. Withdrawal of PTU resulted in marked restoration of these deficits so that, at 90 days, the total number of OMP(+) cells were only 20% less than 90‐day‐old controls. These results indicate that thyroid hormones are essential for maturation of single ORNs and accretion of new mature ORNs in the OE of suckling and post‐weaning rat. Also, the process of maturation and the final number of mature ORNs show remarkable recovery from hypothyroid‐induced growth retardation.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00064-0

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractOlfactory epithelium retains the capacity to recover anatomically after damage well into adult life and perhaps throughout its duration. None the less, olfactory dysfunctions have been reported widely for elderly humans. The present study investigates the effects of aging on the neurophysiological and anatomical status of the olfactory epithelium in barrier‐raised Fischer 344X Brown Norway F 1 hybrid rats at 7, 10, 25 and 32/35 months old. The posterior part of the olfactory epithelium in 32/35‐month‐old rats is well preserved. Globose basal cells are dividing, and new neurons are being born even at this advanced age. None the less, the numbers of proliferating basal cells and immature, GAP‐43 (+) neurons are significantly decreased. Neurophysiological status was evaluated using voltage‐sensitive dye techniques to assess inherent patterns of odorant‐induced activity in the epithelium lining the septum and the medial surface of the turbinates. In middle and posterior zones of the epithelium, there were neither age‐related changes in overall responsivity of this part of the olfactory epithelium to any of five odorants, nor shifts in the location of the odorant‐induced hotspots. The inherent activity patterns elicited by the different odorants do become more distinct as a function of age, which probably reflects the decline in immature neurons and a slight, but not statistically significant, increase in mature neurons as a function of age. In contrast with the excellent preservation of posterior epithelium, the epithelium lining the anterodorsal septum and the corresponding face of the turbinates is damaged in the 32/35‐month‐old animals: in this part, horizontal basal cells are reactive, more basal cells and sustentacular cells are proliferating than in younger animals or in posterior epithelium of the same animals, and the neuronal population is less mature on average. Our findings indicate that degeneration of the olfactory epithelium is not an inevitable or pre‐programmed consequence of the aging process, since the posterior zone of the epithelium is very well preserved in these barrier‐protected animals. However, the deterioration in the anterior epithelium suggests that environmental insults can accumulate or become more severe with age and overwhelm the regenerative capacity of the epithelium. Alternatively, the regenerative capacity of the epithelium may wane somewhat with age. Either of these mechanisms or some combination of them can account for the functional and anatomical deterioration of the sense of smell associated with senescence in humans.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00046-9

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractThe distribution of the adenosine‐producing ecto‐enzyme 5′‐nucleotidase was investigated histochemically in the developing rat olfactory bulb. Rat pups underwent either unilateral surgical occlusion of the right external naris or sham surgery on postnatal day 1. At 10, 20, or 30 days postpartum, horizontal sections of the olfactory bulb were reacted histochemically to reveal the locus and intensity of 5′‐nucleotidase activity. Relative staining levels were determined by optical densitometry in standardized bulb regions. A marked, age‐related increase in staining density was observed. Reaction product was found primarily in neuropil areas. The P10 and P20 control animals did not exhibit right/left differences in bulb staining; however, some laterality was observed in P30 animals. Inter‐glomerular and regional variations were observed throughout the developmental period, including (1) differences between neighboring glomeruli; (2) a gradient in the dorsal‐ventral axis of the bulb; and (3) a higher staining density in the medial‐caudal portion of the bulb. In subjects with occluded nares, asymmetries in right/left bulb 5′‐nucleotidase staining patterns were detected throughout development. Bulbs ipsilateral to the blocked nares exhibited increased staining density, suggesting that the procedure enhanced enzymatic activity. Understanding these variations in 5′‐nucleotidase staining may be important for a complete understanding of the mechanisms of olfactory bulb maturation and may give insight into the possible role of this enzyme in synaptic malleability during nervous system development and regeneration.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00040-8

出版商:Wiley    

年代:1997

数据来源: WILEY

摘要:

AbstractNorepinephrine is supplied to both deep and superficial layers of the olfactory bulb through dense projections from the locus coeruleus.16Beta‐adrenergic receptors are located in nearly all bulb laminae, with high‐density foci of β‐1 and β‐2‐adrenoceptors present in the glomerular layer.29Early olfactory experiences that increase norepinephrine levels in the bulb also decrease the density of β‐1‐ and β‐2‐adrenoceptors, as well as the number of high‐density glomerular foci of β‐2‐receptors.30Changes in bulb norepinephrine levels, therefore, may affect the density of β‐adrenoceptors in the bulb. In the current study, we test this hypothesis by performing unilateral lesions of the locus coeruleus with 6‐hydroxydopamine on postnatal day 4, and examining the density of β‐1‐ and β‐2‐adrenergic receptors in the main olfactory bulb of the rat using125I‐labeled iodopindolol receptor autoradiography on postnatal day 19. Locus coeruleus destruction resulted in a statistically significant increase in the density of adrenergic receptors in the ipsilateral bulb compared to the contralateral bulb. Both β‐1‐ and β‐2‐adrenoceptor subtypes increased in density with this manipulation, although the number of glomerular layer high‐density β‐2 foci was not significantly different between the two bulbs. These results are consistent with the hypothesis that changes in olfactory bulb norepinephrine can regulate the density of β‐adrenergic receptors in the bulb.

ISSN:0736-5748    

DOI:10.1016/S0736-5748(96)00041-X

出版商:Wiley    

年代:1997

数据来源: WILEY