Alterations in expression of the neurotrophic factors glial cell line-derived neurotrophic factor, ciliary neurotrophic factor and brain-derived neurotrophic factor, in the target-deprived olfactory neuroepithelium

Neuronal growth factors play an important role in the development and maintenance of the nervous system. In the olfactory system, neurogenesis and synapse formation occur not only during development but throughout life and it would be expected that growth factors play a significant role in these ongoing processes. We have examined the expression of three neurotrophic factors, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor and brain-derived neurotrophic factor in the normal rat olfactory system and following synaptic target ablation (olfactory bulbectomy). We found that brain-derived neurotrophic factor immunoreactivity was confined to the horizontal basal cells of the olfactory neuroepithelium and was unaltered by bulbectomy. Glial cell line-derived neurotrophic factor immunoreactivity was present in the mature olfactory neurons and also their synaptic target cells in the olfactory bulb. Following bulbectomy, glial cell line-derived neurotrophic factor immunoreactivity was abolished from the neuroepithelium. Ciliary neurotrophic factor was present throughout the olfactory neuronal lineage with strongest immunoreactivity in the horizontal basal cells and mature olfactory neurons as well as several cell types in the olfactory bulb. Postbulbectomy, there was loss of strong ciliary neurotrophic factor immunoreactivity in olfactory neurons, however, low levels persisted in the remaining neuronal population. Horizontal basal cell immunoreactivity persisted over three months. Our results would be consistent with glial cell line-derived neurotrophic factor expression in mature olfactory neurons being dependent upon functional synaptic contact with the olfactory bulb. Alternatively, this factor may be acting as target-derived growth factor for olfactory neurons, a role in keeping with its function in spinal motoneurons and in the nigrostriatal system. Brain-derived neurotrophic factor is implicated in the trophic support of immature neurons. Ciliary neurotrophic factor is clearly important in this unique neuronal system but elucidation of its role awaits further investigation.     

Ciliary neurotrophic factor-immunoreactivity in olfactory sensory neurons

Ciliary neurotrophic factor (CNTF) has been implicated in processes of neuroprotection, axonal regeneration and synaptogenesis in the lesioned CNS. In the olfactory system, which is characterized by particularly robust neuroplasticity throughout life, the concentration of CNTF is high even under physiological conditions. In the present study, the cellular localization of CNTF-immunoreactivity was studied in the rat and mouse olfactory epithelium. In both species, individual olfactory sensory neurons (ONs) displayed intense CNTF-immunoreactivity. The number of CNTF-ir ONs varied interindividually in rats and was lower in mice than in rats. In olfactory epithelia of mice expressing beta-galactosidase under control of the CNTF promoter, cells of the ON layer were immunoreactive for the reporter protein. CNTF-ir ONs were olfactory marker protein-positive and growth associated protein 43-negative. CNTF-ir ONs lacked apoptotic markers, and the number of specifically labeled ONs was apparently unchanged after light chemical lesioning of the epithelium, indicating that CNTF-immunoreactivity was not associated with ON death. Electron microscopy of CNTF-ir ON axons in innervated olfactory bulb glomeruli documented that they formed typical ON axonal synapses with target neurons. Three dimensional reconstructions of bulb pairs showed a striking similarity of the positions of glomeruli innervated by CNTF-ir ON axons in left and right bulbs of individual animals and interindividually. The number of innervated glomeruli differed interindividually in rats and was lower in mice than in rats. The results show that in rodents CNTF-immunoreactivity occurs in a subset of mature, functionally competent ONs. The localization of target glomeruli suggests that CNTF-immunoreactivity may be associated with the expression and/or activation of specific olfactory receptor proteins.     

Ciliary neurotrophic factor (CNTF) in the olfactory system of rats and mice.

The rodent olfactory system is a regarded model for the relationship between neurotrophic factors, their receptors, and their compound influence on the notable lifelong neuroplasticity occurring in this sensory system. It was known that high amounts of ciliary neurotrophic factor (CNTF), a hematopoietic cytokine, can be found in the olfactory bulb. In the awarded work, a detailed cellular characterization of CNTF-localization in the olfactory system was obtained. The results demonstrated CNTF-immunoreactivity in olfactory ensheathing cells, newborn interneurons in the olfactory bulb, and in a subpopulation of mature olfactory sensory neurons in the olfactory epithelium. Three-dimensional reconstructions of CNTF-immunoreactive axonal bulbar projections of these neurons revealed an ordered bilaterally symmetric pattern. This finding implies a potential connection between neuronal CNTF-expression in the olfactory epithelium and olfactory information processing.     

Glial cells from adult rat olfactory bulb: immunocytochemical properties of pure cultures of ensheathing cells.

Three morphologically and immunohistochemically distinct types of cell were present in primary cultures of adult rat olfactory nerve and glomerular layers of the olfactory bulb. One cell type was multipolar and stained positively for glial fibrillary acidic protein; a second type had fried egg-like morphology and stained with antibodies to epitope ED1; the third cell type had fusiform morphology, reacted with antibodies to vimentin and laminin and was glial fibrillary acidic protein- and ED1-negative. Trypsinization of these primary cultures (3 min, 37 degrees C), detached multipolar and fusiform cells only. When detached cells were set up in secondary culture on a glass substrate, fusiform cells did not attach, resulting in a pure culture of multipolar cells. Multipolar cells were glial fibrillary acidic protein- and myelin basic protein-positive and had the properties of so-called ensheathing cells or Blanes' glia. Immunoreactivity with anti-nerve growth factor receptor and anti-fibronectin allowed us to identify four distinct populations of multipolar ensheathing cells. One population was nerve growth factor receptor-positive, fibronectin-negative. A second was nerve growth factor receptor-negative and fibronectin-positive. A third was positive for both markers and the remaining cells did not stain for either of them. The morphological and immunological characteristics of cultured cells from olfactory nerve and glomerular layers were similar to those of Schwann cells and the similarities could account for the permissivity to axonal growth of the olfactory bulb.     

Vasoactive intestinal polypeptide-immunoreactive neurons in the main olfactory bulb of the hedgehog (Erinaceus europaeus)

Vasoactive intestinal polypeptide (VIP) immunoreactivity was localized by the indirect antibody enzyme method (PAP technique) in the main olfactory bulb of the hedgehog. Most VIP-immunoreactive cells were located in the glomerular layer and throughout the external plexiform layer. Fewer cells were observed in the granule cell layer. At the morphological level they exhibit the characteristics of periglomerular, external tufted, superficial short axon, horizontal and Van Gehuchten cells. It should be mentioned that another specific neuronal type was found in the inner third of the external plexiform layer, which is not described in other animals. These results revealed that a high number of intrinsic neuronal types of the olfactory bulb of the hedgehog display a strong VIP immunoreactivity.     

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     

GDNF、GFAP和NGFRp75在成年大鼠和金黄地鼠嗅球成鞘细胞的免疫组织化学研究

目的　研究GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达 ,探索成鞘细胞在中枢神经再生中的作用。　方法　用免疫组织化学ABC法 ,显示GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达和分布 ,同时用NGFRp75和GFAP染色作为阳性对照。　结果　在成年大鼠和金黄地鼠嗅球的纤维层和小球层内均可见深棕色的GDNF免疫组织化学反应的成鞘细胞。在小球层与纤维层分界处和小球层与分子层分界处及嗅小球之间密集分布 ,在嗅小球之内较稀疏。同时在同一嗅球组织的另两组切片的相同部位 ,分别出现GFAP和NGFRp75免疫反应性细胞 ,间接说明GDNF免疫反应的结构是嗅球成鞘细胞。　结论　嗅球成鞘细胞含有胶质细胞源性神经营养因子     

成年大鼠嗅球和鼻腔嗅粘膜成鞘细胞的分离、培养与鉴定

目的　在嗅球成鞘细胞 (OECs)移植到脊髓可有助于损伤神经纤维再生的基础上 ,分别对嗅球和嗅粘膜的OECs进行培养 ,探索自体嗅粘膜作为OECs供体的可能性。　方法　根据OECs、成纤维细胞和星形胶质细胞贴壁时间的不同 ,采用差时贴壁方法分离出OECs,培养 14div后进行NGFRp75和GDNF的免疫细胞化学染色。　结果　按形态学和免疫组织化学特性 ,培养的嗅球和嗅粘膜OECs可分为 3类 :双极细胞、三级细胞和扁圆细胞 ,其中以双极细胞最多。嗅粘膜的双极成鞘细胞的突起更加细长。　结论　差时贴壁细胞分离法是一种简单、经济、实用的成鞘细胞分离方法。鼻腔嗅粘膜OECs的形态学和免疫细胞化学特性与嗅球OECs基本相同 ,本实验为临床开展自体嗅粘膜OECs修复脊髓损伤的研究提供参考     

The glial cells in the nerve fiber layer of the rat olfactory bulb

In mammals the olfactory receptor neurons are the only ones that are known to undergo continuous cell renewal in the adult animal. This means that the axon of each newly formed neuron must grow into the olfactory bulb to find its appropriate target cell. It is presumed that astrocytes ensheath the olfactory axons as they course through the nerve fiber layer of the bulb even though the cells in question differ ultrastructurally from typical astrocytes. The purpose of the present study was to examine the glial cells in the nerve fiber layer of the rat olfactory bulb in an effort to resolve this apparent discrepancy. Two morphologically distinct types of glial cell were found in the nerve fiber layer. One type, which resembled the typical astrocytes that are present in other areas of the cental nervous system, contained bundles of filaments in an electron-lucent cytoplasm. These cells also formed endfeet on blood vessels and formed part of the external glial limiting membrane. They did not, however, ensheath the olfactory axons. The cytoplasm of the other type of glial cell was denser than that of typical astrocytes and contained fewer filaments, which were seldom grouped into bundles. These cells also formed part of the glial limiting membrane at the surface of the bulb and were the only ones that ensheathed the olfactory axons. It is concluded that the cell ensheathing the olfactory axons in the nerve fiber layer of the rat olfactory bulb is a morphological variant of the typical astrocyte. One role of the former cell may be to support or encourage the growth of olfactory axons within the central nervous system.     

Distribution of enkephalin-like immunoreactivity in the rat main olfactory bulb

Enkephalin-like immunoreactivity was localized within the main olfactory bulb of the rat using immunohistochemical techniques. These studies utilized well characterized antisera directed to either leu⁵- or met⁵-enkephalin. Specificity was established by absorption of the antisera with either 10 μM synthetic leu⁵- or met⁵-enkephalin.Specific enkephalin-like immunoreactivity was observed within several different cell populations including (1) periglomerular cells, (2) granule cells and their processes within the external plexiform layer and (3) occasional short-axon (horizontal) cells within the granule and external plaxiform layers. The granule cell layer contained the greatest number of immunoreactive cells. Only a limited number of immunoreactive cells were found in both the periglomerular and granule cell layers, suggesting the enkephalin-containing neurons represent a sub-population within each layer.The absence of immunoreactive processes in the periventribular white matter, as well as the morphologies of immunoreactive bulbar neurons, indicates that enkephalin is found exclusively within intrinsic olfactory bulb neurons.     

Formation of an olfactory glomerulus: morphological aspects of development and organization.

We have studied the development of olfactory nerves in the rat from their first contact with the telencephalic vesicle until the formation of glomerular structures in the olfactory bulb at early postnatal period. The study is based on serial semithin and ultrathin sections of material prepared for electron microscopy and antibodies to label radial glial cells, glial fibrillary acidic protein and Rat-401. Beginning on embryonic day 12, developing olfactory axons from the olfactory placode are accompanied by migratory cells, also derived from the olfactory placode, that reach the prospective olfactory bulb by embryonic day 13. The mass of migratory cells accumulate superficial to the telencephalic vesicle. The cells increase in number by mitotic divisions. The majority of these cells represent precursor elements that will later develop into the ensheathing cells of the olfactory nerves and olfactory nerve layer of the adult. Some migratory cells penetrate into the prospective olfactory bulb early during development. The first synaptic contacts of olfactory axons with dendritic processes in the olfactory bulb were observed at embryonic day 18. Glomerular formation is initiated by penetration of cells from the migratory mass into the prospective glomerular layer by embryonic day 20 to postnatal day 0. These cells form walls surrounding zones of high synaptic density forming protoglomeruli. Postnatally, the peripheral processes of radial glial cells branch profusely delimiting glomerular formations and transform into periglomerular astrocytes. Rat-401 stains radial glial cells from embryonic day 14. Immunoreactivity becomes restricted to the olfactory glomeruli during the first postnatal weeks and it virtually disappears by the end of the first postnatal month. We conclude that the early penetration of cells from the migratory mass into the prospective olfactory bulb, observed immediately after the first synaptic contacts were established, initiates the formation of olfactory glomeruli which becomes completed by the transformation of radial glial cells into periglomerular astrocytes.     

新生大鼠嗅鞘细胞的改良培养方法研究

目的:探讨新生大鼠嗅鞘细胞的分离、培养和纯化方法。方法:取2 d内的新生大鼠嗅球组织,在解剖显微镜下分离取材,原代培养嗅鞘细胞,运用差速贴壁法和差速贴壁+阿糖胞苷+细胞生长刺激因子法两种方法进行纯化,在倒置显微镜下观察不同培养时间嗅鞘细胞的形态、数量和NGFRp75抗体免疫荧光结果,统计嗅鞘细胞的纯度。结果:嗅鞘细胞的纯度分别是差速贴壁组平均为60.41%±4.32%,差速贴壁+阿糖胞苷+细胞生长刺激因子法组平均为84.98%±4.03%。两组数据用t检验进行统计学分析,差异有统计学意义(P<0.05)。结论:取材于新生大鼠;显微镜下分离脑膜;差速贴壁+阿糖胞苷+细胞生长刺激因子法纯化,都是良好的嗅鞘细胞培养方法。     

Encouraging regeneration in the central nervous system: is there a role for olfactory ensheathing cells?

The olfactory system holds a privileged position within the adult mammalian central nervous system; olfactory neurons undergo continual replacement and regeneration of synaptic contacts following injury, a feature shared by only a select few neuronal systems. The olfactory ensheathing cell, a glial cell found only in this system, is thought to play a central role in this regenerative process and has hence been the focus of numerous studies into promoting CNS regeneration following injury, in particular of the spinal cord. In trials, olfactory ensheathing cells have achieved some of the most promising results yet in promoting CNS regeneration, including a degree of functional recovery in humans following CNS injury. Comparatively, numerous other strategies, both those involving cellular transplantation and those examining neutralisation of inhibitory factors of the CNS, have achieved limited success. A combinational strategy, with olfactory ensheathing cells at its centre, is arguably the best way forward in encouraging effective recovery following CNS injury. This review examines the inhibitory environment of the CNS and the research to date on overcoming its effects on the regrowth of injured axons. The efficacy of therapies involving olfactory ensheathing cells, and the place of these therapies among the many other strategies being developed is examined.     

Calbindin-D-28k-like immunoreactive structures in the olfactory bulb and anterior olfactory nucleus of the human adult: distribution and cell typology--partial complementarity with parvalbumin

Calbindin-D-28k and parvalbumin are calcium-binding proteins. The laminar distribution and morphological features of calbindin-D-28k-like immunoreactive structures were studied in 60-microns-thick sections of the human olfactory bulb. Except for the olfactory nerve layer, immunoreactive neurons were present in all layers of the olfactory bulb. They reached highest densities in the external plexiform layer and internal granule cell layer. Considerable numbers of calbindin-like nerve cells were also found in the olfactory tract and in distal portions of the anterior olfactory nucleus. When comparing the distribution of calbindin-positive structures to that of parvalbumin-positive ones a partially complementary distribution pattern was found. Calbindin-like immunoreactive portions of the anterior olfactory nucleus and olfactory tract were mirrored by immunonegative areas in adjacent sections stained for parvalbumin. Using the combined pigment-Nissl procedure we observed the presence of lipofuscin deposits in nearly 80% of all the calbindin-immunoreactive neurons analysed. Moreover, analysis of their lipofuscin deposits rendered the further differentiation of morphologically similar neuronal subpopulations possible. In contrast, all parvalbumin-like immunoreactive neurons remained free of lipofuscin granules.     

Immunocytochemical characterisation of olfactory ensheathing cells of zebrafish

Continuous lifelong neurogenesis is typical of the vertebrate olfactory system. The regenerative ability of olfactory receptor neurons is dependent on the glial cell type specific to the olfactory pathway, designated ‘olfactory ensheathing cells’. Several studies to date have focused on mammalian olfactory ensheathing cells, owing to their potential roles in cell‐based therapy for spinal cord injury repair. However, limited information is available regarding this glial cell type in non‐mammalian vertebrates, particularly anamniotes. In the current immunocytochemical study, we analysed the features of olfactory ensheathing cells in the zebrafish, Danio rerio. Fish provide a good model for studying glial cells associated with the olfactory pathway of non‐mammalian vertebrates. In particular, zebrafish has numerous valuable features that enable its use as a prime model organism for genetic, neurobiological and developmental studies, as well as toxicology and genomics research. Paraffin sections from decalcified heads of zebrafish were processed immunocytochemically to detect proteins used in the research on mammalian olfactory ensheathing cells, including glial fibrillary acid protein (GFAP), S100, neural cell adhesion molecule (NCAM), polysialylated NCAM (PSA‐NCAM), vimentin (VIM), p75NTR and galactin (Gal)‐1. Notably, GFAP, S100, NCAM and Gal‐1 were clearly observed, whereas no vimentin staining was detected. Weak immunostaining for PSA‐NCAM and p75NTR was evident. Moreover the degree of marker expression was not uniform in various tracts of the zebrafish olfactory pathway. The immunostaining patterns of the zebrafish olfactory system are distinct from those of other fish to some extent, suggesting interspecific differences. We also showed that the olfactory pathway of zebrafish expresses markers of mammalian olfactory ensheathing cells. The olfactory systems of vertebrates have similarities but there are also marked variations between them. The issue of whether regional and interspecific differences in immunostaining patterns of olfactory pathway markers have functional significance requires further investigation.     

Immunohistochemical changes in the anterior olfactory nucleus of the developing rat

The anterior olfactory nucleus (AON) is located caudal to the olfactory bulb in the olfactory peduncle. Although this important structure is involved in the bilateral coordination of olfactory information, relatively little is known about its development, structure, or function. The present report details results from an immunohistochemical examination of specific neuronal (microtubule-associated protein-2: MAP2, calbindin D28-k, neuropeptide-Y: NPY) and glial (astrocytes; glial fibrillary-associated protein, or GFAP, oligodendrocytes; RIP) populations in postnatal Days 10, 20, and 30 rats. MAP2-immunoreactivity (-ir), was present throughout the AON, although most dense in the outer plexiform layer. Increases in labeling occurred from Day 10 to Day 30, reflecting the maturation of dendritic processes. Both temporal and regional differences in expression were found with the two neuronal markers. For example, although substantial numbers of calbindin-ir cells were observed as early as Day 10, relatively few cells exhibited NPY-ir. An apparent decline in the number of stained figures was observed from Days 20–30 with both markers. Most cells exhibiting calbindin- or NPY-ir were found in the inner half of the cellular zone of the AON. GFAP-ir was localized mainly to the subependymal zone and the lateral olfactory tract (LOT) at Day 10, with successive increases in staining in the cellular and plexiform layers at Days 20 and 30. Oligodendrocyte-ir was restricted to the anterior commissure and the LOT at Day 10, with dramatic increases in labeling of the cellular and plexiform layers observed by Days 20 and 30. These results represent some of the first analyses of the maturation of specific cellular phenotypes within this large neural region. © 1997 John Wiley & Sons, Inc. Dev Psychobiol 31: 181–192, 1997     

Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization

 This study compares the mRNA expression pattern for the three glutamate transporters EAAC1, GLT1 and GLAST in rat brain, using a sensitive non-radioactive in situ hybridization technique. The results confirm the predominantly neuronal localization of EAAC1 mRNA, the astroglial and ependymal localization of GLAST mRNA and the astroglial and neuronal localization of GLT1 mRNA. Further, we demonstrate, using a novel differential double hybridization protocol, that the presence of GLT1 mRNA in neurons is more widespread than previously thought, and that it encompasses the majority of neurons in the neocortex, neurons in the external plexiform layer in the olfactory bulb, neurons in dorsal and ventral parts of the anterior olfactory nucleus, the majority of neurons in the anteromedial thalamic nuclei, the CA3 pyramidal neurons in the hippocampus and neurons in the inferior olive. In addition, we demonstrate marked variations in the expression levels of GLT1 and GLAST mRNAs in different brain areas, suggesting that their mRNA levels are regulated by different mechanisms. Finally, for EAAC1 we demonstrate also a widespread distribution and a marked heterogeneity in the expression levels. EAAC1 is strongly expressed by a heretofore unrecognized group of cells in white matter tracts such as the corpus callosum, fimbria-fornix or anterior commissure. Also, strong EAAC1 expression is present in groups of scattered cells in grey matter areas of much of the forebrain and the cerebellum. These results provide more detailed information about the precise cellular localization of these three glutamate transporters and their regulation at the mRNA level. Accepted: 29 January 1998     

Changes in mitotic rate and GFAP expression in the primary olfactory axis of streptozotocin-induced diabetic rats

Many diabetic individuals develop anosmia but the mechanism(s) causing the dysfunction in the olfactory system is (are) unknown. Glial fibrillary acidic protein expression is reduced in diabetic retinopathy and is also reduced, with unknown consequences, in other brain regions of diabetic rats. We used immunohistochemistry and immunoblotting from untreated control and streptozotocin-induced type 1 (insulin dependent) diabetic rats to investigate main olfactory epithelial mitotic rate and glial fibrillary acidic protein expression in the lamina propria of the sensory epithelium and in the olfactory bulb. Numbers of bromodeoxyuridine-positive cells were significantly lower in the diabetic sensory epithelium compared to non-diabetic controls. Immunohistochemical observations suggested a qualitative difference in glial fibrillary acidic protein expression in both regions examined especially in the olfactory bulb external plexiform layer and the lamina propria. Immunoblot analysis confirmed that the diabetic olfactory bulb and lamina propria expressed less glial fibrillary acidic protein compared to the non-diabetic control group. The lower expression levels in the olfactory bulb external plexiform layer suggested by immunohistochemistry do not reflect a change in the number of astrocytes since the numbers of S100B(+) cells were not different between the two groups.