Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration.

Netrin-1 is a bifunctional secreted protein that directs axon extension in various groups of developing axonal tracts. The transmembrane DCC (deleted in colorectal cancer) receptor is described as netrin-1 receptor and is involved in the attractive effects of netrin-1. In this study, we examined the spatio-temporal expression patterns of both netrin-1 and DCC in the rat olfactory system at different stages of development and during axonal regeneration following unilateral bulbectomy. High DCC expression was detected on the pioneer olfactory axons as they are extending toward the telencephalon. This expression was transient since from embryonic day 16 onwards, DCC was no longer detected along the olfactory nerve path. From embryonic day 14 until birth, DCC was also expressed within the mesenchyme surrounding the olfactory epithelium. During the same period, netrin-1 protein was detected along the trajectory of olfactory axons up to the olfactory bulb and its expression pattern in the nasal mesenchyme largely overlapped that of DCC. Moreover, netrin-1 continued to be present during the two first post-natal weeks, and a weak protein expression still persisted in the dorso-medial region of the olfactory epithelium in adult rats. While unilateral bulbectomy induced a transient up-regulation of netrin-1 in the lamina propria, particularly in the dorso-medial region of the neuroepithelium, no DCC expression was detected on the regenerating olfactory axons. In the developing olfactory bulb, the extension of mitral cell axons was associated with DCC presence while netrin-1 was absent along this axonal path. DCC was also highly expressed in the newly formed glomeruli after birth, and a weak DCC expression was still detected in the glomerular layer in adult rats. Taken together, these data support the notion that netrin-1, via DCC expressed on axons, may play a role in promoting outgrowth and/or guidance of pioneering olfactory axons toward the olfactory bulb primordium. Moreover, association of netrin-1 with mesenchymal DCC may provide a permissive environment to the growth of both pioneer and later-growing axons. The maintenance of netrin-1 expression in the nasal mesenchyme of adult rats as well as its regional up-regulation following unilateral bulbectomy infer that netrin-1, even in the absence of DCC, may be involved in the process of axonal growth of newly differentiated olfactory receptor neurons probably through the use of other receptors.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Spatio-temporal patterns of ensheathing cell differentiation in the rat olfactory system during development.

An immunocytochemical approach with specific glial markers was used to investigate the temporal and spatial patterns of differentiation of ensheathing glia wrapping axon fascicles along the primary olfactory pathway of the rat during development. The two glial markers tested, the proteins S-100 and glial fibrillary acidic protein, are known to be expressed at different stages of maturation in glial cells. The S-100 protein was first weakly expressed in cells accompanying the olfactory axons at embryonic day 14 (E14), while a first faint glial fibrillary acidic protein staining was detected along the olfactory axons at E15 and along the vomeronasal nerves at E16. A strong S-100 immunoreactivity was already present from E16 onwards along the axon fascicles through their course in both the nasal mesenchyme and the subarachnoid space before entering the olfactory nerve layer of the olfactory bulb. A gradual increase in glial fibrillary acidic protein expression was observed along this part of the developing olfactory pathway from E16 up to E20, when an adult-like pattern of staining intensity was seen. By contrast, most of the ensheathing cells residing in the olfactory nerve layer exhibited some delay in their differentiation timing and also a noticeable delayed maturation. It was only from E20 onwards that a weak to moderate S-100 expression was detected in an increasing number of cells throughout this layer, and only few of them appeared weakly glial fibrillary acidic protein positive at postnatal days 1 and 5. The immunocytochemical data indicate that there is a proximodistal gradient of differentiation of ensheathing cells along the developing olfactory pathway. The prolonged immaturity of ensheathing cells in the olfactory nerve layer, which coincides with the formation of the first glomeruli, might facilitate the sorting out of olfactory axons leading to a radical reorganization of afferents before they end in specific glomeruli.