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Olfactory ensheathing cells (OECs) are candidates for therapeutic approaches for neural regeneration due to their ability to assist axon regrowth in central nervous system lesion models. However, little is understood about the processes and mechanisms underlying migration of these cells. We report here that novel lamellipodial protrusions, termed lamellipodial waves, are integral to OEC migration. Time-lapse imaging of migrating OECs revealed that these highly dynamic waves progress along the shaft of the cells and are crucial for mediating cell-cell adhesion. Without these waves, cell-cell adhesion does not occur and migrational rates decline. The activity of waves is modulated by both glial cell line-derived neurotrophic factor and inhibitors of the JNK and SRC kinases. Furthermore, the activity of lamellipodial waves can be modulated by Mek1, independently of leading edge activity. The ability to selectively regulate cell migration via lamellipodial waves has implications for manipulating the migratory behavior of OECs during neural repair., ((c) 2007 Wiley-Liss, Inc.)

The olfactory system in rodents and many other mammals is classically divided into two anatomically separate, and morphologically distinct, sensory systems: the main olfactory system and the accessory olfactory system. We have now identified a novel third population of olfactory marker protein-expressing sensory neurons that is located in a discrete pocket of the rostral nasal septum, which we refer to as the septal organ of Grüneberg (SOG). Neurons in this region of the septum are located in the submucosa, in small grape-like clusters, rather than in a pseudostratified neuroepithelium, as seen in both the olfactory and vomeronasal neuroepithelia. Despite their unusual location, axons projecting from the SOG neurons fasciculate into several discrete bundles and terminate in a subset of main olfactory bulb glomeruli. These glomeruli most likely represent a subset of atypical glomeruli that are spatially restricted to the caudal main olfactory bulb. The unique rostral position of the SOG suggests that the SOG may be functionally specialized for the early detection of biologically relevant odorants., (Copyright 2005 Wiley-Liss, Inc.)

Olfactory marker protein (OMP) is expressed by mature primary olfactory sensory neurons during development and in adult mice. In mice that lack OMP, olfactory sensory neurons have perturbed electrophysiological activity, and the mice exhibit altered responses and behavior to odor stimulation. To date, defects in axon guidance in mice that lack OMP have not been investigated. During development of the olfactory system in mouse, primary olfactory axons often overshoot their target glomerular layer and project into the deeper external plexiform layer. These aberrant axonal projections are normally detected within the external plexiform layer up to postnatal day 12. We have examined the projections of primary olfactory axons in OMP-tau:LacZ mice and OMP-GFP mice, two independent lines in which the OMP coding region has been replaced by reporter molecules. We found that axons overshoot their target layer and grow into the external plexiform layer in these OMP null mice as they do in wild-type animals. However, in the absence of OMP, overshooting axons are more persistent and remain prominent until 5 weeks postnatally, after which their numbers decrease. Overshooting axons are still present in these mice even at 8 months of age. In heterozygous mice, axons also overshoot into the external plexiform layer; however, there are fewer axons, and they project for shorter distances, compared with those in a homozygous environment. Our results suggest that perturbed electrophysiological responses, caused by loss of OMP in primary olfactory neurons, reduce the ability of primary olfactory axons to recognize their glomerular target., (Copyright 2005 Wiley-Liss, Inc.)

The early axon scaffolding in the embryonic vertebrate brain consists of a series of ventrally projecting axon tracts that grow into a single major longitudinal pathway connected across the midline by commissures. We have investigated the role of Brother of CDO (BOC), an immunoglobulin (Ig) superfamily member distantly related to the Roundabout (Robo) family of axon-guidance receptors, in the development of this embryonic template of axon tracts in the zebrafish brain. A zebrafish homologue of BOC was isolated and shown to be expressed predominantly in the developing neural plate and later in the neural tube and developing brain. Zebrafish boc was initially highly localized to discrete bands in the mid- and hindbrain, but, as the major brain subdivisions emerged, it became more evenly expressed along the rostrocaudal axis, particularly in dorsal regions. The function of zebrafish boc was examined by a loss-of-function approach. Analysis of embryos injected with antisense morpholinos designed against boc revealed highly selective defects in the development of dorsoventrally projecting axon tracts. Loss of boc caused ventrally projecting axons, particularly those arising from the presumptive telencephalon, to follow aberrant trajectories. These data indicate that boc is an axon-guidance molecule playing a fundamental role in pathfinding during the early patterning of the axon scaffold in the embryonic vertebrate brain., (Copyright (c) 2005 Wiley-Liss, Inc.)

Primary olfactory neurons project axons from the olfactory neuroepithelium lining the nasal cavity to the olfactory bulb in the brain. These axons grow within large mixed bundles in the olfactory nerve and then sort out into homotypic fascicles in the nerve fiber layer of the olfactory bulb before terminating in topographically fixed glomeruli. Carbohydrates expressed on the cell surface have been implicated in axon sorting within the nerve fiber layer. We have identified two novel subpopulations of primary olfactory neurons that express distinct alpha-extended lactoseries carbohydrates recognised by monoclonal antibodies LA4 and KH10. Both carbohydrate epitopes are present on novel glycoforms of the neural cell adhesion molecule, which we have named NOC-7 and NOC-8. Primary axon fasciculation is disrupted in vitro when interactions between these cell surface lactoseries carbohydrates and their endogenous binding molecules are inhibited by the LA4 and KH10 antibodies or lactosamine sugars. We report the expression of multiple members of the lactoseries binding galectin family in the primary olfactory system. In particular, galectin-3 is expressed by ensheathing cells surrounding nerve fascicles in the submucosa and nerve fiber layer, where it may mediate cross-linking of axons. Galectin-4, -7, and -8 are expressed by the primary olfactory axons as they grow from the nasal cavity to the olfactory bulb. A putative role for NOC-7 and NOC-8 in axon fasciculation and the expression of multiple galectins in the developing olfactory nerve suggest that these molecules may be involved in the formation of this pathway, particularly in the sorting of axons as they converge towards their target., (Copyright 2004 Wiley-Liss, Inc.)

Primary olfactory axons expressing the same odorant receptor gene sort out and converge to fixed sites in the olfactory bulb. We examined the guidance of axons expressing the P2 odorant receptor when they were challenged with different cellular environments in vivo. In the mutant extratoes mouse, the olfactory bulb is lacking and is replaced by a fibrocellular mass. In these animals, primary olfactory axons form glomerular-like loci despite the absence of normal postsynaptic targets. P2 axons are able to sort out from other axons in this fibrocellular mass and converge to form loci of like axons. The sites of these loci along mediolateral and ventrodorsal axes were highly variable. Similar convergence was observed for larger subpopulations of axons expressing the same cell surface carbohydrates. The sorting out and convergence of like axons also occurred during regeneration following bulbectomy. Olfactory axon behaviour in these models demonstrates that sorting and convergence of axons are independent of the target, which instead provides distinct topographic cues for guidance., (Copyright 2003 Wiley-Liss, Inc.)

Binding of cell surface carbohydrates to their receptors specifically promotes axon growth and synaptogenesis in select regions of the developing nervous system. In some cases these interactions depend upon cell-cell adhesion mediated by the same glycoconjugates present on the surface of apposing cells or their processes. We have previously shown that the plant lectin Dolichos biflorus agglutinin (DBA) binds to a subpopulation of mouse primary olfactory neurons whose axons selectively fasciculate prior to terminating in the olfactory bulb. In the present study, we investigated whether these glycoconjugates were also expressed by postsynaptic olfactory neurons specifically within the olfactory pathway. We show here for the first time that DBA ligands were expressed both by a subset of primary olfactory neurons as well as by the postsynaptic mitral/tufted cells in BALB/C mice. These glycoconjugates were first detected on mitral/tufted cell axons during the early postnatal period, at a time when there is considerable synaptogenesis and synaptic remodelling in the primary olfactory cortex. This is one of the few examples of the selective expression of molecules in contiguous axon tracts in the mammalian nervous system. These results suggest that glycoconjugates recognized by DBA may have a specific role in the formation and maintenance of neural connections within a select functional pathway in the brain., (Copyright 2002 Wiley-Liss, Inc.)

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway., (Copyright 2001 Wiley-Liss, Inc.)

The restricted expression of the low affinity nerve growth factor receptor p75NTR by olfactory ensheathing cells suggests that this molecule is involved in the development of the olfactory nerve pathway. To begin to understand the role of p75NTR, we examined the development of the primary olfactory system in p75NTR(-/-) and wild-type mice. Our results demonstrate that, although p75NTR is not essential for the initial assembly of the olfactory nerve, it plays an important role in the postnatal maturation of the olfactory bulb. In the absence of p75NTR, there is exuberant growth of some primary olfactory axons into the olfactory bulb. These axons either aberrantly bypass the glomerular layer and project into deeper lamina or grow into an abnormal bleb of tissue protruding from the medial surface of the dorsocaudal olfactory bulb. These blebs become apparent in neonatal mice and contain axons expressing olfactory marker protein that form ectopic glomerular-like tufts. Histochemical staining with the plant lectin Dolichos biflorus agglutinin revealed that axons sorted out and selectively converged on glomeruli within these blebs. Our results suggest that p75NTR indirectly influences axon growth but not glomerular targeting and plays a role in the postnatal maturation of laminar cytoarchitecture in the olfactory bulb., (Copyright 2000 Wiley-Liss, Inc.)

The chondroitin sulfate proteoglycans neurocan and phosphacan are believed to modulate neurite outgrowth by binding to cell adhesion molecules, tenascin, and the differentiation factors heparin-binding growth-associated molecule and amphoterin. To assess the role of these chondroitin sulfate proteoglycans in the olfactory system, we describe here their expression patterns during both embryonic and postnatal development in the mouse. Immunoreactivity for neurocan was first detected in primary olfactory neurons at embryonic day 11. 5 (E11.5). Neurocan was expressed by primary olfactory axons as they extended toward the rostral pole of the telencephalon as well as by their arbors in glomeruli after they contacted the olfactory bulb. The role of neurocan was examined by growing olfactory neurons on an extracellular matrix substrate containing neurocan or on extracellular matrix in the presence of soluble neurocan. In both cases, neurocan strongly promoted neurite outgrowth. These results suggest that neurocan supports the growth of primary olfactory axons through the extracellular matrix as they project to the olfactory bulb during development. Phosphacan, unlike neurocan, was present within the mesenchyme surrounding the E11.5 and E12.5 nasal cavity. This expression decreased at E13.5, concomitant with a transient appearance of phosphacan in nerve fascicles. Within the embryonic olfactory bulb, phosphacan was localised to the external and internal plexiform layers. However, during early postnatal development phosphacan was concentrated in the glomerular layer. These results suggest that phosphacan may play a role in delineating the pathway of growing olfactory axons as well as defining the laminar organization of the bulb. Together, the spatiotemporal expression patterns of neurocan and phosphacan indicate that these chondroitin sulfate proteoglycans have diverse in situ roles, which are dependent on context-specific interactions with extracellular and cell adhesion molecules within the developing olfactory nerve pathway., (Copyright 2000 Wiley-Liss, Inc.)

The olfactory neuroepithelium is a highly plastic region of the nervous system that undergoes continual turnover of primary olfactory neurons throughout life. The mechanisms responsible for persistent growth and guidance of primary olfactory axons along the olfactory nerve are unknown. In the present study, we used antibodies against the Eph-related receptor, EphA5, to localise EphA5, and recombinant EphA5-IgG fusion protein to localise its ligands. We found that although both EphA5 and its ligands were both expressed by primary olfactory neurons within the embryonic olfactory nerve pathway, there was no graded or complementary expression pattern. In contrast, the expression patterns altered postnatally such that primary olfactory neurons expressed the ligands, whereas the second-order olfactory neurons, the mitral cells, expressed EphA5. The role of EphA5 was analysed by blocking EphA5-ligand interactions in explant cultures of olfactory neuroepithelium using anti-EphA5 antibodies and recombinant EphA5. These perturbations reduced neurite outgrowth from explant cultures and suggest that intrafascicular axon repulsion may serve to limit adhesion and optimise conditions for axon growth.

In the adult olfactory nerve pathway of rodents, each primary olfactory axon forms a terminal arbor in a single glomerulus in the olfactory bulb. During development, axons are believed to project directly to and terminate precisely within a glomerulus without any exuberant growth or mistargeting. To gain insight into mechanisms underlying this process, the trajectories of primary olfactory axons during glomerular formation were studied in the neonatal period. Histochemical staining of mouse olfactory bulb sections with the lectin Dolichos biflorus-agglutinin revealed that many olfactory axons overshoot the glomerular layer and course into the deeper laminae of the bulb in the early postnatal period. Single primary olfactory axons were anterogradely labelled either with the lipophilic carbocyanine dye, 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), or with horse-radish peroxidase (HRP) by localized microinjections into the nerve fiber layer of the rat olfactory bulb. Five distinct trajectories of primary olfactory axons were observed in DiI-labelled preparations at postnatal day 1.5 (P1.5). Axons either coursed directly to and terminated specifically within a glomerulus, branched before terminating in a glomerulus, bypassed glomeruli and entered the underlying external plexiform layer, passed through the glomerular layer with side branches into glomeruli, or branched into more than one glomerulus. HRP-labelled axon arbors from eight postnatal ages were reconstructed by camera lucida and were used to determine arbor length, arbor area, and arbor branch number. Whereas primary olfactory axons display errors in laminar targeting in the mammalian olfactory bulb, axon arbors typically achieve their adult morphology without exuberant growth. Many olfactory axons appear not to recognize appropriate cues to terminate within the glomerular layer during the early postnatal period. However, primary olfactory axons exhibit precise targeting in the glomerular layer after P5.5, indicating temporal differences in either the presence of guidance cues or the ability of axons to respond to these cues.

The rodent olfactory epithelium consists of a mosaic of primary sensory olfactory neurons (PONs) which express distinct putative olfactory receptor proteins. Recent evidence suggests that individual subsets of these sensory neurons project to separate glomeruli in the olfactory bulb (Vassar et al., [1994] Cell 79:981-991). In the present study we have identified two distinct subsets of primary sensory olfactory neurons (PONs) in the H-OMP-LacZ-6 transgenic mouse. In these transgenic mice, a LacZ reporter gene under the control of a 294 base pair element from the 5' promoter region of the olfactory marker protein (OMP) gene was expressed in a subset of PONs located in a discrete band of neuroepithelium in the nasal cavity. These LacZ positive neurons were not randomly located within this band but were more concentrated within a locus between endoturbinates IIb and III. The axons of these neurons densely innervated three adjacent and bilaterally symmetrical glomeruli present in the ventromedial olfactory bulb. Labeling of tissue sections with the plant lectin Dolichos biflorus (DBA) revealed an independent subset of PONs in the transgenic mice. These neurons were present in a wide region of the nasal cavity that included the neuroepithelial band containing the LacZ expressing neurons. The DBA labeled axons terminated in glomeruli in the rostromedial and dorsolateral olfactory bulb surfaces. Although the glomeruli innervated by the LacZ and DBA positive axons were predominantly non-overlapping there were glomeruli in the ventral olfactory bulb that were labeled by both DBA and LacZ markers. Eight different types of glomeruli were characterized. Most notably, glomeruli were identified which were innervated partially by both or by either subset alone. In these cases, axon subsets were observed to terminate within discrete subregions of a glomerulus. These results support the hypothesis that phenotypically distinct subsets of PONs converge on to the same glomeruli but also indicate that some glomeruli are innervated by more than one subset of sensory neuron. These findings have implications for understanding how the olfactory projection is formed and how olfactory information is processed.

Primary olfactory axons project from the nasal olfactory neuroepithelium to glomeruli in the olfactory bulb where they synapse with mitral cells, the second-order olfactory neurons. We have shown that the heparin-binding growth factor FGF-1 is expressed by olfactory nerve ensheathing cells which surround fascicles of primary olfactory axons en route to the olfactory bulb. These cells are believed to modulate olfactory axon growth between the olfactory neuroepithelium and the olfactory bulb. During late embryogenesis, FGF-1 expression is turned on in the mitral cells, and the FGF-1 peptide becomes confined to layers of synaptic neuropil in the postnatal olfactory bulb. FGF-1 is selectively present in glomeruli and the external plexiform layer. In cultures of olfactory neuroepithelial cells, complexes between FGF-1 and an appropriate activating heparan sulfate proteoglycan stimulated morphological differentiation of both olfactory nerve ensheathing cells and primary sensory olfactory neurons. Thus, the spatiotemporal expression and the functional properties of FGF-1 in this system suggest that this molecule plays an important regulatory role in the formation of the olfactory pathway.

Primary sensory olfactory neurons exhibit a mosaic topographical projection from the olfactory neuroepithelium in the nasal cavity to the olfactory bulb formation of the telencephalon. Axons from primary neurons that are widely scattered in the epithelium terminate in discrete regions of the olfactory bulb. It has been hypothesised that carbohydrates present on the surface of primary olfactory axons mediate selective fasciculation and the formation of the topographical pathway. We examined the expression of the disaccharide N-acetyl-lactosamine in both the developing and the adult rat olfactory system. N-acetyl-lactosamine was expressed by all primary sensory olfactory neurons and by their terminations in the olfactory bulb throughout embryonic development and early postnatal life. In adults, N-acetyl-lactosamine was restricted to a subpopulation of primary sensory olfactory neurons that were dispersed throughout the neuroepithelium but that projected predominantly to the ventrolateral and ventromedial surfaces of the olfactory bulb. The axons of these neurons sort out in the outer layer of the bulb and preferentially self-fasciculate to form distinct axon bundles that terminate within select glomeruli. The role of N-acetyl-lactosamine in axon growth was tested by culturing primary sensory olfactory neurons on substrate-bound carbohydrates. Olfactory neuroepithelium cultures from both embryonic and postnatal rats revealed that substrate-bound N-acetyl-lactosamine was a strong and specific neurite growth-promoting agent. These data suggest that, during development of the olfactory projection, N-acetyl-lactosamine, which is present on all olfactory axons, acts as a nonselective permissive substrate for axon growth. In adults, however, the restricted distribution of N-acetyl-lactosamine on a subpopulation of axons may facilitate sorting out and self-fasciculation, which is necessary for preserving the mosaic nature of the olfactory pathway in this highly plastic region of the nervous system. These results support the hypothesis that cell surface carbohydrates are involved in axon growth in the olfactory system.

Interactions between carbohydrate ligands and their receptors play an important role in cell adhesion and migration in many tissues. Cell-surface carbohydrates that contain terminal galactose have previously been implicated in primary sensory axon growth in the rodent olfactory system. The aim of the present study was to determine whether galectin-1, a galactose-binding receptor, was expressed within the rat primary olfactory pathway. Immunohistochemical and in situ hybridisation analyses revealed expression of galectin-1 by primary sensory olfactory neurons during the major embryonic period of axonogenesis as well as in maturity. In the adult olfactory bulb, galectin-1 was expressed by both second-order projection neurons and interneurons and was selectively localised to the synaptic neuropil layers. Mitral cells, the principal postsynaptic target of primary olfactory axons, began expressing this lectin soon after genesis and maintained high levels into adulthood. The expression of galectin-1 in the primary olfactory pathway and olfactory bulb neuropil suggests a role for this lectin both in the initial formation and in the subsequent maintenance of neuronal connections between the peripheral and the central olfactory neurons as well as between neurons within the bulb.

The axons of the primary sensory olfactory neurons project from the olfactory neuroepithelium lining the nasal cavity, onto glomeruli covering the surface of the olfactory bulb. Neuroanatomical studies have shown previously that individual olfactory glomeruli are innervated by neurons that are dispersed widely within the nasal cavity. The aim of the present study was to test the hypothesis that phenotypically unique subsets of primary sensory olfactory neurons, scattered throughout the nasal cavity, project to a subset of glomeruli in specific olfactory bulb loci. Immunochemical and histochemical analyses in neonatal mice revealed that the plant lectin, Dolichos biflorus agglutinin, bound to a subset of mature primary sensory olfactory neurons which express the olfactory marker protein. This subset of neurons was principally located in the rostromedial and dorsal portions of the nasal cavity and projected specifically to a subset of glomeruli in the rostromedial and caudodorsal portions of the olfactory bulb. Analysis of Dolichos biflorus-reactive axons revealed that these axons coursed randomly, with no evidence of their selective fasciculation, within the olfactory nerve. It was only at the level of the rostral olfactory bulb that a significant reorganisation of their trajectory was observed. Within the outer fibre layer of the bulb, discrete bundles of lectin-reactive axons began to coalesce selectively into fascicles which preferentially oriented toward the medial side of the olfactory bulb. These data demonstrated that a phenotypically distinct subset of primary sensory olfactory neurons exhibits a topographical projection from the olfactory epithelium to the olfactory bulb, and suggests that these, and other subsets, may form the basis of the mosaic nature of this pathway. Moreover, it appears that the outer nerve fibre layer in the rostral olfactory bulb plays an important instructive role in the guidance and fasciculation of olfactory sensory axons.