Mechanisms of axonal plasticity: lessons from the olfactory pathway.

The olfactory pathway has emerged recently as an effective model for studying general principles of axon extension and regeneration. A variety of both trophic as well as repulsive molecules are found in the olfactory pathway and are being characterized for their roles in promoting the high capacity for plasticity and growth in olfactory receptor cell axons. In addition, olfactory ensheathing cells, which line the olfactory nerve, have been shown to promote axon extension not only in the olfactory pathway but also in the injured spinal cord. This review summarizes some of our current knowledge of these mechanisms and how they may function collectively to promote axon plasticity.

G(o) protein-dependent survival of primary accessory olfactory neurons.

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the G(o)-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain G(i2) and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the G(o)alpha gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of G(o)-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of G(o) protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

Glomerular formation in the developing rat olfactory bulb.

Using the confocal microscope together with markers for the cellular components of glomeruli, we examined the spatiotemporal cellular interactions that occur between the axons of olfactory receptor cells, their dendritic targets, and glial cells during the critical period of glomerular formation. We have employed markers of immature and mature olfactory receptor cell axons, mitral/tufted cell dendrites, and glial cells as well as a synapse-associated protein for double- and triple-label immunocytochemistry. Axons of olfactory receptor cells grew into a dense dendritic zone of the olfactory bulb (comprising the dendrites of both mitral and tufted cells) between E17 and E18. At E19, these axons coalesced into protoglomeruli, which continued to develop until birth, when the basic anatomical structure of adult glomeruli emerged. Neither mitral/tufted cell dendrites nor olfactory bulb astrocytes became specifically associated with these protoglomeruli until E21. Ensheathing cells remained restricted to the outer nerve fiber layer and did not appear to contribute to glomerular formation. Finally, the synaptophysin staining has shown that synaptic constituents are expressed as early as E17, prior to the appearance of mature olfactory receptor cell axons. Based on these data, we have established a time line detailing the temporal and spatial interactions that occur between cell types during late embryonic rat olfactory bulb development. We conclude that the initial event in the formation of glomeruli is the penetration of the mitral/tufted cell dendritic zone by olfactory receptor cell axons. The coalescence of dendritic and glial processes into glomerular structures appears secondary to the arrival of the olfactory receptor cell axons.

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform.

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli.

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway.

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb.

Olfactory glomeruli are innervated by more than one distinct subset of primary sensory olfactory neurons in mice.

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.

Expression and localization of FGF-1 in the developing rat olfactory system.

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.