Expression of L1 and N-CAM cell adhesion molecules during development of the mouse olfactory system

The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.     

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. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

Development of a glial network in the olfactory nerve: role of calcium and neuronal activity

In adult olfactory nerves of mammals and moths, a network of glial cells ensheathes small bundles of olfactory receptor axons. In the developing antennal nerve (AN) of the moth Manduca sexta, the axons of olfactory receptor neurons (ORNs) migrate from the olfactory sensory epithelium toward the antennal lobe. Here we explore developmental interactions between ORN axons and AN glial cells. During early stages in AN glial-cell migration, glial cells are highly dye coupled, dividing glia are readily found in the nerve and AN glial cells label strongly for glutamine synthetase. By the end of this period, dye-coupling is rare, glial proliferation has ceased, glutamine synthetase labeling is absent, and glial processes have begun to extend to enwrap bundles of axons, a process that continues throughout the remainder of metamorphic development. Whole-cell and perforated-patch recordings in vivo from AN glia at different stages of network formation revealed two potassium currents and an R-like calcium current. Chronic in vivo exposure to the R-type channel blocker SNX-482 halted or greatly reduced AN glial migration. Chronically blocking spontaneous Na-dependent activity by injection of tetrodotoxin reduced the glial calcium current implicating an activity-dependent interaction between ORNs and glial cells in the development of glial calcium currents.     

Degenerative and regenerative processes in the olfactory system of homing pigeons.

Experimental resection of the olfactory nerve in the homing pigeon induces a total degeneration of the nerve and olfactory epithelium. The orthograde degenerative process starts before the retrograde one. Ten days after resection, new neurons begin to differentiate from the basal cells. The axon forms earlier than the distal dendritic process, and the speed of growth increases slowly. The regenerated axons only reach the bulb in the 5th month. Two months after resection the olfactory epithelium is similar to that of the intact control side. The ultrastructural features of the mucosa and olfactory axons are similar to those of normal ones.     

The neurite growth promoting protease nexin 1 in glial cells of the olfactory bulb of the gerbil: An ultrastructural study

The glia-derived serine protease inhibitor and neurite outgrowth promotor protease nexin-1 (PN-1) is expressed in Schwann cell precursors and astroblasts during embryogenesis. In the adult nervous system, PN-1 persists in the Schwann cells and olfactory glia only. Light-microscopic immunohistochemistry has revealed the presence of PN-1 in the olfactory mucosa and in the nerve fiber layer of the olfactory bulb. The present electron-microscopic study of the gerbil olfactory bulb confirms the occurrence of PN-1 in ensheathing cells of the olfactory nerve fiber layer, a special type of glia which envelops olfactory axons. In addition, PN-1 is contained in typical astrocytes of the nerve fiber layer and of the glomerular layer. It is inferred that synthesis of PN-1 in the olfactory bulbs is maintained throughout adulthood because its neurite outgrowth promoting action is required for the continuous renewal of olfactory receptor neurons.     

Reciprocal interactions between olfactory receptor axons and olfactory nerve glia cultured from the developing moth Manduca sexta

In olfactory systems, neuron-glia interactions have been implicated in the growth and guidance of olfactory receptor axons. In the moth Manduca sexta, developing olfactory receptor axons encounter several types of glia as they grow into the brain. Antennal nerve glia are born in the periphery and enwrap bundles of olfactory receptor axons in the antennal nerve. Although their peripheral origin and relationship with axon bundles suggest that they share features with mammalian olfactory ensheathing cells, the developmental roles of antennal nerve glia remain elusive. When cocultured with antennal nerve glial cells, olfactory receptor growth cones readily advance along glial processes without displaying prolonged changes in morphology. In turn, olfactory receptor axons induce antennal nerve glial cells to form multicellular arrays through proliferation and process extension. In contrast to antennal nerve glia, centrally derived glial cells from the axon sorting zone and antennal lobe never form arrays in vitro, and growth-cone glial-cell encounters with these cells halt axon elongation and cause permanent elaborations in growth cone morphology. We propose that antennal nerve glia play roles similar to olfactory ensheathing cells in supporting axon elongation, yet differ in their capacity to influence axon guidance, sorting, and targeting, roles that could be played by central olfactory glia in Manduca.     

Astroglia demonstrate regional differences in their ability to maintain primary dendritic outgrowth from mouse cortical neurons in vitro

To determine whether glia from different regions of the central nervous system (CNS) initiate or maintain primary dendritic growth, embryonic day 18 mouse cortical neurons were co-cultured with rat (postnatal day 4) astroglial cells derived from retina, spinal cord, mesencephalon, striatum, olfactory bulb, retina, and cortex. Axon and dendrite outgrowth from isolated neurons was quantified using morphological and immunohistochemical techniques at 18 h and 1, 3, and 5 days in vitro. Neurons initially extend the same number of neurites, regardless of the source of glial monolayer; however, glial cells differ in their ability to maintain primary dendrites. Homotypic cortical astrocytes maintain the greatest number of primary dendrites. Glia derived from the olfactory bulb and retina maintained intermediate numbers of dendrites, whereas only a small number of primary dendrites were maintained by glia derived from striatum, spinal cord, or mesencephalon. Longer axons were initially observed from neurons grown on glia that did not maintain dendrite number. Axonal length, however, was similar on the various monolayers after 5 days in vitro. Neurons that were grown in media conditioned by either mesencephalic or cortical glia for the first 24 h followed by culture media from glia of the alternate source for 4 days in vitro confirmed that glia maintained, rather than initiated, the outgrowth of the primary dendritic arbor. These results indicate that glial cells derived from various CNS regions differ in their ability to maintain the primary dendritic arbor from mouse cortical neurons in vitro. © 1995 John Wiley & Sons, Inc.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Expression of the small conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channel,SK3, in the olfactory ensheathing glial cells of rat brain

Olfactory sensory neurons are wrapped by ensheathing glial cells in the olfactory nerve layer (ONL). Neither functional roles nor electrical properties of ensheathing glial cells have been, as yet, fully clarified. Four subunits (SK1–4) of small conductance Ca²⁺-activated K⁺ (SK) channels have been cloned. In the present study, immunohistochemical analyses showed that SK3 channels are expressed in ensheathing glial cells in the rat olfactory bulb, in addition to neuronal cells in other regions. Western blotting analysis demonstrated that SK3 was predominantly expressed in the olfactory bulb, thalamus, moderately in the hippocampus and cerebellum and modestly in the cerebral cortex of the rat brain. SK3 immunoreactivity was detected in the ONL of the olfactory bulb, neural cell body and fibers of the substantia nigra and hypothalamus. SK3 immunoreactivity was quite intense in the outer (superficial) part of the ONL. SK3-immunoreactive structures were overlapped with glial fibrillary acidic protein (GFAP), but not with vimentin, markers for glial cells and olfactory sensory axons, respectively. Immunoelectron microscopy showed that SK3 immunoreactivity was localized in thin processes that enfolded fascicles of immunonegative olfactory nerve axons. These results indicate that SK3 is expressed specifically in the olfactory ensheathing glial cells in olfactory regions.This work was supported in part by a Grant-in-Aid to A.F. for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by scholarship from Ono Pharmaceutical Company, and by Narishige Neuroscience Research Foundation.     

Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice

The olfactory epithelium of mice generally consists of olfactory cells, progenitors of olfactory cells (globose basal cells), supporting cells, and horizontal basal cells. However, in the dorsal fossa (the roof) of the posterior nasal cavity of mice, we found seven epithelial patches consisting of only non-neuronal cell types, i.e., supporting cells and horizontal basal cells, among the normal olfactory epithelium. The supporting cells occupied three or four layers in the apical to middle regions; in the basal region, horizontal basal cells were localized in a single row adjacent to the basement membrane. Bowman's gland ducts were also present in the epithelium. Neuronal cells (olfactory cells and globose basal cells) were totally absent. The ultrastructure of the supporting cells, horizontal basal cells, and Bowman's glands was essentially similar to that in the normal olfactory epithelium. In the early postnatal period (P1-P7), cell types in the epithelium were the same as those in the normal olfactory epithelium. From P10 to P21, olfactory cells and globose basal cells had disappeared from the olfactory epithelium. At this period, the number of TUNEL-positive cells was significantly higher than that in the surrounding olfactory epithelium; ultrastructurally, many apoptotic figures were observed. This suggests that the epithelium consisting of supporting cells and horizontal basal cells is generated by the apoptotic death of olfactory cells and globose basal cells during postnatal development.     

Presence of novel N-CAM glycoforms in the rat olfactory system

The functional activity of the neural cell adhesion molecule N-CAM can be modulated by posttranslational modifications such as glycosylation. For instance, the long polysialic acid side chains of N-CAM alter the adhesion properties of the protein backbone. In the present study, we identified two novel carbohydrates present on N-CAM, NOC-3 and NOC-4. Both carbohydrates were detected on N-CAM glycoforms expressed by subpopulations of primary sensory olfactory neurons in the rat olfactory system. Based on the expression of NOC-3 and NOC-4 and the olfactory marker protein (OMP), four independent subpopulations of primary sensory olfactory neurons were characterized. These neurons expressed: both NOC-3 and NOC-4 but not OMP; both NOC-4 and OMP but not NOC-3; NOC-3, NOC-4, and OMP together; and OMP alone. The NOC-3- and NOC-4-expressing neurons were widely dispersed in the olfactory neuroepithelium lining the nasal cavity. The axons of NOC-4 expressing neurons innervated all glomeruli in the olfactory bulb, whereas the NOC-3 expressing axons terminated in a discrete subset of glomeruli scattered throughout the whole olfactory bulb. We propose that both NOC-3 and NOC-4 are part of a chemical code of olfactory neurons which is used in establishing the topography of connections between the olfactory neuroepithelium and the olfactory bulb. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 659–670, 1997     

Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

Cell Adhesion Molecules and the Migration of LHRH Neurons during Development

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.     

Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex alpha-galactosyl containing glycolipid, as well as N-linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal-NCAM, which contains a blood group B-like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA-NCAM, another NCAM glycoform, Gal-NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA-NCAM expression by immature neurons to the expression of Gal-NCAM by mature neurons.     

Expression of olfactory receptors in the cribriform mesenchyme during prenatal development

Olfactory receptors (ORs) are expressed in sensory neurons of the nasal epithelium, where they are supposed to be involved in the recognition of suitable odorous compounds and in the guidance of outgrowing axons towards the appropriate glomeruli in the olfactory bulb. During development, some olfactory receptor subtypes have also been found in non-sensory tissues, including the cribriform mesenchyme between the prospective olfactory epithelium and the developing telencephalon, but it is elusive if this is a typical phenomenon for ORs. Monitoring the onset and time course of expression for several receptor subtypes revealed that 'extraepithelial' expression of ORs occurs very early and transiently, in particular between embryonic stages E10.25 and E14.0. In later stages, a progressive loss of receptor expressing cells was observed. Molecular phenotyping demonstrated that the receptor expressing cells in the cribriform mesenchyme co-express key elements, including Galpha(olf), ACIII and OMP, characteristic for olfactory neurons in the nasal epithelium. Studies on transgenic OMP/GFP-mice showed that 'extraepithelial' OMP/GFP-positive cells are located in close vicinity to axon bundles projecting from the nasal epithelium to the presumptive olfactory bulb. Moreover, these cells are primarily located where axons fasciculate and change direction towards the anterior part of the forebrain.     

Transregulation of erbB expression in the mouse olfactory bulb.

Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.     

Outgrowing olfactory axons contain the Reelin receptor VLDLR and navigate through the Reelin-rich cribriform mesenchyme

Chemosensory neurons in the olfactory epithelium (OE) project axonal processes to the olfactory bulb (OB) of the brain. During embryonic stages, on their trajectory to the OB, the outgrowing axons traverse the so-called cribriform mesenchyme, which is located between the OE and the OB. The molecular cues guiding these axons through the cribriform mesenchyme are largely unknown. To identify molecules influencing the axonal trajectory in the murine cribriform mesenchyme, we performed microarray analyses focusing on extracellular matrix (ECM) proteins present in this tissue. Thereby, the ECM protein Reelin turned out to be an interesting candidate. Reelin was found to be expressed by numerous cells in the cribriform mesenchyme during the embryonic stages when the first axons navigate from the OE to the OB. These cells were closely associated with olfactory axons and apparently lack glial and neuronal markers. In the mesenchyme underlying the OE, localization of the Reelin protein was not confined to the Reelin-expressing cells, but it was also observed to be widely distributed in the ECM—most prominently in regions traversed by olfactory axons. Importantly, these axons were found to be endowed with the Reelin receptor very-low-density lipoprotein receptor (VLDLR). Finally, Reelin expression was also detectable in neuronal cells of the OB, which are contacted by VLDLR-positive olfactory axons. In summary, the results of the present study suggest that a Reelin/VLDLR signaling pathway might contribute to the formation of olfactory projections to the OB and the establishment of initial contacts between the incoming axons and neurons in the OB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Funding:  This work was supported by the Deutsche Forschungsgemeinschaft.     

Rhinotopy is disrupted during the re-innervation of the olfactory bulb that follows transection of the olfactory nerve

Re-innervation of the olfactory bulb was investigated after transection of the olfactory nerve using monoclonal antibody RB-8 to assess whether rhinotopy of the primary olfactory projection is restored. In normal animals RB-8 heavily stains the axons, and their terminals, that project from the ventrolateral olfactory epithelium onto glomeruli of the ventrolateral bulb (termed RB-8(+)). In contrast, axons from dorsomedial epithelium are unlabeled (RB-8(-)) and normally terminate in the dorsomedial bulb. Sprague-Dawley rats underwent unilateral olfactory nerve transection and survived for 6 weeks prior to perfusion, sectioning and immunostaining with RB-8. Nerve lesion does not shift the position of the boundary between RB-8(+) and RB-8(-) regions of the epithelium. However, following transection and bulb re-innervation, the distribution of RB-8(+) and RB-8(-) axons is markedly abnormal. First, in all 10 experimental animals RB-8(-) axons displace RB-8(+) axons from anterior glomeruli. Furthermore, the usual target of the RB-8(-) fibers, i.e. the dorsomedial bulb at more posterior levels of the bulb, remains denervated, judging by the lack of staining with antibodies that label axons derived from all epithelial zones. Finally, RB-8(+) fibers invade foreign territory in the dorsolateral bulb on the lesioned side in some cases. The shifts in terminal territory in the bulb after transection contrast with the restoration of the normal zonal patterning of the projection after recovery from methyl bromide lesion, but is consistent with reports of mistargeting by a receptor-defined subset of neurons after transection.