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During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve., (© 2014 Wiley Periodicals, Inc.)

Differentiated olfactory sensory neurons express specific presynaptic proteins, including enzymes involved in neurotransmitter transport and proteins involved in the trafficking and release of synaptic vesicles. Studying the regulation of these presynaptic proteins will help to elucidate the presynaptic differentiation process that ultimately leads to synapse formation. It has been postulated that the formation of a synapse between the axons of the sensory neurons and the dendrites of second order neurons in the olfactory bulb is a critical step in the processes of sensory neuron maturation. One approach to study the relationship between synaptogenesis and sensory neuron maturation is to examine the expression patterns of synaptic molecules through the olfactory neuron lineage. To this end we designed specific in situ hybridization probes to target messengers for proteins involved in presynaptic vesicle release. Our findings show that, as they mature, mouse olfactory neurons sequentially express specific presynaptic genes. Furthermore, the different patterns of expression of these presynaptic genes suggest the existence of discrete steps in presynaptic development: genes encoding proteins involved in scaffolding show an early onset of expression, whereas expression of genes encoding proteins involved in the regulation of vesicle release starts later. In particular, the signature molecule for glutamatergic neurons vesicle glutamate transporter 2 shows the latest onset of expression. In addition, contact with the targets in the olfactory bulb is not controlling presynaptic protein gene expression, suggesting that olfactory sensory neurons follow an intrinsic program of development.

Apoptosis of olfactory sensory neurons (OSNs) induced by olfactory bulbectomy (OBX) leads to the activation of resident macrophages within the olfactory epithelium (OE). These macrophages phagocytose degenerating OSNs and secrete chemokines, which recruit additional macrophages into the OE, and cytokines/growth factors, which regulate basal cell proliferation and differentiation and maturation of OSNs. In this study we apply for the first time the use of liposome-encapsulated clodronate to selectively deplete macrophages during the OSN degeneration/regeneration cycle in order to elucidate the role(s) of macrophages in regulating cellular mechanisms that lead to apoptosis and neurogenesis. Mice were injected intranasally and intravenously with either liposome-encapsulated clodronate or empty liposomes prior to and after OBX or sham OBX. At 48 hours after surgery the numbers of macrophages in the OE of both sham and OBX clodronate-treated mice were significantly reduced compared to liposome-treated controls (38% and 35%, respectively, P < 0.05). The reduction in macrophage numbers was accompanied by significant decreases in OE thickness (22% and 21%, P < 0.05), the number of mOSNs (1.2- and 1.9-fold, P < 0.05), and basal cell proliferation (7.6- and 3.8-fold, P < 0.005) in sham and OBX mice, respectively, compared to liposome-treated controls. In OBX mice there was also increased immunoreactivity for active caspase-3 in the OE and olfactory nerves of clodronate-treated OBX mice compared to liposome-treated controls. These results indicate that macrophages modulate the OSN population in the normal and target-ablated murine OE by influencing neuronal survival and basal cell proliferation, resulting in neurogenesis and replacement of mature OSNs., ((c) 2007 Wiley-Liss, Inc.)

The expression of the gap junction subunit connexin 43 was studied in the olfactory epithelium of adult mice. In agreement with conclusions from previous immunohistochemical studies, we observed expression of mRNA encoding for connexin 43 in layers of the epithelium containing nuclei belonging to sustentacular cells. However, we also observed expression of connexin 43 mRNA in the layers containing nuclei belonging to mature olfactory receptor neurons (ORNs), immature ORNs, and basal cells. Connexin 43 mRNA expression was low in dorsomedial regions of the nasal cavity but higher ventrally. This differential regional distribution was consistent with expression in a transgenic mouse of a LacZ reporter gene driven by the proximal 6.5 kb of the connexin 43 promoter. LacZ was expressed in cells colabeled with antibody against olfactory marker protein (OMP), corroborating that mature ORNs express connexin 43. LacZ staining also was observed in sustentacular and basal cells and in immature ORNs. Double-label studies with antibodies against connexin 43 and OMP and expression of connexin 43 in the epithelium of bulbectomized mice were also consistent with expression of connexin 43 in mature ORNs. This is the first report of expression of a connexin subunit in mature ORNs. Our findings of connexin subunits in mature ORNs raise the novel possibility that gap junctions may play a fundamental role in information processing in the olfactory epithelium., (Copyright 2000 Wiley-Liss, Inc.)

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration., (Copyright 2000 Wiley-Liss, Inc.)