Developing Mouse Olfactory System Research Articles

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

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.

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons.

Gamma-aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin-releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools.

Fas-Associated Factor 1 as a Regulator of Olfactory Axon Guidance

Axon guidance is a crucial part of neural circuit formation. While precise axonal targeting forms the basis of accurate information delivery, the mechanisms that regulate this process are still unclear. Apoptotic signaling molecules have been identified in the axon terminal, but their specific role in axon guidance is not well understood. Here we use the mouse olfactory system as an in vivo model to demonstrate that by modulating Fas-associated factor 1 (FAF1), an apoptosis regulatory molecule, we can rewire axonal projections. Interestingly, FAF1 is highly expressed in the developing mouse olfactory system, but its expression is downregulated postnatally. Using a tetracycline-inducible promoter Tet-Off system, we generated transgenic mice in which FAF1 is specifically expressed in immature olfactory sensory neurons (OSNs) and show that overexpression of FAF1 not only misroutes OSN axons to deep layers of the olfactory bulb but also leads to widespread disruption of the glomerular layer. In addition, we also demonstrate that the specific convergence of P2 receptor OSN axons is completely distorted in the FAF1 mice. Strikingly, all of the mutant phenotypes can be recovered by shutting down FAF1 expression through the administration of doxycycline. Together, our study provides clear in vivo evidence that an apoptotic molecule can indeed regulate axon targeting and that OSNs can restore their organization even after broad disruption.

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system

Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure-specific monoclonal antibodies (HepSS-1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS-1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin-A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events.

Cadherin expression in the developing mouse olfactory system

Although odor receptors have been implicated in establishing the topography of olfactory sensory neurons (OSNs) in the olfactory bulb (OB), it is likely other molecules are also involved. The cadherins (CDHs) are a large family of cell adhesion molecules that mediate cell:cell interactions elsewhere in the central nervous system. However, their distribution and role in the olfactory system have remained largely unexplored. We previously demonstrated that intracellular binding partners of cadherins, the catenins, have unique spatiotemporal patterns of expression in the developing olfactory system. To further our understanding of cadherin function within the developing olfactory system, we now report on the localization of 11 classical cadherins-CDH1, 2, 3, 4, 5, 6, 8, 10, 11, 13, and 15. We demonstrate the expression of all but CDH5 and CDH15 in neuronal and/or glial cells in primary olfactory structures. CDH1 and CDH2 are expressed by OSNs; CDH2 expression closely parallels that seen for gamma-catenin in OSN axons. CDH3 and CDH11 are expressed by olfactory ensheathing glia, which surround OSN axons in the outer OB. CDH2, CDH4, and CDH6 are expressed within neuropil. CDH2, CDH4, CDH6, CDH8, CDH10, CDH11, and CDH13 are expressed by projection neurons within the main and accessory OBs. We conclude that cadherin proteins in the developing olfactory system are positioned to underlie the formation of the odorant map and local circuits within the OB.

Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system

Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.

BMP mRNA and protein expression in the developing mouse olfactory system.

The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (-ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6-ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood.

No Neurotrophins Needed

Adult mammalian olfactory neurons can regenerate, but are the mechanisms at work in these mature neurons the same as those that regulate olfactory neuron proliferation and differentiation during development? Nef et al. report that the neurotrophins and their cognate Trk receptors that have been implicated in the regenerative process are not at work in the developing mouse olfactory system. The authors analyzed mice that did not express TrkA, TrkB, or TrkC, or the neurotrophins NT3 and BDNF. Although the olfactory epithelium and olfactory bulb of wild-type mice did show expression of some Trk receptors and neurotrophins at birth, their absence in mutant mice did not affect nasal cavity or olfactory bulb development at this time, nor did it alter axonal projections from the former to the latter. All the mutant mice do die at early postnatal ages. Hence, neurotrophin signaling is critical to the proliferation of mature neurons, but not to the proliferation and development of embryonic neurons. S. Nef, M. E. Lush, T. E. Shipman, L. F. Parada, Neurotrophins are not required for normal embryonic development of olfactory neurons. Dev. Biol. 234 , 80-92 (2001). [Online Journal]

Distinct localization of two closely-related Ig superfamily molecules, BIG-1 and BIG-2, in the developing mouse olfactory system

Distinct localization of two closely-related Ig superfamily molecules, BIG-1 and BIG-2, in the developing mouse olfactory system

Distribution of neuronal intermediate filament proteins in the developing mouse olfactory system.

The distribution of neuronal intermediate filament proteins in the developing mouse olfactory bulb and olfactory epithelium was characterized by immunocytochemical approach. Antibodies against alpha-internexin, neurofilament triplet proteins (NFTPs; NF-L, NF-M, and NF-H) and peripherin were used to determine their expression at different developmental stages. Alpha-internexin and peripherin were first found to be co-localized in the olfactory neuroepithelium during early development. At the perinatal stage, expression patterns of alpha-internexin and peripherin are distinguishable by spatial and temporal manner: peripherin is predominantly expressed in the olfactory nerves; whereas alpha-internexin is expressed in both olfactory nerves and olfactory bulb. Our observation suggests that peripherin as well as alpha-internexin may play some roles in the process formation of olfactory nerves during development. In the developing olfactory periglomerulus, alpha-internexin was found around postnatal Day 3, whereas NFTPs were not observed until postnatal Day 7. Our data showed that the expression of alpha-internexin preceded those of the NFTPs in most neurons of the developing olfactory bulb. Some small neurons in the adult olfactory bulb were uniquely labeled with antibody to alpha-internexin. Our results suggest that alpha-internexin may play a functional role in the neuronal cytoarchitecture of developing olfactory system, and can be a neuronal marker for detecting postmitotic migrating neurons in the adult olfactory bulb.

Distribution of neuronal intermediate filament proteins in the developing mouse olfactory system

Distribution of neuronal intermediate filament proteins in the developing mouse olfactory system

Transneuronal spread of Semliki Forest virus in the developing mouse olfactory system is determined by neuronal maturity

Many neurotropic virus infections have been shown to be virulent in neonatal and suckling mice but avirulent in weaned mice. The neurotropic alphavirus Semliki Forest virus is a well-studied example of this and importantly the age-related change in neurovirulence of this virus has been shown to be independent of specific immune responses. During the first two postnatal weeks many major physiological changes including axonogenesis, synaptogenesis and myelination occur within the rodent CNS. To investigate whether these changes affect virus replication, spread and virulence we have studied the course of infection in the mouse olfactory system. The olfactory system is well-characterized with regard to its development and neuroanatomy and represents an important route of entry of many neurotropic viruses. Following Semliki Forest virus infection, mice younger than 14 days-of-age died from a fulminant panencephalitis, whilst those 15 days and older survived and cleared the infection. Microscopic examination of brains from mice inoculated intranasally either bilaterally or unilaterally and stained by in situ hybridization to detect viral RNA revealed spread of infection along neurites in a circuit-specific manner. Spread in the main olfactory bulb and to primary, secondary and tertiary olfactory connections was observed. In neonatal mice virus rapidly spread throughout the olfactory system and the temporal progress of the infection correlated with the known connectivity patterns of this system. Both anterograde and retrograde axonal spread were observed. During the first three postnatal weeks the rate and extent of virus spread decreased with increasing age. Spread of infection between specific structures was closely related to neuronal maturation. As olfactory system connections matured transmission of virus was curtailed. In mice inoculated at six weeks or six months-of-age infection was minimal in and rarely observed beyond the continually renewed olfactory nerve layer. The ability of this virus to replicate and, or spread in the CNS is clearly linked to neuronal maturation.

Role of Galectin-1 in the Developing Mouse Olfactory System

Primary sensory olfactory neurons reside in a neuroepithelium lining the nasal cavity and project topographically onto the surface of the olfactory bulb, a rostral extension of the telencephalon. Galectin-1, a bivalent galactose-binding vertebrate lectin, is expressed in the developing rodent olfactory system. In the present study, the mouse olfactory neuron cell line 4.4.2 was used to examine the role of galectin-1 in neurite outgrowthin vitro.Recombinant galectin-1 has neurite outgrowth-promoting activity when used as a substrate for 4.4.2 cells. When either galectin-1 or lactose was added to the culture media, the neurite outgrowth-promoting activity was abolished. These results demonstrate that galectin-1 can modulate neurite growthin vitro.Thein vivorole of galectin-1 was investigated by examining the topographical organization of the olfactory pathway in mice carrying a null mutation for galectin-1. UsingDolichos biflorusagglutinin as a convenient histochemical marker of a subpopulation of primary sensory olfactory neurons which project topographically to the dorsomedial olfactory bulb, we show an aberrant topography of olfactory axons in the null mutants. A subset of primary sensory olfactory axons failed to project to their correct target sites in the caudal olfactory bulb. These data indicate that galectin-1 is involved in the growth and/or guidance of primary sensory olfactory axons between the nasal cavity and the olfactory bulb. This is the first demonstration that a lectin has neurite outgrowth-promoting activity and plays a role in neuronal pathfinding in the mammalian nervous system.

Calcitonin gene-related peptide in the developing mouse olfactory system

The development of calcitonin gene-related peptide (CGRP) was investigated in the mouse olfactory system. The peptide was not found in olfactory receptor neurons in embryos examined from gestational day 13 (E13) to E18 but was present in other brain regions and in peripheral tissues. At E18, CGRP-immunoreactive fibers, presumably of trigeminal, not olfactory receptor cell, origin were observed within the lamina propria. These data suggest that in vivo CGRP does not act through olfactory receptor neurons to regulate phenotypic expression in the olfactory bulb.

Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis

Closure of the nostril by electrocauterization on postnatal day (PN) 1 or 2 was used to study effects of olfactory deprivation on developing olfactory epithelium (OE) and bulb (OB) in CD-1 mice. No damage was observed in OE sections 1 or 3 days after closure, and at PN 30 no difference was found in the number of OE receptors between closed and open sides. Odor deprivation and a decrease in functional activity in experimental bulbs was evident from deoxyglucose autoradiographs at PN 21 and PN 30. At PN 30 deprived bulbs appeared smaller than nondeprived bulbs. Nissl stains revealed normal cytoarchitecture, but a protargol stain demonstrated fewer intraglomerular dendrites in deprived bulbs. At PN 30, volumes of deprived bulbs were 26% smaller than nondeprived bulbs. The volume of each bulbar lamina was 13 to 35% smaller than the comparable nondeprived lamina except for the ventricular/subependymal zone which was not significantly different between bulbs. Volumes of bulbs contralateral to the closed naris and the volumes of their laminae were not significantly different from control bulbs, suggesting no hypertrophy of nondeprived laminae. Deprivation did not affect the number of mitral cells seen at PN 30, their nuclear size, or their number of nucleoli. Lateral olfactory tract cross-sectional area was also unaffected by deprivation. Mitral cell perikaryal size, however, was smaller in deprived bulbs. Soma surface areal density of deprived mitral-to-granule cell synapses in deprived bulbs was 65% of the nondeprived density, while the density of granule-to-mitral cell synapses was only 46% of the nondeprived density. It is concluded that neonatal naris closure brings about a functional deprivation of the OB without receptor degeneration. Neonatal olfactory deprivation affects the perikaryal surface area but not the number of mitral cells. Also, deprivation markedly affects the reciprocal synapses between mitral and granule cells. Olfactory sensation thus appears necessary for normal development of OB neurons and synapses.