Immature Olfactory Receptor Neurons Research Articles

Regeneration of olfactory neuroepithelium in 3-methylindole-induced anosmic rats treated with intranasal chitosan

Olfactory dysfunction significantly impairs the life quality of patients but without effective treatments to date. The previous report has demonstrated that chitosan mediates the differentiation of olfactory receptor neurons (ORNs) through insulin-like growth factors and insulin-like growth factor binding protein-2 axis in an in vitro model. However, whether chitosan can further treat olfactory dysfunction in vivo remains unexplored. This study aims to evaluate the therapeutic effect of chitosan on a 3-methylindole-induced anosmic rat model. Intraperitoneal injection of 3-methylindole is performed to induce anosmia in rats. Experimental results demonstrate that the food-finding duration after chitosan treatment gradually decrease to around 80 s, and both the olfactory neuroepithelium (ON) thickness and mature ORNs (expressing olfactory marker protein) are significantly restored. Furthermore, proliferating cells (expressing bromodeoxyuridine) are mainly co-expressed with immature ORNs (expressing βIII tubulin) below the intermediate layer of the ON in the chitosan-treated group on day 28 following 3-methylindole treatment. Conversely, proliferating cells are scattered over the ON, and co-localized with immature ORNs and sustentacular cells (expressing keratin 18) in the sham group, and even immature ORNs go into apoptosis (expressing DNA fragmentation and cleaved caspase-3), possibly causing incomplete regeneration. Consequently, chitosan regenerates the ON by regulating olfactory neural homeostasis and reducing ORN apoptosis, and serves as a potential therapeutic intervention for olfactory dysfunction in the future.

Evidence of SARS-CoV2 Entry Protein ACE2 in the Human Nose and Olfactory Bulb

Usually, pandemic COVID-19 disease, caused by SARS-CoV2, presents with mild respiratory symptoms such as fever, cough, but frequently also with anosmia and neurological symptoms. Virus-cell fusion is mediated by angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) with their organ expression pattern determining viral tropism. Clinical presentation suggests rapid viral dissemination to the central nervous system leading frequently to severe symptoms including viral meningitis. Here, we provide a comprehensive expression landscape of ACE2 and TMPRSS2 proteins across human postmortem nasal and olfactory tissue. Sagittal sections through the human nose complemented with immunolabelling of respective cell types represent different anatomically defined regions including olfactory epithelium, respiratory epithelium of the nasal conchae and the paranasal sinuses along with the hardly accessible human olfactory bulb. ACE2 can be detected in the olfactory epithelium as well as in the respiratory epithelium of the nasal septum, the nasal conchae, and the paranasal sinuses. ACE2 is located in the sustentacular cells and in the glandular cells in the olfactory epithelium as well as in the basal cells, glandular cells, and epithelial cells of the respiratory epithelium. Intriguingly, ACE2 is not expressed in mature or immature olfactory receptor neurons and basal cells in the olfactory epithelium. Similarly, ACE2 is not localized in the olfactory receptor neurons albeit the olfactory bulb is positive. Vice versa, TMPRSS2 can also be detected in the sustentacular cells and the glandular cells of the olfactory epithelium. Our findings provide the basic anatomical evidence for the expression of ACE2 and TMPRSS2 in the human nose, olfactory epithelium, and olfactory bulb. Thus, they are substantial for future studies that aim to elucidate the symptom of SARS-CoV2 induced anosmia via the olfactory pathway.

Detection and localization of the thyroid hormone receptor beta mRNA in the immature olfactory receptor neurons of chum salmon

Thyroid hormone (TH) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in various organs and tissues of vertebrates. It is generally accepted that anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal stream during their seaward migration, and they then use olfaction to discriminate their natal stream during the spawning migration. Both serum TH levels and the specific binding values of TH in the salmon olfactory epithelium were markedly increased during the seaward migration. However, thyroid hormone receptor (TR) expression in the olfactory epithelium has not been confirmed in vertebrates. We investigated gene expression of TR isoforms in chum salmon (O. keta) by both molecular biological and histochemical techniques. Expression of TRβ mRNA was detected in the olfactory epithelium by reverse transcriptase polymerase chain reaction (RT-PCR). Nucleotide sequencing demonstrated the existence of a remarkable homology between the RT-PCR product and part of the ligand-binding domain of other teleost TRβ isoforms. By in situ hybridization using a digoxygenin-labeled salmon olfactory TRβ cRNA probe, signals for salmon olfactory TRβ mRNA were observed preferentially in the perinuclear regions of immature olfactory receptor neurons (ORNs), as protein gene product 9.5 (PGP9.5)-immunopositive ORNs. Our results provide the first detection of TRβ gene expression in the olfactory epithelium, and suggested the possibility that TRβ may be involved in cell maturation and/or cell differentiation of the ORNs in Pacific salmon.

Expression of Nogo receptor 1 in the regeneration process of the mouse olfactory epithelium.

Nogo receptor 1 (NgR1) is the most important Nogo-A receptor. By its interaction with myelin-associated inhibitory proteins, NgR1 inhibits the regeneration of axons and is extensively expressed in the central nervous system. However, the expression of NgR1 in regenerable neurons, such as olfactory neurons, and its expression in the regeneration progress of olfactory neurons have not been reported. In this study, we demonstrated that NgR1 was expressed in the cell bodies of certain mature olfactory receptor neurons (ORNs) but was not expressed in immature ORNs in the olfactory epithelium (OE) of normal adult mice. On day 21 after OE injury, NgR1 was expressed not only in the cell bodies of mature ORNs but also in the cell bodies of glial fibrillary acidic protein (GFAP)-positive cells in the top and submucosal layers of the OE. On day 48 after model establishment, NgR1 expression decreased in the cell bodies of the GFAP-positive cells. On day 56 after model establishment, no NgR1 expression was found in the cell bodies of the GFAP-positive cells, and NgR1 was again expressed only in the mature ORNs. Our results demonstrated that NgR1 expression is upregulated in the OE after injury, which suggests that NgR1 might be involved in the regeneration of the OE.

Reduced amount of olfactory receptor neurons in the rat model of depression

Reduced olfactory sensitivity has been reported in depressive disorder. However, the pathological mechanism is still unclear. The reduced olfactory bulb (OB) volume and reduced hippocampal neurogenesis has been unraveled in major depressive disorder (MDD). However, changes in olfactory epithelium (OE) have not been reported, which might contribute to olfactory deficits in MDD. In the context, we investigated the thickness of OE in a chronic unpredictable mild stress (CUMS) rat model of depression using hematoxylin and eosin (HE) staining. Simultaneously, the basal cells (labeled by nerve growth factor receptor (p75NGFR)), immature olfactory receptor neurons (ORNs) (marked by growth-associated protein 43 (GAP43)) and mature ORNs (labeled by olfactory marker protein (OMP)) in OE were detected by immunohistochemistry. The results showed that the thickness of OE, the number of basal cells, immature ORNs as well as mature ORNs decreased dramatically in the OE of CUMS rats. Those findings indicate that the reduced number of ORNs might induce OE atrophy in CUMS rats and the abnormalities of the OE may be partially responsible for the reduced olfactory sensitivity in MDD.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5−/− olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5–Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Taurine deficiency and apoptosis: Findings from the taurine transporter knockout mouse

Apoptosis is characterized by cell shrinkage, nuclear condensation, DNA-fragmentation and apoptotic body formation. Compatible organic osmolytes, e.g. taurine, modulate the cellular response to anisotonicity and may protect from apoptosis. Taurine transporter knockout mice ( taut−/− mice) show strongly decreased taurine levels in a variety of tissues. They develop clinically important age-dependent diseases and some of them are characterized by apoptosis. Increased photoreceptor apoptosis leads to blindness of taut−/− mice at an early age. The taurine transporter may not be essential for the differentiation of photoreceptor cells, but many mature cells do not survive without an intact taurine transporter. The olfactory epithelium of taut−/− mice also exhibits structural and functional abnormalities. When compared with wild-types, taut−/− mice have a significantly higher proliferative activity of immature olfactory receptor neurons and an increased number of apoptotic cells. This is accompanied by electrophysiological findings indicating a reduced olfactory sensitivity. Furthermore, taut−/− and taut+/− mice develop moderate unspecific hepatitis and liver fibrosis beyond 1 year of age where hepatocyte apoptosis and activation of the CD95 system are pronounced.

Transition of mouse de novo methyltransferases expression from Dnmt3b to Dnmt3a during neural progenitor cell development

Dnmt3a and Dnmt3b, which are known as functional de novo methyltransferases, are responsible for creating genomic methylation patterns during mammalian development. Recently, we have shown that specific expression of Dnmt3b in epiblast, embryonic ectoderm, hematopoietic progenitor cells and spermatogonia cells is followed by Dnmt3a expression (Watanabe D, Suetake I, Tada T, Tajima S (2002) Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev 118:187–190; Watanabe D, Suetake I, Tajima S, Hanaoka K (2004) Expression of Dnmt3b in mouse hematopoietic progenitor cells and spermatogonia at specific stages. Gene Expr Patterns 5:43–49). In this study, we analyzed the expression of mouse de novo methyltransferases during development of the nervous systems. In the embryonic olfactory epithelium (OE), Dnmt3b was specifically expressed in Mash1 positive globose basal cells (i.e. transiently amplifying neural progenitor cells), while Dnmt3a was expressed in immature olfactory receptor neurons. Dnmt3b-positive cells were rarely observed in the adult OE, but were increased in regenerating OE with intranasal ZnSO 4 administration. Dnmt3b was also detected in the E8.5 neural plate, E10.5 spinal cord and retina cells, while Dnmt3a was expressed in postmitotic young neurons. Furthermore, Dnmt3b was specifically expressed in ES cells, while Dnmt3a was transiently expressed during neural cell differentiation of ES cells. Dnmt3b is specifically expressed in progenitor cells during hematopoiesis, spermatogenesis and neurogenesis, suggesting an important role in the initial steps of progenitor cell differentiation. Dnmt3a is expressed in postmitotic young neurons following the Dnmt3b expression. Dnmt3a may be required for the establishment of tissue-specific methylation patterns of the genome. The coordinated expression of de novo methyltransferases from Dnmt3b to Dnmt3a suggests conserved mechanisms of de novo methylation of the genome and different functions for Dnmt3b and Dnmt3a during progenitor cell development.

Neuronal expression of Nogo‐A mRNA and protein during neurite outgrowth in the developing rat olfactory system†

The major impediments to axonal regeneration in the central nervous system are growth-inhibitory proteins present in the myelin sheath, and Nogo-A is one of the most potent inhibitors synthesized by oligodendrocytes. However, neuronal expression of Nogo-A during development suggests that it may have an additional role. The spatio-temporal regulation of both Nogo-A mRNA and protein expression was examined by in situ hybridization and immunohistochemistry in the developing rat olfactory system. During embryonic and postnatal development (from E13 to P6), Nogo-A mRNA and protein were strongly expressed by differentiating neurons in the olfactory epithelium and in the olfactory bulb. From the second postnatal week, a progressive down-regulation of both Nogo-A mRNA and protein occurred, such that only a weak expression persisted in the adult olfactory system. Using double-immunostainings in the adult olfactory epithelium, we determined that Nogo-A was preferentially expressed by immature olfactory receptor neurons extending axonal processes toward the olfactory bulb. At all developmental stages, Nogo-A protein was preferentially targeted in olfactory axons emerging from the olfactory epithelium. Using an in vitro model of olfactory axon growth, we demonstrated that, in addition to its presence along the entire axon length, Nogo-A accumulated in axonal growth cone and at axonal branching points, with a distribution similar to that of microtubule-associated proteins. Moreover, Nogo-A was transiently expressed in dendritic processes in the postnatal olfactory bulb. Together, our data suggest that, in non-pathological conditions, Nogo-A may be involved in the processes of axonal growth and dendritic modeling through the regulation of microtubule dynamics.

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying "microzones" of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.

Expression of TGF-β type II receptors in the olfactory epithelium and their regulation in TGF-α transgenic mice

Numerous in vitro studies of neurogenesis of olfactory receptor neurons (ORNs) suggest that transforming growth factor (TGF)-β promotes the maturation/differentiation of olfactory progenitors. We demonstrate that in vivo both mature and immature ORNs, and possibly a basal neuronal progenitor cell, express the TGF-β type II receptor (TGF-βRII), suggesting that these cells are targets for TGF-β signaling. In a previous study of neurogenesis in the OE of TGF-α overexpressing transgenic (T) mice, we observed an apparent reduction in the expression of olfactory marker protein (OMP), a marker of terminal differentiation in ORNs in T mice compared to nontransgenic (NT) littermate controls; this was confirmed by Western blotting and immunohistochemistry. In contrast, there was no apparent difference between T and NT mice in the intensity of immunoreactivity for a neuronal marker, protein gene product 9.5. Because TGF-α overexpression has been reported to affect TGF-β signaling in other epithelia, we compared the expression of the TGF-β type II receptor (TGF-βRII) in T and NT mice. The intensity of TGF-βRII immunoreactivity on ORNs was substantially reduced in T compared to NT mice. Similar reductions in TGF-βRII expression in vomeronasal receptor neurons and in other epithelia in the nasal cavity of T mice were also observed. Taken together, these results indicate that TGF-β signaling regulates terminal differentiation of ORNs in vivo and suggest ways in which interactions between TGF-α and TGF-β signaling pathways may interact in the OE.

Mature olfactory receptor neurons express connexin 43

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.

Apoptosis in the development of the mouse olfactory epithelium

Apoptotic cells were detected in the mouse olfactory epithelium (OE) at different embryonic and postnatal stages by in situ nick translation (ISNT) and Tdt-mediated dUTP nick end-labeling (TUNEL) techniques. During development, the apoptotic process presented two peaks. One at E12 during the invagination of the olfactory placode and the second at E16 corresponding to olfactory axon synaptogenesis. Then, from E18, a sharp decrease in the number of apoptotic cells was observed and at E19 the apoptotic index reached low values that were maintained in postnatal stages, P1 and P8, and in the adult. Apoptotic nuclei belonged to mature as well as immature olfactory receptor neurons (ORNs). Indeed, double-labeling experiments evidenced apoptotic neurons immunopositive for olfactory marker protein (OMP), carnosine and GAP-43. According to our data, two apoptotic phases occur during early development. One is involved in the morphogenesis of the OE when this last is not yet, or poorly, connected to its target, the olfactory bulb (OB). The second peak of apoptosis is more closely dependent on the interplay between OE and OB.

Alterations in the neurotrophic factors BDNF, GDNF and CNTF in the regenerating olfactory system.

Neurogenesis, axonal outgrowth and synapse formation are usually restricted to specific stages during central nervous system development, but the mature olfactory system maintains these capacities. The cycle of neuronal turnover can be experimentally induced by surgical ablation of the olfactory bulb (OB). We are interested in the growth factor regulation of these processes and the trophic role played by the target tissue, the OB. We studied the immunohistochemical expression of three neurotrophic factors, brain derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the rat olfactory neuroepithelium (ON) and OB and in target-deprived ON at 1, 3 and 12 weeks post unilateral bulbectomy. We found BDNF immunoreactivity (IR) was restricted to the basal cells and did not alter postbulbectomy. GDNF-IR was expressed by mature olfactory receptor neurons (ORNs), their axons and target cells in the OB in controls, but was absent from the ON postbulbectomy. Hence, the expression of GDNF by ORNs was found to be target-dependent. CNTF-IR was present in ORNs and their target cells in the OB, in basal cells and in some immature ORNs. Postbulbectomy, CNTF-IR was unaltered in the basal cells, and very low levels were detectable in maturing ORNs in the ON. Our results indicate that these three factors may contribute to the trophic regulation of this neuronal pathway in a coordinated fashion. Previous work has shown that BDNF promotes survival of ORNs in vitro, and TrkB expression has been found in both immature and mature ORNs. Hence, BDNF produced by basal cells may be acting locally on neurons expressing TrkB. Expression of CNTF by both the basal cells and the ORNs suggests that it may play an integral role in this neuronal differentiation pathway. Finally, the expression of GDNF exclusively by mature ORNs in the ON, its presence in the target cells in the OB and abolition of expression by bulbectomy, suggests that it may be target-derived. This provides a major mechanism by which the bulb could exert trophic influences on ORNs.

Differential expression of lactoseries carbohydrate epitopes HNK-1, CD15, and NALA by olfactory receptor neurons in the developing chick.

The formation of the nasal lining with its sensory and its nonsensitive respiratory epithelium requires a spatially ordered pattern of cellular differentiation. Aiming at identifying cell recognition molecules that may be involved in cellular differentiation steps, we applied a panel of antibodies to terminal carbohydrate sequences of the lactoseries on the developing chick olfactory epithelium. This approach is based on the idea that these terminal sugar residues may be involved in certain steps of maturation. Restricted expression of three epitopes NALA, HNK-1, and CD15 was observed in olfactory receptor neurons. The first immature olfactory receptor neurons were observed by day 3 of incubation, expressing the HNK-1 epitope, whereas a total epithelial staining was observed for NALA. By day 9 of incubation high numbers of HNK-1 positive immature olfactory receptor neurons were observed. At the same time mature olfactory receptor neurons showed immunoreactivity for CD15, whereas NALA was still expressed throughout the whole epithelial cell population. However, there was a pronounced staining in the population of mature olfactory receptor neurons. Around hatching only CD15 was detectable in (mature) olfactory receptor neurons, whereas HNK-1 and NALA immunoreactivity have switched to glandular and sustentacular cells respectively. The differentiation-dependent expression patterns of these three cell surface molecules suggest them as suitable markers to explore mechanisms that determine embryonic olfactory receptor neurogenesis.

Expression of extracellular matrix molecules and cell surface molecules in the olfactory nerve pathway during early development.

The spatial and temporal expression patterns of several extracellular matrix molecules--laminin and fibronectin and cell surface molecules, neural cell adhesion molecule (NCAM), L1, tenascin, chondroitin sulfate proteoglycan, and peanut agglutinin (PNA) binding sites--were investigated during early olfactory nerve development. NCAM and L1 have similar patterns: They are expressed in the olfactory nerve and on the olfactory receptor neurons (ORNs) commencing with the earliest olfactory axon outgrowth (E12-E15). Their expression patterns suggest that both NCAM and L1 are associated with extension and fasciculation of olfactory axons. A comparison of L1 and olfactory marker protein suggests that L1 is expressed predominantly on immature ORNs. Laminin has an unique punctate staining pattern in the developing olfactory pathway as early as E12. These laminin puncta might play a role in olfactory neurite outgrowth and guidance. At E14, when pioneer olfactory axons enter the brain, the laminin-positive meninges on the surface of the olfactory bulb primordium break down but remain intact in the rest of the telencephalon. This suggests a functional interaction between the olfactory axons and the glial-pial barrier. Fibronectin staining is diffuse throughout the cranial mesenchyme but is absent from the olfactory nerve pathway. No specific patterns of tenascin or chondroitin sulfate, were observed during early olfactory development. PNA binding sites were associated with olfactory axon fasciculation. The expression of several extracellular matrix molecules and cell surface molecules is spatially and temporally regulated in the developing olfactory system. These molecules, thus, may play functional roles in olfactory axon outgrowth, fasciculation, and/or guidance.

Expression of extracellular matrix molecules and cell surface molecules in the olfactory nerve pathway during early development

The spatial and temporal expression patterns of several extracellular matrix molecules—laminin and fibronectin and cell surface molecules, neural cell adhesion molecule (NCAM), L1, tenascin, chondroitin sulfate proteoglycan, and peanut agglutinin (PNA) binding sites—were investigated during early olfactory nerve development. NCAM and L1 have similar patterns: They are expressed in the olfactory nerve and on the olfactory receptor neurons (ORNs) commencing with the earliest olfactory axon outgrowth (E12-E15). Their expression patterns suggest that both NCAM and L1 are associated with extension and fasciculation of olfactory axons. A comparison of L1 and olfactory marker protein suggests that L1 is expressed predominantly on immature ORNs. Laminin has an unique punctate staining pattern in the developing olfactory pathway as early as E12. These laminin puncta might play a role in olfactory neurite outgrowth and guidance. At E14, when pioneer olfactory axons enter the brain, the laminin-positive meninges on the surface of the olfactory bulb primordium break down but remain intact in the rest of the telencephalon. This suggests a functional interaction between the olfactory axons and the glial-pial barrier. Fibronectin staining is diffuse throughout the cranial mesenchyme but is absent from the olfactory nerve pathway. No specific patterns of tenascin or chondroitin sulfate, were observed during early olfactory development. PNA binding sites were associated with olfactory axon fasciculation. The expression of several extracellular matrix molecules and cell surface molecules is spatially and temporally regulated in the developing olfactory system. These molecules, thus, may play functional roles in olfactory axon outgrowth, fasciculation, and/or guidance. ©1996 Wiley-Liss, Inc.

Apoptosis in the Neuronal Lineage of the Mouse Olfactory Epithelium: Regulationin Vivoandin Vitro

The olfactory epithelium (OE) of the mouse provides a unique system for understanding how cell birth and cell death interact to regulate neuron number during development and regeneration. We have examined cell death in the OE in normal adult mice; in adult mice subjected to unilateral olfactory bulbectomy (surgical removal of one olfactory bulb, the synaptic target of olfactory receptor neurons (ORNs) of the OE); and in primary cell cultures derived from embryonic mouse OE.In vivo,cells at all stages in the neuronal lineage—proliferating neuronal precursors, immature ORNs, and mature ORNs—displayed signs of apoptotic cell death; nonneuronal cells did not. Bulbectomy dramatically increased the number of apoptotic cells in the OE on the bulbectomized side. Shortly following bulbectomy, increased cell death involved neuronal cells of all stages. Later, cell death remained persistently elevated, but this was due to increased apoptosis by mature ORNs alone.In vitro,apoptotic death of both ORNs and their precursors could be inhibited by agents that prevent apoptosis in other cells: aurintricarboxylic acid (ATA), a membrane-permeant analog of cyclic AMP (CPT-cAMP), and certain members of the neurotrophin family of growth factors (brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 5), although no neurotrophin was as effective at promoting survival as ATA or CPT-cAMP. Consistent with observed effects of neurotrophins, immunohistochemistry localized the neurotrophin receptors trkB and trkC to fractions of ORNs scattered throughout neonatal OE. These results suggest that apoptosis may regulate neuronal number in the OE at multiple stages in the neuronal lineage and that multiple factors—potentially including certain neurotrophins—may be involved in this process.

Neuroplasticity in the olfactory system: Differential effects of central and peripheral lesions of the primary olfactory pathway on the expression of B‐50/GAP43 and the olfactory marker protein

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B-50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X-100 (TX-100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B-50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B-50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP-expressing neurons occurs only after peripheral lesion with TX-100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B-50/GAP43-expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP-expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B-50/GAP43 and OMP at long times after peripheral lesion with TX-100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.

Glial influences on axonal growth in the primary olfactory system

Neurogenesis in the olfactory epithelium continues throughout the entire life of mammals, and it is the axons of these newly formed olfactory receptor neurons that grow into the target tissue after the first cranial nerve is injured, not the regenerating axons of mature cells. These axons are able to enter and grow within the CNS of adult animals, unlike regenerating axons in injured dorsal roots, the majority of which are prevented from penetrating very far into the spinal cord. One reason why the olfactory axons are so successful in entering the CNS may be due, at least partially, to the fact that they are ensheathed by a type of glial cell (the ensheathing cell) that expresses phenotypic features of both astrocyte and Schwann cells. The presence of both L1/Ng-CAM and N-CAM in the plasma membranes of both ensheathing cells and immature olfactory receptor neurons would enable the olfactory axons to use the glial cell surfaces as a substratum on which to grow. It is probably also true that ensheathing cells synthesize and secrete laminin, thus providing an additional adhesive substrate for the olfactory axons, as well as glia-derived nexin and nerve growth factor, both of which are neurite-promoting agents.