Chemokine signaling is required for homeostatic and injury-induced neurogenesis in the olfactory epithelium.

The olfactory epithelium (OE) possesses unique lifelong neuroregenerative capacities and undergoes constitutive neurogenesis throughout mammalian lifespan. Two populations of stem cells, frequently dividing globose basal cells (GBCs) and quiescent horizontal basal cells (HBCs), readily replace olfactory neurons throughout lifetime. Although lineage commitment and neuronal differentiation of stem cells has already been described in terms of transcription factor expression, little is known about external factors balancing between differentiation and self-renewal. We show here that expression of the CXC-motif chemokine receptor 4 (CXCR4) distinguishes both types of stem cells. Extensive colocalization analysis revealed exclusive expression of CXCR4 in proliferating GBCs and their neuronal progenies. Moreover, only neuronal lineage cells were derived from CXCR4-CreER-tdTomato reporter mice in the OE. Furthermore, Cre-tdTomato mice specific for HBCs (Nestin+ and Cytokeratin14+) did not reduce CXCR4 expression when bred to mice bearing floxed CXCR4 alleles, and did not show labeling of the neuronal cells. CXCR4 and its ligand CXCL12 were markedly upregulated upon induction of GBC proliferation during injury-induced regeneration. in vivo overexpression of CXCL12 did downregulate CXCR4 levels, which results in reduced GBC maintenance and neuronal differentiation. We proved that these effects were caused by CXCR4 downregulation rather than over-activation by showing that the phenotypes of CXCL12-overexpressing mice were highly similar to the phenotypes of CXCR4 knockout mice. Our results demonstrate functional CXCR4 signaling in GBCs regulates cell cycle exit and neural differentiation. We propose that CXCR4/CXCL12 signaling is an essential regulator of olfactory neurogenesis and provide new insights into the dynamics of neurogenesis in the OE.

Morphogenesis is transcriptionally coupled to neurogenesis during peripheral olfactory organ development.

Sense organs acquire their distinctive shapes concomitantly with the differentiation of sensory cells and neurons necessary for their function. Although our understanding of the mechanisms controlling morphogenesis and neurogenesis in these structures has grown, how these processes are coordinated remains largely unexplored. Neurogenesis in the zebrafish olfactory epithelium requires the bHLH proneural transcription factor Neurogenin 1 (Neurog1). To address whether Neurog1 also controls morphogenesis, we analysed the migratory behaviour of early olfactory neural progenitors in neurog1 mutant embryos. Our results indicate that the oriented movements of these progenitors are disrupted in this context. Morphogenesis is similarly affected by mutations in the chemokine receptor gene, cxcr4b, suggesting it is a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-box cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development.

Visualizing Cell Cycle Phase Organization and Control During Neural Lineage Elaboration.

In neural precursors, cell cycle regulators simultaneously control both progression through the cell cycle and the probability of a cell fate switch. Precursors act in lineages, where they transition through a series of cell types, each of which has a unique molecular identity and cellular behavior. Thus, investigating links between cell cycle and cell fate control requires simultaneous identification of precursor type and cell cycle phase, as well as an ability to read out additional regulatory factor expression or activity. We use a combined FUCCI-EdU labelling protocol to do this, and then applied it to the embryonic olfactory neural lineage, in which the spatial position of a cell correlates with its precursor identity. Using this integrated model, we find the CDKi p27KIP1 has different regulation relative to cell cycle phase in neural stem cells versus intermediate precursors. In addition, Hes1, which is the principle transcriptional driver of neural stem cell self-renewal, surprisingly does not regulate p27KIP1 in this cell type. Rather, Hes1 indirectly represses p27KIP1 levels in the intermediate precursor cells downstream in the lineage. Overall, the experimental model described here enables investigation of cell cycle and cell fate control linkage from a single precursor through to a lineage systems level.

Comparative growth in the olfactory system of the developing chick with considerations for evolutionary studies.

Despite the long-held assumption that olfaction plays a relatively minor role in the behavioral ecology of birds, crown-group avians exhibit marked phylogenetic variation in the size and form of the olfactory apparatus. As part of a larger effort to better understand the role of olfaction and olfactory tissues in the evolution and development of the avian skull, we present the first quantitative analysis of ontogenetic scaling between olfactory features [olfactory bulbs (OBs) and olfactory turbinates] and neighboring structures (cerebrum, total brain, respiratory turbinates) based on the model organism Gallus gallus. The OB develops under the predictions of a concerted evolutionary model with rapid early growth that is quickly overcome by the longer, sustained growth of the larger cerebrum. A similar pattern is found in the nasal cavity where the morphologically simple (non-scrolled) olfactory turbinates appear and mature early, with extended growth characterizing the larger and scrolled respiratory turbinates. Pairwise regressions largely recover allometric relationships among the examined structures, with a notable exception being the isometric trajectory of the OB and olfactory turbinate. Their parallel growth suggests a unique regulatory pathway that is likely driven by the morphogenesis of the olfactory nerve, which serves as a structural bridge between the two features. Still, isometry was not necessarily expected given that the olfactory epithelium covers more than just the turbinate. These data illuminate a number of evolutionary hypotheses that, moving forward, should inform tradeoffs and constraints between the olfactory and neighboring systems in the avian head.

Sequential Maturation of Olfactory Sensory Neurons in the Mature Olfactory Epithelium.

The formation of the olfactory nerve and olfactory bulb (OB) glomeruli begins embryonically in mice. However, the development of the olfactory system continues throughout life with the addition of new olfactory sensory neurons (OSNs) in the olfactory epithelium (OE). Much attention has been given to the perinatal innervation of the OB by OSN axons, but in the young adult the process of OSN maturation and axon targeting to the OB remains controversial. To address this gap in understanding, we used BrdU to label late-born OSNs in young adult mice at postnatal day 25 (P25-born OSNs) and timed their molecular maturation following basal cell division. We show that OSNs in young adults undergo a sequential molecular development with the expression of GAP 43 (growth-associated protein 43) > AC3 (adenylyl cyclase 3) > OMP (olfactory marker protein), consecutively, in a time frame of ∼8 d. To assess OSN axon development, we implemented an in vivo fate-mapping strategy to label P25-born OSNs with ZsGreen. Using sampling intervals of 24 h, we demonstrate the progressive extension of OSN axons in the OE, through the foramen of the cribriform plate, and onto the surface of the OB. OSN axons reached the OB and began to target and robustly innervate specific glomeruli ∼10 d following basal cell division, a time point at which OMP expression becomes evident. Our data demonstrate a sequential process of correlated axon extension and molecular maturation that is similar to that seen in the neonate, but on a slightly longer timescale and with regional differences in the OE.

Regional differences in mitral cell development in mouse olfactory bulb.

Olfactory sensory neurons (OSNs) located in the dorsomedial and ventromedial regions of the olfactory epithelium (OE) are distinguished from one another based on their molecular expression patterns. This difference is reflected in the separation of the glomerular layer of the olfactory bulb (OB) into dorsomedial and ventrolateral regions. However, it is unclear whether a complementary separation is also evident in the projection neurons that innervate the OB glomeruli. In this study, we compared the development of the OB between different regions by focusing on the transcription factor, Tbx21, which is expressed by mitral and tufted cells in the mature OB. Examining the OB at different developmental ages, we found that Tbx21 expression commenced in the anteromedial region called the tongue-shaped area, followed by the dorsomedial and then ventrolateral areas. We also showed that the tongue-shaped area was innervated by the OSNs located in the most dorsomedial part of the ventrolateral OE, the V-zone:DM. Interestingly, the generation of OSNs occurred first in the dorsomedial zone including the V-zone:DM, suggesting a correlation between the time course of OSN generation in the OE and Tbx21 expression in their target region of the OB. In contrast, expression of vGluT1, which is also found in all mitral cells in the mature OB, was first detected in the ventrolateral region during development. Our findings demonstrate that the development of projection neurons occurs in a compartmentalized manner in the OB; tongue-shaped, dorsomedial, and ventrolateral areas, and that not all projection neurons follow the same developmental pathway.

Potential roles of smell and taste in the orientation behaviour of coral-reef fish larvae: insights from morphology.

An ontogenetic analysis of the olfactory organ and the number and distribution of internal taste buds was carried out in two neon gobies (Elacatinus lori and Elacatinus colini) with the goal of revealing morphological trends that might inform an understanding of the roles of olfaction and taste in larval orientation behaviour. The pattern of development of the olfactory organ is unremarkable and enclosure of the olfactory epithelium occurs concurrently with metamorphosis and settlement in both species. Like other gobies, juvenile and adult E. lori and E. colini lack complex olfactory lamellae, and lack the accessory nasal sacs present in some adult gobies that could facilitate active olfactory ventilation (i.e., sniffing). A small number of internal taste buds are present at hatch with most found in the caudal region of the buccal cavity (on gill arches, roof of buccal cavity). As taste bud number increases, they demonstrate an anterior spread to the lips, buccal valves and tongue (i.e., tissue covering the basihyal). In the absence of an active ventilatory mechanism for the olfactory organs, the water that moves through the buccal cavity with cyclic gill ventilation may provide chemical cues allowing the internal taste buds to play a role in chemical-mediated orientation and reef-seeking behavior in pelagic larval fishes.

G protein γ subunit Gγ13 is essential for olfactory function and aggressive behavior in mice.

Most olfactory receptors in vertebrates are G protein-coupled receptors, whose activation by odorants initiates intracellular signaling cascades through heterotrimeric G proteins consisting of α, ß, and γ subunits. Abolishment of the α subunits such as Gαolf in the main olfactory epithelium and Gαi2 and Gαo in the vomeronasal organ resulted in anosmia and/or impaired behavioral responses. In this study, we report that a G protein γ subunit, Gγ13, is expressed in a spatiotemporal manner similar to those of Gαolf and Gαi2 in the olfactory system and vomeronasal organ, respectively. In addition, Gγ13 was found in the glomeruli of the main olfactory bulb but was largely absent in the glomeruli of the accessory olfactory bulb. Using the Cre-loxP system, the Gγ13's gene Gng13 was nullified in the mature olfactory sensory neurons and apical vomeronasal sensory neurons where the Cre recombinase was expressed under the promoter of the Omp gene for the olfactory marker protein. Immunohistochemistry indicated much reduced expression of Gγ13 in the apical vomeronasal epithelium of the mutant mice. Behavioral experiments showed that the frequency and duration of aggressive encounters in the male mutant mice were significantly lower than in WT male mice. Taken together, these data suggest that the Gγ13 subunit is a critical signaling component in both the main olfactory epithelium and apical vomeronasal epithelium, and it plays an essential role in odor-triggered social behaviors including male-male aggression.

The Neuroregenerative Capacity of Olfactory Stem Cells Is Not Limitless: Implications for Aging.

The olfactory epithelium (OE) of vertebrates is a highly regenerative neuroepithelium that is maintained under normal conditions by a population of stem and progenitor cells, globose basal cells (GBCs), which also contribute to epithelial reconstitution after injury. However, aging of the OE often leads to neurogenic exhaustion, the disappearance of both GBCs and olfactory sensory neurons (OSNs). Aneuronal tissue may remain as olfactory, with an uninterrupted sheet of apically arrayed microvillar-capped sustentacular cell, or may undergo respiratory metaplasia. We have generated a transgenic mouse model for neurogenic exhaustion using olfactory marker protein-driven Tet-off regulation of the A subunit of Diphtheria toxin such that the death of mature OSNs is accelerated. At as early as 2 months of age, the epithelium of transgenic mice, regardless of sex, recapitulates what is seen in the aged OE of humans and rodents. Areas of the epithelium completely lack neurons and GBCs; whereas the horizontal basal cells, a reserve stem cell population, show no evidence of activation. Surprisingly, other areas that were olfactory undergo respiratory metaplasia. The impact of accelerated neuronal death and reduced innervation on the olfactory bulb (OB) was also examined. Constant neuronal turnover leaves glomeruli shrunken and affects the dopaminergic interneurons in the periglomerular layer. Moreover, the acceleration of OSN death can be reversed in those areas where some GBCs persist. However, the projection onto the OB recovers incompletely and the reinnervated glomeruli are markedly altered. Therefore, the capacity for OE regeneration is tempered when GBCs disappear.SIGNIFICANCE STATEMENT A large percentage of humans lose or suffer a significant decline in olfactory function as they age. Therefore, quality of life suffers and safety and nutritional status are put at risk. With age, the OE apparently becomes incapable of fully maintaining the neuronal population of the epithelium despite its well known capacity for recovering from most forms of injury when younger. Efforts to identify the mechanism by which olfactory neurogenesis becomes exhausted with age require a powerful model for accelerating age-related tissue pathology. The current OMP-tTA;TetO-DTA transgenic mouse model, in which olfactory neurons die when they reach maturity and accelerated death can be aborted to assess the capacity for structural recovery, satisfies that need.

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Chronic perinatal odour exposure with heptaldehyde affects odour sensitivity and olfactory system homeostasis in preweaning mice.

Exposure to specific odorants in the womb during pregnancy or in the milk during early nursing is known to impact morpho-functional development of the olfactory circuitry of pups. This can be associated with a modification in olfactory sensitivity and behavioural olfactory-based preferences to the perinatally encountered odorants measured at birth, weaning or adult stage. Effects depend on a multitude of factors, such as odorant type, concentration, administration mode and frequency, as well as timing and mice strain. Here, we examined the effect of perinatal exposure to heptaldehyde on the neuro-anatomical development of the olfactory receptor Olfr2 circuitry, olfactory sensitivity and odour preferences of preweaning pups using mI7-IRES-tau-green fluorescent protein mice. We found that perinatal odour exposure through the feed of the dam reduces the response to heptaldehyde and modulates transcript levels of neuronal transduction proteins in the olfactory epithelium of the pups. Furthermore, the number of I7 glomeruli related to Olfr2-expressing OSN is altered in a way similar to that seen with restricted post-natal exposure, in an age-dependent way. These variations are associated with a modification of olfactory behaviours associated with early post-natal odour preferences at weaning.

The ontogeny of the olfactory system in ceratophryid frogs (Anura, Ceratophryidae).

The aquatic-to-terrestrial shift in the life cycle of most anurans suggests that the differences between the larval and adult morphology of the nose are required for sensory function in two media with different physical characteristics. However, a better controlled test of specialization to medium is to compare adult stages of terrestrial frogs with those that remain fully aquatic as adults. The Ceratophryidae is a monophyletic group of neotropical frogs whose diversification from a common terrestrial ancestor gave rise to both terrestrial (Ceratophrys, Chacophrys) and aquatic (Lepidobatrachus) adults. So, ceratophryids represent an excellent model to analyze the morphology and possible changes related to a secondary aquatic life. We describe the histomorphology of the nose during the ontogeny of the Ceratophryidae, paying particular attention to the condition in adult stages of the recessus olfactorius (a small area of olfactory epithelium that appears to be used for aquatic olfaction) and the eminentia olfactoria (a raised ridge on the floor of the principal cavity correlated with terrestrial olfaction). The species examined (Ceratophrys cranwelli, Chacophrys pierottii, Lepidobatrachus laevis, and L. llanensis) share a common larval olfactory organ composed by the principal cavity, the vomeronasal organ and the lateral appendix. At postmetamorphic stages, ceratophryids present a common morphology of the nose with the principal, middle, and inferior cavities with characteristics similar to other neobatrachians at the end of metamorphosis. However, in advanced adult stages, Lepidobatrachus laevis presents a recessus olfactorius with a heightened (peramorphic) development and a rudimentary (paedomorphic) eminentia olfactoria. Thus, the adult nose in Lepidobatrachus laevis arises from a common developmental 'terrestrial' pathway up to postmetamorphic stages, when its ontogeny leads to a distinctive morphology related to the evolutionarily derived, secondarily aquatic life of adults of this lineage.

Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear.

Sonic hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleost ears. It remains unknown how altered function of Shh relates to morphogenetic changes that coincide with the evolution of limbs and novel auditory organs in the ear. In this study, we used the tetrapod, Xenopus laevis, to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C-start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh function in axis specification. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1385-1400, 2017.

Quantitative comparative analysis of the nasal chemosensory organs of anurans during larval development and metamorphosis highlights the relative importance of chemosensory subsystems in the group.

The anuran peripheral olfactory system is composed of a number of subsystems, represented by distinct neuroepithelia. These include the main olfactory epithelium and vomeronasal organ (found in most tetrapods) and three specialized epithelia of anurans: the buccal-exposed olfactory epithelium of larvae, and the olfactory recess and middle chamber epithelium of postmetamorphic animals. To better characterize the developmental changes in these subsystems across the life cycle, morphometric changes of the nasal chemosensory organs during larval development and metamorphosis were analyzed in three different anuran species (Rhinella arenarum, Hypsiboas pulchellus, and Xenopus laevis). We calculated the volume of the nasal chemosensory organs by measuring the neuroepithelial area from serial histological sections at four different stages. In larvae, the vomeronasal organ was relatively reduced in R. arenarum compared with the other two species; the buccal-exposed olfactory epithelium was absent in X. laevis, and best developed in H. pulchellus. In postmetamorphic animals, the olfactory epithelium (air-sensitive organ) was relatively bigger in terrestrial species (R. arenarum and H. pulchellus), whereas the vomeronasal and the middle chamber epithelia (water-sensitive organs) was best developed in X. laevis. A small olfactory recess (likely homologous with the middle chamber epithelium) was found in R. arenarum juveniles, but not in H. pulchellus. These results support the association of the vomeronasal and middle chamber epithelia with aquatic olfaction, as seen by their enhanced development in the secondarily aquatic juveniles of X. laevis. They also support a role for the larval buccal-exposed olfactory epithelium in assessment of oral contents: it was absent in X. laevis, an obligate suspension feeder, while present in the two grazing species. These initial quantitative results give, for the first time, insight into the functional importance of the peripheral olfactory subsystems across the anuran life cycle.

Trpm5 expression in the olfactory epithelium.

The Ca2+-activated monovalent cation channel Trpm5 is a key element in chemotransduction of taste receptor cells of the tongue, but the extent to which Trpm5 channels are expressed in olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE) of adult mice as part of a specific pheromonal detection system is debated. Here, we used a novel Trpm5-IRES-Cre knockin strain to drive Cre recombinase expression, employed previously validated Trpm5 antibodies, performed in situ hybridization experiments to localize Trpm5 RNA, and searched extensively for Trpm5 splice variants in genetically-labeled, Trpm5-expressing MOE cells. In contrast to previous reports, we find no evidence for the existence in adult mouse OSNs of the classical Trpm5 channel known from taste cells. We show that Trpm5-expressing adult OSNs express a novel Trpm5 splice variant, Trpm5-9, that is unlikely to form a functional cation channel by itself. We also demonstrate that Trpm5 is transiently expressed in a subpopulation of mature OSNs in the embryonic olfactory epithelium, indicating that Trpm5 channels could play a specific role in utero during a narrow developmental time window. Ca2+ imaging with GCaMP3 under the control of the Trpm5-IRES-Cre allele using a newly developed MOE wholemount preparation of the adult olfactory epithelium reveals that Trpm5-GCaMP3 OSNs comprise a heterogeneous group of sensory neurons many of which can detect general odorants. Together, these studies are essential for understanding the role of transient receptor potential channels in mammalian olfaction.

5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system.

The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc.

Transcriptomic profiles of the upper olfactory rosette in cultured and wild Senegalese sole (Solea senegalensis) males.

The aims of this study were the characterization of the upper olfactory epithelium of cultured and wild Senegalese sole mature males at histological and transcriptomic (using RNA-Seq) level. No significant differences in tissue structure, cell types and cellular distribution pattern (olfactory sensory neurons) were identified between cultured and wild specimens. Deep transcriptomic analysis showed 2387 transcripts were differentially expressed between cultured and wild groups. A detailed analysis identified the differentially expressed transcripts included some olfactory receptors (OR, TAAR and V2R-like) and transcripts related with the control of reproduction such as the brain aromatase cytochrome P450 and tachykinin-3. Also a wide set of genes related with lipid sensing, metabolism and transport were differentially expressed and these transcripts were often down-regulated in cultured fish. Furthermore, cultured males presented a higher expression of genes related with goblet cells and mucin production that modulates innate and adaptive immune responses. All these changes in gene expression could be explained by different nutritional status and diet preference. The different expression of transcripts related to olfaction, reproduction, nutrient sensing and immune system demonstrate distinct differences in functionalities between cultured and wild soles providing new clues about the sexual dysfunction in this species.

Neural regeneration dynamics of Xenopus laevis olfactory epithelium after zinc sulfate-induced damage.

Neural stem cells (NSCs) of the olfactory epithelium (OE) are responsible for tissue maintenance and the neural regeneration after severe damage of the tissue. In the normal OE, NSCs are located in the basal layer, olfactory receptor neurons (ORNs) mainly in the middle layer, and sustentacular (SUS) cells in the most apical olfactory layer. In this work, we induced severe damage of the OE through treatment with a zinc sulfate (ZnSO4) solution directly in the medium, which resulted in the loss of ORNs and SUS cells, but retention of the basal layer. During recovery following injury, the OE exhibited increased proliferation of NSCs and rapid neural regeneration. After 24h of recovery, new ORNs and SUS cells were observed. Normal morphology and olfactory function were reached after 168h (7 days) of recovery after ZnSO4 treatment. Taken together, these data support the hypothesis that NSCs in the basal layer activate after OE injury and that these are sufficient for complete neural regeneration and olfactory function restoration. Our analysis provides histological and functional insights into the dynamics between olfactory neurogenesis and the neuronal integration into the neuronal circuitry of the olfactory bulb that restores the function of the olfactory system.

Rabbit olfactory stem cells. Isolation protocol and characterization.

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.