Immunohistochemical Analysis of Olfactory Sensory Neuron Populations in the Developing Olfactory Organ of the Guppy, Poecilia reticulata (Cyprinodontiformes, Poecilidae).

Olfaction is fundamental for sensing environmental chemicals and has obvious adaptive advantages. In fish, the peripheral olfactory organ is composed of lamellae in which the olfactory mucosa contains three main categories of olfactory sensory neurons (OSNs) as follows: ciliated (cOSNs), microvillous (mOSNs), and crypt cells. We studied the appearance of these different OSNs during development of Poecilia reticulata, given its growing use as animal model system. We performed immunohistochemical detection of molecular markers specific for the different OSNs, carrying out image analyses for marked-cell counting and measuring optical density. The P. reticulata olfactory organ did not show change in size during the first weeks of life. The proliferative activity increased at the onset of secondary sexual characters, remaining high until sexual maturity. Then, it decreased in both sexes, but with a recovery in females, probably in relation to their almost double body growth, compared to males. The density of both cOSNs and mOSNs remained constant throughout development, probably due to conserved functions already active in the fry, independently of the sex. The density of calretinin-positive crypt cells decreased progressively until sexual maturity, whereas the increased density of calretinin-negative crypt cell fraction, prevailing in later developmental stages, indicated their probable involvement in reproductive activities.

Response of Olfactory Sensory Neurons to Mercury Ions in Zebrafish: An Immunohistochemical Study.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

Early germline differentiation in bivalves: TDRD7 as a candidate investigational unit for Ruditapes philippinarum germ granule assembly.

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves.

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin.

BACKGROUND: Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. METHODS: MSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). RESULTS: MSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. DISCUSSION: BM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

VASA expression suggests shared germ line dynamics in bivalve molluscs.

Germ line segregation can occur during embryogenesis or after embryogenesis completion, with multipotent cells able to give rise to both germ and somatic cells in the developing juvenile or even in adulthood. These undifferentiated cells, in some animals, are self-renewing stem cells. In all these cell lineages, the same set of genes, among which vasa, appears to be expressed. We traced VASA expression during the peculiar gonad rebuilding of bivalves to verify its presence from undifferentiated germ cells to mature gametes in an animal taxon in which the mechanism of germ line establishment is still under investigation. We utilized antibodies produced against VASPH, VASA homolog of Ruditapes philippinarum (Subclass Heterodonta), to compare the known expression pattern of R. philippinarum to two species of the Subclass Pteriomorphia, Anadara kagoshimensis and Crassostrea gigas, and another species of the Subclass Heterodonta, Mya arenaria. The immunohistological data obtained support a conserved mechanism of proliferation of "primordial stem cells" among the simple columnar epithelium of the gut, as well as in the connective tissue, contributing to the seasonal gonad reconstitution. Given the taxonomic separation of the analyzed species, we suggest that the process could be shared in bivalve molluscs. The presence of germ cell precursors in the gut epithelium appears to be a feature in common with model organisms, such as mouse, fruit fly, and human. Thus, the comparative study of germ line establishment can add details on bivalve development, but can also help to clarify the role that VASA plays during germ cell specification.

Regenerative medicine in hearing recovery.

Hearing loss, or deafness, affects 360 million people worldwide of which about 32 million are children. Deafness is irreversible when it involves sensory hair cell death because the regenerative ability of these cells is lost in mammals after embryo development. The therapeutic strategies for deafness include hearing aids and/or implantable devices. However, not all patients are eligible or truly benefit from these medical devices. Regenerative medicine based on stem cell application could play a role in both improvement of extant medical devices and in vivo recovery of auditory function by regeneration of inner ear cells and neurons. A review of recent literature on the subject indicates that two promising approaches to renewal and differentiation of cochlear tissues are transplantation of stem cells and in situ administration of growth factors. Rather than directly regenerating dead cells, these procedures apparently induce, through various pathways, differentiation of resident cochlear cells. More studies on the possible adverse effects of transplanted cells and the recovery of tonotopic sensorineural activity or required. To date, no reliable clinical results have been obtained in the field of cochlear regeneration.

Histopathological analysis of the olfactory epithelium of zebrafish (Danio rerio) exposed to sublethal doses of urea.

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L(-1)) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan-neuronal labelling with anti-Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of Gα olf-, TRPC2- and TrkA-expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and Gα olf-immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.

Immunocytochemical characterisation of olfactory ensheathing cells of zebrafish.

Continuous lifelong neurogenesis is typical of the vertebrate olfactory system. The regenerative ability of olfactory receptor neurons is dependent on the glial cell type specific to the olfactory pathway, designated 'olfactory ensheathing cells'. Several studies to date have focused on mammalian olfactory ensheathing cells, owing to their potential roles in cell-based therapy for spinal cord injury repair. However, limited information is available regarding this glial cell type in non-mammalian vertebrates, particularly anamniotes. In the current immunocytochemical study, we analysed the features of olfactory ensheathing cells in the zebrafish, Danio rerio. Fish provide a good model for studying glial cells associated with the olfactory pathway of non-mammalian vertebrates. In particular, zebrafish has numerous valuable features that enable its use as a prime model organism for genetic, neurobiological and developmental studies, as well as toxicology and genomics research. Paraffin sections from decalcified heads of zebrafish were processed immunocytochemically to detect proteins used in the research on mammalian olfactory ensheathing cells, including glial fibrillary acid protein (GFAP), S100, neural cell adhesion molecule (NCAM), polysialylated NCAM (PSA-NCAM), vimentin (VIM), p75NTR and galactin (Gal)-1. Notably, GFAP, S100, NCAM and Gal-1 were clearly observed, whereas no vimentin staining was detected. Weak immunostaining for PSA-NCAM and p75NTR was evident. Moreover the degree of marker expression was not uniform in various tracts of the zebrafish olfactory pathway. The immunostaining patterns of the zebrafish olfactory system are distinct from those of other fish to some extent, suggesting interspecific differences. We also showed that the olfactory pathway of zebrafish expresses markers of mammalian olfactory ensheathing cells. The olfactory systems of vertebrates have similarities but there are also marked variations between them. The issue of whether regional and interspecific differences in immunostaining patterns of olfactory pathway markers have functional significance requires further investigation.

Transplanted human adipose tissue-derived stem cells engraft and induce regeneration in mice olfactory neuroepithelium in response to dichlobenil subministration.

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electro-olfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy.

Quantitative analysis of crypt cell population during postnatal development of the olfactory organ of the guppy, Poecilia reticulata (Teleostei, Poecilidae), from birth to sexual maturity.

Crypt cells are one of three types of olfactory sensory neuron, differing from ciliated and microvillar cells in shape, localization and number, and found only in fish. Although crypt cells are morphologically well characterized, their function remains unclear. They were hypothesized to be involved in reproductive behaviours by detecting sex pheromones, but electrophysiological investigations revealed sensitivity to only amino acids. However, the number of crypt cells in adult guppies is not the same in the two sexes. In this study, we compared the size of the crypt cell population in juvenile guppies during the first 90 days after birth. The purpose of our study was to clarify whether a correlation exists between sex and the number of these olfactory neurons. The data show that guppies reach adult crypt cell density when they become sexually mature. Despite a constant increment in volume during development of the olfactory organ, the minimum density of crypt neurons occurs at ~45 days. Moreover, in the early weeks, the density of crypt neurons is greater in males than in females because in females the total number of cells decreases significantly after just 7 days. In adults, however, crypt neurons are found in higher density in females than in males. These findings suggest that the number of crypt cells is sex specific, with independent developmental dynamics between males and females. A role in pheromone detection could explain such a difference, but the early appearance of crypt cells in the first days of life is suggestive of other, not sexually related, functions.

Human cord blood CD133+ stem cells transplanted to nod-scid mice provide conditions for regeneration of olfactory neuroepithelium after permanent damage induced by dichlobenil.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133(+) stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQalpha1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss.

Molecular Markers in the Study of Non-model Vertebrates: Their Significant Contributions to the Current Knowledge of Tetrapod Glial Cells and Fish Olfactory Neurons.

The knowledge of the morphological and functional aspects of mammalian glial cells has greatly increased in the last few decades. Glial cells represent the most diffused cell type in the central nervous system, and they play a critical role in the development and function of the brain. Glial cell dysfunction has recently been shown to contribute to various neurological disorders, such as autism, schizophrenia, pain, and neurodegeneration. For this reason, glia constitutes an interesting area of research because of its clinical, diagnostic, and pharmacological relapses. In this chapter, we present and discuss the cytoarchitecture of glial cells in tetrapods from an evolutive perspective. GFAP and vimentin are main components of the intermediate filaments of glial cells and are used as cytoskeletal molecular markers because of their high degree of conservation in the various vertebrate groups. In the anamniotic tetrapods and their progenitors, Rhipidistia (Dipnoi are the only extant rhipidistian fish), the cytoskeletal markers show a model based exclusively on radial glial cells. In the transition from primitive vertebrates to successively evolved forms, the emergence of a new model has been observed which is believed to support the most complex functional aspects of the nervous system in the vertebrates. In reptiles, radial glial cells are prevalent, but star-shaped astrocytes begin to appear in the midbrain. In endothermic amniotes (birds and mammals), star-shaped astrocytes are predominant. In glial cells, vimentin is indicative of immature cells, while GFAP indicates mature ones.Olfactory receptor neurons undergo continuous turnover, so they are an easy model for neurogenesis studies. Moreover, they are useful in neurotoxicity studies because of the exposed position of their apical pole to the external environment. Among vertebrates, fish represent a valid biological model in this field. In particular, zebrafish, already used in laboratories for embryological, neurobiological, genetic, and pathophysiological studies, is the reference organism in olfactory system research. Smell plays an important role in the reproductive behavior of fish, with direct influences also on the numerical consistency of their populations. Taking into account that a lot of species have considerable economic importance, it is necessary to verify if the model of zebrafish olfactory organ is also directly applicable to other fish. In this chapter, we focus on crypt cells, a morphological type of olfactory cells specific of fish. We describe hypothetical function (probably related with social behavior) and evolutive position of these cells (prior to the appearance of the vomeronasal organ in tetrapods). We also offer the first comparison of the molecular characteristics of these receptors between zebrafish and the guppy. Interestingly, the immunohistochemical expression patterns of known crypt cell markers are not overlapping in the two species.

Differential nickel-induced responses of olfactory sensory neuron populations in zebrafish.

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for Gα olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for Gα olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in Gα olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram.

Natural Heteroplasmy and Mitochondrial Inheritance in Bivalve Molluscs.

Heteroplasmy is the presence of more than one type of mitochondrial genome within an individual, a condition commonly reported as unfavorable and affecting mitonuclear interactions. So far, no study has investigated heteroplasmy at protein level, and whether it occurs within tissues, cells, or even organelles. The only known evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental Inheritance (DUI)-reported so far in ∼100 bivalve species-in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). Because of such segregation, mitochondrial oxidative phosphorylation proteins reach a high amino acid sequence divergence (up to 52%) between the two lineages in the same species. Natural heteroplasmy coupled with high sequence divergence between F- and M-type proteins provides a unique opportunity to study their expression and assess the level and extent of heteroplasmy. Here, for the first time, we immunolocalized F- and M-type variants of three mitochondrially-encoded proteins in the DUI species Ruditapes philippinarum, in germline and somatic tissues at different developmental stages. We found heteroplasmy at organelle level in undifferentiated germ cells of both sexes, and in male soma, whereas gametes were homoplasmic: eggs for the F-type and sperm for the M-type. Thus, during gametogenesis, only the sex-specific mitochondrial variant is maintained, likely due to a process of meiotic drive. We examine the implications of our results for DUI proposing a revised model, and we discuss interactions of mitochondria with germ plasm and their role in germline development. Molecular and phylogenetic evidence suggests that DUI evolved from the common Strictly Maternal Inheritance, so the two systems likely share the same underlying molecular mechanism, making DUI a useful system for studying mitochondrial biology.

Light and transmission electron microscopy study of the peripheral olfactory organ of the guppy, Poecilia reticulata (Teleostei, Poecilidae).

A study of the peripheral olfactory organ, with special attention to the olfactory epithelium, has been carried out in the guppy (Poecilia reticulata). Guppy is well known to have a vision-based sexual behavior. The olfactory chamber caudally opens directly in an accessory nasal sac, which is bent medially and gives rise to two recesses that can be considered secondary accessory nasal sacs, antero-medial and postero-medial, respectively. The sensory epithelium, which lines only the medial wall of the nasal cavity, is basically flat rising in a very low lamella only in the posterior part. The olfactory receptors are not evenly distributed in the olfactory mucosa, but aggregate in shallow folds separated by epithelial cells with evident microridges. Ciliated olfactory sensory neurons and microvillous olfactory sensory neurons are clearly identified by transmission electron microscopy (TEM). Scarce crypt olfactory neurons are found throughout the sensory folds. The nasal sacs indicates the capacity to regulate the flow of odorant molecules over the sensory epithelium, possibly through a pump-like mechanism associated with gill ventilation. The organization of the olfactory organ in guppy is simple and reminds what is found in early posthatching stages of fish which at the adult state have a well developed olfactory organ. This simple organization supports the idea that the guppy rely on olfaction less than other fish species provided with more extended olfactory receptorial surface.

Differential response of olfactory sensory neuron populations to copper ion exposure in zebrafish.

The peripheral olfactory system of fish is in direct contact with the external aqueous environment, so dissolved contaminants can easily impair sensory functions and cause neurobehavioral injuries. The olfactory epithelium of fish is arranged in lamellae forming a rosette in the olfactory cavity and contains three main types of olfactory sensory neurons (OSNs): ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs), common to all vertebrates, and a third minor group of olfactory neurons, crypt cells, absent in tetrapods. Since copper is a ubiquitously diffusing olfactory toxicant and a spreading contaminant in urban runoff, we investigated the effect of low copper concentration on the three different OSNs in the olfactory epithelium of zebrafish, a model system widely used in biological research. Image analysis was applied for morphometry and quantification of immunohistochemically detected OSNs. Copper exposure resulted in an evident decrease in olfactory epithelium thickness. Moreover, after exposure, the lamellae of the dorsal and ventral halves of the olfactory rosettes showed a different increase in their sensory areas, suggesting a lateral migration of new cells into non-sensory regions. The results of the present study provide clear evidence of a differential response of the three neural cell populations of zebrafish olfactory mucosa after 96h of exposure to copper ions at the sublethal concentration of 30µgL-1. Densitometric values of cONS, immunostained with anti-G αolf, decreased of about 60% compared to the control. When the fish were transferred to water without copper addition and examined after 3, 10 and 30days, we observed a partial restoration of anti-G αolf staining intensity to normal condition. The recovery of cOSNs appeared sustained by neuronal proliferation, quantified with anti-PCNA immunostaining, in particular in the early days after exposure. The densitometric analysis applied to mOSNs, immunostained with anti-TRPC2, revealed a statistically significant decrease of about 30% compared to the control. For cOSNs and mOSNs, the decrement in staining intensity may be indicative of cell death, but reduction in antigen expression may not be excluded. In the post-exposure period of 1 month we did not find recovery of mOSNs. We hypothesize that cOSNs are more sensitive than mOSNs to copper treatment, but also more prompted to tissue repair. Anti-TrkA-immunopositive crypt cells appeared not to be affected by copper exposure since statistical analysis excluded any significant difference between the control and treated fish. Comparative studies on OSNs would greatly enhance our understanding of the mechanisms of olfaction.

Crypt cell markers in the olfactory organ of Poecilia reticulata: analysis and comparison with the fish model Danio rerio.

Olfactory crypt neurons have been observed in several bony fishes and chondrichtyans. Although their morphology is uniform in all fish, very few is known about their antigenic properties, usually studied in zebrafish, but quite overlooked in other species. We tested in Poecilia reticulata (guppy) the two antibodies recognized to mark zebrafish crypt cells: while anti-S100 showed an immunohistochemical pattern comparable to what reported in zebrafish, anti-TrkA gave no signal. Western blot analysis revealed that S100-antiserum bound an antigen of expected weight, probably belonging to the S100 family. On the contrary, anti-TrkA detected more bands, but the protein/s might be too much diffused and/or diluted in the tissue to be detected with immunohistochemistry. Because of the high level of conservation in the Trk family proteins of the kinase domain, on which anti-TrkA was produced, we also tested anti-TrkB to exclude cross reactivity. Immunohistochemistry and Western blot confirmed that anti-TrkB displayed high specificity to its target and a different staining pattern compared to anti-TrkA, but, as anti-TrkA, it did not label crypt neurons. Finally, we documented that calretinin, a known marker of zebrafish ciliated and microvillous olfactory cells, in the guppy is expressed also by a subpopulation of S100-positive crypt neurons. These results reveal differences in antigen expression between zebrafish and guppy crypt cells. Together with the already known species-specific projections to the olfactory bulb and a heterogeneous panel of odorants, our findings support the possibility that crypt cells are functionally less uniform as supposed.

Recovery of the olfactory receptor neurons in the African Tilapia mariae following exposure to low copper level.

Low levels of Cu(2+) are known to specifically cause olfactory neuron death in fish olfactory epithelium. This study investigated the morphological changes in the olfactory mucosa of the cichlid Tilapia mariae, after a 4-day exposure to different concentrations of Cu(2+) (20, 40 and 100 microg/l), and the regeneration time-frame, when fish exposed to 20 microg/l were returned to dechlorinated tap water. Light microscopy, combined with Fluoro Jade-B staining, permitted the observation of a dose-dependent damage which became less severe and more circumscribed to receptor cells when Cu(2+) concentration decreased. The regeneration process in the olfactory tissue was examined in fish after 0, 3, and 10 days of recovery in well water. Immunostaining with PCNA showed a massive mitotic activity in the basal region of the mucosa immediately after exposure was terminated. The mitotically produced elements were immature neurons since they expressed the neural growth-associated phosphoprotein GAP-43. After 3 days of recovery the nuclei had already completed their migration to the upper portion of the epithelium and mitotic activity was much less intensive. After 10 days the olfactory tissue did not present differences when compared to the control tissue. These results suggest that after 10 days the regeneration is completed and the integrity of the tissue restored.

Immunocytochemical characterisation of ensheathing glia in the olfactory and vomeronasal systems of Ambystoma mexicanum (Caudata: Ambystomatidae).

The olfactory and vomeronasal systems of vertebrates are characterised by neurogenesis occurring throughout life. The regenerative ability of olfactory receptor neurons relies on specific glial cells, the olfactory and vomeronasal axon-surrounding cells. Numerous studies have examined mammalian olfactory ensheathing cells which are considered potential candidates for spinal cord injury repair using cell-based therapy. With regard to non-mammalian vertebrates, limited information is available on these glial cells in fish, and there is no information on them in terrestrial anamniotes, the amphibians. In the present research, we studied the immunocytochemical characteristics of axon-surrounding cells in Ambystoma mexicanum. Urodeles have relatively simple olfactory and vomeronasal systems, and represent a good model for studying ensheathing cells in extant representatives of basal tetrapods. Sections from the decalcified heads of A. mexicanum were immunocytochemically processed for the detection of proteins used in research on mammalian olfactory-ensheathing cells. S100, GFAP and NCAM were clearly observed. p75NTR, Gal-1 and PSA-NCAM showed weak staining. No vimentin immunopositivity was observed. The corresponding areas of the olfactory and vomeronasal pathways displayed the same staining characteristics, with the exception of Gal-1, p75NTR and PSA-NCAM in the mucosae. The degree of marker expression was not uniform throughout the sensory pathways. In contrast to fish, both olfactory and vomeronasal nerves displayed uniform staining intensity. This study showed that some markers for mammalian and fish-ensheathing glia are also applicable in urodeles. The olfactory systems of vertebrates show similarities, and also clear dissimilarities. Further investigations are required to ascertain the functional significance of these regional and interspecific differences.