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In the olfactory system of vertebrates, neurogenesis occurs throughout life. The regenerating activities of the olfactory receptor neurons are connected to particular glial cells in the olfactory pathway: the olfactory ensheathing cells. A considerable number of studies are available in literature regarding mammalian olfactory ensheathing cells; this is due to their potential role in cell-based therapy for spinal cord injury repair. But very little is known about these cells in non-mammalian vertebrates. In this study we examined the immunocytochemical characteristics of the olfactory ensheathing cells in fish, which provide a good model for the study of glial cells in the olfactory pathway of non-mammalian vertebrates. Paraffin sections from decalcified heads of Poecilia reticulata (microsmatic fish) and Carassius auratus (macrosmatic fish) were processed to immunocytochemically detect ensheathing cell markers used in research on mammals: GFAP, S100, NCAM, PSA-NCAM, vimentin, p75NTR and galectin-1. GFAP, S100 and NCAM were clearly detected in both fish, though the intracranial tract of the primary olfactory pathway of Carassius appears more S100 stained than the extracranial tract. P75NTR staining is more evident in Poecilia, PSA-NCAM positivity in Carassius. A slight vimentin immunostaining was detected only in Carassius. No galectin-1 staining appeared in the olfactory pathways of either fish. This study shows that some markers for mammalian olfactory ensheathing cells also stain the olfactory pathway in fish. Immunocytochemical staining differs in the two fish under examination, even along the various tracts of the olfactory pathway in the same species.

We analyzed the distribution of intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin in the brain and spinal cord of the adult brown anole lizard, Anolis sagrei. The GFAP immunoreactivity is strong and the positive structures are basically represented by fibers of different lengths and thicknesses which are arranged in a regular radial pattern throughout the central nervous system. In the brain regions that have a thicker neural wall, the radial orientation is not so evident as in the thinner areas. These fibers emerge from radial ependymoglia (tanycytes) whose cell bodies are generally GFAP-immunopositive. The glial fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls. In the spinal cord, the optic tectum and the lateroventral regions of the mesencephalon and medulla oblongata, star-shaped astrocytes coexist with radial structures. Vimentin-immunoreactive structures are absent in the brain and spinal cord. In A. sagrei the immunohistochemical response of the astroglial intermediate filaments appears typical of a mature astroglial cell lineage, since they fundamentally express GFAP immunoreactivity. A Western-blot analysis reveals a GFAP-positive single band, common to the different nervous areas. This immunohistochemical study shows that the star-shaped astrocytes have a different distribution in saurians and while the glial pattern of A. sagrei is more evolved than in urodeles it remains immature as compared with crocodilians, avians, and mammals. This condition suggests that reptiles represent a fundamental step in the phylogenetic evolution of the vertebrate glial cells., ((c) 2005 Wiley-Liss, Inc.)

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. [ABSTRACT FROM AUTHOR]

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), p75 NTR 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 p75 NTR 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. [ABSTRACT FROM AUTHOR]

Summary: The GLUT-1 isoform of the glucose transporter is commonly accepted as a reliable molecular marker of blood–brain barrier endothelia in neural vasculature organized in a three-dimensional network of single vessels. The brain of the lizard Podarcis sicula is characterized by a vascular architecture based on a pattern of paired vessels. The presence and distribution of GLUT-1 were studied in adult lizards using both light and transmission electron microscopic techniques. Immunoperoxidase histochemistry was applied to sections from paraffin-embedded brain using gold-conjugated secondary antibodies to localize this antigen on ultrathin sections. The transverse sectioned pairs of vessels did not show the same size and, in particular, the two elements of the same pair often differed in their diameters. Light microscopy revealed immunopositivity in both parenchymal and meningeal vessels. In each transverse-sectioned vascular pair, one element was intensely labelled, and the adjacent one showed only slight or negligible reaction. Colloidal gold particles were restricted to endothelial cells, showing an asymmetric labelling pattern, which was always characterized by markedly higher density of immunolabelling of the abluminal rather than the luminal plasmalemma. Moreover, in every vascular pair, one profile had lower amounts of scantier labelling by gold particles than the adjacent element. This pattern indicates functional differences between the adjacent vascular limbs regarding glucose transport. [Copyright &y& Elsevier]

Highlights • Nickel exposure affects ciliated olfactory sensory neurons more than microvillous olfactory sensory neurons. • Crypt cells are not affected by nickel exposure. • Nickel exposure reduces the thickness of the olfactory epithelium. • The olfactory epithelium of the ventral half of the olfactory rosette appears more sensitive to nickel exposure. Abstract 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 G α 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 G α 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 G α 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. [ABSTRACT FROM AUTHOR]

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 96 h of exposure to copper ions at the sublethal concentration of 30 μg L −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 30 days, 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. [ABSTRACT FROM AUTHOR]