The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula. GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star-shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.

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Olfaction in fish has been studied using preferentially macrosmatic species as models. In the present research, the labelling patterns of different neuronal markers and lectins were analyzed in the olfactory neurons and in their bulbar axonal endings in the guppy Poecilia reticulata, belonging to the group of microsmatic fish. We observed that calretinin immunostaining was confined to a population of olfactory receptor cells localized in the upper layers of the sensory mucosa, probably microvillous neurons innervating the lateral glomerular layer. Immunoreactivity for S100 proteins was mainly evident in crypt cells, but also in other olfactory cells belonging to subtypes projecting in distinct regions of the bulbs. Protein gene product 9.5 (PGP 9.5) was not detected in the olfactory system of the guppy. Lectin binding revealed the presence of N-acetylglucosamine and a-N-acetylgalactosamine residues in the glycoconjugates of numerous olfactory neurons ubiquitously distributed in the mucosa. The low number of sugar types detected suggested a reduced glycosidic variability that could be an index of restricted odorant discrimination, in concordance with guppy visual-based behaviors. Finally, we counted few crypt cells which were immunoreactive for S100 and calretinin. Crypt cells were more abundant in guppy females. This difference is in accordance with guppy gender-specific responses to pheromones. Cells immunoreactive to calretinin showed no evidence of ventral projections in the bulbs. We assumed the hypothesis that their odorant sensitivity is not strictly limited to pheromones or sexual signals in general.

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.

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 Ni 2+ 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 histochemicallydetected 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 Ni 2+ 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 Ni 2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram.

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.

Reconstitution and renewal of tissues are key topics in developmental biology. In this brief work, we analyzed the wintry spent phase of the reproductive cycle in the Manila clam Ruditapes philippinarum (Bivalvia, Veneridae) in order to study the gonad rebuilding that in this species occurs at the beginning of the warmer months. We labeled VASA homolog protein-a germ cell marker-and compared the histological observations of the spent phase with those of the previously analyzed gametogenic phase. In R. philippinarum, during the reproductive season, most of the body mass is represented by sack-like structures (acini) full of developing gametes. In that period, VASA-stained cells are present at the basal pole of the gut epithelium, in the connective tissue, and around the acini. We here show that during the spent phase large portions of the intestine lack such cell type, except for some areas showing a few faintly VASA-stained cells. Cells with similar nuclear morphology are present among loosely organized cells of connective tissue, sometimes as single units, sometimes in small groups, rarely partially organized in primordial gonadic structures. These observations match the findings of RNA-targeting studies that during the spent phase identified the source of bivalve germ cells within the connective tissue in the form of quiescent units and add new information on the possible maintenance of VASA-stained, multipotent cells among the batiprismatic cells of the intestine during the whole life span of these bivalves.

Lectin-binding histochemistry was used to investigate the distribution and density of defined carbohydrate sequences on the cell surface glycoproteins of the olfactory receptors of rat during development. The olfactory and vomeronasal receptors showed a positive labelling after biotinylated Lycopersicum esculentum lectin binding on embryonic day 16 (E16), while horseradish peroxidase-labelled Glycine max, Bandeiraea simplicifolia (BSA-I) and its B4 isomer BSA-I-B4 agglutinins started to label from day 18 (E18). From this stage onward there was a progressive increase in the intensity and number of lectin-binding olfactory receptors. The first lectin-labelled bundles of axons penetrating the olfactory bulb were observed on E20; from E21 it was possible to identify the first labelled glomeruli that, on the first day (P1) of postnatal life, showed a feature very similar to that of the adult. The lectin staining patterns indicate that during development there are differences in the kind and distribution of saccharidic moieties on the surface of rat olfactory neurons. The possible role of carbohydrate-containing glycoproteins in the reception and transduction of the odours and in the modulation of the cell-cell interactions in the olfactory system is discussed.