G. Montalbano

Differential distribution of S100 protein and calretinin in mechanosensory and chemosensory cells of adult zebrafish (Danio rerio).

Calcium-binding proteins play a critical role in vertebrate sensory cells, and some of them have been detected in mechanosensory and chemosensory cells of bony and cartilaginous fishes. In this study immunohistochemistry and Western blot were used to investigate the occurrence and the distribution of S100 protein and calretinin in mechanosensory (neuromasts of the lateral line system; maculae and cristae ampullaris of the inner ear) as well as chemosensory (superficial and oral taste buds; olfactory epithelium) cells in adult zebrafish (Danio rerio). Specific protein bands with an estimated molecular weight of around 10 kDa and 30 kDa were detected by Western blot and were identified with S100 protein and calretinin, respectively. S100 protein and calretinin were observed segregated in mechanosensory and chemosensory cells, and the presence of S100 protein in a cell excluded that of calretinin, and vice versa. As a rule, the mechanosensory cells were S100 protein positive, whereas the chemosensory ones displayed calretinin immunoreactivity. Calretinin was also detected in nerve fibers supplying some of the investigated organs. In the olfactory epithelium, S100 protein immunoreactivity was present in the crypt olfactory sensory neurons, whereas calretinin immunoreactivity was widespread in olfactory sensory neurons and probably other olfactory cells. In this localization the co-expression of S100 protein and calretinin cannot be excluded. These results demonstrate the cell segregation of two specific calcium-binding proteins, and they enable to selectively label these cells by using easily reproducible immunohistochemical techniques associated to well-known antibodies.

S100 protein-like immunoreactivity in the crypt olfactory neurons of the adult zebrafish

The olfactory epithelium of some teleosts, including zebrafish, contains three types of olfactory sensory neurons. Because zebrafish has become an ideal model for the study of neurogenesis in the olfactory system, it is of capital importance the identification of specific markers for different neuronal populations. In this study we used immunohistochemistry to analyze the distribution of S100 protein-like in the adult zebrafish olfactory epithelium. Surprisingly, specific S100 protein-like immunostaining was detected exclusively in crypt neurons, whereas ciliated and microvillous neurons were not reactive, and the supporting glial cells as well. The pattern of immunostaining was exclusively cytoplasmic without apparent polarity within the soma, and the intensity of immunostaining was not related with the maturative stage of the neurons. The role of S100 protein in crypt olfactory neurons is unknown, although it is probably associated with the capacity of these cells to respond to chemical stimuli. In any case, it represents an excellent marker to identify crypt olfactory neurons in zebrafish.

The Oral Cavity of the Adult Zebrafish (Danio rerio)

The zebrafish is a common model for developmental studies including those regarding tooth, palate and tongue. Nevertheless, little information is available about the morphology of the oral cavity in this teleost, especially in adult animals. In this study we used light, scanning and transmission electron microscopy to describe in detail the morphology of the oral cavity of adult zebrafish. The oral cavity could be divided into three different zones: the outer containing the lips, the intermediate corresponding to the internal valves and the internal that corresponds to the tongue. In the upper and lower intermediate zones, there were semilunar shaped valves, more prominent in the upper part. The internal lower zones correspond to the palate and the tongue, which is an individualized structure filled with numerous transversal ridges. Both the intermediate and internal zones were covered by a stratified epithelium containing numerous mucous and rodlet cells. Present data provide the first description of the morphology and structure of the oral cavity in the adult zebrafish and might serve as a baseline for developmental studies of the oral cavity using this teleost as a model.

TrkA and p75NTR in the ovary of adult cow and pig

Neurotrophins play a critical role in the development of the mammalian ovary, oogenesis and folliculogenesis. In this study we investigated the cell localization of the two main receptors for nerve growth factor (NGF), TrkA and p75NTR, in the cow and pig ovary, using immunohistochemistry. Specific immunoreactivity for TrkA and p75NTR was detected in the ovary of both species, but the pattern and intensity of immunostaining were marginally different between them. The follicular cells regularly expressed immunoreactivity for both receptors. Immunoreactivity was also detected in the oocytes, independently of the maturational stage of the follicles, with the exception of primordial and primary follicles of the pig which did not display p75NTR. Taken together, these results suggest a possible direct role of NGF on oocytes expressing TrkA and p75NTR, in addition to the well‐known roles in other ovary functions. The practical relevance of these data remains to be clarified.

Morphology of the Lingual Dorsal Surface and Oral Taste Buds in Italian Lizard (Podarcis sicula)

With 4 figures

The lingual dorsal surface of the blue-tongue skink (Tiliqua scincoides).

The blue‐tongue lizard (Tiliqua scincoides) is a variety of large skink common throughout Australia. There are seven species of Tiliqua and all of them have long bodies, short limbs and short and robust tails. T. scincoides occurs in a wide range of habitats; its diet is omnivorous. When threatened, it opens the mouth and protrudes its characteristic large fleshy cobalt blue tongue. It is currently found as a popular species and also as a pet animal in the European countries. No data are available in literature about the morphology of the tongue of T. scincoides; therefore, the aim of the present study was to investigate by means of scanning electron microscopy and light microscopy, the anatomy of the dorsal lingual surface. Our results demonstrate the presence of a tongue tip with a smooth surface without papillae. The foretongue was characterized by a stratified epithelium with foliate‐like papillae and deep inter‐papillar spaces in the middle part and cylindrical papillae with a flat surface in the lateral parts. All the posterior area of the tongue was characterized by more compacted papillae and the inter‐papillar spaces were very narrow. Light microscopy showed the presence of melanin throughout the tongue. No taste buds were recognized on the lingual dorsal surface. Therefore, the papillae probably have a mechanical function showing an important role in the swallowing phase. The morphology of the tongue surface can be correlated to the diet and, different roles, as in other examined species, can be hypothesized for different areas.

Scanning Electron Microscopical Study of the Lingual Epithelium of Green Iguana (Iguana iguana)

During the last few years, green iguanas (Iguana iguana) have turned out to be one of the most popular pets. They are omnivorous. In their way of feeding, this crucial function is performed by capturing of the preys and mostly, this is carried out by the tongue. The role of the tongue is also fundamental during the intra‐oral transport and during the swallowing of food. This has been reported in several studies about chameleons, agamids and iguanids, nevertheless published data about the mechanisms of capturing and swallowing the prey, and the morphological descriptions about the tongue epithelium, are scarce. Therefore, the aim of this present study was to analyse the morphology of the lingual epithelium in green iguanas by scanning electron microscopy. Three different areas were demonstrated on the tongue surface: the tongue tip, characterized by a smooth epithelium without papillae, a foretongue, completely covered by numerous closely packed cylindriform papillae, and a hindtongue with conical‐like papillae. Some taste buds were recognized on the middle and the posterior parts of the tongue. Different functional roles could be hypothesized for the three tongue areas: the tongue tip could have a role related to the movements of the prey immediately after the capturing, while the middle papillae and the hindtongue could have an important role concerning the swallowing phase.

Calretinin in the peripheral nervous system of the adult zebrafish.

Calretinin is a calcium‐binding protein found widely distributed in the central nervous system and chemosensory cells of the teleosts, but its presence in the peripheral nervous system of fishes is unknown. In this study we used Western blot analysis and immunohistochemistry to investigate the occurrence and distribution of calretinin in the cranial nerve ganglia, dorsal root ganglia, sympathetic ganglia, and enteric nervous system of the adult zebrafish. By Western blotting a unique and specific protein band with an estimated molecular weight of around 30 kDa was detected, and it was identified as calretinin. Immunohistochemistry revealed that calretinin is selectively present in the cytoplasm of the neurons and never in the satellite glial cells. In both sensory and sympathetic ganglia the density of neurons that were immunolabelled, their size and morphology, as well as the intensity of immunostaining developed within the cytoplasm, were heterogeneous. In the enteric nervous system calretinin immunoreactivity was detected in a subset of enteric neurons as well as in a nerve fibre plexus localized inside the muscular layers. The present results demonstrate that in addition to the central nervous system, calretinin is also present in the peripheral nervous system of zebrafish, and contribute to completing the map of the distribution of this protein in the nervous system of teleosts.

Acid-sensing ion channel 2 (ASIC2) in the intestine of adult zebrafish

Acid-sensing ion channels (ASICs) in mammals monitor acid sensing and mechanoreception. They have a widespread expression in the central and peripheral nervous system, including the gut. The distribution of ASICs in zebrafish is known only in larvae and at the mRNA level. Here we have investigated the expression and cell distribution of ASIC2 in the gut of adult zebrafish using PCR, Western blot and immunohistochemistry. ASIC2 mRNA was detected in the gut, and a protein consistent with predicted ASIC2 (64kDa molecular mass) was detected by Western blot. ASIC2 positivity was found in a subpopulation of myenteric neurons in the enteric nervous system, as well in enteroendocrine epithelial cells. These data demonstrate for the first time the occurrence of ASIC2 in the gut of adult zebrafish where it presumably acts as a chemosensor and a mechanosensor.

Expression and anatomical distribution of TrkB in the encephalon of the adult zebrafish (Danio rerio)

Neurotrophins are a family of growth factor primarily acting in the nervous system, throughout two categories of membrane receptors on the basis of their high (Trk receptors) or low (p75NTR) affinity. Both neurotrophins and Trk receptors are phylogenetically conserved and are expressed not only in the central and peripheral nervous system but also in non-nervous tissues of vertebrates and some invertebrates. The brain-derived neurotrophic factor (BDNF)/TrkB system plays an important role in the development, phenotypic maintenance and plasticity of specific neuronal populations. Considering that this system is poorly characterized in the central nervous system of teleosts, the expression and anatomical distribution of TrkB in the brain of the adult zebrafish using reverse transcriptase-polymerase chain reaction (RT-PCR), Western-blot and immunohistochemistry were analysed. Both the riboprobe and the antibody used were designed to map within the catalytic domain of TrkB. RT-PCR detected specific TrkB mRNA in brain homogenates, while Western-blot identified one unique protein band with an estimated molecular weight of 145kDa, thus corresponding with the TrkB full-length isiform of the receptor. Immunohistochemistry showed specific TrkB immunoreactivity in restricted areas of the encephalon, i.e. the hypothalamus and a specific neuronal subpopulation of the reticular formation. The present results demonstrate, for the first time, that, as in mammals, the encephalon of adult zebrafish expresses TrkB in specific zones related to food intake, behaviour or motor activity.