Potential Neuroprotective Role of Calretinin-N18 and Calbindin-D28k in the Retina of Adult Zebrafish Exposed to Different Wavelength Lights.

The incidence rates of light-induced retinopathies have increased significantly in the last decades because of continuous exposure to light from different electronic devices. Recent studies showed that exposure to blue light had been related to the pathogenesis of light-induced retinopathies. However, the pathophysiological mechanisms underlying changes induced by light exposure are not fully known yet. In the present study, the effects of exposure to light at different wavelengths with emission peaks in the blue light range (400-500 nm) on the localization of Calretinin-N18 (CaR-N18) and Calbindin-D28K (CaB-D28K) in adult zebrafish retina are studied using double immunofluorescence with confocal laser microscopy. CaB-D28K and CaR-N18 are two homologous cytosolic calcium-binding proteins (CaBPs) implicated in essential process regulation in central and peripheral nervous systems. CaB-D28K and CaR-N18 distributions are investigated to elucidate their potential role in maintaining retinal homeostasis under distinct light conditions and darkness. The results showed that light influences CaB-D28K and CaR-N18 distribution in the retina of adult zebrafish, suggesting that these CaBPs could be involved in the pathophysiology of retinal damage induced by the short-wavelength visible light spectrum.

Expression and Localization of BDNF/TrkB System in the Zebrafish Inner Ear.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is involved in multiple and fundamental functions of the central and peripheral nervous systems including sensory organs. Despite recent advances in knowledge on the functional significance of BDNF and TrkB in the regulation of the acoustic system of mammals, the localization of BDNF/TrkB system in the inner ear of zebrafish during development, is not well known. Therefore, the goal of the present study is to analyze the age-dependent changes using RT-PCR, Western Blot and single and double immunofluorescence of the BDNF and its specific receptor in the zebrafish inner ear. The results showed the mRNA expression and the cell localization of BDNF and TrkB in the hair cells of the crista ampullaris and in the neuroepithelium of the utricle, saccule and macula lagena, analyzed at different ages. Our results demonstrate that the BDNF/TrkB system is present in the sensory cells of the inner ear, during whole life. Therefore, this system might play a key role in the development and maintenance of the hair cells in adults, suggesting that the zebrafish inner ear represents an interesting model to study the involvement of the neurotrophins in the biology of sensory cells.

The Alimentary Tract of African Bony-Tongue, Heterotis niloticus (Cuvier, 1829): Morphology Study.

A morphological study of the alimentary tract, from the oropharyngeal cavity to the rectum, including the attached glands, of African bony-tongue, Heterotis niloticus (Cuvier, 1829) was carried out by gross anatomy, and light microscope analysis. This study aimed to give a deeper knowledge of the alimentary tract morphological features of this species of commercial interest. H. niloticus is distinguished by individual morphological characteristics showing a digestive tract similar to that of reptiles and birds. Within the oropharyngeal cavity, two tubular structures with digitiform ends are arranged on both lateral sides of the triangular tongue. The oropharyngeal cavity connects the stomach by a short esophagus. This latter is adapted to mechanical trituration, and it is divided into a pars glandularis and a thick-walled pars muscularis. The gizzard flows into the anterior intestine and two blind pyloric appendages, which exhibit specific functions, including immune defense for the presence of secondary lymphoid organs. The anterior intestine continues with the middle and posterior tracts up into the rectum. According to the histological observations, all regions of the alimentary tract have common structural features, typical of hollow organs, with differences in the mucosa structure that reflects the different functions of the apparatus, from mouth to anus. Within this study, we provided the first basis for future studies on optimizing rearing conditions, feed conversion ratio, and the digestive capacity, improving the growth performance of this species, and ensuring its conservation.

Morphology of the Atlantic salmon (Salmo salar) tongue.

The Atlantic salmon (Salmo salar) is a freshwater and marine fish of the family Salmonidae, widely farmed in aquaculture facilities in several countries. The salmon are carnivorous, but in aquaculture, alternative foods have been experienced. It is well known that feeding in captivity should cause adaptation and modifications of the morphological characteristics of the oral cavity, especially of tongue; therefore, the aim of this study was to investigate, by light, laser confocal and scanning electron microscopy, the morphological characteristics of the tongue dorsal surface, considering the importance of the correlations between feeding habits and the anatomy of the tongue. Scanning electron microscopy demonstrates the presence of caniniform teeth with oro-aboral orientation surrounded by numerous filiform papillae, single, fused or arranged in row. Oro-aborally, the papillae show an appearance like a rosette and they disappear at level of the root. Light and laser confocal microscopy demonstrates that the mucosa is covered by a non-keratinized stratified pavement epithelium with, in the deepest layer, the presence of a triangular structure whose apex is cranially directed and base facing aborally. In this structure, spindle-shaped cells are present, with a vimentin immunoreactivity, that for their characteristics could be adult mesenchymal stem cells. The obtained data could be useful not only for further studies on the nutrition, but it is interesting the detection of tissues typical of the embryo-fetal phase in the adult specimens tongue, thus giving a basis for studies of potential applications, if any, regarding cell therapies for different clinical indications.

Expression of Acetylcholine- and G protein coupled Muscarinic receptor in the Neuroepithelial cells (NECs) of the obligated air-breathing fish, Arapaima gigas (Arapaimatidae: Teleostei).

The air-breathing specialization has evolved idependently in vertebrates, as many different organs can perfom gas exchange. The largest obligate air-breathing fish from South America Arapaima gigas breathe air using its gas bladder, and its dependence on air breathing increases during its growth. During its development, gill morphology shows a dramatic change, remodeling with a gradual reduction of gill lamellae during the transition from water breathing to air breathing . It has been suggested that in this species the gills remain the main site of O2 and CO2 sensing. Consistent with this, we demonstrate for the first time the occurrence of the neuroepithelial cells (NECs) in the glottis, and in the gill filament epithelia and their distal halves. These cells contain a broader spectrum of neurotransmitters (5-HT, acetylcholine, nNOS), G-protein subunits and the muscarininic receptors that are coupled to G proteins (G-protein coupled receptors). We report also for the first time the presence of G alpha proteins coupled with muscarinic receptors on the NECs, that are thought as receptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Based on the specific orientation in the epithelia and their closest vicinity to efferent vasculatures, the gill and glottal NECs of A. gigas could be regarded as potential O2 and CO2 sensing receptors. However, future studies are needed to ascertain the neurophysiological characterization of these cells.

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.

Beta-catenin localization and timing of early development of bovine embryos obtained from oocytes matured in the presence of follicle stimulating hormone.

In mammalian species, embryos which grow more rapidly are believed to be more competent and viable than they are slower developing counterparts. Although the most important decrease in development occurs between the zygote and blastocyst stages, there is a growing amount of evidence to suggest that maturation conditions and oocyte quality have a profound influence on the developmental potential of early mammalian embryos. Gene transcripts and polypeptides stored in the oocytes, such as junctional proteins, sustain the initial development of embryos. In the present study we demonstrated a relationship between the timing of the development of in vitro-produced bovine embryos and the distribution and localization of the junctional protein beta-catenin. We further demonstrated that the presence of FSH during IVM supports cleavage and the blastocyst rate, and also has a positive effect on the speed of development, since embryos obtained from oocytes matured with the gonadotropin and observed on days 4, 5 and 6 post-insemination (p.i.) grew faster than those matured in a medium supplemented with BSA. Moreover, the majority of embryos which developed past the 16-cell stage showed a proper distribution of beta-catenin just beneath the membrane surfaces of all blastomeres and an appropriate morphology, as confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. In conclusion, our data suggest that supplementing FSH during in vitro maturation aids the development of bovine embryos and promotes the correct expression of beta-catenin, increasing the likelihood that embryos will develop to the blastocyst stage.

Absence of Meissner corpuscles in the digital pads of mice lacking functional TrkB.

The TrkB-expressing sensory neurons seem to be involved in touch and other discriminative sensibilities. Thus, several slowly and rapidly adapting cutaneous mechanoreceptors, as well as muscle spindles, are reduced or absent in the territory of the trigeminal nerve in functionally TrkB-deficient mice. Whether this also occurs in the cutaneous or muscular territories of dorsal root ganglia has not been analyzed. Here we used immunohistochemistry and transmission-electron microscopy to analyze the impact of a mutation in the gene coding for TrkB on Meissner and Pacinian corpuscles, and muscle spindles. The animals were studied at the post-natal days 15 and 25, because at this time all the mechanoreceptors examined are fully developed. Typical Meissner's corpuscles, displaying S-100 protein immunoreactivity, were found in the digital pads of wild-type and TrkB+/- mice whereas they were absent in the TrkB-/- animals. Regarding Pacinian corpuscles, the mutation in the trkB gene does not alter either the immunohistochemical or the ultrastructural characteristics. Finally, in muscle spindles the arrangement of the intrafusal muscle fibers and nerve fibers was unchanged in the mutated animals. Nevertheless, about 10% of muscle spindles showed increased number of the intrafusal cells (between 6 and 12) and were supplied by more than one large myelinic nerve fiber. The present results strongly suggest that TrkB-expressing sensory neurons in dorsal root ganglia, like those of the trigeminal ganglion, are responsible for the development and maintenance of several rapidly adapting cutaneous mechanoreceptors, i.e. Meissner's corpuscles.

Topographical and drug specific sensitivity of hair cells of the zebrafish larvae to aminoglycoside-induced toxicity.

The hair cells of the lateral line system of fishes are morphologically and physiologically similar to the hair cells of the mammalian inner ear, also sharing its molecular characteristics. For this reason, it has been used as a powerful animal model to analyze in vivo ototoxicity. In this work, we examined the dose-dependent effects of two potent ototoxic aminoglycosides, neomycin and gentamicin, on the hair cells of two selected neuromasts (L1 and T1, the first of the trunk and the terminal located in the fin, respectively) of the lateral line in the ET4 transgenic zebrafish line. The hair cells of this strain selectively and constitutively display fluorescence. The fish were treated for 24 h at different doses (1, 2.5, 5, 10 and 100 µM levels) of both aminoglycosides. Immediately after treatment the morphology and the number of cells in L1 and T were analyzed under a fluorescence microscope. The results show that neomycin and gentamicin have different effects on the hair cell death at the same concentration, showing also different toxicity in L1 and T1 neuromasts. The toxicity observed in the hair cells of T1 neuromast was less than in L1 especially for the gentamicin treatment. These results demonstrate different sensitivity of hair cells of the lateral line to ototoxic drugs according to topographical localization and suggest the in vivo assay of the L1 neuromast of zebrafish larva and low doses of neomycin as an ideal model to study ototoxicity induced by aminoglycosides.

Rodlet cells development in the intestine of sea bass (Dicentrarchus labrax).

The enigmatic rodlet cells (RCs) are characterized by conspicuous inclusions named "rodlets". They were discovered over 100 years ago and were considered as parasites but shortly afterward interpreted as endogenous cells. The RCs have been described in different tissues of marine and freshwater teleosts, but their origin and function remain unknown. This work was designed to an ultrastructural study on RCs development and distribution in intestinal epithelium of Dicentrarchus labrax. Three different stages of RCs development from early precursor cells to mature phase were observed, as well as a migration and finally an extrusion of their contents. In this study, the immature cells were found near the basal epithelium membrane. They were mainly identified by a rough endoplasmic reticulum with dilated cisternae, by developing rodlets and a thin fibrillar coat. The maturing RCs, localized in the middle zone of the epithelium, appeared to be undergoing a reorganization of the cell organelles. The mature RCs, placed near the free surface, showed a thick subplasmalemmar fibrillar coat. Most of the organelles were aggregated at the cell apex with a basally located nucleus. A cellular polarity was more evident. One of the most conspicuous features was the occurrence of mature rodlets club-sac in shape orientated toward the cell apex. Adhesive junctions between surface epithelial cells and RCs, while discharging their contents, were seen. We have connected morphological figures and distribution to different stages of development in RCs, supporting the hypothesis of their secretory function.