Expression of ghrelin and leptin in the chemosensory system of adult zebrafish.

Numerous data show that the chemosensory system seems to be modulated by changes in the circulating levels of different molecules such as ghrelin, orexin, leptin, NPY, CCK. The chemosensory system of the zebrafish is represented by the taste buds (skin, oral and oropharyngeal), the olfactory rosette and the solitary chemosensorial cells (SCCs). The purpose of our study was to analyze the distribution of two peripheral hormones such as ghrelin and leptin in the chemosensory organs of the zebrafish. Our results demonstrated the presence of immunoreaction for all antibodies used in the zebrafish chemosensory organs even if with different distribution. In particular, IR was observed for ghrelin in the olfactory rosette while IR for leptin was found in the olfactory rosette, in the skin and oropharyngeal taste buds and in the gills. Both these hormones were detected in the intestine, used as a control.

Intestinal immunity of dogfish Scyliorhinus canicula spiral valve: A histochemical, immunohistochemical and confocal study.

The present study describes histochemical and immunohistochemical characteristics of the spiral valve and its associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula. The mucosal surface of the spiral valve represents the first line of defense against pathogens coming from the external environment through food. Epithelial, mucus and immune cells play a key role in controlling the inflammatory response. Valve intestine of S. canicula had many folds lined by simple columnar cells and goblet cells, which later reacted positive to PAS, AB and AB-PAS, histochemical stains differentiated the different types of mucins; lectin histochemistry (PNA and WGA), detected neutral and acid mucins secreted that plays an important role in protection against invading pathogens. Integrin α5ß1 was expressed in enterocytes that line the valve's folds with greater marking in the apical part of the cells. Laminin was found on the apical side of the epithelium, in fibrillar and cellular elements of the lamina propria and in the muscularis mucosa. In the spiral valve gut-associated lymphoid tissue (GALT) has been studied. For the first time, massive leucocytes aggregates were identified by confocal immunofluorescence techniques, using the following antibodies: TLR2, S100, Langerin/CD207. Our results expand knowledge about Dogfish valve intestine giving important news in understanding comparative immunology.

Synthesis of bioinspired collagen/alginate/fibrin based hydrogels for soft tissue engineering.

Hydrogels based on natural polymers offer a range of properties to mimic the native extracellular matrix, and provide microenvironments to preserve cellular function and encourage tissue formation. A tri-component hydrogel using collagen, alginate and fibrin (CAF) was developed and investigated at three collagen concentrations for application as a functional extracellular matrix analogue. Physical-chemical characterization of CAF hydrogels demonstrated a thermo-responsive crosslinking capacity at physiological conditions with stiffness similar to native soft tissues. CAF hydrogels were also assessed for cytocompatibility using L929 murine fibroblasts, pancreatic MIN6 ß-cells and human mesenchymal stem cells (hMSCs); and demonstrated good cell viability, proliferation and metabolic activity after 7 days of in vitro culture. CAF hydrogels, especially with 2.5% w/v collagen, increased alkaline phosphatase production in hMSCs indicating potential for the promotion of osteogenic activity. Moreover, CAF hydrogels also increased metabolic activity of MIN6 ß-cells and promoted the reconstitution of spherical pseudoislets with sizes ranging between 50 and 150 µm at day 7, demonstrating potential in diabetic therapeutic applications.

Melatonin treatment suppresses appetite genes and improves adipose tissue plasticity in diet-induced obese zebrafish.

PURPOSE: Overweight and obesity are important risk factors for diabetes, cardiovascular diseases, and premature death in modern society. Recently, numerous natural and synthetic compounds have been tested in diet-induced obese animal models, to counteract obesity. Melatonin is a circadian hormone, produced by pineal gland and extra-pineal sources, involved in processes which have in common a rhythmic expression. In teleost, it can control energy balance by activating or inhibiting appetite-related peptides. The study aims at testing effects of melatonin administration to control-fed and overfed zebrafish, in terms of expression levels of orexigenic (Ghrelin, orexin, NPY) and anorexigenic (leptin, POMC) genes expression and morphometry of visceral and subcutaneous fat depots. METHODS: Adult male zebrafish (n = 56) were divided into four dietary groups: control, overfed, control + melatonin, overfed + melatonin. The treatment lasted 5 weeks and BMI levels of every fish were measured each week. After this period fishes were sacrificed; morphological and morphometric studies have been carried out on histological sections of adipose tissue and adipocytes. Moreover, whole zebrafish brain and intestine were used for qRT-PCR. RESULTS: Our results demonstrate that melatonin supplementation may have an effect in mobilizing fat stores, in increasing basal metabolism and thus in preventing further excess fat accumulation. Melatonin stimulates the anorexigenic and inhibit the orexigenic signals. CONCLUSIONS: It seems that adequate melatonin treatment exerts anti-obesity protective effects, also in a diet-induced obesity zebrafish model, that might be the result of the restoration of many factors: the final endpoint reached is weight loss and stabilization of weight gain.

Neuromast hair cells retain the capacity of regeneration during heavy metal exposure.

The neuromast is the morphological unit of the lateral line of fishes and is composed of a cluster of central sensory cells (hair cells) surrounded by support and mantle cells. Heavy metals exposure leads to disruption of hair cells within the neuromast. It is well known that the zebrafish has the ability to regenerate the hair cells after damage caused by toxicants. The process of regeneration depends on proliferation, differentiation and cellular migration of sensory and non-sensory progenitor cells. Therefore, our study was made in order to identify which cellular types are involved in the complex process of regeneration during heavy metals exposure. For this purpose, adult zebrafish were exposed to various heavy metals (Arsenic, cadmium and zinc) for 72h. After acute (24h) exposure, immunohistochemical localization of S100 (a specific marker for hair cells) in the neuromasts highlighted the hair cells loss. The immunoreaction for Sox2 (a specific marker for stem cells), at the same time, was observed in the support and mantle cells, after exposure to arsenic and cadmium, while only in the support cells after exposure to zinc. After chronic (72h) exposure the hair cells were regenerated, showing an immunoreaction for S100 protein. At the same exposure time to the three metals, a Sox2 immunoreaction was expressed in support and mantle cells. Our results showed for the first time the regenerative capacity of hair cells, not only after, but also during exposure to heavy metals, demonstrated by the presence of different stem cells that can diversify in hair cells.

Localization of cholecystokinin in the zebrafish retina from larval to adult stage.

The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.

Fine structure of the canal neuromasts of the lateral line system in the adult zebrafish.

The mechanosensory lateral line system of fish is responsible for several functions such as balance, hearing, and orientation in water flow and is formed by neuromast receptor organs distributed on head, trunk and tail. Superficial and canal neuromasts can be distinguished for localization and morphological differences. Several information is present regarding the superficial neuromasts of zebrafish and other teleosts especially during larval and juvenile stages, while not as numerous data are so far available about the ultrastructural characteristics of the canal neuromasts in adult zebrafish. Therefore, the aim of this study was to investigate by transmission electron microscopy the ultrastructural aspects of cells present in the canal neuromasts. Besides the typical cellular aspects of the neuromast, different cellular types of hair cells were observed that could be identified as developing hair cells during the physiological turnover. The knowledge of the observed cellular types of the canal neuromasts and their origin could give a contribution to studies carried out on adult zebrafish used as model in neurological and non-neurological damages, such as deafness and vestibular disorders.

Bone marrow failure may be caused by chromosome anomalies exerting effects on RUNX1T1 gene.

BACKGROUND: The majority of the cases of bone marrow failure syndromes/aplastic anaemias (BMFS/AA) are non-hereditary and considered idiopathic (80-85%). The peripheral blood picture is variable, with anaemia, neutropenia and/or thrombocytopenia, and the patients with idiopathic BMFS/AA may have a risk of transformation into a myelodysplastic syndrome (MDS) and/or an acute myeloid leukaemia (AML), as ascertained for all inherited BMFS. We already reported four patients with different forms of BMFS/AA with chromosome anomalies as primary etiologic event: the chromosome changes exerted an effect on specific genes, namely RUNX1, MPL, and FLI1, leading to the disease. RESULTS: We report two further patients with non-hereditary BM failure, with diagnosis of severe aplastic anaemia and pancytopenia caused by two different constitutional structural anomalies involving chromosome 8, and possibly leading to the disorder due to effects on the RUNX1T1 gene, which was hypo-expressed and hyper-expressed, respectively, in the two patients. The chromosome change was unbalanced in one patient, and balanced in the other one. CONCLUSIONS: We analyzed the sequence of events in the pathogenesis of the disease in the two patients, including a number of non-haematological signs present in the one with the unbalanced anomaly. We demonstrated that in these two patients the primary event causing BMFS/AA was the constitutional chromosome anomaly. If we take into account the cohort of 219 patients with a similar diagnosis in whom we made cytogenetic studies in the years 2003-2017, we conclude that cytogenetic investigations were instrumental to reach a diagnosis in 52 of them. We postulate that a chromosome change is the primary cause of BMFS/AA in a not negligible proportion of cases, as it was ascertained in 6 of these patients.

The Tongue Dorsal Surface in Fish: A Comparison Among Three Farmed Species.

The oral cavity of vertebrates has a very important role in many functions strictly related to the food processing. Two species, sea bass Dicentrarchus labrax and seabream Sparus aurata, are mainly involved in the aquaculture industry in the mediterranean area; moreover, the white seabream Diplodus sargus sargus was recently chosen as the best candidate for the diversification of species of commercial interest. This investigation was carried out, using the standard procedures for light and scanning electron microscopy, to analyse the morphology of the tongue dorsal surface in order to show whether relationships are present between the tongue morphology and the nutritional choices of these species. In all the three investigated species, three different areas were observed on the dorsal lingual surface: an apex, a body and a root. In the sea bass D. labrax, numerous caninelike teeth, organized in pads, were present along the dorsal surface. The presence of numerous taste buds, scattered on the tongue, was shown. In the seabream S. aurata, the apical part of the tongue is inserted in a pouch with a characteristic medial ridge on the body surface. Numerous taste buds were scattered on the whole surface. In the white seabream D. sargus sargus, a pouch partially covering the apex was clearly observed. In the pouch, filiform-like and foliate-like papillae can be distinguished. The results show the important role of the fish tongue in the food ingestion process, demonstrating the interaction of food processing, taste and morphological characteristics.

Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds.

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.

Light regulates the expression of the BDNF/TrkB system in the adult zebrafish retina.

The retina of the adult zebrafish express brain-derived neurotrophic factor (BDNF) and its signaling receptor TrkB. This functional system is involved in the biology of the vertebrate retina and its expression is regulated by light. This study was designed to investigate the effects of cyclic (12 h light/12 h darkness) or continuous (24 h) exposure during 10 days to white light, white-blue light, and blue light, as well as of darkness, on the expression of BDNF and TrkB in the retina. BDNF and TrkB were assessed in the retina of adult zebrafish using quantitative real-time polymerase chain reaction and immunohistochemistry. Exposure to white, white-blue, and blue light causes a decrease of BDNF mRNA and of BDNF immunostaining, independently of the pattern of light exposition. Conversely, in the same experimental conditions, the expression of TrkB mRNA was upregulated and TrkB immunostaining increased. Exposition to darkness diminished BDNF and TrkB mRNAs, and abolished the immunostaining for BDNF but not modified that for TrkB. These results demonstrate the regulation of BDNF and TrkB by light in the retina of adult zebrafish and might contribute to explain some aspects of the complex pathophysiology of light-induced retinopathies.

Morphology of the tongue dorsal surface of gilthead seabream (Sparus aurata).

The gilthead seabream is a food fish, one of the most frequently used in aquaculture. In the species of commercial interest, feeding in captivity is very important and this process is strictly related to the morphological characteristics of the oral cavity. The aim of this study is, using the standard procedures for light and scanning electron microscopy, to analyze the morphology of the tongue dorsal surface to show if relationships are present between the tongue morphology and the nutritional habits and choices of this farmed species. The main characteristic of the gilthead seabream oral cavity floor is the presence of an apical pouch, with, probably, a protective role mainly for the apical, free part of the tongue. Three zones, like in other teleosts, an apex, a body and a root, can be clearly distinguished. In the pouch foliate-like papillae were observed, while the whole tongue is characterized by the presence of two types of papillae, respectively with a fungiform and cylindroid aspect, both randomly distributed throughout the whole dorsal surface of the tongue. Scattered and numerous taste buds, with the typical pear-onion shape, together with small and numerous taste pores are also present, distributed throughout the tongue surface. Our results demonstrate that in the gilthead seabream important mechanic and sensory roles are carried out by specific anatomical structures. Our anatomical data could give, together with further biochemical and physiological data, an important support with the aim of improving the nutrition of aquaculture species.

Functional pain in hospitalised and school children.

AIM: Aim of the study was to recognise the role of psychological disagreement in children and adolescents suffering from functional pain. METHODS: Two groups of children, adolescents and their parents were interviewed: group H (hospitalized patients), group S (students, at school). Suitable investigations excluded organic lesions. The following data were analysed: 1) presence of pain in relation with: i) sex and age; ii) relation with parents, brothers, other relatives, schoolfellows; 2) efficacy of possible treatments. RESULTS: Group H: 194 patients, median age 10 years; 134 referred pain: 62 out of 92 males and 72 out of 102 females; location of pain: abdomen, limbs, head, back. Family disagreements: 36, functional pain 32; schoolfellows disagreements 114, functional pain 79. Correlations of pain with sex, increasing age, family and schoolfellows disagreements: non statistically significant. Group S: 246 students, median age: 13 years; 188 referred pain: 78 out of 118 males and 110 out of 128 females; pain was statistically more frequent in females, it increased with age. Location of pain: limbs, abdomen, head, back. Family disagreements: 31, functional pain 28, schoolfellows disagreements 140, functional pain 114. Correlations of pain with family and schoolfellows disagreements: non-statistically significant. Several parents gave answers which were different from their children's. Pharmacological and dietary interventions failed to obtain regression of pain. CONCLUSION: In both groups, the referred disagreements were not statistically different among children with functional pain and those without pain; such psychological distress was not the only factor causing functional pain. The empiric treatment adopted was inefficacious.

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.

Morphology of the lingual dorsal surface and oral taste buds in Italian lizard (Podarcis sicula).

The Italian lizard (Podarcis sicula) is the most diffused reptile in Italy, but it is also present in other European countries. This lizard belongs to the Lacertidae family, lives near walls, slants and along the borders of the paths; its diet includes bugs and aracnids. No data are so far available in literature about the three-dimensional morphology of the tongue of Podarcis sicula, therefore the aim of the present paper was to study by scanning electron and light microscopy the three-dimensional characteristics of the dorsal lingual surface and moreover the presence of chemosensory receptors like the taste buds in the oral cavity. Our results demonstrate that the Podarcis sicula tongue is a triangular muscular membranous organ, dorsoventrally flattened and that three different areas can be observed: a bifid apex, a body and a root. No papillae were observed in the apex, characterized by a flattened mucosa and by two deep median pouches. In the body cylindrical papillae with a flat surface are present, aborally gradually substituted by imbricated papillae. Foliate-like papillae were observed in the lateral parts of the tongue body. No sensory structures were showed on the lingual dorsal surface, while they were numerous in the oral cavity, particularly on the gingival epithelium. The light microscopy shows, on the dorsal surface, a stratified pavimentous not keratinized epithelium, conversely keratinized along the ventral surface. Many caliciform cells on the lateral parts of the papillae, deputed to the secretion of mucus, were also observed. Therefore, the results obtained in this paper could give a contribution to the knowledge of the tongue anatomy in a species widely diffused in different European countries and could be of help for clinical purposes in reptiles.

Expression of brain-derived neurotrophic factor and TrkB in the lateral line system of zebrafish during development.

The neuromasts of the lateral line system are regarded as a model to study the mechanisms of hearing, deafness, and ototoxicity. The neurotrophins (NTs), especially brain-derived neurotrophic factor (BDNF), and its signaling receptor TrkB are involved in the development and maintenance of neuromasts. To know the period in which the BDNF/TrkB complex has more effects in the neuromast biology, the age-related changes were studied. Normal zebrafish from 10 to 180 days post-fertilization (dpf), as well as transgenic ET4 zebrafish 10 and 20 dpf, was analyzed using qRT-PCR, western blot, and immunohistochemistry. BDNF and TrkB mRNAs followed a parallel course, peaking at 20 dpf, and thereafter progressively decreased. Specific immunoreactivity for BDNF and TrkB was found co-localized in all hairy cells of neuromasts in 20 and 30 dpf; then, the number of immunoreactive cells decreased, and by 180 dpf BDNF remains restricted to a subpopulation of hairy cells, and TrkB to a few number of sensory and non-sensory cells. At all ages examined, TrkB immunoreactivity was detected in sensory ganglia innervating the neuromasts. The present results demonstrate that there is a parallel time-related decline in the expression of BDNF and TrkB in zebrafish. Also, the patterns of cell expression suggest that autocrine/paracrine mechanisms for this NT system might occur within the neuromasts. Because TrkB in lateral line ganglia did not vary with age, their neurons are potentially capable to respond to BDNF during the entire lifespan of zebrafish.

Sox-2 in taste bud and lateral line system of zebrafish during development.

The Sox-2 is a transcription factor involved in adult neurogenesis in different vertebrate species, including fishes. Sox-2 also participates in growth and renewal on sensory cells in neuromasts of the fish lateral line system, and it is essential for development of taste buds in mammals. Using immunohistochemistry and Western blot we have investigated the occurrence and localization of Sox-2 taste buds and neuromast of zebrafish from 10 days post-fertilization to adult stage (1 year). The antibody used identifies two protein bands with estimated molecular weights of 34 and 37kDa which are consistent with those predicted for Sox-2. Sensory cells in taste buds displayed Sox-2 immunoreactivity at all the ages sampled, whereas in the neuromasts Sox-2 expression was restricted to the basal non-sensory cells. Interestingly Sox-2 immunoreactivity was observed in epithelial cells associated with both taste buds and neuromasts. Present results demonstrate that Sox-2 expressed in taste buds and neuromasts of zebrafish during the whole lifespan. Nevertheless, whereas the role of Sox-2 in taste buds of zebrafish remains to be established, the results in neuromast suggest that Sox-2 could participate in cell renewal of the mechanosensory cells.

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.