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.

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.

Ultrastructure of Lingual Papillae in Common Chimpanzee (Pan troglodytes) Foetus, Newborn and Adult Specimens.

Among primates, the two recognized species of chimpanzees (common chimpanzee, Pan troglodytes; pygmy chimpanzee, Pan paniscus) are considered to be the most similar to humans. Importantly, in mammals, the food intake behaviour largely determines the tongue morphology, including the type, proportion and distribution of gustatory and non-gustatory tongue papillae. The lingual papillae form during its development and mature in post-natal life depending on the different feeding. In this study, we have used scanning electron microscopy to analyse the age-related changes in the lingual papillae of foetal, newborn and adult P. troglodytes. Four main types of lingual papillae, denominated filiform, fungiform, foliate and vallate, and one subtype of filiform papillae called conical papillae, were found. The main age-related changes observed in all kinds of papillae were a progressive keratinization and morphological complexity along the lifespan. During the foetal period, there was scarce keratinization, which progressively increases in young animals to adulthood. The number of filiform increased with ageing, and both filiform and fungiform papillae in adult tongues are divided into pseudopapillae. On the other hand, the vallate papillae vary from smooth simple surfaces in foetal tongues to irregular surfaces with grooves and pseudopapillae (microscopic papilla-shaped formations within the papilla itself) in adults. These results describe for the first time the age-related variations in the three-dimensional aspect of lingual papillae of the chimpanzee tongue and provide new data to characterize more precisely these structures in the human closest specie.

Expression and distribution of leptin and its receptors in the digestive tract of DIO (diet-induced obese) zebrafish.

The expression and localization of leptin (A and B) and its receptor family in control and diet-induced obese (DIO) adult male zebrafish gut, after 5-weeks overfeeding, administering Artemia nauplii, as fat-rich food, were investigated. Recently, the obese adult zebrafish was considered an experimental model with pathophysiological pathways similar to mammalian obesity. Currently, there are no reports about leptin in fish obesity, or in a state of altered energy balance. By qRT-PCR, leptin A and leptin B expression levels were significantly higher in DIO zebrafish gut than in the control group (CTRL), and the lowest levels of leptin receptor mRNA appeared in DIO zebrafish gut. The presence of leptin and its receptor proteins in the intestinal tract was detected by western blot analysis in both control and DIO zebrafish. By single immunohistochemical staining, leptin and leptin receptor immunoreactive endocrine cells were identified in the intestinal tract either in DIO or control zebrafish. Moreover, leptin immunopositive enteric nervous system elements were observed in both groups. By double immunohistochemical staining, leptin and its receptor were colocalized especially in DIO zebrafish. Thus, our study represents a starting point in the investigation of a possible involvement of leptin in control of energy homeostasis in control and DIO zebrafish.

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.

Acid-sensing ion channel immunoreactivities in the cephalic neuromasts of adult zebrafish.

The neuromasts are the morphofunctional unit of the lateral line system serving as mechanosensors for water flow and movement. The mechanisms underlying the detection of the mechanical stimuli in the vertebrate mechanosensory cells remain poorly understood at the molecular level, and no information is available on neuromasts. Mechanotransduction is the conversion of a mechanical stimulus into an electrical signal via activation of ion channels. The acid-sensing ion channels (ASICs) are presumably involved in mechanosensation, and therefore are expected to be expressed in the mechanoreceptors. Here we used immunohistochemistry to investigate the occurrence and distribution of ASICs in the cephalic neuromasts of the adult zebrafish. Specific immunoreactivity for ASIC1 and ASIC4 was detected in the hair cells while ASIC2 was restricted to the nerves supplying neuromasts. Moreover, supporting and mantle cells; i.e., the non-sensory cells of the neuromasts, also displayed ASIC4. For the first time, these results demonstrate the presence of the putative mechanoproteins ASIC1, ASIC2 and ASIC4 in neuromasts, suggesting a role for these ion channels in mechanosensation.

Orexin A expression in the ovary of dog and cat.

Orexin A and B, also known as hypocretin A and B, are hypothalamic neuropeptides arising from a precursor to the 130 amino acid, called pre-pro orexin. They are synthesized mainly in lateral and posterior hypothalamus and are involved in different functions such as regulation of food intake and energy balance. Orexins and orexin receptors were previously described also in different tissues and organs outside the brain. The aim of this study was to demonstrate by means of the immunofluorescence technique, the presence of orexin A in the ovary of cat and dog, to support the hypothesis of the role of this substance also at the level of the female genital system. The presence of orexin A in the ovary either in dog or in cat is in agreement with previous data on the presence and role of orexins in the female genital system of other species.

Cholecystokinin: how many functions? Observations in seabreams.

A short overview on the regional distribution of the gastro-intestinal peptide hormone cholecystokin (CCK) in fish is presented. In particular, the results of molecular and immunological studies on seabreams, Diplodus puntazzo and Diplodus sargus, are reported, which, by demonstrating CCK in the hindgut, open new questions regarding the functional role of this hormone in that part of the intestine. The putative involvement of hindgut CCK in the feedback control of digestive processes was tested by measuring CCK gene and protein expression in fed and fasted fish. The results of this study led to hypothesize different roles for the two CCK isoforms in D. sargus, one of which related to regulation of digestive processes from pyloric caeca through hindgut. On the other hand, a functional role alternative to regulation of digestive processes may be inferred for the other isoform.

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.

Cholecystokinin in white sea bream: molecular cloning, regional expression, and immunohistochemical localization in the gut after feeding and fasting.

BACKGROUND: The peptide hormone cholecystokinin (CCK), secreted by the midgut, plays a key role in digestive physiology of vertebrates including teleosts, by stimulating pancreatic secretion, gut motility, and gallbladder contraction, as well as by delaying gastric emptying. Moreover, CCK is involved in the regulation of food intake and satiation. Secretion of CCK by the hindgut is controversial, and its biological activity remains to be elucidated. The present paper addresses the regional distribution of intestinal CCK in the white sea bream, Diplodus sargus, as well as the possible involvement of hindgut CCK in digestive processes. METHODOLOGY/PRINCIPAL FINDINGS: Full-lengths mRNAs encoding two CCK isoforms (CCK-1 and CCK-2) were sequenced and phylogenetically analyzed. CCK gene and protein expression levels in the different gut segments were measured 3 h and 72 h after feeding, by quantitative real-time RT-PCR and Western blot, respectively. Moreover, endocrine CCK cells were immunoistochemically detected. Fasting induced a significant decrease in CCK-2 in all intestinal segments, including the hindgut. On the other hand, no significant difference was induced by fasting on hindgut CCK-1. CONCLUSIONS/SIGNIFICANCE: The results demonstrated two CCK isoforms in the hindgut of D.sargus, one of which (CCK-2) may be involved in the feedback control of uncompleted digestive processes. On the other hand, a functional role alternative to regulation of digestive processes may be inferred for D.sargus CCK-1, since its expression was unaffected by feeding or fasting.

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.

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.

Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development.

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS.

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.