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The occurrence and localization of neurotrophins and their specific TrK receptor-like proteins in the adrenal gland of chicken, duck and ostrich were examined by immunohistochemical methods. In all species studied NGF-, TrK A- and TrK C-like immunoreactivity was observed in neurons and fibers of adrenal ganglia. Thin TrK A- and TrK C-like immunoreactive fibers were also observed among chromaffin cells. NT-3-like immunoreactivity was detected in chromaffin cells as revealed by the double immunolabelings NT-3/chromogranin A and NT-3/DbetaH. The interrenal tissue never showed IR to any neurotrophins and TrK tested, and none of the adrenal structures displayed immunoreactivity to BDNF and TrK B. Double immunolabelings NGF/TrK A, NGF/TrK C and TrK A/TrK C showed colocalization in some neurons and fibers in adrenal ganglia. In adrenal glands of the species studied, the distribution of neurotrophins and TrK receptors could suggest an involvement of NT-3 on neuronal populations innervating adrenal ganglia by means of its high affinity receptor TrK C and low affinity receptor TrK A. In addition, NGF could be utilized by neuronal populations of adrenal ganglia through its preferential receptor TrK A by an autocrine or paracrine modality of action.

By means of immunochemistry and immunohistochemistry, we investigated in the gut of teleostean species the presence and localization of three neurotrophins: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin (NT)-3. In all studied species both NGF- and NT-3-like immunoreactivity (IR) were present in the enteric nervous system, while BDNF-like IR was never detected. More in particular, both NGF and NT-3-like IR were detected in neurons of small and large intestine, while only NT3-like IR was also observed in stomach plexuses. Furthermore, Western blot analysis revealed the presence of molecules immunoreactive to NGF and NT-3, which weight were very similar to those of mammalian corresponding neurotrophins. These results extend to teleost species the presence and distribution of NGF- and NT-3-like IR in the enteric nervous system, suggesting a well-preserved presence of these substances in the gut during vertebrate phylogenesis.

This study reports the immunohistochemical localization and distribution of orexin A and B-like and their receptors-like peptides in the gastroenteric tract of chicken. The immunoreactivity is distributed in endocrine cells, nerve fibers and neurons, both in the stomach and intestine, and shows a discrete conformity with the data till now reported for Mammals. Our study suggests a possible participation of orexin-like peptides in the modulation of chicken gastroenteric activities and the preservation of their main distribution compared to Mammals. Western blot analysis has confirmed the presence of prepro-orexin and both receptors in the examined tissues. This survey represents the first evidence of the presence of orexin-like peptides in the gastroenteric tract of non mammalian species, and the results could help to better understand the alimentary control and body weight in domestic birds, which are of relevance to determine the productive factors in breeding animals. This study might also serve as a baseline for future experimental studies on the regulation of the gastroenteric functions in non mammalian Vertebrates.

Expression and distribution of leptin and ghrelin in the digestive ap-paratus of DIO (diet-induced obesity) zebrafish Maruccio L, Russo F, Arcamone N, Mania M, Randazzo B, Ciriaco E In this report we analyzed the expression and distribution of leptin and ghrelin in the digestive apparatus of DIO zebrafish. Leptin is anorexigenic peptide while ghrelin is orexigenic one. They act as antagonists. In this study in order to develop an obesity phenotype adult zebrafish were divided into two dietary groups. The con-trol group was fed with Artemia (5 mg cysts/fish/day) once per day. The overfeeding group was fed three times per day with Artemia (60 mg cysts/fish/day). For calorie restriction, the zebrafish was fed with Artemia (2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. At the end of this period, after 24h of fasting, the zebrafish digestive apparatus were sampled for immunohistochemistry, western blotting and qRT-PCR techniques. Leptin and ghrelin immunoreactivity were found in the enteric nervous system and neuroen-docrine cells in overfeeding and control zebrafish intestine. The number of immunopositive cells is greater in DIO zebrafish than in control ones. In both groups leptin immunoreactive nervous fibers were found around ducts and vessels of liver. The presence of proteins were confirmed by western blotting analysis. By qRT-PCR, leptin and ghrelin mRNA levels are higher in the overfeeding zebrafish intestine and in control zebrafish liver. The immunological detection of ghrelin and leptin in control zebrafish are in agreement with literature data. In DIO zebrafish the results are similar to those found for obese mammals. In conclusion, this study represent a starting point to investigate the mechanisms involved in the regulation of appetite and energy balance in DIO zebrafish.

Leptin is a modulator of food intake and energy homeostasis both in mammals and in some species of nonmammals vertebrates. In this study, we reported for the first time, using an immunohistochemical and immunochemical approach, the presence and distribution of immunoreactivity to leptin-like protein in the gastroenteric tract of Dicentrarchus labrax (bass) and Carassius auratus (goldfish), two teleostean species with different feeding and different adaptative morphological organization of the gastroenteric tract. Bass stomach showed intense immunoreactivity to leptin-like protein in all regions, with immunoreactive cells located at the base of the mucosal plicae and at the apical margin of the gastric crypts. Immunoreactive fibers and neuronal cells were observed close to vascular structures in the pyloric region. In bass and goldfish intestine, rare immunoreactive cells were observed along the mucosal epithelium mostly at the base or the apex of intestinal folds in the proximal and medium intestine; numerous immunoreactive nerve fibers in the circular and longitudinal layers of the tunica muscolaris as well as in the myenteric plexus were observed. Western blot analysis recognized a ∼15 kDa signal with a similar expression pattern for goldfish and sea bass. Our results could contribute to confirm the evolutive conservation of leptin-like proteins and their probably precocious functional diversification in fish. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

KEY WORDS ghrelin; Carassius auratus; Dicentrarchus labrax; immunohistochemistry;gastrointestinal tractABSTRACT The occurrence and localization of ghrelin peptide in the gastrointestinal tract ofCarassius auratus and Dicentrarchus labrax, two fish species that exhibit different feeding behav-ior, different habitats, and different anatomical organizations of the gastroenteric tract, wereexamined by immunohistochemical methods and western blotting analysis. All of the gastrointesti-nal segments studied displayed immunohistochemical localizations of ghrelin peptide. Numeroussingle or clustered immunoreactive cells were found along the gastric folds, particularly in the py-loric region of Dicentrarcus labrax, whereas scattered ghrelin immunoreactive cells were observedin the intestinal epithelium of both fish species. Double immunolabeling PGP 9.5/ghrelin demon-strated the localization of ghrelin peptide also in nerve fibers and neuronal cells of the submucosaland myenteric plexuses, often in association with vascular structures. Western blotting analysisconfirmed the presence of ghrelin peptide in the gatrointestinal tract of both species studied, whosemolecular weight was similar to that of the corresponding mammalian prepro-ghrelin. The findingscould support the hypothesis that this peptide is an important appetite regulator in fish and couldconfirm the presence of the ghrelin peptide, starting from its precursor proteins, in the gastrointes-tinal tract of the goldfish and the sea bass. Microsc. Res. Tech. 00:000–000, 2009.

Leptin is a hormone produced and secreted mainly by adipocytes, but also by other tissues such as placenta, brain, mammary, and pituitary glands. The gastric epithelium has also been reported as a source of leptin in mammals. In this study we examined the presence of leptin in the chicken gastroenteric tract by immunohistochemistry and Western blotting. Strong and widespread leptin-like immunoreactivity was observed in the mucosal epithelium of proventriculus plicae and in the epithelium of the complex compound glands. Numerous leptinimmunoreactive cells were found along lining epithelium of duodenum villi, while few leptin-immunoreactive cells were observed in the basal zone of duodenum glands. Many leptin-immunoreactive cells were found in the caeca at the basal zone of glands, while very few leptinimmunoreactive cells were found in the deep glandular structures of the large intestine. In the homogenates of chicken gastroenteric tract the protein detected using a human leptin-specific antibody had estimated molecular weight of approximately 15–16 kDa. To our knowledge, this is the first report demonstrating leptin-like protein distribution in the whole gastroenteric tract of bird. This finding constitute important data for the further understanding of the mechanism that regulate feeding behaviour in birds, farm animal for which the control of food intake and fatness are a high economic interest.

KEY WORDS neurotrophins; fish; juxtaglomerular cells; immunohistochemistryABSTRACT By means of immunochemistry and immunohistochemistry, we investigated in thekidney of freshwater and marine teleostean species for the presence and localization of three neuro-trophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin(NT)-3. In both species studied, NGF-like and NT-3-like immunoreactivity were present in the kid-ney with different distribution patterns, while BDNF-like immunoreactivity was never detected. Ingoldfish, NGF-like and NT-3-like immunoreactivity were identified extensively in cells along partof the arterial branches adjacent to the afferent arterioles. In scorpion fish, NGF-like and NT-3-likeimmunoreactive cells were observed both on afferent arterioles and on adjacent secondarybranches derived from renal arteries. No immunoreactivity was detected in other renal structures.A staining pattern of immunoreactivity similar to that obtained for NGF and NT-3 was detectedutilizing S100 antibody as a juxtaglomerular (JG) cell marker. Double immunolabellings NGF/S100 and NT-3/S100 evidenced the coexistence of neurotrophin-like proteins and S100-like proteinin the same immunoreactive cells, thus identifying them as juxtaglomerular cells. Western blotanalysis revealed the presence of molecules immunoreactive to NGF and NT-3, whose molecularweights were very similar to those of the corresponding mammalian neurotrophins. These findingsextend the presence and distribution of NGF-like and NT-3-like IR in the kidney to teleost species,suggesting a probable participation of these proteins in the renal functions of freshwater andmarine teleosts. Microsc. Res. Tech. 66:17–24, 2005.

In this study we have clearly demonstrated that ghrelin producing cells exist in the chicken gastrointestinal tract. The distribution pattern and morphological character- istics of ghrelin-ic suggest that they correspond to endocrine type cells. However, further investigations are required in order to identify the specific cell types according to the current classification (Solcia et al., 2000). In the frog stomach ghrelin endocrine cells have been observed in the mucosa and in pit-associated glands (Galas et al., 2002). In the rat and human stomach ghrelin-producing endocrine cells have been identified as X/A like cells of the oxyntic mucosa (Kojima et al., 1999; Date et al., 2000). These results indicate that chicken ghrelin is also evolutionally conserved both in its distribution and in its aminoacidic sequence.

Summary The presence and distribution patterns of ghrelin, a gastric acylated peptide, were studied in the entire gastrointestinal tract of the chicken ( Gallus domesticus ) using the peroxidase–antiperoxidase immunohistochemical method, western blot analysis and a specific antibody against the C-terminal region of rat ghrelin. Ghrelin-immunopositive cells were observed in the mucosal layer of all segments examined. The largest numbers of ghrelin-positive cells were located at the base of lobuli of the proventriculus gland, along villi of the intestines and in crypts of the duodenum. Lower numbers of ghrelin-immunostained cells were located in crypts of jejunum and ileum and only few ghrelin-immunostained cells were detected at the base of crypts of the large intestine. Closed and open types of cells were observed in all segments. Western blot analysis confirmed the presence of ghrelin-like protein in the entire chicken gastrointestinal tract. The anatomical distribution patterns and the morphological characteristics of chicken ghrelin-positive cells suggest that they are endocrine cells. Furthermore, it is concluded that ghrelin shows a high degree of preservation during evolution from non-mammalian vertebrates to mammals.

The present study investigates the occurrence of Trk-like neurotrophin receptor proteins in the lizard and frog kidney. In lizard rare TrkB-like immunoreactive cells in intermediate and distal tubules were found. TrkClike immunoreactive cells were numerous in collecting tubules and became less numerous in collecting ducts. No TrkC-like immunoreactivity was detected in the ureteric duct. In the frog, we observed numerous TrkC-like immunoreactive cells in collecting tubules and ducts while they were scattered among negative epithelial cells in the wolffian duct. TrkB- and TrkA-like immunoreactivity was never found. Western blot analysis demonstrated that the frog and lizard kidney contains TrkC-like protein; TrkB-like protein was present only in the lizard kidney. These results demonstrate for the first time the occurrence of Trk-like proteins in the kidney of amphibians and reptiles, and aid in the assessment of the role of Trk receptor-like proteins in the kidney physiology of vertebrates.

The immunolocalization of S100-like protein was investigated in the kidney of saltwater fishes (Dicentrarchus labrax; Coris julis; Serranus cabrilla; Scorpaena porcus), amphibia (Rana aesculenta), reptiles (Lacerta viridis), and aves (Gallus domesticus; Strutio camelus). S100-like immunoreactivity was detected in the juxtaglomerular cells of all saltwater fishes studied. No immunoreactivity was observed in other tracts of the nephron or in the interstitial tissue. In frog kidney, S100-like immunoreactive cells were localized in the proximal tubule, singly distributed or placed side by side in clusters of two or three cells. S100-like immunoreactive cells were distributed in the distal and in the collecting tubules in lizard, chicken, and ostrich kidney. In the distal tubule of lizard kidney, S100-like immunoreactive cells were numerous and uniformly distributed. In lizard collecting tubules, S100-like immunoreactive cells showed less intense immunoreactivity than in the distal tubule, except for a cluster of cells at the junction with the initial collecting duct. In chicken and ostrich kidney, S100-like immunoreactive cells of the distal tubules were closely packed together. In the collecting tubules, S100-like immunoreactive cells were alternate to negative cells. These results indicate the high conservation degree of S100 proteins through phylogenesis and suggest a functional role for these proteins in the vertebrate kidney.

The presence of neurotrophins and their specific receptors in adult and developing Japanese quail ovaries Nechita E-L, Arcamone N, Maruccio L, Solcan C, Cotea C Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of neurotrophins family, which are involved in growth, maintaining, differentiation and survival of neurons in the central and peripheral nervous system. The activity of these neurotrophins is elicited in systems other than nervous, such as reproductive and endocrine systems. In mammals, neurotrophins and their receptors TrkA, TrkB, TrkC play a critical role in oogenesis and folliculogenesis, promoting the follicular development and oo-cyte maturation, also during early embryonic development. In this study we report the presence of NGF, BDNF, NT-3 and their specific receptors in adult and embryonic quail gonads. The investigations were con-ducted on embryonic stages from 4th to 17th day of incubation and on adult laying quail ovaries. Immunohisto-chemical Envision technique was used. The immunoreactivity (ir) was observed from 6th day of embryonic de-velopment. Neurotrophins and their receptors were differently distributed in cortical and medullary areas. The localization of NGF, NT3 and Trk receptors-ir was differently observed in cytoplasm of oocytes, follicular cells and stromal cells, both in adult and embryonic quail ovaries from the 9th day of incubation, while BDNF ir was observed only in adult. The results obtained confirm the involvement of neurotrophins during the development of gonads in quails, as well as in mammals. Furthermore, the occurrence of neurotrophins and the receptors could provide new insights on their possible paracrine and/or autocrine mode of action in quail ovary during development and folliculogenesis.

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalamus. It is present in neuronal elements of a number of peripheral organs. We have examined whether PACAP occurs in the gill arch of Carassius auratus L. in which our recent studies have shown the presence of VIP-like peptide. Immunohistochemistry has revealed PACAP-like immunoreactivity in the anterior branches of the post-trematic glossopharyngeal and vagus nerves. PACAP-immunoreactive nerve cell bodies and fibers are present in connective tissue on the oral side of the gill arch. Colocalization studies carried out by the application of double immunofluorescence show that a PACAP-like peptide coexists with VIP in the same nerve cell bodies and fibers. The localization pattern of PACAP in the gill arch of goldfish suggests its possible involvement in the regulation of secretory activities.

By immunohistochemistry VIP-like immunoreactive cells were observed in the second segment of some renal proximal tubules of Carassius auratus. Immunoreactivity displayed a granular appearance all over the cells. VIP-like immunoreactive cells showed a shape of an overturned flask whose globe-like part looks like the tubular lumen. No immunoreactive material was observed in other tracts of the nephron and in the interstitial cells. The presence of VIP-like immunoreactive cells is discussed in relation to their possible function.

The localization of vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) nerve cell bodies and fibers has been studied in the gill arches of goldfish (Carassius auratus, L.) using the peroxidase-antiperoxidase (PAP) immunohistochemical method. It was found that VIP-ir nerve cell bodies are localized in connective tissue on the oral side of the gill arch; these cells were present as single cells, in couples or as small clusters. Moreover, a dense network of VIP-ir fibers was observed beneath the lining epithelium of the raker cushion. The possible involvement of this peptide in mucus secretion in the gill arches of teleost is discussed.