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In this study, evidence for leptin receptor (LR) and gastric leptin immunoreactivity along the digestive tract of the rainbow trout (Oncorhynchus mykiss), is reported. Besides this, the regulation of gastric leptin and its transcript by fatty acids was analyzed in vitro. LR was detected mainly in the cells of the stomach gastric glands and in the brush border of the epithelium of the anterior, middle and distal intestine. In the stomach LR was co-distributed with leptin. The regulation of gastric leptin and its transcript by fatty acids was analyzed by in vitro incubations. Rabbit polyclonal antibodies anti rainbow trout leptin were developed and employed to detect leptin concentration in the stomach and in the incubation medium. Stomach slices were incubated with butyric (4:0), oleic (18:1n-9), α-linolenic (18:3n-3) and arachidonic fatty acids (20:4n-6). All fatty acids caused an increase in the protein in both the stomach and culture medium, while leptin transcript was not modified. Overall, the results confirm the gastric leptin release upon nutritional modulation.

Orexin A and B peptides and the receptor OX2R were studied in sea bass and goldfish gastroenteric tract by immunoblotting combined with densitometric analysis using NIH Image J software and immunohistochemical techniques. These teleost species present a different gut organization and diverse feeding habits. Immunoblotting experiments showed one band of 16 kDa corresponding to prepro-orexin, and one band of 38 kDa corresponding to the OX2R receptor. Immunohistochemical localization of OXA and OXB was observed in the enteric nervous system throughout the gastroenteric tract of both species. OXA and OXB immunoreactive cells were found in the gastric and intestinal regions of sea bass, and were mainly found in the basal region of folds in intestinal bulb, and in the midgut and hindgut of goldfish. The distribution of OX2R was mainly detected in the mucosa of the gastroenteric tract of sea bass and goldfish. This distribution suggests an endocrine action of OXA and OXB in the gastrointestinal tract as well as involvement in the peripheral control of food intake and digestive processes in both species. This study might also serve to determine the productive factors in breeding and as a baseline for future experimental studies on the regulation of the gastroenteric functions in non-mammalian vertebrates. Anat Rec, 299:1121-1129, 2016. © 2016 Wiley Periodicals, Inc.

Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.

Orexin A and B peptides and the receptor OX2R were studied in sea bass and goldfish gastroenteric tract by immunoblotting combined with densitometric analysis using NIH Image J software and immunohistochemical techniques. These teleost species present a different gut organization and diverse feeding habits. Immunoblotting experiments showed one band of 16 kDa corresponding to prepro-orexin, and one band of 38 kDa corresponding to the OX2R receptor. Immunohistochemical localization of OXA and OXB was observed in the enteric nervous system throughout the gastroenteric tract of both species. OXA and OXB immunoreactive cells were found in the gastric and intestinal regions of sea bass, and were mainly found in the basal region of folds in intestinal bulb, and in the midgut and hindgut of goldfish. The distribution of OX2R was mainly detected in the mucosa of the gastroenteric tract of sea bass and goldfish. This distribution suggests an endocrine action of OXA and OXB in the gastrointestinal tract as well as involvement in the peripheral control of food intake and digestive processes in both species. This study might also serve to determine the productive factors in breeding and as a baseline for future experimental studies on the regulation of the gastroenteric functions in non-mammalian vertebrates.

The S100 protein in nervous tissue appears to play important roles in regulating neuronal differentiation, glial proliferation, plasticity, development, axonal growth, and in neurogenetic processes. In fish, the adult neurogenic activity is much higher than in mammals. In this study, the localization of S100 protein was investigated in the brain of annual teleost fish, Nothobranchius furzeri, which is an emerging model organism for aging research. By immunohistochemical techniques, S100 immunoreactivity (IR) was detected in glial cells, small neurons, and fibers throughout all regions of central nervous system (CNS) with different pattern of distribution. In the telencephalon, S100 IR was seen in the olphactory bulbs and in different areas of the telencephalic hemispheres. In the diencephalon, S100 positivity was observed in the habenular nuclei of the epithalamus, in the cortical thalamic nucleus, in the dorsal, ventral and caudal portions, the latter with the posterior recessus nucleus, and in the diffuse inferior lobe of the hypothalamus, along the diencephalic ventricle and in the dorsal optic tract. In the mesencephalon, S100 IR was observed in the longitudinal tori, in the optic tectum, and along the mesencephalic ventricle. In the rhombencephalon, S100 IR was shown in valvula and body of the cerebellum, and in some nuclei of the medulla oblongata. The results suggest that S100 may play a key role in the maintenance of the CNS and in neurogenesis processes in the adulthood. The S100 protein in nervous tissue appears to play important roles in regulating neuronal differentiation, glial proliferation, plasticity, development, axonal growth, and in neurogenetic processes. In fish, the adult neurogenic activity is much higher than in mammals. In this study, the localization of S100 protein was investigated in the brain of annual teleost fish, Nothobranchius furzeri, which is an emerging model organism for aging research. By immunohistochemical techniques, S100 immunoreactivity (IR) was detected in glial cells, small neurons, and fibers throughout all regions of central nervous system (CNS) with different pattern of distribution. In the telencephalon, S100 IR was seen in the olphactory bulbs and in different areas of the telencephalic hemispheres. In the diencephalon, S100 positivity was observed in the habenular nuclei of the epithalamus, in the cortical thalamic nucleus, in the dorsal, ventral and caudal portions, the latter with the posterior recessus nucleus, and in the diffuse inferior lobe of the hypothalamus, along the diencephalic ventricle and in the dorsal optic tract. In the mesencephalon, S100 IR was observed in the longitudinal tori, in the optic tectum, and along the mesencephalic ventricle. In the rhombencephalon, S100 IR was shown in valvula and body of the cerebellum, and in some nuclei of the medulla oblongata. The results suggest that S100 may play a key role in the maintenance of the CNS and in neurogenesis processes in the adulthood. © 2011 Wiley-Liss, Inc.

This study provides an immunohistochemical approach to the expression of leptin in the gastrointestinal tract of the monogastric South American sea lion (Otaria flavescens), and the poligastric bottlenose dolphin (Tursiops truncatus). The specific organization of the gastrointestinal tract is examined in relation to the neuroendocrine regulation of the gut exerted by leptin. In the South American sea lion some leptin-like-immunoreactive (ir) cells, and endocrine type cells, were found in the pit of gastric mucosal folds and in the epithelium of duodenum as well as numerous neurons were detected in the submucosal and myenteric plexuses of the stomach. In the bottlenose dolphin, many leptin-like-ir cells, and exocrine type cells, were identified in the mucosal layer of the main stomach as well as several neurons and nervous fibers were detected in nervous plexuses of main stomach, pyloric stomach, proximal, and middle intestine. Our data suggest that the distribution of leptin-like peptides is similar in the two species, notwithstanding the different anatomical organization of the gastrointestinal apparatus of South American sea lion and bottlenose dolphin. These findings "suggest" the presence of a basal plan in the regulation of food intake, body weight, energy balance and of the gastrointestinal functions in general also in marine mammals with different and specific feeding habits.

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.

A progestin receptor (PR) has been detected in the olfactory organ of the trout Salmo trutta fario. The specificity of this receptor was high for 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), but it also bound 17alpha-hydroxy-progesterone (17alpha-OHP) and 21-hydroxyprogesterone (21-OHP), even when present at low concentrations (10-fold in relative binding affinity assay). Progesterone (P) competed effectively at much higher concentrations (1,000-fold in relative binding affinity assay). Immunohistochemical studies carried out with three different monoclonal antibodies against human progesterone receptor (hPR), chicken progesterone receptor hinge region (cPR), and chicken progesterone receptor A/B domain (PR22), revealed that immunoreactivity was present in the epithelium of the olfactory organ of females and males of the trout Salmo trutta fario only against hPR. Western blotting showed two hPR immunoreactive bands of about 62 and 66 kDa. Finally, a portion of the cDNA of about 300 nucleotides extending over the DNA binding domain and the ligand binding domain was cloned and sequenced, revealing a high degree of sequence homology of the PR in Salmo trutta fario with the PR in other teleosts.

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.