N. Arcamone

The effects of cadmium on the gills of the goldfish Carassius auratus L.: Metal uptake and histochemical changes

Abstract 1. Uptake of cadmium and histochemical changes of mucopolysaccharides and of cytochromeoxidase in the gill epithelium of Carassius auratus were examined at different times after 10ppm Cd 2+ exposure in hard water (520 mg/l CaCO 3 ). 2. All animals survived treatment and no significant behavioural changes were observed. 3. Cd 2+ precipitates completely as CdCO 3 in the first 72 hr. Nevertheless, metal uptake in the organ was observed beyond this period. 4. After 96 hr an increase in the amount of sulphate mucins and a reduction in intensity of the cytochrome-oxidase reaction were observed. 5. Cadmium toxicity in very hard water is interpreted as being due to CdCO 3 ingestion which might cause histochemical alterations in the gills.

Immunohistochemical and immunochemical characterization of the distribution of leptin-like proteins in the gastroenteric tract of two teleosts (Dicentrarchus labrax and Carassius auratus L.) with different 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.

PACAP (pituitary adenylate cyclase-activating peptide)-like immunoreactivity in the gill arch of the goldfish, Carassius auratus: distribution and comparison with VIP.

Abstract 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.

Distribution of ghrelin peptide in the gastrointestinal tract of stomachless and stomach-containing teleosts.

The occurrence and localization of ghrelin peptide in the gastrointestinal tract of Carassius auratus and Dicentrarchus labrax, two fish species that exhibit different feeding behavior, different habitats, and different anatomical organizations of the gastroenteric tract, were examined by immunohistochemical methods and western blotting analysis. All of the gastrointestinal segments studied displayed immunohistochemical localizations of ghrelin peptide. Numerous single or clustered immunoreactive cells were found along the gastric folds, particularly in the pyloric region of Dicentrarcus labrax, whereas scattered ghrelin immunoreactive cells were observed in the intestinal epithelium of both fish species. Double immunolabeling PGP 9.5/ghrelin demonstrated the localization of ghrelin peptide also in nerve fibers and neuronal cells of the submucosal and myenteric plexuses, often in association with vascular structures. Western blotting analysis confirmed the presence of ghrelin peptide in the gatrointestinal tract of both species studied, whose molecular weight was similar to that of the corresponding mammalian prepro‐ghrelin. The findings could support the hypothesis that this peptide is an important appetite regulator in fish and could confirm the presence of the ghrelin peptide, starting from its precursor proteins, in the gastrointestinal tract of the goldfish and the sea bass. Microsc. Res. Tech. 2009.

Neurotrophin-like immunoreactivity in the gut of teleost species.

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.

Orexin and orexin receptor like peptides in the gastroenteric tract of Gallus domesticus: An immunohistochemical survey on presence and distribution.

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.

Ghrelin in the Gastroenteric Tract of Birds: Immunoreactivity Expression

S. Neglia*, N. Arcamone, V. Esposito and G. Gargiulo Department of Biological Structures, Functions and T echnology – Faculty of Veterinary Medicine, University of Naples ‘‘Federico II’’ – Italy *Correspondence: Dipartimento di Strutture, Funzioni e T ecnologie Biologiche – Facolta di Medicina Veterinaria, Universita di Napoli ‘‘Federico II’’, V ia Veterinaria, 1, I-80137 Napoli, Italy E-mail: sineglia@unina.it

The teleost kidney expresses Trk Neurotrophin Receptor-Like Proteins

Neurotrophins are involved in the development and maintenance of some neuronal populations, acting through tyrosine protein kinase (Trk) receptors, TrkA, TrkB and TrkC. In addition, recent studies have demonstrated the occurrence of Trk receptors in a wide variety of adult and embryonic non-neuronal tissues in vertebrates, including kidney. Nevertheless no data are available on Trk proteins distribution in teleost kidney. The present study, by using an immunoperoxidase technique, analyses Trk receptor protein distribution in the kidney of three species of freshwater and saltwater teleost. TrkA-like immunoreactivity was the only one detected in all examined species, while TrkB-like immunoreactivity was completely absent and TrkC-like immunoreactivity was detected only in goldfish. The TrkA immunoreactive cells were mainly localised in the collecting duct system, as this system was the only one to also display TrkC. Such data could provide new clues to Trk in fish and aid assessment of the role of Trk protein receptors during vertebrate evolution.

HISTOCHEMICAL STUDY OF THE EFFECTS OF CADMIUM UPTAKE ON OXIDATIVE ENZYMES OF INTERMEDIARY METABOLISM IN KIDNEY OF GOLDFISH (CARASSIUS AURATUS)

Abstract Uptake of cadmium and histochemical changes of oxidative enzymes—succinate dehydrogenase (E.C. 1.3.99.1), cytochrome oxidase (E.C. 1.9.3.1) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) —in the kidney of Carassius auratus were examined at different times after 10 ppm Cd2+ exposure in very alkaline tap water (346 mg/L HCO3−), which determines a fast and strong precipitation of cadmium as carbonate. A low accumulation of cadmium after 7 days of treatment followed by a gradual increase until the 40th day was observed. The oxidative enzyme activity that resulted was: very weak after 7 days, higher after 14 days and lower after 40 days of treatment. The variations in the enzyme activity along with the accumulation of the metal are interpreted as an increased energy requirement followed by an impairment of mitochondria.

Orexins and receptor OX2R in the gastroenteric apparatus of two teleostean species: Dicentrarchus labrax and Carassius auratus.

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.