Analysis of the Expression of Neurotrophins and Their Receptors in Adult Zebrafish Kidney.

Neurotrophins and their receptors are involved in the development and maintenance of neuronal populations. Different reports have shown that all neurotrophin/receptor pathways can also play a role in several non-neuronal tissues in vertebrates, including the kidney. These signaling pathways are involved in different events to ensure the correct functioning of the kidney, such as growth, differentiation, and regulation of renal tubule transport. Previous studies in some fish species have identified the neurotrophins and receptors in the kidney. In this study, for the first time, we compare the expression profiles (mRNA and protein) of all neurotrophin/receptor pathways in the kidney of the adult zebrafish. We quantify the levels of mRNA by using qPCR and identify the expression pattern of each neurotrophin/receptor pathway by in situ hybridization. Next, we detect the proteins using Western blotting and immunohistochemistry. Our results show that among all neurotrophins analyzed, NT-3/TrkC is the most expressed in the glomerule and tubule and in the hematopoietic cells, similar to what has been reported in the mammalian kidney.

Expression and immunohistochemical detection of leptin-like peptide in the gastrointestinal tract of the South American sea lion (Otaria flavescens) and the bottlenose dolphin (Tursiops truncatus).

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.

Neurotrophin Trk receptors in the brain of a teleost fish, Nothobranchius furzeri.

Trk neurotrophin receptors are transmembrane tyrosine kinase proteins known as TrkA, TrkB, and TrkC. TrkA is the high affinity receptor for nerve growth factor, TrkB is the one for both brain-derived neurotrophic factor and neurotrophin-4, and TrkC is the preferred receptor for neurotrophin-3. In the adult mammalian brain, neurotrophins are important regulators of neuronal function and plasticity. This study is based on Nothobranchius furzeri, a teleost fish that is becoming an ideal candidate as animal model for aging studies because its life expectancy in captivity is of just 3 months. In adult N. furzeri, all three investigated neurotrophin Trk receptors were immunohistochemically detected in each brain region. TrkA positive neuronal perikarya were localized in the dorsal and ventral areas of the telencephalon and in the cortical nucleus; TrkB immunoreactivity was observed in neuronal perikarya of the dorsal and ventral areas of the telencephalon, the diffuse inferior lobe of the hypothalamus, and Purkinje cells; TrkC positive neuronal perikarya were detected in the most aboral region of the telencephalon, in the magnocellular preoptic nucleus and in few neurons dispersed in the hypothalamus. Numerous positive fibers were widely distributed throughout the brain. Radial glial cells lining the mesencephalic and rhombencephalic ventricles showed immunoreactivity to all three Trks. These findings suggest an involvement of neurotrophins in many aspects of biology of adult N. furzeri.

GDNF family ligand RET receptor in the brain of adult zebrafish.

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.

Distribution of glial cell line-derived neurotrophic factor receptor alpha-1 in the brain of adult zebrafish.

Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for several types of neurons in the central and peripheral nervous systems. The biological activity of GDNF is mediated by a multicomponent receptor complex that includes a common transmembrane signaling component (the rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor) as well as a GDNF family receptor alpha (GFRalpha) subunit, a high-affinity glycosyl phosphatidylinositol (GPI)-linked binding element. Among the four known GFRalpha subunits, GFRalpha1 preferentially binds to GDNF. In zebrafish (Danio rerio) embryos, the expression of the GFRalpha1a and GFRalpha1b genes has been shown in primary motor neurons, the kidney, and the enteric nervous system. To examine the activity of GFRalpha in the adult brain of a lower vertebrate, we have investigated the localization of GFRalpha1a and GFRalpha1b mRNA and the GFRalpha1 protein in zebrafish. GFRalpha1a and GFRalpha1b transcripts were observed in brain extracts by reverse transcription-polymerase chain reaction. Whole-mount in-situ hybridization experiments revealed a wide distribution of GFRalpha1a and GFRalpha1b mRNAs in various regions of the adult zebrafish brain. These included the olfactory bulbs, dorsal and ventral telencephalic area (telencephalon), preoptic area, dorsal and ventral thalamus, posterior tuberculum and hypothalamus (diencephalon), optic tectum (mesencephalon), cerebellum, and medulla oblongata (rhombencephalon). Finally, expression patterns of the GFRalpha1 protein, detected immunohistochemically, correlated well with the mRNA expression and provided further insights into translational activity at the neuroanatomical level. In conclusion, the current study demonstrated that the presence of GFRalpha1 persists beyond the embryonic development of the zebrafish brain and, together with the GDNF ligand, is probably implicated in the brain physiology of an adult teleost fish.

Glial cell line-derived neurotrophic factor in Purkinje cells of adult zebrafish: an autocrine mode of action?

Glial cell line-derived neurotrophic factor (GDNF) has a neuroprotective role in Purkinje cells of cerebellum, promoting the survival and the differentiation of these cells. Its signalling is mediated by a receptorial complex GFRalpha1/RET. In the brain of adult zebrafish (Danio rerio) we previously investigated GDNF expression and localization, but no data exist regarding GFRalpha1 and RET presence. Thus, the present study was designed to clarify the morphological relation between GDNF and its receptorial complex GFRalpha1/RET immunoreactivity in the cerebellum of adult zebrafish. The expression of gdnf, GFRalpha1 and ret genes was demonstrated in adult zebrafish cerebellum by a standard RT-PCR. The distribution of GDNF and its receptorial complex GFRalpha1/RET was examined by single and double immunocytochemical stainings. In the valvula and corpus cerebelli GDNF, GFRalpha1 and RET immunoreactivity was seen co-localized in Purkinje cells, identified morphologically and by using an antiserum against a specific marker for these cells, aldolase C enzyme. In the vestibulolateralis lobe, Purkinje neurons were lacking in both the eminentiae granulares and medial caudal lobe. These results demonstrated the expression of the GDNF receptorial complex in adult zebrafish cerebellum and suggest an autocrine mode of action of GDNF in Purkinje cells.

Immunoreactivity to glial cell line-derived neurotrophic factor and its receptors in the trout pancreas: a further endocrine-exocrine relationship?

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting the survival of several neuronal populations in the central, peripheral and autonomous nervous system. Outside the nervous system, GDNF functions as a morphogen in kidney development and regulates spermatogonial differentiation. GDNF exerts its roles by binding to glial cell line-derived neurotrophic factor receptor (GFR) a1, which forms a heterotetramic complex with rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor. In this study we report the presence of GDNF-, RET- and GFRa1-like immunoreactivity in the pancreas of juvenile trout. GDNF immunoreactivity was observed in the islet cells, while GFRa1- and RET- immunoreactivity was observed in the exocrine portion. These findings suggest a paracrine role of GDNF towards exocrine cells showing GDNF receptors GFRa1 and RET. The relationship could reflect physiological interactions, as previously indicated in mammalian pancreas, and/or a trophic role by endocrine cells on exocrine parenchyma, which shows a conspicuous increase during animal growth.

Distribution of neurotrophin and TrK receptor-like immunoreactivity in the adrenal gland of birds.

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.