L. Castaldo

Localisation of neurotrophin – containing cells in higher vertebrate intestine

Neurotrophins are structurally related proteins that regulate the development, differentiation and maintenance of many neuronal populations. In higher vertebrates (reptiles, birds and mammals) four neurotrophins have been found: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3 and NT4/5. In the gut, experimental data and the occurrence of neurotrophin receptors in intestinal neurons and endocrine cells suggest neurotrophin involvement in intestinal physiology. However, very few data are available regarding the cellular localization and distribution of neurotrophins in the gut. In this study we report the presence of NGF, BDNF and NT3 in neurons and endocrine cells of mouse, duck and lizard intestine. In particular, immunoreactivity to NGF was observed: (a) in both endocrine and nerve cells of mouse and duck intestine, (b) in endocrine cells of lizard gut. Immunoreactivity to BDNF was seen: (a) in nerve cells of mouse intestine, (b) in very few endocrine cells of mouse and duck intestine. Immunoreactivity to NT3 was detected: (a) in nerve cells of the mouse intestine, (b) in endocrine and nerve cells of duck and lizard gut. Our results, together with data previously reported, on the distribution of specific neurotrophin receptors, seem to suggest a possible paracrine/autocrine mechanism of neurotrophin action in both the enteric nervous system and endocrine cells.

Trk-neurotrophin receptor-like immunoreactivity in the gut of teleost species.

Abstract Neurotrophins, acting through their high-affinity signal-transducing Trk receptors, are involved in the development, differentiation and maintenance of discrete neuron populations in the higher vertebrates. Furthermore, the presence of Trk receptors in some non-neuronal tissues, including the endocrine cells of the gut, could indicate an involvement of neurotrophins also in these tissues. Recently, neurotrophins and neurotrophin receptor proteins have been identified in the lower vertebrates and invertebrates, whose amino acid sequences are highly homologous with those found in mammals. The present study investigates the occurrence and distribution of Trk-like proteins in the neurons and gut endocrine cells in five species of teleost. Single and double immunolabeling was carried out on fresh and paraffin-embedded tissue using commercially available antibodies against sequences of the intracytoplasmic domain of the mammalian Trk. Western-blot analysis, carried out on samples of stomach and intestine of bass, identified proteins whose estimated molecular masses (140 kDa, 145 kDa and 143–145 kDa) were similar to those reported for full-length TrkA, TrkB and TrkC in the higher vertebrates. TrkA-like immunoreactivity was found in the enteric nervous system plexuses of three fish species. Trk-like immunoreactivity was observed in the endocrine cells as follows: sparse TrkA-like immunoreactive endocrine cells were detected only in the intestine; TrkB-like immunoreactive cells were detected only in the stomach; and TrkC-like immunoreactive cells were found both in the intestine of the carp and in the stomach of the bass, where they also showed TrkB-like immunoreactivity. These findings confirm the occurrence and distribution of Trk-like proteins in teleosts. These proteins are closely related to the Trk neurotrophin receptors of mammals. The functional significance of Trk-like proteins in both neuronal and non-neuronal cells of teleosts is still not clear.

Co-localization of Trk neurotrophin receptors and regulatory peptides in the endocrine cells of the teleostean stomach

Recently it has been observed that a subpopulation of gut endocrine cells in vertebrates express Trk‐like proteins, suggesting that neurotrophins could regulate the synthesis and storage of amines and peptides of these cells. Nevertheless, the peptides and amines present in the endocrine cells that express Trks have not been characterized. In this study we used immunohistochemistry to investigate the occurrence of Trk‐like proteins (TrkA‐like, TrkB‐like and TrkC‐like) and the possible co‐localization of these with peptides and/or biogenic amines in the endocrine cells of the stomach of three teleost (bass, gilt‐head and scorpionfish). No TrkA‐like immunoreactivity (IR) was detected in the stomach of these species, whereas TrkB‐like IR and TrkC‐like IR were observed in numerous cells of the gastric epithelium. TrkB‐like immunoreactive cells were present in all three species examined, and were particularly abundant in the blind sac. Conversely, TrkC‐like immunoreactive cells were found only in the bass stomach, apparently co‐localized with TrkB‐like IR. TrkB‐like IR was found co‐localized with somatostatin IR in scorpionfish, and with somatostatin and CGRP IR in gilt‐head and bass. Gastric endocrine cells expressing 5‐HT, glucagon, insulin, met‐, leu‐enkephalin, substance P, PYY, VIP, CCK, NPY, bombesin and motilin were unreactive for Trk‐like proteins. The present results provide direct evidence for the occurrence of Trk‐like neurotrophin receptor proteins in a subpopulation of the teleostean gastric endocrine cells and suggest that neurotrophins could regulate, as in neurons, the expression of some neuropeptides such as somatostatin and CGRP. Anat Rec 256:219–226, 1999.

Brain-derived neurotrophic factor: mRNA expression and protein distribution in the brain of the teleost Nothobranchius furzeri

BDNF (brain‐derived neurotrophic factor) is a member of the neurotrophin family and it is implicated in regulating brain development and function. The BDNF gene organization and coding sequence are conserved in all vertebrates. The present survey was conducted in a teleost fish, Nothobranchius furzeri, because it is an emerging model of aging studies due to its short lifespan and shows the high rate of adult neurogenesis typical of anamniotes. The present survey reports: 1) the identification and characterization of the cDNA fragment encoding BDNF protein, and 2) the localization of BDNF in the whole brain. BDNF mRNA expression was assessed by in situ hybridization, by employing an antisense RNA probe; BDNF protein was detected by employing a sensitive immunohistochemical technique, along with highly specific affinity‐purified antibodies to BDNF. Both BDNF mRNA and protein were detected in neurons and glial cells of all regions of the brain of N. furzeri. Interestingly, BDNF was localized also in brain areas involved in adult neurogenic activities, suggesting a specific role for this neurotrophic factor in controlling cell proliferation. These results provide baseline information for future studies concerning BDNF involvement in the aging processes of the teleost brain. J. Comp. Neurol. 522:1004–1030, 2014.

Brain-derived neurotrophic factor in higher vertebrate pancreas: immunolocalization in glucagon cells

Brain-derived neurotrophic factor (BDNF) is a growth factor that belongs to the group of neurotrophins. Its amino acid sequences are well conserved during vertebrate phylogenesis. Pancreatic tissue has recently been reported to be one of the physiological sources of BDNF in humans and mice. In this study we investigated the presence and localization of BDNF immunoreactivity (IR) in the pancreas of three species of higher vertebrates: mouse, duck and lizard. BDNF IR was present in the islets and in single cells scattered in the exocrine parenchyma of all three species examined. Using double staining, BDNF IR was seen to be colocalized with glucagon IR in all the species studied. There was a total overlap of BDNF and glucagon IR in duck and lizard pancreas, and partial overlap in mouse pancreas. Our findings suggest that, as well as the primary structure, the presence and pattern of distribution of BDNF in higher vertebrates is also well conserved. Moreover, the abundance of BDNF IR in the pancreas of the species studied leads us to the suggestion that these neurotrophins could regulate the function of pancreatic innervation and/or act on pancreatic cells in a paracrine/autocrine fashion.

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. Microsc. Res. Tech., 2012.

Neuronal and non-neuronal Trk neurotrophin receptor-like proteins in Eisenia foetida (Annelida Oligochaeta)

The occurrence and distribution of Trk proteins, which are the high-affinity signal-transducing receptors for neurotrophins, have been investigated in earthworms (Eisenia foetida) using polyclonal antibodies which map within their catalytic domain. Western-blot analysis identified major protein bands whose estimated molecular masses were consistent with those of the full-length Trk proteins in vertebrates. Specific immunoreactivity for TrkA-, TrkB-, and TrkC-like was observed in neuronal populations of the dorsal cerebral, subpharyngeal and ventral cord ganglia. Furthermore, TrkA-like immunoreactivity was observed in subcutaneous neurons and nerve fibers between muscle layers in the peripheral nervous system. TrkB- and TrkC-like immunoreactivity was observed in the gut innervation. Non-neuronal expression of TrkB and TrkC proteins was found in epidermal cells, and TrkC-like immunoreactivity was detected in the gut epithelium.

GDNF family ligand immunoreactivity in the gut of teleostean fish

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRα, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.

Postnatal development of intestinal endocrine cell populations in the water buffalo

The frequency and distribution of 11 endocrine cell populations were studied in the intestine of differently aged buffalo, grouped on the basis of diet: 2‐d‐olds (suckling), 5‐mo‐olds (weaning) and 5‐y‐olds (ruminant adult diet). The endocrine cell populations were identified immunocytochemically using antisera against 5‐hydroxytryptamine (5‐HT), somatostatin, gastrin, cholecystokinin (CCK), COOH‐terminal octapeptide of gastrin/CCK, neurotensin, motilin, gastric inhibitory polypeptide (GIP), secretin, glucagon/glicentin (GLU/GLI) and polypeptide YY (PYY). In adult buffalos the regional distribution of endocrine cells is similar to that of other adult ruminants. During postnatal development, these cell types showed the following changes in their frequency and distribution: (1) 5‐HT, neurotensin and gastrin/CCK immunoreactive cells (i.c.) showed a decrease in frequency with age; (2) somatostatin i.c. frequency remained stable with age; (3) motilin, GIP, secretin and CCK i.c. showed a slight increase in frequency with age; (4) GLU/GLI and PYY i.c. decreased in frequency with age in the small intestine, caecum and proximal colon and an increase in frequency in the rectum. It was hypothesised that the endocrine cell types, whose presence and localisation is substantially stable in all examined ages, probably contain substances that are strictly necessary for intestinal function. In contrast the hormones contained in the cell populations that decreased with age, are probably involved in physiological needs during the milk and weaning diet or play a role in intestinal growth.

Immunolocalization of S100-like protein in the brain of an emerging model organism: Nothobranchius furzeri.

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.