L. Maruccio

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.

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.

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.

Artemin-like immunoreactivity in the zebrafish, Danio rerio

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.

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.

Expression and immunohistochemical detection of Nesfatin-1 in the gastrointestinal tract of Casertana pig

In this study, we report nesfatin-1 immunoreactivity in the gastrointestinal tract of Casertana breed pig. The newly discovered anorexigenic peptide nesfatin-1 has been shown to possess physiological relevance in regulating food intake and energy homeostasis at a central level, although evidence has been accumulating that it may also play important functions at a more local gastroenteric level. Nesfatin-1 immunoreactive endocrine cells have been detected in the gastric fundus and ileocecal valve. Nesfatin-1 immunopositive neurons and nerve fibers have been observed mainly in the enteric plexuses. Western blot analysis confirmed the immunohistochemical observations, showing immunoreactive bands in all analyzed gastrointestinal tracts with the exception of the rectum. Nesfatin-1 immunodetection in the swine digestive system reinforces the importance of the role played by nesfatin-1 at the gastrointestinal level and sustains the necessity to study the role of this peptide in the regulation of food intake in farm species for which weight gain is essential for optimizing production.

Structural, Histochemical and Immunocytochemical Study of the Forestomach Mucosa in Domestic Ruminants

With 8 figures

Orexin 1 receptor in the seminiferous tubules of boar testis: an immunohistochemical study.

Orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R) are two G-protein-coupled receptors that bind their ligands, orexin A (OXA) and B (OXB), with different affinities. The male genital system represents an important target for OXA, which appears to play a role in the control of steroidogenesis and germ cell development in the testis. It is known that among domestic breeding animals, in the boar the number of Leydig cells is very high and OXA appears to have stimulatory activity on testosterone production. In this study, we aimed to evaluate the presence of OX₁R in the boar testis in order to extend our knowledge concerning the distribution and a potential functional role of the orexinergic system in the male reproductive tract of farm animals. The presence of OX₁R immunopositive cells in seminiferous tubules of the boar testis enables us to hypothesize a possible role of OXA on male germ cells cycle in pig. Further investigations, involving functional and ultrastructural analysis, may contribute to our understanding of the role of orexins in the boar genital system.