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The neuromast is the morphological unit of the lateral line of fishes and is composed of a cluster of central sensory cells (hair cells) surrounded by support and mantle cells. Heavy metals exposure leads to disruption of hair cells within the neuromast. It is well known that the zebrafish has the ability to regenerate the hair cells after damage caused by toxicants. The process of regeneration depends on proliferation, differentiation and cellular migration of sensory and non-sensory progenitor cells. Therefore, our study was made in order to identify which cellular types are involved in the complex process of regeneration during heavy metals exposure. For this purpose, adult zebrafish were exposed to various heavy metals (Arsenic, cadmium and zinc) for 72h. After acute (24h) exposure, immunohistochemical localization of S100 (a specific marker for hair cells) in the neuromasts highlighted the hair cells loss. The immunoreaction for Sox2 (a specific marker for stem cells), at the same time, was observed in the support and mantle cells, after exposure to arsenic and cadmium, while only in the support cells after exposure to zinc. After chronic (72h) exposure the hair cells were regenerated, showing an immunoreaction for S100 protein. At the same exposure time to the three metals, a Sox2 immunoreaction was expressed in support and mantle cells. Our results showed for the first time the regenerative capacity of hair cells, not only after, but also during exposure to heavy metals, demonstrated by the presence of different stem cells that can diversify in hair cells.

The hair cells of the lateral line system of fishes are morphologically and physiologically similar to the hair cells of the mammalian inner ear, also sharing its molecular characteristics. For this reason, it has been used as a powerful animal model to analyze in vivo ototoxicity. In this work, we examined the dose-dependent effects of two potent ototoxic aminoglycosides, neomycin and gentamicin, on the hair cells of two selected neuromasts (L1 and T1, the first of the trunk and the terminal located in the fin, respectively) of the lateral line in the ET4 transgenic zebrafish line. The hair cells of this strain selectively and constitutively display fluorescence. The fish were treated for 24 h at different doses (1, 2.5, 5, 10 and 100 μM levels) of both aminoglycosides. Immediately after treatment the morphology and the number of cells in L1 and T were analyzed under a fluorescence microscope. The results show that neomycin and gentamicin have different effects on the hair cell death at the same concentration, showing also different toxicity in L1 and T1 neuromasts. The toxicity observed in the hair cells of T1 neuromast was less than in L1 especially for the gentamicin treatment. These results demonstrate different sensitivity of hair cells of the lateral line to ototoxic drugs according to topographical localization and suggest the in vivo assay of the L1 neuromast of zebrafish larva and low doses of neomycin as an ideal model to study ototoxicity induced by aminoglycosides.

Obesity is a multifactorial disease generated by an alteration in balance between energy intake and expenditure, also dependent on genetic and non-genetic factors. Moreover, various nuclei of the hypothalamus receive and process peripheral stimuli from the gastrointestinal tract, controlling food intake and therefore energy balance. Among anorexigenic molecules, brain-derived neurotrophic factor (BDNF) acts through the tyrosine-kinase receptor TrkB. Numerous data demonstrate that the BDNF/TrkB system has a fundamental role in the control of food intake and body weight. Quantitative PCR and immunohistochemistry for both BDNF and TrkB were used to determine changes in levels in the brain and gastro-intestinal tract of an experimental zebrafish model of diet-induced obesity. Overfed animals showed increased weight and body mass index as well as accumulation of adipose tissue in the visceral, subcutaneous and hepatic areas. These changes were concomitant with decreased levels of BDNF mRNA in the gastro-intestinal tract and increased expression of TrkB mRNA in the brain. Overfeeding did not change the density of cells displaying immunoreactivity for BDNF or TrkB in the brain although both were significantly diminished in the gastro-intestinal tract. These results suggest an involvement of the BDNF/TrkB system in the regulation of food intake and energy balance in zebrafish, as in mammals.

The enigmatic rodlet cells (RCs) are characterized by conspicuous inclusions named “rodlets”. They were discovered over 100 years ago and were considered as parasites but shortly afterward interpreted as endogenous cells. The RCs have been described in different tissues of marine and freshwater teleosts, but their origin and function remain unknown. This work was designed to an ultrastructural study on RCs development and distribution in intestinal epithelium of Dicentrarchus labrax. Three different stages of RCs development from early precursor cells to mature phase were observed, as well as a migration and finally an extrusion of their contents. In this study, the immature cells were found near the basal epithelium membrane. They were mainly identified by a rough endoplasmic reticulum with dilated cisternae, by developing rodlets and a thin fibrillar coat. The maturing RCs, localized in the middle zone of the epithelium, appeared to be undergoing a reorganization of the cell organelles. The mature RCs, placed near the free surface, showed a thick subplasmalemmar fibrillar coat. Most of the organelles were aggregated at the cell apex with a basally located nucleus. A cellular polarity was more evident. One of the most conspicuous features was the occurrence of mature rodlets club-sac in shape orientated toward the cell apex. Adhesive junctions between surface epithelial cells and RCs, while discharging their contents, were seen. We have connected morphological figures and distribution to different stages of development in RCs, supporting the hypothesis of their secretory function. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.

The zebrafish pineal gland plays an important role in different physiological functions including the regulation of the circadian clock. In the fish pineal gland the pinealocytes are made up of different segments: outer segment, inner segment and basal pole. Particularly, in the outer segment the rhodopsin participates in the external environment light reception that represents the first biochemical step in the melatonin production. It is well known that the rhodopsin in the adult zebrafish is well expressed in the pineal gland but both the expression and the cellular localization of this protein during development remain still unclear. In this study using qRT-PCR, sequencing and immunohistochemistry the expression as well as the protein localization of the rhodopsin in the zebrafish from larval (10 dpf) to adult stage (90 dpf) were demonstrated. The rhodopsin mRNA expression presents a peak of expression at 10 dpf, a further reduction to 50 dpf before increasing again in the adult stage. Moreover, the cellular localization of the rhodopsin-like protein was always localized in the pinealocyte at all ages examined. Our results demonstrated the involvement of the rhodopsin in the zebrafish pineal gland physiology particularly in the light capture during the zebrafish lifespan.

Acid-sensing ion channels (ASICs) in mammals monitor acid sensing and mechanoreception. They have a widespread expression in the central and peripheral nervous system, including the gut. The distribution of ASICs in zebrafish is known only in larvae and at the mRNA level. Here we have investigated the expression and cell distribution of ASIC2 in the gut of adult zebrafish using PCR, Western blot and immunohistochemistry. ASIC2 mRNA was detected in the gut, and a protein consistent with predicted ASIC2 (64 kDa molecular mass) was detected by Western blot. ASIC2 positivity was found in a subpopulation of myenteric neurons in the enteric nervous system, as well in enteroendocrine epithelial cells. These data demonstrate for the first time the occurrence of ASIC2 in the gut of adult zebrafish where it presumably acts as a chemosensor and a mechanosensor.

The family of B1 Sox transcription factors plays critical roles in the early stages of development, including the central nervous system. It was demonstrated that Sox2 is expressed in repressed neural stem cells. Therefore, we decided to investigate the expression of Sox2 in the brain of zebrafish at different ages to identify potential neurogenic areas, and to establish the developmental changes they undergo. The brains were assessed by qRT-PCR, western blot, and immunohistochemistry. The maximal expression of Sox2 was found at 15 dpf progressively decreases up to 30 dpf, then increases up to 40 dpf and remains unchanged up to 180 dpf. By western blot three protein bands of 28 kDa, 34 kDa (main band), and 38 kDa were detected in the brain of 180 dpf animals. The immunolocalization of Sox2 revealed that by 15 dpf Sox2 was detected in cells of the olfactory bulb, the walls of the telencephalic and diencephalic ventricles, several nucleus in the diencephalons, and the tectum opticum; by 25-50 dpf the Sox2 positive areas were the same as above, and in the rhombencephalic ventricle and cerebellum. In adult animals Sox2 was restricted to the olfactory bulb and to cells of the telencephalic ventricle walls. Taken together present results demonstrate that the potential neurogenic areas in the brain of zebrafish are widespread than in mammals and change with development, but they are primarily concentrated around the ventricles and olfactory bulb in adults, following a similar localization as in mammals.

The Italian lizard (Podarcis sicula) is the most diffused reptile in Italy, but it is also present in other European countries. This lizard belongs to the Lacertidae family, lives near walls, slants and along the borders of the paths; its diet includes bugs and aracnids. No data are so far available in literature about the three-dimensional morphology of the tongue of Podarcis sicula, therefore the aim of the present paper was to study by scanning electron and light microscopy the three-dimensional characteristics of the dorsal lingual surface and moreover the presence of chemosensory receptors like the taste buds in the oral cavity. Our results demonstrate that the Podarcis sicula tongue is a triangular muscular membranous organ, dorsoventrally flattened and that three different areas can be observed: a bifid apex, a body and a root. No papillae were observed in the apex, characterized by a flattened mucosa and by two deep median pouches. In the body cylindrical papillae with a flat surface are present, aborally gradually substituted by imbricated papillae. Foliate-like papillae were observed in the lateral parts of the tongue body. No sensory structures were showed on the lingual dorsal surface, while they were numerous in the oral cavity, particularly on the gingival epithelium. The light microscopy shows, on the dorsal surface, a stratified pavimentous not keratinized epithelium, conversely keratinized along the ventral surface. Many caliciform cells on the lateral parts of the papillae, deputed to the secretion of mucus, were also observed. Therefore, the results obtained in this paper could give a contribution to the knowledge of the tongue anatomy in a species widely diffused in different European countries and could be of help for clinical purposes in reptiles.

Calretinin is a calcium-binding protein found widely distributed in the central nervous system and chemosensory cells of the teleosts, but its presence in the peripheral nervous system of fishes is unknown. In this study we used Western blot analysis and immunohistochemistry to investigate the occurrence and distribution of calretinin in the cranial nerve ganglia, dorsal root ganglia, sympathetic ganglia, and enteric nervous system of the adult zebrafish. By Western blotting a unique and specific protein band with an estimated molecular weight of around 30 kDa was detected, and it was identified as calretinin. Immunohistochemistry revealed that calretinin is selectively present in the cytoplasm of the neurons and never in the satellite glial cells. In both sensory and sympathetic ganglia the density of neurons that were immunolabelled, their size and morphology, as well as the intensity of immunostaining developed within the cytoplasm, were heterogeneous. In the enteric nervous system calretinin immunoreactivity was detected in a subset of enteric neurons as well as in a nerve fibre plexus localized inside the muscular layers. The present results demonstrate that in addition to the central nervous system, calretinin is also present in the peripheral nervous system of zebrafish, and contribute to completing the map of the distribution of this protein in the nervous system of teleosts.

The present study was carried out to analyse the vascularization of the pigeon bursa cloacalis of Fabricius and to determine whether it undergoes age-dependent changes during its functionally most important growth period after hatching of the pigeon. Morphological assessment of vascular corrosion casts, studied qualitatively and quantitatively, was applied for the first time to investigate the vascularization of the pigeon pigeon bursa of Fabricius. This also allowed us to analyse the microvasculature and morphological aspects of the vessel interrelationships as occurring in the natural state. The casts were compared with histological sections stained by haematoxylin-eosin and by binding of the lectin e-PHA (Phaseolus vulgaris, erythroagglutinin) to blood vessels. The vascular architecture of the bursa of Fabricius of the pigeon revealed that the organ is irrigated via two pathways, first through the terminal capillary system of lymphoid follicles arising from the internal pudendal artery, and secondly through arteries originating from the cloacal vasculature of the collum of the organ supplying the periluminal capillary system of the pigeon bursa of Fabricius. Both systems are drained by a venous system which is collateral to the system of the internal pudendal artery and clearly functions as a direct link between the lumen and vasculature of the cloaca or gut, respectively, and the bursa fabricii. This could allow the lymphocytes to be confronted with antigens from the contents of the gut, and their subsequent transport into the secondary lymphoid organs of the organism. Our results demonstrate that the blood vessels, as major and supplying part of the lymphoid system of the bursa Fabricii, clearly reflect three different phases of development: the evolution phase from about day 20 until day 50 post-hatching, the mature phase from days 50 to 90, and the involution phase after day 90. During the evolution phase the density of the vessel system rapidly increases, while in the mature phase the vascular architecture is maintained. The involution phase is dominated by vascular degeneration combined with shrinkage of the whole organ. Therefore, the morphology of the vasculature distinctly reflects the functional status of this primary lymphoid organ during its lifespan.

Expression and distribution of leptin and ghrelin in the digestive ap-paratus of DIO (diet-induced obesity) zebrafish Maruccio L, Russo F, Arcamone N, Mania M, Randazzo B, Ciriaco E In this report we analyzed the expression and distribution of leptin and ghrelin in the digestive apparatus of DIO zebrafish. Leptin is anorexigenic peptide while ghrelin is orexigenic one. They act as antagonists. In this study in order to develop an obesity phenotype adult zebrafish were divided into two dietary groups. The con-trol group was fed with Artemia (5 mg cysts/fish/day) once per day. The overfeeding group was fed three times per day with Artemia (60 mg cysts/fish/day). For calorie restriction, the zebrafish was fed with Artemia (2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. At the end of this period, after 24h of fasting, the zebrafish digestive apparatus were sampled for immunohistochemistry, western blotting and qRT-PCR techniques. Leptin and ghrelin immunoreactivity were found in the enteric nervous system and neuroen-docrine cells in overfeeding and control zebrafish intestine. The number of immunopositive cells is greater in DIO zebrafish than in control ones. In both groups leptin immunoreactive nervous fibers were found around ducts and vessels of liver. The presence of proteins were confirmed by western blotting analysis. By qRT-PCR, leptin and ghrelin mRNA levels are higher in the overfeeding zebrafish intestine and in control zebrafish liver. The immunological detection of ghrelin and leptin in control zebrafish are in agreement with literature data. In DIO zebrafish the results are similar to those found for obese mammals. In conclusion, this study represent a starting point to investigate the mechanisms involved in the regulation of appetite and energy balance in DIO zebrafish.

In the present study, the tongue dorsal surface of the white sea bream Diplodus sargus sargus (Linnaeus, 1758) (teleosts, Sparidae) was studied, to give a support to the knowledge of the anatomical characteristics of the oral cavity of a species with a future potential use in aquaculture. The feeding habits in captivity are one of the critical steps in the species of commercial interest, strictly related to the morphological characteristics of the oropharyngeal cavity. This study was undertaken, using light and scanning electron microscopy, to analyse the morphology of the dorsal tongue surface to show whether relationships are present between the anatomy and the nutritional habits and choices of this farmed species. Three zones, an apex, a body and a root, can be clearly distinguished, with a pouch partially covering the apex. In the pouch, cylindroid and mucosal ridges were observed. Along the whole tongue surface, from the apex to the root, on the medial and lateral parts, many papillae are present, with a fungiform, cylindroid and cone-like aspect. Taste buds are showed on the dorsal surface of some papillae. © 2014 The Royal Swedish Academy of Sciences.

In addition to their well-known roles within the nervous system, the neurotrophins and their receptors regulate some functions in the reproductive system. In this study we used combined morphological and immunohistochemical techniques to investigate the presence and cellular localization in the rat testicle of the two receptors of nerve growth factor (NGF), i.e. TrkA and p75(NTR). Furthermore, to evaluate whether increased plasma levels of NGF affect the ageing process, 4-methylcathechol (4-MC), an inductor of NGF synthesis, was administered. Both TrkA and p75(NTR) were expressed in rat testicles, but the pattern and intensity of immunoreaction were marginally different between them. In adult rats TrkA was expressed in spermatozoa and spermatids, and p75 was expressed in spermatogonia. In newborn rats TrkA immunoreactivity was found in the Leydig cells, whereas p75 was detected in a cellular layer that surrounds the seminiferous tubules. In adult treated animals the immunoreaction for TrkA and p75(NTR) was also localized in the spermatocytes, whereas in newborn treated rats no changes in the pattern of immunoreaction was observed. The present findings suggest a role of the NGF/TrkA/p75 system in the physiology of reproduction, but the practical relevance of this remains to be established.

Thymocytes and thymic stromal cells cross-talk in a bidirectional manner within the thymus, thus contributing to the generation of mature T-cells. The thymic stromal cells in the rat express the high- (TrkA, TrkB) and low-affinity (p75NTR) receptors for neurotrophins. In this study we analysed the regulation of TrkA, TrkB and p75NTR expression in the rat thymus by thymocytes. We induced thymocyte apoptosis by administration of corticoids in rats, and then analysed the expression and distribution of these receptors 1, 4 and 10 days later. Thymocyte death was assessed by the activation of caspase-3 in cells undergoing apoptosis. We observed massive thymocyte apoptosis 1 day after injection and, to a lesser extent, after 4 days, which was parallel with a reduction in the density of thymic epithelial cells normally expressing TrkA and p75NTR. Furthermore, TrkA expression was found in cortical thymic epithelial cells, which normally lack this receptor. The expression of TrkB was restricted to a subset of macrophage-dendritic cells, and remained unchanged with treatment. The normal pattern of neurotrophin receptor expression was almost completely restored by day 10. The results demonstrate that the expression of neurotrophin receptors by thymic epithelial cells, but not by macrophage-dendritic cells, is regulated by thymocytes.

Whereas it is nowadays clear that neurotrophins are involved in the regulation of various aspects of the functioning of immune system, knowledge of their actual immunomodulatory roles is still fragmentary and incomplete. In this respect, knock-out mouse models remain particularly unexplored. In the present study, the expression of the TrkB neurotrophin receptor in murine spleen was addressed at the mRNA (reverse transcription/polymerase chain reaction) and protein (Western blot) levels. Once the presence of TrkB at both levels was demonstrated, the age-dependent changes in the pattern of expression of the receptor were analyzed and quantified, and TrkB-positive cells were identified by immunohistochemistry. TrkB-immunoreactive cells, identified as red pulp macrophages, were detected in the spleen throughout postnatal development and adult life; their numbers peaked at the age of 15 days. The absence of functional TrkB did not appear to result in morphological changes as assessed by light and electron microscopy of spleens from 15-day-old mice knockout for the trkB gene. The present results support the idea that, in the murine spleen, TrkB and its ligands are involved in macrophage physiology in a developmentally regulated fashion, but they do not seem to be essential for macrophage survival.

The avian primary lymphoid organs, the thymus and the bursa of Fabricius, undergo age-dependent changes leading in some cases to the complete atrophy of the organ. Nevertheless, the timetable of the involutive process as well as the consequences in the structure and functionality of the organs vary largely in the time frame and structural changes among species. On the other hand, and in contrast with the large body of literature reporting the structural and functional changes in mammalian primary lymphoid organs, the age-dependent changes in avian thymus and bursa of Fabricius are scarce, fragmentary, and heterogeneous. This article reviews the current literature on this topic, and focuses primarily on the involution of the bursa of Fabricius.

The olfactory neurons of vertebrates, including teleosts, regenerate through adult life. This ability seems to depend partially on neurotrophic factors, which include some neurotrophins, mainly nerve growth factor (NGF), and their cognate Trk receptors. Zebrafish is an ideal model for studying neurogenesis in the olfactory system, but what kinds of olfactory sensory neurons (ciliated, microvillous or crypt neurons) are under the control of neurotrophins during adult life is unknown. Here we examined the expression of Trk proteins in the olfactory epithelium of adult zebrafish using immunohistochemistry. TrkA-like immunoreactivity was the only Trk detected, and it was restricted to the somata of crypt sensory neurons, their central processes being apparently unreactive. These results provide indirect evidence for a possible role of NGF-like molecules (i.e. neurotrophin-6 and neurotrophin-7) in the peripheral olfactory system of adult zebrafish, acting on TrkA-like receptor expressing crypt neurons. It remains to be established whether or not TrkA-like receptors and their ligands play a function in the neurogenesis or differentiation of these cells.

The occurrence of TrkB in the murine thymus (15-day and 3-month old) was investigated by Northern blot, Western blot and immunohistochemistry. Furthermore, the thymus of 15-day-old mice carrying a non-functional mutation on trkB was analyzed. Both trkB mRNA and 145 kDa TrkB protein were detected. In addition, isolated lymphocytes and stromal cells also expressed this protein. The thymus of homozygous functionally TrkB-deficient animals showed structural and ultrastructural changes consistent with massive death of cortical lymphocytes, confirmed with TUNEL. Present results suggest a role for TrkB in maintaining the survival or preventing massive death of lymphocytes in the mammalian thymus.

The lateral line system, formed of both superficial (pit organs) and canal neuromasts, is one of the major mechanosensory systems in fish. It has always been assumed that this system depends on neurotrophins and their cognate Trk receptors for development and maintenance, as has been shown in other mechanosensitive systems of vertebrates. However, until now this issue has not been specifically addressed. In this study we used immunohistochemistry to investigate the occurrence and localization both of neurotrophins (NGF-, BDNF- and NT-3-like) and of Trk-like proteins (TrkA-, TrkB-, TrkC-like) in alevins of Salmo salar and S. trutta. All cells in the pit organs of S. salar displayed strong immunoreactivity for TrkB-like and BDNF-like, whereas they were restricted to the hair cells in S. trutta. The hair, supporting and mantle cells of S. salar, and the mantle cells of S. trutta, also expressed TrkA-like immunoreactivity. In the canal neuromasts BDNF-, TrkA- and TrkB-like proteins were present in all cells, without differences between species. NGF-, NT-3- and TrkC-like immunoreactivity were never detected. The present results suggest that mechanoreceptive hair cells, as well as supporting cells, in the lateral line system are under the control of the BDNF–TrkB-like complex, and probably of ligands of TrkA-like receptors.

Neurotrophins are a family of growth factor primarily acting in the nervous system, throughout two categories of membrane receptors on the basis of their high (Trk receptors) or low (p75NTR) affinity. Both neurotrophins and Trk receptors are phylogenetically conserved and are expressed not only in the central and peripheral nervous system but also in non-nervous tissues of vertebrates and some invertebrates. The brain-derived neurotrophic factor (BDNF)/TrkB system plays an important role in the development, phenotypic maintenance and plasticity of specific neuronal populations. Considering that this system is poorly characterized in the central nervous system of teleosts, the expression and anatomical distribution of TrkB in the brain of the adult zebrafish using reverse transcriptase-polymerase chain reaction (RT-PCR), Western-blot and immunohistochemistry were analysed. Both the riboprobe and the antibody used were designed to map within the catalytic domain of TrkB. RT-PCR detected specific TrkB mRNA in brain homogenates, while Western-blot identified one unique protein band with an estimated molecular weight of 145 kDa, thus corresponding with the TrkB full-length isiform of the receptor. Immunohistochemistry showed specific TrkB immunoreactivity in restricted areas of the encephalon, i.e. the hypothalamus and a specific neuronal subpopulation of the reticular formation. The present results demonstrate, for the first time, that, as in mammals, the encephalon of adult zebrafish expresses TrkB in specific zones related to food intake, behaviour or motor activity

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease.

Nerve growth factor (NGF) and its signal-transducing receptor TrkA are expressed in the thymus. However, their possible role during thymic organogenesis is unknown. Here we analyze the thymus of trk A-kinase deficient 2-week-old mice. trk A-kinase +/+ and +/− mice had a normal thymus, whereas the thymus of trk A-kinase −/− mice showed lack of delimitation between the cortex and medulla, lower thymocyte density, and the presence of epithelial cell islands and numerous cysts lined with endodermal epithelium. The present results indicate that TrkA is necessary for the normal development of the thymus, and that its absence causes an arrest in the differentiation of endodermal epithelial cells. Whether this lack of differentiation has functional implication has yet to be determined.

Increasing evidence suggests that, in addition to peripheral sensory and sympathetic neurons, the enteric neurons are also under the control of neurotrophins. Recently, neurotrophin receptors have been detected in the developing and adult mammalian enteric nervous system (ENS). Nevertheless, it remains to be established whether neurotrophin receptors are expressed in all enteric neurons and/or in glial cells and whether expression is a common feature in the enteric nervous system of all mammals or if interspecific differences exist. Rabbit polyclonal antibodies against Trk proteins (regarded as essential constituents of the high-affinity signal-transducing neurotrophin receptors) and p75 protein (considered as a low-affinity pan-neurotrophin receptor) were used to investigate the cell localization of these proteins in the ENS of adult man, horse, cow, sheep, pig, rabbit, and rat. Moreover, the percentage of neurons displaying immunoreactivity (IR) for each neurotrophin receptor protein was determined. TrkA-like IR and TrkC-like IR were observed in a neuronal subpopulation in both the myenteric and submucous plexuses, from esophagus to rectum in humans, and in the jejunum-ileum of the other species. Many neurons, and apparently all glial cells, in the human and rat enteric nervous system also displayed p75 IR. TrkB-like IR was found restricted to the glial cells of all species studied, with the exception of humans, in whom IR was mainly in glial cells and a small percentage of enteric neurons (about 5%). These findings indicate that the ENS of adult mammals express neuronal TrkA and TrkC, glial TrkB, and neuronal-glial p75, this pattern of distribution being similar in all examined species. Thus, influence of specific neurotrophins on their cognate receptors may be considered in the physiology and/or pathology of the adult ENS.

Neurotrophins are growth factors which bind to signal-transducing receptors called Trk proteins. The neurotrophins and their receptor proteins are present in the mammalian and avian lymphoid organs, thus suggesting that these factors could act upon cells of the immune system. Nevertheless, little is known about the cellular distribution of neurotrophins and their receptor proteins in avian lymphoid tissues. In this study we use immunohistochemistry to detect the cellular localisation of neurotrophins and their receptor proteins in the pigeon caecal tonsil, used as a model for avian secondary lymphoid organs. Rabbit polyclonal antibodies against neurotrophins (nerve growth factor -NGF-, brain-derived neurotrophic factor -BDNF- and neurotrophin -3 NT-3-) and against specific epitopes of TrkA, TrkB and TrkC proteins were used. Cytokeratins, vimentin, S-100 protein and chromogranin A were studied in parallel to identify cells which seemed to express neurotrophins and Trk proteins. TrkA-like protein was seen in the intestinal epithelium, whereas TrkB-like and TrkC-like proteins was found in cells which we identified as dendritic cells and macrophages. BDNF-like and NT-3-like reactivity was localised in intestinal epithelial cells, especially endocrine cells. Present results add further evidence to the presumptive immune role of neurotrophins and their receptors and the possible functions of these peptides in the caecal tonsil are discussed.

BACKGROUND The neurotrophins are a family of growth factors that act on responsive cells through specific high-affinity signal-transducing receptors called Trk (A, B, and C) proteins. The neurotrophin receptor proteins are widely distributed in both nervous and nonnervous tissues, including the lymphoid organs. The expression of these receptor proteins by a cell population is an indication of responsiveness to the respective binding neurotrophin. The present study investigated the presence and cellular localization of high-affinity neurotrophin receptor proteins in equine and bovine Peyer's patches. METHODS Peyer's patches from horse and cow intestine were fixed in Bouin's fixative, embedded in paraffin cut 10 microns thick, and studied immunohistochemically using rabbit polyclonal antibodies against specific epitopes of the intracellular domain of the Trk receptor proteins. RESULTS Immunoreactivity (IR) for Trk-like proteins was found in specific cell populations in Peyer's patches. TrkA-IR in the horse was localized in dendritic cells of the interfollicular T-cell zones and in follicular dendritic cells of the lymphoid follicles; in the cow, TrkA-ir was present in reticulum cells. TrkB-like IR was present in cells found inside lymphoid follicles of the horse, probably reticulum cells. Furthermore, in both species, TrkB-IR was found in interstitial dendritic cells and/or macrophages of the intestinal lamina propria. No specific TrkC-like immunostaining was found in immunocompetent cells of Peyer's patches. CONCLUSIONS Present findings demonstrate that, as in other lymphoid organs, the accessory nonlymphoid cells express immunoreactivity for high-affinity neurotrophin receptor proteins. These results seem to favor the notion that neurotrophins, especially nerve growth factor, could have a physiological role in secondary lymphoid organs, possibly acting on accessory cells and not directly on lymphocytes.

Neurotrophins are growth factors acting on responsive cells through membrane receptors identified as Trk tyrosine kinase proteins (A, B and C). Trks are present in the mammalian lymphoid organs, and indirect evidence suggests that they are also present in the avian bursa of Fabricius. This study was designed to analyze (a) the occurrence and localization of Trk proteins in the bursa of Fabricius; and (b) whether the post-hatching growth of the organ (from hatching to 75 days) involves cells expressing Trk proteins. We used pigeon bursae of Fabricius, and rabbit polyclonal antibodies against specific epitopes of TrkA, TrkB and TrkC. Cytokeratins and vimentin were studied in parallel with label non-lymphoid cells of the bursal follicles. Immunoreactivity (IR) for all assessed antigens was found in specific non-lymphoid cells. From hatching to 15 days, TrkB-like IR was found outside the follicles in cells localized beneath the follicle associated (FAE) and interfollicular (IFE) epithelium. Between 30–75 days TrkB-like IR labelled the medullary secretory dendritic cells (SDC). The density of SDC displaying IR increased up to 60 days. TrkA-like and TrkC-like IR was primarily observed in FAE and IFE, but also in the medullary reticular epithelial cells (REC) up to 15 days. The present results provide evidence of the occurrence, localization and post-hatching changes in Trk proteins in avian bursa of Fabricius. Trks were localized on non-lymphoid cells which participate in providing the adequate microenvironment for B lymphocyte maturation. Furthermore, the cell segregation in the expression of Trks suggests specific roles for their ligands in controlling the function of medullary SDC and REC, hence bursal lymphoid follicle physiology.

Background The medulla of the avian bursal lymphoid follicles contains heterogeneous cell populations, including the so-called medullary reticular epithelial cells (REC). These cells may contribute to the bursal microenvironment for B-lymphocyte differentiation and maturation. The bursa of Fabricius undergoes well-characterized posthatching developmental changes, but the age-related changes of the medullary REC have not been studied. The present study approaches this topic by analyzing hallmarks of epithelial cells: the occurrence of cytokeratin-type intermediate filaments and of desmosomes and desmoplakins in pigeon medullary REC. Methods The bursae of Fabricius of male king pigeons (Columba livia L.) Morini's strain were examined at different ages (from hatching to 120 days after hatching) by light microscopic immunohistochemistry for pan-cytokeratins and desmoplakins and by transmission electron microscopy. The area occupied by medullary cytokeratin-immunoreactive cells was evaluated with quantitative image analysis. Results At hatching, cytokeratin immunoreactivity was not detected in the bursal lymphoid follicles. During the posthatching growth period of the organ (7–75 days), there was a progressive and significant increase in the area occupied by cytokeratin-immunoreactive medullary REC, in the intermediate filaments filling the cytoplasm of REC, and in the number of desmosomes. Conversely, during the regressive period analyzed (90–120 days), the density of cytokeratin-positive cells progressively decreased, although they retained their ultrastructural characteristics. The evaluation of desmoplakin immunoreactivity paralleled that of cytokeratin. Conclusion The present results demonstrate that the medullary REC of the pigeon bursa of Fabricius undergoes age-dependent changes parallel with that involving the whole organ. The possible contribution of medullary REC to the bursal microenvironment is discussed.

The gilthead seabream is a food fish, one of the most frequently used in aquaculture. In the species of commercial interest, feeding in captivity is very important and this process is strictly related to the morphological characteristics of the oral cavity. The aim of this study is, using the standard procedures for light and scanning electron microscopy, to analyze the morphology of the tongue dorsal surface to show if relationships are present between the tongue morphology and the nutritional habits and choices of this farmed species. The main characteristic of the gilthead seabream oral cavity floor is the presence of an apical pouch, with, probably, a protective role mainly for the apical, free part of the tongue. Three zones, like in other teleosts, an apex, a body and a root, can be clearly distinguished. In the pouch foliate-like papillae were observed, while the whole tongue is characterized by the presence of two types of papillae, respectively with a fungiform and cylindroid aspect, both randomly distributed throughout the whole dorsal surface of the tongue. Scattered and numerous taste buds, with the typical pear-onion shape, together with small and numerous taste pores are also present, distributed throughout the tongue surface. Our results demonstrate that in the gilthead seabream important mechanic and sensory roles are carried out by specific anatomical structures. Our anatomical data could give, together with further biochemical and physiological data, an important support with the aim of improving the nutrition of aquaculture species.

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions.

The European sea bass, a member of the Moronidae family, is a food fish, considered one of the first models for the intensive breeding in salt water. It has nowadays an important and increasing presence in the international fishing markets. Sea basses are carnivorous, feeding on little fishes and invertebrates. Considering the important role of the tongue during the intraoral transport and the swallowing of food, scarce data are present in literature about its morphology. The aim of this study was to analyze the morphology of the tongue by means of scanning electron and light microscopy. Adult sea basses were obtained from the aquarium of the Sicilian Center of Experimental Ichthyiopathology of the University of Messina. The fishes were anaesthetized with MS 222 and the heads were then quickly removed and processed for the paraffin embedding and SEM processing. Three different tongue regions could be distinguished: an apex, a body, and a root. Scanning electron and light microscopy showed the presence of numerous canine-like teeth, surrounded by taste buds and numerous fungiform and conical papillae. The teeth were curved and their tips were posteriorly oriented. The results confirm, in teleosts too, the fundamental role of the tongue in the mechanics of food ingestion. Moreover, the presence of taste buds demonstrates the interaction of food processing and taste. These data could be a potential source to identify new and better methods of nutrition in the breeding of this fish.

The aquaporins (AQPs) are a family (AQP-AQP10) of transmembrane channel proteins that mediate the transport of water, ions, gases, and small molecules across the cell membrane, thus regulating cell homeostasis. AQP4 has the highest water permeability and it is involved in hearing and vision in mammals. Here, we used immunohistochemistry to map the presence of AQP4 in the sensory organs of adult zebrafish. The antibody used detected by Western blot proteins of 34 kDa (equivalent to that of mammalian AQP4) and maps in the sensory cells of taste buds, the hair sensory cells of the neuromast and of the maculae, and cristae ampullaris of the inner ear. Moreover, the retinal photoreceptors display AQP4 immunoreactivity. The non-sensory cells in these organs were AQP4 negative. These results suggest that the AQP4 could play a role in the regulation of water balance and ion transport in the sensory cells of zebrafish, bringing new data for the utilizing of this experimental model in the biology of sensory system.

We have recently demonstrated that monolateral lesions of the Nucleus Basalis Magnocellularis (NBM), which is a nucleus sending cholinergic projections to the fronto-parietal cortex, cause a loss in the intensity of Timm staining in the intrahippocampal pathway of mossy fibres (MF). Moreover, these lesions induce ultrastructural changes consistent with the occurrence of degeneration of presynaptic buttons of MF. The present study was designed to quantify the effects of NBM lesioning on the morphology of the presynaptic buttons of MF. Moreover the effects of 4-week choline alfoscerate (alphaGFC) treatment on the density of Timm staining and on the ultrastructure of presynaptic buttons of MF were assessed, alphaGFC, which was given at an oral daily dose of 100 mg/kg, is a precursor in the biosynthesis of several brain phospholipids which increases the availability of choline in the nervous tissue. Monolateral lesions of NBM cause, 4 weeks after lesioning, a significant decrease in the intensity of Timm staining in the MF area accompanied by a loss of about 23% of presynaptic buttons of MF. Moreover about 40% of presynaptic buttons of MF show an impaired morphology. alphaGFC administration restored the intensity of Timm staining in the MF area. In alphaGFC-treated rats, the loss of presynaptic buttons and the number of impaired buttons were reduced to about 12% and 27%, respectively in comparison with non-treated animals. These results confirm and extend our previous observations indicative of the occurrence of transneuronal degenerations in the MF of the hippocampus after monolateral NBM lesioning. Moreover these findings show that alphaGFC treatment is able to counter in part these degenerative changes.

The neuromasts of the lateral line system are regarded as a model to study the mechanisms of hearing, deafness, and ototoxicity. The neurotrophins (NTs), especially brain-derived neurotrophic factor (BDNF), and its signaling receptor TrkB are involved in the development and maintenance of neuromasts. To know the period in which the BDNF/TrkB complex has more effects in the neuromast biology, the age-related changes were studied. Normal zebrafish from 10 to 180 days post-fertilization (dpf), as well as transgenic ET4 zebrafish 10 and 20 dpf, was analyzed using qRT-PCR, western blot, and immunohistochemistry. BDNF and TrkB mRNAs followed a parallel course, peaking at 20 dpf, and thereafter progressively decreased. Specific immunoreactivity for BDNF and TrkB was found co-localized in all hairy cells of neuromasts in 20 and 30 dpf; then, the number of immunoreactive cells decreased, and by 180 dpf BDNF remains restricted to a subpopulation of hairy cells, and TrkB to a few number of sensory and non-sensory cells. At all ages examined, TrkB immunoreactivity was detected in sensory ganglia innervating the neuromasts. The present results demonstrate that there is a parallel time-related decline in the expression of BDNF and TrkB in zebrafish. Also, the patterns of cell expression suggest that autocrine/paracrine mechanisms for this NT system might occur within the neuromasts. Because TrkB in lateral line ganglia did not vary with age, their neurons are potentially capable to respond to BDNF during the entire lifespan of zebrafish.

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS. © 2009 Elsevier Ireland Ltd. All rights reserved.

The Sox-2 is a transcription factor involved in adult neurogenesis in different vertebrate species, including fishes. Sox-2 also participates in growth and renewal on sensory cells in neuromasts of the fish lateral line system, and it is essential for development of taste buds in mammals. Using immunohistochemistry and Western blot we have investigated the occurrence and localization of Sox-2 taste buds and neuromast of zebrafish from 10 days post-fertilization to adult stage (1 year). The antibody used identifies two protein bands with estimated molecular weights of 34 and 37kDa which are consistent with those predicted for Sox-2. Sensory cells in taste buds displayed Sox-2 immunoreactivity at all the ages sampled, whereas in the neuromasts Sox-2 expression was restricted to the basal non-sensory cells. Interestingly Sox-2 immunoreactivity was observed in epithelial cells associated with both taste buds and neuromasts. Present results demonstrate that Sox-2 expressed in taste buds and neuromasts of zebrafish during the whole lifespan. Nevertheless, whereas the role of Sox-2 in taste buds of zebrafish remains to be established, the results in neuromast suggest that Sox-2 could participate in cell renewal of the mechanosensory cells.

Summary During the last few years, green iguanas (Iguana iguana) have turned out to be one of the most popular pets. They are omnivorous. In their way of feeding, this crucial function is performed by capturing of the preys and mostly, this is carried out by the tongue. The role of the tongue is also fundamental during the intra-oral transport and during the swallowing of food. This has been reported in several studies about chameleons, agamids and iguanids, nevertheless published data about the mechanisms of capturing and swallowing the prey, and the morphological descriptions about the tongue epithelium, are scarce. Therefore, the aim of this present study was to analyse the morphology of the lingual epithelium in green iguanas by scanning electron microscopy. Three different areas were demonstrated on the tongue surface: the tongue tip, characterized by a smooth epithelium without papillae, a foretongue, completely covered by numerous closely packed cylindriform papillae, and a hindtongue with conical-like papillae. Some taste buds were recognized on the middle and the posterior parts of the tongue. Different functional roles could be hypothesized for the three tongue areas: the tongue tip could have a role related to the movements of the prey immediately after the capturing, while the middle papillae and the hindtongue could have an important role concerning the swallowing phase.

S100 proteins are EF-hand calcium-binding protein highly preserved during evolution present in both neuronal and non-neuronal tissues of the higher vertebrates. Data about the expression of S100 protein in fishes are scarce, and no data are available on zebrafish, a common model used in biology to study development but also human diseases. In this study, we have investigated the expression of S100 protein in the central nervous system of adult zebrafish using PCR, Western blot, and immunohistochemistry. The central nervous system of the adult zebrafish express S100 protein mRNA, and contain a protein of approximately 10 kDa identified as S100 protein. S100 protein immunoreactivity was detected widespread distributed in the central nervous system, labeling the cytoplasm of both neuronal and non-neuronal cells. In fact, S100 protein immunoreactivity was primarily found in glial and ependymal cells, whereas the only neurons displaying S100 immunoreactivity were the Purkinje's neurons of the cerebellar cortex and those forming the deep cerebellar nuclei. Outside the central nervous system, S100 protein immunoreactivity was observed in a subpopulation of sensory and sympathetic neurons, and it was absent from the enteric nervous system. The functional role of S100 protein in both neurons and non-neuronal cells of the zebrafish central nervous system remains to be elucidated, but present results might serve as baseline for future experimental studies using this teleost as a model.

Neurotrophins acting through Trk signal-transducing receptors play essential roles in the nervous system, and probably in some non-neuronal tissues. In the present study, we used RT-PCR, Western-blot and immunohistochemistry to investigate the occurrence and cellular localization of TrkB in the mouse liver, from newborns to 6 months. Furthermore, the structure of the liver in mice carrying a mutation in the trkB gene, resulting in a non-functional protein, was studied. The analysis of the DNA sequence showed that hepatic trkB gene is identical to the cerebral one, and TrkB mRNA and TrkB full-length protein (145 kDa) were detected at all the ages sampled. Immunohistochemistry revealed age-dependent changes in the pattern of TrkB expression. From 0 to 15 days, the TrkB was detected in morphologically and immunohistochemically identified monocyte-macrophage-dendric cells scattered throughout the organ, while in animals 3- and 6-months-old it was restricted to nerve fibres. Interestingly, there was a parallelism between TrkB expression by monocyte-macrophage-dendric cells and the presence of hepatic erythroblastic islands. In agreement with a possible role of TrkB on hepatic haematopoiesis, the liver of 15 days old TrkB (-/-) mice still contained erythroblastic islands, whereas they were absent in the wild-type littermates. Another striking finding was the absence of nerve profiles in the TrkB (-/-) animals. All together, present results support the role of TrkB in the murine liver in maintaining the innervation of the organ, and more importantly throughout an unknown mechanism in controlling the hepatic haematopoietic function.

The mechano and chemosensory organs of adult teleosts undergoes a continuous cell renewal and turnover which is regulated in part by growth factors. Here, we investigated the occurrence and the cell localization of epidermal growth factor (EGF) in the lateral line system and taste bud of adult zebrafish, using Western blot and immunohistochemistry associated to a polyclonal antibody against mammalian EGF. Furthermore, the distribution of S100 protein was studied in parallel to label hair sensory cells in the lateral line system. Western blot revealed one unique protein band with an estimated molecular weight of about 13 kDa, equivalent to the EGF of mammals. Specific immunoreactivity for EGF was observed in the epithelial basal and/or supporting cells of the neuromasts of the lateral line system and taste buds. Conversely, the sensory cells in both sensory structures were devoid of immunostaining. Present results demonstrate the occurrence of EGF in mechano and sensory system of adult zebrafish, suggesting a role for this molecule in the cell renewal and turnover of these structures.

The oral cavity in vertebrates plays a very important role in food intake mechanics and also contains chemical sensors for food and environmental water. Comparative studies revealed morphological and functional inter-specific differences that can be correlated to evolutionary events and might represent the adaptation to environmental conditions present in different habitats (Iwasaki, 2002). Particularly these events are strictly dependent on the food intake mechanisms determining the success of the adaptation of vertebrates to their environment and therefore their survival (Roth and Wake, 1989). Since data about the morphology of the zebrafish oral cavity are scarce the aim of the present research was to study it by light, scanning and transmission electron microscopy and immunohistochemistry for some growth factor receptors, i.e. trkA and trkB. The zebrafish was chosen because as a vertebrate it has valuable features as a model for study of vertebrate development (Withfield, 2002). Adult zebrafishes were anesthesized with MS222 and the samples were taken and routinely processed for light and electron microscopy and for indirect peroxidase immunohistochemistry. The topographical and ultrastructural study demonstrated the presence of three different zones. The outer zone is characterized orally by the presence of a papilla and by an epithelium with numerous taste buds that are chemosensory endorgans consisting of modified epithelial cells. Their function is to sample potential food and selecting it according to its edibility (Hansen et al., 2002). Both in upper and lower intermediate zones, the presence of internal valves with a semi-lunar shape was evident, probably able to regulate the mouth closure. The most internal lower zone corresponds to the tongue that in zebrafish is only a thickening of the pavement mucosa with a stratified epithelium. Immunohistochemistry demonstrated both in upper and lower zones the presence of numerous taste buds that were immunoreactive to TrkA and TrkB-like neurotrophin receptors, confirming the role of these receptors in taste buds sensory cells (Germana et al., 2004). The present results give a contribution to the morphological knowledge of the zebrafish oral cavity, an animal model commonly used for developmental studies of vertebrates.

The olfactory epithelium of some teleosts, including zebrafish, contains three types of olfactory sensory neurons. Because zebrafish has become an ideal model for the study of neurogenesis in the olfactory system, it is of capital importance the identification of specific markers for different neuronal populations. In this study we used immunohistochemistry to analyze the distribution of S100 protein-like in the adult zebrafish olfactory epithelium. Surprisingly, specific S100 protein-like immunostaining was detected exclusively in crypt neurons, whereas ciliated and microvillous neurons were not reactive, and the supporting glial cells as well. The pattern of immunostaining was exclusively cytoplasmic without apparent polarity within the soma, and the intensity of immunostaining was not related with the maturative stage of the neurons. The role of S100 protein in crypt olfactory neurons is unknown, although it is probably associated with the capacity of these cells to respond to chemical stimuli. In any case, it represents an excellent marker to identify crypt olfactory neurons in zebrafish.

TrkB plays crucial roles in the development and maintenance of taste buds in mammals. In this study we investigated the presence and cell localization of Trks (TrkA, TrkB and TrkC) in taste buds of the zebrafish (Danio rerio) using Western blot and immunohistochemistry. Proteins of 140 and 145 kDa, identified as full-length TrkA and TrkB, were detected. Conversely, the anti-TrkC antibody recognized a protein lower than expected (100 kDa). In agreement with these results the sensory cells of taste buds, displayed TrkA- and TrkB-like, but not TrkC-like, immunoreactivity. TrkA and TrkB co-existed in the same taste buds, but remains to be clarified whether or not they are co-expressed in the same cells. Present results demonstrate that as for mammals neurotrophins might play a role in sensory cells of the teleostean taste buds.

The avian primary lymphoid organs, the thymus and the bursa of Fabricius, undergo age-dependent changes leading in some cases to the complete atrophy of the organ. Nevertheless, the timetable of the involutive process as well as the consequences in the structure and functionality of the organs vary largely in the time frame and structural changes among species. On the other hand, and in contrast with the large body of literature reporting the structural and functional changes in mammalian primary lymphoid organs, the age-dependent changes in avian thymus and bursa of Fabricius are scarce, fragmentary, and heterogeneous. This article reviews the current literature on this topic, and focuses primarily on the involution of the bursa of Fabricius.

Employing quantitative autoradiography, pineal body glucose utilization (GU) was measured in daytime or at night in prepubertal (aged 1 month), adult (aged 3 months), and mature (over 12 months old) rats. In prepubertal and adult rats, in daytime, GU values within the pineal tissue were homogeneously distributed around 65 mumol glucose/100 g per min. In prepubertal animals no significant variations in GU were observed between daytime and nocturnal measurements. A circadian metabolic rhythmicity was evident in adult rats, with a GU peak measured at 2 a.m. In mature animals, GU also varied between day and night, with an increment in the relative difference between the two values. The present investigation is the first to demonstrate that circadian metabolic rhythmicity is absent before sexual maturation while it is enhanced in 12-month-old rats. These changes in pineal energy metabolism with advancing age are intriguing in view of the concept that the pineal gland may be involved in functional changes occurring during the process of aging.

Increasing evidence suggests that neurotrophins could regulate immune functions acting directly or indirectly on immunocompetent cells. The indirect pathway involves stromal cells of the primary and secondary lymphoid organs. In the present study the occurrence of Trk proteins (TrkA, TrkB and TrkC), regarded as the high-affinity signal-transducing receptors for neurotrophins, was investigated in cow lymphoid organs using immunohistochemistry. The thymus and spleen of both fetal and adult animals, and the palatine tonsils, lymph nodes and Peyer's patches of adult animals, were analysed. Unidentified cells displaying TrkA-like immunoreactivity were found in the fetal thymus, whereas those expressing this protein in the adult gland were identified as epithelial cells. In the spleen, immunoreactive TrkA was observed in cells of the white pulp. TrkB immunoreactivity in both fetal and adult thymus and spleen was localized in monocyte/macrophage cells. As a rule, TrkC was absent from the thymus and the spleen independent of the animal's age. Different types of stromal cells, but never the lymphocytes themselves, displayed TrkA, TrkB, or TrkC immunoreactivity in the other lymphoid organs analysed. As in other vertebrate species, Trk proteins in the lymphoid organs of the cow were localized in the stromal, non-lymphoid cells, thus suggesting that neurotrophins might regulate the immune function acting indirectly on lymphocytes.

Background: The medial wall of the vomeronasal organ (VNO) is lined with a sensory epithelium that is closely related to the olfactory epithelium, which is developed from the olfactory placode. It undergoes continuous replacement during its life span. In other sensory epithelia, cell proliferation is under the control of some trophic factors. Whether these proteins are involved in the continuous turnover of the VNO epithelium is unknown. This study approaches this topic by analyz- ing the occurrence of signal-transducing receptor proteins for neurotroph- ins (Trk proteins) and epidermal growth factor (EGFr). Methods: VNO samples were obtained from adult horses (n 5 9) and processed for Western blot or immunohistochemical detection of TrkA, TrkB, TrkC, and EGFr. For immunohistochemistry, both frozen and forma- lin-fixed, paraffin-embedded sections were used. Antibodies against Trk proteins were polyclonal antibodies that map within the intracytoplasmic domain. Antibodies against EGFr were monoclonal antibodies that map within the external (clone EGFR1) or the cytoplasmic (clone F4) domains. Results: TrkA-like, but not TrkB- or TrkC-like, protein was detected in the VNO. By using immunoblotting, protein bands of TrkA-like protein with estimated molecular weights of 43-45, 55, and 60 kDa were found. In agreement with these findings, the sensory epithelium lining the VNO displayed strong TrkA-like immunoreactivity. On the other hand, regular protein bands with estimated molecular weights of 100 and 170 kDa, corresponding with immature and full-length EGFr, respectively, were found with the clone F4, whereas the clone EGFR1 was ineffective in detecting EGFr with Western blot analysis. Positive EGFr immunolabel- ling was observed regularly in the supranuclear pole of the sensory epithelial cells, and the pattern was identical with both antibodies used. Conclusions: The present results provide evidence for the occurrence of EGFr in the VNO of the adult horse, suggesting a role for their ligands (EGF and transforming growth factor-a) in this organ, probably in continu- ous cell replacement, during the adult life span. However, although immunoreactivity for TrkA-like protein was regularly observed, because the full-length protein was not found, whether or not its putative ligands (nerve growth factor and neurotrophin-3) act on these cells remains to be demonstrated. Anat. Rec. 247:299-306, 1997. r 1997 Wiley-Liss, Inc.

The avian thymus and bursa of Fabricius are the specific organs where the maturation and differentiation of T- and B-lymphocytes, respectively, take place. In the mammalian lymphoid organs mRNAs of the neurotrophins and their receptors have been identified but their localization at the protein level remains still unknown. This study was undertaken to analyze the localization of the Trk family of tyrosine kinase receptors in the avian primary lymphoid organs (thymus and bursa of Fabricius) during the posthatching development using immunohistochemistry. These proteins serve as essential constituents of the high affinity receptors for neurotrophins. In the thymus of all groups of age specific immunoreactivity (IR) was observed for all three Trks: TrkA-like IR was found labelling medullary epithelial cells and a subpopulation of cortical epithelial cells; TrkB-like IR was found in the medullar dendritic cells and cortical macrophages; TrkC-like IR labelled the cortical epithelial cells and scattered medullar clusters of epithelial cells (including Hassal's corpuscles). Quantitative analysis revealed age-dependent decrease in the area occupied by TrkA-like IR in the cortex, and age-dependent increase in the medulla; no changes were detected in the area occupied by TrkB-like IR; the TrkC-like immunoreactive cells increase from 7 to 30 days and then decrease. Regarding to the bursa of Fabricius, TrkA- and TrkC-like IR were exclusively found in the epithelial cells of the follicle associated and the interfollicular epithelia, as well as TrkC-like IR in some medullary reticular epithelial cells of adult animals. Nevertheless, TrkB-like IR labelled extrafollicular unidentified cells in 7 days old animals, and the follicular secretory dendritic cells at 30 and 60 post-hatching. The area occupied by the medullary TrkB-like IR cells increased between 30 and 60 days. No immunostaining of lymphocytes was observed for any of the assessed antigens. The blood vessels of both the thymus and the bursa of Fabricius were immunoreactive for TrkA- and TrkC-like proteins. The present results provide evidence for the localization of Trks in the non-lymphoid cells (epithelial and dendritic) of the avian primary lymphoid organs, suggesting a role for neurotrophins in these cells. Moreover, the selective cell localization of each Trk protein, and the absence of apparent overlapping, claims for a differential role of the specific Trk ligands. Whether or not these findings have functional relevance for T- and B-lymphocytes processing in avian primary lymphoid organs is discussed.