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.     

GDNF and GFRα co-receptor family in the developing feline gut

Glial cell-line derived neurotrophic factor (GDNF) and the GFRα co-receptors play a role in the developing enteric nervous system. The co-receptors elicit their action by binding receptor tyrosine kinase RET.This immunohistochemical study reports the presence of GDNF and its specific co-receptor GFRα1 in the cat gastrointestinal apparatus during development, from stage 9 to 22. At stage 9 and 11, immunoreactivity (IR) to GDNF was observed in the cells of mesenchyme of the anterior gut. From stage 14 to 22, GDNF IR was detected in nervous plexuses; moreover, GDNF and GFRα1 IR appeared localized in gastrointestinal endocrine cells. The presence of GDNF in the enteric nervous system and in the endocrine cells suggests an involvement of this neurotrophic factor in the gastrointestinal development. Moreover, the presence of the co-receptor GFRα1 in endocrine cells and its absence in the enteric nervous system seems to indicate a different mode of transduction of GDNF signal. GFRα2 and GFRα3 co-receptors were not detected.     

GDNF family ligand RET receptor in the brain of adult zebrafish

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

The cellular network of interstitial cells associated with the deep muscular plexus of the guinea pig small intestine

Summary Systematic examination using electron microscopic montages and serial sections has demonstrated that three types of interstitial cell, namely gap junction-rich cells, glycogen-rich cells and fibroblast-like cells, are densely located along the whole extent of the deep muscular plexus of the guinea pig small intestine. They tend to be distributed in an alternating fashion in the cellular network, connected with muscle cells of the outer, circular layer by means of gap junctions. These three types of interstitial cell show close relations to two types of nerve varicosity: one type is characterized by clear round vesicles with diameters of about 50 nm, and the other by flattened vesicles measuring about 35 nm by 70 nm. Electron-dense patches have been observed at the cytoplasmic side of the axonal membranes. Muscle cells of both inner and outer circular layers also show close relations to these two types of nerve varicosity. These morphological features are discussed with the implication that they may have some regulatory role in intestinal movement.     

Evolution of myogenesis in fish: a sturgeon view of the mechanisms of muscle development

Patterns of initial muscle formation are well documented for teleost fish. Here, attention is focused upon sturgeons, which arose close to the base of the actinopterygian radiation and whose early development has remained largely unresearched. We demonstrate that some features of muscle development are common to both groups of fish, the most important being the origin and form of migration of adaxial cells to establish the superficial slow fibre layer. This, together with information on initial innervation and capillarisation, strongly suggests a common basis for muscle developmental mechanisms among fish. An important feature that is different between sturgeons and teleosts is that sturgeons lack any cellular dorsal–ventral separation of the myotome that involves the insertion of muscle pioneer (MP)-like cells at the site of the future horizontal septum. This, and information from other fish and from sarcopterygians, permits the supposition that such MP-defined dorsal–ventral separation is a teleost apomorphism. These and other findings are discussed in relation to their significance for the evolution of fish muscle developmental patterns.     

Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method

Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180 kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia.     

Morphogenesis of the secondary envelope of the oocyte in a teleostean fish of the family Cyprinodontidae: Aphyosemion splendopleure

The follicular epithelium of the oocyte in Aphyosemion splendopleure is made up of prismatic cells in which the density of the cytoplasm is variable. At the end of vitellogenesis the follicular cells show polarity. The rough endoplasmic reticulum is localized in the cytoplasm situated between the nucleus and the basal lamina. This reticulum splits up into vesicles limited by a membrane covered with ribosomes. At the beginning of postvitellogenesis the Golgi apparatus produces numerous secretion granules in the cytoplasmic region contiguous to the surface of the oocyte. These Golgi elements, together with amorphous material whose origin we have not been able to define, form the secondary envelope of the egg. This envelope is made up of two layers: an inner layer formed by an agglomeration with a membranous aspect and a superficial layer made up of tubular elements. The superficial layer forms an ornamentation clearly shown up by scanning electron microscopy.     

Responses to stimulation of marginal fibers in the teleostean optic tectum

Summary The marginal fibers (mf) constitute a major fiber component of the teleostean optic tectum, and this report deals with the physiological properties of these fibers and their postsynaptic elements. The mf are unmyelinated axons which originate at the torus longitudinalis and run lateralwards, parallel to one another, along the tectum's most superficial layer (stratum marginale). Here they synapse upon the dendritic arborizations of the pyramidal (p) neurons. These arborizations originate from a single apical dendritic shaft which, near the soma, receives retinofugal axon terminals. The p-neurons also have a basal dendritic shaft and a descending axon, both of which branch out horizontally at the stratum griseum centrale (SGC). The mf were stimulated through surface microelectrodes, and field potentials were recorded on-beam throughout the tectal thickness by means of micropipettes. The mf action potential (M-spike) may show two negative subpeaks which propagate at 0.20 and 0.16 m/s. Its refractory period is followed by a period of increased amplitude and decreased latency. The M-spike is followed by a series of slow waves, namely: (a) The S-wave, which probably represents the monosynaptic depolarization of the p-neuron's apical arbor; (b) The N-wave which possibly represents an active current sink at the point where these dendritic arborizations merge into the apical dendritic shaft; (c) The D-wave, recorded at the SGC, which possibly represents the activation of the p-neuron's axon and terminals (and perhaps also basal dendritic shaft and branches); and (d) the L-wave, which might represent a late depolarization of the p-neuron's apical arborizations.Morphologically and electrophysiologically, there are several similarities between the tectal mf/p-neuron system and the cerebellar parallel fiber/Purkinje cell system.     

The glial cell line-derived neurotrophic factor (GDNF) does not acutely change acetylcholine release in developing and adult neuromuscular junction

We use immunocytochemistry to show that the trophic molecule glial cell line-derived neurotrophic factor (GDNF) and its receptor GDNF family receptor α-1 (GFRα-1) are present in both neonatal (P6) and adult (P45) rodent neuromuscular junctions (NMJ) colocalized with several synaptic markers. However, incubation with exogenous GDNF (10–200 ng/ml, 1–3 h), does not affect spontaneous ACh release. Moreover, GDNF does not change the size of the evoked ACh release from the weak and the strong axonal inputs on dually innervated postnatal endplates nor in the most developed singly-innervated synapses at P6 and P45. Our findings indicate that GDNF (unlike neurotrophins) does not acutely modulate transmitter release during the developmental process of synapse elimination nor as the NMJ matures.     

Immunohistochemical localisation of neuronal nitric oxide synthase in the rainbow trout kidney

The distribution of nitrergic nervous structures in the trout kidney was studied by peroxidase-linked ABC immunostaining procedures using a polyclonal antibody raised against the neuronal isoform of nitric oxide synthase. The nitrergic plexus reaches the kidney along the vasculature, mainly running with the postcardinal vein where nitrergic fibres, microganglia like cellular clusters and isolated neurones were detected. The atubular head-kidney only showed isolated nitrergic fibres close to the larger arteries. On the other hand, the collecting tubules, collecting ducts, large arteries and glomerular arterioles of the tubular middle and posterior trunks were innervated by nitrergic fibres even though immunoreactive neurones were also observed in close apposition to some tubular elements and large arteries. These results suggest that, according to morphofunctional differences between the fish and mammalian kidneys, nitrergic neural structures may be involved in the control of particular renal functions in the rainbow trout.     

Nerve growth factor in the adult brain of a teleostean model for aging research: Nothobranchius furzeri

Nerve growth factor (NGF) acts on central nervous system neurons, regulating naturally occurring cell death, synaptic connectivity, fiber guidance and dendritic morphology. The dynamically regulated production of NGF beginning in development, extends throughout adult life and aging, exerting numerous roles through a surprising variety of neurons and glial cells.This study analyzes the localization of NGF in the brain of the teleost fish Nothobranchius furzeri, an emerging model for aging research due to its short lifespan. Immunochemical and immunohistochemical experiments were performed by employing an antibody mapping at the N-terminus of the mature chain human origin NGF. Western blot analysis revealed an intense and well defined band of 20 kDa, which corresponds to proNGF of N. furzeri. Immunohistochemistry revealed NGF immunoreactivity (IR) diffused throughout all regions of telencephalon, diencephalon, mesencephalon and rhomboencephalon. It was detected in neurons and in glial cells, the latter mostly lining the mesencephalic and rhomboencephalic ventricles. Particularly in neurons, NGF IR was localized in perikarya and, to a less extent, in fibers.The widespread distribution of proNGF suggests that it might modulate numerous physiological functions in the adult brain of N. furzeri. The present survey constitutes a baseline study to enhance the understanding of the mechanisms underlying the role of NGF during aging processes.     

Transplant of embryonal nervous tissue preserves the responses of rat retinal ganglion cells after section of the optic nerve

Summary We have investigated the effectiveness of embryonal tectal tissue transplants in preserving the physiological activity of lesioned ganglion cells by recording the visual responses from the adult rat retina after section of the optic nerve, with or without transplants of embryonal nervous tissue on the stump. We have found that transplant of embryonal nervous tissue at the level of the optic nerve section has dramatic effects in preserving visual retinal responses to patterned stimuli for times as long as five months after surgery. By this time retinal responses to patterned stimuli have almost completely disappeared in control animals with optic nerve section alone.     

Leptin in teleostean fish,towards the origins of leptin physiology

Teleostean leptin was first cloned in 2005, more than a decade after the discovery of mammalian leptin. The reason for this delay lies in the very poor primary sequence conservation (∼13–25%) between mammalian and fish leptins. These low sequence conservations indicate a high degree of molecular evolvability and warrant a search for different and original functions of leptin in teleosts. Indeed, new and original insights are obtained because of the unique phylogenetic position of teleostean fish as the earliest vertebrates and because of their ectothermy, which means that teleosts are more flexible in changing their metabolism than mammals and leptin could play a role in this flexibility. Research during the last decade reveals that leptin is a truly pleiotropic hormone in fish and mammals alike, with functions among others in the regulation of food intake and body weight, development, but also in the regulation of the stress axis and acclimation processes to for instance low oxygen levels in the water. In this review, we provide an overview of the teleostean leptin work done in the last ten years, and demonstrate that the power of a comparative approach leads to new insights on the origins of leptin physiology.     

Endocrine cells in the gastrointestinal tract of a stomachless teleostean fish

Endocrine cells in the gastrointestinal tract of the stomachless teleostean fish, Notemigonus crysoleucas, were studied using electron microscopy. Located between the absorptive cells of the intestinal epithelium, the enteroendocrine cells were very few in number. While some of the cells had their secretory granules located basally and a long narrow part extending toward the lumen, many appeared rounder and the plane of the section did not indicate that they extended to the lumen. Based upon size and shape of secretory granules, there appear to be several different types of cells: those with the smallest granules distributed throughout the intestine, those with intermediate sized granules more commonly found in the middle and distal segments and a few with large granules seen most often in the distal intestine.     

Dual presence of chromogranin A-like immunoreactivity in a population of endocrine-like cells and in nerve fibers in the human anal canal

Light microscopic immunohistochemistry was used to investigate the presence and distribution of chromogranin A-like immunoreactivity in the human anal canal. In the anal transitional zone (ATZ), anal duct and anal gland epithelium, a varied number of mostly elongated cells strongly stained for CGA, using an antibody directed to a highly species-conserved region of the CGA molecule or the monoclonal antibody LK2H10. The density of CGA-immunoreactive (ir) cells strikingly increased from the ATZ epithelium towards the anal gland epithelium. CGA-ir cells possessed single processes running perpendicularly to reach the epithelial surface and exhibited basal ramifications that extended parallel to the basal lamina. The number of CGA-ir cells in anal glands exceeded CGA-ir cells in the crypt-bearing colorecal-type mucosa. The abundant population of CGA-ir cells in the anal canal most likely represents a population of specialized endocrine or paracrine cells. CGA-like immunoreactivity was also present in anocutaneous Merkel cells. A sparse number of vascular and non-vascular CGA-ir varicose nerve fibers was present throughout the layers and rostrocaudal divisions of the anal canal and in the perianal skin. Proposed functions of CGA in neuroendocrine cells and nerves of the anal canal include calcium binding and regulation, secretory granule matrix formation, and generation of bioactive peptides.     

Ganglion cells in achalasia of the cardia

Summary The histopathology of 40 cases of achalasia of the cardia, 6 cases of oesophageal spasm-incoordination and 4 cases of scleroderma was examined. Three cases of carcinoma and 6 cases of reflux oesophagitis were used as a control group. A nearly complete loss of myenteric ganglion cells was found in the upper thickened segment in achalasia. Some surviving ganglion cells were found in the lower segments in half the cases of achalasia; in two cases counts were normal in this segment. The occurrence of neuronal chromatolysis in 9 biopsies of achalasia supports the view that an active disease process was involved. The preganglionic parasympathetic fibres in two cases of achalasia were normal in appearance and number; this somewhat limited evidence tends to count against a primary disorder of the preganglionic neurone in this condition. The 6 cases of oesophageal spasm-incoordination showed similar neuronal loss to that in the lower segment in achalasia. Possibly oesophageal spasm represents an early stage or incomplete expression of achalasia. One case of scleroderma showed loss of ganglion cells, but the myenteric plexus was here involved by the disease process. None of the 9 cases in the control group showed any loss of ganglion cells or chromatolysis. Acute and chronic inflammation was not convincingly associated with loss of ganglion cells in either achalasia or oesophageal spasm.     

Hypothalamic neurons innervating fat tissue in the pig express leptin receptor immunoreactivity

While leptin receptors have been found in both the autonomic ganglion neurons and the hypothalamic nuclei, studies dealing with the projections from the central nervous system to the adipose tissue have been conducted mainly in laboratory animals. Therefore, the purpose of our study was to establish whether hypothalamic neurons are transsynaptically connected to adipose tissue depots in the pig, and if these neurons express leptin receptor immunoreactivity. Pseudorabies virus (PRV; Bartha's K strain) was introduced in perirenal or subcutaneous adipose tissue depots in domestic pigs. On day 9, animals were euthanized and hypothalami were collected and processed immunohistochemicaly with primary antisera against PRV and leptin receptor (OBR). PRV-labeled neurons were localized in paraventricular nucleus, supraoptic nucleus and arcuate nucleus following injections in both the perirenal and the subcutaneous adipose tissue depots. Ventromedial nucleus, dorsomedial nucleus and preoptic area-labeled neurons were observed after injection of the PRV into the perirenal adipose tissue, while in the lateral hypothalamic area-labeled neurons projected only to the subcutaneous adipose tissue. The majority of the PRV-labeled neurons simultaneously expressed OBR-immunoreactivity. Our results provide the morphological data on multisynaptic projections from hypothalamus to the fat tissue in the pig and demonstrate that these neurons, located in areas involved in reproductive processes and feeding behavior, may regulate fat tissue metabolism.     

HIP/PAP, a member of the reg family, is expressed in glucagon-producing enteropancreatic endocrine cells and tumors

Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) protein, a member of the reg family, is constitutively expressed by some specialized epithelial cell subsets in the digestive tract and the pancreas. We performed a detailed analysis of the expression of HIP/PAP protein in normal digestive endocrine cells according to their localization, lineage, and differentiation stage, and in digestive endocrine tumors according to their site of origin and hormonal profile. In both adult and fetal normal tissues, HIP/PAP expression was detected only in endocrine cells of the small intestine, ascending colon, and pancreas. Two different expression patterns were identified: (a) a strong cytoplasmic labeling observed in the endocrine cells of the digestive mucosa and the outer rim of Langerhans islets specialized in the synthesis of glucagon and glucagon-like peptides; (b) a weak cytoplasmic immunoreactivity observed in the other pancreatic endocrine cell populations. HIP/PAP expression was detected in 36 of the 184 cases of digestive endocrine tumors examined; 32 of these cases (89%) were pancreatic. The 2 patterns observed in the normal state were retained: (a) a strong labeling was observed in 5% to 100% of tumor cells in 26 tumors, all expressing glucagon or glucagon-like peptides; (b) a weak labeling was present in 10 tumors, presenting various hormonal profiles. In conclusion, a strong expression of HIP/PAP is characteristic of glucagon-producing normal and neoplastic enteropancreatic endocrine cells. Our results lend further support to the concept that members of the reg family play regulatory roles in various endocrine cell populations and that their expression in endocrine cells is lineage-specific.