Hormonal control of growth hormone secretion.

Growth hormone secretion by the somatotroph cells depends upon the interaction between hypothalamic regulatory peptides, target gland hormones and a variety of growth factors acting in a paracrine or autocrine fashion. This review will be focused on recent data regarding the mechanism by which growth hormone-releasing hormone (GHRH) influences somatotroph cell function and the physiological role played by Ghrelin and leptin in the regulation of growth hormone (GH) secretion. It is well established that binding of GHRH to its receptor leads to activation of protein kinase A (PKA). More recently, it was found that GHRH can also activate mitogen-activated protein (MAP) kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Whether somatotroph adenomas, either with or without a GS-alpha mutation, have alterations in some of the components of the activation of the MAP kinase pathway remains to be known. The recent isolation of Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, can be considered a landmark in the GH field, which opens up the possibility of gaining greater insight into our understanding of the mechanisms involved in the regulation of GH secretion and somatic growth. Indeed, preliminary evidences indicate that this peptide exerts a marked stimulatory effect on plasma GH levels in both rats and humans. Finally, it is well known that GH secretion is markedly influenced by nutritional status. Leptin has emerged as an important adipose tissue-generated signal that is involved in the regulation of GH secretion, thus providing an integrated regulatory system of growth and metabolism. Although the effects of leptin on GH secretion in humans remain to be clarified, indirect evidences indicate that it may play an inhibitory role.

Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97.

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.

A comparative study of protein kinase C-like immunoreactive cells in the retina.

The present study is a morphological and quantitative analysis of protein kinase C-like immunoreactive (PKC-L ir) bipolar cells in the retinas of five different vertebrate species (chicken, tench, zebrafish, goldfish and rat). The morphology of PKC-L-ir bipolar cell axon terminals in fish differs significantly from those of chicken and rat retinas. Fish have bulky terminals whereas chicken and rat have their terminals in the form of small knob-shaped branches. In tench and goldfish, PKC-L-ir bipolar cells gradually decrease in size from the medial (i.e., in tench: mean +/- SD soma area of 30.09 +/- 5.98 microm2) to the peripheral (i.e., in tench: 19.93 +/- 1.73 microm2) retinal regions. This is not observed in chicken, rat or zebrafish where there is more homogeneity in s oma and axon terminal sizes between different retinal regions. Except in chicken, cell density increases from the central (i.e., in tench: mean +/- SD 1795.88 +/- 242.35 cells/mm2) to the peripheral (i.e., in tench: 4295.41 +/- 279.23 cells/mm2) retina. This study provides data that show relevant differences in the PKC-L-ir bipolar morphology and density among birds, fish and mammals. Moreover, these structural variations could mean not only differences in the cellular physiology, but also in the patterns of development and maintenance of the retina in each species.

Protein kinase C-like immunoreactive cells in embryo and adult chicken retinas.

Morphological evidence of a temporal parallelism between the appearance of the alpha isoform of protein kinase C (PKC) and some processes such as synaptogenesis in the plexiform layers of the chicken retina is offered. Immunostaining experiments were performed throughout embryonic, young and adult chicken life. The results help to understand the development of rod bipolar cells.

Enzyme histochemical identification of microglial cells in the retina of a fish (Tinca tinca).

Histochemistry for nucleoside diphosphatase was used to study the microglial cells in the adult tench retina. An abundant population of microglial cells was located in the vascular membrane, nerve fibre layer, inner and outer plexiform layers and scattered cells were observed in the inner nuclear layer. Rounded and amoeboid cells could be seen close to the vessel in the vascular membrane, bipolar cells in the nerve fibre layer and ramified cells in the rest of the layers. Several microglial forms could correspond to developing cells. The pattern of distribution was similar to that described in other vertebrates, but with several differences, such as the presence of microglial cells in the vascular membrane and inner nuclear layer and the overlap of processes in the plexiform layers.

Neurotrophins and their receptors in the tench retina during optic nerve regeneration.

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.

Increased levels of TrkA in the regenerating retinal ganglion cells of fish.

Retinal ganglion cells of the fish have the spontaneous capacity to regenerate after nerve crush, a phenomenon known to be facilitated by nerve growth factor (NGF). We have studied the high-affinity NGF receptor TrkA, during the regeneration of the tench (Tinca tinca L.) optic nerve, using immunocytochemical techniques. TrkA-like immunoreactivity increased during the regeneration of the retinal ganglion cells. The increase is followed by a change in the subcellular distribution from perinuclear in control cells to cytoplasmic and perinuclear in regenerating ones. This increase was observed when antibodies against the extracellular domain of TrkA were used; no changes in TrkA-like immunoreactivity were observed with antibodies against the intracellular domain of TrkA. We thus conclude that modulation of TrkA is involved in the regeneration of fish retinal ganglion cells.

Immunohistochemical distribution of neurotrophins and their receptors in the rat retina and the effects of ischemia and reperfusion.

1. Neurotrophins are molecules that regulate the survival, development and maintenance of specific functions in different populations of nerve cells. 2. In the present work, we studied the localization, at the cellular level, of the different neurotrophins and their receptors within the rat retina in control and after ischemia-reperfusion of the retina. We found variations in the localization of some of these molecules depending on the reperfusion time of the retina after the ischemic lesion. 3. Thus it is suggested that the changes in the distribution and concentration of neurotrophins and their receptors caused by ischemia are protective reactions related to neuronal damage and synaptic reorganization.

Diencephalic and mesencephalic structures related to the optic nerve organization in tench (Tinca tinca L., 1758). A study using fluoro-gold.

The location of several diencephalic and mesencephalic structures in the teleost fish, Tinca tinca, which have not been described previously, was made possible by injecting Fluoro-Gold, as an anterograde and retrograde tracer, into the optic nerve. In the pretectal area, we found the tractus opticus accessorius and the nucleus opticus dorsolateralis. We have made some specifications about the location and nomenclature of the branches belonging to the optic tracts and two nuclei also related to the visual system (the nucleus commissura posterior and the nucleus pretectalis periventricularis pars dorsalis). This study also presents the retinal projections to the optic tectum and the glial cells in the injected optic nerve of the tench. The laminar nucleus and Edinger-Westphal nucleus are also identified and described in relation to the ciliary pathway.

S-100-positive glial cells are involved in the regeneration of the visual pathway of teleosts.

Glial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) were studied by labelling with anti-S-100 antibody. In normal fish, S-100-positive bipolar cells were found in the optic nerve, optic tract, and in the diencephalic visual pathways. After crushing the left optic nerve, the distribution and the number of S-100-immunoreactive cells were modified. In the injured nerve, 7 to 15 days after crushing no immunoreactive cell bodies were found in the crushed area, but a greater number of S-100-positive cells were found on both sides of the injured area. Sixty days after crushing, positive cells penetrating the crushed area were observed; the normal pattern was almost restored 200 days after crushing. In the diencephalon, 25 days after crushing, the number of S-100-positive cells increased remarkably and the most intense immunostaining of glial processes was observed 60 days after crushing. The density of S-100-labelled cells decreased after 4 months postcrushing. However, in the optic tectum no changes were observed. The increase of glial cells in the lesioned visual pathway suggests that they could play an important role in axonal regeneration after crushing.

Distribution of S100 immunoreactivity in the retina and optic nerve head of the teleost Tinca tinca L.

The distribution of S100 immunoreactivity within the normal and regenerating retina and optic nerve head of the teleost Tinca tinca L. has been investigated using the avidin-biotin complex (ABC) method and a polyclonal antibody against S100. Astrocytes and Müller cells were labeled with this antibody. This represents the first description of astrocytes localized in the optic nerve head and in the nerve fiber layer of the fish retina displaying a typical bipolar morphology. Horizontal cells in the inner nuclear layer were immunolabeled; we also observed species-specific S100 labeling of horizontal cells of the H1 subtype. No significant changes were seen in the S100 immunoreactive Müller cells, astrocytes, or horizontal cells in the tench retina after optic nerve crushing and during regeneration. These results might help to understand the function of glial cells in the normal and experimentally induced regenerating fish visual system.

Microglia in normal and regenerating visual pathways of the tench (Tinca tinca L., 1758; Teleost): a study with tomato lectin.

We have studied the microglial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) by using the lectin from Lycopersicum esculentum (tomato), which, in our case, has been demonstrated as a specific marker for teleost microglia. In the normal fish, there are tomato lectin positive microglial cells in the retina, optic nerve, and optic tectum. Following optic nerve crush, we observed a more extensive labeling of the microglia in the crushed optic nerve and in the contralateral optic tectum affecting the stratum opticum and stratum fibrosum et griseum superficiale. In both cases, there was an increase of rounded and less ramified microglial cells, and granular cells. This response of a more extensive labeling of microglial cells increases to a maximum at 2-3 weeks after the crush; the density of labeled microglial cells decreases after 3 months after crushing. However, in the retina no changes were observed after optic nerve crush. These results suggest that the microglial cells could play an important role in regeneration of fish optic pathway, as other neuroglial cells do.

Plasmodium falciparum: characterization of a 0.7-kbp, moderately repetitive sequence.

A 692-bp-long repetitive sequence of Plasmodium falciparum was cloned. Two contiguous repeats were sequenced. The homology between them was 85%, the differences being due only to base replacements. The sequence was found 60 times repeated in the genome and was detected in 11 of 14 chromosomes. When used as a probe, the cloned fragment detected the parasite with the same sensitivity the 21-bp repeat (pRepHind) did. PCR amplification detected 0.02 pg of DNA, equivalent to a single parasite, in strains from America, Asia, and Africa. The restriction patterns were polymorphic and different among the strains. Analysis of lambda EMBL clones that contain the repetitive sequence confirms that it is present in various genomic contexts and is located subtelomerically.

Expression of brain-derived neurotrophic factor and of its functional receptor in neonatal and adult rat retina.

The expression of mRNA coding for brain-derived neurotrophic factor (BDNF) and for its functional receptor, the full-length tyrosine kinase receptor trkB (trkB mRNA), was examined in early postnatal and adult rat retina by in situ hybridization using digoxygenin and radioactively-labeled oligonucleotide probes. BDNF and trkB mRNAs are expressed in the ganglion cell layer at postnatal-days (PN) 1, 4, 7, 14, 60, in proximal neuroblastic layer (PN 1, 4, 7), and proximal inner nuclear layer (PN 14, 60). Subpopulations of developing and mature retinal cells are thus capable of synthesizing BDNF.

Calbindin D-28K distribution in the retina of the developing trout (Salmo fario L.).

The appearance of calbindin D-28K, a calcium-binding protein, during development of the trout retina was studied by immunohistochemistry. The first calbindin immunoreactive cells appear in the inner nuclear layer at the equator of the embryonic retina at the stage 227 degrees C (around embryonic day 15). Just before hatching, stage 440 degrees C (around embryonic day 30) cells located in the ganglion cell layer and inner nuclear layer, expressed calbindin. This pattern of immunoreactivity was conserved in post-embryonic retinae (alevins 15 days old). In adult retinae the ganglion cells showed a faint immunoreaction; the amacrine cells are markedly fewer and their immunoreaction declined; and the bipolar cells expressed calbindin for the first time. The results obtained in the present work attending to the expression of calbindin, generally conforms with the vitreal to scleral progression of differentiation of the teleost retina. Ganglion, amacrine and bipolar cells undergo further maturation after beginning calbindin expression.