Jean-Michel Danger

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163 +/- 8, 233 +/- 16, 151 +/- 12 and 60 +/- 13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

SummaryThe distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.

Distribution and characterization of neuropeptide Y in the brain of an elasmobranch fish.

Using a specific antiserum raised against synthetic neuropeptide Y (NPY), the distribution of immunoreactivity in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula has been examined with the indirect fluorescence and the peroxidase-antiperoxidase methods. The highest density of NPY-immunoreactive neurons was found in the basal telencephalon and in the hypothalamus. Numerous NPY-containing perikarya were located in the entopeduncular and the preoptic nuclei, in the nucleus lobi lateralis and in the nucleus lateralis tuberis. NPY-immunopositive fibers were observed throughout the fish brain. In particular, dense networks of fibers were present in the entopeduncular and the habenular nuclei, in the nucleus tuberculi posterioris and in the lateral lobes. Scattered fibers were observed in all other parts of the brain except in the cerebellum where no NPY-immunoreactive material could be detected. A plexus of NPY-immunoreactive fibers arising from the preoptic neurosecretory complex appeared to run through the basal hypothalamus and the pituitary stalk. These fibers terminated in the intermediate lobe of the pituitary, suggesting that NPY may be involved in the control of melanotropin secretion. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. Brain and pituitary extracts showed a good cross-reactivity to the NPY antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. HPLC analysis resolved two major forms of immunoreactive NPY in the hypothalamus while the pars intermedia contained only authentic NPY. The widespread distribution of NPY neurons in the fish brain and pituitary suggests the involvement of NPY in a variety of physiological functions, including the neuroendocrine control of the pituitary.

Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an α-MSH-release inhibiting factor

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.

An npy-like peptide may function as msh-release inhibiting factor in xenopus-laevis

This study demonstrates the presence of a rich plexus of neuropeptide Y (NPY) immunoreactive fibers in the hypothalamus and in the intermediate lobe of the pituitary of Xenopus laevis. During superfusion of neurointermediate lobe tissue, synthetic NPY induces a rapid, powerful and dose-dependent inhibition of in vitro release of MSH, endorphin and other proopiomelanocortin (POMC) derived peptides. Therefore, NPY undoubtedly is one of the growing number of neuropeptides that are likely involved in control of the amphibian MSH cells. Although a number of stimulatory neuropeptides have been found, this is the first neuropeptide to apparently function through an inhibitory mechanism. In that a 2-hr treatment with NPY did not influence POMC biosynthesis, nor processing of this prohormone to smaller peptides, we conclude that the primary action of NPY is a direct effect on the secretory process of the MSH cell.

Characterization, cerebral distribution and gonadotropin release activity of neuropeptide Y (NPY) in the goldfish

The presence of a peptide closely related to porcine NPY has been demonstrated in the goldfish brain and pituitary by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). The RIA data demonstrate that displacement curves of brain extracts are parallel to a porcine NPY standard and that in HPLC a compound present in brain extracts is co-eluted with porcine NPY. The distribution of this NPY-like factor within the central nervous system was studied by radioimmunoassay and immunohistochemistry. The results indicated that NPY has a widespread distribution with the highest concentrations being found in the telencephalon and diencephalon. In the pituitary gland, NPY immunoreactive terminals characterized at the electron microscope level were found in the different lobes and, in particular, in close association with the gonadotrophin (GTH) secreting cells. Using anin vitro perifusion system, it was shown that NPY causes a dose dependent increase of GTH release from anterior lobe fragments.These data indicate for the first time in teleosts that NPY is present and widely distributed in the brain and pituitary, and that among other putative functions, could be implicated in the multihormonal release of GTH from the pituitary.

Neuropeptide Y (NPY) modulatesin vitro gonadotropin in release from rainbow trout pituitary glands

This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10−7 M) induced a 20–30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10−7 M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3–7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.

Co-distribution of neuropeptide Y and its C-terminal flanking peptide in the brain and pituitary of the frog Rana ridibunda

By means of the peroxidase-anti-peroxidase technique, the distribution of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON) has been studied on serial sections of the brain and pituitary of the frog Rana ridibunda. Throughout the brain, NPY and C-PON-immunoreactive perikarya exhibited a remarkable co-distribution. These two peptides were found to be co-located within the same cell bodies in various brain regions including the dorsal and ventral pallium, the dorsal and ventral infundibular nuclei and the preoptic nucleus. The distribution of NPY- and C-PON-containing fibers in the brain and pituitary was similar. Sequential double immunohistochemical staining using the indirect immunofluorescence method, showed that NPY and C-PON were actually located within the same nerve processes throughout the frog brain and in the intermediate lobe of the pituitary. These studies indicate that the deduced C-PON sequence is present within the frog precursor to NPY and is formed in vivo in the frog brain. Like NPY, C-PON is transported distally in nerve terminals and is likely released with NPY in various regions of the brain and in the intermediate lobe of the pituitary.

V. Melanotropin release inhibiting activity of neuropeptide Y: Structure-activity relationships

Abstract We have recently shown that the release of α-MSH by the intermediate lobe of the frog pituitary is inhibited by neuropeptide Y (NPY). Using the perifusion technique, we have compared in the present study, the α-MSH release inhibiting activities of NPY, various NPY short chain analogues and two other members of the pancreatic polypeptide family, peptide YY (PYY) and avian pancreatic polypeptide (APP). The order of biological potency was NPY > NPY[2–36] > NPY[16–36] > NPY[25–36] > NPY[1–15]. Among the two pancreatic polypeptides tested, PYY appeared to be almost as potent as NPY while APP was 6 times less active than NPY. Neither NPY[1–15] nor NPY[16–36] could antagonize the inhibitory effect of NPY on α-MSH release. The structure-activity relationship study suggests that the bioactive determinant of NPY is located in the C-terminal part of the molecule.

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1.

Prohormone convertases (PCs) are calcium‐dependent serine endoproteases of the subtilisin/kexin family that play a key role in the posttranslational processing of precursors for biologically active peptides. In this study, we have characterized the cDNA encoding PC1 in the European green frog Rana ridibunda. A frog brain cDNA library was screened by using a heterologous probe at low stringency, and a 2.3‐kb cDNA clone encoding PC1 was isolated. This cDNA encodes a 736‐residue protein with a 26‐amino‐acid signal peptide. Comparative structural analysis revealed that frog PC1 exhibits a high degree of amino acid identity with its mammalian counterparts, in particular in the subtilisin‐like catalytic domain. Northern blot analysis resolved two major transcripts of 3.0 kb and 5.0 kb that were expressed differentially in the brain and pituitary. In situ hybridization studies showed that, in the frog brain, the highest densities of PC1 mRNA are present in the amygdala, the hypothalamus, and the anterior preoptic area. High concentrations of PC1 mRNA also were found in the pars distalis and pars intermedia of the pituitary, whereas the pars nervosa was devoid of hybridization signal. The wide distribution of PC1 mRNA in the brain and pituitary suggests that, in frog, PC1 is involved in the processing of a number of hormone and neuropeptide precursors. J. Comp. Neurol. 405:160–172, 1999.