F. Leboulenger

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)

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.

Direct radioimmunoassays for plasma corticosterone and aldosterone in frog. I. Validation of the methods and evidence for daily rhythms in a natural environment

Abstract Two radioimmunoassay techniques for direct measurement of frog plasma concentrations of corticosterone after prior ethanol extraction for deproteinization, and of aldosterone without prior extraction, are described. Specific antibodies against corticosterone 21-hemisuccinate and aldosterone 18,21-diacetate-3-carboxymethoxime derivatives conjugated to bovine serum albumin are raised in rabbits. The sensitivity threshold of the assays allows the assessment of corticosterone in 10-μl and aldosterone in 5-μl samples of plasma. Sephadex LH-20 chromatography demonstrates the validity of both methods. The intra- and interassay reproducibilities and the accuracy of each assay have been studied. The conditions making it possible to reduce aldosterone fluctuations during blood taking have been ascertained. Using these techniques corticosterone and aldosterone concentrations have been assessed in the plasma of 214 frogs caught in their natural habitat at 4-hr intervals during a 40-hr period in mid-June. The existence of synchronous and reproducible 24-hr rhythms of corticosterone and aldosterone plasma levels has been demonstrated. High concentrations of both corticosteroids are recorded during the night and low concentrations are recorded during daylight. The amplitude of corticosterone fluctuations is 3.5-fold greater than that of aldosterone fluctuations. Corticosteroid rhythms are compared to activity phases of frogs during the day at this period of the year.

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.

In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. II. Influence of adrenocorticotropin upon aldosterone production.

In order to investigate various factors capable of regulating frog adrenal steroidogenesis, Rana ridibunda adrenal fragments were continuously perifused for 10 hr with amphibian culture medium (ACM). Aldosterone concentrations in the effluent medium were assayed without prior extraction by means of a sensitive and highly specific radioimmunoassay method. In all the experiments, large amounts of aldosterone were secreted even in the absence of ACTH stimulation. Aldosterone output paralleled temperature variations (5 to 30°). A highly significant correlation (r = 0.982; P < 0.01) was established between the outputs of corticosterone and aldosterone during this experiment. Infusion of frog distal lobe extract gave a dose-related response, with the highest dose (0.08 distal lobe eq/ml) yielding a 6.7-fold increase in aldosterone output. In this experiment, interrenal tissue produced two times as much aldosterone as corticosterone. When various dilutions of frog intermediate lobe extracts were tested upon interrenal fragments, a linear log-dose response in aldosterone production was observed for the lower doses and a plateau was reached for the higher doses (0.05 and 0.1 intermediate lobe eq/ml). Dibutyryl cyclic AMP, at a dose of 10 mM, led to a 3.18-fold increase in aldosterone output. These results suggest that aldosterone secretion is controlled by ambient temperature and by circulating levels of adrenocorticotropin. They demonstrate that, in frogs, output of aldosterone is two times higher than output of corticosterone. The aldosterone-corticosterone ratio is even larger after stimulation by high doses of ACTH. Finally, they confirm the presence of large concentrations of biologically active corticotropin in the intermediate lobe of frog pituitary.

Localization and identification of α-melanocyte-stimulating hormone (α-MSH) in the frog brain

Abstract The distribution of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. α-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize α-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive α-MSH coeluted with synthetic des-Nα-acetyl α-MSH, authentic α-MSH and their sulfoxide derivatives. The heterogeneity of α-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-Nα-acetyl α-MSH is present. These findings support the existence of discrete α-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic α-MSH.

Thyrotropin-releasing hormone stimulates the release of melanotropin from frog neurointermediate lobes in vitro

Abstract The hypothalamus of Amphibia contains large amounts of tripeptide P-Glu-His-Pro-NH2 (mammalian thyrotropin-releasing hormone, TRH). However, synthetic TRH is unable to stimulate thyrotropin release from frog pituitary gland. The recent discovery of TRH in the skin of the frog suggests a possible role of this peptide in skin-colour adaptation. Thus we have investigated the role of TRH upon melanotropin (α-MSH) release from perifused frog neurointermediate lobes. A dose related increase in α-MSH release was observed when TRH was added to the perifusion medium. Half-maximum stimulation occurred with the 1 × 10−8M dose. Theophylline at a dose of 2 × 10−3M strongly enhanced TRH-induced α-MSH release, indicating that cyclic AMP may be the second messenger. α-MSH releade was not modified by crude homogenates of rat hypothalamus but was significantly reduced when the hypothalamus extracts were preincubated with specific TRH antibodies. As far is known, these results provide the first evidence that P-Glu-His-Pro-NH2 stimulates the release of α-MSH from frog neurointermediate lobes in vitro . The present findings suggest a possible feedback loop between skin TRH and pituitary MSH in Amphibia.

Atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog

The distribution of atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog Rana ridibunda was investigated by indirect immunofluorescence and the immunogold technique, using an antiserum generated in rabbits against synthetic atrial natriuretic factor (Arg 101-Tyr 126). A stereotaxic atlas of neurons containing atrial natriuretic factor-like material was prepared to show the widespread distribution of atrial natriuretic factor-positive cell bodies and fibres in the brain. Appreciable numbers of immunoreactive perikarya were observed in the dorsal and medial pallium, the medial septal nucleus, the anteroventral and ventrolateral areas of the thalamus, the lateral forebrain bundle, the posterocentral and posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus and the anteroventral tegmental nucleus of the mesencephalon. A heavy accumulation of atrial natriuretic factor-like immunoreactive cell bodies and very dense fibre bundles were noted in the interpeduncular nucleus of the mesencephalon. Fibres were generally seen where cell bodies were observed, particularly in all regions of the pallium and septum nuclei, in the ventral thalamus, the infundibular nucleus and the tegmental area. Moderate numbers of fibres were also noted in several regions where cell bodies were absent, mainly in the amygdala and the infundibular nucleus, the median eminence and most mesencephalic regions. At the electron microscopic level, the immunoreactivity was restricted to dense core vesicles and absent in clear vesicles. These results provide the first evidence for the presence of atrial natriuretic factor in the brain of a non-mammalian chordate. The localization of atrial natriuretic factor-positive material in the frog central nervous system suggests that this peptide may act as a neuromodulator or a neurotransmitter in amphibians.

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.

Serotonin stimulates corticosteroid secretion by frog adrenocortical tissue in vitro

The mode of action of serotonin (5-HT) in the regulation of frog adrenal steroidogenesis was studied in vitro using the perifusion system technique. Graded doses of 5-HT (from 10(-8) to 10(-6) M) increased both corticosterone and aldosterone production in a dose-dependent manner. Short pulses (20 min) of 10(-6) M 5-HT, administered at 130 min intervals within the same experiment, did not cause any desensitization phenomenon. Indomethacin (IDM; 5 microM), a cyclooxygenase inhibitor which induced a dramatic decrease in the spontaneous secretion of corticosteroids, did not impair the stimulatory effect of 5-HT on corticosterone and aldosterone production. In the absence of calcium, 5-HT (10(-6) M) was still able to stimulate corticosteroid production. Dantrolene (5 x 10(-5) M), a blocker of calcium mobilization from intracellular pools which significantly inhibited the spontaneous production of corticosteroids, did not suppress 5-HT-evoked corticosteroid secretion. These results show that 5-HT, stored in adrenal chromaffin cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in the frog. Our data also indicate that the mechanism of action of 5-HT does not depend on prostaglandin biosynthesis.