Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Structural analysis of the oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana temporaria

The carbohydrate chains of the mucins which constitute the jelly coat surrounding the eggs of Rana temporaria were released by alkaline borohydride treatment. Neutral and acidic oligosaccharide-alditols were purified by ion-exchange chromatography and HPLC. From the structural analysis, based upon 1H and 13C-NMR spectroscopy in combination with MALDI-TOF, the following glycan units are proposed. Abbreviations: MALDI-TOF, matrix assisted laser desorption ionization - time of flight; HPLC, high performance liquid chromatography; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; HMQC, heteronuclear multiple-quantum coherence spectroscopy; ROESY, rotating-frame overhauser enhancement spectroscopy; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GalNAc-ol, N-acetylgalactosaminitol; GlcA, glucuronic acid     

Middle ear transmission in the grass frog, Rana temporaria

The anuran middle ear serves to transmit eardrum vibrations to the inner ear. In order to do this efficiently, the eardrum and middle ear must operate as an impedance transformer matching the low impedance of air to the higher impedance of the fluid-filled inner ear. In amniotes, one of the mechanisms used to achieve impedance transformation is to have the middle ear work as a force-amplifying lever system. Here, we present evidence that the grass frog middle ear also implements a lever system. The columellar footplate, which sits in the oval window, is firmly connected to the otic capsule along its ventral edge. Therefore, simple in-out movement of the columella is prevented while a rotational movement around the footplate's ventral edge is possible. The latter movement pattern was confirmed by laser vibrometry measurements of eardrum and footplate vibrations. The results showed that the footplate vibrations were 20–30 dB weaker than those of the eardrum and that the two structures vibrated 180° out of phase (at low frequencies). The lever ratio was approximately 6, i.e. somewhat higher than lever ratios reported for amniotes. Hence, the middle ear lever probably makes a significant contribution to impedance matching in frogs. Accepted: 1 July 1997     

Anuran abundance and persistence in agricultural landscapes during a climatic extreme

Climate change in concert with habitat loss and degradation are major threats to global biodiversity. As part of climate change, the occurrence of extreme climatic events is expected to increase. Agricultural intensification has led to the increased homogeneity of agricultural habitats and declines in farmland species diversity. We investigated the abundance of common frog Rana temporaria populations in boreal farmlands in relation to multiple scales of landscape structure during years 2002–2003, and the effect of habitat structure on R. temporaria population persistence during a severe drought. On average, 113 and 24 egg clutches were observed per site in years 2002 and 2003, respectively. This dramatic decline was synchronized over the entire study area (ca. 320 km²), with some local variations. Population persistence was higher in sites where ground water levels decreased less but it was also positively correlated with the heterogeneity of the adjacent landscape. Under normal weather conditions, local habitat characteristics had a dominating role on R. temporaria population abundance, as it was positively correlated with the amount of ditches and ponds within the study sites. After the drought, however, population abundance was related to landscape and regional level factors. The results indicate that landscape homogenization can have negative effects on population persistence during climate change. They also show that the scale at which landscape characteristics affect populations can be strongly dependent of processes functioning at large scales, such as weather. In summary, heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations and serve as sources of recovery in postdisturbance recovery phases. Thus, maintaining such areas should be promoted in long-term biodiversity conservation.     

Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development

Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat α-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II–III tadpoles and in successive larval stages. Soon after hatching, stages II–III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI–XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone. Received: 28 July 1997 / Accepted: 8 December 1997     

Reaktionsmuster verschiedener Mittel-und Zwischenhirnzentren von Rana temporaria L. nach Unterbrechung der Nervenbahnen des Pinealkomplexes

Zusammenfassung Der Einfluß des lichtempfindlichen Pinealkomplexes (Stirnorgan und Epiphysis cerebri) von Rana temporaria auf dorsale diencephale und mesencephale Hirnzentren breitet sich über ein größeres Gebiet aus als man bisher angenommen hat. Nach operativer Unterbrechung des Nervus- und Tractus pinealis wurde mit Punktzählverfahren die Dichte der Zell- und Zellkernvolumina sowie der Kapillaroberfläche im dorsalen Thalamus, mesencephalen (postthalamischen) zentralen Grau, Ganglion habenulae, Tectum opticum, in der Area praetectalis und im Subcommissuralorgan (Sco) bestimmt. Die morphometrischen Meßergebnisse lassen sich wie folgt interpretieren: 1. Der Pinealkomplex wirkt auf das Sco, das zentrale Grau und die Area praetectalis ein (vgl. Paul u. Mitarb., 1971); das Sco wird in erster Linie über das Stirnorgan beeinflußt. 2. Weitere Kerngebiete des Zwischenhirns (Ganglion habenulae und dorsaler Thalamus) gehören funktionell zu dem vom Pinealkomplex kontrollierten System. 3. Die von Kemali und Braitenberg (1970) beschriebene Asymmetrie der Ganglia habenularum zeigt sich auch im funktionellen Zusammenspiel dieser Kerngebiete mit dem Pinealkomplex, dem dorsalen Thalamus und dem dorsalen Abschnitt des zentralen Graus. Die in Stirnorgan und Epiphysis cerebri transformierten Lichtreize werden in Hirnabschnitte weitergeleitet, die olfaktorische und retinale Signalmuster verarbeiten. Über die biologische Bedeutung der nervösen Beziehungen des Stirnorgans zum sekretorischen Sco können noch keine Angaben gemacht werden.

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Study of mesencephalic and diencephalic centers after transection of the centripetal nervous pathways of the pineal complex in Rana temporaria L.

Summary Nervous influences of the pineal complex of the frog, Rana temporaria, exerted upon mesencephalic and diencephalic centers were investigated using morphometric methods after the transection of the nervus or the tractus pinealis. Densities of the cell- and nuclear volumes and of the capillaries were measured by a point-counting method in the dorsal thalamus, the mesencephalic (postthalamic) central gray, the habenular ganglion, the optic tectum, the pretectal area, and the subcommissural organ (sco). The morphometric measurements showed: 1. A functional influence of the pineal complex on the sco, the central gray, and the pretectal area (see Paul et al., 1971); the frontal organ seems to be primarily related to the sco. 2. The habenular ganglia and the dorsal thalamus belong to a system which is controlled by the pineal complex. 3. The morphological asymmetry of the habenular ganglia (Kemali and Braitenberg, 1970) has an influence on the functional interactions between the pineal complex, the dorsal thalamus, and the central gray. The impulses arising in the light-sensitive pineal complex of the frog are conducted to brain regions which are known to integrate olfactory and retinal sensory inputs. The functional significance of the nervous connexion of the frontal organ with the secretory sco is still open to discussion.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Arbeitskreis Prof. Dr. A. Oksche).     

Die Ontogenese der aminhaltigen Neuronensysteme im Gehirn von Rana temporaria

Zusammenfassung Vorkommen und Verteilung biogener Amine im Gehirn von Rana temporaria-Kaulquappen wurden fluoreszenzmikroskopisch untersucht. Catecholaminhaltige Perikaryen erscheinen ab Stadium 20 im Nucleus reticularis mesencephali, im Tuber cinereum und im Bulbus olfactorius, ab Stadium 22 in den Flügelplatten der Medulla oblongata und in der Area praeoptica. Ab Entwicklungsstufe 20 zeigen sich ventrolateral in Medulla oblongata und Mittelhirn, lateral vom Organon vasculosum hypothalami, im Bereich des medialen Vorderhirnbündels und im Striatum catecholaminhaltige Faseranschwellungen, ab Stadium 22 außerdem in der Eminentia mediana und dem Hypophysenzwischenlappen, in der Commissura transversa (bis zur Stufe 26), in der Commissura anterior (bis zur Stufe 26) und in der Pars ventrolateralis nuclei lateralis septi. Im Striatum ist von dieser Entwicklungsstufe an ein zweites Areal mit grün fluoreszierenden Varikositäten nachweisbar. Ab Stadium 26 finden sich auch in der Pars dorsolateralis des lateralen Septumkerns catecholaminhaltige Faseranschwellungen.Ab Entwicklungsstufe 22 sind 5-HT-haltige, gelb fluoreszierende Perikaryen im Nucleus raphes und in seiner Umgebung zu beobachten, gelb fluoreszierende Varikositäten im Nucleus interpeduncularis und zwischen medialem und lateralem Septumkern.

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Ontogeny of the amine-containing nerve cell systems in the brain of Rana temporaria

Summary The occurrence and distribution of biogenic amines in the brain of Rana temporaria tadpoles have been investigated with the fluorescence-microscope. From the embryonic developmental stage 20 onwards catecholamine-containing cell bodies are shown to be present in the nucleus reticularis mesencephali, the tuber cinereum and the olfactory bulb, and from stage 22 onwards also within the dorsolateral areas of the medulla oblongata and within the preoptic area. Catecholamine-containing enlargements of nerve fibres occur in the ventrolateral parts of the medulla oblongata and the midbrain, in an area lateral to the hypothalamic organon vasculosum, within the region of the medial forebrain bundle and within the striatum, in all stages following stage 20. These enlargements also occur in the median eminence and the pars intermedia of the hypophysis, in the commissura transversa (up to stage 26), in the commissura anterior (also up to stage 26) and in the pars ventrolateralis nuclei lateralis septi in all stages after 22. From the same stage onwards a second area of green fluorescent varicosities can be demonstrated within the striatum. After stage 26 catecholamine-containing enlargements of nerve fibres additionally are to be found in the dorsolateral part of the lateral septal nucleus.After appearing at stage 22 5-HT-containing, yellow fluorescent perikarya are to be observed within the nucleus raphes and its neighbourhood, and yellow fluorescent varicosities in the interpeduncular nucleus and in an area between the medial and the lateral septal nucleus.

Herrn Professor Dr. med. W. Bargmann zum 65. Geburtstag gewidmet.     

Scanning electron microscopy of the bullfrog's retina and pigment epithelium

Summary The retina and pigment epithelium of the bullfrog (Rana catesbiana) were studied with the scanning electron microscope. Fixed-dehydrated tissues were critical point dried with CO₂, then cracked in the plane of the long axis of the photoreceptors. The cellular layers of the retina and the lateral surfaces of pigment epithelial cells were visualized. The four major types of frog photoreceptor were identified: red rod, green rod, single cone, and double cone. Cone myoids were observed to be contracted in light-adapted retinas and elongated in more dark adapted retinas.This work was supported by a career development award EY-18,083 to the author and research grant EY 00468 to Dr. Kenneth T. Brown.The author gratefully acknowledges the skillful technical assistance of Ms. Maria T. Maglio.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.