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PubMed2005

Ontogeny of central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui.

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1-15; 1 = oviposition/15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.

Animals↗
PubMed1996

Localization of diazepam-binding inhibitor-related peptides and peripheral type benzodiazepine receptors in the frog adrenal gland.

The adrenal gland of mammals contains high concentrations of peripheral-type benzodiazepine receptors (PBR) and diazepam-binding inhibitor (DBI), a polypeptide which acts as an endogenous ligand for PBR. The aim of the present study was to investigate the localization of DBI and PBR in the adrenal gland of the frog Rana ridibunda. Reverse transcription followed by polymerase chain reaction with specific primers for the frog DBI cDNA showed the presence of DBI mRNA in frog adrenal gland extracts. The cellular distribution of DBI and PBR was investigated using an antiserum against the octadecaneuropeptide DBI [33-50] (ODN) and antibodies against the 18-kDa isoquinoline binding protein subunit of PBR (IBP), respectively. ODN-like immunoreactivity was found in chromaffin cells and in Stilling s cells, but not in adrenocortical cells. IBP-like immunoreactivity was observed in chromaffin cells, in Stilling s cells and in a small proportion (11%) of steroid-secreting cells. The ODN- and IBP-immunoreactive materials were homogeneously distributed in the cytoplasm of chromaffin cells and concentrated at the periphery of large cytoplasmic vesicles in Stilling s cells. The proportion of ODN-positive Stilling s cells showed marked circannual variations with a maximum in July. Similarly, the proportion of IBP-positive Stilling s cells was 17 times higher in July than in December. These results indicate that, in the frog adrenal gland, DBI-related peptides and PBR are simultaneously expressed in chromaffin cells and Stilling s cells, suggesting that endogenous ligands for PBR may play a physiological role in the control of adrenal cell activity.

Adrenal Glands↗
PubMed1998

In situ characterization of mast cells in the frog Rana esculenta.

The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253+/-45 mast cells/mm2) was significantly higher than that of the heart (5.3+/-0. 4/mm2) and kidney (15.3+/-1.4 /mm2). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue+/safranin+ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue-/safranin+. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue+/safranin-. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell ( approximately 0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues ( approximately 3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.

Animals↗
PubMed2005

Thyroid endocrine disruption in stonerollers and cricket frogs from perchlorate-contaminated streams in east-central Texas.

In October 2001 and March 2002, a field survey of central stonerollers (Campostoma anomalum) from perchlorate-contaminated streams in central Texas was conducted to assess thyroid endocrine disruption. A survey of adult male and female cricket frogs (Acris crepitans) was performed at the same site between 2001 and 2003. Perchlorate is an oxidizer primarily used in solid-fuel rockets, and many sites that processed or used perchlorate are now contaminated. Histological analysis revealed that the fish from contaminated sites had increased thyroid follicular hyperplasia, hypertrophy, and colloid depletion. Multivariate analysis was generally found to be more powerful than univariate analysis. Seasonal differences existed in the degree of thyroidal perturbation were discovered, and fish were generally less sensitive to thyroidal perturbations in March compared to October. Thyroidal histological indicators were also correlated to levels of perchlorate in the fish, water, and periphyton. Periphyton was frequently most strongly correlated to thyroidal indices, suggesting that exposure through the food chain may be of import. In addition, one of the presumed reference sites turned out to be contaminated with perchlorate, and this was reflected by thyroidal biomarkers before perchlorate was detected in the stream water or biota. There was no evidence of colloid depletion or hyperplasia in frogs from any of the sites, although frogs from two sites with greatest mean water perchlorate concentrations exhibited significantly greater follicle cell hypertrophy. Furthermore, there was a significant positive correlation between follicle cell height and mean water perchlorate concentrations for frogs collected from all sites. This is the first known published account of perchlorate-induced thyroid disruption in fish under field situations, only the second known published account for amphibians, and also points out the value of biomarkers for contaminant biomonitoring.

Animals↗
PubMed1981

Isolation of frog heart sarcolemma possessing (Ca2+ + Mg2+)-ATPase and Ca2+ pump activities.

The presence of a (Ca2+ + Mg2+)-ATPase and Ca2+ pump in frog heart sarcolemma was investigated. Frog cardiac sarcolemma was isolated by using a modification of a recent procedure for isolating sarcolemma from dog heart (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 17-39). Despite the sparseness of sarcoplasmic reticulum in frog heart, this tissue possessed (Ca2+ + Mg2+)-ATPase activity which appeared endogenous to the sarcolemma. The purified sarcolemmal vesicles also possessed ouabain-sensitive (Na+ + K+)-ATPase, Na+ -Ca2+ exchange activity and Ca2+ pump activity. Inclusion of ionophore A23187 into the assay medium resulted in an approximately 5-fold increase in (Ca2+ + Mg2+)-ATPase which may be due to removal of inhibitory concentrations of Ca2+ accumulated within the membrane vesicles by an ATP-dependent process. Vesicles pre-loaded with 45Ca2+, via Na+-Ca2+ exchange, lost 92% of their Ca2+ when ATP was present and only 60% in the absence of ATP. The results indicate that frog heart sarcolemma possesses a (Ca2+ + Mg2+)-ATPase and an ATP-dependent Ca2+ pump which may be involved in the outward pumping of Ca2+ from the heart cell.

Animals↗
PubMed1981

Acidification and sodium entry in frog skin epithelium.

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na+ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na+ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na+ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO2; and (d) the positive correlation between net Na+ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1975

Regulation of hepatic tyrosine aminotransferase in the frog Rana temporaria.

The regulation of hepatic tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) in the rat has been extensively studied but little is known about the enzyme from other sources. We have studied the regulation of this enzyme in the frog Rana temporaria and in this paper we report that: 1. Cortisone acetate, adrenocorticotropic hormone and alpha-methyl-p-tyrosine, an agent known to induce hepatic tyrosine aminotransferase in the rat via activation of the pituitary-adrenal axis, have no effect on the activity of the enzyme in the frog. 2. Dibutyryl-3',5'-cyclic AMP induces the enzyme to about 2-fold. 3. Injection of tyrosine methyl ester and a protein-rich diet result in an increase in the enzyme activity. This increase is of the same order of magnitude as that caused by dibutyryl cyclic AMP. 4. Glucose significantly reduces tyrosine aminotransferase activity in frog liver. These results suggest that cyclic AMP induces the enzyme via a mechanism independent of glucocorticoids. The frog offers a model for studies on the regulation of hepatic tyrosine aminotransferase in vivo without interference from secondary effects mediated by the adrenals.

Adrenocorticotropic Hormone↗
PubMed1978

Structural properties of frog muscle myosin.

Frog myosin is a labile molecule, undergoing irreversible aggregation and rapid loss of ATPase; however, a procedure is described which provides highly purified myosin, with stable solubility and enzymatic properties, from skeletal muscle of Rana catesbeiana. Frog myosin contains heavy chains and light chains 1, 2, and 3. Light chain 3 is present in excess over light chain 1, and light chain 2 may occur as either, or both, of 2 closely migrating bands. On two-dimensional electrophoresis, light chain 1 generates an isoelectric component with pK 5.60; light chain 2 generates a complex pattern with 3 or 4 major components; and light chain 3 generates 2 major components with pK 5.00 and 4.92. The same subunit composition is obtained for frogs acclimated at 25 and 5 degrees C; however, proteolytic artifacts may occur in myosin preparations purified in the absence of protease inhibitors, especially in warm-acclimated frogs.

Acclimatization↗
PubMed1983

Delayed induction of the cell body response and enhancement of regeneration following a condition/test lesion of frog peripheral nerve at 15 degrees C.

Axons subjected to a prior injury exhibit an enhanced regenerative capability following a subsequent lesion. This phenomenon, known as the 'conditioning effect' has been demonstrated in both central and peripheral axons from a variety of species, but the locus of enhancement has remained in doubt. Several reports have tentatively concluded that the effect involves processes located in the cell body of the injured axon, while other investigators have implicated local processes at the site of the injury. The present experiments made use of a previous observation, the apparent suppression of the cell body response in injured frog neurons when the animals are housed at 15 degrees C, to investigate the location of the conditioning effect. The effect occurred in frog neurons from animals at 25 degrees C in the presence of a cell body response. The prior (conditioning) lesion produced a significant increase in regeneration rate (12-17%) and a significant decrease in the latency to the onset of regenerative outgrowth (12%). In contrast, regeneration was initially unaffected by the earlier conditioning lesion when the frogs were kept at 15 degrees C. After a 26-30 day interval, however, the rate of regeneration increased by 27%, coincident with the appearance of chromatolysis in the injured cell bodies. The 'conditioned' axons also continued to elongate, in contrast to singly-lesioned axons at 15 degrees C, and eventually reinnervated their target muscles. The cell body appeared to be the primary locus of the 'conditioning effect' in these experiments. This conclusion was further examined by measuring regenerative outgrowth in 15 degrees C neurons in which a cell body response had been induced prior to the test lesion. This was accomplished by maintaining frogs at 25 degrees C for 7 days after the conditioning lesion. The animals were then returned to the 15 degrees C environment for an additional 7 days before producing the test lesion. With this paradigm, regenerative outgrowth was enhanced without the 26-30 day delay interval. These results also suggest that the conditioning effect stimulates processes in the cell body of the injured axon, but the experiments do not indicate which processes were affected.

Animals↗
PubMed1985

Pharmacological activation of locomotor patterns in larval and adult frog spinal cords.

The effects of amino acids, catecholamines, and their agonists shown to elicit locomotor activity in several vertebrate species were examined in spinal animals and isolated nervous systems of developing tadpoles (Rana catesbiana) and adult frogs (R. catesbiana and pipiens). Elicited activity was correlated in spinal animals by video and electromyographic analysis, and in in vitro spinal cords by recordings of tail and hindlimb motor activity. Of the agents tested, only N-methyl-DL-aspartate (NMA), an amino acid agonist, was effective in eliciting motor activity in spinal animals. In isolated nervous systems, both NMA and D-glutamate added to the bath activated locomotor activity. NMA injected i.p. into tadpoles with high spinal cord transections elicited coordinated swimming motor activity in axial and hindlimb muscles that was roughly typical for the stage of development of the animal. In late stage tadpoles (st. XX), NMA also elicited wiping and alternating or synchronous (i.e. kicking or jumping) hindlimb movements. Addition of NMA or glutamate to a bath containing an in vitro tadpole spinal cord preparation elicited ventral root motor activity characteristic of swimming, but without a rostrocaudal phase lag. Rhythmic activity thought to underlie stepping and kicking was seen in lateral ventral rootlets innervating the hindlimbs. In adult frogs with high spinal cord transections, injection of NMA elicited a general sequence of spontaneous hindlimb motor functions: reflex wiping, stepping, and kicking or jumping. Isolated frog spinal cords were not responsive to bath applied NMA, under the present conditions. The activation by amino acids or their agonists of different motor functions in both larval and adult frogs, as well as in higher and lower vertebrates, suggests a general significance of amino acid-activated receptors in the neural networks controlling locomotor function.

2-Aminoadipic Acid↗
PubMed1986

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

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. alpha-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 alpha-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive alpha-MSH coeluted with synthetic des-N alpha-acetyl alpha-MSH, authentic alpha-MSH and their sulfoxide derivatives. The heterogeneity of alpha-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-N alpha-acetyl alpha-MSH is present. These findings support the existence of discrete alpha-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic alpha-MSH.

Animals↗
PubMed1990

The asymmetry of the habenular nuclei of female and male frogs in spring and in winter.

Male and female frog brains stained according to the Nissl method and cut transversely, 15 microns thick, at the level of the habenular nuclei, were investigated in spring and winter. Right and left habenular nuclei were examined. The volume and the standard deviations were calculated in each portion of the habenular nuclei investigated. The frog habenula consists of a single cell group: one on the right and two on the left side of the brain which differ, among themselves, both in the volumes of the neuropil and of the cellular ring. Functional corollaries of this striking asymmetry are still unknown. However, female and male frogs' habenular nuclei are longer and larger in spring--when frogs are sexually active--than in winter. We propose that structural brain asymmetries may be sex linked and may be induced by steroid hormonal effect in the central nervous system.

Animals↗
PubMed1990

Human odor intensity perception: correlation with frog epithelial adenylate cyclase activity and transepithelial voltage response.

Although a number of odorants are hypothesized to depolarize frog olfactory receptor cells by binding to ciliary glycoproteins which activate membrane-bound G-proteins to induce adenylate cyclase-mediated increases in intracellular cAMP (cyclic adenosine 3',5'-monophosphate), it is not known whether these odorants influence human odor perception via similar mechanisms. In this paper we present evidence derived from odor attribute ranking and multidimensional scaling procedures that the perceived intensity of such odorants to humans is correlated with (a) the amount of adenylate cyclase activity they induce in an in vitro frog olfactory cilia preparation and (b) the magnitude of their influence on the frog transepithelial voltage response or electro-olfactogram (EOG). These observations are in accord with the hypothesis that the perception of the intensity of some odors by humans is associated with cAMP-related epithelial processes and imply that remarkable homologies exist between the intensity-related olfactory receptor mechanisms of frog and man.

Adenylyl Cyclases↗
PubMed1992

Identification of a B-50-like protein in frog brain synaptosomes.

Vestibular compensation in the frog following unilateral labyrinthectomy is accompanied by distinct changes in the endogenous phosphoprotein patterns in total frog brain homogenate and isolated synaptosomes. The purpose of this study was to characterize one of these proteins, an acidic 45-kDa synaptosomal protein, resembling in some of its features the growth-associated protein GAP-43/B-50. Our results demonstrate by comparative analysis with purified rat B-50/GAP-43 that the 45-kDa protein (IP 4.8) in synaptosomal membranes of frog brain is phosphorylated by added purified PKC, cross-reacts with affinity-purified rabbit antibodies to rat B-50 and exhibits a Staphylococcus aureus V8 protease peptide digestion pattern corresponding to rat B-50. Therefore, we conclude that the acidic 45-kDa synaptosomal protein is a growth-associated B-50-like protein, probably involved in processes responsible for compensatory reorganization of the vestibular structures after hemilabyrinthectomy in the frog.

Animals↗
PubMed1995

Distribution of two molecular forms of gonadotropin-releasing hormone (GnRH) in the central nervous system of the frog Rana ridibunda.

Two molecular forms of gonadotropin-releasing hormone (GnRH) have been recently characterized in the brain of the frog Rana ridibunda i.e. mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). Using highly specific antisera against each form of GnRH, we have investigated the distribution of these two neuropeptides in the frog brain by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. mGnRH-immunoreactive cell bodies were restricted to a well defined region corresponding to the septal-anterior preoptic area. mGnRH-containing fibers projected through the ventral diencephalon and ended in the median eminence. In contrast, cGnRH-II-immunoreactive structures were widely distributed in the frog brain. In the telencephalon cGnRH-II-positive elements formed a ventromedial column extending from the olfactory bulb to the septal area, a pathway which corresponds to the terminal nerve. A dense accumulation of cGnRH-II-immunoreactive cell bodies was also found in the septal-anterior preoptic area; these neurons sent processes towards the median eminence via the hypothalamus. Double immunostaining revealed that, in this area, mGnRH- and cGnRH-II-like immunoreactivity co-existed in the same neurons. In the mid-diencephalon, numerous cGnRH-II-immunoreactive perikarya were found, surrounding the third ventricle, in the posterior preoptic and infundibular areas. Many of these neurons sent processes towards the ventricular cavity. More caudally, a dense population of cGnRH-II-immunoreactive perikarya was also observed in the nucleus of the paraventricular organ and the posterior tubercle. Dorsally, the thalamus, the tegmentum, the tectum and the granular layer of the cerebellum were richly innervated by cGnRH-II-positive fibers. In the medulla oblongata, numerous cGnRH-II-immunoreactive perikarya were seen in several cranial nerve nuclei. Ventrally, a dense plexus of immunoreactive fibers projected rostrocaudally into the spinal cord. The occurrence of mGnRH- and cGnRH-II-like immunoreactivity in the septal-anterior preoptic area and the hypothalamo-pituitary pathway supports the view that both peptides act as hypophysiotropic neurohormones. The widespread distribution of cGnRH-II-immunoreactive elements in the central nervous system of the frog strongly suggests that this peptide may also exert neuromodulator and/or neurotransmitter activities.

Animals↗
PubMed1993

Ganglion-blocking activity of dequalinium in frog and rat sympathetic ganglia in vitro.

The effect of dequalinium on ganglionic transmission and responses to exogenous acetylcholine receptor agonists were studied in frog and rat sympathetic ganglia. In frog ganglia, dequalinium reduced ganglionic transmission (measured using gross extracellular recording) with an EC50 of 2 microM. At 1 microM, dequalinium produced non-surmountable antagonism of the ganglion depolarization evoked by nicotinic receptor activation. Dequalinium reduced the amplitude of evoked and spontaneous excitatory postsynaptic potentials recorded intracellularly from frog neurones, but had no effect on the action potential elicited by injection of a depolarizing current pulse. Hyperpolarizing and depolarizing responses to muscarine recorded extracellularly from frog ganglia were antagonized by 3 microM dequalinium. In rat ganglia, synaptic transmission and depolarization by an exogenous nicotinic agonist were only slightly inhibited by dequalinium at a concentration of 100 microM. At 30 microM, dequalinium produced a 17% reduction in the depolarization of this tissue by muscarine (1 microM). These results add to the evidence of the diversity of nicotinic and muscarinic acetylcholine receptors, and indicate that dequalinium may be a valuable tool for the study of these receptors. However its usefulness as a probe for calcium-activated potassium channels may be limited by its actions at acetylcholine receptors.

Action Potentials↗
PubMed1985

Effects of cold adaptation and starvation on sciatic nerve fibers in the frog.

The conditions under which frogs are kept prior to experimentation were found to have a measurable effect on peripheral nerve structure. Frogs kept for 12 weeks at 4 degree C had markedly shrunken sciatic nerve fibers compared with frogs kept at 19 degrees C. Intermediate fiber shrinkage was found for frogs kept at 19 degrees C without feeding. Counts of neurofilaments and microtubules showed that fiber shrinkage was from a preferential loss of filaments, indicating cold- or starvation-induced atrophy of the axon's cyto-skeleton. This effect, however, was superimposed with additional osmotic axonal shrinkage, causing filament densities to increase per area. There were no changes in myelin sheath thickness due to cold adaptation or fasting.

Adaptation, Physiological↗
PubMed1986

An examination of the ability of inositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea.

We have examined the ability of inositol 1,4,5-trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10-500 microM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impermeant anion by Cl-, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg2+ was lowered to 40 microM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0-0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4 degrees to 22 degrees C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3-induced calcium release from rat liver microsomes, had no effect at 50 microM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.

2,3-Diphosphoglycerate↗