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Plane-specific brainstem commissural inhibition in frog second-order semicircular canal neurons.

Commissural inputs of identified second-order semicircular canal neurons were studied by separate stimulation of each of the three canal nerves on either side in the vitro frog brains. The spatial pattern of these inputs was further investigated in those second-order canal neurons that received a monosynaptic input from only one ipsilateral canal nerve (91%). Since similar results were obtained in the presence as in the absence of the cerebellum, commissural inputs must have been relayed via fibers crossing in the brainstem. Following stimulation of individual semicircular canal nerves, commissural inputs were either inhibitory or excitatory. A commissural inhibition was evoked in the majority of the recorded neurons (79%) by stimulation of the coplanar semicircular canal nerve on the contralateral side. In the remaining neurons, a commissural excitatory input was evoked. A commissural excitation, originating from the two noncoplanar semicircular canals, predominated in most (68%) of the recorded neurons and was independent of the type of second-order canal neuron. The onset latency of the canal plane-specific commissural inhibitory potentials was di- or trisynaptic. Stimulation of the contralateral VIIIth nerve evoked excitatory commissural responses. The canal plane-specific commissural inhibition therefore might have been masked by commissural excitatory responses as in earlier studies. The similar organization of the canal plane-specific commissural inhibition in frog and cat corroborates the notion of a phylogenetically conservative, basic vestibular organization. The presence of a canal plane-unspecific commissural excitation, however, appears to be a feature that is specific to frogs. The functional implications of these similarities and differences are discussed.

Animals↗

Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.

Animals↗

GABAergic inhibition shapes frequency tuning and modifies response properties in the auditory midbrain of the leopard frog.

The functional role of GABAergic inhibition in shaping the frequency tuning of 96 neurons in the torus semicircularis of the leopard frog, Rana pipiens, was studied using microiontophoresis of the GABAA receptor antagonist, bicuculline methiodide. Bicuculline application abolished, or reduced in size, the inhibitory tuning curves of 72 neurons. In each case, there was a concommitant broadening of the excitatory tuning curve such that frequency-intensity combinations that were inhibitory under control conditions, became excitatory during disinhibition with bicuculline methiodide. These effects were observed irrespective of the excitatory tuning curve configuration prior to bicuculline methiodide application. Results indicate an important role for GABA-mediated inhibition in shaping the frequency selectivity of neurons in the torus semicircularis of the leopard frog. Bicuculline application also affected several other response properties of neurons in the leopard frog torus. Disinhibition with bicuculline methiodide increased both the spontaneous firing rate (18 cells) and stimulus-evoked discharge rate (81 cells) of torus neurons, decreased the minimum excitatory threshold for 18 cells, and altered the temporal discharge pattern of 47 neurons. Additional roles for GABAergic inhibition in monaural signal analysis are discussed.

Acoustic Stimulation↗

Effect of aestivation on muscle characteristics and locomotor performance in the green-striped burrowing frog, Cyclorana alboguttata.

The Green-striped burrowing frog, Cyclorana alboguttata survives extended drought periods by burrowing underground and aestivating. These frogs remain immobile within cocoons of shed skin and mucus during aestivation and emerge from their burrows upon heavy rains to feed and reproduce. Extended periods of immobilisation in mammals typically result in muscle atrophy and a decrease in muscle performance. We examined the effect of aestivation and hence prolonged immobilisation, on skeletal muscle mass, in vitro muscle performance, and locomotor performance in C. alboguttata. Frogs were aestivated in soil for 3 months and were compared with control animals that remained active, were fed, and had a continual supply of water. Compared to the controls, the wet mass of the gastrocnemius, sartorius, gracilus major, semimembranosus, peroneus, extensor cruris, tibialis posticus and tibialis anticus longus of aestivators remained unchanged indicating no muscle atrophy. The in-vitro performance characteristics of the gastrocnemius muscle were maintained and burst swimming speed was unaffected, requiring no recovery from the extended period of immobilisation associated with aestivation. This preservation of muscle size, contractile condition and locomotor performance through aestivation enables C. alboguttata to compress their life history into unpredictable windows of opportunity, whenever heavy rains occur.

Animals↗

Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog.

The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4 x 10(-11) to 10(-6) M ANP or 10(-7) to 3 x 10(-4) M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na+-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5 x 10(-7) M KT-5720 and >10(-5) M H-89), but not by a specific PKG inhibitor (5 x 10(-7) M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na+ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na+ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10(-4) M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na+ absorption of the frog urinary bladder.

Animals↗

AVP-independent high osmotic water permeability of frog urinary bladder and autacoids.

In the isolated frog urinary bladder a 20- to 50-fold increase of the osmotic water permeability has been revealed in the absence of arginine vasopressin (AVP) as a result of several successive changes of the serosal Ringer solution. This increase of the osmotic water permeability was of the same magnitude as that of the effect of 1 nM AVP. Similarly to the effect of AVP, the amount of adenosine 3',5'-cyclic monophosphate (cAMP) in the cells rose, and aggregates of intramembraneous particles were formed in the apical plasma membrane of granular cells (as shown by the freeze-fracture method). Immunocytochemical studies using anti-actin monoclonal antibodies indicated depolymerization of F-actin following the AVP-independent change in water permeability. It was possible to decrease the high level of osmotic permeability to the initial level if 10 microl/ml of frog blood serum or a lipid extract of this blood serum, or 1 microM arachidonic acid or 1 nM prostaglandin E2 was added to the serosal Ringer solution. The rapid restoration of the osmotic water impermeability of the epithelium after the AVP- evoked effect was achieved by the addition to the serosal Ringer solution of Ringer solution in which intact frog urinary bladders had been previously incubated for 1 h. The data obtained indicate that the maintenance of the impermeability to water of the osmoregulating epithelium and the restoration of the initial low level of the osmotic permeability after the effect of AVP are due to participation of prostaglandin E2 and other autacoids as well as, probably, some physiologically active substances of a lipid nature that are present in the blood serum.

Animals↗

Examining the timing of miniature endplate potential releases at the frog and mouse neuromuscular junctions for deviations from Poisson expectations.

The intervals between miniature endplate potentials (MEPPs) were measured at frog and mouse neuromuscular junctions in several different solutions. Data sets with monotonic trends in MEPP frequency were discarded. The remaining data sets had between 283 and 5024 MEPPs. The fit to the exponential distribution, which describes a Poisson process, was tested with Sherman's statistic. If the intervals are not distributed exponentially, this statistic indicates whether they deviate because they are more concentrated or more diffuse. In 6 of 20 data sets from the frog and in 3 of 7 data sets from the mouse the intervals between MEPPs were not distributed exponentially. Some of these were clustered, while others were diffuse. In one frog data set release was periodic. In all data sets releases appeared to be independent, judging from the integrated power spectra. One data set may show fractal behavior. Calculations of the approximate entropy gave no indication of an underlying regularity, so there is no evidence for a chaotic process. The lack of fit to the exponential distribution due to either concentrated or diffuse interval distributions is mimicked by a model in which release is Poisson, but with a mean rate that randomly shifts to a different level.

Action Potentials↗

Membrane potential and conductance of frog skin gland acinar cells in resting conditions and during stimulation with agonists of macroscopic secretion.

Frog skin glands were stripped of connective tissue and investigated using the nystatin-permeabilized whole-cell patch-clamp configuration. The membrane potential in unstimulated acinar cells was -69.5+/-0.7 mV, and the conductance was dominated by K+, based on ion substitution experiments. The cells were electrically coupled through heptanol- and halothane-sensitive gap junctions. During application of gap junction blockers, the whole-cell current/voltage relationship displayed strong outward rectification. Outward currents were blocked by barium. Stimulation by agonists known to cause increases in either cytosolic cAMP ([cAMP]c) (isoproterenol, prostaglandin E2, both at 2 microM) or free cellular Ca2+ concentration ([Ca2+]c) (noradrenaline, 10 microM, added with propranolol, 5 microM; carbachol, 100 microM) in the frog skin glands caused reversible depolarization: by 34+/-3 mV, 36+/-3 mV, 25+/-3 mV (plateau-phase), and 20+/-3 mV, respectively. Ion substitution experiments showed that stimulation through either pathway (cAMP or Ca2+) resulted in the activation of a Cl- conductance. Application of noradrenaline or adrenaline resulted in a faster depolarization (rates 22 mV/s, 26 mV/s) than stimulation by isoproterenol or prostaglandin E2 (5.6-5.7 mV/s). Cells that were depolarized by exposure to isoproterenol or prostaglandin E2 partially repolarized when stimulated by noradrenaline. The repolarization was blocked by Ba2+ (5 mM) or prazosine (1 microM), consistent with the activation of Ca(2+)-dependent K+ channels via alpha1-adrenergic receptors. We conclude that in the frog skin gland both Ca(2+)-dependent and cAMP-dependent Cl- channels are present in the apical membrane. Increases in free [Ca2+]c in the cAMP-stimulated gland results in the activation of K+ channels, thereby increasing the driving force for Cl- exit.

Adrenergic alpha-Agonists↗

Mosaic evolution of neural development in anurans: acceleration of spinal cord development in the direct developing frog Eleutherodactylus coqui.

Previous studies have shown that spinal cord development in direct developing frogs of the genus Eleutherodactylus, which have evolutionarily lost the tadpole stage, differs from that in biphasically developing anurans (with the larval and the adult stage separated by metamorphosis). The present study of spinal cord development in Eleutherodactylus coqui provides additional information about neurogenesis, neuronal differentiation and growth analyzed by immunostaining for proliferating cell nuclear antigen (PCNA), in situ hybridization for NeuroD, and morphometric measurements in various developmental stages. Furthermore, spinal cord development in the frogs Discoglossus pictus, Xenopus laevis, and Physalaemus pustulosus, which belong to different anuran families but all exhibit biphasic development, was similarly analyzed. This comparative analysis allows inference of the ancestral anuran pattern of spinal cord development and how it has been modified during the evolution of Eleutherodactylus. All biphasically developing frogs analyzed share a similar pattern of spinal cord development, suggesting that this is ancestral for anurans: after neural tube closure, levels of proliferation and neurogenesis in the spinal cord were low throughout embryogenesis until they were upregulated drastically at early larval stages followed by development of the lateral motor columns. In contrast, no such quiescent embryonic period exists in E. coqui, where rapid growth, high levels of proliferation and neurogenesis, and early formation of lateral motor columns occur shortly after neural tube closure, while other parts of the central nervous system develop more slowly. Thus, spinal cord development has been accelerated during the evolution of Eleutherodactylus relative to the development of other parts of the central nervous system, probably related to the precocious development of limbs in this lineage.

Animals↗

TRK neurotrophin receptor-like proteins in the kidney of frog (Rana esculenta) and lizard (Podarcis sicula): an immunohistochemical study.

The present study investigates the occurrence of Trk-like neurotrophin receptor proteins in the lizard and frog kidney. In lizard rare TrkB-like immunoreactive cells in intermediate and distal tubules were found. TrkC-like immunoreactive cells were numerous in collecting tubules and became less numerous in collecting ducts. No TrkC-like immunoreactivity was detected in the ureteric duct. In the frog, we observed numerous TrkC-like immunoreactive cells in collecting tubules and ducts while they were scattered among negative epithelial cells in the wolffian duct. TrkB- and TrkA-like immunoreactivity was never found. Western blot analysis demonstrated that the frog and lizard kidney contains TrkC-like protein; TrkB-like protein was present only in the lizard kidney. These results demonstrate for the first time the occurrence of Trk-like proteins in the kidney of amphibians and reptiles, and aid in the assessment of the role of Trk receptor-like proteins in the kidney physiology of vertebrates.

Animals↗

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA.

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.

Animals↗

Scheloribatid mites as the source of pumiliotoxins in dendrobatid frogs.

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2'-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2'-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida.

Alkaloids↗

Identification and characterization of protein kinase C-related enzymes in frog retina.

We isolated two types of protein kinase C-related enzymes (fPKC) in frog (Rana catesbeiana) retina. Three sequential steps of column chromatography of DEAE-cellulose, butyl-Toyopearl, and hydroxylapatite were carried out and resulted in the separation of two types of distinct Ca2+/phospholipid-dependent protein kinase activities. These proteins reacted with antibodies raised against synthetic peptides based on the predicted amino acid sequences of rat protein kinase C delta or zeta subspecies, and showed molecular masses of 90 and 75 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Immunocytochemical analysis of 90 and 75K fPKC in frog retina revealed that the 75K fPKC was localized in the outer segment of cone cells and in the microvillar projections of Müller cells. These results suggested that fPKCs play differential roles on the photosignal transduction in frog retina.

Amino Acid Sequence↗

Localization of sodium pump sites in frog urinary bladder.

[3H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and autoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na+ transport was completely reversible in toad bladder whereas, in frog bladder, [3H]ouabain was tightly bound and Na+ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [3H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of certain (Na+ -K+)-ATPase ligands(K+, ATP) to levels known to reduce ouabain binding. High K+ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [3H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na+ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significantly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.

Adenosine Triphosphatases↗

Action of aldosterone on frog skin in the presence and absence of in vitro molting effects.

The molting which occurs in frog skin following exposure to high concentrations of aldosterone interferes with the interpretation of physiological measurements. Exposure of skins from frogs maintained in standard smooth tanks to 5 - 10(-7) M aldosterone caused within a few hours erratic responses in short-circuit current Io and conductance K followed by sustained stimulation of Io and K; 10(-8) M aldosterone caused only stimulation of Io and K. Storage of frogs in "rojgh tanks" eliminated in vitro molting on exposure to 5 - 10(-7) M aldosterone. IO and K were then superimposable for 3 h, after which Io increased far more rapidly than K. These results are consistent with an early effect on permeability of the active pathway and later effects on metabolism, either a direct effect on the pump or enhanced interaction between transport and metabolism.

Aldosterone↗

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at 0 degrees C and 25 degrees C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 degrees C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 degrees C than at 25 degrees C. The steady state data are consistent with an adsorption model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model. Diffusion-limited flux is ruled out on the basis of "influx profile analysis" of the time course of cycloleucine entry at both 0 degrees C and 25 degrees C. At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 degrees C and 25 degrees C. The rate constant of efflux does not vary with cellular concentration. These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflux only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 degrees C and 25 degrees C. Therefore, an adsorption model for cycloleucine accumulation in frog muscle appears to be untenable.

Amino Acids↗

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.

Animals↗

Seasonal variation in the active transporting ability and in the membrane ATPase activity of the frog intestinal epithelium.

The active sugar and amino acid transport in the small intestine of the American leopard frog (Rana pipiens) and a species of European frog (Rana esculenta) decreases during the winter months. Parallel with this the (Na+, K+)-stimulated ("pump") ATPase activity is markedly depressed. No seasonal changes are observed in the intestine of the tropical bullfrog (Rana catesbeiana). It is assumed that the low pump-ATPase activity is caused by the hibernation of the frogs living in moderate or subtropical areas and is connected to a biological clock. The decreased active transport of non-electrolytes appears to be a consequence of the change of the ATPase activity.

Adenosine Triphosphatases↗