Sensitivity of Quails (Coturnix coturnix), Siskins (Carduelis spinus), and Frogs (Rana ridibunda) to West Nile Virus.

The level of viremia and features of the course of experimental infection caused by West Nile virus were studied in two species of migratory birds, siskins Сarduelis spinus and quails Coturnix coturnix, and in one species of amphibians, frogs Rana ridibunda. In quails, the virus caused a fatal disease; histological analysis revealed pathological changes in the heart, kidneys, liver, and brain stem. In siskins and frogs, virus antigen was detected in cloacal smears despite the absence of clinical manifestations, the level of viremia was sufficient to infect insect vectors during bloodsucking. These findings suggest that siskins and frogs can be potential reservoirs of West Nile virus and play a role in its circulation.

Effects of HCN channel blockade on the intensity­response function of electroretinographic ON and OFF responses in dark adapted frogs.

Hyperpolarization­activated and cyclic nucleotide­gated (HCN) channels are well expressed in the vertebrate retina. Their role in formation of electroretinographic (ERG) responses to stimulus onset (b­wave) and stimulus offset (d­wave) are largely unknown. In this study we investigated the effects of pharmacological blockade of HCN channels (with ZD7288 or ivabradine) on the ERG b­ and d­waves in dark adapted frog eyecup preparations. Initially, the dose­response relationship of ZD7288 effects on the b­ and d­waves was investigated. Afterwards, the effects of 75 µM ZD7288 on the stimulus ­ response function of the ERG b­ and d­waves were explored over a wide intensity range (10 log units). Finally, the effects of 30 µM ivabradine on the same function were studied. Perfusion with 75 µM ZD7288 did not change the absolute and relative sensitivity of the ERG ON and OFF responses. It caused an enhancement of the d­wave amplitude at all suprathreshold stimulus intensities, while the b­wave amplitude was slightly enhanced only in the range of higher intensities. As a result of the greater blocker effect on the OFF response amplitude, the b/d amplitude ratio was significantly decreased over the whole intensity range. ZD7288 caused a prolongation of the b­wave half­width duration, but a shortening of the d­wave half­width duration at higher intensities. Similar results were obtained when 30 µM ivabradine was used for HCN channel blockade. Our results clearly demonstrate that the blockade of retinal HCN channels changes the balance between the ON and OFF responses in the distal frog retina. This ON/OFF imbalance may be one of the causes for visual disturbances reported in ivabradine treated patients.

Effects of picrotoxin on light adapted frog electroretinogram are not due entirely to its action in proximal retina.

In order to evaluate the site of action of picrotoxin (antagonist of ionotropic GABA receptors) on the electroretinographic (ERG) b- and d-waves, in this study we compared its effects on the intensity-response function of the ERG waves in intact light adapted frog eyecup preparations with its effects in eyecups, where the activity of proximal neurons was blocked by 1 mMN-methyl-d-aspartate (MNDA). Picrotoxin markedly enhanced the b- and d-wave amplitude and slowed the time course of the responses at all stimulus intensities in the intact eyecups. Perfusion with NMDA alone caused significant enhancement of the b-wave amplitude and diminution of the d-wave amplitude without altering their time course in the entire intensity range. When picrotoxin was applied in combination with NMDA, an enhancement of the b-wave amplitude and slowing of its time course were observed at all stimulus intensities. The increase of the b-wave amplitude was significantly higher than that seen in NMDA group. Combined application of picrotoxin and NMDA caused an enhancement of the d-wave amplitude at the lower stimulus intensities and its diminution at the higher ones, while the d-wave time course was delayed over the entire intensity range. The results obtained indicate that a part of picrotoxin effects on the amplitude and time course of the photopic ERG b- and d-waves are due to its action in the distal frog retina.

Why do green rods of frog and toad retinas look green?

Amphibian "green" rods express a blue-sensitive cone visual pigment, and should look yellow. However,when observing them axially under microscope one sees them as green. We used single-cell microspectrophotometry (MSP) to reveal the basis of the perceived color of these photoreceptors. Conventional side-on MSP recording of the proximal cell segments reveals no selective longwave absorbing pigment explaining the green color. End-on MSP recording shows, in addition to the green rod visual pigment, an extra 2- to 4-fold attenuation being almost flat throughout the visible spectrum. This attenuation is absent in red (rhodopsin) rods, and vanishes in green rods when the retina is bathed in high-refractive media, and at wide illumination aperture. The same treatments change the color from green to yellow. It seems that the non-visual pigment attenuation is a result of slender green rod myoids operating as non-selective light guides. We hypothesize that narrow myoids, combined with photomechanical movements of melanin granules, allow a wide range of sensitivity regulation supporting the operation of green rods as blue receptors at mesopic-to low-photopic illumination levels.End-on transmittance spectrum of green rods looks similar to the reflectance spectrum of khaki military uniforms. So their greenness is the combined result of optics and human color vision.

Ultrastructural study of the lateral line neuromasts in tadpoles of Saudi Bufo dhufarensis and Rana ridibunda.

Neuromast (hair cells) structure in Bufo dhufarensis and Rana ridibunda larvae was observed by Scanning Electron Microscopy (SEM). Neuromasts were found arranged in one well-defined line in the head, body, and tail regions forming the lateral line and also found haphazardly scattered in most of the body parts. Their number was significantly high in the head region, and then it gradually decreased along the posterior end of the body. The structure of neuromasts in these three regions was basically similar for each species. In Rana, neuromasts were found few in number, either spherical or oval in shape lacking hair-like structure except in the tail region where hair cells were found. While in Bufo, neuromasts were numerous. Long kinocilia and many stereocilia were found in the neuromasts. Kinocilia were either solitary or in clusters. In addition to the main functions of the neuromasts we discovered a new function which was not found in previous researches, neuromasts were also used to remove any attached object on the tadpole's skin, by directing the kinocilium to the object thing and rolling onto it then detaching it outwards.

Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain.

"Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

[Membrane cholesterol oxidation effects on synaptic vesicle cycle in frog (RANA ridibunda) motor nerve terminals].

In experiments on frog (Rana ridibunda) neuromuscular junction the influence of cholesterol oxidation on the presynaptic vesicular cycle was investigated. Application of cholesterol oxidase (1 u. a.) during 1/2 hour led to the oxidation of - 0.007 mg cholesterol per 1 g tissue and reduced stability of lipid rafts in the nerve terminals. Using electrophysiological techniques it was shown that the cholesterol oxidation decreases the evoked neurotransmitter release. In experiments with fluorescent FM-dyes the depression of the synaptic vesicles exo-endocytosis and the dispersion of synaptic vesicles clusters were revealed. Comparative analysis of electrophysiological and optical data, as well as experiments with water soluble quencher of FM-dye indicated the possibility of some neurotransmitter release by "kiss-and-run" pathway, when short-lived fusion pore is formed. It was concluded that cholesterol oxidation inhibit evoked exocytosis, and also synaptic vesicle delivery from reserve pool to cites of exocytosis probably by break of the clusterization. Perhaps the synaptic vesicles of recycling pool release the neurotransmitter using the kiss-and-run mechanism.

Curcumin acts as a pro-oxidant inducing apoptosis via JNKs in the isolated perfused Rana ridibunda heart.

Amphibians are known to better tolerate and endure adverse environmental conditions such as redox imbalances conferred by reactive oxygen species (ROS), compared to mammals. Interestingly, the exact adaptation strategies and signaling mechanisms mediating these effects have not been fully elucidated. Therefore, in the present study, we probed into the molecular response of the isolated perfused Rana ridibunda heart to curcumin, in the context of mitogen-activated protein kinases (MAPKs) phosphorylation patterns and apoptotic markers occurrence. In particular, this polyphenol was found to exert a pro-oxidant effect in our model and to significantly upregulate p38-MAPK and JNKs phosphorylation (thus activation). The early apoptosis observed, substantiated by poly(ADP-ribose) polymerase (PARP) cleavage, was established to be JNKs- and ROS-mediated, while no involvement of p38-MAPK was detected. Subsequently, the pro-oxidative activity of curcumin was confirmed to mimic H(2) O(2). Furthermore, NADPH oxidase as well as Na(+) /K(+) -ATPase were found to mediate JNKs phosphorylation as well as PARP proteolytic cleavage. Curcumin exerts pleiotropic actions, both beneficial and detrimental and is currently the subject of intense scientific research. Being a low-molecular-weight antioxidant, it is intriguing to investigate curcumin's role in redox homeostasis in the amphibian heart, under conditions that apparently favor its pro-oxidative properties. Comparative studies of its multifaceted role in different species may contribute to the clarification of the signaling mechanisms it triggers and the terminal physiological response it confers. Collectively, this is to our knowledge, the first time that the signal transduction pathways stimulated by curcumin have been assessed in a non-mammalian species.

[Inhibitory regulation of glutamate receptors in the frog motoneuron].

The interaction of exogenously applied excitatory (glutamate and their agonists NMDA, AMPA, kainate) and inhibitory (glycine and GABA) amino acid effects was studied intracellularly in the motoneurones of the isolated frog spinal cord. During simultaneous glycine or GABA bath applications GLU-, AMPA-, KA- and NMDA-evoked responses were, respectively, decreased up to 45.8 +/- 2.9% (n = 12) and 67.8 +/- 3.9% (n = 16), 13.9 +/- 4.3% (n = 9) and 32.1 +/- 8.3% (n = 12), 36.8 +/- 8.2% (n = 7) and 48.0 +/- 11.8% (n = 6), 7.7 +/- 3.5% (n = 9) and 18.1 +/- 3.8% (n = 14) from the control. Sequential applications of EAA after glycine or GABA as well as the applications of EAA-agonist and glycine (GABA) mixture demonstrated similar results. The decrease of EAA-responses by glycine and GABA was abolished by selective GlyR antagonist strychnine (1 microM) and the selective GABAR antagonist SR95531 (gabazine, 20 MM), respectively. The data revealed differences in inhibitory effect of glycine and GABA on the excitation responses mediated by different types of glutamate receptors in the frog motoneurones: the predominant inhibitory effect of glycine and GABA on NMDA-responses and weak inhibitory effect on KA- and GLU-responses. Inhibitory effect of glycine was twice as much as that of GABA at the same concentration.

[Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca2+-channels].

The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nuclear erythrocyte. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nuclear-cytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are expressed as a decrease of the cell volume. The blockator of Ca2+-channels verapamil produces an alternated increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nuclear-cytoplasmic transfer.

Distribution of prolactin receptor in frog (Rana ridibunda) dorsal skin during hibernation.

The role of prolactin in the regulation of frog skin functions is still unclear particularly during environmental changes. In this study, prolactin receptor (PRLR) was detected in active and hibernating frog dorsal skin using immunohistochemical method. PRLR immunoreactivity in active frogs was observed in the epidermis, in the secretory epithelium of granular glands and the secretory channel cells of the glands. Myoepithelial cells of granular glands that started accumulating secretory material or those with a full lumen were PRLR immunoreactive, while some myoepithelial cells of empty granular glands were negative for PRLR. In hibernating frogs, this immunoreactivity was observed in the same regions; however, immunoreactivity was more intense than that in active frogs. PCNA was employed for detection of proliferative activity of PRL in the dorsal skin, and immunoreactivity was detected in the nuclei of a few epidermis cells and in the duct of glands of active frogs. The number of immunoreactive nuclei in these regions increased in hibernating and in prolactin injected groups. We conclude that prolactin provides morphological and functional integrity of skin stimulating the proliferation and regulating the function of granular glands and plays an important role in the adaptation of amphibians to the long winter period.

Neurotoxic and immunotoxic effects of fenthion and omethoate on frogs at acute exposure.

The study was carried out to investigate the neurotoxic and immunotoxic effects of fenthion- and omethoate-used agricultural areas on frogs (Rana ridibunda) at acute exposure. The neurotoxic effects of the chemicals were evaluated by measuring the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Biomarkers selected for immunotoxic monitoring were the activities of adenosine deaminase (ADA) and myeloperoxidase (MPO) in various tissues of frogs exposed to 10 or 20 parts per million (ppm) dosages of fenthion and omethoate for 24, 48, 72 and 96 hours. Results showed that the administrations of chemicals fluctuated AChE and BChE activities in some tissues of frogs treated with both dosages at all the periods. With regard to the immunotoxic effects, MPO activity was increased in almost all the tissues of frogs after 10 and 20 ppm dosages and for 24, 48, 72 and 96 hours exposure of fenthion and omethoate as compared to those of control whereas ADA activity did not change in all the tissues. This may reflect the potential role of these parameters as useful biomarkers for toxicity of fenthion and omethoate.

[The formation of new characteristics in life cycle of the marsh frog (Rana ridibunda) in thermal pond conditions].

Using mark and recapture approach, the long-term population dynamics in the marsh frog (Rana ridibunda) was studied. Group-marking of metamorphs was conducted in a small thermal pond serving as a sedimentation basin for discharged waters from Nizhny Tagil metallurgic works. Depending on the time of metamorphosis, three groups of individuals could be singled out, namely: early ones (group I), middle ones (group II), and late ones that overwinter as tadpoles and complete metamorphosis in May of the next year (group III). Upon metamorphosis completion, individuals of group I were found to be significantly larger than those of group II, and individuals of both these groups to be significantly smaller than those of group III. After first wintering, immature individuals from group I were significantly larger than either individuals from group II or metamorphs from group III, though a growth rate of the latter was significantly higher than in groups I and II. These discrepancies were observed both between immature and adult individuals. Over the period from metamorphosis completion to the first wintering ending, survivorship in group I was significantly higher and did not differ between groups II and III. In adult frogs, maximum survivorship was registered in group III and minimum one in group II; the detected differences recurred in each age class till the fourth wintering. However, in age classes that overwintered 4 and 5 times, maximum survivorship was observed in group II, which can be treated as a compensation for rather low survivorship of this group at younger ages. All the events of tadpoles of this species overwintering (except in other thermal water bodies) that are described in literature, correspond to rare deviations from normal ontogenesis. Therefore, the revealed formation of a numerous group of overwintering tadpoles in successive generations should be considered as a new adaptation which sense is a decrease of competition between tadpole groups when using the highly productive resources of the thermal pond practically year-round. The advantage in body size and growth rate of not only tadpoles but also of metamorphs, immature and adult individuals of group III indicates that after metamorphosis the strategy of this group still remains successful. The reason for that is unusually large body size of metamorphs which provides higher postmetamorphic survivorship and greater female fecundity.

Three methods for estimation of changes in frequency characteristics of potentials elicited by long-lasting (fatiguing) activity of isolated muscle fibres.

The present study was assigned to compare the applicability and sensitivity of three different methods (discrete wavelet transforms (DWT), median frequency (MDF) - calculated on the basis of fast Fourier transform, and spectral indices) for analysis of frequency content changes in potentials produced by repetitive stimulation from isolated slow (SMFs)- and fast (FMFs)-fatigable muscle fibers during uninterrupted activity. In order to affect fatigue, prior to starting the fatiguing stimulation (5 Hz), some of the fibres were exposed to microwave electromagnetic field and others were sham exposed. All the methods studied demonstrated that during a long-lasting fibre activity, the shift of the potential frequency content to lower frequencies was a consequence of lowering in higher frequency components and increasing in low frequency ones. The changes were faster in FMFs than SMFs and stronger in sham exposed fibres vs. microwave exposed. The MDF alterations in both fibre types and protocols were quite similar to other methods characterizing either individual changes in low and high frequency scales of the signal (discrete wavelet transform - DWT) or changes in their spectral ratio (spectral indices). The spectral indices showed sensitivity to fatigue effect at the final stages that made their changes considerably non-linear. DWT allowed processing the temporal occurrence of frequency content changes that corresponded to the alterations in the separate potential phases as well as earlier detection and precise evaluation of fatigue onset and progressing. All methods are applicable but DWT is a preferable for study of muscle fibre fatigue in clinical and experimental neurophysiology.

The mechanisms of multi-component paired-pulse facilitation of neurotransmitter release at the frog neuromuscular junction.

We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca(2+) concentration ([Ca(2+)](o), 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca(2+)](o) (0.5 mM), both F1 and F2 increased, and a third "early" component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the "fast" Ca(2+) buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the "slow" Ca(2+) buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr(2+) (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 microM) at normal [Ca(2+)](o) produced a marked decrease of F2, and at low [Ca(2+)](o), a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca(2+) binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca(2+) ions and are located farther from the release-controlling Ca(2+) channels than the Ca (2+) sensor that mediates phasic release.

Kinetics of turn-offs of frog rod phototransduction cascade.

The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca(2+)-clamping solution. A flash applied in the dark-adapted state elicits a wave of PDE activity whose rising and decaying phases have characteristic times near 0.5 and 2 seconds, respectively. Nonsaturating steady background shortens both phases roughly to the same extent. The acceleration may exceed fivefold at the backgrounds that suppress approximately 70% of the dark current. The time constant of the process that controls the recovery from super-saturating flashes (so-called dominant time constant) is adaptation independent and, hence, cannot be attributed to either of the processes that shape the main part of the PDE wave. We hypothesize that the dominant time constant in frog rods characterizes arrestin binding to rhodopsin partially inactivated by phosphorylation. A mathematical model of the cascade that considers two-stage rhodopsin quenching and transducin inactivation can mimic experimental PDE activity quite well. The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding. This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

GABAa and GABAc receptor mediated influences on the intensity-response functions of the b- and d-wave in the frog ERG.

We assessed the contribution of GABAa and GABAc receptors to GABAergic effects on b- and d-wave in frog ERG in a wide range of light stimulation conditions. The amplitude of both b- and d-wave was increased during GABAa receptor blockade by bicuculline as well as during additional GABAc receptor blockade by picrotoxin. The effects of GABAa receptor blockade were more pronounced in light adaptation conditions. They strongly depended on stimulus intensity and showed considerable ON/OFF-response asymmetry. The effects of GABAc receptor blockade were more pronounced in dark adaptation conditions. They didn't vary much with stimulus intensity and showed little ON/OFF-response asymmetry.

A skeletochronological study of age, growth and longevity in a population of the frog Rana ridibunda from southern Europe.

Age at sexual maturity and longevity in a population of Rana ridibunda from north-eastern Greece were studied by skeletochronology performed on the phalanges. Analysis of the age structure was based on counting the lines of arrested growth (LAGs). Sexual maturity for both sexes arises during the first year or after the first hibernation. Ages ranged from 1 to 5 years (mean=2.96) among 52 males and from 1 to 5 years (mean=3.73) among 56 females. The mean snout-vent length was 69.03+/-12.6mm in males and 82.38+/-13.27 mm in females. The difference between the sexes in age and size was significant. Growth of individuals was fitted on? The von Bertalanffy model. The growth coefficient (K) was 0.57 in males and 0.54 in females, mainly due to faster male growth between metamorphosis and maturation.

Forces applied by cilia measured on explants from mucociliary tissue.

Forces applied by intact mucus-propelling cilia were measured for the first time that we know of using a combined atomic force microscopy (AFM) and electrooptic system. The AFM probe was dipped into a field of beating cilia and its time-dependent deflection was recorded as it was struck by the cilia while the electrooptic system simultaneously and colocally measured the frequency to ensure that no perturbation was induced by the AFM probe. Using cilia from frog esophagus, we measured forces of approximately 0.21 nN per cilium during the effective stroke. This value, together with the known internal structure of these cilia, leads to the conclusion that most dynein arms along the length of the axoneme contribute to the effective stroke of these cilia.

Peculiarities of nerve conduction block produced by lidocaine and ajmaline.

The development of tonic and use-dependent block of nerve conduction under the effect of antiarrhythmic drug ajmaline and local anesthetic lidocaine was examined on sciatic nerve of Rana ridibunda. Ajmaline and lidocaine attenuated the amplitude of action potential by half during 120 and 13 minutes, respectively. Ajmaline produced virtually immediate use-dependent block of conductance, while it was minor in lidocaine-treated nerve preparation.