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Sustained potential shifts in the toad tectum reflect prey-catching and avoidance behavior.

Sustained potential shifts (SPS) were recorded for 10 s from the surface of the optic tectum of toads presented with live prey and moving artificial prey stimuli. On the anterior tectal surface, a negative SPS was followed by a positive wave; the converse was true for the posterior tectum. Some animals were immobilized, and they exhibited a monophasic negative SPS in the anterior tectum and a positive wave in more posterior regions. The number of orienting responses made by toads to moving "wormlike" stimuli was reflected in the amplitude of the SPS, as was avoidance to stimuli in an "antiworm" configuration. Behavioral activity was most closely related to the negativity of the SPS recording. The SPS of toads responding to live prey showed no direct time relationship between the SPS and behavior, suggesting that the SPS reflects sensory or decision-making activity rather than the consequent behavior.

Animals↗

Bidder's organ in the toad Bufo marinus: effects of orchidectomy on the morphology and expression of lamina-associated polypeptide 2.

The Bidder's organ is an undeveloped ovary located anterior to the testis in male true toads (Bufonidae). The presence of sperm and oocytes, derived from the primordial germ cells of the male toad, provides an exceptional condition for the study of germ cell differentiation in vertebrates. In this study, the effects of orchidectomy on morphology and on lamina-associated polypeptide 2 (LAP2) expression were investigated in the Bidder's organ of Bufo marinus. To characterize bidderian oocytes, oogenesis in the ovary was divided into six stages. It was found that ovarian and bidderian oocytes were morphologically identical. To determine the expression of LAP2 isoforms, oocytes from the ovary and the Bidder's organ were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. In amphibians, LAP2beta is expressed in somatic cells, whereas LAP2omega is expressed in oocytes. The Bidder's organ normally contained previtellogenic oocytes of less than 150 micro m in diameter that expressed LAP2beta. However, the organ in some male toads contained a few larger oocytes, resulting in the faint detection of LAP2omega. After orchidectomy, bidderian oocytes grew and strongly expressed LAP2omega. Moreover, as in the ovary, LAP2omega was upregulated in bidderian oocytes of 240 microm in diameter. This work represents the first demonstration of the molecular similarity between ovarian and bidderian oocytes.

Animals↗

Effect of triiodothyronine and thyroxine on mitochondrial alpha-glycerophosphate dehydrogenase activity and mitochondrial protein content of liver, muscle and brain of toad, Bufo melanostictus.

The responsiveness of the adult toad to triiodothyronine (T3) and thyroxine (T4) was studied by measuring the mitochondrial alpha-glycerophosphate dehydrogenase activity and mitochondrial protein content of liver, muscle and brain of toad. Both T3 and T4 increased the alpha-GPD activity and mitochondrial protein content of liver and muscle of toad. The extent of increase in the alpha-GPD activity and mitochondrial protein content were more pronounced with T3 than with T4. Further that the muscle exhibited more alpha-GPD activity than liver, whenever liver showed greater mitochondrial protein content than that of muscle. Brain showed no significant change in the alpha-GPD activity and mitochondrial protein content. Injections of cycloheximide showed inhibition of T3 induced changes in liver and muscle. Injection of propylthiouracil also counteracted the T4 induced effects of liver and muscle.

Animals↗

Effects of ultraviolet radiation on toad early life stages.

BACKGROUND: Exposure to harmful levels of ultraviolet-B radiation (UVB), a component of solar radiation, has been suggested as a potential cause of amphibian declines. METHODS: We measured solar radiation (UVB, ultraviolet-A, and visible) wavebands in breeding ponds of Bufo boreas (boreal toad, a montane species that has undergone severe population declines) and Bufo woodhousii (Woodhouse's toad, a plains toad that has not experienced declines)and examined tolerances of these species to simulated solar UVB exposures in the laboratory. RESULTS: We found larvae of both species to be tolerant of simulated solar UVB in excess of solar UVB levels observed in their breeding ponds. B. boreas tadpoles were more tolerant of simulated solar UVB exposure than B. woodhousii tadpoles, possibly because of greater amounts of photoprotective melanin in B. boreas skin. CONCLUSIONS: UVB levels observed in B. boreas habitats do not currently appear to constitute a threat to the survival of these animals; however, long-term (> 1 month) exposure to UVB levels comparable to levels associated with the water interface appears to reduce survival in B. woodhousii tadpoles. Therefore, future increases in surface and water column UVB radiation in bufonid habitats might pose significant survival risks to B. boreas or B. woodhousii populations.

Animals↗

Neurohypophyseal hormone-sensitive adenyl cyclase of toad urinary bladder.

An adenyl cyclase preparation derived from epithelial cells of the urinary bladder of the toad, Bufo marinus, is described. This cyclase preparation is specifically stimulated by neurohypophyseal hormones and various synthetic analogs which evoke a hydroosmotic response in the intact bladder. The relative stimulatory effects of these compounds have been compared on the cyclase preparation and in the intact bladder. The peptide concentrations required for half-maximal stimulation (affinity) in the cell-free and intact systems were parallel; however the magnitude of stimulation produced by saturating concentrations of peptides did not correlate. Furthermore, it was found that peptide analogs which inhibit the hydroosmotic effect of [8-arginine]-vasopressin on the intact bladder also inhibit the stimulation of the toad bladder cyclase preparation by vasopressin. Prostaglandin E(1), mercaptans, and disulfides, which inhibit the hormone-induced hydroosmotic response of the intact bladder, did not antagonize the stimulation of the toad bladder cyclase preparation by vasopressin.

Adenylyl Cyclases↗

Regulation of phosphorylation of a specific protein in toad-bladder membrane by antidiuretic hormone and cyclic AMP, and its possible relationship to membrane permeability changes.

Phosphorylation of a specific protein was decreased in intact toad bladders by exposure to either antidiuretic hormone or monobutyryl cyclic AMP. The decrease in phosphorylation caused by these agents preceded the change in electrical potential difference (an indicator of the rate of sodium ion transport) observed in response to the same compounds. The addition of cyclic AMP to homogenates of toad bladder led to a decrease in phosphorylation of the same, or a similar, protein. In subcellular fractionation studies, the effect of cyclic AMP on the phosphorylation of this protein was observed in those fractions rich in membrane fragments, but not in the nuclear or cell-sap fractions. These and other results are compatible with the possibility that the regulation by vasopressin and cyclic AMP of sodium and/or water transport in toad bladder may be mediated through regulation of the phosphorylation of this specific protein.

Animals↗

Aldosterone-induced increase in protein phosphatase activity of toad bladder.

A study has been carried out of the effect of aldosterone on the endogenous phosphorylation and dephosphorylation of membrane-bound and of soluble proteins from toad bladder. Membrane-bound protein D (apparent molecular weight, 49,000), a protein which may possibly be involved in the regulation of sodium transport across the mucosal epithelium of toad bladder, contained a substantial fraction of the radioactive phosphate incorporated into membrane proteins; moreover, it was the only protein to appear consistently in autoradiographs of polyacrylamide gels of phosphorylated membrane proteins. Pretreatment of toad bladder slices with aldosterone caused an increase in the endogenous dephosphorylation of membrane-bound protein D. A half-maximal increase in this dephosphorylation occurred at an aldosterone concentration of 20-40 nM. The increase in protein D phosphatase activity induced by aldosterone was prevented by inhibitors of RNA and protein synthesis as well as by spironolactone, a specific antagonist of aldosterone. The mineralocorticoid, 9alpha-fluorohydrocortisone, also increased protein D phosphatase activity, but testosterone did not. Aldosterone also increased the removal of [(32)P]phosphate from protein D in the cell sap. In contrast to the increase in protein D phosphatase activity, aldosterone had little effect on the phosphorylation of protein D by endogenous protein D kinase. In some experiments, effects of aldosterone and of cAMP, qualitatively similar to those found with protein D, were also observed on the phosphorylation and dephosphorylation of a protein with an apparent molecular weight of 37,000, in both the microsomal and cell sap fractions. No consistent effect of preincubation with aldosterone or of cAMP was observed on any membrane-bound or cell sap protein other than protein D and the 37,000 dalton protein.

Adenosine Triphosphate↗

Translation of hormone-induced messenger RNA in amphibian oocytes: I. Induction by estrogen of messenger RNA encoded for vitellogenic protein in the liver of the male African clawed toad (Xenopus laevis).

Induction of the synthesis of the vitellogenic proteins, lipovitellin and phosvitin, in the liver of the male African clawed toad (Xenopus laevis) was investigated as a function of time after treatment with estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol]. The appearance of mRNAs encoded for lipovitellin and phosvitin in the cytoplasmic fraction of the liver was assayed by microinjections of hepatic mRNA preparation [either polyribosomes or poly(A)-rich RNA] into oocytes obtained from mature female toads. Oocytes were then incubated in the presence of radioactive amino acid(s) at 19 degrees for periods of time varying from 4 to 18 hr after microinjection. The results show that at 2 hr after hormone treatment more mRNA was present in the cytoplasm, and that from 2 to 72 hr after treatment the level of induced mRNA increased almost linearly to 110% above the control values. Experiments employing specific lipovitellin antiserum indicated no radioactive lipovitellin among the proteins synthesized in oocytes microinjected with hepatic mRNAs isolated from 3 to 9 hr after hormone treatment. However, a marked synthesis of immunoprecipitable, radioactive lipovitellin and an enhanced incorporation of [3H]serine occurred in the oocytes microinjected with hepatic mRNA preparations obtained from toads treated with hormone for 12 or more hr. The identities of the proteins encoded by the mRNAs induced early in estrogen action (2-9 hr) in the male amphibian liver are unknown. It is surmised that some of these proteins may function in the regulation of the subsequent synthesis of the vitellogenic proteins.

Animals↗

Characterization and partial purification of solubilized active opiate receptors from toad brain.

Opiate receptors have been solubilized from toad brain membranes in active form by using digitonin. Between 40% and 50% of the stereospecific binding activity present in toad brain membranes is recoverable in the ultracentrifugal supernatant of digitonin extracts. Binding of opiates to the solubilized receptor is enhanced 4- to 5-fold by decreasing digitonin concentration to 0.1% or less prior to binding. The solubilized receptor is similar to the membrane-bound receptor in its affinity for various ligands and its sensitivity to heat, trypsin, and N-ethylmaleimide. Moreover, the sodium effect seen in membrane-bound receptor is retained in the solubilized preparation. Both membrane-bound and soluble toad receptors show weak binding of enkephalins, suggesting that they are predominantly of the mu type. The solubilized opiate receptor has an approximate molecular weight of 350,000-400,000. Purification of up to 20-fold has been achieved by gel filtration on Sepharose CL-6B.

Animals↗

The carcinogenicity of some antimalarial drugs using the Egyptian toad Bufo regularis as a biological test animal.

Feeding Egyptian toads (Bufo regularis) with chloroquine and primaquine separately induced tumor formation in 14% and 19% of the animals, respectively. When chloroquine and primaquine were given in combination, the tumor incidence increased to 23.5%. Chloroquine feeding resulted in tumors located in the liver (lymphosarcomas) and primaquine in tumors in the kidney (histiocytic sarcomas). Toads fed chloroquine plus primaquine developed tumors in the liver, kidney, lung, and urinary bladder, and all the tumors were diagnosed as histiocytic sarcomas. It is speculated that one or more metabolites of chloroquine and primaquine (e.g., quinone) may be responsible for tumor induction in the toads.

Animals↗

Preferential limb use in relation to epicoracoid overlap in the shoulder girdle of toads.

We studied left-right asymmetry in forelimb and hindlimb use in green toads (Bufo viridis) and fire-bellied toads (Bombina bombina) and investigated its possible relationship to the asymmetric structure of the shoulder girdle (epicoracoid overlap) in the latter species. To estimate forelimb use preference we used a modified ''snout-wiping test'' (Bisazza et al., 1997) and to estimate hindlimb use preference we used the ''righting response test'' (Robins et al., 1998). The position of epicoracoids in living animals was determined as proposed earlier (Borkhvardt & Malashichev, 1997). We also examined behavioural laterality in B. viridis to check whether B. viridis is a left-handed species, as it was not clear from the previous study. Our results showed that B. viridis predominantly used the left forelimb and left hindlimb in experimental situations at both the population and individual levels. The same tests were applied to fire-bellied toads, B. bombina, with right or left epicoracoids in the top (dorsal) position. We found that (1) B. bombina is mostly a symmetric species, sometimes using both hands or feet simultaneously; (2) it is ambidextrous in hand and foot use with non-significant left side preference at the population level; (3) there is a positive and significant concordance between the position of epicoracoids and the forelimb preference in B. bombina. Animals with the right epicoracoid in the dorsal position (R. top = L. superficial) prefer to use the right forelimb in the ''snout-wiping test'' and L. top animals predominantly use their left forelimb.

Journal Article↗

Pawedness and motor asymmetries in toads.

Pawedness was investigated in three species of toads, Bufo bufo , Bufo viridis , and Bufo marinus. Samples from natural populations were collected in two successive years and tested during attempts to remove a strip of paper stuck onto the snout ( Bufo bufo and Bufo viridis ) or during attempts to remove an elastic balloon wrapped around the head ( Bufo bufo ). A preferential right forelimb use at the population level was observed in Bufo bufo in both tests, whereas no clear pawedness was observed in Bufo viridis. Bufo marinus also showed no laterality in the paper-strip test, but it appeared to be strongly lateralised in another test that investigated which side the toad rotated when turned on its back underwater. Preferentially turning to the toad's left side as part of a righting response, Bufo marinus released the left forelimb first, using the right forelimb to control the roll to the upright position, and push to the surface. These results suggest that preferential limb use has a long evolutionary history, which dates back to early tetrapods.

Journal Article↗

The effect of calcium withdrawal on the structure and function of the toad bladder.

Previous reports have indicated that calcium is necessary to support active sodium transport by the toad bladder, and may be required as well in the action of vasopressin on both toad bladder and frog skin. The structure and function of the toad bladder has been studied in the absence of calcium, and a reinterpretation of the previous findings now appears possible. When calcium is withdrawn from the bathing medium, epithelial cells detach from one another and eventually from their supporting tissue. The short-circuit current (the conventional means of determining active sodium transport) falls to zero, and vasopressin fails to exert its usual effect on short-circuit current and water permeability. However, employing an indirect method for the estimation of sodium transport (oxygen consumption), it is possible to show that vasopressin exerts its usual effect on Q(oo2) when sodium is present in the bathing medium. Hence, it appears that the epithelial cells maintain active sodium transport when calcium is rigorously excluded from the bathing medium, and continue to respond to vasopressin. The failure of conventional techniques to show this can be attributed to the structural alterations in the epithelial layer in the absence of calcium. These findings may provide a model for the physiologic action of calcium in epithelia such as the renal tubule.

Animals↗

Mucosal surface morphology of the toad urinary bladder. Scanning electron microscope study of the natriferic and hydro-osmotic response to vasopressin.

The mucosal cell surface of the toad urinary bladder was examined by scanning electron microscopy, and changes in the structure of the surface of the granular cell were correlated with specific physiological responses to vasopressin. Survey views of the mucosal surface demonstrated that there was no consistent repeating anatomical relationship between the granular cell and the mitochondria-rich cell that would support the concept of cooperativeness in the response to vasopressin. During base-line states of Na+-transport and water flux, the microvilli on the mucosal surface of the granular cell are arranged in a ridge-like network with occasional individual projections. When water flux is increased by exposing the tissue to vasopressin, in the presence of an osmotic gradient across the tissue the microvilli on the granular cell lose the ridge structure and appear, predominantly, as individual projection. Variability-of this appearance points out the necessity of examining large areas and many samples before the significance of any morphological change can be assessed. Blocking the simultaneously occurring natriferic response of the toad urinary bladder with 10(-2)M ouabain does not prevent these changes in the microvilli. When the hydro-osmotic response is blocked by eliminating the osmotic gradient, the granular cell shows no consistent change in mucosal surface morphology even when fixed at the height of the natriferic response. The mitochondria-rich and mucous cells did not show any change in morphology throughout these studies. We conclude that the changes in the mucosal surface morphology of the toad bladder seen after exposure to vasopressin are a result of the increased water flux that occurs when an osmotic gradient exists across the tissue, and are not related to the natriferic response or any specific alteration in the membrane properties.

Animals↗

The ultrastructure of cardiac desnosomes in the toad and their relationship to the intercalated disc.

The fine structure of desmosomes and intercalated discs in the toad heart is discussed. A definite relationship between the dense components of these structures and the dense region of the Z band is demonstrated. The dense region of the Z band characteristically widens at its approach to the plasma membrane, and often terminates beneath it in a distinct discoidal plaque. Cardiac desmosomes appear to be structures which result from the intimate apposition of plaques of Z band material. These desmosomes retain the Z band function as sites of attachment for myofilaments. The suggestion is made that rotation of a desmosome through 90 degrees and splitting of filaments from the adjacent sarcomere could result in the formation of a simple step-like intercalated disc. Intermediate stages in this process are illustrated. Complex discs present in the toad probably represent the alignment of groups of simple discs produced by contractile forces. Possible physiologic functions of the disc and desmosome are discussed. Other morphologic features of toad cardiac cells include a distinct amorphous outer coat to the sarcolemma, a prominent N band, and a granular sarcoplasm with poorly developed reticulum.

Animals↗

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel.

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.

Animals↗

Effects of temperature and physical activity on blood flow shunts and intracardiac mixing in the toad Bufo marinus.

Blood flow in systemic (.Qsys) and pulmocutaneous (.Qpul) arteries was measured as a function of body temperature (10 degrees, 20 degrees, and 30 degrees C) at rest and following enforced physical activity in conscious, adult cane toads (Bufo marinus). Arterial and mixed venous hemoglobin concentration (CHb) and total oxygen content (Co2, tot) were measured in a separate group under identical conditions. Heart rate (fH) and total flow (.Qtot) increased significantly (P<0.001) with elevated temperature and with activity, whereas stroke volume (VS) increased (P<0.001) only with activity. .Qtot ranged about 10-fold, from 10 degrees C (rest) to 30 degrees C (activity); increases in both fH and VS contributed to the increase in .Qtot. The overall distribution of blood to the pulmocutaneous circuit (net L-R shunt) increased with both temperature and activity and was significantly correlated with .Qtot. These data indicate that blood flow distribution in toads is a direct function of cardiac output, and this is linked to relative changes in resistance in the major outflow vessels. Arterial O2 saturation (Sa) was high (mean=93%) in all conditions except activity at 30 degrees C, when it decreased to 74% and contributed to a decrease in the arteriovenous O2 difference. Venous O2 saturation (Sv) was high at rest (76%) and dropped significantly during activity to about 30% at all temperatures. Intracardiac arterial-venous mixing (systemic mixing index) showed the strongest correlation with variation in fH with minimal mixing (17%) occurring at about 50 beats min-1. The most mixing occurred at the lowest fH (13 beats min-1) and at the highest fH (103 beats min-1). The results indicate that the heart of a 0.25-kg toad becomes more efficient from an oxygen transport perspective from low fH to 50 beats min-1 and then less efficient at higher fH, contributing to an uncoupling of blood flow and metabolic rates at these high rates.

Animals↗

Effects of body temperature and hydration state on organismal performance of toads, Bufo americanus.

Temperature and humidity are dominant environmental variables affecting performance of nocturnal, terrestrial amphibians. Toads are frequently active at body temperatures (T(b)) and hydration states (HS) that yield suboptimal performance. We investigated the combined effects of T(b) and HS on feeding, locomotion, and metabolism of Bufo americanus. More toads responded to the presence of prey when fully hydrated than when dehydrated, and times to orient to prey, maneuver around a barrier, and reach prey were less in hydrated than in dehydrated animals. Time to capture prey decreased with increasing T(b) in fully hydrated, but not dehydrated, toads, and hydrated animals caught prey more rapidly than did dehydrated animals. Distance traveled in 5 min and aerobic scope were affected by T(b). Generally, individuals that performed well in the feeding experiments at a particular T(b) and HS also performed well at a different T(b) and HS. The same was true for distance traveled and aerobic scope. However, within combinations of T(b) and HS, correlations between performance variables were minimal. Specialization of a particular variable resulting in high performance at a certain T(b) and HS does not appear to exact a cost in terms of performance at a different T(b) and HS.

Animals↗