Stanley D. Hillyard

The Water Absorption Response: A Behavioral Assay for Physiological Processes in Terrestrial Amphibians

Terrestrial amphibians take up water by abducting the hind limbs and pressing a specialized portion of the ventral skin to a moist surface, using a characteristic behavior called the water absorption response. An assay of the water absorption response was used to quantify physiological factors associated with thirst and water uptake. Dramatic changes in the water absorption response resulted from subtle changes in hydration state and from altering the reserve water supply in the urinary bladder. The water absorption response could be induced by intraperi‐toneal and intracerebroventricular injection of angiotensin II, demonstrating that components of the renin‐angiotensin system on both sides of the blood‐brain barrier have a dipsogenic function in amphibians. These experiments also demonstrated that the water absorption response could be influenced by changes in barometric pressure. Toads avoided the water absorption response on hyperosmotic substrates, and behavioral experiments showed that the amphibian skin served a sensory function similar to that of the lingual epithelium of mammals. The water absorption response assay has enormous potential as a tool for the investigation of physiological processes and sensory capabilities of amphibians.

BEHAVIORAL, MOLECULAR AND INTEGRATIVE MECHANISMS OF AMPHIBIAN OSMOREGULATION

Amphibian water balance has been studied at many levels of biological order. Terrestrial species must react to environmental cues that relate to water availability while some arboreal species have cutaneous skin secretions that can reduce evaporative water loss. The Indian tree frog. Polypedates maculatus, uses cutaneous secretions and wiping behavior to lower evaporation but also relies on moist microclimates to endure prolonged survival away from water. The related species, P. leucomystax, inhabits wetter forest habitats. Preliminary studies with this species are unable to demonstrate the expression of wiping behavior, indicating that arid habitats may be a powerful selective force for this behavior. Laboratory experiments on rehydrating toads in the genus Bufo indicate that animals are able to detect changes in barometric pressure and humidity that might result in the availability of water under field situations. Experiments with Bufonid species and with spadefoot toads, Scaphiopus couchi, show that the peptide hormone, angiotensin II, stimulates cutaneous drinking in a similar manner seen for oral drinking by other vertebrate classes. Amphibian tissues have long been used as a model for the study of basic physiological principles of epithelial ion and water transport. Recent progress with tissue cultures has provided information on the molecular structure of ion and water channels that can be applied to obtain a better understanding, at the molecular level, of ion and water balance strategies used by the wide variety of amphibian species. Terrestrial amphibians are more tolerant of dehydration than are other vertebrates and are able to store dilute urine in their urinary bladder. Toads appear to be able to detect the presence of water in their bladders in addition to the availability of water in their environment. Dehydrated toads are able to rehydrate very rapidly by the coordination of behavioral and physiological mechanisms to enhance cutaneous water absorption. The integration of behavior with cutaneous water gain, renal handling of ions and water and the role of the lymphatic system in overall water balance involves complex interactions between neural and hormonal factors. Experiments are summarized that describe the contribution of individual factors however much more information is needed before the nature of these interactions are fully understood.

Behavior Associated with the Water Absorption Response by the Toad, Bufo punctatus

water potential of the surface when the toads terminated the WR averaged -1.43 + 0.82 atmospheres (ATM). If the water potential of the moist surface was initially reduced by saturating the tissue with urea solutions of 100, 250 and 500 mM, the percentage of trials in which the WR was observed decreased relative to trials with water-saturated tissues. The WR was not observed at the highest urea concentration, suggesting that toads are able to detect substrate water potential. If the feet were covered, toads placed on tissue saturated with 500 mM urea solutions remained on the tissue significantly longer than toads whose feet were able to contact the solution. Thus, sensory structures in the feet appear to be involved in the detection of substrate water potential. Associated with the WR toads demonstrated three characteristic behaviors: 1) moves, in which toads would systematically walk to different areas of the tissue and, periodically, stop before moving to the next area. On all but the 500 mM urea substrates the moves occurred after the WR had been displayed on successive areas of the tissue; 2) kicks, in which the toads would rub the hind feet over the surface of the ventral skin in order to remove particles adherent to the skin; and 3) settles, in which toads would laterally oscillate their entire bodies while demonstrating the WR. The latter behavior usually resulted in an increase in the area of skin in contact with the substrate. The area-specific rates of water gain from water-saturated tissues were similar to literature values reported for water gain by B. punctatus from free water.

Effects of temperature and salinity on gill Na+-K+ ATPase activity in the pupfish, Cyprinodon salinus

Abstract 1. 1. Pupfish (Cyprinodon salinus) were acclimated to salinities equivalent to fresh-water, 1 2 seawater, seawater and 1 1 2 sea-water at each of three temperatures; 15°, 25° and 30°C. 2. 2. The specific activity of Na+-K+ ATPase in gill homogenates from these animals was lowest in 1 2 SW at each temperature and rose significantly in fresh-water- and sea-water-acclimated fish. 3. 3. The specific activity of Na+-K+ ATPase in both the fresh-water- and sea-water-acclimated fish increased significantly with decreasing acclimation temperature.

Angiotensin II Stimulates Cutaneous Drinking in the Toad Bufo punctatus

The renin-angiotensin system regulates drinking in many vertebrates but has not been thought to serve this function in amphibians because injections of angiotensin II (AII) have failed to stimulate either oral or cutaneous drinking. We tested the effects of intraperitoneal injections of AII on the duration of water absorption behavior and water weight gain of the red-spotted toad, Bufo punctatus. We found that doses of 1, 5, and 200 μg AII/100 g body weight significantly increased both the duration of water absorption behavior and water weight gain. The effect was eliminated by prior injection of saralasin (a specific competitive inhibitor of AII). This suggests that the mechanism that regulates hydration in amphibians is homologous to thirst mechanisms in other vertebrates and may represent an important step in the evolution of thirst in terrestrial vertebrates.

Desert toads discriminate salt taste with chemosensory function of the ventral skin.

Toads obtain water by absorption across their skin. When dehydrated, desert toads exhibit stereotyped hydration behavior in which they press their ventral skin onto a moist surface. However, dehydrated toads avoid surfaces moistened with hyperosmotic NaCl and KCl solutions (Hoff KvS, Hillyard SD. 1993. J. Exp. Biol. 183:347–351). We have studied neural mechanisms for this avoidance with physiologic, behavioral, and morphologic approaches. Spinal nerves innervating the ventral skin could be stimulated by exposure to a hyperosmotic NaCl solution applied to the outer surface of the skin. This neural response occurred with much longer latency than to mechanical stimulation and could be reduced by amiloride, a blocker for Na+ channels known to be responsible for epithelial ion transport and salt taste transduction. In behavioral experiments, avoidance of a NaCl solution was also reduced by adding amiloride to the solution, suggesting involvement of amiloride‐sensitive Na+ channels for detecting the hyperosmotic salt solution. Neural tracing with fluorescent dye revealed spinal nerve endings and connections to putative receptor cells, both located in the deeper layer of the epidermis. Either of these or both may be associated with the transduction of Na+ flowing into the skin. The ability of toads to detect hyperosmotic salt solutions in their environment reveals a previously unknown chemosensory function for spinal nerves in anuran amphibians. J. Comp. Neurol. 408:125–136, 1999.

Central Angiotensin II Induces Thirst-Related Responses in an Amphibian

Angiotensin II (A-II), a potent inducer of thirst-related behavior in many vertebrate species, was injected into the third ventricle of the brain of the spadefoot toad, Scaphiopus couchii. Following injection of 10 ng A-II the animals demonstrated a significant increase in water absorption response (WR) behavior, in which toads press their ventral skin to a moist surface and absorb water by osmosis. This increase in the frequency of WR behavior was positively correlated with an increase in water gain during a 2-hr period indicating that centrally injected A-II stimulates water intake by this amphibian species. We have previously demonstrated that WR behavior is also induced by intraperitoneal (i.p.) injections of A-II in several anuran species, including S. couchii. Thus, amphibians, like other vertebrates, demonstrate an increase in water intake in response to either centrally administered or circulating A-II. A second series of experiments was conducted to determine whether the above response to A-II might be secondary to increases in the circulating levels of aldosterone (ALDO) or antidiuretic hormone because the release of both of these hormones has been shown by others to be stimulated by A-II. Scaphiopus couchii injected i.p. with either ALDO or arginine vasotocin in dosages of 1, 10, and 100 micrograms/100 g body weight showed no increase in WR behavior relative to toads injected with saline alone. These results suggest that A-II acts directly on the brain of S. couchii to induce WR behavior.

Effects of angiotensin II and bladder condition on hydration behavior and water uptake in the toad, Bufo woodhousei

1. Water absorption response (WR) behavior and water weight gain were examined in hydrated toads, Bufo woodhousei, treated with angiotensin II (AII) or with a control Ringers solution. The effects of urinary bladder condition (ad lib. bladder urine or empty bladder) were examined concurrently. 2. Toads treated with AII (100 micrograms/100 g body weight), spent more time in WR posture and absorbed more water than Ringers-injected toads. 3. Toads with empty bladders maintained WR posture for longer periods of time and gained more weight than toads whose bladders were not emptied. 4. The effects of AII and bladder urine on water absorption by B. woodhousei appear to be separate and additive.

The Effects of Temperature and Salinity Acclimation on Metabolic Rate and Osmoregulation in the Pupfish Cyprinodon salinus

Alterations in standard metabolic rate (V02), plasma osmolality and critical thermal maxima were examined in pupfish, Cyprinodon salinus, acclimated to freshwater and to Salt Creek water adjusted to salinities of approximately onehalf sea water (?2 SW; 560 mosm/kg) and sea water (SW; 1,168 mosm/kg). Three acclimation temperatures, 15, 25 and 30 C were maintained at each salinity. The V02 Of 1/2 SW and SW fish was significantly greater than that of FW fish at each acclimation temperature. V02, measured at the acclimation temperature, increased in FW and ? SW fish as acclimation temperature increased. The V02 of SW fish, however, was not significantly different between the 25 and 30 C acclimation groups. Thus, SW fish appear to fully compensate for the effects of increased temperature between 25 and 30 C. Plasma osmolality increased significantly with acclimation to increased salinity. As the acclimation temperature was increased from 25 to 30 C the plasma osmolality of the SW fish dropped significantly, and that of the FW fish rose significantly. Plasma osmolality of the ? SW fish was statistically identical at each acclimation temperature. The ability to osmoregulate in hypersaline environments then requires the elevated temperatures associated with this condition. This is consistent with natural history observations on C. salinus which only encounters high salinities during summer months when high temperatures occur. The critical thermal maximum (CTM) was significantly elevated by increasing acclimation temperature at each salinity. The /2 SW acclimated fish had higher CTM values than FW acclimated fish at each acclimation temperature, however, CTM was not further elevated by acclimation to salinities higher than ? SW.

Effects of Cd++ on short-circuit current across epithelial membranes. I. Interactions with Ca++ and vasopressin on frog skin.

SummaryCadmium ion (Cd++) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringers solution at 10−4, 10−3 and 5×10−3m concentration. Resistance was reduced by 10−4m Cd++ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10−4 and 10−3m Cd++ produced additive stimulation which was reversible by washing with Cd++-free Ringers. If SCC was first stimulated by Cd++, further stimulation by vasopressin was additive with 10−4m Cd++ but completely inhibited by 10−3m Cd++. Elevating the calcium ion (Ca++) concentration of the outer Ringers from 10−3m to 5×10−3m or 10−2m prior to Cd++ treatment did not reduce the magnitude of SCC stimulation by Cd++. Removal of Ca++ from the outside Ringers with 2×10−3m EDTA increased SCC as predicted. Subsequent addition of 5×10−3m Cd++ drastically reduced SCC below control levels while equimolar concentrations of Cd++ and EDTA reduced SCC only to control levels. These results suggest that Cd++ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca++ removal and may be a useful probe for elucidating these components.