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Sodium detection during the water absorption response in Rana pipiens
Physiology & Behavior 68 (2000) 373–376
Sodium detection during the water absorption response in Rana pipiens Spencer J. Kostinsky, Kirk Miller, Charles N. Stewart* Departments of Biology and Psychology, Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604-3003, USA Received 2 September 1998; received in revised form 23 August 1999; accepted 21 September 1999
Abstract Although taste in vertebrates is typically associated with specialized receptors in the lingual epithelium, Hoff and Hillyard reported that the toad, Bufo punctatus, is able to “taste” sodium with the abdominal skin. This was reflected in a differential behavioral response to hypertonic NaCl. The present study tests for the presence of such abdominal chemoreceptors in the frog Rana pipiens. The experiment was a five-condition design in which frogs were placed on filter paper saturated with: deionized water, 250 mM NaCl, 350 mM NaCl, 12.9 M amiloride, or 350 mM NaCl ⫹ 12.9 M amiloride. The time that the frogs remained on the test substrate before moving to a surface of deionized water was recorded. It was necessary to dehydrate the frogs to 80% of their body weight to elicit a behavioral response to the NaCl whereas dehydration to 90% of their body weight has been reported effective in Bufo punctatus. The frogs displayed significantly shorter mean times to move on both concentrations of NaCl compared to deionized water, with the shortest times occurring when 350 mM NaCl was used. Amiloride alone did not have an effect upon times to move to deionized water, but did significantly reduce the response to 350 mM NaCl. Movement to amiloride ⫹ 350 mM NaCl did not differ significantly from that to deionized water. The results indicate that Rana pipiens, like Bufo punctatus, have epithelial chemoreceptors for the detection of NaCl on hydrated surfaces and that these receptors, like those of mammals, are amiloride sensitive. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Amphibians; Sodium detection; Water Absorption response; Rana pipiens
1. Introduction Amphibians living in dry or hot climates experience substantial water loss through their permeable skin as a result of rapid evaporation. To compensate for this, amphibians take in water from the environment. Frogs and toads do not drink [1], but they absorb water through the integument, especially through an area of thin vascularized skin on the posterior underside of the body (known as the pelvic or seat patch; [2]). The seat patch allows semiterrestrial anurans to use minute sources of water, such as soil moisture, to rehydrate. Because anurans rapidly rehydrate by passive movement of water across their epithelia, it is necessary that they avoid contacting solutions that are hypertonic to their body fluids (⬎243–-284 mM) to avoid osmotic dehydration. Brekke, Hillyard, and Winokur [3] showed that desert toads, Bufo punctatus, avoid solutions hypertonic to their body fluids. Dehydrated toads were placed on wetted surfaces saturated with various concentrations of urea, and the duration of time they remained on the surface was recorded. Brekke and her colleagues found significant decreases in the time that the toads remained on the wetted surfaces as the concentration * Corresponding author. Tel.: 717-291-3823; Fax: 717-291-4387 E-mail address: [email protected]
of urea increased. Similar results were obtained using surfaces saturated with NaCl. These results suggest the toads have cutaneous receptors either for detecting osmolality or for detecting specific solutes in hydration sources. Hoff and Hillyard [4] attempted to demonstrate that the toads have cutaneous receptors for detecting Na⫹. Amiloride-sensitive Na⫹ channels are present on the apical membrane of the skin of all amphibians, and mediate the intake of Na⫹ across the epithelium [5]. Amiloride-sensitive Na⫹ channels are also present on the tongues of drinking vertebrates and mediate Na⫹ influx into the taste buds [6,7]. Hoff and Hillyard [4] hypothesized that amiloride-sensitive Na⫹ channels are important in the detection of Na⫹; they hypothesized that, “toads taste sodium with their skin” (p. 347). Bufo punctatus, dehydrated by 10% of their standard weight, were placed on a surface of hyperosmotic NaCl. The mean time until toads moved off the surface was significantly less than when they were placed on a surface of deionized water. To determine if Bufo punctatus detected Na⫹ specifically or if they detected osmolality in general, the investigators added amiloride to the surface saturated with NaCl. They reasoned that, if the toads responded to NaCl because they detect osmolality, adding amiloride to the surface should not affect that response, because the con-
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centrations of solutes on the surfaces with and without amiloride are the same. On the other hand, if their movement off the surface wetted with NaCl solution was a taste phenomenon, the toads should no longer move quickly off the surface because the taste receptors are blocked by the amiloride. They found that, in the presence of amiloride and NaCl, the duration that subjects remained on the hydrated surface increased to a significantly greater time compared to toads placed on a substrate with NaCl alone. Hoff and Hillyard [4] concluded that the cutaneous amiloride-sensitive Na⫹ channels present in Bufo punctatus, “serve in a sensory capacity for Na⫹” (p. 349). Further supporting this conclusion was the observation that amiloride did not affect the time spent on a surface saturated with KCl. Similarly, amiloride reduces humans’ taste perception of NaCl, but has no effect on perception of KCl [8]. The present study is a replication of the study by Hoff and Hillyard [4] examining cutaneous detection of NaCl by the Northern leopard frog (Rana pipiens). Rana pipiens were chosen because they are somewhat terrestrial, suggested by their tolerance for dehydration to 50–55% of their standard weight [9], and by the considerable distances they reside from sources of standing water during the summer (cited in [10]). Also, unlike Bufo punctatus, they are readily available from commercial suppliers year round. We hypothesized that partially dehydrated frogs can detect NaCl on a hydrated surface, and that the detection of NaCl is blockable with amiloride.
2. Materials and method 2.1. Subjects Twenty Rana pipiens, purchased from Charles D. Sullivan Co., Inc., were housed individually in 10 ⫻ 5 ⫻ 5⬙ plastic containers with wire mesh tops. The containers were filled with 400 mL of dechlorinated water and rested on a 9⬚ angle so that the water was pooled at one end of the container. Frogs were kept on a 10-h light:14-h dark photoperiod at an approximate temperature of 22⬚C. They were fed crickets twice a week. The plastic containers were numbered for the purpose of subject identification. Subjects were tested individually and randomly assigned to a condition; 10 frogs were assigned to each condition studied. 2.2. Design This study utilized a five-condition design. The five levels of the independent variable (type of test solution onto which the subject was initially placed) were (1) deionized water, (2) 250 mM NaCl, (3) 350 mM NaCl, (4) 12.9 M amiloride, and (5) 350 M NaCl ⫹ 12.9 mM amiloride. The dependent variable was the ability of Rana pipiens to detect NaCl on hydrated surfaces. This ability was measured according to (1) the percentage of subjects that moved
off the wetted surface of test solution, and (2) the amount of time the subjects spent on the wetted surface of test solution before moving to the surface of deionized water. 2.3. Apparatus The apparatus used for behavioral observation of Rana pipiens on a wetted surface was adopted from the paradigm of Brekke et al. [3] and Hoff and Hillyard [4]. A glass-bottom aquarium (70 ⫻ 36.3 ⫻ 35 cm) was supported 25.5 cm above an angled mirror (70 ⫻ 36.3 cm), and the walls of the aquarium were covered with brown paper so the frogs could not view the experimenter. A smaller aquarium (35 ⫻ 17.5 ⫻ 20.3 cm), also covered with brown paper, was placed inside the larger aquarium. The bottom of this smaller aquarium was lined with two 14.5 ⫻11.3-cm pieces of Kimwipe separated by 0.6 cm. One of these Kimwipes was saturated with test solution, while the other was saturated with deionized water. The apparatus was constructed so that the underside of the subjects could be observed in the mirror, and the observer could not be detected by the subject. 2.4. Procedure The frogs’ bladders were emptied, and the frogs were placed in a dry container, without access to water, for 15–16 h. In that time the frogs were dehydrated by an average of 21.43% (range 10.88–37.24) of their standard weight. (Pilot studies revealed that neither fully hydrated Rana pipiens nor Rana pipiens dehydrated by 10% of their standard weight responded to solutions of NaCl, even at concentrations as high as 450 mM). Each dehydrated frog was placed on the surface wetted with test solution, and was observed in the angled mirror to determine whether or not it moved from the aversive stimulus to the surface of deionized water. The duration of time that each frog spent on each substrate was recorded. Each trial lasted 5 min. The percentage of frogs that moved from the test solution to deionized water and the mean amount of time the subjects remained on the surface saturated with test solution were calculated. Analyses of variance with post hoc Bonferroni t-tests and Fisher exact probability tests were used as appropriate to compare conditions.
3. Results Only 1 of 10 frogs moved from the test surface of deionized water, whereas 8 of the 10 frogs initially placed on a wetted test surface of either 250 or 350 mM NaCl moved to the surface of deionized water (Fig. 1). These differences were significant (Fisher exact probability test, p ⬍ 0.05). Surprisingly, 7 of the 10 frogs tested moved from the 350 mM NaCl ⫹ amiloride test surface. Amiloride alone produced the same effect as deionized water alone with only 1 out of 10 frogs leaving (Fig. 1). The addition of amiloride to 350 mM NaCl significantly increased the time that frogs remained on that solution to a
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Fig. 1. Number of subjects that moved to the adjacent surface of deionized water as a function of the test solution presented on the original surface. Fig. 2. Mean time R. pipiens remained on the initial surface before moving to deionized water, as a function of the test solution presented.
value that was not different from the time that frogs spent on deionized water (Fig. 2). Analysis of variance showed that there was a significant effect of type of test solution on the time animals remained on the test surfaces, F(3, 36) ⫽ 11.476, p ⬍ 0.0001. Bartlett’s test of homogeneity of variance showed no significant differences in the standard deviations for the test conditions. Bonferroni t-tests showed that the differences between deionized water and each of the NaCl solutions was significant (t ⫽ 4.3119 and t ⫽ 5.4158, respectively, p ⬍ 0.001). The difference between deionized water and amiloride ⫹ 350 mM NaCl was not significant, whereas the difference between 350 mM NaCl and amiloride ⫹ 350 mM NaCl was significant (t ⫽ 3.2773, p ⬍ 0.05). The data from a separate control group tested with amiloride alone did not differ significantly from the group tested with deionized water. Finally, some noteworthy observations were made concerning the animals’ behaviors and responses when placed on test surfaces saturated with NaCl solutions. For example, when moving from the NaCl, the frogs did not just leap to the surface of deionized water. Most gradually moved toward the deionized water surface, pressing their seat patches to the NaCl surface for several seconds, and then moving to another region of the surface. This process continued until the subjects crossed to the surface of deionized water. Additionally, some frogs that were initially presented with NaCl would press their bodies up against the wall of the test chamber and extend their legs, an apparent attempt to climb out of the test apparatus.
4. Discussion The results of this study suggest that Rana pipiens, like Bufo punctatus, can detect NaCl with their skin. The frogs distinguished hypertonic solutions of NaCl from pure water, as indicated by the significant increase in the number of
frogs that moved, and the reduction in the mean time to move, when NaCl was present on the wetted surface. Other behavioral observations recorded during the trials also suggest that Rana pipiens can detect NaCl with their skin. First, frogs that were initially placed on a surface wetted with NaCl solution attempted to exit the test chamber by climbing its walls, but frogs that were initially placed on a surface wetted with deionized water did not attempt to exit the test chamber. Second, the frogs shifted position on the surface saturated with NaCl solution, evidently because they were searching for a less aversive region of the surface. Similarly, Brekke et al. [3] observed an increased frequency of movements by toads placed on hyperosmotic urea solutions. That the frogs lowered their seat patch region to the surface for several seconds, and then moved on to a new portion of the surface, provides evidence that the seat patch is the region of the abdominal skin used to detect NaCl. Stebbins and Cohen [2] and Hoff and Hillyard [4] make this suggestion as well, and it seems reasonable, considering the seat patch’s integral contribution to cutaneous water uptake. The results of this study not only suggest that Rana pipiens can detect NaCl on wetted surfaces, but also that the detection of NaCl is mediated, at least in part, by amiloridesensitive channels. Frogs initially placed on the surface wetted with 350 mM NaCl ⫹ amiloride remained on the initial surface longer than did the frogs initially placed on the surface wetted with only 350 mM NaCl (Fig. 2). Although the number of frogs responding to 350 mM NaCl was not affected by amiloride (Fig. 1), the time to move increased, and did not differ from the response to deionized water. Data in Hoff and Hillyard [4] and in the present study are also consistent with an amiloride-insensitive component of salt taste if one considers only the number of animals responding. This is consistent with salt taste by the tongue of numerous mammalian species [11].
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There are two possible explanations for Bufo punctatus’ and Rana pipiens’ amiloride-blockable response to NaCl. The first hypothesis, which is advocated by Hoff and Hillyard [4], is that anurans have a discrete taste mechanism for NaCl that stimulates them to move from salty substrates unless amiloride is present. An alternative hypothesis is that anurans experience osmotic thirst that causes them to escape from salty substrates unless amiloride is present. Several lines of evidence support a discrete taste mechanism on the abdominal epithelium of anurans as the explanation for the anurans’ response to NaCl. Most fundamentally, the fact that the response is inhibited by amiloride suggests a salt-tasting mechanism compatible with our understanding of other salt-tasting systems. Amiloride-sensitive Na⫹ channels on the apical membrane of mammalian tongues mediate the chemoreception of NaCl [6,7] Kinnamon and Getchell [11] explain that amiloride has been shown to drastically reduce the response to NaCl in hamsters, rats, dogs, monkeys, and humans. When amiloride was applied to the tongue of these mammals, a 70% reduction in the afferent nerve response to NaCl was recorded. (The remaining 30% of the afferent nerve response is attributed to amiloride-insensitive Na⫹ channels such as TTXsensitive channels). Similarly, in Hoff and Hillyard’s study [4] and in the present study, the latency of the anurans’ behavioral response to NaCl was increased by 70%. Additional evidence for a salt-tasting mechanism includes Hoff and Hillyard’s demonstration that Bufo punctatus escape from surfaces wetted with KCl solutions, as they do from surfaces wetted with NaCl solutions, but the escape response for KCl is not inhibited by amiloride. (The present study did not include using KCl as a test solution.) The amiloride-insensitive response to KCl is also consistent with a mammalian salt-tasting mechanism, in which the nervous response to KCl is not reduced by amiloride. Further evidence for cutaneous Na⫹ tasting was established by Nagai et al. [12], who confirmed the presence of peripheral nerves, in the deep cell layers of the skin of Bufo alvarius, that respond to NaCl. The alternative hypothesis, however, is that anurans move from a hydrated surface of hypertonic NaCl solution because the hypertonic NaCl solution makes them thirsty.
The thirst hypothesis may explain why subjects must be dehydrated before they will respond to NaCl. Dehydration causes volumetric thirst and an animal’s overall level of thirst is a sum of its osmotic thirst (caused by increases in body fluid solute concentration) and volumetric thirst (caused by decreases in body fluid volume). A direct relationship between plasma osmotic concentration and water absorption response in the toad has been shown [13]. This study provides evidence that Rana pipiens, like Bufo punctatus, is able to detect NaCl on a wet surface. This may be a phenomenon common to anuran amphibians.
References [1] Bentley PJ, Yorio T. Do frogs drink? J Exp Biol 1979;79:41–6. [2] Stebbins RC, Cohen NW. A Natural History of Amphibians. Princeton, NJ: Princeton University Press, 1995. [3] Brekke DR, Hillyard SD, Winokur RM. Behavior associated with the water absorption response by the toad, Bufo punctatus. Copeia 1991; 1991:393–401. [4] Hoff KV, Hillyard SD. Toads taste sodium with their skin: sensory function in a transporting epithelium. J Exp Biol 1993;183:347–51. [5] Lindemann B, Van Driessche W. Sodium-specific membrane channels of frog skin are pores: current fluctuations reveal high turnover. Science 1977;195:292–4. [6] DeSimone JA, Heck GL, DeSimone SK. Active ion transport in dog tongue: a possible role in taste. Science 1981;214:1039–41. [7] DeSimone JA, Heck GL, Mierson S, DeSimone SK. The active ion transport of canine lingual epithelium in vitro. Implications for gustatory transduction. J Gen Physiol 1984;83:633–56. [8] Schiffman SS, Lockhead E, Maes FW. Amiloride reduces the taste intensity of Na⫹ and Li⫹ salts and sweeteners. Proc Natl Acad Sci USA 1983;80:6136–40. [9] Churchill TA, Storey KB. Metabolic effects of dehydration on an aquatic frog, Rana pipiens. J Exp Biol 1994;198:147–54. [10] Lillywhite HB, Licht P. Movement of water over toad skin: functional role of epidermal sculpturing. Copeia 1974;1974:165–71. [11] Kinnamon SC, Getchell TV. Sensory transduction in olfactory receptor neurons and gustatory receptor cells. In: Getchell TV, Doty RL, Bartoshuk LM, Snow JB, editors. Smell and Taste in Health and Disease. New York: Raven Press, Ltd., 1991. pp. 145–74. [12] Nagai T, Koyama H, Hillyard SD. Desert toads discriminate salt taste with chemosensory function of the ventral skin. J Comp Neurol 1999; 408:125–36. [13] Hoffman J, Katz U. Elevated plasma osmotic concentration stimulates water absorption response in a toad. J Exp Zool 1999;284:168–73.
Sodium detection during the water absorption response in Rana pipiens Spencer J. Kostinsky, Kirk Miller, Charles N. Stewart* Departments of Biology and Psychology, Franklin & Marshall College, P.O. Box 3003, Lancaster, PA 17604-3003, USA Received 2 September 1998; received in revised form 23 August 1999; accepted 21 September 1999
Abstract Although taste in vertebrates is typically associated with specialized receptors in the lingual epithelium, Hoff and Hillyard reported that the toad, Bufo punctatus, is able to “taste” sodium with the abdominal skin. This was reflected in a differential behavioral response to hypertonic NaCl. The present study tests for the presence of such abdominal chemoreceptors in the frog Rana pipiens. The experiment was a five-condition design in which frogs were placed on filter paper saturated with: deionized water, 250 mM NaCl, 350 mM NaCl, 12.9 M amiloride, or 350 mM NaCl ⫹ 12.9 M amiloride. The time that the frogs remained on the test substrate before moving to a surface of deionized water was recorded. It was necessary to dehydrate the frogs to 80% of their body weight to elicit a behavioral response to the NaCl whereas dehydration to 90% of their body weight has been reported effective in Bufo punctatus. The frogs displayed significantly shorter mean times to move on both concentrations of NaCl compared to deionized water, with the shortest times occurring when 350 mM NaCl was used. Amiloride alone did not have an effect upon times to move to deionized water, but did significantly reduce the response to 350 mM NaCl. Movement to amiloride ⫹ 350 mM NaCl did not differ significantly from that to deionized water. The results indicate that Rana pipiens, like Bufo punctatus, have epithelial chemoreceptors for the detection of NaCl on hydrated surfaces and that these receptors, like those of mammals, are amiloride sensitive. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Amphibians; Sodium detection; Water Absorption response; Rana pipiens
1. Introduction Amphibians living in dry or hot climates experience substantial water loss through their permeable skin as a result of rapid evaporation. To compensate for this, amphibians take in water from the environment. Frogs and toads do not drink [1], but they absorb water through the integument, especially through an area of thin vascularized skin on the posterior underside of the body (known as the pelvic or seat patch; [2]). The seat patch allows semiterrestrial anurans to use minute sources of water, such as soil moisture, to rehydrate. Because anurans rapidly rehydrate by passive movement of water across their epithelia, it is necessary that they avoid contacting solutions that are hypertonic to their body fluids (⬎243–-284 mM) to avoid osmotic dehydration. Brekke, Hillyard, and Winokur [3] showed that desert toads, Bufo punctatus, avoid solutions hypertonic to their body fluids. Dehydrated toads were placed on wetted surfaces saturated with various concentrations of urea, and the duration of time they remained on the surface was recorded. Brekke and her colleagues found significant decreases in the time that the toads remained on the wetted surfaces as the concentration * Corresponding author. Tel.: 717-291-3823; Fax: 717-291-4387 E-mail address: [email protected]
of urea increased. Similar results were obtained using surfaces saturated with NaCl. These results suggest the toads have cutaneous receptors either for detecting osmolality or for detecting specific solutes in hydration sources. Hoff and Hillyard [4] attempted to demonstrate that the toads have cutaneous receptors for detecting Na⫹. Amiloride-sensitive Na⫹ channels are present on the apical membrane of the skin of all amphibians, and mediate the intake of Na⫹ across the epithelium [5]. Amiloride-sensitive Na⫹ channels are also present on the tongues of drinking vertebrates and mediate Na⫹ influx into the taste buds [6,7]. Hoff and Hillyard [4] hypothesized that amiloride-sensitive Na⫹ channels are important in the detection of Na⫹; they hypothesized that, “toads taste sodium with their skin” (p. 347). Bufo punctatus, dehydrated by 10% of their standard weight, were placed on a surface of hyperosmotic NaCl. The mean time until toads moved off the surface was significantly less than when they were placed on a surface of deionized water. To determine if Bufo punctatus detected Na⫹ specifically or if they detected osmolality in general, the investigators added amiloride to the surface saturated with NaCl. They reasoned that, if the toads responded to NaCl because they detect osmolality, adding amiloride to the surface should not affect that response, because the con-
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centrations of solutes on the surfaces with and without amiloride are the same. On the other hand, if their movement off the surface wetted with NaCl solution was a taste phenomenon, the toads should no longer move quickly off the surface because the taste receptors are blocked by the amiloride. They found that, in the presence of amiloride and NaCl, the duration that subjects remained on the hydrated surface increased to a significantly greater time compared to toads placed on a substrate with NaCl alone. Hoff and Hillyard [4] concluded that the cutaneous amiloride-sensitive Na⫹ channels present in Bufo punctatus, “serve in a sensory capacity for Na⫹” (p. 349). Further supporting this conclusion was the observation that amiloride did not affect the time spent on a surface saturated with KCl. Similarly, amiloride reduces humans’ taste perception of NaCl, but has no effect on perception of KCl [8]. The present study is a replication of the study by Hoff and Hillyard [4] examining cutaneous detection of NaCl by the Northern leopard frog (Rana pipiens). Rana pipiens were chosen because they are somewhat terrestrial, suggested by their tolerance for dehydration to 50–55% of their standard weight [9], and by the considerable distances they reside from sources of standing water during the summer (cited in [10]). Also, unlike Bufo punctatus, they are readily available from commercial suppliers year round. We hypothesized that partially dehydrated frogs can detect NaCl on a hydrated surface, and that the detection of NaCl is blockable with amiloride.
2. Materials and method 2.1. Subjects Twenty Rana pipiens, purchased from Charles D. Sullivan Co., Inc., were housed individually in 10 ⫻ 5 ⫻ 5⬙ plastic containers with wire mesh tops. The containers were filled with 400 mL of dechlorinated water and rested on a 9⬚ angle so that the water was pooled at one end of the container. Frogs were kept on a 10-h light:14-h dark photoperiod at an approximate temperature of 22⬚C. They were fed crickets twice a week. The plastic containers were numbered for the purpose of subject identification. Subjects were tested individually and randomly assigned to a condition; 10 frogs were assigned to each condition studied. 2.2. Design This study utilized a five-condition design. The five levels of the independent variable (type of test solution onto which the subject was initially placed) were (1) deionized water, (2) 250 mM NaCl, (3) 350 mM NaCl, (4) 12.9 M amiloride, and (5) 350 M NaCl ⫹ 12.9 mM amiloride. The dependent variable was the ability of Rana pipiens to detect NaCl on hydrated surfaces. This ability was measured according to (1) the percentage of subjects that moved
off the wetted surface of test solution, and (2) the amount of time the subjects spent on the wetted surface of test solution before moving to the surface of deionized water. 2.3. Apparatus The apparatus used for behavioral observation of Rana pipiens on a wetted surface was adopted from the paradigm of Brekke et al. [3] and Hoff and Hillyard [4]. A glass-bottom aquarium (70 ⫻ 36.3 ⫻ 35 cm) was supported 25.5 cm above an angled mirror (70 ⫻ 36.3 cm), and the walls of the aquarium were covered with brown paper so the frogs could not view the experimenter. A smaller aquarium (35 ⫻ 17.5 ⫻ 20.3 cm), also covered with brown paper, was placed inside the larger aquarium. The bottom of this smaller aquarium was lined with two 14.5 ⫻11.3-cm pieces of Kimwipe separated by 0.6 cm. One of these Kimwipes was saturated with test solution, while the other was saturated with deionized water. The apparatus was constructed so that the underside of the subjects could be observed in the mirror, and the observer could not be detected by the subject. 2.4. Procedure The frogs’ bladders were emptied, and the frogs were placed in a dry container, without access to water, for 15–16 h. In that time the frogs were dehydrated by an average of 21.43% (range 10.88–37.24) of their standard weight. (Pilot studies revealed that neither fully hydrated Rana pipiens nor Rana pipiens dehydrated by 10% of their standard weight responded to solutions of NaCl, even at concentrations as high as 450 mM). Each dehydrated frog was placed on the surface wetted with test solution, and was observed in the angled mirror to determine whether or not it moved from the aversive stimulus to the surface of deionized water. The duration of time that each frog spent on each substrate was recorded. Each trial lasted 5 min. The percentage of frogs that moved from the test solution to deionized water and the mean amount of time the subjects remained on the surface saturated with test solution were calculated. Analyses of variance with post hoc Bonferroni t-tests and Fisher exact probability tests were used as appropriate to compare conditions.
3. Results Only 1 of 10 frogs moved from the test surface of deionized water, whereas 8 of the 10 frogs initially placed on a wetted test surface of either 250 or 350 mM NaCl moved to the surface of deionized water (Fig. 1). These differences were significant (Fisher exact probability test, p ⬍ 0.05). Surprisingly, 7 of the 10 frogs tested moved from the 350 mM NaCl ⫹ amiloride test surface. Amiloride alone produced the same effect as deionized water alone with only 1 out of 10 frogs leaving (Fig. 1). The addition of amiloride to 350 mM NaCl significantly increased the time that frogs remained on that solution to a
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Fig. 1. Number of subjects that moved to the adjacent surface of deionized water as a function of the test solution presented on the original surface. Fig. 2. Mean time R. pipiens remained on the initial surface before moving to deionized water, as a function of the test solution presented.
value that was not different from the time that frogs spent on deionized water (Fig. 2). Analysis of variance showed that there was a significant effect of type of test solution on the time animals remained on the test surfaces, F(3, 36) ⫽ 11.476, p ⬍ 0.0001. Bartlett’s test of homogeneity of variance showed no significant differences in the standard deviations for the test conditions. Bonferroni t-tests showed that the differences between deionized water and each of the NaCl solutions was significant (t ⫽ 4.3119 and t ⫽ 5.4158, respectively, p ⬍ 0.001). The difference between deionized water and amiloride ⫹ 350 mM NaCl was not significant, whereas the difference between 350 mM NaCl and amiloride ⫹ 350 mM NaCl was significant (t ⫽ 3.2773, p ⬍ 0.05). The data from a separate control group tested with amiloride alone did not differ significantly from the group tested with deionized water. Finally, some noteworthy observations were made concerning the animals’ behaviors and responses when placed on test surfaces saturated with NaCl solutions. For example, when moving from the NaCl, the frogs did not just leap to the surface of deionized water. Most gradually moved toward the deionized water surface, pressing their seat patches to the NaCl surface for several seconds, and then moving to another region of the surface. This process continued until the subjects crossed to the surface of deionized water. Additionally, some frogs that were initially presented with NaCl would press their bodies up against the wall of the test chamber and extend their legs, an apparent attempt to climb out of the test apparatus.
4. Discussion The results of this study suggest that Rana pipiens, like Bufo punctatus, can detect NaCl with their skin. The frogs distinguished hypertonic solutions of NaCl from pure water, as indicated by the significant increase in the number of
frogs that moved, and the reduction in the mean time to move, when NaCl was present on the wetted surface. Other behavioral observations recorded during the trials also suggest that Rana pipiens can detect NaCl with their skin. First, frogs that were initially placed on a surface wetted with NaCl solution attempted to exit the test chamber by climbing its walls, but frogs that were initially placed on a surface wetted with deionized water did not attempt to exit the test chamber. Second, the frogs shifted position on the surface saturated with NaCl solution, evidently because they were searching for a less aversive region of the surface. Similarly, Brekke et al. [3] observed an increased frequency of movements by toads placed on hyperosmotic urea solutions. That the frogs lowered their seat patch region to the surface for several seconds, and then moved on to a new portion of the surface, provides evidence that the seat patch is the region of the abdominal skin used to detect NaCl. Stebbins and Cohen [2] and Hoff and Hillyard [4] make this suggestion as well, and it seems reasonable, considering the seat patch’s integral contribution to cutaneous water uptake. The results of this study not only suggest that Rana pipiens can detect NaCl on wetted surfaces, but also that the detection of NaCl is mediated, at least in part, by amiloridesensitive channels. Frogs initially placed on the surface wetted with 350 mM NaCl ⫹ amiloride remained on the initial surface longer than did the frogs initially placed on the surface wetted with only 350 mM NaCl (Fig. 2). Although the number of frogs responding to 350 mM NaCl was not affected by amiloride (Fig. 1), the time to move increased, and did not differ from the response to deionized water. Data in Hoff and Hillyard [4] and in the present study are also consistent with an amiloride-insensitive component of salt taste if one considers only the number of animals responding. This is consistent with salt taste by the tongue of numerous mammalian species [11].
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There are two possible explanations for Bufo punctatus’ and Rana pipiens’ amiloride-blockable response to NaCl. The first hypothesis, which is advocated by Hoff and Hillyard [4], is that anurans have a discrete taste mechanism for NaCl that stimulates them to move from salty substrates unless amiloride is present. An alternative hypothesis is that anurans experience osmotic thirst that causes them to escape from salty substrates unless amiloride is present. Several lines of evidence support a discrete taste mechanism on the abdominal epithelium of anurans as the explanation for the anurans’ response to NaCl. Most fundamentally, the fact that the response is inhibited by amiloride suggests a salt-tasting mechanism compatible with our understanding of other salt-tasting systems. Amiloride-sensitive Na⫹ channels on the apical membrane of mammalian tongues mediate the chemoreception of NaCl [6,7] Kinnamon and Getchell [11] explain that amiloride has been shown to drastically reduce the response to NaCl in hamsters, rats, dogs, monkeys, and humans. When amiloride was applied to the tongue of these mammals, a 70% reduction in the afferent nerve response to NaCl was recorded. (The remaining 30% of the afferent nerve response is attributed to amiloride-insensitive Na⫹ channels such as TTXsensitive channels). Similarly, in Hoff and Hillyard’s study [4] and in the present study, the latency of the anurans’ behavioral response to NaCl was increased by 70%. Additional evidence for a salt-tasting mechanism includes Hoff and Hillyard’s demonstration that Bufo punctatus escape from surfaces wetted with KCl solutions, as they do from surfaces wetted with NaCl solutions, but the escape response for KCl is not inhibited by amiloride. (The present study did not include using KCl as a test solution.) The amiloride-insensitive response to KCl is also consistent with a mammalian salt-tasting mechanism, in which the nervous response to KCl is not reduced by amiloride. Further evidence for cutaneous Na⫹ tasting was established by Nagai et al. [12], who confirmed the presence of peripheral nerves, in the deep cell layers of the skin of Bufo alvarius, that respond to NaCl. The alternative hypothesis, however, is that anurans move from a hydrated surface of hypertonic NaCl solution because the hypertonic NaCl solution makes them thirsty.
The thirst hypothesis may explain why subjects must be dehydrated before they will respond to NaCl. Dehydration causes volumetric thirst and an animal’s overall level of thirst is a sum of its osmotic thirst (caused by increases in body fluid solute concentration) and volumetric thirst (caused by decreases in body fluid volume). A direct relationship between plasma osmotic concentration and water absorption response in the toad has been shown [13]. This study provides evidence that Rana pipiens, like Bufo punctatus, is able to detect NaCl on a wet surface. This may be a phenomenon common to anuran amphibians.
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