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EXPERIMENTAL ZOOLOGY 286:572–584 (2000)
Effects of Dehydration on Plasma Osmolality,
Thirst-Related Behavior, and Plasma and Brain
Angiotensin Concentrations in Couch’s Spadefoot
Toad, Scaphiopus couchii
Department of Biological Sciences, Northern Arizona University, Flagstaff,
Arizona 86011
Under dehydrating conditions, many terrestrial vertebrates species exhibit increases in plasma osmolality and their drinking behavior. Under some circumstances, this behavioral change is accompanied by changes in plasma and central angiotensin concentrations, and it
has been proposed that these changes in angiotensin levels induce the thirst-related behaviors. In
response to dehydration, the spadefoot toad, Scaphiopus couchii, exhibits thirst-related behavior
in the form of cutaneous drinking. This behavior has been termed water absorption response (WR)
behavior. Spadefoot toads live in harsh desert environments and are subject annually to dehydrating conditions that may induce thirst-related behavior. We tested the hypothesis that an increase
in WR behavior is associated with both an increase in plasma osmolality and an increase in plasma
and brain angiotensin concentrations. First, we determined the degree of dehydration that was
necessary to initiate WR behavior. Animals dehydrated to 85% of their standard bladder-empty
weight via deprivation of water exhibited WR behavior more frequently than control toads left in
home containers with water available. Next, using the same dehydration methods, we determined
the plasma osmolality and sodium concentrations of dehydrated toads. Toads dehydrated to 85%
standard weight also had a significant increase in plasma osmolality, but exhibited no overall
change in plasma sodium concentrations, indicating that while an overall increase in plasma osmolality appears to be associated with WR behavior in S. couchii, changes in sodium concentrations alone are not sufficient to induce the behavior. Finally, plasma and brain angiotensin
concentrations were measured in control toads and toads dehydrated to 85% standard weight.
Plasma and brain angiotensin concentrations did not increase in dehydrated toads, indicating
that dehydration-induced WR behavior that is associated with changes in plasma osmolality may
not be induced by changes in endogenous angiotensin concentrations in S. couchii. J. Exp. Zool.
286:572–584, 2000. © 2000 Wiley-Liss, Inc.
Because of their relatively permeable skin, anuran amphibians can become dehydrated faster
than other terrestrial vertebrates (Bentley, ’66).
Accordingly, anurans adapted to xeric environments have mechanisms to limit the impact of living in a dehydrating environment. One adaptive
mechanism involves seasonal estivation. For most
of the year, desert anurans remain underground
and avoid the aboveground dehydrating conditions
(Shoemaker et al., ’69). Also, desert anurans can
store water in their bladders and reabsorb the
water while underground (Ruibal, ’62; McClanahan, ’72). In addition, some species including the
desert toad, Couch’s spadefoot toad (Scaphiopus
couchii), can increase urea production in response
to drying soil, increasing the plasma osmolality
(McClanahan, ’72), which spadefoot toads utilize
to equilibrate with soil water potential (Shoemaker et al., ’69).
As the ambient temperature rises during late
spring, soil water potential diminishes, and S.
couchii may experience net water loss and an increase in plasma osmolality (Shoemaker et al.,
’69). Upon arrival of the first heavy summer monsoon rains, S. couchii emerge from their burrows
and seek ephemeral ponds for reproduction. With
the initial light rains, emergent toads have been
observed rehydrating on moist substrates, utilizGrant sponsor: National Science Foundation; Grant number: IBN
*Correspondence to: William E. Johnson, Box 5640, Dept. of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011.
Received 1 December 1998; Accepted 27 September 1999
ing a form of cutaneous drinking described as water absorption response (WR) behavior (Stille, ’58;
Dimmitt and Ruibal, ’80; Brekke et al., ’91;
Propper and Johnson, ’94). By pressing their
splayed hind limbs against the moist surface, dehydrated S. couchii can regain rapidly the lost
water weight lost (Claussen, ’69; Propper and
Johnson, ’94; Johnson et al., ’95).
Previous laboratory studies have demonstrated
WR behavior in several species of desert-adapted
anurans (Brekke et al., ’91; Propper and Johnson,
’94). Propper and Johnson (’94) determined that
under laboratory conditions, S. couchii dehydrated
for 5 hr exhibited consistent WR behavior associated with an average of 10% body weight loss for
the group. These results showed that dehydration
alone is sufficient to elicit WR behavior, but that
the degree of dehydration necessary to elicit this
behavior, as measured by changes in osmolality,
remains undefined. One purpose of this study is
to quantify the association between the onset of
WR behavior and changes in plasma osmolality
in dehydrated S. couchii. In addition, sodium concentrations were compared between hydrated and
dehydrated toads, in order to test the hypothesis
that an increase in WR behavior in dehydrated
S. couchii is associated with an increases in
plasma sodium concentrations. Increases in sodium concentrations have been associated with
increases in thirst-related behavior in other vertebrates (Fitzsimons, ’79).
One potential neuroendocrine mediator of WR behavior is the hormone angiotensin II (A-II). Angiotensin II has been shown to induce thirst-related
behavior in many taxa of vertebrates (Rolls and
Rolls, ’82; Kobayashi et al., ’79). Peripherally injected angiotensin II increases drinking behavior
in teleosts (Hirano and Hasegawa, ’84; Perrott et
al., ’92), elasmobranchs (Hazon et al., ’89), reptiles
(Fitzsimons and Kaufman, ’77), birds (Snapir et al.,
’76; Fitzsimons, ’79, ’98), and mammals (Fitzsimons,
’79, ’98). In addition, A-II injected intraperitoneally
also stimulates WR behavior in two families of
anurans (Hoff and Hillyard, ’91; Tran et al., ’92;
Propper and Johnson, ’94). The fact that peripherally administered A-II can stimulate robust drinking behavior in many different species supports the
view that endogenous plasma angiotensin, released
under dehydrating conditions, may play a role in
thirst-related behavior in vertebrates.
A substantial body of correlative evidence links
increases in plasma osmolality with both an increase in plasma A-II and thirst-related behavior.
Dehydration-induced increases in serum osmola-
lity, plasma A-II, and plasma sodium concentrations are observed in mammals (Mann et al., ’80;
Yamaguchi, ’81) and birds (Gray and Simon, ’85;
Gray and Erasmus, ’89). Increased plasma concentrations of A-II are also associated with increased thirst-related behavior in mammals
(Johnson et al., ’84), birds (Gray and Erasmus,
’88), and fish (Carrick and Balment, ’83; Perrott
et al., ’92; Tierney et al., ’95). These correlative
data suggest that induction of thirst-related behavior in vertebrates may be caused by increases
in plasma osmolality and/or in plasma A-II concentrations.
There is some preliminary evidence for a peripheral, circulating renin-angiotensin system in
anurans. The presence of angiotensinogen and angiotensin-converting enzyme activity has been established in the plasma of two species of bufonid
amphibians (Nolly and Fasciolo, ’71a, b; FernandezPardal et al., ’86). In addition, the A-II antagonist
saralasin attenuates WR behavior in Bufo punctatus
(Hoff and Hillyard, ’91), but not in S. couchii or
Bufo cognatus (see Propper and Johnson, ’94). The
results from these studies suggest that endogenous
plasma A-II may have a role in thirst-related behavior in some species of anurans. In order to determine the relationship between dehydration,
plasma A-II and thirst-related behavior in amphibians, we examined the effects of dehydration
on plasma osmolality and A-II concentrations in
S. couchii in the laboratory.
There is also evidence that a central renin-angiotensin system may be associated with WR behavior in S. couchii. Angiotensin II injected
intracerebroventricularly (icv) induced WR behavior at a 1000-fold less dose than that injected
ip (10 ng/100g-animal vs. 10 µg/100 g-animal:
Propper and Johnson, ’94; Propper et al., ’95). Angiotensin injected icv can also induce thirst-related
behavior in mammals (Epstein et al., ’70; Wright
et al., ’88), reptiles (Fitzsimons and Kaufman, ’77),
and birds (Wada et al., ’75; Snapir et al., ’76; Takei,
’77), suggesting that regulation of thirst by a central renin-angiotensin system is an evolutionarily
conserved phenomenon among vertebrates.
Because both dehydration and A-II injected icv
can stimulate drinking in rats and other species
(Fitzsimons, ’79), many studies have explored the
possibility that concentrations of central angiotensin increase in response to dehydration (reviewed by Saavedra, ’92). Dehydration, via 48-hr
water deprivation, induced an increased release
of angiotensins from rat paraventricular nuclei
(Harding et al., ’92). However, Yamaguchi (’81) and
Yamaguchi et al. (’81) found no changes in hypothalamic tissue A-II concentrations in water-deprived or nephrectomized rats, compared to
water-replete rats. Because of the limited number
of studies, the association between brain angiotensin
concentrations and thirst-related behavior is not
well defined in any class of vertebrates, including
Amphibia. Therefore, a second goal of this study
was to test the hypothesis that changes of brain
angiotensin concentrations occur in S. couchii exposed to dehydrating conditions previously shown
to induce WR behavior.
Collection and care
Male S. couchii were collected from the Buenos
Aires National Wildlife Refuge in Pima County,
AZ during the monsoon season in July, 1994 and
July, 1995. Following transport to Northern Arizona University, all toads were housed four to five
per container (53 × 41 × 18 cm) filled halfway with
moistened soil. Water was provided in 10-cm water dishes, and was changed three times weekly.
Toads were fed waxworms and crickets three times
a week, and were maintained on a seasonal photoperiod in a temperature-controlled room maintained at 22°C. All experiments were conducted
in a room maintained at a constant 22°C.
Experiment 1: WR behavior
This experiment was conducted in September
of 1994 on toads collected in July, 1994. In order
to insure that all toads were fully hydrated initially, ten male S. couchii were hydrated for 3 hr
in 0.5 liter of water in a 40-liter aquarium, enough
to cover the bottom of the aquarium and the ventral surface of the toads, then returned to their
home containers. Twenty-four hours later and before the toad was weighed, we emptied each toad’s
bladder by cannulation with a micropipette tip inserted into its cloaca. This weight is referred to
as the toad’s standard weight (SW; Ruibal,’62).
Toads were then randomly assigned to one of
four treatments: toads were placed in a dry 40liter container then allowed to dehydrate to 95%,
90%, 85%, and 100% SW (rate of dehydration for
all treatments: 0.63 ± 0.03 g/hr). Control toads
(100% SW) were left in their home container with
water available ad libitum. Each toad received
each treatment once in random order every 4 days.
The experiment room was kept at 22°C and 20–
25% relative humidity. Air flow was kept at a
recommended level of 10 air changes per hour, ac-
cording to Guide to the Care and Use of Laboratory Animals (National Research Council, ’96).
At the end of the treatment period, toads were
placed in a petri dish filled with 25 ml dechlorinated tap water. Toads were scored for behavior
by the method established by Propper and Johnson (’94): toads were scored for WR behavior once
every 10 min for 2 hr, with a 1 given for each
observation period where WR behavior was exhibited (ventral seat patch pressed against wet
substrate), and a 0 for each observation period
where no WR behavior was observed.
The scores were summed for the 2-hr test period for each toad’s four individual treatments.
The combined scores were analyzed by Friedman’s
repeated measures ANOVA by ranks, and post hoc
comparisons were determined by utilizing the
multiple comparison test in Chapter 7 of Siegel
and Castellan (’88).
Experiment 2: plasma osmolality
and angiotensin
This experiment was conducted in June 1995
on toads captured in July 1994. Twenty-nine male
toads were prehydrated as above before determination of their standard weight on the treatment
day. Each toad was then assigned to one of the
four treatments as described above in experiment
1, for a total of seven toads per treatment group
(except for eight toads in 85% SW group). We could
not conduct this study concurrently on the same
toads as in experiment 1, because removal of blood
by heart puncture would produce a hypovolemic
condition which might affect subsequent WR behavior (Taylor, Mayer, and Propper, 1999).
After treatment, a 200- to 500-µl blood sample
was removed from each toad by heart puncture
with a heparin-coated 1-cc syringe, then prepared
for radioimmunoassay as described below. The
sample was transferred to a chilled (4°C) 1.5-µl
polypropylene microcentrifuge tube containing 7.5
µl of a peptidase inhibitor cocktail of the following composition: 4 µg/ml enalpralat maleate
(Sigma), 440 µg/ml 1,10 phenanthroline (Sigma),
40 µg/ml APMSF (Sigma), 1 µg/ml pepstatin
(Sigma), 0.1%-mercaptoethanol (Sigma), and 6.25
mM EDTA (Ledwith et al., ’93). After centrifugation at 2000 g for 5 min, the plasma was decanted
into a cryogenic tube for future peptide analysis.
A 10-µl sample of plasma was removed at this
time for measurement of osmolality with a Wescor
5500 osmometer. In a pilot study, no effect was
noted for the peptidase inhibitor in these measurements (Johnson, unpublished data). Differ-
ences in osmolalities among treatment groups
were determined by one-way ANOVA, and post
hoc comparisons were determined with StudentNeuman-Keuls multiple comparison procedure.
at –20°C until protein determination. Protein concentrations were measured using a modified
Bradford assay (Bradford, ’76) previously utilized
by Propper and Moore (’91).
Experiment 3: plasma osmolality, sodium
and angiotensin, and brain angiotensin
Radioimmunoassay of plasma and
brain angiotensin
This experiment was conducted in October–November, 1995 from toads collected in July, 1995.
After prehydration and bladder cannulation as
described in experiment 1, toads were either dehydrated to 85% SW (n = 10), or left to hydrate (n
= 9) in 0.5 l of dechlorinated tap water in a 40-l
aquarium for an equivalent length of time. Toads
were then sacrificed by decapitation. Trunk blood
was collected and treated with peptidase inhibitors as described previously. Plasma osmolality
was measured on a Wescor 5500 osmometer, and
sodium concentrations were determined by flame
Brains were dissected and removed intact
within 7 to 11 min of decapitation. Brains were
immediately placed in Tissue Tek O.C.T. compound and frozen on dry ice. Frozen brains were
then placed in cryogenic storage tubes and stored
at –80°C.
Cross reactivity was measured for angiotensin
I, angiotensin III (A-III), and arginine vasotocin (Fig. 1A). Since angiotensin III cross-reacted
100% with the antiserum, results were reported
as immunoreactive angiotensin (ir-ANG) concentrations.
Plasma procedures were carried out on ice (4°C).
Unextracted plasma was used because of small
volumes of plasma from some animals (150–500
µl). Also, Mann, Johnson, and Ganten (’80) used
unextracted plasma when measuring dipsogenic
levels of A-II in rats, and Lijnen et al. (’78) found
significant correlation between extracted and
unextracted A-II concentrations. Parallelism was
determined by comparing standard curves to serial
dilutions of unextracted plasma by analysis of covariance (Fig. 1B; Dayton, ’70). There was homogeneity of regression between curves (FANCOVA 1,7 = 3.33,
P > 0.10).
Sixty microliters of unextracted plasma was
brought up to 100 µl with the assay buffer, PEgel buffer (10 mM potassium phosphate, 1 mM
EDTA, 0.1% gelatin, pH = 7.3). Standard curves
were obtained by adding 100 µl of 5Val-A-II (0.25–
1000 pg; BACHEM California, Inc., Torrance, CA)
in assay buffer, 100 µl of the antiserum 1:75,000
in assay buffer (RAS 7002N for experiment 2; for
experiment 1 and 3, IHC-7002; Peninsula Laboratories, Inc., Belmont, CA), and 50 µl of 7000–
8000 cpm of labeled tracer (3-[125I]-(iodotyrosyl4)
angiotensin II (5-leucine), Amersham Corp., Arlington Heights, IL). All standards were run in
duplicates. Tubes were incubated 18 hr at 4°C.
One ml of dextran-coated charcoal (1 g NORIT-A
charcoal with 0.02 g Dextran [Sigma, St. Louis, MO]
in 100 ml PE-Gel assay buffer) was pipetted into
each tube and tubes were immediately centrifuged
at 3000 rpm for 10 min. Supernatants were counted
on a Beckman 5500B gamma counter. The minimum detection limit ranged from 0.39 to 1.2 pg.
The intraassay coefficient of variation was 9.0 ±
1.3%, and the interassay coefficient of variation was
21 ± 0.8% for 2 pg and 20 pg replicates on five assays total with both antisera.
For determination of parallelism in unextracted
brain homegenate, whole brain homogenate was
split into two samples, with one sample spiked
Tissue preparation
Brains were frozen to cryostat chucks with Tissue Tek O.C.T. compound then sectioned in 200µm thick transverse sections in a Leica cryostat
kept at –17°C. Sections were thaw-mounted then
frozen to microscope slides and stored at –80°C
in an airtight slide box until microdissection.
Eight areas of the toad brain were identified
using descriptions from Northcutt and Royce (’75)
and Neary and Northcutt (’83). All brain areas
were removed by the micropunch technique described in Palkovitz and Brownstein (’82) and
Zoeller and Moore (’85). Using a modified stainless-steel hypodermic 26-gauge needle, five to
seven punches were made within each brain area
and placed in a drop of 1 M acetic acid on a chilled
4°C spatula. The punches were then transferred
to a chilled tube containing 250 µl of 1 M acetic
acid. The brain punches were then homogenized
using a Branson 450 sonifier with a double step
microtip for 2 sec. (Output 1 for 10 % cycle 10
watts). The homogenized brain solutions were centrifuged at 11,000g for 20 min, and from each
sample 200 µl of the supernatant was lyophilyzed
and stored dessicated at –80°C until assayed for
angiotensin by RIA. The remaining 50 µl was
dried under airjet in a 37°C water bath and stored
with 1 pg/µl A-II and one sample left unspiked.
Each sample was serially diluted, then run in RIA
standardization. Parallelism was determined by
comparing regressions of serial dilutions of whole
brain homogenates with standard curves by analysis of covariance (Dayton, ’70). There was no difference between slopes (Fig. 2; FANCOVA 1,11 = 0.40,
P > 0.5), indicating that there were no extraneous agents in the unextracted brain homogenate.
Lyophilyzed brain homogenates were reconstituted in 100 µl PE-gel assay buffer (10 mM potassium phosphate, 1 mM EDTA, 0.1% gelatin, pH =
7.3). Standard curves were obtained by similar
methods used in determining plasma standards.
The minimum detection limit ranged from 0.39 to
1.2 pg. The intraassay coefficient of variation was
7.6%, and the interassay coefficient of variation was
12.5%. As noted earlier, the antiserum cross-reacts
with A-III 100% (Peninsula Laboratories, personal
communication) and the results are reported as immunoreactive angiotensin (ir-ANG).
Eight brain areas from each toad were sampled
in the experiment. All results were expressed as
pg angiotensin/µg protein. Because many samples
had protein concentrations below detectable limits, the angiotensin concentrations of five of the
brain areas were not included due to insufficient
sample size for statistical analysis.
Fig. 1. (A) Competitive binding curves of angiotensins I,
III, and arginine vasotocin against the standard curve for AII. Angiotensin II and III competed similarly with labelled AII for the antisera; the other peptides compete at much higher
concentrations. (B) Parallelism among regression lines of serial dilutions (60, 30, 15, and 7.5 µl) of two plasma samples
from different toads compared to the standard curve for A-II,
determined by analysis of covariance (Dayton, ’70). There was
no difference among slopes of the plasma samples and the
standard curve (FANCOVA 1,7 = 3.33, P > 0.10).
Fig. 2. Serial dilutions of whole brain homegenates run
in RIA against standard curves. There was no difference
among slopes (FANCOVA 1,11 = 0.40, P > 0.5).
All results, including osmolalities, sodium concentrations, plasma angiotensin concentrations,
and brain angiotensin concentrations, were analyzed by Student’s t-test for independent samples.
Angiotensin concentrations determined to be outliers by statistical methods described in Sokol and
Rohlf (’81) were removed from the analysis.
Experiment 1: WR behavior
There was a significant effect of dehydration on
WR behavior in S. couchii (Fig. 4A). Toads dehydrated to 85% SW showed significantly more WR
behavior than 0% SW toads (χ2= 13.82, P = 0.003).
Angiotensin III and WR behavior
Because A-III can comprise 10–20% of the circulating angiotensin in mammals (Semple et al.,
’76; Hanssens et al., ’90), and our assay could
not distinguish between A-II and A-III, a repeated measures ANOVA experiment was designed to examine the induction of WR behavior
by A-III. On the first experimental day, hydrated, bladder-emptied toads were injected intraperitoneally with either 0, 10, and 100 µg
A-III/100 g-wt toad. Toads were scored for the
total number of 10 min periods spent in WR
behavior after each treatment (see experiment
1). The experiment was repeated twice four
days apart so each toad received each treatment
in random order. Results were analyzed by Friedman’s repeated measures ANOVA by ranks.
The median number of time periods all toads
spent in WR behavior after each treatment is
shown in Figure 3. Angiotensin III did not induce
an increase in WR behavior in hydrated toads
(P = 0.91).
Fig. 3. Angiotensin III injected intraperitoneally at 0,
10, or 100 µg/100 g-wt toad in hydrated toads did not induce an increase in WR behavior. Vertical lines through
bar graphs represent intraquartile ranges for WR behavior in the three treatments.
Figure 4.
According to the results of the post hoc test, the
90% and 95% SW groups were not different from
either the 85% or 100% SW groups.
To determine whether toads showing WR behavior were more effective in rehydration than
toads not showing the behavior, a Spearman’s correlation analysis was used. The toads that spent
more time in WR behavior gained more weight
(Fig. 4B; r = 0.65, P < 0.005).
Experiment 2: plasma osmolality and
angiotensin from heart puncture
There was a significant effect of dehydration on
the plasma osmolalities of the toads (Fig. 5A).
When dehydrated to 85% SW, toads had significantly higher plasma osmolalities than when left
in home containers (F3,6 = 3.51, P = 0.031). According to the results of the post hoc test, the 90%
and 95% SW groups were not different from either the 85% or 100% SW groups.
There was no change in plasma angiotensin concentrations with dehydration treatment (Fig. 5B;
F3,26 = 0.56, P = 0.65). There was also no correlation between angiotensin concentrations and
plasma osmolality (Fig. 6; Pearson’s r = 0.174, P
= 0.396), indicating that increases in plasma osmolality do not appear associated with circulating angiotensin concentrations.
Experiment 3: plasma osmolality, plasma
sodium concentrations, and plasma
angiotensin concentrations from trunk
blood; brain angiotensin
As in experiment 2, toads dehydrated to 85%
SW had significant increases in plasma osmolality (Fig. 7A; t = 3.51, P = 0.003). However, the
overall increase in osmolality was not augmented
by a concurrent increase in plasma sodium concentrations (Fig. 7A; t = 0.136, P = 0.193).
Fig. 4. (A) Experiment 1: Bar graphs represent median (±
intraquartile ranges) amount of time periods spent by S. couchii
in water absorption response (WR) behavior after four treatments: dehydration to 95%, 90%, or 85% standard weight (SW),
or kept in a container as a control (100% SW). Bars with different letters above them are significantly different. Toads dehydrated to 85% SW spent more time in WR behavior than
control toads. (B) Percent weight change during WR behavior
test plotted against median WR behavior score for each toad
in all treatments. The different dehydration treatment groups
are represented by different symbols. Results from the correlation test indicated that toads regained most, if not all, of the
water that was lost during dehydration treatment, and toads
that spent more time in WR behavior gained more weight.
Fig. 5. (A) Experiment 2: Bar graphs represent mean (± SEM)
plasma osmolalities for toads dehydrated to 95% SW, 90% SW,
or 85% SW, or kept in a container as a control (100% SW) as
in the behavioral experiment. Bars with different letters above
them are significantly different. At 85% SW loss, toads had
higher plasma osmolalities than toads in 100% SW treatment
but not different from either 95% or 90% SW. Error bars are
standard error of the mean. (B) Bar graphs represent mean (±
SEM) plasma angiotensin (ir-ANG) concentration from the
toads’ blood collected by heart puncture. There was no difference in ir-ANG concentrations among the SW groups.
Fig. 6. Experiment 2: Plasma osmolalities versus plasma
ir-ANG concentrations. There was no significant correlation
between the variables, indicating that there was no association between dehydration-induced increases in plasma osmolalities and plasma ir-ANG concentrations.
As with experiment 2, plasma angiotensin concentrations from trunk blood did not increase with
dehydration (Fig. 7B; t = 0.72, P = 0.49), and there
was no correlation between plasma osmolality and
angiotensin concentrations (Fig. 8A; r = –0.22, P
= 0.40). There was no difference between the dehydrated and hydrated groups (Fig. 8B) in ir-ANG
concentrations of the anterior pituitary (T = 76, P
= 0.74), posterior pituitary (t = 0.94, P = 0.37),
and preoptic area (t = 0.89, P = 0.40).
Plasma osmolality and WR behavior
This study demonstrates that dehydration to 85%
SW is sufficient to consistently induce WR behavior in S. couchii. These results concur with the previous results with S. couchii by Propper and
Johnson (’94), except that in the 1994 study toads
were not prehydrated or bladder cannulated. Since
the toads in the 1994 study showed WR behavior
at 10% mass loss, some toads may have started dehydration treatment already at a water deficit.
Brekke et al. (’91) and Tran et al. (’92) also demonstrated that dehydrated bladder-emptied Bufo
punctatus and Bufo woodhouseii exhibit WR behav-
Fig. 7. (A) Experiment 3: Bar graphs indicate mean plasma
osmolalities (± SEM for total osmolality) for toads dehydrated
to 85% SW or hydrated (100% SW). Toads dehydrated to 85%
SW had significantly higher plasma osmolalities than toads
in 100% SW treatment. Plasma sodium concentrations
(shaded areas) were not different between the two treatments.
(B) Bar graphs represent mean (± SEM) plasma ir-ANG concentrations from the toads’ trunk blood. There was no difference in plasma ir-ANG concentrations between the 100% SW
and 85% SW groups.
Fig. 8. (A) Experiment 3: Plasma ir-ANG concentrations
plotted against plasma osmolalities. There was no correlation between plasma osmolalities and plasma ir-ANG concentrations. (B) Bar graphs represent mean (± SEM) ir-ANG
concentrations in three brain areas from dehydrated (85%
SW) or hydrated (100% SW) toads. There was no effect of
dehydration to 85% SW on ir-ANG concentrations in the anterior pituitary, posterior pituitary, and preoptic area. Numbers in parentheses above bar graphs indicate sample sizes.
ior at varied levels of dehydration. Plasma osmolality was not measured in Propper and Johnson (’94)
or Brekke et al. (’91), therefore it is difficult to determine the dehydration level for toads in those experiments. Though plasma osmolality was not
measured in toads exhibiting WR behavior (experiment 1), dehydration to 85% SW resulted in an increase in plasma osmolality for toads in experiments
2 and 3. Considered together, these findings support the hypothesis that an increase in thirst-related behavior is associated with an increase in
plasma osmolality in S. couchii. In other vertebrate
taxa, increases in plasma osmolality are also associated with increases in thirst-related behavior
(Fitzsimons and Kaufman, ’77; Fitzsimons, ’79;
Carrick and Balment, ’83; Perrott et al., ’92). Thus,
an increase in plasma osmolality appears to be associated with increased drinking behavior in vertebrates in general.
Because sodium comprises a large portion of the
total plasma solute concentration (McClanahan,
’67, ’72), changes in plasma osmolality may greatly
affect sodium concentrations and thereby induce
thirst-related behavior. The proposed mammalian
thirst-related osmoreceptors may be sodium-sensitive (Andersson and Westbye, ’70). Rats given
icv injections of hypertonic saline show increased
drinking of both water and saline solutions
(Buggy, ’77). Emergent S. couchii have elevated
plasma sodium levels compared to foraging and
breeding pond toads (McClanahan, ’67), thus it is
possible that plasma sodium concentration changes
may be associated with the induction of WR behavior in S. couchii. Although spadefoot toads injected ip with hypertonic saline showed WR
behavior more often than toads injected with isotonic saline (Taylor, Mayer, and Propper, ’99),
plasma osmolality did not increase (Mayer and
Propper, 2000). In addition, plasma sodium levels
in our study did not increase significantly when
toads were dehydrated to 85% SW. In light of this
recent evidence, sodium concentration changes in
peripheral circulation may not be involved in the
induction of WR behavior in S. couchii.
Alternatively, overall osmolality may be more
important in the induction of WR behavior rather
than any one solute. Injections of hypertonic sucrose solutions induced WR behavior in S. couchii,
an indication that induction of WR behavior may
be associated with cellular dehydration (Taylor et
al., ’99). Toads in our study had overall increases
in osmolality, although when considered separately, sodium concentrations did not increase.
Therefore, increases in osmolality may be due to
increased concentrations of other osmolytes. In
addition, hemorrhaging, a form of extracellular
dehydration, also induced WR behavior in S.
couchii (Taylor et al., ’99), indicating that changes
in plasma volume may have significant impact on
WR behavior as well.
Another factor that underscores the complexity
of thirst-related behavior in anurans is the observation that toads become opportunistic, anticipatory drinkers in response to regular exposure to
dehydrating conditions. Jorgensen (’94) found that
Bufo bufo maintained a steady hydrated state
when water was available. How anticipatory
drinking is related to the water deprivation-induced WR behavior is not clear. Therefore, as with
other vertebrates, regulation of thirst-related behavior in S. couchii probably involves a combination of factors including overall osmolality and
volume level, rather than any one factor.
Angiotensin and WR behavior
The results from our present studies indicate
that although dehydration causes an increase of
plasma osmolality in S. couchii, there is no correlation between changes in osmolalities and concentrations of plasma angiotensin. One possibility
is that endogenous plasma angiotensin alone may
not be a stimulus for WR behavior in S. couchii.
Nolly and Fasciolo (’71b) found that in the toad,
Bufo arenarum, acclimation to hypertonic NaCl
solutions increased plasma osmolalities but
caused little change in plasma renin activity. In
one study in rats (Abdelaal et al., ’76), subcutaneous injection of hypertonic saline induced thirst
but caused no change in plasma angiotensin. In
addition, plasma A-II concentrations did not increase in S. couchii injected ip with hypertonic
saline (Mayer and Propper, 2000). Our initial
studies have shown that dehydration to a threshold of 85% SW mass loss is sufficient to induce
WR behavior and an increase in osmolality, therefore, in S. couchii, osmotically-induced thirst-related behavior may not be associated with changes
in plasma angiotensin concentrations that may invoke thirst.
Another alternative hypothesis to consider is
that plasma angiotensin concentrations may increase to concentrations sufficient to induce thirst,
then rapidly diminish before onset of observed behavior. Hoff and Hillyard (’91) have noted that
the time course of angiotensin activity in toads is
largely undefined. In our present study, angiotensin II concentrations did not increase with 85%
SW dehydration, but an A-II signal to induce
thirst may have already diminished by sample collection time. Angiotensin is a relatively fast-acting compound; four classes of vertebrates respond
to angiotensin injections with drinking after 5–
15 min (reviewed by Fitzsimons, ’79; Mann et al.,
’80). Angiotensin II injected ip induces thirst-re-
lated behavior in S. couchii within 15 min (Propper and Johnson, ’94). There are also reported
differences in the length of time necessary to dehydrate rats in order to produce dipsogenic concentrations of angiotensin (12 vs. 48 hr; Mann et
al., ’80; Van Eekelen and Phillips, ’88). A time
course study with increased sample sizes could
address the timing in dehydration-induced angiotensin release.
The lack of dehydration-induced angiotensin response in S. couchii may provide some evidence
why both saralasin and captopril had no effect in
attenuating WR behavior in S. couchii (Propper
and Johnson, ’94; Propper, unpublished data). In
another anuran species, Bufo arenarum, saralasin
did not block hydrosomotic water uptake responses (Reboreda and Segura, ’89). However, dehydrated Bufo punctatus respond to saralasin, a
nonselective angiotensin antagonist, by decreasing WR behavior (Hoff and Hillyard, ’91). The
desert dwelling toad B. punctatus also had a 10fold greater sensitivity to exogenously administered A-II in the induction of WR behavior than
S. couchii, and a 100-fold greater sensitivity than
Bufo cognatus (Hoff and Hillyard, ’91; Propper and
Johnson, ’94). The WR response for B. punctatus
is also at a lower mass loss threshold than for S.
couchii (Brekke et al., ’91). Hoff and Hillyard (’91)
and Hillyard et al. (’98) have suggested that the
interspecific difference in WR behavior may be due
to adaptation to environments where water availability differs.
Alternatively, there may be a central A-II response involved with thirst-related behavior in S.
couchii and other anurans. The colocalization of
A-II and atrial natriuretic hormone (ANH) receptors in several brain regions of Xenopus laevis
(Kloas and Hanke, ’92) supports the view that AII may have central effects in anuran fluid homeostasis. Although dehydration to 85% SW in
the laboratory did not increase ir-ANG concentrations significantly in the brain regions (anterior
and posterior pituitary, preoptic area) examined
in this study, increases in ir-ANG concentrations
in the periventricular nuclei of the hypothalamus
have been found in toads exhibiting WR behavior
in the field (Johnson, Propper, and Hillyard, ’98).
In addition, angiotensin receptors are present in
the ventral hypothalamus of S. couchii near ventricular areas (Propper and Hillyard, ’96). In rats,
both drinking behavior and A-II concentrations in
the hypothalamus increased significantly after
hemorrhage (Phillips, Heininger, and Toffolo, ’96).
The combined evidence supports the possibility of
a central renin-angiotensin thirst-related system
in S. couchii.
Similar to our peripheral A-II study, where
toads were sampled only once after dehydration,
changes in thirst-related central angiotensin could
have occurred prior to sampling. Harding et al.
(’92) found significant amounts of angiotensin released over several hours from the paraventricular nuclei of water-deprived rats. In rats, a
single bolus of angiotensin infused icv has been
estimated to have a half-life of approximately 30
sec before degradation by peptidases (Harding et
al., ’86). Therefore, central angiotensin in amphibians may be produced, bound, and degraded too
rapidly for a single measurement in an individual
animal to accurately reflect prior changes in angiotensin concentrations related to the elicited
thirst-related behavior.
A third possibility is that toads were not dehydrated sufficiently to produce an increase in central angiotensin concentrations that would invoke
thirst, especially if the thirst-related behavioral
response is elicited by hypovolemia. Spadefoot
toads dehydrated to 70% SW show significant decreases in blood pressure (Hillman, ’80). Central
angiotensin has been demonstrated in mammals
to play a simultaneous role in blood pressure regulation and in thirst behavior (Unger et al., ’88;
Evered, ’91). In support of this hypothesis for
anurans, microinjection of the hypotensive agent αadrenergic antagonist phenoxybenzamine (POB)
into the midbrain tegmentum of the toad B. arenarum increases water uptake (Segura et al., ’82).
Therefore, if a thirst-related response in S. couchii
is associated with central angiotensin control of
blood pressure, then a central angiotensin response
may not occur until the toad is exposed to severe
dehydration and resultant hypotension.
In summary, S. couchii exhibits an increase
in plasma osmolality and in thirst-related behavior when dehydrated to 85% SW in the laboratory, but does not show a concurrent increase in
plasma angiotensin during the sampling intervals
used in these experiments. Immunoreactive angiotensin can be found in the brain of S. couchii,
but concentrations did not increase in the brain
areas examined after exposure to dehydrating
conditions in the laboratory. Therefore, the thirstrelated behavior induced in S. couchii in these
studies is probably not associated with an increase in plasma angiotensin concentrations or
in changes in brain angiotensin concentrations,
but may be induced by an increase in plasma osmolality.
We would like to thank Stan Hillyard of UNLV
for his field help and intellectual guidance; Loretta
Mayer, Randy Wade, and Lisa Rania Gibbons for
laboratory assistance and animal collection help;
and Dr. Robert Manicke of the University of Maryland for statistical advice. We would like to especially thank the staff at the Buenos Aires National
Wildlife Refuge for our housing, collecting permits,
and general assistance.
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