COMPENSATORY BEHAVIORAL RESPONSES
TO COMBINED CHALLENGES TO BODY
SODIUM AND FLUID BALANCE
By
KIMBERLY JEAN LUCIA
Bachelor of Science in Biology
Pennsylvania State University
State College, Pennsylvania
2008
Submitted to the Faculty of the
Graduate College of the
Oklahoma State University
in partial fulfillment of
the requirements for
the Degree of
MASTER OF SCIENCE
December, 2010
COMPENSATORY BEHAVIORAL RESPONSES
TO COMBINED CHALLENGES TO BODY
SODIUM AND FLUID BALANCE
Thesis Approved:
Kathleen S. Curtis, PhD
Thesis Adviser
Alexander J. Rouch, PhD
Bruce A. Benjamin, PhD
Mark E. Payton, PhD
Dean of the Graduate College
ii
TABLE OF CONTENTS
Chapter
Page
I. INTRODUCTION .......................................................................................... 1
II. REVIEW OF LITERATURE ......................................................................... 4
Osmotic Thirst ............................................................................................ 6
Volemic Thirst ............................................................................................ 8
Sodium Appetite ......................................................................................... 8
Combined Thirst and Sodium Appetite ..................................................... 10
Goals ........................................................................................................ 12
III. METHODOLOGY ..................................................................................... 13
Animals .................................................................................................... 13
Experimental Groups ............................................................................... 13
Experiment 1 ............................................................................................ 14
Experiment 2 ............................................................................................ 15
Experiment 3 ............................................................................................ 17
iii
Chapter
Page
IV. FINDINGS ................................................................................................ 19
Experiment 1 ............................................................................................ 19
Experiment 2 ............................................................................................ 25
Experiment 3 ............................................................................................ 28
V. CONCLUSION ......................................................................................... 33
Experiment 1 ............................................................................................ 34
Experiment 2 ............................................................................................ 36
Experiment 3 ............................................................................................ 37
General Discussion .................................................................................. 39
REFERENCES .............................................................................................. 44
iv
LIST OF TABLES
Table
Page
1. Mean body weight and percent change in body weight from day 1 for rats
from Experiment 1 ................................................................................... 20
2. Percent change in body weight from day 1 for rats from Experiment 2 .. 26
3. Percent change in body weight from day 1 for rats from Experiment 3 .. 28
4. Amount of sodium, total fluid ingested, and concentration of the ingested
fluid after 3 hours for the sodium deprived groups and the sodium and water
deprived groups in Experiment 1 and Experiment 3 ............................... 31
v
LIST OF FIGURES
Figure
Page
1. Percent change in body weight from day 1 ............................................. 20
2. Cumulative hourly intakes of 0.5 M NaCl and water ............................... 22
3. Cumulative intakes of 0.5 M NaCl and water during the first hour of testing
................................................................................................................ 24
4. Blood measurements from rats maintained on standard sodium diet or
sodium deficient diet with or without overnight water deprivation............ 27
5. Cumulative 0.5 M NaCl and water intake at 3 hours by rats in Experiment 3
................................................................................................................ 29
6. Blood measurements after 3-hour 0.5 M NaCl and water intake by rats
maintained on standard sodium diet or sodium deficient diet with or without
overnight water deprivation ..................................................................... 32
vi
CHAPTER I
INTRODUCTION
The maintenance of body fluid volume and osmolality within a narrow
range is critical for survival. Both the internal and external environments are
constantly changing and, without mechanisms to maintain body fluid
homeostasis, numerous biological processes such as neural signaling, enzymatic
reactions, and tissue perfusion would be compromised. Given the importance of
maintaining the appropriate volume and concentration of body fluids, it is not
surprising that there are multiple mechanisms for restoring body fluids to the
normal range when deviations occur. Compensatory neural, hormonal, renal,
and behavioral responses correct perturbations of body fluid volume or
osmolality.
Numerous challenges to body fluid balance, both physiological and
pharmacological, can be produced experimentally, but two common physiological
methods are water deprivation and dietary sodium deprivation. Water
deprivation causes a decrease in body fluid volume and an osmotic movement of
water from inside the cells to outside the cells to minimize the change in
1
concentration. In other words, water deprivation produces both intracellular and
extracellular dehydration. Water deprivation leads to hormonal responses,
notably, the release of vasopressin from the posterior pituitary and activation of
the renin-angiotensin system, which increases circulating levels of angiotensin II.
Angiotensin II acts at vascular receptors to cause vasoconstriction, thereby
maintaining pressure in the face of fluid loss, whereas vasopressin acts to
conserve water at the kidneys. In addition, behavioral responses are stimulated
by the familiar feeling of “thirst” -- the desire to seek and consume water. These
responses act together to correct the dehydration and restore body fluids to
normal. Compensatory responses to water deprivation have been studied since
the early 1900’s, but the mechanisms and central pathways that underlie the
behavioral response remain poorly understood.
Dietary sodium deficiency is a different physiological challenge to body
fluid balance that depletes body sodium stores. As with water deprivation,
dietary sodium deficiency causes compensatory neural, hormonal, renal, and
behavioral responses that ultimately correct the imbalance. Similar to water
deprivation leading to thirst and the consumption of water, dietary sodium
deficiency causes a “sodium appetite,” the desire to seek and consume salt, and
the ingestion of salt or salt solutions. While the sensation of sodium appetite
may not be as familiar as thirst, it is nonetheless an important response to
restore body sodium levels. Like thirst, sodium appetite has been studied for
nearly a century, but there is still a considerable amount of information necessary
to fully understand the signals and central pathways involved in sodium appetite.
2
It may not be surprising that progress in understanding both thirst and
sodium appetite has been relatively slow given the complexity of these
behavioral responses. It is largely for this reason that most investigators typically
focus on thirst or sodium appetite individually rather than studying both together.
However, some of the mechanisms underlying thirst and sodium appetite
overlap, and thus, there is information to be gained about the control of
compensatory behavioral responses by combining stimuli for thirst and sodium
appetite. More specifically, better understanding of the mechanisms involved in
behavioral responses to body fluid challenges may be obtained by examination
of mechanisms common to thirst and sodium appetite, as well as those distinct to
each. Accordingly, the goal of these studies was to assess the effect of
combined overnight water deprivation and dietary sodium deficiency on water
intake and salt intake. In addition, we sought to determine whether differences in
fluid volume, as measured by hematocrit or plasma protein concentration, or
osmolality, as measured by plasma sodium concentration, accounted for the
behavioral responses observed. Finally, we wanted to assess the effect of the
compensatory water intake and salt intake on fluid volume and plasma
osmolality.
3
CHAPTER II
REVIEW OF LITERATURE
Maintenance of the appropriate volume and osmolality of body fluids
within a narrow range is critical for many biological processes such as neural
signaling, enzymatic reactions, and tissue perfusion. A striking example of the
vital importance of adequate body sodium levels comes from a case report by
Wilkins and Richter in 1940 [1], describing a three and a half year old boy who
was admitted to the hospital with premature development of secondary sex
characteristics. An interview with the parents revealed that the boy also had an
insatiable desire to consume salt that began when he was 12 months old. The
excess salt intake continued thereafter; however, upon admittance to the
hospital, the boy was exclusively fed the regular hospital diet which had low
levels of sodium. Seven days after being admitted, the boy died. An autopsy
found adrenal pathology, most notably of the zona glomerulosa. This layer is the
source of the mineralocorticoid, aldosterone, which acts at the kidneys to
promote the reabsorption of sodium. Without aldosterone, a large amount of
body sodium is lost in urine. It was concluded that, due to the absence of
aldosterone, the boy’s need for sodium far exceeded that of a normal person with
4
functional adrenal glands. Thus, the boy’s “sodium appetite” was credited for
keeping him alive since, with his adrenal insufficiency, the low salt hospital diet
was inadequate and he died.
The tight regulation of body fluid volume and osmolality are supported by
hormonal, renal, neural, and behavioral mechanisms. In the case study
described above, the lack of the hormone, aldosterone, served as a challenge
that was adequately compensated by the behavioral response of consuming salt.
Unfortunately, when the boy was not allowed to consume salt, he could no longer
compensate for the sodium challenge and that inability proved fatal. Thus,
concerted compensatory hormonal, renal, neural, and behavioral responses to
challenges to body fluid volume or osmotic regulation are paramount to
maintaining body fluid balance. Interestingly, more attention has focused on
renal, neural, and hormonal responses than on the behavioral responses.
Compensatory behaviors, also known as motivated behaviors, are goaldirected behaviors meant to satisfy a physical need in order to maintain
homeostasis. The concept of motivated behaviors traces its origins to
Psychology, and includes reproductive behavior and hunger, as well as the less
familiar thirst and salt appetite. Studies of the latter two, thirst and sodium
appetite, date back to the seminal work of Richter, a Psychobiologist at Johns
Hopkins University in the early 1900’s. Richter was a pioneer in the field of
sodium appetite and developed the depletion-repletion model for studying thirst
and salt appetite that is still used today. This model may involve depriving an
animal of dietary sodium or of water, and then observing behavioral responses
5
when sodium or water is subsequently made available. While many advances in
the study of both thirst and sodium appetite have been made over the last fifty
years, there is still a considerable way to go in our understanding of these
complex processes.
Thirst
Thirst can be defined as the desire to seek and consume water, and is
stimulated via two different mechanisms, osmotic or volemic.
Osmotic Thirst
The concentration of solutes in body fluids is maintained within a range of
1-2% [2], and deviations beyond this threshold generate signals and,
subsequently, responses to correct the imbalance. When the concentration of
non-permeable solutes in the extracellular fluid increases or when body water
decreases, plasma osmolality increases. This increase in plasma osmolality
causes an osmotic movement of water from inside the cells to outside the cells to
minimize the increase in concentration. For example, it is well established that
injection of a hypertonic sodium chloride solution into an animal will increase
plasma osmolality and induce thirst [3]. Other studies used hypertonic solutions
of sucrose or sorbitol to determine whether the signal to produce thirst was
dependent on an increase in extracellular sodium concentration specifically, or if
6
solutes that are unable to traverse the cell membrane can stimulate water intake.
These other solutions, which increase plasma osmolality, also produced thirst [4,
5]. In contrast, solutions containing urea or glucose, which can diffuse through
the cell membrane and therefore do not increase extracellular fluid concentration,
did not produce a marked thirst [4, 5]. Thus, thirst can be stimulated by
increased plasma sodium or by increased plasma osmolality, in general.
While it is established that changes in plasma osmolality can produce
thirst, how and where changes in fluid concentration are detected to stimulate
thirst is still somewhat controversial. In this regard, comprehensive reviews by
Bourque [6, 7] will be summarized here. Verney first coined the term
“osmoreceptor” as the specialized cell type that detects changes in fluid
osmolality and stimulate thirst. It was hypothesized that these osmoreceptor
cells were located in specialized areas of the brain with an incomplete bloodbrain barrier known as circumventricular organs. An incomplete blood-brain
barrier enables circumventricular organs to detect changes in osmolality as well
as in circulating hormone levels. Attention has focused on the forebrain organum
vasculosum of the lamina terminalis (OVLT) as an important circumventricular
organ that contains osmoreceptors, because lesions of this area have been
found to prevent thirst after an increase in osmolality [8]. Later studies by
McKinley showed that neurons within the OVLT are activated by osmotic stimuli
such as hypertonic NaCl [9, 10].
7
Volemic Thirst
Decreased body fluid volume has also been implicated as a stimulus for
thirst. Loss of volume causes a decrease in renal perfusion, which activates the
renin-angiotensin-aldosterone system, ultimately leading to increased circulating
levels of angiotensin II. In addition to acting as a vasoconstrictor, angiotensin II
has been shown to be an important hormonal signal for thirst. Detection of
circulating angiotensin II occurs at the subfornical organ (SFO), a forebrain
circumventricular organ (see ref [11, 12] for reviews). A second mechanism for
stimulating thirst in response to hypovolemia is thought to involve cardiac and
arterial baroreceptor signaling. Baroreceptors are stretch-mediated receptors
located in the heart and great vessels. Baroreceptor afferent signaling is
detected in the nucleus of the solitary tract (NTS) in the hindbrain. It is known
that the inflation of balloons at the junction of the superior vena cava and right
atrium of rats stimulates baroreceptors and decreases the drinking response to
hypovolemia [13], whereas lesions of the NTS increase the drinking response to
hypovolemia [14].
Sodium Appetite
Sodium appetite has not received as much attention as other motivated
behaviors, such as hunger or reproduction, or even thirst. This could be due, in
part, to the fact that humans generally consume adequate amounts of sodium in
their diet, so the feeling of a “sodium appetite” is not as familiar a sensation as
8
“thirst” or “hunger”. This is especially true in the United States where a high salt
diet is common. Nonetheless, sodium is of vital importance for life, and
challenges to body sodium balance produce multiple hormonal, renal, and
behavioral responses to restore sodium levels to normal. In regard to the
behavioral response, sodium appetite can be produced by numerous
experimental manipulations such as dietary sodium deficiency, treatment with
desoxycorticosterone acetate (DOCA; a precursor to aldosterone),
adrenalectomy, or intracerebroventricular (ICV) infusion of angiotensin II, to
name only a few. Many of these experimental manipulations involve increases in
angiotensin II or aldosterone, or both.
Angiotensin II and aldosterone are the primary hormonal signals produced
in response to challenges that decrease body sodium levels. These hormones
are part of the renin-angiotensin-aldosterone system and work together to
promote the retention of sodium by the kidneys, and to stimulate sodium
ingestion via central actions. A study by Fluharty and colleagues [15]
demonstrated a synergistic relationship between angiotensin II and aldosterone
treatment, wherein ICV angiotensin II and peripheral aldosterone together
produce a much greater sodium appetite in rats than either cause individually. A
pronounced sodium appetite was even generated by doses of angiotensin II and
aldosterone that alone would not cause sodium ingestion. In more physiological
methods of stimulating sodium appetite, circulating angiotensin II is thought to act
at forebrain circumventricular organs [16]. Although the site of action for
9
aldosterone remains unclear, recent studies suggest the NTS as one possibility
[17, 18].
In addition to hormonal responses to body sodium challenges, cardiac and
arterial baroreceptors are also important signals for sodium appetite. A study by
Toth and colleagues [19] showed that inflating a balloon at the junction of the
superior vena cava and the right atrium in rats also decreases sodium ingestion
after a sodium appetite was produced experimentally by peritoneal dialysis with
hypertonic colloid or by treatment with DOCA. A later study by De Gobbi and
colleagues in 2008 [20] demonstrated that sodium ingestion was also decreased
after superior vena cava-right atrial balloon inflation upon treatment with the
diuretic-natriuretic furosemide and captopril (an inhibitor of angiotensinconverting enzyme). These studies demonstrate the importance of baroreceptor
signaling in the compensatory behavioral responses to challenges to body
sodium levels.
Combined Thirst and Sodium Appetite
Most studies investigate thirst and sodium appetite separately.
Nonetheless, new information may be obtained from studies that combine
challenges to body fluid regulation, as a number of the mechanisms that underlie
thirst and sodium appetite overlap. By examining both the common and distinct
mechanisms involved in thirst and sodium appetite, new insights into the
pathways involved in behavioral responses to body fluid challenges may be
10
revealed. Studies that investigate thirst and sodium appetite simultaneously are
not common. A series of studies by De Luca and colleagues [21-23] used water
deprivation to elicit a sodium appetite in their water deprivation-partial repletion
protocol. In this protocol, rats are water deprived 24 – 36 hours and are then
given access to water for one hour. After the initial hour of access to only water,
a bottle of hypertonic saline was added to the cages and rats consumed fairly
substantial volumes. The idea is that water deprivation causes both thirst and
sodium appetite, but that the “competing drive” of thirst makes detecting the
sodium appetite difficult. Thus, if rats are permitted to replete their water deficit,
the sodium appetite will emerge. However, these studies used a single stimuluswater deprivation- to study the various signals that cause sodium ingestion.
In contrast, a study by Stricker and colleagues in 2001 [24] showed that
rats that were water deprived overnight would consume hypertonic NaCl if it was
the only solution available. However, despite the voluntary intake of NaCl, this
would not be considered a sodium appetite in the traditional sense. These rats
ingested the hypertonic NaCl and relied on their kidneys to excrete the excess
sodium such that the ingested fluid could correct the dehydration. In fact, the
goal of the study was to determine if the animals could correct the dehydration by
consuming hypertonic saline. Thus, studies that examine the effect of combining
various challenges to body fluid regulation have not yet been conducted.
As a number of the underlying mechanisms of thirst are also involved in
sodium appetite, an examination of combined challenges that could stimulate
both of these compensatory behavioral responses will allow a better
11
understanding of the pathways involved. Both thirst and sodium appetite are
elicited via complex mechanisms, but studying them together may provide useful
information that would not be obtained otherwise. For example, it may be
possible to determine whether one is more salient under the given challenges, or
if the effects of combining challenges to body fluid balance are additive. The
following experiments examine behavioral responses to combined sodium and
water deprivation. The goals of these studies were to assess the behavioral
consequences of these challenges, to determine whether differences in
osmolality, as measured by plasma sodium concentration, or fluid volume, as
measured by hematocrit or plasma protein concentration, could account for the
observed behavioral outcomes, and to ascertain whether the behavioral
responses allowed the rats to compensate for induced deficits in body sodium
and fluid volume.
12
CHAPTER III
METHODOLOGY
General Methods
Animals
Adult male Sprague-Dawley rats (Harlan) weighing 335 – 490 g prior to
the start of experiments were housed individually in plastic cages and given free
access to rodent chow and water unless otherwise specified. Animals were kept
in a temperature-controlled room (21 – 25°C) on a 12 :12 hour light/dark cycle
with lights on at 7:00 A.M. for a minimum of seven days prior to experimental
manipulation. All methods were approved by the Oklahoma State University
Center for Health Sciences Animal Care and Use Committee.
Experimental Groups
In all the experiments to be described, there were four experimental
groups. In the first group, rats were maintained on a standard sodium diet
(Harlan) for ten days with ad libitum access to water (control group). A second
13
group of rats was also maintained on the standard sodium diet for ten days and,
after the tenth day, was water deprived overnight (approximately 22 hours; water
deprived group). The third group of animals was placed on sodium-deficient
rodent chow (background sodium approximately 0.01-0.02%; Harlan) for ten
days with ad libitum access to water (sodium deprived group). The fourth group
of rats was placed on the sodium-deficient rodent chow for ten days and then
water deprived overnight (sodium and water deprived group). Testing began at
approximately 7:30 A.M. on day 11 (test day).
Experiment 1: Behavioral Responses to Dietary Sodium and Water
Manipulations. The goal of Experiment 1 was to assess the behavioral
consequences of combined dietary sodium deprivation and overnight water
deprivation using two-bottle intake tests.
1. Procedures. Prior to diet manipulations, rats were given ad libitum
access to 0.5 M NaCl in graduated tubes, along with water, for two to three days
to adapt them to the availability of the solution. At the conclusion of the three
adaptation days, the 0.5 M NaCl was removed from the cages. Rats were then
randomly assigned to one of the four experimental groups, as described
(Experimental Groups), and were either placed on the sodium deficient diet or
remained on the standard sodium diet. All rats were weighed prior to diet
manipulations, prior to water deprivation on day 10, and on the day of testing. A
subset of rats was also weighed on days 3, 5, and 7 of the diet manipulations.
14
After ten days, water bottles were removed from the cages of rats assigned to
overnight water deprivation groups. On the day of testing, food and water were
removed from all cages and rats were given both water and 0.5 M NaCl in 50 mL
graduated centrifuge tubes. Intakes of each solution were recorded after 5, 10,
15, 30, 45, and 60 minutes and then hourly for a total of seven hours.
2. Statistics. All data are reported as group means +/- standard errors.
Sodium and water intake during hours one through seven of the behavioral test
each were analyzed using a three-way, repeated measures analysis of variance
(ANOVA) with sodium deprivation (sodium deprived or not sodium deprived),
water deprivation (water deprived or not water deprived), and time as the three
factors. Significant main effects or interactions (p<0.05) were further analyzed
using Student-Newman-Keuls tests. In addition, sodium and water intake in the
first hour were analyzed separately for each of the four groups using a two way
repeated measures ANOVA with solution (0.5M NaCl or water) and time as
factors. Significant main effects or interactions (p<0.05) were analyzed using
Student-Newman-Keuls tests.
Experiment 2: Effect of Dietary Sodium and Water Manipulations on Blood
Measurements. The goal of Experiment 2 was to determine whether group
differences in hematocrit, plasma protein concentration, or plasma sodium
concentration might account for any behavioral differences observed during twobottle intake tests in Experiment 1.
15
1. Procedures. A second group of rats was assigned to the four
experimental groups as described (Experimental Groups). All rats were weighed
prior to diet manipulations, on days four and eight of the diet manipulation, and
on the day of testing. After ten days on either the standard sodium diet or the
sodium deficient diet, those rats assigned to the water deprivation groups were
water deprived overnight. On the day of testing, rats were deeply anesthetized
with sodium pentobarbital (1.0 mL I.P.; 50 mg/mL; Fisher) and rapidly
decapitated to collect trunk blood. Two microcapillary tubes were filled and
centrifuged for determination of hematocrit. The remaining trunk blood was
centrifuged and plasma was collected. A small sample of plasma was used to
measure plasma protein concentration using a refractometer (Reichert, Depew,
NY). The remaining plasma was stored at -20°C prior t o analysis of plasma
sodium concentration using an ion sensitive electrode (EasyLyte). Sodium is the
primary osmolyte in plasma; thus, our measures of plasma sodium concentration
are indicative of osmolality.
2. Statistics. All data are reported as group means +/- standard errors.
Group differences in hematocrit, plasma protein concentration, and plasma
sodium concentration each were analyzed using a two-way ANOVA with sodium
deprivation (sodium deprived or not sodium deprived) and water deprivation
(water deprived or not water deprived) as factors. Significant main effects or
interactions (p<0.05) were analyzed using Student-Newman-Keuls tests.
16
Experiment 3: Effect of Behavioral Responses on Blood Measurements.
The goal of Experiment 3 was to assess the effect of salt and water consumed
during the two-bottle intake test after dietary sodium and/or water manipulations
on hematocrit, plasma protein concentration, and plasma sodium concentration.
1. Procedures. A third group of rats was assigned to the four experimental
groups as described (Experimental Groups). Prior to diet manipulations, rats
were given ad libitum access to 0.5 M NaCl for three days to adapt them to the
availability of the solution. At the conclusion of the three adaptation days, the 0.5
M NaCl was removed from each cage. Rats were then placed on either the
sodium deficient diet or remained on the standard sodium diet. All rats were
weighed prior to diet manipulations, on days four and eight of the diet
manipulation, and on the day of testing. After ten days, water bottles were
removed from the cages of rats assigned to overnight water deprivation groups.
On the day of testing, food and water were removed from all cages and rats were
given access to water and 0.5 M NaCl in 50 mL graduated centrifuge tubes.
Intakes of each solution were recorded after three hours.
The amount of sodium (in mMol) consumed at three hours by the sodium
deprived and the sodium and water deprived groups was calculated as (volume
of 0.5 M NaCl (mL) × 0.5). The total fluid consumed at three hours was
calculated as [water intake (mL) + 0.5 M NaCl intake (mL)]. The concentration of
the total ingested fluid (in mMol Na /L) was also calculated for these two groups
as ([(volume of 0.5 M NaCl (mL) × 0.5) / Total fluid consumed (mL)] × 1000). For
purposes of comparison, we also evaluated the amount of sodium consumed, the
17
total fluid ingested, and the concentration of the total fluid consumed at three
hours for the sodium deprived group and the sodium and water deprived group
from Experiment 1 using these equations. At the conclusion of testing, rats were
deeply anesthetized with sodium pentobarbital (1.0 mL I.P.) and rapidly
decapitated to collect trunk blood. Hematocrit, plasma protein concentration, and
plasma sodium concentration were measured as described in Experiment 2.
2. Statistics. All data are presented as group means +/- standard errors.
Cumulative water and sodium intake each were analyzed using two-way
ANOVAs with sodium deprivation (sodium deprived or not sodium deprived) and
water deprivation (water deprived or not water deprived) as the two factors.
Significant main effects (p<0.05) or interactions were analyzed using StudentNewman-Keuls tests. Hematocrit, plasma protein concentration, and plasma
sodium concentration each were analyzed using two-way ANOVAs with the two
factors being sodium deprivation (sodium deprived or not sodium deprived) and
water deprivation (water deprived or not water deprived). Significant main effects
or interactions (p<0.05) were analyzed using Student-Newman-Keuls tests.
18
CHAPTER IV
FINDINGS
Experiment 1:
Body weights were measured on days 1, 10, and 11 for each animal in
Experiment 1 to determine whether the sodium deficient diet had substantial
effects on body weight that may have affected behavior independent of diet
condition. As shown in Table 1, body weights increased from day 1 to day 10 in
all groups, as reflected by the percent change. Although the groups on the
sodium deficient diet appeared to have a more gradual rate of weight gain, they
did not lose weight from day 1 to day 10. As expected, body weight decreased
after overnight water deprivation and thus, the rate of weight gain decreased
abruptly. A subset of animals was also weighed on days 3, 5, and 7. The
percent changes in body weight from day 1 for the four groups are shown in
Figure 1, which illustrates the steady increase in body weight from day 1 to day
10 in all groups, and the abrupt decrease after overnight water deprivation.
Hourly 0.5 M NaCl intakes by the four groups are shown in Figure 2A. A
3-way repeated measures ANOVA of hourly 0.5 M NaCl intake revealed main
19
Table 1: Mean Body Weight and Percent Change in Body Weight from Day 1
for Rats from Experiment 1. Values are means ± s.e.m.
Sodium
Deprived
(n = 8)
Sodium and
Water
Deprived
(n = 8)
Day 1
455.7 ± 18.7
384.3 ± 7.5
407.5 ± 19.5
404.4 ± 16.0
Day 10
509.3 ± 16.8
443.0 ± 9.6
438.3 ± 18.4
438.9 ± 16.3
Day 11
(Test Day)
517.5 ± 17.2
417.3 ± 9.4
440.6 ± 18.2
416.8 ± 16.4
% Change
Days 1-10
12.1 ± 2.0
15.2 ± 0.6
7.7 ± 0.8
8.6 ± 1.3
% Change
Days 1-11
13.8 ± 1.8
8.6 ± 0.5
8.3 ± 0.9
3.1 ± 1.2
Percent Change in Body Weight
from Day 1 (%)
Control
(n = 6)
Water
Deprived
(n = 6)
16
Control (484 ± 8.2)
14
Water Dep (384.5 ± 11.4)
12
Sodium Dep (415.3 ± 25.7)
10
Sodium and Water Dep (406.2 ± 21.7)
8
6
4
2
0
0
1
2
3
4
5
6
7
8
9
10
11
Day of Body Weight Measurement
Figure 1: Percent Change in Body Weight from Day 1 (mean ± s.e.m.). Data
are from a subset of rats in Experiment 1. Diamonds represent the control group
(n=4), squares represent the water deprived group (n=4), triangles represent the
sodium deprived group (n=6), and circles represent the sodium and water
deprived group (n=6). The numbers in the legend are the body weights (mean ±
s.e.m.) on day 1 for each of the four groups.
20
effects of sodium deficiency [F(1,24) = 62.82, p<0.001], water deficiency [F(1,24)
= 12.00, p<0.01], and time [F(6,144) = 29.47, p<0.001]. There was also an
interaction between sodium deficiency and water deficiency [F(1,24) = 4.76,
p<0.05]. Post hoc Student-Newman-Keuls tests of the interaction showed that
the sodium deprived group drank more 0.5 M NaCl than did all of the other
groups. The sodium and water deprived group drank significantly less 0.5 M
NaCl than did the sodium deprived group, but more than both of the groups that
were on the standard sodium diet. The two groups on the standard sodium diet
were not different from each other.
There was also an interaction between sodium deficiency and time
[F(6,144) = 15.41, p<0.001]. Post hoc tests revealed that, regardless of whether
they were water deprived, 0.5 M NaCl intake by sodium deprived animals
increased for the first three hours and then reached an asymptote between three
and four hours. Nonetheless, at all time points, sodium deprived animals
consumed more 0.5 M NaCl than did animals that were on the standard sodium
diet. The overall 0.5 M NaCl intake did not change at any time point for either
group of animals that were on the standard sodium diet.
Hourly water intakes by the four groups are shown in Figure 2B. A 3-way
repeated measures ANOVA revealed main effects of sodium deficiency [F(1,24)
= 22.46, p<0.001], water deficiency [F(1,24) = 99.03, p<0.001], and time
[F(6,144) = 50.37, p<0.001]. There were also significant interactions between
sodium deficiency and time [F(6,144) = 11.01, p<0.001] as well as between water
deficiency and time [F(6,144) = 5.23, p<0.001]. Post-hoc tests on the interaction
21
A.
Cumulative 0.5 M NaCl Intake (mL)
18
Control
Water Dep
16
Sodium Dep
14
Sodium and Water Dep
12
10
8
6
4
2
0
0
60
120
180
240
300
360
420
300
360
420
Time (minutes)
B.
45
Cumulative Water Intake (mL)
Control
Water Dep
40
Sodium Dep
Sodium and Water Dep
35
30
25
20
15
10
5
0
0
60
120
180
240
Time (minutes)
Figure 2: Cumulative Hourly Intake (mean ± s.e.m.) of 0.5 M NaCl and Water.
Diamonds represent the control group (n=6), squares represent the water deprived
group (n=6), triangles represent the sodium deprived group (n=8), and circles represent
the sodium and water deprived group (n=8). Panel A- cumulative hourly intakes of 0.5 M
NaCl. 3-way rm ANOVA revealed main effects of sodium deprivation, water deprivation,
and time. There were also significant interactions between sodium deprivation and
water deprivation [F(1,24) = 4.76, p<0.05] and between sodium deprivation and time
[F(6,144) = 15.41, p<0.001]. Panel B- cumulative hourly intakes of water. 3-way rm
ANOVA revealed main effects of sodium deprivation, water deprivation, and time. There
were also significant interactions between sodium deprivation and time [F(6,144) = 11.01,
p<0.001] and between water deprivation and time [F(6,144) = 5.23, p<0.001].
22
between water deficiency and time showed that, for water deprived animals,
regardless of whether they were also sodium deprived, water intake increased for
the first three hours and then reached an asymptote between three and four
hours. As might be expected, water deprived animals consumed more water
than did animals that were not water deprived at all time points. Water intake did
not change at any time point for animals that were not water deprived except
between the first and second hour.
To more closely evaluate the relationship between 0.5 M NaCl and water,
we also compared intakes of 0.5 M NaCl and water by each of the four groups
during the first hour (Figure 3A-D). A 2-way repeated measures ANOVA of 0.5 M
NaCl and water intake by the control group in the first hour revealed a main effect
of time [F(5,25) = 5.48, p<0.01]. Post-hoc tests revealed that, overall, fluid intake
increased slightly, but significantly, over time for this group. Similar statistical
analyses of intakes by the water deprived group showed main effects of both
solution [F(1,5) = 121.36, p<0.001] and time [F(5,25) = 33.55, p<0.001], and an
interaction between solution and time [F(5,25) = 33.80, p<0.001]. Post-hoc tests
of the interaction revealed that water intake was significantly greater than 0.5 M
NaCl intake at all time points for this group. Furthermore, 0.5 M NaCl intake did
not change over time, whereas, water intake significantly increased at 10, 30,
and 45 minutes relative to the previous time points. For the sodium deprived
group, there were main effects of solution [F(1,7) = 15.16, p<0.01] and time
[F(5,35) = 36.89, p<0.001]. Post-hoc tests revealed that, overall, 0.5 M NaCl
intake was significantly greater than water intake, and that total fluid intake
23
A.
B.
25
Cumulative Intake (mL)
Cumulative Intake (mL)
15
10
5
0
*
*
#
15
*
*
#
*
10
#
5
15
30
45
0
60
15
D.
25
0.5M NaCl Intake
Water Intake
Cumulative Intake (mL)
20
15
10
5
0
0
15
30
45
60
25
Sodium Deprived
0.5M NaCl Intake
30
Time (min)
Time (min)
Cumulative Intake (mL)
*
Water Intake
20
0
0
C.
Water Deprived
0.5M NaCl Intake
Water Intake
20
25
Control
0.5M NaCl Intake
45
Sodium and Water Deprived
*
Water Intake
20
*
*
*
15
#
*
*
10
#
#
5
0
60
0
Time (min)
15
30
45
60
Time (min)
Figure 3: Cumulative Intakes (mean ± s.e.m.) of 0.5 M NaCl and Water During the First Hour of Testing. Squares
represent 0.5 M NaCl intake and circles represent water intake. Panel A- control group (n=6). Panel B- water deprived
group (n=6). Panel C- sodium deprived group (n=8). Panel D- sodium and water deprived group (n=8). * = significantly
greater than 0.5 M NaCl intake at corresponding time, # = significantly greater than preceding time point.
24
increased significantly over time. Finally, for the sodium and water deprived
group, a 2-way repeated measures ANOVA revealed a main effect of solution
[F(1,7) = 10.51, p<0.05] and time [F(5,35) = 24.84, p<0.001], and an interaction
between solution and time [F(5,35) = 10.88, p<0.001]. Post-hoc tests of the
interaction showed that water intake was significantly greater than 0.5 M NaCl
intake at every time point for this group. Intake of 0.5 M NaCl changed only
slightly (from approximately 2 mL to 5 mL) during the first hour, whereas water
intake increased significantly at 10, 15, and 60 minutes relative to the previous
time points.
Experiment 2:
Animals in Experiment 2 were weighed on days 1, 4, 8, and 11. As shown
in Table 2, the percent change in body weight from day 1 increased in all groups,
similar to that observed in Experiment 1. Specifically, the percent change was
somewhat less in the groups that were sodium deprived and the rate of body
weight gain decreased on day 11 for the groups that were water deprived
overnight.
Figure 4 shows hematocrit, plasma protein concentration, and plasma
sodium concentration in the four groups; each was analyzed using a 2-way
ANOVA. Analysis of group differences in hematocrit revealed no main effects
and no interactions. Analysis of group differences in plasma protein
concentration revealed a main effect of sodium deprivation [F(1,24) = 5.898,
25
Table 2: Percent Change in Body Weight from Day 1 for Rats from
Experiment 2. Values are means ± s.e.m.
Control
(n = 6)
Water
Deprived
(n = 6)
Sodium
Deprived
(n = 8)
Sodium and
Water
Deprived
(n = 8)
% Change
Days 1-4
5.0 ± 0.3
4.1 ± 0.2
2.3 ± 0.4
2.5 ± 0.3
% Change
Days 1-8
12.6 ± 0.9
11.5 ± 0.7
7.7 ± 1.0
8.2 ± 0.3
% Change
Days 1-11
17.3 ± 1.3
8.9 ± 0.7
10.2 ± 1.1
5.0 ± 0.3
p<0.05]. Post-hoc tests of the main effect showed that, regardless of whether
they were water deprived overnight, the groups on the sodium deficient diet had
significantly greater plasma protein concentrations than did those on the
standard sodium diet. In contrast, analysis of group differences in plasma
sodium concentration revealed a main effect of water deprivation [F(1,24) =
9.931, p<0.01]. Post-hoc tests of this main effect showed that, regardless of
whether they were sodium deprived, the groups that were water deprived had
significantly greater plasma sodium concentrations than did those that were not
water deprived.
26
A.
50
No Water Dep
Water Dep
Hematocrit (%)
45
40
35
30
25
Standard Sodium Diet
Sodium Deficient Diet
6.5
Plasma Proteins (g/dL)
B.
6
No Water Dep
Water Dep
5.5
5
4.5
4
3.5
Standard Sodium Diet
C.
Sodium Deficient Diet
Plasma Sodium (mMol/L plasma water)
160
150
No Water Dep
Water Dep
140
130
120
110
100
Standard Sodium Diet
Sodium Deficient Diet
Figure 4: Blood Measurements from Rats Maintained on Standard Sodium
Diet or Sodium Deficient Diet with or without Overnight Water Deprivation.
Panel A- Hematocrit. 2-way ANOVA revealed no main effects or interactions.
Panel B- Plasma protein concentrations. 2-way ANOVA revealed a main effect
of sodium deprivation [F(1,24) = 5.898, p<0.05]. Panel C- Plasma sodium
concentrations. 2-way ANOVA revealed a main effect of water deprivation [F(1,24)
= 9.931, p<0.01].
27
Experiment 3:
Animals in Experiment 3 were weighed on days 1, 4, 8, and 11. As shown
in Table 3, the percent change in body weight from day 1 was comparable to that
observed in Experiments 1 and 2 with a somewhat slower rate of increase in the
sodium deprived groups and a reduced rate of increase following water
deprivation.
Table 3: Percent Change in Body Weight from Day 1 for Rats in Experiment
3. Values are means ± s.e.m.
Control
(n = 7)
Water
Deprived
(n = 6)
Sodium
Deprived
(n = 8)
Sodium and
Water
Deprived
(n = 8)
% Change
Days 1-4
5.9 ± 1.0
5.3 ± 0.5
2.3 ± 0.4
2.4 ± 0.4
% Change
Days 1-8
13.9 ± 1.6
11.0 ± 0.9
6.2 ± 0.6
6.4 ± 0.4
% Change
Days 1-11
18.3 ± 2.3
7.7 ± 1.2
8.7 ± 0.6
3.1 ± 0.6
Figure 5 shows cumulative, 3-hour intakes of 0.5 M NaCl (A) and water
(B) by the four groups. A 2-way ANOVA on group differences in the three hour
0.5 M NaCl intake revealed main effects of sodium deprivation [F(1,25) = 30.283,
p<0.001] and water deprivation [F(1,25) = 5.558, p<0.05]. Specifically,
regardless of whether they were water deprived, 0.5 M NaCl intake was
significantly greater in groups that were sodium deprived. At the same time,
28
Cumulative 0.5 M NaCl Intake (mL)
A.
16
Experiment 3
14
Experiment 1
12
10
8
6
4
2
0
Control
Water Deprived Sodium Deprived
Sodium and
Water Deprived
Cumulative Water Intake (mL)
B.
40
35
Experiment 3
Experiment 1
30
25
20
15
10
5
0
Control
Water Deprived
Sodium
Deprived
Sodium and
Water Deprived
Figure 5: Cumulative 0.5 M NaCl and Water Intake (mean ± s.e.m.) at 3
Hours by Rats in Experiment 3. 2-way ANOVAs revealed significant main
effects of sodium deprivation [F(1,25) = 30.283, p<0.001] and water deprivation
[F(1,25) = 5.558, p<0.05] for 0.5 M NaCl intake and main effects of sodium
deprivation [F(1,25) = 13.031, p<0.01] and water deprivation [F(1,25) = 98.686,
p<0.001] for water intake. Horizontal black bars show intakes of 0.5 M NaCl and
water at three hours by rats from Experiment 1 for comparison.
29
groups that were water deprived consumed significantly less 0.5 M NaCl,
regardless of whether they were sodium deprived. Interestingly, however, there
was no interaction between sodium deprivation and water deprivation in 0.5 M
NaCl intake. A 2-way ANOVA on group differences in water intake at three hours
revealed main effects of sodium deprivation [F(1,25) = 13.031, p<0.01] and water
deprivation [F(1,25) = 98.686, p<0.001]. Regardless of sodium deprivation,
water intake was significantly greater in groups that were water deprived. At the
same time, regardless of water deprivation, groups that were sodium deprived
consumed significantly more water than groups that were not sodium deprived.
However, there was no interaction between sodium deprivation and water
deprivation for water intake. To facilitate comparisons, horizontal black lines
indicate the intake of 0.5 M NaCl (Figure 5A) and water (Figure 5B) at three
hours by animals from Experiment 1.
Table 4 shows the amount of sodium ingested at three hours by sodium
deprived groups and the sodium and water deprived groups in Experiment 1 and
Experiment 3, as well as the total volume of fluids consumed, and the
concentration of the ingested fluid. Within experimental groups, the amount of
sodium consumed, total fluid ingested, and the concentration of the ingested fluid
were comparable in the two experiments.
30
Table 4: Amount of Sodium, Total Fluid Ingested, and Concentration of the
Ingested Fluid After 3 Hours for the Sodium Deprived Groups and the
Sodium and Water Deprived Groups in Experiment 1 and Experiment 3.
Experiment 1
Experiment 3
Amount of
Na
(in mMol
Na)
Total Fluid
Ingested
(mL)
Concentration
(in mMol
Na/L)
Amount of
Na
(in mMol
Na)
Total Fluid
Ingested
(mL)
Concentration
(in mMol
Na/L)
Sodium
Deprived
6.8 ± 0.42
27.9 ± 2.7
252.4 ± 14.8
6.3 ± 0.74
22.0 ± 1.7
289.9 ± 32.1
Sodium and
Water
Deprived
3.8 ± 0.66
41.1 ± 3.0
94.4 ± 17.2
3.2 ± 0.98
40.2 ± 4.5
70.3 ± 3.2
Figure 6 shows hematocrit, plasma protein concentration, and plasma
sodium concentration in the four groups after 3 hours of access to 0.5 M NaCl
and water. These data each were analyzed using 2-way ANOVAs. Analysis of
group differences in hematocrit revealed a main effect of water deprivation
[F(1,25) = 4.553, p<0.05]. Post-hoc tests showed that hematocrit was
significantly greater in the groups that were water deprived compared to that in
the groups that were not water deprived. In contrast, analysis of group
differences in plasma protein concentration showed a main effect of sodium
deprivation [F(1,25) = 14.823, p<0.001]. Post-hoc tests revealed that the groups
that were not sodium deprived had significantly greater plasma protein
concentrations than did groups that were sodium deprived. Finally, analyses of
group differences in plasma sodium concentration revealed no main effects and
no interactions.
31
A.
50
No Water Dep
Water Dep
Hematocrit (%)
45
40
35
30
25
Standard Sodium Diet
6.5
Plasma Proteins (g/dL)
B.
6
Sodium Deficient Diet
No Water Dep
Water Dep
5.5
5
4.5
4
3.5
Standard Sodium Diet
Sodium Deficient Diet
Plasma Sodium (mMol/L plasma water)
C.
160
No Water Dep
Water Dep
150
140
130
120
110
100
Standard Sodium Diet
Sodium Deficient Diet
Figure 6: Blood Measurements After 3-Hour 0.5 M NaCl and Water Intake by
Rats Maintained on Standard Sodium Diet or Sodium Deficient Diet with or
without Overnight Water Deprivation. A- Hematocrit. 2-way ANOVA revealed
a main effect of water deprivation [F(1,25) = 4.553, p<0.05]. B- Plasma protein
concentrations. 2-way ANOVA revealed a main effect of sodium deprivation
[F(1,25) = 14.823, p<0.001]. C- Plasma sodium concentrations. 2-way ANOVA
revealed no main effects or interactions.
32
CHAPTER V
CONCLUSION
There is a large body of literature on the behavioral consequences of
sodium deprivation alone, as well as of water deprivation alone (see ref [12, 25,
26] for reviews). However, very little work has been done to investigate the
outcome of combining these two challenges to body fluid balance. In a series of
studies by De Luca and colleagues [21-23], it was found that 24-36 hours of
water deprivation elicits a sodium appetite in rats if thirst is first eliminated by
providing rats with water for one hour before access to a hypertonic NaCl
solution. However, the goal of those studies was to use water deprivation as a
stimulus that allowed the investigators to examine diverse signals that drive
sodium intake. Stricker's group [24] also found that rats will drink hypertonic
NaCl after overnight water deprivation if they are given only hypertonic NaCl to
drink. However, the goal of those studies was to determine whether
consumption of the hypertonic NaCl solution would correct dehydration. Thus,
these investigators looked at thirst and/or sodium appetite after water
deprivation, rather than addressing the question about the combined effect of
water and sodium deprivation. The goals of the present study were to combine
33
dietary sodium deprivation and overnight water deprivation to assess the
behavioral consequences of these challenges, to determine whether differences
in plasma sodium concentration or volume status could account for the observed
behavioral outcomes, and to ascertain whether the behavioral responses allowed
the rats to compensate for induced deficits in body sodium and fluid volume.
Experiment 1:
The results of Experiment 1 revealed that there was not a large disparity in
either the mean body weights (Table 1) or percent change in body weight (Figure
1) among the four groups. All rats gained weight steadily throughout the
protocol. Clearly, the rate of weight gain decreased after water deprivation, both
in rats on the standard sodium diet and in rats on the sodium deficient diet, as
rats lose body fluid volume and may also decrease food intake during water
deprivation. Animals on the sodium deficient diet seemed to gain weight more
gradually than did the animals on the standard sodium diet, but dietary sodium
deficiency did not cause the animals to lose weight. Thus, we conclude that
consequences of the different diets on body weight did not contribute to the
behaviors seen on test day.
In two-bottle intake tests (Figure 2A-B), the control group drank very little
fluid during seven hours, as expected. Not surprisingly, the water deprived group
drank large volumes of water [27] and very little 0.5 M NaCl on test day. In
contrast, the sodium deprived group drank large volumes of 0.5 M NaCl and
34
similar volumes of water [28]. The combined intake of 0.5 M NaCl and water
yielded a hypertonic solution by the end of the drinking test, which is consistent
with previous finding [27]. Rats that were both sodium and water deprived drank
significantly more water and significantly less 0.5 M NaCl than did animals that
were only sodium deprived. In fact, the combination of 0.5 M NaCl and water
ingested by the sodium and water deprived group yielded a slightly hypotonic
solution by the end of the test. Nonetheless, the sodium and water deprived
group drank significantly more 0.5 M NaCl than did the two groups on the
standard sodium diet.
To more closely evaluate the effect of the experimental manipulations on
0.5 M NaCl and water intake, we also assessed the patterns of drinking by the
four groups during the first hour (Figure 3A-D). The sodium deprived group
drank 0.5 M NaCl first; only later in the test did water intake increase and
eventually surpass 0.5 M NaCl consumption. This pattern is consistent with
previous studies of sodium deprived rats [28], but is unlike the pattern that was
seen for rats that were both sodium and water deprived. The sodium and water
deprived group drank significantly more water than 0.5 M NaCl throughout the
first hour of testing. In fact, the pattern of intake for the sodium and water
deprived group in the first hour more closely resembled that of the water deprived
group except, of course, that the sodium and water deprived group drank a
greater amount of 0.5 M NaCl.
35
Experiment 2:
Percent changes in body weight observed in Experiment 2 (Table 2) were
similar to those in Experiment 1. Thus, the experimental manipulations produced
consistent effects on body weight and reinforce the idea that differences in the
behaviors seen on test day in Experiment 1 were not attributable to differences in
body weight or in body weight changes.
There were no differences in hematocrit (Figure 4A) among the groups,
which could lead one to surmise that volume status did not contribute to the
observed behaviors. However, there were differences in plasma protein
concentration (Figure 4B) among the groups. Animals on the sodium deficient
diet had greater plasma protein concentrations than did animals on the standard
sodium diet, which could be the result of sodium deprived animals excreting
greater volumes of urine to conserve plasma sodium and overall plasma
osmolality during sodium deprivation. In any case, it is unlikely that volume
status was solely responsible for the behavioral outcomes on test day. If it had
been, one would have expected the two groups on the sodium deficient diet to
consume similar amounts of both 0.5 M NaCl and water during the intake test
since they had similar plasma protein values prior to the drinking test. Following
this logic, one might make the unreasonable prediction that the two groups on
the standard sodium diet would also have consumed similar amounts of both
solutions due to similar plasma protein concentrations. Clearly, the four groups
behaved very differently on test day, which leads to the conclusion that, although
36
differences in volume status may contribute to the behavioral responses, they
were not the only signal that elicited the behaviors.
The results from Experiment 2 also revealed that, regardless of whether
rats were sodium deprived, animals that were water deprived overnight had
greater plasma sodium concentrations than did animals that were not water
deprived (Figure 4C). On the surface, this finding may seem counterintuitive as
one would have expected the difference to be reduced plasma sodium
concentration in the sodium deprived groups. However, others have shown a
normalization of plasma sodium concentration with prolonged dietary sodium
deprivation in rats [29] and only a slight, albeit significant, decrease in plasma
sodium in humans [30]. Moreover, depriving animals of water decreases body
fluid volume, which likely accounts for the apparent increase in plasma sodium
concentration. In any case, if decreased plasma osmolality (as measured by
plasma sodium concentration) was the primary signal, we would have expected
the groups that were water deprived to behave similarly on test day. Clearly, this
was not observed. Thus, neither changes in volume status nor plasma
osmolality was solely responsible for the observed behavioral outcomes. It is,
however, possible that a combination of these two signals, and/or signals related
to sodium or water deprivation such as angiotensin II or aldosterone, led to the
differences in behavioral responses seen on test day.
Experiment 3:
37
Percent changes in body weight observed in Experiment 3 (Table 3) were
comparable to those in both Experiment 1 and Experiment 2, with the sodium
deprived groups having a more gradual rate of weight gain and the water
deprived groups having a decreased rate of weight gain after water deprivation.
Both sodium and water intake by rats in Experiment 3 (Figure 5 A and B) were
similar to those by rats in Experiment 1. In fact, the amount of sodium
consumed, total fluid ingested, and the overall fluid concentrations at three hours
were comparable within the two experiments (Table 4). Thus, the ingestion of
both 0.5 M NaCl and water are reliable and reproducible responses to the
experimental manipulations.
The results from the blood measurements in Experiment 3 (Figure 6)
showed that, regardless of whether the animals were sodium deprived, rats that
were water deprived had greater hematocrit than those that were not water
deprived. Since the water deprived groups consumed large volumes of fluids in
three hours (~25 mL total fluid by rats that were water deprived; ~41 mL total
fluid by rats that were both water and sodium deprived), this difference could
result from the water deprived groups excreting more of the ingested fluid than
did the groups that were not water deprived. Somewhat counterintuitive, then, is
the observation that plasma protein concentrations did not show a similar trend.
Rather, rats that were sodium deprived had a lower plasma protein concentration
than did rats that were not sodium deprived, regardless of water deprivation.
However, differences in the sensitivity of the methods used to measure
hematocrit and plasma protein concentration may account for the discrepancies
38
observed, particularly when plasma volume and sodium concentration may be
influenced by rapid ingestion of large volumes of water and concentrated NaCl.
Given that red blood cell volume likely is affected by the rapid influx of volume
and/or sodium into systemic circulation upon absorption of the ingested fluids,
hematocrit measurements may be more variable, and thus, plasma protein
concentration may more accurately reflect changes in volume. More interesting,
however, are the measurements of plasma sodium concentrations, which did not
differ among the four groups. Therefore, it seems that the ultimate effect of the
behavioral responses was to restore normal plasma sodium concentration, even
if more variability in body fluid volume occurred as a result.
General Discussion:
Each of the four experimental conditions produced different behavioral
outcomes during the two-bottle intake tests (Figure 2, 3, 5). The control group
ingested very little fluid on the test day, while rats that were water deprived drank
large volumes of water and very little 0.5 M NaCl. In contrast, rats that were
sodium deprived drank large quantities of 0.5 M NaCl and comparable volumes
of water. Rats that were both sodium and water deprived drank significantly
more water and significantly less 0.5 M NaCl than did the sodium deprived group,
but significantly more 0.5 M NaCl than did the control and water deprived groups.
Clearly, then, the behavioral consequences of combined water and sodium
39
deprivation differ from those observed with either water or sodium deprivation
alone.
In fact, while the sodium and water deprived group did not ‘ignore’ sodium
deprivation, these animals responded very quickly and drank large volumes of
water when fluids were made available, suggesting that water deprivation is the
more potent stimulus. Alternatively, the behavioral responses may be due to an
additive effect of combining the two challenges. Given that water deprived rats
drank approximately 30 mL of water and negligible amounts of 0.5 M NaCl (<2
mL) during the seven hour test, and that sodium deprived rats drank ~20 mL of
water and 15 mL of 0.5 M NaCl, one would predict that rats that were both
sodium and water deprived would drink ~50 mL of water and 15 mL of 0.5 M
NaCl (i.e., water intake and 0.5 M NaCl intake in approximately a 3.3:1 ratio) if
the two challenges were simply additive. However, the sodium and water
deprived group drank approximately 42 mL of water and 9 mL of 0.5 M NaCl.
Although these volumes both are somewhat less than would be predicted, the
water intake is proportionally greater, as reflected by the water intake:0.5 M NaCl
intake ratio of ~4.7:1. Similarly, the concentration of the fluid ingested by the
sodium and water deprived group was ~88.2 mMol Na/L vs ~115 mMol Na/L that
would be yielded by the predicted volumes. Finally, the pattern of intake in the
first hour for the sodium and water deprived group differed markedly from the
pattern observed for the sodium deprived group, and actually more closely
resembled the pattern seen for the water deprived group. Together, these
observations suggest that thirst is the more salient of the two drives during
40
combined sodium and water deprivation. Nonetheless, we cannot rule out the
possibility that decreases in body fluid volume and body sodium that occur during
the combination of dietary sodium deficiency and overnight water deprivation
somehow prevented these rats from consuming greater volumes of both
solutions, thereby masking the additive effect of combined water and sodium
deprivation.
On the surface, it may seem surprising that the ingestion of water and 0.5
M NaCl observed on the test day were so poorly predicted by changes in volume
or by changes in plasma osmolality. Given the complexity of the changes in
body fluid volume and plasma osmolality that occurred during our experimental
manipulations, it is possible that the combination of these two factors led to the
observed behaviors. Alternatively, an entirely different set of signals may have
stimulated the behaviors. Hormones such as angiotensin II and aldosterone are
released in response to decreased body fluid volume or body sodium and are
well known to stimulate the intake of water and salt [15, 31]. Another possibility
is altered baroreceptor signaling, such as may occur during decreased fluid
volume, which also has been implicated in the stimulation of both water intake
[13] and sodium intake [19]. Finally, we cannot discount the possibility that these
signals may work in concert to dictate the behavioral responses to combined
water and sodium deprivation, with the specific response determined by the
relative weight of each.
Regardless of the specific signal(s) that produced the behaviors observed
on the test day, it is clear that, as a result, plasma sodium concentrations were
41
comparable in all four groups whereas body fluid volume was more variable. The
observation that a wide range of behavioral responses all led to similar plasma
sodium levels underscores the importance of body sodium regulation, and
dramatically illustrates the effectiveness of the repertoire of compensatory
responses that includes behaviors like water intake and NaCl intake, as well as
hormonal and renal mechanisms. Nonetheless, it was surprising that hematocrit
and plasma protein concentration varied so widely. The obvious interpretation of
these findings is that body fluid volume is less tightly regulated. Though there
may be truth to this position, it cannot be ignored that hematocrit and plasma
protein concentrations are indirect measures of plasma volume. Direct
measurement of body fluid volume (e.g., using dye dilution methods) will provide
more accurate assessment of the volume status of these animals and help to
resolve uncertainty about assessment of body fluid volume based on hematocrit
and plasma protein concentration measurements.
In summary, previous studies have typically focused on the signals and
behavioral responses to sodium deprivation or water deprivation alone, while
those studies that have integrated thirst and sodium appetite [21-24] did so for
reasons different from the goals of the present study. We sought to determine
the effect of combined water and sodium deprivation on water and 0.5 M NaCl
intake, and have demonstrated reliable and reproducible behavioral responses.
We also have begun to deduce the signals that underlie these motivated
behaviors, by showing that neither changes in plasma osmolality, as indicated by
plasma sodium concentration, nor changes in fluid volume, as indicated by
42
hematocrit and plasma protein concentration, alone could account for the
behavioral responses. Additional studies are needed to further investigate
hormonal signals such as angiotensin II and aldosterone, as well as the role of
neural input from baroreceptors and/or the kidneys. Finally, it will be important to
measure urine volume and urine sodium concentration to more fully understand
the compensatory responses that promote the maintenance of appropriate
plasma sodium concentration in the face of combined challenges to body fluid
volume and sodium.
43
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Wolf, A.V., Osmometric analysis of thirst in man and dog. Am J Physiol,
1950. 161(1): p. 75-86.
3.
Jones, A.B. and K.S. Curtis, Differential effects of estradiol on drinking by
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Implications for hyper- vs. hypo-osmotic stimuli for water intake. Physiol
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46
VITA
Kimberly Jean Lucia
Candidate for the Degree of
Master of Science
Thesis: COMPENSATORY BEHAVIORAL RESPONSES TO COMBINED
CHALLENGES TO BODY SODIUM AND FLUID BALANCE
Major Field: Biomedical Sciences
Biographical:
Education:
Completed the requirements for the Master of Science in Biomedical
Sciences at Oklahoma State University Center for Health Sciences,
Tulsa, Oklahoma in December, 2010.
Completed the requirements for the Bachelor of Science in Biology at
Pennsylvania State University, State College, Pennsylvania in May,
2008.
Name: Kimberly Jean Lucia
Date of Degree: December, 2010
Institution: Oklahoma State University
Location: Tulsa, Oklahoma
Title of Study: COMPENSATORY BEHAVIORAL RESPONSES TO COMBINED
CHALLENGES TO BODY SODIUM AND FLUID BALANCE
Pages in Study: 46
Candidate for the Degree of Master of Science
Major Field: Biomedical Sciences
Scope and Method of Study:
To determine the effect of combined dietary sodium deficiency and overnight
water deprivation on 0.5 M NaCl and water intake by rats, and to begin to
characterize the signals that underlie these behavioral responses by measuring
changes in both fluid volume, as indicated by hematocrit and plasma protein
concentration, and plasma osmolality, as indicated by plasma sodium
concentration.
Findings and Conclusions:
The sodium and water deprived group drank significantly more water and
significantly less 0.5 M NaCl than the sodium deprived group, but significantly
more 0.5 M NaCl than the control and water deprived groups. In addition, the
sodium and water deprived rats responded very quickly and ingested large
volumes of water when fluids were made available. The pattern of intake in the
first hour by rats in the sodium and water deprived group more closely resembled
the pattern of intake by water deprived rats. Thus, it is possible that water
deprivation is the more salient of the two stimuli. Neither changes in fluid volume
nor plasma osmolality alone could account for the observed behaviors. While the
behavioral responses led to greater variability in volume measurements, plasma
sodium concentration was restored to normal levels. These findings demonstrate
the importance of body sodium regulation, and dramatically illustrate the
effectiveness of the repertoire of compensatory responses that includes
behaviors like water intake and NaCl intake.
ADVISER’S APPROVAL: Kathleen S. Curtis, PhD