SODEUM APPETITE INDUCED BY
DiETARY DEPRWATQON:
THE PHYSMLOGY 0F 1T8 DEVELOPMENT
ANE} THE BEHAVlORAL RESPONSES
LEADENG T0 HS. SATIATION

Thesis for the Degree 0f M. A.
MiCHlGAN STATE UNIVERSITY -
ROBERT fOHN CONTRERAS
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ABSTRACT

SODIUM APPETITE INDUCED BY DIETARY DEPRIVATION:
THE PHYSIOLOGY OF ITS DEVELOPMENT AND
THE BEHAVIORAL RESPONSES LEADING
TO ITS SATIATION

By

Robert John Contreras

The Specific hunger for salt is an inherited motivation of the
land dwelling animal that is activated in conditions of sodium need.
Sodium is the mainstay of the extracellular fluid compartment. Its
concentration has an effect on body water distribution and therefore
on the proper functioning of the cells of the body. When a nutri-
tional deficit has been incurred, the animal re5ponds in two ways:

(I) physiologically through the adrenal—renal axis via aldosterone to
promote the reabsorption of sodium, and (2) behaviorally to ingest
greater quantities of salt.

In Experiments I and 11, research was directed at determining
the urinary and blood chemical changes associated with sodium defi-
ciency. The results show that urinary sodium excretion is greatly
reduced after one day of sodium deprivation and minimized thereafter.
The fall in urinary sodium paralleled the rise in plasma aldosterone
concentration. Plasma sodium and plasma protein levels were not

associated with sodium appetite; However, a significant increase in

Robert John Contreras

plasma potassium levels was detected after twenty days of sodium
deprivation. It was suggested that sodium appetite might be impor-
tant to combat against hyperkaelemia (high plasma potassium) although
the appetite develops long before plasma potassium increases.

The temporal characteristics of drinking saline and distilled
water by sodium deprived rats were analyzed in Experiment III. These
rats were observed to montonically decrease the proportion of time
spent drinking saline as they approached satiation. The dynamic
aSpects of the behavior underlying this proportional decrease were
that drinking bursts remained constant in length and pauses between
bursts became longer. It was pointed out that satiation of sodium
appetite through saline ingestion was not a stepwise but rather a
continuous process, and arguments were made to explain salt intake

in terms of an adaptation mechanism.

SODIUM APPETITE INDUCED BY DIETARY DEPRIVATION:
THE PHYSIOLOGY OF ITS DEVELOPMENT AND
THE BEHAVIORAL RESPONSES LEADING

TO ITS SATIATION

BY

Robert John Contreras

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF ARTS

Department of Psychology

1974

To My Parents

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Glenn I. Hatton for his invaluable
guidance of this thesis. I also wish to offer my appreciation to
Dr. John 1. Johnson and Dr. James L. Zachs for many valuable sug-
gestions on the manuscript. In addition, I am indebted to Dr. Rudy
Bernard for having the blood samples of Experiment II analyzed, and
to Mr. Bob Holmes for assistance in collecting the data of
Experiment I.

This research was supported by a grant from the National
Institute of Neurological Diseases and Stroke, #N809140, to
Glenn I. Hatton and by a Biomedical Science grant, #71-0803, to

the author.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . vi

LIST OF FIGURES . . . . . . . . . . . . . . . . vii

INTRODUCTION . . . . . . . . . . . . . . . . . 1

Metabolic Control of Salt Appetite . . . . . . . . . 3
Taste Control of Salt Appetite . . . . . . . . . . . 3

EXPERIMENT I . . . . . . . . . . . . . . . . . 8
Procedure . . . . . . . . . . . . . . . . . 8
Subjects . . . . . . . . . . . . . . . . . 8
Metabolism cages . . . . . . . . . . . . . . 8
Diets . . . . . . . . . . . . . . . . . . 9
Blood sample . . . . . . . . . . . . . . . . 13
Solutions . . . . . . . . . . . . . . . . . l3
Preference tests . . . . . . . . . . . . . . . 13
Results . . . . . . . . . . . . . . . . . . l4
EXPERIMENT II . . . . . . . . . . . . . . . . . 30

Procedure . . . . . . . . . . . . . . . . . . 30

Subjects . . . . . . . . . . . . . . . . . 30
Blood sample . . . . . . . . . . . . . . . . 31

Results . . . . . . . . . . . . . . . . . . 31
Discussion of Experiments I and II . . . . . . . . . 31

Body metabolism . . . . . . . . . . . . . . . 31
Preference tests . . . . . . . . . . . . . . . 36

iv

EXPERIMENT III . .
Procedure
Subjects

Apparatus . .
Solutions

Preference tests .

Results

Water replete .
Water deprived

GENERAL DISCUSSION

Blood .

Patterns of Intake .

LIST OF REFERENCES
APPENDICES
Appendix

A. Apparatus .

B. Raw Data .

O

Page
39
39
39
39
40
4O
41

41
59

75

75
76

80

84

86

Table

LIST OF TABLES

Effects of Diet on Metabolic Exchange Variables in
Holtzman Rats . . . . . . . . . . .

Urine Volume and Urine Sodium Concentration Values
From Sodium Deprived and Control Rats . .

The Values of Serum Sodium, Potassium, and Protein
in Sodium Deprived and Control Subjects

The Values of Plasma Sodium, Potassium, and Protein
in Sodium Deprived and Control Subjects

Characteristics of Drinking .4 M Saline by Water
Replete and Water Deprived Rats . . .

Characteristics of Drinking by Water Replete and
Water Deprived Rats . . . . . . . .

Raw Data-~Body Metabolism
Blood Data

Raw Data—-Patterns of Intake

vi

Page

16

20

22

32

42

58

86
88

89

LIST OF FIGURES

Figure

1. This is a flow chart depicting the various stages
of Experiment I . . . . . . .

2. The mean total urinary sodium excreted as a function
of time under conditions of: (A) Mouse Breeder Blox
food diet baseline; (B) Nutritional Biochemicals Test
Diet (plus 1% NaCl) baseline; (C) sodium deprivation;
(D) recovery; (E) sodium deprivation and water
deprivation in sodium deprived (Exptl.) and normal
(Control) rats .

3. The cumulative mean intake of distilled water and .4
molar saline as a function of time. There were two
groups of water replete subjects, those that were
sodium deficient (E-NaCl; E-HZO) and those that
were normal controls (C-NaCl; C-HZO) .

4. The cumulative mean intake of distilled water and .4
molar saline as a function of time. There were
two groups of water deprived subjects, those that
were sodium deficient (E-NaCl; E-HZO) and those
that were normal controls (C-NaCl; C-HZO)

S. The cumulative mean intake of distilled water and .3
molar saline as a function of time. There were
two groups of water deprived subjects, those that
were sodium deficient (E-NaCl; E-H O) and those
that were normal controls (C-NaCl; C-HZO)

6. The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the
first 10 minutes of the preference test. There
were two groups of water replete rats, one group
was fed a sodium deficient diet (Exptl.) and
the other was a normal control (Control) group

vii

Page

11

18

24

27

29

4S

Figure Page

7. The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the
second 10 minute period of the preference test.
There were two groups of water replete rats, one
group was fed a sodium deficient diet (Exptl.)
and the other was a normal control (Control)
group . . . . . . . . . . . . . . . . . 47

8. The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the
last 40 minutes of the preference test. There
were two groups of water replete rats, one group
was fed a sodium deficient diet (Exptl.) and the
other was a normal control (Control) group . . . . 49

9. The mean proportion of time spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water replete, normal controls,
and drank from the distilled water bottle first . . . 51

10. The mean proportion of time Spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water replete, sodium deficient,
and drank from the distilled water bottle first . . . 53

11. The mean proportion of time Spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water replete, sodium deficient,
and drank from the saline bottle first . . . . . . SS

12. The mean proportion of time Spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water replete, normal controls,
and drank from the saline bottle first . . . . . . S7

13. The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the first
10 minutes of the preference test. There were two
groups of water deprived rats, one group was fed a
sodium deficient diet (Exptl.) and the other was a
normal control (Control) group . . . . . . . . 61

14. The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the
second 10 minutes of the preference test. There
were two groups of water deprived rats, one group
was fed a sodium deficient diet (Exptl.) and the
other was a normal control (Control) group . . . . 63

viii

Figure

15.

16.

17.

18.

19.

The cumulative (from right to left) mean frequency
of interdrink intervals occurring within the
last 40 minutes of the preference test. There
were two groups of water deprived rats, one
group was fed a sodium deficient test (Exptl.)

and the other was a normal control (Control) group .

The mean proportion of time Spent drinking distilled
water and .4 molar saline as a function of time.

These animals were water deprived, sodium deficient,

and drank from the saline bottle first .

The mean prOportion of time spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water deprived, normal controls,
and drank from the saline bottle first . . . .

The mean proportion of time spent drinking distilled
water and .4 molar saline as a function of time.
These animals were water deprived, normal controls,
and drank from the distilled water bottle first .

The mean proportion of time Spent drinking distilled
water and .4 molar saline as a function of time.

These animals were water deprived, sodium deficient,

and drank from the distilled water bottle first .

ix

Page

65

67

69

71

73

INTRODUCTION

It would be adaptive for fluctuations in the internal milieu
to have a direct effect upon the gustatory system. In this case,
information about the physiological condition of the organism is
transmitted to the sensing system where it is used to determine the
relative desirability of various substances in the external environ-
ment. Halpern (1967) has summarized the ways that internal changes
may influence taste. He divided them into neural, salivary, and
vascular factors. The Specific appetite for salt is a phenomenon that
lends itself quite readily to this problem of internal-external
environment interaction. The role of blood in information transfer
has not been uncovered, yet.

Several methods have been used to produce the specific hunger
for salt in the laboratory. Bilateral adrenalectomy results in the
loss of aldosterone, the mineralocorticoid hormone which normally
maintains sodium balance by promoting reabsorption of sodium by the
kidney. Bare (1949) showed that an adrenalectomized rat ingests
significantly greater quantities of salt solution compared to normal
controls and to its own preoperative levels. This increase in salt
intake occurs across a wide range of concentrations. In need-free

rats there is an increasing preference (over water) for the salt

solution as its concentration approaches isotonicity (.9%), with a
sharp decline at greater concentrations. Increasing the animal's
salt need elevates total NaCl solution intake and shifts the maximal
preference point out toward the higher concentration. By implanting
a unilateral fistula into the parotid gland of sheep results in a
rapid depletion through salivary loss. Denton and his associates
(1965, 1969) demonstrated that the amount of saline consumed is
quantitatively related to the extent of the animal's deficit. By the
method of intraperitoneal dialysis using a glucose solution large
amounts of sodium are removed from the body (Falk and Herman, 1961).
Subcutaneous injections of formalin (Jalowiec 8 Stricker, 1970)
produced salt appetite in rats by sequestering body fluids at the
injection site. A less widely used method, is to place animals on a
low sodium diet (Nachman, 1962; Wagman, 1963; Jalowiec and Stricker,
1973).

Studies of sodium appetite have generally found that sodium
deficient rats increase their sodium solution intake until the
internal environment reaches hydromineral balance. Two types of
satiation take place during sodium repletion; one type is mediated
through oral and stomach receptors, and the other through a long term
metabolic satiation. An immediate or short term satiation refers to
the neural changes that occur in response to orogastric stimulation.
Long term control arises when the ingestants are absorped into the

blood.

Metabolic Control of Salt Appetite

 

By employing long-term tests, Fregly (1958) found that
adrenalectomy in rats led to an increased preference for sodium salts
(chloride, sulfate, bicarbonate, and nitrate) but did not produce an
increased preference for non-sodium salts such as potassium, lithium,
or calcium chloride. Denton (1965) was able to Show the same thing
in sodium-deficient sheep which developed preferences for NaCl or
NaHCO3 but not for potassium, calcium, or magnesium chloride.

Need-free rats prefer to drink a dilute saline rather than
water. However, Davenport (1973) was able to eliminate palatability
factors in his experiments and demonstrated that need-free rats prefer
to drink water rather than isotonic saline. The ingestion of sodium
salts is more susceptible to learned preferences based on the bene-
ficial or toxic consequences of metabolism in long term rather than

in short term tests.

Taste Control of Salt Appetite

 

C. P. Richter (1939) proposed that sodium deficiency altered
the taste bud membrane in a way that increased its sensitivity to
external stimulation. He proposed a peripheral "on" mechanism that
would enhance taste receptor acuity in salt need. In a free choice
situation, where subjects were allowed to ingest tap water and/or
NaCl solution, preference thresholds were measured (Richter, 1939;
Bare, 1949). Both experiments demonstrated that the preference
threshold for NaCl solution to be significantly lower among adrenalec~

tomized than among normal controls. This result is due to the

increased motivation of sodium deficiency rather than to changes in
absolute sensitivity. When adrenalectomized and normal controls are
highly motivated to discriminate between water and weak concentrations
of saline, psychophysical thresholds for salt detection were the same
for both groups of animals (Carr, 1952; Harriman G Macleod, 1953).

Behaviorally, at least, receptor acuity has not been increased
by physi010gical deprivation. Electrophysiological evidence substan-
tiated the idea that absolute thresholds are not altered by sodium
deficiency. Pfaffmann and Bare (1950) report that the minimum con-
centration necessary to evoke increased discharge of the chorda tympani
nerve to different suprathreshold concentrations of NaCl solutions.
They confirmed that sodium deficiency did not alter the afferent
neural response. The response profile of single units may exhibit
threshold and suprathreshold differences that would not Show up in a
whole nerve recording, however. The results from these behavioral and
electrophysiological experiments are consistent with the notion that
salt preferences are centrally rather than peripherally mediated.

It has been shown that physiological deprivation does not
alter the sensitivity of the taste receptors. However, physiological
deprivation may alter the adaptive state of the taste system and
this can be used to explain salt appetite. This "off" or ”stop"
mechanism proposes that sodium deficient and normal controls adapt
to a salt stimulus at different rates. This mechanism could be
peripheral or central in nature. In the first case, sodium defi-
ciency would alter the sodium receptor membrane in a way that would

prolong its reactivity to a sodium stimulus. In turn, the peripheral

nerve taste response would adapt less readily in a sodium deficient
rat than in a nondeficient one.

A central "stop" mechanism to account for salt appetite
would depend upon the hedonic qualities of sensory stimulation. In
this instance, all concentrations of NaCl solution would initially
taste pleasant to both a normal and sodium deficient rat, but this
hedonic quality would persist longer for the deficient animal. In
order that gustatory stimulation have an affective component, this
input must reach the hypothalamus and/or limbic structures (Pfaffmann,
1960; 1961). Recent evidence (Bernard and Nord, 1971; Norgren and
Leonard (1971) suggested that the central tegmental pathways, which
emanate from the gustatory pontine nucleus, provide a potential route
whereby gustatory inputs, could reach the hypothalamus.

It is believed that information concerning the internal state
of sodium balance is transmitted to the gustatory system to modify
the response during external sodium Stimulation. There is evidence
(Bradley, 1973) to Show that alterations in the chemistry of the blood
perfusing the tongue can produce an identifiable effect on the multi—
fiber response of the gustatory nerve (chorda tympani). It would
seem that the blood may be an important means of communicating the
state of the internal environment to the sensory system of taste.
Through this mechanism, preferences for certain food elements may be
enhanced or attenuated depending upon physiological need. It is
hypothesized, for sodium appetite, that plasma sodium levels perfusing
the tongue region affect the gustatory neural response, a decrease

in plasma sodium resulting in an increase in the response strength,

as measured by degree of adaptation. to external sodium chloride
stimulation.

Before the question could be attacked directly, it was felt
that more basic questions needed to be answered. First, it was
important to know what blood chemical changes were associated with
sodium deficiency. If the blood does indeed alter gustatory neural
responses, what causes these effects and how do they arise? Secondly,
it was important to find some behavioral support for the adaptation
hypothesis. Does the temporal pattern of saline ingestion in a
sodium deficient rat suggest an adaptation-like process?

With these thoughts in mind experiments were designed to
investigate the relationship between dietary sodium deprivation and
salt appetite. Experiment I determined the physiological and be-
havioral responses of water replete and water deprived rats who were
given a dietary sodium depleted diet ad_libitum. The relationship
between blood electrolyte composition and dietary sodium deprivation
was examined in Experiment 11. The purpose of experiment III was to
study changes in the temporal characteristics of salt preference
during a one hour, three bottle test.

In summary, the purpose of these studies was to answer the
following questions:

1. What are the effects on those variables, known to be crucial
in body water and electrolyte balance, of two diets that are
nutritionally balanced, but differ in sodium, potassium,

and chlorine content?

What changes in urinary and blood electrolyte composition
occur in water replete and water deprived rats that are
maintained on a sodium deficient diet?

Can a preference for salt be induced in rats that are
dietary sodium deficient but are not water deprived?

What are the temporal characteristics of sodium chloride
solution intake as a previously dietary sodium deprived

rat drinks to satiation?

EXPERIMENT I

The specific appetite for salt has been produced experimentally

by placing animals on a low sodium diet for an extended period of

time (Nachman, 1962; Nachman G Pfaffmann, 1963). A preference test,

a measure of salt appetite, usually follows this deprivation period.
These preference tests are always given to water deprived rats. It

was the interest of this experiment to compare changes in variables,
known to be crucial in body water and electrolyte balance, in water
replete and water deprived rats that were maintained on a sodium
deficient diet. The major variables were the levels of sodium and

potassium.that remained in the blood or were excreted in the urine.

Procedure

Subjects. Animals were 24 male albino rats of the Holtzman
strain, 90-100 days old at the start of the experiment. They were
individually housed and had Wayne Mouse Breeder Blox and demineralized
water continually present. They were situated in a windowless room
which was maintained at 22-25 C. The fluorescent lighting of the room
was on a 14-10 hour (lights on--500 hour; lights off—-1900 hour)

light-dark cycle for the duration of the experiment.

{getabolism cages. Rats were housed in standard Acme Metal

 

PTOdUCtS :metabolism cages. A 50 cm. tall base supported each cage.

A total of 12 metabolism cages were arranged on two tables, six per
table. The surfaces of the tables were elevated 94 cm. above the
floor.

The week before the experiment began, each rat was handled
for two minutes a day. These rats were tested in squads of size 12;
they were run in the summer and fall of 1972. Depicted by flow chart
in Figure l and proceeding from the top to the bottom are the differ-
ent stages of the experiment. For the entire experiment, 24 hour
measures of body weight, food and water intake, and urine volume were
recorded for each animal. These measures were always recorded between
1000 and 1100 hours. A daily urine Specimen from each animal was
saved for future analysis of sodium and potassium concentration by
flame photometry and total solids by use of a refractometer. Two
100 ml. calibrated drinking tubes were always fixed to the metabolism
cage, one cylinder containing de-mineralized water and the other
empty. These two cylinders were randomly placed between drinking

outlets to discourage position preferences.

Diets: Two powdered laboratory diets, Wayne Mouse Breeder
Blox (BB) and a sodium deficient Nutritional Biochemicals Test Diet
(T0), were used. A superficial comparison of the diets indicated an
odor and texture dissimilarity. When a 1 percent sodium chloride
mixture was added to the TD it contained 1.4% potassium, 1.53%
chlorine, and .42% sodium. On the other hand, BB contained .77%
potassium, .66% chlorine, and .42% sodium. The first nine days
(days 1-9) served as a baseline period for input and output variables

under BB. The following 14 days (days 10-23) served the same

10

Figure 1. This is a flow chart depicting the various stages of
Experiment I.

11

 

Days 1-9

 

Breeder Blox I

 

 

NaCl, Days 10-23

/\

l Test Diet plus 1%

 

 

 

 

 

Sodium deficient diet Control diet

Days 24-33; Days 24-33;
Preference test or Preference test or
Blood sample on Blood sample on
Day 33. Day 33.

 

 

 

 

 

/

      

 

 

 

 

Recovery

Days 34-38
Sodium deficient diet Control diet
Days 39-49; Days 39-49;
Water deprivation Water deprivation
Days 44-49; Days 44-49;
Preference test Preference test
Day 49. Day 49.

 

 

 

 

 

12

function for the TD which was supplemented with a 1% NaCl mixture.

An average body weight was computed for each animal for this 14 day
period. From this average, animals were divided equally in number

and by weight into four conditions by the following method.

Rats were rank ordered from heaviest to lightest and paired
on the basis of body weight similarity. Within each animal pair one
animal was randomly designated as either sodium deprived (D=TD,
no sodium) or normal (N=TD plus 1% NaCl), and as either preference
test (PT) or blood sample (BS). The other member of the pair was
automatically designated conditions Opposite to those of the first.
If the first rat was designated D-PT (sodium deprived-~preference
test), then the second rat was designated as N-BS (Normal--Blood
Sample). Therefore, subjects were divided into two diet groups, 10
days later half of each group were to receive a preference test and
the other half a blood sample.

Although Figure 1 shows that all subjects had a 14 day TD
baseline, some subjects had more. Only the last 14 days were included
in the data. To avoid having preference tests and blood samples fell
on the same day one animal pair per day was started on its diet
condition. From day 24 till the end of the experiment, the first
animal pair was one day ahead of the second pair, two days ahead of
the third, and so on.

Blood samples and preference tests were given on day 33. The
blood sample was acquired after metabolic data collection, and the

preference test immediately followed the blood sample.

13

Blood sample. The rat was removed from his home cage and

 

taken to a separate room. The subject was anesthetized with ether
and a 1.5 to 2 ml blood sample was withdrawn from the left ventricle
by inserting a needle of a 2 cc. glass syringe through the rat's rib
cage. All samples were centrifuged; a sample of the serum was
analyzed for protein concentration in a refractometer, and the

remainder was frozen for subsequent analysis by flame photometry.

Solutions. All saline solutions were made as molar concen-
trations with anhydrous NaCl and distilled water and were kept at

room temperature .

Preference tests. A one hour, two-bottle preference test

 

was given to the second rat in his home cage. He was given a choice
between .4M NaCl solution and distilled water to drink. The metabolism
cage was fixed with two 100 ml gas collecting tubes that were cali-
brated to a .2 ml and contained drinking solutions. The experimenter
sat on a stool behind the metabolism cage so that he could record the
amount of fluid intake on a minute to minute basis. Both bottle
spouts were introduced into the cage simultaneously. A five second
taste sample was allowed from each solution; thereafter, the drinking
spout was quickly withdrawn. After both solutions were tasted the
drinking spouts were concurrently inserted into the cage and the rat
was allowed a one hour access.

A five day recovery period (days 34-38) was instituted such
that a 1% NaCl mixture was added to the T0 of the experimental group.

The control group's diet remained unchanged. Following recovery,

14

experimental subjects were again deprived of salt. On day 43, both
control and experimental subjects were adapted to a 23.0 hour water
deprivation schedule. At 1100 hour subjects were removed from their
metabolism cage and put into drinking boxes for one hour. Each box
was fixed with two 100 m1 gas measuring tubes, graduated in 0.2 ml,
had Plexiglas covers for observations of drinking, and had a moveable
guillotine door which separated the drinking Spouts from the rest of
the box (for detailed description of apparatus, see Appendix A).
Adaptation to this schedule lasted 6 days. The position of each
drinking cylinder was randomized, one bottle contained distilled water
and the other remained empty. A two bottle preference test was given
on day 49. The same method for presenting drinking spouts was used
for this preference test as in the previous one. The amount of fluid
intake from each tube was recorded for the first 15 sec, 30 sec, and
every minute during the 60 min drinking period. Those subjects who
were tested in the summer were given a choice between .4M NaCl solu-
tion and distilled water; however, a weaker concentration (.3M) of

NaCl solution was used to test the second set of animals.

Results

Body metabolism data were grouped into blocks of days in the
following manner: 7-9, 10-13, 14-18, 19-23, 24-28, 29-33, 35-38,
39-43, and days 44-49. To eliminate any carry over effects from the
treatments day 34 was excluded from statistical analysis. Every
metabolic variable was averaged within each block of days for experi-

mental and control groups. A student tftest was used to make between

15

group comparisons and the results of the analysis are located in
Appendix B.

The effects that BB and TD had on the metabolism of the albino
rat are documented in Table 1. The numerical figures in each cell are
the means plus or minus the standard error. These numbers represent
an average measure of 24 animals over a three-day period. Days 7
through 9 and days 21-23 were chosen to represent baseline metabolism
under BB and TD respectively. As indicated by Table 1, the following
variables were significantly different: Water intake (t_= 3.541,

EE.‘ 142, p_< .001), urine sodium concentration (t_= 6.568, df_= 142,
p_< .001), total urinary sodium excretion (£_= 8.750, E£.= 142,
p.< .001), and urinary total solids (2.: 3.453, df_= 142, p_< .001).

In Figure 2 total urinary sodium excretion is graphed across
days. Data points of every third day are plotted through day 39.
Thereafter daily measurements are plotted. These third day data
points are representative of the overall trend of the data as deter-
mined by t-test analysis (see Appendix B). Minor differences that
existed between the two groups during dietary baseline or recovery
phases of the experiment were nonsignificant (see Appendix B). How-
ever, highly significant results were obtained when sodium was removed
from the experimental group's diet (see Appendix B). There was no
overlap in the distribution of the total amount of sodium excreted in
the urine between groups. Every experimental subject excreted less
sodium than any control subject each day of both sodium deprivation
periods. On the first day of each sodium deprivation period the

average urinary sodium 1055 was .209 mEq. on day 24 and .446 mEq. on

16

TABLE 1

Effects of Diet on Metabolic Exchange
Variables in Holtzman Rats

 

 

Metabolic Mouse Breeder Nutritional Biochem
Variables Blox Test diet + 1% NaCl
Food intake 19.777 :T .268 20.319 : .311
(a)
*Water intake 32.708 : .699 37.736 i 1.229
(m1.)
Urine volume 17.208 i .528 19.152 t .965
(m1.)
*Urine Na Conc. 190.180 i 6.280 132.263 i 6.140
(mEq./L.)
Urine K Conc. 168.625 i 6.126 169.875 i 8.093
(mEq./L.)
*Urine Na 3.105 S .073 2.230 i .069
(mEq./Day)
Urine K 2.719 t .063 2.823 t .076
(mEq./Day)
*Urine-total solids 455.375 1 3.965 434.513 i 4.527
(Refractive
Index)

 

Note: Values are X'i S.E., N = 24 in each cell.

*p < .05 in comparison between laboratory diets.

17

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day 39. On subsequent days except on the first day of water depriva-
tion, the average sodium less was .021 mEq. per day. Each subject's
urine sodium output was higher on the first day of water deprivation
when compared with its previous day's output (E_= 4.600, d£_= 22,

p. <.001). Control subjects underwent a similar increase but was
nonsignificant (t_= .457, df_= 22, p_> .50). When water deprivation
days (45-49) were compared with pre-water deprivation days (39-43),
control animals decreased the amount of sodium eliminated in the
urine (t_= 9.650, df_= 118, p_< .001). This dr0p in sodium excretion
neither reached the low levels of the experimental group nor did it
recover to the level that existed prior to water deprivation.

Total urinary sodium excretion (mEq./day) is a derived measure
whose value is determined by the multiplicative relationship: Urine
volume (ml.) X urine sodium concentration (mEq./L.). Urine volume
and urine sodium concentration measures basic to Figure 1 are
summarized in Table 2. A perusal of this table shows that the ex-
perimental group urinated more per day than the control group. The
experimental group should have lower urinary sodium concentrations to
parallel their higher urine volumes. This result is verified in
Table 2. When the urine volume factor is eliminated in the total
urinary sodium excretion measure, differences between groups during
TD baseline and recovery phases of this study were nonsignificant
(see Appendix B).

The average amount of sodium excreted in the urine of experi-
mental animals following 10 days of sodium deficiency was .373 mEq.,

56 percent of that total was eliminated on the first day. As expected,

20

TABLE 2

Urine Volume and Urine Sodium Concentration Values
From Sodium Deprived and Control Rats

 

U . Urine sodium concentration
rine volume ml.

 

 

 

Days (meq./L.)
Experimental Control Experimental Control
19.8 15.2 155.7 193.8
20.3 15.3 154.8 200.8
16.4 15.2 181.7 205.0
12 25.3 17.8 116.1 137.2
15 27.1 18.8 102.3 158.8
18 22.7 15.0 117.6 178.5
21 21.0 16.0 114.7 160.5
24 19.3 15.7 16.0 164.8
27 22.5 16.9 1.1 139.8
30 22.6 17.1 .6 142.3
33 23.4 18.8 .8 143.2
36 27.7 23.3 113.4 125.4
39 25.5 20.1 16.6 144.8
40 29.6 22.1 1.1 133.3
41 26.0 19.7 1.1 147.0
42 24.0 19.3 1.1 141.9
43 28.0 18.3 .8 168.9
44 7.5 9.1 38.8 298.2
45 5.0 6.1 9.4 220.7
46 5.3 6.0 3.2 236.3
47 6.1 6.4 3.2 235.4
48 5.8 7.3 3.6 247.0
49 5.5 7.5 4.0 247.3

 

21

there were no significant differences between experimental and control
subjects' serum sodium levels (t_= .930, df_= 9, p_> .10). In

Table 3 the values of serum sodium, potassium, and protein are
reported for experimental and control groups. The numbers in each
cell represent the mean plus or minus the standard error. Sodium
deficient animals did not have significantly different serum potas-
sium (E_= .130, d£_= 9, p_> .50) and serum protein (£_= 1.70, I

d£_= 9, p_> .10) levels than controls. An insufficient amount of
blood was extracted from one control subject to be analyzed and
included in the data.

Although the experimental groups' serum sodium level was not
appreciably different from that of the controls, sodium deprived
subjects preferred to drink a .4 M NaCl solution rather than distilled
water in a two-bottle preference test situation. Figure 3 shows the
cumulative mean intake of both solutions by water replete subjects.
As soon as they tasted the salt solution, the activity level of most
subjects increased. Experimental subjects drank significantly more
saline than controls (t_= 7.67, d£_= 10, p_< .001). In fact every
subject drank more NaCl solution than any one of the controls. A
preference ratio is the amount of NaCl solution intake divided by the
total amount of fluid intake times 100. The preference ratio for
experimental subjects was 60% whereas it was 38.4% for controls.
Sodium deprived rats drank 12.13 ml. of saline which is equal to 4.35
mEq. of sodium. This amount surpassed their urinary sodium less by
almost four milliequivalents. The supplementary intake of water

diluted the ingested saline to 1.4% (about .25 M NaCl). The control

22

TABLE 3

The Values of Serum Sodium, Potassium, and Protein
in Sodium Deprived and Control Subjects

 

 

Experimental group Control group

N=6 N=5
Serum Na 131.16 S 1.35 133.40 1 2.20
Serum K 4.83 i 0.31 4.80 t .37
Serum protein 6.16 i 0.13 6.33 i .10

 

Note: Values are the X'i S.E.

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group, however, drank 1.25 mEq. of sodium and diluted their ingested
saline to .89% which is isotonic with plasma.

The preference test data from water deprived rats are graphed
in Figures 4 and 5. The cumulative fluid intake of .4M (t.= 5.37,
_f_= 8, p_< .001) and .3 M (t.= 3.49, 9f.= 10, p_< .001) NaCl
solution was significantly greater for experimentals than for con-
trols. Their corresponding preference ratios were 45.1% and 39.4%
for the experimental group and 16.5% and 24.2% for the control group.
Sodium deprived subjects drank 36% more .3 M saline (13.06 ml.) than
.4 M saline (9.63 ml.) but drank 56% more distilled water (33.06 m1.
vs. 21.33 ml.) as well. In contrast with water replete subjects
water deprived rats mix their intakes between saline and water to
lower concentrations. The experimental group ingested substantial
amounts of sodium (3.828 mEq. of .4 M NaCl; 3.898 m Eq. of .3 M NaCl)
but their combined intakes of saline and water were near isotonic
levels (1.05% for .4 M NaCl, .69% for .3 M NaCl). Controls, on the
other hand, ingested less sodium (1.349 mEq. of .4 M NaCl; 1.871
mEq. of .3 M NaCl) at lower dilutional levels (.38% for .4 M NaCl,

.42% for .3 M NaCl).

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EXPERIMENT II

The abnormally low serum sodium values obtained in Experiment I
were suspect. This experiment was carried out to reassess the role
that blood factors have in salt appetite. Blood samples were ob-

tained from rats after 10 and 20 days of sodium deprivation.

Procedure

Subjects. Animals were 12 male albino rats of the Holtzman
strain, 90-100 days old at the start of the experiment. They were
housed and maintained under the same conditions as described for the
animals of Experiment I, with one exception. They were given TD
supplemented with a 1 percent NaCl mixture ad libitum instead of BB.

Rats were adapted to the T0 for two weeks, during which time
individual body weights were recorded daily at 0900 hour. An average
body weight was computed over these 14 days for each rat. From this
average animals were divided equally in number and by weight into
experimental and control groups. The experimental group did not have
their diet supplemented with NaCl, but the diet for the control group
remained unchanged. Two blood samples were obtained from each animal
by heart puncture 10 and 20 days after subjects were divided into

groups.

30

31

Blood sample. The same blood sampling technique employed in

 

Experiment I was used here, except that the experimenter used

heparinized syringes.

Results

Table 4 summarizes the results of Experiment 11, and indicates
that the absolute levels of blood sodium were higher than those
obtained in Experiment I (see Table 3). The experimental group's
plasma sodium and plasma protein values were not significantly dif-
ferent from controls after 10 (E_= .010, d£.= 10, p_> .50; E_= .323,
d£.= 10, p_ >.50) and 20 (£_= .344, d£.= 8, p_> .50; £_= .239, df
df_= 8, p_> .50) days of sodium deficiency, respectively. Unexpec-
tedly plasma sodium increased for both groups; however, when the
data from the two groups were combined no significant increases were
found (t_= 1.590, df_= 21, p_> .10). The overall plasma protein level
marginally decreased from 6.59 to 6.37 when the data from both groups
were combined at 10 and at 20 days, respectively (£_= 1.880,
d£’= 20, .10 < p_< .05). Findings from a between—group comparison
of plasma potassium show insignificant differences after 10 days
(E_= .843, df_= 10, p_< .10), but a significant increase was obtained
in 20 day sodium deficient rats over controls (t_= 2.904, d§_= 9,

.05 < p_< .02).

Discussion of Experiments I and II

 

Body metabolism. Although it was not a crucial aSpect of

 

this thesis, baseline comparisons were made between an unfamiliar

diet with one that had been used extensively. This meant comparing

32

TABLE 4

The Values of Plasma Sodium, Potassium, and Protein
in Sodium Deprived and Control Subjects

 

Experimental group

 

 

 

 

 

10 day 20 day

Plasma Na 143.00 i 1.417 148.20 i 4.771

*Plasma K 4.46 i .077 5.33 i .285

Plasma protein 6.53 i .120 6.350 t .051
Control group

Plasma Na 142.90 i .945 146.40 i 2.616

Plasma K 4.53 i .059 4.45 i .146

Plasma protein 6.58 i .109 6.38 i .108
*Significant difference, p_< .05.

33

changes in the metabolic variables crucial for maintaining hydromineral
balance in rats maintained on a standard diet (BB) with those main-
tained on the experimental test diet (TD + 1% NaCl). Baseline data
revealed elevated water intakes for those rats maintained on TD over
those with BB, although equal volumes of urine were eliminated.
Ostensibly, the data imply that blood volume was higher in T0 fed
rats. This is reasonable since TD had a higher mineral content

(1.4% potassium, 1.53% chlorine versus .77% potassium, .66% chlorine)
so more water was needed to dilute the metabolites to isotonicity.

The flexibility of urinary sodium loss should be underscored because
the urinary levels of potassium were not raised but that of sodium was
lowered to satisfy homeostasis (Table 1). This drop in urinary sodium
excretion was enough to be reflected in the urinary total solids
measurement, too. Potassium and sodium excretion levels are comple-
mentary processes since renal potassium absorption coincides with
sodium reabsorption (Canong, 1971, p. 525). Therefore, an increased
concentration of potassium might be expected to accompany a decreased
sodium output. Although urinary potassium levels were not signifi-
cantly greater in T0 fed rats, the importance of reduced sodium loss
may rest with the need to excrete potassium (Michell, 1972).

The dynamic picture of sodium exchange is understood best by
examining urinary excretion levels under different conditions of
nutrition. Urinary sodium levels are sensitive to subtle changes in
intake since excretion levels fluctuate in order to maintain internal
constancy. Variations of sorts in the amount of sodium excreted per

day are conveyed in Figure 2. As previously mentioned, baseline

34

levels of sodium were different under diets that differed in their
mineral content.

A most important deviation occurs when sodium is subtracted
from the TD. The rat reSponds quickly by decreasing its sodium output
to "obligatory" (Chew, 1965) amounts. This minute sodium loss is a
consequence of the physical environment and energy metabolism of the
rat. These losses are unavoidable and are responsible for the
eventual deficiency. After ten days of sodium deprivation approxi-
mately .3 to .6 mEq. of sodium loss were associated with increased
saline preference. The major portion of these losses occurred within
the first couple of days, thereafter mineralocorticoid levels evidently
increased enough to facilitate virtually complete renal sodium reten-
tion (Bojesen, 1966; Marusic 8 Mulrow, 1967; Jalowiec G Stricker,
1973). When sodium was returned to the deficient diet sodium excre-
tion levels were quick to return to normal. The urinary sodium excre-
tion pattern that was characteristic of the first sodium deprivation
period was also characteristic of the second. These rats re-
experienced a rapid reduction in sodium excretion, although the first
day's loss was larger this time. By the third day of deprivation
sodium losses were again minimal.

A peculiarity of the second sodium deficiency period was the
introduction of water deprivation that offset the ordinarily low
quantities of urinary sodium loss. Water deprivation was signaled by.
an increase in the amount of sodium excreted. This finding is com-
patible with the results obtained with Walters' (1973) investigation.

This occurrence was probably induced by an increase in osmotic stress

35

as a consequence of an increased food to water intake ratio. The
quantity of food ingested was reduced to approximately two-thirds of
their normal amount, but that of water was reduced to about 50%.

In the interest of preserving intracellular fluid volume the concen-
tration of the extracellular fluid was reduced. 0n subsequent days
of the water deprivation regimen, sodium elimination was reduced to
pre-water deprivation levels. These urine sodium losses remained low
although food intake progressively increased but water intake and
urine volume remained constant. In this instance, osmotic stress was
relieved by an increased potassium output. Increased plasma aldos-
terone concentrations probably prevented sodium elimination (Marusic 8
Mulrow, 1967).

Water deprivation only slightly increased the control group's
urinary sodium output. A comparable description of the regulatory
determinants that underlie the increase has already been stated for
sodium deficient rats. Following this increase in sodium output,
urinary sodium dropped to its lowest level and then began to gradually
increase as the animals adapted to their water deprivation schedule.
This increase is due primarily to the fact that they eat more food.
This increased osmotic stress did not result in higher water intakes
as Hatton and Almli (1967) had found. Water intake remained rela-
tively stable, but the concentrations of sodium and potassium increased
to appease the osmotic load. Apparently, the animals were already
ingesting asymptotic amounts of water during the hourly drinking
period. Hatton and Almli (1967) gave their subjects a .5 hour free

access to water .

36

The experimental group's urine output was significantly higher
than control values throughout the study and was attributed to popu-
lation differences in water exchange and not to experimental manipula-
tion. Water intake was also higher in the experimental group.
Although the two groups were equated on the basis of body weight, this
did not insure equivalence in fluid exchange. However, these differ-
ences were overlooked because similar baseline values of total urinary

sodium excretion were found between the two groups.

Preference tests. Short-term preference tests are usually

 

given only to water deprived rats. This procedure makes it difficult
to attribute preference behavior to taste factors because of the added
motivational dimension of thirst confuses the situation. However, in
this study, a short term preference for salt was established in sodium
deficient rats that were water replete (Figure 3). As soon as these
animals tasted the salt solution their general activity level in-
creased substantially. They drank saline quite readily. They drank
sparingly from the distilled water cylinder to supplement their salt
intakes. Control subjects initially showed interest in the salt
solution, but it subsided after three milliliters had been ingested.
Clearly, sodium deficiency altered the acceptability of saline, as
control animals drank more water than saline whereas experimental
subjects did the converse. The combined intake of water and saline
was hypertonic in sodium deficient rats and isotonic in control rats.
This result is in conflict with Jalowiec's and Stricker's (1973)
results as they observed their adrenalectomized rats to supplement

their saline intakes with enough water to dilute their intake to

37

isotonic levels. This difference may be attributable to the elevated
plasma aldosterone concentrations in dietary sodium depleted rats
potentiating their sodium appetite. As in the case with adrenal-
ectomized rats, sodium deficient rats overcompensate their sodium
losses by ingesting much more than they excreted. The reason for
this overshoot may be caused by learning factors which result from
association of sodium taste with recovery from sodium deficiency as
McCutcheon and Levy (1972) submit.

Water deprivation tended to reverse the emphasis in drinking.
On a percentage basis, experimental animals drank more water than
saline. The cumulative volumetric intake curves (Figure 4) for saline
intake and water intake correspond with those of Nachman and Pfaffmann
(1963). The sodium deficient subjects readily ingested .4 M saline
whereas control subjects drank less. For both concentrations (.4 M
and .3 M) of saline, ingestion increased for approximately the first
twenty minutes, thereafter it tapered off. Decreasing the concen-
tration of salt solution to .3 M (Figure 5) increased the rate of
saline ingestion. Since the animal is motivated by a salt need and a
water need, he initially drinks more saline to satisfy both needs.
As he drinks saline, his salt need becomes satisfied, but if he
continues this course his body fluids will become hypertonic to the
plasma. Therefore, his preference for water remains steadfast.
Osmotic stress and sodium satiation are induced more slowly while
drinking a less concentrated salt solution.

These curves of cumulative volumetric intake produced a

negatively accelerating curve. If the assumption is made that

38

volumetric intake is a linear function of the time spent drinking,
then a cumulative curve will show negative acceleration if either

burst duration decreased and/or interdrink interval increased

(Allison G Castellan, 1970).

EXPERIMENT III

The standard measurement in a two-bottle preference test has
been the amount of solution taken in over a given period of time.
The emphasis in most of these experiments has been on the manipula-

tion of the bodily state and how this affects intake. The purpose
‘ of this experiment is to analyze the immediate intake pattern of
rats deprived of sodium and that of control rats, in particular
their preference intake of distilled water and saline. The purpose
of this approach is to elucidate taste factors in the restorative

process of sodium replenishment.

Procedure

Subjects. Animals were 24 male albino rats of the Holtzman
strain, 90-100 days old at the start of the experiment. They were
housed and maintained under the same conditions as described for the
animals of Experiment 1, except that they were given TD supplemented

with 1 percent NaCl mixture ad libitum instead of BB.

 

Apparatus. Three 100 ml graduated cylinders, fitted with
rubber stoppers and glass drinking spouts, were attached to the
front of each cage with broom clamps. The drinking cylinders were

randomly interchanged among the broom clamps each day. Two cylinders

39

40

were filled with demineralized water and the third remained empty.

A drinkometer was connected to each fluid containing cylinder.

Licks on the drinking cylinders were recorded on cumulative recorders
(moved at a Speed of .33 mm/sec).

Two independent groups of rats were run in squads of size six
because there were only 12 drinkometers available. Subjects were
adapted to TD and divided into two groups by the same manner as
described for the animals of Experiment 11. All animals were given
two preference tests under two conditions of water balance following
the same sequence of dietary regimens as described for the animals
of Experiment I (see Table 1). Individual body weights and fluid
intakes were the only metabolic variables recorded each day for the
duration of the experiment. Body weight was recorded at 0800 hour
and a one hour measurement of fluid intake was recorded over the
ensuing hour with drinkometer-recorder circuit turned on. Although
the animals had 24 hour access to the drinking Spouts except during

water deprivation, fluid intake was only measured during this hour.

Solutions. NaCl solutions were made the same way as in

Experiment 1.

Preference tests. Each rat was given a five second taste

 

sample from a drinking cylinder that contained a .4M NaCl solution
and from one containing distilled water; thereafter, the drinking
spouts were withdrawn. A moveable masonite door, one quarter inch
thick, was placed on the inner front surface of each cage which
separated the three drinking spouts from the rest of the cage. The

drinking cylinders were reattached to the cage, the

41

drinkometer-recorder circuit was turned on, and the masonite doors
were removed to give the animals a one hour access. In the water
replete condition, subjects were not given taste samples prior to
preference testing.

The basic datum that was collected was whether the subject
was drinking or not drinking. Drinking was indicated by an upward
deflection of the pen of an event marker. Licking characteristics
of drinking were beyond the resolution of the cumulative recorder.
The criterion for identifying successive drinking periods was the
shortest distance X traveled by the pen of the event marker such that
two successive contacts with a drinking spout can reasonably be
regarded as members of two different bursts. This distance was

approximately .33 mm.

Results

Interdrink intervals (offset to onset, IDI), and burst dura-
tion (onset to offset, 80) are recorded in Table 5 for salt solution
drinking only. Both 101 and BD were analyzed across the first 10
minutes and the last 50 minutes of the one hour preference test for
both water replete (WR) and water deprived (WD) subjects. As can be
seen, the average interval between drinking bursts increased in the
last 50 minutes when compared to the initial 10 minutes of the test
period. This result occurred under both conditions of water

hydration.

Water replete. All but one subject's average IDI was longer

 

during the last 50 minutes of the drinking period compared to the

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first 10 minutes for both control (sodium deficient plus 1% NaCl) and
experimental (sodium deficient diet) animals. The experimental group
exhibited a significantly lower IDI for the first 10 minutes of
drinking than the corresponding value for the control group (3.: 2.418,
d§_= 10, p_< .05). There were no differences between groups in 101
for the last 50 minutes, however. Because of these differences in
101, a cumulative frequency distribution of IDI is graphed in Figs. 6
through 8. There were proportionately more short 1015 and fewer long
1015 for the experimental group than for the control group. Further-
more, the number of long 101 increased proportionately for both groups
as the drinking period progressed. Statistically, 80 remained constant
across the entire drinking session for both groups (5.: .421, d£_= 3/9,
p_> .20). However, it appears to decrease during the last 50 minutes.

The distribution of time Spent drinking salt solution and
distilled water is graphed in Figs. 9 through 12. It is evident that
experimental subjects Spend more time drinking salt solution than
control subjects. Two distributions of the time Spent drinking were
graphed for each group and were based upon whether the subject started
drinking salt solution or distilled water first. Out of 12 rats, 9
started with the salt solution.

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Table 6. Drinking duration (DD) was defined as the amount of succes-
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rat continued drinking from the same solution. Pauses between bursts

were not included in the computation. When the DD for the first

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58

TABLE 6

Characteristics of Drinking by Water
Replete and Water Deprived Rats

 

Water replete

 

 

 

 

 

 

 

Control Experimental
H20 NaCl H20 NaCl
2.008 3.492 2.933 11.158
.794 1.046 1.157 4.598
3.000 2.833 4.167 11.833
1.936 1.115 1.455 1.113
Water deprived
9.344 3.969 8.163 10.188
2.137 .878 2.343 3.182
15.375 6.000 12.500 14.875
1.645 1.512 1.584 1.518
A = Total time spent drinking (min.).
B = Continuous drinking time (min.).
C = Total fluid intake (m1.).
D = Intake to time spent drinking ratio (ml./min.).

59

encounter of salt solution was compared, it was found that it was
significantly longer for the experimental group over the control

group (£_= 2.454, d£_= 10, p_< .05). The average DD across the entire
drinking session for distilled water and salt solution did not differ
significantly between groups. The total amount of time spent drinking
(£_= 4.177, d£_= 10, p_< .01) salt solution and the total amount of
solution intake (£.= 4.989, df_= 10, p_< .001) was significantly

greater for experimentals than for controls.

Water deprived. The behavioral features of drinking peculiar
to water deprived animals in contrast with those of water replete
animals are a matter of degree and not of substance. That is, IDI
increased proportionately for both groups as the drinking period
prOgressed. For the first 10 minutes of drinking 101 was signifi-
cantly shorter for the experimental group (t_= 3.666, d§_= 14,
p_< .01) but not during the last 50 minutes of the drinking session.
The cumulative frequency distributions of IDI as shown in Figs. 13
through 15 indicate the same pattern of proportional differences
described for water replete subjects, although differences between'
the groups were larger. Similarly, BD remained constant throughout
the drinking period for both groups.

A perusal of the distribution of the time spent drinking
both solutions (Figs. 16-19) but focusing on the time spent with
saline, both groups experienced an initial heightened re5ponse
followed by a steady decline in the time that was spent drinking.
The experimental group was characterized by having a larger initial

response and a slower decline than the control group. Proportionally,

60

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74

the experimental group Spent about 95% of its time drinking during
the initial two minutes of saline ingestion. It was after 14 minutes
of drinking before it declined to less than 10%. The correSponding
percentages for the control group were 44% and declined to less than
10% after six minutes of saline ingestion. This aforementioned
description was less apparent for water replete subjects.

The total amount of time spent drinking (E_= 4.332, d£_= l4,
E.< .001) salt solution and the total amount of salt solution intake
(E_= 4.710, d£_= l4, p_< .001) were significantly greater for sodium
deprived animals than for controls. Unlike the water replete condi-
tion, the average DD for the first encounter (t_= 4.261, d£_= l4,
E.< .001) and for the entire drinking session (£_= 2.381, d£_= l4,
E.< .05) for saline were both significantly longer for experimental

animals than controls.

GENERAL DISCUSSION

Elggd, With an average of only .373 mEq. of sodium elimi-
nated over a ten day period of sodium deficiency, a significant
decrease in serum sodium wasunlikely to be obtained. Serum measure-
ments of sodium, potassium, and protein were nonsignificant and
therefore implied that they were not major conditions eliciting sodium
appetite due to diet deficiency. These results are consistent with
those of Denton (1965) and Jalowiec and Stricker (1973) which suggest
that variations in the concentration of sodium in the plasma does not
determine the appetite for sodium. However, plasma aldesterone
concentrations were significantly above baseline values (Marusic G
Mulrow, 1967). It is possible that mineralocorticoids have a two-
fold function: (1) to prevent severe sodium losses through renal
reabsorption; and perhaps (2) to potentiate the Specific hunger for
salt in conditions of sodium deficiency (Jalowiec G Stricker, 1973).
This last premise must be guarded with skepticism, since adrenalec-
tomized rats can have a sodium appetite without mineralocorticoids.
On the other hand, Wolf and Handal (1966) demonstrated that normal
rats administered desexycorticosterone (DOC) or aldosterone deve10ps

a salt appetite. Jalowiec and Stricker (1973) demonstrated that

75

76

despite smaller sodium deficits dietary sodium deprived rats showed
larger NaCl intakes than adrenalectomized rats.

These serum values were generally lower than those reported
in previous investigations (Jalowiec G Stricker, 1973; Wright, 1973).
Secondly, in order to extend the observations of the first experiment
blood samples were also acquired from 20 day sodium deficient rats.
These were the reasons for doing Experiment II. The results from
data taken after ten days of sodium deficiency parallel those in
Experiment I, but the absolute levels of blood sodium compare
favorably with previous investigations. An extended observation of
rats deprived of salt for 20 days show that blood sodium did not
depart from control values. Blood potassium levels of sodium
deficient animals, on the other hand, was significantly higher
compared to normal controls. Potassium levels after 20 days of salt
deprivation was higher than that after 10 days of deprivation; but
the level after 10 days of deprivation was not different from that of
controls. Mitchell (1972) noticed that sheep with higher concen-
trations of plasma potassium tend to show greater sodium preference.
Furthermore, he argues that the augmented sodium intake in sodium

depleted sheep might be important to combat against hyperkalaemia.

Patterns of intake. Both water replete and water deprived

 

rats showed a monotonic decrease in the proportion of the time spent
drinking saline as they approached satiation. The dynamic aspects of
the behavior underlying this prOportional decrease were that drinking
bursts remained constant and pauses became longer. This is in dis-

agreement with the pattern of intake reported for water drinking

77

(Stellar G Hill, 1952), saccharin drinking (Hulse, 1967), and nutri-
tive drinking (Allison 8 Castellan, 1970) as they also found burst
duration to decrease. A procedural difference to account for this
contradiction is that these aforementioned experiments measured
licking characteristics of drinking. Hence, decreasing lick duration
and increasing interlick intervals would appreciably lower burst
duration and go undetected in the method used here. If volumetric
intake was recorded periodically, the ratio of intake to burst
duration should decrease progressively during the drinking session.

Scrutinizing the temporal patterns of drinking and the cumu-
lative frequency distribution of interdrink intervals for water
replete (Figs. 6-12) and water deprived (Figs. 13-19) rats indicate
that satiation through saline intake is not a stepwise process but
rather a continuous process. As the rat begins to drink in his
sodium deficient condition the probability of drinking saline first
is high. If he chooses saline, initially a large proportion of the
time is spent drinking but progressively this proportion decreases.
The proportion of saline drinking decreased because the probability
of initiating a burst decreased.

The drinking behavior in rats is generally intermittent. In
the preference test situation of this experiment, the rats were
observed to drink in short time intervals. Occasionally they would
also alternate in their drinking by switching from saline to dis-
tilled water and vice versa. Meiselman and Halpern (1973) using
human subjects demonstrated an enhancement of taste intensity by

stimulating the tongue with alternating pulses of saline and water.

' ll]

fir T'
‘1...“

 

‘.

78

This was a reverse adaptation affect, since continuous stimulation
with a single solution generally causes decrements in both human
magnitude estimation and in neural firing rates. The drinking
duration data (Table 6) show that sodium deficient rats drank from
the salt solution cylinder successively for longer periods of time
than normal rats. However, they did not switch to water any more
or less frequently than control animals. It seems unlikely that any
differential adaptation effects would arise between groups due to
alternating fluid drinking. The drinking duration data do imply
that sodium deficient rats adapt more slowly to the salt stimulus
than normal controls.

Halpern and Marowitz (1973) demonstrated in the rat that
short lick-duration stimuli generated a consistent phasic response
in the electrical transcript of a multiunit chorda tympani nerve
re5ponse. Rats drink in a discontinuous fashion because they lick
when they drink from a spout. It is possible that the neural re-
sponse one normally sees when a continuous amount of solution is
placed on the tongue, is actually an accumulation of these short
serial phasic responses (Meiselman G Halpern, 1973). These responses
perhaps summate within drinking bursts and perhaps across bursts if
the interval between them is not too long.

The temporal drinking patterns of sodium deficient rats as
Opposed to those of normal rats reveal differences that may be
explained in terms of an adaptation process. Sodium deficient rats
drink more saline than normals because it takes longer for their

gustatory system to adapt to the salty taste. The neural correlates

‘3
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1

79

of such a process perhaps lie in analyzing the adaptive characteris-
tics of the taste system. Keidel, e£_al, (1961) concluded that adap-
tation is a fundamental principle by which the nervous system accumu-
lates information about the external and internal environments.

After Bradley's (1973) demonstration of blood chemical changes
affecting gustatory neural responses, it seems that the first order

neuron is a good place to start looking for adaptation changes.

 

LI ST OF REFERENCES

 

 

LIST OF REFERENCES

Allison, J. 8 Castellan, N. J. Temporal characteristics of nutritive {5]
drinking in rats and humans. Journal of Comparative and '
Physiological Psychology, 1970, 19, ll6J123.

 

Bare, J. K. The specific hunger for sodium chloride in normal and
adrenalectomized rats. Journal of Comparative and ‘
PhysiolOgical Psychology, 1949, 33, 242-253. ‘1

 

 

 

Bernard, R. A. 8 Nord, S. G. A first-order synaptic relay for taste
fibers in the pontine brain stem of the rat. Brain Research,
1971, 39, 349-356.

 

Bojesen, E. Concentrations of aldesterone and corticosterone in
peripheral plasma of rats. The effects of salt depletion,
salt repletion, potassium loading and intravenous injections
of renin and angiotensin II. European Journal of Steroids,
1966, 1, 145-169.

 

Bradley, R. M. ElectrOphysiological investigations of intravascular
taste using perfused rat tongue. American Journal of

Physiology, 1973, 224, p. 300.

 

Carr, W. J. The effect of adrenalectomy upon the NaCl taste threshold
in the rat. Journal of Comparative and Physiological
Psych010g , 1952j'45, 377-380.

 

Chew, R. M. Water metabolism of mammals. In Physiological mammalOg ,
by Mayer, W. V. G Van Gelder, R. G. New York: Academic
Press, 1965.

 

Denton, D. A. Evolutionary aspects of the emergence of aldesterone
secretion and salt appetite. Physiological Review, 1965,
45, 245-295.

 

Denton, D. A. Salt appetite. Nutrition Abstracts and Reviews,
1969, 32, 1043-1049.

 

80

81

Devenport, L. D. Aversion to a palatable saline solution in rats:
Interactions of physiology and experience. Journal of
Comparative and Physiological Psychology, 1973, 83, 98-105.

 

Falk, J. L. G Herman, T. S. Specific appetite for NaCl without
postingestional repletion. Journal of Comparative and
PhysiolOgical Psychology, 1961, 54, 405-408.

 

 

Fregly, M. J. Specificity of the sodium chloride appetite of
adrenalectomized rats; substitution of lithium chloride for
sodium chloride. American Journal of Physiology, 1958,
125, 645-653.

 

Ganong, W. F. Review of medical physiology, Los Altos, Calif.:
Lange Medical Publications, 1971.

 

Halpern, B. P. Some relationships between electrophysiology and
behavior in taste. In M. R. Kare G O. Maller (Eds.), The
chemical senses and nutrition. Baltimore: Johns Hopkins-
Press, 1967.

 

Halpern, B. P. G Marowitz, L. A. Taste responses to lick duration
stimuli. Brain Research, 1973, 52, 473—478.

 

Harriman, A. E. 8 MacLeod, R. B. Discriminative thresholds for
salt for normal and adrenalectomized rats. American
Journal of Psychology, 1953, 66, 465-471.

 

Hatton, G. I. G Almli, C. R. Learned and unlearned components of
the rat's adaptation to water deprivation. Psychonomic
Science, 1967, 9, (ll).

Hulse, S. H. Licking behavior of rats in relation to saccharin
concentration and shifts in fixed ratio reinforcement.
Journal of Comparative and Physiological Psychology,
1967] 64, 478-484.

Jalowiec, J. E. 6 Stricker, E. M. Restoration of body fluid balance
following acute sodium deficiency in rats. Journal of
Comparative and Physiological Psychology, 1970, 29, 94-102.

 

Jalowiec, J. E. 8 Stricker, E. M. Sodium appetite in adrenalectomized
rats following dietary sodium deprivation. Journal of
Comparative and Physiological Psychology, 1973, 83, 66-77.

 

Keidel, §£_§l, Adaptation: Loss or gain of sensory information?
In W. A. Rosenblith (Ed.), Sensory communication. New York:
MIT Press and John Wiley 6 Sons, 1961.

I I! n—fium “Ohio“...tm -4

I.

82

McCutcheon, B. & Levy, C. Relationship between NaCl rewarded
bar-pressing and duration of sodium deficiency. Physiology
and Behavior, 1972, 8, 761-763.

Meiselman, H. L. G Halpern, B. P. Enhancement of taste intensity
through pulsatile stimulation. Physiology and Behavior,
1973, ll, p. 713.

 

Michell, A. R. A relationship between plasma potassium concentration
and salt appetite. Proceedings of the Physiological Society,
3 June 1972. Journal of Physiology, 1972, 225, 50-51.

 

 

Murusic, E. T. G Mulrow, P. J. Stimulation of aldesterone bio-
synthesis in adrenal mitochondria by sodium depletion.
Journal of Clinical Investigation, 1967, 393 2101-2108.

Nachman, M. Taste preferences for sodium salts by adrenalectomized
rats. Journal of Comparative and Physiological Psychology,
1962, 55, 1124-1129.

Nachman, M. 8 Pfaffmann, C. Gustatory nerve discharges in normal
and sodium deficient rats. Journal of Comparative and
Physiological Psychology, 1963, 52, 1007-1011.

 

Norgren, R. 6 Leonard, C. M. Taste pathways in rat brainstem, Science,

1971, 173, no. 4002, p. 1136.

Pfaffmann, C. 5 Bare, J. K. Gustatory nerve discharges in normal
and adrenalectomized rats. Journal of Comparative and
Physiological Psychology, 1950, 43, 320-324.

 

Pfaffmann, C. The pleasures of sensation. Psychology Review,
1960, 95, 253-268.

 

Pfaffmann, C. The sensory and motivating properties of the sense
of taste. In M. R. Jones (Ed.), Nebraska symposium on
motivation. Lincoln: University of NEbraska Press, 1961,
71-108.

 

Richter, C. P. Salt taste thresholds of normal and adrenalectomized
rats. Endocrinology, 1939, 24, 367-371.

 

Stellar, E. G Hill, J. H. The rat's rate of drinking as a function
of water deprivation. Journal of Comparative and
Physiological Psychology, 1952, 45, 96-102.

 

 

Wagman, W. Sodium chloride deprivation: Development of sodium
chloride as a reinforcement. Science, 1963, 140, 1403-1404.

“‘J

83

Walters, J. K. Prolonged acute thirst: Some neural, physiolOgical,
and behavioral correlates in rats. Unpublished master's
thesis, Michigan State University, 1973.

Wolf, G. & Handal, P. J. Aldosterone induced sodium appetite:
Dose-response and specificity. EndocrinolOgy, 1966,
28, 1120-1124.

 

Wright, J. W. Effects of saline consumption and adrenalectomy
on water balance in rats. Behavioral Biology, 1973, 8,
no. 2.

 

APPENDICES

."

APPENDIX A

APPARATUS

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APPENDIX A

APPARATUS

Description of the drinking_box

 

The drinking box was made up of six individual drinking
compartments. Twelve glass gas collecting tubes were attached to the
drinking box. The tubes were calibrated in 0.2 ml. Each compartment
was 25.5 cm. long, 14 cm. wide, and 14.5 cm. deep. The entire bottom
of the drinking box consisted of hardware cloth and stood 4 cm. from
the floor. Two drinking spouts extended into each compartment by
2.54 cm., these holes were six cm. from the bottom and 2.5 cm.
from the side of the compartment. A small guillotine door was built
into each drinking compartment which could be raised to expose the
spouts of the gas-collecting tubes. An aluminum track permitted
verticle mobility by the masonite door. Each compartment had a
plexiglas top, which was hinged at one end and had a magnetic lock

on the other end in which to secure the compartment.

Equipment, suppliers, and unit prices

 

1. Individual metabolism cage with base, Acme Metal Products,
$45.00.

2. Animal balance, Ohaus, $74.75.

3. Flame photometer #143, Instrumentation Laboratories,
$2390.00.

84

 

3

85

Centrifuge, International Equipment Co., $270.00.
Drinkometer, Grason-Stadler Co., Entire unit - $96.00/ea.
Gas measuring tubes, Arthur H. Thomas, $28.12/cs.

100 m1. graduate cylinders, M.S.U. General Stores, $3.28/ea.

AM.‘.-' "'-
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APPENDIX B

RAW DATA

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88

TABLE 8

Blood Data

 

10 days of sodium deficiency

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Serum Na Serum K Serum protein
Exptl. Control Exptl. Control Exptl. Control
134 138 S 4 6.0 6.45
134 134 5 6 6.2 6.3
128 135 5 4 6.0 6.1
127 125 4 5 6.4 6.6
132 135 4 S 6.2 6.2
132 6 6.2 5.9

10 days of sodium deficiency
Plasma Na Plasma K Plasma protein
Exptl. Control Exptl. Control Exptl. Control
137 143 4.5 4.45 6.1 6.1
143 145 4.2 4.5 6.5 6.8
145 140.5 4.3 4.45 6.7 6.7
146 140 4.55 4.8 6.8 6.5
144 143.5 4.5 4.55 6.8 6.7
146.5 145.5 4.7 4.45 6.3 6.7
20 days of sodium deficiency
Plasma Na Plasma K Plasma protein
Exptl. Control Exptl. Control Exptl. Control
146.5 144 5.4 4.55 6.3 5.9
143.5 156 4.6 4.45 6.3 6.5
167.0 151 5.4 4.9 6.5 6.6
142 139 6.3 4.7 6.3
142 147.5 4.95 3.9 6.3 6.5
141 4.2 6.5

 

89

TABLE 9
Raw Data--Patterns of Intake

 

Water replete--Expt1.

 

 

 

 

 

 

 

 

 

 

Total fluid Total drinking Total drinking
intake (ml) time (min) duration (min)
H20 Saline H20 Salt H20 Saline
5 10 2.05 6.90 1.025 3.45
l 8 .90 6.20 .45 3.10
5 18 3.55 13.65 .50 1.95
7 9 4.90 14.00 .45 1.40
4 15 3.95 14.85 3.95 14.85
3 11 2.25 11.35 .57 2.90

Volumetric intake Interdrink interval Burst duration
per minute (ml/min) (min) (min)

H20 Saline 10 min 50 min 10 min 50 min
2.44 1.45 .25 1.38 .31 .21
1.11 1.29 .11 1.91 .14 .14
1.41 1.32 .30 ..92 .30 .32
1.43 .64 .23 .56 .33 .15
1.01 1.01 .05 .08 .75 .40
1.33 .97 .37 1.92 .31

 

Water replete--Control

Drinking Duration Total fluid intake (ml)

 

First encounter (min)

 

H20 Saline
6.75 2 2
1.70 5 7
4.00 5 3
2.65 2 2
14.85 1 1
6.00 3 2-

 

90

TABLE 9--Continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Total drinking Drinking duration Volumetric intake
time (min) (min) per minute (ml/min)

H20 Saline H20 Saline H20 Saline

1.90 .60 1.90 .60 1.05 3.33

3.15 7.60 .79 1.90 1.59 .92
.85 3.85 .43 1.93 p 5.88 .78
3.10 4.80 .78 .96 0.65 .42
1.05 1.30 .21 .33 .95 .77
2.00 2.80 .67 .57 1.50 .71
Interdrink interval Burst duration Drinking duration
(min) (min) first encounter (min)
10 min 50 min 10 min 50 min saline
.54 2.30 .10 .05 .60
.15 1.25 .17 .17 1.35
.33 3.09 .68 .27 2.05
.88 2.44 .77 .18 1.90
.50 .23 .09 .09 .05
1.07 1.45 .21 .55 1.25
Total fluid Water deprived--Expt1. Drinking duration
intake (m1) Total drinking time (min) (min)

H20 Saline H20 Saline H20 Saline
15 8 6.1 4.3 . 1.22 1.08
15 9 8.2 5.35 1.64 .89
14 17 11.0 9.25 5.50 9.25
14 18 10.2 11.75 3.40 3.92
15 11 8.3 11.05 2.08 3.69

23 5.55 16.00 .62 1.78
6 17 4.4 13.15 .44 1.32
13 16 11.55 10.65 3.85 3.55

 

91

TABLE 9--Continued.

 

 

 

 

 

 

 

 

 

 

Volumetric intake Interdrink interval Burst duration
per minute (ml/min) (min) (min)
H20 Saline 10 min 50 min 10 min 50 min
2.46 1.86 .19 9.90 .23 .26
1.83 1.68 .17 3.88 .35 .15
1.27 1.84 .13 .10 .57 .31
1.37 1.53 .11 2.34 1.11 .57
1.81 1.00 .08 .73 .17 .23
1.44 1.44 .10 .83 .53 .36
1.36 1.29 .14 .90 .60 .34
1.13 1.50 .10 1.19 .37 .22
Drinking duration Water-replete-Control Total drinking
first encounter (min) Total fluid intake (m1) time (min)
Saline H20 Saline H20 Saline
1.90 15 4 6.50 2.15
2.20 17 7 7.70 3.70
9.25 14 5 7.30 3.50
9.30 13 4 7.40 2.90
8.05 16 7 10.85 4.25
6.30 19 7 10.85 3.95
3.40 13 8 13.25 5-60
4.85 16 6 10.90 5.70

 

92

TABLE 9--Continued.

 

Water deprived--Control

 

Drinking duration Volumetric intake per Interdrink intervals

 

 

 

 

 

 

 

 

(min) minute (ml/min) (min)
H20 Saline H20 Saline 10 min 50 min
1.63 .67 2.31 1.86 .95 2.67
1.28 .62 2.21 1.89 2.45 4.98
1.04 .58 1.92 1.43 .60 11.53
1.48 .48 1.76 1.38 .80 8.10
5.43 1.42 1.47 1.65 .55 49.05
2.17 .79 1.75 1.77 2.07 4.98
1.89 0.80 .98 1.43 .67 4.34
2.18 1.43 1.47 1.05 .61 5.55
Burst duration Drinking duration
(min) first encounter (min)

10 min 50 min Saline

.54 .24 1.55

.44 .19 1.10

.34 .07 .30

.21 .55 .05

.62 .41 .30

.25 .69 1.25

.27 .46 .05

.49 2.6

 

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01815144