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THE INFLUENCE OF SUBCUTANFOUSLY INJEUTED SODIUM
CHLORIDE SOLUTIONS ON READINESS TO DRINK AND
AMOUNT OF WATER CONSUMED BY ALBINO RATS

By

Larry William Thornton

A THESIS

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

MASTER OF ARTS

Department of Psychology

1966

ACKNOWLEDGMENT

The author expresses appreciation to Dr. Glenn
I. Hatton and Dr. John I. Johnson for their aid in
preparation of this thesis. The author is grateful to

Mrs. Thornton for typing the drafts.

11

ACKNOWLEDGMENT
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES

GENERAL INTRODUCTION

EXPERIMENT I

Method

Subjects
Apparatus
Procedure
Results
Discussion

EXPERIMENT II

Method

Subjects
Apparatus
Procedure
Results
Discussion

TABLE

OF CONTENTS

GENERAL DISCUSSION AND SUMMARY

BIBLIOGRAPHY~

APPENDICES

111

Page
11

iv

vi

LIST OF TABLES

Table Page

F)

Medians and ranges of latency to drink, in
seconds, after injection of saline
SOlutionS O O O O 0 I O O O O O O 8

2. Summary of Analysis of variance (amount
drunk) 0 O O O O O O O O O O O 0 l7

3. Summary of analysis of variance (readiness
to drink) 0 o c 9 c o o o o o 0 o 18

A. Comparison of miliequivalents of NaCl injected
and amount drunk in Wayner's experiment and
the present experiment . . . . . . . 22

5. Summary of analysis of variance (amount
drunk) 9 o . o o o .0 o o o o o o 32

6. Summary of analysis of variance (readiness
to drink) . . . '. . . . . . . . . 32

iv

LIST OF FIGURES
Figures Page

1. Latency to drink of the 2A albino rats
immediately after injection of saline '
solutions for each saline concentration . 9

2. Readiness to drink and amount drunk by
the 2A albino rat after 23 1/2 hour
water deprivation as a function of days
of habituation period and treatment
conditions . . . . . . . . . . ll

3. Percentage change in amount drunk and
readiness to drink of the 2H albino rats .
as a function of concentration of NaCl . 15

u. Latency to drink of 2N albino rats six
hours after injection of saline solutions
for each saline concetration . . . . 26

5. Readiness to drink and amount drunk by 2D
albino rats after 23 1/2 hour water
deprivation as a function of days of
habituation period and as a function of
days on and after treatment conditions . 28

6. Percentage change in amount drunk and
readiness to drink of the 2A albino rats
as a function of concentration of NaCl
(six hour delay) . . . . . . . . 30

APPENDIX A:
APPENDIX B:

APPENDIX C:

LIST OF APPENDICES

Apparatus . . . . .

Raw Data . . . . ,

Individual Comparisons

vi

Page
39
N2
48

GENERAL INTRODUCTION

Essential to a complete understanding of the many
processes of water regulation is knowledge of the factors
leading to the initiation of drinking behavior. Many
investigators have considered changes in blood plasma
osmolarity to be of critical importance for the instiga-
tion of water ingestion as well as for the determination
of the amount of water ingested. Using male hooded rats,
Wayner (196“) found that water consumption varied
directly with the concentration of saline solutions
injected subcutaneously. Corbit (1965) obtained a similar
relationship between amount drunk and concentration of
intravenously infused saline solutions in albino rats.
Also using albino rats, Hatton (1965) has observed changes
in the electrical activity of the lateral hypothalamus
following subcutaneous injections of hypertonic saline
solutions. The latency between injection and electrical
change in hydrated rats was found to be approximately
five minutes. It seems reasonable that this time lapse
is necessary for the occurrence of plasma changes
sufficient to trigger some osmodetcction mechanism. The
electrophysiological changes, then, could be related to
the instigation.of drinking behavior in the intact,

awake animal.

O'Kelly (1963) presented some essential processes
involved in regulatory behavior. The processes and their
definitions are;

(a) "detector" functions, whereby significant

perturbations of relevant system variables

can be discovered and can initiate the
regulatory sequence;

 

(b) selective "orientation and discrimination"
functions that enable the organism to respond
differentially and appropriately to the
environment stimulus possibilities;

(c) "correctional" functions which act in such
a fashion as to restore system steady states,
e.g. motor programs of sucking, chewing,
swallowing, etc., in hunger or thirst; and

(d) "satiety" functions, serving to terminate the
corrective motor activity.

It is possible to hypothesize that some portion
of the lateral hypothalamus serves as one of the structures
underlying the detector function. Supporting data for
this hypothesis, in addition to the electrophysiological
observations mentioned above, are available. Anderson
and McCann (1955) induced drinking in goats with
electrical stimulation of the lateral hypothalamus.
Greer (1955) has shown that electrically stimulating the
lateral hypothalamic area increases drinking in rats.
After removing parts of the lateral hypothalamic area by
lesioning, Montemurro and Stevenson (1957) found that
dehydrated rats did not drink.

In our laboratory, a program of research is directed
towards identifying the neural structures which detect

changes in plasma osmolarity and, thus, participate in the

3

initiation of water ingestion. There are no behavioral
data available which allow direct comparison with the
changes in electrical actiVity which have been observed
following subcutaneous saline injections. It is
necessary to know if the onset of drinking is temporally
correlated with the electrOphysiological changes in the
hypothalamus. The present set of experiments were planned
in part to establish this temporal relationship.

These studies were also designed to provide a partial
answer to the question asked by O'Kelly, page 75;

How well does the vertebrate recognize his
internal state of hydration or dehydration?

Is the rat capable not only of discriminating
between dehydration and satiation, but also discriminating
between different degrees of dehydration? The present
experiments attempt to establish the ability of the rat
to discriminate between different degrees of elevated
plasma osmolarity, using readiness to drink as the index
of discrimination.

Bolles (1962) has defined readiness to drink as the
time lapse between access to water and the initiation of
water ingestion.

Amount of water ingested in a predetermined time
period is the behavioral index used as a post Egg measure
of degree of dehydration. Previous research has demonstrated

this to be a reliable index.

EXPERIMENT I

The variable on which hypothalamic activity and
behavioral activity were compared in this experiment was
the time for the change in plasma osmolarity to be detected
as indicated by readiness to drink. The experiment rests
on two assumptions: (a) that detection is accompanied by
electrical activity in the lateral hypothalamus, and (b)
that detection results in initiation of drinking behavior.

To make the data from this study comparable to
electrOphysiological data, subcutaneous sodium chloride
injections were used to initiate drinking behavior in the
rats. The literature is replete with studies employing I
this technique, e.g. Young, Beyer, and Rickey (1952);
Wayner, Wetrus, and Blonk (1962); and Heyer (1951).

To establish a relationship between degree of
dehydration and readiness to drink and amount drunk, the
rats were injected with different concentrations of
sodium chloride solution. The experiment, then, was a
parametric study of the.initiation and amount of drinking

following injections of different concentrations of saline

solution.
Method
Subjects

Twenty-four male albino rats, obtained from Spartan

Research Animals, were housed in individual cages in a

u

temperature--controlled room, with constant light. The

rats weighed approximately 300 i lOgrams on arrival.

Apparatus
The drinking box with six compartments is described
in the appendix. Six graduated 100 milliliter gas
measuring tubes were used to hold the water and measure
the amount of water consumed by the rat during the drinking
session. A clock, with hundredth second divisions, was
used to measure latencies. A foot pedal switch started
and stopped the clock. Six 1 cubic (one per rat) centimeter
syringes (2N ga.) were used for subcutaneous injections.
An animal restrainer described in the appendix, was used

to hold the rate while giving the salt injections,

Procedure

On the day of arrival, twenty-four rats were assigned
to two treatment groups. The first group received an
ascending series of injections: .87%, A%, 8%, 16% and .87%;
and the other group received a descending series of
injections: .87%, 16%, 8%, us, and .871.

Subjects were adapted to a 23 1/2 hour water
deprivation schedule and to the drinking box. .During this
session, latency to drink after water was made available
and amount of water drunk was recorded. A drinking
response was defined as licking at the spout for three or

more seconds. After all individual rats' readiness to

drink had not systematically increased for five days, the
series of injections was begun.

The rats were placed in the drinking box with water
available for a half hour throughout the adaptation
period and the treatment period. During this period, both
latency to drink and amount drunk were recorded. After
the half hour of drinking on treatment days, the six
animals to be tested were removed from the drinking box,
placed in the restrainer, injected with lcc of salt
solution subcutaneously in the back at the level of the
hind legs, and placed back into the drinking box. The
criterion for subcutaneous placement of the needle was
free movement of the needle between the skin and flesh
of the rat. Time from injection to initiation of drinking
was measured, and Ss were allowed to drink for an
additional 30 minutes, after which, they were removed
from the drinking box, weighed, and placed back into their

home cages. The amount of water consumed after injection

was then recorded.

Results

The readiness to drink data from the treatment
conditions is presented in a histogram for quick meaningful
inspection, and as medians and ranges of latencies to drink
after injections of different concentrations of saline
solutions. The histogram presents the data before

transformations are made in the following statistical

analyses. The latency to drink data for the six
treatment conditions, (first .87% saline injections,

Last .87% saline injection, 16% saline injection, 8%
saline injection, and h% saline injection), are presented
in Figure l.

The medians and ranges of latency to drink, in
seconds, after injection of different concentrations
of saline solutions are presented in Table l.

The overall median latency to drink for all 83
after injections of A%, 8%, and 16%, was 321 seconds.

The data collected from the drinking behavior
during the adaptation stage of the experiment and drinking
behavior after 23 1/2 hour water deprivation throughout
the treatment stage of the experiment are presented in
Figure 2.

A t test was used to determine if the amount drunk
by the 2” rats after 23 1/2 hour deprivation and before
injection of saline solutions on all treatment days was
different from the amount drunk by the same 2A rats after
23 1/2 hour water deprivation and the day after injection
of saline solutions on all treatment days. A t test for
correlated observations was used. A two tailed t test for
correlated observations was used to determine if the amount
of water drunk by the 2A albino rats after injection of
.87% saline solution was different from the amount of
water drunk by the same 2“ albino rats after injection of

4% saline solution.

 

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FIGURE 1

Latency to drink of the 2M albino rats immediately after
injection of saline solutions for each saline concentration.

Number of Subjects

10

10

15

10

15

10

10

V

11111

10

First .87% Saline Concentration

 

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LATENCY T0 DRINK AFTER SALINE INJECTION (IN
SECONDS)

11

FIGURE 2

Readiness to drink and amount drunk by the 2A albino
rats after 23 1/2 hour water deprivation as a function
of days of habituation period and treatment conditions.
The ordinate on the left side of the graph presents
the mean reciprocal latenices to drink. The ordinate
on the right side of the graph presents the mean
mililiters of water drunk. The left half of the
abscissa presents the succeeding days of adaptation.
The right half of the abscissa presents the day of
injection of each concentration of saline solution,
followed by the three days after the day of injection.
The readiness to drink data and amount drunk data are
after 23 1/2 hour water deprivation. The readiness

to drink data and amount drunk data after injections
are presented in another graph (Figure 3). Each data
point is determined from 2“ male albino rats.

12

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In Figure 2 both readiness to drink of the 2A albino
rats and amount of water drunk are plotted as a function of
days.

The data points on Figure 2 show that the amount of
water drunk at normal daily drinking sessions following
injection days is less than the amount of water drunk
during the normal daily drinking session of the injection
day. A t test of the difference between the mean amount
drunk by 2A albino rats after 23 1/2 hour water deprivation
on the treatment day and the mean amount drunk by 2b albino
rats after 23 1/2 hour water deprivation on the day following
the treatment day is significant. (t - 12.13, df - 23,

p < .025).

The mean difference between mean reciprocal latency
to drink after injections of A% and .87% saline solutions
is significant (t - 1.7“, df - 22, p < .05). The means
for amount drunk under these two conditions were not
different. (t - 1.35, df - 23, p < .05).

The sharp decrease in amount drunk on the second
day after 8% injection occurred when the rats had no food.

The data for amount of water drunk after injections
of saline solutions are presented in units of mililiters
of water consumed. The data is transformed by finding
the difference between the amount of water drunk by each
rat after injection of .87% saline solution and the amount
of water drunk by the same rat after injection of A%,

after 8%, and after 16% saline solution. Each difference

1n

was divided by the amount of water drunk by the rat after
injection of .87% saline solution. The resultant member is
called the percent change from control injection.

Using mean percent change from control of 2“ rats,

a two way analysis of variance was used to determine if
concentration effects, order effects, or interaction
effects were reliable. Duncan's test was used for
individual comparisons.

In Figure 3 the water intake and readiness to drink
data for the three injection conditions are plotted as
percentages of physiological saline injected control on
a standardized scale.

Statistical analysis of amount drunk, revealed
that the difference between the means of the ascending and
descending orders was not statistically significant (F 8
1.75, df 8 1/22, p > .05). The means of amount drunk for
the three saline conditions were significantly different
at p < .001 (F a A2.29, df = 2/uu). The interaction of
orders with concentrations was not statistically
significant (F = .A9, df = 2/AA, p > .05).

Duncan's (Winer, 1962) test resulted in the following
information: A% 83 drank less than 8% 83 (p < .05); and
8% Ss drank less than 16% 83 (p < .05).

The data-for readiness to drink after injection of
saline solutions were transformed twice. The reciprocals
of the latencies to drink after injection was found in

order to justify using parametric statistical tests. The

15

FIGURE 3

Percentage change in amount drunk and readiness to drink of
'the 2A albino rats as a function of concentration of NaCl.

One graph is used to present the amount of water drunk by

the 2A albino rats after injection of each saline solution
and readiness to drink by the 2“ rats after injection of each
saline solution. To make the amount drunk and readiness

to drink data comparable, the means of amount drunk (percent
change from control) were divided by the standard deviation
of the pooled data, and the means of readiness to drink
(percent change from control) were divided by the standard
deviation of the pooled data.

The ordinate presents of percent change from control
injection. The abscissa presents the different concentra—
tions of saline solutions injected.

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TABLE 2

Summary of Analysis of Variance
(Amount Drunk).

 

 

 

Between Subjects 23

Order (0) l 15.61 1.75
85 within groups 22 8.92

Within Subjects ”8

Concentration—(C) 2 131.5 A2.29*
O x C 2 1.5“ 0.49
C x 83 within gps. A“ 3.11

 

reciprocal latency to drink for each rat after injection
of .87% saline solution was subtracted from the reciprocal
latency to drink for the same rat after injection of U%,
after 8%, and after 16% saline solution. The difference
for each rat was then divided by the reciprocal latency
to drink for the same rat after injection of .87% saline
solution. The resultant number is called the percent
change from control injection. Using mean percent change
from control for 2H rats, a two way analysis of variance
was used to determine if concentration effects, order
effects, and interaction effects were reliable. Duncan‘s
test was used for individual comparisons.

Statistical analysis of readiness to drink revealed
that the difference between ascending and descending orders
was not statistically significant, (F - 2.20, df = 1/22,

p > .05). Means of readiness to drink for the different

concentrations were likewise not significantly different

18

(F = .68, df = 2/uu, p > .05). The interaction of orders
with concentrations was significant (F 8 3.29, df I 2/AA,

p < .05).

TABLE 3

Summary of Analysis of Variance
(readiness to drink)

Source of

 

 

 

Variation DF MS F
Between 38 23

Order (0) l 9.A0 2.20
Ss within gps 22 H.27

Within 85 “8

Concentration (C) 2 .53 .68
o x c 2 2.57 3.29*
C x 83 within gps AM .78

 

+— 7 *—

Discussion

 

What are the possible causes of delay in time from
injection to initiation of drinking behavior? Latency is
determined by the access of water to the site of injection,
by the ease of transport of solution between plasma and
the site of injection, by the extent of dehydration of
the blood, and by the change in cellular hydration level
in the central neural detector, and by the threshold
characteristics of that detector. Once detection has
occured the effect of the change in osmolarity on be-

havior may be delayed by other factors, such as, knowledge

19

of where water is located, and effectiveness of motor
acts in getting to the water.

Wayner (196A) demonstrated that serum Na in male
hooded rats increased to a maximum 35 minutes after a
subcutaneous injection of 3.2 miliequivalents of 15%
saline solution, while drinking behavior occurred 9
minutes after injections. He also demonstrated that
during the first 15 minutes, after injection of 3.2, 6.5,
and 13 miliequivalents of 15% saline solution, the
increase in serum Na in the rats' blood was similar..
Wayner found that the normal serum Na level in the blood
of the hooded rats was 1A5.9 miliequivalents.

The concentration of 16% (2.7“ miliequivalents)
saline solution in the present experiment is closest to
Wayner's concentration of 3.2 miliequivalents of 15%
saline solution. The median latency to drink after
injection of 16% saline solution was 5 minutes 21 seconds.
Factors which may account for the differences between
Wayner's and the present results are adaptation to drinking
box, handling procedures, lighting, temperature, humidity,
strain differences, and possible site of injection.

The results in the present experiment lead to the
conclusion that the amount of water drunk by albino rats
in the above test situations is a montonically increasing
function of concentration of subcutaneously injected saline
solution. The conclusion is in agreement with other

comparable experiments in the literature.

20

Wayner (1964) demonstrated that male hooded rats
drank increasing amounts of water with increasing
concentrations of subcutaneously injected saline solutions,
when given 4 hours to drink after injections. Table 3,
summarizes the results of the present experiment and
Wayner's results.

Wayner's injections of l and 2 miliequivalents
resulted in greater amounts of water consumed than did
injections of 1.87 and 2.74 miliequivalents in the present
study. The factor which presumably contributed to the
consumatory difference was that Wayner allowed his rats
to drink 3 1/2 hours longer than the rate in the present
experiment.

Although there is no apparent relationship between
the means of readiness to drink.and concentration of saline
solutions, the variability of readiness to drink decreases
with increasing concentration. This conclusion is not in
agreement with Corbit (1965). From his results of 3.66,
2.45, and 1.58 minutes to initiate drinking after
intravenous injections of 5, 10, and 20% saline solutions,
Corbit concluded that latency to drink was inversely
related to the concentration of the injected solution.
Corbit's results are in contrast to the results of the
present experiment, which were 7.29, 5.13, and 5.37 minutes
to drink after subcutaneous injections of 4, 8, and 16%

saline solutions.

21

The differences in experimental procedure and
injection route could explain the differences in results:

1. Corbit injected the saline solution intravenously,
with 1 cc syringe at a rate of 0.2 ml/minute, which
probably resulted in a faster rate of plasma concentration
change than the rate of plasma concentration change after
subcutaneous injection. Corbit‘s rats were tested in their
home cage, whereas, the rats in the present experiment
were tested in a drinking box outside of their home cage.
The above factors could underlie the observation that
Corbit's rats drank in a shorter time than the rats in the
present experiment.

2. Corbit gave injections in an ascending order on
successive days, whereas, in the present experiment, the
latencies came from one group of ascending injections and
another group of descending injections, with a three day
interval between injections. Corbit's procedure of
injecting increasing concentrations of saline solution on
successive days might explain why he obtained decreasing
latencieS-with increasing concentrations.

The final conclusion of the present experiment is
that the two response measures are different. The responses
and their measures, how long rats spend drinking, or amount
drunk, and how long rats take to initiate drinking after
injection of saline solution or latency to drink, were
demonstrated to be different in different stages of the

present experiment. In the habituation stage, total time

22

spent drinking and amount drunk stabilized before time to
approach and initiate drinking. Water intake decreased
significantly the day after a test injection, whereas,
there was no apparent relationship between readiness to
drink and daily drinking sessions after day of injection.
Amount of water drunk increases monotonically with
increases in concentration of injected saline solution,
whereas, the variability of readiness to drink seems to
decrease with increasing concentrations of saline

solutions.
TABLE 4

Comparison of miliequivalents of NaCl injected and amount
drunk in Wayner‘s experiment and the present experiment.

 

 

 

 

Miliequivalents of NaCl injected Milliliters drunk
Present Experiment Wayner Present Experiment Wayner
.15~ 2.29-
.68 3.10 '
l 7.5
1.37 - 6.2
2 10.5
2.74 9.9
4 17.0
6 26.0
8 29.0

 

 

 

EXPERIMENT II

In Experiment I, readiness to drink was measured
immediately after injections of different concentrations
of saline solutions. The results indicated that varying
the concentration of saline solution did not have a
significant effect on readiness to drink. Diffusion
time, and pain might have had an effect, on readiness
to drink, which was differential for the various
concentrations. The present experiment was designed to
control for the above effects on readiness to drink. The
rats were injected as in experiment I and then delayed
for six hours before being given access to water. The
time delay allows the saline to diffuse throughout the
intra and extracellular compartments and the effects of

pain to subside.

Method
Subjects
The 24 rats of Experiment I served as $3 for Experiment
II, with the exception of one which died before Experiment

II uas.begun.

Apparatus
The apparatus from Experiment I was used in
Experiment II.
23

24

Procedure

The 83 were assigned to ascending and descending
conditions in Experiment II such that: one-half of each
group was made up of animals who had been given an
ascending series of injections in the first study; the
other half had received the descending series. It was
after this assignment had been made that one rat,
scheduled to receive a descending series, died, leaving
that group with eleven 85.

Following a period of 46 days on ad lib conditions
after Experiment I, deprivation schedules were instituted
as before. The procedures for habituation for this
second study were similar to those of the first and the
same measurements were taken.

0n treatment days, 85 were removed from their home
cages, allowed to drink for 1/2 hour in the drinking
boxes, during which the food was removed from the home
cages, injected with 0.20 cc of xylocaine and a salt
solution, and returned to their living cages for a delay
period of six hours. Following this delay period, 83
were again placed in the drinking boxes and allowed access
to water for one hour. Treatments were administered
every fourth day. Latency to drink and amount drunk were

measured for each period of access to water.

Statistics and Graphs
The same statistical tests, units, and graph construc—

tion techniques were used in Experiment 11, as in Experiment I.

25

Results

Figure 4 presents latency to drink of the 23 albino
rats for each saline concentration.

In Figure 5, the readiness to drink data and the
water intake data are plotted as a function of days and
treatments.

In Figure 6, the water intake and readiness to
drink data for the three injection conditions are plotted
as percentages of change from the physiological saline
control condition on-a standardized scale. An analysis
of variance on the water intake data showed these means
to differ significantly (F = 29.58, df 8 2/44, p < .05).
Duncan's test reveals that all pairs of means differ
significantly (p < .05).

The difference between the means of the ascending
and descending orders was not statistically significant
(F - .86, df 8 1/22, p > .05). The interaction of orders
with concentrations was not significant (F - 2.63,
df 2'2/4u, p > .05).

Statistical analysis of the readiness to drink
data revealed that the difference ascending and descending
orders was not significant (F - .09, df - 1/22,’p > .05).
The interaction of orders with concentrations was not
significant (F a .18, df - 2/44, p > .05). Means of
readiness to drink for the different concentrations was

significantly differentCF - 3.71, df - 2/44, p < .05).

~~—'—-vV-H_ _

26

FIGURE 4

Latency to drink of 24 albino rats six hours after
injection of saline solutions for each saline concentra-
tion.

Number of Subjects

H
O

U1

0

10

Ul

O

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O

15

10

-15

10

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Z 4 I r'—'—11\\
E 4% Concentration
J—'1 J \—
I l l I l l I l ‘ f

 

0_ .51 1.01 1.51 2.01 2.51_3.01 3.51 4.01 4.51 6.80
.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 +

Latency to Drink (in Seconds)

 

28

FIGURE 5

Readiness to drink and amount drunk by 24 albino rats
after 23 1/2 hour water deprivation as a function of
days of habituation period and as a function of days
on and after treatment conditions. (Before injection)

A

29

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30

FIGURE 6

Percentage change in readiness to drink and amount drunk
of 24 albino rats as a function of concentration of
saline solutions (6 hour delay).

31

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32
TABLE 5

Summary of Analysis of Variance
(Amount drunk)

W

 

 

 

_Source of Variation DF MS F
Between 83 23
Order (0)I l 3.02 .86
Ss within gps. 22 3.52
Withing Se 48
Concentrations (C) 2 11.83 29.58*
0 x C 2 1.05 2.63
C x Ss within gps. 44 .40

TABLE 6

Summary of Analysis of Variance
(Readiness to drink)

W

 

 

 

Source of Variation , DF MS F
Between Ss - 23

Order (07' l 17.11 .09
Ss within groups 22 174.03

Within 85 48 '
Concentrations (C) 2 119.79 3.71*
O x C 2 6.01 .18

C x 88 within groups 44 32.23

 

Duncan's test resulted in the following information:
Ss given 4% injections were less ready to drink than 16%
83 (p < .05). The other pairs of means did not differ

significantly (p > .05).

33

The mean difference between amount drunk after
injection of 4% and .87% saline was significant, (t =
4.04, df - 22, p < .05).

The mean difference between readiness to drink
after injection of 4% and after injection of .87% saline

is significant (t a 4.07, df = 22, p < .05).

Discussion

After delaying the availability of water for six
hours following subcutaneous injections of saline solutions,
the mean amount of water that rats consume increased with
an increase in the concentration of the saline solution.

A similar function was obtained by Wayner (1964).

Given this six hour delay, the mean readiness to
drink after injection of 16% saline solution was greater
than the mean readiness to drink after 4% solution.

Wayner (1964) showed that when l meq. of NaCl was injected,
the mean amount drunk for increasing delays does not
change, but when 2 meq. were injected the mean amount
drunk decreased up to 4 hours, then increased, indicating
that changes in higher concentrations over time might
result in differences after six hours between 4% and 16%.
These injections were closest to those Wayner used.
Actually no prior information is available relating

readiness to drink and delay time following injections.

GENERAL DISCUSSION AND SUMMARY

The relationship between various concentrations of
subcutaneous saline injections and readiness to drink and
amount of water consumed was investigated. 8

In Experiment I, when access to water was given
immediately following injection, amount drunk was an
increasing monotonic function of increasing concentrations
of saline injections. Readiness to drink was constant
across saline injections.

These results were interpreted as indicating that
rats could discriminate degree of dehydration produced by
these means, if amount of water consumed is used as a
measure. Mean readiness to drink as a measure of
discrimination under these conditions did not discriminate
injection conditions, whereas the decreasing variance of
readiness to drink with increasing concentrations indicated
differences.

In Experiment II, readiness to drink and amount
drunk were measured after a six hour delay following
injection and differed somewhat from the findings in
Experiment I. Delay time resulted in a depression of
readiness to drink for 4% NaCl injection, 8 and 16%
injections remained similar in both experiments. These
results are interpreted as evidence that some internal

regulatory mechanism is adequate in the rat to adjust

34

35

injections of low concentrations. This has been shown
to be the case with solutions of low concentrations and
similar delays, when the solutions were administered
intragastrically (Hatton & O'Kelly, 1966). The same
relationship as in Experiment II, that is, these two
injections did not differ on the readiness to drink
measure, but differed in the predicted direction on the
amount drunk measure, was demonstrated.

An indication of a relationship between behavioral
and electrical physiological data was evidenced by the
finding that when conditions of injections were comparable,
the time between injections and drinking approximated the
time between injections and electrical changes in certain
parts of the hypothalamus. Further research is necessary
to establish this as a causal relationship.

Finally, the differences between readiness to drink
and amount of water consumed indicates differences between
two components of drinking behavior: initiation of-

drinking and duration of drinking.

BIBLIOGRAPHY

36

BIBLIOGRAPHY

Belles, R. C. The readiness to eat and drink: The effect
of deprivation conditions. J. Comp. Physiol. Psychol.,
1962, Vol. 55, #2, pp. 230—234.

 

Corbit, J. D. Effect of intravenous sodium chloride on

drinking in the rats. J. Comp. Physiol. Psychol.,
1965. 62. pp. 397-406.

Greer, M. A. Suggestive evidence of a primary "Drinking
center" in the hypothalamus of the rat. Pro. Soc.
Exp. Biol. Med., 1955, 89, pp. 59-62.

Hatton, G. I. Unpublished research, 1965.

Hatton, G. I., and O'Kelly, L. I. Water regulation in the
rat: Consummatory and excretory responses to short-

term osmotic stress. J. Comp. Physiol. Psychol.,
1966,61, pp. 477—479.

Montemurro, D. G., and Stevenson, J. A. F. Adipsia produced
by hypothalamic lesions in the rat. Can. J. Biochem.
and Physiol., 1957, 35, pp. 31-37.

O'Kelly, L. I. The psychophysiology of motivation. Annul
Review of Psychology, 1963, Vol. 14.

Stellar, E., and Hill, J. H. The rat's rate of drinking as
a function of water deprivation. J. Comp. Physiol.
Psychol., 1952, Vol. 45, pp. 96-102.

Wayner, M. J., Wetrus, B., and Blonk, D. Artificial
Thirst, Serum Na, and behavioral implications in
the brooded rat. Psychol. Rpts., 1962, 11: p/ 667.

Wayner, M. J. Effects of intracavotid injections of
hypertonic saline on spinal reflex excitability.
Thirst, The MacMillan Co., New York, 19643

Winer, B. J. Statistical Principles in Experimental Design.
McGraw-Hill Book Co., 1962. .

37

APPENDI CES

38

APPENDIX A

Apparatus

39-

Description of the drinking boxes.

The drinking box was made up of six drinking
compartments. Six glass collecting tubes were attached
to the drinking box. The drinking compartments were
1 3/4 inches above a layer of sawdust. The bottom of
each drinking compartment consisted of 1/2 inch hardware
cloth. Each drinking compartment was 11 3/4 inches long,
5 1/2 inches wide, and 7 3/4 inches deep. The hole through
which the drinking spout extended was 2 1/2 inches from
the bottom of the drinking compartments, and 2 1/4 inches
from the sides of the drinking compartment. The drinking
spout extended one inch into the drinking compartment.
The drinking spout was coupled to the gas collecting tube
by rubber tubing. The tops of the drinking compartments
were hinged to the compartments and were made of plexi-
glass. The gas collecting tubes were 27 inches long.

The tubes were calibrated in .2 mililiters.

Description of the restrainer

The restrainer was a two section wood block. The
top section was hinged to the bottom section. The top
section was shorter than the bottom section. Thick sponges
were glued to the bottom of the top section and to the top
of the bottom section. The rat was placed on the sponge

of the bottom section, and the tOp section was pulled down

no

41

around the rat and secured to the bottom section. A 2 x 2
inch hole in the t0p section permitted access to the rat

for subcutaneous injections.

' APPENDIX B

Raw Data

42

m

Experiment I

Latency to drink after saline injections.

 

 

 

 

Ascending
Rat # .87% 4% 8% 16% .87%
1 2,922 832 337 305 320
2 382 220 1,025 320 595
3 587 284 305 310 315
7 3,079 1,460 1,437 255 800
8 2,676 345 275 400 1,145
9 330 435 300 235 3.600
13 137 451 236 345 1,326
14 292 3,591 1,706 555 19,800
15 2,635 1,237 435 300 504
19 6,176 1,804 307 119 110
20 524 311 365 273 330
21 297 375 294 206 395
Descending

4 690 404 301 315 495
5 2,738 647 1,051 490 2,090
6 1.219 486 463 555 3.855
10 618 279 228 255 5.330
11 1,115 247 341 130 150
12 1,160 496 209 220 215
16 1,308 552, 232 240 306
17 11,708 438 300 270 660
18 1,772 381 433 690 632
22 313 495 232 440 2,775
23 744 258 321 480 400
24 4.350 324 309 175 510

 

43

44

Experiment I

Amount drunk after saline injections.

 

Ascending

 

4% 8% 16% .87%

.87%

Rat

 

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...........
90 7.790 14.170
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868.“. 0.4686680

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Descending

 

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1122111 1 22

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...... .0 O O . O .
21223133323“

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ll
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17
18
22
23
24

 

45

Experiment I

Comparison of amount drunk before injection on treatment
day and amount drunk day after injection for each saline
’ concentration (in mililiters).

 

 

 

 

 

 

 

 

 

2;; = _.
4% 8% 16%

Rat # T Day After T 'Day After T Day After
1 20.8 15.4 18.0 10.2 21.2 14.2
2. 21.8 17.6 21.4 9.8 23.8 15.6
3. 19.2 14.6 15.8 12.4 16.8 15.0
7 18.0 15.4 21.0 18.0 17.0 18.2
8 23.0 15.2 23.2 17.2 18.2 18.0
9 22.0 14.0 20.0 16.9 19.3 19.1

13 22.0 17.4 21.2 5.0 21.6 19.6
14 18.4 14.6 16.4 3.2 21.8 17.8
15 . 26.8 17.8 25.0 20.4 26.8 24.2
19 22.0 20.0 24.0 19.0 19.2 16.6
20 18.4 16.6 13.6 15.8 20.0 15.0
21 25.0 17.0 18.2 13.0 18.6 16.2
4 23.0 14.0 18.0 11.2 17.2 13.6
5 19.5 14.6 16.0 10.2 19.6 13.6
6 24.4 21.0 22.0 14.8 22.2 20.4
10 20.2 14.0 22.6 21.0 19.2 19.4
11 16.2 11.6 22.0 18.6 15.0 16.0
12 20.0 13.8 18.0 17.0 ' 15.2 15.0
16 218.2 15.6 19.0 2.0 20.8 19.4
17 21.6 18.0 19.6 4.6 23.0 21.0
18 18.2 19.0 16.8 3.2 23.2 19.6
22 22.6 18.2 21.6 19.0 21.0 14.2
23 22.4 18.8 21.2 17.0 23.2 21.6
24 26.6 20.0 24.4 16.8 23. 19.2

 

!
4

46

Experiment II (6 hour delay)

Latency to drink after saline injections

M

 

 

 

 

Ascending
Rat # .87% 4% 8% 16% .87%
1 31.201 .20 .65 .60 2.40
2 .60 .60 .50 .50 1.40
3 70.20 38.50 5.00 .45 6.90
7 1.00 .50 .70 .55 .85
8 1.05 .60 1.40 .60- 14.85
9 .70 .40 .75 .55 11.45
13 10.40 24.85 .55 .50 29.00
15 9.90. 24.70 1.50 1.40 2.80
19 2.20' .55 .50. .50 1.10
20 12.30 .70 .50 .55 4.00
21 .80. 6.80 .45 .65 9.20
Descending

4 1.50 .70, .55 .70 27.15
5 1.10 .40 .30‘ .60 2.25
6 15.60 .40 .40_ .50 .75
10 1.20 .35 .50 .50 .75
11 .55' .40 .45 .501 .90
16 11.90- .90 .40 .65 .80
17 13.20 .55 .40 .60 .80
18 .70 .60 .50 .70' .80
23 095 .40 050 .40 1.80
24 095 065 .50 090 17.90

 

47

Experiment II

Amount drunk after saline injections,

Ascending

 

4% 8% 16% .87%

.87%

Rat #

 

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...........

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1 11 1112.1.

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Descending

 

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............

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11

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............

 

APPENDIX C

Individual Comparisons

48

Experiment I

Latency to drink after saline injections in quartiles
(in secondS).

W

 

Rat # 4% Rat # 8% Eat # 16%
2 220 12 209 19 119
11 247 10 228 11 130
23 258 16 232 24 175
10 279 22 232 21 206
3 284 13 236 12 220
20 311 8 275 7 235
24 324 21 294 16 240
8 345 9 300 7 255
21 375 17 300 10 255
18 381 4 301 17 270
4 404 3 305 20 273
9 435 19 307 15 300
17 438 24 309 1 305
13 451 23 321 3 310
6 486 1 337 4 315
22 495 11 341 2 320
12 496 20 365 13 245
16 552 18 433 8 400
5' 647 15 435 22 440
1 832 6 463 23 480
15 1237 2 1025 5 490
7 1460 5 1051 14 555
9 1804 7 1437 6 555
14 3591 14 1706 18 690

 

49

Experiment II (6 hour delay)

Latency to drink after saline selections in quartiles
(in seconds).

 

 

_ Rat # 4% Rat # 81 Rat # 16%
1 .30. 5 .30 23 .40
10 .35- 6 .40 3 .45
12 .35 16 .40 2 .50
9 .40 17 .40 19 .50
5 .40 11 .45 6 .50
6 .40 21 .45 10 .50
11 .40 2 .50 11 .50
23 .40 19 .50 13 .50
7 .50 20 .50 7 .55
19 .55- 4 .50 9 .55
17 .55 10 .50, 20 .55
22 .55 . 18 .50 22 .55
2 .60 23 .50 1 .60
8 .60 12 .50 8 .60
18 .60 13 .55 5 .60
24 .65 22 .55 12 .60
20 .70 24 .55 17 .60
4 .70 1 .65 21 .65
16 .90 7 .70 16 .65
21 6.80 9 .75- 4 .70
15 24.70 8 1.40 18 .70
13 24.85- 15' 1.50. 24 .90
3 38.50 3 5.00 15 1.40

 

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