- Email: [email protected]
Gustatory adaptation as an explanation for dietary-induced sodium appetite
Physiology & Behavior, Vol. 15, pp. 569-576. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A.
Gustatory Adaptation as an Explanation for Dietary-Induced Sodium Appetite R O B E R T J. C O N T R E R A S AND G LEN N I. H A T T O N
Department o f Psychology, Michigan State University, East Lansing MI 48824 (Received 25 November 1974) CONTRERAS, R. J. AND G. I. HATTON. Gustatory adaptation as an explanation for dietary-induced sodium appetite. PHYSIOL. BEHAV. 15(5) 569-576, 1975. - To test the hypothesis that a Na deprived rat takes longer to adapt to a salt stimulus than a normal rat the temporal characteristics of drinking 0.4 M NaC1 and distilled water were investigated. Analysis showed that Na Deprived rats took less time between drinking episodes (interdrink intervals) and drank consecutively for longer periods of time (drinking time) than normal controls. These results were attributed primarily to taste factors because postingestional and thirst influences were at a minimum. Research was also directed at determining the urinary and blood chemical changes associated with dietary sodium deprivation. The levels of sodium in plasma were unchanged because of reduced sodium excretion but the levels of potassium were significantly increased after 20 days of deprivation. Thus, sodium appetite might be important to combat against hyperkaelemia (high plasma potassium) although the appetite develops long before an increase in potassium is detected. Sodium appetite Pattern of intake
Gustatory adaptation Water deprivation Dietary sodium deprivation
THE nature of taste control over sodium intake has been a subject of controversy for almost 40 years. The most prominant theory of this period was proposed by Richter [20]. He suggested that sodium deficiency made the taste receptors more sensitive to salt stimulation. This conclusion arose from the observation that the preference threshold for salt in a two-bottle preference test with water was decreased considerably by sodium deficiency [2]. Richter's notions were challenged by psychophysical [4,8] and electrop.hysiological [ 17,19] experiments, however, as they demonstrated that detection thresholds for NaC1 were the same in adrenalectomized and normal rats. The results from these behavioral and electrophysiological experiments suggest that salt preferences are centrally rather than peripherally mediated. More recently, Bradley [3] showed that the concentration of NaC1 in the blood bathing the taste receptors influences the afferent gustatory input. This has been the only study to substantiate the notion that the changed salt preference in sodium deficiency may be a result of a changed neural response at the receptor level. Even if Richter's theory were correct in asserting that thresholds were lowered, it does not explain why a sodium deficient rat consumes more sodium than a normal rat. An increase in gustatory sensitivity only increases the availability o f sodium to the rat. It does not insure that a rat will consume more salt to meet its physiological needs. The problem is to explain why a Na deprived rat increases its NaC1 intake across a wide range of concentrations [ 1 6 ] . Some gustatory mechanism is needed that permits greater
Preference test
Plasma potassium
than normal intake when the animal's "sodium reservoir" [22] has been depleted. One possibility is a float-valve mechanism which is time dependent like the process of adaptation. The valve shuts off intake once the reservoir has been filled. Thus, if emphasis is shifted from when to start drinking (detection threshold) to when to stop drinking (adaptation) saline, then different experimental questions arise. One logical question is: Will a sodium deficient rat be more resistant to gustatory adaptation than a normal one? The drinking behavior of the rat is characterized as being intermittent because rats drink in bursts, pause, and drink again. According to the adaptation hypothesis, the temporal patterns of intake of Na deprived and normal rats would be different in the following ways: the drinking pattern to NaC1 of a Na deprived rat as compared to a normal rat would consist of longer burst durations, shorter interdrink intervals, longer drinking times, or some combination of the three. Drinking time is a measurement indicating how long the rat stayed with one solution when it has more than one solution to drink from, as in a t w o bottle preference test. Taste explanations are, however, confounded by factors of thirst [7]. Nachman and Pfaffmann [17] and Nachman [15] studied the effects of sodium deprivation on consumption in two-bottle, short-term preference tests. They found that sodium deprivation led to an increase in preference for NaC1 solution over water [17] and other nonsodium salt solutions [15]. The animals of these two experiments were also water deprived. From these experi-
ZThis research was supported by U.S.P.H.S. Research Career Development Award 1K04 GM 22680 from NIGMS and Research Grant NS09140 from NINDS to G.I.H. and by a Biomedical Sciences Support Grant from Michigan State University to R.J.C. We thank James K. Waiters, Stephen R. Overmann, and Thomas L. Kodera for comments and criticisms on an earlier version of the manuscript. 569
570
C O N T R E R A S A N D HATTON
ments it is not clear how much the increase in preference for NaC1 can be attributed to taste factors alone. Therefore, the animals of our experiment were given preference tests under two conditions of hydration: 23 hr water deprived and nondeprived. The purposes of the current study were threefold: (1) to examine NaC1 preference with and without the confounding factor of thirst; (2) to extend the observations of a previous investigation [10] by studying the effects of prolonged sodium deprivation on blood chemistry and urine concentration; and (3) to examine the goodness of fit of the taste adaptation explanation to behavioral data.
always fixed to the cage, one cylinder filled with demineralized water and the other empty. These two cylinders were randomly alternated between the left and fight holes in the front of the metabolism cages to discourage position preferences. The animals were divided equally in n u m b e r and by weight into sodium and control groups. The procedural sequence for this aspect of the experiment is shown in Table 1. TABLE 1 PROCEDURAL SEQUENCE FOR PREFERENCE TESTS Days Conditions
EXPERIMENT 1 Short-term preference tests are used to maximize taste factors and to minimize postingestional influences. These preference tests are usually given to water deprived rats [17, 21, 23]. It was the interest of this experiment to (1) show a salt solution preference in water replete rats that were sodium deprived; (2) compare changes in food and water intake, body weight, urine volume and concentration, in water replete and water deprived rats that were maintained on a sodium deficient diet; and (3) to study the effects of prolonged sodium deprivation on plasma electrolyte levels. Method A n i m a l s . Thirty-six male albino rats of the Holtzman strain, 9 0 - 1 0 0 days old at the start of the experiment, were used. They were individually housed in standard Acme Metal Products metabolism cages and had food and demineralized water present at all times. They were fed a powdered, sodium deficient diet (Nutritional Biochemicals Test Diet). The colony room was kept at 2 2 - 2 5 ° C , with a 1 4 - 1 0 hr light-dark cycle for the duration of the experiment. Of the 36 rats, 12 were used for measures of blood chemistry. The remaining. 24 rats underwent preference tests while water replete and water deprived, and were monitored for changes in food and water intake, body weight, and urine volume and concentration. S o l u t i o n s . All saline solutions were made as molar concentrations with anhydrous NaC1 and distilled water and were kept at room temperature. B l o o d samples. Twelve rats were adapted to the diet (supplemented with 1 percent NaC1, i.e., 1 g NaCI: 99 g diet) for two weeks. Matched on the basis of body weight, the animals were divided into equal-sized sodium deficient and control groups. The NaC1 supplemented to the diet was then terminated for the Na deprived groups. Two blood samples were obtained from each animal by heart puncture (syringes were coated with heparin) I0 and 20 days after subjects were divided into groups. All samples were centrifuged; a sample of plasma was analyzed for protein concentration in a refractometer, and the remainder was frozen for subsequent analysis by flame photometry. The animals were sacrificed following the last blood sample. M e t a b o l i s m . For the remaining 24 animals, daily measures of body weight, food and water intake, and urine volume were recorded for each animal at the same time each day. A daily urine specimen from each animal was saved for future analysis of sodium and potassium concentration by flame photometry and of total solids by refractometry. Two 100 ml calibrated drinking tubes were
1-24 15-24 24 25-29 30-40 35-40 40
Adaptation to Na supplemented diet Sodium deficient diet* Preference test - water replete Recovery, Na supplemented diet Sodium deficient diet* 23.0 hr water deprivation Preference test - water deprived
*Control animals always had diet supplemented with 1 percent NaCI P r e f e r e n c e test - w a t e r replete c o n d i t i o n s . A one hr, two-bottle preference test was given to each rat in his home cage. It was given a choice between 0.4 M NaC1 and distilled water to drink. The metabolism cage was fixed with two 100 ml gas measuring tubes that were calibrated to 0.2 ml and contained drinking solutions. Both spouts were introduced into the cage simultaneously. A 5 sec taste sample was allowed from each solution, after which the drinking spout was quickly withdrawn. After both solutions were tasted the drinking spouts were concurrently re-inserted into the cage and the rat was allowed a one hour access. P r e f e r e n c e test - w a t e r d e p r i v e d c o n d i t i o n s . Animals were adapted to 23 hr water deprivation for 5 days prior to the preference test. Following routine metabolic data collection, the rats were removed from their home cage and put into individual drinking boxes for 1 hr. Water only was available in the drinking boxes and food only was available in the home cages. Each drinking box contained two 100 ml gas measuring tubes, and had Plexiglas covers for observations of drinking, and a movable guillotine door which separated the drinking spouts from the rest of the box. The position of each drinking cylinder was randomized, one bottle contained distilled water and the other remained empty. A two-bottle preference test, similar to the one given under water replete conditions, was given after the 5 day adaptation period in the drinking box. Results B l o o d analysis. The effects of sodium deficiency on three plasma paramaters are presented in Table 2. When rats had been sodium deprived for 10 and 20 days, the concentration of sodium and protein in the plasma were not different from controls. The only change that did occur came after 20 days of sodium deprivation. These Na deprived rats showed a significantly higher concentration of plasma potassium after 20 days in comparison to the values obtained after 10 days of deprivation (t for repeated measures, t = 3.42, d f = 4, p<0.05). Controls, on the other hand, did not show this same increase in the concentration of plasma potassium.
ADAPTATION AND SODIUM APPETITE
571
TABLE 2 THE VALUES OF PLASMA SODIUM, POTASSIUM, AND PROTEIN IN SODIUM DEPRIVED AND CONTROL ANIMALS 10 day Experimental group Plasma Na (mEq/L) 143.00 -+ 1.417 Plasma K* (mEq/L) 4.46 -+ 0.074 Plasma protein (g/100 ml) 6.53 -+ 0.120 Control group Plasma Na (mEq/L) 142.90 -+ 0.945 Plasma K (mEq/L) 4.53 +- 0.056 Plasma protein (g/100 ml) 6.58 +- 0.109
20 day
~_~ 3.2-
¢'~
,~E 2.4
148.20 -+4.771 5.33 -+0.285 6.350 -+ 0.051
1.6"
8
I,IJ J"
146.40 +- 2.616 4.45 -+ 0.146 6.38 +- 0.108
Variations in the amount of sodium excreted per day as a consequence of different conditions of nutrition are conveyed in Fig. 1. The amount of sodium eliminated in the urine decreased rapidly when sodium was removed from the diet. Every sodium deprived animal excreted less sodium than any control animal each day of both sodium deprivation periods (see Phase B and D in Fig. 1). There was a significant increase in urine sodium output for sodium deprived rats (t = 4.6, d f = 22, p < 0 . 0 0 1 ) when water deprivation was introduced. For each animal, sodium output was higher on the first day (Day 35) of water deprivation compared with its previous day's output. The small increase in sodium output for the control group was not significant (t = 0.457, d f = 22, p<0.50). Body weight, food and water intake, and urine volume measurements are recorded in Table 3 for all phases of the experiment. P r e f e r e n c e t e s t - w a t e r r e p l e t e c o n d i t i o n s . For Na deprived animals, regardless of hydration conditions, the mean total intake was significantly higher than control
AVERAGE
B
C
Na
fa 27 DAYS FIG. 1. Mean amounts of sodium excretion in sodium deficient rats (frilled circles) and normal controls (open circles) under conditions of: (A) sodium deficient test diet (plus 1 percent NaCI) baseline; (B) sodium deprivation; (C) recovery; (D) sodium deprivation and, beginning on Day 35, water deprivation. animals (see Fig. 2, all p < 0 . 0 0 1 , two-tailed t test). A preference ratio can be derived from the total amount of NaC1 solution intake divided by the total amount of fluid intake times 100. The preference ratio for sodium deprived rats was 60 % and 38.4% for controls. Sodium deprived animals consumed an amount of sodium equivalent to 4.94 mEq which surpassed their urinary loss by almost 4 mEq. The supplementary intake of water was sufficient to dilute the ingested saline to 1.42 percent. The control group, however, drank 1.29 mEq of sodium and diluted their ingested saline to 0.87 percent which is isotonic with plasma. This concentration difference (1.42 percent versus 0.87 percent) was significant (t = 2.73, d f = I0, p<0.025). Preference test w a t e r d e p r i v e d c o n d i t i o n s . The preference ratios were 45.1 percent for the sodium deprived
TABLE 3 BODY WEIGHT, F O O D AND WATER INTAKE, AND URINE MEASUREMENTS REPRESENTING ALL PHASES OF EXPERIMENT 1
Experimental group Food intake
D
B:
*Significant difference, p<0.05.
Metabolism.
A
VOLUME
Phase A
Phase B
Phase C
Phase D~ *
Phase D2 *
18.45
19.73
20.3
20.0
15.3
42.60
40.77
43.2
41.6
15.1
388.75
418.70
438.70
444.80
426.00
24.8
23.13
27.40
26.70
5.91
18.90
20.3
20.5
19.3
14.7
33.90
33.53
35.7
34.5
15.0
387.75
419.60
440.50
446.9
422.7
18.55
16.73
19.9
7.0
(g) Water intake (ml) Body weight
(g) Urine volume (ml) Control group Food intake
(g) Water intake (ml) Body weight
(g) Urine volume
21.2
*Measurements were divided into prewater deprivation and water deprivation periods.
572
C O N T R E R A S A N D HATTON
~2- Water Replete E v s-
E-NaC~_ _
.
LLI
/
,
~
E-H20
_z Z LIJ
:E
Water Deprived
E_H20
E tu
_z 12Z LLI
:E
/ /
_
i'o
2'o
- .ii _.__.----
3'o
--
~o
--
s'o
s'o
MINUTES
FIG. 2. The cumulative mean intakes of distilled water and 0.4 M NaC1 as a function of time are plotted for rats that were sodium deficient (E-NaCI; E-H20) or normal controls (C-NaCI;C-H20). The top graph represents data collected from rats that were water replete and the bottom graph from rats that were water deprived. group and 16.5 percent for the control group. The sodium deprived group ingested substantial amounts of sodium (3.92 mEq) but their combined total intakes of saline and water were hypotonic (0.73 percent). Controls on the other hand, ingested less sodium (1.38 m E q ) a t weaker dilutional concentrations (0.35 percent). Similar to the water replete condition, this concentration difference (0.73 percent versus 0.35 percent) was significant (t = 3.76, df = I0, p < 0 . 0 1 ) , although the absolute values were much lower.
ment and energy metabolism of the rat [5]. The first five days of both sodium deprivation periods produced similar effects. This indicates that the first deprivation period had no observable consequence on the second and that the aldosterone system controlling sodium reabsorption was functioning optimally. Furthermore, the sodium output observed during the first few days of sodium deprivation was inversely related to the increase in aldosterone biosynthesis reported by Marusic and Mulrow [11]. The only difference between sodium deprivation periods came with the introduction of water deprivation, which triggered a short-rived increase in sodium output. This occurrence was probably induced by an increase in osmotic stress as a result of an increased food to water intake ratio, since on the first day of water deprivation food intake was reduced 33 percent and water intake was reduced 50 percent. Although plasma sodium was maintained at homeostatic levels, the levels of potassium significantly increased above control values. This increase in the concentration of potassium occurred after 20 days of Na deprivation. Thus, the appetite for sodium might be important to combat against hyperkaelemia (high plasma potassium) [14] although increases in saline intake develop long before an increase in potassium is detected. A slaort-term preference for 0.4 M NaC1 was established in sodium deprived rats regardless of whether they were water deprived or water replete. By comparing hydration conditions, it allows one to isolate the stimulatory effects of taste from the stimulatory effects of cellular dehydration and hypovolemia [7]. It was demonstrated that this preference for 0.4 M NaC1 had different behavioral consequences depending on what hydrational state the animals were in. Combining the intakes of water with 0.4 M NaC1, sodium deprived rats that were water replete consumed an overall hypertonic solution (1.42 percent). Placed in a similar situation, adrenalectomized rats were found to supplement their saline intakes with enough water to dilute their total intake to isotonic levels [10]. This concentration difference in total intake between intact and adrenalectomized rats suggests that elevated levels of aldosterone in the blood of the intact rat functions to potentiate sodium appetite [10]. Although the water deprived rat is also sodium deprived, the animal initially drinks water at a faster rate than the 0.4 M saline. After the first 1 5 - 2 0 min of the preference test saline consumption subsides, but water intake remains steadfast for a longer period of time.
Discussion Measurements of plasma sodium and plasma protein of sodium deprived rats were not different from control levels and therefore implied that they were not major factors eliciting sodium appetite. These results are consistent with those of Denton [6], who demonstrated that plasma variation in sodium concentration was not associated with sodium appetite in sheep. Upon examining urine sodium levels u n d e r different conditions of nutrition (see Fig. 1), it is easy to see why variation in plasma sodium was minimized. When sodium was subtracted from the rat's diet, sodium o u t p u t was drastically reduced. It was mainrained at low levels until sodium was returned to the diet. Then it rose to normal levels as abruptly as it dropped when sodium was removed from the diet. The minute sodium losses that did occur during deprivation were unavoidable and were probably a consequence of the physical environ-
EXPERIMENT 2 It was evident from Experiment 1 that sodium deprivation altered the acceptability of 0.4 M NaC1 regardless of whether the animals were water deprived or water replete. When cumulative volumetric intake was examined (see Fig. 2) a negatively accelerating curve was produced. If it is assumed that volumetric intake is a linear function of the time spent drinking, then a cumulative curve will show negative acceleration of either burst duration decreased and/or interdrink interval increased [1]. The approach of this experiment was to compare the temporal characteristics of salt solution drinking in water replete and water deprived rats that were maintained on a sodium-free diet with rats fed a normal diet. In so doing, sodium consumption may be shown to be controlled, in part, by gustatory adaptation.
A D A P T A T I O N AND SODIUM APPETITE
573
Method Animals. Twenty-four male albino rats of the Holtzman strain, 9 0 - 1 0 0 days old at the start of the experiment, were used. They were housed in metabolism cages and maintained under the same conditions as the animals of Experiment 1. Apparatus. Three 100 ml graduated cylinders, fitted with rubber stoppers and glass drinking spouts, were attached to the front of each cage and were randomly interchanged each day. During adaptation two cylinders were filled with demineralized water and the third remained empty, according to the method of Myers and Holman [13]. A drinkometer was connected to each cylinder containing fluid. Licks on the drinking spouts were recorded on a paper tape event marker. Animals were adapted to the test diet and divided into two groups by the same manner as described for the animals of Experiment 1. All animals were given two preference tests under two conditions of water balance following the same sequence of dietary regimens as described for the metabolism animals of Experiment 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 hr and 1 hr measurement of fluid intake was recorded over the ensuing hour with drinkometer-recorder circuit turned on. Although the animals had 24 hr access to the drinking spouts except during water deprivation, fluid intake was only measured during this hour. Solutions. NaC1 solutions were made the same way as in Experiment 1. Preference tests. Preference tests t o o k place in the home cage regardless of whether the rat was water replete or water deprived. A movable masonite door was positioned on the inner front surface of each cage and separated the drinking spouts from the rest of the cage. When the masonite doors were removed the animals were given a one hour free access to 0.4 M NaC1 and distilled water. In the water replete condition, animals were not given taste samples prior to preference testing.
Drinking was indicated by an upward deflection of the pen of an event marker. Two successive contacts with a drinking spout, separated by at least one second, were regarded as members of two different bursts.
Results Total time spent drinking and total amount of fluid intake are recorded in Table 4. The total amount of time spent drinking salt solution and the total amount of salt solution intake were significantly greater for sodium deficient animals under both conditions of hydration (t test, all ps significant by at least p<0.01). Interdrink interval (offset to onset, IDI) and burst duration (onset to offset, BD) are described in Table 5 for salt solution drinking only. Both IDI and BD were analyzed across the first 10 min when absorption is minimal [9,18] and the last 50 min of the 1 hr preference test for both water replete and water deprived animals. F or the first 10 min of drinking IDI was significantly shorter for the two experimental groups (t test: water replete, t = 2.418, d f = 10, p < 0 . 0 5 ; water deprived, t = 3.666, d r = 11, p<0.01). Statistically, BD remained constant across the entire drinking session for both experimental and control groups under both conditions of hydration (water replete: F(3,19) = 0.421, p > 0 . 2 0 ; water deprived: F(3,27) = 1.580, p>0.20). TABLE 4 CHARACTERISTICS OF DRINKING BY WATER REPLETE AND WATER DEPRIVED RATS
H20 Water replete A B Water deprived A B
Control NaC1
Experimental H20 NaC1
2.008 3.000
3.492 2.833
2.933 4.167
11.158 11.833
9.344 15.375
3.969 6.000
8.163 12.500
10.188 14.875
A = Total time spent drinking (min). B = Total fluid intake (ml).
TABLE 5 CHARACTERISTICS OF DRINKING 0.4 M SALINE BY WATER REPLETE AND WATER DEPRIVED RATS Control 10 min Water replete IDI* BDt Water deprived IDI* BDt
50 min
0.575 min 0.335 min
1.790 min 0.219 min
0.218 min 0.364 min
1.128 min 0.251 min
1.087 min 0.395 min
11.399 min 0.373 min
0.125 min 0.492 min
2.483 min 0.304 min
Water replete Control Exptl.
DT~: PR§ * = interdrink interval. t = burst duration.
Experimental 10 min 50 min
1.200 min 51.42%
5.992 min 73.95%
Water deprived Control Expfl. 0.900 min 28.07%
~: = drinking time, 1st encounter. § = preference ratio.
5.656 min 54.33%
574
CONTRERAS AND HATTON
111]
H20 Replete; Na-Dolicient;NaCI 1st
~III]
H20 Replete;Control;NaCI 1st
f--l-H20
i----I-HzO ,,,,+,-+a
,l]iflfi[[jf ,, 1|0-[~
. . . .
H20 Deprived;Na-Deficient;NaCI1st
H20 Deprived;Control; NaCI 1st
I I-H20 iTl-ffq-Nac,
IIIIIE-~
+ " l J I J ~
--
~----lq--H2c~
+'-t;i+i+I+J;rl 2'rl";rl H20 Replete;Na-Deficient;H20 1st
H20 Replete; Control; H20 1st
r--7-H20 ITIT11-NaCI
i i-H20 ffTI-I-I-NaCI
I--
~ 60_z
H20 Deprived;Control;H20 1st
H20 Deprived;Na-Deficient;H20 1st
~-H20 ITIII-I-NaCI
~ - H20 flTIT1-NaCI
A
_z
I~1 40-
Z
<
I
'
'
~'2
'
'
l'a
MINUTES
'""
'
+To
'
I
'
'
w~
'
'
~
'""'
'
~
'
'
'
MINUTES
FIG. 3. The mean proportions of time spent drinking distilled water and 0.4 NaC1 as a function of time are plotted for animals that were sodium deficient or normal controls, and were either water deprived or water replete, and whether they began by drinking 0.4 M NaC1 solution or distilled water first.
ADAPTATION AND SODIUM APPETITE
575
Drinking time (DT) was defined as the amount of successive drinking time from one solution before switching to another. Successive drinking bursts were combined as long as the rat continued drinking from the same solution. Pauses between bursts were not included in the computation. When DT for the first encounter of salt solution was compared, it was found to be significantly longer for experimental animals under both hydration conditions (two-tailed t test: water replete, t = 2.454, dr= 10, p < 0 . 0 5 ; water deprived, t = 4.261, dr= 14, p<0.001). The distribution of time spent drinking salt solution and distilled water is graphed in Fig. 3. Two distributions of time spent drinking were graphed for each group and were based upon whether the rat started drinking salt solution or distilled water first. From a total of 12 water replete rats, 9 started with salt solution. There were 9 water deprived rats out of 16 that did the same. Qualitatively, the a m o u n t of time spent with saline gradually declines during the test period. This decline, generally, was more sudden in controis. To account for the differences in distribution of salt solution drinking, sodium deficient rats drank more often and consecutively for longer stretches of time than controls. DISCUSSION Drinking behavior in rats is intermittent. In the preference test situation of this experiment, they were observed to drink for short time intervals, pause, and either continue drinking from the same solution or switch to the other solution. Any differences in the intermittent pattern of drinking between sodium deficient rats and controls may be attributed to the possibility that sodium deficiency modified the gustatory system of rats and thereby changed their reaction to suprathreshold NaC1 solutions. Previous research was directed at disproving the notion that adrenalectomy and sodium deficiency altered the detection threshold for NaC1 solutions [4, 8, 17, 19]. The data from Experiment 2 suggest that, after sodium deprivation, the resulting change in preference of saline over water is caused by a shift in the adaptation level of the gustatory system. For a sodium deficient rat, it takes more time to adapt to a salt stimulus than it does a normal rat. Both water replete and water deprived rats showed a gradual decline in the proportion of time spent drinking saline during the test hour. The dynamic aspects underlying this proportional decrease were that drinking bursts remained constant and pauses between bursts became longer. This was the general trend for all rats. However, there were certain behavioral features of drinking peculiar
to sodium deficient rats that distinguished them from controls and which support an argument based on differential adaptation. Sodium deprived rats took less time between drinking episodes (IDI) and drank consecutively (DT) for longer durations than controls. It is evident that sodium deficiency altered the acceptability of saline. Most importantly, these results were obtained within the first ten minutes of the drinking period when post-ingestional influences are at a minimum. Meiselman and Halpern [12] using human subjects, demonstrated an enhancement of taste intensity by stimulating the tongue with alternating pulses of saline and water. Since distilled water and saline were available to the animals of this study and they were observed to alternate in their drinking by switching from one solution to another, it could be argued that these rats were alternating solutions in order to enhance the taste quality or intensity of the fluids being consumed. The sodium deprived rats of this study, however, did not switch to water more frequently than controls, and were, therefore, not acting to enhance taste quality or intensity. The mechanism by which dietary deprivation alters the adaptation level of the taste system may be peripheral and/or central in nature. Peripheral mediation of NaC1 preference cannot be disregarded, although the evidence is more supportive to central mediation. Regardless of where it occurs, the difference between homeostasis and the deficient state on some (several) physiological variable(s) determines the degree of NaC1 preference. More NaC1 intake is needed to correct a large deficit than a small deficit. Based on the results from Experiment 2, sodium deprivation alters the acceptability of NaC1. The change in acceptability was attributed primarily to taste factors for two reasons. First, the temporal characteristics of drinking responsible for the increase in NaC1 intake occurred within the first 10 minutes of the preference test. Postingestional influences on drinking were minimal at this time. Secondly, the same results were obtained in animals that were water deprived and nondeprived. Hence, the stimulatory effects of taste were studied in its pure form without the additional motivation of thirst to confuse the situation. There are a n u m b e r of possible physiological variables responsible for determining the adaptation level of the taste system. They have been thoroughly discussed elsewhere [6,10]. Adaptation level would depend upon the concentration of some ion(s) (sodium, potassium) or hormone(s) (aldosterone, angiotensin) within the body (blood, saliva, sodium reservoir). Cessation of NaCI drinking would correspond to a return to homeostasis.
REFERENCES 1. Allison, J. and N. J. CasteUan. Temporal characteristics of nutritive drinking in rats and humans. J. comp. physiol. Psychol. 70: 116-125, 1970. 2. Bare, J. K. The specific hunger for sodium chloride in normal and adrenalectomized white rats. J. comp. physiol. Psychol. 42: 242-253, 1949. 3. Bradley, R. M. Electrophysiological investigation of intravascular taste using perfused rat tongue. Am. J. Physiol. 224: 300-304, 1973. 4. Carr, W. J. The effect of adrenalectomy upon the NaCI taste threshold in the rat. J. comp. physioL Psychol. 45: 377-408, 1952.
5. Chew, R. M. Water metabolism in mammals. In: Physiological Mammalogy, edited by W. V. Mayer and R. G. Van Gelder. New York: Academic Press, 1965, pp. 43-178. 6. Denton, D. A. Evolutionary aspects of the emergence of aldosterone secretion and salt appetite. Physiol. Rev. 45: 245-295, 1965. 7. Emits, T. and D. Corbit. Taste as a dipsogenic stimulus. J. comp. physiot Psychol. 83: 27-31, 1974. 8. Harriman, A. E. and R. B. MacLeod. Discrimination thresholds for salt for normal and adrenalectomized rats. Am. J. Physiol. 66: 465-471, 1953.
576 9. Hatton, G. I. and C. T. Bennett. Satiation of thirst and termination of drinking: Roles of plasma osmolality and absorption. Physiol. Behav. 5: 4 7 9 - 4 8 7 , 1970. 10. Jalowiec, J. E. and E. M. Stricker. Sodium appetite in adrenalectomized rats following dietary sodium deprivation. J. comp. physiol. Psychol. 82: 6 6 - 7 7 , 1973. 11. Marusic, E. F. and P. J. Mulrow. Stimulation of aldosterone biosynthesis in adrenal mitochondria by sodium depletion. J. clin. Invest. 46: 2101-2108, 1967. 12. Meiselman, H. L. and B. P. Halpern. Enhancement of taste intensity through pulsatile stimulation. Physiol. Behav. 11: 7 1 3 - 7 1 6 , 1973. 13. Myers, R. D. and R. B. Holman. A procedure for eliminating position habit in preference-aversion tests for ethanol and other fluids. Psychon. ScL 6: 2 3 5 - 2 3 6 , 1966. 14. Michell, A. R. A relationship between plasma potassium concentration and salt appetite. (Proceedings of the Physiological Society, 3 June 1972)J. Physiol. 225: 5 0 - 5 1 , 1972. 15. Nachman, M. Taste preferences for sodium salts by adrenalectomized rats. J. comp. physiol. Psychol. 55: 1124-1129, 1962. 16. Nachman, M. and L. P. Cole. Role of taste in specific hungers. In: Handbook o f Sensory Physiology, Vol. 1V, Chemical Senses, Part 2, edited by L. Beidler. Berlin: Springer-Verlag, 1971, pp. 337-362.
CONTRERAS AND HATTON 17. Nachman, M. and C. Pfaffmann. Gustatory nerve discharges in normal and sodium-deficient rats. Z comp. physiol. Psychol. 56: 1007-1011, 1963. 18. O'Kelly, L. I., J. L. Falk and D. Flint. Water regulation in the rat: I. Gastrointestinal exchange rates of water and sodium chloride in thirsty animals. J. comp. physioL Psychol. 51: 1 6 - 2 1 , 1958. 19. Pfaffmann, C. and J. K. Bare. Gustatory nerve discharges in normal and adrenalectomized rats. J. comp. physiol. Psychol. 43: 3 2 0 - 3 2 4 , 1950. 20. Richter, C. P. Salt taste thresholds of normal and adrenalectomized rats. Endocrinology 24: 3 6 7 - 3 7 1 , 1939. 21. Smith, D. F., E. M. Stricker and G. R. Morrison. NaCI solution acceptability by sodium-deficient rats. Physiol. Behav. 4: 2 3 4 - 2 4 3 , 1969. 22. Stricker, E. M. and G. Wolf. Blood volume and tonicity in relation to sodium appetite. Z comp. physiol. Psychol. 62: 2 7 5 - 2 7 9 , 1966. 23. Wagman, W. Sodium chloride deprivation: Development of sodium chloride as a reinforcement. Science 140: 1403-1404, 1963.
Gustatory Adaptation as an Explanation for Dietary-Induced Sodium Appetite R O B E R T J. C O N T R E R A S AND G LEN N I. H A T T O N
Department o f Psychology, Michigan State University, East Lansing MI 48824 (Received 25 November 1974) CONTRERAS, R. J. AND G. I. HATTON. Gustatory adaptation as an explanation for dietary-induced sodium appetite. PHYSIOL. BEHAV. 15(5) 569-576, 1975. - To test the hypothesis that a Na deprived rat takes longer to adapt to a salt stimulus than a normal rat the temporal characteristics of drinking 0.4 M NaC1 and distilled water were investigated. Analysis showed that Na Deprived rats took less time between drinking episodes (interdrink intervals) and drank consecutively for longer periods of time (drinking time) than normal controls. These results were attributed primarily to taste factors because postingestional and thirst influences were at a minimum. Research was also directed at determining the urinary and blood chemical changes associated with dietary sodium deprivation. The levels of sodium in plasma were unchanged because of reduced sodium excretion but the levels of potassium were significantly increased after 20 days of deprivation. Thus, sodium appetite might be important to combat against hyperkaelemia (high plasma potassium) although the appetite develops long before an increase in potassium is detected. Sodium appetite Pattern of intake
Gustatory adaptation Water deprivation Dietary sodium deprivation
THE nature of taste control over sodium intake has been a subject of controversy for almost 40 years. The most prominant theory of this period was proposed by Richter [20]. He suggested that sodium deficiency made the taste receptors more sensitive to salt stimulation. This conclusion arose from the observation that the preference threshold for salt in a two-bottle preference test with water was decreased considerably by sodium deficiency [2]. Richter's notions were challenged by psychophysical [4,8] and electrop.hysiological [ 17,19] experiments, however, as they demonstrated that detection thresholds for NaC1 were the same in adrenalectomized and normal rats. The results from these behavioral and electrophysiological experiments suggest that salt preferences are centrally rather than peripherally mediated. More recently, Bradley [3] showed that the concentration of NaC1 in the blood bathing the taste receptors influences the afferent gustatory input. This has been the only study to substantiate the notion that the changed salt preference in sodium deficiency may be a result of a changed neural response at the receptor level. Even if Richter's theory were correct in asserting that thresholds were lowered, it does not explain why a sodium deficient rat consumes more sodium than a normal rat. An increase in gustatory sensitivity only increases the availability o f sodium to the rat. It does not insure that a rat will consume more salt to meet its physiological needs. The problem is to explain why a Na deprived rat increases its NaC1 intake across a wide range of concentrations [ 1 6 ] . Some gustatory mechanism is needed that permits greater
Preference test
Plasma potassium
than normal intake when the animal's "sodium reservoir" [22] has been depleted. One possibility is a float-valve mechanism which is time dependent like the process of adaptation. The valve shuts off intake once the reservoir has been filled. Thus, if emphasis is shifted from when to start drinking (detection threshold) to when to stop drinking (adaptation) saline, then different experimental questions arise. One logical question is: Will a sodium deficient rat be more resistant to gustatory adaptation than a normal one? The drinking behavior of the rat is characterized as being intermittent because rats drink in bursts, pause, and drink again. According to the adaptation hypothesis, the temporal patterns of intake of Na deprived and normal rats would be different in the following ways: the drinking pattern to NaC1 of a Na deprived rat as compared to a normal rat would consist of longer burst durations, shorter interdrink intervals, longer drinking times, or some combination of the three. Drinking time is a measurement indicating how long the rat stayed with one solution when it has more than one solution to drink from, as in a t w o bottle preference test. Taste explanations are, however, confounded by factors of thirst [7]. Nachman and Pfaffmann [17] and Nachman [15] studied the effects of sodium deprivation on consumption in two-bottle, short-term preference tests. They found that sodium deprivation led to an increase in preference for NaC1 solution over water [17] and other nonsodium salt solutions [15]. The animals of these two experiments were also water deprived. From these experi-
ZThis research was supported by U.S.P.H.S. Research Career Development Award 1K04 GM 22680 from NIGMS and Research Grant NS09140 from NINDS to G.I.H. and by a Biomedical Sciences Support Grant from Michigan State University to R.J.C. We thank James K. Waiters, Stephen R. Overmann, and Thomas L. Kodera for comments and criticisms on an earlier version of the manuscript. 569
570
C O N T R E R A S A N D HATTON
ments it is not clear how much the increase in preference for NaC1 can be attributed to taste factors alone. Therefore, the animals of our experiment were given preference tests under two conditions of hydration: 23 hr water deprived and nondeprived. The purposes of the current study were threefold: (1) to examine NaC1 preference with and without the confounding factor of thirst; (2) to extend the observations of a previous investigation [10] by studying the effects of prolonged sodium deprivation on blood chemistry and urine concentration; and (3) to examine the goodness of fit of the taste adaptation explanation to behavioral data.
always fixed to the cage, one cylinder filled with demineralized water and the other empty. These two cylinders were randomly alternated between the left and fight holes in the front of the metabolism cages to discourage position preferences. The animals were divided equally in n u m b e r and by weight into sodium and control groups. The procedural sequence for this aspect of the experiment is shown in Table 1. TABLE 1 PROCEDURAL SEQUENCE FOR PREFERENCE TESTS Days Conditions
EXPERIMENT 1 Short-term preference tests are used to maximize taste factors and to minimize postingestional influences. These preference tests are usually given to water deprived rats [17, 21, 23]. It was the interest of this experiment to (1) show a salt solution preference in water replete rats that were sodium deprived; (2) compare changes in food and water intake, body weight, urine volume and concentration, in water replete and water deprived rats that were maintained on a sodium deficient diet; and (3) to study the effects of prolonged sodium deprivation on plasma electrolyte levels. Method A n i m a l s . Thirty-six male albino rats of the Holtzman strain, 9 0 - 1 0 0 days old at the start of the experiment, were used. They were individually housed in standard Acme Metal Products metabolism cages and had food and demineralized water present at all times. They were fed a powdered, sodium deficient diet (Nutritional Biochemicals Test Diet). The colony room was kept at 2 2 - 2 5 ° C , with a 1 4 - 1 0 hr light-dark cycle for the duration of the experiment. Of the 36 rats, 12 were used for measures of blood chemistry. The remaining. 24 rats underwent preference tests while water replete and water deprived, and were monitored for changes in food and water intake, body weight, and urine volume and concentration. S o l u t i o n s . All saline solutions were made as molar concentrations with anhydrous NaC1 and distilled water and were kept at room temperature. B l o o d samples. Twelve rats were adapted to the diet (supplemented with 1 percent NaC1, i.e., 1 g NaCI: 99 g diet) for two weeks. Matched on the basis of body weight, the animals were divided into equal-sized sodium deficient and control groups. The NaC1 supplemented to the diet was then terminated for the Na deprived groups. Two blood samples were obtained from each animal by heart puncture (syringes were coated with heparin) I0 and 20 days after subjects were divided into groups. All samples were centrifuged; a sample of plasma was analyzed for protein concentration in a refractometer, and the remainder was frozen for subsequent analysis by flame photometry. The animals were sacrificed following the last blood sample. M e t a b o l i s m . For the remaining 24 animals, daily measures of body weight, food and water intake, and urine volume were recorded for each animal at the same time each day. A daily urine specimen from each animal was saved for future analysis of sodium and potassium concentration by flame photometry and of total solids by refractometry. Two 100 ml calibrated drinking tubes were
1-24 15-24 24 25-29 30-40 35-40 40
Adaptation to Na supplemented diet Sodium deficient diet* Preference test - water replete Recovery, Na supplemented diet Sodium deficient diet* 23.0 hr water deprivation Preference test - water deprived
*Control animals always had diet supplemented with 1 percent NaCI P r e f e r e n c e test - w a t e r replete c o n d i t i o n s . A one hr, two-bottle preference test was given to each rat in his home cage. It was given a choice between 0.4 M NaC1 and distilled water to drink. The metabolism cage was fixed with two 100 ml gas measuring tubes that were calibrated to 0.2 ml and contained drinking solutions. Both spouts were introduced into the cage simultaneously. A 5 sec taste sample was allowed from each solution, after which the drinking spout was quickly withdrawn. After both solutions were tasted the drinking spouts were concurrently re-inserted into the cage and the rat was allowed a one hour access. P r e f e r e n c e test - w a t e r d e p r i v e d c o n d i t i o n s . Animals were adapted to 23 hr water deprivation for 5 days prior to the preference test. Following routine metabolic data collection, the rats were removed from their home cage and put into individual drinking boxes for 1 hr. Water only was available in the drinking boxes and food only was available in the home cages. Each drinking box contained two 100 ml gas measuring tubes, and had Plexiglas covers for observations of drinking, and a movable guillotine door which separated the drinking spouts from the rest of the box. The position of each drinking cylinder was randomized, one bottle contained distilled water and the other remained empty. A two-bottle preference test, similar to the one given under water replete conditions, was given after the 5 day adaptation period in the drinking box. Results B l o o d analysis. The effects of sodium deficiency on three plasma paramaters are presented in Table 2. When rats had been sodium deprived for 10 and 20 days, the concentration of sodium and protein in the plasma were not different from controls. The only change that did occur came after 20 days of sodium deprivation. These Na deprived rats showed a significantly higher concentration of plasma potassium after 20 days in comparison to the values obtained after 10 days of deprivation (t for repeated measures, t = 3.42, d f = 4, p<0.05). Controls, on the other hand, did not show this same increase in the concentration of plasma potassium.
ADAPTATION AND SODIUM APPETITE
571
TABLE 2 THE VALUES OF PLASMA SODIUM, POTASSIUM, AND PROTEIN IN SODIUM DEPRIVED AND CONTROL ANIMALS 10 day Experimental group Plasma Na (mEq/L) 143.00 -+ 1.417 Plasma K* (mEq/L) 4.46 -+ 0.074 Plasma protein (g/100 ml) 6.53 -+ 0.120 Control group Plasma Na (mEq/L) 142.90 -+ 0.945 Plasma K (mEq/L) 4.53 +- 0.056 Plasma protein (g/100 ml) 6.58 +- 0.109
20 day
~_~ 3.2-
¢'~
,~E 2.4
148.20 -+4.771 5.33 -+0.285 6.350 -+ 0.051
1.6"
8
I,IJ J"
146.40 +- 2.616 4.45 -+ 0.146 6.38 +- 0.108
Variations in the amount of sodium excreted per day as a consequence of different conditions of nutrition are conveyed in Fig. 1. The amount of sodium eliminated in the urine decreased rapidly when sodium was removed from the diet. Every sodium deprived animal excreted less sodium than any control animal each day of both sodium deprivation periods (see Phase B and D in Fig. 1). There was a significant increase in urine sodium output for sodium deprived rats (t = 4.6, d f = 22, p < 0 . 0 0 1 ) when water deprivation was introduced. For each animal, sodium output was higher on the first day (Day 35) of water deprivation compared with its previous day's output. The small increase in sodium output for the control group was not significant (t = 0.457, d f = 22, p<0.50). Body weight, food and water intake, and urine volume measurements are recorded in Table 3 for all phases of the experiment. P r e f e r e n c e t e s t - w a t e r r e p l e t e c o n d i t i o n s . For Na deprived animals, regardless of hydration conditions, the mean total intake was significantly higher than control
AVERAGE
B
C
Na
fa 27 DAYS FIG. 1. Mean amounts of sodium excretion in sodium deficient rats (frilled circles) and normal controls (open circles) under conditions of: (A) sodium deficient test diet (plus 1 percent NaCI) baseline; (B) sodium deprivation; (C) recovery; (D) sodium deprivation and, beginning on Day 35, water deprivation. animals (see Fig. 2, all p < 0 . 0 0 1 , two-tailed t test). A preference ratio can be derived from the total amount of NaC1 solution intake divided by the total amount of fluid intake times 100. The preference ratio for sodium deprived rats was 60 % and 38.4% for controls. Sodium deprived animals consumed an amount of sodium equivalent to 4.94 mEq which surpassed their urinary loss by almost 4 mEq. The supplementary intake of water was sufficient to dilute the ingested saline to 1.42 percent. The control group, however, drank 1.29 mEq of sodium and diluted their ingested saline to 0.87 percent which is isotonic with plasma. This concentration difference (1.42 percent versus 0.87 percent) was significant (t = 2.73, d f = I0, p<0.025). Preference test w a t e r d e p r i v e d c o n d i t i o n s . The preference ratios were 45.1 percent for the sodium deprived
TABLE 3 BODY WEIGHT, F O O D AND WATER INTAKE, AND URINE MEASUREMENTS REPRESENTING ALL PHASES OF EXPERIMENT 1
Experimental group Food intake
D
B:
*Significant difference, p<0.05.
Metabolism.
A
VOLUME
Phase A
Phase B
Phase C
Phase D~ *
Phase D2 *
18.45
19.73
20.3
20.0
15.3
42.60
40.77
43.2
41.6
15.1
388.75
418.70
438.70
444.80
426.00
24.8
23.13
27.40
26.70
5.91
18.90
20.3
20.5
19.3
14.7
33.90
33.53
35.7
34.5
15.0
387.75
419.60
440.50
446.9
422.7
18.55
16.73
19.9
7.0
(g) Water intake (ml) Body weight
(g) Urine volume (ml) Control group Food intake
(g) Water intake (ml) Body weight
(g) Urine volume
21.2
*Measurements were divided into prewater deprivation and water deprivation periods.
572
C O N T R E R A S A N D HATTON
~2- Water Replete E v s-
E-NaC~_ _
.
LLI
/
,
~
E-H20
_z Z LIJ
:E
Water Deprived
E_H20
E tu
_z 12Z LLI
:E
/ /
_
i'o
2'o
- .ii _.__.----
3'o
--
~o
--
s'o
s'o
MINUTES
FIG. 2. The cumulative mean intakes of distilled water and 0.4 M NaC1 as a function of time are plotted for rats that were sodium deficient (E-NaCI; E-H20) or normal controls (C-NaCI;C-H20). The top graph represents data collected from rats that were water replete and the bottom graph from rats that were water deprived. group and 16.5 percent for the control group. The sodium deprived group ingested substantial amounts of sodium (3.92 mEq) but their combined total intakes of saline and water were hypotonic (0.73 percent). Controls on the other hand, ingested less sodium (1.38 m E q ) a t weaker dilutional concentrations (0.35 percent). Similar to the water replete condition, this concentration difference (0.73 percent versus 0.35 percent) was significant (t = 3.76, df = I0, p < 0 . 0 1 ) , although the absolute values were much lower.
ment and energy metabolism of the rat [5]. The first five days of both sodium deprivation periods produced similar effects. This indicates that the first deprivation period had no observable consequence on the second and that the aldosterone system controlling sodium reabsorption was functioning optimally. Furthermore, the sodium output observed during the first few days of sodium deprivation was inversely related to the increase in aldosterone biosynthesis reported by Marusic and Mulrow [11]. The only difference between sodium deprivation periods came with the introduction of water deprivation, which triggered a short-rived increase in sodium output. This occurrence was probably induced by an increase in osmotic stress as a result of an increased food to water intake ratio, since on the first day of water deprivation food intake was reduced 33 percent and water intake was reduced 50 percent. Although plasma sodium was maintained at homeostatic levels, the levels of potassium significantly increased above control values. This increase in the concentration of potassium occurred after 20 days of Na deprivation. Thus, the appetite for sodium might be important to combat against hyperkaelemia (high plasma potassium) [14] although increases in saline intake develop long before an increase in potassium is detected. A slaort-term preference for 0.4 M NaC1 was established in sodium deprived rats regardless of whether they were water deprived or water replete. By comparing hydration conditions, it allows one to isolate the stimulatory effects of taste from the stimulatory effects of cellular dehydration and hypovolemia [7]. It was demonstrated that this preference for 0.4 M NaC1 had different behavioral consequences depending on what hydrational state the animals were in. Combining the intakes of water with 0.4 M NaC1, sodium deprived rats that were water replete consumed an overall hypertonic solution (1.42 percent). Placed in a similar situation, adrenalectomized rats were found to supplement their saline intakes with enough water to dilute their total intake to isotonic levels [10]. This concentration difference in total intake between intact and adrenalectomized rats suggests that elevated levels of aldosterone in the blood of the intact rat functions to potentiate sodium appetite [10]. Although the water deprived rat is also sodium deprived, the animal initially drinks water at a faster rate than the 0.4 M saline. After the first 1 5 - 2 0 min of the preference test saline consumption subsides, but water intake remains steadfast for a longer period of time.
Discussion Measurements of plasma sodium and plasma protein of sodium deprived rats were not different from control levels and therefore implied that they were not major factors eliciting sodium appetite. These results are consistent with those of Denton [6], who demonstrated that plasma variation in sodium concentration was not associated with sodium appetite in sheep. Upon examining urine sodium levels u n d e r different conditions of nutrition (see Fig. 1), it is easy to see why variation in plasma sodium was minimized. When sodium was subtracted from the rat's diet, sodium o u t p u t was drastically reduced. It was mainrained at low levels until sodium was returned to the diet. Then it rose to normal levels as abruptly as it dropped when sodium was removed from the diet. The minute sodium losses that did occur during deprivation were unavoidable and were probably a consequence of the physical environ-
EXPERIMENT 2 It was evident from Experiment 1 that sodium deprivation altered the acceptability of 0.4 M NaC1 regardless of whether the animals were water deprived or water replete. When cumulative volumetric intake was examined (see Fig. 2) a negatively accelerating curve was produced. If it is assumed that volumetric intake is a linear function of the time spent drinking, then a cumulative curve will show negative acceleration of either burst duration decreased and/or interdrink interval increased [1]. The approach of this experiment was to compare the temporal characteristics of salt solution drinking in water replete and water deprived rats that were maintained on a sodium-free diet with rats fed a normal diet. In so doing, sodium consumption may be shown to be controlled, in part, by gustatory adaptation.
A D A P T A T I O N AND SODIUM APPETITE
573
Method Animals. Twenty-four male albino rats of the Holtzman strain, 9 0 - 1 0 0 days old at the start of the experiment, were used. They were housed in metabolism cages and maintained under the same conditions as the animals of Experiment 1. Apparatus. Three 100 ml graduated cylinders, fitted with rubber stoppers and glass drinking spouts, were attached to the front of each cage and were randomly interchanged each day. During adaptation two cylinders were filled with demineralized water and the third remained empty, according to the method of Myers and Holman [13]. A drinkometer was connected to each cylinder containing fluid. Licks on the drinking spouts were recorded on a paper tape event marker. Animals were adapted to the test diet and divided into two groups by the same manner as described for the animals of Experiment 1. All animals were given two preference tests under two conditions of water balance following the same sequence of dietary regimens as described for the metabolism animals of Experiment 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 hr and 1 hr measurement of fluid intake was recorded over the ensuing hour with drinkometer-recorder circuit turned on. Although the animals had 24 hr access to the drinking spouts except during water deprivation, fluid intake was only measured during this hour. Solutions. NaC1 solutions were made the same way as in Experiment 1. Preference tests. Preference tests t o o k place in the home cage regardless of whether the rat was water replete or water deprived. A movable masonite door was positioned on the inner front surface of each cage and separated the drinking spouts from the rest of the cage. When the masonite doors were removed the animals were given a one hour free access to 0.4 M NaC1 and distilled water. In the water replete condition, animals were not given taste samples prior to preference testing.
Drinking was indicated by an upward deflection of the pen of an event marker. Two successive contacts with a drinking spout, separated by at least one second, were regarded as members of two different bursts.
Results Total time spent drinking and total amount of fluid intake are recorded in Table 4. The total amount of time spent drinking salt solution and the total amount of salt solution intake were significantly greater for sodium deficient animals under both conditions of hydration (t test, all ps significant by at least p<0.01). Interdrink interval (offset to onset, IDI) and burst duration (onset to offset, BD) are described in Table 5 for salt solution drinking only. Both IDI and BD were analyzed across the first 10 min when absorption is minimal [9,18] and the last 50 min of the 1 hr preference test for both water replete and water deprived animals. F or the first 10 min of drinking IDI was significantly shorter for the two experimental groups (t test: water replete, t = 2.418, d f = 10, p < 0 . 0 5 ; water deprived, t = 3.666, d r = 11, p<0.01). Statistically, BD remained constant across the entire drinking session for both experimental and control groups under both conditions of hydration (water replete: F(3,19) = 0.421, p > 0 . 2 0 ; water deprived: F(3,27) = 1.580, p>0.20). TABLE 4 CHARACTERISTICS OF DRINKING BY WATER REPLETE AND WATER DEPRIVED RATS
H20 Water replete A B Water deprived A B
Control NaC1
Experimental H20 NaC1
2.008 3.000
3.492 2.833
2.933 4.167
11.158 11.833
9.344 15.375
3.969 6.000
8.163 12.500
10.188 14.875
A = Total time spent drinking (min). B = Total fluid intake (ml).
TABLE 5 CHARACTERISTICS OF DRINKING 0.4 M SALINE BY WATER REPLETE AND WATER DEPRIVED RATS Control 10 min Water replete IDI* BDt Water deprived IDI* BDt
50 min
0.575 min 0.335 min
1.790 min 0.219 min
0.218 min 0.364 min
1.128 min 0.251 min
1.087 min 0.395 min
11.399 min 0.373 min
0.125 min 0.492 min
2.483 min 0.304 min
Water replete Control Exptl.
DT~: PR§ * = interdrink interval. t = burst duration.
Experimental 10 min 50 min
1.200 min 51.42%
5.992 min 73.95%
Water deprived Control Expfl. 0.900 min 28.07%
~: = drinking time, 1st encounter. § = preference ratio.
5.656 min 54.33%
574
CONTRERAS AND HATTON
111]
H20 Replete; Na-Dolicient;NaCI 1st
~III]
H20 Replete;Control;NaCI 1st
f--l-H20
i----I-HzO ,,,,+,-+a
,l]iflfi[[jf ,, 1|0-[~
. . . .
H20 Deprived;Na-Deficient;NaCI1st
H20 Deprived;Control; NaCI 1st
I I-H20 iTl-ffq-Nac,
IIIIIE-~
+ " l J I J ~
--
~----lq--H2c~
+'-t;i+i+I+J;rl 2'rl";rl H20 Replete;Na-Deficient;H20 1st
H20 Replete; Control; H20 1st
r--7-H20 ITIT11-NaCI
i i-H20 ffTI-I-I-NaCI
I--
~ 60_z
H20 Deprived;Control;H20 1st
H20 Deprived;Na-Deficient;H20 1st
~-H20 ITIII-I-NaCI
~ - H20 flTIT1-NaCI
A
_z
I~1 40-
Z
<
I
'
'
~'2
'
'
l'a
MINUTES
'""
'
+To
'
I
'
'
w~
'
'
~
'""'
'
~
'
'
'
MINUTES
FIG. 3. The mean proportions of time spent drinking distilled water and 0.4 NaC1 as a function of time are plotted for animals that were sodium deficient or normal controls, and were either water deprived or water replete, and whether they began by drinking 0.4 M NaC1 solution or distilled water first.
ADAPTATION AND SODIUM APPETITE
575
Drinking time (DT) was defined as the amount of successive drinking time from one solution before switching to another. Successive drinking bursts were combined as long as the rat continued drinking from the same solution. Pauses between bursts were not included in the computation. When DT for the first encounter of salt solution was compared, it was found to be significantly longer for experimental animals under both hydration conditions (two-tailed t test: water replete, t = 2.454, dr= 10, p < 0 . 0 5 ; water deprived, t = 4.261, dr= 14, p<0.001). The distribution of time spent drinking salt solution and distilled water is graphed in Fig. 3. Two distributions of time spent drinking were graphed for each group and were based upon whether the rat started drinking salt solution or distilled water first. From a total of 12 water replete rats, 9 started with salt solution. There were 9 water deprived rats out of 16 that did the same. Qualitatively, the a m o u n t of time spent with saline gradually declines during the test period. This decline, generally, was more sudden in controis. To account for the differences in distribution of salt solution drinking, sodium deficient rats drank more often and consecutively for longer stretches of time than controls. DISCUSSION Drinking behavior in rats is intermittent. In the preference test situation of this experiment, they were observed to drink for short time intervals, pause, and either continue drinking from the same solution or switch to the other solution. Any differences in the intermittent pattern of drinking between sodium deficient rats and controls may be attributed to the possibility that sodium deficiency modified the gustatory system of rats and thereby changed their reaction to suprathreshold NaC1 solutions. Previous research was directed at disproving the notion that adrenalectomy and sodium deficiency altered the detection threshold for NaC1 solutions [4, 8, 17, 19]. The data from Experiment 2 suggest that, after sodium deprivation, the resulting change in preference of saline over water is caused by a shift in the adaptation level of the gustatory system. For a sodium deficient rat, it takes more time to adapt to a salt stimulus than it does a normal rat. Both water replete and water deprived rats showed a gradual decline in the proportion of time spent drinking saline during the test hour. The dynamic aspects underlying this proportional decrease were that drinking bursts remained constant and pauses between bursts became longer. This was the general trend for all rats. However, there were certain behavioral features of drinking peculiar
to sodium deficient rats that distinguished them from controls and which support an argument based on differential adaptation. Sodium deprived rats took less time between drinking episodes (IDI) and drank consecutively (DT) for longer durations than controls. It is evident that sodium deficiency altered the acceptability of saline. Most importantly, these results were obtained within the first ten minutes of the drinking period when post-ingestional influences are at a minimum. Meiselman and Halpern [12] using human subjects, demonstrated an enhancement of taste intensity by stimulating the tongue with alternating pulses of saline and water. Since distilled water and saline were available to the animals of this study and they were observed to alternate in their drinking by switching from one solution to another, it could be argued that these rats were alternating solutions in order to enhance the taste quality or intensity of the fluids being consumed. The sodium deprived rats of this study, however, did not switch to water more frequently than controls, and were, therefore, not acting to enhance taste quality or intensity. The mechanism by which dietary deprivation alters the adaptation level of the taste system may be peripheral and/or central in nature. Peripheral mediation of NaC1 preference cannot be disregarded, although the evidence is more supportive to central mediation. Regardless of where it occurs, the difference between homeostasis and the deficient state on some (several) physiological variable(s) determines the degree of NaC1 preference. More NaC1 intake is needed to correct a large deficit than a small deficit. Based on the results from Experiment 2, sodium deprivation alters the acceptability of NaC1. The change in acceptability was attributed primarily to taste factors for two reasons. First, the temporal characteristics of drinking responsible for the increase in NaC1 intake occurred within the first 10 minutes of the preference test. Postingestional influences on drinking were minimal at this time. Secondly, the same results were obtained in animals that were water deprived and nondeprived. Hence, the stimulatory effects of taste were studied in its pure form without the additional motivation of thirst to confuse the situation. There are a n u m b e r of possible physiological variables responsible for determining the adaptation level of the taste system. They have been thoroughly discussed elsewhere [6,10]. Adaptation level would depend upon the concentration of some ion(s) (sodium, potassium) or hormone(s) (aldosterone, angiotensin) within the body (blood, saliva, sodium reservoir). Cessation of NaCI drinking would correspond to a return to homeostasis.
REFERENCES 1. Allison, J. and N. J. CasteUan. Temporal characteristics of nutritive drinking in rats and humans. J. comp. physiol. Psychol. 70: 116-125, 1970. 2. Bare, J. K. The specific hunger for sodium chloride in normal and adrenalectomized white rats. J. comp. physiol. Psychol. 42: 242-253, 1949. 3. Bradley, R. M. Electrophysiological investigation of intravascular taste using perfused rat tongue. Am. J. Physiol. 224: 300-304, 1973. 4. Carr, W. J. The effect of adrenalectomy upon the NaCI taste threshold in the rat. J. comp. physioL Psychol. 45: 377-408, 1952.
5. Chew, R. M. Water metabolism in mammals. In: Physiological Mammalogy, edited by W. V. Mayer and R. G. Van Gelder. New York: Academic Press, 1965, pp. 43-178. 6. Denton, D. A. Evolutionary aspects of the emergence of aldosterone secretion and salt appetite. Physiol. Rev. 45: 245-295, 1965. 7. Emits, T. and D. Corbit. Taste as a dipsogenic stimulus. J. comp. physiot Psychol. 83: 27-31, 1974. 8. Harriman, A. E. and R. B. MacLeod. Discrimination thresholds for salt for normal and adrenalectomized rats. Am. J. Physiol. 66: 465-471, 1953.
576 9. Hatton, G. I. and C. T. Bennett. Satiation of thirst and termination of drinking: Roles of plasma osmolality and absorption. Physiol. Behav. 5: 4 7 9 - 4 8 7 , 1970. 10. Jalowiec, J. E. and E. M. Stricker. Sodium appetite in adrenalectomized rats following dietary sodium deprivation. J. comp. physiol. Psychol. 82: 6 6 - 7 7 , 1973. 11. Marusic, E. F. and P. J. Mulrow. Stimulation of aldosterone biosynthesis in adrenal mitochondria by sodium depletion. J. clin. Invest. 46: 2101-2108, 1967. 12. Meiselman, H. L. and B. P. Halpern. Enhancement of taste intensity through pulsatile stimulation. Physiol. Behav. 11: 7 1 3 - 7 1 6 , 1973. 13. Myers, R. D. and R. B. Holman. A procedure for eliminating position habit in preference-aversion tests for ethanol and other fluids. Psychon. ScL 6: 2 3 5 - 2 3 6 , 1966. 14. Michell, A. R. A relationship between plasma potassium concentration and salt appetite. (Proceedings of the Physiological Society, 3 June 1972)J. Physiol. 225: 5 0 - 5 1 , 1972. 15. Nachman, M. Taste preferences for sodium salts by adrenalectomized rats. J. comp. physiol. Psychol. 55: 1124-1129, 1962. 16. Nachman, M. and L. P. Cole. Role of taste in specific hungers. In: Handbook o f Sensory Physiology, Vol. 1V, Chemical Senses, Part 2, edited by L. Beidler. Berlin: Springer-Verlag, 1971, pp. 337-362.
CONTRERAS AND HATTON 17. Nachman, M. and C. Pfaffmann. Gustatory nerve discharges in normal and sodium-deficient rats. Z comp. physiol. Psychol. 56: 1007-1011, 1963. 18. O'Kelly, L. I., J. L. Falk and D. Flint. Water regulation in the rat: I. Gastrointestinal exchange rates of water and sodium chloride in thirsty animals. J. comp. physioL Psychol. 51: 1 6 - 2 1 , 1958. 19. Pfaffmann, C. and J. K. Bare. Gustatory nerve discharges in normal and adrenalectomized rats. J. comp. physiol. Psychol. 43: 3 2 0 - 3 2 4 , 1950. 20. Richter, C. P. Salt taste thresholds of normal and adrenalectomized rats. Endocrinology 24: 3 6 7 - 3 7 1 , 1939. 21. Smith, D. F., E. M. Stricker and G. R. Morrison. NaCI solution acceptability by sodium-deficient rats. Physiol. Behav. 4: 2 3 4 - 2 4 3 , 1969. 22. Stricker, E. M. and G. Wolf. Blood volume and tonicity in relation to sodium appetite. Z comp. physiol. Psychol. 62: 2 7 5 - 2 7 9 , 1966. 23. Wagman, W. Sodium chloride deprivation: Development of sodium chloride as a reinforcement. Science 140: 1403-1404, 1963.