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Feedback control of thirst in rats
Physiology and Behavior, Vol. 8, pp. 1005-101 I, Brain Research Publications Inc., 1972. Printed in Great Britain.
Feedback Control of Thirst in Rats' ALLEN FEIDER 2
Department of Psychology, University qf New Brunswick, Fredericton, New Brunswick, Canada (Received 15 December 1971) FEIDER, A. Feedback controlof thirst in rats. PHYSIOL.BEHAV.8 (6) 1005-1011, 1972.--To compare the effects of stomach distention and fluid concentration changes on thirst, different solutions were injected into the stomachs of water deprived rats. Rapid injections of large volumes of isotonic saline, up to 15 cma in 1 rain, were given to produce stomach distention, but had no effect on drinking. Intragastric injections of water decreased drinking within one to four min and injections of hypertonic saline increased drinking. Bar pressing for water on a continuous reinforcement schedule produced results similar to those for drinking, except that there was a brief, small decrease in bar pressing immediately following injections of large volumes. Fluid concentration changes, but not stomach distention, seem to be an important factor in the negative feedback control of thirst in the rat. Thirst
Intragastric loads
Stomach distention
Hypertonic loads
STOMACH distention has been described as a negative feedback factor controlling thirst in rats, but this conclusion is open to question. In some studies which seem to show that stomach distention reduces thirst, water was placed directly into the stomach at a slow rate which approximated the rate of normal drinking, 1-2 cmS/min [22], and in other studies water was quickly placed into the stomach, but animals were allowed a recovery period before experimental observations started [25]. In both cases it is likely that possible cues from stomach distention were confounded with changes produced by water absorption and cellular hydration which occur soon after water has been ingested [24, 28]. In other studies which seem to show that stomach distention reduces thirst, experimental observations were obtained while rats' stomachs were distended by mechanical means or by rapid injections of large volumes of fluid [1, 3]. Under these conditions however, since water is normally drunk at a slow rate and is rapidly cleared from the stomach, the stomach is abnormally distended and the cues from greater than normal distention may be aversive [13, 21]. The following experiments are an attempt to separate the effects of stomach distention from the effects of water concentration changes in the reduction of thirst as measured both by drinking and by bar pressing for water. Stomach distention was manipulated by injecting different volumes of fluids into the stomachs of rats at varying rates of injection. Fluid concentration changes were controlled by varying the concentration of sodium chloride in the injected fluids. EXPERIMENT 1
Method Animals. The animals were 62 male albino rats of the Sprague-Dawley strain, experimentally naive and 120-150
Bar pressing behavior
days old at the start of the experiment. A chronic intragastric (IG) tube [10] was placed in each animal one week prior to the start of the experiment using combined sodium pentobarbital and ether anesthesia. The rats were housed in individual cages with Purina chow constantly available throughout the experiment, but with water absent for the 22 hr immediately preceding each experimental session. Apparatus. The two Skinner boxes used delivered approximately 0.05 cm s of water for each reinforced response. The end walls of two other Skinner boxes were replaced with modified panels which carried a house light, and which had a 1.3 cm hole through which animals could reach to lick at a glass drinking tube connected to a drinkometer. Sage syringe pumps and Sage swivel joints were used for I G injections, which took place in open boxes, 43 × 43 × 45.5 cm high. Relay operated programming and recording equipment was located in a third room, separate from the room containing the Skinner boxes and the room containing the IG injection equipment. Preliminary training. The rats were randomly divided into two groups of 31, an instrumental response (IR) group and a consummatory response (CR) group. Animals in the IR group were trained to bar press for water in daily 18 min sessions. After two sessions on a continuous reinforcement schedule, IR animals were trained for 10 sessions on a variable interval one-min (VI-I) schedule of reinforcement. Animals in the CR group were placed into the modified Skinner boxes and allowed to drink for seven daily 18 min sessions. Just before each of the last four sessions of preliminary training both CR and IR animals were placed into the large open injection boxes for 12 min where their I G tubes were connected to the injection pumps but no fluid was injected. This sham injection, or 0 cm s injection, was performed by turning the injection pumps on with the syringe
1This research was supported by National Research Council of Canada Grant APA-255. The author expresses his appreciation to Anne Amys, Mary Ann Linseman, and Margaret MacLean for their assistance. 2The author can provide, upon request, a table giving the summary tables for the analyses of variance performed on group data in these studies. 1005
1006
I FIDFR
drive carriage missing from the pumps. After alternate sessions throughout preliminary training an I G injection of approximately 2 cm ~ of water was given to each animal, to prevent blocking of the I G tubes. Throughout the experiment, 4 animals, 2 CR and 2 I R were observed at the same time. Preoperative observations. To make up the six betweenanimals treatment groups described in Table 1 three subgroups were formed within the CR group and within the IR group. Animals were randomly assigned to subgroups with the restriction that at least I0 animals were in each subgroup. Before each daily session, the water deprived animals were placed in the large open boxes for a 12 rain injection period; during this time animals from the different subgroups were injected with either water, 0 . 8 5 ~ NaCI, or 2 . 0 ~ NaCI. Three sets of the 16 possible volume-load time treatment combinations described in Table 1 were administered to each animal, with four daily sessions of 0 c m s injection intervening between successive sets of treatments. Within each set of treatments, the order of treatments was random with the restriction that one of each group of four treatments was a 0 cm 3 injection. When a load time was 1, 3, or 6 rain injections occurred at the end of the 12 min injection period. Immediately after the injection period, animals were placed into the experimental chambers, and allowed to drink for 18 min (CR animals), or allowed to bar press for water on a VI-I schedule of reinforcement for 18 min (IR animals). Records of bar presses and licks were obtained for each successive three rain period of the 18 min sessions. All preoperative treatments were completed by 51 animals, at least eight animals in each of the six between-anhnals treatment groups. Vagotomies. Partial vagotomies were performed on all but four animals, who were exposed to a sham vagotomy. Surgery was done using combined sodium pentobarbital and ether anesthesia, and vagotomies consisted of stripping and removing the vagus nerves from approximately 2 cm of the esophagus next to the stomach. These vagotomies were performed in an attempt to eliminate cues from stomach distention and cues from osmoreceptors in the stomach as possible feedback factors controlling thirst during postoperative observations.
Postoperative observations. After live days recovery from the operations, all animals were retrained for four days with 0 cm 3 injection treatments. Thirty-six animals completed all postoperative observations, which were lhe same as preoperative observations. Post-mortem examinations. Inspection indicated that IG tubes ended in the stomachs of all but a few animals, for whom the tubes ended in the esophagus several mm short of the stomach. The effectiveness of the partial vagotomies was determined from I0~ paraffin sections of the esophagus, alternately stained with Mallory's Collagen Stain [7] and Weil's Myelin Stain [19]. The vagus nerves were clearly absent for 17 animals. Results and Discussion Preoperative observations. Where necessary, animals were randomly excluded from the analysis of preoperative data to form six groups of eight animals each. F o r the purposes of analysis, baseline responding for each animal for each set of 16 treatments was defined as the average total response observed for the four 0 cm 3 injection sessions for that animal. All response measures for each set of 16 treatments were then expressed as percentages of this total session baseline. Similarly, the data points in all the figures are plotted as percentages of this same total session baseline. Figures 1, 2, and 3 show that drinking was not decreased by stomach distention produced by I G loads of isotonic saline. Even when 15 cm ~ of isotonic saline was injected into the stomach in the minute immediately preceding the drinking session, distention had no effect (Fig. 2). A further indication that stomach distention did not affect drinking is that changing the rates of injections had no effect on drinking, no matter what solution was injected. In contrast, Figs. 3 and 4 show that injections of water immediately decreased drinking, so that drinking was reduced within four minutes of the placement of water in the stomach. Injections of hypertonic saline had the opposite effect, producing increased drinking. Since animals with no injections were drinking as rapidly as possible at the beginning of the sessions, the increase due to hypertonic saline was apparent only later in the sessions (Figs. 3
TABLE I DESIGN OF EXPERIMENT 1
Values of variable
Variables Between animals variables Response meamlre
Consummatory response, instrumental respon~
Solution
~5 ,,~ Water, n..... /,, NaCI, 2.0% NaCI
Within animals variables Volume of intragastric load
0, 5, 10, 15 cm'a(cc.)
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and 4). All of the differences described in reference to Figs. 1, 2, 3, and 4 were confirmed by a six way analysis of variance of the preoperative data. The results concerning bar pressing for water on a VI-1 schedule of reinforcement were different from the results for drinking (Figs. 1 and 2), but the consideration of these differences is deferred to Experiment 3. Postoperative observations. Using the 0.01 level of confidence, individual analyses of variance for each animal showed that postoperative drinking and bar pressing for water were the same as preoperative lesponding, even for those cases in which the vagotomies were confirmed histologically. This result is a further indication that distention of the stomach is not an important factor in reducing thirst in rats, since distention cues are carried primarily in the vagus nerves [15, 23, 29, 30]. The failure of the vagotomies to affect behavior is also an indication that vagal afferents from osmoreceptors in the stomach [30] are not necessary for the effects observed. EXPERIMENT 2 The results of Experiment 1 could have been obtained even if stomach distention normally inhibits drinking. Since different concentrations of NaC1 were given to different
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FIG. 2. Drinking (CR subjects) and bar pressing for water (IR subjects) after different volumes of intragastric loads of isotonic saline injected in one minute (standard errors of the means are indicated for IR data).
groups of animals, it is possible that stomach distention cues were consistently paired with particular changes in thirst, and that these changes in thirst became conditioned to distention cues. If this did happen, the normal inhibition of thirst by distention would no longer be apparent. The purpose of Experiment 2 was to eliminate this possibility by giving different concentrations of NaCI to the same subjects.
Method Animals. The animals were five male albino rats of the Sprague-Dawley strain, experimentally naive and 120-150 days old at the start of the experiment. A chronic I G tube was placed in each animal approximately one week prior to the start of the experiment. The rats were housed in individual cages with Purina chow constantly available throughout the experiment, but with water absent for 21 hr before each experimental session. Apparatus. The apparatus used for C R animals in Experiment 1 was used in this experiment. Procedure. The details o f the procedure were the same as those for C R animals in Experiment I, except as indicated below. After pretraining, each animal was observed for four experimental sessions and two control sessions per week for three successive weeks. Animals were allowed water ad lib on the remaining day each week. Experimental sessions were preceded by I G loads of 10 em a of either water, 0.85~o NaCI, or 2.0yo NaCI, injected in the last min of a five min injection period. These treatments were administered to each animal on four occasions in a random order with the restriction that two of each six sessions were control sessions, sessions preceded by a five rain period in the injection boxes
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FIG. 3. Drinking after different volumes of intragastric loads (standard errors of the means are indicated for water and 2.0'~, NaCI).
with 0 cm 3 injections. At the end of the five min injection period, animals were immediately placed into the modified Skinner boxes and their drinking was observed for six successive three min periods. One minute loads of l0 cm a were chosen for the present experiment since 10 cm 3 loads were effective in controlling drinking in Experiment 1 and since this rapid load of a relatively large volume should produce at least as much stomach distention as that normally present in the rat [I, 112, 17, 22]. Results and Discussion For the purposes of analysis, baseline responding for each animal was defined as the average total response observed for the six control sessions for that animal. All response measures and data points were then expressed as percentages of this baseline. The results of the experiment along with the corresponding results from Experiment 1 are shown in Fig. 4. The results of Experiment 2 were essentially the same as those for Experiment 1. There is no indication in Fig. 4 that stomach distention decreased drinking in either experiment, since 10 cm a IG loads of 0.85% NaCI injected in one minute did not decrease drinking. Both experiments show however that drinking was strongly affected by the concentration of NaCI of the I G load; water decreased drinking, isotonic saline had little effect, and hypertonic saline increased drinking. An analysis of variance confirmed these conclusions.
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FIG. 4. Drinking after different intragastric Loads injected in one minute (standard errors of the means are indicated).
These results were obtained in Experiment 2 when there was no opportunity for conditioning of particular thirst states to stomach distention cues, so it is unlikely that the results of Experiment 1 can be attributed to such conditioning. EXPERIMENT 3
In Experiment 1, increased I G volumes of water, 0.85% NaCI, and 2.0% NaCI all depressed bar pressing for water with water available on a VI-1 schedule of reinforcement. Although alternative explanations have been offered by others who have obtained similar results [5, I l, 14, 26], this finding can be interpreted as showing that thirst as measured by an instrumental response is not increased by increasing concentrations of NaCI, or that the increase is small and is masked by decreased thirst due to stomach distention. Others [27, 35] however have found that increased NaC1 concentrations in IG loads increased bar pressing for water, when water was available on a continuous schedule of reinforcement, and it has also been shown [5] that as the average interval of a variable interval schedule of reinforcement is made shorter, increased salt loads more clearly increase bar pressing for water. The present experiment was designed to show that NaC1 concentrations of IG loads, but not stomach distention, determine bar pressing for water when water is available on a continuous schedule of reinforcement. If this could be demonstrated, then the decreased bar pressing which was observed in Experiment I after increased IG
FEEDBACK CONTROL OF THIRST loads of isotonic and hypertonic saline could be attributed to factors introduced by partial reinforcement rather than to factors associated with instrumental responding per se. Method Animals. The animals were five male albino rats of the Sprague-Dawley strain, 180-200 days old at the start of the experiment. The animals had previously been trained in the acquisition and extinction of a bar pressing response for water in a successive brightness discrimination task at the ages of 120-160 days. A chronic I G tube was placed in each animal approximately one week before the start of the present experiment. The rats were housed in individual cages with Purina chow constantly available, but with water absent for the 21 hr immediately prior to each experimental session. Apparatus. The apparatus was the same as that used for I R animals in Experiment 1, except that each bar press was followed by the delivery of 0.02 cm 8 of water rather than 0.05 cm 8. The smaller amount of water per reinforcement was used in this experiment in order to reduce satiation during bar pressing; the effects of the smaller reinforcement were probably small compared to the effects of the change in reinforcement schedule from VI-1 to continuous reinforcement. Procedure. All animals were retrained to bar press for water on a continuous schedule of reinforcement for six daily 18 min sessions. Just before each session in the Skinner boxes, animals were connected to the injection pumps for a one minute 0 cm 8 injection. After pretraining, each animal was observed for three experimental sessions and three control sessions per week for six successive weeks; subjects were allowed water ad lib on the remaining day of each week. Experimental sessions were immediately preceded by I G loads of 10 cm 3 of either water, 0.85% NaC1, or 2.0% NaCI injected in one min, while control sessions were immediately preceded by a one min period in the injection boxes with 0 cm 8 injections. The order of control and experimental sessions was random with the restrictions that there were three control sessions in each six sessions, and that there was one of each type of experimental session in each six sessions. Bar pressing for water on a continuous schedule of reinforcement was recorded for all sessions in six consecutive time periods. For the first three weeks, sessions were 18 rain long, six three-min periods, and for the last three weeks, sessions were 60 min long, six ten-min periods. The I G tube in one animal became nonfunctional after two weeks of observations, so that complete observations were obtained for four animals. Results and Discussion
F o r the purposes of analysis, baseline responding for the six sessions in each week for each animal was defined as the average total response observed for the week's three control sessions for that animal. All response measures and data poin*s were then expressed as percentages of this total session baseline. The results of the experiment are shown in Fig. 5 along with the corresponding results from Experiment 1. The surprising result of Experiment 1, shown in Fig. 5a, was that bar pressing for water decreased rather than increased after I G injections of 2.0 % NaCI (Also see Fig. I). Fig. 5B shows that when the schedule of reinforcement was changed from VI-1 in Experiment 1 to continuous reinforcement in Experiment 3, bar pressing after 2.0~o NaCI increased but was still no greater than bar pressing after no I G load. Since there was little indication of a decrease in responding with C
1009 time for either the no load or the 2.0 % NaCI conditions in Fig. 5B, it seemed likely that animals in both conditions continued to respond at maximum rates throughout the sessions. Differences between the two conditions should be observable in longer sessions however, where satiation and decreased bar pressing should occur sooner for the no load condition than for the 2.0% NaCI load condition [5, 27]. Fig. 5C shows the results for one hour sessions, and it is clear here that 2.0 % NaCI loads increased bar pressing for water as compared to no load conditions.
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FIG. 5. Bar pressing for water after different intragastric loads injected in one minute (standard errors of the means are indicated).
The curves for isotonic saline, 0.85% NaCI, in Figs. 513 and 5C show that isotonic loads may produce some initial suppression of bar pressing for water, which is probably due to the effects of stomach distention rather than to the effects of the salt solution [8, 9]. It appears that stomach distention may produce effects, perhaps aversive or distractive [11, 13, 21], which decrease bar pressing for water without affecting drinking, and it is possible that these factors interact with variables introduced by partial reinforcement to result in the more pronounced decrease in bar pressing observed in the first experiment [5]. Others have suggested that the system responsible for the negative feedback control of consummatory responses is not the same as that for instrumental responses [20], and perhaps this is also demonstrated by the present data. However, the similarities between the I R data of Experiment 3 and the CR data of Experiments 1 and 2
1010
FEIDER
are more striking than the differences. The curves for bar pressing in Fig. 5C are very much like those for drinking in Fig. 4, showing little effect of stomach distention, and clear, large effects of water concentration changes. The necessity for different time scales for the abscissa of the two figures is probably due to the more rapid ingestion of water when subjects were drinking than when they were bar pressing for water. Separate analyses of variance for the 18 min and the 60 min sessions in Experiment 3 confirmed the trends described. GENERAL DISCUSSION In the present studies there was little indication that stomach distention was either a necessary or a sufficient cause of decreased water intake in the rat. Others' results are compatible with the extension of this conclusion to the general case, at least for rats. Ad lib eating and drinking patterns in rats for both solid and liquid diets consist of small, well spaced meals [12, 17, 34] in which stomach contents probably don't reach the volumes used in the present studies. Taking into consideration the stomach clearance times for food and solutions in the rat [18, 28, 32], stomach contents would probably not exceed the volumes used in the present studies by more than a few cubic centimeters even after the large
meals which follow deprivation [6, 12, 16, 31]. It may be that eating results in factors which interact with stomach distention to produce an inhibition of drinking that could not be observed in the present experiments, where only water and salt solutions were placed into the stomach. Such an inhibitory interaction seems unlikely however, since rats normally drink after meals of solid diet, perhaps in response to fluid concentration changes produced by water loss to thc gastrointestinal tract [17]. It may be that drinking in rats is controlled differently in natural environments, but there is no clear indication that this should be so [4]. There are some data to indicate that even for food intake, stomach distention does not act as a simple negative feedback factor [33]. In contrast to the failure of stomach distention to inhibit drinking, changes in fluid concentration clearly affected water intake. Drinking was decreased within one to four minutes by stomach loads of water, and was increased by stomach loads of hypertonic saline. The present studies also showed that vagal afferents from osmoreceptors in the stomach [30] are not necessary for inhibition of drinking, since vagotomies had no clear effect on drinking. Except for an initial small decrease in responding after large stomach load volumes, similar conclusions hold for bar pressing for water on a continuous schedule of reinforcement.
REFERENCES 1. Adolph, E. F. Thirst and its inhibition in the stomach. Ant. J. Physiol. 161: 374-386, 1950. 2. Adolph, E. F. Regulation of water intake in relation to body water content. In: Handbook of Physiology, Section 6: Alimentary Canal, Volume 1 : Food and Water Intake, edited by C. F. Code. Washington, D.C.: American Physiological Society, 1967, pp. 163-171. 3. Adolph, E. F., J. P. Barker and P. A. Hoy. Multiple factors in thirst. Am. J. Physiol. 178: 538-562, 1954. 4. Barnett, S. A. The Rat: A Study in Behaviour. Chicago: Aldine, 1963, pp. 34-71. 5. Beck, R. C. and J. F. McLean. Effect of schedule of reinforcement and stomach loads on bar pressing by thirsty rats. J. comp. physiol. Psychol. 63: 530-535, 1967. 6. Bolles, R. C. The interaction of hunger and thirst in the rat. J. comp. physiol. Psychol. 54- 580-584, 1961. 7. Carleton, H. M. and R. A. Drury. Histological Technique. (3rd ed.) London: Oxford University Press, 1957, p. 107. 8. Corbit, J. D. Effect of intravenous sodium chloride on drinking in the rat. J. comp. physiol. Psychol. 60: 397--406, 1965. 9. Corbit, J. D. and S. Tuchapsky. Gross hypervolemia: Stimulation of diuresis without effect on drinking. J. eomp. physiol. Psychol. 65: 38--41, 1968. 10. Epstein, A. N. Water intake without the act of drinking. Science, 131: 497-498, 1960. 11. Fayu, C. The effects of stomach distention on thirst: The value of using a variety of measures. Acta Psychol. Taiwan. 1 : 144-152, 1958. 12. Fitzsimons, J. T. and J. Le Magnen. Eating as a regulatory control of drinking in the rat. J. comp. physiol. Psychol. 67: 273-283, 1969. 13. Grossman, S. P. A Textbook of Physioiogical Psychology. New York: John Wiley & Sons, 1967, p. 434. 14. Hatton, G. I. Drive shifts during extinction: Effects on extinction and spontaneous recovery of bar-pressing behavior. J. comp. physiol. PsychoL 59: 385-391, 1965. 15. Iggo, A. Tension receptors in the stomach and the urinary bladder. J. Physiol., Lond. 128: 593-607, 1955. 16, Jacobs, H. L. The interaction of hunger and thirst: Experimental separation of osmotic and oral-gastric factors in regulat-
ing caloric intake. In: Thirst, edited by M. J. Wayner. Oxford: Pergamon Press, pp. 117-134. 17. Kisileff, H. R. Food-associated drinking in the rat. J. comp. physiol. Psychol. 67: 284-300, 1969. 18. Lepkovsky, S., R. Lyman, D. Flemming, M. Nagumo and M. M. Dimick. Gastrointestinal regulation of water and its effect on food intake and rate of digestion. Am. J. Physiol. 188: 327-331, 1957. 19. Lillie,R.D. Histopathologic Technique. Philadelphia: Blakiston, 1948, p. 1972. 20. Margules, D. L. and L. Stein. Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psyehol. 67: 327-335, 1969. 21. Miller, N. E. and M. L. Kessen. Reward effects of food via stomach fistula compared with those of food via mouth. J. comp. physiol. Psychol. 45: 555-564, 1952. 22. Miller, N. E., R. I. Sampliner and P. Woodrow. Thirstreducing effects of water by stomach fistula vs. water by mouth measured by both a consummatory and an instrumental response. J. comp. physiol. Psychol. 50: 1-5, 1957. 23. Niijima, A. Afferent impulses in the gastric and oesophageal branch of the vagal nerve of toad. Physiol. Behav. 2: 1-4, 1967. 24. Novin, D., A. Fox and M. Berger. The relation between saline solution ingested and tissue conductivity. Physiol. Behav. 1: 167-170, 1966. 25. O'Kelly, L. The effect of preloads of water and sodium chloride on voluntary water intake of thirsty rats. J. comp. physiol. Psychol. 47: 7-13, 1954. 26. O'Kelly, L., L. T. Crow, J. T. Tapp and G. I. Hatton. Water regulation in the rat: Drive intensity and fixed ratio responding. J. comp. physiol. Psychol. 61: 194-197, 1966. 27. O'Kelly, L. and J. L. Falk. Water regulation in the rat: It. The effects of preloads of water and sodium chloride on the bar-pressing performance of thirsty rats. d. comp. physiol. Psychol. 51: 22-25, 1958. 28. O'Kelly, L., 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: 16-21, 1958.
FEEDBACK CONTROL OF THIRST 29. Paintal, A. S. A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst. J. Physiol. Lond. 126: 255-270, 1954. 30. Sharma, K. N. Receptor mechanisms in the alimentary tract. Their excitation and functions. In" Handbook of Physiology, Section 6: Alimentary Canal, Volume 1: Food and Water Intake, edited by C. F. Code. Washington, D.C.: American Physiological Society, 1967, pp. 225-237. 31. Smith, M. H. Effects of intravenous injections on eating. J. comp. physiol. Psychol. 61 : 11-14, 1966. 32. Snowdon, C. T. Gastrointestinal sensory and motor control of food intake. J. comp. physiol. Psychol. 71: 68-76, 1970.
1011 33. Snowdon, C. T. and A. N. Epstein. Oral and intragastric feeding in vagotomized rats. J. comp. physiol. Psychol. 71: 59-67, 1970. 34. Thomas, D. W. and J. Mayer. Meal taking and regulation of food intake by normal and hypothalamic hyperphagic rats. J. comp. physiol. Psychol. 66: 642-653, 1968. 35. Wayner, M. J., F. M. Brown, M. Kitayama and M. Gray. Effects of salt arousal of drinking and water deprivation on performance of CRF and VI-1 schedules of reinforcement. Physiol. Behav. 5: 99-109, 1970.
Feedback Control of Thirst in Rats' ALLEN FEIDER 2
Department of Psychology, University qf New Brunswick, Fredericton, New Brunswick, Canada (Received 15 December 1971) FEIDER, A. Feedback controlof thirst in rats. PHYSIOL.BEHAV.8 (6) 1005-1011, 1972.--To compare the effects of stomach distention and fluid concentration changes on thirst, different solutions were injected into the stomachs of water deprived rats. Rapid injections of large volumes of isotonic saline, up to 15 cma in 1 rain, were given to produce stomach distention, but had no effect on drinking. Intragastric injections of water decreased drinking within one to four min and injections of hypertonic saline increased drinking. Bar pressing for water on a continuous reinforcement schedule produced results similar to those for drinking, except that there was a brief, small decrease in bar pressing immediately following injections of large volumes. Fluid concentration changes, but not stomach distention, seem to be an important factor in the negative feedback control of thirst in the rat. Thirst
Intragastric loads
Stomach distention
Hypertonic loads
STOMACH distention has been described as a negative feedback factor controlling thirst in rats, but this conclusion is open to question. In some studies which seem to show that stomach distention reduces thirst, water was placed directly into the stomach at a slow rate which approximated the rate of normal drinking, 1-2 cmS/min [22], and in other studies water was quickly placed into the stomach, but animals were allowed a recovery period before experimental observations started [25]. In both cases it is likely that possible cues from stomach distention were confounded with changes produced by water absorption and cellular hydration which occur soon after water has been ingested [24, 28]. In other studies which seem to show that stomach distention reduces thirst, experimental observations were obtained while rats' stomachs were distended by mechanical means or by rapid injections of large volumes of fluid [1, 3]. Under these conditions however, since water is normally drunk at a slow rate and is rapidly cleared from the stomach, the stomach is abnormally distended and the cues from greater than normal distention may be aversive [13, 21]. The following experiments are an attempt to separate the effects of stomach distention from the effects of water concentration changes in the reduction of thirst as measured both by drinking and by bar pressing for water. Stomach distention was manipulated by injecting different volumes of fluids into the stomachs of rats at varying rates of injection. Fluid concentration changes were controlled by varying the concentration of sodium chloride in the injected fluids. EXPERIMENT 1
Method Animals. The animals were 62 male albino rats of the Sprague-Dawley strain, experimentally naive and 120-150
Bar pressing behavior
days old at the start of the experiment. A chronic intragastric (IG) tube [10] was placed in each animal one week prior to the start of the experiment using combined sodium pentobarbital and ether anesthesia. The rats were housed in individual cages with Purina chow constantly available throughout the experiment, but with water absent for the 22 hr immediately preceding each experimental session. Apparatus. The two Skinner boxes used delivered approximately 0.05 cm s of water for each reinforced response. The end walls of two other Skinner boxes were replaced with modified panels which carried a house light, and which had a 1.3 cm hole through which animals could reach to lick at a glass drinking tube connected to a drinkometer. Sage syringe pumps and Sage swivel joints were used for I G injections, which took place in open boxes, 43 × 43 × 45.5 cm high. Relay operated programming and recording equipment was located in a third room, separate from the room containing the Skinner boxes and the room containing the IG injection equipment. Preliminary training. The rats were randomly divided into two groups of 31, an instrumental response (IR) group and a consummatory response (CR) group. Animals in the IR group were trained to bar press for water in daily 18 min sessions. After two sessions on a continuous reinforcement schedule, IR animals were trained for 10 sessions on a variable interval one-min (VI-I) schedule of reinforcement. Animals in the CR group were placed into the modified Skinner boxes and allowed to drink for seven daily 18 min sessions. Just before each of the last four sessions of preliminary training both CR and IR animals were placed into the large open injection boxes for 12 min where their I G tubes were connected to the injection pumps but no fluid was injected. This sham injection, or 0 cm s injection, was performed by turning the injection pumps on with the syringe
1This research was supported by National Research Council of Canada Grant APA-255. The author expresses his appreciation to Anne Amys, Mary Ann Linseman, and Margaret MacLean for their assistance. 2The author can provide, upon request, a table giving the summary tables for the analyses of variance performed on group data in these studies. 1005
1006
I FIDFR
drive carriage missing from the pumps. After alternate sessions throughout preliminary training an I G injection of approximately 2 cm ~ of water was given to each animal, to prevent blocking of the I G tubes. Throughout the experiment, 4 animals, 2 CR and 2 I R were observed at the same time. Preoperative observations. To make up the six betweenanimals treatment groups described in Table 1 three subgroups were formed within the CR group and within the IR group. Animals were randomly assigned to subgroups with the restriction that at least I0 animals were in each subgroup. Before each daily session, the water deprived animals were placed in the large open boxes for a 12 rain injection period; during this time animals from the different subgroups were injected with either water, 0 . 8 5 ~ NaCI, or 2 . 0 ~ NaCI. Three sets of the 16 possible volume-load time treatment combinations described in Table 1 were administered to each animal, with four daily sessions of 0 c m s injection intervening between successive sets of treatments. Within each set of treatments, the order of treatments was random with the restriction that one of each group of four treatments was a 0 cm 3 injection. When a load time was 1, 3, or 6 rain injections occurred at the end of the 12 min injection period. Immediately after the injection period, animals were placed into the experimental chambers, and allowed to drink for 18 min (CR animals), or allowed to bar press for water on a VI-I schedule of reinforcement for 18 min (IR animals). Records of bar presses and licks were obtained for each successive three rain period of the 18 min sessions. All preoperative treatments were completed by 51 animals, at least eight animals in each of the six between-anhnals treatment groups. Vagotomies. Partial vagotomies were performed on all but four animals, who were exposed to a sham vagotomy. Surgery was done using combined sodium pentobarbital and ether anesthesia, and vagotomies consisted of stripping and removing the vagus nerves from approximately 2 cm of the esophagus next to the stomach. These vagotomies were performed in an attempt to eliminate cues from stomach distention and cues from osmoreceptors in the stomach as possible feedback factors controlling thirst during postoperative observations.
Postoperative observations. After live days recovery from the operations, all animals were retrained for four days with 0 cm 3 injection treatments. Thirty-six animals completed all postoperative observations, which were lhe same as preoperative observations. Post-mortem examinations. Inspection indicated that IG tubes ended in the stomachs of all but a few animals, for whom the tubes ended in the esophagus several mm short of the stomach. The effectiveness of the partial vagotomies was determined from I0~ paraffin sections of the esophagus, alternately stained with Mallory's Collagen Stain [7] and Weil's Myelin Stain [19]. The vagus nerves were clearly absent for 17 animals. Results and Discussion Preoperative observations. Where necessary, animals were randomly excluded from the analysis of preoperative data to form six groups of eight animals each. F o r the purposes of analysis, baseline responding for each animal for each set of 16 treatments was defined as the average total response observed for the four 0 cm 3 injection sessions for that animal. All response measures for each set of 16 treatments were then expressed as percentages of this total session baseline. Similarly, the data points in all the figures are plotted as percentages of this same total session baseline. Figures 1, 2, and 3 show that drinking was not decreased by stomach distention produced by I G loads of isotonic saline. Even when 15 cm ~ of isotonic saline was injected into the stomach in the minute immediately preceding the drinking session, distention had no effect (Fig. 2). A further indication that stomach distention did not affect drinking is that changing the rates of injections had no effect on drinking, no matter what solution was injected. In contrast, Figs. 3 and 4 show that injections of water immediately decreased drinking, so that drinking was reduced within four minutes of the placement of water in the stomach. Injections of hypertonic saline had the opposite effect, producing increased drinking. Since animals with no injections were drinking as rapidly as possible at the beginning of the sessions, the increase due to hypertonic saline was apparent only later in the sessions (Figs. 3
TABLE I DESIGN OF EXPERIMENT 1
Values of variable
Variables Between animals variables Response meamlre
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Solution
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and 4). All of the differences described in reference to Figs. 1, 2, 3, and 4 were confirmed by a six way analysis of variance of the preoperative data. The results concerning bar pressing for water on a VI-1 schedule of reinforcement were different from the results for drinking (Figs. 1 and 2), but the consideration of these differences is deferred to Experiment 3. Postoperative observations. Using the 0.01 level of confidence, individual analyses of variance for each animal showed that postoperative drinking and bar pressing for water were the same as preoperative lesponding, even for those cases in which the vagotomies were confirmed histologically. This result is a further indication that distention of the stomach is not an important factor in reducing thirst in rats, since distention cues are carried primarily in the vagus nerves [15, 23, 29, 30]. The failure of the vagotomies to affect behavior is also an indication that vagal afferents from osmoreceptors in the stomach [30] are not necessary for the effects observed. EXPERIMENT 2 The results of Experiment 1 could have been obtained even if stomach distention normally inhibits drinking. Since different concentrations of NaC1 were given to different
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groups of animals, it is possible that stomach distention cues were consistently paired with particular changes in thirst, and that these changes in thirst became conditioned to distention cues. If this did happen, the normal inhibition of thirst by distention would no longer be apparent. The purpose of Experiment 2 was to eliminate this possibility by giving different concentrations of NaCI to the same subjects.
Method Animals. The animals were five male albino rats of the Sprague-Dawley strain, experimentally naive and 120-150 days old at the start of the experiment. A chronic I G tube was placed in each animal approximately one week prior to the start of the experiment. The rats were housed in individual cages with Purina chow constantly available throughout the experiment, but with water absent for 21 hr before each experimental session. Apparatus. The apparatus used for C R animals in Experiment 1 was used in this experiment. Procedure. The details o f the procedure were the same as those for C R animals in Experiment I, except as indicated below. After pretraining, each animal was observed for four experimental sessions and two control sessions per week for three successive weeks. Animals were allowed water ad lib on the remaining day each week. Experimental sessions were preceded by I G loads of 10 em a of either water, 0.85~o NaCI, or 2.0yo NaCI, injected in the last min of a five min injection period. These treatments were administered to each animal on four occasions in a random order with the restriction that two of each six sessions were control sessions, sessions preceded by a five rain period in the injection boxes
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with 0 cm 3 injections. At the end of the five min injection period, animals were immediately placed into the modified Skinner boxes and their drinking was observed for six successive three min periods. One minute loads of l0 cm a were chosen for the present experiment since 10 cm 3 loads were effective in controlling drinking in Experiment 1 and since this rapid load of a relatively large volume should produce at least as much stomach distention as that normally present in the rat [I, 112, 17, 22]. Results and Discussion For the purposes of analysis, baseline responding for each animal was defined as the average total response observed for the six control sessions for that animal. All response measures and data points were then expressed as percentages of this baseline. The results of the experiment along with the corresponding results from Experiment 1 are shown in Fig. 4. The results of Experiment 2 were essentially the same as those for Experiment 1. There is no indication in Fig. 4 that stomach distention decreased drinking in either experiment, since 10 cm a IG loads of 0.85% NaCI injected in one minute did not decrease drinking. Both experiments show however that drinking was strongly affected by the concentration of NaCI of the I G load; water decreased drinking, isotonic saline had little effect, and hypertonic saline increased drinking. An analysis of variance confirmed these conclusions.
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These results were obtained in Experiment 2 when there was no opportunity for conditioning of particular thirst states to stomach distention cues, so it is unlikely that the results of Experiment 1 can be attributed to such conditioning. EXPERIMENT 3
In Experiment 1, increased I G volumes of water, 0.85% NaCI, and 2.0% NaCI all depressed bar pressing for water with water available on a VI-1 schedule of reinforcement. Although alternative explanations have been offered by others who have obtained similar results [5, I l, 14, 26], this finding can be interpreted as showing that thirst as measured by an instrumental response is not increased by increasing concentrations of NaCI, or that the increase is small and is masked by decreased thirst due to stomach distention. Others [27, 35] however have found that increased NaC1 concentrations in IG loads increased bar pressing for water, when water was available on a continuous schedule of reinforcement, and it has also been shown [5] that as the average interval of a variable interval schedule of reinforcement is made shorter, increased salt loads more clearly increase bar pressing for water. The present experiment was designed to show that NaC1 concentrations of IG loads, but not stomach distention, determine bar pressing for water when water is available on a continuous schedule of reinforcement. If this could be demonstrated, then the decreased bar pressing which was observed in Experiment I after increased IG
FEEDBACK CONTROL OF THIRST loads of isotonic and hypertonic saline could be attributed to factors introduced by partial reinforcement rather than to factors associated with instrumental responding per se. Method Animals. The animals were five male albino rats of the Sprague-Dawley strain, 180-200 days old at the start of the experiment. The animals had previously been trained in the acquisition and extinction of a bar pressing response for water in a successive brightness discrimination task at the ages of 120-160 days. A chronic I G tube was placed in each animal approximately one week before the start of the present experiment. The rats were housed in individual cages with Purina chow constantly available, but with water absent for the 21 hr immediately prior to each experimental session. Apparatus. The apparatus was the same as that used for I R animals in Experiment 1, except that each bar press was followed by the delivery of 0.02 cm 8 of water rather than 0.05 cm 8. The smaller amount of water per reinforcement was used in this experiment in order to reduce satiation during bar pressing; the effects of the smaller reinforcement were probably small compared to the effects of the change in reinforcement schedule from VI-1 to continuous reinforcement. Procedure. All animals were retrained to bar press for water on a continuous schedule of reinforcement for six daily 18 min sessions. Just before each session in the Skinner boxes, animals were connected to the injection pumps for a one minute 0 cm 8 injection. After pretraining, each animal was observed for three experimental sessions and three control sessions per week for six successive weeks; subjects were allowed water ad lib on the remaining day of each week. Experimental sessions were immediately preceded by I G loads of 10 cm 3 of either water, 0.85% NaC1, or 2.0% NaCI injected in one min, while control sessions were immediately preceded by a one min period in the injection boxes with 0 cm 8 injections. The order of control and experimental sessions was random with the restrictions that there were three control sessions in each six sessions, and that there was one of each type of experimental session in each six sessions. Bar pressing for water on a continuous schedule of reinforcement was recorded for all sessions in six consecutive time periods. For the first three weeks, sessions were 18 rain long, six three-min periods, and for the last three weeks, sessions were 60 min long, six ten-min periods. The I G tube in one animal became nonfunctional after two weeks of observations, so that complete observations were obtained for four animals. Results and Discussion
F o r the purposes of analysis, baseline responding for the six sessions in each week for each animal was defined as the average total response observed for the week's three control sessions for that animal. All response measures and data poin*s were then expressed as percentages of this total session baseline. The results of the experiment are shown in Fig. 5 along with the corresponding results from Experiment 1. The surprising result of Experiment 1, shown in Fig. 5a, was that bar pressing for water decreased rather than increased after I G injections of 2.0 % NaCI (Also see Fig. I). Fig. 5B shows that when the schedule of reinforcement was changed from VI-1 in Experiment 1 to continuous reinforcement in Experiment 3, bar pressing after 2.0~o NaCI increased but was still no greater than bar pressing after no I G load. Since there was little indication of a decrease in responding with C
1009 time for either the no load or the 2.0 % NaCI conditions in Fig. 5B, it seemed likely that animals in both conditions continued to respond at maximum rates throughout the sessions. Differences between the two conditions should be observable in longer sessions however, where satiation and decreased bar pressing should occur sooner for the no load condition than for the 2.0% NaCI load condition [5, 27]. Fig. 5C shows the results for one hour sessions, and it is clear here that 2.0 % NaCI loads increased bar pressing for water as compared to no load conditions.
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The curves for isotonic saline, 0.85% NaCI, in Figs. 513 and 5C show that isotonic loads may produce some initial suppression of bar pressing for water, which is probably due to the effects of stomach distention rather than to the effects of the salt solution [8, 9]. It appears that stomach distention may produce effects, perhaps aversive or distractive [11, 13, 21], which decrease bar pressing for water without affecting drinking, and it is possible that these factors interact with variables introduced by partial reinforcement to result in the more pronounced decrease in bar pressing observed in the first experiment [5]. Others have suggested that the system responsible for the negative feedback control of consummatory responses is not the same as that for instrumental responses [20], and perhaps this is also demonstrated by the present data. However, the similarities between the I R data of Experiment 3 and the CR data of Experiments 1 and 2
1010
FEIDER
are more striking than the differences. The curves for bar pressing in Fig. 5C are very much like those for drinking in Fig. 4, showing little effect of stomach distention, and clear, large effects of water concentration changes. The necessity for different time scales for the abscissa of the two figures is probably due to the more rapid ingestion of water when subjects were drinking than when they were bar pressing for water. Separate analyses of variance for the 18 min and the 60 min sessions in Experiment 3 confirmed the trends described. GENERAL DISCUSSION In the present studies there was little indication that stomach distention was either a necessary or a sufficient cause of decreased water intake in the rat. Others' results are compatible with the extension of this conclusion to the general case, at least for rats. Ad lib eating and drinking patterns in rats for both solid and liquid diets consist of small, well spaced meals [12, 17, 34] in which stomach contents probably don't reach the volumes used in the present studies. Taking into consideration the stomach clearance times for food and solutions in the rat [18, 28, 32], stomach contents would probably not exceed the volumes used in the present studies by more than a few cubic centimeters even after the large
meals which follow deprivation [6, 12, 16, 31]. It may be that eating results in factors which interact with stomach distention to produce an inhibition of drinking that could not be observed in the present experiments, where only water and salt solutions were placed into the stomach. Such an inhibitory interaction seems unlikely however, since rats normally drink after meals of solid diet, perhaps in response to fluid concentration changes produced by water loss to thc gastrointestinal tract [17]. It may be that drinking in rats is controlled differently in natural environments, but there is no clear indication that this should be so [4]. There are some data to indicate that even for food intake, stomach distention does not act as a simple negative feedback factor [33]. In contrast to the failure of stomach distention to inhibit drinking, changes in fluid concentration clearly affected water intake. Drinking was decreased within one to four minutes by stomach loads of water, and was increased by stomach loads of hypertonic saline. The present studies also showed that vagal afferents from osmoreceptors in the stomach [30] are not necessary for inhibition of drinking, since vagotomies had no clear effect on drinking. Except for an initial small decrease in responding after large stomach load volumes, similar conclusions hold for bar pressing for water on a continuous schedule of reinforcement.
REFERENCES 1. Adolph, E. F. Thirst and its inhibition in the stomach. Ant. J. Physiol. 161: 374-386, 1950. 2. Adolph, E. F. Regulation of water intake in relation to body water content. In: Handbook of Physiology, Section 6: Alimentary Canal, Volume 1 : Food and Water Intake, edited by C. F. Code. Washington, D.C.: American Physiological Society, 1967, pp. 163-171. 3. Adolph, E. F., J. P. Barker and P. A. Hoy. Multiple factors in thirst. Am. J. Physiol. 178: 538-562, 1954. 4. Barnett, S. A. The Rat: A Study in Behaviour. Chicago: Aldine, 1963, pp. 34-71. 5. Beck, R. C. and J. F. McLean. Effect of schedule of reinforcement and stomach loads on bar pressing by thirsty rats. J. comp. physiol. Psychol. 63: 530-535, 1967. 6. Bolles, R. C. The interaction of hunger and thirst in the rat. J. comp. physiol. Psychol. 54- 580-584, 1961. 7. Carleton, H. M. and R. A. Drury. Histological Technique. (3rd ed.) London: Oxford University Press, 1957, p. 107. 8. Corbit, J. D. Effect of intravenous sodium chloride on drinking in the rat. J. comp. physiol. Psychol. 60: 397--406, 1965. 9. Corbit, J. D. and S. Tuchapsky. Gross hypervolemia: Stimulation of diuresis without effect on drinking. J. eomp. physiol. Psychol. 65: 38--41, 1968. 10. Epstein, A. N. Water intake without the act of drinking. Science, 131: 497-498, 1960. 11. Fayu, C. The effects of stomach distention on thirst: The value of using a variety of measures. Acta Psychol. Taiwan. 1 : 144-152, 1958. 12. Fitzsimons, J. T. and J. Le Magnen. Eating as a regulatory control of drinking in the rat. J. comp. physiol. Psychol. 67: 273-283, 1969. 13. Grossman, S. P. A Textbook of Physioiogical Psychology. New York: John Wiley & Sons, 1967, p. 434. 14. Hatton, G. I. Drive shifts during extinction: Effects on extinction and spontaneous recovery of bar-pressing behavior. J. comp. physiol. PsychoL 59: 385-391, 1965. 15. Iggo, A. Tension receptors in the stomach and the urinary bladder. J. Physiol., Lond. 128: 593-607, 1955. 16, Jacobs, H. L. The interaction of hunger and thirst: Experimental separation of osmotic and oral-gastric factors in regulat-
ing caloric intake. In: Thirst, edited by M. J. Wayner. Oxford: Pergamon Press, pp. 117-134. 17. Kisileff, H. R. Food-associated drinking in the rat. J. comp. physiol. Psychol. 67: 284-300, 1969. 18. Lepkovsky, S., R. Lyman, D. Flemming, M. Nagumo and M. M. Dimick. Gastrointestinal regulation of water and its effect on food intake and rate of digestion. Am. J. Physiol. 188: 327-331, 1957. 19. Lillie,R.D. Histopathologic Technique. Philadelphia: Blakiston, 1948, p. 1972. 20. Margules, D. L. and L. Stein. Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psyehol. 67: 327-335, 1969. 21. Miller, N. E. and M. L. Kessen. Reward effects of food via stomach fistula compared with those of food via mouth. J. comp. physiol. Psychol. 45: 555-564, 1952. 22. Miller, N. E., R. I. Sampliner and P. Woodrow. Thirstreducing effects of water by stomach fistula vs. water by mouth measured by both a consummatory and an instrumental response. J. comp. physiol. Psychol. 50: 1-5, 1957. 23. Niijima, A. Afferent impulses in the gastric and oesophageal branch of the vagal nerve of toad. Physiol. Behav. 2: 1-4, 1967. 24. Novin, D., A. Fox and M. Berger. The relation between saline solution ingested and tissue conductivity. Physiol. Behav. 1: 167-170, 1966. 25. O'Kelly, L. The effect of preloads of water and sodium chloride on voluntary water intake of thirsty rats. J. comp. physiol. Psychol. 47: 7-13, 1954. 26. O'Kelly, L., L. T. Crow, J. T. Tapp and G. I. Hatton. Water regulation in the rat: Drive intensity and fixed ratio responding. J. comp. physiol. Psychol. 61: 194-197, 1966. 27. O'Kelly, L. and J. L. Falk. Water regulation in the rat: It. The effects of preloads of water and sodium chloride on the bar-pressing performance of thirsty rats. d. comp. physiol. Psychol. 51: 22-25, 1958. 28. O'Kelly, L., 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: 16-21, 1958.
FEEDBACK CONTROL OF THIRST 29. Paintal, A. S. A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst. J. Physiol. Lond. 126: 255-270, 1954. 30. Sharma, K. N. Receptor mechanisms in the alimentary tract. Their excitation and functions. In" Handbook of Physiology, Section 6: Alimentary Canal, Volume 1: Food and Water Intake, edited by C. F. Code. Washington, D.C.: American Physiological Society, 1967, pp. 225-237. 31. Smith, M. H. Effects of intravenous injections on eating. J. comp. physiol. Psychol. 61 : 11-14, 1966. 32. Snowdon, C. T. Gastrointestinal sensory and motor control of food intake. J. comp. physiol. Psychol. 71: 68-76, 1970.
1011 33. Snowdon, C. T. and A. N. Epstein. Oral and intragastric feeding in vagotomized rats. J. comp. physiol. Psychol. 71: 59-67, 1970. 34. Thomas, D. W. and J. Mayer. Meal taking and regulation of food intake by normal and hypothalamic hyperphagic rats. J. comp. physiol. Psychol. 66: 642-653, 1968. 35. Wayner, M. J., F. M. Brown, M. Kitayama and M. Gray. Effects of salt arousal of drinking and water deprivation on performance of CRF and VI-1 schedules of reinforcement. Physiol. Behav. 5: 99-109, 1970.