PLASMA CQNDETEGNS AT THE ENETIATEQN QT DRENKENG {N SALT ENJECTED RATS Thesis Tar Hm Degree of M. A. MECHEGAN STATE UNIVERSITY Charles Robert Almii 1968 LI B R AR Y 5 Tafffljgan State TH Es|$ - . University FE T '_ . _-.~— 4-_—.—-.m ,m-*... ‘- ....- _. ‘5‘... T deter: initis teres1 adapte Plasma Conditions at the Initiation of Drinking in Salt Injected Rats by Charles Robert Almli Abstract of Master's Thesis Completed Spring Term, 1968 The problem under investigation in this thesis was the determination of blood plasma conditions which lead to the initiation of drinking in water satiated rats. Also of in- terest was the stability of these conditions as rats were adapted to a drinking schedule. Previous research has shown that satiated rats injected subcutaneously with 16% sodium chloride solution, reinitiate drinking between 3 and 11 min. postinjection. However, no data are available to show the reliability of this post- injection latency to drink for a given rat. The reliability of postinjection latency to drink was determined in Ex- periment 1. The determination of blood conditions at the initiation of drinking behavior (Experiment 2), took ad- vantage of this phenomenon as measured in Experiment 1. Accordingly, blood samples were taken when the rat would have normally reinitiated drinking behavior following injection. In Experiment 1, adaptations to recurrent deprivation schedules, separated by ad lib recovery periods, were studied. I? was found that latency to drink decreased over subsequent deprivation periods, indicating that this was a learne body 1 ing 9: ents t The r‘ versy drink Charles Robert Almli learned component of adaptation behavior. Water intake and body weight, in contrast, required the same adjustment dur- ing each deprivation period, indicating that these compon- ents of adaptation were not influenced by prior learning. The results are discussed in the context of a prior contro- versy in the literature. In Experiment 2, latency to drink following subcut- aneous injection of 16% sodium chloride solution was measured on Days 0, 1, 2, 3, 5, and 10, of two equivalent deprivation periods separated by five days of ad lib con- ditions. It was found that across and within treatment days, postinjection latencies to drink were highly consistent (r=0.711). Therefore, a mean was computed for each rat, based on two postinjection latencies. Each mean represented a 'predicted' postinjection latency to drink for that rat. A blood sample was drawn at the 'predicted' post- injection latency to drink, using this as representative of when the rat would normally reinitiate drinking following salt injection. Across days of adaptation (O, 1, 2, 3, 5, and 10), with the exception of transition days 1 and 2, a postinjection increase of 2-3% over non-injected controls, in plasma osmotic pressure was sufficient to instigate drinking behavior. These threshold conditions are discussed in terms of regulatory mechanisms, and in relation to other pertinent research on the subject. Approved: gum J m Date: w/Véf Glenn I. Hatton, Chairman John I. Johnson Jr. Ralph Levine PLASMA CONDITIONS AT THE INITIATION 0F DRINKING IN SALT INJECTED RATS By Charles Robert Almli A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1968 .411TT ACKNOWLEDGEMENT The author expresses appreciation to Dr. Glenn I. Hatton for invaluable assistance in the preparation of this thesis, and to Dr. John I. Johnson Jr. and Dr. Ralph Levine for their pertinent consultation. Special appreciation is ex- pressed to my wife Sheila, who performed her role as a laboratory-widow with the utmost of patience. 11 TABLE OF CONTENTS Page ACKNOWLEDGEMENT . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . v LIST OF APPENDICES . . . . . . . . . . . . vii GENERAL INTRODUCTION . . . . . . . . . . . EXPERIMENT I . . . . . . . . . . . . . . Method . HCDU‘MUIUI U.) H SubJeCtS O O O O O O O O O O O I O 0 Apparatus . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . Results 0 o o o o o o o o o o o o 0 Discussion . . . . . . . . . . . . . 1 EXPERIMENT II . . . . . . . . . . . . . . 1h Method . 19 31113360138 0 o o o o o o o o o o o o o 19 Apparatus . . . . . . . . . . . . . 19 Procedure 0 I O O O O O O O O I O O 20 Results 0 O O O O O O O O O O O 0 C 23 Discussion . . . . . . . . . . . . . 40 REFERENCES 0 O O O O O O G O I O O O O 0 6O APPENDICES O O O O O O O O O O O O O O O 6 3 111 LIST OF TABLES Page Means and standard errors of postinjection latencies to drink . . . . . . . . . 27 Means, medians, and ranges of postinjection latencies to drink following the first and second injection treatments, and for the two injection treatments combined . . . . 32 Mean threshold values of plasma osmolality at the postinjection initiation of drink- ing behavior, and mean latencies to drink for 'postinjection' rats, as a function of days of adaptation to 23.5 hr. water deprivation o o o o o o o o o o o 55 iv Figures 1. 2. 7. LIST OF FIGURES Experimental design for Experiment I . . . Mean reciprocal latency to drink, mean per- cent body weight intake, and mean body weight, during adaptation to 23.5 hr. water deprivation and during ad lib recovery periods, plotted as a function of days of the experiment . . . . . . . . . Experimental design for Experiment II . . Mean percent body weight intake during adapt- ation to 23.5 hr. water deprivation and during ad lib recovery periods, plotted as a function of days of the experiment . . Latency to drink of 35 albino rats immediat- ely after injection of 16% saline solution for the first and second injection treat- ments, and the means obtained from both treatments combined . . . . . . . . A scattergram showing the relationship be- tween the postinjection latency to drink following injection 1 and postinjection latency to drink following injection 2, for 35 albino rats . . . . . . . . Mean plasma osmolality at the postinjection reinitiation of drinking behavior, of 35 albino rats, plotted as a function of days of adaptation to 23.5 hr. water deprivation . . . . . . . . . . Mean plasma protein concentration at the postinjection reinitiation of drinking behavior, of 35 albino rats, plotted as a function of days of adaptation to 23.5 hr. water deprivation . . . . . . . A scattergram showing the lack of rela- tionship between plasma osmolality and plasma protein concentration at the postinjection re-initiation of drink- ing behavior for 35 albino rats . . . . Page 10 25 25 29 31 35 37 39 Figures Page 10. Plasma protein concentration and plasma osmol- ality at the postinjection re-initiation of drinking behavior of 35 albino rats, plotted as a function of time postinjection when the blood samples were withdrawn . . . . b2 11. Mean plasma osmolality obtained predrink, postdrink, and postinjection at the re-initiation of drinking behavior, plotted as a function of days of adapt- ation to 23.5 hr. water deprivation . . . 51 12. Mean plasma protein concentration obtained predrink, postdrink, and postinjection at the re-initiation of drinking be- havior, plotted as a function of days of adaptation to 23.5 hr. water deprivation . 53 vi LIST OF APPENDICES Page APPENDIX A: Apparatus . . . . . . . . . . . 63 APPENDIX B: Procedure . . . . . . . . . . . 67 APPENDIX C: Raw Data vii GENERAL INTRODUCTION When water supply is restricted, profound changes occur in most mammals (extensively reviewed by Chew, 1965). These changes can be observed in the behavior, physiology, and basic chemistry of the animal. Depending upon the regimen of water restriction, there may result distinct short-term and longrterm alterations which have pervasive influence on the animal's.interaction with its environment. In studying animals.as they adapt to some regimen, information is ob- tained regarding both these short-term and long-term con- ditions, as well as transitional states through which the animal may pass in the process of adaptation. 0f specific relevance to this thesis are certain aSpects of water- orientedeehavior, and the blood chemistry closely associ- ated with that behavior, of rats as they adapt to a schedule of water deprivation. The ultimate goal of this thesis was to determine blood plasma conditions at the initiation of drinking be- havior of rats as they adapt to 23.5 hr. water deprivation. However, unlike most behavioral phenomena, this variable is not readily accessible to direct observation. Therefore, an indirect method of determining blood conditions at the initiation of drinking behavior was necessary. The indirect method used in this study consisted of obtaining a 'predicted' 1 postinjection (16% sodium chloride) latency to drink, and withdrawing a blood sample at the time when the rat would normally be initiating drinking behavior. In order to obtain a predicted postinjection latency to drink for rats as they were adapted to 23.5 hr. water dep- rivation, it was necessary to establish that, in terms of water intake, successive adaptations to water deprivation schedules were equivalent and comparable. This problem is presented in Experiment 1. It was also necessary to estab- lish that postinjection latencies to drink at each of several different levels of adaptation to deprivation, for a given rat, were consistent. This problem is presented in Experi- ment 2. Once the previous two preblems were resolved, indicat- ing that successive adaptations to deprivation schedules were equivalent, and that latencies to drink following in- jeetion at eachiof several different levels of adaptation were.consistent,.a 'predicted' postinjection latency to drink could be computed for each rat. The predicted post- injection latency to drink, representing the time when the .rat.would normally initiate drinking, could then be used to determine when the blood sample would be drawn. In this way, bloodrconditions at the initiation of drinking behavior could be determined for each of the different levels of adaptation to deprivation. This was accomplished in Experi- ment 2. EXPERIMENT I The physiological and behavioral changes accompanying adaptation to water deprivation schedules have received limited attention by researchers. There is, however, wide- spread use of animals on such schedules in the fields of learning, motivation, and regulation. This experiment represents an attempt to resolve the first problem toward the determination of blood conditions at the initiation of drinking behavior. It was necessary to establish whether or not rats had to readapt to successive deprivation periods, when the deprivation periods were separ- ated by ad lib recovery periods. This problem was sign- ificant because in Experiment 2, identical treatments would be administered on Specified days of two separate adapt- ation to deprivation periods. Thus, on selected days of separate adaptation periods, it would be necessary to have rats at the same state (as indexed by amount drunk) of adaptation each time. According to Ghent (1957). however, this would not be possible because she has interpreted the increasing amount drunk with successive days of adaptation to deprivation as a learning phenomenon. If this were the case, the rats would not have to readapt to each successive deprivation period because of the learning that had taken place in the 3 previous deprivation periods. In contrast, Beck (1962) has concluded that the increasing water intake over successive days of adaptation to deprivation was due to an increased need for water. His conclusion was based on the finding that when rats were deprived of water one day and allowed two recovery days on ad lib, they drank the same amount of water each time, indicating that the water intakes were not influenced by prior learning. The present experiment attempted to resolve this con- troversy by using; a) 10-day adaptation to water deprivation periods, within which drinking is known to stabilize, and b) constant external stimulus conditions during the ad lib recovery periods, which eliminates confounding factors associated with a novel drinking situation. By continuing to place the rats in the drinking boxes during the ad lib periods, the stimulus complex of the drinking situation would retain its familiarity and the rats would not have to readapt to the stimulus complex during the following dep- rivation periods. Thus, the latency to drink and the amount drunk during successive adaptation to deprivation periods should reflect the degree of dehydration experienced by the rats, and will not be confounded by a novel drinking situation each time. Method S e 8 Eleven male albino rats of the Holtzman strain, were housed in individual wire cages in a temperature controlled room (73°-78° F.), under constant light conditions. Wayne Lab Blox were constantly available in the home cage through- out the experiment. The rats were approximately 100 days old and weighed 360 i 25 grams at the start of the experi- ment. Apparatus Two six-unit drinking boxes were used for the observa- tions of the drinking behavior. The boxes were constructed of wood, with individual Plexiglas covers and hardware cloth floors. Each unit was fixed with a 100 m1. gas collecting tube, graduated in 0.2 ml. (For a more complete description of the drinking boxes, see Appendix). A 0.01-sec. timer was used to measure latency to drink. A foot pedal switch started and stopped the timer. A six compartment carrying-cage was used for tranSport- ing the subjects from the weighing scales to the drinking boxes. (For a description of the carrying-cage, see Appendix). Prggeduze The procedure consisted of three 10-day periods of adaptation to 23.5 hr. water deprivation, with 5-day ad lib periods interpolated between the deprivation periods. The experimental design is presented in Figure 1. 5 AD LIB PERIODS I III DEPRIVATION F— "F PERIODS I n m r 1— DAYS l IO IS 25 4O Figure 1 Experimental design for Experiment 1. Ad lib and Adaptation to deprivation periods are superimposed on days of the experiment. I, II, III: refer to 10-day adaptation to 23.5 hr water deprivation periods. I, II: refer to 5-day ad lib recovery periods. Upon arrival at the laboratory the subjects were random- ly divided, six were assigned to one group and five were assigned to a second group. They were then maintained on ad lib food and water. After three days on ad lib, the subjects were weighed and the water bottles were removed from the home cages. For the next 10 days, the subjects were weighed and given 0.5 hr. free access to water in the drinking boxes. Latency to drink after being placed in the drinking box and water intake were measured daily. Following the tenth day on this schedule, ad lib con- ditions were begun. One group (6 §S) was placed in the drinking boxes for 0.5 hr. at their usual daily drinking time, while the second group (5 S3) was placed in individual compartments in a carrying-cage for that 0.5 hr. This con- tinued for 5 days, during which all subjects were weighed as before and water intakes were recorded for those subjects which were in the drinking boxes. A second 10-day adapt- ation period, identical to the first, followed the five days of ad lib conditions, after which the subjects were again returned to ad lib conditions. The second ad lib period was like the first, except that all subjects were placed in the drinking boxes for 0.5 hr. at their usual daily drinking time. A third 10-day adaptation period was then given, and was identical to the first two described above. On Day 10 of Adaptation periods 2 and 3, a 16% saline injection was administered subcutaneously to all rats immediately following the drinking period. The subjects were then returned to the drinking boxes where postinjection latency to drink was measured. The injections were given in an attempt to determine the reliability of postinjection latency to drink, on two separate occasions, for a given rat. This method was used in Experiment 2 to obtain a 'predicted' postinjection latency to drink. This predicted postinjection latency was used to estimate when the awake rat would drink, and blood samples were drawn at that time under anesthesia. Results The results of the experiment, for both groups combined, are summarized in Figure 2. The top panel of the figure represents the means for reciprocal latency to drink as a function of days of the experiment. There was no over-lap of the distributions of reciprocal latencies for Day 1 and Day 10 of Adaptation 1. No reliable difference was found between the 'carrying-cage' subjects and the 'drinking box' subjects in latency to drink on the first day of Adaptation 2 (t=1.21, df=9), although there was an overall significant difference between Day 10 of Adaptation 1 and Day 1 of Adapt- ation 2 (t=2.08, df=10, p<.05). The latencies continue to shorten during Adaptation 2, but on Day 1 of Adaptation 3, mean reciprocal latency was reliably lower than it was on the last day of the previous Adaptation period (t=2.h6, df=10, p<-025). The center panel of the figure summarizes the water intake, expressed as a percentage of body weight. The differences between Day 1 and Day 10 means for each Adaptation FIGURE 2 Mean reciprocal latency to drink, mean percent body weight intake, and mean body weight, of the 11 albino rats during adaptation to 23.5 hr. water deprivation and during ad lib recovery periods, plotted as a function of days of the experiment. Ad lib periods (I and II), and Adaptation periods (I, II, and III), are as indicated below the baseline. 10 A O 6.. L 0 O .6: .5 sza o» BAD ll 1] 9.5;: 3m 1. z40 o .1 an) m m N _ O 411» “_l n u q d u w—m 0— >404 x o +0.0 m >40 . I o _ >40 o . so; 0 >3... 32 Table 2 Means, medians, and ranges of postinjection latencies to drink following the first and second injection treatments, and for the two injection treatments combined. N=35.* Injection Mean Median Range ‘ ppegtment (nip:sec) (min:§ep) se t :sec Injection I 5:”5 5:28 2:51 to 11:26 Injection II 6:01 5:4h 3:52 to 11:06 Iijections I and II combined 5:53 5:“2 2:51 to 11:26 *Note: Latencies to drink are grouped irreSpective of Day group. 33 The means for blood plasma osmolality-(mOSm./l.) at the initiation of drinking behavior, are plotted as a function of days of adaptation to the deprivation period in Figure 7. An analysis of variance computed on the means indicated that they were significantly different (F=5.787, df=5/29. P<.Ol). Duncan's test (Edwards, 1960), showed that plasma osmolality on Day 0 was reliably higher than on Days 2, 3, 5, and 10 of adaptation (P<.Ol). Plasma protein concentration (grams/100 ml.) was used as an index of plasma volume, increases in plasma protein concentration indicating decreased plasma volume (Stricker, 1966). In Figure 8 are plotted the means for protein con- centration at the initiation of drinking behavior as a function of days of adaptation to the deprivation period. An analysis of variance computed on the means yielded a significant difference in mean protein concentration (F=5.00, df=5/29, p<.01). Duncan's test, indicated that protein concentration at the initiation of drinking be- havior on Day 0 was significantly higher than Days 2, 3, land 5 (p<.01). Also, mean protein concentration for Day 1 Ives significantly higher than the mean concentration for Day 3 (p<.01). The lack of relationship between protein concentration axui osmolality is plotted in scattergram form in Figure 9, Irrotein concentration being plotted as a function of osmol- ality. 3h Figure 7 Mean plasma osmolality at the initiation of drinking be- havior, following injection of 16% saline solution, of 35 albino rats, plotted as a function of days of adapt- ation to 23.5 hr. water deprivation. Standard error indicated by flags. 35 zo_._.<>_mmmo 0... 2033—3204 “.0 m>.mdwo o... zo_._.<._.n__mn_uo o... zo_._.<._.d._._.._<402mo <2m<4d hon non 8N nmN _mN NmN nmN 0. >454 n >400 N >40. _ >40. 0 En:. quIIl-IIIIJJIW oo TH 0.0 S" 5' (D (D I'm OOI/O) NIELOHd VWSV'Id ‘9 IO 9 (D #0 In Figure 10, plasma osmolality and plasma protein con- centration are plotted as a function Of minutes postinjection in which the blood samples were obtained. Discussipp Detection of a state of water imbalance can be indexed by the initiation of corrective behavior, e.g., drinking behavior. Unfortunately, the degree of dehydration neces- sary for the detection process to occur is not as easily indicated. Here, we cannot simply look at the behavioral act of ingestion: the physiological conditions which in- fluence corrective behavior must be tapped. Increased blood plasma concentration, and the cellular changes thereby produced, have been considered to be of critical importance for the instigation and maintenance of water ingestion (Fitzsimons, 1963: Kutscher, 1966: Wolf, 1948). If this is true, latency to drink following a salt injection may reflect the time required for blood con- ditions to change sufficiently to stimulate the detector mechanism. In this experiment, four main questions were asked and attempts were made at their resolution. The questions asked, and the answers obtained from this experiment are presented in the following text. The first question to be resolved was: ”What is the latency to drink of rats, satiated immediately before in- jection of 16% saline solution, while on ad lib and while adapting to 23.5 hr. water deprivation?” 41 Figure 10 Plasma protein concentration and plasma osmolality at the postinjection initiation of drinking behavior of 35 albino rats, plotted as a function of time postinjection when the blood samples were withdrawn. The bar graph represents the number of observations per time period. 42 (O) I "80 N) AII'IV'IOWSO VWSV'Id 3-4 4-5 5-3 6-7 7-3 o-e 9-IOIO-ll II-Ii‘ o o a: a: a: on m m o: o: a: a: I I I I I IIII. E 4 O I I: 01 .. 'at‘t": . .335; d 40 . I: 4 O 1 E 4 g .I L 4 . . L 4 C j I: 4 O - J. I I I I I I I4. I I an“ o: a: co :0 0 <9 ‘I’ e)! "’ ID ID co m 8103‘an (V) (1NOOI/9) .IIO 'ON NIBiOHd VWSV'Id MINUTES POSTINJEOTION 43 Neither mean latencies to drink for the first and the second injection treatments, nor the mean latencies to drink on different treatment days were reliably different. Lat- ency to drink, following salt injection, was not different whether the rat was on ad lib conditions, or on Days 1, 2, 3, 5, or 10, of a 23.5 hr. water deprivation schedule. Thus, it is concluded that latencies to drink, following salt injection, while adapting to 23.5 hr. water deprivation are not different at various stages of the adaptation process. The second question to be answered is: ”How does post- injection latency to drink while adapting (0-10 days on the schedule) to 23.5 hr. water deprivation compare with rats fully adapted (over 20 days On the schedule) to the schedule?" Due to the similarities in procedure, the results of the present experiment can best be compared with the results Obtained by Hatton and Thornton (1968). In both experiments, the rats were habituated to the drinking situation, handling and injection procedures were similar, site and concentration of the injected solution were the same, and injections were administered to satiated animals. The fact that Hatton and Thornton (1968) carried out their manipulations on rats fully adapted to 23.5 hr. water deprivation, whereas in the present experiment the manipulations were carried out on rats during the adaptation process, lends itself to meaning- ful comparisons. Hatton and Thornton (1968) found that the median lat- ency to drink following a 16% saline injection was 44 5 min. 22 sec. for fully adapted rats. This compares quite favorably with the results obtained here for rats during the adaptation process. Here, the median latencies to drink for the two injection treatments were 5 min. 28 sec. and 5 min. 44 sec. The range of the latencies to drink for the Hatton and Thornton experiment were 1 min. 59 sec. to 10 min. 47 sec., while in the present experiment the range of the latencies to drink were 2 min. 51 sec. to 11 min. 26 sec. Thus, it seems that when certain variables are con- trolled (e.g., habituation to the drinking situation, handl- ing procedures, lighting, temperature, and site of injection), latency to drink following a 16% saline injection is highly similar whether administered to rats under ad lib conditions, while adapting to 23.5 hr. water deprivation, or to rats fully adapted to this schedule. The third question put forth was: ”How reliable is latency to drink, in satiated rats following 16% saline injection, over two separate but similar occasions?” It has been demonstrated in Experiment 1, that when deprivation periods are interrupted by ad lib recovery periods, rats must readapt to each ensuing deprivation per- iod. Water intake and body weight require the same adjust- ment process during each adaptation. Due to this recurrent adaptation process, it was possible to inject a rat on two separate occasions and have the animal at approximately the same state of adaptation each time. 45 In the present experiment it was found that only three out of a possible thirty-five rats were more than one min- ute apart in latency to drink following the two injection treatments. A mean shift of 16.53 sec., in latency to drink, from 5 min. 45 sec. to 6 min. 1 see. was not statistically significant. Rats that were above the mean in latency to drink following the first injection, tended to be above the mean in latency to drink following the second injection, and vice versa. This relationship is described by the high correlation coefficient (r=0.711) obtained between the two injection treatments. Rats that initiated drinking extremely fast following the first injection regressed towards the mean on the second injection. On the other hand, rats that were extremely slow to initiate drinking following the first injection were also extremely slow to initiate drinking following the second injection. It is concluded from the above that during adaptation to 23.5 hr. water deprivation, postinjection latencies to drink are highly consistent and reliable. From the answers to the preceding questions it becomes apparent that injection of hypertonic saline solutions have a profound effect on the rat, that of producing the be- havioral act of drinking. The osmotic stress experienced by the rat, following injection, becomes the controlling in- fluence in his behavior. Whether the animal is on ad lib conditions, adapting to 23.5 hr. Water deprivation, or fully adapted to 23.5 hr. water deprivation, the variables which normally control the initiation of drinking behavior are 46 washed-out by the injection of hypertonic saline solutions. Further support for the predominant influence of hyper- tonic saline injections comes from experiments in which water intake postinjection was measured. Hatton and Thorn- ton (1968) found that the amount of water drunk increased monotonically with increased saline concentrations. Oatley (1967b) found that when hypertonic saline injections were administered at different times of the day, there was no potentiation or inhibition of the amount drunk at any of the times when tests were made. The osmotic stimulus swamped the rather small diurnal differences in drinking (found by Oatley, 1967a), and the amount of water drunk was system- atically related only to the salt injections. In order to attain an understanding of the mechanism by which the body detects a state of dehydration, it is necessary to determine bodily conditions when detection and initiation of drinking behavior occur. As has been previously indicated, blood plasma conditions have been considered to be critically involved in the detection process. This brings us to the forth, and most important question: ”What are the plasma conditions (osmolality and protein concentration) at the initiation of drinking be- havior, while on ad lib conditions and while adapting to 23.5 hr. water deprivation?" In an earlier attempt to define some aspects of this problem, Hatton and Thornton (1968) found that 86% of their salt injected rats initiated drinking within 11 min. Later, In using another group of rats, they withdrew blood samples 2, 5, 8, and 11 min. postinjection. Here again, they used rats fully adapted to 23.5 hr. water deprivation and satiated immediately before injection. They found that in response to a 16% saline injection, plasma protein concentration de- creased, and plasma osmolality increased, as a function of minutes postinjection. In the present experiment, by using the mean of the two postinjection latencies to drink, which were shown to be highly consistent, it was possible to calculate a close approximation of a given rats predicted latency to drink. Thus, on a given rats treatment day, the injection was ad- ministered and at the calculated mean latency to drink, the blood sample was drawn. Using this procedure, the results of the present experi- ment do not carry the same relationship reported by Hatton and Thornton (1968). Here, both plasma protein concentration and osmolality were irregular, with no clear-cut increase or decrease as a function of minutes postinjection (Figure 10). The difference between the present experiment and that of Hatton and Thornton was to be anticipated. In the present experiment, time postinjection and blood conditions were de- pendent upon the initiation of drinking behavior, whereas in the Hatton and Thornton experiment the major independent variable was time postinjection. Fitzsimons (1963) attempted to determine the percentage increase in osmotic pressure of body fluids at which rats would start to drink following slow intravenous infusion of 48 hypertonic solutions. The mean percent increase in osmotic pressure at which normal rats, infused with 1-M. sodium chloride, started to drink was 1.6:0.11%. Special emphasis was placed on slow infusion (3.5 to 14.2 micro 1./100 grams body weight/min.) because the increase in osmotic pressure produced by slow infusion is similar to the gradual rise that occurs naturally due to a continuing loss of body water. The percentage increase (1.6%) reported by Fitzsimons is well in accord with Verney's (19u7) estimation of 1-2% in- crease in plasma osmolality being sufficient to trigger cen- tral detector cells. In the present experiment, the dilution of the salt pocket produced by the subcutaneous injection and the sub- sequent elevation in plasma osmolality is probably similar in rate to the osmotic pressure elevations produced by slow intravenous infusion used by Fitzsimons (1963). The results of the present experiment depict plasma conditions at the initiation of drinking behavior while rats adapt to 23.5 hr. water deprivation. The plasma conditions reported here represent threshold values for the initiation of drinking behavior, and can best be appreciated when com- pared with the results of other related experiments conducted in our laboratory. Hatton and Dittrich (1967) withdrew blood samples from rats, while adapting to 23.5 hn water deprivation, at the time when they normally would begin their daily drinking period. These 'predrink' blood samples were drawn on Days 0, 1, 2, 3, 5, and 10, of the adaptation to deprivation period, 49 and represent plasma conditions after 23.5 hrs. of water dep- rivation for successive days of the deprivation period. Mean predrink plasma osmolality is plotted as a function of days of adaptation to 23.5 hr. water deprivation in Figure 11, and mean predrink plasma protein concentration is plotted as a function of days of adaptation to 23.5 hr. water dep- rivation in Figure 12. Using a similar procedure, Hatton and Almli (1967b) withdrew blood samples on Days 0, 1, 2, 3, 5, and 10, of adaptation to 23.5 hr. water deprivation. The blood samples, in this study, were withdrawn immediately following the daily drinking period. These 'postdrink' blood samples represent plasma conditions after 23.5 hrs. of water dep- rivation and drinking to satiation for successive days of the deprivation period. Mean postdrink plasma osmolality (Figure 11) and protein concentration (Figure 12), are plotted as a function of days of adaptation to 23.5 hr. water deprivation. Also in Figures 11 and 12, are the plasma conditions obtained in the present experiment, plotted as a function of days of adaptation to 23.5 hr. water deprivation. These 'postinjection' blood samples represent plasma conditions when rats re-initiate drinking behavior, following drinking to satiation and a salt injection, for successive days of the deprivation period. The combined results of these three experiments define the blood plasma conditions at three points of adaptation to 23.5 hr. water deprivation. They indicate plasma 50 Figure 11 Mean plasma osmolality obtained predrink, postdrink, and postinjection at the initiation of drinking behavior, plotted as a function of days of adaptation to 23.5 hr. water deprivation. Standard error indicated by flags. 'Predrink' data from Hatton and Dittrich (1967), NA36. 'Postdrink' data from Hatton and Almli (1967b), N=36. 'Postinjection' data from the present experiment, N=35. 51 zo_._.<>_mdwo o... 20....4._.n__mn_wc O... 29.523204 “.0 m>HHdoe Hope: .9: m.MN on :oHpopdoum no name no coaponsm m mm .mde .soapooncapmoa. Rom Refine on medosopoa save one .hOabdnon wsAxsanc no sodpoapasd :oHpoonzdpmoa on» no hpAHoHOSmO saunas no mosaob cHOSmOASp zoos m canoe 56 that body weight increases are now seen (Experiment 1). On these days, the initiation of drinking behavior occurs when plasma osmolality increases approximately 2-3% from the 'postdrink' level. The smaller percentage increases in plasma osmolality necessary to produce drinking on Days 1 and 2 are probably due to the fact that the rats are still adjusting to the deprivation. Water intake is not sufficient to maintain body weight (Experiment 1), or to decrease plasma osmolality to the levels found on the days later in the schedule. Thus, the osmotic stress imposed by the salt injection produces drinking with a smaller percentage increase from the 'post- drink' level. However, this phase is highly complex and requires further study. As shown in Figure 12, plasma protein concentration does not reflect a 'threshold' for the initiation of drink- ing behavior as is indicated by plasma osmolality. If changes in plasma protein concentration are interpreted in terms of plasma volume, (e.g., increases in protein concen- tration indicating decrease in plasma volume, and vice versa) on Days 3, 5, and 10, plasma volume at the re-initiation of drinking (postinjection) and following the drinking period (postdrink) are relatively stable with no large differences separating the two conditions. In terms of plasma osmolality on these same days, however, the two conditions are distinct- ly separated. Here, there is a 2-3% increase in osmolality from the postdrink condition to the re-initiation of drink- ing behavior. 57 In contrast, plasma volume at the re-initiation of drink- ing on Days 1 and 2 has increased considerably from the plasma volume following the drinking period. But, the plasma osmolality threshold for these two days represents an in- crease of less than 1% over the 'postdrink' plasma osmol- ality. The ad lib rat, Day 0, does not fit into either of the schemes presented above. At the re-initiation of drinking there is a rather large decrease in plasma volume, with a threshold increase in plasma osmolality of 2.75% over the 'postdrink' condition. Thus, it seems that during adaptation to water dep- rivation there is a process changeover at about the third day of the schedule in regard to the re-initiation of drink- ing following hypertonic injection. Early in the schedule (Day 1 and 2), there are large changes in plasma volume with small changes in plasma osmolality between the 'post- drink' condition and the re-initiation of drinking behavior. Later, (Days 3, 5, and 10), the situation is reversed. Small changes in plasma volume are associated with large changes in plasma osmolality between the 'postdrink' con- dition and the re-initiation of drinking behavior. This seems to indicate that during the early days of adaptation to deprivation, especially Day 1, the rat is not drinking enough water to replenish both the cells and the plasma. When the rat is then injected with hypertonic saline, water is drawn from the cells into the plasma thereby in- creasing plasma volume. Here, it seems likely that the 58 re-initiation of drinking is triggered by dehydration at the cellular level, rather than by changes in plasma osmolality. This interpretation is based on the fact that Day 1 plasma osmolality 'postdrink' and 'postinjection' are not different from plasma osmolality when on ad lib conditions. By Day 3 and beyond, rats drink sufficient amounts of water to rehydrate both the cells and the plasma. At the re-initiation of drinking, plasma volume is not different from plasma volume following the drinking period. Thus, it seems that the injection procedure did not change plasma volume, whereas plasma osmolality at the re-initiation of drinking reflects a distinct threshold, a 2-3% increase in plasma osmolality over the 'postdrink’ condition. The ad lib rat (Day 0) is similar to the rats with more than three days of experience on the deprivation schedule. Although plasma volume at the re-initiation of drinking is decreased from the 'postdrink' condition, it is not differ- ent from the plasma volume at the re-initiation of drinking on Day 10. So here again, the threshold increase in plasma osmolality of 2-3% is indicated at the re-initiation of drinkina behavior. In sum, blood conditions at the re-initiation of drink- ing behavior, following saline injection, seem to be depen- dent on the total degree of hydration experienced by the rat, i.e., at the plasma and cellular level. During the early days on the schedule, when drinking is not sufficient to bring the degree of hydration up to some required level, 59 the rats seem to be reSponding to cellular dehydration rather than elevations in plasma osmolality per se. Later in the schedule, sufficient amounts of water are ingested, and the re-initiation Of drinking following injection, seems to be in response to changes in plasma osmolality. Thus, plasma osmolality seems to be a critical variable for the detection of states of dehydration under the conditions of this experiment. When satiated rats were injected with a hypertonic saline solution on Days 0, 3, 5, and 10, an in- crease in plasma osmolality of approximately 2-3% was sufficient to instigate corrective behavior in the form of drinking. This result corresponds with Verney's (1947) estimation of a 1-2% increase in plasma concentration being sufficient to trigger central detector cells. REFERENCES 60 REFERENCES Bayliss, L. E. L vi C t S stems. San Francisco: W. H. Freeman and Co., 19 6. Beck, R. C. The rats adaptation to a 23.5 hr. water-depriva- tion schedule. J C m h s s chol., 1962, 55, 646-648. Chew, R. H. Water metabolism of mammals. In W. V. Mayer and R. G. Van Gelder (Ed.), Physiplpgical Mammalpgy. New York: Acedemic Press, 1965, 1;, 44-149. . Edwards, A. L. E a D a e ea . New York: Binehart, 1960. Fink, J. B. and Patton, R. M. Decrement of a learned drink- ing response accompanying changes in several stimulus char- acteristicS- W-: 1953. 51.6.. 23-27. Fitzsimons, J. T. The effects of slow infusions of hypertonic solutions on drinking and drinking thresholds in rats. 1. PM” 1963: 1&2: BLILI‘35LI’0 Ghent, Lila. Some effects of deprivation on eating and drink- ing behavior. J C s s ., 1957, 59, 172-176. Gilman, A. The relation between blood osmotic pressure, fluid distribution, and voluntary water intake. e J s .. 1937: 1 0: 323’328- Greenleaf, J. E. Involuntary hypohydration in man and animals: a review. Washington, D. 0.: Nat. Aeronaut. Space Admin., 1966. Hatton, G. I. Unpublished research, 1965. Hatton, G. I. and Almli, C. R. Learned and unlearned com- ponents of the rat's adaptation to water deprivation. Esychpn. m0: 1967: 2: 583-584. (a) Hatton, G. I. and Almli, C. R. Unpublished research, 1967. (b) Hatton, G. I. and Dittrich,R. Unpublished research, 1967. Hatton, G. I. and Thornton, L. W. Hypertonic injections, blood changes, and the initiation of drinking. Q, Cpmp, Bhysipl. Kutscher, C. L. An osmometric analysis of drinking in salt injected rats. Physipl, apd Behav., 1966, ;, 79-83. Oatley, K. Diurnal influences on postdeprivational drinking in rats. J, Cpmp, Rhysipl, Psychpl., 1967, 64, 183-185. (a) 61 62 Oatley, K. Drinking in response to salt injections at diff- erent times of day. Psypnpn, 801., 1967, 9, 439-440. (b) Pitts, R. F. Physiplpgy pf the Kidney and dey Elpids. Chicago: Year Book Medical Pub. Inc., 1965, Page 27 and 32. Ruch, T. C. and Patton, H. D. Physiplpgy and Bipphysips. Philadelphia: W. B. Saunders Co., 1965, Page 891. Stricker, E. M. Extracellular fluid volume and thirst. Thornton, L. W. The influence of subcutaneously injected sodium chloride solutions on readiness to drink and amount of water consumed by albino rats. 1966, Unpublished M.A. thesis, Michigan State University. Verney, E. B. Antidiuretic hormone and the factors which determine its release. 2399, 391, $99, (Lppdpp), Series B, 1947: $35: 25-106. Wayner, M. J.: Wetrus, B.: and Blonk, D. Artifical thirst, serum sodium, and behavioral implications in the hooded rat. W. 1962. 11. 667-674. Wolf, A. V. Estimation of changes in plasma and extracell- ular fluid volume following changes in body content of water and certain solutes by means of an osmometric equation. Ama1i_£i_£h1§igl., 1948, i5}, 499-502. Wolf, A. V. T s 8 he u e d a d wa e c . Springfield: Charles C. Thomas Pub. 1958, Page 20. Young, P. T.: Heyer, A. W.: and Richey, H. W. Drinking patterns in the rat following water deprivation and sub- cutaneous injections of sodium chloride. J, Cpmp, Physipl. EEYQthoo 1952: 4 . 90-95. “ APPENDIX A Apparatus 63 D 8 ti e dr b as The drinking box was made up of six individual drink- ing compartments. Six glass gas collecting tubes were attached to the drinking box, the tubes were 27 in. long and were calibrated in 0.2 ml. The drinking compartments were 1 3/4 in. above a layer of corn-cob chips. The bottom of each drinking compartment consisted of 1/2 in. hardware cloth. Each drinking compartment was 11 3/4 in. long, 5 1/2 in. wide, and 7 3/4 in. deep. The drinking spout ex- tended into the compartment 1 in., through a hole that was 2 1/2 in. from the bottom and 2 1/4 in. from the sides of the compartment. The metal drinking Spout was coupled to the gas collecting tube by rubber tubing. Each compartment was equiped with an individual Plexiglas cover, which was hinged at one end and had magnetic locks on the other end in which to secure the compartment. Des e a -ca e The carrying-cage was made of wood with hardware cloth backing and fiber board doors in front. The carrying-cage consisted of six individual compartments which were 9 in. long, 4 1/8 in. high, and 6 1/4 in. deep. One whole side of the carrying-cage was 1/2 in. hardware cloth, the opposite side was made up of six individual sliding fiber board doors. The doors were equiped with magnetic looks. The overall dimensions of the carrying-cage were 19 in. long, 14 in. high, and 6 1/2 in. deep. A drawer-pull was attached to the top for a handle. 64 Descr t a a e a r The animal restrainer consisted of two wood sections which were hinged together. The base was made of wood and was 11 in. long, 6 in. wide, and 2 in. high. The top section was shorter than the bottom section: 4 1/2 in. long, 6 in. wide, and 2 in. high. Thick sponges were glued to the bottom of the top section and to the top of the bottom section. The rat was placed on the bottom section and the tOp section was pulled down over the rat and secured to the bottom section by a hook. The hind quarters of the rat were exposed, which permitted access to the rat for subcutaneous injection. A diagram of the restrainer is on the following page. 65 WOOD / / y = I/ S PONGE k 6" >|/ ANIMAL RESTRAINER APPENDIX B Procedure 67 Des t e e t r d Each rat, on his treatment day, was taken to an adjoin- ing room in a carrying-cage immediately following the drink- ing period. The rat was etherized in a battery jar, which had wood shavings in the bottom and a hardware cloth plat- form. When the rat was sufficiently immobilized, it was removed from the ether jar and placed on its stomach on a table. The rat was then injected in the hind quarters area and a stop-watch was started at the time of injection. The degree of anesthesia was regulated by means of an ether- filled nose cone. Following the injection the rat was turned on its back. It took approximately 30 sec. to do the heart puncture and draw the blood, therefore, the heart puncture was started 15 sec. before the rat's mean latency to drink. The location of the heart on the rat's left side was determined by touch, the heart was punctured, and approximately 1 1/2 cc. of blood was drawn. At the completion of the heart puncture and drawing of the blood, the whole blood was care- fully injected into a centrifuge tube and centrifuged for about 3 min. During this time the rat was returned to the home cage. When the blood was sufficiently Spun down, the plasma was drawn off. A small drop was analyzed for protein con- centration in a refractometer. The remaining plasma was put into 6 dram vials, which were sealed and immediately frozen. These samples were later analyzed for osmolality 68 69 in a freezing-point osmometer. The blood samples were drawn in heparinized syringes, the heparin was drawn into the syringe until the barrel was coated and then it was squirted out. The Panheprin (1000 U.S.P. units/ml.) was manufactured by Abbott Lab- oratories; Chicago, Ill. The blood samples were Spun down for approximately 3 min. in a centrifuge (Model CL) manufactured by International Equipment Co.; Needham Heights, Mass. Protein concentration (grams/100 ml.) was determined in a Total Solids Meter (American Optical Co., Model 10h01), which measures the refractive index of solutions. The value is read where the sharp boundry between the dark and light fields crosses the scale. The prism was cleaned with dis— tilled water, and dried, after each determination. Osmolality (mOSm) was determined in a freezing-point osmometer (manufactured by Precision Systems; Framingham, Mass., under the brand name of Osmette). Each sample was run through two determinations and the mean was computed as the sample value. If the first two determinations were more than 5 milliosmols apart, a third determination was per- formed and the mean computed on the three determinations. APPENDIX C Raw Data 70 71 o.3« «.«« 3.«« o.3« «.«« «.«« «.«« «.«« «.«« o.o« «.«3 «.«3 33 3.«3 3.«3 «.«3 3.«3 o.«3 «.o« o.«3 3.«3 «.o« 3.«3 3.«3 «.m 03 «.«3 «.3« «.m3 o.o« «.m« «.m« «.o« «.«3 o.m« «.m3 o.3« o.«3 « «.«3 «.«3 «.3 3.33 «.3« «.«3 «.3 «.3 «.3 «.3 3.«3 3.«3 « «.o« 3.«3 «.o« «.«3 «.3« o.m« «.m« «.o« «.3« «.o« «.«3 «.«3 a «.3« «.3 o.3« 3.3 «.o« 3.3« «.3 «.3 «.3 «.«3 «.3 3.3 « «.3« «.3« «.«3 «.m3 o.o« «.«3 «.m3 «.«3 «.«3 3.«3 «.«3 3.33 m «.03 «.3« 3.«« «.«3 3.«3 «.«« «.3« «.«3 «.o« «.«3 «.«3 3.33 3. «.33 «.«3 «.3 «.33 3.3 «.3 «.3 «.«3 «.«3 «.3 «.3 0.3 « o.o« o.«3 «.«3 «.33 o.«3 «.«3 o.«3 3.33 o.«3 o.«3 3.«3 «.03 « «.3« «.o« «.«3 3.«3 «.«3 «.3« o.«3 3.«3 o.3« 3.«3 o.«3 «.33 3 mmm awn awn hwn «mm awn awn awn hmm awn «MG hMQ «M? 333 «H .3 mmqamumlzqmadamqm4 .33Lz .HHH was .HH .H mooahom coapdpmocd wcfihso .nodpmbdh umoo Rooms .3: m.mm mo .03 «mm .m .N .3 made so A.Ha 23V oxwp23 Honda H pzoa3hooxm .HH .H moo3noa :03pwpaouw 303 .co3pmb3naoo nevus .3: m.m~ on no3pdpmdcm mo .Hauz .HHH dad «««.3 ««. ««0.3 «0. ««0.3 «0. «««3. «n.« «««. ««.« «30. n«.«« 33 ««0.3 «0. 003. «0.« «03.3 3«. «on. 3«.« «««. «0.3 «00. 0«.0«3 03 ««0.3 «0. «3«. 33.3 «««.3 «a. ««0.3 «0. «««. ««.3 «00. «3.03 0 ««0.3 00. «00.3 30. «03.3 3«. ««0.3 3«. «««. 33.0 «30. «3.«« « «««. ««. ««0.3 «0. 030.3 00. 333. ««.m «3«. 3«.« «30. ««.3« a ««3.3 ««. ««0.3 «0. 3m«.3 3«. ««0.3 «0. «««. 3«.« «00. 0m.a«3 « «««.3 «a. «««. «0.3 «««.3 «a. 000.3 ««.3 3mm. 3«.3 «no. «3.«« m ««3.3 «a. ««3.3 ««. «««.3 «a. 030.3 00. «3«. «0.3 «00. «n.««« 3 3m«.3 3«. «««.3 ««. «««.3 ««. ««3.3 00. «««. «3.3 «00. ««.333 m «««.3 ««. 030.3 00. «03.3 3«. «33. 03.« 3mm. 3«.3 «00. 33.033 « «««.3 «a» «««.3 «a. ««3.3 «a. «3«. 3«.3 «3«. ««.3 «00. 3«.0mm 3 IIIMMHWIIHquIIIMMHWIIHmAILIIMMMWIIHquImwmllllquqllJMMHIIIHuqlnJMMHIIIIIHdw mmz 03 .Im 3mm 313mm 03 3mm. 3 «mm «alnmm‘ 3+mumw| « 03 20 03 «no 3 mama so 3.00m :3v xCHHU op monopofl Haoonn3oon «no 32330 on hocopwq H psoe3honxm 72 «m3 «m3 «m3 «m3 3«3 «33 «03 «mm 003 «m3 3«m «mm «mm «mm «mm 33 «03 «mm «mm «mm «33 «an «an «an 350 «33 3«« «mm «mm «30 0mm 03 «33 «33 033 003 ««3 «00 «an «an «an «03 ««n «3« 33m «3m 3«m 0 003 303 «on 303 ««3 ««m «an «mm ««m 033 «mm «3m 33m 03m ««m « 033 «03 303 «03 «m3 «0m «00 ««m 3«« 003 ««m «30 0mm «30 ««m a «33 3«3 ««3 «m3 3«3 333 «00 «00 «33 «m3 «um 3mm «mm ««m «an « 003 «mm «on «03 ««3 ««m «an «an 3«« «03 «mm «mm 03m «30 «mm m 000 ««m ««m ««m 333 ««m «mm «mm «mm «an 3«« 030 ««m ««m 03« 3 003 «mm «mm «mm 333 ««m ««m «an ««m 303 «mm «3m 33m «30 «mm m 00m 33m «mm ««m «03 3«« «mm «mm «mm «mm «3m «0m «3« 33« ««m « 3«3 333 «03 «33 3«3 «mm ««m ««m 00« «33 ««m «3m «3m 3«« ««m 3 {WHO «we awn 3M9 3mm mwo «we hmo awn hwm mwn 3WD mwn «mm «mm mz 333 «««3mmmwwaa3amqmdu 3 .33uz .HHH «cw .HH .H «0033oa co3pmpamom you .c03pm>33nou Honda .33 m.mm Op co3pwpowod mo .03 «co .m .N .3 .0 «man so Amamhw 23V 35w3os poom H psoa33oaxm 73 Experiment II Postinjection latency to drink, in min. and sec. and reciprocals (in sec.), for injection 1 and injection 2, for days of adapt- ation to 23.5 hr. water deprivation. (16% sodium chloride). .1. Lat. (Min83ec) Lat. (in sec.) Day _s_ Inj. ‘53. 3(- Inj. Inj. i Group No. I II I II Day 08 1 4827 5819 4853 .0037 .0031 .0034 2 7836 6838 7807 .0021 .0025 .0023 3 6823 5857 6810 .0026 .0027 .0027 4 10816 d0805 10810 .0016 .0016 .0016 5 4820 3852 4806 .0038 .0042 .0040 6 6821 6802 6811 .0026 .0027 .0027 Day 18 1 4854 5807 5800 .0033 .0032 .0033 2 7839 6859 7819 .0021 .0023 .0022 3 6801 6819 6810 .0027 .0026 .0027 4 6810 6831 6820 .0027 .0025 .0026 5 5818 5844 5831 .0031 .0028 .0030 6 4822 5800 4841 .0038 .0033 .0036 Day 28 1 2851 5827 4809 .0058 .0032 .0045 2 3828 4814 3851 .0048 .0039 .0044 3 7807 6831 6849 .0023 .0025 .0024 4 6838 5856 6817 .0025 .0028I .0027 5 5808 5818 5813 .0032 .0031 .0032 6 11826 11806 11816 .0014 .0015 .0015 Day 38 1 5825 6822 5853 .0030 .0026 .0028 2 6801 5821 5841 .0027 .0031 .0029 3 7804 6841 6852 .0023 .0024 .0024 4 5850 6849 6819 .0028 .0024 .0026 5 7835 8841 8808 .0021 .0019 .0020 6 5828 6825 5856 .0030 .0025 .0028 Day 58 1 ---- ---- ---- --------------- 2 5841 6820 6800 .0029 .0026 .0028 g 6807 6858 6832 .0027 .0023 .0025 5828 4854 5811 .0030 .0034 .0032 5 3841 3857 3849 .0045 .0042 .0044 6 4820 5801 4840 .0038 .0033 .0036 Day 108 1 4819 4849 4834 .0038 .0034 .0036 2 4839 5826 5802 .0035 .0030 .0033 3 3800 4858 3859 .0055 .0033 .0044 4 6829 5845 6807 .0025 .0028 .0027 5 4849 4831 4840 .0034 .0036 .0035 6 4843 5837 5810 .0035 .0029 .0032 7L. Experiment II Plasma osmolality and protein concentration at the postinject- ion initiation of drinking behavior, for successive days of adaptation to 23.5 hr. water deprivation. N=35. Day § Mean Postinjection Protein Osmolality Group: No. lat. to drink (g/100 ml.) (mOsm.) ___ M1n8Sec Day 08 71 4853 6.5 .299.3 2 7807 6.6 300.0 3 6810 6.6 302.0 5 ”306 6.6 29805 6 6311 6.7 298.0 Day 18 1 5800 6.6 286.7 2 7319 60“ 29105 3 6810 6.4 290.5 L" 6820 6.“ 30105 5 5831 6.5 299.0 6 14‘3“]. 605 29100 Day 28 1 4809 6.4 286.5 2 3851 6.6 289.0 3 6:49 6.3 283.5 4 6817 6.5 286.0 5 5813 6.2 287.5 6 11816 6.1 293.5 Day 38 1 5853 6.1 286.5 2 5841 60"“ 28605 3 6852 6.3 296.0 4 6819 6.4 291.0 6 5856 6.1 291.5 Day 58 1 ---- --- ----- 2 6800 603 28805 3 6332 601‘“ 29105 1+ 5311 69"" 29605 5 3849 6.2 28300 6 4840 6.5 291.5 Day 10. 1 4.34 6.5 293-5 2 5302 6.5 29205 a 3859 6.6 286.0 6807 6.3 29205 5 4840 6.2 29007 6 5310 603 29203 75 Experiment II Water intake (in ml.) for successive days of adaptation to 23.5 hr. water deprivation during Adaptation IV. N=35. TREATMENT DAIS 1 0.2 ‘1u.2 17.2 20.2 ---- 21.2 2.8 10.6 17.0 18.4 20.6 19.4 3.6 17.8 15.0 17.6 16.6 22.8 4.8 15.8 17.8 20.6 18.8 15.4 1.2 15.2 18.0 19.2 24.8 24.4 3.8 16.8 22.2 19.6 19.2 18.8 Oxkn FUD N 76 000.0 00.0 000.0 00.0 000.0 00.0 00.0 00.0 000.0 00.0 0 000.0 00.0 000.0 00.0 000.0 00.0 1000.0 00.0 000.0 00.0 0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 000.0 00.0 0 000.0 00.0 -8--- ...u 000.0 00.0 000.0 00.0 000.0 00.0 0 mmm 000 mmm 000 .wmm 080 mmm. 000 www. 080 mu w” .m01 0. mm: m 0 00H modpwpmmu< wC0nsu codpwbannou Honda .9: n.mm on :OHpMDQGUw mo 0000 o>0mmooo:m Mom .A.oom QHV MSHHU on hocopmH Hmoonmaomu 0:0 homopmq HH pama0hmmum Experiment II Body weight (in grams), for successive days of adaptation to 23.5 hr. water deprivation during Adaptation IV. TREATMENT DAYS 0 1 2 3 5 10 469 431 433 426 --- 462 086* 030 028 058 001 052 078 052 039 053 021 7025 065 050 062 050 060 055 006 038 021 020 077 000 050 065 072 052 078 031 78 N=35. llalIWIIHHII!\llmlfllllfllllINN1|||W||H|W|Wll 31293 03037 9824