Physiology & Behavior, Vol. 17, pp. 837--839. Pergamon
Press and Brain Research Publ., 1976. Printed in the U.S.A.
Genetic Analysis of Schedule Induced Polydipsia JAMES P. SYMONS AND RICHARD L.SPROTT
The Jackson Laboratory, Bar Harbor, ME 04609 (Received 26 December 1975) SYMONS, J. P. AND R. L. SPROTT. Genetic analysis of schedule induced polydipsia. PHYSIOL. BEHAV. 17(5) 837-839, 1976. - Susceptibility to schedule induced polydipsia (SIP) was assessed in mice of the C57BL/6J and DBA/2J strains, in the Ft hybrid between these two strains, and in three segregating generations. The results provided clear evidence that susceptibility is inherited. Two alleles have been demonstrated; Sip d, the dominant (susceptible) allele, and Sip b, the recessive (nonsusceptible) allele. Inbred mice
SCHEDULE induced polydipsia (SIP) is a phenomenon which is obtained in rats food deprived to 80% of their ad lib weight and provided with periodic food reinforcement at intervals greater than 45 sec, but less than 180 sec. Following food reinforcement, bursts of drinking occur which result in consumption of abnormal amounts of water (up to 50% of the animal's body weight in 3 hr test sessions). Little or no water is consumed in the animal's home cage during the 21 hr intersession interval [2, 3, 4]. Investigations of possible causes for the SIP phenomenon have centered on physiological and behavioral mechanisms. An example of the approach used to test for physiological mechanisms is the hypothesis that SIP is a result of a dry mouth caused by water being pulled into the stomach by the intermittent presentation of dry food [ 15], as well as by the absorption of water in the mouth by the food itself. If this were the case then animals should exhibit SIP on high density reinforcement schedules which they do not [ 3]. In addition, animals probably should stop working for food when water is not available. However, animals continue to bar press for food when water is not present
number of other behaviors which are schedule induced, e.g., schedule induced aggression, wheel running, and air licking . Recently, Wayner  has advanced a general physiological hypothesis to account for SIP and other adjunctive behaviors as well. Essentially, the Wayner hypothesis supposes that there is a causal relationship between food deprivation and the adjunctive behavior. Food deprivation leads to increased motor excitability and activity, possibly controlled by the lateral hypothalamus. This excitability then becomes conditioned to stimuli associated with intermittent scheduled food delivery. As conditioning develops the adjunctive behavior becomes stereotyped. SIP has been obtained in a number of species, e.g., rhesus monkey , white Swiss mice , chimpanzee , and pigeon . The only reported species in which an attempt to induce SIP was not successful was the golden hamster [ 17]. The research reported here was generated from observations that one inbred mouse strain (C57BL/6J) did not develop SIP after being tested for approximately seven months with 16 different schedules and two different size pellets. This suggested to us that the SIP phenomenon might have a genetic component. The purpose of the present study was to clearly establish the nature of such a genetic component if it could be shown to exist.
[4,8]. An example of behavioral hypotheses is one which supposes that an animal could better predict the appearance of the next pellet by licking at the tube between reinforcements [ 1, 10, 11, 12]. It has been demonstrated, however, that if an empty tube is available rather than water, licking ceases although it would still serve as a timing cue . In addition, if a 50 sec delay is introduced between the last lick and the next reinforcement SIP still developes [ 6]. The most current hypothesis is that SIP is an example of adjunctive behavior. This hypothesis suggests that SIP is an extraneous behavior, which is induced by certain schedules, and which is concurrent with food delivery. There are a
Animals Mice of six genotypes were used in the experiment. Mice were obtained from the Production Department of the Jackson Laboratory, Bar Harbor, Maine, unless otherwise noted. Only male mice were used. The genotypes and sample sizes were C57BL/6J, N = 4; DBA/2J, N = 3;
This investigation was supported by NIH Research Grant GM 21266 from the National Institute of General Medical Sciences. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. 837
SYMONS AND SPROTT on rats [2, 3, 4]. Criteria used to determine polydipsia were: that the drinking be schedule dependent, i.e., more drinking occurred in low density reinforcement schedules than in high density schedules; that the drinking occur postreinforcement rather than scattered throughout the reinforcement intervals, and that the drinking appear to be excessive, i.e., as much as or more water consumed in a number of the 3 hr test sessions than is normally consumed in 24 hr in the home cage, with a minimum mean session intake of 5.5 ml in the last week of testing. A genetic analysis of a single locus model of inheritance requires the use of six genotypic groups: parental types (C57BL/6J and DBA/2J), F~ hybrid (B6D2F1), backcrosses (C 57BL/6J × B6D2F1 and DBA/2J × B6D2F1), and the F2 hybrid (B6D2F1 x B6D2F1). Data from all six groups are then used to assess the adequacy of the model.
C57BL/6J x DBA/2J (B6D2F1), N = 3; C57BL/6J x B6D2F1, N = l l ; B 6 D 2 F 1 x B6D2F1 (B6D2F2), N = 27 (all hybrids bred in experimenter's colony room). All mice were at least 8 weeks of age at the beginning of the test procedure and were housed singly in clear plastic cages. The colony room was maintained at 72 ° -+ 2°F with a 12 hr light/dark cycle.
Apparatus and Procedure Two identical 7.64 x 9.525 x 12.065 cm deep operant conditioning chambers constructed of clear 0.3175 cm Plexiglas were used in the experiment. Both chambers had a grid floor of 0.238 cm diameter stainless steel rods. A mouse lever (Lehigh Valley Model No. 1535)was mounted on one wall and to the left of the lever a food tray was located. 20 mg Noyes food pellets (Formula M) were provided by means of a Gebrands pellet dispenser (Model D). On the wall adjacent to the food tray a 15 cc drinking tube was mounted. The reinforcement schedules were programmed using standard relay circuity. Responses and reinforcements were recorded on counters as well as displayed on cumulative recorders (Gebrands-Harvard Model C-3). In addition, licks were counted by drinkometers (Lehigh Valley Model 2 2 1 - 0 5 ) which counted down a predeterming counter (GS Model 1176). Every 50 licks resulted in a hatch mark on the event line of the cumulative record. At the end of each session these hatch marks were totalled and multiplied by 50 to give the number of licks during a test session. The test chambers were housed in 1.524 x 0.762 x 0.914 meter plywood cubicles. One 25 W bulb was used to light the cubicle and a blower was attached to the cubicle to provide air circulation. Ad lib body weights were obtained for each animal one week prior to the first conditioning session. Three days before conditioning began all but 6 grams of food were removed from the home cage. This procedure usually resulted in a weight loss to about 80% of the ad lib body weight by the first day of conditioning. Animals were maintained at the 80% deprivation level by small supplements of food in the home cage following the daily test session. Each animal was tested 5 days a week for 5 or 6 weeks. The first 2 C57BL/6J mice were tested on the following schedules: Continuous Reinforcement (CRF), Fixed Ratio (FR) 5, 10, 15, 25, 40, 60, 120 and 200 and Fixed Interval (FI) 21, 45, 60, 90, 120, and 150 sec with 45 and 20 mg pellets. For subsequent animals the first 2 weeks consisted of conditioning trials in which the following schedules of reinforcement were used: CRF, FI 30, 60, 90 sec. In weeks 3 through 5 or 6 animals were tested on the FI 150 sec schedule. The determination to test a group of animals 3 or 4 weeks on the FI 150 schedule usually was based on whether there were equipment problems or if a week's data were incomplete. In the initial conditioning sessions animals were tested for 2 hr a day which provided ample time to consume large numbers of food pellets. During the FI 150 sessions animals were allowed to work for 70 reinforcements which resulted in trials lasting approximately 3 hr. Total amount of water consumed, bar presses and licks were recorded daily. The final week's water intake on the FI 150 sec schedule was used in deciding whether an animal exhibited the SIP behavior. We used criteria like those employed in research
RESULTS Animals consuming the greatest and least amount of water for both parental genotypes are shown in Fig. 1. During the test sessions on the FI 150 sec schedule DBA/2J mice consumed up to 11.0 ml of water in 3 hr for the best performance and as low as 7.0 ml of water for the animal showing the least intake. The dips in intake (e.g. Day 19 for DBA's) occurred on Mondays after a weekend of ad lib access to water. Even under these conditions the mice are clearly polydipsic. C57BL/6J mice, on the other hand, ranged from about 2.0 to 0.8 ml of water for the best and least intakes during the test sessions on the FI 150 sec schedule. There was no overlap between the two parental strains. The intake for the F] hybrid (B6D2F1) showed the same range of intake as the DBA/2J genotype.
FI 1.50 SEC
IO.O 9.0 8.0 7.C
5.£ 4.C 3£ 2.C I.C 0
FIG.1.Daily water intakes (greatest and least) of DBA/2J and C57BL/6J mice under experimental conditions favorable to the development of schedule induced polydipsia. Table 1 presents expected and observed ratios for the backcrosses and F2 generations for a single locus model. If a single locus model applied to SIP then all backcross animals to DBA/2J (DBA/2J × B 6 D 2 F I ) would be poly-
GENETIC ANALYSIS OF POLYDIPSIA
839 TABLE 1
A SUMMARY OF SUSCEPTIBILITY TO SCHEDULE INDUCED POLYDIPS1A (SIP). SUSCEPTIBILITY IS EXPRESSED AS THE NUMBER OF SUSCEPTIBLE SUBJECTS TO THE TOTAL NUMBER OF SUBJECTS IN EACH GROUP. EXPECTED RATIOS WERE GENERATED USING MENDELIAN PRINCIPLES AND THE ASSUMPTION OF SINGLE LOCUS INHERITANCE. AN ANIMAL WAS CLASSIFIED AS POLYDIPSIC IF THE MEAN INTAKE DURING THE LAST WEEK OF TESTING WAS 5.5 ML OR GREATER
dipsic. The o b s e r v e d result is 10 o u t of 11 a n i m a l s were polydipsic. F o r t h e b a c k c r o s s to C 5 7 B L / 6 J it w o u l d be e x p e c t e d t h a t half of t h e mice w o u l d show SIP. T h e o b s e r v e d result was 6 a n i m a l s o u t o f 1 1 were polydipsic. F o r the F 2 h y b r i d ( B 6 D 2 F 1 x B 6 D 2 F 1 ) 75% o f t h e animals w o u l d be e x p e c t e d t o be polydipsic or for this sample size 20.25 o u t o f 27. The result we o b s e r v e d was t h a t 18 o u t o f 27 a n i m a l s s h o w e d SIP. T h e X2 b e t w e e n t h e b a c k c r o s s e s was 3 . 6 6 6 7 , p = 0 . 0 5 6 while n o n e of t h e X2 tests for t h e b a c k c r o s s e s a n d F2 g e n o t y p e b e t w e e n o b s e r v e d a n d e x p e c t e d values were significant. DISCUSSION T h e results p r o v i d e s t r o n g e v i d e n c e t h a t SIP is u n d e r t h e i n f l u e n c e of a single locus, provisionally d e s i g n a t e d Sip ( s c h e d u l e i n d u c e d polydipsia). T w o alleles have b e e n d e m o n s t r a t e d : Sip d, t h e d o m i n a n t ( D B A / 2 J ) allele w h i c h results in s c h e d u l e i n d u c e d p o l y d i p s i a susceptibility, and Sipb, t h e recessive ( C 5 7 B L / 6 J ) allele, w h i c h results in
Genotype DBA/2JxFx C57BL/6JxF1 10/I 1 11/11
F2 18/27 20.25/27
n o n s u s c e p t i b i l i t y to polydipsia u n d e r t h e e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this e x p e r i m e n t . We ( u n p u b l i s h e d o b s e r v a t i o n s ) have o b t a i n e d data o n w a t e r c o n s u m p t i o n of f o o d d e p r i v e d animals of t h e p a r e n t a l g e n o t y p e s w h i c h s h o w e d similar intakes. B o t h strains decrease in 24 h r w a t e r c o n s u m p t i o n a f t e r i n t r o d u c t i o n o f f o o d d e p r i v a t i o n to 80% of ad lib b o d y weight, a n d gradually increase i n t a k e s t o near baseline level d u r i n g 19 days o f t h e f o o d d e p r i v a t i o n r e g i m e n ( 3 . 5 - 5 c c / 2 4 hr). T h u s , it w o u l d seem t h a t t h e f o o d d e p r i v a t i o n c o n d i t i o n does n o t cause polydipsia in e i t h e r g e n o t y p e . It is possible t h a t t h e SIP effect is a pleitropic e f f e c t of some k n o w n locus, w i t h k n o w n physiological consequences. This possibility c a n n o t be tested u n t i l t h e Sip locus is l o c a t e d o n a linkage g r o u p a n d c o m p a r e d t o k n o w n loci o f t h e group. However, t h e k n o w l e d g e t h a t genetic variance for t h e trait exists, a n d t h a t susceptible a n d n o n s u s c e p t i b l e animals are readily available, s h o u l d provide t h e i m p e t u s for a f u r t h e r search for physiological correlates of SIP susceptibility.
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