Drinking induced by parenteral injections of pilocarpine

Pharmacology Biochemistry & Behavior, Vol. 5, pp. 633-638. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Drinking Induced by Parenteral Injections of Pilocarpine P A T R I C I A E. G A Y , 2 S A M U E L C. B E N N E R A N D R U S S E L L C. L E A F

Rutgers University, Camden and N e w Brunswick, N.J. (Received 6 October 1976) GAY, P. E., S. C. BENNER, AND R. C. LEAF. DrinMng induced by parenteral injections ofpilocarpine. PHARMAC. BIOCHEM. BEHAV. 5(6) 633-638, 1976. - Parenteral (IP) injections of pilocarpine, in doses from 3.75-30 mg/kg, reliably produced drinking in water-satiated rats. This effect was not diminished by pretreatment with either centrally active (scopolamine, atropine) or peripherally active (methyl scopolamine, methyl atropine) cholinergic blocking agents, suggesting that pilocarpine does not induce drinking via a cholinergic mechanism. Repeated injections of low doses, but not high doses, of pilocarpine augmented drinking over trials. Drinking

Pilocarpine

Scopolamine

Atropine

IN P R E V I O U S studies on the effects of pilocarpine on mouse killing by rats [ 1 , 2 ] , it was noted that pilocarpinetreated animals often drank when water bottles were replaced following mouse killing tests. When water consumption was measured in the course of another mouse killing study [ 3 ] , animals with a history of repeated pilocarpine injections drank significantly m o r e water in a 1-hr test, when 1-hr water deprived and administered 7.5 mg/kg pilocarpine, than did similarly deprived vehicleinjected controls. Animals injected with 15 mg/kg pilocarpine, however, did not increase intake over control levels and 24-hr water deprivation decreased water intake in b o t h pilocarpine dose groups. Because pilocarpine is a c h o l i n o m i m e t i c drug [5] and because central application of cholinergic substances can produce drinking (see review by Myers, [ 6 ] ) , it seemed possible that parenteral injections of pilocarpine might induce drinking by stimulation of cholinergic brain systems involved in drinking. On the o t h e r hand, pilocarpine also activates the parasympathetic system, producing water loss by salivation, lacrimation, defecation, and urination. This latter, peripheral action of pilocarpine might also provide a mechanism for thirst induction. The purpose of the following experiments was two-fold: (1) to explore further the relationship between parenteral pilocarpine administration and water c o n s u m p t i o n , and (2) to determine whether the drinking resulting from pilocarpine administration is related to the c h o l i n o m i m e t i c action of the drug.

EXPERIMENT 1 Previous work [3] suggested that, with short (1-hr) drinking periods, only low doses of pilocarpine elicited drinking in nondeprived rats. It seemed possible that a longer drinking period might identify a more dose-related function.

Animals The animals were 30 adult male H o l t z m a n rats ( 2 8 5 - 3 7 4 g). T h e y were housed and tested in single Wahmann laboratory cages.

Procedure All animals were adapted to a f o o d and water schedule for 3 days in which water was continuously available from water tubes (graduated cylinders stoppered with rubber and a stainless steel drinking tube), but f o o d (Purina pelleted chow) was removed for 3 hours each day. At the end of the 3-day adaptation period, animals were r a n d o m l y assigned to 5 i n d e p e n d e n t drug dose groups of 6 rats each. On Day 4, each group of rats received IP injections of 0.0, 3.75, 7.5, 15.0, or 30.0 mg/kg pilocarpine HC1 (in 1 cc/kg 0.9% NaC1) at the beginning of the 3-hr drinking period. Water intake was measured every 15 min for the first hr and every 30 min thereafter. All tests were administered between 10 a.m. and 1 p.m.

~This research was supported by USPHS Grant MH21247 to Russell C. Leaf, Patricia E. Gay and Sherwood O. Cole, Principal Investigators, and by funds provided by the Rutgers University Research Council. The authors wish to thank Maisy Tang and John L. Falk for their advice on the design and interpretation of these studies and Sandra Colsher for her technical assistance. This paper was presented, in part, at a meeting of the Eastern Psychological Association, New York, N.Y., 1976. 2 Reprint requests to: Patricia E. Gay, Department of Psychiatry, University of Utah Medical Center, 50 North Medical Drive, Salt Lake City, Utah 84132 633

634

GAY, B E N N E R AND L E A F

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150 rain.

180 rnin.

FIG. 1. Cumulative water intake as a function of 5 doses of pilocarpine and time postinjection.

Results and Discussion

Figure 1 shows c u m u l a t i v e water i n t a k e as a f u n c t i o n of dose a n d t i m e p o s t i n j e c t i o n . Because the 15 min and 45 rain readings did n o t differ f r o m the general t r e n d of the data, these data were o m i t t e d . The data for each time period were a n a l y z e d separately using analysis of variance followed b y the following o r t h o g o n a l c o m p a r i s o n s : (1) vehicle c o n t r o l versus pilocarpine dose groups c o m b i n e d , and (2) low doses of p i l o c a r p i n e (3.75 a n d 7.5 mg/kg) versus high doses of p i l o c a r p i n e (15 and 30 mg/kg). On the basis of previous data [ 3 ] , it was p r e d i c t e d t h a t the pilocarpine groups would differ from the vehicle c o n t r o l and t h a t there m i g h t be a t i m e - d e p e n d e n t difference in the effects of the low and high dose groups. As predicted, the p i l o c a r p i n e - t r e a t e d animals d r a n k significantly more t h a n t h e vehicle-injected controls. A t 30 rain p o s t i n j e c t i o n , this difference first b e c a m e statistically d e t e c t a b l e (drug dose effect, F ( 4 , 2 5 ) = 3.15, p < 0 . 0 5 , o r t h o g o n a l c o m p a r i s o n vehicle vs pilocarpine dose groups c o m b i n e d , F ( 1 , 2 5 ) = 7.15, p < 0 . 0 5 ) a n d the increased drinking only b e c a m e m o r e p r o n o u n c e d during successive time periods (drug dose effect, 180 rain, F ( 4 , 2 5 ) = 6.52, p < 0 . 0 1 ; same o r t h o g o n a l c o m p a r i s o n , F ( 1 , 2 5 ) = 16.85, p < 0 . 0 1 ). The t i m e course for d r i n k i n g was dose-related. While vehicle-treated animals d r a n k little and usually only at the b e g i n n i n g of the test session, animals t r e a t e d with low doses of p i l o c a r p i n e d r a n k m o s t during the earlier segments of the drinking period and by 60 90 rain p o s t i n j e c t i o n , the cumulative milliliters c o n s u m e d was inversely related to pilocarpine dose ( o r t h o g o n a l c o m p a r i s o n low vs high dose, F ( 1 , 2 5 ) = 7.20, p < 0 . 0 5 ) . With higher doses of pilocarpine, however, d r i n k i n g was delayed u n t i l later in the drinking period, so t h a t by 180 m i n p o s t i n j e c t i o n , t o t a l c o n s u m p tion was more directly related to drug dose ( o r t h o g o n a l

c o m p a r i s o n , low vs high dose, F ( 1 , 2 5 ) = 6.42, p < 0 . 0 5 ) . O b s e r v a t i o n s of the animals suggested t h a t d r i n k i n g occurred only after the peripheral effects of pilocarpine injections had subsided. EXPERIMENT 2 In E x p e r i m e n t 1, w a t e r c o n s u m p t i o n for the 7.5 m g / k g group was n o t as high as t h a t observed previously [ 3 ] . However, the earlier s t u d y used rats w i t h a history of repeated p i l o c a r p i n e injections. Moreover, it is established that e x c e p t at high doses, r e p e a t e d injections of pilocarpine are necessary to reliably i n d u c e m o u s e killing b y rats [2, 7, 8 ] . The following e x p e r i m e n t e x p l o r e d the possibility t h a t repeated injections of pilocarpine would increase water consumption. In a d d i t i o n , this e x p e r i m e n t had as a s e c o n d purpose e x p l o r a t i o n of the d u r a t i o n a n d n a t u r e of the pilocarpine effect. For one-half of the animals the d r i n k i n g period was e x t e n d e d to 4 hr. The r e m a i n i n g animals were given a 1-hr d r i n k i n g period c o m m e n c i n g 3 hr after p i l o c a r p i n e administration. Animals

The animals were 36 adult, male H o l t z m a n ( 3 0 0 - 5 7 6 g). They were h o u s e d as in E x p e r i m e n t 1.

rats

Procedure

At the b e g i n n i n g of the e x p e r i m e n t , rats were r a n d o m l y assigned to two groups. One group (N = 1 8 ) w a s never w a t e r deprived ( i m m e d i a t e access group), while the o t h e r group was deprived of water for 3 hr daily (delayed access group). All animals were a d a p t e d to a f o o d a n d water schedule for 3 days in w h i c h water was available as d e t e r m i n e d by group a s s i g n m e n t , b u t f o o d was r e m o v e d for 4 hr each day.

PILOCARPINE DRINKING

635

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12345 210 rain.

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FIG. 2. Cumulative water intake as a function of 5 treatments with pilocarpine. Functions on the left are for animals which had the opportunity to drink immediately following pilocarpine injection. Functions on the right are for animals which were not allowed access to water until 3 hr after injection. At the e n d of the 3-day a d a p t a t i o n period, animals in each access group were r a n d o m l y assigned t o 3 i n d e p e n d e n t drug dose c o n d i t i o n s (0.0, 7.5, or 15 m g / k g p i l o c a r p i n e ) of 6 rats each. On Days 4 - 8 , rats received IP i n j e c t i o n s o f t h e a p p r o p r i a t e drug dose at the b e g i n n i n g of the 4-hr test period. F o r animals in t h e i m m e d i a t e access group, w a t e r was available i m m e d i a t e l y f o l l o w i n g i n j e c t i o n a n d i n t a k e was m e a s u r e d every 30 min. F o r animals in t h e delayed access group, w a t e r was m a d e available 3 hr after i n j e c t i o n ant] i n t a k e was m e a s u r e d every 30 m i n for 1 hr. All tests were a d m i n i s t e r e d b e t w e e n 9 : 3 0 a.m. a n d 1:30 p.m. Results and Discussion

Analysis of variance p e r f o r m e d o n t h e t o t a l a m o u n t of water c o n s u m e d i d e n t i f i e d significant dose, F ( 2 , 3 0 ) = 3.96, p < 0 . 0 5 , a n d access group, F ( 1 , 3 0 ) = 19.09, p < 0 . 0 1 , effects as well as significant trials x dose, F ( 8 , 1 2 0 ) = 2.11, p < 0 . 0 5 , and trials x dose x access group, F ( 8 , 1 2 0 ) = 2.14, p < 0 . 0 1 , i n t e r a c t i o n s . As in E x p e r i m e n t 1, p i l o c a r p i n e i n j e c t i o n s increased drinking. The i m m e d i a t e access g r o u p d r a n k significantly m o r e w a t e r t h a n t h e delayed access g r o u p , a l t h o u g h the 1-hr access p e r i o d allowed was m o r e t h a n • a d e q u a t e to drink t h e average 4 5 ml of w a t e r c o n s u m e d by t h e i m m e d i a t e access g r o u p (see Fig. 2). To f u r t h e r explicate the i n t e r a c t i o n s , the data were a n a l y z e d separately for each access group. Analysis of vaiiance p e r f o r m e d o n the d e l a y e d access group data identified o n l y a significant drug dose effect, F ( 2 , 1 5 ) = 4.62, p < 0 . 0 5 . Thus, while delaying access to w a t e r for 3 hr following p i l o c a r p i n e i n j e c t i o n decreases w a t e r c o n s u m p tion s u b s t a n t i a l l y ( f r o m a p p r o x i m a t e l y 5 ml to 1 ml), a small dose effect can still be i d e n t i f i e d . At the same t i m e p o s t i n j e c t i o n , t h e i m m e d i a t e access g r o u p was d e m o n strating nearly zero c o n s u m p t i o n . This suggests t h a t pilo-

carpine has, at m o s t , a weak p h a r m a c o l o g i c a l a c t i o n at 3 - 4 hr p o s t i n j e c t i o n and t h a t w a t e r loss, due to the peripheral effects of p i l o c a r p i n e , m a y c o n t r i b u t e to a m i n o r e x t e n t to the increased water c o n s u m p t i o n seen in the delayed access group. While n o n e of the delayed access groups, s h o w e d significant changes in w a t e r c o n s u m p t i o n over trials, t h e r e was such a change in o n e i m m e d i a t e access group (trials x drug dose i n t e r a c t i o n : F ( 8 , 1 5 ) = 2.82 p < 0 . 0 1 ) . The 7.5 m g / k g p i l o c a r p i n e g r o u p increased w a t e r i n t a k e over trials, b u t i n t a k e r e m a i n e d relatively c o n s t a n t for the 15 m g / k g group. This is similar to the p h e n o m e n o n of pilocarpinei n d u c e d m o u s e killing where, particularly at lower doses, r e p e a t e d i n j e c t i o n s are necessary b e f o r e killing occurs [2, 7,

81. Over trials, m a x i m u m rate of i n t a k e a p p e a r e d to shift to earlier s e g m e n t s o f the d r i n k i n g p e r i o d (see Fig. 2). Ratings of the severity of side effects i n d i c a t e d t h a t t h e peripheral effects of p i l o c a r p i n e also decreased in d u r a t i o n w i t h successive i n j e c t i o n s a n d t h a t m a x i m u m d r i n k i n g o c c u r r e d following cessation of these p e r i p h e r a l effects. Thus, the malaise p r o d u c e d b y p i l o c a r p i n e m a y be i n h i b i t o r y to drinking behavior. EXPERIMENT

3

E x p e r i m e n t 2 suggested t h a t water loss due to the peripheral effects of p i l o c a r p i n e m i g h t c o n t r i b u t e , at least to a m i n o r e x t e n t , to the drug's a c t i o n o n drinking. O b s e r v a t i o n s o n the r e l a t i o n s h i p b e t w e e n the side effects of pilocarpine a n d o n s e t of drinking, h o w e v e r , suggested t h a t the p e r i p h e r a l effects m i g h t i n h i b i t drinking. The following e x p e r i m e n t e x p l o r e d the effects of the cholinergic b l o c k i n g agents, s c o p o l a m i n e and m e t h y l s c o p o l a m i n e , o n piloc a r p i n e - i n d u c e d drinking. It was h o p e d t h a t by these m e a n s

636

GAY, BENNER AND LEAF

we could evaluate the relative contribution of the central and peripheral effects of pilocarpine to pilocarpine-induced drinking. Animals

The animals were 180 adult mate Holtzman ( 3 0 0 - 5 0 0 g). They were housed as in Experiment 1.

rats

injected with the appropriate pretreatment drug (the handling group received an injection of zero volume because preliminary results suggested that volume of pretreatment agent might be an important factor), followed 15 min later by the appropriate dose of pilocarpine. The 3-hr drinking test began immediately after the pilocarpine injection. All tests were administered between 10 a.m. and 1 p.m.

Procedure

All animals were adapted to a food and water schedule for 2 - 3 days as in Experiment 1. At the end of the adaptation period, animals were randomly assigned to one of 6 IP pretreatment conditions (handling, saline vehicle, 0.5 mg/kg scopolamine HBr, 0.5 mg/kg scopolamine methyl Br, 1.0 mg/kg scopolamine HBr, or 1.0 mg/kg scopolamine methyl Br) and one of 5 IP doses of pitocarpine (0.0, 3.75, 7.5, 15.0, or 30.0 mg/kg) to create 30 independent groups of 6 animals each. On the following day, each animal was

z_

Neither scopolamine nor methyl scopolamine reduced total water consumption (see Fig. 3), and, in fact, both doses of scopolamine appeared to facilitate such drinking. Analysis of variance performed on the total intake data identified a significant pretreatment effect, F(5,150) ; 4.16, p<0.01, and a pilocarpine dose effect, F(4,150) = 11.39, p<0.01, but no interaction. T-tests demonstrated that both doses of scopolamine increased drinking beyond

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PILOCARPINE DRINKING

637

control levels. While pretreatment with the quaternary scopolamine analogue did not significantly enhance drinking, neither was a decrement in pilocarpine drinking observed. Both pretreatment drugs also abolished the inverse dose-response relationship of pilocarpine to cumulative water intake usually seen at 90 min postinjection. Analysis of the cumulative intake at 90 rain identified a significant pretreatment effect, F(5,150) = 3.34, p<0.01, a significant pilocarpine dose effect, F(4,150) = 4.26, p<0.01, and a significant pretreatment x pilocarpine dose interaction, F(20,150) = 2.16, p<0.01. As in the case of the 180 min data, t-tests indicated that scopolamine sigmificantly enhanced (p<0.01) drinking beyond control levels, but methyl scopolamine did not. These data suggest that the inverse dose-response function seen 90 min postpilocarpine is due to an inhibitory effect of the parasympathomimetic actions of pilocarpine. When these side effects are blocked, drinking becomes more dose-related. Moreover, these data suggest that pilocarpine-induced drinking is not totally due to water loss generated by the parasympathomimetic effects of pilocarpine. EXPERIMENT 4

To test the hypothesis that the results of Experiment 3 were due to scopolamine's cholinergic blocking properties orL sites involved in drinking and not to some other action of the drug (e.g., general activation), an attempt was made to partially replicate that experiment using atropine and methyl atropine as the cholinergic blocking agents. Animals The animals ( 2 8 0 - 3 3 1 g).

were

29

adult,

male

Holtzman

rats

Pro cedure The animals were adapted to the food and water schedule for 3 days as in Experiments 1 and 3. They were then randomly assigned to 3 pretreatment groups: 2.0 mg/kg atropine SO4 (N = 8), 2.0 mg/kg atropine methylnitrate (N = 8), and saline vehicle (N = 8). (All doses of atropine are expressed as mg/kg of the base.) The next day, all animals were injected with the appropriate pretreatment drag, followed 15 min later by an IP injection of 30 mg/kg, pilocarpine. A fourth group (N = 5) received 2.0 mg/kg atropine and vehicle. Drinking test were conducted as in Experiment 3. Results and Discussion Atropine and methyl atropine also failed to block pilocarpine-induced drinking (see Fig. 4). Animals in the atropine pretreatment group had a mean total intake similar to that of the methyl atropine pretreatment group and the saline pretreatment group. The combined pilocarpine groups, however, did drink significantly more than the group which received atropine and saline, but no pilocarpine (orthogonal comparison: F ( 1 , 2 5 ) = 5.64, p<0.05).

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FIG. 4. Effects of pretreatment with atropine (A), methyl atropine (MA) or saline (S) on drinking induced by 30 mg/kg pilocarpine. The atropine control (AC) group received atropine and saline, but no pilocarpine. GENERAL DISCUSSION In these experiments, drinking was reliably produced by intraperitoneal injections of pilocarpine. Mean total consumption (in a 3-hr period) ranged from approximately 3 . 5 - 7 ml for the highest dose group. The time course of drinking was dose related, with animals receiving low doses of pilocarpine initiating drinking earlier in the test session than those receiving higher doses, although high doses eventually produced more drinking than low doses. Initiation of drinking seemed related to the disappearance of peripheral drug effects. Moreover, blocking these effects (Experiment 3) resulted in earlier initiation of drinking in animals receiving high pilocarpine doses. Increased water consumption following pilocarpine does not appear to be a result of the drug's cholinomimetic activity. The peripheral parasympathetic effects of the drug seem to be primarily inhibitory to drinking. Blocking these effects enhances drinking and does not reduce it as would be expected if the drinking were due to water loss or a peripheral cholinergic thirst mechanism [4]. Blockade of both the central and peripheral effects of the drug also does not reduce drinking, suggesting that the drug is not acting directly on cholinergic brain systems for drinking. The mechanism by which pilocarpine does induce drinking remains unclear, although previous studies [3] demonstrating that amygdala lesions block such drinking suggest that the effect may be a central one. While scopolamine significantly enhanced pilocarpineinduced drinking in Experiment 3, atropine failed to do so in Experiment 4. This suggests that the augmentation of drinking by scopolamine was due to some action of the drug other than its cholinergic blocking properties. As for mouse killing, repeated administration of low doses of pilocarpine appeared to increase drinking behavior. This action may be due to habituation to peripheral effects of the drug [2,8] or possibly to the sensitization of some site involved in pilocarpine-induced drinking [7]. Further research is necessary to elucidate this issue and others concerning pilocarpine-induced drinking.

REFERENCES 1. Gay, P. E., R. C. Leaf and F. B. Arble. Inhibitory effects of preand posttest drugs on mouse-killing by rats. Pharmac. Biochem. Behav. 3: 33-45, 1975.

2. Gay, P. E. and R. C. Leaf. Rat strain differences in pilocarpineinduced mouse killing. Physiol. Psychol. 4: 28-32, 1976.

638 3. Gay, P. E., S. O. Cole and R. C. Leaf. Interactions of amygdala lesions with effects of pilocarpine and d-amphetamine on mouse-killing, feeding, and drinking in the rat. J. comp. physiol. Psychol. 90" 630 642, 1976. 4. Gerald, M. C. and R. P. Maickel. Evidence for peripheral cholinergic components in thirst-induced water consumption. Int. J. Neuropharmac. 8" 337 346, 1969. 5. Goodman, L. S. and A. Gilman (Eds.) The Pharmacological Basis of Therapeutics. New York: The Macmillan Co., 1970.

GAY, B E N N E R AND L E A F 6. Myers, R. D. Handbook o f Drug and Chemical Stimulation of the Brain. New York: Van Nostrand Reinhold, 1974. 7. Vogel, J. R. and R. C. Leaf. Initiation of mouse killing in non-killer rats by repeated pilocarpine treatment. Physiol. Behav. 8:421 424, 1972. 8. Wnek, D. J. and R. C. Leaf. Effects of cholinergic drugs on prey-killing by rodents. Physiol. Behav. 10:1107 1113, 1973.