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Anticholinergic blockade of schedule-induced polydipsia
Physiology and Behavior. Vol. 5, pp. 635-640. Pergamon Press, 1970. Printed in Great Britain
Anticholinergic Blockade of ScheduleInduced Polydipsia C H A R L E S D. B U R K S A N D A L A N E. F I S H E R
Psychology Department, University of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A. (Received 28 August 1969) BURGS,C. D. ANDA. E. FISHER. Anticholinergicblockadeofschedule-inducedpolydipsia. PHYSIOL.BEHAV.5 (6) 635-640, 1970.--Intraperitoneal administration of atropine sulfate and atropine methyl nitrate (3, 6, 9 mg/kg) decreased scheduleinduced polydipsia as a function of increasing drug dosage. Physiological saline had no effect. Of the two anticholinergic drugs administered, atropine sulfate attenuated the polydipsic response more effectively. It was concluded that scheduleinduced polydipsia, like other types of excessive drinking (e.g. chemical, deprivation and salt induced drinking) is mediated, at least in part, by cholinergic neural pathways. Some drug effect on eating was observed, particularly following atropine methyl nitrate, but control data indicated that any effects on eating were secondary, and related to anticholinergic effect upon water intake and salivation. Thirst
Schedule-induced polydipsia
Anticholinergic effects on drinking
Anticholinergic sites of action
presentation of food pellets is a sufficient condition for producing schedule-induced polydipsia. Although the aforementioned physiological and behavioral techniques are quite divergent, the behavioral result of these manipulations is to induce water ingestion in the satiated rat. If water intake initiated by cholinergic stimulation and terminated by anticholinergic stimulation can be conceptualized as the outcome of neural activity in functionally related cholinergic pathways in the CNS, then it is reasonable to ask if schedule-induced drinking is also mediated by cholinergic pathways.
IN R~CENT YEARStWO quite different experimental manipulations have been discovered which induce increased water intake in the laboratory rat. In 1960, Grossman [6] reported that direct stimulation of the lateral hypothalamus with small amounts (3-5 ~tg) of crystalline cholinergic drugs induced rats to initiate drinking in the absence of body water deficits. Grossman's findings were extended by Fisher and Coury [5] who identified a number of brain sites from which cholinergic stimulation could trigger a drinking response. These sites correlated closely with the system of limbic structures which Papez [11] and Nauta [10] have proposed as potential substrates for emotional and motivational processes. Evidence for a system of cholinergic neurons which may monitor thirst-related events and mediate thirst-related behavior has been further strengthened by work with anticholinergic agents. Tertiary anticholinergic drugs such as atropine sulfate arid scopolamine have blocked cholinergically induced drinking when applied centrally [8, 14] and peripherally [7]. The same drugs have depressed deprivation [13] and salt-induced drinking [1] following peripheral injection but quaternary anticholinergics (such as atropine methyl nitrate) which pass the blood brain barrier with difficulty, have been virtually ineffective. Independent of investigations using chemical stimulation techniques, Falk [3] reported that when food pellets were distributed on a temporal reinforcement schedule (variable interval 1 min), rats ingested inordinate amounts of water (up to ½ their body weight) during 3.17 hr sessions when water was freely available in the experimental chamber. The phenomenon, known as psychogenic or schedule-induced polydipsia, has been reproduced in a number of studies aimed at specifying the controlling variables. While agreement has not been reached in this regard, it has been verified that spaced
EXPERIMENT 1
The purpose of the present investigation was to superimpose intraperitoneal injections of the anticholinergic drug, atropine sulfate and its quaternary analog, atropine methyl nitrate upon ongoing schedule-induced polydipsia to assess the extent to which injections of these drugs terminate or attenuate schedule-induced polydipsia and to seek further evidence concerning the role of cholinergic pathways in the mediation of drinking. It should be pointed out that the testing conditions for schedule-induced polydipsia afford an important control not provided for in previous investigations of anticholinergic effects on drinking behavior. If the rat decreases or stops drinking but continues to ingest the food pellets during testing, the evidence for specific drug action becomes less tenuous. METHOD
Subjects The subjects were 12 Marland, male, hooded rats mainmined throughout the experiment at 80 per cent of their mean
1This research was supported in part by Public Health Service Grant MH-1951 to the second author and by a post-doctoral fellowship to the first author received under a Public Health Service Psychobiology Training Grant MH-I 1114 to the University of Pittsburgh. 635
¢~36
BURKS AND f=IStlER food pellets and ad lib water'. During these sessions the animals learned the location of food and water and baseline water intake was measured. Home cage water intake (21:! hr/day) was also recorded. Following the baseline procedure, the animals were tested on a Fixed Time (FT) 60 sec food reinforcement schedule in which one pellet was dispensed every 60 sec independently of the animal's behavior. The animals received 150 pellets during the 150 rain experimental session and water was freely available. Experimental injection procedures were not initiated until fluid intake during test sessions had stabilized. Following stabilization of the schedule-induced polydipsic response, each animal received each dose level of atropine sulfate on separated test days in different orders, as well as a control injection of physiological saline. All four solutions (0, 3, 6, 9 mg/kg) were represented on each test day. Following this series, all dose levels of atropine methyl nitrate were administered to each rat in different orders. On drug test days animals received their first 15 pellets in the test cage before drug injection. Pellet dispensing was then stopped, the 15 min pre-injection water intake was recorded, and injections were administered. The session was then resumed with pellets being dispensed each minute for the remaining 135 min. There was at least one standard test day between tests for drug effects. Animals were required to return to pre-injection water intake levels prior to further drug testing.
starting weights of 300 g by limiting their food intake. Sufficient Purina Lab Chow was given daily following testing to maintain the animals at this level.
Apparatus Eight (8) rat testing chambers were equipped with a Davis pellet dispenser and with a food cup positioned 1 ~ in. above a grid floor. Glass water bottles with metal drinking spouts were affixed to the exterior of the chamber. The lip of the spout was kept in. behind a drinking aperture positioned 4~ in. to the right of the food cup. All experimental conditions were programmed with appropriate relay-switching circuitry. Tongue-tube contacts were sensed by standard Grason-Stadler Model E46904 drinkometer circuits, passed through pulse formers, and recorded by electro-mechanical counters and cumulative recorders (Scientific Prototype and Gerbrands). Forty-five (45) mg Noyes lab rat food pellets were used as reinforcers.
Anticholinergic Solutions Solutions were prepared containing 0, 3, 6 or 9 mg of atropine sulfate (Mallinckrodt Chemical Co.) or atropine methyl nitrate (Sigma Chemical Co.) per 1 ml of physiological saline (Abbott Laboratories). The volume of solution administered was 1 ml of either atropine sulfate, atropine methyl nitrate or physiological saline per kg of animal body weight. Pilot work had indicated that doses of atropine sulfate of 10mg/kg or above terminated schedule-induced drinking within 5 min after administration. The 3, 6 and 9 mg/kg concentrations were chosen to determine whether a doseresponse relationship could be demonstrated.
RESULTS
Water intake following administration of physiological saline as well as the varying dosages of atropine sulfate and atropine methyl nitrate is presented in Fig. 1 along with preand post-drug day test session data. It can be seen that drinking decreased progressively as the dose level of atropine sulfate or atropine methyl nitrate increased. While both atropine sulfate and atropine methyl nitrate were effective in reducing schedule-induced polydipsia below control levels, atropine methyl nitrate was less effective. Animals ingested mean
PROCEDURE
The animals were reduced to 80 per cent of their free feeding weight. They were then confined in an experimental chamber for 150 min per day for 5 days and given free access to 150
I00
90
I
---I---
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I
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I
I
I
I
I
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EXPERIMENTAL HOME CAGE H ATROPINE SULFATE O O 0---0 ATROPINE METHYL NITRATE O - - . - - ' C )
80 '-' u
70
== 6 o
z
50
0
~ 4o Z
'~ 30 2o
,A,
IO I PRE DRUG
POST DRUG NoCI
3m9/~
6mg/k q
9mglkq
DOSAGE
FIG. 1. Mean experimental session and home cage water intake for pre-drug, drug and post-drug days.
SCHEDULE, INDUCED POLYDIPSIA
637
values of 38, 31 and 26 cm s of water on days in which 3, 6 and 9 mg of atropine sulfate was administered and 46, 39 and 32 cm 8 for dosages of 3, 6 and 9 mg of atropine methyl nitrate. A mean intake of 66 cm 3 was recorded for days on which physiological saline was administered. Pre-injection intake levels were approximated by the first post-injection test session and were fully recovered by the second post-injection test session. An average of 11 cm 3 of water was consumed in the 15 rain pre-injection baseline period in each drug injection session. The post-injection data presented in Fig. 2 shows a mean of 56 cm 3 consumed following injections of physiological saline; 27, 20 and 15 cm s following 3, 6 and 9 mg injections of atropine sulfate; and 34, 29 and 20 cm 3 following injections of 3, 6 and 9 mg of atropine methyl nitrate. These differences are all statistically significant. A Dunnett's Test [2] performed on the post-injection water intake measures presented in Fig. 2 confirmed that each dose of atropine sulfate or atropine methyl nitrate produced significantly greater decreases in water intake than did physiological saline (p < 0.01). Further, an analysis of variance indicated that the previously mentioned difference in the effectiveness of atropine sulfate and atropine methyl nitrate in reducing schedule-induced polydipsia is statistically significant (p < 0.05) and that the differential intakes following the administration of the 3, 6 and 9 mg doses are also statistically significant under both drug conditions (p < 0.05). Figure 1 documents an increase in home cage water intake during the 21½ hr period following anticholinergic drug administration. As may be seen in Fig. 1, home cage water intake increased by a mean of 7, 7 and 12 cm 3 following administration of 3, 6 and 9 mg of atropine sulfate. Similarly, under atropine methyl nitrate the mean home cage water intake increased by 3, 4 and 12 cm 3 following administration of 3, 6 and 9 mg dosages. Home cage intake levels, however, returned to pre-injection levels by the first post-injection day. Following physiological saline injections, the mean home cage intake increased by only 1 cm 3.
60
I
i
Representative records of the typical patterns of drinking observed following anticholinergic and physiological saline injections are presented in Fig. 3. Licks were cumulatively recorded following each pellet delivery with each subsequent pellet delivery resetting the recording pen to baseline. Although there were individual differences in lick burst lengths, a pattern consisting of pellet delivery closely followed by extended lick bursts was typical for all the animals. On drug test days a normal licking pattern was maintained during the 15 min pre-injection period. The pattern of drinking established in the 15 min pre-injection period continued throughout the session when physiological saline was administered (see Fig. 3). However following administration of an anticholinergic drug, three differentiable drinking patterns emerged. F o r one pattern, drinking continued for 5-25 min following drug administration, stopped abruptly and failed to reappear during the remainder of the session. An equally dramatic pattern differed only in that drinking reappeared near the end of the experimental session. The time of total suppression of drinking did not appear to be consistently related to dose level. If total post-injection intake alone were considered in such cases, the effect of the drug on water intake was often rather minimal. However, analysis of the pattern of responding left no doubt that the drug had a total but transient suppressive effect on the drinking. A third but less frequently observed pattern did not involve a cessation of post-pellet licking but water intake was markedly attenuated. Patterns of drinking observed under anticholinergic stimulation were not found to be specific to atropine sulfate or atropine methyl nitrate but occurred under both conditions and at all dosage levels. Furthermore, none of the animals showed a uniform pattern under all drug conditions. As in previous investigations using peripheral injections of anticholinergic agents [8, 13], there was a minor drug effect on food intake. Under atropine sulfate 14 per cent of the drug administrations was followed by reductions in eating while 39 per cent of the atropine methyl nitrate administrations resulted
I
~ \
M 0---0
i
ATROPINE SULFATE ATROPINE METHYL NITRATE
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40
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iO
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I 0
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I 6 DOSAGE (mglk~l)
I 9
FIG. 2. Mean water intake as a function of drug dosage level. Fifteen rain. pre-injection intakes are excluded.
638
BURKS AND ]f:ISHFP.
in decreased food intake. However, food intake was only effected when drinking was totally suppressed and the total effect upon eating relative to drinking was slight. Table I shows the mean number of pellets left unconsumed at each dosage level. Under 3, 6 and 9 mg dosages of atropine sulfate, there was a 3, 1 and 6 per cent decrease in eating which corn-
pared with a 45, 58 and 63 per cent decrease in drinking. Similarly, 3, 6 and 9 mg dosages of atropine methyl nitrate resulted in 5, 13 and 15 per cent decreases in eating as compared to 35, 48 and 56 per cent decreases in drinking. Occasionally rats allowed pellets to accumulate intermittently after drinking had terminated (particularly under
3OO
(a)
0
t SALINE
(b)
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DRUG
3oo1 (d) ~
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oUffr rW DRUG
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FIG. 3. Patterns of licking observed under physiological saline (a) and drug administrationswherelicking was totally suppressed (b)licking was temporarily suppressed (c) and licking was virtually unchanged but water intake was minimal (d).
SCHEDULE-INDUCED POLYDIPSIA
639
atropine methyl nitrate), but during most tests animals consumed each pellet as it became available whether water was consumed or not.
Mean Pellets Unconsumed
injections of 9 rng/kg of atropine sulfate and two animals received 9 mg/kg of atropine methyl nitrate on the first test day. Three days later the animals that initially received the atropine sulfate received the atropine methyl nitrate and vice versa. On each test day, drugs were administered 10 min before presentation of wet mash. Following the feeding tests the animals were adapted to a 23 hr water deprivation schedule. The animals were given access to water 1 hr daily. Dry food was available at all times. After daily water intake had stabilized atropine sulfate and atropine methyl nitrate injections were administered in the same manner described for the food tests.
Atropine Sulfate Atropine Methyl Nitrate
RESULTS
TABLE 1 MEAN NUMBER OF THE 135 PELLETS UNCONSUMED FOLLOWING ADMINISTRATION OF 3, 6, AND 9 MG/KG OF ATROPINE SULFATE OR ATROPINE METHYL NITRATE
Dosage (mg/kg) 3 6 9
3.75 2.00 8.50
6.76 17.33 20.25
EXPERIMENT 2
One aspect of Experiment 1 makes interpretation of the data equivocal. In that study rats on a food pellet delivery schedule which induced polydipsia were maintained on a chronic food deprivation schedule between tests. Under such conditions eating has to be considered a more probable response than drinking. It follows, then, that the eating response could have been more resistant to any non-specific depressant actions of the drug and that this could explain why drinking seemed to be inhibited selectively. The purpose of Experiment 2 was to test the effects of anticholinergic agents on eating and drinking under conditions which permitted a more rigid test for selective drug action. Unfortunately, it was not feasible to utilize testing situations related directly to psychogenic polydipsia since any major shift in conditions or motivational states would itself disrupt or eliminate the phenomenon under study. Consequently, we chose to measure the effect of anticholinergic agents following equivalent periods of food and water deprivation. METHOD
Table 2 shows that the 9 mg/kg dosages of atropine sulfate and atropine methyl nitrate were ineffective in reducing the intake of wet mash of 23 hr food deprived animals. However, similar drug injections markedly affected water intake under 23 hr water deprivation conditions. Atropine sulfate injections produced a 66 per cent decrease in water intake (p < 0.02) while injections of atropine methyl nitrate reduced water intake by 46 per cent (p < 0.05) when compared to pre-drug intake. These percentage decreases are very similar to the values obtained with the same drug dosage in the scheduleinduced polydipsia tests of Experiment l, although one is starting from different baselines.
TABLE 2 MEAN FOOD AND WATER INTAKE UNDER PRE-DRUG AND DRUG
CONDITIONS 1 hr Food Intake* (g) Drug
Atropine sulfate Atropine methyl nitrate
1 hr Water Intaket (cm3)
Pre-drug Dmg Day Pre-drug Drug Day Day Day
42.75
41.50
31.90
10.75~/
41.25
40.25
31.97
17.40"*
Subjects
The animals were four male, Marland, hooded rats permanently housed in round Plexiglas cages equipped with a glass buret which permitted measurement of water intake to an accuracy of 0.1 ml. Anticholinergic Solutions
*Animals food deprived for 23 hr prior to tests. tAnimals water deprived for 23 hr prior to tests. ~:Significantly different from pre-dmg session (t-test for difference between two correlated sample, d f : 3 ; t = 5.11; p < 0.02). **Significantly different from pre-drug session (t-test for difference between two correlated samples, d r = 3; t = 4.07; p < 0.05).
The highest dosage of anticholinergic drugs utilized in Experiment 1 was selected for use in this study. DISCUSSION PROCEDURE
The animals were first adapted to a 23 hr food deprivation schedule during which water was continually available. During a 1 hr daily feeding period, wet mash consisting of two parts water to one part powdered Purina lab chow was presented in metal food cups which were weighed before and after each feeding period. Once food intake had stabilized, drug injections were administered. Two of the animals received intraperitoneal
Atropine sulfate, previously found to be effective in blocking chemical, deprivation and salt-induced drinking [1, 7, 13] was also effective in Experiment 1 in attenuating scheduleinduced drinking. The data suggest that schedule-induced polydipsia may be mediated, at least in part, by cholinergic mechanisms. Factors in Experiment 1 which might favor food ingestion over water ingestion, however, preclude a clear interpretation on the basis of that data alone. In Experiment 2 the concept of selective anticholinergic
640
Bt)RKS AND t=ISHI::R
action was put to a more stringent test. Since no chronic food deprivation condition was operating in Experiment 2 and since food and water deprivation periods were of equal duration, the drugs had as great an opportunity to disrupt eating as drinking. Nevertheless~ there was a highly selective depression of water intake and no effect on food intake. These results are of interest for two reasons. First, the probability is increased that the selective anticholinergic depression of psychogenic polydipsia in Experiment 1 is a real effect rather than an artifact. Second, these data make it more likely that the slight reduction in eating of dry food noted in Experiment 1 and in previous investigations of ingestion following anticholinergic administration [7, 12, 13] is not related to a general depression of behavior but to dry-mouth conditions resulting from both decreased water consumption and anticholinergic effects on salivation which make it more difficult to swallow dry food. The eating of wet mash was not affected at all by administration of anticholinergic agents in Experiment 2. The finding that atropine methyl nitrate is almost as effective as atropine sulfate in depressing schedule-induced drinking or deprivation drinking is a surprising one. There was a statistically significant difference in the effectiveness of the two drugs in Experiment 1 but the difference was small relative to drug(s) vs. saline differences. Previous investigators [1, 13] have shown the quaternary form (AtMNO3) to be quite ineffective in blocking deprivation or salt-induced drinking. Such studies have strongly suggested a central action for atropine sulfate since atropine methyl nitrate does not pass the blood brain barrier readily, but is a more potent peripheral antimuscarinic agent than atropine sulfate. The present data make it necessary to consider alternative hypotheses relative to the site of action problem. First, the major effects of both atropine sulfate and atropine methyl nitrate in the present studies could be peripheral. Schmidt [12] suggested that atropine blocks acetylcholine action along the entire gastrointestinal tract with concomitant hypomotility of the stomach and intestines. Although these and other peripheral actions could certainly affect behavior, such a theory does not explain why most animals continued to eat in both experiments or why a purely peripheral effect of either form of atropine on salivary secretion should not have actually increased drinking. A second alternative is that atropine sulfate has a predominately central action while atropine methyl nitrate has a
major effect in the periphery. Certainly, atropine methyl nitrate had a much greater effect on food intake than atropine sulfate in Experiment 1, suggesting that this more potent antimuscarinic drug might be having a general, disruptive effect on the animal's ongoing behavior. However, the data from Experiment 2 speak against the general disruption hypothesis, and there is again no obvious reason why drinking should be attenuated selectively by a peripheral action of atropine methyl nitrate. On the contrary. there is more evidence to suggest, as discussed previously, that the effects of both drugs on eating are due to a peripheral action on salivary secretions. Finally, one must consider the possibility that both atropine sulfate and atropine methyl nitrate had a major central effect. Although quaternary nitrogen compounds do not readily penetrate the brain, there is evidence that at least some of the drinking controlled by cholinergic mechanisms (cholinergically-induced drinking) is a low threshold response which can be blocked by extremely small quantities of centrally applied anticholinergic drugs [8, 9]. Firemark [4] has found evidence that a quaternary nitrogen compound (Pyridine-2aldoxime methyl iodide) can enter the brain in small quantities with a selective distribution to specific brain areas. Although direct evidence is lacking, one cannot rule out the possibility that atropine methyl nitrate could have a central anticholinergic action on a very low threshold response. Such an alternative may seem at odds with data reported by Stein [13] and DeWied [1 ] but both investigators found some attenuation of drinking under atropine methyl nitrate and both were dealing with more drastic physiological manipulations. Scheduleinduced drinking may be more sensitive to anticholinergic action than either deprivation-induced drinking or saltinduced drinking. Our present inclination is to favor the hypothesis that both drugs act centrally to inhibit drinking, while atropine methyl nitrate has a more potent peripheral dry-mouth action which partially disrupts eating as well. The data also suggest the hypothesis that cholinergicallyinduced drinking and schedule-induced drinking could be mediated by a common cholinergic mechanism. Further studies are underway to test this possibility, and preliminary results indicate that centrally applied atropine can block either cholinergically-induced drinking or scbedule-induced polydipsia.
REFERENCES
I. De Wied, D. Effect of autonomic blocking agents and structurally related substances on the "salt arousal of drinking." Physiol. Behav. 1: 193-197, 1966. 2. Edwards, A. L. Experimental Design in Psychological Research. New York: Rinehart, 1960. 3. Falk, J. L. Production of polydipsia in normal rats by an intermittent food schedule. Science 133: 195-196, 1961. 4. Firemark, H., C. F. Barlow and L. J. Roth. The penetration of 2-PAM-C14into brain and the effect of cholinesterase inhibitors on its transport. J. Pharmae. exp. Ther. 145: 252-265, 1964. 5. Fisher, A. E. and J. N. Coury. Cholinergic tracing of a central neural circuit underlying the thirst drive. Science 138: 691--693, 1962. 6. Grossman, S. P. Eating or drinking elicited by direct adrenergic or cholinergic stimulation of the hypothalamus. Science 132: 301-302, 1960. 7. Grossman, S. P. Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms. Am. J. Physiol. 202: 1230-1236, 1962.
8. Levitt, R. A. and A. E. Fisher. Anticholinergic blockade of centrally induced thirst. Science 154: 520--522, 1966. 9. Levitt, R. A. and A. E. Fisher. Failure of central anticholinergic brain stimulation to block natural thirst. Physiol. Behaw 2" 425--428, 1967. 10. Nauta, W. J. H. Central nervous organization and the endocrine motor system. In: Advances in Neuroendocrinology, edited by A. R. Nalbondov. Urbana, Illinois: University of Illinois Press, 1963. I 1. Papez, J. W. Proposed mechanism of emotion. Arch. Neurol. Psychiat. 38: 725-743, 1937. 12. Schmidt, H. Jr., S. J. Moak and W. G. van Meter. Atropine depression of food and water intake in the rat. Am. J. Physiol. 192: 543-545, 1958. 13. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46--48, 1963. 14. Stein, L. and J. Seifter. Musearinic synapses in the hypothalamus. Am. J. PhysioL 202: 751-756, 1962.
Anticholinergic Blockade of ScheduleInduced Polydipsia C H A R L E S D. B U R K S A N D A L A N E. F I S H E R
Psychology Department, University of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A. (Received 28 August 1969) BURGS,C. D. ANDA. E. FISHER. Anticholinergicblockadeofschedule-inducedpolydipsia. PHYSIOL.BEHAV.5 (6) 635-640, 1970.--Intraperitoneal administration of atropine sulfate and atropine methyl nitrate (3, 6, 9 mg/kg) decreased scheduleinduced polydipsia as a function of increasing drug dosage. Physiological saline had no effect. Of the two anticholinergic drugs administered, atropine sulfate attenuated the polydipsic response more effectively. It was concluded that scheduleinduced polydipsia, like other types of excessive drinking (e.g. chemical, deprivation and salt induced drinking) is mediated, at least in part, by cholinergic neural pathways. Some drug effect on eating was observed, particularly following atropine methyl nitrate, but control data indicated that any effects on eating were secondary, and related to anticholinergic effect upon water intake and salivation. Thirst
Schedule-induced polydipsia
Anticholinergic effects on drinking
Anticholinergic sites of action
presentation of food pellets is a sufficient condition for producing schedule-induced polydipsia. Although the aforementioned physiological and behavioral techniques are quite divergent, the behavioral result of these manipulations is to induce water ingestion in the satiated rat. If water intake initiated by cholinergic stimulation and terminated by anticholinergic stimulation can be conceptualized as the outcome of neural activity in functionally related cholinergic pathways in the CNS, then it is reasonable to ask if schedule-induced drinking is also mediated by cholinergic pathways.
IN R~CENT YEARStWO quite different experimental manipulations have been discovered which induce increased water intake in the laboratory rat. In 1960, Grossman [6] reported that direct stimulation of the lateral hypothalamus with small amounts (3-5 ~tg) of crystalline cholinergic drugs induced rats to initiate drinking in the absence of body water deficits. Grossman's findings were extended by Fisher and Coury [5] who identified a number of brain sites from which cholinergic stimulation could trigger a drinking response. These sites correlated closely with the system of limbic structures which Papez [11] and Nauta [10] have proposed as potential substrates for emotional and motivational processes. Evidence for a system of cholinergic neurons which may monitor thirst-related events and mediate thirst-related behavior has been further strengthened by work with anticholinergic agents. Tertiary anticholinergic drugs such as atropine sulfate arid scopolamine have blocked cholinergically induced drinking when applied centrally [8, 14] and peripherally [7]. The same drugs have depressed deprivation [13] and salt-induced drinking [1] following peripheral injection but quaternary anticholinergics (such as atropine methyl nitrate) which pass the blood brain barrier with difficulty, have been virtually ineffective. Independent of investigations using chemical stimulation techniques, Falk [3] reported that when food pellets were distributed on a temporal reinforcement schedule (variable interval 1 min), rats ingested inordinate amounts of water (up to ½ their body weight) during 3.17 hr sessions when water was freely available in the experimental chamber. The phenomenon, known as psychogenic or schedule-induced polydipsia, has been reproduced in a number of studies aimed at specifying the controlling variables. While agreement has not been reached in this regard, it has been verified that spaced
EXPERIMENT 1
The purpose of the present investigation was to superimpose intraperitoneal injections of the anticholinergic drug, atropine sulfate and its quaternary analog, atropine methyl nitrate upon ongoing schedule-induced polydipsia to assess the extent to which injections of these drugs terminate or attenuate schedule-induced polydipsia and to seek further evidence concerning the role of cholinergic pathways in the mediation of drinking. It should be pointed out that the testing conditions for schedule-induced polydipsia afford an important control not provided for in previous investigations of anticholinergic effects on drinking behavior. If the rat decreases or stops drinking but continues to ingest the food pellets during testing, the evidence for specific drug action becomes less tenuous. METHOD
Subjects The subjects were 12 Marland, male, hooded rats mainmined throughout the experiment at 80 per cent of their mean
1This research was supported in part by Public Health Service Grant MH-1951 to the second author and by a post-doctoral fellowship to the first author received under a Public Health Service Psychobiology Training Grant MH-I 1114 to the University of Pittsburgh. 635
¢~36
BURKS AND f=IStlER food pellets and ad lib water'. During these sessions the animals learned the location of food and water and baseline water intake was measured. Home cage water intake (21:! hr/day) was also recorded. Following the baseline procedure, the animals were tested on a Fixed Time (FT) 60 sec food reinforcement schedule in which one pellet was dispensed every 60 sec independently of the animal's behavior. The animals received 150 pellets during the 150 rain experimental session and water was freely available. Experimental injection procedures were not initiated until fluid intake during test sessions had stabilized. Following stabilization of the schedule-induced polydipsic response, each animal received each dose level of atropine sulfate on separated test days in different orders, as well as a control injection of physiological saline. All four solutions (0, 3, 6, 9 mg/kg) were represented on each test day. Following this series, all dose levels of atropine methyl nitrate were administered to each rat in different orders. On drug test days animals received their first 15 pellets in the test cage before drug injection. Pellet dispensing was then stopped, the 15 min pre-injection water intake was recorded, and injections were administered. The session was then resumed with pellets being dispensed each minute for the remaining 135 min. There was at least one standard test day between tests for drug effects. Animals were required to return to pre-injection water intake levels prior to further drug testing.
starting weights of 300 g by limiting their food intake. Sufficient Purina Lab Chow was given daily following testing to maintain the animals at this level.
Apparatus Eight (8) rat testing chambers were equipped with a Davis pellet dispenser and with a food cup positioned 1 ~ in. above a grid floor. Glass water bottles with metal drinking spouts were affixed to the exterior of the chamber. The lip of the spout was kept in. behind a drinking aperture positioned 4~ in. to the right of the food cup. All experimental conditions were programmed with appropriate relay-switching circuitry. Tongue-tube contacts were sensed by standard Grason-Stadler Model E46904 drinkometer circuits, passed through pulse formers, and recorded by electro-mechanical counters and cumulative recorders (Scientific Prototype and Gerbrands). Forty-five (45) mg Noyes lab rat food pellets were used as reinforcers.
Anticholinergic Solutions Solutions were prepared containing 0, 3, 6 or 9 mg of atropine sulfate (Mallinckrodt Chemical Co.) or atropine methyl nitrate (Sigma Chemical Co.) per 1 ml of physiological saline (Abbott Laboratories). The volume of solution administered was 1 ml of either atropine sulfate, atropine methyl nitrate or physiological saline per kg of animal body weight. Pilot work had indicated that doses of atropine sulfate of 10mg/kg or above terminated schedule-induced drinking within 5 min after administration. The 3, 6 and 9 mg/kg concentrations were chosen to determine whether a doseresponse relationship could be demonstrated.
RESULTS
Water intake following administration of physiological saline as well as the varying dosages of atropine sulfate and atropine methyl nitrate is presented in Fig. 1 along with preand post-drug day test session data. It can be seen that drinking decreased progressively as the dose level of atropine sulfate or atropine methyl nitrate increased. While both atropine sulfate and atropine methyl nitrate were effective in reducing schedule-induced polydipsia below control levels, atropine methyl nitrate was less effective. Animals ingested mean
PROCEDURE
The animals were reduced to 80 per cent of their free feeding weight. They were then confined in an experimental chamber for 150 min per day for 5 days and given free access to 150
I00
90
I
---I---
~
I
I
I
I
I
I
I
I
I
EXPERIMENTAL HOME CAGE H ATROPINE SULFATE O O 0---0 ATROPINE METHYL NITRATE O - - . - - ' C )
80 '-' u
70
== 6 o
z
50
0
~ 4o Z
'~ 30 2o
,A,
IO I PRE DRUG
POST DRUG NoCI
3m9/~
6mg/k q
9mglkq
DOSAGE
FIG. 1. Mean experimental session and home cage water intake for pre-drug, drug and post-drug days.
SCHEDULE, INDUCED POLYDIPSIA
637
values of 38, 31 and 26 cm s of water on days in which 3, 6 and 9 mg of atropine sulfate was administered and 46, 39 and 32 cm 8 for dosages of 3, 6 and 9 mg of atropine methyl nitrate. A mean intake of 66 cm 3 was recorded for days on which physiological saline was administered. Pre-injection intake levels were approximated by the first post-injection test session and were fully recovered by the second post-injection test session. An average of 11 cm 3 of water was consumed in the 15 rain pre-injection baseline period in each drug injection session. The post-injection data presented in Fig. 2 shows a mean of 56 cm 3 consumed following injections of physiological saline; 27, 20 and 15 cm s following 3, 6 and 9 mg injections of atropine sulfate; and 34, 29 and 20 cm 3 following injections of 3, 6 and 9 mg of atropine methyl nitrate. These differences are all statistically significant. A Dunnett's Test [2] performed on the post-injection water intake measures presented in Fig. 2 confirmed that each dose of atropine sulfate or atropine methyl nitrate produced significantly greater decreases in water intake than did physiological saline (p < 0.01). Further, an analysis of variance indicated that the previously mentioned difference in the effectiveness of atropine sulfate and atropine methyl nitrate in reducing schedule-induced polydipsia is statistically significant (p < 0.05) and that the differential intakes following the administration of the 3, 6 and 9 mg doses are also statistically significant under both drug conditions (p < 0.05). Figure 1 documents an increase in home cage water intake during the 21½ hr period following anticholinergic drug administration. As may be seen in Fig. 1, home cage water intake increased by a mean of 7, 7 and 12 cm 3 following administration of 3, 6 and 9 mg of atropine sulfate. Similarly, under atropine methyl nitrate the mean home cage water intake increased by 3, 4 and 12 cm 3 following administration of 3, 6 and 9 mg dosages. Home cage intake levels, however, returned to pre-injection levels by the first post-injection day. Following physiological saline injections, the mean home cage intake increased by only 1 cm 3.
60
I
i
Representative records of the typical patterns of drinking observed following anticholinergic and physiological saline injections are presented in Fig. 3. Licks were cumulatively recorded following each pellet delivery with each subsequent pellet delivery resetting the recording pen to baseline. Although there were individual differences in lick burst lengths, a pattern consisting of pellet delivery closely followed by extended lick bursts was typical for all the animals. On drug test days a normal licking pattern was maintained during the 15 min pre-injection period. The pattern of drinking established in the 15 min pre-injection period continued throughout the session when physiological saline was administered (see Fig. 3). However following administration of an anticholinergic drug, three differentiable drinking patterns emerged. F o r one pattern, drinking continued for 5-25 min following drug administration, stopped abruptly and failed to reappear during the remainder of the session. An equally dramatic pattern differed only in that drinking reappeared near the end of the experimental session. The time of total suppression of drinking did not appear to be consistently related to dose level. If total post-injection intake alone were considered in such cases, the effect of the drug on water intake was often rather minimal. However, analysis of the pattern of responding left no doubt that the drug had a total but transient suppressive effect on the drinking. A third but less frequently observed pattern did not involve a cessation of post-pellet licking but water intake was markedly attenuated. Patterns of drinking observed under anticholinergic stimulation were not found to be specific to atropine sulfate or atropine methyl nitrate but occurred under both conditions and at all dosage levels. Furthermore, none of the animals showed a uniform pattern under all drug conditions. As in previous investigations using peripheral injections of anticholinergic agents [8, 13], there was a minor drug effect on food intake. Under atropine sulfate 14 per cent of the drug administrations was followed by reductions in eating while 39 per cent of the atropine methyl nitrate administrations resulted
I
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ATROPINE SULFATE ATROPINE METHYL NITRATE
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FIG. 2. Mean water intake as a function of drug dosage level. Fifteen rain. pre-injection intakes are excluded.
638
BURKS AND ]f:ISHFP.
in decreased food intake. However, food intake was only effected when drinking was totally suppressed and the total effect upon eating relative to drinking was slight. Table I shows the mean number of pellets left unconsumed at each dosage level. Under 3, 6 and 9 mg dosages of atropine sulfate, there was a 3, 1 and 6 per cent decrease in eating which corn-
pared with a 45, 58 and 63 per cent decrease in drinking. Similarly, 3, 6 and 9 mg dosages of atropine methyl nitrate resulted in 5, 13 and 15 per cent decreases in eating as compared to 35, 48 and 56 per cent decreases in drinking. Occasionally rats allowed pellets to accumulate intermittently after drinking had terminated (particularly under
3OO
(a)
0
t SALINE
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# DRUG
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DRUG
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FIG. 3. Patterns of licking observed under physiological saline (a) and drug administrationswherelicking was totally suppressed (b)licking was temporarily suppressed (c) and licking was virtually unchanged but water intake was minimal (d).
SCHEDULE-INDUCED POLYDIPSIA
639
atropine methyl nitrate), but during most tests animals consumed each pellet as it became available whether water was consumed or not.
Mean Pellets Unconsumed
injections of 9 rng/kg of atropine sulfate and two animals received 9 mg/kg of atropine methyl nitrate on the first test day. Three days later the animals that initially received the atropine sulfate received the atropine methyl nitrate and vice versa. On each test day, drugs were administered 10 min before presentation of wet mash. Following the feeding tests the animals were adapted to a 23 hr water deprivation schedule. The animals were given access to water 1 hr daily. Dry food was available at all times. After daily water intake had stabilized atropine sulfate and atropine methyl nitrate injections were administered in the same manner described for the food tests.
Atropine Sulfate Atropine Methyl Nitrate
RESULTS
TABLE 1 MEAN NUMBER OF THE 135 PELLETS UNCONSUMED FOLLOWING ADMINISTRATION OF 3, 6, AND 9 MG/KG OF ATROPINE SULFATE OR ATROPINE METHYL NITRATE
Dosage (mg/kg) 3 6 9
3.75 2.00 8.50
6.76 17.33 20.25
EXPERIMENT 2
One aspect of Experiment 1 makes interpretation of the data equivocal. In that study rats on a food pellet delivery schedule which induced polydipsia were maintained on a chronic food deprivation schedule between tests. Under such conditions eating has to be considered a more probable response than drinking. It follows, then, that the eating response could have been more resistant to any non-specific depressant actions of the drug and that this could explain why drinking seemed to be inhibited selectively. The purpose of Experiment 2 was to test the effects of anticholinergic agents on eating and drinking under conditions which permitted a more rigid test for selective drug action. Unfortunately, it was not feasible to utilize testing situations related directly to psychogenic polydipsia since any major shift in conditions or motivational states would itself disrupt or eliminate the phenomenon under study. Consequently, we chose to measure the effect of anticholinergic agents following equivalent periods of food and water deprivation. METHOD
Table 2 shows that the 9 mg/kg dosages of atropine sulfate and atropine methyl nitrate were ineffective in reducing the intake of wet mash of 23 hr food deprived animals. However, similar drug injections markedly affected water intake under 23 hr water deprivation conditions. Atropine sulfate injections produced a 66 per cent decrease in water intake (p < 0.02) while injections of atropine methyl nitrate reduced water intake by 46 per cent (p < 0.05) when compared to pre-drug intake. These percentage decreases are very similar to the values obtained with the same drug dosage in the scheduleinduced polydipsia tests of Experiment l, although one is starting from different baselines.
TABLE 2 MEAN FOOD AND WATER INTAKE UNDER PRE-DRUG AND DRUG
CONDITIONS 1 hr Food Intake* (g) Drug
Atropine sulfate Atropine methyl nitrate
1 hr Water Intaket (cm3)
Pre-drug Dmg Day Pre-drug Drug Day Day Day
42.75
41.50
31.90
10.75~/
41.25
40.25
31.97
17.40"*
Subjects
The animals were four male, Marland, hooded rats permanently housed in round Plexiglas cages equipped with a glass buret which permitted measurement of water intake to an accuracy of 0.1 ml. Anticholinergic Solutions
*Animals food deprived for 23 hr prior to tests. tAnimals water deprived for 23 hr prior to tests. ~:Significantly different from pre-dmg session (t-test for difference between two correlated sample, d f : 3 ; t = 5.11; p < 0.02). **Significantly different from pre-drug session (t-test for difference between two correlated samples, d r = 3; t = 4.07; p < 0.05).
The highest dosage of anticholinergic drugs utilized in Experiment 1 was selected for use in this study. DISCUSSION PROCEDURE
The animals were first adapted to a 23 hr food deprivation schedule during which water was continually available. During a 1 hr daily feeding period, wet mash consisting of two parts water to one part powdered Purina lab chow was presented in metal food cups which were weighed before and after each feeding period. Once food intake had stabilized, drug injections were administered. Two of the animals received intraperitoneal
Atropine sulfate, previously found to be effective in blocking chemical, deprivation and salt-induced drinking [1, 7, 13] was also effective in Experiment 1 in attenuating scheduleinduced drinking. The data suggest that schedule-induced polydipsia may be mediated, at least in part, by cholinergic mechanisms. Factors in Experiment 1 which might favor food ingestion over water ingestion, however, preclude a clear interpretation on the basis of that data alone. In Experiment 2 the concept of selective anticholinergic
640
Bt)RKS AND t=ISHI::R
action was put to a more stringent test. Since no chronic food deprivation condition was operating in Experiment 2 and since food and water deprivation periods were of equal duration, the drugs had as great an opportunity to disrupt eating as drinking. Nevertheless~ there was a highly selective depression of water intake and no effect on food intake. These results are of interest for two reasons. First, the probability is increased that the selective anticholinergic depression of psychogenic polydipsia in Experiment 1 is a real effect rather than an artifact. Second, these data make it more likely that the slight reduction in eating of dry food noted in Experiment 1 and in previous investigations of ingestion following anticholinergic administration [7, 12, 13] is not related to a general depression of behavior but to dry-mouth conditions resulting from both decreased water consumption and anticholinergic effects on salivation which make it more difficult to swallow dry food. The eating of wet mash was not affected at all by administration of anticholinergic agents in Experiment 2. The finding that atropine methyl nitrate is almost as effective as atropine sulfate in depressing schedule-induced drinking or deprivation drinking is a surprising one. There was a statistically significant difference in the effectiveness of the two drugs in Experiment 1 but the difference was small relative to drug(s) vs. saline differences. Previous investigators [1, 13] have shown the quaternary form (AtMNO3) to be quite ineffective in blocking deprivation or salt-induced drinking. Such studies have strongly suggested a central action for atropine sulfate since atropine methyl nitrate does not pass the blood brain barrier readily, but is a more potent peripheral antimuscarinic agent than atropine sulfate. The present data make it necessary to consider alternative hypotheses relative to the site of action problem. First, the major effects of both atropine sulfate and atropine methyl nitrate in the present studies could be peripheral. Schmidt [12] suggested that atropine blocks acetylcholine action along the entire gastrointestinal tract with concomitant hypomotility of the stomach and intestines. Although these and other peripheral actions could certainly affect behavior, such a theory does not explain why most animals continued to eat in both experiments or why a purely peripheral effect of either form of atropine on salivary secretion should not have actually increased drinking. A second alternative is that atropine sulfate has a predominately central action while atropine methyl nitrate has a
major effect in the periphery. Certainly, atropine methyl nitrate had a much greater effect on food intake than atropine sulfate in Experiment 1, suggesting that this more potent antimuscarinic drug might be having a general, disruptive effect on the animal's ongoing behavior. However, the data from Experiment 2 speak against the general disruption hypothesis, and there is again no obvious reason why drinking should be attenuated selectively by a peripheral action of atropine methyl nitrate. On the contrary. there is more evidence to suggest, as discussed previously, that the effects of both drugs on eating are due to a peripheral action on salivary secretions. Finally, one must consider the possibility that both atropine sulfate and atropine methyl nitrate had a major central effect. Although quaternary nitrogen compounds do not readily penetrate the brain, there is evidence that at least some of the drinking controlled by cholinergic mechanisms (cholinergically-induced drinking) is a low threshold response which can be blocked by extremely small quantities of centrally applied anticholinergic drugs [8, 9]. Firemark [4] has found evidence that a quaternary nitrogen compound (Pyridine-2aldoxime methyl iodide) can enter the brain in small quantities with a selective distribution to specific brain areas. Although direct evidence is lacking, one cannot rule out the possibility that atropine methyl nitrate could have a central anticholinergic action on a very low threshold response. Such an alternative may seem at odds with data reported by Stein [13] and DeWied [1 ] but both investigators found some attenuation of drinking under atropine methyl nitrate and both were dealing with more drastic physiological manipulations. Scheduleinduced drinking may be more sensitive to anticholinergic action than either deprivation-induced drinking or saltinduced drinking. Our present inclination is to favor the hypothesis that both drugs act centrally to inhibit drinking, while atropine methyl nitrate has a more potent peripheral dry-mouth action which partially disrupts eating as well. The data also suggest the hypothesis that cholinergicallyinduced drinking and schedule-induced drinking could be mediated by a common cholinergic mechanism. Further studies are underway to test this possibility, and preliminary results indicate that centrally applied atropine can block either cholinergically-induced drinking or scbedule-induced polydipsia.
REFERENCES
I. De Wied, D. Effect of autonomic blocking agents and structurally related substances on the "salt arousal of drinking." Physiol. Behav. 1: 193-197, 1966. 2. Edwards, A. L. Experimental Design in Psychological Research. New York: Rinehart, 1960. 3. Falk, J. L. Production of polydipsia in normal rats by an intermittent food schedule. Science 133: 195-196, 1961. 4. Firemark, H., C. F. Barlow and L. J. Roth. The penetration of 2-PAM-C14into brain and the effect of cholinesterase inhibitors on its transport. J. Pharmae. exp. Ther. 145: 252-265, 1964. 5. Fisher, A. E. and J. N. Coury. Cholinergic tracing of a central neural circuit underlying the thirst drive. Science 138: 691--693, 1962. 6. Grossman, S. P. Eating or drinking elicited by direct adrenergic or cholinergic stimulation of the hypothalamus. Science 132: 301-302, 1960. 7. Grossman, S. P. Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms. Am. J. Physiol. 202: 1230-1236, 1962.
8. Levitt, R. A. and A. E. Fisher. Anticholinergic blockade of centrally induced thirst. Science 154: 520--522, 1966. 9. Levitt, R. A. and A. E. Fisher. Failure of central anticholinergic brain stimulation to block natural thirst. Physiol. Behaw 2" 425--428, 1967. 10. Nauta, W. J. H. Central nervous organization and the endocrine motor system. In: Advances in Neuroendocrinology, edited by A. R. Nalbondov. Urbana, Illinois: University of Illinois Press, 1963. I 1. Papez, J. W. Proposed mechanism of emotion. Arch. Neurol. Psychiat. 38: 725-743, 1937. 12. Schmidt, H. Jr., S. J. Moak and W. G. van Meter. Atropine depression of food and water intake in the rat. Am. J. Physiol. 192: 543-545, 1958. 13. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46--48, 1963. 14. Stein, L. and J. Seifter. Musearinic synapses in the hypothalamus. Am. J. PhysioL 202: 751-756, 1962.