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Prandial drinking induced by atropine
Physiology and Behavior. Vol. 5, pp. 549-554. Pergamon Press, 1970. Printed in Great Britain
Prandial Drinking Induced by Atropine H A R O L D W. C H A P M A N -~ A N D A L A N N. E P S T E I N
Department of Biology and Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, Pa., U.S.A. (Received 2 June 1969) H. W. ANn A. N. EpS~IN. Prandialdrinking induced by atropine. PHYSIOL.BEHAV.,~(5) 549-554, 1970.Rats injected with atropine (1-2.25 mg/kg) before daily 1 hr feeding-drinking sessions acquired the prandial drinking characteristic of surgically desalivate rats. Two to three sessions were required for the full development of prandial drinking. As a consequence of the acquisition of prandial drinking, atropine increased total water intake. Atropine methyl nitrate (which has little central activity) was as effective as atropine sulfate. In 4 of the 6 rats prandial drinking persisted after atropine injections ceased. Persistent prandial drinking was suppressed by pilocarpine and by intra-oral infusions of water with each food pellet. Temporary (pharmacological) as well as permanent (surgical) desalivation induced prandial drinking, supporting the idea that this drinking is generated by the necessity for lubrication when dry food must be swallowed from a dry mouth. Prandial drinking is therefore under peripheral control.
CHAPMAN,
Prandial drinking
Atropine
Drinking
WATER INGESTIONis not necessarily related to the systemic water needs of the rat [2]. It may occur despite gross overhydration [11] and may be maintained by peripheral mechanisms when the ability of the rat to respond to systemic needs has been destroyed by brain lesions [6]. Desalivate rats drink small draughts of water after each pellet even when their systemic needs are met by intragastric hydration [5]. This drinking, which has been termed prandial drinking [12], is gradually acquired after desalivation and is believed to be a learned solution to the problem of swallowing dry food from a dry mouth [4]. Prandial drinking has been distinguished from the mealrelated drinking of normal rats by Kissileff [4]. Normal rats drink large draughts (0.5-2.5 ml) close to meals (84 per cent within 20 min before or after meals of dry food). Only 0.3 per cent of their total intake is in draughts which both occur within a meal and are smaller than 0.5 ml. In contrast, all the drinking of desalivate rats occurs within meals and is in small draughts, of which 80 per cent are less than 0.2 ml. The meals of desalivate rats are therefore repeated sequences of morsels of dry food immediately followed by small draughts of water. Previous investigators of prandial drinking have used surgical procedures. Temporary, reversible desalivation by atropine provides a useful means to confirm and extend the earlier findings. The use of atropine for pharmacological desalivation is of particular interest since the central effect of atropine is to depress water intake [9]. Increased water intake produced by the peripheral action of atropine would emphasize the dichotomy between the peripheral and the central controls of drinking. In addition the occurrence of
prandial drinking in rats with functional salivary glands would permit the converse experiment, the abolition of prandial drinking by drugs which increase salivary flow, such as pilocarpine. GENERAL METHODS
Sprague-Dawley male rats (300-350 g) were trained to press a bar for food pellets and were adapted to a schedule of 23 hr deprivation of food and water. They were fed for 1 hr in a box fitted with a water bottle (with a glass spout and connected to a drinkometer circuit) and a bar which delivered 45 mg Noyes pellets (on a schedule that imposed a minimum interval of 1.5 see between pellet deliveries in order to minimize accumulation of pellets in the hopper). Wasted food was weighed and subtracted from the amount of food delivered. Licking was recorded by the stepping pen of a cumulative recorder (vertical excursions in the accompanying figures) and pellet delivery by deflections of the pen (hatch marks in the figures). A complete description of the apparatus and the method has been published elsewhere [4]. I. ACQUISITIONAND PERSISTENCEOF PRANDIALDRINKING
Method Six rats were injected intraperitoneally with 1.0, 1.5 or 2.25 mg/kg atropine sulfate or atropine methyl nitrate (the latter has little central activity [10]) in a solution of 0.4 mg/ml water. (The drugs were: atropine sulfate injection, U.S.P., with 0.5 per cent chlorobutanol preservative and atropine methyl
1Supported by USPHS Grant NB 03469 and a grant-in-aid from the Nutrition Foundation (to A.N.E.) and by USPHS Grant MH 15767 (to the Institute of Neurological Sciences). This work was briefly reported at the American Physiological Society meeting in August, 1967 (abstracted in Physiologist, Wash. 10: 140, 1967) and was a portion of the thesis submitted by H. W. Chapman for the Ph.D. degree of the University of P0nnsylvania, 1969. sPrescnt address: Biology Department, McMaster University, Hamilton, Ontario, Canada. 549
550
CHAPMAN AND EPSTEIN
nitrate (Inland Alkaloids, Tipton, Ind.) of which solutions were made for each experiment. The solutions were refrigerated after use.) These doses were based on the results of pilot experiments. The injections were made with a minimum of restraint within 30 sec before the start of the session. The rats were given two injections six days apart followed after a recovery day by a course of four consecutive daily injections (in an attempt to maximize the number of persistent prandial drinkers produced; see Results). Twelve days later each rat was injected once with atropine of the other type. The criterion for prandial drinking was that of Kissileff [4] and was that the rats drank minute (rarely more than 0.5 ml) draughts of water immediately after the ingestion of each morsel (one or two pellets) of food throughout the feeding period. In addition, ten rats (supplementary group) which were persistent prandial drinkers after atropine injections were given ad lib food and water for three months. They were then readapted to the feeding schedule and tested for persistence of prandial drinking. Results and Discussion Both types of atropine at all three doses produced prandial drinking. In no rat did prandial drinking occur before the
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first atropine injection. In four of the six cases the prandial drinking persisted after the cessation of atropine injections. Figure l shows the acquisition of prandial drinking in a typical rat. Each day's intake is shown by a pair of bars, solid for water intake and hatched for food intake. In addition, eight complete feeding-drinking records are shown for the days indicated. The first record (that of the day before the first atropine injection) shows the ingestive pattern typical of rats on a 23 hr food and water deprivation schedule. The alternation of eating and drinking contrasts with the ingestive pattern of the rat fed ad lib which [4] drinks larger draughts and seldom drinks within a meal. On the first day of atropine injection (second record) the rat wasted most of the food delivered. The adoption of prandial drinking on the second atropine day resulted in an increase of draught frequency and a decrease in draught size (and in the rat wasting less food). On the last control day before the drug injections the rat drank 25 draughts (mean size 0.52 ml) while on the second atropine day it drank 165 draughts, only one of which was greater than 0.5 ml (mean size 0.13 ml). This ingestive pattern, resulting from the rat's drinking after every one or two pellets, was never seen in untreated rats maintained on this schedule, and in this respect prandial
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FIG. 1. The development of prandial drinking by rat No. 6 on the second day of atropine treatment (Day 11). Dose rate was 1 mg/kg. Histogram above shows daily food (hatched bars) and water (solid bars) intake. Atropine sulfate (solid triangles) injected on Days 5, 11, 13-16, atropine methyl nitrate (open triangles) on Day 28 only. Complete feeding records below for the days indicated. Upper traces of records: licks prodLmed cumulative vertical movement of pen, pellet delivery caused hatch mark. Lower traces: every 20th pellet caused hatch mark. Anorexia produced by atropine methyl nitrate was atypical. Most rats responded similarly to both types of atropine.
PRANDIAL DRINKING INDUCED BY ATROPINE
551
drinking is therefore unlike schedule-induced polydipsia [2] although the behavioral patterns are similar. As a result of the acquisition of prandial drinking the water intake was elevated. After the atropine injections stopped, the prandial drinking and hyperdipsia persisted for one day. Then the rat showed a diminishing tendency to drink prandially at the beginning of each session. Table 1 summarizes the results for all six rats. They all acquired prandial drinking within two to three sessions. Before prandial drinking was acquired the atropinized rats pressed for fewer pellets, accumulated or scattered many pellets, and chewed wastefully. After prandial drinking was acquired the rate of pressing was increased and little food was wasted. In four of the rats prandial drinking persisted for at least two weeks following the consecutive atropine injections. There was no detectable difference in the responses to the two types of atropine, nor was there any effect of the different levels of deprivation (as reflected by the weight ratios) on the persistence of prandial drinking. Rat 10 failed to eat or drink during the first four atropine sulfate injections and on the
lI. EFFECT ON WATER INTAKE Method The second part of the study measured the effect of atropine on the total water intake and the water/food ratio uncomplicated by the anorexia and learning effects of the acquisition studies. All the rats used had previously acquired prandial drinking but were not persistent prandial drinkers at the time the water intake data were collected. Atropine sulfate and atropine nitrate were injected on randomly selected days, the intakes on the day before injection being used as control data. At least four days elapsed between injections in order to allow recovery. Lower doses of atropine (0.25-1.0 mg/kg) were used in order to minimize anorexia. The eight rats used included four used in the acquisition experiment and four from pilot experiments, in which eight out of nine rats exposed to a variety of schedules of food deprivation and atropine treatment had acquired prandial drinking. Three of the latter four had been persistent prandial drinkers and had been used in the experiments described in part III.
TABLE 1 AC'QUISITION OF PRANDIAL DRINKING BY ATROPINIZED RATS
Rat
Atropine
Dose (mg/kg)
Treatment Days to Prandial Drinking
Persistence without Atropine
Body Wt during Testing as ~o of Starting Wt
6 9 8 5 10 11
Sulfate Methyl nitrate Sulfate Methyl nitrate Sulfate Methyl nitrate
1 1 1.5 1.5 2.25 2.25
2 1 3 3 --* 3
no yes yes partial yes no
83 89 80 75 82 80
*Rat was prostrated by drug treatment, showed prandial drinking on subsequent no-treatment days.
third occasion was fed a supplement of dry food in its home cage at the end of the session. When returned to the experimental schedule the rat rapidly became an extremely regular prandial drinker and remained one for the rest of its laboratory life (six months). During this period the only atropine injection it received was one of atropine methyl nitrate which also caused a severe depression of food and water intake. The degree to which prandial drinking persisted without atropine was variable. Persistent prandial drinking usually occurred only at the beginning of a feeding session in the box and did not result in any increase in the water/food ratio. In two rats (9 and 10) the persistent prandial drinking was extremely regular and occurred throughout the remainder of their laboratory lives (six months) during which they were maintained on a 1 hr food-access schedule with ad lib water (Purina pellets were fed on schedule in the home cage between tests). Lastly, persistent prandial drinking was observed in six of the ten rats in the supplementary group when they were tested on schedule after a three month rest period with ad lib food and water.
Results and Discussion Both types of atropine, at all doses, produced increased water intakes as a consequence of prandial drinking. The water intake data for each rat are shown in Fig. 2 which shows the intake on all atropine days as a percentage of the intake on the previous no atropine day. The absolute water intake was increased on 42/50 atropine days (as shown in the figure) while the relative water intake (water/food ratio) was increased on 47/50 days. The mean increases were 46.2 per cent (absolute intakes) and 166.8 per cent (relative intakes). Within the range used there was no apparent dose effect, and it seems probable that the effect is a threshold one. The similarity of the effects produced by the two types of atropine at low dose rates is strong evidence that the drugs act peripherally in producing prandial drinking and the resulting increase in water intake. It is possible that schedule effects [2] contribute to the increased water intakes especially in light of the finding [5] that prandial drinking can be abolished by the intraoral injection of amounts of water smaller than those that the rat
552
CHAPMAN AND EPSTEIN
drinks, implying that the desalivate rat drinks more than is necessary to swallow the dry food. The mean food intake on atropine days was 10.6 g (equivalent to 235 pellets). This (self-imposed) rate of feeding is considerably greater than the (experimenter-imposed) rates of feeding which have been reported [2] as resulting in schedule-induced polydipsia.
in Fig. 4. Note that prandial drinking immediately returned when the infusions ceased. This reappearance of prandial drinking makes it likely that the oral infusion suppressed the drinking directly by replacing the ingested water rather than indirectly by altering the situation. In all six rats persistent prandial drinking disappeared
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4
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FIG. 2. Increased water intake as a result of prandial drinking in atropinized rats. Data shown as percentages of intakes on previous (no drug) days, on a log. scale beginning with 35 per cent. Note similarity of effects of atropine sulfate (solid triangles) and methyl nitrate (open triangles).
I11. SUPPRESSION OF PERSISTENT PRANDIAL DRINKING
Method
Six rats which persisted as prandial drinkers after the termination of atropine treatment were used in the third part of the study. Two were from the acquisition experiment and four were from the pilot experiments. The general method of the tests was as described above. 4 mg/ml Pilocarpine nitrate (U.S.P.) solutions were made for each experiment and refrigerated after use. The Pilocarpine was injected intraperitoneally, in doses of 1-4 mg per rat and at pre-feeding intervals in the range of 5-60 min. In addition, two of the rats were fitted with oral cannulae (the apparatus and procedure has been described by Kissileff [6]) and water was injected into the mouth, just after the delivery of each pellet, in amounts approximately equal to the rat's mean draught size. In other trials the rats were allowed to feed ad lib overnight in the test box or were fed Purina pellets in their home cages (on schedule) in order to assess the effect of the deprivation conditions and the feeding situation on persistent prandial drinking.
Results and Discussion
Pilocarpine suppressed persistent prandial drinking in each of the six rats. Typical records of complete control and drug sessions are shown for each of three rats in Fig. 3. In each case pilocarpine greatly reduced the number of occasions on which drinking followed pellet delivery and ingestion. Oral infusion of water also effectively suppressed prandial drinking in both rats in which this procedure was carried out, as shown
gradually when the rats were allowed to feed in the test chamber overnight. Within 24 hr the feeding patterns resembled those of normal rats. A sample record of such an experiment is shown in Fig. 3 C (24 hr access excerpt). In none of the rats did prandial drinking occur in the home cages. These results show that persistent prandial drinking was not the result of severe, permanent impairment of the glands.
GENERAL DISCUSSION
Atropinized rats eating dry food resemble surgically desalirate rats both in their prandial style of drinking and in the gradual acquisition of this behavior. This phenomenological similarity and the fact that atropine methyl nitrate, which has little central effect, was as effective as atropine sulfate indicate that the effect was mediated by salivary inhibition, a wellestablished peripheral effect of atropine. These data therefore provide support for the peripheral explanation of prandial drinking advanced by Kissileff [5]. Increased water intake was produced by both types of atropine and was secondary to the adoption of prandiat drinking. It is therefore an indirect peripheral effect of atropine in contrast to the central blockade of thirst reported by Stein [9]. Previous investigators of the effect of atropine on the ingestive behavior of rats have avoided frequent and prolonged drug exposures or have given the drug when food or water were available separately [7, 9]. Thus prandial drinking was unlikely or impossible and only the central, de~essant, effect of atropine on water intake was observed. The only exception is a recent report [8] of an increase in water intake resulting from the administration of atropine to rats with ad
PRANDIAL DRINKING INDUCED BY ATROPINE
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FIG. 3, Suppression of persistent prandial drinking by pilocarpine and ad lib feeding (24 hr access). Piiocarpine effect for rats 12 (a, above) and 3 (b, above) and both piloearpine and ad lib feeding effects for rat 10 (c, below). Pilocarpine records are complete one hour sessions. Control sessions are previous (no drugs) days. Excerpt from ad lib day is 75 rain segment 24 hr after access to food on a FI 1.5 see schedule. Control record is of first hour of access. Recording method as in Fig. 1.
lib access to food and water. This increase was attributed to the central diuretic effect of atropine and the feeding behavior of the rats was not recorded. In view of the data reported here it seems likely that these rats were prandial drinkers. These experiments do not support Cannon's theory [1 ] that thirst is an innate reflex for which a dry mouth is the stimulus and drinking the response. On the contrary, since naive rats never drank in response to the first pellets ingested after atropinization we may conclude that dry-mouth drinking is not part of the rat's normal behavioral repertoire. Prandial drinking is almost certainly an operant behavior acquired by rats in response to the novel problem of having to swallow dry food from a dry mouth. The increased drinking of desalivate dogs in the heat [3] may have a similar basis and should
be re-investigated. The fact that this peripherally-controlled, operant-type drinking was induced by a drug that depresses centrally-controlled drinking indicates that the neurological systems for regulatory and prandial drinking are relatively independent despite the identity of their output. The persistence of prandial drinking after it has been induced by atropine remains unexplained. It is, however, of interest as a possible link between prandial drinking, which occurs whenever a desalivate rat is eating dry food, and schedule-induced polydipsia [2], which occurs in normal rats but only under a limited range of conditions (enforced delays between small morsels of dry food). It may be that marginal salivary insufficiency and schedule factors are summating to produce persistent prandial drinking.
554
CHAPMAN AND EPSTEIN
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FIG. 4. Suppression of persistent prandial drinking by intra-oral infusion of water. A 5 sec infusion followed each pellet except for the few pellets obtained during pump operation. During control periods rat drank 0.07 ml/pellet. Mean volume of infusion was 0.08 ml/pellet. Recording method as in Fig. 1 except lower trace not shown and recorder at faster speed (Rat No. 10).
REFERENCES 1. Cannon, W. B. Hunger and thirst. In: Handbook of General Experimental Psychology, edited by C. Murchison. Worcester, Mass,: Clark Univ. Press, 1934, pp. 247-263. 2. Falk, J. L. Conditions producing psychogenic polydipsia in animals. Annls N. Y. Acad. ScL 157: 569-589, 1969. 3. Gregersen, M. I. and W. B. Cannon. The effect of extirpation of the salivary glands on the water intake of dogs while panting. Am. J. Physiol. 102: 336-343, 1932. 4. Kissileff, H. R. Food-associated drinking in the rat. J. comp. physiol. Psychol. 67: 284-300, 1969. 5. Kissileff, H. R. Oropharyngeal control of prandial drinking. J. comp. physiol. Psychol. 67: 309-319, 1969. 6. Kissileff, H. R. and A. N. Epstein. Exaggerated prandial drinking in the "recovered lateral" rat without saliva. J. comp. physiol. Psychol. 67: 301-308, 1969.
7. 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. 8. Soulairac, A. and M. L. Soulairac. Dissociation experimentale de la soif et de la diurcse sous l'action de l'atropine. C. R. Acad. Sci. Paris 266:908-911, 1968. 9. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46-48, 1963. 10. Stoll, H.C. Pharmacodynamic considerations of atropine and related compounds. Am. J. _,Wed.Sci. 215: 577-592, 1948. 11. Stricker, E. M. and E. R. Adair. Body fluid balance, taste and postprandial factors in schedule-induced polydipsia. J. comp. physiol. Psychol. 65: 449--454, 1966. 12. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome. Psychol. Rev. 69: 74-90, 1962.
Prandial Drinking Induced by Atropine H A R O L D W. C H A P M A N -~ A N D A L A N N. E P S T E I N
Department of Biology and Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, Pa., U.S.A. (Received 2 June 1969) H. W. ANn A. N. EpS~IN. Prandialdrinking induced by atropine. PHYSIOL.BEHAV.,~(5) 549-554, 1970.Rats injected with atropine (1-2.25 mg/kg) before daily 1 hr feeding-drinking sessions acquired the prandial drinking characteristic of surgically desalivate rats. Two to three sessions were required for the full development of prandial drinking. As a consequence of the acquisition of prandial drinking, atropine increased total water intake. Atropine methyl nitrate (which has little central activity) was as effective as atropine sulfate. In 4 of the 6 rats prandial drinking persisted after atropine injections ceased. Persistent prandial drinking was suppressed by pilocarpine and by intra-oral infusions of water with each food pellet. Temporary (pharmacological) as well as permanent (surgical) desalivation induced prandial drinking, supporting the idea that this drinking is generated by the necessity for lubrication when dry food must be swallowed from a dry mouth. Prandial drinking is therefore under peripheral control.
CHAPMAN,
Prandial drinking
Atropine
Drinking
WATER INGESTIONis not necessarily related to the systemic water needs of the rat [2]. It may occur despite gross overhydration [11] and may be maintained by peripheral mechanisms when the ability of the rat to respond to systemic needs has been destroyed by brain lesions [6]. Desalivate rats drink small draughts of water after each pellet even when their systemic needs are met by intragastric hydration [5]. This drinking, which has been termed prandial drinking [12], is gradually acquired after desalivation and is believed to be a learned solution to the problem of swallowing dry food from a dry mouth [4]. Prandial drinking has been distinguished from the mealrelated drinking of normal rats by Kissileff [4]. Normal rats drink large draughts (0.5-2.5 ml) close to meals (84 per cent within 20 min before or after meals of dry food). Only 0.3 per cent of their total intake is in draughts which both occur within a meal and are smaller than 0.5 ml. In contrast, all the drinking of desalivate rats occurs within meals and is in small draughts, of which 80 per cent are less than 0.2 ml. The meals of desalivate rats are therefore repeated sequences of morsels of dry food immediately followed by small draughts of water. Previous investigators of prandial drinking have used surgical procedures. Temporary, reversible desalivation by atropine provides a useful means to confirm and extend the earlier findings. The use of atropine for pharmacological desalivation is of particular interest since the central effect of atropine is to depress water intake [9]. Increased water intake produced by the peripheral action of atropine would emphasize the dichotomy between the peripheral and the central controls of drinking. In addition the occurrence of
prandial drinking in rats with functional salivary glands would permit the converse experiment, the abolition of prandial drinking by drugs which increase salivary flow, such as pilocarpine. GENERAL METHODS
Sprague-Dawley male rats (300-350 g) were trained to press a bar for food pellets and were adapted to a schedule of 23 hr deprivation of food and water. They were fed for 1 hr in a box fitted with a water bottle (with a glass spout and connected to a drinkometer circuit) and a bar which delivered 45 mg Noyes pellets (on a schedule that imposed a minimum interval of 1.5 see between pellet deliveries in order to minimize accumulation of pellets in the hopper). Wasted food was weighed and subtracted from the amount of food delivered. Licking was recorded by the stepping pen of a cumulative recorder (vertical excursions in the accompanying figures) and pellet delivery by deflections of the pen (hatch marks in the figures). A complete description of the apparatus and the method has been published elsewhere [4]. I. ACQUISITIONAND PERSISTENCEOF PRANDIALDRINKING
Method Six rats were injected intraperitoneally with 1.0, 1.5 or 2.25 mg/kg atropine sulfate or atropine methyl nitrate (the latter has little central activity [10]) in a solution of 0.4 mg/ml water. (The drugs were: atropine sulfate injection, U.S.P., with 0.5 per cent chlorobutanol preservative and atropine methyl
1Supported by USPHS Grant NB 03469 and a grant-in-aid from the Nutrition Foundation (to A.N.E.) and by USPHS Grant MH 15767 (to the Institute of Neurological Sciences). This work was briefly reported at the American Physiological Society meeting in August, 1967 (abstracted in Physiologist, Wash. 10: 140, 1967) and was a portion of the thesis submitted by H. W. Chapman for the Ph.D. degree of the University of P0nnsylvania, 1969. sPrescnt address: Biology Department, McMaster University, Hamilton, Ontario, Canada. 549
550
CHAPMAN AND EPSTEIN
nitrate (Inland Alkaloids, Tipton, Ind.) of which solutions were made for each experiment. The solutions were refrigerated after use.) These doses were based on the results of pilot experiments. The injections were made with a minimum of restraint within 30 sec before the start of the session. The rats were given two injections six days apart followed after a recovery day by a course of four consecutive daily injections (in an attempt to maximize the number of persistent prandial drinkers produced; see Results). Twelve days later each rat was injected once with atropine of the other type. The criterion for prandial drinking was that of Kissileff [4] and was that the rats drank minute (rarely more than 0.5 ml) draughts of water immediately after the ingestion of each morsel (one or two pellets) of food throughout the feeding period. In addition, ten rats (supplementary group) which were persistent prandial drinkers after atropine injections were given ad lib food and water for three months. They were then readapted to the feeding schedule and tested for persistence of prandial drinking. Results and Discussion Both types of atropine at all three doses produced prandial drinking. In no rat did prandial drinking occur before the
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first atropine injection. In four of the six cases the prandial drinking persisted after the cessation of atropine injections. Figure l shows the acquisition of prandial drinking in a typical rat. Each day's intake is shown by a pair of bars, solid for water intake and hatched for food intake. In addition, eight complete feeding-drinking records are shown for the days indicated. The first record (that of the day before the first atropine injection) shows the ingestive pattern typical of rats on a 23 hr food and water deprivation schedule. The alternation of eating and drinking contrasts with the ingestive pattern of the rat fed ad lib which [4] drinks larger draughts and seldom drinks within a meal. On the first day of atropine injection (second record) the rat wasted most of the food delivered. The adoption of prandial drinking on the second atropine day resulted in an increase of draught frequency and a decrease in draught size (and in the rat wasting less food). On the last control day before the drug injections the rat drank 25 draughts (mean size 0.52 ml) while on the second atropine day it drank 165 draughts, only one of which was greater than 0.5 ml (mean size 0.13 ml). This ingestive pattern, resulting from the rat's drinking after every one or two pellets, was never seen in untreated rats maintained on this schedule, and in this respect prandial
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FIG. 1. The development of prandial drinking by rat No. 6 on the second day of atropine treatment (Day 11). Dose rate was 1 mg/kg. Histogram above shows daily food (hatched bars) and water (solid bars) intake. Atropine sulfate (solid triangles) injected on Days 5, 11, 13-16, atropine methyl nitrate (open triangles) on Day 28 only. Complete feeding records below for the days indicated. Upper traces of records: licks prodLmed cumulative vertical movement of pen, pellet delivery caused hatch mark. Lower traces: every 20th pellet caused hatch mark. Anorexia produced by atropine methyl nitrate was atypical. Most rats responded similarly to both types of atropine.
PRANDIAL DRINKING INDUCED BY ATROPINE
551
drinking is therefore unlike schedule-induced polydipsia [2] although the behavioral patterns are similar. As a result of the acquisition of prandial drinking the water intake was elevated. After the atropine injections stopped, the prandial drinking and hyperdipsia persisted for one day. Then the rat showed a diminishing tendency to drink prandially at the beginning of each session. Table 1 summarizes the results for all six rats. They all acquired prandial drinking within two to three sessions. Before prandial drinking was acquired the atropinized rats pressed for fewer pellets, accumulated or scattered many pellets, and chewed wastefully. After prandial drinking was acquired the rate of pressing was increased and little food was wasted. In four of the rats prandial drinking persisted for at least two weeks following the consecutive atropine injections. There was no detectable difference in the responses to the two types of atropine, nor was there any effect of the different levels of deprivation (as reflected by the weight ratios) on the persistence of prandial drinking. Rat 10 failed to eat or drink during the first four atropine sulfate injections and on the
lI. EFFECT ON WATER INTAKE Method The second part of the study measured the effect of atropine on the total water intake and the water/food ratio uncomplicated by the anorexia and learning effects of the acquisition studies. All the rats used had previously acquired prandial drinking but were not persistent prandial drinkers at the time the water intake data were collected. Atropine sulfate and atropine nitrate were injected on randomly selected days, the intakes on the day before injection being used as control data. At least four days elapsed between injections in order to allow recovery. Lower doses of atropine (0.25-1.0 mg/kg) were used in order to minimize anorexia. The eight rats used included four used in the acquisition experiment and four from pilot experiments, in which eight out of nine rats exposed to a variety of schedules of food deprivation and atropine treatment had acquired prandial drinking. Three of the latter four had been persistent prandial drinkers and had been used in the experiments described in part III.
TABLE 1 AC'QUISITION OF PRANDIAL DRINKING BY ATROPINIZED RATS
Rat
Atropine
Dose (mg/kg)
Treatment Days to Prandial Drinking
Persistence without Atropine
Body Wt during Testing as ~o of Starting Wt
6 9 8 5 10 11
Sulfate Methyl nitrate Sulfate Methyl nitrate Sulfate Methyl nitrate
1 1 1.5 1.5 2.25 2.25
2 1 3 3 --* 3
no yes yes partial yes no
83 89 80 75 82 80
*Rat was prostrated by drug treatment, showed prandial drinking on subsequent no-treatment days.
third occasion was fed a supplement of dry food in its home cage at the end of the session. When returned to the experimental schedule the rat rapidly became an extremely regular prandial drinker and remained one for the rest of its laboratory life (six months). During this period the only atropine injection it received was one of atropine methyl nitrate which also caused a severe depression of food and water intake. The degree to which prandial drinking persisted without atropine was variable. Persistent prandial drinking usually occurred only at the beginning of a feeding session in the box and did not result in any increase in the water/food ratio. In two rats (9 and 10) the persistent prandial drinking was extremely regular and occurred throughout the remainder of their laboratory lives (six months) during which they were maintained on a 1 hr food-access schedule with ad lib water (Purina pellets were fed on schedule in the home cage between tests). Lastly, persistent prandial drinking was observed in six of the ten rats in the supplementary group when they were tested on schedule after a three month rest period with ad lib food and water.
Results and Discussion Both types of atropine, at all doses, produced increased water intakes as a consequence of prandial drinking. The water intake data for each rat are shown in Fig. 2 which shows the intake on all atropine days as a percentage of the intake on the previous no atropine day. The absolute water intake was increased on 42/50 atropine days (as shown in the figure) while the relative water intake (water/food ratio) was increased on 47/50 days. The mean increases were 46.2 per cent (absolute intakes) and 166.8 per cent (relative intakes). Within the range used there was no apparent dose effect, and it seems probable that the effect is a threshold one. The similarity of the effects produced by the two types of atropine at low dose rates is strong evidence that the drugs act peripherally in producing prandial drinking and the resulting increase in water intake. It is possible that schedule effects [2] contribute to the increased water intakes especially in light of the finding [5] that prandial drinking can be abolished by the intraoral injection of amounts of water smaller than those that the rat
552
CHAPMAN AND EPSTEIN
drinks, implying that the desalivate rat drinks more than is necessary to swallow the dry food. The mean food intake on atropine days was 10.6 g (equivalent to 235 pellets). This (self-imposed) rate of feeding is considerably greater than the (experimenter-imposed) rates of feeding which have been reported [2] as resulting in schedule-induced polydipsia.
in Fig. 4. Note that prandial drinking immediately returned when the infusions ceased. This reappearance of prandial drinking makes it likely that the oral infusion suppressed the drinking directly by replacing the ingested water rather than indirectly by altering the situation. In all six rats persistent prandial drinking disappeared
400
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2
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FIG. 2. Increased water intake as a result of prandial drinking in atropinized rats. Data shown as percentages of intakes on previous (no drug) days, on a log. scale beginning with 35 per cent. Note similarity of effects of atropine sulfate (solid triangles) and methyl nitrate (open triangles).
I11. SUPPRESSION OF PERSISTENT PRANDIAL DRINKING
Method
Six rats which persisted as prandial drinkers after the termination of atropine treatment were used in the third part of the study. Two were from the acquisition experiment and four were from the pilot experiments. The general method of the tests was as described above. 4 mg/ml Pilocarpine nitrate (U.S.P.) solutions were made for each experiment and refrigerated after use. The Pilocarpine was injected intraperitoneally, in doses of 1-4 mg per rat and at pre-feeding intervals in the range of 5-60 min. In addition, two of the rats were fitted with oral cannulae (the apparatus and procedure has been described by Kissileff [6]) and water was injected into the mouth, just after the delivery of each pellet, in amounts approximately equal to the rat's mean draught size. In other trials the rats were allowed to feed ad lib overnight in the test box or were fed Purina pellets in their home cages (on schedule) in order to assess the effect of the deprivation conditions and the feeding situation on persistent prandial drinking.
Results and Discussion
Pilocarpine suppressed persistent prandial drinking in each of the six rats. Typical records of complete control and drug sessions are shown for each of three rats in Fig. 3. In each case pilocarpine greatly reduced the number of occasions on which drinking followed pellet delivery and ingestion. Oral infusion of water also effectively suppressed prandial drinking in both rats in which this procedure was carried out, as shown
gradually when the rats were allowed to feed in the test chamber overnight. Within 24 hr the feeding patterns resembled those of normal rats. A sample record of such an experiment is shown in Fig. 3 C (24 hr access excerpt). In none of the rats did prandial drinking occur in the home cages. These results show that persistent prandial drinking was not the result of severe, permanent impairment of the glands.
GENERAL DISCUSSION
Atropinized rats eating dry food resemble surgically desalirate rats both in their prandial style of drinking and in the gradual acquisition of this behavior. This phenomenological similarity and the fact that atropine methyl nitrate, which has little central effect, was as effective as atropine sulfate indicate that the effect was mediated by salivary inhibition, a wellestablished peripheral effect of atropine. These data therefore provide support for the peripheral explanation of prandial drinking advanced by Kissileff [5]. Increased water intake was produced by both types of atropine and was secondary to the adoption of prandiat drinking. It is therefore an indirect peripheral effect of atropine in contrast to the central blockade of thirst reported by Stein [9]. Previous investigators of the effect of atropine on the ingestive behavior of rats have avoided frequent and prolonged drug exposures or have given the drug when food or water were available separately [7, 9]. Thus prandial drinking was unlikely or impossible and only the central, de~essant, effect of atropine on water intake was observed. The only exception is a recent report [8] of an increase in water intake resulting from the administration of atropine to rats with ad
PRANDIAL DRINKING INDUCED BY ATROPINE
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FIG. 3, Suppression of persistent prandial drinking by pilocarpine and ad lib feeding (24 hr access). Piiocarpine effect for rats 12 (a, above) and 3 (b, above) and both piloearpine and ad lib feeding effects for rat 10 (c, below). Pilocarpine records are complete one hour sessions. Control sessions are previous (no drugs) days. Excerpt from ad lib day is 75 rain segment 24 hr after access to food on a FI 1.5 see schedule. Control record is of first hour of access. Recording method as in Fig. 1.
lib access to food and water. This increase was attributed to the central diuretic effect of atropine and the feeding behavior of the rats was not recorded. In view of the data reported here it seems likely that these rats were prandial drinkers. These experiments do not support Cannon's theory [1 ] that thirst is an innate reflex for which a dry mouth is the stimulus and drinking the response. On the contrary, since naive rats never drank in response to the first pellets ingested after atropinization we may conclude that dry-mouth drinking is not part of the rat's normal behavioral repertoire. Prandial drinking is almost certainly an operant behavior acquired by rats in response to the novel problem of having to swallow dry food from a dry mouth. The increased drinking of desalivate dogs in the heat [3] may have a similar basis and should
be re-investigated. The fact that this peripherally-controlled, operant-type drinking was induced by a drug that depresses centrally-controlled drinking indicates that the neurological systems for regulatory and prandial drinking are relatively independent despite the identity of their output. The persistence of prandial drinking after it has been induced by atropine remains unexplained. It is, however, of interest as a possible link between prandial drinking, which occurs whenever a desalivate rat is eating dry food, and schedule-induced polydipsia [2], which occurs in normal rats but only under a limited range of conditions (enforced delays between small morsels of dry food). It may be that marginal salivary insufficiency and schedule factors are summating to produce persistent prandial drinking.
554
CHAPMAN AND EPSTEIN
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oral infusion
control
FIG. 4. Suppression of persistent prandial drinking by intra-oral infusion of water. A 5 sec infusion followed each pellet except for the few pellets obtained during pump operation. During control periods rat drank 0.07 ml/pellet. Mean volume of infusion was 0.08 ml/pellet. Recording method as in Fig. 1 except lower trace not shown and recorder at faster speed (Rat No. 10).
REFERENCES 1. Cannon, W. B. Hunger and thirst. In: Handbook of General Experimental Psychology, edited by C. Murchison. Worcester, Mass,: Clark Univ. Press, 1934, pp. 247-263. 2. Falk, J. L. Conditions producing psychogenic polydipsia in animals. Annls N. Y. Acad. ScL 157: 569-589, 1969. 3. Gregersen, M. I. and W. B. Cannon. The effect of extirpation of the salivary glands on the water intake of dogs while panting. Am. J. Physiol. 102: 336-343, 1932. 4. Kissileff, H. R. Food-associated drinking in the rat. J. comp. physiol. Psychol. 67: 284-300, 1969. 5. Kissileff, H. R. Oropharyngeal control of prandial drinking. J. comp. physiol. Psychol. 67: 309-319, 1969. 6. Kissileff, H. R. and A. N. Epstein. Exaggerated prandial drinking in the "recovered lateral" rat without saliva. J. comp. physiol. Psychol. 67: 301-308, 1969.
7. 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. 8. Soulairac, A. and M. L. Soulairac. Dissociation experimentale de la soif et de la diurcse sous l'action de l'atropine. C. R. Acad. Sci. Paris 266:908-911, 1968. 9. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46-48, 1963. 10. Stoll, H.C. Pharmacodynamic considerations of atropine and related compounds. Am. J. _,Wed.Sci. 215: 577-592, 1948. 11. Stricker, E. M. and E. R. Adair. Body fluid balance, taste and postprandial factors in schedule-induced polydipsia. J. comp. physiol. Psychol. 65: 449--454, 1966. 12. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome. Psychol. Rev. 69: 74-90, 1962.