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Effect of scopolamine on drinking elicited by hypothalamic stimulation
Physiology and Behavior. Vol. 7, pp. 639--641. Pergamon Press, 1971. Printed in Great Britain
BRIEF COMMUNICATION Effect of Scopolamine on Drinking Elicited by Hypothalamic Stimulation I C. G. G E N T I L ' , J. A. F. S T E V E N S O N 8 A N D G. J. M O G E N S O N
Departments of Physiology and Psychology, University of Western Ontario, London, Ontario, Canada (Received 23 December 1970) GENTIL, C. G., J. A.. F. STEVENSONAND G. J. MooENSON. Effect of scopolamine on drinking elicited by hypothalamic stimulation. PHYSIOL.BmtAV. 7 (4) 639--641, 1971.--Small doses of scopolamine (0.5-2.0 mg/kg, I P), which reduced water intake in rats water-deprived for 23 hr, did not reduce drinking elicited by electrical stimulation of the hypothalamus. Higher doses (3.0-4.0 mg/kg) were needed to produce a significant effect on elicited drinking. This difference in effective dosage may be attributed to the more intense central drive induced by hypothalamic stimulation as compared to 23 hr water deprivation. It is proposed that the results are consistent with the hypothesis that scopolamine reduces water intake by influencing a central thirst mechanism. Scopolamine
Water intake
Lateral hypothalamus
Electrical stimulation
ATROPrr,m OR scopolamine, administered systemically or centrally, depresses water intake initiated either by water deprivation [4, 6, 10, 11], by the subcutaneous injection of hypertonic saline [1, 2], or by the central administration of carbachol [4, 6]. These anticholinergic agents also depress feeding in rats that have been food deprived [10, 11]. It has been suggested that the peripheral effect of atropine and scopolamine on salivary secretion is responsible for reduced feeding whereas a central neural effect of these drugs depresses drinking [5, 11]. In the present study the effects of scopolamine on food and water intakes were studied when feeding and drinking were elicited by electrical stimulation of the hypothalamus [9].
Cholinergic pathways
tap water were attached to the walls. The animals received 10 see trains of biphasic rectangular pulses from a Grass SD-5 stimulator spaced 10 sec apart for 20 rain per day (frequency: 60 Hz, pulse duration: 0.2 msee, current: 100-300 tLA). The occurrence of feeding and drinking, the quantity of food and the volume of water ingested were recorded. The test session of 20 rain was begun 30 rain after intraperitoneal injections of a solution of scopolamine hydrobromide in saline (range 0.4-6.0 mg/kg body weight). Physiological saline (0.9 per cent) was used for control tests. The effect of scopolamine on water intake was also measured in animals adapted to drink water for 1 hr following 23 hr water deprivation. The injections of scopolamine were usually two days apart. At the completion of the experiments the animals were anesthetized with sodium pentobarbital and perfused with saline (0.9 per cent) and formalin (10 per cent). The brains were removed and fixed in formalin. Frozen sections showing the electrode tract were cut at a thickness of 35 a and stained with cresyl violet. The electrodes terminated in the vicinity of the lateral hypothalamus in a region extending from the fornix to the zona incerta.
METHODS
Sixteen male Wistar rats, weighing between 400-500 g, were used. Ten of the animals while anesthetized with sodium pentoharbital (50 mg/kg) had bipolar electrodes (Plastic Products Company, MS 303-0.018), insulated except at the tips, implanted under stereotaxic control in the lateral hypothalamus. In preliminary tests these animals were induced by electrical stimulation of the hypothalamus to feed and drink. The animals were adapted to a plexiglass chamber measuring 20 cm by 30 cm and 30 cm high, open at the top. A food jar containing a complete synthetic diet (casein--20.0 per cent, white sugar--63.7 per cent, corn oil--10.0 per cent, s a l t 3.8 per cent, vitamins--2.5 per cent) was placed in the chamber and two bottles fitted with spill-proof metal spouts containing
RESULTS AND DISCUSSION
The frequency of drinking and the volume of water intake induced by hypothalamic stimulation in 10 rats following injections of saline (0.9 per cen0 or scopolamine in different doses are shown in Fig. 1. No change of induced drinking
1Supported by grants from the Medical Research Council. The assistance of Mary Dinning with the histological material and of Blanche Box and R. Woodside with the illustrations is acknowledged. spresent Address: Department of Physiology, School of Medicine, Ribeirao Preto, Sao Paulo, Brazil. Supported by F.A.P.E,S.P., Sao Panlo, Brazil. aDeceased 23 July 1971. 639
640
GENTIL, STEVENSON AND MOGENSON
4
IS-
i
I
15-
O.I
0,4
/I
1.2
2.5
3.0
4.0
FIG. 1. The occurrence of drinking (upper panel) and the volume of water intake (lower panel) during 60 presentations of hypothalamic stimulation (N = 10). Open columns are for control sessions when physiological saline was administered by intraperitoneal injection. Crossed hatched columns are for sessions that followed injections of scopolamine hydrobromide (0.4-4.0 mg/kg body weight). Differences significant at the 5 per cent level are marked by a single X and differences significant at the 1 per cent level by double X.
behavior was observed with injections of scopolamine from 0.4--2.5 mg/kg. A significant reduction in water intake was observed when the dose was 3.0 mg/kg (t = 2.39, p < 0.05) or 4.0 mg/kg (t = 3.08,p < 0.01). The decrease in the frequency of drinking was also statistically significant with 4.0 mg/kg (t = 3.49, p < 0.01), but not with the lower doses. In four of the animals rate of lapping was recorded and it was found that following the administration of scopolamine (4 mg/kg) the lap rate remained unchanged (approximately 7/sec). The blocking effect of scopolamine on thirst induced by 23 hr of water deprivation was tested in 16 animals (including the 10 referred to above) given access to water for 1 hr a day for 5 days. On day four, half of the animals were injected with saline (0.9 per cent) and the other half received scopolamine (1.0 mg/kg). On the fifth day, the groups were reversed and
received either saline (0.9 per cen0 or scopolamine (2 mg]kg). Water intake was significantly reduced at both dose levels (1 mg/kg: t = 4.08,p < 0.01; 2mg/kg: t = 4.53,p < 0.001), consistent with previous observations [8, 10, 11]. Electrical stimulation of the hypothalamus induces theintake of large volumes of water [8]. In the present study, two rats that received hypothalamic stimulation for a 10 hr session drank more than 200 ml of water as compared to less than 15 ml in the same period of time without stimulation and approximately 15 ml during one hour after 23 hr of water deprivation. This apparent very strong central drive elicited by the stimulation may be the reason why a higher dose of scopolamine was necessary to block water intake under these conditions than after 23 hr of water deprivation. To test this possibility the effects of scopolamine (6 mg/kg) on water
TABLE 1 EFFECTOF SCOPOLAMINEON WATERDRINKINGINDUCEDBY HYPOTHALAMICSTIMULATIONUSING Low AND HIGH CURRENTS DURING 10 MIN SESSIONS
Rat
Low Current* Intake Drinking
LH 9 LH 50 LH 53 LH 52 LH 59 LH 1 Mean 4- S.E.M.
ml 5.0 9.0 7.5 6.5 5.0 8.5 6.91 5:0.70
25 30 30 28 25 30 28.0 4-1.0
With Scopolamine Low Current* Intake Drinking ml 0.0 2.0 3.0 3.0 0.2 5.0 2.2t 4-0.7
0 7 17 18 1 24 11.It 4-4.0
6 mg/kg High Current* Intake Drinking ml 4.0 9.0 8.0 7.5 0.0 6.0 5.75 5:1.3
*Low current varied from 100 to 150 IzA.(Mean 120 btA) and high current varied from 150 to 280 (Mean 195 ~tA). tP < 0.01 as compared with same value during control period.
28 29 30 28 0 30 24.1 4-4.8
SCOPOLAMINE AND HYPOTHALAMIC STIMULATION
50,
641
X
:llml CONTROL
O.S
CONTROL
~.o
CONTROL
2.o
FIG. 2. The occurrence of drinking (open columns) and feeding (cross hatched columns) shown in the upper panel and the intakes of water (open columns) and food (cross hatched columns) shown in the lower panel for 60 presentations of hypothalamic stimulation in 10 rats. Control refers to sessions when physiological saline was administered by intraperitoneal injection. Injections of 0.5, 1.0,2.0 mg of scopolamine hydrobromide per kg. body weight significantlyreduced (p < 0.01) the frequency of feeding and the intake of food but did not reduce drinking or the volume of water intake.
intake elicited during 10 min sessions with hypothalamic stimulation were compared using low and high current levels (see Table 1). Although at the lower current level, the frequency and volume of water intake were both significantly reduced by scopolamine, there was no reduction when the current level was increased. The effects o f scopolamine on elicited feeding were measured in a further experiment (see Fig. 2). With doses up to 2 mg/kg, scopolamine did not significantly reduce water intake, confirming the previous experiment. On the other hand, feeding was significantly reduced (0.5 mg/kg: t = 6.07, p < 0.001; 1.0 mg/kg: t = 6.99, p < 0.001; 2.0 mg/kg: t = 5.96, p < 0.001) in proportion to the dose used. Elicited food intake was also suppressed by scopolamine when only food was available (0.5 mg/kg: t = 5.78, p < 0.001 ; 1.0 mg/kg: t = 8.36,p < 0,001; 2.0 mg/kg: t = 7.81,p < 0.001).
One of the prominent effects of scopolamine is to reduce the secretion of saliva and this effect could be responsible for the suppression of feeding. On the other hand, a dry mouth should increase rather than reduce water intake. Stein [11] has suggested that anticholinergic drugs such as scopolamine reduce water intake by a central action. One possibility is that these drugs block cholinergic synapses in the thirst system [3]. In support of this hypothesis Levitt and Fisher [7] showed that the intra-cerebral administration of atropine blocked drinking induced by the intra-cerebral application of carbachol. Drinking elicited by subcutaneous injections of hypertonlc saline is also blocked by the intra-cerebral application of atropine according to a recent report [1]. The evidence of the present study indicates that appropriate hypothalamic stimulation can produce a very strong drive for water intake and that the inhibition of this drive by scopolamine probably occurs within the central mechanism subserving it.
REFERENCES
1. Block, M. L. and A. E. Fisher. Anticholinergic central blockade of salt-aroused and deprivation-induced drinking. Physiol, Behav. 5: 525-527, 1970. 2. De Wied, D. Effect of autonomic blocking agents and structurally related substances on the salt arousal of drinking. PhysioL Behav. 1: 193-197, 1966. 3. Fisher, A. E. Chemical stimulation of the brain. Scient. Am. 210: 60--68, 1964. 4. Grossman, S. P. Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms. Am. J. Physiol. 202: 1230-1236, 1962. 5. Houser, V. P. The effects of adrenergic and cholinergic agents upon eating and drinking in deprived rats. Psychonom. ScL 20: 153-155, 1970. 6. Levitt, R. A. Anticholinergic brain stimulation and thirst
7. 8. 9. 10. 11.
induced by 23 hour water deprivation. Psychonom. Sci. 12: 21-22, 1968. Levitt, R. A. and A. E. Fisher. Anticholinergic blockade of centrally induced thirst. Science 154: 520-522, 1966. Mogenson, G. J. General and specific reinforcement system for drinking behavior. Ann. N.Y. Acad. Sci. 157: 779-795, 1969. Mogenson, G. J. and J. A. F. Stevenson. Drinking and self stimulation with electrical stimulation of lateral hypothalamus. Physiol. Behav. 1: 251-254, 1966. Schmidt, H., Jr., S. J. Moak and W. G. Van Meter. Atropine depression of food and water intake in the rat. Am. Y. Physiol. 192: 543-545, 1958. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46-48, 1963.
BRIEF COMMUNICATION Effect of Scopolamine on Drinking Elicited by Hypothalamic Stimulation I C. G. G E N T I L ' , J. A. F. S T E V E N S O N 8 A N D G. J. M O G E N S O N
Departments of Physiology and Psychology, University of Western Ontario, London, Ontario, Canada (Received 23 December 1970) GENTIL, C. G., J. A.. F. STEVENSONAND G. J. MooENSON. Effect of scopolamine on drinking elicited by hypothalamic stimulation. PHYSIOL.BmtAV. 7 (4) 639--641, 1971.--Small doses of scopolamine (0.5-2.0 mg/kg, I P), which reduced water intake in rats water-deprived for 23 hr, did not reduce drinking elicited by electrical stimulation of the hypothalamus. Higher doses (3.0-4.0 mg/kg) were needed to produce a significant effect on elicited drinking. This difference in effective dosage may be attributed to the more intense central drive induced by hypothalamic stimulation as compared to 23 hr water deprivation. It is proposed that the results are consistent with the hypothesis that scopolamine reduces water intake by influencing a central thirst mechanism. Scopolamine
Water intake
Lateral hypothalamus
Electrical stimulation
ATROPrr,m OR scopolamine, administered systemically or centrally, depresses water intake initiated either by water deprivation [4, 6, 10, 11], by the subcutaneous injection of hypertonic saline [1, 2], or by the central administration of carbachol [4, 6]. These anticholinergic agents also depress feeding in rats that have been food deprived [10, 11]. It has been suggested that the peripheral effect of atropine and scopolamine on salivary secretion is responsible for reduced feeding whereas a central neural effect of these drugs depresses drinking [5, 11]. In the present study the effects of scopolamine on food and water intakes were studied when feeding and drinking were elicited by electrical stimulation of the hypothalamus [9].
Cholinergic pathways
tap water were attached to the walls. The animals received 10 see trains of biphasic rectangular pulses from a Grass SD-5 stimulator spaced 10 sec apart for 20 rain per day (frequency: 60 Hz, pulse duration: 0.2 msee, current: 100-300 tLA). The occurrence of feeding and drinking, the quantity of food and the volume of water ingested were recorded. The test session of 20 rain was begun 30 rain after intraperitoneal injections of a solution of scopolamine hydrobromide in saline (range 0.4-6.0 mg/kg body weight). Physiological saline (0.9 per cent) was used for control tests. The effect of scopolamine on water intake was also measured in animals adapted to drink water for 1 hr following 23 hr water deprivation. The injections of scopolamine were usually two days apart. At the completion of the experiments the animals were anesthetized with sodium pentobarbital and perfused with saline (0.9 per cent) and formalin (10 per cent). The brains were removed and fixed in formalin. Frozen sections showing the electrode tract were cut at a thickness of 35 a and stained with cresyl violet. The electrodes terminated in the vicinity of the lateral hypothalamus in a region extending from the fornix to the zona incerta.
METHODS
Sixteen male Wistar rats, weighing between 400-500 g, were used. Ten of the animals while anesthetized with sodium pentoharbital (50 mg/kg) had bipolar electrodes (Plastic Products Company, MS 303-0.018), insulated except at the tips, implanted under stereotaxic control in the lateral hypothalamus. In preliminary tests these animals were induced by electrical stimulation of the hypothalamus to feed and drink. The animals were adapted to a plexiglass chamber measuring 20 cm by 30 cm and 30 cm high, open at the top. A food jar containing a complete synthetic diet (casein--20.0 per cent, white sugar--63.7 per cent, corn oil--10.0 per cent, s a l t 3.8 per cent, vitamins--2.5 per cent) was placed in the chamber and two bottles fitted with spill-proof metal spouts containing
RESULTS AND DISCUSSION
The frequency of drinking and the volume of water intake induced by hypothalamic stimulation in 10 rats following injections of saline (0.9 per cen0 or scopolamine in different doses are shown in Fig. 1. No change of induced drinking
1Supported by grants from the Medical Research Council. The assistance of Mary Dinning with the histological material and of Blanche Box and R. Woodside with the illustrations is acknowledged. spresent Address: Department of Physiology, School of Medicine, Ribeirao Preto, Sao Paulo, Brazil. Supported by F.A.P.E,S.P., Sao Panlo, Brazil. aDeceased 23 July 1971. 639
640
GENTIL, STEVENSON AND MOGENSON
4
IS-
i
I
15-
O.I
0,4
/I
1.2
2.5
3.0
4.0
FIG. 1. The occurrence of drinking (upper panel) and the volume of water intake (lower panel) during 60 presentations of hypothalamic stimulation (N = 10). Open columns are for control sessions when physiological saline was administered by intraperitoneal injection. Crossed hatched columns are for sessions that followed injections of scopolamine hydrobromide (0.4-4.0 mg/kg body weight). Differences significant at the 5 per cent level are marked by a single X and differences significant at the 1 per cent level by double X.
behavior was observed with injections of scopolamine from 0.4--2.5 mg/kg. A significant reduction in water intake was observed when the dose was 3.0 mg/kg (t = 2.39, p < 0.05) or 4.0 mg/kg (t = 3.08,p < 0.01). The decrease in the frequency of drinking was also statistically significant with 4.0 mg/kg (t = 3.49, p < 0.01), but not with the lower doses. In four of the animals rate of lapping was recorded and it was found that following the administration of scopolamine (4 mg/kg) the lap rate remained unchanged (approximately 7/sec). The blocking effect of scopolamine on thirst induced by 23 hr of water deprivation was tested in 16 animals (including the 10 referred to above) given access to water for 1 hr a day for 5 days. On day four, half of the animals were injected with saline (0.9 per cent) and the other half received scopolamine (1.0 mg/kg). On the fifth day, the groups were reversed and
received either saline (0.9 per cen0 or scopolamine (2 mg]kg). Water intake was significantly reduced at both dose levels (1 mg/kg: t = 4.08,p < 0.01; 2mg/kg: t = 4.53,p < 0.001), consistent with previous observations [8, 10, 11]. Electrical stimulation of the hypothalamus induces theintake of large volumes of water [8]. In the present study, two rats that received hypothalamic stimulation for a 10 hr session drank more than 200 ml of water as compared to less than 15 ml in the same period of time without stimulation and approximately 15 ml during one hour after 23 hr of water deprivation. This apparent very strong central drive elicited by the stimulation may be the reason why a higher dose of scopolamine was necessary to block water intake under these conditions than after 23 hr of water deprivation. To test this possibility the effects of scopolamine (6 mg/kg) on water
TABLE 1 EFFECTOF SCOPOLAMINEON WATERDRINKINGINDUCEDBY HYPOTHALAMICSTIMULATIONUSING Low AND HIGH CURRENTS DURING 10 MIN SESSIONS
Rat
Low Current* Intake Drinking
LH 9 LH 50 LH 53 LH 52 LH 59 LH 1 Mean 4- S.E.M.
ml 5.0 9.0 7.5 6.5 5.0 8.5 6.91 5:0.70
25 30 30 28 25 30 28.0 4-1.0
With Scopolamine Low Current* Intake Drinking ml 0.0 2.0 3.0 3.0 0.2 5.0 2.2t 4-0.7
0 7 17 18 1 24 11.It 4-4.0
6 mg/kg High Current* Intake Drinking ml 4.0 9.0 8.0 7.5 0.0 6.0 5.75 5:1.3
*Low current varied from 100 to 150 IzA.(Mean 120 btA) and high current varied from 150 to 280 (Mean 195 ~tA). tP < 0.01 as compared with same value during control period.
28 29 30 28 0 30 24.1 4-4.8
SCOPOLAMINE AND HYPOTHALAMIC STIMULATION
50,
641
X
:llml CONTROL
O.S
CONTROL
~.o
CONTROL
2.o
FIG. 2. The occurrence of drinking (open columns) and feeding (cross hatched columns) shown in the upper panel and the intakes of water (open columns) and food (cross hatched columns) shown in the lower panel for 60 presentations of hypothalamic stimulation in 10 rats. Control refers to sessions when physiological saline was administered by intraperitoneal injection. Injections of 0.5, 1.0,2.0 mg of scopolamine hydrobromide per kg. body weight significantlyreduced (p < 0.01) the frequency of feeding and the intake of food but did not reduce drinking or the volume of water intake.
intake elicited during 10 min sessions with hypothalamic stimulation were compared using low and high current levels (see Table 1). Although at the lower current level, the frequency and volume of water intake were both significantly reduced by scopolamine, there was no reduction when the current level was increased. The effects o f scopolamine on elicited feeding were measured in a further experiment (see Fig. 2). With doses up to 2 mg/kg, scopolamine did not significantly reduce water intake, confirming the previous experiment. On the other hand, feeding was significantly reduced (0.5 mg/kg: t = 6.07, p < 0.001; 1.0 mg/kg: t = 6.99, p < 0.001; 2.0 mg/kg: t = 5.96, p < 0.001) in proportion to the dose used. Elicited food intake was also suppressed by scopolamine when only food was available (0.5 mg/kg: t = 5.78, p < 0.001 ; 1.0 mg/kg: t = 8.36,p < 0,001; 2.0 mg/kg: t = 7.81,p < 0.001).
One of the prominent effects of scopolamine is to reduce the secretion of saliva and this effect could be responsible for the suppression of feeding. On the other hand, a dry mouth should increase rather than reduce water intake. Stein [11] has suggested that anticholinergic drugs such as scopolamine reduce water intake by a central action. One possibility is that these drugs block cholinergic synapses in the thirst system [3]. In support of this hypothesis Levitt and Fisher [7] showed that the intra-cerebral administration of atropine blocked drinking induced by the intra-cerebral application of carbachol. Drinking elicited by subcutaneous injections of hypertonlc saline is also blocked by the intra-cerebral application of atropine according to a recent report [1]. The evidence of the present study indicates that appropriate hypothalamic stimulation can produce a very strong drive for water intake and that the inhibition of this drive by scopolamine probably occurs within the central mechanism subserving it.
REFERENCES
1. Block, M. L. and A. E. Fisher. Anticholinergic central blockade of salt-aroused and deprivation-induced drinking. Physiol, Behav. 5: 525-527, 1970. 2. De Wied, D. Effect of autonomic blocking agents and structurally related substances on the salt arousal of drinking. PhysioL Behav. 1: 193-197, 1966. 3. Fisher, A. E. Chemical stimulation of the brain. Scient. Am. 210: 60--68, 1964. 4. Grossman, S. P. Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms. Am. J. Physiol. 202: 1230-1236, 1962. 5. Houser, V. P. The effects of adrenergic and cholinergic agents upon eating and drinking in deprived rats. Psychonom. ScL 20: 153-155, 1970. 6. Levitt, R. A. Anticholinergic brain stimulation and thirst
7. 8. 9. 10. 11.
induced by 23 hour water deprivation. Psychonom. Sci. 12: 21-22, 1968. Levitt, R. A. and A. E. Fisher. Anticholinergic blockade of centrally induced thirst. Science 154: 520-522, 1966. Mogenson, G. J. General and specific reinforcement system for drinking behavior. Ann. N.Y. Acad. Sci. 157: 779-795, 1969. Mogenson, G. J. and J. A. F. Stevenson. Drinking and self stimulation with electrical stimulation of lateral hypothalamus. Physiol. Behav. 1: 251-254, 1966. Schmidt, H., Jr., S. J. Moak and W. G. Van Meter. Atropine depression of food and water intake in the rat. Am. Y. Physiol. 192: 543-545, 1958. Stein, L. Anticholinergic drugs and the central control of thirst. Science 139: 46-48, 1963.