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Effects of naloxone and its quaternary analogue on fluid consumption in water-deprived rats
0028-3908/83/060797-04$03.00/O Copyright :.Q 1983 Pergamon Press Ltd
NeuropharmacologyVol. 22, No. 6, pp. 797-800.1983 Printed in Great Britain. All rights reserved EFFECTS
OF NALOKONE
AND ITS QUATERNARY
ON FLUID CONSUMPTION
S.J. Cooper Department
of Psychology,
University
[Acc~p~cxf
ANALOGUE
IN WATER-DEPRIVED
and Suzanne
Turkish
of Birmingham, 3 I Mm&
RATS
Birmingham
B15 2TT, T1.K.
1983 J
Summary - The effects of subcutaneous administration of naloxone and its quaternary analogue on the consumption of water, saline and saccharin solution were investigate In the water-deprived rat. -d 1, effective in blocking peripheral Quaternary naloxone (0.01-10 mg.kg opiate receptors, hfd no effect on fluid intake. In contrast, naloxone (0.01-10 mg.kg ), produced dose-dependent attenuation of intake for each of the three fluids, indicating a central location of the relevant opiate receptors. Access to saline induced additional drinking, compared with water intake, due to an extension of the initial avid consumption which follows the period of water-deprivation. This enhancement of drinking was blocked by naloxone, but not by its quaternary analogue. Interactions between naloxone and taste factors in drinking may, therefore, depend on blockade of central opiate receptors. There is good evidence that endogenous opioid peptides are directly involved in thirst mechanisms in several mammalian species. In recent years, a large body of data has been accumulated, which show that opiate receptor antagonists consistently attenuate water consumption which is induced by a wide variety of thirst stimuli (review by Sanger, 1983). Opiate peptides and receptors are found not only in high concentrations in numerous regions of the brain, but also in several peripheral tissues, including the pituitary, pancreas, adrenal medulla and gastrointestinal tract (Pasternak and Childers, 1983). Selective antagonism of peripheral opiate receptors can be achieved using systemic administration of quaternary snalogues of opiate receptor antagonists, which do not readily cross the blood-brain barrier (Carr and Simon, 1983; Bianchi et al., 1982; Schulz et al., 1979). Comparisons between naloxone and its quaternary analogue therefore provide a means to assess a contribution of ¢ral and/or peripheral receptors to the suppression of drinking responses. Using this approach, Brown and Holtzman (1981) recently demonstrated that quaternary naloxone and naltrexone failed to affect water consumption in water-deprived-fats, whereas the corresponding tertiary compounds produced dose-dependent (0.1-10.0 mg.kg 1 reductions They concluded that blockade of central opiate receptors was instrumental in in drinking. the effects of naloxone on water intake. Taste factors can have major effects on drinking (Emits and Corbit, 1973), and it has been repeatedly suggested that opiate antagonists interact with taste factors in fluid consumption (Cooper, 1982, 1983; Levine et al., 1982; The purpose of the present experiment, therefore, was to confirm and extend Rowland, 1982). the results of the Brown and Holtzman study, by comparing the effects of naloxone and its quaternary analogue, on the consumption of water, saline and saccharin solution, respectiverat. ly, in the water-deprived METHODS The subjects were 60 male hooded rats of the general strain (weight range 250-350 g) bred in our laboratory. They were housed in individual stainless steel cages, with food pellets (modified Diet 41B) and tap water available. Room temperature was maintained at 21oci.1, and a 12hr light--12hr dark cycle was in operation. All animals were accustomed to handling and injection procedures, and were adapted to a deprivation schedule of 22 hr without water, on alternate days. The rats were allocated to three equal groups, according to the type of fluid which was available during the drinking tests. For the first 30 min after each deprivation period, food was removed, and the test fluid was available in a calibrated cylinder, which was clipped to the front of the cage. The groups were given access to water, 0.005 M sodium saccharin solution, and 3.9% sodium chloride solution, respectively. On intervening non-deprivation days, food and water were available ad --* lib Within each taste condition, the animals were subdivided into two groups, one receiving naloxone hydrochloride or isotonic saline vehicle, the other receiving methyl-naloxone hydrochloride or vehicle. Within each drug group, each animal was t sted on 5 occasions, follow-f ing subcutaneous administration of 0.01, 0.1, 1.0 or 10.0 mg.kg , or vehicle, respectively. Order of injection was randomised within each animal, and at least 48hr intervened between 197
798
PreliminaryNotes
successive injections. Doses of drug refer to the base, and injections were given 15-20 min before the drinking test. During the 30 min test (carried out in the light phase), fluid consumption (ml) was determined at 6 min intervals. The data analysed using 3-way analysis of variance (ANOVA) with repeated measures on two factors, separately for naloxone and its quaternary analogue. The factors were dose of drug, fluid condition, and timecourse of drinking. The Dunnett test (one-taiLed) was used for comparisons between individual groups and the corresponding control group, and the t-statistic was employed for planned comparisons between fluid conditions (Miner, 1971). RESULTS Naloxone. In the naloxone-treated groups, there were significant differences between the consumption over 30 min of the three avail ble fluids, F (2,27) = 13.60, P
r
IOL
WATER
VW0
SACCHARIN
j
SALINE
“b-Th+kiOvk5kdi-#o NALOXONE mg.kg-1
FIG. 1 -1 ) reduced intake of water, 0.005M sodium saccharin solution, and Naloxone (0.01-10.0 mg.kg Results are shown as the mean with S.E. 0.09% saline respectively, in water-deprived rats. Statistical comparisons of individual dose conditions against bars. N=lO per group. **pco.o1. *p
199
PreliminaryNotes
VEHICLE
NALOXONE
B
. 0 9%SALINE 0 WATER
Y 2
z6E 3 u_ 4-
TIME COURSE -SIX MINUTE INTERVALS
FIG. 2 Time-course of drinking over a 30 min test for water and saline intake, after injection of vehicle (A) or 1.0 mg.kg-1 naloxone (B). Results are shown as the mean with S.E. bars. N = 10 per group. Comparison of saline intake against water intake: **p
1 after quaternary
naloxone
____^_
-----_.ll-_l-._.._-.--_~.^_I__I__ Methyl-Naloxone 0 _ll-___-l-li
administration
(mg.kg -l)
1.0 0.1 0.01 l--._-.lli~-.-.-l.l~___-__-.___--.---____
10.0 _^________
Water
19.2 21.1
19.1 f1.3
19.0 f1.2
18.2 f1.2
18.8 fO.9
Saccharin
23.2 f2.0
21.2 f2.0
24.2 22.1
21.7 +1.3
20.4 t1.0
Saline
24.6 t2.9
23.5 +1.8
28.1 f2.2
27.8 +2.5
26.0 21.6
Results are shown as mean t SEM. naloxone treatment.
N = 10 per group.
Quaternary naloxone. Whilst there were significant taste condition, as in the naloxone-treated animals, nificant overall main effect of quaternary naloxone, time-course (Table 1).
-_--~ No significant
effects
of quaternary
differences in fluid intake due to analysis of variance revealed no sigor interaction with taste condition or
DISCUSSION Although naloxone produced dose-related reductions of fluid intake in each of the three taste conditions, it? quaternary analogue did not. This result confirms previous findings (Brown and Holtzman, 1981; Sanger, 1983), and indicates that reduction in drinking in waterdeprived rats was due to antagonism at opiate receptors in the brain. The consumption of isotonic saline was enhanced compared with that of water. Hall and Blass (1975) showed that rats which drank isotonic saline had a longer initial uninterrupted bout of drinking, and returned more frequently to drink at later stages, compared with rats which drank water. Fig. 2A confirms the extended initial high level of drinking in rats which had access to isotonic saline. The important finding of the present study was that the increased consumption in the isotonic saline group of animals was selectively abolished by naloxone in doses of 0.1 mg.kg-' and more. Since quaternary naloxone did not attenuate the hyperdipsia associated with saline drinking, peripheral opiate receptors do not appear
800
PreliminaryNotes
to have been involved. The present results nervous system are involved in the increased highly palatable salt solution.
suggest that opioid peptides in the central consumption of fluid which is induced by a
ACKNOWLEDGEMENTS We would like to thank Endo Laboratories for the gift of naloxone, and Francopia (Paris) for the gift of methyl-naloxone hydrochloride. S.T. is a recipient of a postgraduate studentship from the Medical Research Council (U.K.). REFERENCES Quaternary narcotic antagonBianchi, G., Fiocchi, R., Tavani, A. and Manara, L. (1982) and gastrointestinal transit inhibiists' relative ability to prevent antinociception tion in morphine-treated rats as an index of peripheral selectivity. Life Sci., 30: 1875-1884. Opiate antagonists: central Brown, D.R. and Holtzman, S.G. (1981) Brain Res., 221: 432-436. suppressing water intake of the rat. Carr, K.D. and Simon, E.J. (1983) stimulation-induced feeding.
sites of action
Effects of naloxone and its quaternary Neuropharmacology, 22: 127-130.
Palatability-induced drinking after administration Cooper, S.J. (1982) Substance Alcohol naltrexone and diazepam in the non-deprived rat. 3: 259-265. Cooper, S.J. (1983) saccharin choice
analogue
in
on
of morphine, Actions/Misuse,
Effects of opiate agonists and antagonists on fluid intake and Neuropharmacology, 22: 323-328. in the rat.
Emits, T. and Corbit, J.D. Psychol., 83: 37-31.
(1973)
Taste as a dipsogenic
stimulus.
J. camp. physiol.
Orogastric, hydrational, and behavioral controls of Hall, W.G. and Blass, E.M. (1975) drinking following water deprivation in rats. J. camp. physiol. Psychol., 89: 939-954. _I_ Flavor enhances Levine, A.S., Murray, S.S., Kneip, J., Grace, M. and Morley, J.E. (1982) Physiol. Psychol., 28: 23-25. the antidipsogenic effect of naloxone. The actions of opiates and opioid peptides. Pasternak, G.W. and Childers, S.R. (1983) 3 (Turner, P. and Shand, D.G., Eds.), In: Recent --1111 Advances in Clinical Pharmacology, pp.252-279, Churchill, Livingstone, Edinburgh. Comparison of the suppression by naloxone of water intake induced in Rowland, N. (1982) Pharmac. Biochem. Behav., 16: rats by hyperosmolarity, hypovolemia, and angiotensin. 87-91. Opiates and ingestive behavior. Sanger, D.L. (1983) VOl . 2 (Cooper, S.J. Ed.), Academic Press, London
In: Theory (in press).
in Psychopharmacology
Centrally and peripherally mediated Schulz, R., Wuster, M. and Hers, A. (1979) Naunyn-Schmiedeberg's Arch. Pharmacol., of intestinal motility by opioids. -255-260. Winer, B.J. (1971) New York.
Statistical
Principles
in Experimental --- Design,
2nd ed.
inhibition 308:
McGraw-Hill,
NeuropharmacologyVol. 22, No. 6, pp. 797-800.1983 Printed in Great Britain. All rights reserved EFFECTS
OF NALOKONE
AND ITS QUATERNARY
ON FLUID CONSUMPTION
S.J. Cooper Department
of Psychology,
University
[Acc~p~cxf
ANALOGUE
IN WATER-DEPRIVED
and Suzanne
Turkish
of Birmingham, 3 I Mm&
RATS
Birmingham
B15 2TT, T1.K.
1983 J
Summary - The effects of subcutaneous administration of naloxone and its quaternary analogue on the consumption of water, saline and saccharin solution were investigate In the water-deprived rat. -d 1, effective in blocking peripheral Quaternary naloxone (0.01-10 mg.kg opiate receptors, hfd no effect on fluid intake. In contrast, naloxone (0.01-10 mg.kg ), produced dose-dependent attenuation of intake for each of the three fluids, indicating a central location of the relevant opiate receptors. Access to saline induced additional drinking, compared with water intake, due to an extension of the initial avid consumption which follows the period of water-deprivation. This enhancement of drinking was blocked by naloxone, but not by its quaternary analogue. Interactions between naloxone and taste factors in drinking may, therefore, depend on blockade of central opiate receptors. There is good evidence that endogenous opioid peptides are directly involved in thirst mechanisms in several mammalian species. In recent years, a large body of data has been accumulated, which show that opiate receptor antagonists consistently attenuate water consumption which is induced by a wide variety of thirst stimuli (review by Sanger, 1983). Opiate peptides and receptors are found not only in high concentrations in numerous regions of the brain, but also in several peripheral tissues, including the pituitary, pancreas, adrenal medulla and gastrointestinal tract (Pasternak and Childers, 1983). Selective antagonism of peripheral opiate receptors can be achieved using systemic administration of quaternary snalogues of opiate receptor antagonists, which do not readily cross the blood-brain barrier (Carr and Simon, 1983; Bianchi et al., 1982; Schulz et al., 1979). Comparisons between naloxone and its quaternary analogue therefore provide a means to assess a contribution of ¢ral and/or peripheral receptors to the suppression of drinking responses. Using this approach, Brown and Holtzman (1981) recently demonstrated that quaternary naloxone and naltrexone failed to affect water consumption in water-deprived-fats, whereas the corresponding tertiary compounds produced dose-dependent (0.1-10.0 mg.kg 1 reductions They concluded that blockade of central opiate receptors was instrumental in in drinking. the effects of naloxone on water intake. Taste factors can have major effects on drinking (Emits and Corbit, 1973), and it has been repeatedly suggested that opiate antagonists interact with taste factors in fluid consumption (Cooper, 1982, 1983; Levine et al., 1982; The purpose of the present experiment, therefore, was to confirm and extend Rowland, 1982). the results of the Brown and Holtzman study, by comparing the effects of naloxone and its quaternary analogue, on the consumption of water, saline and saccharin solution, respectiverat. ly, in the water-deprived METHODS The subjects were 60 male hooded rats of the general strain (weight range 250-350 g) bred in our laboratory. They were housed in individual stainless steel cages, with food pellets (modified Diet 41B) and tap water available. Room temperature was maintained at 21oci.1, and a 12hr light--12hr dark cycle was in operation. All animals were accustomed to handling and injection procedures, and were adapted to a deprivation schedule of 22 hr without water, on alternate days. The rats were allocated to three equal groups, according to the type of fluid which was available during the drinking tests. For the first 30 min after each deprivation period, food was removed, and the test fluid was available in a calibrated cylinder, which was clipped to the front of the cage. The groups were given access to water, 0.005 M sodium saccharin solution, and 3.9% sodium chloride solution, respectively. On intervening non-deprivation days, food and water were available ad --* lib Within each taste condition, the animals were subdivided into two groups, one receiving naloxone hydrochloride or isotonic saline vehicle, the other receiving methyl-naloxone hydrochloride or vehicle. Within each drug group, each animal was t sted on 5 occasions, follow-f ing subcutaneous administration of 0.01, 0.1, 1.0 or 10.0 mg.kg , or vehicle, respectively. Order of injection was randomised within each animal, and at least 48hr intervened between 197
798
PreliminaryNotes
successive injections. Doses of drug refer to the base, and injections were given 15-20 min before the drinking test. During the 30 min test (carried out in the light phase), fluid consumption (ml) was determined at 6 min intervals. The data analysed using 3-way analysis of variance (ANOVA) with repeated measures on two factors, separately for naloxone and its quaternary analogue. The factors were dose of drug, fluid condition, and timecourse of drinking. The Dunnett test (one-taiLed) was used for comparisons between individual groups and the corresponding control group, and the t-statistic was employed for planned comparisons between fluid conditions (Miner, 1971). RESULTS Naloxone. In the naloxone-treated groups, there were significant differences between the consumption over 30 min of the three avail ble fluids, F (2,27) = 13.60, P
r
IOL
WATER
VW0
SACCHARIN
j
SALINE
“b-Th+kiOvk5kdi-#o NALOXONE mg.kg-1
FIG. 1 -1 ) reduced intake of water, 0.005M sodium saccharin solution, and Naloxone (0.01-10.0 mg.kg Results are shown as the mean with S.E. 0.09% saline respectively, in water-deprived rats. Statistical comparisons of individual dose conditions against bars. N=lO per group. **pco.o1. *p
199
PreliminaryNotes
VEHICLE
NALOXONE
B
. 0 9%SALINE 0 WATER
Y 2
z6E 3 u_ 4-
TIME COURSE -SIX MINUTE INTERVALS
FIG. 2 Time-course of drinking over a 30 min test for water and saline intake, after injection of vehicle (A) or 1.0 mg.kg-1 naloxone (B). Results are shown as the mean with S.E. bars. N = 10 per group. Comparison of saline intake against water intake: **p
1 after quaternary
naloxone
____^_
-----_.ll-_l-._.._-.--_~.^_I__I__ Methyl-Naloxone 0 _ll-___-l-li
administration
(mg.kg -l)
1.0 0.1 0.01 l--._-.lli~-.-.-l.l~___-__-.___--.---____
10.0 _^________
Water
19.2 21.1
19.1 f1.3
19.0 f1.2
18.2 f1.2
18.8 fO.9
Saccharin
23.2 f2.0
21.2 f2.0
24.2 22.1
21.7 +1.3
20.4 t1.0
Saline
24.6 t2.9
23.5 +1.8
28.1 f2.2
27.8 +2.5
26.0 21.6
Results are shown as mean t SEM. naloxone treatment.
N = 10 per group.
Quaternary naloxone. Whilst there were significant taste condition, as in the naloxone-treated animals, nificant overall main effect of quaternary naloxone, time-course (Table 1).
-_--~ No significant
effects
of quaternary
differences in fluid intake due to analysis of variance revealed no sigor interaction with taste condition or
DISCUSSION Although naloxone produced dose-related reductions of fluid intake in each of the three taste conditions, it? quaternary analogue did not. This result confirms previous findings (Brown and Holtzman, 1981; Sanger, 1983), and indicates that reduction in drinking in waterdeprived rats was due to antagonism at opiate receptors in the brain. The consumption of isotonic saline was enhanced compared with that of water. Hall and Blass (1975) showed that rats which drank isotonic saline had a longer initial uninterrupted bout of drinking, and returned more frequently to drink at later stages, compared with rats which drank water. Fig. 2A confirms the extended initial high level of drinking in rats which had access to isotonic saline. The important finding of the present study was that the increased consumption in the isotonic saline group of animals was selectively abolished by naloxone in doses of 0.1 mg.kg-' and more. Since quaternary naloxone did not attenuate the hyperdipsia associated with saline drinking, peripheral opiate receptors do not appear
800
PreliminaryNotes
to have been involved. The present results nervous system are involved in the increased highly palatable salt solution.
suggest that opioid peptides in the central consumption of fluid which is induced by a
ACKNOWLEDGEMENTS We would like to thank Endo Laboratories for the gift of naloxone, and Francopia (Paris) for the gift of methyl-naloxone hydrochloride. S.T. is a recipient of a postgraduate studentship from the Medical Research Council (U.K.). REFERENCES Quaternary narcotic antagonBianchi, G., Fiocchi, R., Tavani, A. and Manara, L. (1982) and gastrointestinal transit inhibiists' relative ability to prevent antinociception tion in morphine-treated rats as an index of peripheral selectivity. Life Sci., 30: 1875-1884. Opiate antagonists: central Brown, D.R. and Holtzman, S.G. (1981) Brain Res., 221: 432-436. suppressing water intake of the rat. Carr, K.D. and Simon, E.J. (1983) stimulation-induced feeding.
sites of action
Effects of naloxone and its quaternary Neuropharmacology, 22: 127-130.
Palatability-induced drinking after administration Cooper, S.J. (1982) Substance Alcohol naltrexone and diazepam in the non-deprived rat. 3: 259-265. Cooper, S.J. (1983) saccharin choice
analogue
in
on
of morphine, Actions/Misuse,
Effects of opiate agonists and antagonists on fluid intake and Neuropharmacology, 22: 323-328. in the rat.
Emits, T. and Corbit, J.D. Psychol., 83: 37-31.
(1973)
Taste as a dipsogenic
stimulus.
J. camp. physiol.
Orogastric, hydrational, and behavioral controls of Hall, W.G. and Blass, E.M. (1975) drinking following water deprivation in rats. J. camp. physiol. Psychol., 89: 939-954. _I_ Flavor enhances Levine, A.S., Murray, S.S., Kneip, J., Grace, M. and Morley, J.E. (1982) Physiol. Psychol., 28: 23-25. the antidipsogenic effect of naloxone. The actions of opiates and opioid peptides. Pasternak, G.W. and Childers, S.R. (1983) 3 (Turner, P. and Shand, D.G., Eds.), In: Recent --1111 Advances in Clinical Pharmacology, pp.252-279, Churchill, Livingstone, Edinburgh. Comparison of the suppression by naloxone of water intake induced in Rowland, N. (1982) Pharmac. Biochem. Behav., 16: rats by hyperosmolarity, hypovolemia, and angiotensin. 87-91. Opiates and ingestive behavior. Sanger, D.L. (1983) VOl . 2 (Cooper, S.J. Ed.), Academic Press, London
In: Theory (in press).
in Psychopharmacology
Centrally and peripherally mediated Schulz, R., Wuster, M. and Hers, A. (1979) Naunyn-Schmiedeberg's Arch. Pharmacol., of intestinal motility by opioids. -255-260. Winer, B.J. (1971) New York.
Statistical
Principles
in Experimental --- Design,
2nd ed.
inhibition 308:
McGraw-Hill,