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Rapid induction of dependence to morphine in rats
Neuropharmacology Vol. 25, No. 3, pp. 31X-307, Printed in Great Britain. All rights reserved
1986
RAPID INDUCTION
Copyright
OF DEPENDENCE IN RATS
0
0028.3908/86 $3.00 + 0.00 1986 PergamonPressLtd
TO MORPHINE
C. M. K. LEUNG, S. DAI and C. W. OGLE Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong (Accepted
13 July 1985)
Summary-The rate of development of dependence to morphine was studied in female rats which were given increasing concentrations of morphine sulphate in their drinking fluid (5% sucrose solution). The occurrence of physical dependence was determined by the naloxone-precipitated withdrawal syndrome at various times during the 3-week experimental period. It was found that a significant degree of the withdrawal syndrome precipitated by naloxone was evident at 24 hr after starting administ~tion of morphine; the syndrome reached its greatest intensity after the rats had received the opiate for 7 days. This study shows that deuendence on morohine can be induced in rats by administration of the opiate in drinking fluid for a period shorter than 7 days. Key words: morphine, dependence, rats.
Most investigators have produced dependence to morphine in experimental animals by repeated injections of the drug or by implanting it in pellet form (Furmkin, 1974; Lange, Roerig, Fujimoto and Busse, 1983). A non-invasive method by oral administration of morphine has also been reported to be successful (Khavari and Risner, 1973; Fuentes, Hunt and Crossland, 1978; Gellert and Holtzman, 1978; Badawy, Evans and Evans, 1982; Dai, Hui and Ogle, 1984). Regardless of the route of administration, long induction periods, lasting up to several weeks, were employed in these investigations. However, the necessity for these long treatment periods was not explained and no attempt was made to determine the minimum time of exposure required for development of opiate dependence. It has, indeed, been reported that signs of physical dependence can be detected in rats as early as 24 hr after only one injection of a substained-release morphine emulsion (Collier, Francis and Schneider, 1972). Thus, it is possible that shorter exposure periods may also be sufficient for producing opiate dependence. The present study was carried out to determine the rate of development of dependence to morphine in rats given the drug in the drinking fluid. METHODS
Female Sprague-Dawley rats, weighing 14&160 g, were housed 3-4 per cage in a room where the temperature was maintained at 23 + 1‘C and relative humidity at 60-70%, and were exposed to a 12-hr lightdark cycle. The animals received rat chow (Ralston Purina Co., U.S.A.) and drinking fluid ad libitum. Naloxone HCl was a gift from Endo Lab. (New York, U.S.A.). Morphone sulphate was obtained from Macfarlan-Smith Ltd (Middlesex). 305
Sucrose, 5% w/v in ordinary tap water, was used to mask the bitter taste of morphine sulphate (Dai et
al., 1984) which was chronically administered in this drinking fluid. The opiate was given in increasing concentrations (48 hr apart) of 0.1,0.2,0.3 and finally 0,4mg/ml (expressed as the salt). The last concentration was used until the end of the 3-week experimental period. Control animals received either tap water or just sucrose 5% w/v in tap water as drinking fluid. Tests for the development of physical dependence on morphine were performed in different groups of animals on days 1, 2,4, 7, 14 and 21 after starting treatment with the drug. The body weight of all rats was measured every 2 days. Daily intake of fluid for each animal was determined by dividing the total consumption of the 3 or 4 rats in each cage by their number. The withdrawal syndrome precipitated by naloxone was used as an indicator of the development of physical dependence on morphine. Naloxone HCl (1 mg/kg; expressed as the salt) was administered intraperitoneally to controls treated with both tap water and sucrose and to rats treated chronically with morphine (Collier, Francis, Henderson and Schneider, 1974). Immediately after the injection of naloxone, the following behavioural parameters (withdrawal signs) were observed for 20 min: wet-dog shakes, head shakes, diarrhoea, ptosis, chattering teeth, writhing, chewing, paw tremor and irritability to touch and handling. The number of wi~drawal signs exhibited by each rat was recorded. The amount of faeces excreted during the observation period, and the percentage of loss of body weight at 4 hr after administration of naloxone, were also measured. Food and fluids were withheld until the completion of all measurements. Probabilities were determined by Student’s g-test
C. M. K. LEUNGet al.
306
6 0 ‘O-
fo-,,,,
I,,
2
4
,I
6
8
I,
I,
10
I,
12
14
1
16
I,
I,,
16
20
Days
Fig. 1. Daily fluid intakes of (A-A) tap water-treated (n = 24), (O-----O) morphine-treated (n = 30) and (a----0) sucrose-treated (n = 30) rats during the 3-week induction period. Values plotted are the means f SE. *P < 0.05, **P -c0.001 when compared with the corresponding values of the sucrose-treated rats. tP < 0.001 when compared with the corresponding values of the sucrose- or morphine-treated animals. for unpaired means. The upper limit for statistical significance was set at P < 0.05. RESULTS
Figure 1 shows the daily intake of fluid throughout the 3-week experimental period. The daily consumption of fluid of the controls given tap water was consistently and significantly less than that of the controls treated with sucrose and the rats treated with morphine (P < 0.001). The intake of the controls treated with sucrose tended to be greater than those
of the animals treated with morphine and statistical significance was reached on days 2, 8, 10, 12, 16 and 18. A transient decrease in intake was also observed in the group treated with morphine after each increment of the concentration of morphine in the drinking fluid (on days 2, 4, 6 and 8). The body weights of the rats in each group showed steady increases during the 3-week period and there were no significant differences between them (data not shown). Intraperitoneal injection of naloxone (1 mg/kg) evoked only a few withdrawal signs and a small percentage body weight loss and amount of faecal excretion in rats treated with sucrose (Fig. 2). A similar finding was made in the controls treated with tap water and injected with naloxone, and in the animals treated chronically with morphine and injected with saline (data not shown). In contrast, naloxone precipitated marked withdrawal signs in the rats treated chronically with morphine. The values for the three withdrawal parameters were already significantly greater than those of the controls treated with sucrose even on day 1. The severity of these parameters tended to increase with longer periods of administration of morphine, but appeared to reach a maximum after 7 days of treatment.
method of inducing dependence on morphine in experimental animals, because a more consistent concentration of morphine can be maintained in the animals and the procedure is not stressful (Fuentes et al., 1978; Badawy, Punjani and Evans, 1981; Badawy et al., 1982). However, animals often refuse to consume significant amount of food or water containing morphine, presumably because of the unpleasant taste. Attempts have been made to overcome these problems, such as by preloading the animals with paranterally-administered morphine (Kumar, Steinberg and Stolerman, 1969) by scheduled provision of morphine in the drinking solution (Gellert and Holtzman, 1978) by masking the taste of the solution
P z .P $ 0
0 6-
1
2
It has been suggested that chronic administration of morphine in drinking water may be a better
7
14
*32
5
21 "32
43
*a
"6
Days
Fig. 2. Naloxone-precipitated DISCUSSION
4
(withdrawal syndrome in (m)
morphine-treated and (0) sucrose-treated rats. The number of animals is shown on the top of each column. Values plotted are the means & SE. *P < 0.05, **P < 0.001 when compared with the corresponding values of the sucrosetreated controls.
Morphine-dependent
of morphine with sucrose (Fuentes et al., 1978; Dai et al., 1984), or by a regimen of starting with a small concentration of morphine and gradually increasing the content of narcotic in the drinking solution (Badawy et al., 1981; Dai et al., 1984). All of these methods, however, generally require several weeks to induce dependence to morphine. A combination of the latter two methods, was employed in the present study. The results not only confirm that dependence on morphine can successfully be induced by oral administration of the drug, but also show that it can develop in a much shorter period of time. If the values for the parameters of the withdrawal syndrome precipitated by naloxone, such as the number of withdrawal signs, percentage loss of body weight and amount of faeces excreted, faithfully reflect the degree of physical dependence on morphine, the present results indicate that the rats do indeed develop physical dependence to the narcotic after only 24 hr of drinking a solution of morphine. However, the degree of dependence appears to be proportional to the duration of exposure to the opiate; a peak level of dependence was achieved after 7 days. It is, therefore, suggested that the present method of administering morphine in a 5% sucrose drinking solution requires an induction period of only 1 week to produce dependence to morphine in rats.
Acknowledgements-The authors wish to thank Endo Lab. (New York, U.S.A.) for the gift of naloxone, and are grateful to Mr G. S. K. Man for his technical assistance.
rats
307 REFERENCES
Badawy A. A.-B., Funjani N. F. and Evans M. (1981) The role of liver tryptophan pyrrolase in the opposite effects of chronic administration and subsequent withdrawal of drugs of dependence on rat brain tryptophan metabolism. Biochem. J. 1%: 161-170. Badawy A. A.-B., Evans C. M. and Evans M. (1982) Production of tolerance and physical dependence in the rat by simple administration of morphine in drinking water. Br. J. Pharmac. 75: 485491. Collier H. 0. J., Francis D. L. and Schneider C. (1972) Modification of morphine withdrawal by drugs interacting with humoral mechanisms: some contradictions and their interpretation. Nature 237: 22&223. Collier H. 0. J., Francis D. L., Henderson G. and Schneider C. (1974) Quasi morphine-abstinence syndrome. Nature 249: 471473. Dai S., Hui S-C. G. and Ogle C. W. (1984) Morphine preference in rats previously morphine dependent. Pharmat. Res. Commun. 16: 495-511. Fuentes V. O., Hunt W. B. and Crossland J. (1978) The production of morphine tolerance and physical dependence by the oral route in the rat. Psychopharmacology 59: 65-69. Furmkin K. (1974) Physical dependence in rats after low morphine doses. Life Sci. 15: 455462. Gellert V. F. and Holtzman S. G. (1978) Development and maintenance of morphine tolerance and dependence in the rat by scheduled access to morphine drinking solution. J. iharmac. exp. Ther. 205: 536546. Khavari K. A. and Risner M. E. (1973) Concentrationingestion relation of morphine-aduheraied food and morphine solution. Psychopharmacology 30: 291-302. Kumar R., Steinberg H. and Stolerman I. P. (1969) Scienrific Basis of Drug Dependence (Steinberg H., Ed.), pp. 209-220. Churchill Press, London. Lange D. G., Roerig S. C., Fujimoto J. M. and Busse L. W. (1983) Withdrawal tolerance and unidirectional noncross-tolerance in narcotic pellet-implanted mice. J. Pharmat. exp. Ther. 224: 13-20.
1986
RAPID INDUCTION
Copyright
OF DEPENDENCE IN RATS
0
0028.3908/86 $3.00 + 0.00 1986 PergamonPressLtd
TO MORPHINE
C. M. K. LEUNG, S. DAI and C. W. OGLE Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong (Accepted
13 July 1985)
Summary-The rate of development of dependence to morphine was studied in female rats which were given increasing concentrations of morphine sulphate in their drinking fluid (5% sucrose solution). The occurrence of physical dependence was determined by the naloxone-precipitated withdrawal syndrome at various times during the 3-week experimental period. It was found that a significant degree of the withdrawal syndrome precipitated by naloxone was evident at 24 hr after starting administ~tion of morphine; the syndrome reached its greatest intensity after the rats had received the opiate for 7 days. This study shows that deuendence on morohine can be induced in rats by administration of the opiate in drinking fluid for a period shorter than 7 days. Key words: morphine, dependence, rats.
Most investigators have produced dependence to morphine in experimental animals by repeated injections of the drug or by implanting it in pellet form (Furmkin, 1974; Lange, Roerig, Fujimoto and Busse, 1983). A non-invasive method by oral administration of morphine has also been reported to be successful (Khavari and Risner, 1973; Fuentes, Hunt and Crossland, 1978; Gellert and Holtzman, 1978; Badawy, Evans and Evans, 1982; Dai, Hui and Ogle, 1984). Regardless of the route of administration, long induction periods, lasting up to several weeks, were employed in these investigations. However, the necessity for these long treatment periods was not explained and no attempt was made to determine the minimum time of exposure required for development of opiate dependence. It has, indeed, been reported that signs of physical dependence can be detected in rats as early as 24 hr after only one injection of a substained-release morphine emulsion (Collier, Francis and Schneider, 1972). Thus, it is possible that shorter exposure periods may also be sufficient for producing opiate dependence. The present study was carried out to determine the rate of development of dependence to morphine in rats given the drug in the drinking fluid. METHODS
Female Sprague-Dawley rats, weighing 14&160 g, were housed 3-4 per cage in a room where the temperature was maintained at 23 + 1‘C and relative humidity at 60-70%, and were exposed to a 12-hr lightdark cycle. The animals received rat chow (Ralston Purina Co., U.S.A.) and drinking fluid ad libitum. Naloxone HCl was a gift from Endo Lab. (New York, U.S.A.). Morphone sulphate was obtained from Macfarlan-Smith Ltd (Middlesex). 305
Sucrose, 5% w/v in ordinary tap water, was used to mask the bitter taste of morphine sulphate (Dai et
al., 1984) which was chronically administered in this drinking fluid. The opiate was given in increasing concentrations (48 hr apart) of 0.1,0.2,0.3 and finally 0,4mg/ml (expressed as the salt). The last concentration was used until the end of the 3-week experimental period. Control animals received either tap water or just sucrose 5% w/v in tap water as drinking fluid. Tests for the development of physical dependence on morphine were performed in different groups of animals on days 1, 2,4, 7, 14 and 21 after starting treatment with the drug. The body weight of all rats was measured every 2 days. Daily intake of fluid for each animal was determined by dividing the total consumption of the 3 or 4 rats in each cage by their number. The withdrawal syndrome precipitated by naloxone was used as an indicator of the development of physical dependence on morphine. Naloxone HCl (1 mg/kg; expressed as the salt) was administered intraperitoneally to controls treated with both tap water and sucrose and to rats treated chronically with morphine (Collier, Francis, Henderson and Schneider, 1974). Immediately after the injection of naloxone, the following behavioural parameters (withdrawal signs) were observed for 20 min: wet-dog shakes, head shakes, diarrhoea, ptosis, chattering teeth, writhing, chewing, paw tremor and irritability to touch and handling. The number of wi~drawal signs exhibited by each rat was recorded. The amount of faeces excreted during the observation period, and the percentage of loss of body weight at 4 hr after administration of naloxone, were also measured. Food and fluids were withheld until the completion of all measurements. Probabilities were determined by Student’s g-test
C. M. K. LEUNGet al.
306
6 0 ‘O-
fo-,,,,
I,,
2
4
,I
6
8
I,
I,
10
I,
12
14
1
16
I,
I,,
16
20
Days
Fig. 1. Daily fluid intakes of (A-A) tap water-treated (n = 24), (O-----O) morphine-treated (n = 30) and (a----0) sucrose-treated (n = 30) rats during the 3-week induction period. Values plotted are the means f SE. *P < 0.05, **P -c0.001 when compared with the corresponding values of the sucrose-treated rats. tP < 0.001 when compared with the corresponding values of the sucrose- or morphine-treated animals. for unpaired means. The upper limit for statistical significance was set at P < 0.05. RESULTS
Figure 1 shows the daily intake of fluid throughout the 3-week experimental period. The daily consumption of fluid of the controls given tap water was consistently and significantly less than that of the controls treated with sucrose and the rats treated with morphine (P < 0.001). The intake of the controls treated with sucrose tended to be greater than those
of the animals treated with morphine and statistical significance was reached on days 2, 8, 10, 12, 16 and 18. A transient decrease in intake was also observed in the group treated with morphine after each increment of the concentration of morphine in the drinking fluid (on days 2, 4, 6 and 8). The body weights of the rats in each group showed steady increases during the 3-week period and there were no significant differences between them (data not shown). Intraperitoneal injection of naloxone (1 mg/kg) evoked only a few withdrawal signs and a small percentage body weight loss and amount of faecal excretion in rats treated with sucrose (Fig. 2). A similar finding was made in the controls treated with tap water and injected with naloxone, and in the animals treated chronically with morphine and injected with saline (data not shown). In contrast, naloxone precipitated marked withdrawal signs in the rats treated chronically with morphine. The values for the three withdrawal parameters were already significantly greater than those of the controls treated with sucrose even on day 1. The severity of these parameters tended to increase with longer periods of administration of morphine, but appeared to reach a maximum after 7 days of treatment.
method of inducing dependence on morphine in experimental animals, because a more consistent concentration of morphine can be maintained in the animals and the procedure is not stressful (Fuentes et al., 1978; Badawy, Punjani and Evans, 1981; Badawy et al., 1982). However, animals often refuse to consume significant amount of food or water containing morphine, presumably because of the unpleasant taste. Attempts have been made to overcome these problems, such as by preloading the animals with paranterally-administered morphine (Kumar, Steinberg and Stolerman, 1969) by scheduled provision of morphine in the drinking solution (Gellert and Holtzman, 1978) by masking the taste of the solution
P z .P $ 0
0 6-
1
2
It has been suggested that chronic administration of morphine in drinking water may be a better
7
14
*32
5
21 "32
43
*a
"6
Days
Fig. 2. Naloxone-precipitated DISCUSSION
4
(withdrawal syndrome in (m)
morphine-treated and (0) sucrose-treated rats. The number of animals is shown on the top of each column. Values plotted are the means & SE. *P < 0.05, **P < 0.001 when compared with the corresponding values of the sucrosetreated controls.
Morphine-dependent
of morphine with sucrose (Fuentes et al., 1978; Dai et al., 1984), or by a regimen of starting with a small concentration of morphine and gradually increasing the content of narcotic in the drinking solution (Badawy et al., 1981; Dai et al., 1984). All of these methods, however, generally require several weeks to induce dependence to morphine. A combination of the latter two methods, was employed in the present study. The results not only confirm that dependence on morphine can successfully be induced by oral administration of the drug, but also show that it can develop in a much shorter period of time. If the values for the parameters of the withdrawal syndrome precipitated by naloxone, such as the number of withdrawal signs, percentage loss of body weight and amount of faeces excreted, faithfully reflect the degree of physical dependence on morphine, the present results indicate that the rats do indeed develop physical dependence to the narcotic after only 24 hr of drinking a solution of morphine. However, the degree of dependence appears to be proportional to the duration of exposure to the opiate; a peak level of dependence was achieved after 7 days. It is, therefore, suggested that the present method of administering morphine in a 5% sucrose drinking solution requires an induction period of only 1 week to produce dependence to morphine in rats.
Acknowledgements-The authors wish to thank Endo Lab. (New York, U.S.A.) for the gift of naloxone, and are grateful to Mr G. S. K. Man for his technical assistance.
rats
307 REFERENCES
Badawy A. A.-B., Funjani N. F. and Evans M. (1981) The role of liver tryptophan pyrrolase in the opposite effects of chronic administration and subsequent withdrawal of drugs of dependence on rat brain tryptophan metabolism. Biochem. J. 1%: 161-170. Badawy A. A.-B., Evans C. M. and Evans M. (1982) Production of tolerance and physical dependence in the rat by simple administration of morphine in drinking water. Br. J. Pharmac. 75: 485491. Collier H. 0. J., Francis D. L. and Schneider C. (1972) Modification of morphine withdrawal by drugs interacting with humoral mechanisms: some contradictions and their interpretation. Nature 237: 22&223. Collier H. 0. J., Francis D. L., Henderson G. and Schneider C. (1974) Quasi morphine-abstinence syndrome. Nature 249: 471473. Dai S., Hui S-C. G. and Ogle C. W. (1984) Morphine preference in rats previously morphine dependent. Pharmat. Res. Commun. 16: 495-511. Fuentes V. O., Hunt W. B. and Crossland J. (1978) The production of morphine tolerance and physical dependence by the oral route in the rat. Psychopharmacology 59: 65-69. Furmkin K. (1974) Physical dependence in rats after low morphine doses. Life Sci. 15: 455462. Gellert V. F. and Holtzman S. G. (1978) Development and maintenance of morphine tolerance and dependence in the rat by scheduled access to morphine drinking solution. J. iharmac. exp. Ther. 205: 536546. Khavari K. A. and Risner M. E. (1973) Concentrationingestion relation of morphine-aduheraied food and morphine solution. Psychopharmacology 30: 291-302. Kumar R., Steinberg H. and Stolerman I. P. (1969) Scienrific Basis of Drug Dependence (Steinberg H., Ed.), pp. 209-220. Churchill Press, London. Lange D. G., Roerig S. C., Fujimoto J. M. and Busse L. W. (1983) Withdrawal tolerance and unidirectional noncross-tolerance in narcotic pellet-implanted mice. J. Pharmat. exp. Ther. 224: 13-20.