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Withdrawal from morphine generalizes to a pentylenetetrazol stimulus
Neuropeptides 5: 37-40,1984
WITHDRAWAL FROM MORPHINE GENERALIZES TO A PENTYLENETETRAZOL STIMULUS M.W. Emmett-Oglesby, C.M. Harris, J.D. Lane and H.Lal Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107, USA (reprint requests to MWE-0) ABSTRACT Rats trained to discriminate pentylenetetrazol (PTZ) from saline in a two-lever food-reinforced operant task were given a three-day course of morphine, 15 to 45 mg/kg tid, ip. On the third day naloxone produced dose-dependent generalization to the PTZ stimulus, with 66% of subjects selecting the PTZ lever after the highest dose (0.32 mg/kg). Following termination of morphine injections, generalization of spontaneous withdrawal was tested. Approximately 50% of subjects selected the PTZ lever at 24 and 48 hrs after the last morphine, and by 96 hrs the percentage of subjects selecting the PTZ lever had dropped to 11%. Rats that chose the PTZ lever at 48 hrs were given diazepam, 5.0 mg/kg, which blocked the PTZ-like stimulus. These data demonstrate that morphine withdrawal produces a stimulus with PTZ-like characteristics which can be blocked by an anxiolytic, and they suggest that the PTZ discrimination may have general utility for investigating drug dependence and withdrawal in animals. INTRODUCTION Withdrawal from many drugs of dependence is characterized, in humans, first by early symptoms such as craving for the drug, anxiety and irritability, and later by overt physical signs. Because of the subjective nature of these symptoms, it has not been possible, until recently, to use animals in experiments designed to explore the role of these symptoms in drug dependence. Recently, however, the use of drug discrimination methodology has provided a novel approach to determining subjective events during drug-withdrawal in nonhuman species (1). This methodology involves training animals to detect a drug induced internal stimulus, and then testing whether other stimuli are detected as similar to the training drug. In this report we describe the utility of the pentylenetetrazol (PTZ) discrimination method (1, 2) for detecting 37
withdrawal in morphine-dependent animals. The rationale for this study is that the pharmacology of PTZ in animals parallels human anxiety (for review, see 2) and that withdrawal from drugs of dependence produces anxiety in man. Therefore, if the discriminative stimuli produced by PTZ are related to anxiety, they should generalize to the drug withdrawal. Previous work from this laboratory has shown that rats trained to detect the discriminative stimulus properties of PTZ would detect a PTZ-like stimulus produced by withdrawal from benzodiazepine dependence, and this stimulus was blocked by an anxiolytic drug (3,4,5). The present experiment was designed to determine if withdrawal from morphine would also produce a stimulus with PTZ-like properties which could be blocked by diazepam. METHODS Male Long Evans hooded rats were trained to perform a two lever operant task, as described previously (1). Drug discrimination training was carried out in Coulbourn behavioral chambers controlled by the Operant Program for the Neurosciences (6). Presses on only one lever were rewarded when subjects were injected with PTZ, 20 mg/kg, and presses on the other lever were rewarded when subjects received saline, 1 ml/kg. Ten correct lever presses were rewarded with one food pellet (FRlO), while lever presses on the incorrect lever were recorded but not reinforced. Subjects received saline or PTZ daily in an irregular sequence during training. They were placed into the behavioral chambers 15 min after the injection, and allowed to press for up to 10 min or to 100 reinforcements. After meeting the criterion of selecting correct lever on 10 consecutive days, rats were used in drug discrimination tests, although training continued on non-test days. Test sessions were terminated immediately after a lever was selected (FRlO completed) and a single reinforcement was delivered, or after 10 min if no lever-choice was made. All injections were intraperitoneal. To test whether morphine or naloxone would produce a PTZ-like stimulus when given acutely, these drugs were tested in the following manner. Morphine, 5 mg/kg, was given 30 min before the test session. Naloxone, 0.32 and 40 mg/kg, was given 10 min before the test session. To test whether morphine could block the PTZ stimulus, morphine, 5 mg/kg, was given 30 min before PTZ, 20 mg/kg, and the rats were tested 15 min later. In a group of 21 rats, discrimination training was halted, and they were treated with morphine at 8-hr intervals for 3 days. On the first day they received 15 mg/kg tid, and on the second day they received 30 mg/kg tid. On the third day, they were injected with 45 mg/kg morphine, followed by an injection of naloxone hydrochloride, and were tested for generalization to the PTZ stimulus 10 min later. Morphine injections were then continued at 45 mg/kg for an additional day. Spontaneous 38
withdrawal was then assessed by testing for generalization 15 min after saline injections at 24, 48 and 96 hrs after the last dose of morphine. Those rats choosing the PTZ lever at 48 hrs post-morphine were given diazepam, 5 mg/kg, and tested again 15 min later. Stimulus control was assessed at 96 hrs after the last morphine administration by testing after PTZ, 20 mg/kg. RESULTS Before chronic morphine administration, rats discriminated PTZ from saline with a high degree of accuracy. Neither morphine nor naloxone generalized to PTZ, and morphine did not block detection of the PTZ stimulus (Table I). Naloxone did not cause diarrhea in rats before chronic morphine. During chronic morphine, naloxone generalized to the PTZ stimulus TABLE I: Generalization of VariOUS in a dose-dependent manner Treatments to the Pentylenetetrazol (see Figure, below) with Stimulus Before Repeated Morphine. 66% of the subjects selletting the PTZ lever Dose % Selecting after the highest dose PTZ Lever Treatment (mu/kg) N tested. Naloxone treatment produced diarrhea in PTZ 20.00 21 100 Saline 21 0 7Or Morphine 5.00 z 22 Naloxone 0.32 17 40.00 8 0 PTZ 6 Morbhine
20.00 5.00
13
85
TABLE II: Spontaneous Withdrawal from Morphine Hours After Last Morphine
N
24
17
% Selecting PTZ Lever 47
4,
1
.04
48
19
42
96
9
11
.08
NALOXONE
.16
.32
(mg/kg)
FIGURE: Generalization of Naloxone to the Pentylenetetrazol Stimulus After Repeated Morphine.
chronic morphine treated rats. Termination of chronic morphine produced no overt signs of withdrawal, such as "wet dog shakes." However approximately 50% of subjects selected the PTZ lever at 24 and 48 hrs following termination of chronic morphine (Table II). The rats selecting the PTZ lever at 48 hrs were given diazepam, 5 mg/kg, and retested. Of these rats 90% selected the saline lever and one made no selection after diazepam. At 96 39
hrs after the last morphine dose the percentage of subjects selecting the PTZ lever had dropped to 11%. When given PTZ at 96 hrs, 90% of the rats selected the PTZ lever. DISCUSSION These results demonstrate that withdrawal from morphine elicits an internal stimulus similar to that of pentylenetetrazol. This PTZ-like withdrawal stimulus 1) is seen early in the course of development of dependence on morphine, 2) can be precipitated in a dose-dependent manner by naloxone and 3) can be blocked by diazepam. Neither saline nor naloxone generalized to the PTZ stimulus before chronic morphine treatment, but after repeated morphine injections, naloxone produced a dose-dependent generalization to the PTZ stimulus (precipitated withdrawal). After termination of chronic morphine, saline also produced a partial generalization to the PTZ stimulus (spontaneous withdrawal). This generalization reflected accurate detection of a weak stimulus because the rats exhibited 90% correct lever choice when given PTZ at the end of the experiment. The experimental subjects showed withdrawal generalized to the PTZ stimulus after only ten injections of morphine given over a 72 hr period: the only physical sign of dependence observed was diarrhea produced by naloxone. Thus the PTZ-like stimulus can be detected in an early state of physical dependence on These findings indicate that detection of withdrawal morphine. as a PTZ-like stimulus is a more sensitive measure than the physical signs of withdrawal from morphine. Our data with diazepam in this experiment (and in 3, 4 and 5) suggest that this withdrawal stimulus is related to anxiety reported by humans undergoing narcotic withdrawal. REFERENCES 1.
2. 3. 4.
5.
6.
Lal, H. and Shearman, G.T. (1980). Interoceptive discriminative stimuli in development of CNS drugs and a case of an animal model of anxiety. Annual Reports in Medicinal Chemistry 15: 51-58. Lal, H. and Emmett-Oglesby, M.W. (1983). Behavioral analogues of anxiety. 22: 1423-1441. Emmett-Oglesby, M.W., Spencer, D.G., Jr., Elmesallamy F. and Lal, H. (1983) The pentylenetetrazol model of anxiety detects withdrawal from diazepam in rats. Life Sciences 33: 161-168. Emmett-Oglesby, M.W., Spencer, D.G., Lewis, M.W., Elmesallamy, F. and H. La1 (1983). Anxiogenic aspects of diazepam withdrawal can be detected in animals. European Journal of Pharmacology 92: 127-130. Lal, H. Emmett-Oglesby, M.W., Spencer, D.G., Jr. and Elmesallamy, F. (1983). Evidence for behavioral signs analogous to anxiety caused by withdrawal from diazepam in the rat. Federation Proceedings 42: 1166. Emmett-Oglesby, M.W., Spencer, D.G. and Arnoult, D.E. (1982). TRS-80-based system for the control of behavioral experiments. Pharmacology Biochemistry and Behavior 17: 583-587. 40
WITHDRAWAL FROM MORPHINE GENERALIZES TO A PENTYLENETETRAZOL STIMULUS M.W. Emmett-Oglesby, C.M. Harris, J.D. Lane and H.Lal Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107, USA (reprint requests to MWE-0) ABSTRACT Rats trained to discriminate pentylenetetrazol (PTZ) from saline in a two-lever food-reinforced operant task were given a three-day course of morphine, 15 to 45 mg/kg tid, ip. On the third day naloxone produced dose-dependent generalization to the PTZ stimulus, with 66% of subjects selecting the PTZ lever after the highest dose (0.32 mg/kg). Following termination of morphine injections, generalization of spontaneous withdrawal was tested. Approximately 50% of subjects selected the PTZ lever at 24 and 48 hrs after the last morphine, and by 96 hrs the percentage of subjects selecting the PTZ lever had dropped to 11%. Rats that chose the PTZ lever at 48 hrs were given diazepam, 5.0 mg/kg, which blocked the PTZ-like stimulus. These data demonstrate that morphine withdrawal produces a stimulus with PTZ-like characteristics which can be blocked by an anxiolytic, and they suggest that the PTZ discrimination may have general utility for investigating drug dependence and withdrawal in animals. INTRODUCTION Withdrawal from many drugs of dependence is characterized, in humans, first by early symptoms such as craving for the drug, anxiety and irritability, and later by overt physical signs. Because of the subjective nature of these symptoms, it has not been possible, until recently, to use animals in experiments designed to explore the role of these symptoms in drug dependence. Recently, however, the use of drug discrimination methodology has provided a novel approach to determining subjective events during drug-withdrawal in nonhuman species (1). This methodology involves training animals to detect a drug induced internal stimulus, and then testing whether other stimuli are detected as similar to the training drug. In this report we describe the utility of the pentylenetetrazol (PTZ) discrimination method (1, 2) for detecting 37
withdrawal in morphine-dependent animals. The rationale for this study is that the pharmacology of PTZ in animals parallels human anxiety (for review, see 2) and that withdrawal from drugs of dependence produces anxiety in man. Therefore, if the discriminative stimuli produced by PTZ are related to anxiety, they should generalize to the drug withdrawal. Previous work from this laboratory has shown that rats trained to detect the discriminative stimulus properties of PTZ would detect a PTZ-like stimulus produced by withdrawal from benzodiazepine dependence, and this stimulus was blocked by an anxiolytic drug (3,4,5). The present experiment was designed to determine if withdrawal from morphine would also produce a stimulus with PTZ-like properties which could be blocked by diazepam. METHODS Male Long Evans hooded rats were trained to perform a two lever operant task, as described previously (1). Drug discrimination training was carried out in Coulbourn behavioral chambers controlled by the Operant Program for the Neurosciences (6). Presses on only one lever were rewarded when subjects were injected with PTZ, 20 mg/kg, and presses on the other lever were rewarded when subjects received saline, 1 ml/kg. Ten correct lever presses were rewarded with one food pellet (FRlO), while lever presses on the incorrect lever were recorded but not reinforced. Subjects received saline or PTZ daily in an irregular sequence during training. They were placed into the behavioral chambers 15 min after the injection, and allowed to press for up to 10 min or to 100 reinforcements. After meeting the criterion of selecting correct lever on 10 consecutive days, rats were used in drug discrimination tests, although training continued on non-test days. Test sessions were terminated immediately after a lever was selected (FRlO completed) and a single reinforcement was delivered, or after 10 min if no lever-choice was made. All injections were intraperitoneal. To test whether morphine or naloxone would produce a PTZ-like stimulus when given acutely, these drugs were tested in the following manner. Morphine, 5 mg/kg, was given 30 min before the test session. Naloxone, 0.32 and 40 mg/kg, was given 10 min before the test session. To test whether morphine could block the PTZ stimulus, morphine, 5 mg/kg, was given 30 min before PTZ, 20 mg/kg, and the rats were tested 15 min later. In a group of 21 rats, discrimination training was halted, and they were treated with morphine at 8-hr intervals for 3 days. On the first day they received 15 mg/kg tid, and on the second day they received 30 mg/kg tid. On the third day, they were injected with 45 mg/kg morphine, followed by an injection of naloxone hydrochloride, and were tested for generalization to the PTZ stimulus 10 min later. Morphine injections were then continued at 45 mg/kg for an additional day. Spontaneous 38
withdrawal was then assessed by testing for generalization 15 min after saline injections at 24, 48 and 96 hrs after the last dose of morphine. Those rats choosing the PTZ lever at 48 hrs post-morphine were given diazepam, 5 mg/kg, and tested again 15 min later. Stimulus control was assessed at 96 hrs after the last morphine administration by testing after PTZ, 20 mg/kg. RESULTS Before chronic morphine administration, rats discriminated PTZ from saline with a high degree of accuracy. Neither morphine nor naloxone generalized to PTZ, and morphine did not block detection of the PTZ stimulus (Table I). Naloxone did not cause diarrhea in rats before chronic morphine. During chronic morphine, naloxone generalized to the PTZ stimulus TABLE I: Generalization of VariOUS in a dose-dependent manner Treatments to the Pentylenetetrazol (see Figure, below) with Stimulus Before Repeated Morphine. 66% of the subjects selletting the PTZ lever Dose % Selecting after the highest dose PTZ Lever Treatment (mu/kg) N tested. Naloxone treatment produced diarrhea in PTZ 20.00 21 100 Saline 21 0 7Or Morphine 5.00 z 22 Naloxone 0.32 17 40.00 8 0 PTZ 6 Morbhine
20.00 5.00
13
85
TABLE II: Spontaneous Withdrawal from Morphine Hours After Last Morphine
N
24
17
% Selecting PTZ Lever 47
4,
1
.04
48
19
42
96
9
11
.08
NALOXONE
.16
.32
(mg/kg)
FIGURE: Generalization of Naloxone to the Pentylenetetrazol Stimulus After Repeated Morphine.
chronic morphine treated rats. Termination of chronic morphine produced no overt signs of withdrawal, such as "wet dog shakes." However approximately 50% of subjects selected the PTZ lever at 24 and 48 hrs following termination of chronic morphine (Table II). The rats selecting the PTZ lever at 48 hrs were given diazepam, 5 mg/kg, and retested. Of these rats 90% selected the saline lever and one made no selection after diazepam. At 96 39
hrs after the last morphine dose the percentage of subjects selecting the PTZ lever had dropped to 11%. When given PTZ at 96 hrs, 90% of the rats selected the PTZ lever. DISCUSSION These results demonstrate that withdrawal from morphine elicits an internal stimulus similar to that of pentylenetetrazol. This PTZ-like withdrawal stimulus 1) is seen early in the course of development of dependence on morphine, 2) can be precipitated in a dose-dependent manner by naloxone and 3) can be blocked by diazepam. Neither saline nor naloxone generalized to the PTZ stimulus before chronic morphine treatment, but after repeated morphine injections, naloxone produced a dose-dependent generalization to the PTZ stimulus (precipitated withdrawal). After termination of chronic morphine, saline also produced a partial generalization to the PTZ stimulus (spontaneous withdrawal). This generalization reflected accurate detection of a weak stimulus because the rats exhibited 90% correct lever choice when given PTZ at the end of the experiment. The experimental subjects showed withdrawal generalized to the PTZ stimulus after only ten injections of morphine given over a 72 hr period: the only physical sign of dependence observed was diarrhea produced by naloxone. Thus the PTZ-like stimulus can be detected in an early state of physical dependence on These findings indicate that detection of withdrawal morphine. as a PTZ-like stimulus is a more sensitive measure than the physical signs of withdrawal from morphine. Our data with diazepam in this experiment (and in 3, 4 and 5) suggest that this withdrawal stimulus is related to anxiety reported by humans undergoing narcotic withdrawal. REFERENCES 1.
2. 3. 4.
5.
6.
Lal, H. and Shearman, G.T. (1980). Interoceptive discriminative stimuli in development of CNS drugs and a case of an animal model of anxiety. Annual Reports in Medicinal Chemistry 15: 51-58. Lal, H. and Emmett-Oglesby, M.W. (1983). Behavioral analogues of anxiety. 22: 1423-1441. Emmett-Oglesby, M.W., Spencer, D.G., Jr., Elmesallamy F. and Lal, H. (1983) The pentylenetetrazol model of anxiety detects withdrawal from diazepam in rats. Life Sciences 33: 161-168. Emmett-Oglesby, M.W., Spencer, D.G., Lewis, M.W., Elmesallamy, F. and H. La1 (1983). Anxiogenic aspects of diazepam withdrawal can be detected in animals. European Journal of Pharmacology 92: 127-130. Lal, H. Emmett-Oglesby, M.W., Spencer, D.G., Jr. and Elmesallamy, F. (1983). Evidence for behavioral signs analogous to anxiety caused by withdrawal from diazepam in the rat. Federation Proceedings 42: 1166. Emmett-Oglesby, M.W., Spencer, D.G. and Arnoult, D.E. (1982). TRS-80-based system for the control of behavioral experiments. Pharmacology Biochemistry and Behavior 17: 583-587. 40