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Naloxone blockade of apomorphine-induced stereotyped behavior
Journal of the NeurologicalSciences, 1979, 43 : 13-17 © Elsevier/North-Holland BiomedicalPress
13
NALOXONE BLOCKADE OF APOMORPHINE-INDUCED STEREOTYPED BEHAVIOR Interaction of Endogenous Opiates with Dopamine
DAVID I. MARGOLIN and BYONG H. MOON Department of Pharmacology, Rush-Presbyterian St. Luke's Medical Center, Chicago, 1L (U.S.A.)
(Received 30 January, 1979) (Accepted 16 March, 1979)
SUMMARY The specific opiate antagonist, naloxone, inhibits the in vivo and in vitro activity of the endogenous opiate compounds which have heretofore been identified. In this study systemic naloxone administration successfully blocked the production of stereotyped behavior induced by the direct dopamine agonist apomorphine. This implies that the endogenous opiates contribute to the production of stereotyped behavior initiated by dopaminergic stimulation and that endogenous opiates may function as central neurotransmitters with dopaminergic activity.
INTRODUCTION The identification of endogenous peptide compounds with in vivo and in vitro activity similar to morphine (Snyder 1977) has prompted investigations to elucidate the function of these peptides. The known effects of exogenous opiates, particularly morphine, provide logical starting points for these studies. One route of investigation concerns the relationship between opiates and brain catecholamines. The acute administration of morphine influences the stereotyped behavior (S.B.) induced by the dopamine agonists, apomorphine or amphetamine. This dopaminergic-induced S.B. is predominantly due to the supranormal activity of dopamine agonists at dopamine receptor sites in the corpus striatum (Fog 1972). Unfortunately, the direction of morphine influence on S.B. is unclear since potentiation (McKenzie and Sadoff 1974) and diminution of dopaminergic-induced S.B. have both been reported (Fog 1970; Puri et al. 1973). Requests for reprints: Byong H. Moon, Ph.D., Department of Pharmacology, Rush-Presbyterian St. Luke's Medical Center, 1753West Congress Parkway, Chicago, IL 60612, U.S.A.
14 The acute administration of morphine causes an increased turnover of brain dopamine, characteristic of agents which antagonize dopamine activity in the central nervous system (CNS), such as the neuroleptics (Puri et al. 1973). Chronic morphine administration produces a progressive increase in spontaneous S.B. (Fog 1970; Smee and Overstreet 1976). Following cessation of chronic morphine treatment, a hypersensitivity to apomorphine- and amphetamine-induced stereotyped behavior is observed (Smee and Overstreet 1976). These effects on S.B. are nonspecific, however, and may accompany the use of either chronic dopamine agonists (Klawans and Margolin 1975) or antagonists (Smith and Davis 1975). The ability of morphine to modify dopaminergic S.B. and brain dopamine turnover suggests an interaction between brain dopamine and exogenous opiates, although the mechanism of this interaction is unclear. In this study we evaluate the effects of systemically administered naloxone on S.B. in order to elucidate whether a relationship exists between brain dopamine and the endogenous opiates as well. METHODS Subjects were 300-350 g, young, male albino guinea pigs. All animals were housed identically with two per plastic cage and permitted free access to food and water. Group A (n -~ 11) animals received sequential injections of 0.1, 0.15, 0.2, and 0.25 mg/kg apomorphine hydrochloride (Merck and Company). Group B (n ~ 12) animals received sequential injections of 0.1, 0.15, 0.2, and 0.25 mg/kg apomorphine hydrochloride with simultaneous injection of 0.4 mg/kg naloxone (Endo). All drugs were administered subcutaneously. Apomorphine hydrochloride was prepared freshly no longer than 15 min prior to injection to minimize oxidation. Naloxone was obtained from the commercial vial containing 0.4 mg/ml. Animals received only one injection per day with no less than 24 hr nor more than 48 hr separating sequential injections. The concentration of solution was prepared so that both groups received an equal volume of solution/ kg body weight. Immediately following injection 3 animals were placed in each metal observation cage. These cages contained metal grids on each side and the bottom to facilitate the observation of stereotyped behavior. Animals were rated for stereotyped behavior according to the following scale: 1-~ ----occasional licking of the grid and exploration; 2 + -~ occasional biting and gnawing at the grid, easily distracted by movement or sound in the room; 3+ -~ sustained moderate-intense biting and gnawing, animal easily distracted by noise; 4 + = persistent and continuous gnawing at one location. Each observation period lasted for one hour during which time 7 observations were recorded, the first two being 5 min apart and the last five 10 min apart. Each reading reflected the predominant degree of stereotyped behavior during the preceding interval, and all ratings were made by a blind observer. A median stereotypy score was computed for each dose of apomorphine, and the number of animals in each group scoring above and below this median was compared statistically. Due to the ordinal nature of the rating scale, the Fisher Exact Probability Test, a non-parametric technique, was utilized.
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APOMORPHINE(mg/kg) Fig. 1. Mean stereotypy scores for group A (n = 11) and group B (n = 12) animals during the 60-min observation period. * P = 0.002, t P < 0.001, ¢t P = 0.001 (Fisher Exact Probability Test). RESULTS Apomorphine-induced stereotyped behavior was effectively blocked by naloxone. Figure 1 graphically illustrates the dose response curve for apomorphine-induced stereotyped behavior. The numerical values corresponding to the points in Fig. 1 are found in Table 1. A comparison of the median stereotypy scores for the two groups demonstrates the statistically significant diminution of stereotypy in the naloxone-treated group at each dose of apomorphine (Table 2). DISCUSSION These data reflect the ability of naloxone to block apomorphine-induced S.B. These agents were chosen because naloxone is a specific opiate blocker and apomorphine is a specific dopamine agonist predominantly acting directly at the dopamine receptor site. Naloxone can block the analgesia induced by the administration of enTABLE 1 MEAN STEREOTYPY SCORES Apomorphine (mg/kg)
Mean 4- SE Group A stereotypy s c o r e
Mean 4- SE Group B stereotypy score
0.10
0.9 4- 0.08 1.89 4- 0.10
0.27 4- 0.08 0.82 4- 0.04
1.93 4- 0.09 2.30 -4- 0.08
1.01 4- 0.11 1.48 -4- 0.13
0.15 0.20 0.25
16 TABLE 2 COMPARISON OF MEDIAN STEREOTYPY SCORES All statistics p e r f o r m e d via the Fisher Exact Probability Test. M e d i a n -~ 0.57
M e d i a n = 1.04
0.1 m g / k g APO
Above
Below
0.15 m g / k g APO
Above
Below
Group A Group B
8 I
I 8
Group A Group B
11 0
0 12
P 0.002 Median -
P < 0.001 1.52
Median = 1.90
0.2 m g / k g APO
Above
Below
0.25 m g / k g APO
Above
Below
Group A Group B
11 1
0 11
Group A Group B
10 1
l 11
P 0.00l
P 0.001
dogenous opiates (Jacquet and Marks 1976) as well as their inhibitory effect on smooth muscle contraction (Puig et al. 1977). When administered alone, naloxone decreases the pain threshold, presumably by blocking endogenous opiate activity (Walker et al. 1977). Similarly, the blockade of stereotyped behavior by naloxone suggests an interaction between endogenous opiates and striatal dopamine activity. Opiate receptors have been identified in high concentration in the corpus striatum, paralleling the high concentration of endogenous opiates contained within the synaptosomal fraction of striatal neurons (Pasternak et al. 1975). Furthermore, enkephalins decrease the firing rate of neurons in several brain regions including the caudate nucleus (Frederickson and Norris 1976). These findings indicate that endogenous opiates may function as neurotransmitters in the striatum. Although the neuropharmacological mechanism of naloxone inhibition of S.B. is unknown, it appears to involve more than the blockade of apomorphine activity at the dopamine receptor site. Kuschinsky and Hornykiewicz (1972) found that morphine and chlorpromazine produced dose-dependent catalepsy and increased striatal homovanillic acid (H.V.A.) in rats. Only the morphine effects, however, were blocked by naloxone administration; naloxone alone produced neither catalepsy nor increase in striatal H.V.A. concentration. Wilkenring et al. (1976) found that morphine inhibited either dopamine or sodium fluoride-induced increase in adenylate cyclase activity in primate amygdaloid complex. Morphine alone, however, had no effect on basal adenylate cyclase activity. Furthermore, morphine blockade of dopamine sensitive adenylate cyclase was blocked by naloxone. The reversal of the morphine effect by naloxone as well as the inhibition of sodium fluoride and dopamine stimulation indicate an action of morphine at a site pharmacologically distinct from the dopamine receptor.
17 Biggio et al. (1978) d e m o n s t r a t e d t h a t a d m i n i s t r a t i o n o f the e n k e p h a l i n a n a l o g ( D - A l a 2 ) - m e t h i o n i n e - e n k e p h a l i n a m i d e caused a m a r k e d d o s e - d e p e n d e n t increase in H . V . A . c o n c e n t r a t i o n in the r a t striatum. This a n a l o g also increased d o p a a c c u m u l a t i o n following d o p a d e c a r b o x y l a s e inhibition, indicating t h a t it increased d o p a m i n e synthesis. This synthesis was p r e v e n t e d by n a l o x o n e a d m i n i s t r a t i o n b u t n o t by destruction o f p o s t s y n a p t i c d o p a m i n e receptors with kainic acid. Thus the o p i a t e d o p a m i n e interaction does n o t a p p e a r to involve the activity of opiates directly at the d o p a m i n e receptor site. A n e x p l a n a t i o n consistent with available d a t a is t h a t e n d o g e n o u s opiates facilitate s t e r e o t y p e d b e h a v i o r initiated b y d o p a m i n e r gic s t i m u l a t i o n via their activity at striatal opiate receptor sites. This m o d e l m a y help to elucidate the role o f e n d o g e n o u s opiates in o t h e r d o p a m i n e - r e l a t e d behaviors such as s c h i z o p h r e n i a a n d e ; : t r a p y r a m i d a l m o v e m e n t disorders.
REFERENCES Biggio, G., M. Casu, M. G. Corda, C. Dibello and G. L. Gessa (1978) Stimulation of dopamine synthesis in caudate nucleus by intrastriatal enkephalins and antagonism by naloxone, Science, 200: 552-554. Fog, R. (1970) Behavioral effects in rats of morphine and amphetamine and a combination of the two drugs, Psychopharmacologia, 16: 305-312. Fog, R. (1972) On stereotypy and catalepsy - - Studies on the effect of amphetamines and neuroleptics in rats, Acta neurol, scand., 48: 1456. Frederickson, R. C. A. and F. H. Norris (1976) Enkephalin-induced depression of single neurons in brain areas with opiate receptors - - Antagonism by naloxone, Science, 194: 440-442. Jacquet, Y. F. and N. Marks (1976) The C-fragment of fl-lipoprotein - - An endogenous neuroleptic or antipsychotogen ? Science, 194: 632-634. Klawans, H. L. and D. I. Margolin (1975) Amphetamine-induced hypersensitivity in guinea pigs - Implications in psychosis and human movement disorders, Arch. gen. Psychiat., 32: 725-732. Kuschinsky, K. and O. Hornykiewicz (1972) Morphine catalepsy in the rat - - Relation to striatal dopamine metabolism, Europ. J. PharmacoL, 19: 119-122. McKenzie, G. M. and M. Sadoff (1974) Effect of morphine and chlorpromazine on apomorphineinduced stereotyped behavior, J. Pharm. Pharmacol., 26: 280-282. Pasternak, G. W., R. Goodman and S. H. Snyder (1975) An endogenous morphine-like factor in mammalian brain, Life Sci., 16: 1765-1769. Puig, M. M., D. Gascon, G. L. Gravisco and J. M. Musacchio (1977) Endogenous opiate receptor ligand - - Electrically induced released in guinea pig ileum, Science, 195: 419-420. Puri, S. K., C. Reddy and H. Lal (1973) Blockade of central dopaminergic receptors by morphine - Effect of haloperidol, apomorphine, or benztropine. Res. Commun. chem. Path. Pharmacol., 5: 389-401. Smee, M. L. and D. H. Overstreet (1976) Alterations in the effects of dopamine agonists and antagonists on general activity in rats following chronic morphine treatment, Psychopharmacology, 49: 125-130. Smith, R. C. and J. M. Davis (1975) Behavioral supersensitivity to apomorphine and amphetamine after chronic high-dose haloperidol treatment, Psychopharm. Comm., 1 : 285-293. Snyder, S. H. (1977) Opiate receptors in the brain, N. EngL J. Med., 296: 266--271. Walker, J. M., G. G. Berntson and C. A. Sandman (1977) An analog of enkephalin having prolonged opiate-like effects in vivo, Science, 196: 85-87. Wilkenring, D., R. K. Mishra and M. H. Makman (1976) Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus, Life Sci., 19: 1129-1138.
13
NALOXONE BLOCKADE OF APOMORPHINE-INDUCED STEREOTYPED BEHAVIOR Interaction of Endogenous Opiates with Dopamine
DAVID I. MARGOLIN and BYONG H. MOON Department of Pharmacology, Rush-Presbyterian St. Luke's Medical Center, Chicago, 1L (U.S.A.)
(Received 30 January, 1979) (Accepted 16 March, 1979)
SUMMARY The specific opiate antagonist, naloxone, inhibits the in vivo and in vitro activity of the endogenous opiate compounds which have heretofore been identified. In this study systemic naloxone administration successfully blocked the production of stereotyped behavior induced by the direct dopamine agonist apomorphine. This implies that the endogenous opiates contribute to the production of stereotyped behavior initiated by dopaminergic stimulation and that endogenous opiates may function as central neurotransmitters with dopaminergic activity.
INTRODUCTION The identification of endogenous peptide compounds with in vivo and in vitro activity similar to morphine (Snyder 1977) has prompted investigations to elucidate the function of these peptides. The known effects of exogenous opiates, particularly morphine, provide logical starting points for these studies. One route of investigation concerns the relationship between opiates and brain catecholamines. The acute administration of morphine influences the stereotyped behavior (S.B.) induced by the dopamine agonists, apomorphine or amphetamine. This dopaminergic-induced S.B. is predominantly due to the supranormal activity of dopamine agonists at dopamine receptor sites in the corpus striatum (Fog 1972). Unfortunately, the direction of morphine influence on S.B. is unclear since potentiation (McKenzie and Sadoff 1974) and diminution of dopaminergic-induced S.B. have both been reported (Fog 1970; Puri et al. 1973). Requests for reprints: Byong H. Moon, Ph.D., Department of Pharmacology, Rush-Presbyterian St. Luke's Medical Center, 1753West Congress Parkway, Chicago, IL 60612, U.S.A.
14 The acute administration of morphine causes an increased turnover of brain dopamine, characteristic of agents which antagonize dopamine activity in the central nervous system (CNS), such as the neuroleptics (Puri et al. 1973). Chronic morphine administration produces a progressive increase in spontaneous S.B. (Fog 1970; Smee and Overstreet 1976). Following cessation of chronic morphine treatment, a hypersensitivity to apomorphine- and amphetamine-induced stereotyped behavior is observed (Smee and Overstreet 1976). These effects on S.B. are nonspecific, however, and may accompany the use of either chronic dopamine agonists (Klawans and Margolin 1975) or antagonists (Smith and Davis 1975). The ability of morphine to modify dopaminergic S.B. and brain dopamine turnover suggests an interaction between brain dopamine and exogenous opiates, although the mechanism of this interaction is unclear. In this study we evaluate the effects of systemically administered naloxone on S.B. in order to elucidate whether a relationship exists between brain dopamine and the endogenous opiates as well. METHODS Subjects were 300-350 g, young, male albino guinea pigs. All animals were housed identically with two per plastic cage and permitted free access to food and water. Group A (n -~ 11) animals received sequential injections of 0.1, 0.15, 0.2, and 0.25 mg/kg apomorphine hydrochloride (Merck and Company). Group B (n ~ 12) animals received sequential injections of 0.1, 0.15, 0.2, and 0.25 mg/kg apomorphine hydrochloride with simultaneous injection of 0.4 mg/kg naloxone (Endo). All drugs were administered subcutaneously. Apomorphine hydrochloride was prepared freshly no longer than 15 min prior to injection to minimize oxidation. Naloxone was obtained from the commercial vial containing 0.4 mg/ml. Animals received only one injection per day with no less than 24 hr nor more than 48 hr separating sequential injections. The concentration of solution was prepared so that both groups received an equal volume of solution/ kg body weight. Immediately following injection 3 animals were placed in each metal observation cage. These cages contained metal grids on each side and the bottom to facilitate the observation of stereotyped behavior. Animals were rated for stereotyped behavior according to the following scale: 1-~ ----occasional licking of the grid and exploration; 2 + -~ occasional biting and gnawing at the grid, easily distracted by movement or sound in the room; 3+ -~ sustained moderate-intense biting and gnawing, animal easily distracted by noise; 4 + = persistent and continuous gnawing at one location. Each observation period lasted for one hour during which time 7 observations were recorded, the first two being 5 min apart and the last five 10 min apart. Each reading reflected the predominant degree of stereotyped behavior during the preceding interval, and all ratings were made by a blind observer. A median stereotypy score was computed for each dose of apomorphine, and the number of animals in each group scoring above and below this median was compared statistically. Due to the ordinal nature of the rating scale, the Fisher Exact Probability Test, a non-parametric technique, was utilized.
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to 0 5 0 OZS-
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APOMORPHINE(mg/kg) Fig. 1. Mean stereotypy scores for group A (n = 11) and group B (n = 12) animals during the 60-min observation period. * P = 0.002, t P < 0.001, ¢t P = 0.001 (Fisher Exact Probability Test). RESULTS Apomorphine-induced stereotyped behavior was effectively blocked by naloxone. Figure 1 graphically illustrates the dose response curve for apomorphine-induced stereotyped behavior. The numerical values corresponding to the points in Fig. 1 are found in Table 1. A comparison of the median stereotypy scores for the two groups demonstrates the statistically significant diminution of stereotypy in the naloxone-treated group at each dose of apomorphine (Table 2). DISCUSSION These data reflect the ability of naloxone to block apomorphine-induced S.B. These agents were chosen because naloxone is a specific opiate blocker and apomorphine is a specific dopamine agonist predominantly acting directly at the dopamine receptor site. Naloxone can block the analgesia induced by the administration of enTABLE 1 MEAN STEREOTYPY SCORES Apomorphine (mg/kg)
Mean 4- SE Group A stereotypy s c o r e
Mean 4- SE Group B stereotypy score
0.10
0.9 4- 0.08 1.89 4- 0.10
0.27 4- 0.08 0.82 4- 0.04
1.93 4- 0.09 2.30 -4- 0.08
1.01 4- 0.11 1.48 -4- 0.13
0.15 0.20 0.25
16 TABLE 2 COMPARISON OF MEDIAN STEREOTYPY SCORES All statistics p e r f o r m e d via the Fisher Exact Probability Test. M e d i a n -~ 0.57
M e d i a n = 1.04
0.1 m g / k g APO
Above
Below
0.15 m g / k g APO
Above
Below
Group A Group B
8 I
I 8
Group A Group B
11 0
0 12
P 0.002 Median -
P < 0.001 1.52
Median = 1.90
0.2 m g / k g APO
Above
Below
0.25 m g / k g APO
Above
Below
Group A Group B
11 1
0 11
Group A Group B
10 1
l 11
P 0.00l
P 0.001
dogenous opiates (Jacquet and Marks 1976) as well as their inhibitory effect on smooth muscle contraction (Puig et al. 1977). When administered alone, naloxone decreases the pain threshold, presumably by blocking endogenous opiate activity (Walker et al. 1977). Similarly, the blockade of stereotyped behavior by naloxone suggests an interaction between endogenous opiates and striatal dopamine activity. Opiate receptors have been identified in high concentration in the corpus striatum, paralleling the high concentration of endogenous opiates contained within the synaptosomal fraction of striatal neurons (Pasternak et al. 1975). Furthermore, enkephalins decrease the firing rate of neurons in several brain regions including the caudate nucleus (Frederickson and Norris 1976). These findings indicate that endogenous opiates may function as neurotransmitters in the striatum. Although the neuropharmacological mechanism of naloxone inhibition of S.B. is unknown, it appears to involve more than the blockade of apomorphine activity at the dopamine receptor site. Kuschinsky and Hornykiewicz (1972) found that morphine and chlorpromazine produced dose-dependent catalepsy and increased striatal homovanillic acid (H.V.A.) in rats. Only the morphine effects, however, were blocked by naloxone administration; naloxone alone produced neither catalepsy nor increase in striatal H.V.A. concentration. Wilkenring et al. (1976) found that morphine inhibited either dopamine or sodium fluoride-induced increase in adenylate cyclase activity in primate amygdaloid complex. Morphine alone, however, had no effect on basal adenylate cyclase activity. Furthermore, morphine blockade of dopamine sensitive adenylate cyclase was blocked by naloxone. The reversal of the morphine effect by naloxone as well as the inhibition of sodium fluoride and dopamine stimulation indicate an action of morphine at a site pharmacologically distinct from the dopamine receptor.
17 Biggio et al. (1978) d e m o n s t r a t e d t h a t a d m i n i s t r a t i o n o f the e n k e p h a l i n a n a l o g ( D - A l a 2 ) - m e t h i o n i n e - e n k e p h a l i n a m i d e caused a m a r k e d d o s e - d e p e n d e n t increase in H . V . A . c o n c e n t r a t i o n in the r a t striatum. This a n a l o g also increased d o p a a c c u m u l a t i o n following d o p a d e c a r b o x y l a s e inhibition, indicating t h a t it increased d o p a m i n e synthesis. This synthesis was p r e v e n t e d by n a l o x o n e a d m i n i s t r a t i o n b u t n o t by destruction o f p o s t s y n a p t i c d o p a m i n e receptors with kainic acid. Thus the o p i a t e d o p a m i n e interaction does n o t a p p e a r to involve the activity of opiates directly at the d o p a m i n e receptor site. A n e x p l a n a t i o n consistent with available d a t a is t h a t e n d o g e n o u s opiates facilitate s t e r e o t y p e d b e h a v i o r initiated b y d o p a m i n e r gic s t i m u l a t i o n via their activity at striatal opiate receptor sites. This m o d e l m a y help to elucidate the role o f e n d o g e n o u s opiates in o t h e r d o p a m i n e - r e l a t e d behaviors such as s c h i z o p h r e n i a a n d e ; : t r a p y r a m i d a l m o v e m e n t disorders.
REFERENCES Biggio, G., M. Casu, M. G. Corda, C. Dibello and G. L. Gessa (1978) Stimulation of dopamine synthesis in caudate nucleus by intrastriatal enkephalins and antagonism by naloxone, Science, 200: 552-554. Fog, R. (1970) Behavioral effects in rats of morphine and amphetamine and a combination of the two drugs, Psychopharmacologia, 16: 305-312. Fog, R. (1972) On stereotypy and catalepsy - - Studies on the effect of amphetamines and neuroleptics in rats, Acta neurol, scand., 48: 1456. Frederickson, R. C. A. and F. H. Norris (1976) Enkephalin-induced depression of single neurons in brain areas with opiate receptors - - Antagonism by naloxone, Science, 194: 440-442. Jacquet, Y. F. and N. Marks (1976) The C-fragment of fl-lipoprotein - - An endogenous neuroleptic or antipsychotogen ? Science, 194: 632-634. Klawans, H. L. and D. I. Margolin (1975) Amphetamine-induced hypersensitivity in guinea pigs - Implications in psychosis and human movement disorders, Arch. gen. Psychiat., 32: 725-732. Kuschinsky, K. and O. Hornykiewicz (1972) Morphine catalepsy in the rat - - Relation to striatal dopamine metabolism, Europ. J. PharmacoL, 19: 119-122. McKenzie, G. M. and M. Sadoff (1974) Effect of morphine and chlorpromazine on apomorphineinduced stereotyped behavior, J. Pharm. Pharmacol., 26: 280-282. Pasternak, G. W., R. Goodman and S. H. Snyder (1975) An endogenous morphine-like factor in mammalian brain, Life Sci., 16: 1765-1769. Puig, M. M., D. Gascon, G. L. Gravisco and J. M. Musacchio (1977) Endogenous opiate receptor ligand - - Electrically induced released in guinea pig ileum, Science, 195: 419-420. Puri, S. K., C. Reddy and H. Lal (1973) Blockade of central dopaminergic receptors by morphine - Effect of haloperidol, apomorphine, or benztropine. Res. Commun. chem. Path. Pharmacol., 5: 389-401. Smee, M. L. and D. H. Overstreet (1976) Alterations in the effects of dopamine agonists and antagonists on general activity in rats following chronic morphine treatment, Psychopharmacology, 49: 125-130. Smith, R. C. and J. M. Davis (1975) Behavioral supersensitivity to apomorphine and amphetamine after chronic high-dose haloperidol treatment, Psychopharm. Comm., 1 : 285-293. Snyder, S. H. (1977) Opiate receptors in the brain, N. EngL J. Med., 296: 266--271. Walker, J. M., G. G. Berntson and C. A. Sandman (1977) An analog of enkephalin having prolonged opiate-like effects in vivo, Science, 196: 85-87. Wilkenring, D., R. K. Mishra and M. H. Makman (1976) Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus, Life Sci., 19: 1129-1138.