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Chronic phencyclidine, like amphetamine, produces a decrease in [3H]spiroperidol binding in rat striatum
European Journal of Pharmacology, 78 (1982) 363-365 Elsevier Biomedical Press
363
Short communication
CHRONIC PHENCYCLIDINE, LIKE AMPHETAMINE, PRODUCES A DECREASE IN [3H]SPIROPERIDOL BINDING IN RAT STRIATUM HAROLD A. ROBERTSON
Department of Pharmacology, Faculty of Medicine, Dalhousie Universi(v, Halifax, Nova Scotia, Canada B3H 4H7 Received 12 January 1982, accepted 13 January 1982
H.A. ROBERTSON, Chronic phencyclidine, like amphetamine, produces a decrease in ["~H]spiroperidol binding in rat striatum, European J. Pharmacol. 78 (1982) 363-365. Rats were treated with d-amphetamine sulfate (5 and l0 mg/kg i.p.) and phencyclidine (PCP) (5 mg/kg i.p.) twice per day. After 21 days, [3H]spiroperidol binding in striatum was reduced by all treatments; receptor number (Bma~) and not affinity (KD) was affected. These results suggest that the psychotic effect of PCP may, like those of amphetamine, involve changes in dopamine receptors. Phencyclidine
Amphetamine
Dopamine receptors
[ 3H]Spiroperidol
I. Introduction
Phencyclidine (PCP) produces psychotomimetic effects in humans. These effects mimic the primary symptoms of schizophrenia and it has been suggested that PCP may be superior to amphetamine as drug model for schizophrenia (Allen and Young, 1978; Snyder, 1980). A variety of evidence suggests that the behavioral effects of PCP are, like those of amphetamine, mediated by dopaminergic mechanisms (Schlemmer et al., 1978; Doherty et al., 1980). In this present investigation, I show that chronic treatment of rats with PCP produces changes in dopamine antagonist (neuroleptic) binding that are similar to those seen after chronic amphetamine treatment.
2. Materials and methods
Male Wistar rats (200-350 g) were treated twice daily (10 mg/kg i.p.) with either d-amphetamine sulphate (gift of Smith, Kline and French, Ltd. Downsview, Ontario) or phencychdine (obtained from Bureau of Dangerous Drugs, Ottawa). Control animals received saline injections. After 21 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
Striatum
days treatment, animals were left drug free for at least 24 h prior to sacrifice. The animals were then killed and striata were removed and homogenized with a glass homogenizer and teflon pestle in 10 vol (w/v) of ice-cold 0.32 M sucrose and centrifuged 755 × g for 15 min. The supernatant was centrifuged 15 min at 40000 × g, the pellet resuspended in 10 vol 50 mM Tris-HC1 pH 7.4 using a polytron (setting 5). This suspension was then centrifuged 40000 × g for 15 rain and the supernatant discarded. The pellet was finally suspended in 25 vol of incubation buffer (120 mM NaC1, 2mM CaCI 2, 0.5 mM MgC12, 3.6 mM KC1, 50 mM Tris, 10 /~M pargyline and 5.6 mM ascorbic acid). Each experiment involved at least 6 animals in each treatment group and the procedure has been repeated 5 times. Aliquots of tissue (200-300 /~g protein) were incubated in incubation buffer for 30 min at 37°C with [3H]spiroperidol (New England Nuclear, spec. act. 35.9 Ci/mmol) (final concentrations 2, 1, 0.5, 0.25, 0.125 and 0.062 nM) in a final vol of l ml. Specific binding was defined as that displaced by 10/tM domperidone (Janssen Pharmaceutica NV) and, at 0.25 mM, was always greater than 80%. Incubations were terminated by filtration on Whatman G F / B glass fibre filters
364 which were washed with 2 X 5 ml of i n c u b a t i o n buffer. B o u n d r a d i o a c t i v i t y was d e t e r m i n e d by liquid scintillation c o u n t i n g in 3.6 ml Biofluor ( N e w E n g l a n d N u c l e a r ) with an efficiency of a b o u t 40%.
500>-
~
400-
CONTROL
~
.
~ ~ 200
BMAX CONTROL 494
I00
3. Results
:::
0
PCP
' 0'25 01~
KD 023
401
0 38
D AMPHET 337
0.29
(
3H SPIROPERIDOL ( nM }
A s p r e v i o u s l y r e p o r t e d , chronic t r e a t m e n t of a n i m a l s with d - a m p h e t a m i n e p r o d u c e d decreases in [ 3 H ] s p i r o p e r i d o l b i n d i n g in rat striatal h o m o genates (fig. 1). T h e decrease in [3H]spiroperidol b i n d i n g was the result of a decrease in the d e n s i t y o f r e c e p t o r sites (Bmax). The changes in a p p a r e n t a f f i n i t y ( K o ) of [ 3 H ] s p i r o p e r i d o l for the neuroleptic r e c e p t o r were always small a n d not significant. The decrease in Bm~, after chronic da m p h e t a m i n e was dose-related; a dose of 10 m g / k g p r o d u c e d a greater decrease in Bm~x than 5 m g / k g (fig. 1). C h r o n i c P C P t r e a t m e n t , like d - a m p h e t a m i n e
400-
///,/•u/f
E 300-
-
~t-CONTROL
Fig. 2. Effect of d-amphetamine and phencyclidine (PCP) on [3H]spiroperidol binding in rat striatum. A typical experiment is shown. The entire experiment (for the 5 mg PCP/kg twice daily dosage group) has been repeated 4 times (i.e. on 4 separate groups of rats, treated for 21 days) with similar results. The average decrease in the density of [ 3H]spiroperidol binding sites (Bmax) after chronic PCP was 17.84-4.0% (mean ± S,E.M., 4 experiments). In this experiment, the effect of chronic damphetamine (10 mg/kg twice daily) is shown for comparison. All determinations were performed in triplicate and typical S.E.M. are shown for the 2 nM points (* P<0.05 compared with control, *** P<0.001 compared with control). Inset shows K D and B..... for data illustrated.
t r e a t m e n t , also resulted in a decrease in r e c e p t o r d e n s i t y (Bin,x) with no a p p a r e n t effect on affinity (fig. 2).
o - 5 mg/kg
4. D i s c u s s i o n • 10 mg/kg
> 200-
~: 100-
0
I1~ r
o,~5o'2s 0'5
CONTROL 5mg/kg
438 379
0.29 0.29
lOmg/kg
343
0.29
;
~H-SPIROPERIDOL
( nM )
Fig. 1. Effect of chronic d-amphetamine on [3H]spiroperidol binding in rat striatum. A typical experiment is shown. The entire experiment (for the 10 mg/kg twice daily dosage group) has been repeated 4 times with similar results. The average decrease in the density of [3H]spiroperidol binding sites (Bm,x) after chronic d-amphetamine was 23.0 J=4.5% (mean ± S.E.M., 5 experiments). Inset shows values for Bin,, and Kr~ obtained from Scatchard plots of data shown.
P h e n c y c l i d i n e (PCP) was originally i n t r o d u c e d as an a n a e s t h e t i c a n d was useful because it prod u c e d a n a e s t h e s i a with g o o d analgesia a n d no r e s p i r a t o r y or c a r d i o v a s c u l a r depression. O n rec o v e r y f r o m anaesthesia, however, p a t i e n t s rep o r t e d vivid d r e a m s a n d h a l l u c i n a t i o n s a n d the d r u g was w i t h d r a w n f r o m use in h u m a n s (Snyder, 1980). P C P psychosis resembles s c h i z o p h r e n i a even to a greater extent than does a m p h e t a m i n e psychosis (Allen a n d Young, 1978; Snyder, 1980). A m p h e t a m i n e psychosis has long been thought to be the best e x p e r i m e n t a l m o d e l for s c h i z o p h r e n i a (Snyder, 1973). C h r o n i c t r e a t m e n t (20 days) of rats with a m p h e t a m i n e p r o d u c e d a decrease in the n u m b e r of [3H]spiroperidol b i n d i n g sites in striat u m but resulted in no changes in d o p a m i n e s t i m u l a t e d a d e n y l a t e cyclase activity ( H o w l e t t a n d N a h o r s k i , 1979). Increases in the b i n d i n g of
365 [3H]spiroperidol and [3H]haloperidol to sites in the caudate nucleus, putamen and nucleus accumhens of schizophrenic patients have been reported (Lee et al., 1978) but this finding is currently controversial because of the possible interfering effects of neuroleptic treatment in these patients (Snyder, 1981). A number of workers have reported that PCP appears to be operating, at least in part, by producing dopamine release in a manner similar to amphetamine (Schlemmer et al., 1978) or the non-amphetamine stimulants such as methylphenidate and cocaine (Doherty et al., 1980). The findings presented here show that PCP, like amphetamine, produces a decrease in the number of [3H]spiroperidol binding sites after chronic treatment. The best explanation for this finding would appear to be that chronic hyperactivity of dopaminergic neurotransmission leads to a downregulation of D 2-dopamine (or neuroleptic) receptors. There is now evidence to suggest that dopamine and dopamine agonists bind to a receptor site (the DI site) which is'distinct from the site at which neuroleptics bind (the D 2 site) (Seeman, 1981). Chronic hyperactivity of dopaminergic neurons with amphetamine (Howlett and Nahorski, 1978; Nielsen et al., 1980) or PCP (this paper) produces down-regulation in D 2 (neuroleptic) sites. What are the effects on dopamine agonist sites? This question has not been properly studied yet, partially because of difficulties with dopamine receptor agonist binding and its interpretation. However, evidence from behavioral studies suggests that chronic amphetamine treatment produces a supersensitivity in dopamine agonist receptors (Klawans and Margolin, 1975). It remains to be demonstrated whether this behavioral supersensitivity to chronic amphetamine treatment is accompanied by increased dopamine agonist binding. Chronic amphetamine treatment does not produce any detectable change in dopamine-sensitive adenylate cyclase (Howlett and Nahorski, 1978). The effects of chronic PCP on the behavioral effects of dopamine agonists also remain to be investigated.
In conclusion, these results show that chronic PCP or amphetamine treatment produce similar down-regulation in neuroleptic [3H]spiroperidol receptor density in rat striatum. This suggests it is possible that the production of psychoses in man by these two agents may involve an alteration in D2-dopamine receptor mechanisms.
Acknowledgements Supported by the Medical Research Council of Canada (Grant MA6454). I thank M.R. Peterson for expert technical assistance.
References Allen, R.M. and S.J. Young, 1978, Phencyclidine-induced psychosis, Am. J. Psychiat. 135, 1081. Doherty, J.D., M. Simonovic, R. So and H.Y. Meltzer, 1980, The effect of phencyclidine on dopamine synthesis and metabolism in rat striatum, European J. Pharmacol. 65, 139. Howlett, D.R. and S.R. Nahorski, 1979, Acute and chronic amphetamine treatments modulate striatal dopamine receptor binding sites, Brain Res. 161, 173. Klawans, H.L. and D.I. Margolin, 1975, Amphetamine-induced dopaminergic hyper-sensitivityin guinea pigs. Implications in psychosis and human movement disorders, Arch. Gen. Psychiat. 32, 725. Lee, T., P. Seeman, W.W. Tourtellotte, I.J. Farley and O. Hornykiewicz, 1978, Binding of [3H]neuroleptics and [3H]apomorphine in schizophrenic brains, Nature 274, 897. Nielsen, E.B., M. Neilsen, G. Elhson and C. Braestrup, 1980, Decreased spiroperidol and LSD binding in rat brain after continuous amphetamine, European J. Pharmacol. 66, 149. Schlemmer, R.F. Jr., J.A. Jackson, K.L. Preston, J.P. Bederka, Jr., D.L. Garver and J.M. Davis, 1978, Phencyclidineinduced stereotyped behavior in monkeys: antagonism by pimozide, European J. Pharmacol. 52, 379. Seeman, P., 1981, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Snyder, S.H., 1973, Amphetamine psychosis: a 'model' schizophrenia mediated by catecholamines, Am. J. Psychiat. 130, 61. Snyder, S.H., 1980, Phencyclidine, Nature 285, 355. Snyder, S.H., 1981, Dopamine receptors, neuroleptics and schizophrenia, Am. J. Psychiat. 138, 460.
363
Short communication
CHRONIC PHENCYCLIDINE, LIKE AMPHETAMINE, PRODUCES A DECREASE IN [3H]SPIROPERIDOL BINDING IN RAT STRIATUM HAROLD A. ROBERTSON
Department of Pharmacology, Faculty of Medicine, Dalhousie Universi(v, Halifax, Nova Scotia, Canada B3H 4H7 Received 12 January 1982, accepted 13 January 1982
H.A. ROBERTSON, Chronic phencyclidine, like amphetamine, produces a decrease in ["~H]spiroperidol binding in rat striatum, European J. Pharmacol. 78 (1982) 363-365. Rats were treated with d-amphetamine sulfate (5 and l0 mg/kg i.p.) and phencyclidine (PCP) (5 mg/kg i.p.) twice per day. After 21 days, [3H]spiroperidol binding in striatum was reduced by all treatments; receptor number (Bma~) and not affinity (KD) was affected. These results suggest that the psychotic effect of PCP may, like those of amphetamine, involve changes in dopamine receptors. Phencyclidine
Amphetamine
Dopamine receptors
[ 3H]Spiroperidol
I. Introduction
Phencyclidine (PCP) produces psychotomimetic effects in humans. These effects mimic the primary symptoms of schizophrenia and it has been suggested that PCP may be superior to amphetamine as drug model for schizophrenia (Allen and Young, 1978; Snyder, 1980). A variety of evidence suggests that the behavioral effects of PCP are, like those of amphetamine, mediated by dopaminergic mechanisms (Schlemmer et al., 1978; Doherty et al., 1980). In this present investigation, I show that chronic treatment of rats with PCP produces changes in dopamine antagonist (neuroleptic) binding that are similar to those seen after chronic amphetamine treatment.
2. Materials and methods
Male Wistar rats (200-350 g) were treated twice daily (10 mg/kg i.p.) with either d-amphetamine sulphate (gift of Smith, Kline and French, Ltd. Downsview, Ontario) or phencychdine (obtained from Bureau of Dangerous Drugs, Ottawa). Control animals received saline injections. After 21 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
Striatum
days treatment, animals were left drug free for at least 24 h prior to sacrifice. The animals were then killed and striata were removed and homogenized with a glass homogenizer and teflon pestle in 10 vol (w/v) of ice-cold 0.32 M sucrose and centrifuged 755 × g for 15 min. The supernatant was centrifuged 15 min at 40000 × g, the pellet resuspended in 10 vol 50 mM Tris-HC1 pH 7.4 using a polytron (setting 5). This suspension was then centrifuged 40000 × g for 15 rain and the supernatant discarded. The pellet was finally suspended in 25 vol of incubation buffer (120 mM NaC1, 2mM CaCI 2, 0.5 mM MgC12, 3.6 mM KC1, 50 mM Tris, 10 /~M pargyline and 5.6 mM ascorbic acid). Each experiment involved at least 6 animals in each treatment group and the procedure has been repeated 5 times. Aliquots of tissue (200-300 /~g protein) were incubated in incubation buffer for 30 min at 37°C with [3H]spiroperidol (New England Nuclear, spec. act. 35.9 Ci/mmol) (final concentrations 2, 1, 0.5, 0.25, 0.125 and 0.062 nM) in a final vol of l ml. Specific binding was defined as that displaced by 10/tM domperidone (Janssen Pharmaceutica NV) and, at 0.25 mM, was always greater than 80%. Incubations were terminated by filtration on Whatman G F / B glass fibre filters
364 which were washed with 2 X 5 ml of i n c u b a t i o n buffer. B o u n d r a d i o a c t i v i t y was d e t e r m i n e d by liquid scintillation c o u n t i n g in 3.6 ml Biofluor ( N e w E n g l a n d N u c l e a r ) with an efficiency of a b o u t 40%.
500>-
~
400-
CONTROL
~
.
~ ~ 200
BMAX CONTROL 494
I00
3. Results
:::
0
PCP
' 0'25 01~
KD 023
401
0 38
D AMPHET 337
0.29
(
3H SPIROPERIDOL ( nM }
A s p r e v i o u s l y r e p o r t e d , chronic t r e a t m e n t of a n i m a l s with d - a m p h e t a m i n e p r o d u c e d decreases in [ 3 H ] s p i r o p e r i d o l b i n d i n g in rat striatal h o m o genates (fig. 1). T h e decrease in [3H]spiroperidol b i n d i n g was the result of a decrease in the d e n s i t y o f r e c e p t o r sites (Bmax). The changes in a p p a r e n t a f f i n i t y ( K o ) of [ 3 H ] s p i r o p e r i d o l for the neuroleptic r e c e p t o r were always small a n d not significant. The decrease in Bm~, after chronic da m p h e t a m i n e was dose-related; a dose of 10 m g / k g p r o d u c e d a greater decrease in Bm~x than 5 m g / k g (fig. 1). C h r o n i c P C P t r e a t m e n t , like d - a m p h e t a m i n e
400-
///,/•u/f
E 300-
-
~t-CONTROL
Fig. 2. Effect of d-amphetamine and phencyclidine (PCP) on [3H]spiroperidol binding in rat striatum. A typical experiment is shown. The entire experiment (for the 5 mg PCP/kg twice daily dosage group) has been repeated 4 times (i.e. on 4 separate groups of rats, treated for 21 days) with similar results. The average decrease in the density of [ 3H]spiroperidol binding sites (Bmax) after chronic PCP was 17.84-4.0% (mean ± S,E.M., 4 experiments). In this experiment, the effect of chronic damphetamine (10 mg/kg twice daily) is shown for comparison. All determinations were performed in triplicate and typical S.E.M. are shown for the 2 nM points (* P<0.05 compared with control, *** P<0.001 compared with control). Inset shows K D and B..... for data illustrated.
t r e a t m e n t , also resulted in a decrease in r e c e p t o r d e n s i t y (Bin,x) with no a p p a r e n t effect on affinity (fig. 2).
o - 5 mg/kg
4. D i s c u s s i o n • 10 mg/kg
> 200-
~: 100-
0
I1~ r
o,~5o'2s 0'5
CONTROL 5mg/kg
438 379
0.29 0.29
lOmg/kg
343
0.29
;
~H-SPIROPERIDOL
( nM )
Fig. 1. Effect of chronic d-amphetamine on [3H]spiroperidol binding in rat striatum. A typical experiment is shown. The entire experiment (for the 10 mg/kg twice daily dosage group) has been repeated 4 times with similar results. The average decrease in the density of [3H]spiroperidol binding sites (Bm,x) after chronic d-amphetamine was 23.0 J=4.5% (mean ± S.E.M., 5 experiments). Inset shows values for Bin,, and Kr~ obtained from Scatchard plots of data shown.
P h e n c y c l i d i n e (PCP) was originally i n t r o d u c e d as an a n a e s t h e t i c a n d was useful because it prod u c e d a n a e s t h e s i a with g o o d analgesia a n d no r e s p i r a t o r y or c a r d i o v a s c u l a r depression. O n rec o v e r y f r o m anaesthesia, however, p a t i e n t s rep o r t e d vivid d r e a m s a n d h a l l u c i n a t i o n s a n d the d r u g was w i t h d r a w n f r o m use in h u m a n s (Snyder, 1980). P C P psychosis resembles s c h i z o p h r e n i a even to a greater extent than does a m p h e t a m i n e psychosis (Allen a n d Young, 1978; Snyder, 1980). A m p h e t a m i n e psychosis has long been thought to be the best e x p e r i m e n t a l m o d e l for s c h i z o p h r e n i a (Snyder, 1973). C h r o n i c t r e a t m e n t (20 days) of rats with a m p h e t a m i n e p r o d u c e d a decrease in the n u m b e r of [3H]spiroperidol b i n d i n g sites in striat u m but resulted in no changes in d o p a m i n e s t i m u l a t e d a d e n y l a t e cyclase activity ( H o w l e t t a n d N a h o r s k i , 1979). Increases in the b i n d i n g of
365 [3H]spiroperidol and [3H]haloperidol to sites in the caudate nucleus, putamen and nucleus accumhens of schizophrenic patients have been reported (Lee et al., 1978) but this finding is currently controversial because of the possible interfering effects of neuroleptic treatment in these patients (Snyder, 1981). A number of workers have reported that PCP appears to be operating, at least in part, by producing dopamine release in a manner similar to amphetamine (Schlemmer et al., 1978) or the non-amphetamine stimulants such as methylphenidate and cocaine (Doherty et al., 1980). The findings presented here show that PCP, like amphetamine, produces a decrease in the number of [3H]spiroperidol binding sites after chronic treatment. The best explanation for this finding would appear to be that chronic hyperactivity of dopaminergic neurotransmission leads to a downregulation of D 2-dopamine (or neuroleptic) receptors. There is now evidence to suggest that dopamine and dopamine agonists bind to a receptor site (the DI site) which is'distinct from the site at which neuroleptics bind (the D 2 site) (Seeman, 1981). Chronic hyperactivity of dopaminergic neurons with amphetamine (Howlett and Nahorski, 1978; Nielsen et al., 1980) or PCP (this paper) produces down-regulation in D 2 (neuroleptic) sites. What are the effects on dopamine agonist sites? This question has not been properly studied yet, partially because of difficulties with dopamine receptor agonist binding and its interpretation. However, evidence from behavioral studies suggests that chronic amphetamine treatment produces a supersensitivity in dopamine agonist receptors (Klawans and Margolin, 1975). It remains to be demonstrated whether this behavioral supersensitivity to chronic amphetamine treatment is accompanied by increased dopamine agonist binding. Chronic amphetamine treatment does not produce any detectable change in dopamine-sensitive adenylate cyclase (Howlett and Nahorski, 1978). The effects of chronic PCP on the behavioral effects of dopamine agonists also remain to be investigated.
In conclusion, these results show that chronic PCP or amphetamine treatment produce similar down-regulation in neuroleptic [3H]spiroperidol receptor density in rat striatum. This suggests it is possible that the production of psychoses in man by these two agents may involve an alteration in D2-dopamine receptor mechanisms.
Acknowledgements Supported by the Medical Research Council of Canada (Grant MA6454). I thank M.R. Peterson for expert technical assistance.
References Allen, R.M. and S.J. Young, 1978, Phencyclidine-induced psychosis, Am. J. Psychiat. 135, 1081. Doherty, J.D., M. Simonovic, R. So and H.Y. Meltzer, 1980, The effect of phencyclidine on dopamine synthesis and metabolism in rat striatum, European J. Pharmacol. 65, 139. Howlett, D.R. and S.R. Nahorski, 1979, Acute and chronic amphetamine treatments modulate striatal dopamine receptor binding sites, Brain Res. 161, 173. Klawans, H.L. and D.I. Margolin, 1975, Amphetamine-induced dopaminergic hyper-sensitivityin guinea pigs. Implications in psychosis and human movement disorders, Arch. Gen. Psychiat. 32, 725. Lee, T., P. Seeman, W.W. Tourtellotte, I.J. Farley and O. Hornykiewicz, 1978, Binding of [3H]neuroleptics and [3H]apomorphine in schizophrenic brains, Nature 274, 897. Nielsen, E.B., M. Neilsen, G. Elhson and C. Braestrup, 1980, Decreased spiroperidol and LSD binding in rat brain after continuous amphetamine, European J. Pharmacol. 66, 149. Schlemmer, R.F. Jr., J.A. Jackson, K.L. Preston, J.P. Bederka, Jr., D.L. Garver and J.M. Davis, 1978, Phencyclidineinduced stereotyped behavior in monkeys: antagonism by pimozide, European J. Pharmacol. 52, 379. Seeman, P., 1981, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Snyder, S.H., 1973, Amphetamine psychosis: a 'model' schizophrenia mediated by catecholamines, Am. J. Psychiat. 130, 61. Snyder, S.H., 1980, Phencyclidine, Nature 285, 355. Snyder, S.H., 1981, Dopamine receptors, neuroleptics and schizophrenia, Am. J. Psychiat. 138, 460.