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Phencyclidine-induced ipsilateral rotation in rats with unilateral 6-hydroxydopamine-induced lesions of the substantia nigra
Life Sciences, Vol . 24, pp . 1281-1288 Printed in the U .S .A .
Pergamon Press
PHENCYCLIDINE-INDUCED IPSILATERAL ROTATION IN RATS WITH UNILATERAL 6-HYDROXYDOPAMINE-INDUCED LESIONS OF THE SUBSTANTIA NIGRA Richard G . Fessler,l R . David Sturgeon,l and Herbert Y . Meltzerl ,2 Laboratory of Biological Psychiatry,l Illinois State Psychiatric Institute, and Dept . of Psychiatry, 2 University of Chicago Pritzker School of Medicine (Received in final form February 20, 1979) SUMMARY The effect of phencyclidine (PCP) on rotational behavior in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra was examined and compared to the effects of d-amphetamine and apomorphine . PCP, like d-amphetamine, induced ipsilateral rotation indicating a presynaptic effect on dopamine (DA) neurons whereas apomorphine, a direct acting agonist, caused contralateral rotation . Pretreatment with alpha-methylparatyrosine inhibited PCP-induced rotation approximately to the same extent as it inhibited d-amphetamine-induced rotation, but did not significantly reduce apomorphine-induced contralateral turning, further indicating that PCP has a presynaptic effect on DA neurons . Anti-cholinergic effects of PCP may also contribute to the ipsilateral rotation noted . The administration of low doses of phencyclidine (PCP) to rats and mice induces hyperactivity and stereotypy (1,2) . The observation that low dose haloperidol, a dopamine (DA) receptor blocker, antagonizes the locomotor activity and stereotypy produced by PCP suggests that these effects of PCP are mediated by increased dopaminergic activity (2) . Inhibition of PCP-induced locomotor activity by alpha-methylparatyrosine (AMPT), an inhibitor of tyrosine hydroxylase, the rate limiting step in DA synthesis, further suggests that its actions on dopamine mechanisms might be mediated pre-synaptically (2) . In previous publications, we demonstrated that systemic administration of PCP induces dose-dependent ipsilateral rotation in rats with unilateral electrolytic lesions of the substantia nigra (2,3) . As with its effects on hyperactivity, PCP-induced rotation was affected by drugs which affect both dopaminergic and cholinergic systems . AMPT and the DA receptor blockers, haloperidol and pimozide, blocked PCP-induced rotation, as did the cholinomimetic drug, arecoline . Trihexyphenidyl, an anticholinergic agent, potentiated rotation (3) . Although unilateral electrolytic lesion of the substantia nigra provides a quantitative model for examining the intensity and duration of rotation induced by various drugs, it does not provide an adequate model for examining the pre- vs . post-synaptic effects of such agents . Animals with these lesions rotate ipsilaterally in response to drugs which increase DA activity by pre-synaptic and post-synaptic mechanisms (4) . 0024-3205/79/141281-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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An alternative method of preparing animals which will rotate in ;~ response to drug administration, and which allows analysis of pre- vs . post-synaptic effects, is to lesion the substantia nigra with 6-hydroxy dopamine (6-OHDA) . This technique results in unilateral degeneration of nigrostriatal catecholamine neurons and relatively selective loss of catecholamines ipsilateral to the lesion . Animals with such lesions respond with ipsilateral rotation to drugs which have a pre-synaptic mechanism of action, but rotate contralaterally in response to direct or post-synaptically active DA-agonists . We report here that PCP induced ipsilateral rotation in rats with such lesions . METHODS Male Sprague-Dawley rats (Sprague-Dawley, Inc ., Madison, WI .) weighing Rats 150-175 gms at the time of surgery were used in this experiment . wane group housed (7 per cage) in a roam kept at 26°C with lights on from 5 AM to 7 PM, and were allowed Purina rat chow and water ad libitum . The animals were operated in a David Kopf stereotaxic instrument Desipramine HC1 (25 mg/kg ip) under nembutal (40 mg/kg ip) anesthesia . was administered 15 minutes prior to surgery to reduce norepinephrine depletion . Unilateral intra-nigral injections of 6-OHDA were made via a 25 ul Hamilton syringe (approximately 0 .2 mm in diameter at the tip) 6-OHDA was dissolved in saline attached to a Hamilton PB600 dispenser . 8 .0 u9 of 6-OHDA was injected in a volume and ascorbic acid (0 .2 mg/ml) . of 4 .0 ul at a rate of 1 .0 ul/min . Coordinates calculated from the stereotaxic atlas of Konig and Klippel (7) were AP +2 .4, L + 2 .0, DV +2 .4 . Dose response data for PCP was collected on 6 randomly chosen, previously proven "turners ." Six doses of PCP ranging from 1 .0 mg/kg to 10 .0 mg/kg were randomly administered at 3 day intervals . Comparison of PCP with d-amphetamine and apomorphine began 7-14 days Each subject was tested following 6-OHDA lesion of a new group of rats . During the first under each experimental condition at one week intervals . three weeks, the time course of drug effects and the direction of rotation induced by PCP, d-amphetamine, and apomorphine, were determined by injecting each rat with one of these drugs . The sequence in which each rat received each drug was varied such that one-third of the subjects received each drug During the following two weeks each rat received the second each week . and third drugs such that by the end of session three, each rat had received each drug . Thus, the sequence of drug administration was counterbalanced to control for order effects . The criteria of 100 contralateral turns to apomorphine (1 .0 mg/kg sc) and 50 ipsilateral turns to amphetamine (2 .0 mg/kg ip) within the first 30 min was used to indicate a successful lesion . Subjects which did not meet these criteria were discarded after the third week of testing . Only two subjects, from different groups, were discarded through this criterion, thus the counter-balanced design was not substantially violated . For the fourth session all subjects received physiological saline . During the fifth through seventh weeks, AMPT and saline, AMPT and d-amphetamine, and AMPT and PCP were run in a counterbalanced design similar to that described above . Finally, during week eight each subject again received PCP . On each test day all subjects were moved into the experimental laboratory, and allowed to habituate to the environment . Apomorphine (1 .0 mg/kg, sc), PCP (3 .0 mg/kg ip), and saline were injected immediately prior to
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beginning the session while d-amphetamine (2 .0 mg/kg ip) was injected 30 minutes earlier . These doses and times were chosen on the basis of previous research indicating them as effective parameters for eliciting turning and/or locomotor activity and stereotypy (2,5,8) . Subjects pretreated with AMPT were given two injections of 125 mg/kg ip, at 4 and 2 hours prior to the experimental session . Volumes were calculated to inject all drugs as 1 .0 ml/kg . At the start of an experimental session, each subject was placed in a plexiglass semi-sphere (17 .4 cm deep X 36 .5 cm in diameter), and a light harness (made of thin steel wire) was fitted around the animal's chest . Each full turn the subject made was registered on an Esterline-Angus event recorder . One subject died during the fourth week of testing . Tests of significance for direction and rate of turning were performed with a paired t-test . Based upon previous reports that AMPT did not significantly reduce apomorphine-induced rotational behavior (9,10), the effect of AMPT on apomorphine-induced rotation was not examined in the study reported in Fig . 3 . However, to be certain that AMPT-induced inhibition of PCP- and d-amphetamine-induced rotation was the result of presynaptic inhibition of tyrosine hydroxylase, a group of randomly chosen lesioned rats which had been used in a previous turning experiment were tested with saline, apomorphine, AMPT, and A~~iPT and apomorphine in a randomly assigned manner with 3 day intervals between drug administrations . All other procedures were as previously described . RESULTS The dose response of the effect of PCP on rotational behavior is shown in Fig . l . Rotation increases linearly to a maximum at 3 .0 mg/kg ip . Beyond this dose, increasing ataxia induced by PCP interferes with the subjects ability to move freely and reduces the number of turns completed . The large standard error of the mean, especially at the higher concentrations of PCP, reflects the variation of behavioral response introduced by PCPinduced ataxia . Fig . 2 reports the time course of the effects of apomorphine (1 .0 mg/kg sc), d-amphetamine (2 .0 mg/kg ip), and PCP (3 .0 mg/kg ip) on rotational behavior in the lesioned rats . Apomorphine induced maximal contralateral rotation within ten minutes of administration, after which the response gradually but steadily decreased to baseline over approximately 100 minutes . PCP produced near maximal ipsilateral responding also within ten minutes . The rotational response to PCP decreased during the next 30 minutes, followed by a second period of maximum responding which peaked at 60 minutes . The reduced responding between 20 and 40 minutes following PCP administration might be due to the effects of ataxia on locomotor activity . After the second peak, rotational responding slowly decreased to baseline . Consistent with the work of Ungerstedt (5) the rate of ipsilateral turning induced by d-amphetamine appeared to increase gradually over 40 to 50 minutes, and then to decrease over the next 70 to 80 minutes . In consideration of the data in Fig . 2, and of the report of Ungerstedt (5), 30 minutes were allowed to elapse between the injection of d-amphetamine and the recording of rotation for the data in Fig . 3 . Although the PCPinduced rotational response decreased after its initial peak, the reduced rotation was still quite strong and behavioral recording was begun immediately after its injection . d-Amphetamine induced strong and consistent rotation ipsilateral to the 6-OHDA-induced lesion (p< .001) ; apomorphine induced
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Iß0 PCP DOSE (Wlq/Kg1 FIG . 1 The dose-response function of the effect of PCP on rotational behavior in unilateral 6-OHDA substantia njgra lesioned rats .
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Time course of the effects of apomorphine (1 .0 mq/kg sc), amphetamine (2 .0 mg/kg ip) and PCP (3 .0 mg/kg ip) on the rotational response in unilaterally 6-OHDA substantia nigra rats . Each point represents the average number of turns/10 minutes .
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equally consistent contralateral rotation (p< .001) . PCP-like d-amphetamine, induced strong ipsilateral rotation (p< .025) suggesting that the dopaminergic effects of PCP are presynaptic . These subjects All subjects were tested with saline Burin week four . responded with weak (less than 1 turn/3 minutes , but predominantly ipsilateral rotation (p< .025) . The ipsilateral rotation induced by d-amphetamine and PCP was significantly greater than that in response to saline (p< .O1 and p< .05, respectively) .
FIG . 3 Direction and intensity of turning induced in rats unilaterally lesioned in the left substantia nigra with 6-OHDA . Weeks one through three were run on a counterbalanced design . All subjects were administered saline during week four . Weeks five through seven again were counterbalanced and all subjects received PCP during week eight . Data are reported as total turns in 30 minutes ± S .E .M . AMPT administration alone (125 mg/kg ip 4 and 2 hours prior to testing) resulted in virtually no rotation ; even the weak rotational response to saline was blocked . AMPT pretreatment completely eliminated d-amphetamine induced rotationap< .001) and reduced PCP-induced rotation by 94% (p< .025) . Table 1 indicates that AMPT-pretreatment did not significantly reduce apomorphine-induced rotation . During the final week of testing all subjects were again tested with PCP . Because the subjects again responded with significant ipsilateral rotation (p< .001) it seems unlikely that the low rate of responding seen in rats with AMPT pretreatment could have been due to a recovery from the effects of the lesion .
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Interestingly, one animal which had previously rotated strongly to the ipsilateral side in response to PCP responded with equally strong contralateral rotation to PCP when given AMPT followed by PCP . The reason for this result is uncertain, but is probably not due to recovery from the lesion effect since this same animal again rotated ipsilaterally in response to PCP alone during week eight . TABLE I Intensity of turning induced by saline, apomorphine, AMPT and AMPT plus apomorphine in 6-OHDA lesioned rats . Data are reported as total turns in 30 minutes ± S .E .M . Refer to text .for details . Number of Turns
Saline
L R
7 .3±4 .4 5 .0±2 .1
Apomorphine 0 .0±0 .0 135 .3±6 .3
AMPT 4 .3±3 .8 2 .3±2 .3
AMPT + Apomorphine 0 .0±0 .0 112 .0±8 .0
DISCUSSION The direction, intensity .and duration of rotation reported here for apomorphine and d-amphetamine agree with previous reports (5,6), and indicate successful 6-OHDA lesions . The ipsilateral rotation induced by PCP suggests that it has a presynaptic mechanism~of action, and this suggestion is supported by our finding that AMPT inhibited PCP-induced rotation . Although multiple drug administration can make data interpretation difficult, inspection of the data for individual subjects suggests that drug interactions did not play a major role in the rotational behavior observed in the counterb~lanced administrations . The small decrease in rotation observed during the PCP retest session (week 8) resulted from a moderate decrease in rotation in two subjects which displayed extremely high rotation on the first administration of PCP . Also since the data from the first administration of PCP were not significantly different from those of the second PCP administration, it appears as though the neurochemical lesion was stable throughout the duration of the experiment . This finding is consistent with data reported by Ungerstedt (6) . Multiple and repeated drug administration designs have been used previously for rotation experiments (3,11,12) . Continuous DA release by d-amphetamine is believed to depend unpon "newly synthesized" DA (13) . Reduction of the available pool of newly synthesized DA by AMPT blocks d-amphetamine-induced release of DA and inhibits rotation (14) but does not affect apomorphine-induced rotation (9,10) . Because AMPT also blocked PCP-induced rotation in this experiment it is suggested that PCP also induces rotation through a presynaptic mechanism . Previous behavioral experiments using locomotor activity and rotation in unilateral, electrolytic substantia nigra-lesioned rats, reported only 50Â inhibition of PCP-induced responding with AMPT (2,3), which would indicate that the relative dependence of PCP on newly synthesized DA is less than that of d-amphetamine . The reason for the apparently greater effect of AMPT pretreatment on PCP-induced rotation in unilateral 6-OHDA lesioned rats is, at present, unclear, but may be related to the difference between the schedules in the two studies . In this study, AMPT was administered in two doses of 125 mg/kg ip at 4 and 2 hours prior to PCP . In the electrolytic lesion study, the same dose of AMPT was administered 8 and 4 hours before PCP . Moreover, in the previous studies, behavior was monitored for 1 hour, whereas in this experiment behavior was monitored for only 50 minutes . Fig . 2 indicates that PCP-induced turning during this interval, although
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approximately equivalent to d-amphetamine-induced turning, is not at its maximal response rate as a result of interference from behavioral ataxia . It is possible that under periods of higher behavioral responding, AMPT would not inhibit PCP-induced turning as thoroughly as seen in this experiment . Although the results of this experiment indicate that PCP induces its hyperactivating and stereotypic effects through an indirect dopaminergic mechanism, it cannot be deduced from this measure exactly how this indirect effect is exerted . Blockade of dopamine reuptake (15,16) or an effect on granular stores (17) appear to be more likely than increased DA release . Anti-cholinergic effects of PCP may also contribute to its ability to_ induce rotation in 6-OHDA-lesioned rats . PCP has been reported to have anti-cholinergic and acetylcholinesterase inhibitory effects (18) . In rats with electrolytically-made unilateral lesions of the substantia nigra, anti-cholinergics potentiate and cholinomimetic agents inhibit PCP-induced rotation (3) . In unilateral 6-OHDA striatal lesioned rats, anti-cholinergics cause ipsilateral rotation when administered alone and potentiated d-amphetamine-induced ipsilateral rotation, but do not affect apomorphine-induced contralateral rotation (12) . Cholinomimetics produce no rotation when administered alone, but inhibit both d-amphetamine and apomorphine-induced rotation (12) . Since, the direction of rotation induced by PCP is consistent with the effects of anti-cholinergics, it is conceivable that PCP-induced rotation involves an anti-cholinergic influence as well as an indirect dopaminergic mechanism . AMPT would suppress the latter action but not the former . Our observation that AMPT nearly completely inhibited the effects of PCP on rotation in rats with unilateral 6-OHDA-induced lesions of the substantia nigra suggests that any cholinergic influence of PCP on this behavior is probably less important than its dopaminergic effects . PCP has recently been reported to significantly interfere with 3 H-quinuclidinyl benzilate (QNB) binding in rat brain (concentration which induces 50% dissociation of 3 H-QNB = 30 uM) and to displace the opiate receptor ligand 3 H-dihydromorphine (DHM) (19) (concentration which induced 50% dissociation of 3 H-DHM = 26 uM) . PCP did not displace 3 H-dopamine even at a 1 mM concentration . These results further support the hypothesis that PCP is not a direct acting dopamine agonist but may have an effect at muscarinic and opiate receptors . ACKNOWLEDGEMENTS Supported in part by USPHS MHCRC 30,938 and DA 02081 . recipient of USPHS RCSA MH 47,808 .
HYM is
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
G . CHEN, C .R . ENSOR, D . RUSSELL, and B . BOHNER, J . Pharm . Exp . Ther . 12 7 241-250 (1959) . .KANNER, H .Y . MELTZER, and J .M . DAVIS, Neuroscience Abstracts _1 M 237 (1975) . K .T . FINNEGAN, M .I . KANNER, and H .Y . MELTZER, Pharm . Biochem . Beh . 5 651-660 (1976) . B. COSTAL and R .J . ~üIYLOR, Ps cho harmacolo is 41 57-64 (1975) . U . UNGERSTEDT, Ac ta Physiol . Scand . Suppl . 67 49-68 (1971a) . U . UNGERSTEDT, Acta Physiol . Scand . Suppl . 367 69-93 (1971b) . J .F .R . KÖNIG and R .A . KLIPPEL, The Rat Brain : A Stereotaxic Atlas, Williams and Wilkins Co ., Baltimore 1963 . U . UNGERSTEDT and G .W . ARBUTHNOTT, Brain Research 24 485-493 (1970) .
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P .F . VON VOIGTLANDER and K .E . MOORE, Neuropharmacology _12 451-462 (1973) . T .P . JERUSSI and S .D . GLICK, Psychopharmacologia (Berl .) _40 329-334 (1975) . S .M . ANTELMAN, D .J . EDWARDS, and M . LIN, Brain Research _127 317-322 (1977) . C . PYCOCK, J . MILSON, D . TARSY, and C .D . MARSDEN, Neuropharmacology 17 175-183 (1978) . C.C . CHIEUEH and K .E . MOORE, J . Pharm . Exp . Ther . 19 2 642-653 (1975) . C .A . MARSDEN and H .C . GULDBERG, Neuropharmacology 12195-211 (1973) . R .C . SMITH, H .Y . MELTZER, H . DEKERMENJIAN, and J .M.DAVIS, Neuroscience Abstracts 1 300 (1975) . R .C . SMITH, H .Y . MELTZER, R .C . ARORA, and J .M . DAVIS, Biochem . Pharmacol . 26 1435-1439 (1977) . J.D . DOHERTY, M . SIMONOVIC, R . S0, and H .Y . MELTZER, in preparation . S . MAAYANI, H . WEINSTEIN, S . COHEN, and M . LOKOLOSKY, Proc . Natl . Acad . Sci . 7 0 3103-3107 (1973) . J.P . VINCENT, D . CAVEY, J .M . KAMENKA, P . GENESTE, and M . LAZDUNSKI, Brain Research 152 176-182 (1978) .
Pergamon Press
PHENCYCLIDINE-INDUCED IPSILATERAL ROTATION IN RATS WITH UNILATERAL 6-HYDROXYDOPAMINE-INDUCED LESIONS OF THE SUBSTANTIA NIGRA Richard G . Fessler,l R . David Sturgeon,l and Herbert Y . Meltzerl ,2 Laboratory of Biological Psychiatry,l Illinois State Psychiatric Institute, and Dept . of Psychiatry, 2 University of Chicago Pritzker School of Medicine (Received in final form February 20, 1979) SUMMARY The effect of phencyclidine (PCP) on rotational behavior in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra was examined and compared to the effects of d-amphetamine and apomorphine . PCP, like d-amphetamine, induced ipsilateral rotation indicating a presynaptic effect on dopamine (DA) neurons whereas apomorphine, a direct acting agonist, caused contralateral rotation . Pretreatment with alpha-methylparatyrosine inhibited PCP-induced rotation approximately to the same extent as it inhibited d-amphetamine-induced rotation, but did not significantly reduce apomorphine-induced contralateral turning, further indicating that PCP has a presynaptic effect on DA neurons . Anti-cholinergic effects of PCP may also contribute to the ipsilateral rotation noted . The administration of low doses of phencyclidine (PCP) to rats and mice induces hyperactivity and stereotypy (1,2) . The observation that low dose haloperidol, a dopamine (DA) receptor blocker, antagonizes the locomotor activity and stereotypy produced by PCP suggests that these effects of PCP are mediated by increased dopaminergic activity (2) . Inhibition of PCP-induced locomotor activity by alpha-methylparatyrosine (AMPT), an inhibitor of tyrosine hydroxylase, the rate limiting step in DA synthesis, further suggests that its actions on dopamine mechanisms might be mediated pre-synaptically (2) . In previous publications, we demonstrated that systemic administration of PCP induces dose-dependent ipsilateral rotation in rats with unilateral electrolytic lesions of the substantia nigra (2,3) . As with its effects on hyperactivity, PCP-induced rotation was affected by drugs which affect both dopaminergic and cholinergic systems . AMPT and the DA receptor blockers, haloperidol and pimozide, blocked PCP-induced rotation, as did the cholinomimetic drug, arecoline . Trihexyphenidyl, an anticholinergic agent, potentiated rotation (3) . Although unilateral electrolytic lesion of the substantia nigra provides a quantitative model for examining the intensity and duration of rotation induced by various drugs, it does not provide an adequate model for examining the pre- vs . post-synaptic effects of such agents . Animals with these lesions rotate ipsilaterally in response to drugs which increase DA activity by pre-synaptic and post-synaptic mechanisms (4) . 0024-3205/79/141281-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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An alternative method of preparing animals which will rotate in ;~ response to drug administration, and which allows analysis of pre- vs . post-synaptic effects, is to lesion the substantia nigra with 6-hydroxy dopamine (6-OHDA) . This technique results in unilateral degeneration of nigrostriatal catecholamine neurons and relatively selective loss of catecholamines ipsilateral to the lesion . Animals with such lesions respond with ipsilateral rotation to drugs which have a pre-synaptic mechanism of action, but rotate contralaterally in response to direct or post-synaptically active DA-agonists . We report here that PCP induced ipsilateral rotation in rats with such lesions . METHODS Male Sprague-Dawley rats (Sprague-Dawley, Inc ., Madison, WI .) weighing Rats 150-175 gms at the time of surgery were used in this experiment . wane group housed (7 per cage) in a roam kept at 26°C with lights on from 5 AM to 7 PM, and were allowed Purina rat chow and water ad libitum . The animals were operated in a David Kopf stereotaxic instrument Desipramine HC1 (25 mg/kg ip) under nembutal (40 mg/kg ip) anesthesia . was administered 15 minutes prior to surgery to reduce norepinephrine depletion . Unilateral intra-nigral injections of 6-OHDA were made via a 25 ul Hamilton syringe (approximately 0 .2 mm in diameter at the tip) 6-OHDA was dissolved in saline attached to a Hamilton PB600 dispenser . 8 .0 u9 of 6-OHDA was injected in a volume and ascorbic acid (0 .2 mg/ml) . of 4 .0 ul at a rate of 1 .0 ul/min . Coordinates calculated from the stereotaxic atlas of Konig and Klippel (7) were AP +2 .4, L + 2 .0, DV +2 .4 . Dose response data for PCP was collected on 6 randomly chosen, previously proven "turners ." Six doses of PCP ranging from 1 .0 mg/kg to 10 .0 mg/kg were randomly administered at 3 day intervals . Comparison of PCP with d-amphetamine and apomorphine began 7-14 days Each subject was tested following 6-OHDA lesion of a new group of rats . During the first under each experimental condition at one week intervals . three weeks, the time course of drug effects and the direction of rotation induced by PCP, d-amphetamine, and apomorphine, were determined by injecting each rat with one of these drugs . The sequence in which each rat received each drug was varied such that one-third of the subjects received each drug During the following two weeks each rat received the second each week . and third drugs such that by the end of session three, each rat had received each drug . Thus, the sequence of drug administration was counterbalanced to control for order effects . The criteria of 100 contralateral turns to apomorphine (1 .0 mg/kg sc) and 50 ipsilateral turns to amphetamine (2 .0 mg/kg ip) within the first 30 min was used to indicate a successful lesion . Subjects which did not meet these criteria were discarded after the third week of testing . Only two subjects, from different groups, were discarded through this criterion, thus the counter-balanced design was not substantially violated . For the fourth session all subjects received physiological saline . During the fifth through seventh weeks, AMPT and saline, AMPT and d-amphetamine, and AMPT and PCP were run in a counterbalanced design similar to that described above . Finally, during week eight each subject again received PCP . On each test day all subjects were moved into the experimental laboratory, and allowed to habituate to the environment . Apomorphine (1 .0 mg/kg, sc), PCP (3 .0 mg/kg ip), and saline were injected immediately prior to
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beginning the session while d-amphetamine (2 .0 mg/kg ip) was injected 30 minutes earlier . These doses and times were chosen on the basis of previous research indicating them as effective parameters for eliciting turning and/or locomotor activity and stereotypy (2,5,8) . Subjects pretreated with AMPT were given two injections of 125 mg/kg ip, at 4 and 2 hours prior to the experimental session . Volumes were calculated to inject all drugs as 1 .0 ml/kg . At the start of an experimental session, each subject was placed in a plexiglass semi-sphere (17 .4 cm deep X 36 .5 cm in diameter), and a light harness (made of thin steel wire) was fitted around the animal's chest . Each full turn the subject made was registered on an Esterline-Angus event recorder . One subject died during the fourth week of testing . Tests of significance for direction and rate of turning were performed with a paired t-test . Based upon previous reports that AMPT did not significantly reduce apomorphine-induced rotational behavior (9,10), the effect of AMPT on apomorphine-induced rotation was not examined in the study reported in Fig . 3 . However, to be certain that AMPT-induced inhibition of PCP- and d-amphetamine-induced rotation was the result of presynaptic inhibition of tyrosine hydroxylase, a group of randomly chosen lesioned rats which had been used in a previous turning experiment were tested with saline, apomorphine, AMPT, and A~~iPT and apomorphine in a randomly assigned manner with 3 day intervals between drug administrations . All other procedures were as previously described . RESULTS The dose response of the effect of PCP on rotational behavior is shown in Fig . l . Rotation increases linearly to a maximum at 3 .0 mg/kg ip . Beyond this dose, increasing ataxia induced by PCP interferes with the subjects ability to move freely and reduces the number of turns completed . The large standard error of the mean, especially at the higher concentrations of PCP, reflects the variation of behavioral response introduced by PCPinduced ataxia . Fig . 2 reports the time course of the effects of apomorphine (1 .0 mg/kg sc), d-amphetamine (2 .0 mg/kg ip), and PCP (3 .0 mg/kg ip) on rotational behavior in the lesioned rats . Apomorphine induced maximal contralateral rotation within ten minutes of administration, after which the response gradually but steadily decreased to baseline over approximately 100 minutes . PCP produced near maximal ipsilateral responding also within ten minutes . The rotational response to PCP decreased during the next 30 minutes, followed by a second period of maximum responding which peaked at 60 minutes . The reduced responding between 20 and 40 minutes following PCP administration might be due to the effects of ataxia on locomotor activity . After the second peak, rotational responding slowly decreased to baseline . Consistent with the work of Ungerstedt (5) the rate of ipsilateral turning induced by d-amphetamine appeared to increase gradually over 40 to 50 minutes, and then to decrease over the next 70 to 80 minutes . In consideration of the data in Fig . 2, and of the report of Ungerstedt (5), 30 minutes were allowed to elapse between the injection of d-amphetamine and the recording of rotation for the data in Fig . 3 . Although the PCPinduced rotational response decreased after its initial peak, the reduced rotation was still quite strong and behavioral recording was begun immediately after its injection . d-Amphetamine induced strong and consistent rotation ipsilateral to the 6-OHDA-induced lesion (p< .001) ; apomorphine induced
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Time course of the effects of apomorphine (1 .0 mq/kg sc), amphetamine (2 .0 mg/kg ip) and PCP (3 .0 mg/kg ip) on the rotational response in unilaterally 6-OHDA substantia nigra rats . Each point represents the average number of turns/10 minutes .
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equally consistent contralateral rotation (p< .001) . PCP-like d-amphetamine, induced strong ipsilateral rotation (p< .025) suggesting that the dopaminergic effects of PCP are presynaptic . These subjects All subjects were tested with saline Burin week four . responded with weak (less than 1 turn/3 minutes , but predominantly ipsilateral rotation (p< .025) . The ipsilateral rotation induced by d-amphetamine and PCP was significantly greater than that in response to saline (p< .O1 and p< .05, respectively) .
FIG . 3 Direction and intensity of turning induced in rats unilaterally lesioned in the left substantia nigra with 6-OHDA . Weeks one through three were run on a counterbalanced design . All subjects were administered saline during week four . Weeks five through seven again were counterbalanced and all subjects received PCP during week eight . Data are reported as total turns in 30 minutes ± S .E .M . AMPT administration alone (125 mg/kg ip 4 and 2 hours prior to testing) resulted in virtually no rotation ; even the weak rotational response to saline was blocked . AMPT pretreatment completely eliminated d-amphetamine induced rotationap< .001) and reduced PCP-induced rotation by 94% (p< .025) . Table 1 indicates that AMPT-pretreatment did not significantly reduce apomorphine-induced rotation . During the final week of testing all subjects were again tested with PCP . Because the subjects again responded with significant ipsilateral rotation (p< .001) it seems unlikely that the low rate of responding seen in rats with AMPT pretreatment could have been due to a recovery from the effects of the lesion .
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Interestingly, one animal which had previously rotated strongly to the ipsilateral side in response to PCP responded with equally strong contralateral rotation to PCP when given AMPT followed by PCP . The reason for this result is uncertain, but is probably not due to recovery from the lesion effect since this same animal again rotated ipsilaterally in response to PCP alone during week eight . TABLE I Intensity of turning induced by saline, apomorphine, AMPT and AMPT plus apomorphine in 6-OHDA lesioned rats . Data are reported as total turns in 30 minutes ± S .E .M . Refer to text .for details . Number of Turns
Saline
L R
7 .3±4 .4 5 .0±2 .1
Apomorphine 0 .0±0 .0 135 .3±6 .3
AMPT 4 .3±3 .8 2 .3±2 .3
AMPT + Apomorphine 0 .0±0 .0 112 .0±8 .0
DISCUSSION The direction, intensity .and duration of rotation reported here for apomorphine and d-amphetamine agree with previous reports (5,6), and indicate successful 6-OHDA lesions . The ipsilateral rotation induced by PCP suggests that it has a presynaptic mechanism~of action, and this suggestion is supported by our finding that AMPT inhibited PCP-induced rotation . Although multiple drug administration can make data interpretation difficult, inspection of the data for individual subjects suggests that drug interactions did not play a major role in the rotational behavior observed in the counterb~lanced administrations . The small decrease in rotation observed during the PCP retest session (week 8) resulted from a moderate decrease in rotation in two subjects which displayed extremely high rotation on the first administration of PCP . Also since the data from the first administration of PCP were not significantly different from those of the second PCP administration, it appears as though the neurochemical lesion was stable throughout the duration of the experiment . This finding is consistent with data reported by Ungerstedt (6) . Multiple and repeated drug administration designs have been used previously for rotation experiments (3,11,12) . Continuous DA release by d-amphetamine is believed to depend unpon "newly synthesized" DA (13) . Reduction of the available pool of newly synthesized DA by AMPT blocks d-amphetamine-induced release of DA and inhibits rotation (14) but does not affect apomorphine-induced rotation (9,10) . Because AMPT also blocked PCP-induced rotation in this experiment it is suggested that PCP also induces rotation through a presynaptic mechanism . Previous behavioral experiments using locomotor activity and rotation in unilateral, electrolytic substantia nigra-lesioned rats, reported only 50Â inhibition of PCP-induced responding with AMPT (2,3), which would indicate that the relative dependence of PCP on newly synthesized DA is less than that of d-amphetamine . The reason for the apparently greater effect of AMPT pretreatment on PCP-induced rotation in unilateral 6-OHDA lesioned rats is, at present, unclear, but may be related to the difference between the schedules in the two studies . In this study, AMPT was administered in two doses of 125 mg/kg ip at 4 and 2 hours prior to PCP . In the electrolytic lesion study, the same dose of AMPT was administered 8 and 4 hours before PCP . Moreover, in the previous studies, behavior was monitored for 1 hour, whereas in this experiment behavior was monitored for only 50 minutes . Fig . 2 indicates that PCP-induced turning during this interval, although
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approximately equivalent to d-amphetamine-induced turning, is not at its maximal response rate as a result of interference from behavioral ataxia . It is possible that under periods of higher behavioral responding, AMPT would not inhibit PCP-induced turning as thoroughly as seen in this experiment . Although the results of this experiment indicate that PCP induces its hyperactivating and stereotypic effects through an indirect dopaminergic mechanism, it cannot be deduced from this measure exactly how this indirect effect is exerted . Blockade of dopamine reuptake (15,16) or an effect on granular stores (17) appear to be more likely than increased DA release . Anti-cholinergic effects of PCP may also contribute to its ability to_ induce rotation in 6-OHDA-lesioned rats . PCP has been reported to have anti-cholinergic and acetylcholinesterase inhibitory effects (18) . In rats with electrolytically-made unilateral lesions of the substantia nigra, anti-cholinergics potentiate and cholinomimetic agents inhibit PCP-induced rotation (3) . In unilateral 6-OHDA striatal lesioned rats, anti-cholinergics cause ipsilateral rotation when administered alone and potentiated d-amphetamine-induced ipsilateral rotation, but do not affect apomorphine-induced contralateral rotation (12) . Cholinomimetics produce no rotation when administered alone, but inhibit both d-amphetamine and apomorphine-induced rotation (12) . Since, the direction of rotation induced by PCP is consistent with the effects of anti-cholinergics, it is conceivable that PCP-induced rotation involves an anti-cholinergic influence as well as an indirect dopaminergic mechanism . AMPT would suppress the latter action but not the former . Our observation that AMPT nearly completely inhibited the effects of PCP on rotation in rats with unilateral 6-OHDA-induced lesions of the substantia nigra suggests that any cholinergic influence of PCP on this behavior is probably less important than its dopaminergic effects . PCP has recently been reported to significantly interfere with 3 H-quinuclidinyl benzilate (QNB) binding in rat brain (concentration which induces 50% dissociation of 3 H-QNB = 30 uM) and to displace the opiate receptor ligand 3 H-dihydromorphine (DHM) (19) (concentration which induced 50% dissociation of 3 H-DHM = 26 uM) . PCP did not displace 3 H-dopamine even at a 1 mM concentration . These results further support the hypothesis that PCP is not a direct acting dopamine agonist but may have an effect at muscarinic and opiate receptors . ACKNOWLEDGEMENTS Supported in part by USPHS MHCRC 30,938 and DA 02081 . recipient of USPHS RCSA MH 47,808 .
HYM is
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