Brain Research, 578 (1992) 161-167 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
161
BRES 17637
Evidence for presynaptic dopamine mechanisms underlying amphetamine-conditioned locomotion Sherry L. DiLullo and Mathew T. Martin-Iverson Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton. Alta. (Canada) (Accepted 10 December 1991)
Key words: Amphetamine; Reserpine; ct-Methyl-para-tyrosine; Conditioned locomotion; Dopamine; Rat
Rats with a history of receiving (+)-amphetamine in a specific environment exhibit a conditioned psychomotor response when subsequently placed in that environment without drug treatment. Previous work has shown that while the unconditioned effects of amphetamine can be blocked by dopamine D 1 o r D 2 receptor antagonists or with a-methyl-p-tyrosine, conditioned locomotion is not influenced by these treatments. In the present experiment, a-methyl-p-tyrosine (50 mg/kg, s.c.) was given in conjunction with amphetamine (1.5 mg/kg, s.c.) for 8 days before testing for conditioned locomotion, a-Methyl-p-tyrosine completely blocked amphetamine-induced locomotion but only attenuated amphetamine-conditioned locomotion. Reserpine (reduced over the 8 days from 2.5 to 1.25 mg/kg, i.p.) did not block amphetamineinduced locomotion; indeed, potentiation of amphetamine-induced locomotor activity was observed on the last 3 days of treatment. Reserpine treatment in conjunction with a-methyl-p-tyrosine treatment blocked amphetamine-induced locomotion for the first 4 days only, with full recovery of amphetamine-induced unconditioned locomotion by the last treatment day. Reserpine alone had no effect on amphetamineconditioned locomotion, but completely blocked amphetamine-conditioned locomotion when given with a-methyl-p-tyrosine. It is concluded that the ct-methyl-p-tyrosine-sensitive pool of dopamine mediates the immediate psychomotor effects of amphetamine, but that both the a-methyl-p-tyrosine- and reserpine-sensitive pools of dopamine are involved in the establishment of amphetamine-conditioned locomotion. In addition, the occurrence of amphetamine-conditioned locomotion is independent of the direct effects of amphetamine on locomotion.INTRODUCTION Rats exhibit classically conditioned p s y c h o m o t o r stimulant responses when placed into an environment in which they have a history of r e p e a t e d ( + ) - a m p h e t a m i n e ( A M P ) t r e a t m e n t 8'2°'29. In humans, both the euphoric and physiological effects of stimulants can be conditioned to situational stimuli 26-28. F o r m e r cocaine addicts continue to exhibit strong 'cravings' and drug-like physiological responses when presented with drug-related cues 26"28. These a p p a r e n t conditioned responses are not affected by drug-detoxification or drug-rehabilitation programs 28. Therefore, conditioned stimulant effects likely contribute to the resistance of addicts to these treatments or to psychosocial therapy and m a y account for the high rate of relapse among addicts of cocaine and A M P - l i k e compounds. Similarly, stimulant-induced euphoria in chronic cocaine abusers 13 has b e e n shown to be unaffected by d o p a m i n e ( D A ) r e c e p t o r antagonists. This observation seems to complicate the role of D A in the m e d i a t i o n of the euphoric 31'37 and activating 8'18 effects of stimulants established in the non-human literature. The conditioned euphoric effects of cocaine may be m e d i a t e d by a non-dopaminergic mechanism. This would
account for the a p p a r e n t lack of effect of neuroleptics on cocaine-induced euphoria by experienced stimulant users in this study. This hypothesis is s u p p o r t e d by the observations that neuroleptics block the unconditioned but not the conditioned effects of stimulants in rats 4"2°, and that cocaine-, methylphenidate- and nomifensineconditioned place preferences in rats are not blocked by neuroleptics or D A depletions 21'24'36. A l t h o u g h many of the direct behavioral effects of psyc h o m o t o r stimulants are d e p e n d e n t on dopaminergic mechanisms, the role of D A neurotransmission in conditioned stimulant effects is not well understood. Beninger and H a h n 1 and Beninger and Herz 2 initially investigated the role of D A in A M P - and cocaineconditioned locomotion in rats in an elegant series of experiments. Pimozide, a selective D A D2 r e c e p t o r antagonist blocked the 'establishment' of conditioning when it was administered concomitantly with A M P 1 or cocaine 2 during a 10-day training period but did not block the 'expression' of conditioning when it was administered only on the stimulant-free test days. While the ability of pimozide to block the establishment 15 and not the expression 15"3° of stimulant-conditioned locomotion has been replicated, the mechanism
Correspondence: M.T. Martin-Iverson, Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Mackenzie Centre, Edmonton, Alta., Canada, T6G 2B7. Fax: (1) (403) 492-6841.
162 which this has been attributed to, D A D 2 r e c e p t o r antagonism, m a y be incorrect. Pimozide is an equipotent antagonist of L-type calcium channels 7'1°'4° and has o t h e r antagonistic effects on serotonin z receptors 7,4°, a~-receptors 7, prolactin secretion 1° and calmodulin 7. There is strong evidence against the interpretation that the effects of pimozide on stimulant-conditioning are due to D A D2 r e c e p t o r blockade; namely, o t h e r D A D 2 antagonists do not have similar actions. D A D 2 antagonists such as met o c l o p r a m i d e 9, haloperido125"3°'34, chlorpromazine 25 and sulpiride 3° block the expression of stimulant-conditioned behaviors. H o w e v e r , conclusions as to the role of D A D 2 receptors in the expression of conditioned behaviors cannot be confidently drawn. Non-specific sedating effects of D A r e c e p t o r antagonists were not ruled out in at least one investigation 9. In addition, C a r e y 4 recently d e m o n s t r a t e d that haloperidol does not block the expression of a p o m o r p h i n e - c o n d i t i o n e d contralateral rotation in rats with unilateral striatal D A depletions. Pimozide has been shown to block the establishment of A M P - and a p o m o r p h i n e - c o n d i t i o n e d stereotypy and to block the expression of a p o m o r p h i n e - but not A M P - c o n ditioned stereotypy 15. In contrast, haloperidol does not block the establishment of A M P - c o n d i t i o n e d locomotion 2°, but does attenuate it when given with a calcium channel b l o c k e r (DiLullo and Martin-Iverson, submitted). This indicates that pimozide's unique profile is likely due to its c o m b i n e d D A D z r e c e p t o r antagonism and calcium channel blockade. The unique profile of pimozide in this behavioral p r o c e d u r e indicates that the rather large n u m b e r of behavioral studies utilizing pimozide to draw conclusions concerning the role of D A in mediating behaviors should be re-evaluated. In addition to the evidence against a D A D 2 recept o r - m e d i a t e d postsynaptic process being conditioned with stimulants, there is evidence against involvement of postsynaptic D A D~ receptors, alone or in combination with D A D 2 receptors 4'2°. F u r t h e r m o r e , there is evidence against the view that some presynaptic D A - r e l a t e d process is conditioned after r e p e a t e d pairings of A M P with a unique environment, a - M e t h y l - p a r a - t y r o s i n e ( a M P T ) , a catecholamine synthesis inhibitor, co-administered with A M P during environment-specific conditioning to inhibit the A M P - i n d u c e d release of D A , significantly a t t e n u a t e d AMP-induced locomotion and defecation, but did not block either A M P - c o n d i t i o n e d locomotion or defecation 8. Recently, Hiroi and W h i t e 16 blocked the expression of A M P - c o n d i t i o n e d place-preferences with systemic reserpine ( R E S ) . The possible role of D A from granular stores, which may be released via a calcium-dependent exocytotic mechanism, in A M P - c o n d i t i o n e d behaviors fits logically with a recent finding by DiLullo and Mar-
tin-Iverson (submitted). N e i t h e r haloperidol, a D A D z antagonist, nor nimodipine, an L-type calcium channel blocker, when given independently, influenced A M P conditioned locomotion. H o w e v e r , in conjunction with haloperidol, nimodipine attenuated A M P - c o n d i t i o n e d locomotion, an effect similar to that seen with pimozide, a drug which combines D A D2 r e c e p t o r antagonism with calcium channel blockade. These data suggest that behaviors conditioned with A M P are d e p e n d e n t , in part, on a calcium-mediated mechanism, which could be exocytotic D A release from a RES-sensitive pool. The objective of this study was to examine the roles of the aMPT-sensitive and the RES-sensitive D A pools in A M P - c o n d i t i o n e d and unconditioned behaviors in the rat. This was accomplished by measuring the effects of these agents given during the conditioning process on A M P - i n d u c e d conditioned locomotion. MATERIALS AND METHODS Experimentally naive male Sprague-Dawley rats (n = 10-12 for each group) weighing 250-300 g at the start of the experiment were maintained on a 12:12 h light cycle (lights on 07.00-19.00) with ad libitum access to dry food, wet food mash and water. Locomotor activity (infrared photobeam interruptions) was measured in conditioning boxes following a procedure described previouslys'2°. (+)AMP sulfate, provided courtesy of SmithKline Beecham Pharma, and aMPT methyl ester HCI, purchased from Sigma, were dissolved with double-distilled water into 1.5 mg/ml and 50 mg/ml solutions, respectively. RES, purchased from Aldrich, was dissolved using a few drops of glacial acetic acid, and then diluted with a 50 mM glucose solution into a 2.5 or 1.25 mg/ml solution. The pH was adjusted to 4.0 with 10 N sodium hydroxide. All drug weights are expressed as salts. Doses of the drugs used were chosen on the basis of the literature. The dose of AMP was chosen to produce optimal photobeam interruptions over the course of several days and to elicit conditioned locomotions'2°. The dose of aMPT was chosen to block catecholamine synthesis, based on acute effects 33. The 170-rain time-interval between aMPT and AMP administration was chosen on the basis of previous experience in this laboratory with aMPT during AMP-conditionings and the literature3"33 which indicates that although maximal inhibition of tyrosine hydroxylase occurs 30 min after aMPT administration3, the psychomotor stimulant effects of AMP are maximally reduced after a longer interval. The dose of RES was chosen to deplete the granular stores of DA 22.
Procedures The experiment consisted of two phases: a drug conditioning phase and a drug-free test phase. During the drug conditioning phase (lasting 8 consecutive days) the rats received three daily injections (VEH a or RES, VEH a or aMPT and VEH A or AMP) after which they were placed into the conditioning boxes for 60 min. The test day occurred on the sixth day after the last conditioningday injection to allow for drug clearance and recovery from DA depletions. On this day, all animals received two consecutive injections of vehicle (VEH R i.p. and VEH A, s.c.) prior to placement for 60 rain into the conditioning boxes. There were 8 treatment groups with each animal receiving three treatments daily. The dosing schedule for RES was quite complex because the repeated injections produced marked aphagia, adipsia and akinesia, especially in those rats co-administered aMPT. RES or VEH R (i.p.) was administered 24 h before placement into the conditioning boxes. For all rats given RES, this drug was given at
163 a dose of 2.5 mg/kg for the first 4 days (Day 0-3), and 1.25 mg/kg for Days 4, 5, 7 and 8. VEH R was injected in all groups on Day 6, due to the marked and life-threatening effects of the prior RES treatments. The criterion whereby the dose of RES was reduced was a decrease in body weight to 85% of pre-experimental weight; when this occurred on Day 4, for all except 2 RES-treated rats, the dose of RES was reduced by half for all rats. On Day 6, a number of animals' weights dropped to 80% of their pre-experimental weights, the dose of RES for all rats was missed for this one day. aMPT (50 mg/kg, s.c.) or VEH a (s.c.) was injected 180 min before being placed into the conditioning boxes and AMP (1.5 mg/kg, s.c.) or VEH A (s.c.) was injected 10 min before placement into the conditioning boxes. The groups were as follows: VEH RVEHa-VEH A, VEHR-VEHa-AMP, RES-VEHa-VEHA, RESVEH,~-AMP, VEHR-aMPT-VEHA, VEHR-aMPT-AMP, RESaMPT-VEH A and RES-aMPT-AME Three rats (1 from the RESaMPT-VEH A and 2 from the RES-aMPT-AMP treatment groups) were killed during the experiment (prior to the test phase) because their weights dropped below our criteria for euthanasia. Rats exhibiting weight loss were fed wet mash to encourage feeding.
RESULTS T h e results o f the c o n d i t i o n i n g phase of the experim e n t w e r e s u b j e c t e d to A N O V A with 3 i n d e p e n d e n t factors: (1) R E S p r e t r e a t m e n t , consisting of 2 levels ( V E H R o r R E S ) ; (2) a M P T p r e t r e a t m e n t , consisting o f 2 levels ( V E H a o r a M P T ) ; and (3) A M P t r e a t m e n t , consisting of 2 levels ( V E H A o r A M P ) . Fig. 1 r e p r e s e n t s the unc o n d i t i o n e d l o c o m o t i o n by groups not r e c e i v i n g A M P . O n D a y s 3 and 4, l o c o m o t o r activity in the R E S - p r e t r e a t e d groups was significantly l o w e r t h a n the V E H RV E H a - V E H A g r o u p , but did not differ significantly on o t h e r days. A M P significantly i n c r e a s e d l o c o m o t i o n dep e n d i n g o n the p r e t r e a t m e n t , as can be s e e n in Fig. 2
F7,616 =
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11.2, P < 0.001; a M P T
x A M P × Days: F7,616 = 3.41, P < 0.01). R E S signifStatistics
Locomotor activity for each rat during both phases of the experiment was recorded in 5-min blocks and summed to produce individual daily totals. The means of the individual rat's daily totals were subjected to analysis of variance (ANOVA) with planned comparisons between individual groups. Since ANOVA is not reliable with more than two repeated measures unless there is homogeneity of variances and covariances (a condition that is seldom met due to order effects), in all cases in which repeated measures were used the data were further subjected to a variety of multivariate tests (Pillais Trace, Hotellings t-test and Wilks Lambda; these tests are automatically run in cases of repeated measures by the software package used). The results of the ANOVA are reported only if they were in agreement with the multivariate tests.
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CONSECUTIVE DAYS Fig. 1. The effects of daily injections before placement into conditioning boxes of VEH R (VEH) or RES (2.5-1,25 mg/kg, i.p.), VEHa (VEH) or aMPT (MPT, 50 mg/kg, s.c.) and VEH A (VEH, 1.0 ml/kg, s.c.) on locomotor activity (photobeam interruptions) over 8 consecutive days of 60-rain periods. The critical difference (planned comparisons, P < 0.05) from the VEH-VEH-VEH control group is 100 photobeam interruptions.
Fig. 2. The effects of daily injections before placement into conditioning boxes of VEH R (VEH) or RES (2.5-1.25 mg/kg, i.p.), VEH~ (VEH) or aMPT (MPT, 50 mg/kg, s.c.) and VEH A (VEH) or AMP (1.5 mg/kg, s.c.) on locomotor activity (photobeam interruptions) over 8 consecutive days of 60-min periods, aMPT (VEHMPT-AMP) completely blocked AMP-induced locomotion. RES significantly attenuated AMP-induced locomotion for the first 3 days (RES-VEH-AMP), and then augmented it for the last 3 days of treatment. Combined RES treatment with aMPT (RES-MPTAMP) blocked AMP-induced locomotion for the first 4 days after which locomotor activity recovered to VEH-VEH-AMP levels. The critical difference (planned comparisons, P < 0.05) from the VEHVEH-AMP control group and from the VEH-MPT-AMP group is 100 photobeam interruptions. See Fig. 1 for treatment legends.
164 dicate that AMP-induced locomotion is augmented by repeated RES and blocked by repeated aMPT, and that the aMPT blockade can be reversed with repeated RES treatments. The test phase of the experiment was subjected to A N O V A with the same independent factors as in the conditioning phase. Fig. 3 displays the conditioned locomotor activity induced by exposure to the boxes after two placebo injections. All rats previously treated with AMP, except the group treated with both RES and aMPT, displayed AMP-conditioned locomotion, relative to the VEHR-VEHa-VEH A control group and relative to the percent mean locomotion across all VEH groups which were not conditioned with AMP. Overall, RES pretreatment decreased locomotion on this day (main effect: F1,85 = 9.5, P < 0.005) but did not influence AMP-conditioned locomotion. There was a significant interaction between aMPT and AMP (FI,85 = 9.1, P < 0.003). aMPT significantly decreased (but did not block) AMP-conditioned locomotion. AMP-conditioned locomotion was blocked completely by combined RES and aMPT treatment during conditioning.
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Fig. 3. The effects of 2 vehicle injections to the same groups of rats as in Fig. 2 (those receiving AMP), after a 5-day drug-free rest, on conditioned locomotion (photobeam interruptions) during a 60-min period. Since there were no significant differences among the VEH preinjected groups (VEH-VEH-VEH, VEH-MPT-VEH, RESVEH-VEH and RES-MPT-VEH), locomotionfrom the AMP treatment groups are displayed as the percent of mean locomotion across all VEH groups, aMPT (MPT) attenuated AMP-conditioned locomotion. Combined MPT and RES treatment blocked AMPconditioned locomotion. Error bars represent the standard errors of the means. * Significantly different from the combined VEH control group, P < 0.05. + Significantly different from the VEHVEH-AMP group, P < 0.05.
DISCUSSION Several investigations have demonstrated that the psychomotor stimulant effects of AMP-like compounds are mediated by the aMPT-sensitive pool of newly synthesized DA 3'8"11'23'41, potentiated by depletion of the RESsensitive granular storage pools of DA 23"33"38'41, and can be conditioned to a unique environment in which the drugs are administered 1"2'8'2°'29"3°. This experiment verified these observations: catecholamine synthesis inhibition with aMPT over 8 days completely blocked AMPinduced locomotion. AMP-induced locomotion was ultimately potentiated by repeated RES injections, and repeated AMP injections prior to placement in a unique environment produced the ability of the environment itself to induce AMP-like effects. The results of a previous report, that blockade of AMP-Induced locomotion with aMPT failed to block AMP-conditioned locomotion 8, was also replicated. In addition, two novel observations were made. RES failed to block AMP-conditioned locomotion, but did block AMP-conditioned locomotion when combined with aMPT. This latter finding is especially interesting since the combination of RES and aMPT did not block the locomotor effects of AMP on the last 4 days of treatment. Depletion of granular storage pools of D A with repeated RES injections initially decreased but ultimately potentiated AMP-induced locomotion. This biphasic action may be due to the dose of AMP chosen; maximal locomotion is found with this dose in this laboratory such that initial potentiation of AMP's effects may produce stereotyped licking or sniffing in one location at the expense of locomotion 12. The emergence of a potentiation of AMP-induced locomotion may reflect a tolerance to some of reserpine's effects. A similar mechanism may explain the gradual decrease in the effect of AMP-induced locomotion. Daily injections produce a gradual augmentation of the effects of AMp32; since a maximal locomotor dose was used, the observed decrease in locomotion over days likely represents the emergence of stereotyped behaviors. AMP injections paired with exposure to a unique environment resulted in an AMP-like locomotor response when rats were placed into the environment without AMP treatment. Although a pseudo-conditioning control was not included in this report, previous work in this laboratory following the same AMP-conditioning procedures but with pseudo-conditioning controls, determined that the conditioned AMP-response is unique to the testing environment. Rats which receive AMP while in their home cages 3 h after conditioning trials do not exhibit increases of locomotion when exposed to the conditioning boxes 2°. The present results are therefore not likely
165 a function of an unconditioned spontaneous increase in activity due to previous AMP treatment. There are at least two pharmacologically distinct D A pools. One pool is quickly depleted by aMPT, due to its rapid turnover rate. This 'aMPT-sensitive' pool of D A is mobilized and released calcium-independently 4° by the AMP class of stimulants 3'23, probably via a carrier mechanism 42. The psychomotor stimulant effects of AMP have been demonstrated by several investigators to be blocked by ctMPT 8'33'39. A second pool of D A is depleted by RES and its impulse-dependent release is potentiated by the methylphenidate class (i.e. methylphenidate, cocaine, nomifensine, amfonelic acid) of stimulants 3"23 probably via calcium-dependent exocytosis 35. The psychomotor stimulant effects of non-AMP stimulants are blocked by RES. Rats administered RES prior to AMP show an enhanced behavioral response to AMP 38. The potentiation of AMP by RES may be attributed to supersensitivity of postsynaptic D A receptors, the re-routing of D A that normally fills RES-sensitive vesicles into the AMP-releasable pool 22 and/or alteration in agonistsensitivity of postsynaptic receptors as a function of history of receptor occupancy 5"6. Rats treated with both RES and a M P T displayed a recovery from the initial aMPT-induced blockade of locomotion. This may have resulted from the RES-induced augmentation of the AMP response, and implicates another D A pool or neurotransmitter system in the mechanism through which RES potentiates AMP's effects. Previous work has shown that D A D 1 and D 2 receptor antagonists 2° and a M P T 8 block the behavioral effects of AMP, but fail to block AMP-conditioned locomotion. This indicates that neither postsynaptic events nor the direct presynaptic actions of AMP are responsible for the classical conditioning of AMP-induced locomotion. However, recent findings have provided evidence for a synergistic role of impulse-dependent calcium channels with D A D 2 receptor antagonism in AMP-conditioned locomotion (DiLullo and Martin-Iverson, submitted). In addition, it has been reported that while a M P T injected directly into the nucleus accumbens fails to block AMPconditioned place preferences, a-flupenthixol, a D A receptor blocker injected directly into the nucleus accumbens and systemic RES blocks this conditioned effect 16. These observations led us to hypothesize that the critical event conditioned with AMP is the impulse-dependent release of D A from presynaptic granular storage pools or a synergistic action between the aMPT- and RES-sensitive D A pools. These two possibilities were tested in the present experiment. aMPT attenuated (but did not completely block) the establishment of AMP-conditioned locomotion. This observation is somewhat different from that in an earlier
report from this laboratory, using the same dose of a M P T during AMP-conditioning in which no attenuation was observed 8. In the present case, this dose of a M P T completely blocked AMP-induced locomotion, while in the previous report the blockade was not complete. This differential affect was likely due largely to some recovery of AMP effects on the last 2 days of the 10 treatment days. In the present experiment, conditioning occurred over only 8 days, and no recovery was apparent. Thus, when AMP-induced locomotion was completely blocked some attenuation of conditioned-locomotion occurred. RES treatment alone did not affect AMP-conditioned behaviors. However, in combination with aMPT, RES completely blocked the establishment of AMP-conditioned locomotion. This blockade occurred in spite of the recovery of AMP-induced locomotion over the last 4 days in this treatment group. Previous publications have shown intact conditioning with blockade of the behavioral effects of stimulants 4"8'2°. In the present case, the psychomotor effects of AMP were observed in the absence of conditioned effects. In this report we observed a complete block of AMP-induced locomotion by aMPT, without a corresponding block of conditioned locomotion, and secondly, the ultimate recovery of AMPinduced locomotion with combined RES and aMPT treatment with complete blockade of conditioned locomotion. This double-dissociation indicates that the direct and the conditioned locomotor effects of AMP are subserved by different neural mechanisms. The results from this and other investigations 16 suggest that the psychomotor stimulant effects induced by AMP are expressed when D A from the aMPT-sensitive pool is released and that the establishment of AMP-conditioned behaviors is mediated by the RES and aMPTsensitive pools of DA. Blockade of one of these pools of D A is not sufficient to block the establishment of stimulant-conditioning. RES disrupts vesicles containing noradrenaline (NA) and serotonin (5-HT), as well as those containing DA. It is possible therefore that the blockade of conditioning is dependent upon actions on one of these neurotransmitter systems. Since destruction of forebrain NA-containing terminals with the neurotoxin, DSP4, does not affect the establishment of AMP-conditioned locomotion 8, it is unlikely that the effects of combined treatment with a M P T and RES are mediated by actions on NA systems. Effects on 5-HT cannot presently be ruled out. Decreases in 5-HT generally augment the behavioral effects of AMp19; the possibility that RES-induced depletion of 5-HT mediates the blockade of AMP-conditioned locomotion is not consistent with this observation. On the other hand, destruction of DA-containing
166 terminals with 6 - O H D A has been reported to block A M P - c o n d i t i o n e d locomotion 14. It is therefore most likely that the blockade of A M P - c o n d i t i o n e d locomotion with combined treatment with a M P T and RES is a function of effects on D A - c o n t a i n i n g nerve terminals. O n e question that remains is whether or not the conditioned effects mediated by the RES-sensitive pool are impulse-dependent. The observation that an impulse-dep e n d e n t calcium channel blocker, nimodipine, in combination with a D A D 2 receptor antagonist, haloperidol, attenuates A M P - c o n d i t i o n e d locomotion (DiLullo and Martin-Iverson, submitted) indicates that this may be true, since this c o m p o u n d should block impulse-dependent D A release. To summarize, neither a M P T nor RES given indepen-
REFERENCES I Beninger, R.J. and Hahn, B., Pimozide blocks establishment but not expression of amphetamine-produced environment-specific conditioning, Science, 220 (1983) 1304-1306. 2 Beninger, R.J. and Herz, R.S., Pimozide blocks establishment but not expression of cocaine-produced environment-specific conditioning, Life Sci., 38 (1986) 1425-1431. 3 Braestrup, C., Biochemical differentiation of amphetamine vs. methylphenidate and nomifensine in rats, J. Pharm. Pharmacol., 29 (1977) 463-470. 4 Carey, R.J., Dopamine receptors mediate drug-induced but not Pavlovian conditioned contralateral rotation in the unilateral 6-OHDA animal model, Brain Res., 515 (1990) 292-298. 5 Clark, D., Hjorth, S. and Carlsson, A., Dopamine-receptor agonists: mechanisms underlying autoreceptor selectivity. I: review of the evidence, J. Neural Transm., 62 (1985) 1-52. 6 Clark, D., Hjorth, S. and Carlsson, A., Dopamine-receptor agonists: mechanisms underlying autoreceptor selectivity. II: theoretical considerations, J. Neural Transm., 62 (1985) 171207. 7 Cohen, M.L., Carpenter, R., Schenck, K., Wittenauer, L. and Mason, N., Effect of nitrendipine, diltiazem, trifluoperazine and pimozide on serotonin2 (5-HT2) receptor activation in the rat uterus and iugular vein, J. Pharmacol. Exp. Ther., 238 (1986) 860-867. 8 DiLullo, S.L. and Martin-Iverson, M.T., Presynaptic dopaminergic neurotransmission mediates amphetamine-induced unconditioned but not amphetamine-conditioned locomotion and defecation in the rat, Brain Res., 568 (1991) 45-54. 9 Drew, K.L. and Glick, S.D., Role of D-1 and D-2 receptor stimulation in sensitization to amphetamine-induced circling behavior and in expression and extinction of the Pavlovian conditioned response, Psychopharmacology, 101 (1990) 465-471. 10 Enyeart, J.J., Sheu, S.-S. and Hinkle, P.M., Pituitary Ca2÷ channels: blockade by conventional and novel Ca2+ antagonists, Am. J. Physiol., 253 (1987) C162-C170. 11 Finn, I.B., Iuvone, P.M. and Holtzman, S.G., Depletion of catecholamines in the brain of rats differentially affects stimulation of locomotor activity by caffeine, o-amphetamine, and methylphenidate, Neuropharmacology, 29 (7) (1990) 625-631. 12 Fray, P.J., Sahakian, B.J., Robbins, T.W., Koob, G.E and Iversen, S.D., An observational method for quantifying the behavioral effects of dopamine agonists: contrasting effects of D-amphetamine and apomorphine, Psychopharrnacology, 69 (1980) 253-259. 13 Gawin, EH., Neuroleptic reduction of cocaine-induced paranoia but not euphoria?, Psychopharmacology, 90 (1986) 142-
dently during conditioning of A M P ' s locomotor effects to e n v i r o n m e n t a l stimuli completely blocked the establishment of A M P - c o n d i t i o n e d locomotion. The two drugs given together did block the establishment of AMP-conditioned locomotion. Together with other evidence, this observation indicates that A M P effects on either newly synthesized D A or on vesicular stores of D A are sufficient for conditioning of A M P ' s effects. Both processes must be blocked to block conditioning.
Acknowledgements. This research was supported by the Alberta Mental Health Research Fund (AMHRF) and the Medical Research Council of Canada. S.L.D.L was supported by an AMHRF Studentship. M.T.M.-I. is supported by an Alberta Heritage Foundation for Medical Research Grant and Scholarship.
143. 14 Gold, L.H., Swerdlow, N.R. and Koob, G.E, The role of mesolimbic dopamine in conditioned locomotion produced by amphetamine, Behav. Neurosci., 102 (1988) 544-552. 15 Hiroi, N. and White, N.M., Conditioned stereotypy: behavioral specification of the UCS and pharmacological investigation of the neural change, Pharmacol. Biochem. Behav., 32 (1989) 249-258 16 Hiroi, N. and White, N.M., The reserpine-sensitive dopamine pool mediates (+)-amphetamine-conditioned reward in the place preference paradigm, Brain Res., 510 (1990) 33-42. 17 Kang, S.-Y., Kleinman, P.H., Woody, G.E., Millman, R.B., Todd, T.C., Kemp, J. and Lipton, D.S., Outcomes for cocaine abusers after once-a-week psychosocial therapy, Am. J. Psychiatry, 148 (1991) 630-635. 18 Kelly, P.H. and Iversen, S.D., Selective 6-OHDA-induced destruction of mesolimbic DA neurons: abolition of psychostimulant induced locomotor activity in rats, Eur. J. Pharmacol., 40 (1976) 45-56. 19 Martin-Iverson, M.T., Leclere, J.E and Fibiger, H.C., Cholinergic-dopaminergic interactions and the mechanisms of action of antidepressants, Eur. J. Pharmacol., 94 (1983) 193-201. 20 Martin-Iverson, M.T. and McManus, D.J., Stimulant-conditioned locomotion is not affected by blockade of D1 and/or D2 dopamine receptors during conditioning, Brain Res., 521 (1990) 175-184. 21 Martin-Iverson, M.T., Ortmann, R. and Fibiger, H.C., Place preference conditioning with methylphenidate and nomifensine, Brain Res., 332 (1985) 59-67. 22 Martin-Iverson, M.T., Yamada, N., By, A.W. and Lodge, B.A., 'Designer' amphetamines: effects on behavior and monoamines with or without reserpine and/or MPT pretreatment, J. Psychiat. Neurosci., 16 (1991) 253-261. 23 McMillan, B.A., CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs, Trends Pharmacol. Sci., 4 (1983) 429-432 24 Mithani, S., Martin-Iverson, M.T., Phillips, A.G. and Fibiger, H.C., The effects of haloperidol on amphetamine- and methylphenidate-induced conditioned place preference and locomotor activity, Psychopharmacology, 90 (1986) 247-252. 25 Miyamoto, K. and Hada, H., Effects of pimozide, haloperidol and chlorpromazine on methamphetamine-induced behaviors, Int. J. Neurosci., 32 (1987) 408. 26 Muntaner, C., Cascella, N.G., Kumor, K.M., Nagoshi, C., Herning, R. and Jaffe, J., Placebo responses to cocaine administration in humans: effects of prior administrations and verbal instructions, Psychopharmacology, 99 (1989) 282-286. 27 O'Brien, C.P., Ehrman, R. and Ternes, J., Classical condition-
167 ing in human opioid dependence. In S.G. Goldberg and I.P. Stolerman (Eds.), Behavioral Analysis of Drug Dependence, Academic Press, Orlando, FL, 1986, pp. 329-356. 28 O'Brien, C.P., Childress, A.R., Arndt, I.O., McLennan, A.T., Woody, G.E. and Maany, I., Pharmacological and behavioral treatments of cocaine dependence: controlled studies, J. Clin. Psychiatry, 49 (1988) 17-22. 29 Pickens, R.W. and Crowder, W.E, Effects of CS-US interval on conditioning of drug response, with assessment of speed of conditioning. Psychopharrnacologia, 11 (1967) 88-94. 30 Poncelet, M., Dangoumau, L., Soubrie, P. and Simon, P., Effects of neuroleptic drugs, clonidine and lithium on the expression of conditioned behavioral excitation in rats, Psychopharmacology, 92 (1987) 393-397. 31 Roberts, D.C.S., Koob, G.E, Klonoff, P. and Fibiger, H.C., Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens, Pharmacol. Biochem. Behav., 12 (1980) 781-787. 32 Robinson, T,E., and Becket, J.B., Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev., 11 (1986) 157-198. 33 Scheel-Kruger, J., Comparative studies of various amphetamine analogues demonstrating different interactions with the metabolism of the catecholamines of the brain, Eur. J. Pharmacol., 14 (1971) 47-59. 34 Schiff, S.R., Conditioned dopaminergic activity, Biol. Psychiatry, 17 (1982) 135-155.
35 Shore, P.A., Actions of amfonelic acid and other non-amphetamine stimulants on the dopamine neuron, J. Pharm. Pharmacol., 28 (1976) 855-857. 36 Spyraki, C., Fibiger, H.C. and Phillips, A.G., Cocaine-induced place preference conditioning: lack of effects of neuroleptics and 6-hydroxydopamine lesions, Brain Res., 253 (1982) 195203. 37 Spyraki, C., Fibiger, H.C. and Phillips, A.G., Dopaminergic substrates of amphetamine-induced place preference conditioning, Brain Res., 253 (1982) 185-193. 38 Stolk, J.M. and Rech, R.H., Enhanced stimulant effects of o-amphetamine in rats treated chronically with reserpine, J. Pharmacol. Exp. Ther., 161 (1968) 75-83. 39 Stolk, J.M. and Rech, R.H., Antagonism of o-amphetamine by alpha-methyl-L-tyrosine: behavioral evidence for the participation of catecholamine stores and synthesis in the amphetaminestimulant response, Neuropharmacology, 9 (1970) 249-263. 40 Tecott, L.H., Kwong, L.L., Uhr, S. and Peroutka, S.J., Differential modulation of dopamine D2 receptors by chronic haloperidol, nitrendripine and pimozide, Biol. Psychiatry, 21 (1986) 1114-1122. 41 Weissman, A., Koe, B.K. and Tenen, S.S., Anti-amphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. Exp. Ther., 151 (1966) 339-352, 42 Westerink, B.H.C., Hofsteede, R.M,, Tuntler, J. and de Vries, J.B., Use of calcium antagonism for the characterization of drug-evoked dopamine release from the brain of conscious rats determined by microdialysis, J. Neurochem., 52 (1989) 722-729.
161
BRES 17637
Evidence for presynaptic dopamine mechanisms underlying amphetamine-conditioned locomotion Sherry L. DiLullo and Mathew T. Martin-Iverson Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton. Alta. (Canada) (Accepted 10 December 1991)
Key words: Amphetamine; Reserpine; ct-Methyl-para-tyrosine; Conditioned locomotion; Dopamine; Rat
Rats with a history of receiving (+)-amphetamine in a specific environment exhibit a conditioned psychomotor response when subsequently placed in that environment without drug treatment. Previous work has shown that while the unconditioned effects of amphetamine can be blocked by dopamine D 1 o r D 2 receptor antagonists or with a-methyl-p-tyrosine, conditioned locomotion is not influenced by these treatments. In the present experiment, a-methyl-p-tyrosine (50 mg/kg, s.c.) was given in conjunction with amphetamine (1.5 mg/kg, s.c.) for 8 days before testing for conditioned locomotion, a-Methyl-p-tyrosine completely blocked amphetamine-induced locomotion but only attenuated amphetamine-conditioned locomotion. Reserpine (reduced over the 8 days from 2.5 to 1.25 mg/kg, i.p.) did not block amphetamineinduced locomotion; indeed, potentiation of amphetamine-induced locomotor activity was observed on the last 3 days of treatment. Reserpine treatment in conjunction with a-methyl-p-tyrosine treatment blocked amphetamine-induced locomotion for the first 4 days only, with full recovery of amphetamine-induced unconditioned locomotion by the last treatment day. Reserpine alone had no effect on amphetamineconditioned locomotion, but completely blocked amphetamine-conditioned locomotion when given with a-methyl-p-tyrosine. It is concluded that the ct-methyl-p-tyrosine-sensitive pool of dopamine mediates the immediate psychomotor effects of amphetamine, but that both the a-methyl-p-tyrosine- and reserpine-sensitive pools of dopamine are involved in the establishment of amphetamine-conditioned locomotion. In addition, the occurrence of amphetamine-conditioned locomotion is independent of the direct effects of amphetamine on locomotion.INTRODUCTION Rats exhibit classically conditioned p s y c h o m o t o r stimulant responses when placed into an environment in which they have a history of r e p e a t e d ( + ) - a m p h e t a m i n e ( A M P ) t r e a t m e n t 8'2°'29. In humans, both the euphoric and physiological effects of stimulants can be conditioned to situational stimuli 26-28. F o r m e r cocaine addicts continue to exhibit strong 'cravings' and drug-like physiological responses when presented with drug-related cues 26"28. These a p p a r e n t conditioned responses are not affected by drug-detoxification or drug-rehabilitation programs 28. Therefore, conditioned stimulant effects likely contribute to the resistance of addicts to these treatments or to psychosocial therapy and m a y account for the high rate of relapse among addicts of cocaine and A M P - l i k e compounds. Similarly, stimulant-induced euphoria in chronic cocaine abusers 13 has b e e n shown to be unaffected by d o p a m i n e ( D A ) r e c e p t o r antagonists. This observation seems to complicate the role of D A in the m e d i a t i o n of the euphoric 31'37 and activating 8'18 effects of stimulants established in the non-human literature. The conditioned euphoric effects of cocaine may be m e d i a t e d by a non-dopaminergic mechanism. This would
account for the a p p a r e n t lack of effect of neuroleptics on cocaine-induced euphoria by experienced stimulant users in this study. This hypothesis is s u p p o r t e d by the observations that neuroleptics block the unconditioned but not the conditioned effects of stimulants in rats 4"2°, and that cocaine-, methylphenidate- and nomifensineconditioned place preferences in rats are not blocked by neuroleptics or D A depletions 21'24'36. A l t h o u g h many of the direct behavioral effects of psyc h o m o t o r stimulants are d e p e n d e n t on dopaminergic mechanisms, the role of D A neurotransmission in conditioned stimulant effects is not well understood. Beninger and H a h n 1 and Beninger and Herz 2 initially investigated the role of D A in A M P - and cocaineconditioned locomotion in rats in an elegant series of experiments. Pimozide, a selective D A D2 r e c e p t o r antagonist blocked the 'establishment' of conditioning when it was administered concomitantly with A M P 1 or cocaine 2 during a 10-day training period but did not block the 'expression' of conditioning when it was administered only on the stimulant-free test days. While the ability of pimozide to block the establishment 15 and not the expression 15"3° of stimulant-conditioned locomotion has been replicated, the mechanism
Correspondence: M.T. Martin-Iverson, Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Mackenzie Centre, Edmonton, Alta., Canada, T6G 2B7. Fax: (1) (403) 492-6841.
162 which this has been attributed to, D A D 2 r e c e p t o r antagonism, m a y be incorrect. Pimozide is an equipotent antagonist of L-type calcium channels 7'1°'4° and has o t h e r antagonistic effects on serotonin z receptors 7,4°, a~-receptors 7, prolactin secretion 1° and calmodulin 7. There is strong evidence against the interpretation that the effects of pimozide on stimulant-conditioning are due to D A D2 r e c e p t o r blockade; namely, o t h e r D A D 2 antagonists do not have similar actions. D A D 2 antagonists such as met o c l o p r a m i d e 9, haloperido125"3°'34, chlorpromazine 25 and sulpiride 3° block the expression of stimulant-conditioned behaviors. H o w e v e r , conclusions as to the role of D A D 2 receptors in the expression of conditioned behaviors cannot be confidently drawn. Non-specific sedating effects of D A r e c e p t o r antagonists were not ruled out in at least one investigation 9. In addition, C a r e y 4 recently d e m o n s t r a t e d that haloperidol does not block the expression of a p o m o r p h i n e - c o n d i t i o n e d contralateral rotation in rats with unilateral striatal D A depletions. Pimozide has been shown to block the establishment of A M P - and a p o m o r p h i n e - c o n d i t i o n e d stereotypy and to block the expression of a p o m o r p h i n e - but not A M P - c o n ditioned stereotypy 15. In contrast, haloperidol does not block the establishment of A M P - c o n d i t i o n e d locomotion 2°, but does attenuate it when given with a calcium channel b l o c k e r (DiLullo and Martin-Iverson, submitted). This indicates that pimozide's unique profile is likely due to its c o m b i n e d D A D z r e c e p t o r antagonism and calcium channel blockade. The unique profile of pimozide in this behavioral p r o c e d u r e indicates that the rather large n u m b e r of behavioral studies utilizing pimozide to draw conclusions concerning the role of D A in mediating behaviors should be re-evaluated. In addition to the evidence against a D A D 2 recept o r - m e d i a t e d postsynaptic process being conditioned with stimulants, there is evidence against involvement of postsynaptic D A D~ receptors, alone or in combination with D A D 2 receptors 4'2°. F u r t h e r m o r e , there is evidence against the view that some presynaptic D A - r e l a t e d process is conditioned after r e p e a t e d pairings of A M P with a unique environment, a - M e t h y l - p a r a - t y r o s i n e ( a M P T ) , a catecholamine synthesis inhibitor, co-administered with A M P during environment-specific conditioning to inhibit the A M P - i n d u c e d release of D A , significantly a t t e n u a t e d AMP-induced locomotion and defecation, but did not block either A M P - c o n d i t i o n e d locomotion or defecation 8. Recently, Hiroi and W h i t e 16 blocked the expression of A M P - c o n d i t i o n e d place-preferences with systemic reserpine ( R E S ) . The possible role of D A from granular stores, which may be released via a calcium-dependent exocytotic mechanism, in A M P - c o n d i t i o n e d behaviors fits logically with a recent finding by DiLullo and Mar-
tin-Iverson (submitted). N e i t h e r haloperidol, a D A D z antagonist, nor nimodipine, an L-type calcium channel blocker, when given independently, influenced A M P conditioned locomotion. H o w e v e r , in conjunction with haloperidol, nimodipine attenuated A M P - c o n d i t i o n e d locomotion, an effect similar to that seen with pimozide, a drug which combines D A D2 r e c e p t o r antagonism with calcium channel blockade. These data suggest that behaviors conditioned with A M P are d e p e n d e n t , in part, on a calcium-mediated mechanism, which could be exocytotic D A release from a RES-sensitive pool. The objective of this study was to examine the roles of the aMPT-sensitive and the RES-sensitive D A pools in A M P - c o n d i t i o n e d and unconditioned behaviors in the rat. This was accomplished by measuring the effects of these agents given during the conditioning process on A M P - i n d u c e d conditioned locomotion. MATERIALS AND METHODS Experimentally naive male Sprague-Dawley rats (n = 10-12 for each group) weighing 250-300 g at the start of the experiment were maintained on a 12:12 h light cycle (lights on 07.00-19.00) with ad libitum access to dry food, wet food mash and water. Locomotor activity (infrared photobeam interruptions) was measured in conditioning boxes following a procedure described previouslys'2°. (+)AMP sulfate, provided courtesy of SmithKline Beecham Pharma, and aMPT methyl ester HCI, purchased from Sigma, were dissolved with double-distilled water into 1.5 mg/ml and 50 mg/ml solutions, respectively. RES, purchased from Aldrich, was dissolved using a few drops of glacial acetic acid, and then diluted with a 50 mM glucose solution into a 2.5 or 1.25 mg/ml solution. The pH was adjusted to 4.0 with 10 N sodium hydroxide. All drug weights are expressed as salts. Doses of the drugs used were chosen on the basis of the literature. The dose of AMP was chosen to produce optimal photobeam interruptions over the course of several days and to elicit conditioned locomotions'2°. The dose of aMPT was chosen to block catecholamine synthesis, based on acute effects 33. The 170-rain time-interval between aMPT and AMP administration was chosen on the basis of previous experience in this laboratory with aMPT during AMP-conditionings and the literature3"33 which indicates that although maximal inhibition of tyrosine hydroxylase occurs 30 min after aMPT administration3, the psychomotor stimulant effects of AMP are maximally reduced after a longer interval. The dose of RES was chosen to deplete the granular stores of DA 22.
Procedures The experiment consisted of two phases: a drug conditioning phase and a drug-free test phase. During the drug conditioning phase (lasting 8 consecutive days) the rats received three daily injections (VEH a or RES, VEH a or aMPT and VEH A or AMP) after which they were placed into the conditioning boxes for 60 min. The test day occurred on the sixth day after the last conditioningday injection to allow for drug clearance and recovery from DA depletions. On this day, all animals received two consecutive injections of vehicle (VEH R i.p. and VEH A, s.c.) prior to placement for 60 rain into the conditioning boxes. There were 8 treatment groups with each animal receiving three treatments daily. The dosing schedule for RES was quite complex because the repeated injections produced marked aphagia, adipsia and akinesia, especially in those rats co-administered aMPT. RES or VEH R (i.p.) was administered 24 h before placement into the conditioning boxes. For all rats given RES, this drug was given at
163 a dose of 2.5 mg/kg for the first 4 days (Day 0-3), and 1.25 mg/kg for Days 4, 5, 7 and 8. VEH R was injected in all groups on Day 6, due to the marked and life-threatening effects of the prior RES treatments. The criterion whereby the dose of RES was reduced was a decrease in body weight to 85% of pre-experimental weight; when this occurred on Day 4, for all except 2 RES-treated rats, the dose of RES was reduced by half for all rats. On Day 6, a number of animals' weights dropped to 80% of their pre-experimental weights, the dose of RES for all rats was missed for this one day. aMPT (50 mg/kg, s.c.) or VEH a (s.c.) was injected 180 min before being placed into the conditioning boxes and AMP (1.5 mg/kg, s.c.) or VEH A (s.c.) was injected 10 min before placement into the conditioning boxes. The groups were as follows: VEH RVEHa-VEH A, VEHR-VEHa-AMP, RES-VEHa-VEHA, RESVEH,~-AMP, VEHR-aMPT-VEHA, VEHR-aMPT-AMP, RESaMPT-VEH A and RES-aMPT-AME Three rats (1 from the RESaMPT-VEH A and 2 from the RES-aMPT-AMP treatment groups) were killed during the experiment (prior to the test phase) because their weights dropped below our criteria for euthanasia. Rats exhibiting weight loss were fed wet mash to encourage feeding.
RESULTS T h e results o f the c o n d i t i o n i n g phase of the experim e n t w e r e s u b j e c t e d to A N O V A with 3 i n d e p e n d e n t factors: (1) R E S p r e t r e a t m e n t , consisting of 2 levels ( V E H R o r R E S ) ; (2) a M P T p r e t r e a t m e n t , consisting o f 2 levels ( V E H a o r a M P T ) ; and (3) A M P t r e a t m e n t , consisting of 2 levels ( V E H A o r A M P ) . Fig. 1 r e p r e s e n t s the unc o n d i t i o n e d l o c o m o t i o n by groups not r e c e i v i n g A M P . O n D a y s 3 and 4, l o c o m o t o r activity in the R E S - p r e t r e a t e d groups was significantly l o w e r t h a n the V E H RV E H a - V E H A g r o u p , but did not differ significantly on o t h e r days. A M P significantly i n c r e a s e d l o c o m o t i o n dep e n d i n g o n the p r e t r e a t m e n t , as can be s e e n in Fig. 2
F7,616 =
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x A M P × Days: F7,616 = 3.41, P < 0.01). R E S signifStatistics
Locomotor activity for each rat during both phases of the experiment was recorded in 5-min blocks and summed to produce individual daily totals. The means of the individual rat's daily totals were subjected to analysis of variance (ANOVA) with planned comparisons between individual groups. Since ANOVA is not reliable with more than two repeated measures unless there is homogeneity of variances and covariances (a condition that is seldom met due to order effects), in all cases in which repeated measures were used the data were further subjected to a variety of multivariate tests (Pillais Trace, Hotellings t-test and Wilks Lambda; these tests are automatically run in cases of repeated measures by the software package used). The results of the ANOVA are reported only if they were in agreement with the multivariate tests.
icantly d e c r e a s e d
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CONSECUTIVE DAYS Fig. 1. The effects of daily injections before placement into conditioning boxes of VEH R (VEH) or RES (2.5-1,25 mg/kg, i.p.), VEHa (VEH) or aMPT (MPT, 50 mg/kg, s.c.) and VEH A (VEH, 1.0 ml/kg, s.c.) on locomotor activity (photobeam interruptions) over 8 consecutive days of 60-rain periods. The critical difference (planned comparisons, P < 0.05) from the VEH-VEH-VEH control group is 100 photobeam interruptions.
Fig. 2. The effects of daily injections before placement into conditioning boxes of VEH R (VEH) or RES (2.5-1.25 mg/kg, i.p.), VEH~ (VEH) or aMPT (MPT, 50 mg/kg, s.c.) and VEH A (VEH) or AMP (1.5 mg/kg, s.c.) on locomotor activity (photobeam interruptions) over 8 consecutive days of 60-min periods, aMPT (VEHMPT-AMP) completely blocked AMP-induced locomotion. RES significantly attenuated AMP-induced locomotion for the first 3 days (RES-VEH-AMP), and then augmented it for the last 3 days of treatment. Combined RES treatment with aMPT (RES-MPTAMP) blocked AMP-induced locomotion for the first 4 days after which locomotor activity recovered to VEH-VEH-AMP levels. The critical difference (planned comparisons, P < 0.05) from the VEHVEH-AMP control group and from the VEH-MPT-AMP group is 100 photobeam interruptions. See Fig. 1 for treatment legends.
164 dicate that AMP-induced locomotion is augmented by repeated RES and blocked by repeated aMPT, and that the aMPT blockade can be reversed with repeated RES treatments. The test phase of the experiment was subjected to A N O V A with the same independent factors as in the conditioning phase. Fig. 3 displays the conditioned locomotor activity induced by exposure to the boxes after two placebo injections. All rats previously treated with AMP, except the group treated with both RES and aMPT, displayed AMP-conditioned locomotion, relative to the VEHR-VEHa-VEH A control group and relative to the percent mean locomotion across all VEH groups which were not conditioned with AMP. Overall, RES pretreatment decreased locomotion on this day (main effect: F1,85 = 9.5, P < 0.005) but did not influence AMP-conditioned locomotion. There was a significant interaction between aMPT and AMP (FI,85 = 9.1, P < 0.003). aMPT significantly decreased (but did not block) AMP-conditioned locomotion. AMP-conditioned locomotion was blocked completely by combined RES and aMPT treatment during conditioning.
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Fig. 3. The effects of 2 vehicle injections to the same groups of rats as in Fig. 2 (those receiving AMP), after a 5-day drug-free rest, on conditioned locomotion (photobeam interruptions) during a 60-min period. Since there were no significant differences among the VEH preinjected groups (VEH-VEH-VEH, VEH-MPT-VEH, RESVEH-VEH and RES-MPT-VEH), locomotionfrom the AMP treatment groups are displayed as the percent of mean locomotion across all VEH groups, aMPT (MPT) attenuated AMP-conditioned locomotion. Combined MPT and RES treatment blocked AMPconditioned locomotion. Error bars represent the standard errors of the means. * Significantly different from the combined VEH control group, P < 0.05. + Significantly different from the VEHVEH-AMP group, P < 0.05.
DISCUSSION Several investigations have demonstrated that the psychomotor stimulant effects of AMP-like compounds are mediated by the aMPT-sensitive pool of newly synthesized DA 3'8"11'23'41, potentiated by depletion of the RESsensitive granular storage pools of DA 23"33"38'41, and can be conditioned to a unique environment in which the drugs are administered 1"2'8'2°'29"3°. This experiment verified these observations: catecholamine synthesis inhibition with aMPT over 8 days completely blocked AMPinduced locomotion. AMP-induced locomotion was ultimately potentiated by repeated RES injections, and repeated AMP injections prior to placement in a unique environment produced the ability of the environment itself to induce AMP-like effects. The results of a previous report, that blockade of AMP-Induced locomotion with aMPT failed to block AMP-conditioned locomotion 8, was also replicated. In addition, two novel observations were made. RES failed to block AMP-conditioned locomotion, but did block AMP-conditioned locomotion when combined with aMPT. This latter finding is especially interesting since the combination of RES and aMPT did not block the locomotor effects of AMP on the last 4 days of treatment. Depletion of granular storage pools of D A with repeated RES injections initially decreased but ultimately potentiated AMP-induced locomotion. This biphasic action may be due to the dose of AMP chosen; maximal locomotion is found with this dose in this laboratory such that initial potentiation of AMP's effects may produce stereotyped licking or sniffing in one location at the expense of locomotion 12. The emergence of a potentiation of AMP-induced locomotion may reflect a tolerance to some of reserpine's effects. A similar mechanism may explain the gradual decrease in the effect of AMP-induced locomotion. Daily injections produce a gradual augmentation of the effects of AMp32; since a maximal locomotor dose was used, the observed decrease in locomotion over days likely represents the emergence of stereotyped behaviors. AMP injections paired with exposure to a unique environment resulted in an AMP-like locomotor response when rats were placed into the environment without AMP treatment. Although a pseudo-conditioning control was not included in this report, previous work in this laboratory following the same AMP-conditioning procedures but with pseudo-conditioning controls, determined that the conditioned AMP-response is unique to the testing environment. Rats which receive AMP while in their home cages 3 h after conditioning trials do not exhibit increases of locomotion when exposed to the conditioning boxes 2°. The present results are therefore not likely
165 a function of an unconditioned spontaneous increase in activity due to previous AMP treatment. There are at least two pharmacologically distinct D A pools. One pool is quickly depleted by aMPT, due to its rapid turnover rate. This 'aMPT-sensitive' pool of D A is mobilized and released calcium-independently 4° by the AMP class of stimulants 3'23, probably via a carrier mechanism 42. The psychomotor stimulant effects of AMP have been demonstrated by several investigators to be blocked by ctMPT 8'33'39. A second pool of D A is depleted by RES and its impulse-dependent release is potentiated by the methylphenidate class (i.e. methylphenidate, cocaine, nomifensine, amfonelic acid) of stimulants 3"23 probably via calcium-dependent exocytosis 35. The psychomotor stimulant effects of non-AMP stimulants are blocked by RES. Rats administered RES prior to AMP show an enhanced behavioral response to AMP 38. The potentiation of AMP by RES may be attributed to supersensitivity of postsynaptic D A receptors, the re-routing of D A that normally fills RES-sensitive vesicles into the AMP-releasable pool 22 and/or alteration in agonistsensitivity of postsynaptic receptors as a function of history of receptor occupancy 5"6. Rats treated with both RES and a M P T displayed a recovery from the initial aMPT-induced blockade of locomotion. This may have resulted from the RES-induced augmentation of the AMP response, and implicates another D A pool or neurotransmitter system in the mechanism through which RES potentiates AMP's effects. Previous work has shown that D A D 1 and D 2 receptor antagonists 2° and a M P T 8 block the behavioral effects of AMP, but fail to block AMP-conditioned locomotion. This indicates that neither postsynaptic events nor the direct presynaptic actions of AMP are responsible for the classical conditioning of AMP-induced locomotion. However, recent findings have provided evidence for a synergistic role of impulse-dependent calcium channels with D A D 2 receptor antagonism in AMP-conditioned locomotion (DiLullo and Martin-Iverson, submitted). In addition, it has been reported that while a M P T injected directly into the nucleus accumbens fails to block AMPconditioned place preferences, a-flupenthixol, a D A receptor blocker injected directly into the nucleus accumbens and systemic RES blocks this conditioned effect 16. These observations led us to hypothesize that the critical event conditioned with AMP is the impulse-dependent release of D A from presynaptic granular storage pools or a synergistic action between the aMPT- and RES-sensitive D A pools. These two possibilities were tested in the present experiment. aMPT attenuated (but did not completely block) the establishment of AMP-conditioned locomotion. This observation is somewhat different from that in an earlier
report from this laboratory, using the same dose of a M P T during AMP-conditioning in which no attenuation was observed 8. In the present case, this dose of a M P T completely blocked AMP-induced locomotion, while in the previous report the blockade was not complete. This differential affect was likely due largely to some recovery of AMP effects on the last 2 days of the 10 treatment days. In the present experiment, conditioning occurred over only 8 days, and no recovery was apparent. Thus, when AMP-induced locomotion was completely blocked some attenuation of conditioned-locomotion occurred. RES treatment alone did not affect AMP-conditioned behaviors. However, in combination with aMPT, RES completely blocked the establishment of AMP-conditioned locomotion. This blockade occurred in spite of the recovery of AMP-induced locomotion over the last 4 days in this treatment group. Previous publications have shown intact conditioning with blockade of the behavioral effects of stimulants 4"8'2°. In the present case, the psychomotor effects of AMP were observed in the absence of conditioned effects. In this report we observed a complete block of AMP-induced locomotion by aMPT, without a corresponding block of conditioned locomotion, and secondly, the ultimate recovery of AMPinduced locomotion with combined RES and aMPT treatment with complete blockade of conditioned locomotion. This double-dissociation indicates that the direct and the conditioned locomotor effects of AMP are subserved by different neural mechanisms. The results from this and other investigations 16 suggest that the psychomotor stimulant effects induced by AMP are expressed when D A from the aMPT-sensitive pool is released and that the establishment of AMP-conditioned behaviors is mediated by the RES and aMPTsensitive pools of DA. Blockade of one of these pools of D A is not sufficient to block the establishment of stimulant-conditioning. RES disrupts vesicles containing noradrenaline (NA) and serotonin (5-HT), as well as those containing DA. It is possible therefore that the blockade of conditioning is dependent upon actions on one of these neurotransmitter systems. Since destruction of forebrain NA-containing terminals with the neurotoxin, DSP4, does not affect the establishment of AMP-conditioned locomotion 8, it is unlikely that the effects of combined treatment with a M P T and RES are mediated by actions on NA systems. Effects on 5-HT cannot presently be ruled out. Decreases in 5-HT generally augment the behavioral effects of AMp19; the possibility that RES-induced depletion of 5-HT mediates the blockade of AMP-conditioned locomotion is not consistent with this observation. On the other hand, destruction of DA-containing
166 terminals with 6 - O H D A has been reported to block A M P - c o n d i t i o n e d locomotion 14. It is therefore most likely that the blockade of A M P - c o n d i t i o n e d locomotion with combined treatment with a M P T and RES is a function of effects on D A - c o n t a i n i n g nerve terminals. O n e question that remains is whether or not the conditioned effects mediated by the RES-sensitive pool are impulse-dependent. The observation that an impulse-dep e n d e n t calcium channel blocker, nimodipine, in combination with a D A D 2 receptor antagonist, haloperidol, attenuates A M P - c o n d i t i o n e d locomotion (DiLullo and Martin-Iverson, submitted) indicates that this may be true, since this c o m p o u n d should block impulse-dependent D A release. To summarize, neither a M P T nor RES given indepen-
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