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Increased occupation of D1 and D2 dopamine receptors accompanies cocaine-induced behavioral sensitization
BRAIN RESEARCH ELSEVIER
Brain Research 639 (1994) 228-232
Research Report
Increased occupation of D 1 and D 2 dopamine receptors accompanies cocaine-induced behavioral sensitization Lynn Y. Burger *, Mathew T. Martin-Iverson Neurochemical Research Unit, Department of Psychiatry, 1EZ 44 Mackenzie Health Sciences Centre, Unit'ersity of Alberta, Edmonton, Alta. T6E 2B7, Canada (Accepted 2 November 1993)
Abstract
In rats exhibiting behavioural sensitization after daily cocaine (10 mg/kg, i.p.) injections, the occupation of D 1 and D 2 dopamine receptors by dopamine, measured as protection from N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) receptor denaturation, was increased by about 100% compared to animals receiving cocaine in a treatment regimen that produced behavioural tolerance. Co-administration with nimodipine, an agent that blocks the impulse-dependent increase in synaptic concentrations of dopamine caused by cocaine, not only blocked sensitization but also blocked the increase in occupation of receptors. These findings strongly support the hypothesis that enhanced dopamine release and subsequent interaction with dopamine receptors is a substrate for behavioural sensitization to cocaine and have implications for the pharmacotherapy of cocaine abuse. Key words: Sensitization; Cocaine; Dopamine; Dopamine D 1 receptor; Dopamine D 2 receptor; Nimodipine; EEDQ
I. Introduction
Psychomotor stimulant drugs, including cocaine, have been associated with different behavioural effects in rats depending on the regimen of administration. R e p e a t e d daily injections produces sensitization, a gradual increase in drug-induced locomotion and other behaviours such as sniffing, rearing and head movements [1,36]. This intermittent drug delivery has been proposed as an animal model of stimulant-induced psychosis [1,36]. Continuous infusion, on the other hand, leads to the development of behavioural tolerance, even with doses equivalent to those producing sensitization [17,21,19,27]. Sensitization is therefore a function of factors related to the treatment regimen. Several neurochemical substrates have been proposed to account for sensitization. As cocaine is thought to act at the dopamine (DA) transporter to block uptake into presynaptic terminals, its effect is to enhance D A neurotransmission via increasing D A levels in the extraceUular space [11,14,26]. The development
*Corresponding author. Fax: (1) (403) 492-6841. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 0 4 - V
of behavioural sensitization with repeated administration has thus been associated with many different aspects of the D A system including changes in prea n d / o r post-synaptic receptor density, neurotransmitter synthesis, metabolism and spontaneous release (see refs. 12, 29 for reviews on this area). However, the most consistent finding is one of an increase in D A release stimulated by KCI, electrical stimulation or amphetamine, observed in both in vitro release and in vivo microdialysis studies [2,4,28,30-32]. This research is purely correlational, however and does not convincingly establish a role for enhanced stimulated D A release in sensitization to either cocaine or amphetamine. R e p e a t e d treatments of stimulants to animals produce a variety of effects in a variety of brain regions, any one (or more) of which may be responsible for behavioural sensitization. The hypothesis that behavioural sensitization results from increased D A release after cocaine would be strengthened if rats given cocaine continuously, to produce tolerance, fail to exhibit increased stimulated D A release. Additional support would also come from a treatment regimen involving a pharmacological blockade of sensitization that also blocks increased D A
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release. Research using the drug nimodipine, an L-type c a l c i u m c h a n n e l a n t a g o n i s t , has p r e v i o u s l y d e m o n s t r a t e d its ability to b l o c k s e n s i t i z a t i o n [33] a n d t h e i m p u l s e - d e p e n d e n t i n c r e a s e in e x t r a c e l l u l a r D A c o n c e n t r a t i o n s by c o c a i n e [23]. T h e p r e s e n t e x p e r i m e n t is d e s i g n e d to fulfill b o t h o f t h e s e c o n d i t i o n s by m e a s u r ing t h e o c c u p a t i o n o f D A r e c e p t o r s in a n i m a l s g i v e n t r e a t m e n t r e g i m e n s w i t h c o c a i n e t h a t p r o d u c e sensitization, tolerance or the pharmacological blockade of s e n s i t i z a t i o n w i t h n i m o d i p i n e . M o r e i m p o r t a n t l y , this s t u d y u t i l i z e s a n e w a p p r o a c h to i n d i r e c t l y m e a s u r e D A r e l e a s e by a s s e s s i n g t h e a m o u n t o f D A t h a t b i n d s to its r e c e p t o r s . T h i s is a c h i e v e d via t h e c o m p o u n d Nethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline (EEDQ) w h i c h acts by b i n d i n g to D A r e c e p t o r s to i r r e v e r s i b l y d e n a t u r e t h e m via a l k y l a t i o n . B e c a u s e it has a r e l a tively m o d e r a t e a f f i n i t y for t h e r e c e p t o r c o m p l e x , o n l y t h o s e sites n o t b o u n d w i t h D A (or any o t h e r l i g a n d w i t h h i g h affinity) a r e v u l n e r a b l e to E E D Q d e n a t u r a t i o n [7,22,34]. T h e r e f o r e , any o c c u p i e d r e c e p t o r site is left i n t a c t f o r m e a s u r e m e n t w i t h s t a n d a r d b i n d i n g assays. T h i s m e t h o d p r o v i d e s i n f o r m a t i o n a b o u t t h e a m o u n t o f D A b o u n d to its r e c e p t o r s f o l l o w i n g a g i v e n t r e a t m e n t r e g i m e n a n d has b e e n s h o w n to b e s e n s i t i v e to t h e e f f e c t s o f c o c a i n e at e l e v a t i n g t h e o c c u p a t i o n o f D t a n d D 2 r e c e p t o r s [3].
removed for dissection on ice. Left and right sides of the nucleus accumbens and striatum were removed and stored at -80°C for further neurochemical analysis. 2.3. DA receptor binding
Unilateral striatal tissue from each rat (left and right sides randomized across assays) was subjected to radioligand binding assays for both D 1 and D 2 receptor labelling as previously described [3]. [3H]SCH 23390 and [3H]spiperone were used to label D I and D 2 receptors, respectively (with the addition of ketanserin in the D 2 assay to control for 5-HT 2 receptor binding). Due to the small amount of tissue obtained with dissection of the nucleus accumbens, only single point binding for D 1 and D 2 receptor assessment was performed. Ligand concentrations were 0.678 nM for D 1 and 0.049 nM for D 2 receptor analysis (based on the respective K d values for each receptor). All ligands and assay procedure were otherwise the same as for the striatal binding. 2.4. Statistics
The 5 min blocks of photobeam interruptions were added together to produce daily 60 min totals which were then subjected to ANOVA with four factors. These factors were: (1) nimodipine treatment (NIM) with two levels (vehicle or 10 mg/kg); (2) cocaine treatment (COC) with two levels (vehicle or 10 mg/kg/day); (3) regimen of cocaine adminstration (TREAT) being either continuous or intermittent and (4) duration of treatment (DAYS) being 14 days in total. Receptor densities (Bmax) and affinities (K d) were calculated from the data from the receptor binding assays using LIGAND and were subjected to ANOVA with the first three factors listed above. Multiple F tests [15] were applied for individual comparisons
2. Materials and methods 500 2.1. Animals
Forty-eight male Sprague-Dawley rats (Charles River Canada) weighing 250-350 g were individually housed in colony rooms maintained on a 12 h light-dark cycle (lights on at 08:00 h) at 23°C. Food and water was available ad libitum. All subjects were weighed every second day throughout the course of the experiment.
INTERMITTENT COCAINE
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z- 400 _o ha. ~'
i 300~ i
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INTERMITTENT COCAINE WITH NIMODIPINE
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2.2. Drug administration procedure E
After 10 days of habituation following arrival, the rats were assigned to one of eight drug groups (n = 8 in each with vehicle groups divided into intermittent and continuous with n = 4 in each) in a counterbalanced fashion. Cocaine hydrochloride (BDH, 10 m g / kg/day) or vehicle (double distilled water) was delivered either continuously via Alzet osmotic minipumps (Alza, model 2 ML2) or intermittently with daily intraperitoneal (i.p.) injections for 14 days. Surgical implantation of the minipumps (method described elsewhere [3]) was performedon day 0. Nimodipine (Miles Pharmaceuticals Ltd., 10 mg/kg) or its vehicle, polyethylene glycol 400 (100%), was injected i.p. 70 min prior to cocaine each day, beginning on Day 1. Immediately following the daily cocaine injection (vehicle was injected for those animals with minipumps) each rat was placed in its own box equipped with photocell beams (as described previously [20]) to assess activity levels for 1 h. Each photocell beam interruption is represented as one count of gross body movement since any rapid successive actions are not detected. On day 14, 30 min postcocaine, all subjects were administered EEDQ (5 mg/kg, i.p.) dissolved in ethanol, propylene glycol and distilled water (2:1:2). Twenty-four hours later, the rats were decapitated and their brains
CONTROLS 0 0
! 1
I 2
i 3
4
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I 6
7
I 8
9
10
I 11
1 12
I 13
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CONSECUTIVE DAYS
Fig. 1. Activity levels represented as photocell beam interruptions over successive days. Rats were placed in these boxes for one h each day immediately following 10 mg/kg intraperitoneally (i.p.) injected cocaine (intermittent cocaine) or distilled water (controls and continuous cocaine). Continuous cocaine animals received equivalent daily doses (10 mg/kg/day) via Alzet osmotic minipumps subcutaneously implanted as previously described [3]. For the sake of clarity, vehicle groups (intermittent and continuous), nimodipine with vehicle and nimodipine with continuous cocaine rats were pooled together as controls, since there were no significant differences among these groups in terms of activity levels. Intermittent animals exhibited a gradual augmentation of locomotor activity that was blocked by nimodipine co-administration. Continuous drug delivery resulted in tolerance.
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L. E Burger, M. T. Martin-h:erson / Brain Research 639 (1994) 22,'~' -232
where appropriate. Pearson's multiple correlations analysis between locomotor counts and binding results were also calculated.
3. Results
3.1. Locomotor activity Fig. 1 displays the effects of cocaine on locomotor behaviour when delivered either as daily injections for 14 days or continuously with osmotic minipumps. The intermittent cocaine group exhibited behavioural sensitization with continuous cocaine producing an initial stimulant effect followed by the development of toler-
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ance. Intermittently treated cocaine rats failed to develop sensitized locomotion if they were pretreated with nimodipine over the two week period. These findings are supported by ANOVA with multivariatc tests of significance over repeated days which revealed a significant NIM × COC × T R E A T × DAYS effect (Hotellings T~3.2s = 1.1)1, P < 0.(15).
3.2. DA receptor binding Intermittent cocaine delivery produced about a 100% increase in the occupation of both Dl-like and D2-1ike receptors in both striatum (Fig. 2A) and nucleus accumbens (Fig. 2B). That is, increased DA receptor protection from E E D Q denaturation was observed in this group. Continuous infusions of the same quantity of cocaine as given by injection produced behavioural tolerance and also failed to increase the occupation of DA receptors. As with the aforementioned locomotor data, nimodipine blocked the increased occupation of DA receptors observed in the sensitized intermittent cocaine animals. A significant NIM × COC × T R E A T interaction was revealed with ANOVA for both the striatum (FI,40 = 7.98, P < 0.01) and the nucleus accumbens (FI,40 = 5.78, P < 0.05). Correlational analysis of behaviour with receptor binding revealed a significant correlation between locomotor activity and increased occupation of D~ (but not D 2) receptors in the striatum (r = 0.41, P < 0.01). In the nucleus accumbens, however, locomotion correlated with D 2 (but not D,) receptor occupation (r = 0.34, P < 0.05).
4. Discussion ~..~ _Zo Z z
e8
Fig. 2. Receptor binding results for rat striatum (A) and nucleus accumbens (B) 24 h post-EEDQ injection (5 m g / k g , i.p.). Radioligand binding assays for D 1 and D 2 receptors performed with full scatchard plot analysis represented for striatal tissue and single point binding for nucleus accumbens. Both Bma× values are represented as percent of control. Drug groups (n = 8) include intermittent cocaine (VCI), continuous cocaine (VCC), intermittent cocaine with nimodipine (NCI) and continuous cocaine with nimodipine (NCC). VCI animals showed increased occupation of D 1 and D 2 receptors relative to controls. This effect was blocked by nimodipine and continuous cocaine delivery. Standard errors are shown by the bars. *Significantly different from controls.
The present experiment strengthens previous data suggesting increased stimulated release of DA as a neural mechanism of behavioural sensitization. Repeated injections of cocaine produce increased activity levels in the latter half of a two week period. These animals exhibited a greater occupation of D l and D 2 receptor sites in both the striatal and nucleus accumbens terminal fields. If nimodipine is co-administered with cocaine, both behavioural sensitization and increased occupation of DA receptors are blocked. Since the treatment regimen resulting in behavioural tolerance failed to enhance D 1 and D 2 receptor occupation and did not significantly differ from vehicle controls, sensitization appears to be associated with increased binding of DA with its receptors. Nimodipine has previously been shown to block cocaine-induced behavioural sensitization when administered chronically via daily injections [33]. Pani and associates have also demonstrated the ability of nimodipine and other dihydropyridines to prevent the
L.Y. Burger, M.T. Martin-lverson / Brain Research 639 (1994) 228-232
locomotor stimulant effect of cocaine [23] as well as inhibiting the reinforcing properties of cocaine [24]. These authors attribute these effects to the blockade of L-type voltage-sensitive calcium channels in the central nervous system. Recent research has shown that cocaine-induced DA release is dependent on the influx of calcium through these channels which, in addition to uptake blockade, ultimately leads to an increase in extracellular DA [11,14,26]. The present data indicate that blocking the impulse-dependent release of DA induced by cocaine prevents the subsequent increase in DA receptor occupation after repeated intermittent cocaine treatments. This suggests that behavioural sensitization may be a consequence of increased impulse flow in DA neurons. Further support for this concept comes from reports of increased firing in A10 DA neurons following repeated daily injections of cocaine [8,10]. Ultimately, the end result is increased DA available for occupation at D~ and D 2 sites in the striatum and nucleus accumbens. It is interesting to note that cocaine-induced behavioural sensitization correlates with the occupation of different subtypes of the DA receptor system depending on the anatomical location. We report that increased D 1 receptor occupation in the striatum but increased D E receptor occupation in the nucleus accumbens correlates with sensitized locomotor activity. This differentiation of cocaine's effects on DA terminal fields has been reported elsewhere with respect to changes in receptor density [6,16,25]. These various studies have suggested that chronic cocaine decreases the density of D E sites in the striatum while increasing D E receptor sites in the nucleus accumbens. Within the striatum, evidence exists to indicate that DA projections from the A8 - A 9- A 10 region project preferentially to either the Dl-rich striosomes or D2-rich matrix [13]. It is therefore possible that the D 1 pathways are correlated with behavioural sensitization in this brain region. The correlation between D E receptor occupation in the nucleus accumbens and sensitization is less clear in light of the reports of D 1 supersensitivity in this brain region with cocaine-induced sensitization [9,10]. Behavioural sensitization has been proposed to be largely DI receptor mediated since D 1 antagonists in the ventral tegmental area block this phenomenon [35]. In addition, the D 1 supersensitivity in the nucleus accumbens with repeated cocaine has been shown to persist for at least 1 month adding further support for it as a mechanism of behavioural sensitization, itself a long-lasting phenomenon [10]. The present study, however, indicates that augmented DA release which increases the occupation of both D 1 and D z subtypes in the striatum and nucleus accumbens contributes significantly to behavioural sensitization. However, the occupation of DA receptors in other DA terminal fields (e.g. amygdala, medial prefrontal cortex) was not as-
231
sessed in the present experiment. Therefore, contributions to behavioral sensitization from these regions cannot be excluded or included at this time. These data indicate that measuring DA receptor occupation, presumably by DA, provides more information about the DA system alterations that contribute to sensitization than simply measuring changes in DA receptor density or sensitivity. The use of EEDQ as a tool for assessing the functioning DA system also seems more appropriate in light of recent research suggesting that changes in DA receptor density and behavioural sensitization are, essentially, unrelated [18]. Finally, the results reported in this experiment have implications for the problems associated with cocaine abuse. Given the efficacy of nimodipine to block sensitization, to the extent that sensitization in rats models psychoses in humans, this drug may provide a novel therapy for psychoses. Furthermore, the present results support previous reports that nimodipine may be efficacious in the treatment of cocaine addiction [5], since it can block the cocaine-induced increase in DA available for binding to receptors. Acknowledgements.
We acknowledge the excellent technical assistance of Richard Strel. Funding was provided by Miles Pharmaceutical Ltd. L.Y.B. is supported by an Alberta Mental Health Research Fund Studentship. M.T. M-I is an Alberta Heritage Foundation Scholar.
5. References [1] Angrist, B., Psychoses induced by central nervous system stimulants and related drugs. In I. Creese (Ed.), Stimulants." Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 1-30. [2] Antelman, S.M., Eichler, A.J., Black, C.A. and Kocan, D., Interchangeability of stress and amphetamine in sensitization, Science, 207 (1980) 329-331. [3] Burger, L.Y. and Martin-Iverson, M.T., Day/night differences in D1 but not D2 DA receptor protection from EEDQ denaturation in rats treated with continuous cocaine, Synapse, 13 (1993) 20-29. [4] Castaneda, E., Becker, J.B., Robinson, T.E., The long-term effects of amphetamine treatment in vivo on amphetamine, KC1 and electrical stimulation evoked striatal dopamine release in vitro, Life Sci., 42 (1988) 2447-2456. [5] DiLullo, S.L. and Martin-Iverson, M.T., Calcium channel blockade interacts with a neuroleptic to attenuate the conditioning of amphetamine's behavioral effects in the rat, Biol. Psychiatry, 31 (1992) 1143-1150. [6] Goeders, N.E., The effects of chronic cocaine administration on brain neurotransmitter receptors, Drug Dev. Res., 20 (1990) 349-357. [7] Hamblin, M.W. and Creese, I., Behavioral and radioligand binding evidence irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, Life Sci., 32 (1983) 2247-2255. 18] Henry, D.J., Greene, M.A. and White, F.J., Electrophysiological effects of cocaine in the mesoaccumbens dopamine system:
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Repeated administration, J. Pharmacol. Exp. Ther.. 251 (1989) 833-839. [9] Henry, D.J. and White, F.J., Repeated cocaine administration causes persistent enhancement of DI dopamine receptor supersensitivity within the rat nucleus accumbens, Z Pharmacol. Exp. Ther., 258 (1991) 882-890. [10] Henry, D.J. and White, F.J., Electrophysiological correlates of psychomotor stimulant-induced sensitization, Ann. N Y Acad. Sci., 654 (1992) 88-100. [11] Hurd, Y.L. and Ungerstedt, U., Cocaine: an in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum, Synapse, 3 (1989) 48-54. [12] Johanson, C.-E. and Fischman, M.W., The pharmacology of cocaine related to its abuse, Pharmacol. Rev., 41 (1989) 3-52. [13] Jimenez-Castellanos, J. and Graybiel, A.M., Subdivisions of the dopamine-containing A8-A9-A10 complex identified by their differential mesostriatal innervation of striosomes and extrastriosomal matrix, Neuroscience, 23 (1987) 223-242. [14] Kalivas, P.W. and Duffy, P., Effect of acute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens, Synapse, 5 (1990) 48-58. [15] Kiess, H.O., Statistical Concepts for the Behavioral Sciences, Allyn and Bacon, Boston, 1989, pp. 326-331. [16] Kleven, M.S., Perry, B.D., Woolverton, W.L. and Selden, L.S., Effects of repeated injections of cocaine on D 1 and D 2 dopamine receptors in rat brain, Brain Res., 532 (1990) 265-270. [17] Kokkinidis, L., Effects of chronic intermittent and continuous amphetamine administration on acoustic startle, Pharmacol. Biochem. Behav., 20 (1984) 367-372. [18] LaHoste, G.J. and Marshall, J.F., Dopamine supersensitivity and D1/D2 synergism are unrelated to changes in striatal receptor density, Synapse, 12 (1992) 14-26. [19] Martin-Iverson, M.T., Stahl, S.M. and Iversen, S.D., Chronic administration of a selective dopamine D2 agonist: factors determining behavioral tolerance and sensitization. Psychopharmacology, 95 (1988) 534-539. [20] Martin-Iverson, M.T. and McManus, D., Stimulant-conditioned locomotion is not affected by blockade of D I a n d / o r D e dopamine receptors during conditioning, Brain Res., 521 (1990) 175-184. [21] Martin-Iverson, M.T., Stahl, S.M. and Iversen, S.D., Factors determining the behavioral consequences of continuous treatment with 4-propyl-9-hydroxynaphthoxazine, a selective dopamine D2 agonist. In F.C. Rose lEd.), Parkinson's Disease: Clinical and Experimental Advances, J. Libbey, London, 1987, pp. 169-177. [22] Meller, E., Bohmaker, K., Goldstein, M. and Friedhoff, A.J., Inactivation of D1 and d2 dopamine receptors by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinolinein vivo: selective protection by neuroleptics, J. Pharmacol. Exp. Ther., 233 (1985) 656662. [23] Pani, L., Carboni, S., Kusmin, A., Gessa, G.L. and Rossetti, Z.L., Nimodipine inhibits cocaine-induced dopamine release and motor stimulation, Eur. J. PharmacoL, 176 (1990) 245-246.
[24] Pani, L., Kuzmin, A., Martellotta, M.C., Gessa, (i.h. and Fratta. W., The calcium antagonist PN 200-1111 inhibits the reinforcing properties of cocaine, Brain Res. Bull., 26 11991) 445-447. [25] Peris, J., Boyson, S.J., Cass, W.A., Curella, P., Dwoskim LP., Larson, G. Lin, L., Yasuda, R.P. and Zahniser. N.R., Persis tence of neurochemical changes in dopaminc systems afte~ repeated cocaine administration, J. Pharmacol. Exp. Ther., 253 (1990) 38-44. [26] Pettit, lt.O., Pan, H.-T., Parson, Ell. and Justice, ,1.B. Jr.. Extracellular concentratkms of cocaine and dopaminc are enhanced during chronic cocaine administration. J. Neuroehem.. 55 (1990) 798-804. [27] Post, R.M., Intermittent versus continuous stimulation: effect of time interval on the development of sensitization or tolerance, Life Sci., 26 (1980) 1275-1282. [28] Robinson, T.E. and Becket, J.B., Behavioral sensitization is accompanied by an enhancement in amphetamine-stimulated dopamine release from striatal tissue in vitro, Eur. J. Pharmacol., 85 (1982) 253-254. [29] Robinson, T.E. and Becker, 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. [30] Robinson, T.E., Becker, J.B. and Presty, S.K., Long-term facilitation of amphetamine-induced rotational behavior and striatal dopamine release produced by a single exposure to amphetamine: sex differences, Brain Res., 253 (1982) 231-241. [31] Robinson, T.E., Becker, J.B., Young, E.A., Akil, H. and Castaneda, E., The effects of footshock stress on regional brain dopamine metabolism and pituitary/3-endorphin release in rats previously sensitized to amphetamine, Neuropharmacology. 26 (1987) 679-691. [32] Robinson, T.E., Jurson, P.A., Bennett, J.A. and Bentgen, K.M., Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)amphetamine: a microdialysis study in freely moving rats, Brain Res., 462 (1988) 211-222. [33] Reimer, A.R. and Martin-lverson, M.T., Nimodipine and haloperidol attenuate behavioral sensitization to cocaine but only nimodipine blocks the establishment of conditioned locomotion induced by cocaine, Psychopharmacology, in press. [34] Sailer, C.F., Kreamer, L.D., Adamovage, L.A. and Salama, A.I., Dopamine receptor occupancy in vivo: measurement using Nethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ), Life Sci., 45 (1989) 917-929. [35] Stewart, J. and Vezina, P., Microinjections of SCH 23390 into the ventral tegmental area and substantia nigra pars reticulata attenuate the development of sensitization to the locomotor activating effects of systemic amphetamine, Brain Res., 495 (1989) 401-406. [36] Segal, D.S. and Schuckit, M.A., Animal models of stimulant induced psychosis. In I. Creese lEd.), Stimulants: Neurochemieal, Behavioral, and Clincal Perspectives, Raven. New York, 1983, pp. 131-168.
Brain Research 639 (1994) 228-232
Research Report
Increased occupation of D 1 and D 2 dopamine receptors accompanies cocaine-induced behavioral sensitization Lynn Y. Burger *, Mathew T. Martin-Iverson Neurochemical Research Unit, Department of Psychiatry, 1EZ 44 Mackenzie Health Sciences Centre, Unit'ersity of Alberta, Edmonton, Alta. T6E 2B7, Canada (Accepted 2 November 1993)
Abstract
In rats exhibiting behavioural sensitization after daily cocaine (10 mg/kg, i.p.) injections, the occupation of D 1 and D 2 dopamine receptors by dopamine, measured as protection from N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) receptor denaturation, was increased by about 100% compared to animals receiving cocaine in a treatment regimen that produced behavioural tolerance. Co-administration with nimodipine, an agent that blocks the impulse-dependent increase in synaptic concentrations of dopamine caused by cocaine, not only blocked sensitization but also blocked the increase in occupation of receptors. These findings strongly support the hypothesis that enhanced dopamine release and subsequent interaction with dopamine receptors is a substrate for behavioural sensitization to cocaine and have implications for the pharmacotherapy of cocaine abuse. Key words: Sensitization; Cocaine; Dopamine; Dopamine D 1 receptor; Dopamine D 2 receptor; Nimodipine; EEDQ
I. Introduction
Psychomotor stimulant drugs, including cocaine, have been associated with different behavioural effects in rats depending on the regimen of administration. R e p e a t e d daily injections produces sensitization, a gradual increase in drug-induced locomotion and other behaviours such as sniffing, rearing and head movements [1,36]. This intermittent drug delivery has been proposed as an animal model of stimulant-induced psychosis [1,36]. Continuous infusion, on the other hand, leads to the development of behavioural tolerance, even with doses equivalent to those producing sensitization [17,21,19,27]. Sensitization is therefore a function of factors related to the treatment regimen. Several neurochemical substrates have been proposed to account for sensitization. As cocaine is thought to act at the dopamine (DA) transporter to block uptake into presynaptic terminals, its effect is to enhance D A neurotransmission via increasing D A levels in the extraceUular space [11,14,26]. The development
*Corresponding author. Fax: (1) (403) 492-6841. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 0 4 - V
of behavioural sensitization with repeated administration has thus been associated with many different aspects of the D A system including changes in prea n d / o r post-synaptic receptor density, neurotransmitter synthesis, metabolism and spontaneous release (see refs. 12, 29 for reviews on this area). However, the most consistent finding is one of an increase in D A release stimulated by KCI, electrical stimulation or amphetamine, observed in both in vitro release and in vivo microdialysis studies [2,4,28,30-32]. This research is purely correlational, however and does not convincingly establish a role for enhanced stimulated D A release in sensitization to either cocaine or amphetamine. R e p e a t e d treatments of stimulants to animals produce a variety of effects in a variety of brain regions, any one (or more) of which may be responsible for behavioural sensitization. The hypothesis that behavioural sensitization results from increased D A release after cocaine would be strengthened if rats given cocaine continuously, to produce tolerance, fail to exhibit increased stimulated D A release. Additional support would also come from a treatment regimen involving a pharmacological blockade of sensitization that also blocks increased D A
229
L.Y. Burger, M.T. Martin-lverson / Brain Research 639 (1994) 228-232
release. Research using the drug nimodipine, an L-type c a l c i u m c h a n n e l a n t a g o n i s t , has p r e v i o u s l y d e m o n s t r a t e d its ability to b l o c k s e n s i t i z a t i o n [33] a n d t h e i m p u l s e - d e p e n d e n t i n c r e a s e in e x t r a c e l l u l a r D A c o n c e n t r a t i o n s by c o c a i n e [23]. T h e p r e s e n t e x p e r i m e n t is d e s i g n e d to fulfill b o t h o f t h e s e c o n d i t i o n s by m e a s u r ing t h e o c c u p a t i o n o f D A r e c e p t o r s in a n i m a l s g i v e n t r e a t m e n t r e g i m e n s w i t h c o c a i n e t h a t p r o d u c e sensitization, tolerance or the pharmacological blockade of s e n s i t i z a t i o n w i t h n i m o d i p i n e . M o r e i m p o r t a n t l y , this s t u d y u t i l i z e s a n e w a p p r o a c h to i n d i r e c t l y m e a s u r e D A r e l e a s e by a s s e s s i n g t h e a m o u n t o f D A t h a t b i n d s to its r e c e p t o r s . T h i s is a c h i e v e d via t h e c o m p o u n d Nethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline (EEDQ) w h i c h acts by b i n d i n g to D A r e c e p t o r s to i r r e v e r s i b l y d e n a t u r e t h e m via a l k y l a t i o n . B e c a u s e it has a r e l a tively m o d e r a t e a f f i n i t y for t h e r e c e p t o r c o m p l e x , o n l y t h o s e sites n o t b o u n d w i t h D A (or any o t h e r l i g a n d w i t h h i g h affinity) a r e v u l n e r a b l e to E E D Q d e n a t u r a t i o n [7,22,34]. T h e r e f o r e , any o c c u p i e d r e c e p t o r site is left i n t a c t f o r m e a s u r e m e n t w i t h s t a n d a r d b i n d i n g assays. T h i s m e t h o d p r o v i d e s i n f o r m a t i o n a b o u t t h e a m o u n t o f D A b o u n d to its r e c e p t o r s f o l l o w i n g a g i v e n t r e a t m e n t r e g i m e n a n d has b e e n s h o w n to b e s e n s i t i v e to t h e e f f e c t s o f c o c a i n e at e l e v a t i n g t h e o c c u p a t i o n o f D t a n d D 2 r e c e p t o r s [3].
removed for dissection on ice. Left and right sides of the nucleus accumbens and striatum were removed and stored at -80°C for further neurochemical analysis. 2.3. DA receptor binding
Unilateral striatal tissue from each rat (left and right sides randomized across assays) was subjected to radioligand binding assays for both D 1 and D 2 receptor labelling as previously described [3]. [3H]SCH 23390 and [3H]spiperone were used to label D I and D 2 receptors, respectively (with the addition of ketanserin in the D 2 assay to control for 5-HT 2 receptor binding). Due to the small amount of tissue obtained with dissection of the nucleus accumbens, only single point binding for D 1 and D 2 receptor assessment was performed. Ligand concentrations were 0.678 nM for D 1 and 0.049 nM for D 2 receptor analysis (based on the respective K d values for each receptor). All ligands and assay procedure were otherwise the same as for the striatal binding. 2.4. Statistics
The 5 min blocks of photobeam interruptions were added together to produce daily 60 min totals which were then subjected to ANOVA with four factors. These factors were: (1) nimodipine treatment (NIM) with two levels (vehicle or 10 mg/kg); (2) cocaine treatment (COC) with two levels (vehicle or 10 mg/kg/day); (3) regimen of cocaine adminstration (TREAT) being either continuous or intermittent and (4) duration of treatment (DAYS) being 14 days in total. Receptor densities (Bmax) and affinities (K d) were calculated from the data from the receptor binding assays using LIGAND and were subjected to ANOVA with the first three factors listed above. Multiple F tests [15] were applied for individual comparisons
2. Materials and methods 500 2.1. Animals
Forty-eight male Sprague-Dawley rats (Charles River Canada) weighing 250-350 g were individually housed in colony rooms maintained on a 12 h light-dark cycle (lights on at 08:00 h) at 23°C. Food and water was available ad libitum. All subjects were weighed every second day throughout the course of the experiment.
INTERMITTENT COCAINE
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2.2. Drug administration procedure E
After 10 days of habituation following arrival, the rats were assigned to one of eight drug groups (n = 8 in each with vehicle groups divided into intermittent and continuous with n = 4 in each) in a counterbalanced fashion. Cocaine hydrochloride (BDH, 10 m g / kg/day) or vehicle (double distilled water) was delivered either continuously via Alzet osmotic minipumps (Alza, model 2 ML2) or intermittently with daily intraperitoneal (i.p.) injections for 14 days. Surgical implantation of the minipumps (method described elsewhere [3]) was performedon day 0. Nimodipine (Miles Pharmaceuticals Ltd., 10 mg/kg) or its vehicle, polyethylene glycol 400 (100%), was injected i.p. 70 min prior to cocaine each day, beginning on Day 1. Immediately following the daily cocaine injection (vehicle was injected for those animals with minipumps) each rat was placed in its own box equipped with photocell beams (as described previously [20]) to assess activity levels for 1 h. Each photocell beam interruption is represented as one count of gross body movement since any rapid successive actions are not detected. On day 14, 30 min postcocaine, all subjects were administered EEDQ (5 mg/kg, i.p.) dissolved in ethanol, propylene glycol and distilled water (2:1:2). Twenty-four hours later, the rats were decapitated and their brains
CONTROLS 0 0
! 1
I 2
i 3
4
I 5
I 6
7
I 8
9
10
I 11
1 12
I 13
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CONSECUTIVE DAYS
Fig. 1. Activity levels represented as photocell beam interruptions over successive days. Rats were placed in these boxes for one h each day immediately following 10 mg/kg intraperitoneally (i.p.) injected cocaine (intermittent cocaine) or distilled water (controls and continuous cocaine). Continuous cocaine animals received equivalent daily doses (10 mg/kg/day) via Alzet osmotic minipumps subcutaneously implanted as previously described [3]. For the sake of clarity, vehicle groups (intermittent and continuous), nimodipine with vehicle and nimodipine with continuous cocaine rats were pooled together as controls, since there were no significant differences among these groups in terms of activity levels. Intermittent animals exhibited a gradual augmentation of locomotor activity that was blocked by nimodipine co-administration. Continuous drug delivery resulted in tolerance.
231)
L. E Burger, M. T. Martin-h:erson / Brain Research 639 (1994) 22,'~' -232
where appropriate. Pearson's multiple correlations analysis between locomotor counts and binding results were also calculated.
3. Results
3.1. Locomotor activity Fig. 1 displays the effects of cocaine on locomotor behaviour when delivered either as daily injections for 14 days or continuously with osmotic minipumps. The intermittent cocaine group exhibited behavioural sensitization with continuous cocaine producing an initial stimulant effect followed by the development of toler-
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ance. Intermittently treated cocaine rats failed to develop sensitized locomotion if they were pretreated with nimodipine over the two week period. These findings are supported by ANOVA with multivariatc tests of significance over repeated days which revealed a significant NIM × COC × T R E A T × DAYS effect (Hotellings T~3.2s = 1.1)1, P < 0.(15).
3.2. DA receptor binding Intermittent cocaine delivery produced about a 100% increase in the occupation of both Dl-like and D2-1ike receptors in both striatum (Fig. 2A) and nucleus accumbens (Fig. 2B). That is, increased DA receptor protection from E E D Q denaturation was observed in this group. Continuous infusions of the same quantity of cocaine as given by injection produced behavioural tolerance and also failed to increase the occupation of DA receptors. As with the aforementioned locomotor data, nimodipine blocked the increased occupation of DA receptors observed in the sensitized intermittent cocaine animals. A significant NIM × COC × T R E A T interaction was revealed with ANOVA for both the striatum (FI,40 = 7.98, P < 0.01) and the nucleus accumbens (FI,40 = 5.78, P < 0.05). Correlational analysis of behaviour with receptor binding revealed a significant correlation between locomotor activity and increased occupation of D~ (but not D 2) receptors in the striatum (r = 0.41, P < 0.01). In the nucleus accumbens, however, locomotion correlated with D 2 (but not D,) receptor occupation (r = 0.34, P < 0.05).
4. Discussion ~..~ _Zo Z z
e8
Fig. 2. Receptor binding results for rat striatum (A) and nucleus accumbens (B) 24 h post-EEDQ injection (5 m g / k g , i.p.). Radioligand binding assays for D 1 and D 2 receptors performed with full scatchard plot analysis represented for striatal tissue and single point binding for nucleus accumbens. Both Bma× values are represented as percent of control. Drug groups (n = 8) include intermittent cocaine (VCI), continuous cocaine (VCC), intermittent cocaine with nimodipine (NCI) and continuous cocaine with nimodipine (NCC). VCI animals showed increased occupation of D 1 and D 2 receptors relative to controls. This effect was blocked by nimodipine and continuous cocaine delivery. Standard errors are shown by the bars. *Significantly different from controls.
The present experiment strengthens previous data suggesting increased stimulated release of DA as a neural mechanism of behavioural sensitization. Repeated injections of cocaine produce increased activity levels in the latter half of a two week period. These animals exhibited a greater occupation of D l and D 2 receptor sites in both the striatal and nucleus accumbens terminal fields. If nimodipine is co-administered with cocaine, both behavioural sensitization and increased occupation of DA receptors are blocked. Since the treatment regimen resulting in behavioural tolerance failed to enhance D 1 and D 2 receptor occupation and did not significantly differ from vehicle controls, sensitization appears to be associated with increased binding of DA with its receptors. Nimodipine has previously been shown to block cocaine-induced behavioural sensitization when administered chronically via daily injections [33]. Pani and associates have also demonstrated the ability of nimodipine and other dihydropyridines to prevent the
L.Y. Burger, M.T. Martin-lverson / Brain Research 639 (1994) 228-232
locomotor stimulant effect of cocaine [23] as well as inhibiting the reinforcing properties of cocaine [24]. These authors attribute these effects to the blockade of L-type voltage-sensitive calcium channels in the central nervous system. Recent research has shown that cocaine-induced DA release is dependent on the influx of calcium through these channels which, in addition to uptake blockade, ultimately leads to an increase in extracellular DA [11,14,26]. The present data indicate that blocking the impulse-dependent release of DA induced by cocaine prevents the subsequent increase in DA receptor occupation after repeated intermittent cocaine treatments. This suggests that behavioural sensitization may be a consequence of increased impulse flow in DA neurons. Further support for this concept comes from reports of increased firing in A10 DA neurons following repeated daily injections of cocaine [8,10]. Ultimately, the end result is increased DA available for occupation at D~ and D 2 sites in the striatum and nucleus accumbens. It is interesting to note that cocaine-induced behavioural sensitization correlates with the occupation of different subtypes of the DA receptor system depending on the anatomical location. We report that increased D 1 receptor occupation in the striatum but increased D E receptor occupation in the nucleus accumbens correlates with sensitized locomotor activity. This differentiation of cocaine's effects on DA terminal fields has been reported elsewhere with respect to changes in receptor density [6,16,25]. These various studies have suggested that chronic cocaine decreases the density of D E sites in the striatum while increasing D E receptor sites in the nucleus accumbens. Within the striatum, evidence exists to indicate that DA projections from the A8 - A 9- A 10 region project preferentially to either the Dl-rich striosomes or D2-rich matrix [13]. It is therefore possible that the D 1 pathways are correlated with behavioural sensitization in this brain region. The correlation between D E receptor occupation in the nucleus accumbens and sensitization is less clear in light of the reports of D 1 supersensitivity in this brain region with cocaine-induced sensitization [9,10]. Behavioural sensitization has been proposed to be largely DI receptor mediated since D 1 antagonists in the ventral tegmental area block this phenomenon [35]. In addition, the D 1 supersensitivity in the nucleus accumbens with repeated cocaine has been shown to persist for at least 1 month adding further support for it as a mechanism of behavioural sensitization, itself a long-lasting phenomenon [10]. The present study, however, indicates that augmented DA release which increases the occupation of both D 1 and D z subtypes in the striatum and nucleus accumbens contributes significantly to behavioural sensitization. However, the occupation of DA receptors in other DA terminal fields (e.g. amygdala, medial prefrontal cortex) was not as-
231
sessed in the present experiment. Therefore, contributions to behavioral sensitization from these regions cannot be excluded or included at this time. These data indicate that measuring DA receptor occupation, presumably by DA, provides more information about the DA system alterations that contribute to sensitization than simply measuring changes in DA receptor density or sensitivity. The use of EEDQ as a tool for assessing the functioning DA system also seems more appropriate in light of recent research suggesting that changes in DA receptor density and behavioural sensitization are, essentially, unrelated [18]. Finally, the results reported in this experiment have implications for the problems associated with cocaine abuse. Given the efficacy of nimodipine to block sensitization, to the extent that sensitization in rats models psychoses in humans, this drug may provide a novel therapy for psychoses. Furthermore, the present results support previous reports that nimodipine may be efficacious in the treatment of cocaine addiction [5], since it can block the cocaine-induced increase in DA available for binding to receptors. Acknowledgements.
We acknowledge the excellent technical assistance of Richard Strel. Funding was provided by Miles Pharmaceutical Ltd. L.Y.B. is supported by an Alberta Mental Health Research Fund Studentship. M.T. M-I is an Alberta Heritage Foundation Scholar.
5. References [1] Angrist, B., Psychoses induced by central nervous system stimulants and related drugs. In I. Creese (Ed.), Stimulants." Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 1-30. [2] Antelman, S.M., Eichler, A.J., Black, C.A. and Kocan, D., Interchangeability of stress and amphetamine in sensitization, Science, 207 (1980) 329-331. [3] Burger, L.Y. and Martin-Iverson, M.T., Day/night differences in D1 but not D2 DA receptor protection from EEDQ denaturation in rats treated with continuous cocaine, Synapse, 13 (1993) 20-29. [4] Castaneda, E., Becker, J.B., Robinson, T.E., The long-term effects of amphetamine treatment in vivo on amphetamine, KC1 and electrical stimulation evoked striatal dopamine release in vitro, Life Sci., 42 (1988) 2447-2456. [5] DiLullo, S.L. and Martin-Iverson, M.T., Calcium channel blockade interacts with a neuroleptic to attenuate the conditioning of amphetamine's behavioral effects in the rat, Biol. Psychiatry, 31 (1992) 1143-1150. [6] Goeders, N.E., The effects of chronic cocaine administration on brain neurotransmitter receptors, Drug Dev. Res., 20 (1990) 349-357. [7] Hamblin, M.W. and Creese, I., Behavioral and radioligand binding evidence irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, Life Sci., 32 (1983) 2247-2255. 18] Henry, D.J., Greene, M.A. and White, F.J., Electrophysiological effects of cocaine in the mesoaccumbens dopamine system:
232
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Repeated administration, J. Pharmacol. Exp. Ther.. 251 (1989) 833-839. [9] Henry, D.J. and White, F.J., Repeated cocaine administration causes persistent enhancement of DI dopamine receptor supersensitivity within the rat nucleus accumbens, Z Pharmacol. Exp. Ther., 258 (1991) 882-890. [10] Henry, D.J. and White, F.J., Electrophysiological correlates of psychomotor stimulant-induced sensitization, Ann. N Y Acad. Sci., 654 (1992) 88-100. [11] Hurd, Y.L. and Ungerstedt, U., Cocaine: an in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum, Synapse, 3 (1989) 48-54. [12] Johanson, C.-E. and Fischman, M.W., The pharmacology of cocaine related to its abuse, Pharmacol. Rev., 41 (1989) 3-52. [13] Jimenez-Castellanos, J. and Graybiel, A.M., Subdivisions of the dopamine-containing A8-A9-A10 complex identified by their differential mesostriatal innervation of striosomes and extrastriosomal matrix, Neuroscience, 23 (1987) 223-242. [14] Kalivas, P.W. and Duffy, P., Effect of acute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens, Synapse, 5 (1990) 48-58. [15] Kiess, H.O., Statistical Concepts for the Behavioral Sciences, Allyn and Bacon, Boston, 1989, pp. 326-331. [16] Kleven, M.S., Perry, B.D., Woolverton, W.L. and Selden, L.S., Effects of repeated injections of cocaine on D 1 and D 2 dopamine receptors in rat brain, Brain Res., 532 (1990) 265-270. [17] Kokkinidis, L., Effects of chronic intermittent and continuous amphetamine administration on acoustic startle, Pharmacol. Biochem. Behav., 20 (1984) 367-372. [18] LaHoste, G.J. and Marshall, J.F., Dopamine supersensitivity and D1/D2 synergism are unrelated to changes in striatal receptor density, Synapse, 12 (1992) 14-26. [19] Martin-Iverson, M.T., Stahl, S.M. and Iversen, S.D., Chronic administration of a selective dopamine D2 agonist: factors determining behavioral tolerance and sensitization. Psychopharmacology, 95 (1988) 534-539. [20] Martin-Iverson, M.T. and McManus, D., Stimulant-conditioned locomotion is not affected by blockade of D I a n d / o r D e dopamine receptors during conditioning, Brain Res., 521 (1990) 175-184. [21] Martin-Iverson, M.T., Stahl, S.M. and Iversen, S.D., Factors determining the behavioral consequences of continuous treatment with 4-propyl-9-hydroxynaphthoxazine, a selective dopamine D2 agonist. In F.C. Rose lEd.), Parkinson's Disease: Clinical and Experimental Advances, J. Libbey, London, 1987, pp. 169-177. [22] Meller, E., Bohmaker, K., Goldstein, M. and Friedhoff, A.J., Inactivation of D1 and d2 dopamine receptors by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinolinein vivo: selective protection by neuroleptics, J. Pharmacol. Exp. Ther., 233 (1985) 656662. [23] Pani, L., Carboni, S., Kusmin, A., Gessa, G.L. and Rossetti, Z.L., Nimodipine inhibits cocaine-induced dopamine release and motor stimulation, Eur. J. PharmacoL, 176 (1990) 245-246.
[24] Pani, L., Kuzmin, A., Martellotta, M.C., Gessa, (i.h. and Fratta. W., The calcium antagonist PN 200-1111 inhibits the reinforcing properties of cocaine, Brain Res. Bull., 26 11991) 445-447. [25] Peris, J., Boyson, S.J., Cass, W.A., Curella, P., Dwoskim LP., Larson, G. Lin, L., Yasuda, R.P. and Zahniser. N.R., Persis tence of neurochemical changes in dopaminc systems afte~ repeated cocaine administration, J. Pharmacol. Exp. Ther., 253 (1990) 38-44. [26] Pettit, lt.O., Pan, H.-T., Parson, Ell. and Justice, ,1.B. Jr.. Extracellular concentratkms of cocaine and dopaminc are enhanced during chronic cocaine administration. J. Neuroehem.. 55 (1990) 798-804. [27] Post, R.M., Intermittent versus continuous stimulation: effect of time interval on the development of sensitization or tolerance, Life Sci., 26 (1980) 1275-1282. [28] Robinson, T.E. and Becket, J.B., Behavioral sensitization is accompanied by an enhancement in amphetamine-stimulated dopamine release from striatal tissue in vitro, Eur. J. Pharmacol., 85 (1982) 253-254. [29] Robinson, T.E. and Becker, 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. [30] Robinson, T.E., Becker, J.B. and Presty, S.K., Long-term facilitation of amphetamine-induced rotational behavior and striatal dopamine release produced by a single exposure to amphetamine: sex differences, Brain Res., 253 (1982) 231-241. [31] Robinson, T.E., Becker, J.B., Young, E.A., Akil, H. and Castaneda, E., The effects of footshock stress on regional brain dopamine metabolism and pituitary/3-endorphin release in rats previously sensitized to amphetamine, Neuropharmacology. 26 (1987) 679-691. [32] Robinson, T.E., Jurson, P.A., Bennett, J.A. and Bentgen, K.M., Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)amphetamine: a microdialysis study in freely moving rats, Brain Res., 462 (1988) 211-222. [33] Reimer, A.R. and Martin-lverson, M.T., Nimodipine and haloperidol attenuate behavioral sensitization to cocaine but only nimodipine blocks the establishment of conditioned locomotion induced by cocaine, Psychopharmacology, in press. [34] Sailer, C.F., Kreamer, L.D., Adamovage, L.A. and Salama, A.I., Dopamine receptor occupancy in vivo: measurement using Nethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ), Life Sci., 45 (1989) 917-929. [35] Stewart, J. and Vezina, P., Microinjections of SCH 23390 into the ventral tegmental area and substantia nigra pars reticulata attenuate the development of sensitization to the locomotor activating effects of systemic amphetamine, Brain Res., 495 (1989) 401-406. [36] Segal, D.S. and Schuckit, M.A., Animal models of stimulant induced psychosis. In I. Creese lEd.), Stimulants: Neurochemieal, Behavioral, and Clincal Perspectives, Raven. New York, 1983, pp. 131-168.