Brain Research, 573 (1992) 331-335 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
331
BRES 25065
Cocaine-induced behavioral sensitization and D 1 dopamine receptor function in rat nucleus accumbens and striatum R. Dayne Mayfield, Gaynor Larson and Nancy R. Zahniser Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262 (USA)
(Accepted 19 November 1991) Key words: Adenylyl (adenylate) cyclase; Cocaine; Dopamine D 1 receptor; Nucleus accumbens; Striatum; Behavioral sensitization;
Quantitative autoradiography
Based on electrophysiological data showing that repeated cocaine administration produces persistent enhancement of D 1 dopamine (DA) receptor-mediated responses in nucleus accumbens (NAc), we investigated whether changes in neurochemical properties of these receptors resulted when rats were injected with cocaine (15 mg/kg) for 6 days followed by a 7-day abstinence period. D 1 DA receptor density and affinities for either [aH]SCH 23390 or DA were similar between NAc and striatum and between saline and cocaine treatment groups. DAstimulated adenylyl cyclase activity was 1.5-fold higher in striatum than in NAc; however, repeated cocaine treatment produced no persistent changes in enzyme activity in either brain area. Repeated administration of cocaine, as well as other psychomotor stimulants, results in an increased behavioral response to subsequent drug challenge i°'ls'19. An intriguing aspect of behavioral sensitization is that the phenomenon can be detected for weeks to months after repeated drug administration has been discontinued. Behaviors mediated by both the nigrostriatal and mesolimbic dopamine (DA) systems show sensitization. However, current research has focused on the mesolimbic D A system because of its role in mediating the reinforcing effects of cocaine 12"24. Repeated cocaine treatment produces long-term enhancement of D 1, but not 02, D A receptor responsiveness in nucleus accumbens (NAc), suggesting that D i D A receptors in the terminal region of the mesolimbic pathway may be involved in mediating the persistent effects of repeated cocaine administration 7, One mechanism for this increased responsiveness might be enhanced D1 D A receptor binding and/or signal transduction. Long-lasting changes in D~ D A receptors, as measured by antagonist binding, have not been observed in NAc after repeated cocaine administration; however, in striatum there are conflicting reports ~1'~7. Potential changes in the affinity of D~ D A receptors for agonists and the coupling of D 1 D A receptors to second messenger systems have not been investigated. The goal of the present investigation was to determine if persistent changes in the affinity of D 1 receptors for D A and/or D 1 stimulated adenylyl cyclase (AC) activity occurred in response to repeated cocaine treatment.
Male Sprague-Dawley rats (Sasco, Omaha, NE; 180300 g) were housed 4-6 per cage in a colony room maintained at 25°C on a 12-h light/dark cycle with ad libitum access to rat chow and water. Saline (1 ml/kg, i.p.) or cocaine (15 mg/kg, i.p.) was administered once daily at approximately 11.00 h for 6 consecutive days. Locomotor and stereotypic activities were rated according to the method of Barr et al. 2 with minor modifications. The animals were rated on the first and last treatment day by two experienced observers who were blind to the drug treatments. Rats were taken to a testing room and placed into cylindrical wire mesh chambers (diameter = 30 cm, height = 38 cm) and allowed a 1-h habituation period. Subsequently, each animal was rated for 2 min at 10-min intervals beginning 10 min prior to injection and continuing 60 min post-injection. Stereotypic activity (head bobbing) was defined as the occurrence of repetitive up-and-down or side-to-side movements of the head (not including movements during breathing or grooming) and was scored as the number of 10-s intervals in each 2-min rating period in which heading bobbing occurred (maximum score possible = 12). Locomotor activity (quadrant crossing) was defined as the number of times the head and forepaws of the animal crossed a quadrant boundary during each 2-rain rating period. Data were analyzed by repeated measures analysis of variance (ANOVA). Subsequent comparisons were made with univariate F-tests using the error term from the highest order interaction of the overall A N O V A
Correspondence: R.D. Mayfield, Department of Pharmacology, University of Colorado Health Sciences Center, C-236, 4200 East 9th Avenue, Denver, CO 80262, U.S.A. Fax: (1) (303) 270-7097.
332 as the error for each comparison. A f t e r a 7-day abstinence from drug treatment, the rats were killed by decapitation and the brains removed. In initial experiments both quantitative a u t o r a d i o g r a p h y ( Q A R ) of D 1 D A receptors and A C activity were measured in N A c and striatum from saline and cocainetreated rats. In a second set of experiments only A C activity was d e t e r m i n e d in these brain regions following
A.
behavioral testing. Q A R analysis was p e r f o r m e d on coronal brain hemisections (10/~m) cut between 2.2 and 0.70 m m from b r e g m a 16. Assays and analyses were carried out as described previously 17 with the following changes. Ketanserin (100 nM; gift from Janssen Research Foundation, Beerse, Belgium) was included in all assays to inhibit binding of [3H]SCH 23390 to 5-HT 2 receptors 23. Saturation curves were generated with 6 concentrations of [3H]SCH 23390 (0.1-10 nM). A d j a c e n t sections were used to determine total and non-specific binding. S C H 39166 (1 ~M; gift from Schering-Plough Research, Bloomfield, NJ) was used to define non-specific binding. Competition curves were generated with 8 concentrations of D A (100 n M - 1 m M ) in the presence of 1.3 nM [3H]SCH 23390. Preliminary experiments using 10 concentrations of D A , did not indicate high and low affinity states of the receptor. G r a p h P a d ( G r a p h P a d Software, San Diego, C A ) was used to d e t e r m i n e kinetic p a r a m e ter estimates. A C activity was m e a s u r e d in the N A c and striatum according to the m e t h o d of Soloman et al. 2~. N A c was dissected from coronal sections according to H o r n 8, and dorsal striatum was dissected from the same section of tissue. Crude m e m b r a n e s were isolated by homogenization in 2 m M H E P E S buffer (pH 7.5) containing 2 m M E G T A , centrifugation at 27,000× g for 20 min and resuspension in 2.5 ml buffer p e r 100 mg wet weight tissue. Assays contained 20-30/~g protein from N A c and 3 5 - 4 5 / t g from striatum. Protein content was d e t e r m i n e d
~
~ I. g.~ ~~ ~'~'4. ~'~
Head Bobbing
8 I n Saline 7' O Cocaine 6' 5. 4. 3. 2. 1. 0. Day 1
B.
Day 6
Quadrant Crossing
30 -~ ~ 25 "- 20 .=_ E. 15 10 ~
5 0 Day 1
Day 6
Fig. 1. Stereotypic behavior (A) and locomotor activity (B) produced by the first and last injection of saline or cocaine (15 mg/kg, i.p.) in rats treated once-daily for 6 days. Head bobbing was scored as the number of 10-s intervals in each 2-min rating period in which head bobbing occurred (maximum score possible = 12). Quadrant crossing scores represent the number of times the head and forepaws of the animal crossed a quadrant boundary during each 2-min rating period. Repeated measures ANOVA indicated that the time course of the drug response was not altered by repeated cocaine treatment; therefore, the data were collapsed across the time of peak drug response (10-30 min post-injection) for presentation. Mean data + S.E.M. are shown for n = 16 animals/group. *P < 0.05 cocaine day 1 compared with cocaine day 6.
TABLE I No differences in D 1 DA receptor binding characteristics in nucleus accumbens (NAc) and striatum of rats 7 days after receiving 6 oncedaily injections of saline or cocaine (15 mg/kg, i.p.)
KA (1/Kd; Kd = affinity) and Bmax values were determined from [3H]SCH 23390 saturation curves and Ki values were determined from DA competition curves. [3H]SCH 23390
Dopamine
KA values (L/nmol)
Bmax (fmol/mg protein)
K i values (itM)
NA c
S triatum
NA c
S triaturn
NA c
Saline (n = 8)
3.4 + 0.2
3.2 + 0.1
320 + 7
328 + 10
9.7 + 0.9
11.0 + 0.7
Cocaine (n = 8)
3.3 + 0.2
3.2 + 0.2
296 + 10 .
322 + 8
10.7 + 0.7
11.i + 0.6
Striatum
333 by the method of Bradford 4. Assay conditions according to Zahniser et al. 25 were used with the following changes. GTP (0.1 mM) was included, and 1 m M isobutylmethylxanthine (dissolved in 2.5% D M S O ) was substituted for 1 m M theophylline. Basal and DA-stimulated (1 /zM-1 mM) activities were measured. Estimates of kinetic parameters were determined from concentrationresponse curves with the non-linear curve fitting routine A L L F I T 6. Behavioral responses to repeated cocaine treatment are illustrated in Fig. 1. Cocaine increased both stereotyped head bobbing and locomotor activity over saline controls. Repeated cocaine treatment resulted in a sensitization of stereotypic head bobbing as indicated by the increased rating of this behavior on day 6 compared to day 1 (Fig. 1A; Ft,30 29.9, P < 0.05). However, locomotor activity was not further enhanced by repeated cocaine treatment (Fig. 1B; Ft,30 0.33, n.s.). Although an augmented locomotor response to repeated cocaine and other psychomotor stimulants is expected, our experimental conditions may have masked observance of this type of sensitization. First, locomotor activity as defined by quadrant crossing is subject to an artificial ceiling; thus, the locomotor response to 15 mg/kg may have produced near maximal activation according to our rating protocol. Secondly, the increase in focused stereotypies that was produced by our dosing schedule tended to interfere with locomotor activity, placing a further limit on our ability to detect increases in this behavioral response. Analysis of [3H]SCH 23390 saturation curves in N A c and striatum detected no differences in D 1 D A receptor density between control and cocaine-treated rats (Table I). These findings agree with our previous observations, using only a single saturating concentration of radioligand, in which Sprague-Dawley rats were treated oncedaily for 8 days with 10 mg/kg cocaine followed by one day of withdrawal 17. Furthermore, the results presented here are in agreement with those of Kleven et al. 11 who observed no change in the density of D1 D A receptors in N A c of Sprague-Dawley rats treated with 10 mg/kg cocaine once daily for 15 days and then withdrawn for 2 weeks. However, in striatum, a dramatic decrease in D1 D A receptor density was reported with this treatment protocol 11. In contrast, Lim et al. ~3 found that the density of D~ D A receptors was increased in the striatum of W i s t a r - K y o t o rats 16-18 h after twice-daily cocaine treatment (20 mg/kg, s.c.) for 3 or 7 days. The variable effects on D x D A receptor regulation in striatum could be attributed to differences in rat strains, cocaine treatment/withdrawal protocols or radioligand binding conditions. Despite inconsistent effects on D~ D A receptor density in striatum, there is general agreement that no persistent changes occur in the density of D 1 D A recep-
tors in N A c in response to repeated cocaine administration. Measurements of D t D A receptor affinity using antagonist radioligands have consistently shown that repeated cocaine administration produces no change in either N A c or striatum 11'13 (Table I). However, potential changes in agonist affinity had not been investigated prior to this study. Analysis of competition curves indicated that the affinity of D1 D A receptors for D A was approximately 10/zM in both the N A c and striatum (Table I). These results suggest that repeated cocaine administration causes no persistent alterations in D 1 D A
A.
NUCLEUS ACCUMBENS 150 125
----o-
Saline Cocaine
100 m
+i
75 50250"
f 10 °
101
10 2
10 3
10 4
[Dopamine] (t,tM)
B.
STRIATUM 150' ~ "< L.
125 100 75 5o
~o
Cocaine
25 ~J . 10°
.
. 101
.
.
10 2
10 3
104
[Dopamine] (ktM) Fig. 2. DA-stimulated AC activity in NAc (A) and striatum (B) following a 7-day abstinence period from repeated saline or cocaine administration. The drug administration protocol was described in Fig. 1. Basal activities (pmol/mg.min) in NAc were 70.7 ___6.5 (saline) and 64.4 + 6.4 (cocaine) and in striatum were 71.7 + 7.5 (saline) and 66.5 + 8.0 (cocaine). Parameter estimates were obtained using ALLFIT6 with the minimum response constrained to zero. ECs0 estimates (~M) in NAc were 10.0 (saline) and 12.2 (cocaine) and in striatum were 14.3 (saline) and 14.1 (cocaine). VmaX estimates (% over basal) in NAc were 85 (saline) and 93 (cocaine) and in striatum were 140 (saline) and 128 (cocaine). Data points represent the mean + S.E.M. for n = 16 animals/group.
334 receptor affinity for either agonists or antagonists in N A c or striatum. In addition to behavioral and Q A R studies, a functional measure of D 1 D A r e c e p t o r - m e d i a t e d signal transduction was investigated. Basal A C activity was similar in both N A c and striatum, and D A p r o d u c e d a concentration-related increase in this activity (Fig. 2). In both brain regions co-incubation with selective D 1 D A receptor antagonists, 1/~M S C H 23390 or 39166, completely abolished the activity p r o d u c e d by 0.1 m M D A and attenuated the activity p r o d u c e d by 1 m M D A by 60% (data not shown). A l t h o u g h ECs0 estimates for D A stimulation of A C activity did not differ in N A c and striatum, the maximal response to D A was 1.5-fold higher in striatum (Fig. 2). This finding is interesting because D 1 D A receptor density is similar in these two brain regions 3'5 (Table I) and suggests that a population of D 1 D A receptors exists in N A c which is not coupled to the A C system. D o s e - r e s p o n s e analysis indicated that neither the Vmax nor ECs0 estimates for D A - s t i m u l a t e d A C activity were altered in N A c and striatum 7 days after r e p e a t e d cocaine or saline t r e a t m e n t was terminated (Fig. 2). Only one study had previously investigated the effect of cocaine on D A - s t i m u l a t e d A C activity in N A c and striatum, and activity was measured a short time after a single cocaine injection 15. While there are reports of long-term changes in striatal D 1 D A receptor-stimulated A C activity in response to r e p e a t e d exposure to amphetamine, these studies showed that an acute challenge dose of a m p h e t a m i n e a p p e a r e d to be required after withdrawal from r e p e a t e d a m p h e t a m i n e in o r d e r to observe changes in A C activity induced by r e p e a t e d drug treat-
This work was supported by USPHS Grant DA 04216. Dr. Mayfield was supported by training Grant AA 07464. The authors wish to thank Drs. S.J. Boyson and J.E. Dildy-Mayfietd for their careful review of this manuscript.
1 Barnett, J.W., Segal, D.S. and Kuczenski, R., Repeated amphetamine pretreatment alters the responsiveness of striatal dopamine-stimulated adenylate cyclase to amphetamine-induced desensitization, J. Pharmacol. Exp. Ther., 242 (1987) 40-47. 2 Barr, G.A., Sharpless, N.S., Cooper, S., Schiff, S.R., Paredes, W. and Bridger, W.H., Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy, Life Sci., 33 (1983) 1341-1351. 3 Boyson, S.J., McGonigle, P. and Molinoff, P.B., Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain, J. Neurosci., 6 (1986) 3177-3188. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 5 Dawson, T.M., Gehlert, D.R., McCabe, R.T., Barnett, A. and Wamsley, J.K., D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis, J. Neurosci., 6 (1986) 2352-2365. 6 De Leaa, A., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 7 Henry, D.J. and White, F.J., Repeated cocaine administration causes persistent enhancement of D1 dopamine receptor sensitivity within the rat nucleus accumbens, J. Pharmacol. Exp.
Ther., 258 (1991) 882-890. 8 Horn, A.S., Cuello, A.C. and Miller, R.J., Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase, J. Neurochem., 22 (1974) 265-270. 9 Johansen, P.A., Hu, X.-T. and White, F.J., Relationship between D1 dopamine receptors, adenylate cyclase and the electrophysiological responses of rat nucleus accumbens neurons, J. Neural Trans., 86 (1991) 97-113. 10 Kalivas, P.W., Dully, P., DuMars, L.A. and Skinner, C., Behavioral and neurochemical effects of acute and daily cocaine adminstration in rats, J. Pharmacol. Exp. Ther., 245 (1988) 485-492. 11 Kleven, M.S., Perry, B.D., Woolverton, W.L. and Seiden, L.S., Effects of repeated injections of cocaine on DI and D2 dopamine receptors in rat brain, Brain Research, 532 (1990) 265-270. 12 Kuhar, M.J., Ritz, M.C. and Boja, J.W., The dopamine hypothesis of the reinforcing properties of cocaine, Trends Neurosci., 14 (1991) 299-302. 13 Lim, D.K., Yu, Z.J., Hoskins, B., Rockhold, R.W. and Ho, I.K., Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: II. Dopamine receptors, Neurochem. Res., 15 (1990) 621-627.
ment 1'2°. O u r protocol did not e m p l o y a cocaine challenge which m a y explain the lack of functional changes in D 1 D A receptor-stimulated A C activity after r e p e a t e d cocaine. Nonetheless, the electrophysiological studies showing a persistent increase in D1 D A r e c e p t o r sensitivity in N A c following r e p e a t e d cocaine administration also used no cocaine challenge 7. O u r results suggest that it is unlikely that changes in D1 D A r e c e p t o r properties and increases in D 1 D A receptor-stimulated A C activity explain the D~ r e c e p t o r sensitization in N A c observed in electrophysiological studies. The enhanced sensitivity may still be m e d i a t e d by a D~ D A r e c e p t o r that is coupled to A C but may reflect changes b e y o n d the r e c e p t o r itself. Recently, it has been d e m o n s t r a t e d that r e p e a t e d cocaine administration increases the activities of forskolin-stimulated A C and cyclic A M P - d e p e n d e n t protein kinase selectively in N A c 22. These results, together with the results from the present experiments, indicate persistent effects of cocaine on the A C system may be m e d i a t e d d o w n s t r e a m from the D 1 D A receptor at the level of the catalytic subunit of A C and the protein kinase. Alternatively, the r e c e p t o r - m e d i a t e d changes measured electrophysiologically may be m e d i a t e d by a D 1 D A r e c e p t o r subtype that is not coupled t o A C 9'14 or a more general change in neuronal excitability.
335 14 Mahan, L.C., Burch, R.M., Monsma, Jr., F.J., and Sibley, D.R., Expression of striatal DI dopamine receptors coupled to inositol phosphate production and Ca2+ mobilization in Xenopus oocytes, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 21962200. 15 Memo, M., Pradhan, S. and Hanbauer, I., Cocaine-induced supersensitivity of striatal dopamine receptors: role of endogenous calmodulin, Neuropharmacology, 20 (1981) 1145-1150. 16 Paxinos, G. and Watson, C., The Bat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. 17 Peris, L, Boyson, S.J., Cass, W.A., CureUa, P., Dwoskin, L.P., Larson, G., Lin, L.-H., Yasuda, R.P. and Zahniser, N.R., Persistence of neurochemical changes in dopamine systems after repeated cocaine administration, Y. Pharmacol. Exp. Ther., 253 (1990) 38-44. 18 Post, R.M. and Contel, N.R., Human and animal studies of cocaine: implications for development of behavioral pathology. In I. Creese (Ed.), Stimulants: Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 169-203. 19 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 psy-
chosis, Brain Res. Rev., 11 (1986) 157-198. 20 Roseboom, P.H., Hewlett, G.H.K. and Gnegy, M.E., Repeated amphetamine administration alters the interaction between D-Istimulated adenylyl cyclase activity and calmodulin in rat striatum, J. Pharmacol. Exp. Ther., 255 (1990) 197-203. 21 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylyl cyclase assay, Anal. Biochem., 58 (1974) 541-548. 22 Terwilliger, R.Z., Beitner-Johnson, D., Sevarino, K.A., Crain, S.M. and Nestler, E.J., A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function, Brain Research, 548 (1991) 100-110. 23 Wamsley, J.K., Hunt, M.E., McQuade, R.D. and Alburges, M.E., [3H]SCH39166, a D1 dopamine receptor antagonist: binding characteristics and localization, Exp. Neurol., 111 (1991) 145-151. 24 Wise, R.A. and Rompr6, P.-E, Brain dopamine and reward, Annu. Rev. PsychoL, 40 (1989) 191-225. 25 Zahniser, N.R., Heidenreich, K.A. and Molinoff, P.B., Binding of 3H-amino-6,7-dihydroxy-l,2,3,4-tetrahydronapthalene to rat striatal membranes: effects of purine nucleotides and ultraviolet irradiation, Mol. Pharmacol., 19 (1981) 371-378.
331
BRES 25065
Cocaine-induced behavioral sensitization and D 1 dopamine receptor function in rat nucleus accumbens and striatum R. Dayne Mayfield, Gaynor Larson and Nancy R. Zahniser Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262 (USA)
(Accepted 19 November 1991) Key words: Adenylyl (adenylate) cyclase; Cocaine; Dopamine D 1 receptor; Nucleus accumbens; Striatum; Behavioral sensitization;
Quantitative autoradiography
Based on electrophysiological data showing that repeated cocaine administration produces persistent enhancement of D 1 dopamine (DA) receptor-mediated responses in nucleus accumbens (NAc), we investigated whether changes in neurochemical properties of these receptors resulted when rats were injected with cocaine (15 mg/kg) for 6 days followed by a 7-day abstinence period. D 1 DA receptor density and affinities for either [aH]SCH 23390 or DA were similar between NAc and striatum and between saline and cocaine treatment groups. DAstimulated adenylyl cyclase activity was 1.5-fold higher in striatum than in NAc; however, repeated cocaine treatment produced no persistent changes in enzyme activity in either brain area. Repeated administration of cocaine, as well as other psychomotor stimulants, results in an increased behavioral response to subsequent drug challenge i°'ls'19. An intriguing aspect of behavioral sensitization is that the phenomenon can be detected for weeks to months after repeated drug administration has been discontinued. Behaviors mediated by both the nigrostriatal and mesolimbic dopamine (DA) systems show sensitization. However, current research has focused on the mesolimbic D A system because of its role in mediating the reinforcing effects of cocaine 12"24. Repeated cocaine treatment produces long-term enhancement of D 1, but not 02, D A receptor responsiveness in nucleus accumbens (NAc), suggesting that D i D A receptors in the terminal region of the mesolimbic pathway may be involved in mediating the persistent effects of repeated cocaine administration 7, One mechanism for this increased responsiveness might be enhanced D1 D A receptor binding and/or signal transduction. Long-lasting changes in D~ D A receptors, as measured by antagonist binding, have not been observed in NAc after repeated cocaine administration; however, in striatum there are conflicting reports ~1'~7. Potential changes in the affinity of D~ D A receptors for agonists and the coupling of D 1 D A receptors to second messenger systems have not been investigated. The goal of the present investigation was to determine if persistent changes in the affinity of D 1 receptors for D A and/or D 1 stimulated adenylyl cyclase (AC) activity occurred in response to repeated cocaine treatment.
Male Sprague-Dawley rats (Sasco, Omaha, NE; 180300 g) were housed 4-6 per cage in a colony room maintained at 25°C on a 12-h light/dark cycle with ad libitum access to rat chow and water. Saline (1 ml/kg, i.p.) or cocaine (15 mg/kg, i.p.) was administered once daily at approximately 11.00 h for 6 consecutive days. Locomotor and stereotypic activities were rated according to the method of Barr et al. 2 with minor modifications. The animals were rated on the first and last treatment day by two experienced observers who were blind to the drug treatments. Rats were taken to a testing room and placed into cylindrical wire mesh chambers (diameter = 30 cm, height = 38 cm) and allowed a 1-h habituation period. Subsequently, each animal was rated for 2 min at 10-min intervals beginning 10 min prior to injection and continuing 60 min post-injection. Stereotypic activity (head bobbing) was defined as the occurrence of repetitive up-and-down or side-to-side movements of the head (not including movements during breathing or grooming) and was scored as the number of 10-s intervals in each 2-min rating period in which heading bobbing occurred (maximum score possible = 12). Locomotor activity (quadrant crossing) was defined as the number of times the head and forepaws of the animal crossed a quadrant boundary during each 2-rain rating period. Data were analyzed by repeated measures analysis of variance (ANOVA). Subsequent comparisons were made with univariate F-tests using the error term from the highest order interaction of the overall A N O V A
Correspondence: R.D. Mayfield, Department of Pharmacology, University of Colorado Health Sciences Center, C-236, 4200 East 9th Avenue, Denver, CO 80262, U.S.A. Fax: (1) (303) 270-7097.
332 as the error for each comparison. A f t e r a 7-day abstinence from drug treatment, the rats were killed by decapitation and the brains removed. In initial experiments both quantitative a u t o r a d i o g r a p h y ( Q A R ) of D 1 D A receptors and A C activity were measured in N A c and striatum from saline and cocainetreated rats. In a second set of experiments only A C activity was d e t e r m i n e d in these brain regions following
A.
behavioral testing. Q A R analysis was p e r f o r m e d on coronal brain hemisections (10/~m) cut between 2.2 and 0.70 m m from b r e g m a 16. Assays and analyses were carried out as described previously 17 with the following changes. Ketanserin (100 nM; gift from Janssen Research Foundation, Beerse, Belgium) was included in all assays to inhibit binding of [3H]SCH 23390 to 5-HT 2 receptors 23. Saturation curves were generated with 6 concentrations of [3H]SCH 23390 (0.1-10 nM). A d j a c e n t sections were used to determine total and non-specific binding. S C H 39166 (1 ~M; gift from Schering-Plough Research, Bloomfield, NJ) was used to define non-specific binding. Competition curves were generated with 8 concentrations of D A (100 n M - 1 m M ) in the presence of 1.3 nM [3H]SCH 23390. Preliminary experiments using 10 concentrations of D A , did not indicate high and low affinity states of the receptor. G r a p h P a d ( G r a p h P a d Software, San Diego, C A ) was used to d e t e r m i n e kinetic p a r a m e ter estimates. A C activity was m e a s u r e d in the N A c and striatum according to the m e t h o d of Soloman et al. 2~. N A c was dissected from coronal sections according to H o r n 8, and dorsal striatum was dissected from the same section of tissue. Crude m e m b r a n e s were isolated by homogenization in 2 m M H E P E S buffer (pH 7.5) containing 2 m M E G T A , centrifugation at 27,000× g for 20 min and resuspension in 2.5 ml buffer p e r 100 mg wet weight tissue. Assays contained 20-30/~g protein from N A c and 3 5 - 4 5 / t g from striatum. Protein content was d e t e r m i n e d
~
~ I. g.~ ~~ ~'~'4. ~'~
Head Bobbing
8 I n Saline 7' O Cocaine 6' 5. 4. 3. 2. 1. 0. Day 1
B.
Day 6
Quadrant Crossing
30 -~ ~ 25 "- 20 .=_ E. 15 10 ~
5 0 Day 1
Day 6
Fig. 1. Stereotypic behavior (A) and locomotor activity (B) produced by the first and last injection of saline or cocaine (15 mg/kg, i.p.) in rats treated once-daily for 6 days. Head bobbing was scored as the number of 10-s intervals in each 2-min rating period in which head bobbing occurred (maximum score possible = 12). Quadrant crossing scores represent the number of times the head and forepaws of the animal crossed a quadrant boundary during each 2-min rating period. Repeated measures ANOVA indicated that the time course of the drug response was not altered by repeated cocaine treatment; therefore, the data were collapsed across the time of peak drug response (10-30 min post-injection) for presentation. Mean data + S.E.M. are shown for n = 16 animals/group. *P < 0.05 cocaine day 1 compared with cocaine day 6.
TABLE I No differences in D 1 DA receptor binding characteristics in nucleus accumbens (NAc) and striatum of rats 7 days after receiving 6 oncedaily injections of saline or cocaine (15 mg/kg, i.p.)
KA (1/Kd; Kd = affinity) and Bmax values were determined from [3H]SCH 23390 saturation curves and Ki values were determined from DA competition curves. [3H]SCH 23390
Dopamine
KA values (L/nmol)
Bmax (fmol/mg protein)
K i values (itM)
NA c
S triatum
NA c
S triaturn
NA c
Saline (n = 8)
3.4 + 0.2
3.2 + 0.1
320 + 7
328 + 10
9.7 + 0.9
11.0 + 0.7
Cocaine (n = 8)
3.3 + 0.2
3.2 + 0.2
296 + 10 .
322 + 8
10.7 + 0.7
11.i + 0.6
Striatum
333 by the method of Bradford 4. Assay conditions according to Zahniser et al. 25 were used with the following changes. GTP (0.1 mM) was included, and 1 m M isobutylmethylxanthine (dissolved in 2.5% D M S O ) was substituted for 1 m M theophylline. Basal and DA-stimulated (1 /zM-1 mM) activities were measured. Estimates of kinetic parameters were determined from concentrationresponse curves with the non-linear curve fitting routine A L L F I T 6. Behavioral responses to repeated cocaine treatment are illustrated in Fig. 1. Cocaine increased both stereotyped head bobbing and locomotor activity over saline controls. Repeated cocaine treatment resulted in a sensitization of stereotypic head bobbing as indicated by the increased rating of this behavior on day 6 compared to day 1 (Fig. 1A; Ft,30 29.9, P < 0.05). However, locomotor activity was not further enhanced by repeated cocaine treatment (Fig. 1B; Ft,30 0.33, n.s.). Although an augmented locomotor response to repeated cocaine and other psychomotor stimulants is expected, our experimental conditions may have masked observance of this type of sensitization. First, locomotor activity as defined by quadrant crossing is subject to an artificial ceiling; thus, the locomotor response to 15 mg/kg may have produced near maximal activation according to our rating protocol. Secondly, the increase in focused stereotypies that was produced by our dosing schedule tended to interfere with locomotor activity, placing a further limit on our ability to detect increases in this behavioral response. Analysis of [3H]SCH 23390 saturation curves in N A c and striatum detected no differences in D 1 D A receptor density between control and cocaine-treated rats (Table I). These findings agree with our previous observations, using only a single saturating concentration of radioligand, in which Sprague-Dawley rats were treated oncedaily for 8 days with 10 mg/kg cocaine followed by one day of withdrawal 17. Furthermore, the results presented here are in agreement with those of Kleven et al. 11 who observed no change in the density of D1 D A receptors in N A c of Sprague-Dawley rats treated with 10 mg/kg cocaine once daily for 15 days and then withdrawn for 2 weeks. However, in striatum, a dramatic decrease in D1 D A receptor density was reported with this treatment protocol 11. In contrast, Lim et al. ~3 found that the density of D~ D A receptors was increased in the striatum of W i s t a r - K y o t o rats 16-18 h after twice-daily cocaine treatment (20 mg/kg, s.c.) for 3 or 7 days. The variable effects on D x D A receptor regulation in striatum could be attributed to differences in rat strains, cocaine treatment/withdrawal protocols or radioligand binding conditions. Despite inconsistent effects on D~ D A receptor density in striatum, there is general agreement that no persistent changes occur in the density of D 1 D A recep-
tors in N A c in response to repeated cocaine administration. Measurements of D t D A receptor affinity using antagonist radioligands have consistently shown that repeated cocaine administration produces no change in either N A c or striatum 11'13 (Table I). However, potential changes in agonist affinity had not been investigated prior to this study. Analysis of competition curves indicated that the affinity of D1 D A receptors for D A was approximately 10/zM in both the N A c and striatum (Table I). These results suggest that repeated cocaine administration causes no persistent alterations in D 1 D A
A.
NUCLEUS ACCUMBENS 150 125
----o-
Saline Cocaine
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f 10 °
101
10 2
10 3
10 4
[Dopamine] (t,tM)
B.
STRIATUM 150' ~ "< L.
125 100 75 5o
~o
Cocaine
25 ~J . 10°
.
. 101
.
.
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104
[Dopamine] (ktM) Fig. 2. DA-stimulated AC activity in NAc (A) and striatum (B) following a 7-day abstinence period from repeated saline or cocaine administration. The drug administration protocol was described in Fig. 1. Basal activities (pmol/mg.min) in NAc were 70.7 ___6.5 (saline) and 64.4 + 6.4 (cocaine) and in striatum were 71.7 + 7.5 (saline) and 66.5 + 8.0 (cocaine). Parameter estimates were obtained using ALLFIT6 with the minimum response constrained to zero. ECs0 estimates (~M) in NAc were 10.0 (saline) and 12.2 (cocaine) and in striatum were 14.3 (saline) and 14.1 (cocaine). VmaX estimates (% over basal) in NAc were 85 (saline) and 93 (cocaine) and in striatum were 140 (saline) and 128 (cocaine). Data points represent the mean + S.E.M. for n = 16 animals/group.
334 receptor affinity for either agonists or antagonists in N A c or striatum. In addition to behavioral and Q A R studies, a functional measure of D 1 D A r e c e p t o r - m e d i a t e d signal transduction was investigated. Basal A C activity was similar in both N A c and striatum, and D A p r o d u c e d a concentration-related increase in this activity (Fig. 2). In both brain regions co-incubation with selective D 1 D A receptor antagonists, 1/~M S C H 23390 or 39166, completely abolished the activity p r o d u c e d by 0.1 m M D A and attenuated the activity p r o d u c e d by 1 m M D A by 60% (data not shown). A l t h o u g h ECs0 estimates for D A stimulation of A C activity did not differ in N A c and striatum, the maximal response to D A was 1.5-fold higher in striatum (Fig. 2). This finding is interesting because D 1 D A receptor density is similar in these two brain regions 3'5 (Table I) and suggests that a population of D 1 D A receptors exists in N A c which is not coupled to the A C system. D o s e - r e s p o n s e analysis indicated that neither the Vmax nor ECs0 estimates for D A - s t i m u l a t e d A C activity were altered in N A c and striatum 7 days after r e p e a t e d cocaine or saline t r e a t m e n t was terminated (Fig. 2). Only one study had previously investigated the effect of cocaine on D A - s t i m u l a t e d A C activity in N A c and striatum, and activity was measured a short time after a single cocaine injection 15. While there are reports of long-term changes in striatal D 1 D A receptor-stimulated A C activity in response to r e p e a t e d exposure to amphetamine, these studies showed that an acute challenge dose of a m p h e t a m i n e a p p e a r e d to be required after withdrawal from r e p e a t e d a m p h e t a m i n e in o r d e r to observe changes in A C activity induced by r e p e a t e d drug treat-
This work was supported by USPHS Grant DA 04216. Dr. Mayfield was supported by training Grant AA 07464. The authors wish to thank Drs. S.J. Boyson and J.E. Dildy-Mayfietd for their careful review of this manuscript.
1 Barnett, J.W., Segal, D.S. and Kuczenski, R., Repeated amphetamine pretreatment alters the responsiveness of striatal dopamine-stimulated adenylate cyclase to amphetamine-induced desensitization, J. Pharmacol. Exp. Ther., 242 (1987) 40-47. 2 Barr, G.A., Sharpless, N.S., Cooper, S., Schiff, S.R., Paredes, W. and Bridger, W.H., Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy, Life Sci., 33 (1983) 1341-1351. 3 Boyson, S.J., McGonigle, P. and Molinoff, P.B., Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain, J. Neurosci., 6 (1986) 3177-3188. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 5 Dawson, T.M., Gehlert, D.R., McCabe, R.T., Barnett, A. and Wamsley, J.K., D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis, J. Neurosci., 6 (1986) 2352-2365. 6 De Leaa, A., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 7 Henry, D.J. and White, F.J., Repeated cocaine administration causes persistent enhancement of D1 dopamine receptor sensitivity within the rat nucleus accumbens, J. Pharmacol. Exp.
Ther., 258 (1991) 882-890. 8 Horn, A.S., Cuello, A.C. and Miller, R.J., Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase, J. Neurochem., 22 (1974) 265-270. 9 Johansen, P.A., Hu, X.-T. and White, F.J., Relationship between D1 dopamine receptors, adenylate cyclase and the electrophysiological responses of rat nucleus accumbens neurons, J. Neural Trans., 86 (1991) 97-113. 10 Kalivas, P.W., Dully, P., DuMars, L.A. and Skinner, C., Behavioral and neurochemical effects of acute and daily cocaine adminstration in rats, J. Pharmacol. Exp. Ther., 245 (1988) 485-492. 11 Kleven, M.S., Perry, B.D., Woolverton, W.L. and Seiden, L.S., Effects of repeated injections of cocaine on DI and D2 dopamine receptors in rat brain, Brain Research, 532 (1990) 265-270. 12 Kuhar, M.J., Ritz, M.C. and Boja, J.W., The dopamine hypothesis of the reinforcing properties of cocaine, Trends Neurosci., 14 (1991) 299-302. 13 Lim, D.K., Yu, Z.J., Hoskins, B., Rockhold, R.W. and Ho, I.K., Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats: II. Dopamine receptors, Neurochem. Res., 15 (1990) 621-627.
ment 1'2°. O u r protocol did not e m p l o y a cocaine challenge which m a y explain the lack of functional changes in D 1 D A receptor-stimulated A C activity after r e p e a t e d cocaine. Nonetheless, the electrophysiological studies showing a persistent increase in D1 D A r e c e p t o r sensitivity in N A c following r e p e a t e d cocaine administration also used no cocaine challenge 7. O u r results suggest that it is unlikely that changes in D1 D A r e c e p t o r properties and increases in D 1 D A receptor-stimulated A C activity explain the D~ r e c e p t o r sensitization in N A c observed in electrophysiological studies. The enhanced sensitivity may still be m e d i a t e d by a D~ D A r e c e p t o r that is coupled to A C but may reflect changes b e y o n d the r e c e p t o r itself. Recently, it has been d e m o n s t r a t e d that r e p e a t e d cocaine administration increases the activities of forskolin-stimulated A C and cyclic A M P - d e p e n d e n t protein kinase selectively in N A c 22. These results, together with the results from the present experiments, indicate persistent effects of cocaine on the A C system may be m e d i a t e d d o w n s t r e a m from the D 1 D A receptor at the level of the catalytic subunit of A C and the protein kinase. Alternatively, the r e c e p t o r - m e d i a t e d changes measured electrophysiologically may be m e d i a t e d by a D 1 D A r e c e p t o r subtype that is not coupled t o A C 9'14 or a more general change in neuronal excitability.
335 14 Mahan, L.C., Burch, R.M., Monsma, Jr., F.J., and Sibley, D.R., Expression of striatal DI dopamine receptors coupled to inositol phosphate production and Ca2+ mobilization in Xenopus oocytes, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 21962200. 15 Memo, M., Pradhan, S. and Hanbauer, I., Cocaine-induced supersensitivity of striatal dopamine receptors: role of endogenous calmodulin, Neuropharmacology, 20 (1981) 1145-1150. 16 Paxinos, G. and Watson, C., The Bat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. 17 Peris, L, Boyson, S.J., Cass, W.A., CureUa, P., Dwoskin, L.P., Larson, G., Lin, L.-H., Yasuda, R.P. and Zahniser, N.R., Persistence of neurochemical changes in dopamine systems after repeated cocaine administration, Y. Pharmacol. Exp. Ther., 253 (1990) 38-44. 18 Post, R.M. and Contel, N.R., Human and animal studies of cocaine: implications for development of behavioral pathology. In I. Creese (Ed.), Stimulants: Neurochemical, Behavioral, and Clinical Perspectives, Raven, New York, 1983, pp. 169-203. 19 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 psy-
chosis, Brain Res. Rev., 11 (1986) 157-198. 20 Roseboom, P.H., Hewlett, G.H.K. and Gnegy, M.E., Repeated amphetamine administration alters the interaction between D-Istimulated adenylyl cyclase activity and calmodulin in rat striatum, J. Pharmacol. Exp. Ther., 255 (1990) 197-203. 21 Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylyl cyclase assay, Anal. Biochem., 58 (1974) 541-548. 22 Terwilliger, R.Z., Beitner-Johnson, D., Sevarino, K.A., Crain, S.M. and Nestler, E.J., A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function, Brain Research, 548 (1991) 100-110. 23 Wamsley, J.K., Hunt, M.E., McQuade, R.D. and Alburges, M.E., [3H]SCH39166, a D1 dopamine receptor antagonist: binding characteristics and localization, Exp. Neurol., 111 (1991) 145-151. 24 Wise, R.A. and Rompr6, P.-E, Brain dopamine and reward, Annu. Rev. PsychoL, 40 (1989) 191-225. 25 Zahniser, N.R., Heidenreich, K.A. and Molinoff, P.B., Binding of 3H-amino-6,7-dihydroxy-l,2,3,4-tetrahydronapthalene to rat striatal membranes: effects of purine nucleotides and ultraviolet irradiation, Mol. Pharmacol., 19 (1981) 371-378.
