Chronic Treatment with Dopamine Receptor Antagonists: Behavioral and Pharmacologic Effects on D, and D, Dopamine Receptors

The Journal of Neuroscience, July 1988, 8(7): 2381-2370

Ellen J. Hesqa Andrew B. Norman,b and Ian Creese Center for Molecular and Behavioral Neuroscience, Rutgers, The State University of New Jersey, Newark, New Jersey 07102

Rats were treated for 21 d with the selective D, dopamine ministered drug over the course of the chronic drug treat- receptor antagonist SCH23390, the selective D, dopamine ment. Catalepsy scores of rats receiving spiperone de- receptor antagonist spiperone, the nonselective dopamine creased over the course of treatment, with a significant receptor antagonist cis-flupentixol, or a combination of reduction in catalepsy occurring by treatment day 5. The SCH23390 and spiperone. In addition, a group of rats re- profound catalepsy observed in rats receiving SCH23390 did ceived L-prolyl-L-leucyl-glycinamide (PLG) for 5 d after the not change over the 21 d of treatment. Rats receiving cis- 21 d chronic spiperone treatment. Chronic treatment with flupentixol demonstrated tolerance to its cataleptogenic ef- SCH23390 resulted in a significant increase in D, dopamine fects, with a significant reduction in catalepsy observed by receptor density with no change in the D, dopamine receptor treatment day 7. During the 3 week treatment, the time be- density. Conversely, spiperone treatment resulted in a sig- tween drug injection and a full cataleptic response to cis- nificant increase in D, dopamine receptors and no change flupentixol increased from 20 to 60 min, suggesting a tol- in D, dopamine receptor density. PLG treatment had no ef- erance to the D,, but not D,, dopamine receptor antagonism fect. SCH23390 plus spiperone treatment resulted in a sig- by cis-flupentixol. A group of rats treated with SCH23390 for nificant increase in both D, and D, dopamine receptor den- 21 d received a final acute injection of spiperone on day 22. sities. However, although in vitro cis-flupentixol has an equal These rats were as tolerant to the cataleptogenic effects of affinity for D, and D, dopamine receptors, only the D, do- spiperone as were those rats tested on day 21 of chronic pamine receptor density increased after chronic treatment spiperone treatment. Conversely, rats challenged with an with cis-flupentixol. acute dose of SCH23390 after 21 d spiperone treatment were In vivotreatment with the protein-modifying reagent N-eth- profoundly cataleptic. Thus, D, dopamine receptor up-reg- oxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which ulation is not necessary for the development of behavioral irreversibly inactivates D, and D, dopamine receptors, was tolerance to D, dopamine receptor antagonism, whereas be- used to investigate the paradoxical, selective D, dopamine havioral tolerance to D, dopamine receptor antagonism was receptor up-regulation induced by cis-flupentixol treatment. not observed in these studies in spite of a D, dopamine In vivo treatment with cis-flupentixol before EEDQ admin- receptor up-regulation. istration prevented the D, and D, dopamine receptor reductions induced by EEDQ. However, cis-flupentixol protected, Two distinct dopamine receptor subtypes coexist in the CNS in a dose-dependent manner, a greater percentage of D, (Kebabian and Calne, 1979; Creeseet al., 1983): D, dopamine dopamine receptors than of D, dopamine receptors from receptors mediate the stimulation of adenylate cyclaseactivity EEDQ-induced modification. These data indicate that, in viva, (Hyttel, 1978), while D, dopamine receptorsinhibit the activity cis-flupentixol preferentially interacts with D, dopamine re- of this enzyme (Stoof and Kebabian, 1981; Onali et al., 1984; ceptors and could explain why only D, dopamine receptors Battaglia et al., 1985). Although many neuroleptics block both were up-regulated following chronic treatment with cis-flu- dopamine receptor subtypes, previous researchhas focusedon pentixol. the role of D, dopamine receptorsin the mechanismof action Rats were tested for their cataleptic response to the ad- of neuroleptic drugs. This was due primarily to the high cor- relation between the affinities of neuroleptic drugs at the D, Received May 27, 1987; revised Oct. 26, 1987; accepted Oct. 30, 1987. dopamine receptor and their clinical dosagesas antipsychotic This investigation was supported by NIMH Grant MH 32990, an RSDA MH 00316 (to I.C.), an NIMH predoctoral fellowship MH09535 (to E.J.H.) and the agents (Creeseet al., 1976; Seemanet al., 1976). In addition, Scottish Rite Schizophrenia Research Program, NMJ, USA. The authors would selective D, dopamine receptor antagonistssuch as sulpiride are like to thank Dr. George Koob for discussions and help with statistical analyses. antipsychotic. Investigations of the D, dopamine receptor were We also thank Paula Martin and Christine Strand for manuscript preparation and Andrew Chen, Patricia Newton, Lesley Su, and Diane Swick for technical assis- hampered becauseof the lack of compounds selective for this tance. receptor. Correspondence should be addressed to Dr. Creese, Center for Molecular and Tardive dyskinesia is a major complication of long-term neu- Behavioral Neuroscience, Rutgers, The State University of New Jersey, 195 Uni- versity Avenue, Newark, NJ 07 102. roleptic drug administration usedin treating schizophrenia. This = Present address: Molecular Biology Building, Research Institute of Scripps disorder is characterized by abnormal movements of the facial Clinic, 10666 N. Torrey Pines Road, La Jolla, CA 92037. b Present address: Department of Psychiatry, Laboratory of Behavioral Neu- muscles,tongue, and extremities (for review, see Baldessarini roscience, University of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267. and Tarsy, 1980). To provide an animal model of tardive dyski- Copyright 0 1988 Society for Neuroscience 0270-6474/88/072361-10$02.00/O nesia, rodents have been chronically treated with neuroleptic 2362 Hess et al. - Chronic DJD, Receptor Agonist Treatment drugs (Tarsy and Baldessarini, 1974). The chronic treatment of prevented by prior administration of receptor-selective antag- rodents with neuroleptics does not usually result in the obvious onists (Meller et al., 1985; Battaglia et al., 1986; Hess et al., dyskinetic movements of tardive dyskinesia, but does result in 1987a). Meller et al. (1985) have demonstrated that flupentixol an increased sensitivity to the motor stimulatory effects of do- administration prior to EEDQ administration will “protect” pamine agonists, accompanied by an increase in dopamine re- both D, and D, dopamine receptors from in vivo EEDQ-induced ceptor density. For example, chronic treatment with the D, modification. We have examined the time course of the inter- dopamine receptor-selective antagonist haloperidol results in action of flupentixol at D, and D, dopamine receptors in vivo an increase in D, dopamine receptor density (Burt et al., 1977). by assessing flupentixol protection of D, and D, dopamine re- It has been suggested that “pharmacological” denervation of ceptors from EEDQ-induced inactivation. dopamine receptors occurs during chronic antagonist treatment, Additionally, we have investigated the possible desensitizing which results in the supersensitive response to subsequent ag- effects of the compound L-prolyl-L-leucyl-glycinamide (PLG) on onist administration (Creese and Sibley, 198 1). chronic neuroleptic-induced D, dopamine receptor up-regula- In humans and in rodents, the acute administration of neu- tion. PLG is thought to be a hypothalamic factor that inhibits roleptics results in Parkinsonian or cataleptic extrapyramidal the release of melanocyte-stimulating hormone (MSH) from the motor side effects, respectively. However, tolerance develops to pituitary (Nair et al., 1971). This tripeptide has been reported the extrapyramidal side effects of the classic neuroleptics, such to reverse the increased D, dopamine receptor density that oc- as haloperidol or spiperone, after several weeks of repeated curs after chronic D, dopamine receptor blockade in rats (Chiu administration (Klett and Caffey, 1972; Ezrin-Waters and See- et al., 198 1, 1985). It has been suggested that PLG may be useful man, 1977; Hess et al., 1986a). Similar to the receptor sub- in ameliorating the symptoms of tardive dyskinesia in man type-selective up-regulation seen following chronic D, dopa- (Bhargava, 1984). mine receptor blockade, chronic treatment with the D, dopamine receptor-selective antagonist SCH23390 leads to a selective Materials and Methods up-regulation in D, dopamine receptors (Creese and Chen, 1985). Chronic drug treatment and behavioral testing. Male Sprague-Dawley However, rats receiving chronic SCH23390 treatment demon- rats(180 gm; Simonsen,Gilroy, CA) were maintainedin groupcages strate no tolerance to the cataleptogenic effects of this drug (Hess on ad lib food and water and a 12 hr light/dark cycle (6 AM/6PM). et al., 1986a). Paradoxially, long-term treatment with antago- Rats were injected daily (s.c.) for 21 d at 10 AM with the following nists such as fluphenazine or flupentixol, which have equal af- drugs dissolved in saline and a minimal amount of 0.1 N HCl (5.0 ~1 HCl/ml saline): 0.5 mg/kg SCH23390 (n = 12), 1 mg/kg cis-flupentixol finities for the D, and D, dopamine receptors in vitro, has been (n = 6), 0.2 mg/kg spiperone (n = 12), 0.5 mg/kg SCH23390 plus 0.2 reported to up-regulate only D, dopamine receptors (Murugaiah mg’kg spiperone (n = 6). In addition, 6 control rats were injected with etal., 1984; Mackenzieandzigmond, 1985; Boysonetal., 1986). the saline vehicle. Rats were tested on days 1, 3, 5, 7, 14, and 21 for a It is unclear whether tolerance develops to the cataleptogenic cataleptic response to their administered drug. Also, on day 22, half the effects of the mixed D,/D, dopamine receptor antagonists (Mu- group of chrdnic spiperone-treated animals were injected &h 0.5 mg/ kg SCH23390 andhalf the grouuof chronicSCH23390-treated animals rugaiah et al., 1984). were injected with 0.2 mg/kgspiperone and observed for cataleptic Because D, and D, dopamine receptors interact in the mod- responses. To test for catalepsy, observations were made every 20 min ulation of behavior (Arnt and Hyttel, 1985; Breese and Mueller, for 2 hr following drug injection. Rats were individually placed in a box 1985; Molloy and Waddington, 1985; Hess et al., 1986a), it is (20 x 20 x 30 cm) with a small bar diagonally placed across one comer, possible that the simultaneous antagonism of D, and D, do- 10 cm above the floor. The rat’s front paws were placed on the bar and the time taken for the rat to remove both paws was measured, with a pamine receptors by drugs such as flupentixol may modulate or cutoff time of 120 set (Sanberg et al., 1984). Two days following final prevent the predicted D, dopamine receptor up-regulation. Al- drug injection, the rats were killed by decapitation and their brains ternatively, the paradoxical effects of mixed D,/D, dopamine rapidly removed to chilled saline. The striata were dissected, frozen in receptor antagonists may be due to differences in the interactions liquid nitrogen, and stored (l-2 weeks) at -70°C until radioligand binding assay. Those rats receiving an acute antagonist challenge on day 22 of these drugs at D, and D, dopamine receptors in vivo. For were not used in the radioligand binding assays. example, D, dopamine receptors may be situated in a manner To examine the effects of PLG on neuroleptic-induced D, dopamine that allows a more rapid drug equilibration and prolonged block- receptorup-regulation, rats wereinjected daily (s.c.)with saline(n = ade in vivo. 12) or 0.2 mg/kg spiperone (n = 12) for 2 1 d. On the 5 d following this In order to investigate these phenomena further, we have chronic treatment, half the rats receiving saline (n = 6) and spiperone (n = 6) treatment were administered saline (s.c.). The remaining 6 an- assessed the effects of chronic treatment with a variety of do- imals from each group were injected daily (s.c.) with 20 mg/kg PLG for pamine receptor antagonists on D, and D, dopamine receptor 5 d. Two days following final drug injection, rats were killed and striata densities and have determined the time course of the catalep- dissected as described above. togenic effects of these drugs throughout the chronic treatment Radioligand binding assays. 3H-SCH23390saturation analyses to determine D, dopamine receptor density and 3H-spiperone saturation regimen. We have also examined the dopamine receptor-block- analyses, to determine D, dopamine receptor density were performed ing properties of the mixed D,/D, dopamine receptor antagonist in parallel in the striata of each rat. Flupentixol co&petitions for 0.25 flupentixol at D, and D, dopamine receptors in vivo using the nM 3H-SCH23390 or 0.1 nM 3H-spiperone were performed in parallel protein-modifying reagent N-ethoxycarbonyl-2-ethoxy- 1,2-di- in control rat striata. Tissue from each animal was homogenized (Tek- hydroquinoline (EEDQ). Peripherally administered EEDQ has mar Tissumizersetting 7, 10set) individually in 40 volumesof ice-cold 50 mM Tris-HCl (pH 7.4 at 37°C). The tissue was centrifuged (35,000 x been shown to irreversibly inactivate a number of neurotrans- g, 10 min) and washed once more in the same buffer, followed by a mitter receptors, including D, (Hamblin and Creese, 1983; Nor- final resuspension in assay buffer consisting of 50 mM Tris-HCl (pH 7.4 man et al., 1987) and D, (Hamblin and Creese, 1983; Hess et at 37°C). 5 mM MESO,. and 0.5 mM EDTA. Incubations for each ra- al., 1987a) dopamine receptors, S, serotonin (Battaglia et al., dioliganh were initiate; by adding tissue (2 mg wet weight tissue/tube) to duplicate tubes containing 3H-antagonists to yield a 2.5 ml final assay 1986), and ol,-adrenergic receptors (Adler et al., 1985). EEDQ- volume. All saturation analyses included 6 concentrations of radioli- induced inactivation of these receptors appears to occur at the gand. Competition and saturation studies of 3H-SCH23390 (0.125-2 ligand-recognition site, as EEDQ-induced inactivation can be nM) were conducted in the presence or absence of 100 nM cis-flupentixol The Journal of Neuroscience, July 1988, 8(7) 2363

Figure I. Cataleptic response of rats treated chronically with spiperone (n = 6) or saline (n = 6). Spiperone treatment days 1, 7, and 2 1, and saline treatment day 2 1 are presented. Additionally, data from rats treated with SCH23390 for 2 1 d and then challenged with an acute injection of spiperone on day 22 are presented (n = 6). All drug-treated rats were significantly more cataleptic than 6 saline-treated rats on all testing days t? 60 (p < 0.001). Catalepsy scores on days m 7 and 21 of spiperone treatment and E acute spiperone challenge after chronic 40 SCH23390 treatment were significantly lower than scores on spiperone treatment day 10, < 0.00 1). Catalepsy scores 20 Acute Spiperone from acute spiperone challenge after After 21 days chronic SCH23390 treatment did not Chronic SCH23390 differ significantly from those after day 40 60 80 21 of spiperone treatment. Data represent means + SEM, with a maximum Minutes Post-Injection catalepsy score cutoff of 120 sec.

in order to distinguish the specific from nonspecific binding. All ‘H- Dr. J. Hyttel of H. Lundbeck and Co. (Denmark); SCH23390, Drs. Iorio spiperone assays (0.025-4 nM) were conducted in the presence of 40 nM and Bamett of Schering Corp. (Bloomfield, NJ); spiroperidol, Dr. J. ketanserin to preclude binding of 3H-spiperone to S, serotonin receptors; Leysen, Janssen Pharmaceutics (Beerse, Belgium). Other reagents were nonspecific binding was defined by 1 PM (+)butaclamol. Competition obtained from standard commercial sources. experiments included 2 1 concentrations of flupentixol. Tubes were in- cubated for 40 min at 37”C, filtered over Whatman GF/C glass-fiber filters that were then washed rapidly with 15 ml (3 x 5 ml) of ice-cold Results Tris buffer and counted by scintillation spectroscopy at an efficiency of 50%. Flupentixol competitionfor jH-SCH23390 or ‘H-spiperone -In- vivo ji’upentixol specificity. Male Sprague-Dawley rats (160-l 80 binding gm; Simonsen) were iniected (i.n.) with EEDO -. (8 mrr/ka)-II freshlv dis- Computer-fitted curves for cis-flupentixol competition for 0.25 solved in ethr&ol/wate; (1:l voi/vol) or vehicle alone. To determine nM 3H-SCH23390binding modeledbest to a singlehomogenous whether EEDQ had the same EC,, for D, and D, dopamine receptors, a dose-response for EEDQ was performed, in which rats received 1 mg/ population of binding sites with a K, of 0.69 -t 0.01 nM (n = kg, 2 mg/kg, or 4 mg/kg EEDQ. For protection experiments testing the 3). Computer-fitted curves for cis-flupentixol competition for specificity of flupentixol in vivo, rats were injected (s.c.) with 0.25, 0.5, 0.1 nM 3H-spiperonebinding modeled best to a single homo- 0.75, or 1 mg/kg flupentixol dissolved in saline, or the saline vehicle geneouspopulation of binding sites with a K, of 0.88 k 0.03 40 min before EEDQ (8 mg/kg) injection. Additionally, a second group nM (n = 3). The affinity of flupentixol for D, dopaminereceptors of rats was injected with 6.5 mg/kg flupentixol or vehicle 20, 40, 66, was not significantly different from that of flupentixol for D,

80, 100, or 120 min nrior to EEDO. (8. mp/ka)-II- iniection. Four hours after EEDQ treatment, rats were decapitated, the striata removed, frozen dopamine receptors0, > 0.05). rapidly in liquid nitrogen, and stored (- 70°C) for 2-4 weeks. Saturation analyses of ‘H-antagonist binding were performed in the striata as de- Behavioral effectsof chronic drug administration scribed above, with the following tissue preparation to facilitate flupentixol washout: Tissue from each animal was individually homoge- The overall catalepsy score, collapsedover each 2 hr sessionof nized in 40 volumes of ice-cold 50 mM Tris-HCl (nH 7.4 at 37°C). The each daily treatment for rats treated with the D, dopamine tissue was centrifuged (35,000 x g, 10 min) and &suspended to i0 mg receptor-specificantagonist spiperone,decreased over the course tissue wet weight/ml in this same biffer, followed by a lb min incubation of the 21 d treatment (Fig. 1). A significant reduction in the (37°C). The incubation was stopped by the addition of 30 ml ice-cold overall catalepsy scorerecorded on day 1 of spiperonetreatment 50 mM Tris-HCI. Tissue was centrifuged (35,000 x g, 10 min) and washed once more in this buffer, followed by a final resuspension in occurred by day 5 0, < 0.01) and progressively increasedover assay buffer consisting of 50 mM Tris-HCl (PH 7.4 at 37”C), 5mM MgSO,, the 21 d period (p < 0.001). However, complete tolerance to and 0.5 mM EDTA. The saturation analyses were then comuleted as the cataleptogenic effects of spiperone was not observed: the described above with 3H-SCH23390 a& 3H-spiperone bin&ng per- overall catalepsy score on testing day 2 1 of rats treated chron- formed in parallel on striata from each rat. Data analysis. ‘H-Antagonist saturation data were analyzed by the ically with spiperone was significantly greater 0, < 0.01) than method of Scatchard (1949). A weighted, nonlinear, least-squares curve- the day 2 1 catalepsyscores ofrats treated chronically with saline. fitting program, LIGAND (Munson and Rodbard, 1980) was used in the In contrast to the rats treated with spiperone(Fig. 1) and other computer analysis of competition curves. All data were fitted while classic neuroleptics, in which tolerance to their cataleptogenic constraining the nonspecific binding parameter defined by 100 nM cis- effectsdevelops after chronic treatment (Ezrin-Waters and See- flupentixol or 1 FM (+)butaclamol. Data obtained in the catalepsy- testing sessions were analyzed using arcsine transform analysis of vari- man, 1977), rats receiving chronic administration of the D, ance with repeated measures and the Newman-Keuls test. dopamine receptor-selective antagonist SCH23390 for 21 d Materials. 3H-SCH23390 (87 Ci/mmol) and 3H-spiperone (88 Ci/ demonstratedno tolerance to its cataleptogenicaction over the mmol) were obtained from DuPont-New England Nuclear (Boston, 21 d administration (p > 0.05) (Fig. 2). The cataleptic effects MA). EEDQ was obtained from Aldrich Chemical Co. (Milwaukee, WI), and L-prolyl-L-leucyl-glycinamide from Sigma Chemical Co. (St. Louis, of SCH23390 did diminish over the 2 hr testing session,but MO). The following drugs were generous gifts from the following sources: the time course of this responsedid not change from that ob- butaclamol, Ayerst Laboratories (Montreal, Canada); cis-flupentixol, served on day 1 over the 21 d treatment period. 2364 Hess et al. * Chronic D,/D, Receptor Agonist Treatment h “- 120 r 120 r t.h

100 - 100 - 72 ZT at6 80 - ii 80 - 27 *Day1 fit B ADay 21 -Z5 60- l Day1 B 60- OSaline (day 21) A Day 21 0 Saline (day 21) 8 40 - 40 -

20 - 20 - I 0 I n T Y P I 1 T * Q 20 40 60 80 100 120 20 40 60 80 100 120

Minutes Post-lniection Minutes Post-Injection

Figure 2. Cataleptic response of rats treated chronically with SCH23390 Figure 4. Cataleptic response of rats treated chronically with SCH23390 (n = 6) on days 1 and 21 of treatment, or saline (n = 6) on day 21 of plus spiperone (n = 6) on days 1 and 2 1 of treatment, or saline (n = 6) treatment. SCH23390-treated rats were significantly more cataleptic on day 21 of treatment. SCH23390 plus spiperone-treated rats were than saline-treated rats (p < 0.001). The response to SCH23390 treat- significantly more cataleptic than saline-treated rats (p < 0.001). The ment did not change over the 21 d treatment regimen. Data represent response to SCH23390 plus spiperone treatment did not change over means ? SEM, with a maximum catalepsy score of 120 sec. the 21 d treatment regimen. Data represent means t SEM, with a maximum catalepsy score of 120 sec. Rats receiving chronic administration of the mixed D,/D, dopamine receptor antagonist flupentixol for 2 1 d demonstrated over the 2 hr catalepsy-scoring session did not change for the tolerance to its cataleptogenic action over the 2 1 d administra- 2 1 d duration of either SCH23390 or spiperone treatment. Those tion period, with a significant reduction (p < 0.01) in the overall rats receiving SCH23390 plus spiperone demonstrated no tol- catalepsy score collapsed over each 2 hr session occurring by erance to the cataleptic effect of this combination of D, and D, day 7 of treatment (Fig. 3). This was accompanied by a change dopamine receptor antagonists (Fig. 4). in the pattern of catalepsy observed during the 2 hr catalepsy When rats were treated with SCH23390 for 21 d and then scoring session over the duration of cis-flupentixol treatment. administered an acute injection of spiperone, tolerance to the Over the 3 week treatment, in the first 60 min of the scoring cataleptogenic effects of spiperone was observed (Fig. 1). In fact, session, these animals appeared to become significantly less cat- the catalepsy scores, collapsed over the 2 hr session following aleptic (p < 0.01) after cis-flupentixol administration. That is, acute challenge with spiperone after chronic 21 d SCH23390 the time between injection and a full cataleptogenic response to treatment, were not different (p > 0.05) from the scores of cis-flupentixol increased from 20 to 60 min over the 2 1 d admin- chronic spiperone-treated rats on spiperone injection day 2 1. istration period. In contrast, the pattern of catalepsy observed Conversely, rats challenged with an acute injection of SCH23390

120 -

120 - 100 - 100 - 2 " 80- 1 l Saline mject~on following 80 - 6 21 day chronic spiperone 1 1 $ treatment m 60 - A SCH23390 injection 60 - emy1 following 21 day chronic ADay5 5 saline treatment 0 Day 7 40 - 0 SCH23390 injecton ADay 21 followmg 21 day chronic 40 - 0 Salme spiperone treatment 20 20 - tL w1 + 4 P 20 40 60 80 100 120 40 60 80 100 120 Minutes Post-Injection Minutes Post-Injection Figure 5. Cataleptic response of rats treated chronically with spiperone Figure 3. Cataleptic response of rats treated chronically with cis-flu- for 2 1 d and challenged with an acute injection of either saline (n = 6) pentixol (n = 6) on days 1, 5, 7, and 21, or saline (n = 6) on day 21. or SCH23390 (n = 6) and rats treated with saline for 21 days and Flupentixol-treated rats were significantly more cataleptic than saline- challenged with SCH23390. Catalepsy scores for a final SCH23390 treated rats on all days tested (p < 0.001). Catalepsy scores on days 7 injection were significantly greater than for the final saline injection and 2 1 were significantly lower (p < 0.0 1) than scores on day 1, owing (p < 0.00 1). Catalepsy scores for chronic 2 1 d spiperone treatment and to a reduction in catalepsy scores at 20,40, and 60 min after flupentixol chronic saline treatment with a final SCH23390 challenge did not differ injection (p < 0.05). Data represent means -I SEM, with a maximum significantly. Data represent means GEM, with a maximum catalepsy catalepsy score of 120 sec. score of 120 sec. The Journal of Neuroscience, July 1988, 8(7) 2366

Table 1. Effects of 21 d chronic drug treatments on striatal D, and D, dopamine receptor binding parameters

3H-SCH23390binding 3H-Spiperonebinding B m2.x B mai Chronicdrug (pmol/gm (pmol/gm Kd treatment tissue) ZM) tissue) bf) Control 53.5 + 1.3 0.44 + 0.03 21.7 + 0.7 0.08 k 0.01 Flupentixol 56.1 + 1.8 0.44 i- 0.03 27.9 k 0.8*** 0.10 -t 0.01 Spiperone 54.7 +- 1.7 0.46 k 0.07 26.8 k 0.6*** 0.10 + 0.02 SCH23390 62.0 k 2.0* 0.41 It 0.01 23.6 k 0.6 0.08 + 0.01 SCH23390+ spiperone 63.6 + 2.2** 0.46 k 0.01 28. 6 + 0. 8***,0 0.11 & 0.01 Data represent means & SEM (n = 6) for the affinities (KJ and densities (Bmax) of D, and D, dopamine receptors, determined by saturation analyses of ‘H-SCH23390 and ‘H-spiperone binding, respectively. Rats were treated with cis- flupentixol (lmg/kg per day), spiperone (0.2mgIkg per day), SCH23390 (0.5mg/kg per day) or spiperone (0.2mg/kg per day) plus SCH23390 (0.5mg/kg per day) for 21 d. Asterisks denote values significantly greater than control values: *p < 0.01, **p < 0.005, ***p < 0.001. y SCH23390 plus spiperone value significantly greater than spiperone alone (p < 0.01); l-way ANOVA and Duncan’s test.

after 2 1 d spiperonetreatment were as profoundly cataleptic as Kd of 3H-spiperonebinding was unchangedfrom control values rats that had received the 21 d chronic SCH23390 treatment (Table 2). (Fig. 5). Efects of in vivo jlupentixol pretreatment on EEDQ-induced Effect of chronic drug treatment on striatal dopamine receptor reductions of D, and D, dopamine receptors radioligand binding 3H-SCH23390 and 3H-spiperone binding were markedly re- Consistentwith previous reports by this laboratory (Creeseand duced by in vivo EEDQ treatment in a dose-dependentmanner. Chen, 1985; Hess et al., 1986a), chronic treatment with In vivo EEDQ-induced reductions in D, and D, dopamine re- SCH23390 resulted in a highly significant increase (+ 16%) in ceptors were comparable at all dosesof EEDQ; EEDQ has an D, dopaminereceptor-specific 3H-SCH23390 binding (p < O.Ol), apparent EC,, of approximately 1.5 mg/kg (i.p.) for both the D, but no significant changein D, dopamine receptor-specific3H- and D, dopamine receptors (Table 3). The magnitude of the spiperonebinding was observed (p > 0.05) (Table 1). Chronic EEDQ-induced reductions in D, and D, dopamine receptor treatment with the D, dopamine receptor-selective antagonist binding could be reducedby treating rats with flupentixol before spiperoneresulted in a no significant changein 3H-SCH23390 EEDQ administration. However, at all doses, flupentixol pro- binding (p > 0.05), while 3H-spiperonebinding was significantly tected D, dopamine receptor binding to a greater extent than it increasedby 24% (p < 0.001) (Table 1). Chronic treatment with did D, dopamine receptor binding when flupentixol wasinjected SCH23390 plus spiperone resulted in a significant increase in 40 min before 8 mg/kg EEDQ administration (Table 4). In fact, both 3H-SCH23390(+ 19%)and 3H-spiperonebinding (+ 32%) administration of 0.25 mg/kg flupentixol prior to EEDQ treat- (Table 1). The increasein 3H-spiperonebinding in the animals ment protected 66% of D, dopamine receptors, while only 2 1% treated with SCH23390 plus spiperonewas significantly greater of D, dopamine receptorswere protected by this dose.Admin- (p < 0.05) than that observed in 3H-spiperonebinding in rats treated with spiperone alone. However, the increase observed in 3H-SCH23390 binding in rats treated with SCH23390 plus Table 2. Effects of L-prolyl-L-leucyl-glycinamide (PLG) treatment on spiperonedid not differ significantly from that in 3H-SCH23390 striatal D, dopamine receptor binding after 21 d spiperone binding in rats treated with SCH23390 alone. Chronic treatment administration with the mixed D,/D, dopamine receptor antagonist cis-flupentixol resulted in a significant increase in 3H-spiperonebinding B nmx only (+24%) (J,J< 0.001) with no change observed in 3H- (pmol/gm K* SCH23390 binding (Table 1). The Kdsof3H-SCH23390 binding Treatment(d) tissue) bf) and of 3H-spiperonebinding were unchanged by chronic drug Saline(26) 21.7 + 0.5 0.068 k 0.011 treatment in any experimental condition (Table 1). Saline(21) followedby PLG (5) 21.1 t O.@ 0.072 k 0.002 Spiperone(2 1) followedby Effects of PLG administration following D, dopamine receptor saline(5) 26.13 +* 0.072 + 0.002 up-regulation Spiperone(21) followedby The chronic 21 d spiperone treatment resulted in a significant PLG (5) 27.18 & 0.8*.0 0.075 It 0.003 20% increasein 3H-spiperonebinding 0, < 0.0 1). Administra- Data represent means ? SEM (n = 6) for the affinity (&) and density (B,,,) of D, tion of PLG for 5 d following chronic spiperone treatment did dopamine receptors, determined by saturation analyses of ‘H-spiperone binding. not affect the antagonist-inducedD, dopamine receptor up-reg- Rats were treated with saline or spiperone (0.2 mg/kg) for 2 1 d and then treated with either saline or PLG (20 mg/kg) for the 5 d following chronic treatment. ulation (Table 2). In addition, 5 d PLG administration following Asterisk denotes values significantly different from saline treatment (p < 0.01). 21 d saline treatment did not affect the B,,, of the normosen- y Values not significantly different (p z 0.05) from comparable non-PLG-treated sitive D, dopamine receptor population. In all treatments, the group. 2366 Hess et al. * Chronic D,/D, Receptor Agonist Treatment

Table 3. Dose-dependent reductions in striatal D, and D, dopamine receptor binding after peripheral administration of EEDQ

‘H-SCH23390binding ‘H-Spiperonebinding EEDQs Kd B,,, (% reduction) K* B,,, (O/oreduction) dose @M) (pmol/gmtissue) bf) (pmol/gmtissue) Control 0.47 k 0.02 89.3 * 1.3 0.095 5 0.005 28.1 f 0.2 1 mg/kg 0.48 zk 0.02 61.8 k 5.3 (-31%) 0.103 + 0.010 20.2 + 1.6 (-28%) 2 m&z 0.51 + 0.02 34.2 + 2.2 (-62%) 0.101 & 0.005 11.5 & 0.7 (-59%) 4mg/kg 0.49 * 0.05 16.3 f 1.2 (-82%) 0.123 IL 0.007 6.6 f 0.3 (-77%) Data represent means I!Y SEM (n = 4-5) for affinities (KJ and densities (E,,,) of D, and D, dopamine receptors determined by ‘H-SCH23390 and )H-spiperone saturation analyses. Values in parentheses represent percentage loss ofcontrol receptor densities. Radioligand binding assays for both D, and D, dopamine receptors were performed in parallel on tissue from individual rats.

istration of 1 mg/kg flupentixol was necessaryto achieve a degree tection of D, dopamine receptorswas - 97% during the second of protection of the D, dopamine receptor comparableto that hour and had only declined to 82% protection at the 120 min afforded the D, dopaminereceptor with 0.25 mg/kg flupentixol. point. In some instances,the Kd of 3H-SCH23390 and 3H-spi- A dose of 0.75 mg/kg flupentixol saturated the D, dopamine perone binding was significantly greater than control values, receptor, since the degreeof protection reacheda plateau at this presumably becauseof residual flupentixol remaining after the dose,with 86% of D, dopamine receptors protected from irre- wash procedures, as EEDQ itself does not alter the affinity of versible modification by EEDQ. Becausethe flupentixol-do- 3H-SCH23390or 3H-spiperonebinding (Table 3). pamine receptor interaction is a reversible process,with some flupentixol free and some bound at any given instant, it is not Discussion surprising that 100% protection was never achieved, especially As we have previously reported (Creeseand Chen, 1985; Hess when using a close to saturating dose of EEDQ, as in these et al., i986a), chronic treatment with the D, receptor-selective experiments. antagonist SCH23390 resulted in a significant and selective in- To determine whether the differential protection was a time- creasein the D, dopaminereceptor density. Likewise, consistent dependent phenomenon basedon the accessibility of the drug with previous reports, chronic treatment with the D, dopamine to each dopamine receptor subtype (i.e., flupentixol had more receptor-selectiveantagonist spiperoneselectively increasedthe rapid accessibility to D, dopamine receptors than to D, dopa- D, dopaminereceptor density. However, chronic treatment with mine receptors in vivo), a time course of flupentixol protection the mixed D,/D, receptor antagonist flupentixol resulted in the of D, and D, dopamine receptors from EEDQ-induced inacti- up-regulation of D, dopamine receptors only while the D, do- vation was measured.Protection of D, dopamine receptorsby pamine receptor density was unchanged, consistent with pre- 0.5 mg/kg flupentixol wasbetween 82 and 97% at all time points vious reports (Murugaiah et al., 1984; Mackenzie and Zigmond, tested except the 20 min point, when flupentixol had probably 1985). As we have demonstrated herein, flupentixol has com- not yet reached steady-state levels. The protection afforded D, parable affinities for the D, and D, dopamine receptorsin vitro. dopaminereceptors by flupentixol did not appearto differ from Since many investigators have reported an interaction between the time course of flupentixol protection of D, dopamine re- D, and D, dopamine receptors in the regulation of behavioral ceptors within the time period that had been examined for the (Arnt and Hyttel, 1985; Molloy and Waddington, 1985; Breese catalepsy measures.Maximal protection of D, dopamine re- and Mueller, 1985; Hesset al., 1986a)and electrophysiological ceptors, which occurred 60 min after flupentixol administration, (Carlson et al., 1987) responses,the lack of an increasein D, never exceeded 54% of control levels (Table 5). Furthermore, dopamine receptor density following chronic flupentixol treat- during the second hour of the experiment, protection of D, ment may be due to a modulatory receptor interaction occurring dopamine receptors declined to approximately 45%, while pro- with concomitant D, and D, dopamine receptor blockade. To

Table 4. Effects of flupentixol pretreatment on in viva EEDQ-induced reductions in striatal D, and D, dopamine receptor densities

3H-SCH23390binding 3H-Spiperonebinding Kd B,,, (O/aprotection) B,,, (O/aprotection) Drug treatment NM) (pmol/gmtissue) $M) (pmol/gmtissue)

Control 0.42 I!Z 0.05 63.1 f 1.9 0.06 + 0.003 21.5 + 0.9 EEDQ (8 mdk) 0.53 k 0.04 2.9 + O.l** 0.094 k 0.005 1.3 + 0.1** Flupentixol(O.25mg/kg) 0.52 + 0.04 15.4 2 1.3 (21%)** 0.10 2 0.005 14.6 -t 0.3 (66%)** Flupentixol(0.5 mg/kg) 0.12 + 0.08 20 . 9 k 1 . 1 (30%)** 0.17 + 0.006 17.6 ? 2.3 (81%)* Flupentixol(O.75 mg/kg) 0.68 + 0.05 28.6 + 2.9 (43%)** 0.18 + 0.012 18.7 + 1.3 (86%) Flupentixol (1 mg/kg) 0.72 k 0.06 40.5 + 3.8 (62%)** 0.18 + 0.009 18.6 + 0.9 (86%) Data represent means f SEM (n = 4-7) for affinities (&) and densities (B,,,) of D, and D, dopamine receptors determined by ‘H-SCH23390 and ‘H-spiperone saturation analyses. Values in parentheses represent percentage protection of the receptor-inactivating action of EEDQ by flupentixol, calculated using total receptors inactivated by EEDQ and subtracting receptors remaining after 8 mg/kg EEDQ treatment from control values and from receptors protected by flupentixol. EEDQ (8 mg/kg) was administered (i.p.) 40 min after flupentixol injection (s.c.). Asterisks represent values significantly lower than control values: * p < 0.05, ** p < 0.001. The Journal of Neuroscience, July 1988, 8(7) 2367

Table 5. Time course of flupentixol protection of striatal D, and D, dopamine receptors from EEDQ- induced modification in viva

‘H-SCH23390binding ‘H-Spiperonebinding B,,, (% protection) Kd B,,, (% protection) (pmol/gmtissue) b4 (pmol/gmtissue) Control 0.64 k 0.07 71.1 k 2.1 0.104 k 0.010 22.2 * 1.1 EEDQ 0.69 2 0.14 2.4 f 0.5 0.328 + 0.105 1.8 k 0.4 Time after flupentixoladministration’(min) 20 1.15 + 0.12* 27 7 + 3 9 (37%)** 0.28 k 0.02 15.0 k 0.8 (65%)** 40 1.12 + 0.12 29:0 e 1:4 (39%)** 0.37 k 0.08* 20.4 + 0.9 (91%) 60 1.16 + O.lS* 39.8 2 0.6 (54%)** 0.26 k 0.02 19.7 + 0.3 (88%) 80 0.87 k 0.05 29.8 k 1.8 (40%)** 0.32 +- 0.04 21.5 + 1.0 (97%) 100 1.00 +- 0.10 34.9 * 3.1 (47%)** 0.28 k 0.02 21.4 * 0.6 (96%) 120 1.36 f 0.19* 33.2 f 2.9 (45%)** 0.24 f 0.02 18.5 ? 1.3 (82%)* Data represent meanst SEM (n = 4-6) for affinities(KJ and densities (B,,,) of D, and D, dopamine receptors determined by ‘H-SCH23390 and ‘H-spiperone saturation analyses. Values in parentheses represent percentage protection by flupentixol of EEDQ-induced receptor inactivation. EEDQ (8 mg/kg) was administered (i.p.) 20, 40, 60, 80, 100, or 120 min after Aupentixol injection (0.5 mg/kg, s.c.). Asterisks indicate values significantly different from control values: * p < 0.05, **p < 0.01. test this hypothesis, rats received simultaneoustreatment with D, and D, dopamine receptors. Flupentixol protection of D, a D, receptor-selective antagonist (SCH23390) and a D, do- and D, dopamine receptors from EEDQ-induced inactivation pamine receptor-selective antagonist (spiperone)to mimic the was usedto test this hypothesis. To insure that EEDQ did not potential dual-receptor subtype-blocking activity of the mixed have a greater potency at D, or D, dopamine receptorsin vivo, D,/D, dopamine receptor antagonist flupentixol. Simultaneous an EEDQ dose-responseexperiment assessingthe effects of D, and D, dopamine receptor antagonismresulted in an increase EEDQ on D, and D, dopamine receptorswas performed: in vivo in the density of both D, and D, dopamine receptors using the administration of EEDQ induced comparable percentagere- drug dosesemployed in this study. Thus, simultaneousD, and ductions in D, and D, dopamine receptors. That EEDQ induced D, dopamine receptor antagonism does not appear to reduce equal reductions in D, and D, dopamine receptor binding at all the ability of D, dopamine receptors to up-regulate. dosesadministered in vivo is not surprising. The mechanismof The increase in D, dopamine receptors observed after action of EEDQ is thought to be due to the creation of highly SCH23390 plus spiperone treatment was significantly greater reactive mixed carbonic anhydrides from carboxyl groups that, than the increasein D, dopamine receptorsafter spiperonetreat- in turn, interact with nucleophilic groups such as free a-amino ment alone. Thus, chronic blockageof D, dopamine receptors, groups (Belleau et al., 1968). EEDQ also has the capacity to which alone does not result in a statistically significant D, do- modify enzymes such as serine proteases(Belleau et al., 1968) pamine receptor up-regulation, potentiated the D, dopamine and adenylate cyclase(Hess et al., 1987a), in addition to neu- receptor up-regulation resulting from chronic D, dopamine re- rotransmitter receptors. In fact, we have demonstratedthat in ceptor blockade. Saller and Salama (1986) have recently dem- vitro the carboxyl groups of the adenylate cyclasemolecule ap- onstrated that SCH23390 reduces the in vivo increase in do- pear to be as sensitive to EEDQ-induced alterations as those pamine metabolites elicited by D, dopamine receptor blockade. carboxyl groups modified at the D, dopamine receptor. Thus, Thus, it is possiblethat the potentiation in D, dopaminereceptor EEDQ-induced modifications seem to be dependent on the up-regulation is due to a reduction in dopamine releaseonto availability of carboxyl groupsin a given protein. That is, EEDQ- D, receptors causedby the D, dopamine receptor blockade by induced reductions in the D, and D, dopamine receptor density SCH23390. The reduction in dopamine releasewould result in may be due to nonreceptor-specific carboxyl group modifica- an effective increasein D, dopamine receptor blockade by spi- tions rather than to any specific “affinity” EEDQ might have perone. Alternatively, another possibility is that the enhanced for thesereceptors. D, dopamine receptor up-regulation after SCH23390 plus spi- SinceEEDQ induces comparablepercentage reductions at D, perone treatment may be the result of additional direct D, do- and D, dopamine receptors, it may be usedas a tool to assess pamine receptor blockade by SCH23390. Although SCH23390 the relative interactions of drugs administered in vivo at these has minimal D, dopamine receptor affinity in in vitro radioli- receptors. Treatment with flupentixol before the peripheral gand binding assays(Hess et al., 1986b), any small, additional administration of EEDQ protected both D, and D, dopamine direction D, dopamine receptor blockade causedby SCH23390 receptors from irreversible blockade by EEDQ, as previously in vivo (Stoof and Kebabian, 1981; Andersen and Nielsen, 1986) demonstrated (Meller et al., 1985). However, at all dosesof on top of the large blockade by spiperone may have been suf- flupentixol tested and at all time points after flupentixol injec- ficient to induce the greaterD, dopaminereceptor up-regulation. tion, flupentixol protected a greater percentageof D, dopamine Clearly, more extensive experimentation is necessarybefore this receptorsthan D, dopamine receptors. That is, in vivo, flupen- question can be answered. tixol demonstrateda preferencefor the D, dopamine receptor. An hypothesis to explain the differential effect of chronic flu- The lipophilicity of flupentixol, which can result in free ligand pentixol administration on D, and D, dopamine receptors is depletion, could mask such a small, but functionally significant, that, in contrast to its equal affinity at D, and D, dopamine difference in the determination of flupentixol affinities in vitro. receptorsin vitro, in vivo flupentixol interacts differentially with Conversely, in vivo drug-receptor interactions, assessedfrom 2366 Hess et al. * Chronic D,/D, Receptor Agonist Treatment the use of drug treatment followed by EEDQ treatment, would alepsy over the 2 hr testing sessions may reflect the behavioral reveal these small differences in affinity provided the drug was variability seen when different groups of rats are tested on dif- administered in a subsaturating dose. The in vivo procedure ferent days. In contrast to the pattern of catalepsy observed over assesses relative drug interactions at 2 or more receptors si- the course of chronic SCH23390 treatment, rats became pro- multaneously by measuring fractional occupancy. Thus, small gressively tolerant to the cataleptic effects of spiperone; behav- differences in the affinity of a drug for 2 receptors would become ioral tolerance to these effects became obvious on treatment day obvious because fractional occupancy would never be equal at 5, and the rats became progressively more tolerant over the 2 1 the different receptors, provided the drug is not at saturating d treatment period. Tolerance was not observed to the com- concentrations for the examined receptors. Although this meth- bination of SCH23390 plus spiperone, with maximal catalepsy od of drug-induced protection from EEDQ-induced modifica- scores recorded at all time points on all testing days. Rats treated tion in vivo is not suitable for the quantitative determination of chronically with flupentixol appeared to become less sensitive drug affinities at receptors, it should provide, with ease, a good to the immediate cataleptic effects of this drug over the course reflection of relative drug interactions at specific receptors in of treatment, but continued to demonstrate profound catalepsy vivo. These results are consistent with the results of P. H. An- in the second hour after flupentixol administration. Since the dersen (personal communication), who reports that when using EEDQ protection experiments indicated that flupentixol has a in vivo radioligand binding for a direct quantitative assessment greater D, than D, dopamine receptor-blocking activity, it is of in vivo receptor affinity, flupentixol in vivo has a higher affinity possible that the behavioral tolerance to flupentixol observed for D, dopamine receptors than for D, dopamine receptors. In during the first hour after drug administration is a reflection of our experiments, flupentixol appears to equilibrate at each do- the development of behavioral tolerance to the D, dopamine pamine receptor subtype within 40 min after injection, so the receptor antagonism by flupentixol. behavioral experiments were not confounded by any possible The discovery of antagonist drug-induced receptor up-regu- time-dependent differences in the accessibility of the drug to the lation provided an attractive explanation for the behavioral receptor. Thus, it seems from these results, as well as from the tolerance to chronic antagonist administration (for review, see report of P. H. Andersen (personal communication), that chron- Creese and Sibley, 198 1). However, the results described herein ic flupentixol treatment does not block a sufficient percentage demonstrate that this mechanism cannot explain the tolerance of D, dopamine receptors to induce up-regulation. These results or lack of tolerance of the cataleptic response to the chronic are noteworthy, as they demonstrate that, just as behavioral administration of dopamine receptor antagonists. It is not yet drug studies may not always provide an accurate assessment of clear why rats become tolerant to the cataleptogenic effects of specific drug-receptor interactions (e.g., the once-accepted hy- D, dopamine receptor antagonists, but do not become tolerant pothesis that apomorphine-induced stereotypies were due solely to the D, dopamine receptor antagonist SCH23390. Tolerance to D, dopamine receptor activation), extrapolating specific in to chronic spiperone administration occurs concomitant with a vivo drug-receptor interactions from in vitro radioligand binding D, dopamine receptor up-regulation but does not occur to chronic data may be imprecise. SCH23390 administration in spite of a similar concomitant D, Twenty-one day chronic spiperone treatment, followed by 5 dopamine receptor increase. Conversely, in spite of a normal d PLG treatment, had no effect on the D, dopamine receptor complement of D, dopamine receptors following chronic density. Previous studies (Bhargava, 1984; Chiu et al., 1985) SCH23390 treatment, rats are just as tolerant to spiperone as have reported a PLG-induced reversal in the D, dopamine re- they are following chronic spiperone administration. It is pos- ceptor up-regulation following chronic neuroleptic treatment. sible that in vivo SCH23390 may interact with D, dopamine Although the experiments described in those reports were fun- receptors to induce tolerance to an acute spiperone injection damentally similar to the protocol described herein, those in- after chronic SCH23390 treatment. However, we have previ- vestigators administered haloperidol rather than spiperone; the ously demonstrated that treatment with SCH23390 (0.5 mg/kg, difference in chronic drug treatment may account for the striking s.c.) before peripheral administration of EEDQ protects D, do- differences in the putative D, dopamine receptor density-reg- pamine receptors from irreversible blockade by EEDQ, but that ulatory properties of PGL. PLG may also exert its regulatory neither D, nor S, serotonin receptors were protected from EEDQ- effects, preventing neuroleptic-induced D, dopamine receptor induced modifications by SCH23390 (Hess et al., 1986a). This up-regulation, if it is administered prior to neuroleptic treat- indicates that SCH23390 at the dose administered in this study ment, as previous reports have suggested (Chiu et al., 198 1, interacts in vivo exclusively with D, dopamine receptors. Thus, 1985). Clearly, the prophylactic effects of PLG on dopamine behavioral tolerance to an acute injection of spiperone following receptor up-regulation require further examination. 21 d chronic SCH23390 treatment is probably not due to any As we have previously reported (Hess et al., 1986a), after nonspecific effects of SCH23390 at the D, dopamine receptor. chronic treatment with SCH23390 for 21 d, rats demonstrated It appears then that D, dopamine receptor up-regulation is not no tolerance to the cataleptogenic effects of SCH23390. The necessary for the induction of behavioral tolerance. Although decrease observed in the cataleptic response to SCH23390 over the D, dopamine receptor recognition site may not be up-reg- the 2 hr testing session (Fig. 2) was probably due to rapid me- ulated, alterations in its second-messenger system may have tabolism of the drug, rather than a tolerance phenomenon, since occurred. For example, in a previous study we demonstrated it was present on the first administration of the drug. It might that chronic SCH23390 treatment increased the ability of GTP be suggested that the prolonged catalepsy response to acute to activate adenylate cyclase in rat striatal homogenate (Hess et SCH23390 injection following 2 1 d chronic spiperone treatment al., 1986a). results from a modulatory effect of the increased D, dopamine Many studies have demonstrated that D, and D, dopamine receptor density (compare Figs. 2 and 5). However, rats treated receptors are interactive in their modulation of electrophysio- with saline for 21 d also demonstrated a prolonged catalepsy logical responses, as well as motor behavior. It has recently been response to acute SCH23390 injection (Fig. 5). These results suggested that drug-induced changes in the tonic electrical ac- suggest that the differences observed in SCH23390-induced cat- tivity of the basal ganglia, particularly the globus pallidus, may The Journal of Neuroscience, July 1988, 8(7) 2369 reflect drug-induced behavioral effects (Carlson et al., 1987). effects in clinical trials. That tolerance to the cataleptogenicity That is, agonist-induced increases in pallidal electrical activity of SCH23390 plus spiperone treatment was not observed in this seem to correlate approximately with agonist-induced increases study also suggests that a complete D, plus D, dopamine re- in behavioral responses, while the converse is also true. Thus, ceptor blockade may also produce unremitting Parkinsonian an increase in D, dopamine receptors induced by chronic side effects in the clinical setting. However, the doses of the SCH23390 treatment may result in an overall increase in the drugs used in this study were rather high and the testing extended tonic activity of the basal ganglia owing to the enhanced action only over 2 1 d. It would be useful to test treatments using lower of endogenous dopamine at the up-regulated D, dopamine re- doses of SCH23390 plus spiperone over a longer test period to ceptors. This overall activation imparted by increased D, do- determine whether the development of tolerance to the extra- pamine receptor activity may be great enough to impart what pyramidal side effects of stimultaneous D, and D, dopamine appears to be behavioral tolerance to a D, dopamine receptor receptor blockade can occur. antagonist essentially by “overriding” the D, antagonist signal. These results parallel our earlier findings, where the behavioral effects of the D, receptor agonist LY 17 1555 were potentiated References after a selective D, dopamine receptor up-regulation (Hess et Adler, C. H., E. Meller, and M. Goldstein (1985) Recovery of cu-ad- al., 1986a). In fact, we have also previously demonstrated that renoceptor binding and function after irreversible inactivation by after D, dopamine receptor up-regulation, rats exhibit an in- N-etboxycarbonyl-2-ethoxy- 1,2-dihydroquinoline (EEDQ). Eur. J. crease in spontaneous locomotor activity (Hess et al., 1986a). Pharmacol. 116: 175-178. Following D, dopamine receptor up-regulation, however, nei- Andersen,P. H.,andE. B. Nielsen (1986) Thebenzazepine, SCH23390, inhibits ‘H-NPA binding in mouse brain in vivo. Acta Pharmacol. ther an increase nor decrease in spontaneous activity has been Toxicol. 59: 3 15-3 18. observed (Tarsy and Baldessarini, 1974). Thus, enhanced D, Andersen,P. H., E. B. Nielsen,F. C. 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Neurosci. 3: 23-4 1. the hypothesis (Barone et al., 1986; Waddington, 1986) that D, Barone, P., T. A. Davis, A. R. Braun, and T. N. Chase (1986) Do- dopamine receptor activation may provide a tonic background paminergic mechanisms and motor function: Characterization of D- 1 activation that may be equated with an overall level of arousal. and D-2 recentor interactions. Eur. J. Pharmacol. 123: 109-l 14. In this model, additional D, dopamine receptor stimulation may Battaglia, G., A. B. Norman, E. J. Hess, and I. Creese (1985) D-2 be associated with the development of overt stereotyped be- dopamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in rat striatum. Neurosci. Lett. 59: 177-182. havior, which may be regulated by the level of D, dopamine Battaglia, G., A. B. Norman, P. L. Newton, and I. 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