completely blocked the effect of apomorphine. These results indicate that DA D-1 and D-2 receptors mediate circling behaviour through separate mechanisms ...
Psychopharmacology
Psychopharmacology (1985) 85:346-352
springer-Verlag 1985
Differential involvement of dopamine D-1 and D-2 receptors in the circling behaviour induced by apomorphine, SK & F 38393, pergolide and LY 171555 in 6-hydroxydopamine-lesioned rats Jorn Arnt and John Hyttei
Department of Pharmacology and Toxicology, H. Lundbeck A/S, Ottiliavej 7-9, DK-2500 Copenhagen-Valby, Denmark Abstract. The antagonistic effect of dopamine (DA) D-1 and D-2 antagonists against circling behaviour induced by various DA agonists in 6-OHDA-lesioned rats has been investigated. DA D-l/D-2 selectivity of agonists in vitro was measured by the stimulatory effect on DA-sensitive adenylate cyclase in rat striatal homogenates (D-l), the inhibitory effect on electrically-induced release of 3H-DA in rabbit striatal slices (D-2) and the affinity to 3H-piflutixol (D-l) and 3H-spiroperidol (D-2) binding sites in rat striatal membranes. The contralateral circling behaviour induced by the DA D-1 agonist SK & F 38393 was blocked by the DA D-1 antagonist, SCH 23390, and by the mixed DA D-l/D-2 antagonist cis(Z)-flupentixol, but was not influenced by the DA D-2 antagonists spiroperidol and clebopride. In contrast, circling behaviour induced by the preferential DA D-2 agonists pergolide and LY 171555 was blocked by clebopride, spiroperidol, and cis(Z)-flupentixol, but weakly or not influenced by SCH 23390. Apomorphine-induced circling behaviour was blocked by cis(Z)-flupentixol, partially antagonized by SCH 23390 and clebopride but not inhibited by spiroperidol, although the time-course of circling was changed. Combinations of SCH 23390 with spiroperidol or clebopride in low doses completely blocked the effect of apomorphine. These results indicate that DA D-1 and D-2 receptors mediate circling behaviour through separate mechanisms which can be independently manipulated with respective agonists and antagonists. Furthermore, the results indicate that both DA D-1 and D-2 receptors are involved in the effect of apomorphine, since selective antagonists induced maximally 50% inhibition. Complete blockade was only found in combination experiments and by the mixed D-l/D-2 antagonists cis(Z)-flupentixol, cis(Z)-clopenthixol, and clozapine. Key words: DA D-1 receptors - DA D-2 receptors Circling behaviour - Dopamine agonists - Dopamine antagonists - 6-OHDA lesions - Rat
In vitro experiments indicate the presence of at least two populations of dopamine (DA) receptors, D-1 and D-2, according to the nomenclature proposed by Kebabian and Calne (1979). The D-1 receptor is associated with adenylate cyclase in a stimulatory manner and can be labelled with the neuroleptic thioxanthene ligands 3H-piflutixol or 3H-cis(Z)-flupentixol, whereas D-2 receptors are indepen-
Offprint requests to: J. Arnt
dent of adenylate cyclase or coupled in an inhibitory manner (Stool and Kebabian 1982) and can be labelled with the butyrophenone ligands 3H-spiroperidol or 3H-haloperidol (Hyttel 1978, 1980, 1981a, b, 1983). Using this classification, it has been shown that the standard DA agonist apomorphine has effects on both types of DA receptors in vitro (Kebabian and Calne 1979; Hyttel 1980), but the functional consequences in vivo of this mixed effect are unclear. As no stimulant effects of the selective DA D-1 agonist SK & F 38393 (Tsuruta et al. 1981; Stoof and Kebabian 1982) have been reported in normal rats (Setler et al. 1978), whereas preferential D-2 agonists such as the ergot derivatives LY 141865 and pergolide (Stoof and Kebabian 1982) have potent stimulant activity (Koller et al. 1980; Fuller et al. 1982, 1983; Titus et al. 1983), the D-2 agonistic effect of apomorphine has been considered as most important. This view is also supported by the complete apomorphine-antagonistic effect of selective DA D-2 antagonists (e.g., benzamides) in studies of stereotypy (e.g., Fleminger et al. 1983; Usuda et al. 1981). However, recent studies using the selective DA D-1 antagonist SCH 23390 (Hyttel 1983; Iorio et al. 1983) have complicated the latter argument, since this compound also had potent apomorphine-antagonistic activity in stereotypy experiments (Iorio et al. 1983; Christensen et al. 1984). After unilateral 6-hydroxy-DA (6-OHDA) lesions both DA D-1 and D-2 receptor agonists induce contralateral circling behaviour in similar intensity to that of apomorphine (Setler et al. 1978; Gower and Marriott 1982; Gershanik et al. 1983; Herrera-Marschitz and Ungerstedt 1984; Arnt and Hyttel 1984), but it has been suggested that the DA receptor may lose its structural specificity following 6-OHDA pretreatment (Costall and Naylor 1976), However, recent antagonist studies indicate that antagonist specificity is retained for inhibition of SK & F 38393 and pergolide-induced circling behaviour using SCH23390 (Arnt and Hyttel 1984) and the selective butyrophenone and benzamide D-2 blocking neuroleptics, respectively (Arnt and Hyttel 1984; Herrera-Marschitz and Ungerstedt 1984). This specificity opens the possibility of evaluating the relative importance of DA D-l/D-2 receptors in the effects of apomorphine on circling behaviour, which was the subject of this investigation. Materials and methods
Animals. Male Wistar (Mol 9Wist) SPF rats (170-210 g at the time of operation) were used. They were housed
347 conventionally in Macrolon type III cages in groups of four to five in animal rooms at 21 _+ 1~ C with a relative humidity of 55 + 5%, air exchanges (16 times/h) and day/night cycle (6 a . m . - 6 p.m.). They had free access to commercial food pellets and tap water. Rabbits (both sexes, Danish Landrace 1.5-3 kg) were housed individually under similar conditions, except that the temperature was kept at 18 + 1~ C.
Circling behaviour in 6-hydroxydopamine-lesioned rats. Unilateral lesions were made in pentobarbital-anaesthetized rats by injection of 6-OHDA, HC1 9.7~tg/4~d (equivalent to 8 gg free base) per 4 rain into the median forebrain bundle rostrally to substantia nigra. The saline solution contained ascorbic acid 0.2 mg/ml, was bubbled with N 2 and kept ice-cold. Stereotaxic coordinates were A 3 . 5 - 4 . 0 L 1.0-1.2 V - 2 . 5 - 3 . 0 (K6nig and Klippel 1963) with incisor bar 3 mm below the interaural line. The experiments were made 1 - 5 months after lesioning when stable contralateral circling responses to SK & F 38393 (4.3 ~tmol/kg SC), pergolide (0.05 gmol/kg SC), LY 171555 (0.31 ~tmol/kg SC), and apomorphine (0.26 ~tmol/kg SC) were obtained. Injections were made in the neck. These doses of agonists were chosen as the lowest inducing maximal response and were 2 . 5 - 4 times higher than the ED50 values of each agonist. The circling response was measured in automated rotometer bowls and was recorded every 5 - 1 0 rain during 1 - 2 h, except in selected experiments showing the time-course of agonists, where circling was measured every 2 rain. Only rats showing more than 400 complete turns in 60rain (apomorphine), 90rain (pergolide), or 2 h (SK & F 38393 and LY 171555) control sessions were used. Potencies of agonists were determined by substituting apomorphine (0.26 ~mol/kg SC) with different doses of test compounds using the apomorphine response as the 100% effect level. Inhibition dose-response curves were obtained by alternating test and control sessions on a weekly basis. Antagonists were injected SC 2 h before the agonist except for clozapine and SCH 23390 (1 h). The effect of individual test drug doses was calculated as per cent of the mean effect of control sessions 1 week before and after the test session. At least three doses were tested using 4 to 12 rats per dose. EDso values were calculated by log-probit analysis.
DA receptor binding. Inhibition of binding of 3H-piflutixol (Nuclear Research Centre, Negev, Israel: 8 - 1 2 Ci/mmol) and 3H-spiroperidol (New England Nuclear. Dreieich, FRG, 2 7 - 3 0 Ci/mmol) to rat striata! membranes was estimated by methods very similar to previously described ones (Hyttel 1978, 1981a, b, 1982). In both methods the final pellet was homogenized in 200 volumes of buffer (4 mg original wet weight per sample) and the ligand concentration was 0.5 riM. Incubation times were 30 and 10 rain for 3H-piflutixoI and 3H-spiroperidol, respectively. In the 3H-piflutixol assay all samples contained 30 nM spiroperidol to exclude any binding to DA D-2 receptors (Hyttel 1981b, 1982). IC50-values were determined and transformed to Ki-values by the Cheng and Prusoff equation (1973).
Stimulation of DA-sensitive adenylate cyclase. DA-stimulated adenylate cyclase activity was determined in rat striatal homogenate as described in detail by Hyttel (1978)
except that DA was replaced by drugs in concentrations from 10 -s to 10 -4 M. The increase in c-AMP over basal level was calculated and EDs0-values were determined as the concentration causing half of this increase.
Basal and stimulation evoked release of 3H-DA from preloaded rabbit striatal slices. The caudal part of nucleus caudatus was cut in cross 0.4 mm thick sections with a McIlwain mechanical tissue chopper. Approximately 10 slices were incubated for 30 rain at 37~ in 1 ml buffer containing 0.2 ~M 3H-DA. The buffer was composed as follows: 118mM CaC1, 4.80raM KC1, 1.30raM CaCI2, 1.20 mM MgSO4, 20.0 mM NaHCO3, 1.20 mM KH2PO4, 0.600raM ascorbic acid, 30.0 gM EDTA-Na2, l l . 0 m M glucose, saturated with 5% CO2 in Oz; pH 7.4 at 37 ~ C. The slices were placed on a polypropylene mesh in order to optimize the access of solutes and O2 to the slices. After incubation the slices were rinsed three times with 3 ml buffer and transferred to superfusion chambers, where they were placed on a polypropylene mesh (Tetex PP 500) between platinum electrodes 20ram apart. Superfusion with prewarmed (37~ C) buffer at a rate of 1.0 ml/min was carried out by means of peristaltic pump (Desaga, 132100) placed after the superfusion chambers. Tubing consisted of teflon except ih the pump, where silicone tubes were used. After passage through the chambers, the buffer was collected in 8-rain samples. At the end of superfusion, the slices were solubilized in 1.0ml Soluene-350 (Packard) (30 rain, 60 ~ C). The content of tritium in the eluates and in the tissue solution was determined by scintillation counting after addition of 10 ml Lumagel. Normally eight superfusion chambers were run in parallel. For electrical stimulation, rectangular pulses of 2 ms duration, a frequency of 2 Hz and a current of 10 mA were applied twice for 2 rain each. The first stimulation (Sa) was applied 112 min (14 fractions) and the second ($2) 224 rain (28 fractions) after the start of elution. Stimulation was checked on an oscilloscope. Drug was added between $I and $2, 168 min (21 fractions) after the start of elution. Basal outflow of tritium was expressed as fractional release rate (FRR), i.e., (nCi efflux per 8 min)/(nCi tritium in the slice at the beginning of this 8-min period). The stimulation-evoked overflow of tritium was calculated by subtraction of the basal outflow. The peak of outflow covered five fractions. The calculation for DA release is thus given by (amount of tritium in the five fractions minus the mean of tritium in the preceeding and the following fraction)/(amount of tritium in the fraction at the onset of stimulation). The ratio $2/S~ was calculated and an effect of drug was seen as a deviation from the ratio in control experiments. Effects of agonists were expressed as the concentrations causing a diminution of $2/$1 to 50% of $2/$1 for control experiments (ECs0-values). For each drug three to five concentrations were tested three to seven times.
Drugs. The following drugs were dissolved in saline: SK & F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-lH3-benzazepine, HC1) (Smith, Kline and French, USA); cis(Z)-flupentixol, 2 HC1 (Lundbeck), cis(Z)-clopenthixol, 2 HC1 (Lundbeck), SCH 23390 [(R)-(+)-8-chloro-2,3,4,5tetrahydro-3-methyl-5-phenyl-lH-3-benzazepin_7_ol, hemimaleate, Schering, USA, LY 171555 (trans-(-)4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo [3,4-g] quinoline, HC1 (Lilly, USA) and apomorphine, HC1
348 (Ph.N.: containing 0.02% ascorbic acid)]. Pergolide, mesylate (Lilly, USA) and clebopride, maleate (AlmiraU, Spain) were dissolved in distilled water. Spiroperidol (Janssen, Belgium) and clozapine (Wander, Switzerland) were dissolved in minimal amounts of 0,1 N tartaric or hydrochloric acid, respectively, and diluted with saline. Injection volumes were 5 ml/kg body weight.
Results
r"
Effects ofagonists.
The affinity of DA agonists for DA D-1 and D-2 receptors is shown in Table 1. Also shown are results in the two functional tests for D-1 and D-2 receptors, stimulation of adenylate cyclase and reduction of electrically-induced release of 3H-DA from preloaded striatal slices, respectively. It is seen that SK & F 38393 is a relatively potent displacer of 3H-piflutixol and has 150 times weaker affinity for 3H-spiroperidol binding sites. The opposite profile was obtained with pergolide and LY 171555 [the active enantiomer of LY 141865 (Titus et al. 1983; Wong et al. 1983)] which were 270 and 19 times more potent against 3H-spiroperidol than 3H-piflutixol binding, respectively. Apomorphine had affinity for both binding sites, but with a preference for D A D-2 sites. Striatal D A sensitive adenylate cyclase was stimulated by SK & F 38393 and apomorphine, whereas pergolide and LY 171555 were ineffective. 3H-DA release induced by electric stimulation of rabbit caudate slices was potently inhibited by LY pergolide, 171555 and apomorphine, whereas SK & F 38393 was ineffective. The D A agonists all induced contralateral circling behaviour in rats with unilateral 6-OHDA lesions. Apomorphine, pergolide and LY 171555 were almost equipotent, whereas SK & F 38393 was less potent (Table 1). Examples of the time-effect curves after a maximally effective dose are shown in Fig. 1. Pergolide (0.05 ~tmol/kg SC), LY 171555 (0.31 ~tmol/kg SC) and SK & F 38393 (4.3 ~tmol/kg SC) induced circling with a single maximum. SK & F 38393 had always delayed onset of action of about 20-30 min. In contrast, apomorphine (0.26 ~tmol/kg SC) induced a complex two-peak rotational pattern with one maximum observed within the first 10 min and another one after 4 0 - 5 0 min. The duration of action (not shown) was about 4 h for SK & F 38393, more than 4 h for pergolide and LY 171555 and 60-70 min for apomorphine.
Effect of antagonists. The
inhibitory effect of the D A D-1 antagonist SCH 23390, the DA D-2 antagonists spiroperidol and clebobride, and of the mixed DA D-l/D-2 antagonist cis(Z)-flupentixol against the agonist-induced contralateral circling behaviour was studied in the next series of experiments. The total number of contralateral turns induced in the absence and presence of antagonist during the test period was used for determination of percentage inhibition as shown in Fig. 2. The circling induced by SK & F 38393 (4.3 ~tmol/kg SC) was dose-dependently antagonized by SCH 23390 (EDs0 0.006 ~tmol/kg SC) and cis(Z)-flupentixol (ED50 0.15 ~mol/kg SC) but inhibited only by high and supramaximal cataleptogenic doses of clebopride (EDs019 jxmol/kg SC) or spiroperidol (EDs0 14 ~tmol/kg SC). Apomorphine (0.26 ~tmol/kg SC) was partially (about 50%) inhibited by SCH 23390 (0.23-3.6 ~tmol/kg SC) without dose-dependency. A similar partial effect was observed after clebopride pretreatment in high doses (2.6-10 gmol/kg SC), whereas spiroperidol (0.10-3.2 ~tmol/kg SC) had no significant effect. In contrast, cis(Z)-flupentixol (EDs0 0.23 ~tmol/kg SC) dose-dependently antagonized the effect of apomorphine (Fig. 2). TURNS/2
MIN
0
30
to 60
90
0
30
60
SK &F
5(~
at= . . . .
g ~
phine
3 ; - ..... 6-~
96
120 MIN
38393
y ~ f ~ U~@~
SO
0~--~6
'
' .'8 ' - '
9; ' - '
,~o
MIN
Fig. 1. Examples of time-effect curves of different dopamine agonists inducing contralateral circling behaviour in 6-OHDAlesioned rats. Pergolide (0.05 ~xmol/kg SC), LY 171555 (0.31 Ixmol/kg SC), apomorphine (0.26 ~tmol/kg SC), or SK & F 38393 (4.3 ~xmol/kgSC)was injected immediately before the start of circling recording in 2-min intervals for 1-2 h
Table 1. Effect of dopamine (DA) agonists in test models detecting DA D-1 and D-2 receptor activity in vitro compared with the behavioural effect in vivo Compound
Adenylate cyclase D-1 EC50 (nM)
3H-DA-release D-2 ECs0 (nM)
3H-PIF D-1 Ki (nM)
3H-SPI D-2 Ki (nM)
Circling behaviour in vivo EDs0 (~tmol/kg SC)
Apomophine SK & F 38393 Pergolide LY 171555
6.800 74 >100.000 >100.000
59 > 10.000 5.6 53
360 72 1.600 13.000
23 11.000 6.0 670
0.058 1.6 0.021 0.078
For details, see Methods
oo1
349
SK & F 38393
\
50
O
.004.01.06 s .9 APOMORPHINE
4
.01 .04 .2 ,6
,01 .05
.01 .04 .16 .7 3 l 0 4 0
.2 .8 3
13
\ 1r
I-
Z 0 o IZ
w IO~ t U n,
Fig. 2. Inhibition by dopamine antagonists of circling behaviour in 6-OHDA-lesioned rats. Each vertical column shows the effect of each antagonist (indicated in the bottom together with the doses in ~tmol/kg SC along the abscissa) against SK & F 38393 (4.3 ~tmol/kg SC), apomorphine (0.26 ~tmol/kg SC), pergolide (0.05 ~xmol/kg SC), or LY 171555 (0.31 ~tmol/kg). The ordinate indicates the mean -+ SEM (n = 4-12)% of the circling induced by the agonist alone. The mean 100% level circling behaviour for each group was 1,500-2,000 turns for SK & F 38393 (2-h test period), 625-800 for apomorphine (1 h test period), 1,000-1,200 for pergolide (90-rain test period) and 1,200-1,700 for LY 171555 (2-h test period)
P E R G O L IDE
L
5
~
J LY 173,555
~176 t,
\
\
,004.01.06 .2 .9 4
SCH 23390
.dl .o'5.2 .:s :~ 13
.O| .04.16 .7 3 10 4 0 C I$(Z)-tFPT
SPIROPERIDOL
CLEBOPRIDE
TURNS. MIN-1 2o
2o
IO
lo
--
10
CIS
2"0 3 0 4"0 (Z) F L U P E N T I X O L
50 ~ 1 ~
I ~
-~qr
1
0
~
~
5
'
~
'
~
ClS (Z)-CLO PENTH IX O L
10
20 30 40 CLOZAPIN E
50
60MIN
Fig. 3. Inhibition of apomorphine-induced circling behaviour in 6-OHDA-lesioned rats. Cis(Z)-flupentixol [0.16 ( e - - - e ) or 0.31 (~ ~) Bxmol/kg SC] and cis(Z)-clopenthixol [0.65 ( e - - - e ) or 2.6 (I, ~) ~tmol/kg SC] were injected 2 h before apomorphine (0.26 ~tmol/kg SC) whereas clozapine [3.8 ( e - - - e ) or 15 (~---I,) ~tmol/kg SC] was injected 1 h before apomorphine. Control responses with apomorphine alone are indicated in each figure (e e). The values indicate the mean __+SEM circling frequency of eight rats
Pergolide (0.05 ~tmol/kg SC)-induced circling was only partially inhibited by a highly cataleptogenic dose of SCH 23390 (3.6 ~mol/kg SC), whereas cis(Z)-flupentixol (EDs0 0.03 gmol/kg SC), clebopride (EDs0 0,03 ~tmol/kg SC) and spiroperidol (EDs0 0.04 ~tmol/kg SC) dose-dependently blocked the effect of pergolide (Fig. 2). A similar antagonist profile was obtained with the other reference D A D-2 agonist, LY 171555 (0.31 ~tmoI/kg SC). SCH 23390 had only weak antagonistic effect after a high dose (3,6 [xmol/kg SC), whereas cis(Z)-flupentixol, clebopride, and spiroperidol showed high antagonistic potency (ED50 values 0.04, 0.2, and 0.03 ~tmol/kg SC, respectively) (Fig. 2). The time-effect pattern for antagonist inhibition of circling behaviour was also considered, For SK & F 38393,
pergolide and LY 171555, which all induced regular time-effect curves, no further differentiation between antagonists was found compared with that obtained above using cumulated number of turns (data not shown). However, for apomorphine, which showed two-peak circling behaviour, determination of time-effect curves revealed further differences between the antagonists: Mixed D A D-l/D-2 antagonists, such as cis(Z)-flupentixol, cis(Z)-clopenthixol, and clozapine antagonized apomorphine-induced circling behaviour during the total test period as shown in Fig. 3. SCH 23390 also decreased circling frequency during the whole experiment, but without dose-dependent antagonism (Fig. 4). In contrast, both low and high doses of the DA D-2 antagonists clebopride and spiroperidol modified the
350 TURNS.MIN
-1
20
20
1(
10
6
I0
20 30 40 SCH 23390
5"0
20
l
0"-
60
10-
\
2'0 3~--- 40 SPIROPERIDOL
5'0
6'0
1'0
2'0
3'0
4'0
5'0
6'OMIN
S C H 23390~. SPI ROPE R I DOL
20
20
'~
10
o
10
o
~'~--~'o~'6--g'~g"o CLEBOPRIDE
0~
10 20 30 4'0 50 S C H 23390 + C L E B O P R I D E
60'MIN
Fig. 4. Inhibition of apomorphine-induced circling behaviour in 6-OHDA-lesioned rats. SCH 23390 [0.23 ( 0 - - - 0 ) or 3.6 (k k) ~tmol/kg SC], spiroperidol [0.2 ( O - - - 0 ) or 0.8 (~ F) ~mol/kg SC] and clebopride [0.63 (0 0) or 2.5 (~ ~) gmol/kg SC] were injected 1 h before apomorphine (0.26 ~mol/kg SC). In the combination experiments SCH 23390 [0.06 (O- - - 0) or 0.23 (F ~) ~mol/kg SC] was injected together with spiroperidol (0.05 and 0.2 ~tmol/kg SC, respectively) or clebopride (0.16 and 0.63 ~tmol/kg SC, respectively) 1 h before apomorphine. Control responses with apomorphine alone are also indicated in each figure (0 0). The values indicate the mean + SEM circling frequency of eight rats two-peak circling pattern induced by apomorphine into a single peak of high circling frequency situated between the two peaks obtained in control sessions (Fig. 4). When the DA D-1 antagonist SCH 23390 was combined with equieffective doses of the DA D-2 antagonists, spiroperidol ( 1 : 1 combination with SCH 23390 on mgbasis) or clebopride (4 : 1 combination with SCH 23390 on mg-basis) the effect of apomorphine was now dose-dependently blocked by low doses which did not inhibit or only partially inhibited apomorphine when given alone (compare Figs. 2 and 4).
Discussion Our results indicate that contralateral circling behaviour induced at supersensitive DA receptors in 6-OHDAlesioned rats can be used to differentiate agonists or antagonists acting on D A D-1 and D-2 receptors. The effect of SK & F 38393 was blocked by the selective DA D-1 antagonist, SCH 23390 (Hyttel 1983; Iorio et al. 1983; Christensen et al. 1984) and by other DA antagonists with high D A D-1 receptor affinity (Hyttel 1978, 1980, 1981a, 1983), such as cis(Z)-flupentixol cis(Z)-clopenthixol, clozapine, loxapine, and clothiapine (present results; Arnt and Hyttel 1984; Gower and Marriott 1982). Compounds with selective D A D-2 receptor affinity, such as clebopride, spiroperidol, pimozide, and haloperidol (Hytte11980, 1983) were weak or ineffective (present results, Gower and Marriott 1982). This is consistent with the in vitro selectivity ratio of about 150 of SK & F 38393 at D-1 versus D-2 receptors (Table 1). The reverse antagonist specificity was obtained in the pergolide and LY 171555 experiments, where all neuroleptics with high DA D-2 receptor affinity were potent
blockers, whereas SCH 23390 showed only partial inhibition even at high dosages. This indicates that the D-l/D-2 selectivity ratio of SCH 23390 (a factor 700) obtained in vitro (Hyttel 1983) is retained in vivo. LY 171555 (or rather its racemate, LY 141865) has been considered as the most selective D A D-2 agonist in vitro (Tsuruta et al. 1981), whereas pergolide is considered as an agonist at both DA D-1 and D-2 receptors, since it has some, albeit weak, stimulating activity on adenylate cyclase (Goldstein et al. 1980; Wong and Reid 1980). However, when considering relative binding affinities for DA D-1 and D-2 receptor sites, a D-2/D-1 selectivity ratio of 250 is obtained for pergolide which is higher than that of LY 171555. The lower ratio of LY 171555 is primarily caued by its relatively weak in vitro binding potency, which does not correspond to the potent effect in vivo (Wong et al. 1983; Titus et al. 1983; present results). If the functional in vitro tests for DA D-1 and D-2 receptor activity (adenylate cyclase and 3H-DA release) are considered, both LY 171555 (present results) and pergolide (Starke et al. 1983; Lehmann et al. 1983; present results) can be classified as selective DA D-2 agonists. This is consistent with the behavioural experiments, where pergolide and LY 171555 seem to be equally selective agonists on DA D-2 receptors, and is in agreement with other authors considering pergolide as a D A D-2 agonist in circling experiments (Herrera-Marschitz and Ungerstedt 1984). The results obtained with apomorphine were more complex. First of all, apomorphine - but not the other agonists - had a time-effect curve with two peaks, as also demonstrated previously by others (Ungerstedt 1971; Oberlander et al. 1980; Coward 1983; Herrera-Marschitz and Ungerstedt 1984). This has been taken as an indication of a dual mechanism of action of apomorphine and also involves a pharmacologic kindling phenomenon developing
351 after several apomorphine injections (Coward 1983). Antagonist studies indicated the complexity of apomorphine-induced circling, except when using unselective D A D - l / D - 2 antagonists, e.g., cis(Z)-flupentixol and c/s(Z)-clopenthixol. Such compounds completely antagonized the effect of apomorphine after relatively low doses (present results; Herrera-Marschitz and Ungerstedt 1984; Coward, personal communication). In contrast, selective D A D-2 antagonists, e.g., haloperidol, spiroperidol, and clebopride, had only weak or no antagonistic effect on apomorphine even after high doses, and furthermore converted the two-peak time-effect curve into a single peak of high circling intensity situated in between of the two circling peaks in control experiments (Oberlander et al. 1980; Coward 1983 and personal communication; Herrera-Marschitz and Ungerstedt 1984; present results). The D A D-1 antagonist SCH 23390 abolished the second circling peak of apomorphine but induced only about 50% inhibition of circling even at doses far above those necessary for blockade of SK & F 38393-induced circling. More direct evidence for a mixed D A D-l/D-2 receptor involvement in apomorphine-induced circling came from experiments using the D A D-1 antagonist SCH 23390 in combination with a D A D-2 antagonist in equieffective doses, as shown with clebopride and spiroperidol. Apomorphine-induced circling was dose-dependently blocked by low doses of SCH 23390 plus spiroperidol (1 : 1 dose on mg-basis) or of SCH 23390 + clebopride (1 : 4 dose on mg-basis). The effect of these combinations was greater than the maximally possible effect of any of the compounds alone and the potency was increased to a level comparable with or higher than that of cis(Z)-flupentixol. This indicates that the antagonist combinations act by more than an additive effect, and that apomorphine in the dose used in the present experiment stimulates both D A D-1 and D-2 receptors, although the in vitro data indicate preferential D A D-2 receptor stimulation. Motor impairment does not contribute to the differentiation of the antagonists, but may be responsible for the inhibitory effects seen after very high antagonist doses in Fig. 2, e.g., clebopride and spiroperidol against SK & F 38393. The D A D-1 and D-2 antagonists used in this study induce catalepsy of similar intensity and potency, with EDs0 values ranging between 0.15 and 0.6 ~mol/kg SC (Arnt and Hyttel 1984), and catalepsy would therefore interfere to a similar extent in all circling models. It was remarkable that rats pretreated with supramaximal cataleptogenic doses of antagonists in many instances could be circling vigorously within few minutes after agonist administration (see, e.g., the effect of apomorphine in spiroperidol or clebopride-pretreated rats in Fig. 4 and compare the above mentioned EDs0 values for catalepsy with the doseresponse curves in Fig. 2. It should be noted that the differential effects obtained with D A D-1 and D-2 receptor antagonists, alone and in combination, depend on the 6-OHDA-induced denervation. No differentiation between SCH 23390 and D A D-2 antagonists has been seen in normal rats when apomorphine, pergolide, LY 171555, R U 24213 or amphetamine are used for induction of stereotyped behaviour: SCH 23390 and D A D-2 antagonists potently block the effect of either agonist equally well (Usuda et al. 1981; Fleminger et al. 1983; Iorio et al. 1983; Christensen et al. 1984; O'Boyle et al. 1984; Arnt unpublished observations),
indicating that blockade of either D A D-1 or D-2 receptors is sufficient for antagonism of D A agonist (even a D-2 agonist)-induced stereotypy. This suggests close connections between D A D-1 and D-2 receptor-mediated effects. As mentioned above, both D-1 and D-2 antagonists also induce catalepsy. The reason for this difference between normal and denervated D A receptors is unknown. Previously, it has been suggested that striatal D A receptors involved in circling lose their structural specificity for agonists after denervation (Costall et al. 1976), but this is inconsistent with our results. One possible explanation of the denervation-induced D-l/D-2 selectivity may be an uncoupling of D A D-1 and D-2 receptors which in normal, innervated synapses may be closely connected. This would imply that the D A D-l/D-2 specificity should be present within the same striatal site and is presently being investigated using local injection of selective D A agonists and antagonists into 6-OHDA-denervated striata. Alternatively, the circling behaviour induced by D A D-1 and D-2 agonists may be mediated by different brain sites, from which circling is induced by D A or D A agonists only after denervation. Apomorphine has been shown to induce circling in denervated rats at a number of sites normally having D A terminals (Kozlowski et al. 1980; Starr and Summerhayes 1982) but this has not been investigated with selective D A D-1 and D-2 agonists and antagonists. In conclusion, our data indicate that apomorphine acts at two independent sites in 6 - O H D A denervated rats. Therefore, caution is needed when using apomorphine as a reference D A agonist in the evaluation of D A antagonists with different D A D-l/D-2 selectivity. More reliable results may be obtained by parallel use of selective agonists.
Acknowledgements.
We thank Sonja Jacobsen, Birgit Smedegaard, Tom Halborg, Birgitte Sander Nielsen, Jytte Pedersen and Arne Engholm Pedersen for technical assistance and Kirsten Lindgren for secretarial help. The companies listed in Materials and methods are thanked for providing test compounds.
References Arnt J, Hyttel J (1984) Differential inhibition by dopamine D-1 and D-2 antagonists of circling behaviour induced by dopamine agonists in rats with unilateral 6-hydroxydopamine lesions. Eur J Pharmacol 102:349-354 Cheng, Y-C, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099-3108 Christensen AV, Arnt J, Hyttel J, Larsen J-J Svendsen O (1984) Pharmacological effects of a specific dopamine D-1 antagonist SCH 23390 in comparison with neuroleptics. Life Sci 34 : 1529-1540 Costall B, Naylor RJ, Pycock C (1976) Non-specific supersensitivity of striatal dopamine receptors after 6-hydroxydopamine lesion of the nigrostriatal pathway. Eur J Pharmacol 35 : 275-283 Coward DM (1983) Apomorphine-induced biphasic circling behaviour in 6-hydroxydopamine-lesioned rats. A pharmacological kindling phenomenon. Naunyn-Schmiedeberg's Arch Pharmacol 323:49-53 Fleminger S, Van de Waterbeemd H, Rupniak NMJ, Reavill C, Testa B, Jenner P, Marsden CD (1983) Potent lipophilic substituted benzamide drugs are not selective D-1 dopamine receptor antagonists in the rat. J Pharm Pharmacol 35 : 363-368
352 Fuller RW, Clemens JA, Hynes III MD (1982) Degree of selectivity of pergolide as an agonist at presynaptic versus postsynaptic dopamine receptors: Implications for prevention or treatment of tardive dyskinesia. J Clin Psychopharmacol 2:371-375 Fuller RW, Hemrick-Luecke SK, Wong DT, Pearson D, Threlkeld PG, Hynes III MD (1983) Altered behavioral response to a D2 agonist, LY 141865, in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normotensive rats. J Pharmacol Exp Ther 227: 354- 359 Gershanik O, Heikkila RE, Duvoisin RC (1983) Behavioral correlations of dopamine receptor activation. Neurology 33 : 1489-1492 Goldstein M, Lieberman A, Lew JY, Asano K, Rosenfeld MR, Makman MH (1980) Interaction of pergolide with central dopaminergic receptors. Proc Natl Acad Sci USA 77: 3725- 3728 Gower AJ, Marriott AS (1982) Pharmacological evidence for the subclassification of central dopamine receptors in the rat. Br J Pharmacol 77: 185-194 Herrera-Marschitz M, Ungerstedt U (1984) Evidence that apomorphine and pergolide induce rotation in rats by different actions on D1 and D2 receptor sites. Eur J Pharmacol 98:165-176 Hyttel J (1978) Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro. Life Sci 23:551-556 Hyttel J (1980) Further evidence that 3H-cis(Z)-flupenthixol binds to the adenylate cyclase-associated dopamine receptor (D-l) in rat corpus striatum. Psychopharmacology 67:107-109 Hyttel J (1981a) Similarities between the binding of 3H-pifiutixol and 3H-flupentixol to rat striatal dopamine receptors in vitro. Life Sci 28:563-569 Hyttel J (1981b) Flupentixol and dopamine receptor selectivity. Psychopharmacology 75 : 217 Hyttel J (1982) Preferential labelling of adenylate cyclase coupled dopamine receptors with thioxanthene neuroleptics. In: Kohsaka M, Shohmori T, Tsukuda Y, Woodruff GN (eds) Advances in dopamine research. Pergamon Press, Oxford, Advances in the Biosciences. pp 147-152 Hyttel J (1983) SCH 23390 - the first selective dopamine D-1 antagonist. Eur J Pharmacol 91:153-154 Iorio LC, Barnett A, Leitz FH, Houser VP, Korduba CA (1983) SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. J Pharmacol Exp Ther 226 : 462-468 Kebabian JW, Calne DB (1979) Multiple receptors for dopamine. Nature 277 : 93-96 Koller WC, Weiner WJ, Diamon BI, Nausieda PA, Klawans HL (1980) The pharmacological evaluation of pergolide mesylate as a potential anti-parkinson agent. Neuropharmacology 19:831-837 K6nig JFR, Klippel RA (1963) The rat brain. Williams and Wilkins, Baltimore
Kozlowski MR, Sawyer S, Marshall JF (1980) Behavioural effects and supersensitivity following nigral dopamine receptor stimulation. Nature 287: 52-54 Lehmann J, Briley M, Langer SZ (1983) Characterization of dopamine autoreceptor and [3H]-spiperone binding sites in vitro with classical and novel receptor agonists. Eur J Pharmacol 88 : 11-26 Oberlander C, Dumont C, Boissier JR (1980) Time course of apomorphine-induced circling behaviour after striatal dopamine receptor denervation. Eur J Pharmacol 62:107-110 O'Boyle KM, Pugh M, Waddington JL (1984) Stereotypy induced by the D2 dopamine agonist RU24213 is blocked by the D2 antagonist RO22-2586 and the Da antagonist SCH 23390. Br J Pharmacol 82:242 Setler PE, Sarau HM, Zirkle CL, Saunders HL (1978) The central effects of a novel dopamine agonist. Eur J Pharmacol 50 : 419-430 Starke K, Spfith L, Lang JD, Adelung C (1983) Further functional in vitro comparison of pre- and postsynaptic dopamine receptors in the rabbit caudate nucleus. Naunyn Schmiedeberg's Arch Pharmacol 323:298-306 Starr MS, Summerhayes M (1982) Multifocal brain sites for apomorphine-induced circling and other stereotyped motor behaviour in the 6-hydroxydopamine-lesioned rat. Neurosci Lett 34 : 277-282 Stool JC, Kebabian JW (1982) Independent in vitro regulation by the D-2 dopamine receptor of dopamine-stimulated effiux of cyclic AMP and K+-stimulated release of acetylcholine from rat neostriatum. Brain Res 250:263-270 Titus RD, Kornfeld EC, Jones ND, Clemens JA, Smalstig EB, Fuller RW, Hahn RA, Hynes III MD, Mason NR, Wong DT, Foreman MM (1983) Resolution and absolute configuration of an ergoline-related dopamine agonist, trans-4,4a,5,6,7,8,8a,9octahydro-5-propyl-lH (or 2H)-pyrazolo[3,4-g]quinoline. J Med Chem 26:1112-1116 Tsuruta K, Frey EA, Grewe CN, Cote TE, Eskay RL, Kebabian JW (1981) Evidence that LY 141865 specifically stimulates the D-2 dopamine receptor. Nature 292:463-465 Ungerstedt U (1971) Postsynaptic supersensitivity after 6hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand Suppl 367:69-93 Usuda S, Nishikori K, Noshiro O, Maeno H (1981) Neuroleptic properties of c/s-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5chloro-2-methoxy-4-methylaminobenzamide (YM-09151-2) with selective antidopaminergic activity. Psychopharmacology 73 : 103-109 Wong DT, Reid LR (1980) Activation of adenylate cyclase in rat striatum by an ergoline dopamine agonist, pergolide. Commun Psychopharmacol 4: 269-275 Wong DT, Bymaster FP, Reid LR, Fuller RW, Perry KW, Kornfeld EC (1983) Effect of a stereospecific D2-dopamine agonist on acetylcholine concentration in corpus striatum of rat brain. J Neural Transm 58:55-67 Received July 3, 1984; Final version October 23, 1984
