European Journal of Pharmacology, 186 (1990) 79-86
79
Elsevier EJP 51470
The interaction of c|ozapine with dopamine D 1 versus dopamine Dz receptor-mediated function: behavioural indices A n g e l a M. M u r r a y a n d J o h n L. W a d d i n g t o n Department of ClinicalPharmacology, Royal Collegeof Surgeons in Ireland St. Stephen's Green, Dublin 2, Ireland Received 4 May 1990, accepted 19 June 1990
Studies were undertaken to clarify further the mechanism(s) of action of the atypica~ neuroleptic clozapine, using a behavioural model with the ability to distinguish between relative antagonism of D~ vs. De dopamine receptor-mediated function. Pretreatment with low doses of clozapine (2.5-25.0 mg/kg) readily antagonised intense groomLng induced by the selective D 1 agonist SK&F 77434 (0.75 mg/kg), and in a less consistent manner antagonised hyperactivities induced by the selective De agonist LY 163502 (0.05 mg/kg). In animals whose typical responses to SK&I" 77434 were antagonised by clozapine, no atypical behaviours such as vacuous chewing emerged. However, in animals whose typical responses to LY 163502 were antagonised by clozapine, a syndrome of atypical limb/body jerking was released. Despite clozapine showing comparable affinities for D~ and D e receptors in vitro, this behavioural profile shows similarities to that seen when these agonists are given after pretreatment with a selective D~ antagonist, rather than with a selective D z antagonist or with non-selechve neuroleptics. These results suggest that clozapine has some preferential though not selective action in vivo to antagonise D~ dopamine receptor-mediated function. Clozapine; Dopamine D1 receptors; Dopamine De receptors; Jerking behaviour; Grooming behaviour: (Rat)
1. Introduction
The dibenzodiazepine clozapine is an atypical neuroleptic that is attracting renewed interest. This stems from recent evidence suggesting that clozapine shows antipsychotic efficacy against both positive and negative symptoms in some schizophrenic patients who are resistant to treatment with typical neuroleptics (Kane et al., 1988), while having long been recognised for its reduced liability to induce at least acute extrapyramidal side effects (Casey, 1989). Pharmacologically, clozapine acts at more than one level of synaptic function and can block receptors for a variety of
Correspondence to: J.L. Waddhngton, Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland.
neurotransmitters (Richelson and Nelson, 1984; Hyttel et al., 1985). In relation to dopaminergic (DAergic) neurotransmission, radioligand binding studies in rodent, non-human primate and human brain, both in vitro (Andersen, 1988; Madras et alo~ 19Rg; ~t , i , ,~o,~, . . Hal! . .et .al,,. 1987; . . l::,ecld~ . . t~c-~; and in vivo (Andersen, 1988; Farde et al., 1989), indicate that clozapine exhibits similar affinities for D1 and D2 receptors. Though some neurochemical (Andersen and Braestrup, 1986, Coward et al., 1989) and pharmacological (Chipkin and Latranyi, 1987; CrisweU et al., 1989) studies have suggested that clozapine may exert some preferential blockade of Dl-mediated function, these were carried out at a non-behavioural level or under non-physiological conditions of reserpinisation or 6-hydroxydopamine lesion. This issue has yet to be investigated in terms of behaviour in the intact animal.
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
80 Because of well-established functional interactions between Dt and I)2 receptor systems (Waddington and O'Boyle, 1989; Waddington, 1989), selective D~ antagonists and selective D2 antagonists can each block the behavioural effects both of D~ and of D2 agonists in the whole animal. Thus, any antagonist action of a given neuroleptic drug on behavioural responses induced either by selective D1 or 1)2 agonists would not in itself indicate any preferential action at one subtype of DA receptor. However, we have recently identified an experimental situation with the capacAty to distinguish between blockade of D~ o r D 2 receptors, on the basis of two directions of D~:D2 interaction which appear to regulate distinct elements of typical and atypical behaviour (Murray and Waddington, 1989a,b). We describe here the application of these procedures to the in vivo study of any preferential action of clozapine at D l vs. D 2 receptor-mediated function in the intact adult rat.
2. Materials and methods
2.1. Animals Young adult male Sprague-Dawley rats (Biolabs, Ballina) were used in all experiments. The rats were housed in groups of five per cage with food and water available ad libitum and were maintained on a 12/12 h (6 a.m. on, 6 p.m. off) light/dark cycle.
2.2. Behavioural studies Rats were placed individually in perspex cages 52 x 3 9 x 18 cm and left undisturbed for a habituation period of 2.5 h. Behavioural assessments were carried out in a manner shnilar to that previously described (Murray and Waddington, 1989a,b). Immediately before and at intervals after s.c. injections of drug or vehicle, animals were assessed using a rapid timesampling behavioural check list technique. For this procedure, each tat ~vas observed hldividually for 5 s periods at 1 rain intervals over 5 consecutive rain, using an extended behavioural check list.
This vllowed the presence or absence of the following individual behaviours (occurring alone or in combination) to be determined in each 5 s period: sniffing (Sn); locomotion (L); rearing (R); grooming (Gr, of any form); intense grooming (Gri; a characteristic pattern of grooming of the face with the forepaws followed by vigorous grooming of the hind flank with the snout); chewing (Ch; directed onto any physical material); vacuous chewing (VCh; not directed onto any physical matefial)~ jerking (J; brief jerking movements of the limbs or whole body); stillness (St; motionless, with no behaviour evident). After assessment using the behavioural check list, animals were assessed using a conventional 0-6 point stereotypy rating scale: 0 = asleep or inactive; 1 = episodes of normal activities; 2 = discontinuous activity with bursts of prominent sniffing or rearing; 3 - contiauous stereotyped activity such as sniffing or rearing along a fixed path; 4 = stereotyped sniffing or rearing fixated in one location; 5 - stereotyped behaviour with bursts of licking or gnawing; 6fficontinuous licking or gnawing. This cycle was repeated at 10 min intervals. Rats were used on two occasions only, separated by a drug free inte~al of one week; on each occasion rats were randomly allocated to one of the various treatment groups..Antagonist or vehicle was given 45 min prior to agonist challenge. All assessments were made by an observer unaware of the treatment given to each animal.
2.3. Radioligand binding studies Using methods similar to those previously described (Murray and Waddington, 1989a,b), striata from similar male Sprague-Dawley rats were 11omogenised in 30 volumes of 50 mM Tris-HCl buffer, pH 7.6 at 25 ° C, and centrifuged at 10000 × g at 4 ° C for 5 min. The pellet was tv4ce resuspended, diluted and centrifuged as above. The membrane preparation was finally resuspended at 4-8 mg original wet weight/ml in TrisHCI buffer containing (raM): 120 NaCI, 5 KCI, 1 MgCI2, 2 CaCI2, 0.2 Na2S2Os (as antioxidant) and 10 p m pargyline (as monoamine o~idase inhibitor). The binding of [3H]SCH 23390 (83 Ci/mmol,
8]
Amersham) to D1 receptors was determined by incubating 0.5 ml of membrane suspension with 0.3 nM ligand and unlabelled drugs at 37°C for 20 min in a total volume of 1 mi. Specific binding was defined as that displaced by 100 nM piflutixol (Lundbeck), and represented 90-9570 of total binding. Incubations were stopped by filtration through G F / B filters followed by three 5 ml washes with ice-cold buffer. Radioactivity trapped on the filters was quantified by liquid scintillation spectroscopy after addition of 5 ml of liquiscint (Med Labs) using a Searle model Delta 300 counter with 2370 counting efficiency for tritium. The binding of [3H]spiperone (15 Ci/mmol, Amersham) to D 2 receptors was determined using membranes prepared as above. Incubations contained I ml of membrane suspension with 0.18 nM ligand and unlabelled drugs in a total volume of 5 ml. Specific binding was defined as that displaced by 1 pM domperidone (Janssen) and represented 65-8570 of total binding. Incubation and filtration were as described above.
2.4. Drugs The following investigational drugs were used: S K & F 77434 (3-allyl-2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-lH-3-benzazepine; Sn~th Kline & French, U.S.A.); LY 163502 (trans-[-]-5,5a,6,7,8, 9,9a,10-octahydro-6-propyl-pyrimido-[4,5-g]-quhiolin-2-amine; Lilly, U.S.A.); clozapine (Sandoz, Switzerland); apomorphine (Sigma, U.K.). Clozapine was dissolved in 0.1 N HCI; other reagents were dissolved in distilled water, which contained 0.170 sodium metabisulphite for apomorphine. In behavioural studies~ drugs were given s.c. into the flank, in a volume of 2 ml/kg; control animals received similar injections of the respective vehicle alone.
2.5. Data analysis
averaged over the 1 h period (30 rain for apomorphine), and expressed similarly. Data were analysed using analysis of variance (ANOVA) or the Kruskal-Wallis non-parametric ANOVA, and Student's t-test or Mann-Whitney U-test. Data from radioligand displacement experiments were analysed by a computer-assisted nonlinear iterative curve fitting procedure (Barlow, 1983). The resulting ICso was converted to a Ki value using the Cheng-Prusoff equation: Ki = ICs0/(1 + C / K D ) where C is iigand concentration and KD is the apparent dissociation constant.
3. Results
3.1. Effects of clozapine on behavioural responses to apomorphine As shown in fig. 1, apomorphine (2.5 mg/kg) induced a classical syndrome of compulsive stereotyped behaviour which was not influenced by pretreatment with 2.5-10.0 mg/kg clozapine; a higher dose of clozapine (25.0 mg/kg) tended to
Stereotypy scores
6-
5t
4. 3 2 1 0
r--'-! |
VEHICLE APO 2.5
CLOZ 2.5
CLOZ 10.0
I
I
|
CLOZ 25.0 I
+APO 2.5
From the application of the behavioural check list, the total "counts~ for each individual behaviour was determined as the number of 5 s observation windows in which a given behaviour was evident, summed over a 1 h period, and expressed as means ± S.E.; stereotypy scores were
Dose n~/kg Fig. 1. Effects of pretreatment with clozapine (CLOZ) on stereotyped behaviour induced I~y apomorphine (APO) in comparison with control animals given vehicle. Data are means + S.E. of stereotypy scores for n -- 8 animals per group. * P ~- 0.05 vs. controls; Mann-Whitney U-test.
TABLE I F~fe~t of pceue.aunent with ck~zapine on behaviour~ responses to the D, agonist SK&F 77434 and the D, agonist LY 163502. Data ~e ~ ± S.E. of beha~our~ counts or stereotypy scores for n -- 8 animals per group. Sn, sniffing; L, locomotion; R, rearing; Gr, ~ ; Gfi, intense grooming; Ch, chewing; VCh, vacuous chewing; St. stillness. Dr¢:o Vehicle SK&F77434 +~_ne
Vehicle LY 163502 + ~
mg/kg Sn
L
R
Gr
0.75 2.5 10.0 250
15.6+3.2 22.9+3.2 17.1+3.4 12.1±3.0 b 4.0+1.1 b
2.1+1.2 2.4+1.3 1.7+0.8 6.0+1.6 11.0:1:3.0 a 8.2+1.5 a 2.6+1.4 4.0±2.4 3.1+0.8b 2.4±0.9 1.0+0.8 b 1.14"0.6 b 0.7:!:0.5 b 0.44-0.3 b 0.4+0.3 b
0.05 2.5 10.0 25.0
14.8-1-3.6 21.6+2.1 18.3+2.5 14.54-2.7 6.5+1.3 b
1.5±0.6 5.9+1.1 a 1.1+0.5 b 4.0+2.4 0.2+0.2 b
2.4+1.5 1.84-0.9 0.54-0.4 1.9+1.1 0.4"!"0.3
0.54-0.3 0.64-0.2 0.04-0.0 b 0.14-0.1 b
Gri
Ch
VCh
St
Stereotypy score
0.5+0.4 6.0+1.0 a 1.6+0.5 b 0.4-1"0.4 b 0.0+0.0 b
0.04-0.0 2.6+1.9 1.7+1.1 0.9+0.7 0.04-0.0
0.14-0.1 0.14-0.1 0.54-0A 0.94-0.4 0.44-0.4
26.84-1.1 15.5+3.1 a 22.1-t-2.0 27.5+1.3 b 29.6+0.4 b
0.6+0.1 1.3+0.2 a 0.9+0.2 0.6+0.2 b 0.2-1-0.1 b
0.24-0.2 0.14-0.1 27.8+0.8 2.24-0.6 a 0.64-0.3 19.94-2.0 a 1.04-0.5 1.14-0.7 22.54-1.5 1.04-0.6 1.14-0.7 23.5"1-3.1 0.44-0.4 b 0.94-0.6 29.44-0.6 b
0.6±0.1 1.3+0.2 a 1.1+0.1 0.9+0.2 0.4+0.1 b
0.04-0.0 0.04-0.0 0.04-0.0 0.04-0.0 0.0"4-0.0 b 0.0-F0.0
P < 0.05 vs. vehic~; b p < 0.05 vs. SK&F 77434 or LY 163502; Student's t-test for behavioural counts and Mann-Whitney U-test for stereotypy scores.
reduce stereotypy scores. The principal behaviours induced by apomorphine were compulsive sniffing, with some locomotion and the essential abolition of stillness; these were uninfluenced by 2.510.0 mg/kg clozapine, while 25.0 mg/kg reduced episodes of sniffing and increased episodes of stillness (P < 0.01). No atypical behaviours were evident in animals given vehicle or apomorphine, following pretreatmerit either with vehicle or with any dose of clozapine. 3.2. Effects of clozapine on behavioural responses to S K & F 77434
SK&F 77434 (0.75 mg/kg; table 1) induced episodes of general grooming, intense grooming and ~ with some sniffing and locomotion, and reduced episodes of stillness; there was no classical stereotypy syndrome, and frequent episodes of stillness remained interpolated amongst mere behaviours. The lowest dose of clozapine (2.5 mg/kg) readily antagonised the general grooming and intense grooming responses; higher doses (10.0-25.0 mg/kg) continued this effect in a do'~'-dependent manner, additionally reduced episodes of rearing~ sniffing and locomotion, and increased episodes of stillness. No atypical behaviours were evident in animals given vehicle or SK&F 77434, following pretreat-
ment either with vehicle or with any dose of clozapine. 3.3. Effects of clozapine on behavioural responses to L Y 163502
LY 163502 (0.05 mg/kg; table 1) induced episodes of locomotion and chewing, with some sniffing, and reduced episodes of stillness; there was no cl~sical stereotypy syndrome, and frequent episodes of stillness remained interpolated amongst these behaviours. Clozapine (2.5-25.0 mg/kg) antagonised the locomotion and chewing responses, and increased episodes of stillness, though some of these effects were variably related to dose and only robust at the highest dose utilised. No atypical behaviours were induced by LY 1.63502 given as sole treatment; however, following clozapine pretreatment, LY 163502 induced episodes of jerking of the limbs or whole body in one of the eight animals given 2.5 mg/kg clozapine, two of eight animals given 10.0 mg/kg clozapine and four of eight animals given 25.0 mg/kg clozapine. Behavioural counts for this variable response were zero in animals injected with vehicle or with LY 163502 after vehicle pretreatment, but increased with the dose of clozapine given before LY 163502 (fig. 2; non-parametric ANOVA, P < 0.05).
83
[3H]spiperone-labelled D 2 receptor were essentially indistinguishable (taHe 2). These affinities were modest in comparison with R-SK&F 83566, which selectively displaced only [3H]SCH 23390, and with R-piquindone, which selectively displaced only [3H]spiperone (Murray and Waddington, 1989b).
LIMB/BODY JERKING
Behavioural counts
4. Discussion
V SK&FCL0Z CI~OZ(3LOZ 0.75 2.5 10.0 25.0
I
+ SKF 0.75
V
LY CLOZ CLOZCLOZ 0.05 2.5 10.0 25.0
I
I
+ LY 0.05
Dose mg/kg Fig. 2. Effects of pretreatment with clozapine (CLOZ) on behaviouralresponsesto SK&F77434(SKF) and to LY 163502 (LY) in comparison with control animals given vehicle (V). Data are means+ S.E. of behaviouralcounts for jerking, n -- 8 animals per group. Significantincrease in jerking with dose of clozapine given before LY 163502, P < 0.05; Kruskal-Wa]lis non-parametric ANOVA.
3.4. Radioligand binding studies The in vitro affinities of clozapine for the [3H]SCH 23390-labelled D~ receptor and for the TABLE 2 Displacement of ['~H]SCH 23390 ([3H]SCH) and of ~3H]spiperone ([3H]SPlP) from striatal D ! and D 2 receptors by clozapine and reference compounds. Values are geo.m_.e*.dc means of at least three independent determinations, each performed in duplicate.
R-SK&F 83566 a R.piquindone a Clozapine
K, ( n ~ [3H]SCH (DI)
[3H]SPIP (D2)
DI/D2
1 6610 542
2080 7 449
0.0005 945 1.21
a From Murray and Waddington (1989b).
Any action of a neuroleptic drug to block behavioural responsivity to an agonist acting selectively at one subtype of DA receptor does not, in the 'intact' animal, indicate specific antagonism of that receptor subtype. A number of studies have now established functional interactions between D1 and D2 receptor systems (Molloy and Waddington, 1984; Christensen et al., 1984), such tb~t typical behaviours induced by selective stimulation of one DA receptor subtype are influenced by manipulation of tonic DAergic activity through its counterpart (Waddington, 1989; Waddington and O'Boyle, 1989). For example, both a selective D2 antagonist and a selective D1 antagonist will block typical behaviours induced by a selective De agonist (Pugh e t a l . , 1985; Breese and Mueller, 1985); similarly, typical behavioural responses to a selective D~ agonist are blocked b~,th by a selective D1 antagonist and a selective D2 antagon~,st (Molloy and Waddington, 1987; Gandolfi et al., 1988). However, such antagonist effects can be distinguished by differing actions to concurrently release atypical behaviours, as these appear to have ~eir basis in a second form of D1 : D2 interaction distinct from that regulating typical behaviours (Waddington, 1989; Waddington and O'Boyle, 1989). Thus, while both the selective D1 antagonist R - S K & F 83566 and the selective D2 antagonist R-piquindone will block typical intense grooming induced by the selective D1 agonist SK & F 77434, only pret.reatment with *.he D2 antagonist releases an atypical vacuous chewing response to the D~ agonist (Murray and Waddington, 1989a); similarly, while both R-piquindone and R-SK&F 83566 will block hyl:eractivity responses to the selective D 2 agonist LY 163502,
$4 only pretreatment with the D~ antagonist releases an atypical jerking response to the D 2 agonist (Murray and Waddington, 1989b). On this basis, a neuroleptic acting to antagonise both D~ and D 2 receptors would be expected to block typical responses to selective agonists of either subtype, without rele~ing either atypical behaviour; converse]y, a preferential antagonist of Dl-mediated function would be expected to block both agonists, but with some release of atypical jerking, while a preferential antagonist of D-mediated function would be expected also to block both agonists, but with some release of atypical vacuous chewing. In this study, clozapine had little action to antagonise classical stereotyped behaviour induced by the non-selective DA agonist apomorphine, in agreement with numerous previous reports (Coward et al., 1989). However, clozapine readily blocked typical grooming induced by the Dz agonist SK&F 77434, and has recently been noted to block SK& F 38393-induced ~rooming in :nice (Vasse and Protais, 1988). In the present study, clozapine did not release atypical vacuous chewing in response to SK&F 77434, suggesting that during clozapine treatment, D2-mediated processes are not prefe~ntially attenuated. Conversely, clozapine both blocked typical hyperactivity responses to the D 2 agonist LY 163502 and released episodes of atypical limb/body jerking; this suggests that during clozapine treatment, D~mediated processes are preferentially attenuated. It must be emphasized that clozapine's ability to release jerking behaviour in response to LY 163502 was not identical to that previously reported for a selective D~ receptor antagonist (Murray and Waddington, 1989b); the action of clozapine was weaker and more variable. It was not possible to examine in a meaningful way whether higher doses of clozapine might release stronger or more consistent responses to these agonists, as in preliminary studies a dose of 50 mg/kg produced marked sedation. Release of this jerking response to LY 163502 appears unrelated to blockade of 5-hydroxytryptamine (5-HT) receptors by clozapine (Richelson and Nelson, 1984; Hyttel et al., 1985); such behaviour is released enantioselectively by R- but not S-SK&F 83566, and these enantiomers have similar antagonist affinities for
5-HT receptors (Cross et al., 1987) but show enantioselective blockade of D1 receptors (Murray and Waddington, 1989b). However, as we and others have seen such jerking behaviour only under the combined condition of D 2 receptor stimulation and diminished tonic activity through D l receptors (Waddington et al., 1986; Grabowska- And~n and And6n, 1987; Murray and Waddington, 1989a, b), we conclude that in this in vivo test paradigm, clozapine appears to exert some preferential (but not selective) antagonism of D~-mediated function. These effects in the intact animal are reminiscertt of the report that, in the reserpinised rat, clozapine can antagonise apomorphine-induced stereotyped behaviour in a manner similar to the selective D~ antagonist SCH 23390 (Chipkin and Latranyi, 1987). However, such results appear at variance with several reports that, using both in vitro and in vivo binding techniques, clozapine shows similar affinities for D~ and I)2 receptors (see Introduction); this lack of selectivity at the level of these binding sites is confirmed by the present study. There is some evidence (Andersen and Braestrup, 1986) that clozapine may antagonise the stimulation of adenylate cyclase by DA more potently than it displaces [~H]SCH 23390 binding, suggesting a particular action to inhibit D~ receptor function at this level. Furthermore, the failure of clozapine to reliably elevate plasma prolactin levels (Coward et al., 1989) suggests that functional blockade of !)2 receptors may be less than would be expected on the basis of its affinity for D2 binding sites. The similar affinities of clozapine for DI and D 2 binding sites, but its apparent preferential antagonism of D~-mediated function, should be compared with the properties of the new D~ agonist CY 208-243; this agent also shows similar affinities for Dt and D2 (and several non-DAergic) binding sites, but in functional studies little or no effect attributable to its 1)2 affinity is evident (Markstehl et al., 1988; Murray and Waddington, 1990). Thus, clozapine and CY 208-243 may be antagonist arid agonist counterparts of a previously unrecognised drug series which displays non-selective affL-.i~ies for both D 1 and 1)2 binding sites but, paradoxically, exerts preferential ef-
85
fects on Dt receptor-mediated function by an as yet unspecified mechanism. Irrespective of whether these notions are sustained, clozapine will continue to attract considerable attention. Antipsychotic doses of clozapine produce the greatest in vivo occupation of Dt receptors amongst those neuroleptics so far examined by positron emission tomography in living patients, by virtue of other neuroleptics appearing selective or preferential D 2 antagonists under such conditions (Farde et al., 1989). Also, recent studies have demonstrated clozapine to show antipsychotic efficacy in a significant proportion of schizophrenic patients who do not respond to typical neuroleptic drugs, and it does so without inducing prominent extrapyramidal side effects (Claghorn et al., 1987; Kane et al., 1988). Such studies will prompt further efforts to establish the extent to which these atypical properties of clozapine may or may not be attributable to its effects on Dt receptor-mediated function.
Acknowledgements This work was supported by the Health Research Board of Ireland and the Royal College of Surgeons in Ireland. We thank Lilly, Sandoz and Smith Kline & French for gifts of drugs.
References Andersen, P.H., 1988, Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding to dopamine receptors in vivo in mouse brain, European J. Pharmacol. 146, 113. Andersen, P.H. and C. Braestr~_~p, 1986, Evidence for different states of the dopamine D! receptor: clozapine and fluperlapine may preferentially label an adenylate cyclasecoupled state of the D 1 receptor, J. Neurochem. 47, 1822. Badow, R.B., 1983, Biodata Handling with Microcomputers (Elsevier, Amsterdam). Breese, G.R. and R.A. Mueller, i985, SCH 23390 antagonism of D-2 dopamine agonist depends upon intact catecholaminergic neurons, European J. Pharmacol. 113, i09. Casey, D.E., 2989, C]ozapine: neuroleptic-induced EPS and tardive dyskinesia, Psychopharmacology 99 (Suppl.), $47. Chipkin, R.E. and M.B. Latranyi, 1987, Similarity of clozapine and SCH 23390 in reserpinized rats suggests a common mechanism ,.,f action, European Jo Pharmacol. 136, 371.
Christensen, A.V., J. Arnt, J. Hyttel and O. Svendsen, 1984, Pharmacological effects of a specific dopamine D 1 antagonist SCH 23390 in comparison with neuroleptics, Life Sci. 34, 1529. Claghorn, J., G. Honigfeld, F.S. Abuzzahab, R. Wang, R. Steinbook, V. Tuason and G. Klerman, 1987, The risks and benefits of clozapine versus chlorpromazine, J. Clin. Psychopharmacoi. 7, 377. Coward, D.M., A. lmperato, S. Urwyler and T.G. White, 1989, Biochemical and behavioural properties of clozapine, Psychopharmacology 99 (Suppl.), $6. Criswell, H.E., R.A. Mueiler and G.R. Breese, 1989, Clozapine antagonism of D-1 and D-2 dopamine receptor-mediated behaviours, European J. Pharmacol. 159, 141. Cross, A.J., L. Hewitt, P. Slater and J.L. Waddington, 1987, Interaction of benzazepine enantiomers with rat brain serotonin receptors, Br. J. Pharmacol. 90 (Suppl.), 244P. Faedda, G., N.S. Kula and R.J. Baldessarini, 1989, Pharmacology of binding of 3H-SCH 23390 to D i dopaminergic receptor sites in rat striatal tissue, Biochem. Pharmacol. 38, 473. Farde, L., F.A. Wiesel, A.-L. Nordstrom and G. Sedvall, 1989, D-1 and D-2 dopamine receptor occupancy during treatment with conventional and atypical neuroleptics, Psychopharmacology 99 (Suppl.), $28. Gandolfi, O., R. DalrOlio, A. Vaccheri, P. Roncada and N. Montanaro, 1988, Responses to selective Di and D2 agonists after repeated treatment with selective D! and 1)2 antagonists, Pharmacol. Biochem. Behav. 30, 463. Grabowska-And~n, M. and N,E. And~n, 1987, Inhibito R ~'ole of D1 dopamine receptors for the jerks induced by B-HT 920 in rats, J. Pharm. Pharmacol. 39, 660. Hall, H., L. Farde and G. Sedvall, 1987, Human dopamir~e receptor subtypes - in vitro binding analysis using 3H-SCH 23390 and 3H-racloprt
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Elsevier EJP 51470
The interaction of c|ozapine with dopamine D 1 versus dopamine Dz receptor-mediated function: behavioural indices A n g e l a M. M u r r a y a n d J o h n L. W a d d i n g t o n Department of ClinicalPharmacology, Royal Collegeof Surgeons in Ireland St. Stephen's Green, Dublin 2, Ireland Received 4 May 1990, accepted 19 June 1990
Studies were undertaken to clarify further the mechanism(s) of action of the atypica~ neuroleptic clozapine, using a behavioural model with the ability to distinguish between relative antagonism of D~ vs. De dopamine receptor-mediated function. Pretreatment with low doses of clozapine (2.5-25.0 mg/kg) readily antagonised intense groomLng induced by the selective D 1 agonist SK&F 77434 (0.75 mg/kg), and in a less consistent manner antagonised hyperactivities induced by the selective De agonist LY 163502 (0.05 mg/kg). In animals whose typical responses to SK&I" 77434 were antagonised by clozapine, no atypical behaviours such as vacuous chewing emerged. However, in animals whose typical responses to LY 163502 were antagonised by clozapine, a syndrome of atypical limb/body jerking was released. Despite clozapine showing comparable affinities for D~ and D e receptors in vitro, this behavioural profile shows similarities to that seen when these agonists are given after pretreatment with a selective D~ antagonist, rather than with a selective D z antagonist or with non-selechve neuroleptics. These results suggest that clozapine has some preferential though not selective action in vivo to antagonise D~ dopamine receptor-mediated function. Clozapine; Dopamine D1 receptors; Dopamine De receptors; Jerking behaviour; Grooming behaviour: (Rat)
1. Introduction
The dibenzodiazepine clozapine is an atypical neuroleptic that is attracting renewed interest. This stems from recent evidence suggesting that clozapine shows antipsychotic efficacy against both positive and negative symptoms in some schizophrenic patients who are resistant to treatment with typical neuroleptics (Kane et al., 1988), while having long been recognised for its reduced liability to induce at least acute extrapyramidal side effects (Casey, 1989). Pharmacologically, clozapine acts at more than one level of synaptic function and can block receptors for a variety of
Correspondence to: J.L. Waddhngton, Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2, Ireland.
neurotransmitters (Richelson and Nelson, 1984; Hyttel et al., 1985). In relation to dopaminergic (DAergic) neurotransmission, radioligand binding studies in rodent, non-human primate and human brain, both in vitro (Andersen, 1988; Madras et alo~ 19Rg; ~t , i , ,~o,~, . . Hal! . .et .al,,. 1987; . . l::,ecld~ . . t~c-~; and in vivo (Andersen, 1988; Farde et al., 1989), indicate that clozapine exhibits similar affinities for D1 and D2 receptors. Though some neurochemical (Andersen and Braestrup, 1986, Coward et al., 1989) and pharmacological (Chipkin and Latranyi, 1987; CrisweU et al., 1989) studies have suggested that clozapine may exert some preferential blockade of Dl-mediated function, these were carried out at a non-behavioural level or under non-physiological conditions of reserpinisation or 6-hydroxydopamine lesion. This issue has yet to be investigated in terms of behaviour in the intact animal.
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
80 Because of well-established functional interactions between Dt and I)2 receptor systems (Waddington and O'Boyle, 1989; Waddington, 1989), selective D~ antagonists and selective D2 antagonists can each block the behavioural effects both of D~ and of D2 agonists in the whole animal. Thus, any antagonist action of a given neuroleptic drug on behavioural responses induced either by selective D1 or 1)2 agonists would not in itself indicate any preferential action at one subtype of DA receptor. However, we have recently identified an experimental situation with the capacAty to distinguish between blockade of D~ o r D 2 receptors, on the basis of two directions of D~:D2 interaction which appear to regulate distinct elements of typical and atypical behaviour (Murray and Waddington, 1989a,b). We describe here the application of these procedures to the in vivo study of any preferential action of clozapine at D l vs. D 2 receptor-mediated function in the intact adult rat.
2. Materials and methods
2.1. Animals Young adult male Sprague-Dawley rats (Biolabs, Ballina) were used in all experiments. The rats were housed in groups of five per cage with food and water available ad libitum and were maintained on a 12/12 h (6 a.m. on, 6 p.m. off) light/dark cycle.
2.2. Behavioural studies Rats were placed individually in perspex cages 52 x 3 9 x 18 cm and left undisturbed for a habituation period of 2.5 h. Behavioural assessments were carried out in a manner shnilar to that previously described (Murray and Waddington, 1989a,b). Immediately before and at intervals after s.c. injections of drug or vehicle, animals were assessed using a rapid timesampling behavioural check list technique. For this procedure, each tat ~vas observed hldividually for 5 s periods at 1 rain intervals over 5 consecutive rain, using an extended behavioural check list.
This vllowed the presence or absence of the following individual behaviours (occurring alone or in combination) to be determined in each 5 s period: sniffing (Sn); locomotion (L); rearing (R); grooming (Gr, of any form); intense grooming (Gri; a characteristic pattern of grooming of the face with the forepaws followed by vigorous grooming of the hind flank with the snout); chewing (Ch; directed onto any physical material); vacuous chewing (VCh; not directed onto any physical matefial)~ jerking (J; brief jerking movements of the limbs or whole body); stillness (St; motionless, with no behaviour evident). After assessment using the behavioural check list, animals were assessed using a conventional 0-6 point stereotypy rating scale: 0 = asleep or inactive; 1 = episodes of normal activities; 2 = discontinuous activity with bursts of prominent sniffing or rearing; 3 - contiauous stereotyped activity such as sniffing or rearing along a fixed path; 4 = stereotyped sniffing or rearing fixated in one location; 5 - stereotyped behaviour with bursts of licking or gnawing; 6fficontinuous licking or gnawing. This cycle was repeated at 10 min intervals. Rats were used on two occasions only, separated by a drug free inte~al of one week; on each occasion rats were randomly allocated to one of the various treatment groups..Antagonist or vehicle was given 45 min prior to agonist challenge. All assessments were made by an observer unaware of the treatment given to each animal.
2.3. Radioligand binding studies Using methods similar to those previously described (Murray and Waddington, 1989a,b), striata from similar male Sprague-Dawley rats were 11omogenised in 30 volumes of 50 mM Tris-HCl buffer, pH 7.6 at 25 ° C, and centrifuged at 10000 × g at 4 ° C for 5 min. The pellet was tv4ce resuspended, diluted and centrifuged as above. The membrane preparation was finally resuspended at 4-8 mg original wet weight/ml in TrisHCI buffer containing (raM): 120 NaCI, 5 KCI, 1 MgCI2, 2 CaCI2, 0.2 Na2S2Os (as antioxidant) and 10 p m pargyline (as monoamine o~idase inhibitor). The binding of [3H]SCH 23390 (83 Ci/mmol,
8]
Amersham) to D1 receptors was determined by incubating 0.5 ml of membrane suspension with 0.3 nM ligand and unlabelled drugs at 37°C for 20 min in a total volume of 1 mi. Specific binding was defined as that displaced by 100 nM piflutixol (Lundbeck), and represented 90-9570 of total binding. Incubations were stopped by filtration through G F / B filters followed by three 5 ml washes with ice-cold buffer. Radioactivity trapped on the filters was quantified by liquid scintillation spectroscopy after addition of 5 ml of liquiscint (Med Labs) using a Searle model Delta 300 counter with 2370 counting efficiency for tritium. The binding of [3H]spiperone (15 Ci/mmol, Amersham) to D 2 receptors was determined using membranes prepared as above. Incubations contained I ml of membrane suspension with 0.18 nM ligand and unlabelled drugs in a total volume of 5 ml. Specific binding was defined as that displaced by 1 pM domperidone (Janssen) and represented 65-8570 of total binding. Incubation and filtration were as described above.
2.4. Drugs The following investigational drugs were used: S K & F 77434 (3-allyl-2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-lH-3-benzazepine; Sn~th Kline & French, U.S.A.); LY 163502 (trans-[-]-5,5a,6,7,8, 9,9a,10-octahydro-6-propyl-pyrimido-[4,5-g]-quhiolin-2-amine; Lilly, U.S.A.); clozapine (Sandoz, Switzerland); apomorphine (Sigma, U.K.). Clozapine was dissolved in 0.1 N HCI; other reagents were dissolved in distilled water, which contained 0.170 sodium metabisulphite for apomorphine. In behavioural studies~ drugs were given s.c. into the flank, in a volume of 2 ml/kg; control animals received similar injections of the respective vehicle alone.
2.5. Data analysis
averaged over the 1 h period (30 rain for apomorphine), and expressed similarly. Data were analysed using analysis of variance (ANOVA) or the Kruskal-Wallis non-parametric ANOVA, and Student's t-test or Mann-Whitney U-test. Data from radioligand displacement experiments were analysed by a computer-assisted nonlinear iterative curve fitting procedure (Barlow, 1983). The resulting ICso was converted to a Ki value using the Cheng-Prusoff equation: Ki = ICs0/(1 + C / K D ) where C is iigand concentration and KD is the apparent dissociation constant.
3. Results
3.1. Effects of clozapine on behavioural responses to apomorphine As shown in fig. 1, apomorphine (2.5 mg/kg) induced a classical syndrome of compulsive stereotyped behaviour which was not influenced by pretreatment with 2.5-10.0 mg/kg clozapine; a higher dose of clozapine (25.0 mg/kg) tended to
Stereotypy scores
6-
5t
4. 3 2 1 0
r--'-! |
VEHICLE APO 2.5
CLOZ 2.5
CLOZ 10.0
I
I
|
CLOZ 25.0 I
+APO 2.5
From the application of the behavioural check list, the total "counts~ for each individual behaviour was determined as the number of 5 s observation windows in which a given behaviour was evident, summed over a 1 h period, and expressed as means ± S.E.; stereotypy scores were
Dose n~/kg Fig. 1. Effects of pretreatment with clozapine (CLOZ) on stereotyped behaviour induced I~y apomorphine (APO) in comparison with control animals given vehicle. Data are means + S.E. of stereotypy scores for n -- 8 animals per group. * P ~- 0.05 vs. controls; Mann-Whitney U-test.
TABLE I F~fe~t of pceue.aunent with ck~zapine on behaviour~ responses to the D, agonist SK&F 77434 and the D, agonist LY 163502. Data ~e ~ ± S.E. of beha~our~ counts or stereotypy scores for n -- 8 animals per group. Sn, sniffing; L, locomotion; R, rearing; Gr, ~ ; Gfi, intense grooming; Ch, chewing; VCh, vacuous chewing; St. stillness. Dr¢:o Vehicle SK&F77434 +~_ne
Vehicle LY 163502 + ~
mg/kg Sn
L
R
Gr
0.75 2.5 10.0 250
15.6+3.2 22.9+3.2 17.1+3.4 12.1±3.0 b 4.0+1.1 b
2.1+1.2 2.4+1.3 1.7+0.8 6.0+1.6 11.0:1:3.0 a 8.2+1.5 a 2.6+1.4 4.0±2.4 3.1+0.8b 2.4±0.9 1.0+0.8 b 1.14"0.6 b 0.7:!:0.5 b 0.44-0.3 b 0.4+0.3 b
0.05 2.5 10.0 25.0
14.8-1-3.6 21.6+2.1 18.3+2.5 14.54-2.7 6.5+1.3 b
1.5±0.6 5.9+1.1 a 1.1+0.5 b 4.0+2.4 0.2+0.2 b
2.4+1.5 1.84-0.9 0.54-0.4 1.9+1.1 0.4"!"0.3
0.54-0.3 0.64-0.2 0.04-0.0 b 0.14-0.1 b
Gri
Ch
VCh
St
Stereotypy score
0.5+0.4 6.0+1.0 a 1.6+0.5 b 0.4-1"0.4 b 0.0+0.0 b
0.04-0.0 2.6+1.9 1.7+1.1 0.9+0.7 0.04-0.0
0.14-0.1 0.14-0.1 0.54-0A 0.94-0.4 0.44-0.4
26.84-1.1 15.5+3.1 a 22.1-t-2.0 27.5+1.3 b 29.6+0.4 b
0.6+0.1 1.3+0.2 a 0.9+0.2 0.6+0.2 b 0.2-1-0.1 b
0.24-0.2 0.14-0.1 27.8+0.8 2.24-0.6 a 0.64-0.3 19.94-2.0 a 1.04-0.5 1.14-0.7 22.54-1.5 1.04-0.6 1.14-0.7 23.5"1-3.1 0.44-0.4 b 0.94-0.6 29.44-0.6 b
0.6±0.1 1.3+0.2 a 1.1+0.1 0.9+0.2 0.4+0.1 b
0.04-0.0 0.04-0.0 0.04-0.0 0.04-0.0 0.0"4-0.0 b 0.0-F0.0
P < 0.05 vs. vehic~; b p < 0.05 vs. SK&F 77434 or LY 163502; Student's t-test for behavioural counts and Mann-Whitney U-test for stereotypy scores.
reduce stereotypy scores. The principal behaviours induced by apomorphine were compulsive sniffing, with some locomotion and the essential abolition of stillness; these were uninfluenced by 2.510.0 mg/kg clozapine, while 25.0 mg/kg reduced episodes of sniffing and increased episodes of stillness (P < 0.01). No atypical behaviours were evident in animals given vehicle or apomorphine, following pretreatmerit either with vehicle or with any dose of clozapine. 3.2. Effects of clozapine on behavioural responses to S K & F 77434
SK&F 77434 (0.75 mg/kg; table 1) induced episodes of general grooming, intense grooming and ~ with some sniffing and locomotion, and reduced episodes of stillness; there was no classical stereotypy syndrome, and frequent episodes of stillness remained interpolated amongst mere behaviours. The lowest dose of clozapine (2.5 mg/kg) readily antagonised the general grooming and intense grooming responses; higher doses (10.0-25.0 mg/kg) continued this effect in a do'~'-dependent manner, additionally reduced episodes of rearing~ sniffing and locomotion, and increased episodes of stillness. No atypical behaviours were evident in animals given vehicle or SK&F 77434, following pretreat-
ment either with vehicle or with any dose of clozapine. 3.3. Effects of clozapine on behavioural responses to L Y 163502
LY 163502 (0.05 mg/kg; table 1) induced episodes of locomotion and chewing, with some sniffing, and reduced episodes of stillness; there was no cl~sical stereotypy syndrome, and frequent episodes of stillness remained interpolated amongst these behaviours. Clozapine (2.5-25.0 mg/kg) antagonised the locomotion and chewing responses, and increased episodes of stillness, though some of these effects were variably related to dose and only robust at the highest dose utilised. No atypical behaviours were induced by LY 1.63502 given as sole treatment; however, following clozapine pretreatment, LY 163502 induced episodes of jerking of the limbs or whole body in one of the eight animals given 2.5 mg/kg clozapine, two of eight animals given 10.0 mg/kg clozapine and four of eight animals given 25.0 mg/kg clozapine. Behavioural counts for this variable response were zero in animals injected with vehicle or with LY 163502 after vehicle pretreatment, but increased with the dose of clozapine given before LY 163502 (fig. 2; non-parametric ANOVA, P < 0.05).
83
[3H]spiperone-labelled D 2 receptor were essentially indistinguishable (taHe 2). These affinities were modest in comparison with R-SK&F 83566, which selectively displaced only [3H]SCH 23390, and with R-piquindone, which selectively displaced only [3H]spiperone (Murray and Waddington, 1989b).
LIMB/BODY JERKING
Behavioural counts
4. Discussion
V SK&FCL0Z CI~OZ(3LOZ 0.75 2.5 10.0 25.0
I
+ SKF 0.75
V
LY CLOZ CLOZCLOZ 0.05 2.5 10.0 25.0
I
I
+ LY 0.05
Dose mg/kg Fig. 2. Effects of pretreatment with clozapine (CLOZ) on behaviouralresponsesto SK&F77434(SKF) and to LY 163502 (LY) in comparison with control animals given vehicle (V). Data are means+ S.E. of behaviouralcounts for jerking, n -- 8 animals per group. Significantincrease in jerking with dose of clozapine given before LY 163502, P < 0.05; Kruskal-Wa]lis non-parametric ANOVA.
3.4. Radioligand binding studies The in vitro affinities of clozapine for the [3H]SCH 23390-labelled D~ receptor and for the TABLE 2 Displacement of ['~H]SCH 23390 ([3H]SCH) and of ~3H]spiperone ([3H]SPlP) from striatal D ! and D 2 receptors by clozapine and reference compounds. Values are geo.m_.e*.dc means of at least three independent determinations, each performed in duplicate.
R-SK&F 83566 a R.piquindone a Clozapine
K, ( n ~ [3H]SCH (DI)
[3H]SPIP (D2)
DI/D2
1 6610 542
2080 7 449
0.0005 945 1.21
a From Murray and Waddington (1989b).
Any action of a neuroleptic drug to block behavioural responsivity to an agonist acting selectively at one subtype of DA receptor does not, in the 'intact' animal, indicate specific antagonism of that receptor subtype. A number of studies have now established functional interactions between D1 and D2 receptor systems (Molloy and Waddington, 1984; Christensen et al., 1984), such tb~t typical behaviours induced by selective stimulation of one DA receptor subtype are influenced by manipulation of tonic DAergic activity through its counterpart (Waddington, 1989; Waddington and O'Boyle, 1989). For example, both a selective D2 antagonist and a selective D1 antagonist will block typical behaviours induced by a selective De agonist (Pugh e t a l . , 1985; Breese and Mueller, 1985); similarly, typical behavioural responses to a selective D~ agonist are blocked b~,th by a selective D1 antagonist and a selective D2 antagon~,st (Molloy and Waddington, 1987; Gandolfi et al., 1988). However, such antagonist effects can be distinguished by differing actions to concurrently release atypical behaviours, as these appear to have ~eir basis in a second form of D1 : D2 interaction distinct from that regulating typical behaviours (Waddington, 1989; Waddington and O'Boyle, 1989). Thus, while both the selective D1 antagonist R - S K & F 83566 and the selective D2 antagonist R-piquindone will block typical intense grooming induced by the selective D1 agonist SK & F 77434, only pret.reatment with *.he D2 antagonist releases an atypical vacuous chewing response to the D~ agonist (Murray and Waddington, 1989a); similarly, while both R-piquindone and R-SK&F 83566 will block hyl:eractivity responses to the selective D 2 agonist LY 163502,
$4 only pretreatment with the D~ antagonist releases an atypical jerking response to the D 2 agonist (Murray and Waddington, 1989b). On this basis, a neuroleptic acting to antagonise both D~ and D 2 receptors would be expected to block typical responses to selective agonists of either subtype, without rele~ing either atypical behaviour; converse]y, a preferential antagonist of Dl-mediated function would be expected to block both agonists, but with some release of atypical jerking, while a preferential antagonist of D-mediated function would be expected also to block both agonists, but with some release of atypical vacuous chewing. In this study, clozapine had little action to antagonise classical stereotyped behaviour induced by the non-selective DA agonist apomorphine, in agreement with numerous previous reports (Coward et al., 1989). However, clozapine readily blocked typical grooming induced by the Dz agonist SK&F 77434, and has recently been noted to block SK& F 38393-induced ~rooming in :nice (Vasse and Protais, 1988). In the present study, clozapine did not release atypical vacuous chewing in response to SK&F 77434, suggesting that during clozapine treatment, D2-mediated processes are not prefe~ntially attenuated. Conversely, clozapine both blocked typical hyperactivity responses to the D 2 agonist LY 163502 and released episodes of atypical limb/body jerking; this suggests that during clozapine treatment, D~mediated processes are preferentially attenuated. It must be emphasized that clozapine's ability to release jerking behaviour in response to LY 163502 was not identical to that previously reported for a selective D~ receptor antagonist (Murray and Waddington, 1989b); the action of clozapine was weaker and more variable. It was not possible to examine in a meaningful way whether higher doses of clozapine might release stronger or more consistent responses to these agonists, as in preliminary studies a dose of 50 mg/kg produced marked sedation. Release of this jerking response to LY 163502 appears unrelated to blockade of 5-hydroxytryptamine (5-HT) receptors by clozapine (Richelson and Nelson, 1984; Hyttel et al., 1985); such behaviour is released enantioselectively by R- but not S-SK&F 83566, and these enantiomers have similar antagonist affinities for
5-HT receptors (Cross et al., 1987) but show enantioselective blockade of D1 receptors (Murray and Waddington, 1989b). However, as we and others have seen such jerking behaviour only under the combined condition of D 2 receptor stimulation and diminished tonic activity through D l receptors (Waddington et al., 1986; Grabowska- And~n and And6n, 1987; Murray and Waddington, 1989a, b), we conclude that in this in vivo test paradigm, clozapine appears to exert some preferential (but not selective) antagonism of D~-mediated function. These effects in the intact animal are reminiscertt of the report that, in the reserpinised rat, clozapine can antagonise apomorphine-induced stereotyped behaviour in a manner similar to the selective D~ antagonist SCH 23390 (Chipkin and Latranyi, 1987). However, such results appear at variance with several reports that, using both in vitro and in vivo binding techniques, clozapine shows similar affinities for D~ and I)2 receptors (see Introduction); this lack of selectivity at the level of these binding sites is confirmed by the present study. There is some evidence (Andersen and Braestrup, 1986) that clozapine may antagonise the stimulation of adenylate cyclase by DA more potently than it displaces [~H]SCH 23390 binding, suggesting a particular action to inhibit D~ receptor function at this level. Furthermore, the failure of clozapine to reliably elevate plasma prolactin levels (Coward et al., 1989) suggests that functional blockade of !)2 receptors may be less than would be expected on the basis of its affinity for D2 binding sites. The similar affinities of clozapine for DI and D 2 binding sites, but its apparent preferential antagonism of D~-mediated function, should be compared with the properties of the new D~ agonist CY 208-243; this agent also shows similar affinities for Dt and D2 (and several non-DAergic) binding sites, but in functional studies little or no effect attributable to its 1)2 affinity is evident (Markstehl et al., 1988; Murray and Waddington, 1990). Thus, clozapine and CY 208-243 may be antagonist arid agonist counterparts of a previously unrecognised drug series which displays non-selective affL-.i~ies for both D 1 and 1)2 binding sites but, paradoxically, exerts preferential ef-
85
fects on Dt receptor-mediated function by an as yet unspecified mechanism. Irrespective of whether these notions are sustained, clozapine will continue to attract considerable attention. Antipsychotic doses of clozapine produce the greatest in vivo occupation of Dt receptors amongst those neuroleptics so far examined by positron emission tomography in living patients, by virtue of other neuroleptics appearing selective or preferential D 2 antagonists under such conditions (Farde et al., 1989). Also, recent studies have demonstrated clozapine to show antipsychotic efficacy in a significant proportion of schizophrenic patients who do not respond to typical neuroleptic drugs, and it does so without inducing prominent extrapyramidal side effects (Claghorn et al., 1987; Kane et al., 1988). Such studies will prompt further efforts to establish the extent to which these atypical properties of clozapine may or may not be attributable to its effects on Dt receptor-mediated function.
Acknowledgements This work was supported by the Health Research Board of Ireland and the Royal College of Surgeons in Ireland. We thank Lilly, Sandoz and Smith Kline & French for gifts of drugs.
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Christensen, A.V., J. Arnt, J. Hyttel and O. Svendsen, 1984, Pharmacological effects of a specific dopamine D 1 antagonist SCH 23390 in comparison with neuroleptics, Life Sci. 34, 1529. Claghorn, J., G. Honigfeld, F.S. Abuzzahab, R. Wang, R. Steinbook, V. Tuason and G. Klerman, 1987, The risks and benefits of clozapine versus chlorpromazine, J. Clin. Psychopharmacoi. 7, 377. Coward, D.M., A. lmperato, S. Urwyler and T.G. White, 1989, Biochemical and behavioural properties of clozapine, Psychopharmacology 99 (Suppl.), $6. Criswell, H.E., R.A. Mueiler and G.R. Breese, 1989, Clozapine antagonism of D-1 and D-2 dopamine receptor-mediated behaviours, European J. Pharmacol. 159, 141. Cross, A.J., L. Hewitt, P. Slater and J.L. Waddington, 1987, Interaction of benzazepine enantiomers with rat brain serotonin receptors, Br. J. Pharmacol. 90 (Suppl.), 244P. Faedda, G., N.S. Kula and R.J. Baldessarini, 1989, Pharmacology of binding of 3H-SCH 23390 to D i dopaminergic receptor sites in rat striatal tissue, Biochem. Pharmacol. 38, 473. Farde, L., F.A. Wiesel, A.-L. Nordstrom and G. Sedvall, 1989, D-1 and D-2 dopamine receptor occupancy during treatment with conventional and atypical neuroleptics, Psychopharmacology 99 (Suppl.), $28. Gandolfi, O., R. DalrOlio, A. Vaccheri, P. Roncada and N. Montanaro, 1988, Responses to selective Di and D2 agonists after repeated treatment with selective D! and 1)2 antagonists, Pharmacol. Biochem. Behav. 30, 463. Grabowska-And~n, M. and N,E. And~n, 1987, Inhibito R ~'ole of D1 dopamine receptors for the jerks induced by B-HT 920 in rats, J. Pharm. Pharmacol. 39, 660. Hall, H., L. Farde and G. Sedvall, 1987, Human dopamir~e receptor subtypes - in vitro binding analysis using 3H-SCH 23390 and 3H-racloprt
