- Email: [email protected]
Receptor mechanisms mediating clozapine-inducedc-fos expression in the forebrain
Neuroscience Vol. 65, No. 3, pp. 747-756, 1995
~
Pergamon
0306-4522(94)00552-4
Elsevier ScienceLtd Copyright © 1995IBRO Printed in Great Britain.All rights reserved 0306-4522/95 $9.50+ 0.00
RECEPTOR MECHANISMS MEDIATING CLOZAPINE-INDUCED c-fos EXPRESSION IN THE FOREBRAIN N. G U O , M. A. K L I T E N I C K , C.-S. T H A M and H. C. F I B I G E R * Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 A~tract--The atypical antipsychotic clozapine produces distinctly different regional patterns of c-Jbs expression in rat forebrain than does the prototypical neuroleptic haloperidol. While haloperidoMnduced c-fos expression appears to be mediated by its D 2 dopamine receptor antagonist properties, the mechanisms by which clozapine increases c-fos expression remain uncertain. Using a combination of brain lesion, pharmacological and immunohistochemical techniques, the present study sought to determine the receptor mechanisms by which clozapine increases the number of Fos-like immunoreactive neurons in various regions of the forebrain. To test whether serotonergic and/or noradrenergic systems are involved in clozapine-induced c-fos expression, rats received either 5,7-dihydroxytryptamine lesions of the medial forebrain bundle or 6-hydroxydopamine lesions of the dorsal noradrenergic bundle two weeks prior to clozapine (20 mg/kg) injections. Neither type of lesion affected clozapine-induced c-los expression in the rat forebrain, suggesting that neither serotonergic nor noradrenergic mechanisms are involved in this action of clozapine. In another experiment, the 5-hydroxytryptamine2 receptor antagonist ritanserin (5 mg/kg), either alone or in combination with haloperidol (1 mg/kg), failed to mimic the pattern of c-Jbs expression produced by clozapine. This suggests that clozapine's antagonist actions at 5-hydroxytryptamine2 receptors cannot explain the unique pattern of regional c-fos expression produced by this compound. To determine whether the blockade of subtypes of the D: dopamine receptor family may contribute to clozapine's effects, the dopamine receptor agonists quinpirole and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin(7-OH-DPAT) were injected 15min prior to clozapine. Quinpirole produced a small but significant decrease in clozapine-induced c-fos expression in the medial prefrontal cortex, had larger effects in the lateral septum, and blocked clozapine's actions in the nucleus accumbens and major island of Calleja. Pretreatment with 7-OH-DPAT attenuated clozapine-induced c-fos expression in the nucleus accumbens and lateral septum, completely blocked the expression in the major island of Calleja, but was without effect in the medial prefrontal cortex. Given the different affinities of quinpirole and 7-OH-DPAT for D2, D3 and D4 receptors, these data suggest that clozapine-induced increases in c-fos expression in the nucleus accumbens, major island of Cajella and lateral septal nucleus are due to antagonist actions of this antipsychotic at Da dopamine receptors. They also indicate that while antagonist actions at D4 receptors may contribute, the primary mechanisms by which clozapine increases c-fos expression in the medial prefrontal cortex remain to be determined.
The mechanisms by which clozapine exerts therapeutic effects without producing concomitant extrapyramidal side effects remain elusive. ~'6'13'15 The inability to differentiate atypical (e.g. clozapine) from typical antipsychotic drugs (e.g. haloperidol) on the basis of their actions at dopaminergic, cholinergic, serotonergic, adrenergic, and opiate sigma receptors, 6 implies that there might be more than one mechanism through which neuroleptics exert their actions. Since
the interaction of neuroleptic drugs with neurotransmitter receptors is only the initial step in their actions, recent attention has shifted to investigations of intracellular postreceptor mechanisms. Immediate early genes are useful markers to map changes in neuronal activity, 28'29 and recently immediate early gene m R N A expression (such as c-fos and zlf/268) and Fos immunohistochemistry have been used to examine the effects of neuroleptics in the CNS. 8'27'30Several studies have shown that typical and atypical antipsychotics have regionally different effects on c-fos *To whom correspondence should be addressed. Abbrev&tions: DA, dopamine; 5,7-DHT, 5,7-dihydroxy- expression in the brain: haloperidol induces c-fos tryptamine; DNB, dorsal noradrenergic bundle; EDTA, expression in the nucleus accumbens (NAc), lateral ethylenediaminetetra-acetate; 5-HT, 5-hydroxytrypta- septal nucleus and striatum, while clozapine increases mine (serotonin): MFB, medial forebrain bundle; the number of Fos-positive neurons in the NAc, mPFC, medial prefrontal cortex; NA, noradrenaline; NAc, nucleus accumbens; 6-OHDA, 6-hydroxydopa- lateral septal nucleus and medial prefrontal cortex mine; 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2- (mPFC). 7'3°'36 This regional specificity of neurolepticaminotetralin; PFC, prefrontal cortex. induced immediate early gene expression suggests 747
748
N. Guo et al.
that different patterns of neuronal activity are evoked by the two classes of antipsychotic drugs, and raises the possibility that these regionally different effects are related to the different clinical profiles of these drugs. 36 Therefore, it is important to elucidate how such patterns are generated. Haloperidol-induced striatal c-fos~expression is thought to be D 2 receptor mediated inasmuch as co-administration of a D2 agonist prevents the induct i o n s and the selective D2 antagonist raclopride produces a regional pattern of Fos-like immunoreactivity that is indistinguishable from that produced by haloperidol. 36 Because of the regional differences between clozapine- and haloperidol-induced c-fos expression in the brain, D2 receptor antagonism is not sufficient to account for the unique pattern produced by clozapine, even though D 2 receptors are a c o m m o n site of action for haloperidol and clozapineY Previous work has demonstrated that the antimuscarinic agent scopolamine attenuates haloperidol-induced c-fos expression in the striatum, suggesting that antimuscarinic actions of clozapine may contribute to the failure of this drug to induce c-fos expression in this structure. ~4 However, the receptor mechanisms underlying clozapine-induced c-fos expression in the m P F C and other brain regions are unknown. Because clozapine is also an antagonist at 5-hydroxytryptamine 2 (5-HT~) 24'25 and ~1 noradrenergic receptors, 3'16'34it is possible that it induces c-fos expression in some regions via actions at these receptors. Indeed, Meltzer and colleagues 25,42 have advanced the hypothesis that the ratio of 5-HT 2 to D 2 receptor blockade is a key variable that discriminates between typical and atypical antipsychotic drugs, while Baldessarini et al. 3 have pointed out that a distinguishing feature of clozapine is its relatively potent central antiadrenergic actions and low antidopaminergic activity. In addition, two recently discovered D2-1ike receptor subtypes, D3 and D 4 receptors, are also candidate mechanisms for clozapine-induced c-Jbs expression in the brain. D 3 receptors are expressed mainly in limbic brain areas, including the olfactory tubercle, NAc, islands of Calleja and hypothalamus, 4° but are also present in the striatum. H'~9'2° Their predominant limbic distribution has led to the hypothesis that D 3 receptors are involved in the regulation of cognitive and emotional functions and may thus be relevant to the antipsychotic effects of neuroleptics. 39'4° Like the D 3 receptor, the D 4 receptor has a unique regional distribution, the highest levels of expression apparently occurring in the prefrontal c o r t e x ( P F C ) . 22'31'39 In addition, the affinity of the D4 receptor for clozapine is an order of magnitude higher than for the D 2 receptor, 44 raising the possibility that actions at D4 receptors contribute to clozapine's unique therapeutic profile. In the experiments reported here, the extent to which serotonergic and/or noradrenergic mechanisms are involved in clozapine-induced c-fos expression
was determined by examining the effects of prior lesions of either 5-HT- or noradrenaline (NA)containing neurons on clozapine-induced c-fos expression in the brain. In addition, the 5-HT2 receptor antagonist ritanserin was combined with haloperidol administration to determine if concomitant blockade at D 2 and 5-HT 2 receptors would mimic clozapine's actions on c-fos expression. To test the involvement of subtypes of the D2 receptor family in the actions of clozapine, the somewhat selective D 3 agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OHDPAT), which has about 75- and 1000-fold lower affinity for D 2 and D 4 receptors respectively than for the D 3 receptor, 2° and quinpirole, which has approximately equal affinity for D 3 and D4 receptors and about 100-fold lower affinity for the D2 receptor, 12'2°'37 were combined with clozapine administration.
EXPERIMENTAL PROCEDURES
Animals Adult male Wistar rats Charles River, Quebec (280-3 I0 g) were kept under a 12 h light/12 h dark cycle, with free access to food and water. The rats were handled periodically for at least three to four days prior to the experiment.
Brain lesions Unilateral lesions of the medial forebrain bundle (MFB) were made in 22 rats following surgical procedures detailed elsewhere. 32 In brief, the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic apparatus. The rats were then pretreated with the NA and dopamine (DA) reuptake inhibitor, nomifensine (Research Biochemicals International, Natick, MA; 15mg/kg i.p.), 30~J,0 min prior to the neurotoxin infusion. With the incisor bar set 3.3 mm below the interaural plane, the coordinates were: AP -3.8 mm; ML + 1.7 mm; DV -8.7 mm, relative to bregma. 33 The lesion side received an infusion of 5,7dihydroxytryptamine (5,7-DHT; Sigma, St Louis, MO; 8.0 mg in 3.0 ml of 0.I % ascorbic acid in 0.9% saline), and on the sham side a cannula was lowered to DV - 1.5 mm without drug injection. The drug was infused via a 30-gauge cannula over I0 min (0.3 ml/min), and the cannula was left in place for an additional 5 min to allow for diffusion. For bilateral lesions of the dorsal noradrenergic bundle (DNB) the following coordinates were used: A P + 2.6mm from interaural line, ML + 1.1 mm from midline, DV + 3.7 mm from the interaural line, with the incisor bar set 3.3 mm below the interaural plane. 43 Twenty-two rats received 6hydroxydopamine (6-OHDA; Sigma; 4 mg in 2 ml of 0.3% ascorbic acid in 0.9% saline), and the drug was infused bilaterally through 30-gauge cannulae at 1.0 ml/min for 2 min. Twenty-two rats served as operated controls in which the cannulae were lowered bilaterally in the overlying cortex without infusion.
Neurochemical analyses Two to three weeks after surgery, some of the lesioned rats were killed to determine the effects of 5,7-DHT MFB lesions on 5-HT concentrations and 6-OHDA DNB lesions on NA concentrations in the PFC and other brain regions. The brains were removed and dissected on a cold Petri dish. Brain samples of the PFC, striatum and hippocampus were weighed and placed in a cold homogenizing solution (0.22 N perchloric acid, 0.05% EDTA-Na2, and 0.15% sodium bisulfite). The tissues were sonicated for 30 s and centrifuged at 32,500g for 20min (4°C), and the supernatants were stored at -80°C. Regional brain analysis of 5-HT, NA
Receptor mechanisms of clozapine-induced c-fos expression and DA was performed by reverse phase high-pressure liquid chromatography with electrochemical detection. 4 The flow rate of the mobile phase (33 mM sodium acetate, 0.9mM octanesulfonic acid, 13% methanol, 0.036% EDTA, pH 3.6) was 0.7 ml/min. Standards (10-TM of uric acid, adrenaline, NA, dihydroxyphenylacetic acid, DA, 5hydroxyindoleacetic acid, 3-methoxytyramine, 5-HT) were injected into the high-pressure liquid chromotographyelectrochemical detection system before and after running tissue samples. The tissue levels of 5-HT, NA and DA were determined by comparing sample peaks with those of known standards. Each measure was taken as the average of two separate elutions of each sample, and was utilized for subsequent statistical analysis. The detection limit of the system was approximately 0.5 pmol/injection.
Drug administration To determine whether serotonergic or noradrenergic mechanisms contribute to clozapine-induced c-fos expression in the mPFC and other brain regions, clozapine (H. Lundbeck, Kobenhavn-Valby; 20 mg/kg) or vehicle (40#1 20% acetic acid in 1 ml 0.9% saline) was injected subcutaneously (s.c.) into the 5,7-DHT- or 6-OHDAlesioned rats and sham controls. In addition, some unlesioned rats received ritanserin (Research Biochemicals International, Natick, MA; 5 mg/kg, s.c.) or saline respectively 30 rain before haloperidol (McNeil Pharmaceutical Canada LTD., Stouffville, Ont; l mg/kg, s.c.) or vehicle injections. In other experiments, quinpirole or 7-OH-DPAT were co-administered with clozapine. Rats were injected with clozapine (20mg/kg, s.c.) 15min after the injection of quinpirole (RBI; 1.0mg/kg, s.c.) or 7-OH-DPAT (RBI; 0.5mg/kg, s.c.). Control groups received quinpirole (1.0 mg/kg, s.c.), 7-OH-DPAT (0.5 mg/kg, s.c.) or vehicle respectively following saline injection.
Fos immunohistochemistry Two hours after the final drug injection, the rats were anesthetized and perfused with 0.9% saline followed by 4% paraformaldehyde. The brains were removed and placed in fresh fixative for at least 12 h. Thirty-micrometer sections were cut from each brain using a Vibratome. The sections were preincubated with 0.5% H202 for 15 min to remove endogenous peroxidase activity, and then incubated with the Fos primary antibody (Cambridge Research Biochemicals, CRB OA-I 1-823; diluted 1:3000), a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA; diluted 1 : 200) and avidin-biotinylated horseradish peroxidase complex (Vector Laboratories; 1:200). The reaction product was visualized using the glucose oxidase~zliaminobenzidine-nickel method of Shu et al.38 The sections were mounted on chrome-alum-coated slides, dehydrated and prepared for microscopic examination. The number of
749
Fos-positive nuclei in the mPFC, medial and lateral striatum, NAc and lateral septal nucleus were counted within a 500 x 500/~m grid placed over the area at x79 magnification. Fos-positive nuclei in the major island of Calleja were counted within a 63.5 × 190/tm grid at × 125 magnification. The number of Fos-positive neurons was counted by two individuals unaware of the animals treatment and averaged from four separate sections per animal, and utilized for subsequent statistical analysis.
Statistical analysis For neurochemical data, statistical analysis was performed using a Student's two-tailed t-test. Between-group differences in the number of Fos-positive nuclei within specified brain regions from animals for both lesion experiments and different drug administrations were evaluated by one-way ANOVA. Newman-Keuls' post hoc test was used to compare drug-induced changes in Fos induction in each brain area. RESULTS
Effect o f medial forebrain bundle and dorsal noradrenergic bundle lesions The c o n c e n t r a t i o n s of 5-HT and D A in the P F C a n d striatum are s h o w n in Table 1. The 5,7-DHT lesions significantly reduced 5-HT in the two brain regions assayed, without affecting D A c o n c e n t r a t i o n s in the PFC; 5-HT was decreased in b o t h the P F C a n d the striatum by more t h a n 91%. Table 2 shows the effects of bilateral 6 - O H D A D N B lesions o n forebrain N A a n d D A levels. The lesion caused extensive reductions in N A in the forebrain, with a loss of 93.6% in the P F C a n d 94.7% in the hippocampus. In contrast, the 6 - O H D A lesions did not affect D A c o n c e n t r a t i o n s in the hippocampus, but did produce some reduction in the PFC.
Clozapine-induced Fos-like immunoreactivity Clozapine produced significant increases in Foslike immunoreactivity in the m P F C , N A c a n d lateral spetal nucleus (Figs 1, 2, 4, 7). Fos-positive nuclei were scattered in a h o m o g e n e o u s m a n n e r t h r o u g h o u t each of these structures (Figs 5A, C, E, 8A, C, E) with the exception of the m a j o r island o f Calleja. W i t h i n the latter structure, the vast majority of nuclei were Fos-positive following clozapine (Fig. 6A).
Table 1. Effects of unilateral medial forebrain bundle 5,7-dihydroxytryptamine lesions on forebrain 5-hydroxytryptamine and dopamine levels Region group
5-HT (pmol/mg)
DA (pmol/mg)
Prefrontal cortex control lesion
0.78 + 0.10 0.07 + 0.0l*
(-91.1%)
0.41 _ 0.05 0.48 + 0.07
( + 16.2%)
Striatum control lesion
0.90 + 0.09 0.07 +__0.01"
(-91.8%)
32.26 _ 2.25 25.98 + 2.0
(-- 19.5%)
Data represent the mean (+S.E.M.) tissue concentrations of 5-HT and DA following unilateral MFB 5,7-DHT lesion and sham surgery; numbers in parentheses show % change from corresponding control; n = 6. *Significant (P < 0.01) difference from corresponding control.
N. Guo et al.
750
Table 2. Effects of bilateral noradrenergic bundle 6-hydroxydopamine lesions on forebrain noradrenaline and dopamine levels NA (pmol/mg)
Region group
DA (pmol/mg)
Prefrontal cortex control lesion
1.44 + 0.08 0.09 __+0.01"
(-93.6%)
0.36 _+0.04 0.27 + 0.03
(-24.3%)
Hippocampus control lesion
1.85 __+0.10 0.10 + 0.01'
(-94.7%)
0.022 _+0.00 0.023 + 0.00
(+4.3%)
Data are the mean (±S.E.M.) tissue concentrations of NA and DA after bilateral DNB 6-OHDA lesion or sham surgery; numbers in parentheses represent % change from corresponding control; n = 10 per group. *Significant (P < 0.01) difference from corresponding control.
Effects of 5-hydroxytryptamine and noradrenaline depletions on clozapine-induced c-los expression in the forebrain Unilateral 5,7-DHT M F B lesions did not affect the n u m b e r of clozapine-induced Fos-positive neurons in the mPFC, NAc and lateral septal nucleus, compared to the control side (Fig. 1). As shown in Fig. 2, depletion of N A in the forebrain produced by bilateral 6 - O H D A DNB lesions also failed to affect clozapine-induced c-fos expression in these brain regions. Neither 5-HT nor N A depletions affected baseline levels of c-fos expression (Figs 1, 2).
Effect of ritanserin on haloperidol-induced expression
c-los
Haloperidol (1 mg/kg, s.c.) increased the n u m b e r of Fos-positive neurons in the NAc, medial and lateral striatum, and lateral septal nucleus, while ritanserin (5mg/kg) failed to influence Fos-like immunoreactivity in these structures, as shown in Fig. 3. When combined with haloperidol, ritanserin
180 160 ~
140
z
12o 100 8O
G
.~ O I-=
~ o u_
[] [] II []
Vehicle { Vehicle ( Clozapine Clozapine
sham ] lesion ) ( sham ] (lesion)
failed to influence haloperidol-induced c-fos expression in the NAc, medial and lateral striatum, and lateral septal nucleus, and did not mimic clozapineinduced c-fos expression in the m P F C (Fig. 3).
Effects of quinpirole on clozapine-induced expression
Quinpirole (1 mg/kg, s.c.), which by itself did not affect c-fos expression (Fig. 4), produced small but statistically significant decreases in clozapine-induced Fos-like immunoreactivity in the m P F C (Figs 4, 5A, B). Quinpirole also blocked the clozapineinduced expression in the NAc (Figs 4, 5C, D) and the major island of Calleja (Figs 4, 6B), and had a large, although incomplete, effect in the lateral septal nucleus (Figs 4, 5E, F).
Effects of 7-OH-DPAT on clozapine-induced c-fos expression 7-OH-DPAT (0.5 mg/kg, s.c.) significantly reduced (78.7% and 51.6%) clozapine-induced increases in
180 .u ,~m @ >
0
160 140 120 100
:9=,0 8o
[] [] •
Vehicle ( lesion ) Vehicle ( sham ) Clozapine ( lesion )
[]
Clozapine
u.
.. ~.
4020 F_~F
~
O mPFC
NAc
M.CP
L.CP
LS
Brain Region
Fig. 1. Effect of 5,7-DHT lesion in the MFB on clozapineinduced Fos expression in the brain. 5-HT depletion in the forebrain produced by the lesion did not alter the number of Fos-positive nuclei induced by clozapine (20 mg/kg) in the mPFC, NAc and lateral septal nucleus (LS). Neither sham nor neurotoxic lesion affected baseline levels of Foslike immunoreactivity in the brain. Data represented as the mean + S.E.M. *P < 0.001 vs vehicle; n = 6 per treatment. M.CP, medial striatum; L.CP, lateral striatum.
( sham ) .it. "ie
60
8o 40 20
c-fos
mPFC
NAc
,.-,--p=mr~ ~ M.CP L.CP
LS
Brain Region
Fig. 2. Effect of 6-OHDA lesion of the DNB on clozapineinduced Fos-like immunoreactivity in the brain. NA depletion in the forebrain produced by neurotoxic lesion did not alter the number of Fos-positive nuclei induced by clozapine (20 mg/kg) in the mPFC, NAc and lateral septal nucleus (LS). Baseline levels of c-los expression were not affected by either sham or neurotoxic lesion. Data represented as the mean _ S.E.M. *P < 0.001 vs vehicle; n = 7 per group. M.CP, medial striatum; L.CP, lateral striatum.
Receptor mechanisms of clozapine-induced c-fos expression
180 160 i
[] Saline [] Ritanserin • Haloperidol
140 H a l ° p e r iE~ d ° tRitanserin 1 0**0 1 2 0+8 0
**
**
4O 20
o rmPFC t~
NAc
M.CP L.CP
LS
Brain Region Fig. 3. Effects of saline, ritanserin (5 mg/kg), haloperidol (lmg/kg) and ritanserin (5mg/kg) plus haloperidol (1 mg/kg) on the number of Fos-positive nuclei (mean + SEM) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP) and lateral septal nucleus (LS). *P < 0.01 vs saline; n = 6 per treatment.
the number of Fos-positive neurons in the NAc (Figs 7, 8C, D) and lateral septal nucleus, respectively (Figs 7, 8E, F). 7-OH-DPAT also blocked clozapineinduced c-fos expression in the major island of Calleja (Figs 6C, 7). However, this DA receptor agonist failed to influence the number of clozapineinduced Fos-positive neurons in the mPFC (Figs 7, 8A, B). DISCUSSION
Because clozapine is a more potent antagonist at 5-HT2 receptors than at D 2 receptors, Meltzer and colleagues25'26'42 have proposed that strong blockade at 5-HT 2 receptors in the presence of weak blockade at D2 receptors contributes to the unique clinical
[] Vehicle • [] Ouinpirole []
Clozapine Quinpirole + Clozapine
+
180 t 160 1 '~ 140 t Z"~ 120 1 =
loo~
'~o
80
~
40
.~,
*
+
.
*
*
8o
÷
.+
2O
0 rnPFC
NAc M.CP L.CP LS
ICjM
Brain Region Fig. 4. Effects of vehicle (1 ml/kg), quinpirole (1 mg/kg) clozapine (20 mg/kg), and quinpirole (1 mg/kg) plus clozapine (20mg/kg) on the number of Fos-positive nuclei (mean + SEM) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP), lateral septal nucleus (LS) and the major island of Calleja (ICjM). *P < 0.01 vs quinpirole, fP < 0.01 vs quinpirole + clozapine.
751
profiles of clozapine and other atypical antipsychotic drugs. It has also been suggested that the antagonist actions of clozapine at ~12'3A6 or ct2 noradrenergic receptors 35 contribute to the atypical profile of clozapine. In order to determine if serotonergic or noradrenergic mechanisms play a role in the unique pattern of c-fos expression produced by clozapine, the effects of extensive prior lesions of ascending serotonergic or noradrenergic projections to the telencephalon were examined. These lesions failed to influence the extent or distribution of clozapineinduced Fos-like immunoreactivity in the mPFC, NAc or lateral septal nucleus (Figs 1, 2). This contrasts with earlier observations that 6-OHDA lesions of the mesotelencephalic DA system block haloperidol-induced c-fos expression in the striatum and haloperidol- and clozapine-induced c-fos expression in the NAc. 36 The present lesion results provide no support, therefore, for the hypothesis that clozapineinduced c-fos expression in the mPFC or lateral septal nucleus is mediated by 5-HT2 or ~ 1-noradrenergic receptor blockade. In agreement with the 5-HT lesion study, the 5-HT: antagonist ritanserin failed to mimic clozapine-induced increases in Fos-like immunoreactivity in the mPFC either by itself or when combined with haloperidol, suggesting that 5-HT: antagonism does not contribute to clozapineinduced c-fos expression in the mPFC or other brain regions. Nevertheless, it remains possible that a certain ratio of 5-HT: to D 2 receptor blockade is critical with respect to c-fos expression in different brain regions. This will require an examination of the effects of a range of doses of ritanserin on haloperidol-induced Fos-like immunoreactivity. The failure of haloperidol, a DA receptor antagonist with a five- to 10-fold higher affinity for D2 than D 3 or D4 receptors, 12to induce c-fos expression in the mPFC 36 suggests that D2 receptor antagonism does not account for the effects of clozapine in this structure. The data reported here suggest that actions at subtypes of the D 2 family of receptors, including perhaps D3 and D4 receptors, may contribute to clozapine's effects on c-fos expression in the forebrain. Thus, the dopamine receptor agonist quinpirole, which in vitro has approximately equal affinities for D 3 and D4 receptors but 100-fold lower affinity for D 2 receptors, 12'2°'37 produced small but significant decreases in clozapine-induced c-Jos expression in the mPFC and had large effects in the NAc, major island of Calleja and lateral septal nucleus. In contrast, the somewhat selective D 3 receptor agonist 7-OH-DPAT, which in vitro has about 75-fold lower affinity for D 2 receptors and 1000-fold lower affinity for D 4 receptors, 2°'23 than for D 3 receptors, significantly reduced clozapine-induced increases in the number of Fos-like immunoreactive neurons in the major island of Calleja, NAc and lateral septal nucleus without having any effect in the mPFC. These data suggest that actions at D 3 receptors may mediate clozapine-induced c-)Cos expression
752
N. Guo et al.
Fig. 5. Photomicrographs of Fos-like immunoreactivity in the mPFC (A, B), NAc (C, D) and lateral septal nucleus (E, F) after clozapine (A, C, E) and quinpirole plus clozapine (B, D, F) treatment. Scale bar = 100#m.
in the NAc, major island of Calleja and lateral septal nucleus, while its actions at D4 receptors may contribute to its actions in the mPFC. These working hypotheses are consistent with current anatomical information concerning the distributions of D 3 and D4 receptors, the former being expressed primarily in limbic brain regions such as the NAc and the islands of Calleja, TM and the latter being most highly expressed in the frontal cortex and amygdala. 2z'3t'44 However, several caveats should be emphasized with respect to these interpretations. First,
quinpirole produced only a small attenuation of clozapine-induced c-fos expression in the mPFC (Fig. 4). This suggests that while antagonist actions at D4 receptors may contribute to clozapine's actions in the mPFC, the full spectrum of mechanisms by which it increases c-fos expression in this structure remains to be determined. Second, despite its considerable selectivity in in vitro assays, the extent to which the in vivo actions of 7-OH-DPAT can be attributed to selective actions at D 3 receptors is uncertain.10,17,TM
Receptor mechanisms of clozapine-induced c-fos expression
753
Fig. 6. Photomicrographs of Fos-like immunoreactivity in the major island of Calleja (ICjM) after clozapine (A), quinpirole plus clozapine (B) and 7-OH-DPAT plus clozapine (C) treatment. MS, medial septal nucleus. Scale bar = 46 pm. Some data are not easily accommodated by the hypothesis that clozapine increases c-fos expression in the NAc, major island of Calleja and lateral septal nucleus via actions at D3 receptors and in the mPFC via 0 4 receptor mechanisms. For example, in vitro studies have shown that clozapine has two to threefold higher affinity for D2 receptors than for D3 receptors. 12'4°This appears to be inconsistent with the present results, which suggest that clozapine increases c-fos expression in the NAc, major island of Calleja and lateral septal nucleus via D3 receptor antagonism, because at the same time it failed to produce the expected D2 receptor antagonist-mediated increases in the number of Fos-like positive neurons in the striatum. Given clozapine's higher affinity for D2 than m Clozapine 180
El 7-OHDPAT + Clozapine
.$
160
~
140t
i
"° I
II II
=
=
loo I
'~
~o
~"
6o 4o
~ M.
20 0
mPFC NAc
M.CP L.CP
LS
ICjM
Brain Region Fig. 7. Effects of c]ozapine (20mg/kg) and 7-OH-DPAT
(0.5 mg/kg) plus clozapine (20 mg/kg) on the number of Fos-positive nuclei (mean + S.E.M.) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP), lateral septal nucleus (LS) and major island of Calleja (IcjM). *P < 0.001 vs clozapine; n = 6 per group.
D 3 receptors, its positive effects on c-fos expression in the NAc, major island of Calleja and lateral septal nucleus indicate that its inability to increase the number of Fos-like immunoreactive neurons in the striatum by blocking D2 receptors was not due to insufficient dosing. The disparities between the results of previous receptor binding studies and the present results indicate that in vitro affinities of typical and atypical antipsychotics for DA receptors cannot by themselves account for the patterns of c-los expression produced by these compounds. A number of other contributing factors must therefore be considered. First, it is possible that active metabolites that have affinities for the dopamine receptor subtypes that are different from the parent compounds contribute to the patterns of c-fos expression reported here. Second, the in vivo potencies of antipsychotic drugs in occupying central neurotransmitter receptors do not always correspond to their in vitro affinities for these receptors. 21'26'42It is worth noting in this regard that the conditions under which in vitro affinities are determined no doubt differ very substantially from those that exist in vivo (see also Refs 10, 17 and 18). Finally, multiple neurotransmitter receptors are probably involved in regulating c-los expression in individual neurons. For example, the antimuscarinic drug scopolamine attenuates haloperidol-induced czfos expression in the striatum. TM Similarly, haloperidol-induced c-los expression in the striatum can be reduced by the glutamate receptor antagonist dizocilpine maleate? Robertson and Fibiger36 reported that while 6-OHDA lesions of the mesotelencephalic DA system blocked clozapine-induced increases in the number of Fos-like immunoreactive neurons in the NAc, these
754
N. Guo et al.
Fig. 8. Photomicrographs of Fos-like immunoreactivity in the mPFC (A, B), NAc (C, D) and lateral septal nucleus (E, F) following clozapine (A, C, E) and 7-OH-DPAT plus clozapine treatment (B, D, F) treatment. Scale bar = 100 #m.
lesions had no effect on clozapine's actions in the mPFC and lateral septal nucleus. Indeed, it was on the basis of this observation that a non-dopaminergic mechanism was proposed for clozapine in these structures. 36 The fact that quinpirole significantly attenuates clozapine-induced increases in c-los expression in the mPFC and lateral septal nucleus (Fig. 4) suggests
that dopaminergic mechanisms do contribute, at least in part, to clozapine's actions in these structures. While explanations for this discrepancy are not obvious, it is worth noting that there is precedence for lesion-induced depletions of DA failing to block neurochemical actions of neuroleptics. For example, extensive ( ~ 9 5 % ) 6-OHDA lesions of the nigro-
Receptor mechanisms of clozapine-induced c-fos expression striatal projection fail to affect the ability of haloperidol to reduce striatal c o n c e n t r a t i o n s of acetylcholine. 9 This raises the possibility t h a t the effects o f neuroleptics m a y n o t simply be due to blocking the actions of D A at D A receptors b u t t h a t these c o m p o u n d s have a level o f intrinsic ac-
755
tivity, this perhaps only occurring in the denervated condition. Acknowledgements--This work was supported by the Medical Research Council of Canada and an unrestricted grant from Bristol-Myers Squibb. The authors wish to thank C. Wilson for excellent technical assistance.
REFERENCES
1. Angst J., Bente D., Berner P., Heimann H., Helmchen H. and Hippius H. (1971) Das klinische irkungsbild von clozapine (untersuchung mit dem AMP-system). Pharmakopsychiatrie 4, 201-21 I. 2. Baldessarini R. J. and Frankenburg F. R. (1991) Clozapine: a novel antipsychotic agent. New Engl. J. Med. 324, 746-754. 3. Baldessarini R. J., Huston-Lyons D., Campbell A., Marsh E. and Cohen B. M. (1992) Do central antiadrenergic actions contribute to the atypical properties of clozapine? Br. J. Psychiat. 160, Suppl. 17, 12 16. 4. Blackburn J. R., Phillips A. G., Jakubovic A. and Fibiger H. C. (1986) Increased dopamine metabolism in the nucleus accumbens and striatum following consumption of a nutritive meal but not a palatable non-nutritive saccharine solution. Pharmac. Biochern. Behav. 25, 1095-1100. 5. Bouthenet M.-L., Souil E., Martres M.-P., Sokoloff P., Giros B. and Schwartz J.-C. (1991) Localization of dopamine D 3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D 2 receptor mRNA. Brain Res. 564, 203-219. 6. Deutch A. Y., Moghaddam B., Innis R. B., Krystal J. H., Aghajanian G. K., Bunney B. S. and Charney D. S. (1991) Mechanisms of atypical antipsychotic drugs: implications for novel therapeutic strategies for schizophrenia. Schizophrenia Res. 4, 121-156. 7. Deutch A. Y., Lee M. C. and Iadarola M. J. (1992) Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Molec. Cell Neurosci. 3, 332 341. 8. Dragunow M., Robertson G. S., Faull R. L. M., Robertson H. A. and Jansen K. (1990) D2 dopamine receptor antagonists induce Fos and related proteins in rat striatal neurons. Neuroscience 37, 287-294. 9. Fibiger H. C. and Grewaal D. S. (1974) Neurochemical evidence for denervation supersensitivity: the effect of unilateral substantia nigra lesions on apomorphine-induced increases in neostriatal acetylcholine levels. Life Sci. 15, 57~53. 10. Freedman J. E., Waszczak B. L., Cox R. F. and Liu J.-C. (1994) The dopamine D3 receptor and 7-OH-DPAT. Trends pharmac. Sci. 15, 173. 11. Gehlert D. R., Gackenheimer S. L., Seeman P. and Schaus J. (1992) Autoradiographic localization of [3H]quinpirole binding to dopamine D2 and D3 receptors in rat brain. Eur. J. Pharmac. 211, 189-194. 12. Gingrich J. A. and Caron M. G. (1993) Recent advances in the molecular biology of dopamine receptors. A. Rev. Neurosci 16, 299-321. 13. Gross H. and Langner E. (1966) Das wirkungsprofil eines chemisch neuartigen breitbandneuroleptikums der dibenzodiazepingruppe. Wien. Med. Wochenschr. 116, 814-816. 14. Guo N., Robertson G. S. and Fibiger H. C. (1992) Scopolamine attenuates haloperidol-induced c-los expression in the striatum. Brain Res. 588, 164-167. 15. Kane J. M., Honigfeld G., Singer J., Meltzer H. and the Clozaril Collaborative Study Group (1988) Clozapine for the treatment-resistant schizophrenic. Arch. gen. Psychiat. 45, 789 796. 16. Lane R. F., Blaha C. D. and Rivet J. N. (1988) Selective inhibition of mesolimbic dopamine release following chronic administration ofclozapine: involvement of cd-noradrenergic receptors demonstrated by in vivo voltammetry. Brain Res. 460, 398~401. 17. Large C. H. and Stubbs C. M. (1994) The dopamine D3 receptor: Chinese hamsters or Chinese whispers? Trends pharmac. Sci. 15, 46~,7. 18. Large C. H. and Stubbs C. M. (1994) Large and Stubbs reply. Trends pharmac. Sci. 15, 173 174. 19. Levant B., Grigoriadis D. E. and DeSouza E. B. (1993) [3H]Quinpirole binding to putative D2 and D3 dopamine receptors in rat brain and pituitary gland: a quantitative autoradiographic study. J. Pharmac. exp. Ther. 264, 991-1001. 20. Levesque D. L., Diaz J., Pilon C., Martres M.-P., Giros B., Souil E., Schott D., Morgat J.-L., Schwartz J.-C. and Sokoloff P. (1992) Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin. Proc. natn. Aead. Sci U.S.A. 89, 8155-8159. 21. Leysen J. E., Janssen P. M. F., Schotte A., Luyten W. H. M. L. and Megens A. A. H. P. (1993) Interaction of antipsychotic drugs with neurotransmitter receptor sites in vitro and in vivo in relation to pharmacological and clinical effects: role of 5HT2 receptors. Psyehopharmacology 112, $40-$54. 22. Mansour A., Meador-Woodruff J., Burke S., Bunzow J., Akil H., Van Tol H. H. M., Civelli O. and Watson S. J. (1991) Differential distribution of D 2 and 04 dopamine receptor mRNAs in the rat brain: an in situ hybridization study. Soc. Neurosci Abstr. 17, 599. 23. McElroy J. F., Amy K. A., Ward K. A., Zeller K. L., Cawley J. F. and Mazzola A. L. (1993) In vivo agonist properties of 7-OH-DPAT, a dopamine D3-selective receptor ligand. Soc. Neurosci. Abstr. 19, 1064. 24. Meltzer H. Y. (1989) Clinical studies on the mechanism of action of clozapine: the dopamine serotonin hypothesis of schizophrenia. Psychopharmacology 99, S18~27. 25. Meltzer H. Y., Matsubara S. and Lee J.-C. (1989) Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-l, D-2, and serotonin 2 pK~ values. J. Pharmac. exp. Ther. 251, 238 246. 26. Meltzer H. Y., Zhang Y. and Stockmeier C. A. (1992) Effect of amperozide on rat cortical 5-HT2 and striatal and limbic dopamine D2 receptor occupancy: implications for antipsychotic action. Eur. J. Pharmac. 216, 67-71. 27. Miller J. C. (1990) Induction of c-fos mRNA expression in rat striatum by neuroleptic drugs. J. Neurochem. 54, 1453-1455.
756
N. Guo et al.
28. Morgan J. I. and Curran T. (1989) Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci. 12, 459-462. 29. Morgan J. I. and Curran T. (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. A. Rev. Neurosci 14, 421-451. 30. Nguyen T. Y., Kosofsky B. E., Birnbaum R., Cohen B. M. and Hyman S. E. (1992) Differential expression of c-fos and zif268 in rat striatum after haloperidol, clozapine, and amphetamine. Proc. natn. Acad Sci U.S.A. 89, 4270-4274. 31. O'Malley K. L., Harmon S., Tang L. and Todd R. D. (1992) The rat dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol. 4, 137-146. 32. Paris J. M., Mitsushio H. and Lorens S. L. (1989) Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions. Brain Res. 476, 183-188. 33. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, San Diego. 34. Peroutka S. J. and Snyder S. H. (1980) Relationship of neuroleptic drug effects at brain dopamine, serotonin, ct-adrenergic and histamine receptors to clinical potency. Am. J. Psychiat. 137, 1518-1522. 35. Pickar D., Owen R. R., Litman R. E., Konicki E., Gutierrez R. and Rapaport M. H. (1992) Clinical and biologic response to clozapine in patients with schizophrenia: crossover comparison with fluphenazine. Arch. gen. Psychiat. 49, 345-353. 36. Robertson G. S. and Fibiger H. C. (1992) Neuroleptics increase c-los expression in the forebrain: contrasting effects of haloperidol and clozapine. Neuroscience 46, 315-328. 37. Schwartz J.-C., Giros B., Martres M.-P. and Sokoloff P. (1992) The dopamine receptor family: molecular biology and pharmacology. Sere. Neurosci 4, 99-108. 38. Shu S., Ju G. and Fan L. (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett 85, 169-171. 39. Sibley D. R. and Monsma F. J. Jr. (1992) Molecular biology of dopamine receptors. Trends pharmac. Sci 13, 61-68. 40. Sokoloff P., Giros B., Martres M.-P., Bouthenet M.-C. and Schwartz J.-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 14(~151. 41. Sokoloff P., Giros B., Martres M. P., Andrieux M., Besancon R., Pilon C., Bouthenet M. L., Souil E. and Schwartz J. C. (1992) Localization and function of the D 3 dopamine receptor. Drug Res. 42, 224 230. 42. Stockmeier C. A., DiCarlo J. J., Zhang Y., Thompson P. and Meltzer H. Y. (1993) Characterization of typical and atypical antipsychotic drugs based on in vivo occupancy of serotonin 2 and dopamine 2 receptors. J. Pharmac. exp. Ther. 266, 1374~1384. 43. U'Prichard D. C., Reisine T. D., Mason S. T., Fibiger H. C. and Yamamura H. (1980) Modulation of rat brain orand fl-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle. Brain Res. 187, 143-154. 44. Van Tol H. H. M., Bunzow J. R., Guan H.-C., Sunahara R. K., Seeman P., Niznik H. B. and Civelli O. (1991) Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 6104514. (Accepted 24 October 1994)
~
Pergamon
0306-4522(94)00552-4
Elsevier ScienceLtd Copyright © 1995IBRO Printed in Great Britain.All rights reserved 0306-4522/95 $9.50+ 0.00
RECEPTOR MECHANISMS MEDIATING CLOZAPINE-INDUCED c-fos EXPRESSION IN THE FOREBRAIN N. G U O , M. A. K L I T E N I C K , C.-S. T H A M and H. C. F I B I G E R * Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 A~tract--The atypical antipsychotic clozapine produces distinctly different regional patterns of c-Jbs expression in rat forebrain than does the prototypical neuroleptic haloperidol. While haloperidoMnduced c-fos expression appears to be mediated by its D 2 dopamine receptor antagonist properties, the mechanisms by which clozapine increases c-fos expression remain uncertain. Using a combination of brain lesion, pharmacological and immunohistochemical techniques, the present study sought to determine the receptor mechanisms by which clozapine increases the number of Fos-like immunoreactive neurons in various regions of the forebrain. To test whether serotonergic and/or noradrenergic systems are involved in clozapine-induced c-fos expression, rats received either 5,7-dihydroxytryptamine lesions of the medial forebrain bundle or 6-hydroxydopamine lesions of the dorsal noradrenergic bundle two weeks prior to clozapine (20 mg/kg) injections. Neither type of lesion affected clozapine-induced c-los expression in the rat forebrain, suggesting that neither serotonergic nor noradrenergic mechanisms are involved in this action of clozapine. In another experiment, the 5-hydroxytryptamine2 receptor antagonist ritanserin (5 mg/kg), either alone or in combination with haloperidol (1 mg/kg), failed to mimic the pattern of c-Jbs expression produced by clozapine. This suggests that clozapine's antagonist actions at 5-hydroxytryptamine2 receptors cannot explain the unique pattern of regional c-fos expression produced by this compound. To determine whether the blockade of subtypes of the D: dopamine receptor family may contribute to clozapine's effects, the dopamine receptor agonists quinpirole and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin(7-OH-DPAT) were injected 15min prior to clozapine. Quinpirole produced a small but significant decrease in clozapine-induced c-fos expression in the medial prefrontal cortex, had larger effects in the lateral septum, and blocked clozapine's actions in the nucleus accumbens and major island of Calleja. Pretreatment with 7-OH-DPAT attenuated clozapine-induced c-fos expression in the nucleus accumbens and lateral septum, completely blocked the expression in the major island of Calleja, but was without effect in the medial prefrontal cortex. Given the different affinities of quinpirole and 7-OH-DPAT for D2, D3 and D4 receptors, these data suggest that clozapine-induced increases in c-fos expression in the nucleus accumbens, major island of Cajella and lateral septal nucleus are due to antagonist actions of this antipsychotic at Da dopamine receptors. They also indicate that while antagonist actions at D4 receptors may contribute, the primary mechanisms by which clozapine increases c-fos expression in the medial prefrontal cortex remain to be determined.
The mechanisms by which clozapine exerts therapeutic effects without producing concomitant extrapyramidal side effects remain elusive. ~'6'13'15 The inability to differentiate atypical (e.g. clozapine) from typical antipsychotic drugs (e.g. haloperidol) on the basis of their actions at dopaminergic, cholinergic, serotonergic, adrenergic, and opiate sigma receptors, 6 implies that there might be more than one mechanism through which neuroleptics exert their actions. Since
the interaction of neuroleptic drugs with neurotransmitter receptors is only the initial step in their actions, recent attention has shifted to investigations of intracellular postreceptor mechanisms. Immediate early genes are useful markers to map changes in neuronal activity, 28'29 and recently immediate early gene m R N A expression (such as c-fos and zlf/268) and Fos immunohistochemistry have been used to examine the effects of neuroleptics in the CNS. 8'27'30Several studies have shown that typical and atypical antipsychotics have regionally different effects on c-fos *To whom correspondence should be addressed. Abbrev&tions: DA, dopamine; 5,7-DHT, 5,7-dihydroxy- expression in the brain: haloperidol induces c-fos tryptamine; DNB, dorsal noradrenergic bundle; EDTA, expression in the nucleus accumbens (NAc), lateral ethylenediaminetetra-acetate; 5-HT, 5-hydroxytrypta- septal nucleus and striatum, while clozapine increases mine (serotonin): MFB, medial forebrain bundle; the number of Fos-positive neurons in the NAc, mPFC, medial prefrontal cortex; NA, noradrenaline; NAc, nucleus accumbens; 6-OHDA, 6-hydroxydopa- lateral septal nucleus and medial prefrontal cortex mine; 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2- (mPFC). 7'3°'36 This regional specificity of neurolepticaminotetralin; PFC, prefrontal cortex. induced immediate early gene expression suggests 747
748
N. Guo et al.
that different patterns of neuronal activity are evoked by the two classes of antipsychotic drugs, and raises the possibility that these regionally different effects are related to the different clinical profiles of these drugs. 36 Therefore, it is important to elucidate how such patterns are generated. Haloperidol-induced striatal c-fos~expression is thought to be D 2 receptor mediated inasmuch as co-administration of a D2 agonist prevents the induct i o n s and the selective D2 antagonist raclopride produces a regional pattern of Fos-like immunoreactivity that is indistinguishable from that produced by haloperidol. 36 Because of the regional differences between clozapine- and haloperidol-induced c-fos expression in the brain, D2 receptor antagonism is not sufficient to account for the unique pattern produced by clozapine, even though D 2 receptors are a c o m m o n site of action for haloperidol and clozapineY Previous work has demonstrated that the antimuscarinic agent scopolamine attenuates haloperidol-induced c-fos expression in the striatum, suggesting that antimuscarinic actions of clozapine may contribute to the failure of this drug to induce c-fos expression in this structure. ~4 However, the receptor mechanisms underlying clozapine-induced c-fos expression in the m P F C and other brain regions are unknown. Because clozapine is also an antagonist at 5-hydroxytryptamine 2 (5-HT~) 24'25 and ~1 noradrenergic receptors, 3'16'34it is possible that it induces c-fos expression in some regions via actions at these receptors. Indeed, Meltzer and colleagues 25,42 have advanced the hypothesis that the ratio of 5-HT 2 to D 2 receptor blockade is a key variable that discriminates between typical and atypical antipsychotic drugs, while Baldessarini et al. 3 have pointed out that a distinguishing feature of clozapine is its relatively potent central antiadrenergic actions and low antidopaminergic activity. In addition, two recently discovered D2-1ike receptor subtypes, D3 and D 4 receptors, are also candidate mechanisms for clozapine-induced c-Jbs expression in the brain. D 3 receptors are expressed mainly in limbic brain areas, including the olfactory tubercle, NAc, islands of Calleja and hypothalamus, 4° but are also present in the striatum. H'~9'2° Their predominant limbic distribution has led to the hypothesis that D 3 receptors are involved in the regulation of cognitive and emotional functions and may thus be relevant to the antipsychotic effects of neuroleptics. 39'4° Like the D 3 receptor, the D 4 receptor has a unique regional distribution, the highest levels of expression apparently occurring in the prefrontal c o r t e x ( P F C ) . 22'31'39 In addition, the affinity of the D4 receptor for clozapine is an order of magnitude higher than for the D 2 receptor, 44 raising the possibility that actions at D4 receptors contribute to clozapine's unique therapeutic profile. In the experiments reported here, the extent to which serotonergic and/or noradrenergic mechanisms are involved in clozapine-induced c-fos expression
was determined by examining the effects of prior lesions of either 5-HT- or noradrenaline (NA)containing neurons on clozapine-induced c-fos expression in the brain. In addition, the 5-HT2 receptor antagonist ritanserin was combined with haloperidol administration to determine if concomitant blockade at D 2 and 5-HT 2 receptors would mimic clozapine's actions on c-fos expression. To test the involvement of subtypes of the D2 receptor family in the actions of clozapine, the somewhat selective D 3 agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OHDPAT), which has about 75- and 1000-fold lower affinity for D 2 and D 4 receptors respectively than for the D 3 receptor, 2° and quinpirole, which has approximately equal affinity for D 3 and D4 receptors and about 100-fold lower affinity for the D2 receptor, 12'2°'37 were combined with clozapine administration.
EXPERIMENTAL PROCEDURES
Animals Adult male Wistar rats Charles River, Quebec (280-3 I0 g) were kept under a 12 h light/12 h dark cycle, with free access to food and water. The rats were handled periodically for at least three to four days prior to the experiment.
Brain lesions Unilateral lesions of the medial forebrain bundle (MFB) were made in 22 rats following surgical procedures detailed elsewhere. 32 In brief, the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic apparatus. The rats were then pretreated with the NA and dopamine (DA) reuptake inhibitor, nomifensine (Research Biochemicals International, Natick, MA; 15mg/kg i.p.), 30~J,0 min prior to the neurotoxin infusion. With the incisor bar set 3.3 mm below the interaural plane, the coordinates were: AP -3.8 mm; ML + 1.7 mm; DV -8.7 mm, relative to bregma. 33 The lesion side received an infusion of 5,7dihydroxytryptamine (5,7-DHT; Sigma, St Louis, MO; 8.0 mg in 3.0 ml of 0.I % ascorbic acid in 0.9% saline), and on the sham side a cannula was lowered to DV - 1.5 mm without drug injection. The drug was infused via a 30-gauge cannula over I0 min (0.3 ml/min), and the cannula was left in place for an additional 5 min to allow for diffusion. For bilateral lesions of the dorsal noradrenergic bundle (DNB) the following coordinates were used: A P + 2.6mm from interaural line, ML + 1.1 mm from midline, DV + 3.7 mm from the interaural line, with the incisor bar set 3.3 mm below the interaural plane. 43 Twenty-two rats received 6hydroxydopamine (6-OHDA; Sigma; 4 mg in 2 ml of 0.3% ascorbic acid in 0.9% saline), and the drug was infused bilaterally through 30-gauge cannulae at 1.0 ml/min for 2 min. Twenty-two rats served as operated controls in which the cannulae were lowered bilaterally in the overlying cortex without infusion.
Neurochemical analyses Two to three weeks after surgery, some of the lesioned rats were killed to determine the effects of 5,7-DHT MFB lesions on 5-HT concentrations and 6-OHDA DNB lesions on NA concentrations in the PFC and other brain regions. The brains were removed and dissected on a cold Petri dish. Brain samples of the PFC, striatum and hippocampus were weighed and placed in a cold homogenizing solution (0.22 N perchloric acid, 0.05% EDTA-Na2, and 0.15% sodium bisulfite). The tissues were sonicated for 30 s and centrifuged at 32,500g for 20min (4°C), and the supernatants were stored at -80°C. Regional brain analysis of 5-HT, NA
Receptor mechanisms of clozapine-induced c-fos expression and DA was performed by reverse phase high-pressure liquid chromatography with electrochemical detection. 4 The flow rate of the mobile phase (33 mM sodium acetate, 0.9mM octanesulfonic acid, 13% methanol, 0.036% EDTA, pH 3.6) was 0.7 ml/min. Standards (10-TM of uric acid, adrenaline, NA, dihydroxyphenylacetic acid, DA, 5hydroxyindoleacetic acid, 3-methoxytyramine, 5-HT) were injected into the high-pressure liquid chromotographyelectrochemical detection system before and after running tissue samples. The tissue levels of 5-HT, NA and DA were determined by comparing sample peaks with those of known standards. Each measure was taken as the average of two separate elutions of each sample, and was utilized for subsequent statistical analysis. The detection limit of the system was approximately 0.5 pmol/injection.
Drug administration To determine whether serotonergic or noradrenergic mechanisms contribute to clozapine-induced c-fos expression in the mPFC and other brain regions, clozapine (H. Lundbeck, Kobenhavn-Valby; 20 mg/kg) or vehicle (40#1 20% acetic acid in 1 ml 0.9% saline) was injected subcutaneously (s.c.) into the 5,7-DHT- or 6-OHDAlesioned rats and sham controls. In addition, some unlesioned rats received ritanserin (Research Biochemicals International, Natick, MA; 5 mg/kg, s.c.) or saline respectively 30 rain before haloperidol (McNeil Pharmaceutical Canada LTD., Stouffville, Ont; l mg/kg, s.c.) or vehicle injections. In other experiments, quinpirole or 7-OH-DPAT were co-administered with clozapine. Rats were injected with clozapine (20mg/kg, s.c.) 15min after the injection of quinpirole (RBI; 1.0mg/kg, s.c.) or 7-OH-DPAT (RBI; 0.5mg/kg, s.c.). Control groups received quinpirole (1.0 mg/kg, s.c.), 7-OH-DPAT (0.5 mg/kg, s.c.) or vehicle respectively following saline injection.
Fos immunohistochemistry Two hours after the final drug injection, the rats were anesthetized and perfused with 0.9% saline followed by 4% paraformaldehyde. The brains were removed and placed in fresh fixative for at least 12 h. Thirty-micrometer sections were cut from each brain using a Vibratome. The sections were preincubated with 0.5% H202 for 15 min to remove endogenous peroxidase activity, and then incubated with the Fos primary antibody (Cambridge Research Biochemicals, CRB OA-I 1-823; diluted 1:3000), a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA; diluted 1 : 200) and avidin-biotinylated horseradish peroxidase complex (Vector Laboratories; 1:200). The reaction product was visualized using the glucose oxidase~zliaminobenzidine-nickel method of Shu et al.38 The sections were mounted on chrome-alum-coated slides, dehydrated and prepared for microscopic examination. The number of
749
Fos-positive nuclei in the mPFC, medial and lateral striatum, NAc and lateral septal nucleus were counted within a 500 x 500/~m grid placed over the area at x79 magnification. Fos-positive nuclei in the major island of Calleja were counted within a 63.5 × 190/tm grid at × 125 magnification. The number of Fos-positive neurons was counted by two individuals unaware of the animals treatment and averaged from four separate sections per animal, and utilized for subsequent statistical analysis.
Statistical analysis For neurochemical data, statistical analysis was performed using a Student's two-tailed t-test. Between-group differences in the number of Fos-positive nuclei within specified brain regions from animals for both lesion experiments and different drug administrations were evaluated by one-way ANOVA. Newman-Keuls' post hoc test was used to compare drug-induced changes in Fos induction in each brain area. RESULTS
Effect o f medial forebrain bundle and dorsal noradrenergic bundle lesions The c o n c e n t r a t i o n s of 5-HT and D A in the P F C a n d striatum are s h o w n in Table 1. The 5,7-DHT lesions significantly reduced 5-HT in the two brain regions assayed, without affecting D A c o n c e n t r a t i o n s in the PFC; 5-HT was decreased in b o t h the P F C a n d the striatum by more t h a n 91%. Table 2 shows the effects of bilateral 6 - O H D A D N B lesions o n forebrain N A a n d D A levels. The lesion caused extensive reductions in N A in the forebrain, with a loss of 93.6% in the P F C a n d 94.7% in the hippocampus. In contrast, the 6 - O H D A lesions did not affect D A c o n c e n t r a t i o n s in the hippocampus, but did produce some reduction in the PFC.
Clozapine-induced Fos-like immunoreactivity Clozapine produced significant increases in Foslike immunoreactivity in the m P F C , N A c a n d lateral spetal nucleus (Figs 1, 2, 4, 7). Fos-positive nuclei were scattered in a h o m o g e n e o u s m a n n e r t h r o u g h o u t each of these structures (Figs 5A, C, E, 8A, C, E) with the exception of the m a j o r island o f Calleja. W i t h i n the latter structure, the vast majority of nuclei were Fos-positive following clozapine (Fig. 6A).
Table 1. Effects of unilateral medial forebrain bundle 5,7-dihydroxytryptamine lesions on forebrain 5-hydroxytryptamine and dopamine levels Region group
5-HT (pmol/mg)
DA (pmol/mg)
Prefrontal cortex control lesion
0.78 + 0.10 0.07 + 0.0l*
(-91.1%)
0.41 _ 0.05 0.48 + 0.07
( + 16.2%)
Striatum control lesion
0.90 + 0.09 0.07 +__0.01"
(-91.8%)
32.26 _ 2.25 25.98 + 2.0
(-- 19.5%)
Data represent the mean (+S.E.M.) tissue concentrations of 5-HT and DA following unilateral MFB 5,7-DHT lesion and sham surgery; numbers in parentheses show % change from corresponding control; n = 6. *Significant (P < 0.01) difference from corresponding control.
N. Guo et al.
750
Table 2. Effects of bilateral noradrenergic bundle 6-hydroxydopamine lesions on forebrain noradrenaline and dopamine levels NA (pmol/mg)
Region group
DA (pmol/mg)
Prefrontal cortex control lesion
1.44 + 0.08 0.09 __+0.01"
(-93.6%)
0.36 _+0.04 0.27 + 0.03
(-24.3%)
Hippocampus control lesion
1.85 __+0.10 0.10 + 0.01'
(-94.7%)
0.022 _+0.00 0.023 + 0.00
(+4.3%)
Data are the mean (±S.E.M.) tissue concentrations of NA and DA after bilateral DNB 6-OHDA lesion or sham surgery; numbers in parentheses represent % change from corresponding control; n = 10 per group. *Significant (P < 0.01) difference from corresponding control.
Effects of 5-hydroxytryptamine and noradrenaline depletions on clozapine-induced c-los expression in the forebrain Unilateral 5,7-DHT M F B lesions did not affect the n u m b e r of clozapine-induced Fos-positive neurons in the mPFC, NAc and lateral septal nucleus, compared to the control side (Fig. 1). As shown in Fig. 2, depletion of N A in the forebrain produced by bilateral 6 - O H D A DNB lesions also failed to affect clozapine-induced c-fos expression in these brain regions. Neither 5-HT nor N A depletions affected baseline levels of c-fos expression (Figs 1, 2).
Effect of ritanserin on haloperidol-induced expression
c-los
Haloperidol (1 mg/kg, s.c.) increased the n u m b e r of Fos-positive neurons in the NAc, medial and lateral striatum, and lateral septal nucleus, while ritanserin (5mg/kg) failed to influence Fos-like immunoreactivity in these structures, as shown in Fig. 3. When combined with haloperidol, ritanserin
180 160 ~
140
z
12o 100 8O
G
.~ O I-=
~ o u_
[] [] II []
Vehicle { Vehicle ( Clozapine Clozapine
sham ] lesion ) ( sham ] (lesion)
failed to influence haloperidol-induced c-fos expression in the NAc, medial and lateral striatum, and lateral septal nucleus, and did not mimic clozapineinduced c-fos expression in the m P F C (Fig. 3).
Effects of quinpirole on clozapine-induced expression
Quinpirole (1 mg/kg, s.c.), which by itself did not affect c-fos expression (Fig. 4), produced small but statistically significant decreases in clozapine-induced Fos-like immunoreactivity in the m P F C (Figs 4, 5A, B). Quinpirole also blocked the clozapineinduced expression in the NAc (Figs 4, 5C, D) and the major island of Calleja (Figs 4, 6B), and had a large, although incomplete, effect in the lateral septal nucleus (Figs 4, 5E, F).
Effects of 7-OH-DPAT on clozapine-induced c-fos expression 7-OH-DPAT (0.5 mg/kg, s.c.) significantly reduced (78.7% and 51.6%) clozapine-induced increases in
180 .u ,~m @ >
0
160 140 120 100
:9=,0 8o
[] [] •
Vehicle ( lesion ) Vehicle ( sham ) Clozapine ( lesion )
[]
Clozapine
u.
.. ~.
4020 F_~F
~
O mPFC
NAc
M.CP
L.CP
LS
Brain Region
Fig. 1. Effect of 5,7-DHT lesion in the MFB on clozapineinduced Fos expression in the brain. 5-HT depletion in the forebrain produced by the lesion did not alter the number of Fos-positive nuclei induced by clozapine (20 mg/kg) in the mPFC, NAc and lateral septal nucleus (LS). Neither sham nor neurotoxic lesion affected baseline levels of Foslike immunoreactivity in the brain. Data represented as the mean + S.E.M. *P < 0.001 vs vehicle; n = 6 per treatment. M.CP, medial striatum; L.CP, lateral striatum.
( sham ) .it. "ie
60
8o 40 20
c-fos
mPFC
NAc
,.-,--p=mr~ ~ M.CP L.CP
LS
Brain Region
Fig. 2. Effect of 6-OHDA lesion of the DNB on clozapineinduced Fos-like immunoreactivity in the brain. NA depletion in the forebrain produced by neurotoxic lesion did not alter the number of Fos-positive nuclei induced by clozapine (20 mg/kg) in the mPFC, NAc and lateral septal nucleus (LS). Baseline levels of c-los expression were not affected by either sham or neurotoxic lesion. Data represented as the mean _ S.E.M. *P < 0.001 vs vehicle; n = 7 per group. M.CP, medial striatum; L.CP, lateral striatum.
Receptor mechanisms of clozapine-induced c-fos expression
180 160 i
[] Saline [] Ritanserin • Haloperidol
140 H a l ° p e r iE~ d ° tRitanserin 1 0**0 1 2 0+8 0
**
**
4O 20
o rmPFC t~
NAc
M.CP L.CP
LS
Brain Region Fig. 3. Effects of saline, ritanserin (5 mg/kg), haloperidol (lmg/kg) and ritanserin (5mg/kg) plus haloperidol (1 mg/kg) on the number of Fos-positive nuclei (mean + SEM) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP) and lateral septal nucleus (LS). *P < 0.01 vs saline; n = 6 per treatment.
the number of Fos-positive neurons in the NAc (Figs 7, 8C, D) and lateral septal nucleus, respectively (Figs 7, 8E, F). 7-OH-DPAT also blocked clozapineinduced c-fos expression in the major island of Calleja (Figs 6C, 7). However, this DA receptor agonist failed to influence the number of clozapineinduced Fos-positive neurons in the mPFC (Figs 7, 8A, B). DISCUSSION
Because clozapine is a more potent antagonist at 5-HT2 receptors than at D 2 receptors, Meltzer and colleagues25'26'42 have proposed that strong blockade at 5-HT 2 receptors in the presence of weak blockade at D2 receptors contributes to the unique clinical
[] Vehicle • [] Ouinpirole []
Clozapine Quinpirole + Clozapine
+
180 t 160 1 '~ 140 t Z"~ 120 1 =
loo~
'~o
80
~
40
.~,
*
+
.
*
*
8o
÷
.+
2O
0 rnPFC
NAc M.CP L.CP LS
ICjM
Brain Region Fig. 4. Effects of vehicle (1 ml/kg), quinpirole (1 mg/kg) clozapine (20 mg/kg), and quinpirole (1 mg/kg) plus clozapine (20mg/kg) on the number of Fos-positive nuclei (mean + SEM) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP), lateral septal nucleus (LS) and the major island of Calleja (ICjM). *P < 0.01 vs quinpirole, fP < 0.01 vs quinpirole + clozapine.
751
profiles of clozapine and other atypical antipsychotic drugs. It has also been suggested that the antagonist actions of clozapine at ~12'3A6 or ct2 noradrenergic receptors 35 contribute to the atypical profile of clozapine. In order to determine if serotonergic or noradrenergic mechanisms play a role in the unique pattern of c-fos expression produced by clozapine, the effects of extensive prior lesions of ascending serotonergic or noradrenergic projections to the telencephalon were examined. These lesions failed to influence the extent or distribution of clozapineinduced Fos-like immunoreactivity in the mPFC, NAc or lateral septal nucleus (Figs 1, 2). This contrasts with earlier observations that 6-OHDA lesions of the mesotelencephalic DA system block haloperidol-induced c-fos expression in the striatum and haloperidol- and clozapine-induced c-fos expression in the NAc. 36 The present lesion results provide no support, therefore, for the hypothesis that clozapineinduced c-fos expression in the mPFC or lateral septal nucleus is mediated by 5-HT2 or ~ 1-noradrenergic receptor blockade. In agreement with the 5-HT lesion study, the 5-HT: antagonist ritanserin failed to mimic clozapine-induced increases in Fos-like immunoreactivity in the mPFC either by itself or when combined with haloperidol, suggesting that 5-HT: antagonism does not contribute to clozapineinduced c-fos expression in the mPFC or other brain regions. Nevertheless, it remains possible that a certain ratio of 5-HT: to D 2 receptor blockade is critical with respect to c-fos expression in different brain regions. This will require an examination of the effects of a range of doses of ritanserin on haloperidol-induced Fos-like immunoreactivity. The failure of haloperidol, a DA receptor antagonist with a five- to 10-fold higher affinity for D2 than D 3 or D4 receptors, 12to induce c-fos expression in the mPFC 36 suggests that D2 receptor antagonism does not account for the effects of clozapine in this structure. The data reported here suggest that actions at subtypes of the D 2 family of receptors, including perhaps D3 and D4 receptors, may contribute to clozapine's effects on c-fos expression in the forebrain. Thus, the dopamine receptor agonist quinpirole, which in vitro has approximately equal affinities for D 3 and D4 receptors but 100-fold lower affinity for D 2 receptors, 12'2°'37 produced small but significant decreases in clozapine-induced c-Jos expression in the mPFC and had large effects in the NAc, major island of Calleja and lateral septal nucleus. In contrast, the somewhat selective D 3 receptor agonist 7-OH-DPAT, which in vitro has about 75-fold lower affinity for D 2 receptors and 1000-fold lower affinity for D 4 receptors, 2°'23 than for D 3 receptors, significantly reduced clozapine-induced increases in the number of Fos-like immunoreactive neurons in the major island of Calleja, NAc and lateral septal nucleus without having any effect in the mPFC. These data suggest that actions at D 3 receptors may mediate clozapine-induced c-)Cos expression
752
N. Guo et al.
Fig. 5. Photomicrographs of Fos-like immunoreactivity in the mPFC (A, B), NAc (C, D) and lateral septal nucleus (E, F) after clozapine (A, C, E) and quinpirole plus clozapine (B, D, F) treatment. Scale bar = 100#m.
in the NAc, major island of Calleja and lateral septal nucleus, while its actions at D4 receptors may contribute to its actions in the mPFC. These working hypotheses are consistent with current anatomical information concerning the distributions of D 3 and D4 receptors, the former being expressed primarily in limbic brain regions such as the NAc and the islands of Calleja, TM and the latter being most highly expressed in the frontal cortex and amygdala. 2z'3t'44 However, several caveats should be emphasized with respect to these interpretations. First,
quinpirole produced only a small attenuation of clozapine-induced c-fos expression in the mPFC (Fig. 4). This suggests that while antagonist actions at D4 receptors may contribute to clozapine's actions in the mPFC, the full spectrum of mechanisms by which it increases c-fos expression in this structure remains to be determined. Second, despite its considerable selectivity in in vitro assays, the extent to which the in vivo actions of 7-OH-DPAT can be attributed to selective actions at D 3 receptors is uncertain.10,17,TM
Receptor mechanisms of clozapine-induced c-fos expression
753
Fig. 6. Photomicrographs of Fos-like immunoreactivity in the major island of Calleja (ICjM) after clozapine (A), quinpirole plus clozapine (B) and 7-OH-DPAT plus clozapine (C) treatment. MS, medial septal nucleus. Scale bar = 46 pm. Some data are not easily accommodated by the hypothesis that clozapine increases c-fos expression in the NAc, major island of Calleja and lateral septal nucleus via actions at D3 receptors and in the mPFC via 0 4 receptor mechanisms. For example, in vitro studies have shown that clozapine has two to threefold higher affinity for D2 receptors than for D3 receptors. 12'4°This appears to be inconsistent with the present results, which suggest that clozapine increases c-fos expression in the NAc, major island of Calleja and lateral septal nucleus via D3 receptor antagonism, because at the same time it failed to produce the expected D2 receptor antagonist-mediated increases in the number of Fos-like positive neurons in the striatum. Given clozapine's higher affinity for D2 than m Clozapine 180
El 7-OHDPAT + Clozapine
.$
160
~
140t
i
"° I
II II
=
=
loo I
'~
~o
~"
6o 4o
~ M.
20 0
mPFC NAc
M.CP L.CP
LS
ICjM
Brain Region Fig. 7. Effects of c]ozapine (20mg/kg) and 7-OH-DPAT
(0.5 mg/kg) plus clozapine (20 mg/kg) on the number of Fos-positive nuclei (mean + S.E.M.) in the mPFC, NAc, medial striatum (M.CP), lateral striatum (L.CP), lateral septal nucleus (LS) and major island of Calleja (IcjM). *P < 0.001 vs clozapine; n = 6 per group.
D 3 receptors, its positive effects on c-fos expression in the NAc, major island of Calleja and lateral septal nucleus indicate that its inability to increase the number of Fos-like immunoreactive neurons in the striatum by blocking D2 receptors was not due to insufficient dosing. The disparities between the results of previous receptor binding studies and the present results indicate that in vitro affinities of typical and atypical antipsychotics for DA receptors cannot by themselves account for the patterns of c-los expression produced by these compounds. A number of other contributing factors must therefore be considered. First, it is possible that active metabolites that have affinities for the dopamine receptor subtypes that are different from the parent compounds contribute to the patterns of c-fos expression reported here. Second, the in vivo potencies of antipsychotic drugs in occupying central neurotransmitter receptors do not always correspond to their in vitro affinities for these receptors. 21'26'42It is worth noting in this regard that the conditions under which in vitro affinities are determined no doubt differ very substantially from those that exist in vivo (see also Refs 10, 17 and 18). Finally, multiple neurotransmitter receptors are probably involved in regulating c-los expression in individual neurons. For example, the antimuscarinic drug scopolamine attenuates haloperidol-induced czfos expression in the striatum. TM Similarly, haloperidol-induced c-los expression in the striatum can be reduced by the glutamate receptor antagonist dizocilpine maleate? Robertson and Fibiger36 reported that while 6-OHDA lesions of the mesotelencephalic DA system blocked clozapine-induced increases in the number of Fos-like immunoreactive neurons in the NAc, these
754
N. Guo et al.
Fig. 8. Photomicrographs of Fos-like immunoreactivity in the mPFC (A, B), NAc (C, D) and lateral septal nucleus (E, F) following clozapine (A, C, E) and 7-OH-DPAT plus clozapine treatment (B, D, F) treatment. Scale bar = 100 #m.
lesions had no effect on clozapine's actions in the mPFC and lateral septal nucleus. Indeed, it was on the basis of this observation that a non-dopaminergic mechanism was proposed for clozapine in these structures. 36 The fact that quinpirole significantly attenuates clozapine-induced increases in c-los expression in the mPFC and lateral septal nucleus (Fig. 4) suggests
that dopaminergic mechanisms do contribute, at least in part, to clozapine's actions in these structures. While explanations for this discrepancy are not obvious, it is worth noting that there is precedence for lesion-induced depletions of DA failing to block neurochemical actions of neuroleptics. For example, extensive ( ~ 9 5 % ) 6-OHDA lesions of the nigro-
Receptor mechanisms of clozapine-induced c-fos expression striatal projection fail to affect the ability of haloperidol to reduce striatal c o n c e n t r a t i o n s of acetylcholine. 9 This raises the possibility t h a t the effects o f neuroleptics m a y n o t simply be due to blocking the actions of D A at D A receptors b u t t h a t these c o m p o u n d s have a level o f intrinsic ac-
755
tivity, this perhaps only occurring in the denervated condition. Acknowledgements--This work was supported by the Medical Research Council of Canada and an unrestricted grant from Bristol-Myers Squibb. The authors wish to thank C. Wilson for excellent technical assistance.
REFERENCES
1. Angst J., Bente D., Berner P., Heimann H., Helmchen H. and Hippius H. (1971) Das klinische irkungsbild von clozapine (untersuchung mit dem AMP-system). Pharmakopsychiatrie 4, 201-21 I. 2. Baldessarini R. J. and Frankenburg F. R. (1991) Clozapine: a novel antipsychotic agent. New Engl. J. Med. 324, 746-754. 3. Baldessarini R. J., Huston-Lyons D., Campbell A., Marsh E. and Cohen B. M. (1992) Do central antiadrenergic actions contribute to the atypical properties of clozapine? Br. J. Psychiat. 160, Suppl. 17, 12 16. 4. Blackburn J. R., Phillips A. G., Jakubovic A. and Fibiger H. C. (1986) Increased dopamine metabolism in the nucleus accumbens and striatum following consumption of a nutritive meal but not a palatable non-nutritive saccharine solution. Pharmac. Biochern. Behav. 25, 1095-1100. 5. Bouthenet M.-L., Souil E., Martres M.-P., Sokoloff P., Giros B. and Schwartz J.-C. (1991) Localization of dopamine D 3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D 2 receptor mRNA. Brain Res. 564, 203-219. 6. Deutch A. Y., Moghaddam B., Innis R. B., Krystal J. H., Aghajanian G. K., Bunney B. S. and Charney D. S. (1991) Mechanisms of atypical antipsychotic drugs: implications for novel therapeutic strategies for schizophrenia. Schizophrenia Res. 4, 121-156. 7. Deutch A. Y., Lee M. C. and Iadarola M. J. (1992) Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Molec. Cell Neurosci. 3, 332 341. 8. Dragunow M., Robertson G. S., Faull R. L. M., Robertson H. A. and Jansen K. (1990) D2 dopamine receptor antagonists induce Fos and related proteins in rat striatal neurons. Neuroscience 37, 287-294. 9. Fibiger H. C. and Grewaal D. S. (1974) Neurochemical evidence for denervation supersensitivity: the effect of unilateral substantia nigra lesions on apomorphine-induced increases in neostriatal acetylcholine levels. Life Sci. 15, 57~53. 10. Freedman J. E., Waszczak B. L., Cox R. F. and Liu J.-C. (1994) The dopamine D3 receptor and 7-OH-DPAT. Trends pharmac. Sci. 15, 173. 11. Gehlert D. R., Gackenheimer S. L., Seeman P. and Schaus J. (1992) Autoradiographic localization of [3H]quinpirole binding to dopamine D2 and D3 receptors in rat brain. Eur. J. Pharmac. 211, 189-194. 12. Gingrich J. A. and Caron M. G. (1993) Recent advances in the molecular biology of dopamine receptors. A. Rev. Neurosci 16, 299-321. 13. Gross H. and Langner E. (1966) Das wirkungsprofil eines chemisch neuartigen breitbandneuroleptikums der dibenzodiazepingruppe. Wien. Med. Wochenschr. 116, 814-816. 14. Guo N., Robertson G. S. and Fibiger H. C. (1992) Scopolamine attenuates haloperidol-induced c-los expression in the striatum. Brain Res. 588, 164-167. 15. Kane J. M., Honigfeld G., Singer J., Meltzer H. and the Clozaril Collaborative Study Group (1988) Clozapine for the treatment-resistant schizophrenic. Arch. gen. Psychiat. 45, 789 796. 16. Lane R. F., Blaha C. D. and Rivet J. N. (1988) Selective inhibition of mesolimbic dopamine release following chronic administration ofclozapine: involvement of cd-noradrenergic receptors demonstrated by in vivo voltammetry. Brain Res. 460, 398~401. 17. Large C. H. and Stubbs C. M. (1994) The dopamine D3 receptor: Chinese hamsters or Chinese whispers? Trends pharmac. Sci. 15, 46~,7. 18. Large C. H. and Stubbs C. M. (1994) Large and Stubbs reply. Trends pharmac. Sci. 15, 173 174. 19. Levant B., Grigoriadis D. E. and DeSouza E. B. (1993) [3H]Quinpirole binding to putative D2 and D3 dopamine receptors in rat brain and pituitary gland: a quantitative autoradiographic study. J. Pharmac. exp. Ther. 264, 991-1001. 20. Levesque D. L., Diaz J., Pilon C., Martres M.-P., Giros B., Souil E., Schott D., Morgat J.-L., Schwartz J.-C. and Sokoloff P. (1992) Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin. Proc. natn. Aead. Sci U.S.A. 89, 8155-8159. 21. Leysen J. E., Janssen P. M. F., Schotte A., Luyten W. H. M. L. and Megens A. A. H. P. (1993) Interaction of antipsychotic drugs with neurotransmitter receptor sites in vitro and in vivo in relation to pharmacological and clinical effects: role of 5HT2 receptors. Psyehopharmacology 112, $40-$54. 22. Mansour A., Meador-Woodruff J., Burke S., Bunzow J., Akil H., Van Tol H. H. M., Civelli O. and Watson S. J. (1991) Differential distribution of D 2 and 04 dopamine receptor mRNAs in the rat brain: an in situ hybridization study. Soc. Neurosci Abstr. 17, 599. 23. McElroy J. F., Amy K. A., Ward K. A., Zeller K. L., Cawley J. F. and Mazzola A. L. (1993) In vivo agonist properties of 7-OH-DPAT, a dopamine D3-selective receptor ligand. Soc. Neurosci. Abstr. 19, 1064. 24. Meltzer H. Y. (1989) Clinical studies on the mechanism of action of clozapine: the dopamine serotonin hypothesis of schizophrenia. Psychopharmacology 99, S18~27. 25. Meltzer H. Y., Matsubara S. and Lee J.-C. (1989) Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-l, D-2, and serotonin 2 pK~ values. J. Pharmac. exp. Ther. 251, 238 246. 26. Meltzer H. Y., Zhang Y. and Stockmeier C. A. (1992) Effect of amperozide on rat cortical 5-HT2 and striatal and limbic dopamine D2 receptor occupancy: implications for antipsychotic action. Eur. J. Pharmac. 216, 67-71. 27. Miller J. C. (1990) Induction of c-fos mRNA expression in rat striatum by neuroleptic drugs. J. Neurochem. 54, 1453-1455.
756
N. Guo et al.
28. Morgan J. I. and Curran T. (1989) Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci. 12, 459-462. 29. Morgan J. I. and Curran T. (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. A. Rev. Neurosci 14, 421-451. 30. Nguyen T. Y., Kosofsky B. E., Birnbaum R., Cohen B. M. and Hyman S. E. (1992) Differential expression of c-fos and zif268 in rat striatum after haloperidol, clozapine, and amphetamine. Proc. natn. Acad Sci U.S.A. 89, 4270-4274. 31. O'Malley K. L., Harmon S., Tang L. and Todd R. D. (1992) The rat dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol. 4, 137-146. 32. Paris J. M., Mitsushio H. and Lorens S. L. (1989) Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions. Brain Res. 476, 183-188. 33. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, San Diego. 34. Peroutka S. J. and Snyder S. H. (1980) Relationship of neuroleptic drug effects at brain dopamine, serotonin, ct-adrenergic and histamine receptors to clinical potency. Am. J. Psychiat. 137, 1518-1522. 35. Pickar D., Owen R. R., Litman R. E., Konicki E., Gutierrez R. and Rapaport M. H. (1992) Clinical and biologic response to clozapine in patients with schizophrenia: crossover comparison with fluphenazine. Arch. gen. Psychiat. 49, 345-353. 36. Robertson G. S. and Fibiger H. C. (1992) Neuroleptics increase c-los expression in the forebrain: contrasting effects of haloperidol and clozapine. Neuroscience 46, 315-328. 37. Schwartz J.-C., Giros B., Martres M.-P. and Sokoloff P. (1992) The dopamine receptor family: molecular biology and pharmacology. Sere. Neurosci 4, 99-108. 38. Shu S., Ju G. and Fan L. (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett 85, 169-171. 39. Sibley D. R. and Monsma F. J. Jr. (1992) Molecular biology of dopamine receptors. Trends pharmac. Sci 13, 61-68. 40. Sokoloff P., Giros B., Martres M.-P., Bouthenet M.-C. and Schwartz J.-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 14(~151. 41. Sokoloff P., Giros B., Martres M. P., Andrieux M., Besancon R., Pilon C., Bouthenet M. L., Souil E. and Schwartz J. C. (1992) Localization and function of the D 3 dopamine receptor. Drug Res. 42, 224 230. 42. Stockmeier C. A., DiCarlo J. J., Zhang Y., Thompson P. and Meltzer H. Y. (1993) Characterization of typical and atypical antipsychotic drugs based on in vivo occupancy of serotonin 2 and dopamine 2 receptors. J. Pharmac. exp. Ther. 266, 1374~1384. 43. U'Prichard D. C., Reisine T. D., Mason S. T., Fibiger H. C. and Yamamura H. (1980) Modulation of rat brain orand fl-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle. Brain Res. 187, 143-154. 44. Van Tol H. H. M., Bunzow J. R., Guan H.-C., Sunahara R. K., Seeman P., Niznik H. B. and Civelli O. (1991) Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 6104514. (Accepted 24 October 1994)