Dopamine D2S and D2L receptors may differentially ... - Nature

Molecular Psychiatry (2002) 7, 1075–1082  2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

Dopamine D2S and D2L receptors may differentially contribute to the actions of antipsychotic and psychotic agents in mice R Xu1, D Hranilovic2,3, LA Fetsko1, M Bucan2 and Y Wang1 1

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; 2Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; 3Ruder Bos˘kovic Institute, Department of Molecular Genetics, Zagreb, Croatia Regulation of dopamine D2 receptor (D2) function plays an important role in alleviating either the motor deficits of Parkinson’s disease or psychotic symptoms of schizophrenia. D2 also plays a critical role in sensorimotor gating which can be measured by monitoring the prepulse inhibition of the startle response. Alternative splicing of the D2 gene generates two isoforms, D2S and D2L. Here we investigated the role of D2S and D2L in the mechanisms of action of dopaminergic drugs, using mice lacking D2L (D2L−/−) but expressing D2S as a model system. We found that the typical antipsychotic raclopride was much less potent in inhibiting locomotor activity and eliciting catalepsy (or parkinsonism) in D2L−/− mice, whereas the atypical antipsychotic clozapine was equally effective in D2L−/− and wild-type mice. These suggest that the deletion of D2L diminishes drug-induced parkinsonism. Furthermore, two dopamine agonists, amphetamine and apomorphine, reduced prepulse inhibition to a similar degree in D2L−/− and wild-type mice. These results together suggest that D2S alone can mediate the action of clozapine and the dopamine agonist-induced disruption of prepulse inhibition. The differential binding affinities of these agents for D2S vs D2L were not sufficient to explain the divergent effects of typical vs atypical antipsychotics in D2L−/− mice. These findings suggest that D2S and D2L may differentially contribute to the therapeutic actions and side effects of antipsychotic agents, and may have implications for developing better antipsychotic agents. Molecular Psychiatry (2002) 7, 1075–1082. doi:10.1038/sj.mp.4001145 Keywords: dopamine D2L knockout mice; clozapine; raclopride; haloperidol; locomotion; catalepsy; prepulse inhibition; amphetamine; apomorphine; dissociation constant

Introduction The drugs that are clinically used to treat schizophrenia are generally classified into two broad categories: typical and atypical antipsychotics. It is generally believed that blockade of the central dopamine D2 receptor contributes to the therapeutic efficacy of antipsychotic drugs.1 Despite this commonality, the typical and atypical antipsychotic drugs have been shown to have distinct clinical and behavioral profiles. Typical antipsychotics, such as raclopride and haloperidol, produce extrapyramidal side effects (EPS), including a parkinsonian-like syndrome and tardive dyskinesia.2 EPS (or parkinsonism) are thought to be attributed, at least in part, to blockade of D2 in the striatum.3 In contrast, atypical antipsychotics, such as clozapine, are generally associated with a low incidence of EPS. The

*Correspondence: Y Wang, Department of Pharmacology, University of Pennsylvania School of Medicine, M102 John Morgan building, Philadelphia, PA 19104-6084, USA. E-mail: yywang@ pharm.med.upenn.edu Received 10 October 2001; revised 1 February 2002 and 11 March 2002; accepted 15 March 2002

cellular and molecular mechanisms for these divergent actions of typical vs atypical antipsychotics remain elusive. The dopamine (DA) precursor levodopa or D2 agonists (eg, bromocriptine) effectively relieve the motor symptoms of Parkinson’s disease, but they can also produce psychotic side effects.4 Since regulation of D2 receptor function plays an important role in alleviating either the motor symptoms of Parkinson’s disease or the psychotic symptoms of schizophrenia, there is a possibility that the D2L and D2S receptors may differentially contribute to the therapeutic ratio of these clinically used dopaminergic drugs. Sensorimotor gating represents the ability to gate or filter out extraneous stimuli. This process can be monitored by prepulse inhibition of the startle response (PPI). PPI is a phenomenon that a weak stimulus (prepulse) when presented before a startle (strong) stimulus can attenuate the amplitude of the startle reflex to the strong stimulus. Abnormalities in sensorimotor gating or deficits in PPI have been observed in several neuropsychiatric disorders, such as schizophrenia and amphetamine abuse.5,6 PPI deficits may reflect cognitive dysfunctions in these neuropsychiatric disorders. Studies using pharmacological manipu-

Dopamine D2L knockout mice and dopaminergic drugs R Xu et al

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lations and gene knockout (KO) technology have provided evidence indicating that D2, but not other DA receptors (eg, D1, D3 and D4), plays a critical role in the modulation of DA agonist-induced disruption of PPI or sensorimotor gating.7-10 The D2 receptor exists in two isoforms: the long form (D2L) and the short form (D2S). The two isoforms are generated from the same gene by alternative splicing. D2L differs from D2S by the addition of 29 amino acids in the third intracellular loop of its protein structure. No isoform-specific pharmacological ligands have yet been identified. As an initial step to dissect the distinct functions of either D2 isoform, we have generated mice that are selectively deficient in D2L (D2L−/−).11 The mutant mice still express functional D2S at the level similar to the total D2 in wild-type (WT) mice .11 Thus, D2L−/− mice, which express only D2S, will allow us to determine the function of D2S and to examine the effects of drugs on behaviors mediated by D2S. D2L is the predominant isoform expressed in the brain of WT mice. For example, in WT mice, D2L mRNA comprises approximately 90% of total D2 in the striatum (including the caudate-putamen and nucleus accumbens).11,12 Thus, the comparisons made between D2L−/− and WT mice may reveal the functional roles of D2L.11,13 The purposes of this study were to investigate the involvement of D2S and D2L in the mechanisms of action of typical vs atypical antipsychotic drugs and in the effects of DA agonists on prepulse inhibition of the startle response. The present study was also designed to assess whether dopaminergic ligands exhibit differential binding affinities for D2S vs D2L in brain tissue. These experiments would enable us to evaluate the impact of pharmacological selectivity for two receptor isoforms on the behavioral effects of these drugs in mice.

Materials and methods Animals Dopamine D2L receptor knockout mice (D2L−/−) were produced as previously described in detail.11 Heterozygous mice (D2L+/−) created on a hybrid background (129/Sv × C57BL/6) were backcrossed to the C57BL/6 strain for six generations to establish an incipient congenic B6, to minimize the variability in genetic background among the mice. Animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Behavioral experiments were conducted in an isolated room under dim light between 10:00 am and 5:00 pm. Male mice (3–6 months old) on a congenic B6 genetic background were used. Mice were brought to the experimental room at least 1 h before experiments began, to allow adaptation to the environment. Experiments were conducted blind with respect to the genotype of the mice. Locomotor activity The effects of the drugs on locomotor activity were evaluated using an automated Open Field Activity Molecular Psychiatry

Monitor (MED Associates, St Albans, VT, USA). Mice were injected with either raclopride (0.03–0.3 mg kg−1) or clozapine (0.3–3 mg kg−1). Twenty min after raclopride injection or 30 min after clozapine injection, locomotion (or ambulation) of the mice was measured as the number of beam breaks during a 30 min period.11 All mice received a single injection of vehicle a few days prior to drug treatment (ie, initial vehicle treatment). Three to seven days later, mice received either drug injection or another vehicle injection. Catalepsy Catalepsy was measured using the bar test.11 Twenty min after raclopride treatment or 30 min after haloperidol treatment, the forepaws of each mouse were placed on the horizontal bar 5 cm above the base. The duration that the mouse held the bar without any voluntary movement was recorded, with a cut-off time of 3 min. Acoustic startle response (ASR) and prepulse inhibition of the startle response (PPI) ASR and PPI were measured in startle chambers (San Diego Instruments, San Diego, CA, USA) and using our previously established protocols.14,15 Each mouse was tested twice in the same chamber: without treatment (baseline response) and with drug or saline treatment with 1-week interval. In most cases, mice were tested a third time at 1 week after drug treatment to ensure that there was no change in baseline response. Mice were placed in the startle chambers without treatment or immediately after drug administration and allowed a 10-min acclimation period, during which they were exposed to a continuous 70 decibels (dB) white noise. The mice were then given 20 trials spaced 15 s apart, with one of four different startle intensities (90, 100, 110, or 120 dB) presented as 40-ms bursts of white noise on each trial. Each startle intensity was presented five times in a pseudo-randomized order. Average startle magnitude over a 100-ms recording period (expressed in arbitrary units) was used as a measure of the startle response. The PPI session consisted of 20 trials spaced 15 s apart. Each trial consisted of a 40-ms startle intensity of 120 dB delivered either alone, or 60ms after a 20-ms prepulse stimulus of either 90 dB or 95 dB intensity. The magnitude of PPI was expressed as the percentage of decrease in startle response following exposure to a prepulse (90 or 95 dB), as compared to the startle response in response to the pulse alone (120 dB). Percentage prepulse inhibition (% PPI) was calculated using the following formula: % PPI = 100 − [(startle response for pulse with prepulse/startle response for pulse alone) × 100], where ‘startle response for pulse with prepulse’ is the startle response in prepulse trials (90 dB or 95 dB prepulse). Ninety dB and 95 dB prepulses were used because basal PPI was more pronounced at these two intensities in the strain of mice we used.15 This condition was suitable for examining the effects of DA agonists on PPI


since the pharmacological treatment reduced the magnitude of PPI. Our pilot studies indicated that 10 mg kg−1 was the optimal dose for amphetamine and 1 mg kg−1 was the optimal dose for apomorphine to disrupt PPI in the strain of mice we used. Drugs Raclopride, amphetamine and apomorphine were dissolved in 0.9% saline. Clozapine and haloperidol were dissolved in 0.6% lactic acid and adjusted to pH 5.3 by adding NaOH. All drugs were purchased from SigmaAldrich-Research Biochemicals (St Louis, MO, USA). The vehicle solution injected was either 0.9% saline or 0.6% lactic acid. Apomorphine was administered subcutaneously (s.c.) and other drugs were administered intraperitoneally (i.p.) in a volume of 5 ml kg−1 body weight.

Data analysis Data from locomotor activity were analyzed by twoway analysis of variance (two-way ANOVA). ED50 values were calculated using Prism program (GraphPad, San Diego, CA, USA). Data from catalepsy were compared using Mann–Whitney U test. The comparisons within each genotype on ASR and PPI before and after drug treatment and the data from basal PPI and ASR were evaluated using Student’s t-test. The

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Receptor binding assay Competitive binding assays were performed under conditions similar to those of saturation experiments.11 Briefly, striatal tissue from WT and D2L−/− mice was dissected out and homogenized in ice-cold homogenization buffer. The homogenates were centrifuged twice at 35 000 g for 40 min at 4°C. The final membrane pellets were resuspended in binding buffer and stored in aliquots at −20°C. The protein concentration of membranes was determined using the Bradford Reagent (Sigma). Aliquots of striatal membranes containing 60 ␮g of protein were incubated in duplicate with 0.08 nM of [3H]-YM-09151-2 and 12 concentrations of the competing ligand in a volume of 1.6 ml of binding buffer at 25°C for 40 min. The competing ligands (and their concentrations) used were raclopride (10−5.25– 10−10.25 M), haloperidol (10−5.75–10−11.25 M), clozapine (10−3.75–10−9.25 M), and dopamine (10−4.25–10−9.75 M). [3H]-YM-09151-2 (specific activity, 85.5 Ci mmol−1) was purchased from NEN Life Science Products (Boston, MA, USA). Figure 1 Comparison of the effect of raclopride and clozapine on locomotor activity in D2L knockout (−/−) and WT (+/+) mice. (a) Effects of raclopride on locomotion. There was a significant difference in the effects of raclopride on locomotion between the two genotypes (F1,41 = 4.55, P = 0.0389). The response to initial vehicle treatment was normalized as 100%, and locomotor activity in response to drug treatment was expressed as percentage of the initial vehicle (veh) treatment. Eight mice per genotype were used for each dose. (b) Effects of clozapine on locomotion. There was effect of dose (F2,47 = 30.19, P ⬍ 0.0001), but no effect of genotype (F1,47 = 0.01, P = 0.9249). Eight to ten mice of each genotype were used for each dose. (c) Effects of vehicle injection. Separate groups of mice received repeated vehicle injections (ie, veh-1, veh-2 and veh-3) and were tested on the same days as drug-treated mice. A percentage of 0.9 saline (sal) was used in vehicle injection for raclopride and 0.6% lactic acid was used in vehicle injection for clozapine. There was neither effect of injection (P = 0.9143 for saline; P = 0.3851 for lactic acid) nor effect of genotype (P = 0.1767 for groups receiving saline injection; P = 0.7932 for groups receiving lactic acid injection). Seven to nine mice of each genotype were used for each injection. Molecular Psychiatry


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effects of genotype on ASR and PPI were compared using two-way ANOVA. Dissociation constants (Ki) of dopaminergic ligands were calculated by nonlinear regression with the Prism program and compared using Student’s t-test. A P value ⬍ 0.05 was considered statistically significant. Data presented in figures and tables are mean ± SEM.

Results D2L knockout mice are less sensitive than wild-type mice to raclopride treatment, but the two genotypes are equally sensitive to clozapine treatment To assess the role of D2S and D2L in the actions of typical vs atypical antipsychotics, we compared the effects of the typical antipsychotic raclopride with those of the atypical antipsychotic clozapine in D2L−/− and WT mice. Raclopride was significantly less potent in inhibiting locomotor activity in D2L−/− than in WT mice (Figure 1a). In contrast, clozapine produced a similar dose-dependent decrease in locomotor activity in both D2L−/− and WT mice (Figure 1b). Vehicle injections did not have a significant effect on locomotion in either genotype (Figure 1c). We also used the bar test to evaluate raclopride-induced catalepsy, since this behavioral measure is considered to be a rodent model for assessing EPS of antipsychotics. Raclopride elicited much less cataleptic behavior in D2L−/− than in WT mice (Figure 2a). As a control, haloperidol-induced catalepsy was re-examined. Consistent with our previous study,11 the cataleptic effect of haloperidol was largely reduced in D2L−/− mice (Figure 2b). Other studies have shown that clozapine does not elicit catalepsy in animals or EPS in humans16 and we found that clozapine-induced locomotor inhibition was similar in D2L−/− and WT mice. Thus, the effect of clozapine on catalepsy was not evaluated. Above results suggest that D2L may play a prominent role in D2 antagonistinduced catalepsy. Amphetamine- and apomorphine-induced reduction in prepulse inhibition is similar in D2L knockout and wild-type mice It has been shown that amphetamine-induced reduction of PPI is retained in D3 KO and D4 KO mice, but absent in D2 KO mice which lack both D2L and D2S.9 Moreover, deficient PPI exhibited by DA transporter KO mice can be reversed by the D2 antagonist raclopride, but not the D1 antagonist SCH23390.10 These results indicate that a disruption of PPI or sensorimotor gating results from the stimulation of D2. The D2L KO mouse is a useful research tool for deciphering which D2 isoform, D2L or D2S, plays the major role in modulating DA agonist-induced reduction of PPI. We first examined the baseline levels of ASR and PPI in D2L−/− and WT mice. Figure 3 showed that there were no significant differences between these two genotypes of mice in either basal ASR or basal PPI prior to drug administration. We then examined the effects of DA agonists on PPI in D2L−/− vs WT mice to determine the role of either D2 isoform Molecular Psychiatry

Figure 2 The effect of raclopride and haloperidol on cataleptic behavior of D2L knockout (−/−) and WT (+/+) mice. (a) Raclopride-induced catalepsy. Catalepsy is expressed as the duration of immobility in seconds (sec). Eight to ten mice of each genotype were used for each dose. (b) Haloperidolinduced catalepsy. Eight mice of each genotype were used for each dose. The data shown for 1 mg kg−1 dose of haloperidol were taken from our previous study11 (copyright 2000 by the Society for Neuroscience). **P ⬍ 0.001; ***P ⬍ 0.0005 (Mann–Whitney test).

in drug-induced disruption of PPI. Treatment with amphetamine (10 mg kg−1), an indirect DA agonist, significantly lowered PPI in both genotypes at both 90 dB and 95 dB prepulses (Figure 4a). There was no significant difference in amphetamine-induced reduction of PPI between D2L−/− and WT mice. Amphetamine treatment did not significantly affect ASR in either genotype (not shown). Amphetamine can affect other monoamine systems in addition to causing DA release. Thus, we also evaluated the effects of apomorphine, a direct DA agonist, on PPI. Administration of apomorphine at 1 mg kg−1 significantly reduced PPI in both genotypes (Figure 4b). There was no significant difference in apomorphine-induced reduction of PPI between D2L−/− and WT mice. Apomorphine also reduced ASR in both genotypes to a similar degree (not shown). Mice treated with saline did not exhibit any significant changes in either ASR (not shown) or PPI


(Figure 4c). These results suggest that either D2S can substitute for the function of D2L or D2S is the receptor isoform that normally mediates DA agonist-induced reduction of PPI.

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Binding affinities of dopaminergic ligands for D2S and D2L receptors in striatal tissue In transfected cell lines, several D2 antagonists have been shown to have differential binding affinities for D2S vs D2L.17,18 However, it is uncertain whether the findings in cultured cell lines can be applied directly to mammalian brain tissue. Here we performed competitive binding assays to determine the ability of dopamine and D2 antagonists to displace [3H]-YM09151-2 binding in striatal membranes from both D2L−/− and WT mice. Striatum was chosen because D2L

Figure 3 Baseline levels of acoustic startle response (ASR) and prepulse inhibition of the startle response (PPI) in WT (+/+) and D2L knockout (−/−) mice. (a) Acoustic startle response at 120 decibel (dB) of auditory stimulation. The vertical axis represents the magnitude of startle in arbitrary units. The genotypes of mice are indicated at the bottom of the corresponding bars. There was no effect of genotype (P ⬍ 0.7). (b) Percentage prepulse inhibition (% PPI) shown on the vertical axis was obtained at 90 dB or 95 dB prepulse which are indicated at the bottom of the corresponding bars. There was no effect of genotype at either 90 dB prepulse (P ⬎ 0.09) or 95 dB prepulse (P ⬎0.5). n = 20 per genotype.

Figure 4 Effects of amphetamine and apomorphine on prepulse inhibition of the startle response (PPI) in WT (+/+) and D2L knockout (−/−) mice. (a) % PPI values before (open bars) and after (crossed bars) amphetamine treatment (10 mg kg−1) are compared for each genotype (*P ⬍ 0.05, **P ⬍ 0.01, *** P ⬍ 0.001). The genotypes and the intensities of prepulse stimulus (90 or 95 dB) are indicated below the corresponding bars. n = 12 for +/+ and n = 11 for −/−. There was no effect of genotype with respect to amphetamine-induced PPI reduction at either 90 dB prepulse (F1,44 = 1.5940, P = 0.2136) or 95 dB prepulse (F1,44 = 0.0065, P = 0.9361). (b) % PPI values before (open bars) and after (crossed bars) apomorphine treatment (1 mg kg−1) are compared for each genotype (**P ⬍ 0.01). There was no effect of genotype with respect to apomorphineinduced PPI reduction at either 90 dB prepulse (F1,30 = 0.0047, P = 0.9458) or 95 dB prepulse (F1,30 = 1.4549, P = 0.2375). n = 8 per genotype. (c) Saline treatment (crossed bars) had no effect on % PPI compared to the values before the treatment (open bars) in either +/+ (P ⬎ 0.2) or −/− (P ⬎ 0.9) mice. n = 8 per genotype. Molecular Psychiatry


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Table 1 Dissociation constants (Ki) of dopaminergic ligands in WT (+/+) and D2L knockout (−/−) mice

Table 3 Comparison of ratio of dissociation constant (Ki) from binding assays and ratio of ED50 for locomotor inhibition in WT (+/+) and D2L knockout (−/−) mice

Ki (nM)

Dopamine Raclopride Haloperidol Clozapine

+/+ (D2L)

−/− (D2S)

18.15 ± 7.03a (3) 3.97 ± 0.43 (3) 1.34 ± 0.06 (7) 77.14 ± 3.67 (4)

15.67 ± 2.43a (3) 2.65 ± 0.02* (3) 2.27 ± 0.08* (3) 105.00 ± 7.75* (4)

Typical antipsychotic Raclopride Haloperidol Atypical antipsychotic Clozapine

Ki(−/−)/Ki(+/+)a

ED50(−/−)/ED50(+/+)

0.67 1.69

3.75 6.20

1.36

1.09

Ki(−/−) = Ki(D2S); Ki(+/+) is approximately equal to Ki(D2L) (see Table 1).

a

Ki of dopamine at the high-affinity state. *P ⬍ 0.05. Numbers of experiments are shown in parentheses.

a

is the predominant D2 isoform in this region (苲90% of total D2) in WT mice, whereas D2S is the only isoform in D2L−/− mice. There was no significant difference in the dissociation constants (Ki) of DA for D2S and D2L (Table 1). Raclopride and haloperidol bound to both D2 isoforms with a higher affinity than did DA, whereas clozapine bound to both D2 isoforms with a lower affinity than did DA. Raclopride had a higher affinity (ie, lower Ki) for D2S than for D2L, whereas haloperidol and clozapine had a higher affinity for D2L than for D2S (Table 1).

motor inhibition was over 6.0. The ratio of Ki(−/−) to Ki(+/+) for raclopride was less than 1.0, whereas the ratio of ED50(−/−) to ED50(+/+) for raclopride was over 3.5. These results suggest that the divergent effects of typical vs atypical antipsychotics in D2L−/− mice could not be primarily attributed to the differential binding affinities of these drugs for D2S vs D2L.

Comparison of locomotor inhibition induced by D2 antagonists and their binding affinities for D2S and D2L Raclopride and haloperidol exhibited significantly higher ED50 values (ie, lower potencies) for inhibiting locomotor activity in D2L−/− mice as compared to WT mice (Table 2). In contrast, the ED50 values for clozapine-elicited locomotor inhibition were similar in D2L−/− and WT mice (Table 2). The ratios of ED50 values for drug-induced locomotor inhibition and the ratios of Ki of these drugs for the two D2 isoforms are shown in Table 3. The ratios of Ki(−/−) to Ki(+/+) were similar for clozapine and for haloperidol. However, the ratio of ED50(−/−) to ED50(+/+) for clozapine-induced locomotor inhibition was close to 1.0, whereas the ratio of ED50(−/−) to ED50(+/+) for haloperidol-induced locoTable 2 ED50 values (mg kg−1) for inhibition of locomotor activity in WT (+/+) and D2L knockout (−/−) mice

Typical antipsychotic Raclopride Haloperidol Atypical antipsychotic Clozapine

+/+

−/−

0.08 ± 1.01 0.38 ± 1.23b

⬎0.3a 2.33 ± 1.32b

1.49 ± 1.41

1.62 ± 1.45

a Based on the data for dose-response curve of raclopride in −/− mice, the Prism program generated an approximate value for the ED50(−/−) of raclopride. b ED50 values for haloperidol were from our previous study11 (copyright 2000 by the Society for Neuroscience).

Molecular Psychiatry

Discussion The present study showed that the potency of the typical antipsychotic raclopride on inhibiting locomotor activity and eliciting catalepsy (or parkinsonism) was markedly reduced in mice lacking D2L. In contrast, there was no effect of deletion of D2L on locomotor inhibition induced by the atypical antipsychotic clozapine. We and others previously showed that the cataleptic effects of the typical antipsychotic haloperidol were attenuated in D2L−/− mice.11,19 These results together suggest that the deletion of D2L diminishes the drug-induced EPS (or parkinsonism) that are commonly associated with the use of typical antipsychotic drugs. Since catalepsy is considered to be a useful index for measuring the EPS of antipsychotic drugs,20 we considered the possibility that a compound with less influence on the D2L system might have reduced extrapyramidal side effects. In a previous study, we measured basal immobility (or catalepsy) using both the ring test and bar test.11 Most of D2L−/− mice on a hybrid background (14 out of 17 mice tested) exhibited significant catalepsy in the ring test, whereas WT mice did not. Interestingly, most of these mutant mice (11 out of 17) did not exhibit catalepsy in the bar test, suggesting that the bar test is a less sensitive method of detecting mild catalepsy. When mutant mice were backcrossed to a congenic B6 background, D2L−/− mice still showed longer periods of basal immobility than did WT mice in the ring test, but the difference did not reach statistical significance. One possible explanation is that the B6 genetic background inhibits the expression of basal catalepsy. Another possibility is that developmental adaptation to the absence of certain gene expression may cause some compensatory changes. As a result, such adaptation may obscure the expression of basal catalepsy


caused by the targeted mutation. These possibilities may also explain why D2KO mice that lack both D2L and D2S on a congenic B6 background also do not show any basal catalepsy.21 Furthermore, it is known that D1 antagonists can induce strong catalepsy. However, D1 knockout mice do not show basal catalepsy at all when measured with the ring test.22 The facts that D2L−/− mice displayed reduced spontaneous locomotion and rearing and performed poorly in the rotarod test prior to training, as compared to WT mice suggest that D2L is important for mediating certain types of motor function.11 It had been suggested that D4 rather than D2 is the primary target for the antipsychotic actions of clozapine and this may explain why clozapine is associated with a low incidence of EPS.23 However, this notion has since been revised. Studies using imaging technology and the concentration measurement method have shown that all antipsychotics (including clozapine) occupy high levels of brain D2 at the therapeutic doses, whereas the efficacy of antipsychotics is inconsistent with the percent occupancy of D3 and D4 by these drugs.16 These findings suggest that occupancy of D2 is important for the antipsychotic efficacy of clozapine. In addition, it has been reported that L745870, a selective D4 antagonist, has no antipsychotic property in humans,24 suggesting that D4 is not a target for the therapeutic actions of the existing antipsychotics, including clozapine. Our finding that clozapine was equally potent in D2L−/− and WT mice suggests that in the absence of D2L, D2S functions well in mediating the action of clozapine and may play an important role in the therapeutic efficacy of clozapine. It has been shown that D2 KO mice, which lack both D2L and D2S, are deficient in amphetamine-induced reduction of PPI.9 In the present study, D2L−/− mice, which still express D2S, exhibited amphetamine- and apomorphine-induced reduction of PPI comparable to that of WT mice. These results indicate that D2S alone can mediate the effects of DA agonists on PPI. It has been proposed that DA agonist-induced disruption of PPI in rodents is similar to PPI abnormality in schizophrenia. In addition, antipsychotic drugs acting as D2 antagonists have been shown to, at least partially, correct PPI deficits observed in schizophrenic patients.6 Thus, our findings suggest that an agent which selectively antagonizes D2S may be effective in restoring PPI in schizophrenic patients. The effects of DA agonists on the acoustic startle response have been shown to vary, depending on the species and strains of animals. In rats, administration of amphetamine increases ASR.25,26 In mice, administration of amphetamine or apomorphine can either increase27 or decrease ASR,9 or has no effect on it,28 depending on the mouse strains used. In our study, ASR was not significantly reduced in response to administration of 10 mg kg−1 amphetamine in the strain of mice we used. However, we observed that ASR was significantly reduced in response to a higher dose of amphetamine (ie, 15 mg kg−1) in both WT (43% reduction) and D2L−/− mice (58% reduction)

(F1,30 = 14.6574, P = 0.0006; two-way ANOVA; n = 8 per genotype). There was no significant difference in 15 mg kg−1 amphetamine-induced ASR reduction between WT and D2L−/− mice (F1,30 = 0.1949, P = 0.6621). The magnitude of reduction in PPI induced by 15 mg kg−1 amphetamine was comparable with that induced by 10 mg kg−1 amphetamine in both genotypes of mice. We did not include the above data in the Results section because it has been suggested that a reduction in sensorimotor gating (eg, PPI) is unequivocal only if ASR is not significantly altered by the drug.29 Taken together, these results suggest that the effect of amphetamine on ASR is dependent both on the species or strain difference and on the dose of amphetamine employed. The relative potency for raclopride- and haloperidolinduced locomotor inhibition in WT vs D2L−/− mice was greater than the relative affinity of raclopride and haloperidol for D2L vs D2S. Thus, the reduced potency of raclopride and haloperidol in D2L−/− mice could not be primarily attributed to the differential affinities of these drugs for D2L vs D2S. However, the differential binding affinities of haloperidol and raclopride for individual D2 isoforms may have some impact on their potency for regulating animal behaviors. For example, haloperidol, but not raclopride, had a significantly higher affinity for D2L than for D2S. Correspondingly, the difference in ED50 values for haloperidol-induced inhibition of locomotion between D2L−/− and WT mice was larger than that of raclopride. These results suggest that deletion of D2L more strongly attenuates the effects of haloperidol than the effects of raclopride. Thus, there is a possibility that an antipsychotic drug with lower affinity for D2L than for D2S might have a reduced frequency of EPS. The D2L−/− mouse is a valuable research tool for assessing the pharmacological selectivity of dopaminergic drugs for individual D2 isoforms in brain tissue and whole animal. Our results suggest that the D2L−/− mouse may be a useful model system for differentiating typical antipsychotics from atypical antipsychotics and for examining other properties of dopaminergic drugs. Two typical antipsychotics, raclopride and haloperidol, had markedly attenuated potency on motor activity in mice lacking D2L, suggesting that D2L may play a prominent role in contributing to parkinsonian-like syndromes, at least in animals. The atypical antipsychotic clozapine and the DA agonists amphetamine and apomorphine were equally effective in mice expressing purely D2S (D2L−/−) and in WT mice, suggesting that D2S alone is capable of mediating the action of clozapine and drug effects on PPI. It is often the unpleasant EPS that causes schizophrenic patients to stop taking their medications, which can lead to disastrous consequences. Thus, a better understanding of the mechanisms underlying the side effects (eg, EPS) and the therapeutic actions (eg, reversal of PPI deficits) of antipsychotic drugs could lead to important breakthroughs in developing antipsychotic drugs that have a higher rate of patient compliance.

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Acknowledgments This work was supported by grants from the National Alliance for Research on Schizophrenia and Depression and Whitehall Foundation. We thank Ms E-B Sankoorikal for technical assistance.

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