RIS 0.1-100uM细胞凋亡曲线

DOI10.1007/s00702-004-0109-zJ Neural Transm(2004)111:667–681Neurotoxic potential of haloperidol in comparisonwith risperidone:implication of Akt-mediatedsignal changes by haloperidolai1,H.Ozawa2,M.Tateno1,E.Hashimoto1,and T.Saito11Department of Neuropsychiatry,School of Medicine,Sapporo Medical University,Sapporo,and2Division of Neuropsychiatry,Department of Neurosensory Medicine,Nagasaki University School of Medicine,Nagasaki,JapanReceived November24,2003;accepted January9,2004Published online April2,2004;#Springer-Verlag2004Summary.The neurotoxicity of conventional antipsychotic drugs has emerged as a potential pathogenic event in extrapyramidal side effects(EPS)and in their limited efficacy for negative-cognitive symptoms in schizophrenic patients.The atypical antipsychotics,recently developed,have superior therapeutic efficacy to treat not only positive symptoms but negative symptoms and cognitive dysfunc-tions with much lower potentials of side effects,although the influence of atypical antipsychotics on the regulation of neuronal survival has been less investigated.It is important to clarify the effects of typical and atypical antipsychotics on neuronal survival and their contributions to the therapeutic development and understanding of the pathophysiology of schizophrenia.We measured the neurotoxicity of two antipsychotic drug treatments,haloperidol and risperidone,in primary cultured rat cortical neurons.Immunoblotting and pharmacological agent analyses were used to determine the signal transduction changes implicated in the mechanisms of the neurotoxicity.Haloperidol induced apoptotic injury in cultured cortical neurons, but risperidone showed weak potential to injure the neurons.Treatment with halo-peridol also led the reduction of phosphorylation levels of Akt,and activated caspase-3.The D2agonist bromocriptine and5-HT2A antagonist,ketanserin atten-uated the haloperidol-induced neuronal toxicity.Moreover,brain-derived neuro-trophic factor(BDNF)reduced the caspase-3activity and protected neurons from haloperidol-induced apoptosis.BDNF also reversed the reduced levels of phos-phorylation of Akt caused by treatment with haloperidol.Haloperidol but not risperidone induces caspase-dependent apoptosis by reducing cellular survival signaling,which possibly contributes to the differential clinical therapeutic effi-cacy and expression of side effects in schizophrenia.Keywords:Schizophrenia,extrapyramidal side effects,negative symptoms, haloperidol,risperidone,Akt,BDNF.ai et al.IntroductionConventional antipsychotics such as haloperidol have been widely used to treat schizophrenic patients,but it is well known that the typical antipsychotics often produce extrapyramidal side effects(EPS),including parkinsonism,akathisia and tardive dyskinesia(TD),and are less effective for negative-cognitive symp-toms rather than positive symptoms in such patients.The production of EPS is the major limitation of the use of this class of drugs(Diederich et al.,1998).Recently developed atypical antipsychotics such as serotonin-dopamine antagonists(SDA)have superior therapeutic efficacy for negative symptoms, cognitive dysfunctions and treatment-resistant schizophrenia with lower poten-tials of side effects especially in EPS(Peuskens,1995;Kinon et al.,1996;Song, 1997).It is known that typical antipsychotics have high affinity for dopamine D2 receptors in the brain(Farde et al.,1992).However,atypical antipsychotics have relatively low affinity for D2receptors but higher affinity for5-HT2receptors (Kapur et al.,1999).These pharmacological differences are basically thought to be responsible for the differences of therapeutic effects and side effects profiles of typical and atypical antipsychotics(Meltzer et al.,1989),but the neurochem-ical mechanisms of the differences have not yet been understood.Recently,as a potential pathogenic event in EPS,the neurotoxicity of antipsy-chotics has attracted attention.Treatment with haloperidol has been reported to cause necrotic and apoptotic cell death(Behl et al.,1995;Noh et al.,2000).It was demonstrated that haloperidol caused oxidative stress,resulting from alternations of mitochondrial function,and that vitamin E could protect neurons from halo peridol-induced toxicity(Cadet et al.,1994;Galili et al.,2000).However,the pre-cise mechanism of the neuronal toxicity is poorly understood,and little has been done to investigate the changes of intracellular signaling related to the toxicity.In the history of psychiatric research,the neurodevelopmental hypothesis has been presented as an etiologic and pathophysiologic theory(Weinberger et al.,1987).In addition to this hypothesis,the recent development of molecular neurobiology and neuroimaging and postmortemfindings has given rise to the neurodegenerative hypothesis as a new pathophysiological model of schizo-phrenia(Lieberman et al.,1999).Both hypotheses regard morphological and structural abnormalities to be important in the pathophysiology of this illness. On the other hand,it is also reported that the volume and morphology of the prefrontal cortex and superior temporal gyrus are changed by pretreatment with antipsychotics and that their changes differ between typical and atypical anti-psychotics(Matsumoto et al.,2001).Postmortem study of TD brains showed ventricular dilatation and atrophy in several brain areas,as well as neurodegen-eration and gliosis(Mion et al.,1991;Dalgalarrondo et al.,1994).Furthermore, chronic treatment with haloperidol caused brain damage and TD in psychotic patients(Sunderland et al.,1987).All things considered,it is possible that typical and atypical antipsychotic differently affects neuronal survive and death,and that these effects considerably contribute to the differences in EPS and improvements of negative-cognitive symptoms in schizophrenic patients.We thus,in the present work,carried out a direct comparison study of two kinds of antipsychotics and their potentials for neuronal toxicity in the culturedImplication of Akt-mediated signal changes by haloperidol669 cells to clarify how the neurotoxic potentials of antipsychotics are related to the expression of EPS and the pathophysiology of schizophrenia.We used cortical neurons to investigate the involving cholinergic activity changes,which are related to the cognitive deficits and production of TD.We especially analyzed the effects of antipsychotics on the regulation of the cellular survival signaling pathways,including the PI3-K=Akt pathway,on the basis of the apoptosis-related cascade.Material and methodsNeuronal cell culturesThe animals used in this study were housed and treated according to the guidelines for care and use of experimental animals of the Ethics Committee of Sapporo Medical University.Primary cultures of cortical neurons were prepared from embryonic day18(E18)fetal rats.Jcl:Wistar rats were purchased from CLEAR Japan(Tokyo,Japan).Cortices were dissected from embryonic brains microscopically,and dissociated by incubation in trypsin(Invitrogen,Carlsbad,CA USA) for20min at37 C.Then the trypsin was inactivated by suspending the cells in serum-containing medium(DMEM=10%FBS),and triturated with a glass pipette followed byfiltration.DMEM was obtained from Nissui Pharmaceutical(Tokyo,Japan),and added20mM glucose,2mM L-glutamine1mM sodium bicarbonate and100U=ml gentamycin reagent(Invitrogen).Fetal bovine serum(FBS)was obtained from Sigma(St.Louis,MO USA).The cells were pelleted by centrifugation(400Âg for5min at4 C)and washed once in DMEM=10%FBS.Then the cells were resuspended in a chemically defined plating medium(DMEM=B27[Invitrogen])and viable cells were determined by trypan blue exclusion.The cells were plated at0.5Â105cells=cm2on poly-L-lysine(Sigma)coated tissue culture dishes.For the assessment of neurotoxicity,the cells were plated on24-well dishes(IW AKI,Japan)in0.5ml of plating medium.For the western blot analysis,the cells were plated on35mm dishes(IW AKI,Japan)in3ml of plating medium,and for the measurement of caspase-3activity,the cells were plated on100mm dishes(IW AKI, Japan)in10ml of plating medium.Cultures were maintained in a humidified incubator with an atmosphere of5%CO2and95%air at37 C.Serum free culture with B27supplementation yields nearly pure neuronal cultures(90%>),as judged by the immunocytochemistry for glial fibrillary acidic protein(GFAP)and microtubule-associated-protein2(MAP2).Experiments were performed on culture days8–12in vitro(DIV).Drug treatmentOn the day of the experiment,the medium was removed and replaced by DMEM=B27containing 0.1–100m M haloperidol(Wako,Osaka Japan),0.1–100m M risperidone(gift from Janssen Phar-maceutical,Beerse Belgium),3–10m M bromocriptine(Sigma),1–3m M domperidone(Sigma), 0.1–1m M ketanserin(BIOMOL,Plymouth Meeting,PA USA)or10–30m M BDNF(PeproTech, London UK).Drugs were dissolved in dimethyl sulfoxide(DMSO),except for the BDNF.Thefinal concentration of DMSO was below0.4%.BDNF was dissolved in PBS containing0.1%bovine serum albumin.The duration of each drug treatment is described in detail in thefigure legends.DNA ladder assayTo examine DNA cleavage,soluble cytoplasmic DNA was isolated from5Â106cells and sub-jected to1.8%agarose gel electrophoresis for DNA ladder analysis(Hockenbery et al.,1990) Quantitation of apoptosis by MTT and EIAusing antibodies against MAP2The mitochondrial dehydrogenase activity that cleaves3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)was used to determine cell survival in quantitative colorimetricai et al.assays.The cortical neurons were incubated with MTT(0.5mg=ml)for2hours at37 C,and blue-colored formazan cleaved by dehydrogenase enzymes in active mitochondria was dissolved in DMSO,and quantified spectrophotometrically at570nm.The results are expressed as a percentage of control culture viability.ED50values and95%confidence limits were determined by the method of Fieller with linear regression analysis of log-logit plots(logit conversion:log (p=(1-p));p¼neuronal viability reduction(%))using Yukms Statistical Library(Y ukms,Tokyo Japan).Neuronal death was also evaluated by the enzymatic immunoassay(EIA)using an anti-body against microtubule-associated-protein2(MAP2).Immunocytochemistry and EIAFollowingfixation in4%paraformaldehyde,cultures were rinsed with phosphate-buffered saline (PBS),and initially treated with a solution of3%aqueous H2O2for3min.The cultures were then rinsed in buffer and preincubated in PBS containing5%normal horse serum for30min at room temperature.Next they were incubated with mouse monoclonal IgG against MAP2(1:1000)for 30min at room temperature and then rinsed in buffer and incubated with the biotinylated anti-mouse IgG for30min at room temperature.After a buffer rinse,cultures were incubated with avidin-biotin-horseradish peroxidase complex(V ector Laboratories,Burlingame,CA USA)for 30min at room temperature.Finally,rinsed cultures were incubated with0.05%diaminobenzi-dine tetrahydrochloride(DAB)(Vector Laboratories)and0.01%hydrogen peroxide for3–5min. Reactions were stopped by rinsing the cultures with PBS.Quantitative analysis was performed using0.2mg=ml o-phenylenediamine dihydrochloride(OPD)(Wako)in0.2M Na2HPO3and 0.1M citric acid containing0.1%H2O2as a horseradish peroxidase substrate.In this EIA method, cultures incubated with avidin-biotin-horseradish peroxidase complex,were reacted with OPD solution for3min and then stopped with1N H2S04solution and aliquots were read in a micro-plate at450nm within30min of adding the stop solution.Analysis of caspase-3activityThe activity of caspase-3,which cleaves the substrate acetyl-Asp-Glu-V al-Asp p-nitroanilide (Ac-DEVD-pNA)in the cortical neurons,was analyzed using a caspase-3assay kit(Sigma), following the manufacturer’s instructions.In brief,after the appropriate drug treatment,cortical neurons were scraped off the dish,and the cells were pelleted by centrifugation at600Âg for5min at4 C.After one wash with ice-cold PBS,the cell pellets were suspended in lysis buffer containing 50mM HEPES(pH7.4),5mM CHAPS and5mM DTT.The lysates were incubated on ice for 20min,and centrifuged at18,000Âg for15min at4 C.The reaction was started by adding caspase-3substrate Ac-DEVD-pNA to the cell lysates in96-well plates with assaying buffer(20mM HEPES[pH7.4],0.1%CHAPS,5mM DTT and2mM EDTA).The plates were incubated at 37 C for1–2hours,and the concentrations of pNA released from the substrates by caspase-3 cleaving were calculated,using colorimetrical detection of the absorbance value at405nM.The specificity of the enzyme activity measured was assessed using a selective inhibitor of caspase-3, Ac-DEVD-CHO.The ability to block staurosporin induced caspase-3activation was determined by treating the cells with Ac-DEVD-CHO(10m M)before the treatment with staurosporine(1m M).Western blot analysisFor analysis of the levels of phosphorylated Akt and total Akt,cortical neurons treated with drugs for appropriate times were washed twice with ice-cold PBS and harvested in200m l of lysis buffer containing50mM Tris-HCl,pH7.5,100mM NaCl,1%SDS,20mM EDTA.After sonication for 30sec,the amount of protein in each sample was estimated by the bicinchoninic acid method (Pierce,Rockford,IL),followed by addition of1=5volume of sample buffer(200mM Tris-HCl, pH6.8,10%SDS,25%b-mercaptoethanol,25%glycerol and0.01%bromophenol blue).The samples were boiled for5min,and20m g aliquots were subjected to SDS-PAGE on10%poly-acrylamide gels and transferred to polyvinylidene difluoride(PVDF)membranes(Millipore, Bedford,MA).After blocking with5%nonfat dry milk in Tris-buffered saline containing 0.05%Tween-20overnight at4 C,blots were probed with specific antibodies(anti-Akt,1:5000;anti-phosphorylated Akt,1:1000)for 1h at room temperature,and washed,then incu-bated with Rabbit Ig,HRP-Linked F (ab’)2Fragment (Amersham Pharmacia Biotech,Buckin-ghamshire,England)diluted to 1:5000for 1h at room temperature.Immunoreactive bands were detected with the enhanced chemiluminescence system (ECL).Statistical analysisAll values are presented as mean ÆSEM.Data were analyzed using one-way ANOBA between subjects,and post hoc comparisons were made using the Tukey’s HSD test.In all cases,statistical significance was set at p <0.05.ResultsCortical neurons undergo apoptosis after exposure to haloperidolCortical neuron apoptosis was induced by exposure to haloperidol.In the absence of haloperidol,the cultured neurons exhibited normal cellular morphology with extending neurites (Fig.1).Haloperidol caused morphological changes charac-teristic of apoptosis,including degeneration of neurites and shrinkage of cell bodies.The morphological changes of neurons were evident over 24hr treatment with 30m M haloperidol.To determine whether haloperidol-induced toxicitywas Fig.1.Haloperidol exposure induces apoptosis in cortical neurons.A Representative photo-micrographs of cortical neurons (8DIV)treated with vehicle (0.3%DMSO)or 30m M haloper-idol for 16h and 72hr.The top panels are phase-contrast photomicrographs and the bottom panels are neurons stained with MAP2to clearly visualize apoptotic neuron morphology,including shrunken cell bodies,and fragmented processes.B DNA fragmentation manifests as a DNA ladder.Cortical neurons were treated with vehicle (C)or 30m M haloperidol (H)for 24hr.Positions of molecular size markers (MW)are indicated on the left in base pairs Implication of Akt-mediated signal changes by haloperidol 671apoptosis,a DNA laddering assay was conformed.DNA cleavage into oligonu-cleosome fragments was performed 24hr after 30m M haloperidol exposure in cortical neurons.The quantity of neuronal toxicity was evaluated by the MTT metabolism assay (Fig.2).Haloperidol reduced MTT metabolism in a dose-and time-depen-dent manner.The haloperidol concentration dependence and kinetics for neuro-nal viability assay were comparable with those seen when the cells were assayed for neuronal death by the ELISA method using an antibody against MAP2.Effects of haloperidol and risperidone on cortical neuron survivalThe neurotoxic potentials of haloperidol and risperidone were compared by MTT survival assays.The severity of neuronal toxicity was demonstrated by the evaluation of IC 50values for each drug.The conventional antipsychotic drug haloperidol had the higher toxicity on cortical neurons,with an IC 50value of 5.7m M (Fig.3),serotonin-dopamine antagonist (SDA)risperidone,showed neurotoxicity only with large doses (IC 50>30mM).Fig.2.Quantitation of dose response and kinetics for neuronal death induced by haloperidol exposure.Cortical neurons (8DIV)were treated with 0.1–100m M haloperidol for 72hr,or 30m M haloperidol for 0–96hr.Neuronal viability was determined by MTT metabolism (A ),and immunoreactivity of MAP2(B ),at each point after the initial treatment.Data are theaverages of three independent experimentsai et al.Implication of D2receptor function in the neuronalapoptosis induced by haloperidolWe tested whether the D2receptor agonist bromocriptine could also block cortical neuron toxicity after haloperidol exposure.Cortical neurons were treat-ed with 30m M haloperidol to induce neuronal toxicity (Fig.4).Approximately Fig.4.Suppression of neuronal death,which correlates with D2receptors in haloperidol exposured neurons.Cortical neurons were pretreated with vehicle (C)or 3,10m M bromocrip-tine (Bro 3,Bro 10)for 24hr and then exposure to 30m M haloperidol for 48hr to induce neuronal toxicity.Neuronal toxicity was evaluated by MTT assay.Addition of 1or 3m M domperidone (dom 1,dom 3)reversed the protective effect of bromocriptine.Data are theaverage of three independentexperimentsFig.3.Evaluation of neuronal toxicity induced by different antipsychotics.Cortical neurons were treated with the indicated concentrations of haloperidol or risperidone for each 72hr.ED 50values and 95%confidence limits were determined by the method of Fieller with linear regres-sion analysis as described in Materials and methodsImplication of Akt-mediated signal changes by haloperidol 67350%of cells survived 48hr after the treatment.However,90%of the cells survived in the presence of 10m M bromocriptine.Moreover,the neural protec-tion afforded by bromocriptine was primarily preserved by cotreatment with another D2receptor antagonist,domperidone,suggesting a role for the D2receptors and their signal transduction in bromocriptine protection against neu-ronal toxicity induced by haloperidol.Haloperidol but not Risperidone activates caspase-3in cortical neuronsTo investigate what mechanism is distinct in the neuronal toxicity induced by the haloperidol and risperidone,we evaluated the activity of caspase-3in the cortical neurons.We found that 24hr exposure of the cells to 30m M haloperidol significantly increased caspase-3activity compared with control cells,whereas after treatment with risperidone,it remained at the control level (Fig.5A).The role of caspase-3,DEVD-sensitive activity in cortical neuron apoptosis was also supported by the finding of a significant 6-fold elevation of caspase-3after treatment with the potent apoptosis inducer staurosporine,and its almost com-plete reduction by cotreatment with the specific caspase-3inhibitor DEVD-CHO,similar to a previous report (D’Mello et al.,1998).Haloperidol increased caspase-3activity in a dose-dependent manner (Fig.5B)BDNF suppresses caspase-3activation and protects neuronsfrom apoptosis after exposure to haloperidolTo define signaling pathways that reduce survival of cortical neurons,we tested the effectiveness of neurotrophin,BDNF,against haloperidol-induced caspase-3acti-vation and neuronal apoptosis.Cortical neurons were treated with 30m M haloper-idol for 24hr for caspase-3activity analysis,and for 48hr for survival assay,in the presence or absence of varying concentrations of BDNF,in respective examina-tions.BDNF decreased haloperidol-elevated caspase-3activity in cortical neurons in a dose-dependent manner.For example,after 24hr of treatment,30ng =mlFig.5.Activation of caspase-3in cortical neurons after exposure to haloperidol.A Cortical neurons were treated with vehicle (C)or 30m M haloperidol (Hal),30m M risperidone (Ris),1m M Staurosporine (Sta)or 1m M Staurosporine þAc-DEVD CHO (Inh)for 24hr.Exposure to haloperidol and staurosporine significantly activated caspase-3compared with control.B Dose-dependent activation of caspase-3was observed by haloperidol (24h)treatment.Results inpanels are averages of three different experiments.Error bars are SEMai et al.BDNF decreased haloperidol-elevated caspase-3activity by 65%(Fig.6A).Simi-lar levels of neuroprotection were also observed,for example,after 48hr of treat-ment with haloperidol alone 60%of neurons survived,and 90%of neurons survived after cotreatment with 30ng =ml BDNF (Fig.6B).Inhibition of the PI 3-Kinase-Akt pathway is criticalfor cortical neuron apoptosis induced by haloperidolBDNF can activate several signal transduction systems including the PI 3-kinase pathway (Segal et al.,1996;Yamada et al.,1997).We assayed PI 3-kinase activity by western blot analysis using a phospho-Akt antibody that specifically recognizes activated Akt.The same samples were also probed with an anti-Akt antibody to ensure an equal amount of protein loading in each lane.Phosphorylation of Akt was detectable in cortical neurons maintained in regular culture condition;probably because of PI 3-kinae activation by growth factors in the B27supplement.Treatment with 30m M haloperidol at 4,8,and 18hr caused large decrease in Akt phosphorylation (Fig.7A).To determine whether activation of the PI 3-kinase pathway contributes to BDNF protection against haloperidol,we added BDNF to block PI 3-kinase inhibition by haloperidol.BDNF at 30ng =ml completely reversed the Akt phos-phorylation after its reduction by haloperidol (Fig.7B).5-HT 2A antagonist suppresses neuronal apoptosisinduced by haloperidolThe atypical antipsychotic drug risperidone is known for its high affinity to 5-HT 2A receptors as an antagonist.To obtain further insights into thelimited Fig.6.BDNF protects neurons from haloperidol-induced apoptosis through caspase-3inhibi-tion.Cortical neurons were treated with 30m M haloperidol in the presence or absence of 10or 30ng =ml BDNF.BDNF significantly inhibited caspase-3activation (A ),and protected cortical neurons (B )against 30m M haloperidol exposure.Results in panels are averages of threedifferent experiments.Error bars are SEMImplication of Akt-mediated signal changes by haloperidol 675neurotoxicity induced by risperidone,we tested whether 5-HT 2A antagonism influenced the neuronal toxicity after haloperidol exposure.Cortical neurons were treated with 30m M haloperidol to induce neuronal toxicity (Fig.8).Approximately 55%of cells survived 48hr after the treatment.However,80%of the cells survived in the presence of 1m M 5-HT 2A specific antagonist ketanserin.This suggested a role for 5-HT 2A receptor antagonism on the pro-tective effect against neuronal apoptosis induced by haloperidol.DiscussionIn the current work,we investigated neuronal toxicity caused by the two kinds of antipsychotics,haloperidol and risperidone,to clarify the molecular basisof Fig.7.BDNF reversed the haloperidol-induced PI 3-K =Akt reduction.Cortical neurons were treated with 30m M haloperidol for 4,8or 18hr.Haloperidol reduced the level of Ser-473phosphorylated Akt at all exposure time points (A ).BDNF (10,30m M)increased levels of phosphorylated Akt reduced by exposure to 30m M haloperidol (B ).Akt phosphorylation (pAkt)was examined by Western blot analysis at the indicated times after haloperidol treat-ments.The blots show that the total level of Akt (Akt)remainedconstantFig.8.Suppression of neuronal death by treatment with a 5-HT2A antagonist.Cortical neurons were pretreated with vehicle (C),0.1or 1m M ketanserin (Ket 0.1,Ket 1)for 24hr and then exposed to 30m M haloperidol for 48hr to induce neuronal toxicity.Neuronal toxicity was evaluated byMTT assay.Data are the average of three independent experiments.Error bars are SEM ai et al.the differential expression of motor side effects,and differential efficacy on negative-cognitive symptoms.We found that treatment with haloperidol pro-duced apoptosis of cortical neurons accompanied by shrinkage of the cell body, and prominent DNA ladders,hallmarks of apoptosis(Raff et al.,1993;Stefanis et al.,1997).Haloperidol caused MTT metabolism reduction in a dose and time-dependent manner.We confirmed that neuronal death paralleled MTT reduction using an established MAP2-ELISA.We confirmed the reliability of this method by comparison of the immunoreactivity of MAP2with the number of neurons in cultured cortical neurons previously(data not shown).Recently,neurotoxic effects of antipsychotic drugs have emerged as potential pathogenic events of TD.One of the objectives of this study was to test the hypothesis that neurotoxic properties of antipsychotics are closely linked to the occurrence of EPS in schizo-phrenic patients.Haloperidol,a representative typical antipsychotic drug,showed about6-times higher neurotoxicity than risperidone,one of the atypical antipsy-chotic drugs.These differences suggest that haloperidol may have a stronger inhibitory effect on neuronal survival mechanisms than risperidone.If haloperidol-induced neuronal toxicity is mediated by inhibition of D2 receptor function and subsequent disruption of signaling pathways underlying D2receptors,then agonists of D2receptors might prevent haloperidol-induced neuronal toxicity.In our experiment on cortical neurons,the D2receptor ago-nist bromocriptine blocked haloperidol-induced neurotoxicity.In addition, another specific D2antagonist,domperidone(Sokoloff et al.,1980)prevented the protective effect of bromocriptine against haloperidol-induced neurotoxi-city.Since it is reported that bromocriptine has neuroprotective effect against6-hydoxydopamine mediated by its hydroxyl free radical scavenging activity (Ogawa et al.,1994),the effect of bromocriptine here may relate to the pre-ventions of mitochondrial dysfunction and increasing hydroxyl free radicals by haloperidol.These data suggest that the haloperidol-induced neuronal apoptosis in cortical neurons is mediated by D2receptors,and that the inhibition of D2 receptor regulating signaling pathways plays a key role in the neurotoxicity of haloperidol.To clarify the differences of cellular molecular changes,we next evaluated the activity of caspase-3,in the cortical neurons,because it is known as promi-nent apoptosis-inducing molecule acting in thefinal stages in the cell apoptosis program(Armstrong et al.,1997;Marks et al.,1998).We found that haloperidol activated caspase-3in cortical neurons.In contrast to haloperidol,risperidone exposure had no significant effect on control caspase-3activity.These data sug-gested that the regulation of cellular survival signaling underlying the D2recep-tor inhibition differed between these two kinds of antipsychotics,and that caspase-3activation might contribute to neuronal apoptosis in cortical neurons induced by the antipsychotics,or at least by haloperidol exposure.The active tissue brain level of risperidone at clinical dosage was estimated 1–50nM(Aravagiri et al.,1988,1998).That was about1000-times lower than that concentration of risperidone resulted in neither significant toxicity nor caspase-3activation tested in the present study.The haloperidol concentration in the brain tissue of patients was detected at25–600nM(Kornhuber et al., 1999),and reported maximally to be10m M(Korpi et al.,1984).The concentration。