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  • Expression of Brain-Derived NeurotrophicFactor mRNA in Rat Hippocampus AfterTreatment With Antipsychotic Drugs

    Ou Bai, Jennifer Chlan-Fourney, Rudy Bowen, David Keegan, and Xin-Min Li*Neuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon,Saskatchewan, Canada

    Typical and atypical antipsychotic drugs, though botheffective, act on different neurotransmitter receptors andare dissimilar in some clinical effects and side effects.The typical antipsychotic drug haloperidol has beenshown to cause a decrease in the expression of brain-derived neurotrophic factor (BDNF), which plays an im-portant role in neuronal cell survival, differentiation, andneuronal connectivity. However, it is still unknownwhether atypical antipsychotic drugs similarly regulateBDNF expression. We examined the effects of chronic(28 days) administration of typical and atypical antipsy-chotic drugs on BDNF mRNA expression in the rat hip-pocampus using in situ hybridization. Quantitative anal-ysis revealed that the typical antipsychotic drughaloperidol (1 mg/kg) down-regulated BDNF mRNA ex-pression in both CA1 (P 0.05) and dentate gyrus (P 0.01) regions compared with vehicle control. In contrast,the atypical antipsychotic agents clozapine (10 mg/kg)and olanzapine (2.7 mg/kg) up-regulated BDNF mRNAexpression in CA1, CA3, and dentate gyrus regions of therat hippocampus compared with their respective con-trols (P 0.01). These ndings demonstrate that thetypical and atypical antipsychotic drugs differentially reg-ulate BDNF mRNA expression in rat hippocampus. 2002 Wiley-Liss, Inc.

    Key words: haloperidol; clozapine; olanzapine; BDNF;hippocampus

    The antipsychotic drugs, which are effective in treat-ing several psychiatric illnesses, such as schizophrenia, aredivided into two classes. Atypical antipsychotic drugs, suchas clozapine and olanzapine, block both dopamine andserotonin receptors, improve both the positive and thenegative symptoms of schizophrenia, and have a lowerpropensity to induce extrapyramidal side effects. Typicalantipsychotic drugs, such as haloperidol, block dopaminereceptors and improve positive symptoms of schizophreniabut have a high propensity to cause motor side effects(Meltzer, 1995). Clinical evidence demonstrates that earlyand prolonged intervention with these drugs will improvethe long-term therapeutic outcome (Wyatt and Henter,1998) and that the clinical improvements are not observeduntil 23 weeks following antipsychotic drug administra-

    tion (Kinon and Liebermann, 1996). These ndings indi-cate that the clinical effects are not solely due to theblockade of neurotransmitters. Recent studies showed thatthe typical antipsychotic agent haloperidol increased nervegrowth factor (NGF) expression in the hippocampus, piri-form cortex, striatum, and nucleus accumbens of mice(Ozaki, 2000); however, other studies demonstrated thathaloperidol signicantly decreased BDNF protein expres-sion in the frontal cortex, occipital cortex, and hippocam-pus of rats (Angelucci et al., 2000; Lipska et al., 2001).

    Brain-derived neurotrophic factor (BDNF) is widelyexpressed in the CNS; its highest levels are found in thehippocampus and cortex (Hofer et al., 1990), whereBDNF supports neuronal cell survival, differentiation,connectivity, and morphology (Ghosh et al., 1994;Thoenen, 1995), particularly of dopaminergic neurons(Hyman et al., 1991). Functional and anatomical evidenceimplicates hippocampal involvement in schizophrenia(Falkai and Bogerts, 1986; Weinberger, 1999) and BDNFin hippocampal neuron function. Because haloperidoldown-regulates BDNF expression, it is possible thatBDNF is also involved in the action of atypical antipsy-chotic drugs. The aim of this study was to measure BDNFmRNA expression in the hippocampus of rats that hadbeen treated with the atypical antipsychotic drugs cloza-pine or olanzapine and the typical antipsychotic drughaloperidol using in situ hybridization.

    MATERIALS AND METHODS

    Animals

    Male Wistar rats (200350 g; Charles River Canada,Montreal, Quebec, Canada) were individually housed under a12 hr light-dark cycle at a temperature of 1920C, with free

    Contract grant sponsor: Health Services Utilization and Research Com-mission; Contract grant sponsor: Canadian Psychiatric Research Founda-tion and the Schizophrenia Society of Saskatchewan.

    *Correspondence to: Dr. Xin-Min Li, Neuropsychiatry Research Unit,Department of Psychiatry, University of Saskatchewan, A114 MedicalResearch Building 103 Wiggins Road, Saskatoon, Saskatchewan S7N 5E4,Canada. E-mail: xin-min.li@usask.ca

    Received 30 April 2002; Revised 22 July 2002; Accepted 23 July 2002

    Journal of Neuroscience Research 71:127131 (2003)

    2002 Wiley-Liss, Inc.

  • access to both food and water. All procedures involving animalswere performed in accordance with the Canadian Council onAnimal Care Guidelines and approved by the University ofSaskatchewans Committee on Animal Care and Supply. After1 week of acclimatization, rats were randomly divided into fourgroups (n 3) and were given daily drug injections (i.p.) for 28days. Group 1 received vehicle control (0.4% acetic acid in 0.9%saline); group 2 received haloperidol (1 mg/kg); group 3 re-ceived clozapine (10 mg/kg); group 4 received olanzapine(2.7 mg/kg). The rats were sacriced 18 hr after the last injec-tion. The doses for haloperidol and clozapine were chosen onthe basis of 5-HT2 and D2 ex vivo receptor occupancies in therat brain as determined by Schotte et al. (1993), and the dose forolanzapine was calculated based on a study by Beasley et al.(1997). The treatments did not induce changes in weight.

    In Situ Hybridization

    Brains to be used for in situ hybridization were removedand immediately frozen in isopentane at 45C and stored at 70C. Twelve micrometer coronal sections through the hip-pocampus were thaw mounted on slides doubly coated withpoly-L-lysine and stored at 20C. The prehybridization wascarried out within 72 hr. Mounted sections were xed with 4%paraformaldehyde, acetylated in acetic anhydride, dehydratedthrough an increasing ethanol gradient, delipidated in chloro-form for 10 min, rehydrated to 95% ethanol, air dried, andstored at 20C until the actual hybridization was performed.

    BDNF sense and antisense RNA probes were labeled with35S-CTP, and 2 106 c.p.m. were applied to each slide. Slideswere then incubated at 54C overnight. On the following day,slides were rinsed in 2 SSC buffer (sodium chloride andsodium citrate) four times for 5 min each and treated with10 mg/ml RNase for 30 min, followed by a rinse in RNasebuffer for 30 min at room temperature. Subsequently, the sec-tions were washed in 2 SSC at room temperature for 4 min,2 SSC at 50C for 30 min, and 0.2 SSC at 55C for 30 min.Finally, sections were dried in ascending ethanol concentrationsand allowed to air dry for 1 hr before exposure to photographicemulsion (Li et al., 1998).

    Photographic Emulsion

    Kodak NTB2 photographic emulsion was diluted 1:1with nanopure water and warmed to 42C. The slides weredipped for 3 sec and left to dry for at least 2 hr and were thenplaced in dark boxes with dessicant at 4C for 26 weeks. Theslides were developed for 2 min in Kodak D-19 developer, xedfor 30 sec in Kodak xer, rinsed in water, and air dried. Thesections were lightly counterstained with cresyl violet, dehy-drated, cleared with xylene, and mounted for analysis.

    Quantitative Analysis of BDNF mRNA

    The emulsion-dipped slides were quantitatively analyzedwith image-analysis software (Image-Pro Plus version 4.0). Thearea covered with silver grains was measured using an Olympus(BH-2) microscope with darkeld illumination, and cell numberwas counted with lighteld illumination. The background, anarea of negative expression, was subtracted. Silver grain den-sity area of (positive expression negative expression)/cellnumber. Three elds from each section and three sections from

    each animal were counted. Average percentage area values wereexpressed as percentage of vehicle control values.

    Data Analysis

    The data are presented as mean SD. The groups werecompared using one-way ANOVA, followed by the post hocScheffes test. Signicance is reported as follows: *P 0.05,**P 0.01.

    RESULTSBrain sections from the four treatment groups hy-

    bridized with 35S-labeled BDNF RNA probes are shownin Figure 1. Low-magnication (2.5 lighteld) photo-graphs (top row) show strong expression of BDNFmRNA on hippocampal sections from rats treated withclozapine or olanzapine. High-magnication (40 dark-eld) photographs (three lower rows) show BDNFmRNA expression with silver grain density in the CA1,CA3, and dentate gyrus regions of the hippocampus.Lower silver grain densities are seen in the CA1, CA3, anddentate gyrus regions of hippocampus in rats treated withhaloperidol and the highest density on sections from clo-zapine or olanzapine groups. The decreased expression ofBDNF mRNA is indicated by a lower density of silvergrains, and the increased expression of BDNF is shown bya higher density.

    Quantitative analysis of BDNF mRNA expressionwas performed on three regions of the hippocampus.Analysis by one-way ANOVA showed signicant effectson BDNF expression in CA1 [F(3,8) 67.7], CA3[F(3,8) 33.7], and dentate gyrus regions [F(3,8) 98.6]after drug treatments. Post hoc analysis revealed signi-cance in all three treatment groups (Fig. 2). Haloperidoldecreased BDNF mRNA expression in both CA1 (P 0.05) and dentate gyrus (P 0.01) regions compared withvehicle control. In contrast, clozapine and olanzapine sig-nicantly increased gene expression in all three regions ofthe hippocampus compared with control (P 0.01).

    DISCUSSIONThe present study demonstrates that chronic admin-

    istration of the typical antipsychotic drug haloperidol re-sults in a reduction of BDNF mRNA expression in theCA