Chronic treatment with aripiprazole induces differential gene expression in the rat frontal cortex

Abstract

Chronic treatment of antipsychotic drugs can modulate gene expression in the brain, which may underscore their clinical efficacy. Aripiprazole is the first approved antipsychotic drug of the class of dopamine D₂ receptor partial agonist, which has been shown to have similar efficacy and favourable side-effects profile compared to other antipsychotic drugs. This study aimed to identify differential gene expression induced by chronic treatment of aripiprazole. We used microarray-based gene expression profiling technology, real-time quantitative PCR and Western blot analysis to identify differentially expressed genes in the frontal cortex of rats under 4 wk treatment of aripiprazole (10 mg/kg). We were able to detect ten up-regulated genes, including early growth response gene 1, 2, 4 (Egr1, Egr2, Egr4), chromobox homolog 7 (Cbx7), cannabinoid receptor (Cnr1), catechol-O-methyltransferase (Comt), protein phosphatase 2c, magnesium dependent (Ppm2c), tachykinin receptor 3 (Tacr3), Wiscott–Aldrich syndrome-like gene (Wasl) and DNA methyltransferase 3a (Dnmt3a). Our data indicate that chronic administration of aripiprazole can induce differential expression of genes involved in transcriptional regulation and chromatin remodelling and genes implicated in the pathogenesis of psychosis.

Introduction

Pharmacotherapy is the treatment of choice for psychotic disorders, however, it usually takes several weeks to see clinical improvement after repeated administration of antipsychotic drugs. Accumulating evidence suggests that neural plasticity changes mediated by differential gene expression in the brain may underscore the clinical efficacy of antipsychotic drugs (Hyman and Nestler, 1996; Konradi and Heckers, 2001). Hence, study of the differentially expressed genes after chronic treatment of antipsychotic drugs would be helpful in understanding the molecular mechanism of the action of antipsychotic drugs, identifying the drug target molecules and bringing new insight into the pathogenesis of psychotic disorders.

The molecular underpinnings of the clinical efficacy of antipsychotic drugs are complex and involve many genes. Microarray-based gene expression profiling is a functional genomics method that allows simultaneous assessment of the expression of thousands of genes in a DNA chip, which is suitable for studying complex biological phenomenon and has a broad application in biomedical science research (Brown and Botstein, 1999; Schena et al., 1995). There is an increasing use of this technology to study the molecular mechanism of action of a variety of antipsychotic drugs (Palfreyman et al., 2002). For example, chronic treatment of antipsychotic drugs such as haloperidol, clozapine and risperidone, was found to alter the expression of genes involved in neurotransmission, signal transduction, oxidative stress, cell adhesion, apoptosis, proteolysis and neuroplasticity (Chong et al., 2002; Feher et al., 2005; Kontkanen et al., 2002; MacDonald et al., 2005; Thomas et al., 2003). Recently, Fatemi and colleagues reported that chronic administration of the atypical antipsychotic drug, olanzapine, can induce differential expression of genes involved with multiple cellular functions such as signal transduction, cell communication and metabolism (Fatemi et al., 2006). Our group has identified several differentially expressed genes involved in neurotransmission, synaptic plasticity and proteolysis in the rat frontal cortex under chronic administration of another atypical antipsychotic drug, risperidone, using microarray technology (Chen and Chen, 2005).

Aripiprazole is a novel antipsychotic drug with unique pharmacological properties. It is characterized as a partial agonist of the dopamine D₂ receptor and is the first approved antipsychotic drug of such class (Grunder et al., 2003; Lieberman, 2004). Aripiprazole was reported to have equivalent clinical efficacy and favourable side-effect profile (e.g. extrapyramidal symptoms, cardiovascular side-effects, prolactin elevation and sedation) compared to other typical and atypical antipsychotic drugs (Hirose et al., 2004; Naber and Lambert, 2004). Aripiprazole has high affinity to dopamine D₂/D₃ receptors and also reserves some intrinsic activity of dopamine, which makes it distinct from other typical and atypical antipsychotics (Burris et al., 2002; Kikuchi et al., 1995). Moreover, aripiprazole is also a partial agonist of the 5-HT_1A receptor, hence, it is also proposed as dopamine/serotonin system stabilizer (Jordan et al., 2002).

Nevertheless, except for its unique receptor-binding profile, little is known about the molecular mechanism of action of aripiprazole. The aim of this study was to employ the microarray-based gene expression profiling technology, real-time quantitative PCR (RT-qPCR) and Western blot analysis to identify differentially expressed genes in the frontal cortex of rats under chronic administration of aripiprazole. The frontal cortex was chosen because of its implication in the pathophysiology of psychosis and site of action of antipsychotic drugs (Bunney and Bunney, 2000).

Materials and methods

Animals and drug treatment

Male Sprague–Dawley rats were housed in a temperature- and humidity-controlled environment with a 12-h light/dark cycle and had free access to food and water. The Institutional Animal Care and Use Committee of the Institute approved all the experimental procedures. Twenty animals weighing about 150–200 g were randomly assigned to two groups: the experimental group received an intraperitoneal injection of aripiprazole (10 mg/kg) (kindly provided by Otsuka Pharmaceutical, Tokushima, Japan) once daily for 4 wk; the control group received an intraperitoneal injection of vehicle once daily for 4 wk. Aripiprazole was suspended in 5% gum Arabic–saline acid. Animals were sacrificed under CO₂ anesthesia 24 h after the last injection. The frontal cortex was dissected and stored in RNAlater^® solution (Ambion Inc., Austin, TX, USA) at 4°C for 1 d and stored at −80°C until required.

Total RNA and cDNA preparation

Total RNA from the frontal cortex of each rat was extracted using TRIzol Total RNA Isolation Reagent according to the manufacturers instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). In brief, rat frontal cortex was homogenized directly in 1 ml of TRIzol denaturing reagent per 50–100 mg tissue. Homogenates were incubated on ice for 5 min, vortexed vigorously, and centrifuged at 12 000 g for 10 min at 4°C. For each ml of supernatant, 200 µl chloroform was added, mixed vigorously, incubated on ice for 5 min, and centrifuged at 12 000 g for 10 min at 4°C to collect the aqueous phase. The procedure was repeated once and the aqueous phase was collected, mixed with 0.7 vol. of isopropyl alcohol. The mixtures were incubated at −20°C for 1 h, centrifuged at 12 000 g for 30 min at 4°C. RNA pellets were washed with 75% ethanol twice, air dried, and dissolved in RNase-free water.

Aliquots of total RNA were digested with RQ1 RNase-Free DNase (Promega Corporation, Madison, WI, USA) to remove genomic DNA. After digestion, the total RNA was further purified by chloroform extraction twice and ethanol precipitation. RNA pellets were washed with 75% ethanol, air-dried, and dissolved in the RNA Storage Solution (Ambion Inc.). RNA concentration was determined using spectrophotometry. To ensure the total RNA is free from genomic DNA after DNase digestion, aliquots of total RNA were subjected to PCR using a pair of primers (5′-accacagtccatgccatcac-3′ and 5′-tccaccaccctgttgctgta-3′) to amplify the genomic DNA of rat glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene. The presence of a 450 bp PCR product indicates incomplete DNase digestion. The integrity of 28S and 18S rRNA were checked by electrophoresis of 2 µg total RNA in 1.2% agarose gel containing 2.2 m formaldehyde and in a running buffer containing 0.2 m MOPS (pH 7.0), 20 mm sodium acetate and 10 mm EDTA (pH 8.0).

Microarray analysis of gene expression

Applied Biosystems Rat Genome Survey Microarray (Applied Biosystems, Foster City, CA, USA) containing 26 587 probes representing 27 088 individual genes was used for expression profiling in this study. Five aliquots of RNA from experimental groups and five aliquots of RNA from control animals were randomly chosen for microarry experiment. They were tested individually. In brief, digoxigenin (DIG)-labelled cRNA was prepared from 2 µg total RNA using Applied Biosystems Chemiluminescent RT-IVT Labelling kit according to the manufacturer's protocol. The DIG-labelled cRNA (10 µg) was injected into each array chamber for hybridization at 55°C for 16 h. After washing, the spot image of array was visualized using Applied Biosystems Chemiluminescence Detection kit and Applied Biosystems 1700 Microarray Analyzer according to the manufacturer's protocol (Applied Biosystems). The image was auto-gridded and the signal was quantified, corrected for background and spatially normalized using Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software v. 1.1. The signal data were subjected to further analysis using BRB-ArrayTools v3.4, which was developed by Dr Richard Simon and Amy Peng Lam (available from http://linus.nci.nih.gov/BRB-ArraryTool.html). Differentially expressed genes between control and experimental animals were assessed using class comparison function of the BRB-ArrayTools v3.4, which computes an F test separately for each gene using the normalized data. Genes showing significantly differential expression (p<0.05) were further validated using RT-qPCR and Western blot analysis.

RT-qPCR

For quantitative PCR, cDNA was first prepared by reverse transcription using Superscript II RNase H⁻ Reverse Transcriptase (Invitrogen Life Technologies). A 12 µl reaction mixture containing 2 µg total RNA and 2 µl (50 µm) of random hexamers were first heated at 65°C for 5 min, and quickly chilled on ice. After brief centrifugation to collect the content, 4 µl of 5x first-strand buffer, 2 µl of 0.1 m DTT, and 1 µl (200 U) Superscript II reverse transcriptase were added into the tube. The mixtures were incubated at 42°C for 50 min, and the reaction was stopped by heat inactivation at 70°C for 15 min. RT-qPCR was performed using Applied Biosystems PRISM 7300 Sequence Detection System in combination with continuous SYBR Green detection (Applied Biosystems). The experiment was performed in a 25 µl reaction volume containing 2.5 µl cDNA, 12.5 µl SYBR Green PCR Master Mix, 2.5 µl each of sense and antisense primers (5 µm), and 5 µl H₂O. The general PCR conditions were as follows: polymerase activation at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s. After amplification, a melting curve was acquired to determine the optimal PCR conditions. Primer sequences for PCR amplification were designed using online Primer3 software (http://biocore.unl.edu/cgi-bin/primer3/primer3_www.cgi).

Relative quantification with the standard curve method was used to determine the expression level of gene of interest. At first, serial dilutions of the known amount of cDNA from a reference sample were used to generate the external standard curve. For each unknown sample, the relative amount was calculated using linear regression analysis from their respective standard curves. The expression level of each gene was normalized by the geometric mean of the expression of three housekeeping genes, i.e. Gapdh, cyclophilin A and 18S rRNA in each sample, which is more accurate than data normalized by just one housekeeping gene (Vandesompele et al., 2002). The expression levels of Gapdh and cyclophilin A were assayed using RT-qPCR and SYBR Green detection as described. The 18S rRNA reference gene was measured using Pre-Developed TaqMan Assay Reagents 18S rRNA MGB according to the manufacturer's protocol (Applied Biosystems). All experiments were performed in duplicate.

Protein extraction and quantification

Rat frontal cortex tissue was homogenized in 500 µl cell lyses solution containing 1% Triton X-100, 40 mm Tris–HCl (pH 8.0), 65 mm DTT per 50–100 mg tissue. The cell lyses solution also contained one protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) per 10 ml. The protease inhibitor cocktail tablet comprised antithrombin III, aprotonin, 3,4-dichloroisococemarin, APMSF, E-64, EDTA-Na2, phosphoramidon, bestatin, TIMP-2 and pepstatin. The homogenates were centrifuged at 15 000 g for 15 min at 4°C, and the supernatant was precipitated by 100% of trichloroacetic acid (TCA, final concentration 10%) on ice for 10 min. After centrifugation at 15 000 g for 15 min, the pellets were washed with 10% TCA once, and ice-cold acetone three times. The pellets were air-dried at room temperature and dissolved in 300 µl sample preparation solution containing 8 m urea, 4% Chaps, 2% IPG buffer, 40 mm DTT. Protein concentrations were determined using Coomassie protein assay (Pierce Biotechnology Inc., Rockford, IL, USA).

Western blot analysis

Aliquots of protein (2 µg) were mixed with sample buffer (62.5 mm Tris–HCl (pH 6.8), 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95°C for 10 min. The mixtures were separated on 7.5% SDS–PAGE gels at constant voltage (100 V) for 1 h, and then transferred onto an Immobilon-P Membrane, PVDF for 2 h at constant voltage (100 V) at 4°C using Tank Transfer Systems (Mini Trans-Blot Cell, Bio-Rad Laboratories Inc., Hercules, CA, USA). After blocking with 5% skim-milk for 2 h, the membrane was incubated with 1:1000 rabbit anti-Egr1 polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight with gentle shaking at 4°C. Goat anti-rabbit IgG (1:2000) conjugated with horseradish peroxidase (Santa Cruz Biotechnology Inc.) was used as second antibody. The chemiluminescence signal was visualized using the ECL detection system (GE Health Care Bio-Sciences AB, Uppsala, Sweden) and by exposing the membrane to Kodak BioMax light film for 30 s. The intensities of the bands were assessed using NIHimage software.

Statistical analysis

For RT-qPCR data, the scatter plot of the expression level of each gene in both control and aripiprazole-treated animals were examined first. Extreme outliers were excluded for further comparative analysis. Differences of the normalized mRNA expression levels of selected genes between experimental and control animals were assessed using Student's t test. For Western blot image analysis, Student's t test was used to assess the mean differences of image intensity between control and experimental animals. Statistically significant differences are defined as those with a p value <0.05. All the calculations were implemented using SPSS for Windows version 11.5 (SPSS Inc., Chicago, IL, USA).

Results

Among the 10 animals of each group, we randomly chose five samples from each group for microarray-based gene expression experiments. Each sample was tested individually. We were able to detect 157 gene probes that showed significant differential expression using the class comparison function of the BRB-ArrayTools v3.4 (see Supplementary Table S1; the Supplementary tables are available in the online version of the paper). Because we were not able to validate all the 157 genes, 40 genes were selected for verification based on a review of the literature and our previous knowledge; genes with neurobiological function were taken into consideration first. We compared the mRNA expression levels of these 40 genes in 10 animals from each group (including the original five samples of each group for microarray analysis) using RT-qPCR. The fold difference between experimental group and control animals in microarray experiment and qPCR experiment of these 40 genes are listed in Table 1. The sequences of primers, optimal annealing temperature and the length of amplicon of each gene assessed in qPCR are listed in Supplementary Table S2. Among the 40 genes assayed, we were able to verify 10 genes that were up-regulated significantly by the chronic treatment of aripiprazole, including DNA methyltransferase 3a (Dnmt3a), early growth response 1, 2, 4 (Egr1, Egr2, Egr4), chromobox homolog 7 (Cbx7), cannabinoid receptor 1 (Cnr1), catechol-O-methyltransferase (Comt), protein phosphatase 2c, magnesium dependent (Ppm2c), tachykinin receptor 3 (Tacr3) and Wiskott–Aldrich syndrome-like gene (Wasl). The mean expression levels of each gene as normalized by the geometric mean of 18S rRNA, cyclophilin and Gapdh in both experiment and control animals are summarized in Supplementary Table S3. Representative equations of simple linear regression describing the standard curve of these genes are listed in Supplementary Table S4. To further verify the differentially expressed gene at protein level, we selectively performed Western blot analysis on Egr1 in the frontal cortex of rats. As shown in Figure 1, there is a significantly increased expression of Egr1 protein in the frontal cortex of rats treated with aripiprazole compared to control animals (p<0.05).

Discussion

In line with the findings from our group and other research groups, we were able to identify ten genes that are differentially expressed under chronic administration of aripiprazole in this study. DNA methyltransferase 3a (Dnmt3a) encodes one of the de-novo DNA methyltransferases that play an essential role in mammalian embryonic development, imprinting and X-chromosome inactivation (Okano et al., 1999). Dnmt3a was found to have wide expression in postnatal brain until adulthood, suggesting that Dnmt3a probably plays an important role in the brain maturation (Feng et al., 2005). Previous studies have shown that aberrant methylation of brain genes is associated with certain neuropsychiatric disorders (Amir et al., 1999; Veldic et al., 2004). Recent study also showed that brain DNA methyltransferase regulates synaptic plasticity in the hippocampus (Levenson et al., 2006). Thus, the reduced expression of Dnmt3a by aripiprazole suggests that aripiprazole may exert its effect via epigenetic regulation of certain gene expression that is related to synaptic plasticity. In this study, we found another gene, chromobox homolog 7 (Cbx7) that is involved in chromatin remodelling, is up-regulated by aripiprazole. Cbx7 is one of the polycomb group of genes that are important regulators of body segmentation and cell growth during development and is essential for controlling the lifespan of normal and tumour cells (Gil et al., 2004). Cbx7 is also highly expressed in the brain, and is a member of the polycomb repression complex 1 (PRC1) that plays an important role in neurogenesis during development and neuronal maturation in the adult brain (Vogel et al., 2006). Polycomb proteins were found to bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin (Bernstein et al., 2006), suggesting their involvement in epigenetic regulation of gene expression. Taken together, the up-regulation of Dnmt3a and Cbx7 by chronic treatment of aripiprazole indicates that aripiprazole may influence the epigenetic machinery and consequently regulate some gene expression in the brain through this mechanism.

Three genes of the early growth response gene family, i.e. Egr1, Egr2 and Egr4, were up-regulated by chronic administration of aripiprazole. The increased expression of Egr1 as induced by chronic treatment of aripiprazole was further supported by Western blot analysis. Egr family genes encode inducible transcription regulatory factors that contain the zinc-finger DNA-binding domain and have been implicated in neuronal activity and plasticity (Beckmann and Wilce, 1997; Knapska and Kaczmarek, 2004; O'Donovan et al., 1999), learning and memory (Davis et al., 2003; Toscano et al., 2006). Egr2 is involved in the regulation of myelination of neurons (Nagarajan et al., 2001; Parkinson et al., 2003), while abnormal myelination has been considered as part of the pathophysiology of schizophrenia (Flynn et al., 2003; Woo and Crowell, 2005). Thus, the increased expression of Egr2 gene by chronic treatment of aripiprazole suggests that the clinical efficacy of aripiprazole may be related to myelination of neurons in the brain of patients. Egr family genes function as transcription factors, and several target genes of Egr proteins have been identified (Knapska and Kaczmarek, 2004; James et al., 2005), it would be interesting to see which genes are influenced by aripiprazole through the induction of Egr family genes by aripiprazole, and that may bring new insight into the molecular mechanism of aripiprazole. In a recent report, down-regulation of EGR family gene expression was found in the dorsolateral prefrontal cortex of patients with schizophrenia, and there is a genetic association of EGR family genes and schizophrenia (Yamada et al., 2007), suggesting EGR family genes are involved in the pathophysiology of schizophrenia. Thus, the aripiprazole-induced up-regulation of Egr genes as shown in this study may be relevant to its clinical efficacy.

Catechol-O-methyltransferase (COMT) is involved in degradation of biogenic amine neurotransmitters by catalysing the transfer of a methyl group to the catecholamine neurotransmitters such as dopamine, epinephrine and norepinephrine. The human COMT gene has been repeatedly reported to be associated with schizophrenia, although the results are not conclusive. Studies reported that COMT genotype is associated with the regulation of dopamine neurotransmission in the human brain (Akil et al., 2003) and frontal lobe function (Egan et al., 2001). In addition, schizophrenic patients were found to have aberrant COMT mRNA expression patterns in their dorsolateral prefrontal cortex (Matsumoto et al., 2003). Together, these data suggest that aberrant COMT may be associated with the pathophysiology of schizophrenia. We previously reported that long-term treatment of risperidone or olanzapine results in increased expression of the Comt gene in the rat frontal cortex (Chen and Chen, 2007). In this study, we also found that 4 wk treatment with aripiprazole also increases the expression of Comt gene in rat frontal cortex. Hence, we suggest that the increased expression of Comt following long-term treatment of risperidone, olanzapine and now aripiprazole may help restore the aberrant biogenic amine neurotransmission to normal status, which may partly account for the molecular mechanism of the clinical efficacy of these antipsychotic drugs.

Neuronal Wiskott–Aldrich Syndrome Protein (N-WASP) is one of the five members of the WASP protein family that plays a key role in the regulation of actin polymerization involved with cytoskeletal reorganization (Miki and Takenawa, 2003), it is a housekeeping gene and is also highly expressed in neuronal cells (Fukuoka et al., 1997). N-WASP is also involved in regulating the vesicle formation and traffic (Kessels and Qualmann, 2004), and presynaptic cytomatrix (Dresbach et al., 2001). Recent study showed that N-WASP participates in the reorganization of the presynaptic actin network that underlies the molecular mechanism of learning-related synaptic growth (Udo et al., 2005). Thus, the up-regulation of the expression of the Wasl gene that encodes the rat N-WASP may contribute to the improved cognitive function under long-term treatment of aripiprazole.

Cannabinoid receptor 1, mainly expressed in the brain, has been implicated in the pathogenesis of schizophrenia, because abuse of cannabis can induce psychotic symptoms in normal individuals and worsen the psychotic symptoms in schizophrenia (Ujike and Morita, 2004). Many studies have provided evidence to suggest that there is a functional interaction between the endocannabinoid system and dopaminergic system in the brain (Houchi et al., 2005). Subchronic treatment of antipsychotics such as haloperidol has been reported to result in increased cannabinoid receptor 1 binding in basal ganglia (Andersson et al., 2005; Mailleux and Vanderhaeghen, 1993), while chronic clozapine treatment decreases the cannabinoid receptor 1 binding in the nucleus accumbens (Sundram et al., 2005). In this study, we found chronic treatment with aripiprazole induces increased expression of the Cnr1 gene. This finding suggests that the interaction between the dopamine D₂ receptor and cannabinoid receptor 1 may be of relevance to the therapeutic mechanism of aripiprazole.

Recent clinical trials of antagonists of neurokinin 3 (NK3) receptor showed improvement of positive symptoms in schizophrenia (Meltzer et al., 2004; Spooren et al., 2005), suggesting that antagonists of NK3 might be a new class of antipsychotics and NK3 signalling might be involved in the pathophysiology of schizophrenia (Chahl, 2006). Studies have shown that there are interactions between NK3 receptors and biogenic amine release. Agonists of NK3 receptors induce the release of biogenic amine neurotransmitters such as dopamine, serotonin and norepinephrine in the brain, whereas antagonists of NK3 receptors reduce their release (Spooren et al., 2005). In this study, we found that the Tacr3 gene that encodes the neurokinin receptor 3 of rats is up-regulated by chronic administration of aripiprazole. The reason for the up-regulation needs further study, however, this finding provides a clue to suggest that the mechanism of action of aripiprazole may be of relevance to the neurokinin receptor-mediated signalling pathway.

Serine/threonine protein phosphatases play crucial roles in the intracellular signalling transduction related to synaptic plasticity (Winder and Sweatt, 2001). There are four major serine/threonine protein phosphatases, including PP1, PP2A, PP2B and PP2C. Previous studies have suggested that PP2B (calcineurin) is a susceptible gene for schizophrenia (Gerber et al., 2003; Miyakawa et al., 2003), while PP2A was reported to be part of the complex that mediates the dopaminergic neurotransmission (Beaulieu et al., 2005). Studies also showed that PP1 is one of the targets of the common signalling pathway of a variety of psychotomimetics (Svenningsson et al., 2003). In addition, a variety of antipsychotics were found to increase the activity of calcineurin, indicating that calcineurin may be one of the drug targets of antipsychotics (Rushlow et al., 2005). Taken together, these findings suggest the involvement of serine/threonine protein phosphatase in the pathophysiology of schizophrenia. PP2C family members play key roles in the regulation of cell survival and apoptosis (Tamura et al., 2006); however, there is little information about their role in neuropsychiatric disorders and psychopharmacology. In this study, we found increased gene expression of the Ppm2c gene that encodes the magnesium-dependent protein phosphatase 2C in the frontal cortex of rats under chronic administration of aripiprazole, which suggests that PP2C, like the other members of serine/threonine protein phosphatase, may also be involved in the molecular mechanism of psychoactive drugs and the pathophysiology of psychiatric disorders.

In summary, in this study, we observed that chronic treatment with aripiprazole induces differential expression of several genes in the frontal cortex. The elucidation of these genes sheds some lights on the molecular mechanism of action and helps identify several target molecules of aripiprazole. This study also demonstrates that microarray-based gene expression profiling technology is a useful tool to uncover novel molecular mechanisms of psychotropic drugs. Nevertheless, there are several limitations in this study. First, we only tested 40 genes out of 157 genes that show differential expression in microarray experiments and only 10 out of 40 genes showing differential expression in microarray studies were verified in RT-qPCR study. Hence, to obtain more insight into the molecular mechanism of aripiprazole, we need to verify other genes in the future. Second, we only investigated differentially expressed genes in the frontal cortex; it would be more informative to study the expression pattern of these genes in other brain regions. Third, we only selectively verified the differentially expressed gene Egr1 at the protein level in this study, further studies are needed to assay the other genes at the protein level to consolidate the findings in this study.

Note

Supplementary information accompanies this paper on the Journal's website.

Acknowledgements

The study was supported by grants from the National Science Council, National Bureau of Controlled Drugs, Department of Health and National Health Research Institutes, Taiwan, R. O. C.

Statement of Interest

None.

References