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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: J Neurochem. 2010 Jan 13;113(1):175–187. doi: 10.1111/j.1471-4159.2010.06585.x

Antipsychotic Induced Gene Regulation in Multiple Brain Regions

Matthew James Girgenti †, Laura K Nisenbaum ‡, Franklin Bymaster ‡, Rosemarie Terwilliger †, Ronald S Duman †, Samuel Sathyanesan Newton †,*
PMCID: PMC2921913  NIHMSID: NIHMS220366  PMID: 20070867

Abstract

The molecular mechanism of action of antipsychotic drugs is not well understood. Their complex receptor affinity profiles indicate that their action could extend beyond dopamine receptor blockade. Single gene expression studies and high-throughput gene profiling have shown the induction of genes from several molecular classes and functional categories. Using a focused microarray approach we investigated gene regulation in rat striatum, frontal cortex and hippocampus after chronic administration of haloperidol or olanzapine. Regulated genes were validated by in-situ hybridization, realtime PCR and immunohistochemistry. Only limited overlap was observed in genes regulated by haloperidol and olanzapine. Both drugs elicited maximal gene regulation in the striatum and least in the hippocampus. Striatal gene induction by haloperidol was predominantly in neurotransmitter signaling, G-protein coupled receptors and transcription factors. Olanzapine prominently induced retinoic acid and trophic factor signaling genes in the frontal cortex. The data also revealed the induction of several genes that could be targeted in future drug development efforts. The study uncovered the induction of several novel genes, including somatostatin receptors and metabotropic glutamate receptors. The results demonstrating the regulation of multiple receptors and transcription factors suggests that both typical and atypical antipsychotics could possess a complex molecular mechanism of action.

Keywords: Olanzapine, haloperidol, gene expression, microarray, drug target, striatum

Introduction

Despite significant advances made in the neurobiology and neuropharmacology of antipsychotics, the precise molecular mechanisms underlying the actions of antipsychotic drug treatment have not been identified. Typical or first generation antipsychotic drugs (APDs) such as haloperidol have been used successfully to treat schizophrenia and psychotic symptoms, but extrapyramidal side effects have been reported with treatment (Kane 2001). The advent of atypical or second generation APDs such as clozapine and olanzapine shifted focus away from haloperidol as they exhibited superior efficacy and lower incidence of adverse events. Furthermore, in contrast to lack of improvement or worsening of cognitive performance associated with haloperidol, atypicals are effective against cognitive decline and also improve (Newton & Duman 2007) function. However, the extensive Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study which compared first and second generation APDs showed that the therapeutic efficacy of both classes of APDs did not differ significantly (Lieberman et al. 2005).

Although the D2 receptor blockade appears to be central to antipsychotic effects, receptor affinity studies have shown that several additional receptors are involved in the molecular actions of APDs (Roth et al. 2004). This suggests that multiple intracellular signal transduction cascades could be involved in antipsychotic action. We hypothesize that the spectrum of therapeutic effects observed with chronic treatment, beyond those explainable by sheer D2 blockade, result from chronic modulation of intracellular signaling cascades, in part via changes in the activity of transcription factors and subsequent alterations in gene expression. Microarray analysis of gene expression in rodent models allows us to experimentally catalog antipsychotic induced gene expression in order to obtain insight into their mechanism of action. Earlier array studies showed that haloperidol and clozapine induced numerous presynaptic genes in the frontal cortex (Kontkanen et al. 2002, MacDonald et al. 2005). cDNA array analysis of haloperidol induced genes in the striatum identified synapsin II as an important target gene in antipsychotic action (Chong et al. 2002, Dyck et al. 2007). A comparison of haloperidol and risperidone revealed acute and chronic changes in neural plasticity and calcium homeostasis genes but not dopamine signaling (Feher et al. 2005). Olanzapine altered the regulation of signal transduction and metabolic pathway genes in rat frontal cortex (Fatemi et al. 2006). In-situ hybridization analysis of the Nur family of transcription factors showed that these genes were differentially regulated by typical and atypical antipsychotics and also exhibited distinct striatal expression patterns (Maheux et al. 2005). The involvement of the hippocampus in antipsychotic action has been suggested by studies demonstrating enhanced hippocampal cell proliferation after chronic administration of olanzapine (Kodama et al. 2004). Also, hippocampal expression of NMDA receptor was reduced in schizophrenic brain tissue, lending support to the hypoglutamatergic hypothesis of schizophrenia (Gao et al. 2000). Employing a focused array containing ~3000 genes pertaining to trophic factor, neurotransmitter signaling (Hunsberger et al. 2007) and transcription factors we investigated gene regulation by olanzapine and haloperidol in the striatum, cortex and hippocampus.

Methods

Animals

Male Sprague Dawley rats (170–190 gm; Harlan Sprague-Dawley, Indianapolis, IN) were housed, four per cage, under standard illumination parameters (12 hr light/dark cycle) and were given free access to water and food. Animals were acclimated for three days prior to the initiation of drug treatment. Animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Eli Lilly and Company.

Drug treatment

Rats (N=6) were injected subcutaneously with haloperidol decanoate (21 mg/kg;) (El-Mallakh et al. 2006), olanzapine pamoate (240 mg/kg; Eli Lilly) or vehicle once per week for four weeks. This depot dosing regimen was chosen based on studies in which striatal dopamine D2 receptor occupancy levels of 60–70% were achieved for both drugs across the four weeks of treatment (Barth et al. 2006) and unpublished results. This regimen attempts to simulate the constant D2 receptor blockade that is achieved in the clinic. Animals were sacrificed by rapid decapitation and the brains hemisected, one hemisphere for array analysis and the other for in-situ hybridization analysis. The frontal cortex, hippocampus and striatum were manually dissected and frozen on dry ice.

RNA isolation

RNA was isolated using the RNAqueous kit (Ambion), followed by cleanup with RNAeasy kit (Qiagen). RNA quality was verified by gel electrophoresis and realtime PCR. Independent aliquots were prepared for array hybridization and secondary validation by PCR.

In-situ hybridization (ISH)

Gene expression analysis using radiolabled riboprobes was performed as previously described (Newton et al. 2003). Briefly, riboprobes were generated by PCR amplification using gene-specific primers. Primer sequences are given in Supplement 1. The reverse primer included a T7 template sequence () on the 5′end. Whole rat brain cDNA was used as the template for PCR, which was performed in a realtime PCR instrument (SmartCycler; Cepheid, Sunnyvale, CA) using the Quantitect Sybr Green PCR kit (Qiagen). PCR product was purified by ethanol precipitation and was resuspended in TE buffer. One microgram of the 300 bp PCR product was used to produce radiolabeled riboprobe using a T7-based in vitro transcription kit (Megashortscript; Ambion). All riboprobes were verified by sequencing of the PCR product. Sections were opposed to autoradiographic film (Kodak BioMax MR film, Cat # 870 1302). ISH images were quantified using NIH Image software, and statistical analysis was performed using Microsoft Excel software.

Microarray analysis of gene expression

Array analysis was performed as previously described (Newton et al. 2003, Hunsberger et al. 2007). Briefly, a focused array containing 3000 targets (300 bp open-reading frame PCR products) pertaining to growth factor signaling, transcription factors and kinases was used. Five micrograms of total RNA from vehicle, haloperidol and olanzapine-treated rat striatum, cortex and hippocampus were reverse-transcribed into cDNA using oligo-dT primers. Arrays were hybridized overnight followed by stringent washing and then stained using fluorescent dendrimers (Genisphere Inc., Hatfield, PA). Scanned slide images were analyzed using GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA).

Statistical Analysis of microarray data

Microarray image files were subjected to statistical analysis as previously described (Newton et al. 2003, Ploski et al. 2006). Only spots with signal intensity at least twice above background were utilized for analysis. Channel intensities were normalized using intensity- dependent Lowess normalization. Up-regulated genes were defined as having an average expression ratio of >1.3, and the down-regulated genes were defined as having an average expression ratio of <0.7. Regulated targets (antipsychotic drug treatment versus vehicle treated) were filtered for genes that had a t-test P-value of 0.01 with the false discovery rate (FDR) as the multiple testing correction. GeneSpring 7.3 software (Agilent Technologies, Santa Clara, CA, USA) was used for the statistical analysis and comparison of genes regulated by antipsychotic drugs in multiple brain regions. Bioinformatics analysis was performed by Panther software (Applied Biosystems) and literature searches of gene function.

Realtime PCR

RNA (1 μg) was reverse-transcribed into cDNA using oligo dT primers and reverse-transcriptase. RNA was then hydrolyzed, cDNA precipitated (linear acrylamide, Ambion) and re-suspended in nuclease-free water. Gene specific primers were designed and tested for efficiency and specificity by serial dilutions and melt curve analysis. Sybr Green mix (Sigma) was used to amplify cDNA. Primer sequences are in Supplement 1.

Immunohistochemistry

Cryocut coronal sections (14 μm) were used for slide-based immunohistochemical (IHC) analysis of protein expression as per (Newton et al. 2002) with minor modifications. Sections were fixed in Histochoice fixative (Sigma) for 10 minutes followed by a 5 minute wash in cold 1x phosphate buffered saline (PBS). Slides were then immersed in 0.75% hydrogen peroxide (molecular biology grade, Sigma) in PBS for 5 minutes followed by two 5 minute rinses in 1x PBS. Incubations were performed on slide by demarcating sections with an ImmEdge pen (Vector Laboratories, Burlingame, CA, USA), prior to addition of blocking and antibody solutions. Sections were blocked with 5.0% BSA (w/v) in PBS (Molecular biology grade lyophilized bovine serum albumin, Sigma) at 4°C for 30 minutes. Sections were incubated with primary antibody (anti-Nur77, abcam, 1:100 dilution) in antibody solution (2.5% BSA in PBS) at 4°C overnight. Following primary antibody incubation, slides were washed in 1xPBS three times for 5 minutes each at room temperature. Slides were then incubated in biotinylated secondary antibody (1:400, Vector Laboratories) in 2.5% BSA in PBS for 2 hours at room temperature. Unbound secondary antibody was removed followed by 1 hr incubation in the avidin-biotin complex (ABC) reagent. Antigen detection was performed by diaminobenzidine (DAB) staining according to manufacturer’s instructions (Vector). Vehicle and antipsychotic treated sections were stained for the colored reaction product in parallel and used identical color development timings. Slides were dehydrated by a series of alcohol rinses and coverslipped in DPX mountant (Fluka, Buchs, Switzerland). Sections were viewed under bright field microscopy and photographed using a digital camera (Micropublisher, QImaging).

Results

Antipsychotic-induced gene regulation was investigated using a focused and optimized microarray that contained ~3000 genes representing trophic factor signaling, neurotransmitter signaling and transcription factors. Three brain regions, striatum, frontal cortex and hippocampus were examined after chronic (28 days) dosing with haloperidol or olanzapine. With both drugs, the highest number of regulated genes was in the striatum (haloperidol – 153; olanzapine – 120), followed closely by the frontal cortex (haloperidol – 140; olanzapine – 102). The hippocampus exhibited the fewest gene expression changes (haloperidol – 22; olanzapine - 27) and did not overlap with striatal or cortical profiles.

A GeneSpring comparison of genes regulated by olanzapine and haloperidol in the frontal cortex and striatum revealed limited overlap (Fig. 1). The level of overlap was similar among the regions, 17 in striatum (Fig. 1A) and 12 in frontal cortex (Fig. 1C). The genes that were induced by both APDs are shown in Fig. 1B and D.

Figure 1.

Figure 1

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Overlap of haloperidol and olanzapine induced gene regulation. Venn diagram shows the number of genes regulated by haloperidol and olanzapine in the striatum (A). Fold regulation for upregulated genes was set at 1.3 and down regulated at 0.7. Striatal genes that were regulated by both drugs are listed (B). Venn diagram of genes regulated in the frontal cortex is shown in C. Cortical genes regulated by both haloperidol and olanzapine are listed in D. CamKIIB-calcium/calmodulin-dependent protein kinase II beta, CDT6- cornea derived transcript 6 angiopoietin-like, CRHBP- corticotropin releasing hormone binding protein, Gpr-G-protein coupled receptor, OR51B2- olfactory receptor family, PDGFRA- platelet derived growth factor receptor alpha, SFMBT1- Scm-like 4 mbt domains, SIM1- single-minded 1, TMSB4X- thymosin beta 4, TRH- thyrotropin releasing hormone, VEGF- vascular endothelial growth factor, BMP2-bone morphogenetic protein 2, JUP- junction plakoglobin, LRRN3- leucine rich repeat neuronal 3, PBX1- pre-B-cell leukemia homeobox 1, TAL1- T-cell acute lymphocytic leukemia, GST A3-glutathione S-transferase A3 subunit.

Neurotransmitter signaling and G-protein coupled receptors (GPRs)

Haloperidol and olanzapine induced several neurotransmitter (NT) signaling genes in the striatum (Figs. 2 & 3). NT signaling genes were the predominant class of molecules regulated in the striatum, and involved both antipsychotics. However, the G-protein coupled receptors induced by haloperidol (Fig. 2B) were more numerous and had a greater diversity than those increased by olanzapine (Fig. 3B). Neurotensin, Nur77, corticotropin releasing hormone binding protein (CRHBP), dopamine transporter (DAT) and enkephalin were regulated in the striatum by both drugs (Figs. 2A & 3A). The striatal induction of the dopamine D2 receptor (1.8 fold) by haloperidol is noteworthy. Somatostatin receptors 2 and 5 (SSTR 2, 5) were elevated 1.7 and 1.6 fold respectively by haloperidol (Fig. 2B). Enkephalin and putative enkephalin-induced GPRs (SNSR2, SNSR5) were upregulated (2.3 and 1.7 fold respectively) by haloperidol, indicating potential amplification of enkephalin mediated signaling. Striatal acetylcholinesterase (Ache) gene expression was increased (1.5 fold) by haloperidol, implying a decrease in cholinergic function. Gria 2/GluR2 was induced (1.5 fold) in the striatum by haloperidol and similarly elevated in the hippocampus by olanzapine.

Figure 2.

Figure 2

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Neurotransmitter signaling genes induced in the striatum by haloperidol are shown (A). Bars represent level of gene regulation, mean ratio of (haloperidol:vehicle) fold change, 1 indicates no regulation. Error bars indicate SEM of six replicates. GHRH- growth hormone releasing hormone, Ccr 9- chemokine receptor 9, Grm 4 – metabotropic glutamate receptor 4, Ccr 4-chemokine receptor 4, CrhBP- corticotrophin releasing hormone binding protein, DAT – dopamine transporter, Gad 67 – glutamic acid decarboxylase, Gria 2- ionotropic glutamate receptor 2, Ache- acetylcholinesterase. G-protein coupled receptors (GPRs) induced by haloperidol is shown (B). SnsR2- putative enkephalin-induced GPR 2, Gpr – G protein coupled receptor, TRHR- thyrotropin-releasing hormone receptor, GalR2- galanin receptor 2, D2R-dopamine receptor 2, Oxer 1- oxoeicosanoid receptor 1, SnsR5- putative enkephalin-induced GPR 5, Sstr2- somatostatin receptor 2, Sstr5- somatostatin receptor 5, AR- androgen receptor, Tacr2- tachykinin receptor 2.

Figure 3.

Figure 3

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Neurotransmitter signaling genes induced in the striatum by olanzapine are shown (A). CRHBP- corticotrophin releasing hormone binding protein, S100A1- S100 calcium binding protein A1, DAT- dopamine transporter, TRH- thyrotropin releasing hormone, Cck-cholecytokinin, HRH2- histamine receptor H2, Grm 5- metabotropic glutamate receptor 5, S100A4- S100 calcium binding protein A4, CRH- corticotrophin releasing hormone, Gad 65-glutamic acid decarboxylase. G-protein coupled receptors (GPRs) induced by olanzapine is shown (B). Gpr – G protein coupled receptor, Ptgd R- Prostaglandin D receptor. Bars represent level of gene regulation, mean ratio of (olanzapine:vehicle) fold change, 1 indicates no regulation. Error bars indicate SEM of six replicates.

Transcription factors

Multiple classes of transcription factors (TFs) were induced by haloperidol and olanzapine (Fig. 4). The regulated TFs were categorized into 4 classes, Zinc finger, homeobox, basic helix-loop-helix and kruppel-associated box (KRAB). TFs that did not fall into these classes are indicated as “other”. KRAB domain TFs are a subgroup of zinc finger TFs and are therefore also included as zinc finger TFs. A full listing of the TF genes is in supplement 2. Haloperidol induced 31 TFs in the striatum and 27 in the frontal cortex, while olanzapine elevated 31 in the frontal cortex and 27 in the striatum. The regulation of multiple classes of TFs suggests that several signaling cascades are activated by these APDs and likely lead to changes in diverse gene expression programs.

Figure 4.

Figure 4

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Haloperidol and olanzapine induced transcription factors are shown classified into their DNA binding domains. Transcription factors that did not belong to major structural categories are termed “other”. The number of transcription factor induced by haloperidol or olanzapine and brain region (striatum and cortex) is shown. Length of the bars corresponds to number of transcription factors in each category. KRAB – kruppel-associated box.

Retinoic acid signaling

Several members of the retinoic acid (RA) signaling cascades were regulated by olanzapine. These genes include the retinoid X receptors, RXR β and RXR γ (Fig. 5C). RXR β was induced in both the striatum and cortex. Pre-B cell leukemia (PBX) family transcription factors, myeloid ecotropic viral integration site (MEIS2), bone morphogenetic protein (BMP) and retinoic acid inducible protein (BRINP2) are known downstream targets of the RA cascade, indicating that the RA signaling network is influenced by olanzapine. Furthermore, the elevation of BMPs (Fig. 5A) supports the known mechanism of BRINP2 induction, which occurs in response to simultaneous stimulation by RA and BMPs (Kawano et al. 2004).

Figure 5.

Figure 5

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Trophic factors and cell proliferation genes regulated by olanzapine (A) and haloperidol (B) in the frontal cortex are shown. The retinoic acid signaling cascade was prominently regulated only by olanzapine (C), and are shown corresponding to brain region, frontal cortex and striatum. Bars represent level of gene regulation, mean ratio of (olanzapine/haloperidol:vehicle) fold change, 1 indicates no regulation. Error bars indicate SEM of six replicates. NGFR-nerve growth factor receptor; BMP-bone morphogenetic protein; SCF-stem cell factor; Frzb-frizzled related protein; CNTF-ciliary neurotrophic factor; PBX-pre-b cell leukemia; MEIS-myeloid ecotropic viral integration site; RAI-retinoic acid inducible; BRINP-BMP & retinoic acid inducible; RXR-retinoic acid X receptor.

Trophic factors and Wnt signaling

Activation of the Wnt signaling cascade by olanzapine is indicated by the cortical regulation of several genes from the Wnt pathway (Wnt 7, Frizzled 1, Dishevelled 1 and 2). Trophic factors (Bone morphogenetic proteins (BMPs), Stem cell factor, Insulin-like growth factor (IGF), Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF) were regulated by both olanzapine and haloperidol while markers of cell proliferation (Notch1 and TOAD 64) were induced only by olanzapine.

Drug development candidates

The regulation of molecules that have been recently considered as prospective targets for novel antipsychotic or add-on therapies are shown in Fig. 6. Therapeutic potential of these genes is based on published papers, molecular function and prevailing hypotheses. All candidates shown were regulated in the striatum by haloperidol or olanzapine. Dopamine transporter (DAT) and neurotensin were induced by both drugs but at higher levels by olanzapine (haloperidol – 1.7, 1.3; olanzapine – 2.3, 1.8 fold respectively). The D2 receptor mRNA was increased by haloperidol (1.8 fold) but not olanzapine, and was specific to the striatum. The group I metabotropic glutamate receptor, mGluR5 was elevated (1.6 fold) by olanzapine while the group III glutamate receptor, mGluR4 was increased (1.8 fold) by haloperidol.

Figure 6.

Figure 6

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Potential drug target genes regulated by olanzapine or haloperidol. Candidate genes induced by haloperidol and olanzapine are shown in bars representing fold of regulation. Error bars indicate SEM and data are from N=6. DAT-dopamine transporter; D2R-dopamine D2 receptor; SstR5-somatostatin receptor 5; mGluR-metabotropic glutamate receptor; RXR-retinoic acid X receptor.

Secondary Validation

The results of array analysis were confirmed by independent secondary validation, employing in-situ hybridization or realtime PCR. Shown in Fig. 7 are in-situ hybridization analyses validating olanzapine (Nur77 and enkephalin) and haloperidol (D2 receptor and enkephalin) induced gene regulation. Two striatal regions (dorso-medial and ventro-lateral) were quantified and are shown in bar graphs (Fig. 7). Nurr77 exhibited a somewhat punctuate induction pattern by olanzapine (Fig. 7A) and was elevated at least 1.5 fold in both striatal regions (Fig. 7B). Enkephalin was expressed in the deep and outer cortical layers and robustly induced by olanzapine, with a 1.7 fold increase in the striatum (Fig. 7C,D). The dopamine D2 receptor was significantly elevated by haloperidol and showed a higher expression in the ventrolateral striatum. Haloperidol also induced striatal enkephalin, with a slightly stronger effect in the ventrolateral striatum than the dorsomedial region.

Figure 7.

Figure 7

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Secondary confirmation of microarray data by in-situ hybridization analysis of gene expression. Representative photomicrographs of striatal sections from in-situ hybridization using radiolabeled riboprobes are shown. Regulation of Nurr77 and enkephalin by olanzapine is shown in A and B respectively. Induction of the D2 receptor and enkephalin by haloperidol is shown in C and D. The dorsomedial striatum and ventrolateral striatum (area in circles, indicated by arrows, A) were quantified for all genes, and are shown in bar graphs on the right. Results are expressed as optical measurements from Oln (olanzapine), hal (haloperidol) or veh (vehicle) treated rats. Error bars represent SEM from N=4. (* = p ≤ 0.05).

Several regulated genes were also independently validated by realtime PCR. A list of these genes and corresponding array regulation is shown in Table 1. Protein level confirmation was performed by immunohistochemical analysis of haloperidol-induced Nur77 elevation in the striatum (Fig. 8). Nur77 was detected at low levels in the control sections but clearly discernible in haloperidol treated sections in a neuronal expression pattern.

Table 1.

Secondary confirmation of array data by realtime PCR.

Gene Name Annotation (RefSeq) qRT-PCR Regulation Array Regulation
Dopamine Transporter NM_012694.2 1.24 1.71
Neurotensin NM_001102381.1 4.10 2.58
TOAD 64 Z46882 1.29 1.71
LRRN3 NM_030856 1.30 1.83
TCF20 NM_013836 1.45 1.65
KLHL10 NM_152467 1.62 1.56
GPR56 NM_152242 1.81 1.92
EPAS 1 NM_023090.1 2.03 1.88
HES1 NM_024360.1 2.35 1.64
SOD2 NM_017051 2.49 1.50
MLLT6 NM_139311 2.60 1.64
ATP5A1 NP_004037 2.70 1.54
RXRB NM_021976 3.02 1.82
NFE2 NM_006163 3.05 1.90
JUNDP2 NM_053894.1 3.16 1.67
NGGN U31203 3.28 1.77
BRINP2 NM_173115 3.30 1.62
NCOA1 NM_010881 3.88 1.55
GPR20 NM_005293 4.15 1.69
NMDA2A NM_012573 4.53 1.68
BMP2 NM_017178 4.54 1.81
CREBBP NM_133381.1 4.74 1.71
MEIS2 NM_170675 5.58 1.61
Nur77 NM_024388 1.24 2.37
mGluR5 NM_017012 1.41 1.65
TRH AH002262 1.42 1.84
MSX2 NM_012982.2 1.69 1.81
Zif268 NM_012551 1.96 2.19
Neurotensin NM_001102381.1 4.47 2.31
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List of array regulated genes subjected to secondary validation are shown. Array and PCR fold regulation are shown in adjacent columns. Array regulation data are from N=6 and p ≤ 0.01. Quantitative PCR data are from N=4 and p ≤ 0.05.

Figure 8.

Figure 8

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Validation of Nurr 77 induction by haloperidol. Representative photomicrographs of striatal sections from immunohistochemical analysis of Nurr77 expression after haloperidol administration. Low magnification views of vehicle (left) and haloperidol (right) striatum is shown in the top panel and higher magnification view is shown in the lower panel. AC- anterior commissure, N=3.

Discussion

Our gene expression analysis of antipsychotic-induced gene regulation confirmed the results of earlier single gene studies and also identified the regulation of several novel genes that have not been previously reported.

The genes induced by haloperidol and olanzapine in multiple brain regions provide insight into their mechanism of action and also reveal candidates that can be explored individually as drug targets. It is interesting to note that the highest number of regulated genes, for both APDs, was identified in the striatum, followed by the cortex and then the hippocampus. This distribution in gene induction in the 3 regions appears to reflect the relative involvement of these brain structures in antipsychotic action. This could also suggest that the receptors and signaling molecules influenced by APDs are expressed at higher levels in the striatum and cortex rather than the hippocampus regions.

Dopamine and somatostatin receptor induction

The haloperidol-induced increase in striatal expression of D2 receptor is somewhat surprising at first as D2 blockade is centrally involved in the action of typical APDs. An increase in D2 binding after chronic haloperidol administration has been previously reported (Laruelle et al. 1992, Vasconcelos et al. 2003). This compensatory neuroleptic mediated increase in D2 receptor density has been suggested as the underlying mechanism for withdrawal associated dopamine super sensitivity and the failure of antipsychotic treatment (Samaha et al. 2007). The increase in somatostatin receptors (SSTR) is similarly intriguing as dopamine facilitates somatostatin release (Grunder et al. 1999) and increases receptor density (Rodriguez-Sanchez et al. 1997). Dopamine antagonists on the other hand inhibit somatostatin release and downregulate somatostatin receptors (Rodriguez-Sanchez et al. 1997). Although D2 and SSTR5 receptors have been shown to form hetero-oligomers that exhibit higher functional activity in reducing cAMP, the hetero-oligomerization is understood to be ligand initiated, by either somatostatin or dopamine (Rocheville et al. 2000). Two potential mechanisms could cause the observed increase in D2 and SSTR5 mRNA; 1) the elevation is caused by higher levels of dopamine in particular subcellular compartments (Fuchs & Hauber 2004) or 2) an elevation in D2 density caused a parallel increase in SSTR5, implying a coupling relationship between the two receptors. Further work is necessary to know if functional hetero-oligomers are formed as a result of APD administration as the association can be involved in the psychomotor side effects of neuroleptic treatment (Rocheville et al. 2000). It is however possible that somatostatin signaling is independently regulated by antipsychotics and could have potential therapeutic applicability as it was recently shown that somatostatin mRNA is decreased in postmortem schizophrenic brain (Morris et al. 2008).

Retinoic acid signaling

Several genes pertaining to the retinoic acid (RA) signaling cascade (RXR-β, RXR-γ, BRINP2, MEIS, PBX, RAI1) are induced by olanzapine in the frontal cortex and striatum. Active RA signaling by olanzapine is also indicated by the increase in BMP/RA-inducible neural-specific protein-2 (BRINP2), which is induced jointly by bone morphogenetic protein (BMP) and RA (Kawano et al. 2004). This is an interesting result for at least two reasons; 1) RA signaling is now emerging as an important component of neural plasticity in the adult brain, with hyposignaling contributing to aging-induced memory deficits (Etchamendy et al. 2001, Mingaud et al. 2008), 2) RA signaling intertwines with dopamine signaling and strongly influences neuronal adaptation (Levesque & Rouillard 2007). Furthermore, docosohexaenoic acid (DHA) which reduces haloperidol-induced dyskinesia (Ethier et al. 2004b) was demonstrated to be a retinoid X receptor ligand (de Urquiza et al. 2000). These and other additional lines of evidence (Bremner & McCaffery 2008) make a compelling case to further investigate RA in the context of psychiatric disorders.

The regulation of retinoid receptors by antipsychotics has been reported earlier by in-situ hybridization (Ethier et al. 2004a). There are 3 known retinoid X receptor (RXR) isoforms, α, β and γ. RXR-β and RXR- γ were regulated by olanzapine. Significant body of previous work has shown that the Nur family of transcription factors heterodimerize with retinoid receptors and modulate dopamine neurotransmission (Levesque & Rouillard 2007). Interestingly, the beneficial effects of DHA on haloperidol- induced dyskinesia is lost in Nur77 null mice (Ethier et al. 2004b), despite DHA being a ligand for RXR. A similar relationship between Nur77 and RXR was also observed in amphetamine-induced locomotor activity, where RXR anatagonists blocked ambulatory activity but the effect was absent in Nurr77 deficient mice (Bourhis et al. 2009). This suggests that Nur77 actively regulates RXR signaling. Transgenic mice deficient for retinoid receptors exhibit impaired locomotion and dopamine signaling, including reduced expression of dopamine receptors (Krezel et al. 1998). While a precise understanding of the relationship between retinoid and dopamine signaling is yet to emerge it is likely that the presence of functional RARE (retinoic acid response element, promoter sequence occupied by retinoid receptors) in the D2 receptor regulatory region plays an important role in the modulation of dopamine pathways by RXR (Samad et al. 1997). The elevation of Nur77 and RXR genes in olanzapine striatum and the responsiveness of neurotransmitter pathways to retinoid signaling provide the opportunity to mechanistically test the role of retinoid and dopamine pathways in the actions of an atypical antipsychotic.

Drug development targets

Although improvements have been made in antipsychotic drug development, the incidence of adverse events and metabolic side effects make it necessary to focus towards developing safe and efficacious agents to treat schizophrenia. Lack of clear understanding of disease pathophysiology and mechanism of action of APDs makes this challenging. However, candidate drug targets can be identified from testing available APDs against evidence based hypotheses. The striatal upregulation of mGluR5 by olanzapine emerges as an attractive choice given the hypoglutamatergic hypothesis of schizophrenia (Coyle 2006) and the recent success of mGluR 2/3 agonists in clinical trials (Patil et al. 2007). The potential of mGluR5 as an antipsychotic drug target has already been demonstrated by 2 positive modulator studies (Kinney et al. 2005, Liu et al. 2008), reporting antipsychotic efficacy. Rodent studies using mGluR5 agonists have produced promising results using animal behavioral models (Kinney et al. 2003, Kinney et al. 2005). The induction of mGluR4 by haloperidol is intriguing as it has been known to regulate striatal neurotransmission and has led to successful testing of allosteric modulators of mGluR4 in the reversal of haloperidol-induced cataplexy and treatment of Parkinson’s disease (Niswender et al. 2008).

Neurotensin has long been implicated in schizophrenia pathophysiology and mechanism of antipsychotic action (Gariano & Groves 1989, Binder et al. 2001). It has been suggested as a mediator of antipsychotic effects due to its effective modulation of dopamine neurotransmission and neurotensin receptor agonists are being tested as therapeutic agents to treat schizophrenia (Boules et al. 2005). The striatal induction of neurotensin by both haloperidol and olanzapine confirms the results of previous single gene studies that reported enhanced neurotensin mRNA and peptide release in response to typical and atypical APDs (Binder et al. 2001). It is interesting to note that prepulse inhibition deficits in neurotensin null mice are not reversed by haloperidol or olanzapine, indicating that the elevation of neurotensin by APDs is significantly involved in their action (Kinkead et al. 2005). The biochemical and behavioral similarity between the actions of neurotensin and antipsychotics is striking (Kinkead & Nemeroff 2004, Caceda et al. 2005) and is the basis for targeting the neurotensin system for novel antipsychotic efficacy (Kinkead & Nemeroff 2006).

The dopamine transporter (DAT) has been investigated as a drug target for the treatment of attention deficit/hyperactivity disorder (ADHD) as DAT is strongly implicated in ADHD disease mechanisms (Madras et al. 2005). However, given the importance of dopaminergic tone to schizophrenia, it seems likely that the functional role of DAT in controlling dopamine clearance could be relevant to antipsychotic action. Decreased DAT expression has been reported in schizophrenics (Laakso et al. 2001) while DAT knockdown mice are hyperdopaminergic and hyperactive when presented with novel stimuli (Zhuang et al. 2001). It was recently shown that DAT is regulated by the D2 receptor via a direct physical interaction (Lee et al. 2007), indicating this novel coupling mechanism could significantly influence dopamine neurotransmission. As DAT is elevated by both haloperidol and olanzapine it appears that even modest blockade of D2 can influence DAT function. An elevation in DAT could serve to normalize dopaminergic tone in cases where schizophrenia is due to hyperdopaminergic neurotransmission. While the precise contribution of DAT to antipsychotic action must await further experimentation, it is likely that APD-induced DAT is protective against dopamine spill over. Manipulating DAT expression levels could serve as another potential mechanism by which dopaminergic tone can be modulated.

Trophic factor and Wnt signaling

The olanzapine-induced elevation of several trophic signaling molecules involved in cell proliferation likely reflects non-neuronal cell proliferation that has been reported with chronic administration paradigms (Kodama et al. 2004, Newton & Duman 2007). The induction of these genes could be protective against the cortical cell loss that frequently accompanies schizophrenia progression. As adult neurogenesis has not been shown to occur in the cortex, it is likely that these changes occur in non-neuronal cell types. Indeed, earlier work has reported that olanzapine increases the proliferation of non-neuronal cells in the frontal cortex (Kodama et al. 2004). As trophic factors are effective mediators of plasticity in the brain, their cortical elevation could be potentially involved in reversing or protecting against compromised structural plasticity and impaired cellular resilience in schizophrenia (Palfi et al. 2002, Manji et al. 2003).

Multitude of receptors and mechanisms

It is important to point out that the induction of several neurotransmitters, receptors and transcription factors resonate with the findings of Roth et al (Roth et al. 2004), highlighting the receptor promiscuity of first and second generation antipsychotics. The challenge in obtaining valuable mechanistic insight from gene profiles could lie partly with how these results are interpreted. Rather than subscribing to the viewpoint that drugs with complex pharmacology are better than those with high selectivity (Roth et al. 2004) we feel it is useful to consider the regulated genes in the light of “hypothesis confirming” and “hypothesis generating” data. This will enable us to sift through this rich profile in a meaningful and productive manner, while providing the framework for future testing in preclinical models. The induction of histamine receptor, leptin, IGF and ghrelin points to emergent metabolic events, but could also be involved in therapeutic outcome (Girgis et al. 2008). Since the goal in the field is to develop drugs that result in a more favorable safety and efficacy profile, being able to suppress the adverse phenomena in existing drugs are just as welcome as completely novel candidates. Therefore identifying the source and mechanism of adverse events can also be a promising avenue of research. The gene profiles of olanzapine and haloperidol provide insight into their mechanism of action and also suggest gene targets that require further testing at the level of protein activation and pharmacological functionality. Progress in developing or identifying pharmacologic agents that can activate these targets with high specificity will enable us to address whether targeted drug design can provide superior results to currently available antipsychotics.

Supplementary Material

Supp Table S1
Supp Table S2

Acknowledgments

This work was supported by funding from the Connecticut Mental Health Center and the Department of Mental Health and Addiction Services, National Institute of Health Grants NS051869-01, MH078132 (SSN). We thank Monica Sathyanesan for design of PCR primers and probes.

Footnotes

Disclosure/Conflicts of interest

SSN and RSD declare that this work was funded in part by Lilly. RSD has also received compensation from Lilly, the manufacturer of olanzapine, for speaking on antidepressant related research. LKN is a full-time employee of Eli Lilly and Company, the manufacturer of olanzapine. FB was an employee of Eli Lilly and Company during some of the time these studies were conducted.

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Supplementary Materials

Supp Table S1
NIHMS220366-supplement-Supp_Table_S1.xls (39.5KB, xls)
Supp Table S2
NIHMS220366-supplement-Supp_Table_S2.xls (23.5KB, xls)

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