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FFECTS OF SHORT-TERM AND CHRONIC OLANZAPINE TREATMENTN IMMEDIATE EARLY GENE PROTEIN AND TYROSINEYDROXYLASE IMMUNOREACTIVITY IN THE RAT LOCUS
OERULEUS AND MEDIAL PREFRONTAL CORTEX
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. VERMA,a K. RASMUSSENb AND G. S. DAWEa*

Department of Pharmacology, Yong Loo Lin School of Medicine,ational University of Singapore, Building MD2, 18 Medical Drive,ingapore 117597

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN6285, USA

bstract—Atypical antipsychotic drugs, such as olanzapine,ave been reported to activate the locus coeruleus (LC) and

ead to acute expression of the Fos-like immediate early geneIEG) protein in the LC and medial prefrontal cortex (mPFC).timuli that activate the LC have been reported to increasexpression of tyrosine hydroxylase (TH), the rate-limiting en-yme in catecholamine synthesis. However, the effects ofhronic treatment with olanzapine on IEG expression and theose-dependence of the effects of olanzapine on IEG and THxpression are not known. Thus, we examined Fos-like,-Jun, activating transcription factor 2 (ATF-2), early growthesponse 1 (Egr-1), early growth response 2 (Egr-2), and THmmunoreactivity expression in the LC and mPFC in ratseceiving 2, 4, 8, or 15 mg/kg/day olanzapine by s.c. osmoticinipump for 4 h, 1 week, 2 weeks, or 4 weeks. ATF-2 expres-

ion was up-regulated at all treatment durations, while Egr-1nd Egr-2 were down-regulated in both the LC and mPFC.os-like expression was up-regulated through 2 weeks, butot 4 weeks, in both the LC and mPFC. C-Jun expression wasp-regulated for 4 weeks in the LC and for 2 weeks, but not 4eeks, in the mPFC. At all doses, there were rapid and sus-

ained increases in TH immunoreactivity in the LC, but onlyelayed increases in the mPFC. These data indicate thatlanzapine has rapid effects on IEG in the LC and mPFC,any of which are sustained through four weeks of treat-ent. Further, these data indicate that the delayed increase inH expression in the mPFC parallels, and may play an impor-ant role in, the increased efficacy of olanzapine that emergesver time in humans. © 2006 IBRO. Published by Elsevier Ltd.ll rights reserved.

ey words: antipsychotic agents, genes, immediate-early,yrosine 3-monooxygenase, prefrontal cortex, locus coer-leus.

Corresponding author. Tel: �65-6516-8864; fax: �65-6873-7690.-mail address: [email protected] (G. S. Dawe).bbreviations: ANOVA, analysis of variance; ATF-2, activating tran-cription factor 2; Egr-1, early growth response 1; Egr-2, early growthesponse 2; ELISA, enzyme-linked immunosorbent assay; HSD, hon-stly significantly different; IEG, immediate early gene; LC, locusoeruleus; mPFC, medial prefrontal cortex; �-NADH, �-nicotinamide

wdenine dinucleotide; PBS, phosphate-buffered saline; TH, tyrosineydroxylase.

306-4522/06$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.08.010

573

typical antipsychotic drugs not only produce less extra-yramidal side effects than typical antipsychotics but alsohow better efficacy against the negative and cognitiveymptoms of schizophrenia (Meltzer and McGurk, 1999;andon and Jibson, 2003; Kasper and Resinger, 2003). Inats, acute treatment with the atypical antipsychotic, olan-apine, has been reported to increase expression of the-Fos immediate early gene (IEG) protein in the medialrefrontal cortex (mPFC) (Robertson and Fibiger, 1996;hashi et al., 2000), and locus coeruleus (LC) (Ohashi etl., 2000; Dawe et al., 2001). The activation of c-Fosxpression in the prefrontal cortex, in particular in therelimbic area of the mPFC, the region of the rodent pre-rontal cortex most functionally analogous to the humanorsolateral prefrontal cortex (Otani, 2003; Dalley et al.,004), has been proposed as a marker of atypical antipsy-hotic activity (Robertson and Fibiger, 1992, 1996; Rob-rtson et al., 1994; Deutch and Duman, 1996; Ananth etl., 2001). As the prefrontal cortex is involved in workingemory and executive function (Robbins, 1996; Goldman-akic, 1996; Callicott et al., 1999; Dalley et al., 2004) and

s well characterized as a site of abnormal brain function inchizophrenia (Goldman-Rakic and Selemon, 1997; Calli-ott and Weinberger, 1999; Goldman-Rakic, 1999; Bunneynd Bunney, 2000; Weinberger et al., 2001), it may be thathe greater ability of atypical antipsychotics to activateos-like immunoreactivity in the prefrontal cortex relates to

heir greater efficacy against the cognitive deficits inchizophrenia.

Atypical antipsychotics, including olanzapine, increaseelease of noradrenaline in the prefrontal cortex (Westerinkt al., 1998; Li et al., 1998). Stimulation of the LC increases-Fos expression in the cortex by a �-adrenoceptor-depen-ent mechanism (Bing et al., 1991, 1992; Stone andhang, 1995; Stone et al., 1995). The increased expres-ion of Fos-like immunoreactivity in the prefrontal cortex byoth clozapine and olanzapine appears to be mediated by-adrenoceptors (Ohashi et al., 2000). This suggests thattypical antipsychotic activation of c-Fos expression in theC and prefrontal cortex may be functionally linked. Theresent study explored the relationship between IEG ex-ression in the LC and prefrontal cortex.

Expression of c-Fos immunoreactivity has been con-idered a marker of neuronal activation (Sagar et al., 1988;ragunow and Faull, 1989; Morgan and Curran, 1991).onsistent with this, acute administration of olanzapine
as also shown to increase the activity of LC cells (Dawe
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V. Verma et al. / Neuroscience 143 (2006) 573–585574

t al., 2001; Seager et al., 2004). However, while doses oflanzapine as low as 0.3 mg/kg increased the firing rate ofC cells (Seager et al., 2004), the effects of doses oflanzapine lower than 5 mg/kg on Fos-like immunoreac-ivity in the LC and prefrontal cortex are not known and arenvestigated in the present study.

Recently, it was shown that chronic treatment with aigh dose of olanzapine (10 mg/kg/day), but not a low dosef olanzapine (1 mg/kg/day), for 3 weeks still increased thering rate and burst firing of LC cells (Seager et al., 2005).n the prefrontal cortex, although acute or sub-chronicdministration of olanzapine did not change neuronal firingates, chronic treatment for 2 weeks with a high dose oflanzapine (20 mg/kg per day, s.c.) increased neuronalring rates (Gronier and Rasmussen, 2003). Thus, if IEGxpression is a marker of neuronal activation, it would bexpected that chronic treatment with olanzapine shouldesult in expression of IEGs in the LC and mPFC. Theffects of chronic antipsychotic drug treatments on IEGxpression have been little explored. However, one study

nvestigating cross-tolerance with haloperidol and cloza-ine, reported a reduction in c-Fos expression in the fore-rain, including the prefrontal cortex, in a group of ratsreated with 5 mg/kg/day olanzapine for 3 weeks and sub-equently challenged with 5 mg/kg olanzapine (Sebens etl., 1998). The effects of chronic treatment with olanzapinen IEG expression in the LC and the effects of other dosesnd durations of antipsychotic treatment on IEG expres-ion in the mPFC are not known. In the present study, wenvestigated the effects of various doses and durations ofreatment on IEG expression in both the LC and mPFC.

Stimuli that activate the LC and produce sustainedelease of noradrenaline (e.g. immobilization stress andhronic nicotine administration), up-regulate expression ofyrosine hydroxylase (TH), the rate-limiting enzyme in theatecholamine synthesis pathway (Smith et al., 1991a;itchell et al., 1993; Kvetnansky and Sabban, 1998; Se-

ova et al., 1999; Sabban et al., 2004; Sun et al., 2004).imilarly, chronic treatment with high doses of olanzapineas recently been reported to up-regulate TH expression inhe LC (Ordway and Szebeni, 2004). As studies on nico-ine-induced TH expression found that increased expres-ion in the LC was followed by time-dependent transport tohe terminal fields of the LC projections associated withelayed increases in noradrenaline release (Mitchell et al.,993), we sought to investigate whether increases in THxpression in the mPFC followed the reported increases inhe LC.

Previously, studies of the effects of olanzapine on IEGxpression in the LC have been focused on Fos-like im-unoreactivity. However, a range of IEGs has been sug-ested to act as transcription factors in pathways regulat-

ng the expression of TH including, the AP-1 complex Fosamily proteins and the AP-1 complex Jun family proteins,ctivating transcription factor 2 protein (ATF-2), and earlyrowth response 1 protein (Egr-1) (Guo et al., 1998; Su-uki et al., 2002; Sabban et al., 2004). We therefore inves-igated the effects of short-term and chronic treatment with
lanzapine over a range of doses and treatment durations o
n expression of immunoreactivity for a range of IEGsATF-2, Fos-like, c-Jun, Egr-1, and early growth response(Egr-2)) and TH in the LC and mPFC of rats.

EXPERIMENTAL PROCEDURES

nimals

dult male Sprague–Dawley rats (180–200 g) were obtained fromhe Laboratory Animals Centre, National University of Singapore,ingapore. They were group-housed with free access to food andater. A 12-h light/dark cycle was maintained. All efforts wereade to minimize the number of animals used and their suffering.ll experiments were approved by the institutional animal ethics

eview board of the National University of Singapore and wereonducted in accordance with the International Guiding Principlesor Animal Research (Howard-Jones, 1985).

rugs

lanzapine (Eli Lilly and Company, Indianapolis, IN, USA) wasissolved in distilled water acidified to pH 5.5 by application of 1 MCl and adjusted back to pH 6.0 with 1 M NaOH. The vehicleontrol was saline (0.9% NaCl in distilled water) acidified to pH 6.0y application of 1 M HCl.

Chronic dosing was administered by minipump in order toetter mimic receptor occupancy during clinical dosing by avoiding

nappropriately low trough occupancies (Kapur et al., 2003). Ourose of 8 mg/kg/day olanzapine administered by minipump is

ikely to best approximate to clinically comparable receptor occu-ancy (Kapur et al., 2003). To confirm the effects of short-termlanzapine treatment on TH expression in the LC, acute injectionsf 4 mg/kg olanzapine (s.c.) and acidified saline (n�8 rats perroup) were also made.

mplantation of osmotic minipumps

ats were anesthetized with sevoflurane (8% for induction and–4% for maintenance) and osmotic minipumps (Alzet ModelML2 or 2ML4, Durect Corporation, Cupertino, CA, USA) were

mplanted s.c. The rats (n�80) received acidified saline as aehicle control, 2, 4, 8, or 15 mg/kg/day of olanzapine for durationsf either 4 h, 1 week, 2 weeks, or 4 weeks (n�4 rats for each doset each treatment duration).

erfusion fixation and tissue processing

t the end of the treatment durations, the rats were transcardiallyerfused with 0.9% saline followed by 4% paraformaldehyde inhosphate buffer (pH�7.4) on anesthetic overdose with sodiumentobarbital. The brains were recovered, postfixed for 2–3 days,

mm coronal blocks were embedded in paraffin wax (LeicaP1020, Leica Microsystems, Wetzlar, Germany) and 6 �m serialections were cut on a rotary microtome (Leitz 1512, Leica Micro-ystems) and mounted on slides.

mmunocytochemistry

equential sections were immunostained with rabbit polyclonalntibodies to IEGs and TH. The paraffin was removed with xylenend the sections were rehydrated through ethanol. Endogenouseroxidase was quenched by incubating the sections in 0.3%2O2 for 30 min. The sections were washed thrice in distilledater, washed in phosphate-buffered saline (PBS), and incubatedith normal blocking serum (rabbit ABC staining system, Santaruz Biotechnology, Santa Cruz, CA, USA) in PBS for 1 h at room

emperature (approximately 22 °C). The sections were then incu-ated with the primary antibodies. The primary antibody was
mitted in negative controls. After a wash in PBS, the sections

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V. Verma et al. / Neuroscience 143 (2006) 573–585 575

ere then incubated with a biotinylated anti-rabbit secondary an-ibody for 30 min, washed thrice in PBS, incubated with an avidin-iotinylated–horseradish peroxidase (HRP) complex, and the im-unoreactivity was visualized by development with the diamino-enzidine (DAB) chromogen (rabbit ABC staining system, Santaruz Biotechnology). The slides were then dehydrated throughthanol, cleared in xylene, and coverslipped.

The primary antibodies were initially titrated from 1:50–1:1000ith incubations of 12–72 h, both at room temperature and in a

efrigerator at 4 °C, and with and without a pepsin antigen recoverytep. All the primary antibodies against IEG proteins were from Santaruz Biotechnology: anti-c-Fos (sc-52), anti-c-Jun (sc-1694), anti-TF-2 (sc-187), anti-Egr-1 (sc-189) and anti-Egr-1 (sc-190). The

ollowing incubation protocols were adopted: anti-c-Fos (1:100, 72 ht room temperature), anti-c-Jun (1:100, 48 h at 4 °C), anti-ATF-21:150, 16 h at 4 °C), anti-Egr-1 (1:100, 48 h at room temperature),nd anti-Egr-2 (1:50, 24 h at 4 °C). Sections were also stained withabbit anti-TH (1:150, 24 h at room temperature; AB 152, Chemiconnternational, Temecula, CA, USA).

mage analysis

mmunoreactive nuclei were counted with minor modifications torocedures previously described (Mize, 1994; Nagy et al., 1998;taiger et al., 2000; Dawe et al., 2001). Briefly, images of00�400 �m areas of the prelimbic area of the mPFC at 2.7 mmnterior to bregma and the LC at 9.8 mm posterior to bregmaPaxinos and Watson, 1997) were captured using a light microscopeBX51, Olympus, Tokyo, Japan) and a digital camera (Magnafire SP,ptronics, Goleta, CA, USA) as illustrated in Fig. 1. The immunore-ctive nuclei in the region of interest were detected by binary seg-entation to a fixed threshold and application of a binary dilation–rosion filter to remove artifacts (Image Pro Plus, Media Cyberneticsnc., Silver Spring, MD, USA). The segmentation threshold was fixed

ig. 1. The brain regions within which IEG immunoreactive nuclei andf the mPFC in the rat brain. The areas sampled are denoted by blackf immunostaining for (c) Fos-like protein and (d) c-Jun in the mPFC fo
nd (g) Egr-2 in the mPFC, (h) ATF-2 in the LC, and TH in the (i) LC and (j) mPFars�100 �m (c) to (g), (h) 150 �m, and (i) and (j) 10 �m.
or each antibody across all samples. Immunoreactive nuclei (Fig.c–h) were counted with the application of fixed object size andoundness filters. For quantification of TH immunoreactivity, the ob-ect size and roundness (round to linear) parameters were adjustedo count immunoreactive profiles of cellular elements in the LC (Fig.i) and fiber profiles in the mPFC (Fig. 1j). For both mPFC and LC,our regions of interest were sampled bilaterally from two sections2 �m apart in each brain and the mean number of immunoreactiveuclei or profiles per �m2 was calculated. The data are expressed as

he mean percentage change in the number of immunoreactiveuclei or profiles relative to the pooled mean count for the vehicle-reated control groups (mean�std). Data are likewise expressed ashe mean percentage relative to the count for the 4 h post vehicleontrol group (mean�std).

issue preparation for assay of TH

ats received acute s.c. injections of 4 mg/kg olanzapine orcidified saline (n�8 per group). Four hours after injection, theats were killed by overdose of sodium pentobarbitone�200 mg/kg i.p.) followed by cervical dislocation and decapita-ion. The brain was rapidly removed on ice and placed in a brainatrix, in which a 1 mm thick slice between bregma �9.30 mmnd bregma �10.30 mm (Paxinos and Watson, 1997) was takenetween glass coverslips. The cerebellum was removed and theemaining brainstem tissue containing the LC was homogenized,rozen in liquid nitrogen and stored in a �80 °C freezer until assay.ll tissue was assayed within 2 weeks of sampling.

ndirect antibody enzyme-linked immunosorbentssay (ELISA) for TH

ndirect antibody ELISA for TH protein was performed accordingo the manufacturer’s instructions with an alkaline phosphatase

unoreactive profiles were counted in the (a) LC and (b) prelimbic area. Drawings are adapted from Paxinos and Watson (1997). Examples

eatment with 4 mg/kg/day olanzapine for 2 weeks; (e) ATF-2, (f) Egr-1

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LISA kit (55-81-50 Protein Detector ELISA Kit, AP Blue Phosystem, Kierkegaard & Perry Laboratories, Inc., Gaithersburg,D, USA). All incubations were performed at room temperatureith continuous gentle orbital shaking. Briefly, the antigen waspplied by diluting tissue sample in the coating buffer provided inhe kit and incubating 100 �l of the resulting coating solution in theells of a 96 well optical bottom plate (NUNC, Roskilde, Denmark)

or 1 h. The plate was tapped out and 300 �l per well of the bovineerum albumin blocking solution provided in the kit was applied for5 min. The plate was tapped out and 100 �l of mouse monoclo-al primary antibody to TH (MAB318, Chemicon International)iluted in the blocking solution was applied and incubated for 1 h.egative control wells received blocking solution without primaryntibody. During assay optimization, the primary antibody wasitrated at dilutions from 1:100–1:10,000. A dilution of 1:1000 waselected for the assay. The plate was tapped out and all the wellsere washed three times with the wash solution provided in theit. The phosphatase conjugated goat anti-mouse IgG (H�L) sec-ndary antibody provided in the kit was diluted 1:500 (0.2 �g/ml)

n the blocking solution and applied for 1 h. The plate was tappedut and washed three times with the wash solution; 100 �l of theubstrate solution provided in the kit was applied to each well formin and then 100 �l stop solution was applied to each well.

bsorbance at 630 nm was read (Infinite M200, Tecan, Durham,C, USA) within 10 min of applying the stop solution.

Optimization of the assay compared tissue sample dilutionactors of 1:1, 1:10, 1:100, 1:1000, and 1:10,000. As the assayas linear for dilutions between 1:10 and 1:1000, the assay waserformed by linear fitting to the dilutions in this range to estimatehe expect absorbance for the undiluted sample. The assay waserformed on three independent replicates of each sample. Inach replicate, samples from all vehicle control- and olanzapine-reated rats were compared within the same plate. On each plate,our blank background controls were treated with coating solutionithout tissue sample and four negative control wells for eachample dilution (1:10, 1:100, and 1:1000) received no primaryntibody. The absorbance at 630 nm was corrected for the totalrotein content of the samples, measured as described below,nd expressed as absorbance at 630 nm/mg of total protein.

nzymatic assay for TH

nzymatic assay for TH was performed according to a modifica-ion of the procedures of Kaufman and colleagues (Shiman et al.,971; Craine et al., 1972); 150 �l of 100 mM Tris–HCl buffer,djusted to pH 7.0 at 37 °C with 1 M HCl; 6 �l of 15 mM L-tyrosine,djusted to pH 7.0 at 37 °C with 1 M NaOH; 5 �l of 10 mM,7-dimethyl-5,6,7,8-tetrahydropterine (DMTHP); 100 �l of.43 mM �-nicotinamide adenine dinucleotide (�-NADH), reducedorm; 5 �l of 10,000 units/ml of catalase in cold 100 mM Tris–HCluffer adjusted to pH 7.0 at 37 °C with 1 M HCl; and 10 �l of 5nits/ml of dihydropteridine reductase enzyme in cold 100 mMris–HCl buffer adjusted to pH 7.0 at 37 °C with 1 M HCl. Allhemicals and reagents were purchased from Sigma (St. Louis,O, USA) and the solutions were prepared fresh immediatelyefore use. The plate was immediately orbitally shaken for 1 s and

ncubated to 37�0.5 °C. Absorbance at 340 nm was monitorednce per minute followed by orbital shaking for 1 s (Infinite M200,ecan) until the absorbance had stabilized (approximately 5 min);0 �l of homogenized tissue samples diluted 1:1, 1:10, 1:100,:1000 and 1:10,000 by volume with 100 mM Tris buffer adjusted

o pH 7.0 at 37 °C with 1 M HCl was added to the sample assayells. Deionized water (30 �l) was added to four blank wells perlate. Each sample was assayed in three independent replicatesnd in each replicate samples from all vehicle control- and olan-apine-treated rats were compared within the same plate. Thelate was shaken again for 1 s and the cycles of absorbanceeading and shaking continued for 10 min. The rate of change of
he absorbance at 340 nm corrected for the absorbance of the i
lanks was calculated for each dilution. For each sample, theilution closest to the linear phase of the curve of rate of changef absorbance plotted against dilution was selected for calculationf enzymatic activity. Enzymatic activity was calculated as: en-yme units/ml�(� absorbance at 340 nm/min of the test sam-le�� absorbance at 340 nm/min of the blanks)�0.306 ml assayolume�dilution factor�1000 conversion factor from micromoleso nanomoles�6.22 millimolar extinction coefficient of �-NADH at40 nm�0.3 ml volume of enzyme used. One unit will form.0 nmol of L-DOPA from tyrosine per minute at pH 7.0 at 37 °C.he enzyme activity was corrected for the total protein content of

he samples, measured as described below, and expressed asnits/mg of total protein.

easurement of total protein

rotein determination was performed on the tissue samples as-ayed for TH by ELISA and enzymatic assay by 96-well platepectrophotometric assay with a total protein kit (TP0100, Totalrotein Kit, Micro, Sigma) based on the Bradford assay (Bradford,976); 250 �l of the protein dye reagent (Brilliant Blue G, 0.35g/ml, in phosphoric acid and methanol) was added to each well.he tissue samples and the protein standard (0.3 mg/ml humanlbumin) were diluted 1:1, 1:10, 1:100, 1:1000 and 1:10,000 byolume in 0.85% NaCl and 5 �l was added per well; 5 �l of 0.85%aCl was added to four blank wells per plate. Each sample wasssayed in three independent replicates and in each replicateamples from all vehicle control- and olanzapine-treated rats wereompared within the same plate. Measurements were made be-ween 5 and 10 min of applying the protein. The plate was orbitallyhaken for 1 s, allowed to settle for 60 s, and absorbance waseasured at 595 nm (Infinite M200, Tecan) and corrected for theverage absorbance of the blanks. For each sample, the proteinoncentration was calculated from the test sample dilution closesto the linear phase of the curve of absorbance plotted againstilution factor as: protein concentration (mg/ml)�absorbance ofhe test sample at 595 nm�concentration of the standard/absor-ance of the dilution standard at 595 nm.

tatistical analysis

he IEG and TH immunoreactivity data were analyzed by two-waynalysis of variance (ANOVA) for between-subjects effects ofreatment duration and dose followed by post hoc analysis withukey’s honestly significantly different (HSD) test. The effects ofcute administration of olanzapine and the acidified saline vehicleontrol on TH were compared by two-tailed criteria with Student’s-test for two-samples. An alpha level of 0.05 was applied.

RESULTS

os-like immunoreactivity

ontrol levels of Fos-like immunoreactivity did not signifi-antly differ across the different time points in both the LCone-way ANOVA, F3,12�2.28, n.s.) and the mPFC (one-ay ANOVA, F3,12�1.01, n.s.). Two-way ANOVA of Fos-

ike immunoreactivity revealed significant main effects ofose and duration of olanzapine treatment, as well as an

nteraction of these two factors, in both the LC (dose,

4,60�68.0, P�0.0001; duration, F3,60�465, P�0.0001;ose�duration interaction, F12,60�43.2, P�0.0001) andPFC (dose, F4,60�19.45, P�0.0001; duration, F3,60�2.5, P�0.0001; dose�duration interaction, F12,60�.54, P�0.0001). Post hoc Tukey HSD tests confirmedhat in both LC and mPFC there was a dose-dependent
ncrease in Fos-like immunoreactivity for 4-hour, 1-week,

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nd 2-week durations of treatment (Fig. 2). In the LC, thencrease in Fos-like immunoreactivity induced by the low-st dose, 2 mg/kg/day, did not reach significance until-week treatment. However, on 4-week treatment, Fos-like

mmunoreactivity returned to control levels in the mPFCnd a down regulation of Fos-like immunoreactivity wasbserved in the LC (Fig. 2).

-Jun immunoreactivity

ontrol levels of c-Jun immunoreactivity did not signifi-antly differ across the different time points in both the LCone-way ANOVA, F3,12�1.07, n.s.) and the mPFC (one-ay ANOVA, F3,12�2.86, n.s.). Two-way ANOVA of c-Jun

mmunoreactivity revealed significant main effects of dosend duration of olanzapine treatment, as well as an inter-ction of these two factors, in both the LC (dose,

4,60�122, P�0.0001; duration, F3,60�15.2, P�0.0001;ose�duration interaction, F12,60�7.89, P�0.0001) andhe mPFC (dose, F4,60�649, P�0.0001; duration, F3,60�87, P�0.0001; dose�duration interaction, F12,60�112,�0.0001). Post hoc Tukey HSD tests confirmed that inoth LC and mPFC there was a dose-dependent increase

n c-Jun immunoreactivity for 4 h, 1 week, and 2 weekurations of treatment (Fig. 3). For the lowest dose, 2 mg/g/day, consistent increases in the number of c-Jun immu-

ig. 2. Effects of olanzapine dose and treatment duration on Fos-likemmunoreactivity in (a) the LC and (b) the mPFC. Fos-like immunore-ctivity is expressed as the number of Fos-like immunopositive nucleis a percentage of the pooled mean of the acidified saline vehicleontrol groups (mean�SEM). Following two-way ANOVA, effects oflanzapine dose were compared with the acidified saline controlsithin each treatment duration by Tukey’s HSD post hoc tests

* P�0.05, *** P�0.0005).

oreactive cells were only detected after 2-week treat-t*

ent. The pattern of dose-dependent increases in c-Junmmunoreactivity continued with 4-week treatment in theC, but in the mPFC on 4-week treatment a down-regula-ion of c-Jun immunoreactivity was observed which signif-cantly reduced immunoreactivity below the control levelor the highest dose, 15 mg/kg/day (Fig. 3).

TF-2 immunoreactivity

ontrol levels of ATF-2 immunoreactivity did not signifi-antly differ across the different time points in both the LCone-way ANOVA, F3,12�0.54, n.s.) and the mPFC (one-ay ANOVA, F3,12�1.24, n.s.). Two-way ANOVA of ATF-2


4,60�1402, P�0.0001; duration, F3,60�13.9, P�0.0001;ose�duration interaction, F12,60�4.6, P�0.0001) and thePFC (dose, F4,60�208, P�0.0001; duration, F3,60�50.7,�0.0001; dose�duration interaction, F12,60�63.8, P�.0001). Post hoc Tukey HSD tests confirmed that in theC all doses had increased ATF-2 immunoreactivity by 4 hnd that the number of ATF-2 immunoreactive cells wasustained at all treatment durations (Fig. 4). In the mPFC,ost hoc Tukey HSD tests revealed that the lowest dose,mg/kg/day, decreased the number of cells expressing

TF-2 following 4 h and 1 week treatments but increased

ig. 3. Effects of olanzapine dose and treatment duration on c-Junmmunoreactivity in (a) the LC and (b) the mPFC. c-Jun immunoreac-ivity is expressed as the number of c-Jun immunopositive nuclei as aercentage of the pooled mean of the acidified saline vehicle controlroups (mean�SEM). Following two-way ANOVA, effects of olanza-ine dose were compared with the acidified saline controls within each

reatment duration by Tukey’s HSD post hoc tests (* P�0.05,* P�0.005, *** P�0.0005).

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TF-2 immunoreactivity at 2 week and 4 week treatmentsFig. 4). Higher doses increased ATF-2 immunoreactivityven at the 4 h time-point, but on treatment with theighest dose, 15 mg/kg/day, ATF-2 expression reducedo below control levels at 2 weeks’ treatment (Fig. 4).

gr-1 immunoreactivity

ontrol levels of Egr-1 immunoreactivity did not signifi-antly differ across the different time points in both the LCone-way ANOVA, F3,12�2.00, n.s.) and the mPFC (one-ay ANOVA, F3,12�0.94, n.s.). Two-way ANOVA of Egr-1


4,60�271, P�0.0001; duration, F3,60�12.7, P�0.0001;ose�duration interaction, F12,60�17.2, P�0.0001) andhe mPFC (dose, F4,60�309, P�0.0001; duration,

3,60�41.9, P�0.0001; dose�duration interaction,

12,60�5.80, P�0.0001). Post hoc Tukey HSD tests con-rmed that in both LC and mPFC there was a down-egulation of Egr-1 immunoreactivity at all treatment dura-ions (Fig. 5). In both the LC and the mPFC, the down-egulation of Egr-1 immunoreactivity was inversely relatedo dose and greatest at lower doses. In the LC, the down-

ig. 4. Effects of olanzapine dose and treatment duration on ATF-2mmunoreactivity in (a) the LC and (b) the mPFC. ATF-2 immunore-ctivity is expressed as the number of ATF-2 immunopositive nuclei aspercentage of the pooled mean of the acidified saline vehicle controlroups (mean�SEM). Following two-way ANOVA, effects of olanza-ine dose were compared with the acidified saline controls within each


egulation was greatest on 1-week treatment with 2 mg/an

g/day and decreased with longer treatment durations athis dose (Fig. 5). As for all the IEG immunostaining,omparison with sequential serial sections immunostainedor TH confirmed that the Egr-1 staining counted wasithin the LC (Fig. 6). Egr-1 staining was greatest in thecidified saline vehicle-treated controls but, even in the

ig. 5. Effects of olanzapine dose and treatment duration on Egr-1mmunoreactivity in (a) the LC and (b) the mPFC. Egr-1 immunoreac-ivity is expressed as the number of Egr-1 immunopositive nuclei as aercentage of the pooled mean of the acidified saline vehicle controlroups (mean�SEM). Following two-way ANOVA, effects of olanza-ine dose were compared with the acidified saline controls within each


ig. 6. Representative photomicrographs of serial sections throughhe LC immunostained for (a) Egr-1 and (b) TH after administration of
cidified saline for 2 weeks. IV, IVth ventricle; Me5, mesencephalicucleus of the Vth nerve. The scale bar�200 �m.

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ontrols, only a small subpopulation of the LC cells wasgr-1 immunopositive (Fig. 6). In the mPFC, the down-

egulation was also greatest on treatment with 2 mg/kg/ay, but was similar with 4 h, 1-week and 2-week treat-ents, and slightly greater with 4-week treatment.

gr-2 immunoreactivity

ontrol levels of Egr-1 immunoreactivity did not signifi-antly differ across the different time points in both the LCone-way ANOVA, F3,12�2.07, n.s.) and the mPFC (one-ay ANOVA, F3,12�3.04, n.s.). Two-way ANOVA of Egr-2


4,60�526, P�0.0001; duration, F3,60�11.0, P�0.0001;ose�duration interaction, F12,60�12.6, P�0.0001) andhe mPFC (dose, F4,60�916, P�0.0001; duration, F3,60�8.3, P�0.0001; dose�duration interaction, F12,60�2.1, P�0.0001). Post hoc Tukey HSD tests confirmedhat in both LC and mPFC there was a dose-dependentown-regulation of Egr-2 immunoreactivity at all treatmenturations (Fig. 7). In the LC the down-regulation was sig-ificant at all doses and treatment durations. In the mPFC,he down-regulation was significant only at the higheroses (8 and 12 mg/kg/day) for all but the 4-week treat-ent duration (Fig. 7).

ig. 7. Effects of olanzapine dose and treatment duration on Egr-2mmunoreactivity in (a) the LC and (b) the mPFC. Egr-2 immunoreac-ivity is expressed as the number of Egr-2 immunopositive nuclei as aercentage of the pooled mean of the acidified saline vehicle controlroups (mean�SEM). Following two-way ANOVA, effects of olanza-ine dose were compared with the acidified saline controls within each

kreatment duration by Tukey’s HSD post hoc tests (** P�0.005,** P�0.0005).

H immunoreactivity

ontrol levels of TH immunoreactivity did not significantlyiffer across the different time points in both the LC (one-ay ANOVA, F3,12�2.35, n.s.) and the mPFC (one-wayNOVA, F3,12�0.89, n.s.). Two-way ANOVA of TH immu-oreactivity revealed significant main effects of dose anduration of olanzapine treatment, as well as an interactionf these two factors, in both the LC (dose, F4,60�447,�0.0001; duration, F3,60�13.0, P�0.0001; dose�uration interaction, F12,60�4.6, P�0.0001) and the mPFCdose, F4,60�61.2, P�0.0001; duration, F3,60�1.4, P�0.0001; dose�duration interaction, F12,60�.4, P�0.0001).

Post hoc Tukey HSD tests confirmed that in the LC alloses and durations of treatment increased TH immuno-eactivity (Fig. 8). The increases in TH immunoreactivityere most marked for treatment with 4 mg/kg/day, while

ower and higher doses produced less significant changes.hile doses of 4 mg/kg/day and above produced similar

ncreases in TH immunoreactivity at all treatment durationsFig. 8), post hoc Tukey HSD tests suggest that the signif-cance of the dose�duration interaction in the LC may bettributable to a duration-dependent reduction in the in-reased TH immunoreactivity seen on treatment with 2 mg/

ig. 8. Effects of olanzapine dose and treatment duration on THmmunoreactivity in (a) the LC and (b) the mPFC. TH immunoreactivitys expressed as the number of TH immunopositive nuclei as a per-entage of the pooled mean of the acidified saline vehicle controlroups (mean�SEM). Following two-way ANOVA, effects of olanza-ine dose were compared with the acidified saline controls within each

reatment duration by Tukey’s HSD post hoc tests (** P�0.005,** P�0.0005).

g/day olanzapine (320�24.3% increase at 4 h compared

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ith 151�7.0% increase at 4 weeks, P�0.001). The in-reases in TH immunoreactivity were evident in increasedntensity of staining, especially in the cellular processesFig. 9).

Post hoc Tukey HSD tests showed that in mPFC thencreased TH immunoreactivity was both duration-depen-ent and dose-dependent (Fig. 8). The greatest increases

n TH immunoreactivity were seen on treatment with 8 mg/g/day olanzapine. With doses of 8 and 15 mg/kg/day oflanzapine there were significant increases in TH immu-oreactivity only after 2 weeks’ treatment. Treatmentith lower doses (2 and 4 mg/kg/day) only producedignificant increase in immunoreactivity after 4 weeks’reatment (Fig. 8).

ndirect antibody ELISA and enzymatic assay for TH

reatment with olanzapine produced a substantial andapid increase in TH immunoreactivity. We thereforeought to check this finding with other techniques. Indirectntibody ELISA for TH confirmed that an acute injection ofmg/kg olanzapine s.c. increased TH protein levels in a

rainstem tissue sample containing the LC 4 h after injec-ion (t-test, P�0.0005; Fig. 10). Likewise, enzymatic assayonfirmed increased TH activity in brainstem tissue sam-les containing the LC 4 h after acute administration ofmg/kg olanzapine s.c. (t-test, P�0.01; Fig. 11).

DISCUSSION

e examined Fos-like, c-Jun, ATF-2, Egr-1, Egr-2, and THmmunoreactivity expression in the LC and mPFC in ratseceiving 2, 4, 8, or 15 mg/kg/day olanzapine by s.c. os-otic minipump for 4 h, 1 week, 2 weeks, or 4 weeks.TF-2 expression was up-regulated at all treatment dura-

ions, while Egr-1 and Egr-2 were down-regulated in bothhe LC and mPFC. Fos-like expression was up-regulatedhrough 2 weeks, but not 4 weeks, in both the LC andPFC, although in the LC significant up-regulation at thearlier time points was only observed with 15 mg/kg/daylanzapine. c-Jun expression was up-regulated for 4

ig. 9. Representative photomicrographs of TH immunoreactivity inhe LC after administration of (a) acidified saline and (b) 8 mg/kg/daylanzapine for 1 week. The scale bar�200 �m.

eeks in the LC and for 2 weeks, but not 4 weeks, in thepb

PFC. Table 1 summarizes the pattern of changes acrossime for 8 mg/kg/day olanzapine, the dose in our studyost likely to correspond to clinical dosing (Kapur et al.,003). At all doses, there were rapid and sustained in-reases in TH immunoreactivity in the LC, but only delayed

ncreases in the mPFC. Indirect antibody ELISA for TH andn enzymatic activity assay confirmed that acute admin-

stration of 4 mg/kg olanzapine can up-regulate THithin 4 h.

In the LC and mPFC, short-term (4 h) treatment withigh-doses of olanzapine increased the number of nuclei

mmunoreactive for Fos-like protein. This is consistent withrevious reports (Robertson and Fibiger, 1996; Ohashi etl., 2000; Dawe et al., 2001). Acute administration of olan-apine activates the LC (Dawe et al., 2001; Seager et al.,004). As neuronal activity is associated with c-Fos ex-

ig. 10. Effects of 4 mg/kg olanzapine treatment on TH total proteinontent as measured by indirect antibody ELISA in brainstem tissueamples containing the LC 4 h after injection. Data are expressed asean�SEM. The effects of olanzapine treatment were compared withcidified saline vehicle treatment by two-tailed Student’s t-test com-arisons (*** P�0.0005).

ig. 11. Effects of 4 mg/kg olanzapine treatment on TH enzymaticctivity in brainstem tissue samples containing the LC at 4 h after

njection. Data are expressed as mean�SEM. The effects of olanza-
ine treatment were compared with acidified saline vehicle treatmenty two-tailed Student’s t-test comparisons (* P�0.01).

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ression (Sagar et al., 1988; Dragunow and Faull, 1989;organ and Curran, 1991), activation of LC cells may be

ufficient to induce c-Fos expression. Acute administrationf olanzapine also induces noradrenaline release in therefrontal cortex (Westerink et al., 1998; Li et al., 1998). AsC activation can lead to �-adrenoceptor-dependent in-reases in Fos-like immunoreactivity in the cortex (Bing etl., 1991, 1992; Stone and Zhang, 1995; Stone et al.,995) and the induction of Fos-like immunoreactivity in thePFC by acute administration of olanzapine was blockedy a �-adrenoceptor antagonist (Ohashi et al., 2000), it is

ikely that the increase in Fos-like expression seen in thePFC is, at least in part, a consequence of increasedoradrenaline release and perhaps the action of noradren-line at �-adrenoceptors. Alternatively, olanzapine may

ead to activation of the mPFC, which in turn leads toctivation of the LC. However, this is unlikely as self-timulation of the mPFC did not lead to c-Fos expression inhe LC (Arvanitogiannis et al., 2000).

It has been reported that acute administration of theypical antipsychotic, haloperidol, likewise increases LCctivity (Dinan and Aston-Jones, 1984; Nilsson et al.,005) and induces �-adrenoceptor-dependent increases

n Fos-like immunoreactivity in the cortex (Ohashi et al.,998), although arguably less so than the atypical antipsy-hotic, clozapine, at clinically equivalent doses. In contrast,hronic administration of haloperidol for 3 weeks is re-orted to inactivate the LC (Dinan and Aston-Jones, 1985).

On treatment with olanzapine for 1 week and 2 weeks,ncreases in the numbers of Fos-like immunoreactive nu-lei were sustained in both the LC and mPFC. However,ith 4 weeks’ treatment the numbers of Fos-like immuno-

eactive nuclei in the mPFC of olanzapine-treated animalsere not significantly different from those in saline-treatedontrols. Likewise in the LC, at all but the highest dose oflanzapine, there was no difference from saline-treatedontrols with 4 weeks’ treatment. Treatment with 15 mg/g/day olanzapine for 4 weeks reduced Fos-like immuno-eactivity in the LC. This dose is likely to be well above thelinically comparable range as minipump administration of

able 1. Summary of the pattern of changes in IEG and TH expressi

egion IEG Tre

4 h

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.5 mg/kg/day olanzapine has been reported to best ap- b

roximate the clinically comparable range of D2 receptorccupancy (Kapur et al., 2003). In our study, our dose ofmg/kg/day is most likely to correspond to clinically com-

arable doses. The patterns of change across the variousime points for treatment with 8 mg/kg/day are summarizedn Table 1. Sebens et al. (1998) had found a down-regu-ation of c-Fos expression in the prefrontal cortex of ratsreated daily for 3 weeks with 5 mg/kg olanzapine and thenhallenged with 5 mg/kg olanzapine as a control in study ofaloperidol and clozapine cross-tolerance. In contrast, weaw no evidence of significant down-regulation of Fos-like

n the mPFC. The difference may be due to the dosingegimen. Sebens et al. (1998) administered once daily i.p.njections and perfused the animals 2 h after a challengenjection, whereas we administered the olanzapine by con-inuous infusion by s.c. osmotic minipump. Sebens et al.1998) attributed the down-regulation of c-Fos expressiono the development of tolerance. Development of toleranceay also explain the reduction of Fos-like expression to

ontrol levels observed in the present study.In both the mPFC and LC, at most of the time-points

tudied from 4 h to 4 weeks, the patterns of change inos-like and c-Jun immunoreactivity were similar. This is toe expected as c-Fos and c-Jun are both intimately in-olved in forming the AP-1 complex and is consistent withrevious reports that their expression in response to vari-us stimuli is congruent (Herdegen and Leah, 1998). Aotable exception in the present study, is the pattern ofxpression in the LC on 4 weeks’ treatment with olanzap-

ne. At this time-point, the increase in c-Jun expression isaintained but the expression of Fos-like immunoreactivity

eturns to control levels or less at the highest dose. Thisay be evidence that, at least in the LC, different signalingathways regulate the expression of c-Fos and c-Jun.

ATF-2 is constitutively expressed in many cells through-ut the mammalian nervous system (Herdegen and Leah,998) but nevertheless treatment with olanzapine still in-reased the number of ATF-2-positive nuclei detected. Ourhreshold detection method of counting immunoreactiveuclei may not be detecting de novo expression of ATF-2

ing treatment with 8 mg/kg/day olanzapine

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TF-2. In the LC, olanzapine treatment rapidly and persis-ently up-regulated the number of ATF-2 immunoreactiveuclei. The ATF-2 expression was up-regulated at all treat-ent durations. Intermediate doses of olanzapine (4 mg/

g/day and 8 mg/kg/day) were most effective at inducingoth ATF-2 and TH expression. Administration of doses inhis range by osmotic minipump in rats is likely to mimiclasma concentrations of olanzapine seen in humansBergstrom et al., 2000; Seager et al., 2004, 2005). It maye that the expression of ATF-2 leads to induction of

ncreased TH expression as Suzuki et al. (2002) showedhat ATF-2 is a transcription factor involved in the regula-ion of expression of the TH gene.

In the mPFC, the intermediate doses of olanzapinencreased ATF-2 immunoreactivity across all treatment du-ations, although less robustly than in the LC. However, theowest dose (2 mg/kg/day) initially suppressed ATF-2 ex-ression after treatment for 4 h and 1 week but later

ncreased ATF-2 expression. The highest dose of olanza-ine (15 mg/kg/day) was less effective than intermediateoses in inducing ATF-2 expression, especially on longer-erm treatment. This tendency toward an inverse U-shapedose-dependence suggests that multiple mechanisms trig-ered by the complex pharmacology of olanzapine may beontributing to regulation of ATF-2 expression.

ATF-2 may play a role in the induction of c-Jun. Inome systems, it induces c-Jun in an AP-1 independentanner (van Dam et al., 1995). Also c-jun and ATF-2 cane acted upon by the same c-Jun N-terminal kinases,uggesting involvement of the same secondary messen-er pathways in regulation of their expression (Derijard etl., 1994). The increase in the expression of ATF-2 in theC is much more robust than in the mPFC. It may be that

he increased ATF-2 expression in the LC at 4 weeks helpsn the direct induction of c-Jun. This may contribute to theustained expression of c-Jun, while the expression ofos-like immunoreactivity drops to control levels.

There is an overall down-regulation of both Egr-1 (alsonown as Krox-24) and Egr-2 (also known as Krox-20) inoth the mPFC and LC across all treatment durations. In

he present study, the Egr-1 expression was inverselyroportional to the dose of olanzapine, while the expres-ion of Egr-2 was directly proportional to the dose of olan-apine. The explanation for this is at present unclear.owever, we note that Egr-1 can auto-regulate its tran-cription by binding with high affinity to the response ele-ent in its own promoter, while Egr-2 transcription is sup-ressed by c-Fos in some systems (Gius et al., 1990). Thisay contribute to the observed pattern of expression.

Similarly, Hebert et al. (2005) have recently reportedhat immobilization stress did not induce increased Egr-1 inhe LC, suggesting that Egr-1 may not be involved intress-induced stimulation of TH expression in the LC.owever, although they did find low levels of Egr-1 protein

n the LC in immunoblots of control and immobilized ani-als, they did not find any Egr-1 expression within the LCn immunostaining after immobilization stress (Hebert etl., 2005). We found Egr-1 immunostaining within the LC
s defined by TH immunostaining in consecutive serial s
ections. However, even in the vehicle-controls, where theumber of Egr-1 immunopositive nuclei is greatest, only amall subpopulation of LC cells is Egr-1 immunopositive.his may imply that the immobilization stress applied byerbert et al. (2005) suppresses Egr-1 expression moreompletely than olanzapine administration.

Olanzapine increased the number of TH immunoreac-ive profiles counted in both the LC and mPFC. In the LC,he TH immunoreactive profiles counted most probablyepresent mainly somatodendritic and axonal elements ofC cells. In the mPFC, the TH immunoreactive profilesounted likely represent mostly axonal projections from theC and the dopaminergic cells of the ventral tegmentalrea. In the LC, the increase in the number of TH immu-oreactive profiles appeared with only 4 h of olanzapinereatment suggesting that the increase is unlikely to be dueo growth of new dendrites or axons by TH expressingells. Acute administration of 4 mg/kg olanzapine pro-uced increases in TH protein as measured by indirectntibody ELISA in brainstem tissue samples including LCithin 4 h. This suggests that there may be induction of deovo expression of TH. However, the magnitude of thehange in the TH protein expression as measured byLISA (approximately 4.5-fold) and enzymatic assay (ap-roximately 2.7-fold) did not match the magnitude of thehange observed by immunostaining (approximately six-old). A limitation of our detection of TH by ELISA andnzymatic assay is that the assay was performed in brain-tem tissue samples containing LC, but not selectivelyithin LC tissue. The greater increase observed in the

mmunostaining may reflect increases in TH expression inendritic or axonal profiles that would have otherwiseallen below our detection threshold. This could be due toedistribution of TH or, as TH mRNA may be present inendrites (Dumas et al., 1990), rapid dendritic translationf protein. As there are high levels of TH expression in cellodies in the LC many of these cell bodies are above ouretection threshold even in the vehicle-treated controls.hus, although TH within the cell bodies most probablyepresents the greatest concentration of TH, informationn changes in TH in these cell bodies is lost in our imagenalysis method, which is more sensitive to the increase inhe number of dendritic and axonal elements crossing theetection threshold after olanzapine-treatment. Likewise, it

s important to note that further increases in IEG expres-ion in cells initially expressing IEGs above the detectionhreshold would also be lost with the detection thresholdmage analysis technique applied in this study.

Ordway and Szebeni (2004) found similar effects oflanzapine treatment on TH immunoreactive protein in

he LC measured by Western blotting. However, in theirtudy the increase in TH was more modest (approxi-ately 1.8-fold) and, although the trend toward in-

reased TH levels was present at their earliest 24 h timeoint, the increase was only significant with 18 days oflanzapine treatment at 5 mg/kg bid but not with treat-ent for 12 days or with 3 mg/kg/day for 18 days (Ord-ay and Szebeni, 2004). Perhaps, the continuous infu-
ion of olanzapine by osmotic minipump in the present

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tudy explains the earlier and greater increases in THxpression. Evidence that TH mRNA may be present inendrites (Dumas et al., 1990) and that the induction ofH in the LC is dependent primarily on post-transcrip-

ional mechanisms (Osterhout et al., 2005), is consistentith the possibility of rapid induction of increased THxpression in the LC. Other stimuli that trigger LC cellctivation, for example stress, have been reported toesult in rapid (less than 24 h) induction of TH expres-ion in LC cells (Zigmond et al., 1974; Melia and Duman,991; Smith et al., 1991b; Serova et al., 1999). Aslanzapine activates the LC (Dawe et al., 2001), it mighte expected to rapidly induce TH expression in the LC,specially if activation is sustained by continuous infu-ion. As there is release of noradrenaline within the LConsistent with somatodendritic release (van Gaalen etl., 1997; Pudovkina et al., 2001), early increases inomatodendritic expression of TH may influence LC ac-ivity through increased local release of noradrenalineithin the LC.

In the mPFC, increases in the number of TH immuno-eactive profiles relative to the saline treated control groupere evident only after 2- and 4-week administration oflanzapine. This is consistent with studies of TH expres-ion induced in the LC by other stimuli, such as nicotinedministration, which suggest that it takes several weeks

or newly synthesized TH to be transported along therojections of the LC to the forebrain (Mitchell et al., 1993).he TH immunoreactive profiles detected in the mPFC in

he present study could also arise from the dopaminergicnnervation of the mPFC by the ventral tegmental area,owever levels of noradrenaline in the mPFC exceedhose of dopamine (Fadda et al., 1984) so it is likely thatany of the TH immunoreactive profiles detected repre-

ent noradrenergic fibers arising from the LC. Togetherith the finding that chronic olanzapine treatment for 3eeks produced sustained increases in LC firing andursting (Seager et al., 2005), this may suggest a mech-nism to support further sustained increases in noradren-line release in the mPFC on chronic treatment with olan-apine. However, while TH immunoreactivity in the mPFContinued to increase even with 4 weeks’ treatment, Fos-ike immunoreactivity dropped again after 4 weeks ofdministration of olanzapine. This suggests that on pro-

onged treatment tolerance or other compensatory mech-nisms are activated that suppress the noradrenaline-in-uced activation of Fos-like immunoreactivity in the mPFC.erhaps these mechanisms include suppression of theoradrenaline release despite sustained increases in LCring. To date microdialysis studies have focused on theffects of acute treatment with olanzapine on noradrena-

ine release in the mPFC. The present data suggest theeed for further microdialysis studies to investigate theelease of noradrenaline on chronic treatment withlanzapine.

Although for many years it has been generally as-umed that antipsychotics have a delayed onset of action,ecent studies have questioned this delayed onset hypoth-
sis and suggest that antipsychotics can have clinical ef-
ects as early as 2 h after initiation of treatment but that theagnitude of the actions can increase over time (Agid etl., 2003; Kapur et al., 2005). The results from this study

ndicate that the rapid effects of olanzapine on IEG in theC and mPFC may play an important role in the “early”linical efficacy of antipsychotics. Further, these data in-icate that the delayed increase in TH expression in thePFC may play an important role in the increased clin-

cal efficacy of olanzapine that emerges with continuedreatment.

Future investigation of the effects of other typical andtypical antipsychotics on changes in IEG and TH expres-ion in the LC and mPFC, and how these changes mayelate to mPFC dependent behaviors, may further ournderstanding of how expression of these proteins relateso the mechanisms of action of olanzapine and other atyp-cal antipsychotic drugs.

cknowledgments—This study was supported by a grant from thecademic Research Fund of the National University of Singapore

o Dr. Gavin S. Dawe (R-184-000-045-112). The olanzapine wasrovided by Eli Lilly and Company Indianapolis, IN, USA. Wehank Mrs. Rajini Nagarajah and Ms. Siew Ping Han for theirxcellent administrative and technical assistance. We thank Dr.aura K. Nisenbaum for valuable comments on the manuscript.r. Kurt Rasmussen is an employee of Eli Lilly and Company,

ndianapolis, IN, USA.

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(Accepted 7 August 2006)(Available online 18 September 2006)

Effects of short-term and chronic olanzapine treatment on immediate early gene protein and tyrosine...

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