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Research Report

Overlapping regional distribution of CCK and TPPII mRNAs inCynomolgus monkey brain and correlated levels in humancerebral cortex (BA 10)

Diana Radua,⁎, Birgitta Tomkinsonb, Olof Zachrissonc, Günther Weberd,Jacqueline de Belleroche e, Steven Hirsch f, Nils Lindeforsa

aDepartment of Clinical Neuroscience, Psychiatry Section, Karolinska Institutet, Stockholm, SwedenbDepartment of Biochemistry, Uppsala University, BMC, Uppsala, SwedencNeuronova AB, Stockholm, SwedendCenter for Molecular Medicine, Karolinska Institutet, Stockholm, SwedeneDepartment of Neuromuscular Diseases, Division of Neuroscience and Psychological Medicine, Imperial College of Science,Technology and Medicine, Charring Cross Hospital, London, UKfDepartment of Neuropathology, Division of Neuroscience and Psychological Medicine, Imperial College, London, UK

A R T I C L E I N F O

⁎ Corresponding author. Fax: +46 8 346563.E-mail address: diana.radu@ki.se (D. Radu

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.05.059

A B S T R A C T

Article history:Accepted 19 May 2006Available online 5 July 2006

Tripeptidyl peptidase II (TPPII) is a high molecular weight exopeptidase important ininactivating extracellular cholecystokinin (CCK). Our aims were to study the anatomicallocalization of TPPII and CCK mRNA in the Cynomolgus monkey brain as a basis for apossible functional anatomical connection between enzyme (TPPII) and substrate (CCK) andexamine if indications of changes in substrate availability in the human brain might bereflected in changes of levels of TPPII mRNA. Methods: mRNA in situ hybridization onpostmortem brain from patients having had a schizophrenia diagnosis as compared tocontrols and on monkey and rat brain slices. Results: overlapping distribution patterns ofmRNAs for TPPII and CCK in rat and monkey. High amounts of TPPII mRNA are seen in theneocortex, especially in the frontal region and the hippocampus. TPPII mRNA is also presentin the basal ganglia and cerebellum where CCK immunoreactivity and/or CCK B receptorshave been found in earlier studies, suggesting presence of CCK-ergic afferents from otherbrain regions. Levels of mRNAs for CCK and TPPII show a positive correlation in postmortemhuman cerebral cortex Brodmann area (BA) 10. TPPII mRNA might be affected followingschizophrenia. Discussion: overall TPPII and CCK mRNA show a similar distribution in ratand monkey brain, confirming and extending earlier studies in rodents. In addition,correlated levels of TPPII and CCK mRNA in human BA 10 corroborate a functional linkbetween CCK and TPPII in the human brain.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Tripeptidyl peptidase IICCKBrainSchizophreniaIn situ hybridizationmRNA

Abbreviations:TPP, tripeptidyl peptidaseCCK, cholecystokininBA, Brodmann areaGAPDH, glyceraldehyde-3-phosphatase

).

er B.V. All rights reserved.

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1. Introduction

Tripeptidyl peptidase II (TPPII) is a putative cholecystokinin(CCK) inactivating peptidase (Rose et al., 1996). TPPII is a serineexopeptidase of high molecular weight, which removestripeptides from the N-terminus of longer peptides. It hasbeen purified from liver and red blood cells from differentspecies and the amino acid sequence of TPPII is highlyconserved (Tomkinson, 1999). Two isoforms of TPPII havebeen identified, a cytosolic form and a membrane-boundform, the latter accounting for 30–40% of brain TPPII (Rose etal., 1996; Tomkinson, 1999). The membrane-bound variant ofTPPII has been purified from rat brain and is involved in thedegradation of CCK (Rose et al., 1996; Tomkinson and Nyberg,1995). This is evident since butabindide, a specific inhibitor ofTPPII, decreased food intake in rodents by a CCK-dependentmechanism (Rose et al., 1996).

The neuropeptide CCK is widely distributed in the centralnervous system (Emson et al., 1982; Larsson and Rehfeld, 1979;Savasta et al., 1990). CCK is involved in physiological processessuch as satiety, memory, analgesia and in psychiatricdisorders as anxiety, psychosis, mood disorders and eatingdisorders (Noble et al., 1999).

Gene expression for CCK shows a similar distribution as thepeptide and CCK B receptor except for regions such as thecaudate-putamen and cerebellum, where scarce or no detect-able levels of CCK mRNA can be found (Lindefors et al., 1991;Lindefors et al., 1993; Schiffmann and Vanderhaeghen, 1991).The CCK-like immunoreactive material detected in thecaudate-putamen originates from projecting neurons locatedelsewhere (cortical regions andmesolimbic neurons) (Burgun-der and Young, 1990; Hokfelt et al., 1980; Morino et al., 1994b).CCK-8 is the main form of CCK in mammalian brain (Dockray,1980), but both larger and smaller biologically active C-terminal fragments such as CCK-58, CCK-33 CCK-5 and CCK-4 are also detectable in different species including human(Larsson and Rehfeld, 1979; Lindefors et al., 1993).

Other peptidases degrading CCK have been described.Beside TPPII, aminopeptidases are involved in extra cellularCCK-8 degradation (Konkoy and Davis, 1996; Migaud et al.,1996). Intracellularly, CCK is degraded in the endosomal/lysosomal system after internalization of CCK receptor-ligandcomplex (Roettger et al., 1997; Tarasova et al., 1997), thelysosomal enzyme TPPI being essential for this degradation(Warburton and Bernardini, 2002). In this study, we concen-trate on TPPII as it has the potential to inactivate extracellularCCK-8 and CCK-5, a fragment known to have an affinitycomparable to CCK-8 to the CCK B receptor (Gaudreau et al.,1983) (predominant in the brain). Distribution of TPPII hasbeen mostly studied in rat brain (Facchinetti et al., 1999; Roseet al., 1996; Tomkinson and Nyberg, 1995), while distributionand gene expression of CCK (Burgunder and Young, 1990;Lindefors et al., 1991, 1993; Schiffmann and Vanderhaeghen,1991) and CCK B receptor (Cross et al., 1988; Dietl et al., 1987;Durieux et al., 1988; Mercer et al., 1996; Van Dijk et al., 1984) arealso studied in primate and human brain. In rat brain TPPIIimmunoreactivity is localized to regions rich in CCK(Facchinetti et al., 1999; Rose et al., 1996). Tissue levels ofCCK and CCK mRNA are affected in schizophrenia (Bachus

et al., 1997; Ferrier et al., 1983; Kerwin et al., 1992; Kerwinand Murray, 1992; Virgo et al., 1995). In addition, lowerdensities of CCK B receptor ligand binding have been found infrontal cortex and hippocampus of patients with schizophre-nia as compared to controls (Farmery et al., 1985). Geneexpression of different isoforms of CCK B receptor in theprefrontal cortex are also decreased in schizophrenia(Zachrisson et al., 1999). Our aim was to study the expressionprofile of mRNAs encoding TPPII and CCK, respectively, inprimate brain, as similar localization would be the ground fora possible functional connection between enzyme andsubstrate. In addition, correlated levels of enzyme andsubstrate mRNAs are studied in human brains to furtherdetermine indications of a functional correlation.

2. Results

The probe used for TPPII in this investigation is expected torecognize both the cytosolic and membrane-bound forms ofthe enzyme. In addition, an attempt to study the distributionof a splicing variant of TPPII (Tomkinson et al., 1997) wasperformed using a construct detecting the specific 39 bp of thesplicing variant. This splicing variant contains 13 extra aminoacids near the C-terminal of the enzyme and forms a largercomplex but does not appear to be membrane-bound (Tom-kinson et al., 1997). However, this attempt was unsuccessfuldue to high binding of the sense control (data not shown).

Studies of mRNA encoding TPPII in rat and Cynomolgusmonkey horizontal and sagittal brain sections show similardistribution and overlap inmany regions with the distributionof mRNA encoding its substrate CCK (Figs. 1 and 2). Relativelevels of TPPII and CCK mRNAs are shown in Table 1.

High levels are seen in neocortex (particularly in frontalregions), cerebellar cortex and hippocampus. Lower levels aredetected in caudate, putamen, claustrum, amygdala, andsubstantia nigra. Expression of CCK mRNA is high in frontalcortex and hippocampus, moderate in amygdala, claustrumand substantia nigra. No detectable levels are seen in caudate,putamen and cerebellar cortex. In human BA 10 TPPII mRNA isseen over all cortical layers with less obvious laminationcompared to CCK mRNA (Fig. 3). Labeled cells in human BA 10are shown in Fig. 4.

In the patient material studied, we found 20.17% lowerlevels in actin-corrected CCK mRNA levels and 50.86%lower levels in actin-corrected TPPII mRNA in BA 10 of human postmortem cortex as compared to the control group(% calculated using median values). These differences didnot reach significance at the level of P < 0.05 (Table 2 andFig. 5). The levels of actin (schizophrenia group median 675,range 365.60–890, control group median 460.5, range275–690, NS) and actin-corrected GAPDH mRNA (Fig. 5)were not changed in the schizophrenia group as comparedto the control group.

The mean postmortem delay in the control group was13.2 h (range 3.75–25) and in the schizophrenia group 5.4(range 3–9). Actin mRNA was regarded as an unspecificmarker for mRNA levels used for normalizing data fordifferences in postmortem delay and possible architecturaldifferences.

Fig. 1 – Distribution of mRNA for TPPII and CCK in monkey brain. Scale bar = 1 cm; A sagittal sections 1 cm resp. 0.5 cm frommidline; B horizontal sections at levels parallel to the intercomissural plane. First level through lower temporal lobe, throughamygdaloid region, mesencephalon and cerebellum, second level through upper part of caudate-putamen. Abbreviations:frontal cortex FC, putamen Put, claustrum Cl, caudate nucleus Caud, amygdala Amg, hippocampus Hip, dentate gyrus DG,substantia nigra SN, cerebellum Cer.

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GAPDH mRNA was included as a control mRNA in theanalysis together with CCK and TPPII mRNAs. There was ahigh positive correlation for levels of actin and GAPDH

Fig. 2 – Distribution of mRNA for TPPII and CCK in rat brain.Horizontal sections, scale bar = 1 cm. Abbreviations: nucleusaccumbens N Ac, cerebral cortex CC, caudate/putamen C/Pu,thalamus Thal, hippocampus Hip, dentate gyrus DG,cerebellum Cer.

(Kendall's tau = 0.71, P < 0.001) consistent with both beingunspecific measures of mRNA levels. The actin-correctedlevels of TPPII and CCK mRNA showed a positive correlationin the studiedmaterial (Kendall's tau 0.51, P < 0.05) (Fig. 6). Thisfinding was specific as TPPII or CCK mRNA levels did notcorrelate to GAPDH mRNA levels with or without actincorrection (data not shown).

3. Discussion

TPPII gene expression is widely distributed over cortical andsub-cortical structures in monkey and rat brain, supportingand extending earlier findings in rat (Rose et al., 1996;Tomkinson and Nyberg, 1995). The present findings are alsoin line with immunolocalization of TPPII in rat (Facchinettiet al., 1999). In this study, we see similar distribution ofmRNAfor TPPII and CCK in the species studied, although not a perfectoverlap. This suggests that TPPII might be synthesized andavailable as adegradingenzyme in brain regionswhere there isCCK synthesis or CCK-ergic transmission. There are two mainregionswherewe find a possiblemismatch in distribution: the

Tab

le1

–Distribution

ofTPP

IIm

RNA

inm

onke

ybr

ain;c

ompa

riso

nto

distribu

tion

ofCCK;+

++highleve

ls,+

+m

oderateleve

ls,

+low

leve

ls,−

unde

tectab

le,m

easu

redas

calib

ratedOD,a

rbitrary

units

Fron

talco

rtex

Cor

tex

Putamen

Cau

date

Nac

cumbe

ns

Claustru

mAmyg

dala

Hippo

campu

sDen

tate

gyru

sSu

bstantianigra

Cereb

ellarco

rtex

TPP

IIm

RNA

+++

++

++

+++

+++

+++

+CCK

mRNA

++++

−−

−+

+++

++/−

−

178 B R A I N R E S E A R C H 1 1 0 4 ( 2 0 0 6 ) 1 7 5 – 1 8 2

caudate-putamen and the cerebellum. The caudate andputamen nuclei, where no visible signal for CCK mRNA isdetected, display signal for TPPII mRNA. However, this regionseems to have CCK terminals and receptors, as bothimmunoreactivity for CCK peptide (Hokfelt et al., 1988;Lindefors et al., 1991; Morino et al., 1994a; Schiffmann andVanderhaeghen, 1991) and ligand binding for CCK B receptoris seen in this region in different species (Cross et al., 1988;Dietl et al., 1987; Durieux et al., 1988; Gaudreau et al., 1983;Mercer et al., 1996; Van Dijk et al., 1984), including monkey(Gaudreau et al., 1985; Mercer et al., 1996). Moreover, striatalneurons have been shown to have the capacity to producemRNA encoding CCK in adult rats as up regulation of geneexpression for CCK in the striatum can be induced bydisruption of the nigro-striatal pathway (Schiffmann andVanderhaeghen, 1992). Expression of CCK and CCK B receptorin the cerebellum seems to differ between species. CCKmRNAand CCK peptide are detected in the cerebellum in differentspecies, though mainly during prenatal phase or shortly afterbirth, the levels declining soon thereafter (King and Bishop,1992; Rehfeld et al., 1992). In rodents, no signal for CCK isdetected in the cerebellum by radioimmunoassay (RIA)(Larsson and Rehfeld, 1979; Beinfeld et al., 1981) or ligandbinding studies of the CCK B receptor (Van Dijk et al., 1984)while primate and human studies show high densities ofCCK B receptors or its mRNA in this region (Cross et al., 1988;Dietl et al., 1987; Kinze et al., 1996; Mercer et al., 1996; Sherrinet al., 2004).

The wide spread abundance of TPPII mRNA is compatiblewith other possible functions of this enzyme than degrada-tion of CCK. TPPII preferentially degrades CCK-5 and CCK-8 but could also be involved in degrading other brain peptidessuch as neurokinin A, somatostatin and vasopressin, asshown by in vitro studies (Rose et al., 1996). However, thesepeptides are compared to CCK present in much lower levelsin most brain regions such as, e.g., the cerebral cortex (Harlanet al., 1989; McEwen, 2004; Mengod et al., 1992). The probedesigned for this study recognizes both the membrane-bondand the cytosolic form of the enzyme, the latter one mostlikely involved in general intracellular protein turnover(Tomkinson, 1999). Recently, it has also been demonstratedthat TPPII is involved in antigen processing (Kloetzel, 2004).These results suggest that a broad distribution of TPPIIshould be expected. Ideally, since the membrane-boundform of the enzyme is expected to be involved in CCKdegradation (Rose et al., 1996), a probe specific for this formof the enzyme should be used. However, so far no specificfeatures of the corresponding mRNA for this form of TPPIIhave been established.

Among other regions, the frontal cortex has been impli-cated in the pathophysiology of schizophrenia. In this region,the expression of both CCK mRNA and TPPII is high. Infrontal regions of human postmortem brain (BA 10), we couldidentify similar localization for CCK and TPPII mRNAs, CCKmRNA displaying a more marked lamination. In the smallsample of human BA 10 studied, results suggest that TPPIImRNA and possibly also CCK levels might be affected inschizophrenia. The results should be interpreted withcaution as the studied group is small and the spread forboth TPPII and CCK data is quite high. Moreover, the mean

Fig. 3 – Distribution of mRNA for TPPII and CCK in human BA 10. Scale bar = 1 cm.

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postmortem delay in the control group was 13.2 h and in theschizophrenia group 5.4, a fact we tried to correct for usingactin-normalization of the results. As schizophrenia seems tobe a heterogeneous group of disorders and several hypoth-eses exist around its etiology (Buchanan et al., 2000), differentfindings in postmortem studies may be expected. This studycould not reproduce the findings of Virgo et al. (1995) on adifferent material, where significant decreases of 50–70%were seen using both in situ hybridization and northern blotanalysis of CCK mRNA in BA 10 in a total of 32 individualshaving more homogenous postmortem delay. In that largerstudy, Virgo et al. describe two atypical subjects that did notfollow the same trend, and the methodology and correctionsused were different. Negative findings in schizophreniaregarding CCK in frontal regions have been described earlier,in BA 9 (dorsolateral prefrontal cortex) (Bachus et al., 1997),adjacent to BA 10, and in BA 4 (frontal/motor cortex) (Ferrieret al., 1985) where no significant changes in gene expressionand immunoreactivity for CCK, respectively, were found.Earlier findings show lower gene expression for CCK Breceptors in this region following schizophrenia (Zachrissonet al., 1999). Changes in levels of neuropeptides can beassociated to certain symptomatology in the studied mate-rial, e.g., Ferrier et al. (1983) described reduced levels of CCKimmunoreactivity in temporal cortex, hippocampus and

Fig. 4 – 40× Magnification of emulsion dipped Nissl-stained slidhuman CCK mRNA, respectively. Arrows indicate examples of p

amygdala only in schizophrenic subjects having negativesymptoms. The size of our sample is too small to studycorrelations of levels of CCK mRNA to symptomatology. Thetendency to decreased levels of TPPII mRNA in BA 10 could bean adaptive response to decreased availability of thesubstrate CCK as suggested by other studies (Virgo et al.,1995). TPPII gene expression may be affected by neuroleptictreatment, as reduced activity of other peptidases have beendescribed in rat after haloperidol treatment (Konkoy et al.,1996).

We found TPPII mRNA in the basal ganglia of monkey andrat, in line with earlier findings of TPPII mRNA and enzyme inrat striatum (Rose et al., 1996; Tomkinson andNyberg, 1995). Inthis region, CCK acting on CCK B receptors has an anti-dopaminergic effect on protein phosphorylation, by inducingdephosphorylation of DARPP32 (Snyder et al., 1993). Byaffecting degradation of CCK, TPPII might indirectly interactwith dopaminergic transmission in this area. Decreased tissuelevels of substrate, receptor and/or CCK-degrading enzymesmay affect neurotransmission in prefrontal cortex. A specificTPPII inhibitor such as butabindide has been shown to changefeeding behavior in rats (Rose et al., 1996), suggesting TPPIIinhibition as a possibility to increase CCK-ergic transmission.The functional connection suggested by the correlationsbetween expression of TPPII and CCK mRNAs in human BA

es hybridized with antisense probes for TPPII mRNA andositive cells in human BA10.

Fig. 5 – Actin corrected levels of CCK, TPPII and GAPDHmRNA; C = control group; S = schizophrenia group. Median;box: 25%-75%, whiskers: non-outliermin/max. Levels are notcomparable between analyzed mRNAs as measurementsderive from different sets of hybridization. For TPPII max10.68, one outlier.

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10 opens new possibilities of studying CCK-ergic mechanismsusing enzyme inhibitors in schizophrenia research.

Fig. 6 – Correlations between actin-corrected levels of TPPIIand CCKmRNAs in humanBA 10. The levels of TPPII and CCKmRNA showed a positive correlation in the material studied,Kendalls tau 0.51, P < 0.05.

4. Experimental procedures

4.1. Brain tissue

Horizontal brain slices (14 μm) from male SD rats used forother experiments (for details see Radu et al., 2001)corresponding to a level situated at Bregma −7.1 mm (Paxinosand Watson, 1986) stored at −30 °C were used.

Mature normal Cynomolgus monkeys used for otherexperiments (housed at Swedish Institute for InfectiousDisease Control, Sweden) were decapitated following a lethaldose barbiturate. The hemispheres were separated, frozenlaying on the medial surface and stored at −70 °C untilcryosectioning. Tissue blocks from BA 10 from consentedtissue donors, 7 schizophrenic patients and 6 control indivi-duals (6 patients and 6 controls for TPPII studies, as thematerial from subject no. 14 could not be used), were dissectedat autopsy, frozen and stored at −70 °C. More information onthe human material used is presented elsewhere (Zachrissonet al., 1999).

Whole hemispheres of monkey brains were cryosec-tioned into 16 μm sagittal and horizontal tissue sectionsand human tissue blocks in 14 μm sections using a LeicaFrigocut 2800F, thawed onto slides pre-treated with 2% 3-aminopropyl-triethoxysilane and stored at −30 °C untilanalysis.

4.2. Plasmids and probe preparation

A 293 bp cDNA (bp 263–535, acc no. X01032) of rat CCK wascloned into a pGEM-T easy vector (Promega) linearized withPstI (Pharmacia) and transcribed with T7 polymerase (Phar-macia). The distribution of mRNA encoding CCK peptide inmonkey brain was studied using a 406 bp riboprobe comple-mentary for RNA encoding Cynomolgus monkey CCK (bp 247–650, acc no. M60458 (Bernal et al., 1990)), cloned into a pGEM-Teasy vector (Promega), linearized with Sal 1 (Pharmacia) andtranscribed with T7 polymerase (Pharmacia). The human CCKriboprobe consisted of 150 bp (bp 195–344, acc no. L29400(Takahashi et al., 1985)) cloned into a pBluescript-KS, linear-izedwith Pst1 and transcribedwith T3. For TPPII, we used a 560bp Stu-1 fragment of the non-coding region of humanTPPII (bp4176–4626, acc no. M73047 (Tomkinson and Jonsson, 1991))(pB-SH1). The TPPII probe was cloned into pBluescript-KSplasmids. The human TPPII probe was linearized using EcoR1and transcribed using T7 for obtaining a 260 bp complemen-tary probe. Sense probeswere used as negative controls. For alltranscriptions we used 35S-UTP (Amersham) to obtain labeledcomplementary or sense riboprobes. To control for possiblecytoarchitectural changes due to schizophrenia and postmor-tem delay, we used actin and glyceraldehyde-3-phosphatase(GAPDH) mRNA (Promega) as controls. Commercial plasmidsTriactin and GAPDH (Ambion) were used to obtain 35S-UTP(Amersham) labeled antisense probes for human actin andGAPDH.

4.3. In situ hybridization

Sections were fixed in 4% formaldehyde for 5 min, deprotei-nated for 15min in 0.2MHCl, treated in 0.25% acetic anhydridesolution for 20 min and dehydrated in a series of ethanolincluding a 5 min chloroform step prior to hybridization. Thesections were incubated at 55 °C for 16–20 h with ahybridization buffer containing 50% formamide, 20% 50×dextran sulphate, 6.7% 5 M sodium chloride, 4% 5 M DTT, 2%1 M Tris–HCl pH 7.6, 2% 50× Denhardt's solution, 2% yeast

181B R A I N R E S E A R C H 1 1 0 4 ( 2 0 0 6 ) 1 7 5 – 1 8 2

tRNA (25 mg/ml), 13.3 μl H2O containing 107 cpm probe per mlhybridization buffer. After hybridization, the sections werewashed in 4× SSC, treated with RNase 10 μg/ml at 37 °C for30 min and washed in 2× SSC, 1× SSC and 0.5× SSC for 10 mineach, 0.1× SSC at 60 °C, and 0.1× SSC at room temperature for5 min. The sections were dehydrated in graded series ofethanol, air dried and exposed to X-ray film (B-Max, Amer-sham) including a strip with known amounts of radioactivity.This strip was used for calibration of optical density measure-ments. The developed films were scanned using a flat bedscanner ScanJet 3970 (Hewlett Packard) and analyzed usingImage Gauge 4.0 (FujiFilm, Tokyo, Japan). The results analyzedderive from 3 different hybridizations for CCK and TPPII. Actincorrection was performed by dividing the individual values ofthe studied mRNAs to corresponding values measured foractin mRNA. After X-ray exposure, the slides were dipped inNTB 2 emulsion (Kodak).

Statistica 5.0 (StatSoft, Upsala, Sweden) was used forstatistical analysis. Due to the distribution of our data, weused non-parametric statistics to analyze the results (Mann-Whitney U test and Spearman's rank order correlationstogether with Bonferroni correction when appropriate).

5. Conclusion

We can detect wide spread expression of TPPII mRNA inpostmortem rat and monkey brain and human BA 10, over-lapping regions expressing CCKmRNA. New data are providedon the regional distribution of TPPII and CCK mRNA in theCynomolgus monkey brain with an extensive overlap. Ourfindings are in linewith a functional connection betweenTPPIIand CCK in the human brain.

Acknowledgments

This work was supported by the Swedish Research Council,grant no. 8653, funds at Karolinska Institutet and by stipendsfrom the Swedish Lundbeck Foundation.

Ethical approval: Nr 00-167, 2001-01-08 The KarolinskaInstitutet Research Ethics Committee North.

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