Schizophrenia Research 106 (2008) 229–236

Contents lists available at ScienceDirect

Schizophrenia Research

j ourna l homepage: www.e lsev ie r.com/ locate /schres

Levels of [3H]pirenzepine binding in Brodmann's area 6 from subjects withschizophrenia is not associated with changes in the transcription factor SP1or BACE1

Brian Dean a,b,c,f,⁎, Andrew Soulby a,d,g, Geneviève M. Evin a,c, Elizabeth Scarr a,e

a The Rebecca L. Cooper Research Laboratories, The Mental Health Research Institute, Parkville, Victoria, Australiab The University of Melbourne Department of Psychiatry, Parkville, Victoria, Australiac The University of Melbourne Department of Pathology, Parkville, Victoria, Australiad The University of Melbourne Department of Anatomy, Parkville, Victoria, Australiae The University of Melbourne Centre for Neuroscience, Parkville, Victoria, Australiaf Department of Psychological Medicine, Monash University, Clayton, Victoria, Australiag School of Biomedical Sciences, University of Nottingham, Nottingham, Nottinghamshire, UK

a r t i c l e i n f o

⁎ Corresponding author. The Mental Health Research11, Parkville, Victoria, 3052, Australia. Tel.: +61 3 93895071.

E-mail address: anddali@unimelb.edu.au (B. Dean)

0920-9964/$ – see front matter © 2008 Elsevier B.V.doi:10.1016/j.schres.2008.08.003

a b s t r a c t

Article history:Received 15 April 2008Received in revised form 25 July 2008Accepted 7 August 2008Available online 13 September 2008

Decreased muscarinic M1 receptor (CHRM1) mRNA has been reported in Brodmann's area (BA)6 from subjects with schizophrenia. We have extended this study by measuring levels ofCHRM1 ([3H]pirenzepine binding), CHRM3 ([3H]4-DAMP binding), the transcription factor SP1and the CHRM1 downstream target beta-site APP-cleaving enzyme 1 (BACE1) in BA 6 from 19subjects with schizophrenia and 19 control subjects. Radioligand binding was quantified usingeither in situ radioligand binding with autoradiography or, in cohorts of 10 control subjects and10 subjects with schizophrenia, membrane enriched fraction (MEF) CNS ([3H]pirenzepinebinding only). Levels of SP1 and BACE1 were measured by Western blotting. [3H]pirenzepinebinding to tissue sections was in two layers, binding to tissue sections (Binding layer 1: pb0.01;Binding layer 2: pb0.001) and MEF (pb0.05) were decreased in schizophrenia. Levels of [3H]4-DAMP binding, SP1 and BACE1 were not altered in subjects with the disorder. This study showsa decrease in levels of CHRM1 in BA 6 from subjects with schizophrenia; as CHRM1 and BA 6 areimportant in maintaining normal cognitive function, these data support the hypothesis thatdecreased levels of cortical CHRM1 may contribute to the cognitive deficits associated withschizophrenia. Our findings on BACE1 suggest that the schizophrenia phenotype reported inBACE−/− mice is not simply due to lack of that protein in the cortex.

© 2008 Elsevier B.V. All rights reserved.

Keywords:BACE1CHRM1CHRM3CortexSchizophreniaSP1

1. Introduction

Neuroimaging (Raedler et al., 2003) and postmortem(Newell et al., 2007; Zavitsanou et al., 2004; Zavitsanouet al., 2005; Crook et al., 2001; Mancama et al., 2003; Deanet al., 2002; Deng and Huang, 2005) studies show decreasedcortical muscarinic receptors (CHRM) in schizophrenia. In theprefrontal cortex, it has been shown that it is the expression of

Institute, Locked Bag2940; fax: +61 3 9387

.

All rights reserved.

the CHRM1 (Dean et al., 2002; Mancama et al., 2003; Scarret al., 2006) and not the CHRM2, CHRM3 (Scarr et al., 2006) orCHRM4 (Dean et al., 2002), that is decreased in subjects withthe disorder.

CHRM1 (Wess, 2003) and the cortex (MacDonald et al., 2000)are important in cognition and thus decreased cortical CHRM1may contribute to the cognitive deficits associated with schizo-phrenia (Raedler et al., 2003; Dean et al., 2003). Hence, thefinding of decreased CHRM1 in BA 6, the agranular frontal cortex(Mancama et al., 2003), and BA 9, the granular frontal cortex(Dean et al., 2002), is significant as co-activation of these tworegions is required to achieve certain cognitive tasks (Naghaviand Nyberg, 2005). Significantly, only CHRM1 mRNA data has

230 B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

been thus far reported in BA 6 from subjects with schizophrenia(Mancama et al., 2003) and thus we decided to measure thebindingof [3H]pirenzepine in the sameCNS region from subjectsinwhom radioligand binding had already beenmeasured in BA9(Crook et al., 2001; Dean et al., 2002; Scarr et al., 2006). No studyhad determined if the affinity of [3H]pirenzepine binding wasaltered in schizophrenia, this is important as a change in bindingaffinity could affect the estimation of receptor density usingautoradiography (Deanet al.,1997).Hence,wealsomeasured theaffinity of [3H]pirenzepine binding in BA 6 from subjects withschizophrenia and their matched controls. To determine ifchanges in [3H]pirenzepine binding in BA 6 might be selectivewe also measured (N-methyl)-3H-4-diphenylacetoxy-N-methy-piperidone methiodide ([3H]4-DAMP) binding, as this radioli-gand predominantly binds to cortical CHRM3 (Michel et al.,1989).

We have recently shown that changes in levels of corticalCHRM1 in schizophrenia are not associated with CHRM1genotype (Scarr et al., in press) and may, therefore, be due tochanges in transcription factors that regulate CHRM1 expres-sion. The promoter region of the CHRM1 gene contains an SP1binding site (Pepitoni et al., 1997; Wood et al., 1999; Klett andBonner, 1999) and, at least in a cellular model system (Woodet al., 1999), SP1 regulates CHRM1 expression. Hence we havemeasured levels of SP1 in BA 6 from subjects with schizo-phrenia. Finally, CHRM1hasbeen shown to regulate the levels ofbeta-site APP-cleaving enzyme 1 (BACE1) (Zuchner et al., 2004)and theBACE1−/−mouse has recently been suggested as amodelfor the study of schizophrenia (Savonenko et al., 2008). Hencewemeasured levels BACE1 to see if had been affected by chronicdeficits in CHRM1 signalling in schizophrenia.

2. Materials and methods

2.1. Human tissue collection, clinical follow up and diagnosis

Following approval from the Ethics Committees of theVictorian Institute of Forensic Medicine and the North WesternMental Health Program Behavioural and Psychiatric Researchand Ethics Committee, tissuewas collected from subjects with alikely history of psychiatric disease and from subjects with noapparent history of a neurological or psychiatric disease. Thewhole CNS was removed and the right hemisphere retained forpathological examination. The left hemisphere was sliced witheach 1 cm slice being photographed and macroscopicallyexamined to ensure the absence of any obvious pathologicallesions before being frozen to −70 °C (Dean et al., 1999).

Case history reviews, completed with the Diagnostic Instru-ment for Brain Studies (DIBS) (Hill et al., 1996), allowed aconsensus diagnosis according to DSM-IV criteria (Roberts et al.,1998). Duration of illness (DOI) was calculated as the time fromfirst contactwith a psychiatric service to death, thefinal recordedantipsychotic drug dose (FRADD) was converted to chlorproma-zine equivalents (Foster, 1989; Woods, 2003) and the lifetimeexposure to antipsychotic drugs (LTEA) was calculated as gchlorpromazine equivalents (Knable et al., 2002). Where deathwaswitnessed, the timebetweendeathandautopsywas takenasthe postmortem interval (PMI). Where death was not witnessed,tissue was only taken from individuals who had been seen aliveup to 5 h before being found dead. PMI was calculated as beinghalf way between the donor last been seen alive and being found

dead to autopsy. All cadavers were refrigerated within 5 h ofbeing found and tissue was rapidly frozen to −70 °C within30 min of autopsy. The pH of the CNS tissue was measured asdescribed previously (Kingsbury et al., 1995) as an indication ofagonal status (Torrey et al., 2000; Stan et al., 2006).

2.1.1. Tissue preparationBrodmann's area (BA) 6, the caudal portion of the superior

frontal gyrus and the middle frontal gyrus extending from thecingulate sulcus on the medial surface to the lateral sulcus onthe lateral surface, was dissected from the appropriate CNSslice from 19 subjects with schizophrenia and 19 subjectswith no history of psychiatric or neurological disorders(controls: Table 1). Age (p=0.97), sex ratio (p=1.00), PMI(p=0.96) and CNS pH (p=0.85) did not differ between cohorts.

2.1.1.1. Preparation of crude homogenate and membraneenriched tissue fractions. Due to limited tissue availability,radioligand binding isotherms was completed on sub-sets of10 subjects with schizophrenia and their controls (schizo-phrenia: 8 M / 2F, controls: 7 M / 3F; p=1.00), age (40±4.3 vs.38±3.6 yr; p=0.70), PMI (37±4.6 vs. 41±4.8 h; p=0.61) andCNS pH (6.23±0.09 vs. 6.18±0.07; p=0.71).

Grey matter from the same block of BA 6 used for cuttingfrozen sections was homogenised on ice into either 5.0 ml ofice-cold 10 mM KH2PO4, 10 mM Na2PO4, pH 7.4 (assay buffer:membrane binding assay) or 10 mM Tris–HCl (pH7.4) 1% SDSand 1 mM Na3VO4 (Western blots). The homogenates forradioligand binding were centrifuged at 21,500 g in aBeckmann J2-HS centrifuge, using a JA 20.1 rotor, for 10 minat 4 °C and the pellet suspended in 2.0 ml of assay buffer,centrifuged under identical conditions and suspended in1.0 ml of assay buffer (membrane enriched fraction: MEF).

The protein concentration of the MEF and crude homo-genates were determined using the BioRad protein assayadapted for the microplate reader.

2.2. Radioligand binding

2.2.1. In Situ radioligand binding with autoradiography[3H]pirenzepine (Crook et al., 2001; Dean et al., 2002) and

[3H]4-DAMP (Levey et al., 1994; Araujo et al., 1991) bindingwas essentially as described previously.

Tissue sections (20 μm) were incubated with [3H]pirenze-pine (15 nM) in the presence (non-specific binding (NSB): 2sections) or absence (total binding (TB): 3 sections) of 10−6 Mquinuclidinyl xanthene-9-carboxylate hemioxalate (QNX) inassay buffer at room temperature for 30 min. [3H]4-DAMP wasincubated for 2 h in assay buffer containing the radioligand(3 nM) in the presence (2 sections: NSB) or absence (3 sections:total binding) of 10−5 M 4-DAMP mustard. All sections werewashed twice (2min in ice-cold assaybuffer), dipped in ice-coldwater and thoroughly dried. The dried sections were fixedovernight in paraformaldehyde fumes in a desiccator and then,in conjunction with sets of [3H]micro-scales™, apposed to aBAS-TR2025 imaging plate until an image of appropriateintensity was obtained for scanning in the BAS 5000 highresolution phosphoimager. Exposure time related to both thedensity of binding sites and the specific activity of theradioligand utilized. The intensity of the phosphoimages werethenmeasuredby comparison to the intensity of the [3H]micro-

Table 1Demographic and CNS collection data for subjects from whom BA 6 was collected

Schizophrenia ID Sex Age pH PMI Cause of death Sui DOI FRADD FRACDD a LTEA b FRACD FRACDD

Year h Year mg / day

1 c M 32 6.21 17.0 CO poisoning Y 15 Haloperidol 670 3.67 Benztropine 22 c M 22 6.17 37.0 Combined drug toxicity Y 3 Pimozide 200 0.22 Nil3 M 55 6.10 25.0 Coronary artery atheroma N 33 Thioridazine 400 4.81 Benztropine 2

4 c F 48 6.21 52.5 Pulmonarythromboembolus

N 22 Fluphenazinechlorpromazine

700 5.62 Nil

5 M 65 6.57 41.0 Ischemic heart disease N 35 Fluphenazine 150 5.25 Nil6 M 65 6.29 42.0 Bronchopneumonia N 36 Trifluoperazine haloperidol 460 6.04 Benhexol 47 F 65 6.35 50.0 Rupture aortic aneurysm N 18 Fluphenazine haloperidol 550 3.61 Benztropine 28 M 38 5.52 40.0 Mediastinitis N 15 Haloperidol 160 2.40 Procyclidine 15

9 c M 42 6.44 47.0 Hanging Y 9 Haloperidol 128 0.42 Benztropine 210 c M 41 6.20 31.0 Combined drug toxicity Y 10 Fluphenazine trifluoperazine 500 1.83 Nil11 c M 53 6.29 9.0 Coronary artery atheroma N 9 Trifluoperazine

chlorpromazine300 0.99 Nil

12 c F 26 6.11 41.0 Combined drug toxicity Y 3 Remoxipride 750 0.82 Nil13 c M 19 6.22 43.0 Unascertained Y 11 Nil 0 0.00 Benztropine 214 M 69 6.44 48.0 CO poisoning N b1 Nil 0 0.00

15 c M 41 6.64 48.0 Hanging Y 15 Fluphenazine 166 0.91 Nil16 F 30 6.37 48.0 Hanging Y 11 Flupenthixol thiothixene 600 2.41 Benztropine 217 c M 48 6.62 30.0 Bronchopneumonia 0 24 Flupenthixol thioridazine 1250 10.95 Nil18 F 35 6.36 39.0 Incised wrist injury 1 8 Risperidone 150 0.44 Nil19 F 79 6.27 26.0 Hypothermia 1 6 Fluphenazine 330 0.72 Benhexol 2

Mean 46 6.28 38 16 393 2.69SEM 3.9 0.06 2.7 2.5 72 0.65

Controls ID Sex Age pH PMI Cause of death

Year h

1 M 65 6.47 20.5 Acute myocardial infarct2 F 62 6.45 40.0 Ischemic heart disease3 F 36 6.40 60.0 Dilated cardiomyopathy4 M 60 6.11 27.5 Pulmonary embolus

5 c M 26 6.37 46.4 Electrocution6 c F 21 6.03 58.0 Myocarditis7 c M 43 6.25 45.0 Drowning8 c M 27 6.46 45.0 Drowning9 F 33 6.41 42.0 Multiple injuries

10 c M 30 5.86 27.0 Coronary artery atheroma11 M 48 6.37 24.0 Coronary artery atheroma12 M 57 6.43 27.0 Ischemic heart disease13 c F 56 5.88 24.0 Pericardial tapenade14 M 68 6.06 41.0 Aortic stenosis15 M 68 6.59 27.0 Coronary artery atheroma

16 c M 52 5.98 22.0 Pulmonary thromboembolus17 c M 43 6.43 51.0 Coronary artery atheroma18 c F 39 6.23 65.0 Mitral valve prolapse19 c M 42 6.32 26.0 Coronary artery atheroma

Mean 46 6.27 38SEM 3.4 0.05 3.2

a mg chlorpromazine equivalents / day, LTEA=lifetime exposure to antipsychotic drugs.b kg chlorpromazine equivalents, FRACD=final recorded anticholinergic drug, FRACDD=final recorded anticholinergic drug dose.c subjects used in radioligand binding isotherm studies, PMI=postmortem interval, Sui=suicide, DOI=duration of illness, FRADD=final recorded antipsychotic drug dose.

231B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

scales™ using AIS image analysis software and then the totalminus non-specific binding (specific binding) was expressed asfmol / mg estimated wet weight tissue equivalents (ETE).

To compare the distribution of radioligand binding to classiccortical structure, one section from each subject was stainedwith cresyl violet. Hence each section was first fixed in 10%formalin in phosphate buffered saline (0.15 M NaH2PO4.2H2O,0.15 M Na2HPO4 made to 1 L with 0.9% NaCl, pH=7.4) for 1 h atroom temperature. The sectionswere then placed in 0.1% cresylviolet in 1% acetic acid for 15 min at 37 °C and then rinse indistilled water. Each section was then dehydrated by beingplaced twice in sequential baths of absolute ethanol for 1min at

room temperature and then cleared by being placed twice inxylene for 1 min temperature. Each section was then mountedwith DePeX before being examined under the lightmicroscope.

2.2.2. Saturation isothermsMEFs (0.2mgof protein)were incubated inassay bufferwith

[3H]pirenzepine at a range of concentrations (0.1–15 nM) in theabsence (triplicate, TB) or presence (duplicate, NSB) of 1.0 μMQNX for 30 min in assay buffer at room temperature. Theincubationwas stopped by placing the tubes in an ice bath andfiltration through Whatman GF/B filter paper using a BrandelCell-harvester. After washing the filters ×3 with 3.0 ml ice-cold

Fig. 1. A: A typical autoradiograph of [3H]pirenzepine binding to humanBA6with non-specific binding shownas an inset. Two layers of radioligand binding (layer 1 andlayer 2) could be delineated in some subjects. Comparison of autoradiographic images with cresyl violet stained sections suggests that radioligand binding layer 1overlayed cortical laminae I to III whereas layer 2 overlayed laminae IV to VI. The location of the cortical laminae are defined and labelledwith roman numerals. Wheretwo layers were not visible a template defining the binding layers were used to ensure a constant division of the cortex prior to measuring radioligand binding. B: Thebinding (mean±SEM) of radioligands to frozen tissue sections from Brodmann's area 6 from subjects with schizophrenia and their age / sex matched control subjects.Data are shownwith (n=19 pairs) and without (n=18 pairs) the outlier identified using Grubb's test and the age / sexed matched subjects with schizophrenia. C: Theaffinity and density (mean±SEM) of [3H]pirenzepine binding to a membrane enriched fraction of BA 6 from 10 subjects with schizophrenia and 10 age / sex matchedcontrols (see Section 2.1.1.1). ⁎ pb0.05, ⁎⁎ pb0.01 and ⁎⁎⁎ pb0.001.

232 B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

saline eachfilter paperwasplaced into a scintillationvial, 4.0mlof Beckman Ready-Protein® scintillation cocktail added and theradioactivity on each filter paper counted using a PackardTricarb 2100 TR liquid scintillation analyser. Scatchard analyseswere completed using GraphPad Prism (version 4.01 forWindows, (GraphPad Software Inc., San Diego, California, USAwww.GraphPad.com).

2.2.3. Electrophoresis and western blottingWestern blot analyses of SP1 and BACE1 were performed

using optimised protocols; hence BA 6 homogenates containing30 μg total proteinwere separated on an 8.5% polyacrylamide gel

(each gel contained samples from subjects with schizophreniaand theirmatched control subjects aswell as a sample of internalcontrol (IC: see below)). The proteins on these gels weretransferred onto nitrocellulose membranes and stained with0.2% Ponceau solution and, after confirming optimal transfer, themembranes were destained prior to being probed for either SP1or BACE 1.

All nitrocellulose membranes were blocked with 1% non-fat milk powder in Tris buffered saline and 0.1% Tween-20(TTBS) for one h. For SP1, membranes were incubated inblocking buffer with a 1: 1000 dilution of anti-human SP-1antibody, overnight at 4 °C, washed 4 times for 10min in TTBS

233B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

and then incubated with a 1:2000 dilution of goat anti-rabbitantibody conjugated to HRP for 1 h at room temperature(Holsinger et al., 2002). For BACE1, the nitrocellulosemembranes were blocked with 5% non-fat milk powder and1% BSA in Tris buffered saline containing 0.1% Tween-20 for1 h, incubated in blocking buffer with a 1:1500 dilution ofanti-human BACE-1 antibody overnight at 4 °C, washed 4times for 10 min in TTBS and then incubated with a goat anti-rabbit antibody conjugated to horse radish peroxidase (HRP)(dilution of 1:2500) for 2 h at room temperature.

To measure the intensity of immunogenic protein bands,membranes were washed 4 times for 10 min in TTBS andincubated for 5 min in Pierce ECL reagent (50% Peroxide 50%Luminol) at room temperature. Excess ECL solution wasremoved and the immunogenic bands on each membraneimaged using the Kodak CF 440 image station. The summedintensity of the desired band was then determined fromthe image for each subject in the cohort and converted to aratio of IC.

The IC for western blots was prepared by generating anhomogenate of BA 6 from a subject that was not included inthis study and used to control for gel to gel variation (Deanet al., 2007).

2.3. Statistics

Outlying data was identified with a Grubb's test (Grubb,1969). D'Agostino & Pearson omnibus normality test was usedto determine if the data was normally distributed (Poitras,2006). Numerical demographic, tissue collection, [3H]4-DAMP, [3H]pirenzepine binding isotherm and Western blotdata were compared using a two-tailed student t-test. Non-numerical data were compared using Fisher's exact test todetermine if the frequencies of these variables changed withdiagnosis. [3H]pirenzepine binding to tissue sections, as wellas the effects of suicide and anticholinergic drug status onthose parameters, were analysed using a two-way ANOVAwith diagnosis, suicide or anticholinergic drug status andradioligand binding layer as variables; posthoc Bonferronitests were utilised to identify the source of any significantvariance. Relationships between experimental variables wereanalysed using Pearson-product moment correlation coeffi-

Table 2Comparisons of the potential confounding variables, suicide and anticholinergic drugof [3H]4-DAMP binding, SP1 and BACE 1 in subjects with schizophrenia

[3H]pirenzepine bindingHomogenate binding Tissue sections

Suicide n 6 10

KD Bmax a Layer 1 b L

(nM)

Confounding factor X–

SEM p X–

SEM p X–

SEM p X–

Suicide 4.7 1.33 0.52 163 35 0.54 74 17 0.85 1Non-suicide 5.9 1.20 196 37 71 15

Antichol n 6 11 11 11+ anticholinergic 4.2 0.58 0.22 187 22 0.60 86 14 0.14 1− anticholinergic 6.6 2.10 159 56 54 16

Abbreviations: Antichol=anticholinergica fmol / mg protein.b fmol / mg estimated tissue equivalents.

cient assuming a straight line best fit. Reflecting the size of thecohorts in this study, a significant correlation of r2N0.36 wastaken as reflecting a strong relationship (Franzblau, 1958) of amagnitude that had the potential to influence primarystatistical analyses. In such cases an ANCOVA was completedto ensure the confounding factor had not impacted on theanalyses of experimental data in relation to diagnoses. If datawas shown to be non-parametric an equivalent non-para-metric analyses would be completed.

3. Results

In some subjects, two layers of [3H]pirenzepine bindingwere observed in BA 6 (Fig. 1A); a comparison with cresylviolet stained sections showed that the outer binding layer(layer 1) overlayed cortical laminae I to III whilst the innerlayer (layer 2) covered laminae IV to VI.

3.1. Radioligand binding

Grubb's test showed one outlier in the data from [3H]pirenzepine binding to tissue sections from a control sub-ject (control subject # 19: Table 1); therefore analyses ofthese data are reported with and without the outlier andtheir matched subject with schizophrenia. The diagnosticcohorts without the outlier did not differ with regards toage (p=0.94), sex ratio (p=1.0), PMI (p=0.84) or CNS pH(p=0.82).

[3H]pirenzepine binding varied with diagnosis and bindinglayer whether (diagnosis: F=22.2, df=1,72, pb0.0001; layer:F=8.7, df=1,72, pb0.005) or not (diagnoses: F=29.29, df=1,70,pb0.0001; layer: F=10.0, df=1,70, pb0.005) the outlier andtheir matched pair were included (Fig. 1B). There was nosignificant interaction between these variables (F=0.09,df=1,72, p=0.76; F=0.16, df=1,70, p=0.68). Post hoc analysesshowed significant decreases in [3H]pirenzepine binding inboth layers, whether (layer 1 and layer 2 pb0.01) or not (layer 1pb0.01; layer 2 pb0.001) the outlying data was included.Whether or not the outlying data was included, there was asignificantly lower level of [3H]pirenzepine binding in layer 1compared to layer 2 in tissues from subjectswith schizophrenia(pb0.05) but not in controls.

status, on the affinity and density of [3H]pirenzepine binding as well as levels

Western blots

10 10

ayer 2 b [3H]4DAMP2 SP1 BACE1

(OD) (OD)

SEM p X–

SEM p X–

SEM p X–

SEM p

03 22 0.85 82 9.6 0.53 1.24 0.12 0.58 1.60 0.21 0.2598 21 75 5.0 1.14 0.12 1.27 0.18

19 18 0.14 83 8.2 0.48 1.25 0.12 0.30 1.56 0.22 0.3276 23 74 6.8 1.08 0.13 1.27 0.14

234 B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

[3H]4-DAMP binding was homogenous across the cortex;there was no significant difference between [3H]4-DAMPbinding between subjects with schizophrenia and their agesex matched controls (Mean±SEM: 79±5.5 vs. 85±2.3 fmol /mg ETE; p=0.34). [3H]4-DAMP binding did not correlate withage, PMI, DOI or CNS pH (r2 fromb0.0005 to 0.1).

[3H]pirenzepine to MEF fitted to a single binding sitemodel (y=Bmax⁎x / (KD+x: r2 0.9984 to 0.9217). There was asmall but significant increase in the KD of [3H]pirenzepinebinding to MEF from the subjects with schizophrenia (mean±SEM: 5.1±0.9 nM vs. 3.0±0.16 nM; p=0.03) whilst bindingdensity was decreased in schizophrenia (Fig. 1C).

3.2. Western blot analyses

SP1 (mean±SEM: schizophrenia=1.19±0.09 vs. control1.29±0.09 O.D.; p=0.44) and BACE 1 (schizophrenia=1.41±0.15 vs. control 1.63±0.14 O.D.; p=0.37) did not differ withdiagnoses.

3.3. Potential confounds

There was a meaningful correlation between PMI and theaffinity of [3H]pirenzepinebinding in the controls (r2=0.39) andclose to ameaningful correlation in subjectswith schizophrenia(r2=0.34); in neither case did the slope of the regression linedeviate significantly from zero. There were no meaningfulrelationships between age, PMI, DOI or CNS pHwhether or notthe outlying datawas included in the analyses (r2 from b0.0001to 0.13) and any other experimental measure.

Suicide and treatment with anticholinergic drugs did notsignificantly affect the affinity or density of [3H]pirenzepinebinding to MEF or tissue sections, [3H]4-DAMP binding orlevels of SP1 and BACE 1 (Table 2).

No experimental parameter correlatedwith FRADD (r2 fromb0.002 to 0.02) or LTEA (r2b0.05 to 0.30).

4. Discussion

This study has shown that [3H]pirenzepine binds to a singlesite in BA 6 which is decreased in subjects with schizophrenia.[3H]pirenzepine binds with high affinity to CHRM1 (Watsonet al., 1983), the most abundant CHRM in the human cortex(Flynn et al., 1995). Thus, our data from radioligand binding addsto a previous study (Mancama et al., 2003) that showed thatCHRM1 mRNA was decreased in BA 6 from subjects withschizophrenia. As BA 6 (MacDonald et al., 2000) and CHRM1(Wess, 2003) are important in maintaining cognitive functionour data supports the hypotheses that decreased cortical CHRM1contribute to the cognitive deficits of schizophrenia (Dean, 2004;Dean et al., 2003). Strengthening this is notion are neuroimagingdata showing that BA 6 and BA 9 co-activate during cognitiveprocessing (Naghavi andNyberg, 2005) and that [3H]pirenzepinebinding is decreased in these two regions in schizophrenia ([3H]pirenzepine binding in BA 9 for current cohorts: mean±SEM:schizophrenia=134±16 vs. controls=197±8.6; pb0.002). More-over, [3H]pirenzepine binding in BA 6 and BA 9 shows a highlysignificant correlation (BA 9 vs. BA6 layer 1 (r2=0.88; pb0.0001)and layer 2 (r2=0.89; pb0.0001) in subjects with schizophrenia.This strong relationship between levels of [3H]pirenzepinebinding in subjects with schizophrenia add to the notion that

changes in these two regions may act in concert to contribute tochanges in cortical functioningwhichhas been shown tooccur inschizophrenia (Mitelman et al., 2005).

This study has shown a small significant decrease in theaffinity of [3H]pirenzepine binding in tissue from subjectswith schizophrenia which is of too small a magnitude toinvalidate the use of single-point saturation analyses. Inaddition, this is the first study to show [3H]4-DAMP binding isnot altered in BA 6 from subjects with schizophrenia. [3H]4-DAMP is a selective CHRM3 ligand (Garcia et al., 2005; Michelet al., 1989) and therefore these data suggest that, as in BA 9(Scarr et al., 2006), there are no differences in CHRM3 levels inthe cortex of subjects with schizophrenia.

We postulated that the differences in levels of CHRM1 intissue of subjects with schizophrenia could be associated withaltered levels of SP1, a transcription factor that regulatesCHRM1 expression (Wood et al.,1999; Klett and Bonner,1999).Our data in this study shows no differences in levels of SP 1 inschizophrenia and therefore does not support our hypotheses.However, this does not exclude the possibility that a change inSP 1 activitymight be associatedwith altered CHRM1 levels insubjects with the disorder. In addition, studies indicate thereare a number of transcription factors for the CHRM1 gene(Wood et al., 1999; Klett and Bonner, 1999) and thereforechanges in these other factors could also be a cause of alteredCHRM1 expression in subjects with schizophrenia.

In model systems, activating CHRM1 up-regulates BACE 1expression (Zuchner et al., 2004); therefore decreased corticalCHRM1 could have reduced BACE1 in subjects with schizo-phrenia. Such a change in BACE1 levels would have beensignificant given the recent report of a behavioural schizo-phrenia phenotype in the BACE1−/− mouse (Savonenko et al.,2008). However, our data showing no change in the levels ofBACE1−/− would suggest that the schizophrenia phenotypeobserved in these animals is not simply due to the reducedlevels of cortical BACE1 per se. Our findings do not exclude thepossibility that the phenotypic behaviour may result from theabsence of BACE1 in other CNS regions.

Giventhemethodologyused inourstudies,wewouldmeasurelevels all available CHRM1, CHRM3, SP1 and BACE1, independentof cellular localisation. Significantly, currentevidencesuggests thatCHRM1 (Disney and Aoki, 2008; Elhusseiny et al., 1999; Guizzettiand Costa, 1996), SP1 (Okamoto et al., 2002; Borges et al., 2004)and BACE1 (Rossner et al., 2005) are expressed by neurons andneuroglia. Thus, our data cannot rule out the possibility that cell-type specific changes in CHRM1 and SP1 or BACE1 could beoccurring in the CNS of subjects with schizophrenia and that themagnitudeof changes in SP1 or BACE1 in that cell typemaynot bedetectable by our omnibus measures. Moreover, CHRM1 areexpressed by parvalbumin, calbinding and calretinin immunor-eactive neurons (Disney and Aoki, 2008) and therefore furtherinvestigation is required to determine if levels of CHRM1 aredecreased on all of these cell types as well as on neuroglia.

The consequences of decreased cortical CHRM1 are widereaching. CHRM1 appears to have a role in development of theembryonic CNS prior to the onset of synaptogenesis (Williamset al., 2004), therefore it is possible that early deficits inCHRM1 in subjects who eventually develop schizophreniacould impact on cortical development per se. CHRM1 knockout mice have been reported as having disrupted corticaldevelopment (Zhang et al., 2005) which could support such a

235B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

hypothesis. CHRM1 has recently been shown to be amodulatorof synaptic pruning (Shen et al., 2007) and abnormalities in thisprocess during childhood and adolescence has been suggestedto underlie the pathology of schizophrenia (Hoffman andDobscha, 1989). In addition, it has recently been shown thatenvironmental factors can modulate levels of [3H]pirenzepinebinding in the phospholipase C β1 knockout mouse (McOmishet al., 2008). These data suggest that levels of CHRM1have beeninfluenced by a combination of a genetic modification andenvironmental factors, a combination that is now thought to becritical in precipitating schizophrenia (Tsuang, 2000). Inconclusion, whilst there are multiple mechanisms by whichaberrant CHRM1 might precipitate the symptoms of thedisorder our data suggest these mechanisms do not involvechanges in levels of the SP 1, BACE 1 or CHRM3 in the CNS ofsubjects with schizophrenia.

Role of funding source

The study sponsors had no role in the study design, data collection, writingthe report or decisions on journal submission.

Contributors

Associate Professor Dean was involved in the conceptual design of the studyand took the lead role in preparing the manuscript for publication. Mr. AndrewSoulby had a major role in data collection and analyses and contributed to thepreparation of the final manuscript. Dr Evin provided BACE1 antibody for thisstudyandplayeda significant role inpreparationof themanuscript for submission.Dr Scarrwas involved in data collection and analyses aswell as contributing to thepreparation of the manuscript for publication.

Conflict of interest

Associate Professor Dean has no conflicts of interests regarding this study.Associate Professor Dean has received an unrestricted travel grant from GSK(2007) and an Education Symposium Honorarium from Eli Lilly (2008).

Mr. Soulby has no conflicts of interest or financial disclosures.Dr. Evin has no conflicts of interest or financial disclosures.Dr Scarr reports no conflicts of interest regarding this study.However, in the

past she has received travel support to a conference from GSK (2007) and anhonorarium for a clinical presentation from Astra-Zeneca (2005).

Acknowledgements

BD is a Senior NHMRC Research Fellow (Level B)(400016). ES is the RoyceAbbey Postdoctoral Fellow (Mental Illness). This research was funded in partby NHMRC Project Grants # 192399 and 509333, NIH Grant # R01MH069696and the Rebecca L. Cooper Medical Research Foundation.

References

Araujo, D.M., Lapchak, P.A., Quirion, R.,1991. Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence frommembrane binding and autoradiographic studies. Synapse 9, 165–176.

Borges, K., Myers, S.J., Zhang, S., Dingledine, R., 2004. Activity of the rat GluR4promotor in transfected cortical neurons and glia. Journal of Neuro-chemistry 86, 1162–1173.

Crook, J.M., Tomaskovic-Crook, E., Copolov, D.L., Dean, B., 2001. Lowmuscarinicreceptor binding in prefrontal cortex from subjects with schizophrenia: astudy of Brodmann's areas 8, 9, 10, and 46 and the effects of neurolepticdrug treatment. American Journal of Psychiatry 158, 918–925.

Dean, B., 2004. M1 receptor agonism, a possible treatment for cognitivedeficits in schizophrenia. Neuropsychopharmacology 29, 1583–1584.

Dean, B., Pavey, G., Opeskin, K., 1997. [3H]raclopride binding to brain tissuefrom subjects with schizophrenia: methodological aspects. Neurophar-macology 36, 779–786.

Dean, B., Pavey, G., Chai, S.Y., Mendelsohn, F.A.O., 1999. The localisation andquantification of molecular changes in the human brain using in situradioligand binding and autoradiography. In: Dean, B., Kleinman, J.E.,Hyde, T.M. (Eds.), Using CNS Tissue in Psychiatric Research: A PracticalGuide. Harwood Academic Press, Amsterdam, pp. 67–83.

Dean, B., McLeod, M., Keriakous, D., McKenzie, J., Scarr, E., 2002. Decreasedmuscarinic1 receptors in the dorsolateral prefrontal cortex of subjectswith schizophrenia. Molecular Psychiatry 7, 1083–1091.

Dean, B., Bymaster, F.P., Scarr, E., 2003. Muscarinic receptors in schizophrenia.Current Molecular Medicine 3, 419–426.

Dean, B., Boer, S.A., Mackinnon, A., Berk, M., 2007. CNS 14-3-3zeta: changeswith sex but not psychiatric diagnoses or psychotropic drug treatment.Schizophrenia Research 93, 51–57.

Deng, C., Huang, X.F., 2005. Decreased density of muscarinic receptors in thesuperior temporal gyrus in schizophrenia. Journal of NeuroscienceResearch 81, 883–890.

Disney, A.A., Aoki, C., 2008. Muscarinic acetylcholine receptors in macaqueV1 are most frequently expressed by parvalbumin-immunoreactiveneurons. Journal of Comparative Neurology 707, 1748–1762.

Elhusseiny, A., Cohen, Z., Olivier, A., Stanimirovic, D.B., Hamel, E., 1999.Functional acetylcholine muscarinic receptor subtypes in human brain:identification and cellular localisation. Journal of Cerebral Blood Flowand Metabolism 19, 794–802.

Flynn, D.D., Ferrari-Dileo, G., Mash, D.C., Levey, A.I., 1995. Differentialregulation of molecular subtypes of muscarinic receptors in Alzheimer'sdisease. Journal of Neurochemistry 64, 1888–1891.

Foster, P.,1989.Neuroleptic equivalence. ThePharmaceutical Journal 243, 431–432.Franzblau, A., 1958. A Primer of Statistics for Non-statisticians. Harcourt,

Brace and World, New York.Garcia, N., Santafe, M.M., Salon, I., Lanuza, M.A., Tomas, J., 2005. Expression of

muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in theneuromuscular junction of the newborn and adult rat. Histology andHistopathology 20, 733–743.

Grubb, F., 1969. Procedures for detecting outlying observations in samples.Technometrics 11, 1–21.

Guizzetti,M., Costa, L.G.,1996. Inhibitionofmuscarinic receptor-stimulated glialcell proliferation by ethanol. Journal of Neurochemistry 67, 2236–2245.

Hill, C., Keks, N., Roberts, S., Opeskin, K., Dean, B., Mackinnon, A., Copolov, D.,1996. Problem of diagnosis in postmortem brain studies of schizo-phrenia. American Journal of Psychiatry 153, 533–537.

Hoffman, R.E., Dobscha, S.K., 1989. Cortical pruning and the development ofschizophrenia: a computer model. Schizophrenia Bulletin 15, 477–490.

Holsinger, R.M., McLean, C.A., Beyreuther, K., Masters, C.L., Evin, G., 2002.Increased expression of the amyloid precursor beta-secretase inAlzheimer's disease. Annals of Neurology 51, 783–786.

Kingsbury, A.E., Foster, O.J., Nisbet, A.P., Cairns, N., Bray, L., Eve, D.J., Lees, A.J.,Marsden,C.D.,1995. TissuepHasan indicatorofmRNApreservation inhumanpost-mortem brain. Brain Research Molecular Brain Research 28, 311–318.

Klett, C.P., Bonner, T.I., 1999. Identification and characterization of the rat M1muscarinic receptor promoter. Journal of Neurochemistry 72, 900–909.

Knable, M.B., Barci, B.M., Bartko, J.J., Webster, M.J., Torrey, E.F., 2002.Molecular abnormalities in the major psychiatric illnesses: Classificationand Regression Tree (CRT) analysis of post-mortem prefrontal markers.Molecular Psychiatry 7, 392–404.

Levey, A.I., Edmunds, S.M., Heilman, C.J., Desmond, T.J., Frey, K.A., 1994.Localization of muscarinic m3 receptor protein and M3 receptor bindingin rat brain. Neuroscience 63, 207–221.

MacDonald III, A.W., Cohen, J.D., Stenger, V.A., Carter, C.S., 2000. Dissociatingthe role of the dorsolateral prefrontal and anterior cingulate cortex incognitive control. Science 288, 1835–1838.

Mancama, D., Arranz, M.J., Landau, S., Kerwin, R., 2003. Reduced expression ofthe muscarinic 1 receptor cortical subtype in schizophrenia. AmericanJournal of Medical Genetics B Neuropsychiatry Genetics 119, 2–6.

McOmish, C.E., Burrows, E., Howard,M., Scarr, E., Kim,D., Shin, H.S., Dean, B., vanden, B.M., Hannan, A.J., 2008. Phospholipase C-beta1 knockoutmice exhibitendophenotypes modeling schizophrenia which are rescued by environ-mental enrichment and clozapine administration.Molecular Psychiatry 13,661–672.

Michel, A.D., Stefanich, E., Whiting, R.L., 1989. Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP. European Journal of Pharmacology166, 459–466.

Mitelman, S.A., Buchsbaum, M.S., Brickman, A.M., Shihabuddin, L., 2005.Cortical intercorrelations of frontal area volumes in schizophrenia.Neuroimage 27, 753–770.

Naghavi, H.R., Nyberg, L., 2005. Common fronto-parietal activity in attention,memory, and consciousness: shared demands on integration? Con-sciousness and Cognition 14, 390–425.

Newell, K.A., Zavitsanou, K., Jew, S.K., Huang, X.F., 2007. Alterations ofmuscarinic and GABA receptor binding in the posterior cingulate cortexin schizophrenia. Progress in Neuropsychopharmacology and BiologicalPsychiatry 31, 225–233.

Okamoto, S., Sherman, K., Bai, G., Lipton, S.A., 2002. Effect of the ubiquitoustranscription factors, SP1 and MAZ, on NMDA receptor subunit type 1(NR1) expression during neuronal differentiation. Brain ResearchMolecular Brain Research 107, 89–96.

236 B. Dean et al. / Schizophrenia Research 106 (2008) 229–236

Pepitoni, S., Wood, I.C., Buckley, N.J., 1997. Structure of the m1 muscarinicacetylcholine receptor gene and its promoter. Journal of BiologicalChemistry 272, 17112–17117.

Poitras, G., 2006. More on the correct use of omnibus tests for normality.Economic Letters 90, 304–305.

Raedler, T.J., Knable, M.B., Jones, D.W., Urbina, R.A., Gorey, J.G., Lee, K.S., Egan,M.F., Coppola, R., Weinberger, D.R., 2003. In vivo determination ofmuscarinic acetylcholine receptor availability in schizophrenia. Amer-ican Journal of Psychiatry 160, 118–127.

Roberts, S.B., Hill, C.A., Dean, B., Keks, N.A., Opeskin, K., Copolov, D.L., 1998.Confirmation of the diagnosis of schizophrenia after death using DSM-IV:a Victorian experience. Australian and New Zealand Journal of Psychiatry32, 73–76.

Rossner, S., Lange-Dohna, C., Zeitschel, U., Perez-Polo, J.R., 2005. Alzheimer'sdisease beta-secretase BACE1 is not a neuron-specific enzyme. Journal ofNeurochemistry 92, 226–234.

Savonenko, A.V., Melnikova, T., Laird, F.M., Stewart, K.A., Price, D.L., Wong, P.C.,2008. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizo-phrenia-like phenotypes in BACE1-null mice. Proceedings of the NationalAcademy of Sciences of the United States of America 105, 5585–5590.

Scarr, E., Keriakous, D., Crossland, N., Dean, B., 2006. No change in corticalmuscarinic M2, M3 receptors or [35S]GTPgammaS binding in schizo-phrenia. Life Sciences 78, 1231–1237.

Scarr, E., Cowie, T.F., Kanellakis, S., Sundram, S., Pantelis, C. and Dean, B., inpress.Decreased corticalmuscarinic receptors definea sub-group of subjectswithschizophrenia. Molecular Psychiatry. doi:10.1038/mp.2008.28.

Shen, W., Tian, X., Day, M., Ulrich, S., Tkatch, T., Nathanson, N.M., Surmeier, D.J.,2007. CholinergicmodulationofKir2 channels selectivelyelevatesdendriticexcitability in striatopallidal neurons. Nature Neuroscience 10, 1458–1466.

Stan, A.D., Ghose, S., Gao, X.M., Roberts, R.C., Lewis-Amezcua, K., Hatanpaa, K.J.,Tamminga, C.A., 2006. Human postmortem tissue: what quality markersmatter? Brain Research 1123, 1–11.

Torrey, E.F., Webster, M., Knable, M., Johnston, N., Yolken, R.H., 2000. TheStanley foundation brain collection and neuropathology consortium.Schizophrenia Research 44, 151–155.

Tsuang, M., 2000. Schizophrenia: genes and environment. BiologicalPsychiatry 47, 210–220.

Watson, M., Yamamura, H.I., Roeske, W.R., 1983. A unique regulatory profileand regional distribution of [3H]pirenzepine binding in the rat provideevidence for distinct M1 and M2 muscarinic receptor subtypes. LifeSciences 32, 3001–3011.

Wess, J., 2003. Novel insights into muscarinic acetylcholine receptor functionusing gene targeting technology. Trends in Pharmacological Science 24,414–420.

Williams, B.P., Milligan, C.J., Street, M., Hornby, F.M., Deuchars, J., Buckley, N.J.,2004. Transcription of the M1 muscarinic receptor gene in neurons andneuronal progenitors of the embryonic rat forebrain. Journal ofNeurochemistry 88, 70–77.

Wood, I.C., Garriga, M., Palmer, C.L., Pepitoni, S., Buckley, N.J., 1999. Neuronalexpression of the rat M1 muscarinic acetylcholine receptor gene isregulated by elements in the first exon. Biochemistry Journal 340 (Pt 2),475–483.

Woods, S.W., 2003. Chlorpromazine equivalent doses for the newer atypicalantipsychotics. Journal of Clinical Psychiatry 64, 663–667.

Zavitsanou, K., Katsifis, A., Mattner, F., Huang, X.F., 2004. Investigation of m1/m4 muscarinic receptors in the anterior cingulate cortex in schizo-phrenia, bipolar disorder, and major depression disorder. Neuropsycho-pharmacology 29, 619–625.

Zavitsanou, K., Katsifis, A., Yu, Y., Huang, X.F., 2005. M2/M4 muscarinicreceptor binding in the anterior cingulate cortex in schizophrenia andmood disorders. Brain Research Bulletin 65, 397–403.

Zhang, Y., Dyck, R.H., Hamilton, S.E., Nathanson, N.M., Yan, J., 2005. Disruptedtonotopy of the auditory cortex in mice lacking M1 muscarinicacetylcholine receptor. Hearing Research 201, 145–155.

Zuchner, T., Perez-Polo, J.R., Schliebs, R., 2004. Beta-secretase BACE1 isdifferentially controlled through muscarinic acetylcholine receptorsignaling. Journal of Neuroscience Research 77, 250–257.