Accepted Manuscript

ACCEPTED MANUSCRIPT

The use of a modified [3H]4-DAMP radioligand binding assay with increased selectivity for muscarinic M3 receptor shows that cortical CHRM3 levels are not altered in mood disorders.

Won Je Jeon1,2, Andrew S. Gibbons1,2*, Brian Dean1,2

1Molecular Psychiatry Laboratory, The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia

2Department of Psychiatry, the University of Melbourne, Parkville, Victoria, Australia

Abstract

[3H]4-DAMP is a radioligand that has been used to quantify levels of the muscarinic receptor CHRM3 protein in situ. However, in addition to high affinity binding to CHRM3, [3H]4-DAMP binds with low affinity to CHRM1 confounding the potential to discriminate between changes in these two muscarinic receptors. We have developed a [3H]4-DAMP binding assay, optimised for measuring CHRM3 protein levels in the cortex, with minimal selectivity towards CHRM1. The selectivity of our assay towards CHRM3 was confirmed using recombinant receptor-expressing, cell lysate preparations. [3H]4-DAMP binding levels were similar between wildtype and CHRM1 knockout mice, confirming that the amount of [3H]4-DAMP binding to CHRM1 was negligible. We used this assay to measure CHRM3 protein levels in the frontal pole, obtained post-mortem from subjects with bipolar disorder (n = 15), major depressive disorder (n = 15) and matched controls (n = 20) and showed that [3H]4- DAMP binding was not altered in either bipolar disorder or major depressive disorder. Western BlottingACCEPTED confirmed that CHRM3 MANUSCRIPT protein levels were unchanged in these subjects.

Keywords: Radioligand binding; [3H]4-DAMP; muscarinic receptors; bipolar disorder; major depressive disorder ACCEPTED MANUSCRIPT

*Corresponding Author

Andrew Gibbons Molecular Psychiatry Laboratory The Florey Institute of Neuroscience and Mental Health Melbourne Brain Centre The University of Melbourne Parkville VIC 3010 Email: [email protected]

Abbreviations: BPD: bipolar disorder CHRM: cholinergic muscarinic receptor CON: healthy control ETE: estimated tissue equivalent IC: internal control MDD: major depressive disorder 4-DAMP: 4-diphenylacetoxy-N-methylpiperidine

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

Introduction

Radioligand binding assays are widely used to measure levels of neurotransmitter receptors in the post-mortem central nervous system (CNS) (Dean et al., 2001, Pralong et al., 2000, Zavitsanou et al., 2004). The cholinergic muscarinic receptors (CHRM) have been implicated in the pathophysiology of several psychiatric illnesses, including bipolar disorders (BPD) (Cannon et al., 2006), major depressive disorders (MDD) (Comings et al., 2002) and schizophrenia (Raedler et al., 2003), and several radio-labelled antagonists have been used to measure levels of the different CHRMs (Crook et al., 1999, Crook et al., 2001, Gibbons et al., 2009). One such radioligand, [3H]4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) ([3H]4-DAMP) has been used to measure CHRM3 levels in a number of studies (Gibbons et al., 2009, Tsang et al., 2008). However, the study of the pharmacology of these receptors has thrown doubt on the selectivity of [3H]4-DAMP for CHRM3 (Egan, 2012).

[3H]4-DAMP binds with high affinity to CHRM3 but competition binding studies using the CHRM1 antagonist pirenzepine have shown that it also binds to CHRM1 (Araujo et al., 1991, Michel et al., 1989, Pinter et al., 1999). Araujo et al (1991) reported Kd binding affinities of 0.14 to 0.53nM for CHRM3 and 50 to 59 nM for CHRM1 in the in cortical and subcortical reions of the rodent brain suggestive of high and low affinity [3H]4-DAMP binding sites, respectively. However, other studies suggest CHRM3 and CHRM1 bind with similar affinity reporting Kd values of 4nM for both receptors in the rodent frontal cortex (Zubieta and Frey, 1993). Such differences may be a result of the differing approaches to characterising the binding sites which examined competition binding at a single (Zubieta and Frey, 1993) vs both high and low affinity concentrationsACCEPTED of [3H]4-DAMP MANUSCRIPT (Araujo et al., 1991). Thus estimation of CHRM3 levels using [3H]4-DAMP may be confounded by CHRM1 binding when examining such tissue as human cortex which contains high levels of CHRM1 (Levey, 1993, Oki et al., 2005, Wei et al., 1994).

Within the frontal cortex, CHRM1 and CHRM3 show similar levels of RNA expression with CHRM3 expression predominantly localised to the superficial of deep layers of the cortex while only slight of expression levels is apparent for CHRM1 (Buckley et al., 1988, Wei et al., 1994). However, CHRM1 and CHRM3 protein expression in the frontal cortex are reported to be 25% and 7% of population of CHRMs, respectively (Buckley et al., 1988). (Levey et al., 1994). Thus, there is the ACCEPTED MANUSCRIPT

potential for changes in low affinity binding to these targets radioligands to affect receptor density estimates based on high affinity binding alone.

We have previously reported a reduction in [3H]4-DAMP binding density in Brodmann Area (BA 10) of the frontal pole from subjects with BPD but not subjects with MDD (Gibbons et al., 2009). Having become concerned about the lack of specificity of [3H]4-DAMP binding, we decided to revisit our previous study after developing a modified [3H]4-DAMP assay that had increased selectivity for CHRM3.

2. Materials and Methods

Human and animal tissue

All human tissue used in this study was obtained from the Victorian Brain Bank Network, Florey Institute for Neuroscience and Mental Health, Parkville, Australia. Approval for the study was obtained from both the Ethics Committee of the Victorian Institute of Forensic Medicine and the Mental Health Research and Ethics Committee of Melbourne Health.

Tissue from BA 10 was obtained post-mortem from 15 subjects with MDD, 15 subjects with BPD and 20 subjects with no history of psychiatric illness. This cohort was expanded from the cohort used in Gibbons et al 2009 and included all subjects previously examined in that study. BA 10 was taken as the most rostral portions of the superior frontalACCEPTED gyrus and middle frontal MANUSCRIPT gyrus, bounded ventrally by the superior rostral sulcus. For each subject, clinical case histories were used to enable a senior psychologist and psychiatrist to reach a diagnostic consensus using the Diagnostic Instrument for Brain Studies (DIBS) (Keks et al., 1999). The DIBS is a semi- structured protocol for post-mortem assessment allowing psychiatric diagnosis according to DSM-IV criteria (American Psychiatric Association, 1994). Demographic factors and tissue quality markers, including gender (CON = 13 male: 7 female; BPD = 8 male: 7 female; MDD = 8 male: 7 female), age (mean ± SD: CON = 49 ± 16 years; BPD = 57± 14 years; MDD = 56 ± 17 years), duration of illness (mean ± SD: BPD = 17 ± 11 years; MDD = 15 ± 10 years), incidence of suicide (CON = 0/19; BPD = 10/5; MDD = 3/12), post-mortem interval (PMI) (mean ± SD: CON = 36.8 ± 15.0 h; BPD = 37.0 ± 15.6 h; MDD = 43.4 ± 17.6 h) and brain pH (mean ± SD: CON = 6.30 ± ACCEPTED MANUSCRIPT

0.21; BPD = 6.25 ± 0.24; MDD = 6.52 ± 0.18) were used to assess the impact of subject variability on the experiment.

Frozen CNS tissue from 5 CHRM1-/- mice and 5 wild type (WT: same genetic background) was supplied by Lilly Research Laboratories (Indiana, IN, USA) from mice bred under contract by Taconic (Indiana, IN, USA) in accordance with NIH Guide for the Care and Use of Laboratory Animals. All mice had been euthanized by cervical dislocation. The tissue had been rapidly frozen to -70 °C immediately following dissection.

Cloned human muscarinic receptors (M1, M2, M3, and M4) CHO-K1 cell homogenates were obtained from PerkinElmer (Boston, MA, USA). The membrane homogenates stored at -80 °C until required.

Optimisation of [3H]4-DAMP binding in human and mouse tissue and cell membrane homogenates

The studies to investigate the specificity of [3H]4-DAMP binding to human cortex were completed using tissue from 3 non-psychiatric subjects. Hence, 100 μg of human membrane homogenate, in triplicates, were incubated at room temperature for 60 min in ACCEPTED500 μl.volumes of assay bufferMANUSCRIPT 1 (50 mM Tris–HCl (pH 7.4)) containing 0.05 nM, 0.1 nM, 0.5 nM, 1nM, 2.5 nM, 5 nM or 10 nM [3H]4-DAMP (PerkinElmer, Boston, MA, USA) without (total binding) and with (non-specific binding (NSB) 10 μM 4-DAMP mustard (Santa Cruz Biotechnology, Santz Cruz, CA, USA and Sigma- Aldrich, St. Louis, MO, USA). The incubation was terminated by filtration over Whatman GF/B filter paper, that had been pre-soaked in 0.1% polyethyleneimine (Sigma, St. Louis, MO, USA), using a Brandell Cell Harvester. The filters were rapidly washed three times with cold 0.9% NaCl and the radioactivity was quantified using a scintillation counter. Specific binding was calculated as the difference between the radioactivity bound in the absence (total binding) and in the presence (non-specific bind) of 10 μM 4-DAMP mustard. ACCEPTED MANUSCRIPT

All cell membrane radioligand binding assays were performed in triplicate with 15 μg of CHO-K1 control, CHRM1, CHRM2, CHRM3 and CHRM4 expressing cell homogenates being incubated in 500 μl volumes of assay buffer containing 6 nM [3H]4-DAMP with and without 1 μM pirenzepine dihydrochloride. Nonspecific binding was determined via the inclusion of 10 μM 4-DAMP mustard into the aforementioned assay volumes. The incubations were terminated by filtration Brandell Cell Harvester and processed as descried above.

[3H]4-DAMP and [3H]pirenzepine radioligand binding: in situ radioligand binding with autoradiography

To measure levels of [3H]4-DAMP and [3H]pirenzepine binding, frozen sections (20 μm) were cut from human or mouse brain tissue with a cryostat and mounted onto gelatinised slides. For each tissue sample, 3 sections/subject were used to measure total radioligand binding and 2 sections/subject were used to measure non-specific binding. To measure [3H]4-DAMP binding the sections were incubated in assay buffer 1 for 15 min at room temperature, then rinsed in water and dried. The sections were incubated for 1 h at room temperature in 6 nM [3H]4-DAMP and 1 μM pirenzepine dihydrochloride in assay buffer 1 in the presence (total binding) or absence (non-specific binding) of 10 μM 4-DAMP mustard. Following radioligand binding, the slides were washed twice for 5 min with ice cold assay buffer 1, rinsed in ice cold water, dried and partially fixed overnight in paraformaldehyde vapour prior to autoradiographyACCEPTED. MANUSCRIPT [3H]pirenzepine binding was performed using established protocols that have been previously optimised to produce highly selective binding to CHRM1 (Crook, 1998,

Scarr and Dean, 2008). Slides were incubated in a assay buffer 2 (10mM KH2PO4, 3 10 mM Na2HPO4 (pH 7.4) containing 15nM [ H]pirenzepine in the presence or absence of 1µM quinuclidinyl xanthene-9-carboxylate hemioxilate (Sigma-Aldrich, St. Louis, MO, USA) for 30 min Following radioligand binding, the slides were washed twice for 2 min in assay buffer 2 and rinsed in water dried and fixed in paraformaldehyde vapour as described above.

ACCEPTED MANUSCRIPT

Autoradiography

The fixed slides were apposed to a BAS-TR2025 plate (Fujifilm, Tokyo, Japan) with autoradiographic [3H]microscales™ (Amersham Biosciences, Little Chalfort, UK). [3H]4-DAMP labelled slides were apposed for 3 days. The plates were scanned in a BAS 5000 high resolution phosphoimager (Fujifilm, Tokyo, Japan) and the resulting images were analysed using AIS imaging software (Imaging Research, St. Catharines, ON, Canada). Radioligand binding was measured as an integrated measurement of signal intensity across the entire region of binding. Signal intensities were calibrated against the microscales and expressed as the average amount of total bound radioligand/estimated tissue equivalent (ETE) subtracted from the average non-specific binding for each subject.

Western Blotting

Homogenates were prepared from the cortical tissue at 5% w/v in 10 mM Tris (pH

7.4), 1% sodium dodecylsulfate, 1 mM Na3VO4. Protein concentrations were determined using a modified Lowry method (DC Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA) adapted for microplates. Duplicate 30 μg protein samples were electrophoresed on a 7.5% polyacrylamide gel and transferred onto nitrocellulose membranes. Protein from an independent cortical sample (Internal control) was used to standardise protein levels to control for gel to gel variation as described previously (Gibbons et al., 2008)

ACCEPTED MANUSCRIPT

Nitrocellulose membranes were blocked with 5% non-fat milk powder in Tris buffered saline and 0.1% Tween-20 (TTBS) for 1 h. Membranes were incubated in blocking buffer with a 1:1000 dilution of anti-human CHRM3 antibody (Santa Cruz Biotechnology, Santz Cruz, CA, USA) overnight at 4 °C, washed 3 times for 10 min in TTBS and then incubated with a 1:2000 dilution of donkey anti-goat secondary antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 90 min at room temperature. The membranes were washed 3 times for 10 min in TTBS and incubated for 5 min in Pierce ECL reagent (50% Peroxide 50% Luminol) at room temperature. Excess ECL solution was removed and the immunogenic bands on each membrane imaged using the Kodak CF 440 image ACCEPTED MANUSCRIPT

station. The summed intensity of the immunogenic bands were measured on the chemiluminescent image and expressed as a ratio of the optical density (OD) of the internal control to accommodate gel-to-gel variation.

RNA extraction and reverse transcription

Total RNA was be extracted from 100 mg human post-mortem cortex using Trizol® reagent (Invitrogen, Carlsbad, CA, USA). The RNA was treated with RNase-free DNase 1 (Ambion, Austin, Tx, USA) at 37 °C for 30 min to remove residual genomic DNA. RNA integrity was measured using a bioanalyser (Agilent Technologies, Santa Clara, CA, USA) with RIN values >6 deemed to be of suitable quality for qPCR.

2 μg total RNA was reverse transcribed 44 °C for 1 h in 100U M-MLV RT (Ambion, Austin, Tx, USA), 0.5 mM dNTP, 2.5 μM oligo dT primer, 2.5 μM random decamers and 1 U/μl SUPERase In RNase inhibitor (Ambion, Austin, Tx, USA) in 1 X First-

Strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT) (Ambion, Austin, Tx, USA) and then inactivated at 92 °C for 10 min. The cDNAs stored at -20 °C until required.

Quantitative real-time PCR

Quantitative PCR was performed using the iQ5 real-time PCR detection system and analysed usingACCEPTED IQ5 optical system 2.0 softwareMANUSCRIPT (Bio-Rad Laboratories, Hercules CA, USA). 50 μl reactions containing 1:125 diluted cDNA, 0.4 nM CHRM3 primers (F 5’- AGCAGCAGTGACAGTTGG-3’; R 5’-GAGCACGATGGAGTAGATGG-3’) and 1×IQ SYBR green supermix (Bio-Rad Laboratories, Hercules CA, USA) were denatured at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 30 s and 72 °C for 30 s. Samples were run in triplicate and the mRNA levels calculated from the log 2 of the average triplicate value corrected for primer efficiency. Expression was normalised against the geometric mean of the levels of three stably expressed reference genes, as previously described (Dean et al., 2012).

Statistical Analysis ACCEPTED MANUSCRIPT

Statistical analyses were performed using Prism 5.01 software (Graphpad Software, La Jolla, CA, USA) and Minitab 16.1.0 (Minitab, State College, PA, USA). The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) values for [3H]4-DAMP to membrane were measured by analysis of the saturation data, and Scatchard plots were derived from full saturation analysis of [3H]4-DAMP binding. Data sets were analysed using the D’Agostino & Pearson omnibus test to assess the normaility of the distribution followed by a Grubbs test to detect outliers. One-way ANOVA followed by Tukey's multiple comparison test were used to analyse the levels of CHRM3 protein and mRNA in [3H]4-DAMP binding, Western blot and qPCR in BA10. Kusskal-Wallis test was used to analyse [3H]prienzepine binding. The relationship between demographic and tissue condition data were assessed using Pearson product moment correlation. Relationships with r2 values greater than 0.70 were considered strong enough to have a significant impact on our data, as is appropriate for the small sample sizes used in these experiments (Gliner et al., 2002).

3. Results

[3H]4-DAMP binding assay for CHRM3 selectivity in human frontal cortex, mouse brain and CHRM transfected CHO-K1 cells

Scatchard analysis of our original [3H]4-DAMP binding assay used in previous studies revealed the presence of a high (Kd1 = 0.37 ± 0.06 nM; Bmax1 = 208.7 ± 16.03 fmol/mg) andACCEPTED low (Kd2 = 1.56 ± 0.26 nMMANUSCRIPT; Bmax2 = 455.5 ± 24.4 fmol/mg) affinity binding site following a 60 min incubation period (Fig. 1A). Extending the incubation time to 2hrs did not significantly affect this data confirming that the binding sites were saturated after 1 hr. Thus, in subsequent assays the level of [3H]4-DAMP was set at a concentration (6 nM: 3-fold greater) that would saturate all available binding studies.

Building on known the known pharmacology of [3H]4-DAMP (Araujo et al., 1991), we investigated whether the CHRM1-selective antagonist pirenzepine, which has low affinity for CHRM3 (Zubieta and Frey, 1993), could be used to displace [3H]4-DAMP binding from cortical homogenate. Competition binding analysis showed the addition ACCEPTED MANUSCRIPT

of >10 μM 4-DAMP mustard displaced 60% of [3H]4-DAMP binding in human cortical tissue while the addition of pirenzepine at >1 μM concentration reduced binding by a further 30% (Fig. 1B) with the remaining binding likely to reflect cumulative binding to low affinity targets. From these data we postulated that in the presence of 1 μM pirenzepine the displacement of [3H]4-DAMP with 10 μM 4-DAMP mustard would be highly CHRM3 selective.

To challenge our hypothesis that we could develop a highly CHRM3 selective [3H]4- DAMP binding assay we measured the binding of that radioligand in the absence and presence of 4-DAMP mustard and in the absence or presence of pirenzepine to i) the cortex from wild type and CHRM1-/- mice and ii) membrane CHO-K1 cell homogenates containing either human CHRM1, CHRM2, CHRM3 or CHRM4 receptor. There was a 59% loss of [3H]4-DAMP binding to the cortex from CHRM1-/- mice compared to wildtype (Fig 2A), supporting the notion that the 60% pirenzepine binding to human from cortex was to the CHRM1. Significantly, similar to human cortex, pirenzepine reduced [3H]4-DAMP binding by 51% (p < 0.001) to the cortex of wildtype mice but did not significantly affect the level of the binding of [3H]4-DAMP binding to the cortex from CHRM1-/- (p > 0.05) (Fig. 2A). The rank order of [3H]4- DAMP binding to cloned human CHRMs was CHRM3>>CHRM1=CHRM4>CHRM2 (Fig. 2B). Pirenzepine (1μM) reduced binding of [3H]4-DAMP to CHRM1(70%)= CHRM4 (70%)>CHRM2 (39%)>>CHRM3 (11%).

Radioligand binding, Western blot and qPCR in psychiatric cases ACCEPTED MANUSCRIPT The modified [3H]4-DAMP binding assay, carried out in the presence of 1μM pirenzepine, was used to measure [3H]4-DAMP binding in BA 10 from subjects with mood disorders and age and sex matched controls. In addition, CHRM3 protein levels were measured using Western blotting whilst levels of CHRM3 mRNA were measured using. The distribution of all data sets were normally distributed (K2 = 0.285 to 5.840; p = 0.054 to 0.867) and, therefore, parametric statistical tests were used for all analyses. Despite a lower, albeit not significant (F = 1.37, df = 2,47, p = 0.26) difference between the age of the control group and the BPD and MDD groups, there was no strong correlation between age and the radioligand binding (0.07 < r2 < 0.18), Western blotting (0.01 < r2 < 0.22) and qPCR (0.01 < r2 < 0.35) data. There were also no strong correlations between our data and PMI, CNS pH, DOI or RIN ACCEPTED MANUSCRIPT

(0.00 < r2 < 0.40). The binding density [3H]4-DAMP was homogeneous across the entire grey matter. The levels of binding was not altered in BPD or MDD compared to controls (F = 0.45, df = 2,47, p = 0.64). Western blotting confirmed that there was no significant change in CHRM3 protein with diagnoses (F = 0.42, df = 2,47, p = 0.66). Furthermore, qPCR showed there was no significant change in the levels of CHRM3 mRNA with diagnoses (F = 1.47, df = 2,46, p = 0.24), providing no evidence that CHRM3 expression is altered in either BPD or MDD (Table 1).

We performed [3H]pirenzepine binding in our cohort, under assay conditions that were previously optimised produce highly selective binding to CHRM1 in the frontal cortex with minimal binding to other CHRMs (Crook, 1998, Scarr and Dean, 2008), to determine whether the discrepancy between the data produced with our modified [3H]4-DAMP binding assay and our previous findings of decreased [3H]4-DAMP binding in BPD were due to a decrease in CHRM1 levels. Non-parametric analyses were performed as the distribution of the [3H]pirenzepine data was not normally distributed across all data sets (K2 = 0.644 to 6.320; p = 0.725 to 0.042). There was a trend towards decreased [3H]pirenzepine binding in both MDD and BPD, however, this failed to reach significance (F = 0.45, df = 2,47, p = 0.07) (Table 1). These findings could not be further explored using western blotting or qPCR due to the limited availability of tissue from our cohort.

4. Discussion

Radioligand bindingACCEPTED has become a staple MANUSCRIPT tool in post-mortem research for studying receptor systems. However, interpretation of radioligand binding data can be difficult when the available radioligands do not selectively target individual receptors. In this study we have modified an existing [3H]4-DAMP binding methodology in a way that, under the conditions we describe, is highly selective for CHRM3. This posit is supported by data from CHRM1-/- mice and cloned human CHRMs (1-4). We argue this modified methodology overcomes concerns about [3H]4-DAMP binding to CHRM1 (Araujo et al., 1991) and the potential for the radioligand to bind to CHRM2 and CHRM4 (Tsang et al., 2008). Although we did not determine if [3H]4-DAMP, under the conditions we use, binds to CHRM5, in the human cortex levels of CHRM5 are so low (Vilaro et al., 1990, Yasuda et al., 1993) it is unlikely that measurable ACCEPTED MANUSCRIPT

changes in 3H]4-DAMP binding would result from a loss of all these receptors in human cortex.

Using our modified [3H]4-DAMP binding protocol we did not show a difference in radioligand binding in BA 10 from subjects with BPD, which contradicts our finding using a non-modified [3H]4-DAMP assay (Gibbons et al., 2009). Importantly, in this study we use larger cohorts and have shown that levels of CHRM3 protein and mRNA are not altered in BA10 from subjects with BPD and MDD compared to controls. These data are consistent with the conclusion that levels of CHRM3 are not altered in BA 10 from people with BPD. We used the CHRM1 selective radioligand [3H]pirenzepine to assess the contribution of possible chenges in CHRM1 levels to the decreased in [3H]4-DAMP binding seen in our previous study (Gibbons et al., 2009). The relative Kd’s of [3H]pirenzepine for CHRM1 and CHRM3 in the rat cortex have been reported as 20nm and 500nm, respectively (Zubieta and Frey, 1993), and we have previously optimised our [3H]pirenzepine binding assay to be highly selective for CHRM1 (Crook, 1998, Scarr and Dean, 2008). A trend towards decreased [3H]pirenzepine binding density, was seen in both BPD and MDD compared to controls. In agreement with our previous study (Gibbons et al., 2009), this decrease in [3H]pirenzepine binding was not significant, however, the failure to see this trend in our previous data is likely due to the lower statistical power in that study caused by a smaller sample size. However, our current data suggests that the decrease in [3H]4-DAMP binding in our previous study could have been caused by a cumulative effect of marginal decreases in the level of multiple targets of [3H]4- DAMP, including CHRM1 as well as CHRM2 and CHRM4 which also appear to be displaced in ACCEPTED CHRM overexpressing CHO MANUSCRIPT-K1 cells following the addition of 1M pirenzepine to our assay. A limitation of this study is that we were unable to ascertain the incidence of tobacco use amongst our cohort. It is possible that differing rates of nicotine use between the cohort groups may have influenced our data. However, previous studies have shown that repeated administration of nicotine to rats does not alter binding of the CHRM radioligand [3H]N-methylscopolamine in the cortex (Yamamoto et al., 2011) suggesting our data is unlikely to have been affected by smoking history.

In addition to our previous study in mood disorders (Gibbons et al., 2009), [3H]4- DAMP binding assays have been used to assess differences in CHRM3 protein ACCEPTED MANUSCRIPT

expression in a number of studies. Much of our understanding of CHRM3 distribution within the brain and other organs has been based on spatial analysis of [3H]4-DAMP binding densities (Araujo et al., 1991, Vanwaarde et al., 1994). A loss of [3H]4-DAMP binding density has been reported in the orbitofrontal cortex from subjects with Alzheimers disease who displayed psychotic symptoms (Tsang et al., 2008). [3H]4- DAMP is not altered in the premotor cortex of subjects with schizophrenia (Dean et al., 2008). However, widespread cortical reductions in [3H]4-DAMP binding have been reported in a subpopulation subjects with schizophrenia characterised by a loss of CHRM1 in the dorsolateral prefrontal cortex (Gibbons et al., 2012). Importantly, reduced [3H]pirenzepine binding was also reported in the regions displaying reduced [3H]4-DAMP binding levels (Gibbons et al., 2012). Therefore, it is unclear whether these changes in radioligand binding densities reflect a loss of both CHRM1 and CHRM3 expression or just CHRM1. In light of the potential for low affinity CHRM1 binding to confound [3H]4-DAMP binding data, these findings may need to be revisited to clarify the role of CHRM3 in their findings.

Conclusions

We have developed a radioligand binding assay for the selective measurement of CHRM3 in the cortex and used this to clarify whether CHRM3 is involved in the pathophysiology of mood disorders. Our findings of unaltered CHRM3 protein and mRNA in the CNS of subjects with MDD and BPD do not support a role for this receptor in mood disorders. However, our findings do not exclude the possibility the functional pool of CHRM3 receptors or the activity of the receptors may be altered in ACCEPTEDmood disorders compared MANUSCRIPT to controls. Furthermore our data does not preclude a role for the cholinergic system in mood disorders, as decreased binding of the CHRM2/CHRM4 selective radioligand [3H]AF DX-384 binding has also been reported in the post-mortem CNS of subjects with MDD and BPD (Gibbons et al., 2009); a finding supported by a neuroimaging data showing decreased CHRM2 in the anterior cingulate of patients with BPD (Cannon et al., 2006, Cannon et al., 2011). Thus, further study is needed to understand the role of other CHRMs in the pathophysiology of mood disorders.

ACCEPTED MANUSCRIPT

Acknowledgements

Preparation of the CHO-K1 cells and post-mortem tissue was performed by Madhara Udawela and Geoffrey Pavey, respectively. Brian Dean is an NHMRC Senior Research Fellow. This work was supported in part by NHMRC project grant #628699 and the Victorian Government’s Operational Infrastructure Support.

References

American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington, DC: American Psychiatric Press; 1994. Araujo DM, Lapchak PA, Quirion R. Heterogenous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: Evidence from membrane binding and autoradiographic studies. Synapse. 1991;9:165-76. Buckley NJ, Bonner TI, Brann MR. Localization of a family of muscarinic receptor messenger-RNAs in rat-brain. J Neurosci. 1988;8:4646-52. Cannon DM, Carson RE, Nugent AC, Eckelman WC, Kiesewetter DO, Williams J, et al. Reduced muscarinic type 2 receptor binding in subjects with bipolar disorder. Arch Gen Psychiatry. 2006;63:741-7. Cannon DM, Klaver JK, Gandhi SK, Solorio G, Peck SA, Erickson K, et al. Genetic variation in cholinergic muscarinic-2 receptor gene modulates M-2 receptor binding in vivo and accounts for reduced binding in bipolar disorder. Mol Psychiatr. 2011;16:407-18. Comings DE, Wu S, Rostamkhani M, McGue M, Iacono WG, MacMurray JP. Association of the muscarinic cholinergic 2 receptor (CHRM2) gene with major depression in women. Am J Med Genet. 2002;114:527-9. Crook JM. A possible role for muscarinic cholinergic receptors in the pathology and treatment of schizophrenia. : University of Melbourne; 1998. Crook JM, Dean B, Pavey G, Copolov D. The binding of H-3 AF-DX 384 is reduced in the Caudate-Putamen of subjects with schizophrenia. Life Sci. 1999;64:1761-71. Crook JM, TomaskovicACCEPTED-Crook E, Copolov MANUSCRIPT DL, Dean B. Low muscarinic receptor binding in prefrontal cortex from subjects with schizophrenia: A study of Brodmann's areas 8, 9, 10, and 46 and the effects of neuroleptic drug treatment. Am J Psychiat. 2001;158:918-25. Dean B, Gibbons AS, Tawadros N, Brooks L, Everall IP, Scarr E. Different changes in cortical tumor necrosis factor-[alpha]-related pathways in schizophrenia and mood disorders. Mol Psychiatry. 2012. Dean B, Pavey G, McLeod M, Opeskin K, Keks N, Copolov D. A change in the density of [H-3]flumazenil, but not [H-3]muscimol binding, in Brodmann's Area 9 from subjects with bipolar disorder. J Affect Disord. 2001;66:147-58. Dean B, Soulby A, Evin GM, Scarr E. Levels of [H-3]pirenzepine binding in Brodmann's area 6 from subjects with schizophrenia is not associated with changes in the transcription factor SP1 or BACE1. Schizophr Res. 2008;106:229-36. ACCEPTED MANUSCRIPT

Egan RM. Muscarinic Receptors. In: Fryer AD, Christopoulos A, Nathanson NM, editors. Handbook of Experimental Pharmacology. Germany: Springer; 2012. p. 3- 28. Gibbons AS, Scarr E, Boer S, Money T, Jeon WJ, Felder C, et al. Widespread decreases in cortical muscarinic receptors in a subset of people with schizophrenia. The International Journal of Neuropsychopharmacology. 2012:1-10. Gibbons AS, Scarr E, McLean C, Sundram S, Dean B. Decreased muscarinic receptor binding in the frontal cortex of bipolar disorder and major depressive disorder subjects. J Affect Disord. 2009;116:184-91. Gibbons AS, Scarr E, McOmish CE, Hannan AJ, Thomas EA, Dean B. Regulator of G-protein signalling 4 expression is not altered in the prefrontal cortex in schizophrenia. Aust N Z J Psych. 2008;42:740-5. Gliner JA, Morgan GA, Harmon RJ. Basic associated designs: analysis and interpretation. Journal of the American Academy of Child and Adolescent Psychiatry. 2002;41:1256-8. Keks N, Hill C, Opeskin K, Copolov DL, Dean B. Psychiatric diagnosis after death: the problems of accurate diagnosis. In: Dean B, Hyde TM, Klienman JE, editors. Using CNS Tissue in Psychiatry Research: a Practical Guide. Sydney: J. Gordon & Breach Science Publishers; 1999. p. 19– 37. Levey AI. Immunological localization of m1-m5 muscarinic acetylcholine receptors in peripheral tissues and brain. Life Sci. 1993;52:441-8. Levey AI, Edmunds SM, Heilman CJ, Desmond TJ, Frey KA. LOCALIZATION OF MUSCARINIC M3 RECEPTOR PROTEIN AND M3 RECEPTOR-BINDING IN RAT- BRAIN. Neuroscience. 1994;63:207-21. Michel AD, Stefanich E, Whiting RL. Direct Labeling of Rat M3-Muscarinic Receptors by H-3 4damp. Eur J Pharmacol. 1989;166:459-66. Oki T, Takagi Y, Inagaki S, Taketo MM, Manabe T, Matsui M, et al. Quantitative analysis of binding parameters of [3H]N-methylscopolamine in central nervous system of muscarinic acetylcholine receptor knockout mice. Brain Res Mol Brain Res. 2005;133:6-11. Pinter I, Moszkovszkin G, Nemethy Z, Makara GB. Muscarinic M-1 and M-3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland. Brain RACCEPTEDes Bull. 1999;48:449-56. MANUSCRIPT Pralong D, Tomaskovic-Crook E, Opeskin K, Copolov D, Dean B. Serotonin(2A) receptors are reduced in the planum temporale from subjects with schizophrenia. Schizophr Res. 2000;44:35-45. Raedler TJ, Knable MB, Jones DW, Urbina RA, Gorey JG, Lee KS, et al. In vivo determination of muscarinic acetylcholine receptor availability in schizophrenia. Am J Psychiat. 2003;160:118-27. Scarr E, Dean B. Muscarinic receptors: do they have a role in the pathology and treatment of schizophrenia? J Neurochem. 2008;107:1188-95. Tsang SWY, Francis PT, Esiri MM, Wong PTH, Chen C, Lai MKP. Loss of H-3 4- DAMP binding to muscarinic receptors in the orbitofrontal cortex of Alzheimer's disease patients with psychosis. Psychopharmacology. 2008;198:251-9. Vanwaarde A, Visser GM, Visser TJ, Bouwer J, Paans AM, Vaalburg W. Rodent biodistribution and metabolism of tritiated 4-DAMP, a M(3) subtype-selective cholinoceptor ligand. Nucl Med Biol. 1994;21:41-7. ACCEPTED MANUSCRIPT

Vilaro MT, Palacios JM, Mengod G. Localization of m5 muscarinic receptor mRNA in rat brain examined by in situ hybridization histochemistry. Neuroscience letters. 1990;114:154-9. Wei J, Walton EA, Milici A, Buccafusco JJ. m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC. J Neurochem. 1994;63:815-21. Yamamoto N, Yoshida A, Taki Y, Onoue S, Kagawa Y, Yamada S. Up-regulation of nicotinic and muscarinic receptor mRNA in rat bladder by repeated administration of nicotine in relation to the pharmacokinetics. Life Sci. 2011;89:343-8. Yasuda RP, Ciesla W, Flores LR, Wall SJ, Li M, Satkus SA, et al. Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: distribution of m4 and m5 receptors in rat brain. Molecular pharmacology. 1993;43:149-57. Zavitsanou K, Katsifis A, Mattner F, Huang X-F. Investigation of M1/M4 muscarinic receptors in the anterior cingulate cortex in schizophrenia, bipolar disorder, and major depression disorder. Neuropsychopharmacology. 2004;29:619–25. Zubieta JK, Frey KA. Autoradiographic mapping of M3-muscarinic-receptors in the rat-brain. J Pharmacol Exp Ther. 1993;264:415-22.

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

CHRM3 Control BPD MDD p value

[3H]4-DAMP 62.24 58.76 63.01 F = 0.45, df = 2,47, binding ± 10.98 ± 13.88 ± 15.19 p = 0.64

1.26 1.19 1.25 F = 0.42, df = 2,47, Western Blotting ± 0.21 ± 0.30 ± 0.24 p = 0.66

1.15 1.03 1.17 F = 1.47, df = 2,46, qPCR ± 0.29 ± 0.21 ± 0.19 p = 0.24

CHRM1

[3H]pirenzepine 77.23 ± 59.94 ± 60.17 ± F = 0.45, df = 2,47, binding 13.12 25.46 27.36 p = 0.07

Table 1: The levels of CHRM3 and CHRM1 binding in Brodmanns Area 10 from subjects with bipolar disorder (BPD) or major depressive disorder (MDD). CHRM3 levels have been measured by [3H]4-DAMP binding, Western blotting and qPCR, whilst CHRM1 levels have been measured by [3H]pirenzepine binding. Neither CHRM3 protein or mRNA expression was significantly altered in BPD or MDD compared to control subjects. A trend towards decreased CHRM1 levels failed to reach significance. All measurements are presented as mean ± SD. [3H]4-DAMP and [3H]pirenzepine binding data are expressed in fmol/mg estimated tissue equivalents, Western blotting data as a ratio of the internal control and qPCR data as the relative expression to 3 reference genes.

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

A B

Figure 1. [3H]4-DAMP binding to human cortex homogenates, sections of mouse brain and cloned human muscarinic receptors (M1 and M3) CHO-K1 cell homogenates. (A) Saturation curves of [3H]4-DAMP binding to three different human cortexes and a the scatchard plot derived from full saturation analysis of [3H]4-DAMP binding (inset). Specific binding was determined by substracting the non-specific binding from the total binding. (B) The displacement of [3H]4-DAMP binding by 4- DAMP mustard and pirenzepine at varying concentrations in human cortical tissue. Sections were incubated with 6 nM [3H]4-DAMP and 10-9 to 10-4 M 4-DAMP mustard or 10-6 M 4-DAMP mustard and 10-9 to 10-4 M pirenzepine.

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

A B

***

*** ** ***

[3H]4-DAMP alone

[3H]4-DAMP + 1M pirenzepine

Figure 2. The muscarinic receptor selectivity of the modified [3H]4-DAMP binding assay. (A) Densities of [3H]4-DAMP binding in the presence or absence of 1 μM pirenzepine in sections of WT and CHRM1 KO mice brain. (B) 6 nM radioligand [3H]4-DAMP in the presence or absence of 1 μM pirenzepine was incubated for 60 min at room temperature with and homogenates with CHRM1, CHRM2, CHRM3 and CHRM4 over-expressing CHO-K1 cells. Graphs show mean ± SD, *** = P<0.001, ** = P<0.01

ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT

Research Highlights

We have developed a modified, [3H]4-DAMP radioligand binding assay that is highly selective towards CHRM3.

We have shown that [3H]4-DAMP binding is not altered in bipolar disorder or major depressive disorders.

Using western blotting we have confirmed that CHRM3 protein levels are not altered in these mood disorders.

We have shown that CHRM3 mRNA levels are not altered in bipolar disorder or major depressive disorders suggesting CHRM3 is not involved in the pathophysiology of mood disorders.

ACCEPTED MANUSCRIPT

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Jeon, WJ; Gibbons, AS; Dean, B

Title: The use of a modified [H-3]4-DAMP radioligand binding assay with increased selectivity for muscarinic M3 receptor shows that cortical CHRM3 levels are not altered in mood disorders

Date: 2013-12-02

Citation: Jeon, W. J., Gibbons, A. S. & Dean, B. (2013). The use of a modified [H-3]4-DAMP radioligand binding assay with increased selectivity for muscarinic M3 receptor shows that cortical CHRM3 levels are not altered in mood disorders. PROGRESS IN NEURO- PSYCHOPHARMACOLOGY & BIOLOGICAL PSYCHIATRY, 47, pp.7-12. https://doi.org/10.1016/j.pnpbp.2013.08.001.

Publication Status: Accepted manuscript

Persistent Link: http://hdl.handle.net/11343/43997