M Muscarinic Acetylcholine Receptor Modulation of Dopamine Receptor … · 2017. 3. 28. · M 4 Muscarinic Acetylcholine Receptor Modulation of Dopamine Receptor Functions Nae-Yng

M4 Muscarinic Acetylcholine Receptor Modulation

of Dopamine Receptor Functions

Nae-Yng Amy Chen

BPharmSc (Hons)

A thesis submitted for the degree of Doctor of Philosophy

at Monash University in 2016

Drug Discovery Biology

Monash Institute of Pharmaceutical Sciences

Faculty of Pharmacy and Pharmaceutical Sciences

Monash University

Parkville, Victoria, Australia

Copyright notice

© Nae-Yng Amy Chen (2016). Except as provided in the Copyright Act 1968, this

thesis may not be reproduced in any form without the written permission of the

author.

I CERTIFY THAT I HAVE MADE ALL REASONABLE EFFORTS TO SECURE

COPYRIGHT PERMISSIONS FOR THIRD-PARTY CONTENT INCLUDED IN

THIS THESIS AND HAVE NOT KNOWINGLY ADDED COPYRIGHT CONTENT

TO MY WORK WITHOUT THE OWNER'S PERMISSION.

Table of Contents

i

Table of Contents

List of Figures .................................................................................................................... viii

List of Tables ....................................................................................................................... xii

List of Abbreviations ......................................................................................................... xiii

Abstract ............................................................................................................................... xvi

Publications ..................................................................................................................... xviii

Declaration.......................................................................................................................... xix

Acknowledgements ............................................................................................................. xx

Chapter 1: General Introduction ........................................................................................... 1

1.1 G Protein-Coupled Receptors ................................................................................. 2

1.1.1 GPCR Classification ........................................................................................ 2

1.1.2 GPCR Signalling ............................................................................................. 3

1.1.3 GPCR Allostery ............................................................................................... 6

1.2 Schizophrenia ............................................................................................................ 11

1.2.1 GPCRs in Schizophrenia ............................................................................... 13

1.2.2 Muscarinic Acetylcholine Hypothesis of Schizophrenia............................... 16

1.3 Muscarinic Acetylcholine Receptors ......................................................................... 19

1.3.1 M1 mAChR .................................................................................................... 19

1.3.2 M2 mAChR .................................................................................................... 20

1.3.3 M3 mAChR .................................................................................................... 21

Table of Contents

ii

1.3.4 M4 mAChR .................................................................................................... 21

1.3.5 M5 mAChR .................................................................................................... 23

1.4 Challenges in Translational Research ................................................................... 25

1.5 Prepulse Inhibition and Locomotor Activity ........................................................ 28

1.5.1 Prepulse Inhibition of the Startle Reflex ...................................................... 28

1.5.2 Locomotor Activity ...................................................................................... 31

1.6 Scope of Thesis ..................................................................................................... 33

Chapter 2: Detection and Quantification of Allosteric Modulation of Endogenous M4

Muscarinic Acetylcholine Receptor Using Impedance-Based Label-Free Technology in a

Neuronal Cell Line .............................................................................................................. 35

Chapter 3: Determination of Signalling Cross-Talk between M4 Muscarinic Acetylcholine

and Dopamine Receptors Endogenously Expressed in a Neuronal Cell Line ..................... 45

3.1 Introduction ........................................................................................................... 46

3.2 Materials and Methods .......................................................................................... 49

3.2.1 Materials ........................................................................................................ 49

3.2.2 Cell Culture ................................................................................................... 49

3.2.3 cAMP Bioluminescence Resonance Energy Transfer Biosensor Assay ....... 50

3.2.4 ERK1/2 Phosphorylation Assay .................................................................... 50

3.2.5 Data Analysis ................................................................................................. 52

3.3 Results ................................................................................................................... 53

3.3.1 NG108-15 Cells Endogenously Express D2-like, but Not D1-like, Dopamine

Receptors ..................................................................................................................... 53

Table of Contents

iii

3.3.2 Interaction Studies Reveal a Lack of Signalling Cross-Talk between M4

Muscarinic Acetylcholine and D2-like Dopamine Receptor Ligands ......................... 58

3.4 Discussion ............................................................................................................. 66

Chapter 4: Studying the Effect of Positive Allosteric Modulation of M4 Muscarinic

Acetylcholine Receptors on Psychosis-like Behaviours Induced by a D1 Dopamine

Receptor-selective Agonist in Mice .................................................................................... 70

4.1 Introduction ........................................................................................................... 71

4.2 Material and Methods ........................................................................................... 75

4.2.1 Materials ........................................................................................................ 75

4.2.2 Cell Culture ................................................................................................... 75

4.2.3 Preparation of Cell Membranes ..................................................................... 76

4.2.4 Radioligand Binding Assays in Membrane Preparations .............................. 76

4.2.5 ERK1/2 Phosphorylation Assays................................................................... 77

4.2.6 Animals .......................................................................................................... 78

4.2.7 Drugs ............................................................................................................. 79

4.2.8 Prepulse Inhibition of the Acoustic Startle Response (PPI) .......................... 80

4.2.9 Locomotor Activity (LMA) ........................................................................... 82

4.2.10 Assessment of Compound Exposure in Brain and Plasma ............................ 82

4.2.11 Data and Statistical Analysis ......................................................................... 85

4.3 Results ................................................................................................................... 90

Table of Contents

iv

4.3.1 Potentiation of ACh Function at M4 mAChRs by a Next Generation M4

Muscarinic Receptor Positive Allosteric Modulator, ML253, is Subject to Species

Variability .................................................................................................................... 90

4.3.2 In Vitro and In Vivo Characterisation of R(+)-6-Br-APB, a Selective D1

Dopamine Receptor Agonist ....................................................................................... 95

4.3.3 Drug Vehicles do not Affect Prepulse Inhibition and Locomotor Activity

compared to Saline and Water for Injection in Mice................................................. 102

4.3.4 Assessment of Compound Exposure in Plasma and Brain .......................... 104

4.3.5 Treatments of LY2033298 alone or with Donepezil, an Acetylcholinesterase

Inhibitor, Showed a Trend to Reverse Disruption of Prepulse Inhibition Induced by

R(+)-6-Br-APB .......................................................................................................... 106

4.3.6 Combined Treatment of LY2033298 and Donepezil Reversed

Hyperlocomotor Activity Induced by R(+)-6-Br-APB ............................................. 108

4.4 Discussion ........................................................................................................... 111

Chapter 5: Studying the Role of M4 Muscarinic Acetylcholine Receptors in the

Modulation of D1 Dopamine Receptor Function Using Whole-body Knockout Mice ..... 118

5.1 Introduction ......................................................................................................... 119

5.2 Material and Methods ......................................................................................... 121

5.2.1 Animals ........................................................................................................ 121

5.2.2 Drugs ........................................................................................................... 123

5.2.3 Prepulse Inhibition of the Acoustic Startle Response (PPI) ........................ 123

5.2.4 Locomotor Activity (LMA) ......................................................................... 123

5.2.5 Data and Statistical Analysis ....................................................................... 123

Table of Contents

v

5.3 Results ................................................................................................................. 126

5.3.1 When Re-tested, M4-/- Mice Exhibited Reduced Startle Amplitude and

Improved Prepulse Inhibition, but Exhibited No Change in Locomotor Activity .... 126

5.3.2 M4-/- Mice Exhibit Phenotypic Differences in PPI and LMA Compared to

M4+/+ Mice ................................................................................................................. 129

5.3.3 Determination of the Role of M4 mAChRs in the Modest Reversal of R(+)-6-

Br-APB Treatment-Induced Disruption of Prepulse Inhibition by LY2033298 and

Donepezil Treatments Using M4-/- Mice was Inconclusive ....................................... 132

5.3.4 R(+)-6-Br-APB 1 mg/kg Dose Induces Stereotypic Behaviour in

C57Bl/6NTac Wildtype Mice Not Seen in C57Bl/6J Mice ...................................... 135

5.3.5 LY2033298, Donepezil or LY2033298 and Donepezil Combined Treatment

Decreased Hyperlocomotor Activity Induced by R(+)-6-Br-APB in M4-/- Mice ...... 137

5.4 Discussion ........................................................................................................... 140

Chapter 6: General Discussion ......................................................................................... 147

Appendix 1: Chapter 3 Supporting Information ............................................................... 156

Appendix 1.1: Parameters for functional interaction between M4 mAChR and D2 DR

ligands ............................................................................................................................ 157


Appendix 2.1: Effect of V1 + V2 + R(+)-6-Br-APB 0.1 – 1 mg/kg on acoustic startle

and PPI at 100 and 110 dB pulse intensities in C57Bl/6J mice ..................................... 160

Appendix 2.2: LMA after V1 + V2 + R(+)-6-Br-APB administration in C57Bl/6J mice

....................................................................................................................................... 161

Table of Contents

vi

Appendix 2.3: Comparison between saline + saline + V3 and V1 + V2 + V3 treatments

on acoustic startle and PPI at 100 and 110 dB pulse intensities in C57Bl/6J mice ....... 162

Appendix 2.4: Comparison between saline + saline + V3 and V1 + V2 + V3 treatments

on LMA over time in C57Bl/6J mice ............................................................................ 163

Appendix 2.5: Effect of LY2033298 treatment, with or without donepezil, on

hyperlocomotor activity induced by R(+)-6-Br-APB in C57Bl/6J mice ....................... 164

Appendix 2.6: Snake plot of the mouse M4 mAChR, with residues different from the

human receptor highlighted in red ................................................................................. 166


Appendix 3.1: Effect of re-testing on LMA in C57Bl/6NTac M4-/- mice ..................... 168

Appendix 3.2: Comparison of PPI at 100, 110 and 120 dB pulse intensities between V1

+ V2 + V3 treated M4+/+ and M4

-/- mice on a C57Bl/6NTac background ..................... 169

Appendix 3.3: Comparison of baseline LMA between V1+V2+V3 treated M4+/+ and M4

-

/- mice ............................................................................................................................. 170

Appendix 3.4: Effect of LY2033298 treatment, with or without donepezil, on disruption

of PPI induced by R(+)-6-Br-APB in M4+/+ and M4

-/- mice on a C57Bl/6NTac

background at 120 dB pulse intensity ........................................................................... 171

Appendix 3.5: LMA post R(+)-6-Br-APB administration in C57Bl/6NTac M4+/+ mice

....................................................................................................................................... 172


hyperlocomotor activity induced by R(+)-6-Br-APB in C57Bl/6NTac M4+/+ mice ...... 173


hyperlocomotor activity induced by R(+)-6-Br-APB in C57Bl/6NTac M4-/- mice ....... 174

Table of Contents

vii

References ......................................................................................................................... 175

List of Figures

viii

List of Figures

Chapter 1

Figure 1.1 G protein activation/deactivation cycle............................................................4

Figure 1.2 Simplified signal transduction pathways of Gαs, Gαi and Gβγ proteins...........5

Figure 1.3 Schematic diagram of allosteric modulation of drug action and the

operational model of allosterism......................................................................8

Figure 1.4 Schematic representation of the dopaminergic and cholinergic systems in the

rodent brain.....................................................................................................15

Figure 1.5 Neural network of startle reflex and PPI........................................................30

Figure 1.6 Schematic representation of the direct and indirect pathways of the basal

ganglia circuit in the rodent brain...................................................................32

Chapter 2

Figure 1 LY2033298 potentiates inhibition of forskolin-induced cAMP by

acetylcholine...................................................................................................37

Figure 2 LY2033298 potentiates acetylcholine-induced ERK1/2 phosphorylation.....40

Figure 3 Positive allosteric modulation of acetylcholine by LY2033298 can be

detected with xCELLigence...........................................................................42

Figure 4 Overall change in impedance induced by acetylcholine is predominately Gαi

protein dependent............................................................................................43

List of Figures

ix

Chapter 3

Figure 3.1 D2-like DRs are endogenously expressed in undifferentiated NG108-15

cells.................................................................................................................54

Figure 3.2 DR ligands and ACh inhibit cAMP accumulation in NG108-15 cells

differentiated via different methods................................................................56

Figure 3.3 Chemical structures of M4 mAChR and D2 DR ligands used in interaction

studies.............................................................................................................58

Figure 3.4 M4 mAChR and D2 DR ligands are selective for their respective receptors..59

Figure 3.5 Interaction study between M4 mAChR and D2 DR agonists and antagonists in

NG108-15 cells did not reveal functional interaction between these two

receptors in cAMP accumulation and ERK1/2 phosphorylation assays........61

Figure 3.6 Interaction study between DA and ACh in NG108-15 cells showed additive

agonist effects in ERK1/2 phosphorylation assay..........................................62

Figure 3.7 Interaction study between M4 mAChR PAM, LY2033298, and DA in

NG108-15 cells did not reveal functional interaction between these two

receptors in cAMP accumulation and ERK1/2 phosphorylation assays........63

Figure 3.8 Interaction study between DA or ACh and LY2033298 in the presence of low

concentration of the other orthosteric agonist in NG108-15 cells..................65

Chapter 4

Figure 4.1 Timeline of behavioural experiments.............................................................81

List of Figures

x

Figure 4.2 Chemical structures of LY2033298 and ML253 (M4 mAChR PAMs),

donepezil (acetylcholinesterase inhibitor) and R(+)-6-Br-APB (D1 DR-

selective agonist)............................................................................................91

Figure 4.3 LY2033298 and ML253 potentiation of ACh-induced ERK1/2

phosphorylation in CHO cells stably expressing human or mouse M4

mAChRs are subject to species variability.....................................................91

Figure 4.4 R(+)-6-Br-APB has higher potency at the mouse D1 DR than human D2 DR

in ERK1/2 phosphorylation............................................................................96

Figure 4.5 Brain exposure of R(+)-6-Br-APB in C57Bl/6J mice post i.p.

administration.................................................................................................98

Figure 4.6 R(+)-6-Br-APB at 0.3 mg/kg dose was optimum in disrupting PPI...............99

Figure 4.7 R(+)-6-Br-APB displays a bell-shaped dose-response profile in LMA, with 1

mg/kg dose the most efficient in increasing LMA.......................................101

Figure 4.8 Drug vehicles do not affect PPI or LMA compared to saline+saline+V3

treatment.......................................................................................................103

Figure 4.9 Plasma and brain exposure of LY2033298 and donepezil in C57Bl/6J mice

post i.p. administration.................................................................................105

Figure 4.10 Treatment of LY2033298 alone and in combination with donepezil reverse

disruption of PPI induced by R(+)-6-Br-APB, reaching significance at

P120pp12......................................................................................................107

Figure 4.11 Co-treatment of LY2033298 and donepezil reverses hyperlocomotor activity

induced by R(+)-6-Br-APB..........................................................................110

List of Figures

xi

Chapter 5

Figure 5.1 Timeline of behavioural experiments...........................................................122

Figure 5.2 Re-testing PPI in M4-/- mice decreased startle amplitude and increased PPI

values............................................................................................................127

Figure 5.3 Re-testing LMA in M4-/- mice did not significantly change LMA overall...128

Figure 5.4 M4-/- mice have significantly reduced PPI compared to M4

+/+ mice.............130

Figure 5.5 Spontaneous LMA of M4-/- mice was significantly increased compared to

M4+/+ mice, though this difference was reduced in the testing phase...........131

Figure 5.6 M4+/+ mice showed similar PPI data to the C57Bl/6J mice (Chapter 4), though

effect of the drug were less clear in M4-/- mice, likely due to floor effect....133

Figure 5.7 Treatment of R(+)-6-Br-APB 1 mg/kg caused a decrease in LMA between 40

and 55 min after the first two injections. R(+)-6-Br-APB 0.6 mg/kg dose was

the most effect in increasing LMA in M4+/+ mice.........................................136

Figure 5.8 Co-treatment of LY2033298 and donepezil reverses hyperlocomotor activity

induced by R(+)-6-Br-APB in M4+/+ mice. Treatments of LY2033298 and

donepezil either alone or in combination reduced R(+)-6-Br-APB-induced

hyperlocomotor activity in M4-/- mice..........................................................138

Figure 5.9 Simplified schematic representation of reported localisation of DRs,

mAChRs and nAChRs in the striatum..........................................................141

List of Tables

xii

List of Tables

Chapter 1

Table 1.1 GPCR targets of key neurotransmitter systems indicated in the

pathophysiology of schizophrenia..................................................................16

Table 1.2 In vivo efficacy of some CNS-penetrant M4 mAChR PAMs.........................24

Chapter 2

Table 1 Operational model parameters for functional interaction between ACh and

LY2033298 at the M4 muscarinic acetylcholine receptor..............................41

Chapter 4

Table 4.1 Operational model parameters for functional interaction between ACh and

LY2033298 or ML253 at human and mouse M4 mAChRs............................93

Table 4.2 Pharmacokinetic analysis of LY2033298.....................................................105

List of Abbreviations

xiii


[3H]NMS [3H]N-methyl-scopolamine

AC adenylate cyclase

ACh acetylcholine

ATCM allosteric ternary complex model

ANOVA analysis of variance

BRET bioluminescence resonance energy transfer

cAMP cyclic adenosine monophosphate

CAMYEL cAMP sensor using YFP-Epac-RLuc

CAR conditioned avoidance response

CHO Chinese hamster ovary

CNS central nervous system

DA dopamine

DAG diacylglycerol

DMEM Dulbecco’s modified Eagle’s medium

DMSO dimethyl sulfoxide

DR dopamine receptor

ECL extracellular loop

EDTA ethylenediaminetetraacetic acid

Epac exchange protein directly activated by cAMP

EPS extrapyramidal side effects

ERK1/2 extracellular signal-regulated kinase 1 & 2

FBS fetal bovine serum

FRET Förster/fluorescence resonance energy transfer

GABA γ -aminobutyric acid

GIRK channel G protein-coupled inwardly-rectifying potassium channel

GPCR G protein-coupled receptor

GDP guanosine diphosphate

GRK G protein-coupled receptor kinase


xiv

GTP guanosine triphosphate

HAT sodium hypoxanthine, aminopterin and thymidine

HBSS Hank’s Balanced Salt Solution

HEPES 2-[4-(2-hydroxyethyl)piperazine-1-piperazinyl]ethanesulfonic acid

ICL intracellular loop

i.p. intraperitoneal

IP3 inositol triphosphate

LC liquid chromatography

LMA locomotor activity

LY2033298 3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-

carboxylic acid cyclopropylamide

mAChR muscarinic acetylcholine receptor

MAPK mitogen-activated protein kinase

MEK mitogen-activated protein kinase kinase

ML253 3-amino-5-chloro-4,6-dimethyl-N-(pyridinyl-4-methyl)thieno[2,3-

b]pyridine-2-carboxamide

mRNA messenger ribonucleic acid

MS mass spectrometry

MSN medium spiny neuron

nAChR nicotinic acetylcholine receptor

NAL neutral allosteric ligand

NAM negative allosteric modulator

NOR novel object recognition

PAM positive allosteric modulator

PBS phosphate buffered saline

PEI polyethyleneimine

PIP2 phosphatidylinositol 4,5-bisphosphate

PKA protein kinase A

PLCβ phospholipase Cβ

PPI prepulse inhibition

PTX pertussis toxin


xv

RAF rapidly accelerated fibrosarcoma kinase

RhoGEFs guanine nucleotide exchange factors for Rho

Rluc Renilla luciferase

RT room temperature

TM transmembrane helix

UPLC ultra performance liquid chromatography

w/ with

w/o without

YFP yellow fluorescent protein

Abstract

xvi

Abstract

M4 muscarinic acetylcholine receptors (mAChRs) belong to the Rhodopsin family of G

protein-couple receptors. These receptors are found most abundantly in the striatum and

are implicated in a number of central nervous system disorders, including schizophrenia.

Indeed, a M1/M4 mAChR subtype-preferring agonist, xanomeline, has been shown in

clinical trials to alleviate psychotic symptoms and improve cognitive deficits associated

with both Alzheimer’s disease and schizophrenia. The antipsychotic effects of xanomeline

were found to be predominantly M4 mAChR-mediated, which is in contrast with the multi-

targeted mode of action of current antipsychotics, which display poly-pharmacology but

have the D2 dopamine receptor (DR) as a common therapeutic target. In the striatum, M4

mAChRs are co-expressed with D1 DRs in direct GABAergic output medium spiny

neurons and with D2 DRs in cholinergic interneurons. M4 mAChRs have been shown to

modulate striatal dopaminergic activity, and many M4 mAChR positive allosteric

modulators have been developed as potential antipsychotics.

In Chapter 2, the ability of a label-free technology to detect and quantify the positive

allosteric modulation of endogenous M4 mAChR in a rodent neuronal cell line was

established. The allosteric parameters estimated using this approach are comparable to

those estimated from endpoint-based assays, demonstrating that label-free technologies

can be used to screen for allosteric modulators, including those with no known G protein-

coupling preferences.

Chapters 3 and 4 explored the modulation of endogenous D2 DRs and D1 DRs by M4

mAChRs in vitro and in vivo, respectively. In Chapter 3, it was first established that the

NG108-15 cell line endogenously expresses both M4 mAChRs and D2 DRs, and that

Abstract

xvii

allosteric modulation of ACh by LY2033298, a M4 mAChR-selective positive allosteric

modulator in the presence of ACh, can be detected with end-point based signalling assays,

as well as with label-free technology. The presence of functional cross-talk between M4

mAChRs and D2 DRs was determined by performing interaction studies with an M4

mAChR orthosteric agonist, inverse agonist and positive allosteric modulator combined

with D2 DR ligands in two end-point based signalling assays. Though some small changes

to efficacy were observed in some interactions, overall, there was no apparent functional

cross-talk between these two receptors. This suggests that the cell line and the assays used

for this study was unsuitable for detecting functional cross-talk between M4 mAChRs and

D2 DRs.

In Chapter 4, the cross-talk between M4 mAChRs and D1 DRs in vivo was investigated,

using mouse models of aspects of psychosis. R(+)-6-Br-APB, a selective D1 DR agonist,

was used to induce D1 DR-mediated disruption of prepulse inhibition and increases in

locomotor activity in C57Bl/6J mice. LY2033298 in combination with donepezil, an

acetylcholinesterase inhibitor, showed a trend to reverse the R(+)-6-Br-APB-induced

disruption of prepulse inhibition. In locomotor activity experiments, combined

LY2033298 and donepezil treatment significantly reduced the R(+)-6-Br-APB-induced

increase in locomotor activity.

Chapter 5 describes the investigation of the role of M4 mAChRs in the reversal effects of

LY2033298 and donepezil using whole-body M4 mAChR knockout mice. However, the

results were inconclusive.

Finally, Chapter 6 provides a summary of the findings and discusses the potential future

directions of this study.

Publications

xviii

Publications

Journal Articles

Chen ANY, Malone DT, Pabreja K, Sexton PM, Christopoulos A, Canals M (2015)

Detection and quantification of allosteric modulation of endogenous M4 muscarinic

acetylcholine receptor using impedance-based label-free technology in a neuronal cell line.

J Biomol Screen 20(5):646–654

Conference Abstracts

Oral Presentations

Chen ANY, Christopoulos A, Canals M, Malone DT (2014) Muscarinic acetylcholine M4

receptor regulation of psychosis-like behaviours induced by a dopamine D1 receptor-

selective agonist in mice. ASCEPT-MPGPCR Joint Scientific Meeting 2014, December

2014, Melbourne, Australia

Poster Presentations



selective agonist in mice. ASCEPT-MPGPCR Joint Scientific Meeting 2014, December

2014, Melbourne, Australia



selective agonist in mice. Society for Neuroscience Annual Meeting 2014, November 2014,

Washington, DC, USA

Chen ANY, Picard E, Christopoulos A, Canals M, Malone DT (2014) Effect of positive

allosteric modulators of the M4 muscarinic acetylcholine receptor on the pharmacological

disruption of prepulse inhibition in mice. Australasian Neuroscience Society Conference

2014, January 2014, Adelaide, Australia

Declaration

xix

Declaration

I hereby declare that this thesis contains no material which has been accepted for the award

of any other degree or diploma at any university or equivalent institution and that, to the

best of my knowledge and belief, this thesis contains no material previously published or

written by another person, except where due reference is made in the text of the thesis.

This thesis includes one original paper published in a peer reviewed journal. The core

theme of the thesis is “investigation of the modulation of dopamine receptor function by

M4 muscarinic acetylcholine receptors”. The ideas, development and writing up of all the

papers in the thesis were the principal responsibility of myself, the candidate, working

within the Drug Discovery Biology theme of the Monash Institute of Pharmaceutical

Sciences under the supervision of Dr Daniel Malone, Dr Meritxell Canals and Prof Arthur

Christopoulos.

In the case of Chapter 2, my contribution to the work involved the following:

Thesis

Chapter Publication Title

Publication

Status

Nature and % of

student contribution

2

Detection and Quantification of

Allosteric Modulation of Endogenous

M4 Muscarinic Acetylcholine Receptor

Using Impedance-Based Label-Free

Technology in a Neuronal Cell Line

Published

Performed all of the

experiments, analysed

data and wrote the

manuscript (80%)

Student signature: Date: 10/11/2016

The undersigned hereby certify that the above declaration correctly reflects the nature and

extent of the student’s contributions to this work.

Main Supervisor signature: Date: 10/11/2016

Acknowledgements

xx

Acknowledgements

When I first started this journey, I did not anticipate that while pursuing a PhD, I would

learn much more than just scientific research. All of the people that I had the privilege to

meet throughout this journey and all of the experiences that I encountered have helped

shape me into the person I am today, and for that, I am forever grateful.

***

First of all, I would like to thank my supervisors, Dr Daniel Malone, Dr Meritxell Canals

and Prof Arthur Christopoulos, for giving me the opportunity to undertake this PhD. This

PhD was not without its challenges, and I am very grateful for their continued support,

encouragement and guidance. In particular, I would like to thank Meri and Dan for our

weekly meetings, and for your helpful feedback, patience and confidence in me, especially

when experiments didn’t go as planned. Arthur, thank you for your wisdom and invaluable

advice, your enthusiasm for research was a great source of motivation for me.

I would like to thank my thesis panel members, Dr Robert Lane, Dr Ben Capuano and Prof

Maarten van den Buuse. Rob, thank you for mentorship in the first initial months of my

candidature, and for your insightful suggestions throughout my PhD. Ben, thank you for

your warm encouragement during the panel meetings, and along with Tracey Huynh and

Dr Monika Szabo, for making some of the compounds used in this thesis. Maarten, thank

you for your valuable feedback on the animal research chapters, I really appreciate the

time and effort you have put into helping me.

I would like to extend my thanks to Dr Chris Choy and Leigh Howard for their help in the

MDMF and for the breeding of the knockout mice used in this thesis. Additionally, I am

grateful for the help of Dr David Shackleford and the Biopharmaceutics Section of the

Centre for Drug Candidate Optimisation at MIPS in the pharmacokinetics study of this

thesis. I am also very thankful for Dr Anand Gururajan for periodically checking in on me

and for his feedback on the animal research chapters.

I am eternally grateful to all of my friends, including Alex Shuen, Alice Berizzi, Arisbel

Batista, Basu Chakrabarty, Carmen Klein Herenbrink, Durgesh Tiwari, Elva Zhao, Gemma

Nassta, Georgina Thompson, Joan Ho, Linzi Lim, Lubna Freihat, Nicole Eise, Syahir

Acknowledgements

xxi

Mohd Soffi and TinaMarie Lieu, who were never short of encouraging words or listening

ears. Thank you for creating so many happy memories with me. I would especially like to

thank Briana Davie/Kelsang Tara, Elizabeth Vecchio, Nilushi Karunaratne and Thomas

Coudrat. BriTara, sitting next to you in the office was one of the highlights of my PhD

journey. We have come a long way since that first day, and I will always cherish our

conversations and our reflections on life. Liz, thank you for all the lunches (and occasional

dinners) where we shared our sorrows and frustrations, and celebrated our successes, big

and small. I always walk away from our lunches feeling a million times lighter! Nel, to

quote what I wrote in my Honours thesis, “I feel like you’re my adopted sister”. It still

stands true. Thank you for your amazing friendship, for always being there for me, and for

being the voice of reason when things get rough. Thomas, my co-conspirator on Parkville

Career Forum, thank you for all the “power lunches” and brainstorming sessions, and

being the sounding board when I am stuck on a problem. I am so grateful and proud that

we were brave enough to start up a new student organisation, and this experience has

really helped me grow as a person.

I would also like to thank the irreplaceable David Jaboor. Thank you for your unwavering

belief in me, for your loving support and encouragement, and for providing me with a

healthy dose of downtime when I needed it most. You have kept me grounded (and sane),

especially in the last few months of my PhD. Thank you for everything.

Lastly, I would like to thank my Mom, Dad and Alice. Thank you for your faith in me and

for your support in my pursuit of a PhD. Although we are oceans apart, I feel your love

every day.

Chapter 1:

General Introduction

Chapter 1

2

1.1 G Protein-Coupled Receptors

The guanine nucleotide-binding (G) protein-coupled receptor (GPCR) superfamily

represents the largest family of mammalian cell-surface receptors (Lagerstrom and Schioth,

2008). GPCRs are membrane proteins containing seven transmembrane helix domains

(TM1-TM7) linked by three extracellular (ECL1-ECL3) and three intracellular loops

(ICL1-ICL3), with an extracellular N-terminus and an intracellular C-tail at either ends.

They interact with G proteins to transduce information received from extracellular ligands

into cellular changes (Fredriksson et al., 2003). Approximately 800 functioning GPCRs are

encoded in the human genome (Fredriksson et al., 2003). These receptors are important in

the function of all organ systems, with around 30% of marketed drugs binding GPCRs in

order to exert their therapeutic effect (Muller et al., 2012).

1.1.1 GPCR Classification

The first GPCR classification method, the A-F classification system, divided GPCRs

found in both invertebrates and vertebrates into six families – A (rhodopsin-like), B

(secretin-like), C (metabotropic glutamate-like), D (fungal mating pheromone receptors), E

(cyclic adenosine monophosphate (cAMP) receptors) and F (frizzled/smoothened)

(Attwood and Findlay, 1994; Kolakowski, 1994). This classification is based on the

receptor sequence homology, and of these receptor families, D and E are only found in

invertebrates (Alexander et al., 2015). A more recent alternative classification method

(termed “GRAFS”) divided human GPCRs into five main families based on sequence

homology and receptor function (the overlapped A-F classification nomenclature in

parenthesis) – Glutamate (Family C), Rhodopsin (Family A), Adhesion (Family B),

Frizzled/Taste2 (Family F) and Secretin (Family B) (Alexander et al., 2015; Fredriksson et

al., 2003). Of the receptor families, the Rhodopsin (Family A) family is the largest,

Chapter 1

3

consisting of over 700 receptor proteins, which include olfaction, taste, pheromone and

vision sensory receptors, as well as receptors for neurotransmitters, peptides and hormones

(Fredriksson et al., 2003; Koster et al., 2014). The receptors of interest in this thesis, the

muscarinic acetylcholine receptors (mAChRs) and the dopamine receptors (DRs), belong

to this family.

1.1.2 GPCR Signalling

When an agonist ligand binds to a GPCR, a conformational change in the GPCR protein

structure occurs, shifting the GPCR structure from an inactive to an active state. The active

conformation of the receptor triggers the activation of heterotrimeric G proteins,

comprised of α-, β- and γ-subunits that are coupled to the receptor (Figure 1.1) (Neer,

1995; Wess, 1997). Once activated by the GPCR, the Gα subunit exchanges guanosine

diphosphate (GDP) for guanosine triphosphate (GTP), and then dissociates from the

ligand-bound GPCR and the Gβγ subunits. The dissociated GTP-bound Gα subunit and

Gβγ subunits are then free to activate intracellular effector molecules, leading to

downstream signalling effects (Cabrera-Vera et al., 2003; Neer, 1995). The Gα subunit is

deactivated when GTP is hydrolysed to GDP through its intrinsic GTPase activity, upon

which the subunits reassemble to form the inactive heterotrimeric G protein complex

(Cabrera-Vera et al., 2003; Neer, 1995).

Based on their function and sequence homology, Gα proteins are divided into four major

classes – Gαs, Gαi/o, Gαq/11 and Gα12/13 (Milligan and Kostenis, 2006). In this thesis, the

receptors of interest couple predominantly to Gαs or Gαi/o proteins (Figure 1.2). Gαs

proteins promote cAMP production by activation of the enzyme adenylate cyclase (AC),

whereas Gαi/o proteins inhibit the activity of AC (Ferre, 2015). A major downstream

effector of cAMP is protein kinase A (PKA), which in turn, phosphorylates an array of

Chapter 1

4

Figure 1.1: G protein activation/deactivation cycle. Abbreviations: GDP, guanine

diphosphate; GTP, guanine triphosphate.

Agonist

Gα GβγGDP

(1)

GβγGα

GDPGTP

(2)

GβγGαGTP

(3)

GβγGα

GTP

(4)

GDP

Signal transduction

Chapter 1

5

cytosolic and nuclear proteins (Smith et al., 2011; Walsh et al., 1968). Gαq/11 proteins

activate phospholipase Cβ (PLCβ) to catalyse the hydrolysis of a plasma membrane lipid,

phosphatidylinositol 4,5-bisphosphate (PIP2). The hydrolysis of PIP2 releases inositol

trisphosphate (IP3) and diacylglycerol (DAG), which leads to Ca2+ mobilisation (Hubbard

and Hepler, 2006). Lastly, Gα12/13 proteins are typically involved in the activation of

guanine nucleotide exchange factors for Rho (RhoGEFs), which in turn catalyses the

exchange of GDP for GTP on Rho GTPases (Kozasa et al., 2011).

In addition to Gα proteins, five Gβ (Gβ1-5) and twelve Gγ (Gγ1-12) proteins have been

described, and together can form different combinations of Gβγ subunits, which can also

exert a diverse range of signalling effects (Milligan and Kostenis, 2006). Gβγ subunits

have been associated with the activation of several downstream effectors, including G

protein-coupled inwardly rectifying K+ (GIRK) channels, voltage-gated Ca2+ channels,

ACs and mitogen-activated protein kinases (MAPKs), such as extracellular signal

regulated kinase (ERK; Figure 1.2) (Khan et al., 2013). Furthermore, GPCRs can signal

Gαi

GTP

Gαs

GTP

Figure 1.2: Simplified signal transduction pathways of Gαs, Gαi and Gβγ proteins.

Abbreviations: AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; ERK,

extracellular signal-regulated kinase; GTP, guanine triphosphate; MEK, mitogen-activated

protein kinase kinase; PKA, protein kinase A; RAF, rapidly accelerated fibrosarcoma kinase.

Gβγ

AC

cAMP

PKA

RAF

MEK

ERK

iCa2+

iK+

Chapter 1

6

through G protein-independent mechanisms, such as via regulatory proteins GPCR kinases

(GRKs) and β-arrestins. For detailed reviews regarding these mechanisms, see Ribas et al.

(2007) and Luttrell and Lefkowitz (2002), respectively.

It is now known that certain ligands can stabilise a GPCR active conformation towards

signalling through one pathway over another, or even preferentially signal through G

protein-independent mechanisms over the canonical G protein-dependent signalling

pathways. For a detailed review about this phenomenon, known as biased agonism or

functional selectivity, see Rankovic et al. (2016).

1.1.3 GPCR Allostery

Endogenous ligands of GPCRs bind to a site on the receptor termed the “orthosteric

binding site”. Traditional efforts to develop GPCR drugs have focused on designing small

molecules that bind to the orthosteric site either to directly activate the receptor (agonists),

or to prevent the endogenous ligand from binding (antagonists). However, due to

evolutionary pressure, the orthosteric binding site is highly conserved within receptor

family subtypes (Christopoulos, 2002). As a result, orthosteric ligands often have poor

receptor subtype selectivity, which can lead to side effects due to off-target activity.

In recent years, there has been an increased focus of drug discovery efforts to develop

“allosteric ligands” as therapeutic agents. It is recognised that most, if not all, GPCRs have

allosteric binding sites, which are topographically distinct from the orthosteric site

(Christopoulos, 2014). Allosteric binding sites are under less evolutionary pressure, and

therefore, are generally less conserved between receptor subtypes within a receptor family

(Christopoulos, 2014; Gregory et al., 2010). This allows allosteric ligands the potential to

possess increased receptor subtype selectivity previously not seen with orthosteric ligands.

Chapter 1

7

Another mechanism for allosteric ligands to obtain receptor subtype selectivity is through

“selective cooperativity” (Christopoulos, 2014). As the allosteric and orthosteric binding

sites do not overlap, allosteric and orthosteric ligands can be bound to the same receptor at

the same time. The binding of allosteric ligands can induce a conformational change

within the receptor, which may result in the modulation of the orthosteric binding affinity

and/or signal transduction (Figure 1.3A) (May et al., 2007). Selective cooperativity is a

combined result of the divergence in amino acid sequences and the differences in the

magnitude of the interaction between the orthosteric and the allosteric sites, or

cooperativity, across receptor subtypes (Christopoulos, 2014).

To quantify allosteric modulation, the operational model of allosterism is widely used. The

operational model of allosterism is based on the simple allosteric ternary complex model

(ATCM) proposed by Ehlert (1988) and extended to incorporate the Black and Leff

operational model for functional agonist response (Black and Leff, 1983; Leach et al.,

2007; May et al., 2007) (Figure 1.3B). The affinities of the orthosteric (A) and allosteric

(B) ligands to the unbound receptor (R) are denoted by the dissociation constants KA and

KB, respectively. The extent to which the affinities are modified when both ligands are

bound to the receptor is defined by the cooperativity factor, α. The efficacies of the

orthosteric and the allosteric ligands are denoted as τA and τB, respectively, and the

magnitude by which the allosteric ligand modulates the co-bound orthosteric ligand

efficacy is defined by the cooperativity factor, β (Leach et al., 2007; May et al., 2007).

Chapter 1

8

Orthosteric ligand, A Allosteric ligand, B

Receptor, R

Effector coupling

Affinity modulation

Allosteric

agonism

τB

Orthosteric

agonism

τA

α

Orthosteric ligand

binding affinity

KA

Allosteric ligand

binding affinity

KB

Figure 1.3: (A) Schematic diagram of allosteric modulation of drug action (adapted from

Langmead and Christopoulos, 2006) and (B) the operational model of allosterism (adapted from

Leach et al., 2007).

A

B

R AR

RB ARB

KA

KB

B+

A +

A +

B+

KB/α

KA/α

τA

τA x β

τB

Chapter 1

9

Allosteric ligands that enhance the orthosteric ligand response (binding affinity and/or

functional efficacy; α and/or β > 1) are termed “positive allosteric modulators” (PAMs),

whereas those that diminish the orthosteric ligand response are called “negative allosteric

modulators” (NAMs; 0 < α and/or β < 1). Additionally, some allosteric ligands may also

display agonistic activity on their own (“allosteric agonists”; τB > 0), while some occupy

the allosteric binding site without altering the properties of the co-bound orthosteric ligand

(“neutral allosteric ligands”; NALs; α and β = 1) (Christopoulos, 2014; Kenakin, 2012). In

addition, it is possible for an allosteric ligand to have separate effects on affinity and

efficacy of the orthosteric ligand. For a detailed review on the allosteric effects on

orthosteric agonist actions, see Kenakin (2012).

A unique property of allosteric modulators is that any modulatory effect they have on the

orthosteric ligand is saturable – that is, the modulation reaches a ceiling once all the

allosteric binding sites are occupied. Therapeutically speaking, the saturability of allosteric

interaction allows allosteric modulators, in the absence of off-target effects, to be

administered in a larger dose without the dangers of inducing toxicity or overdose, effects

that are normally seen with high-dose orthosteric ligands (May et al., 2007). Another

property of allosteric modulators is the phenomenon termed “probe dependence”, where

the effects of an allosteric modulator at the same receptor may change depending on the

orthosteric ligand that is co-bound (Leach et al., 2007; Suratman et al., 2011; Valant et al.,

2012). Consequently, this property allows a single allosteric modulator to produce

different effects in the presence of different orthosteric ligands, offering the potential to

use different combinations of allosteric and orthosteric ligands to obtain distinct

pharmacological effects. However, this also poses a challenge for allosteric drug discovery,

as it can complicate the detection and classification of allosteric ligands. While the

endogenous ligand is the ideal orthosteric probe to use, it is not always practical, for

Chapter 1

10

example, in cases where the receptor of interest has multiple endogenous ligands or if the

stability of these compounds is low. Therefore, the orthosteric probe used in screening

programs must be carefully chosen, and when possible, promising allosteric compounds

should be tested against all endogenous ligands (Leach et al., 2007; Suratman et al., 2011;

Valant et al., 2012).

Finally, a key property of allosteric modulators is that they maintain the spatiotemporal

control of receptor activity, as the binding of the orthosteric ligand is required for the

allosteric modulators to exhibit their modulation (Christopoulos, 2014). This characteristic,

in addition to the increased receptor subtype selectivity, make allosteric ligands attractive

therapeutic agents, especially in disorders that are currently sub-optimally treated, such as

schizophrenia.

Chapter 1

11

1.2 Schizophrenia

Schizophrenia is one of the most severe and disabling mental disorders, with a recent

global survey scoring acute schizophrenia the highest disability weight compared to 219

other disorders or disease states (Salomon et al., 2012). The onset of schizophrenia is

typically during the late adolescence and early adulthood, and while approximately only 7

in 1000 individuals will be affected by this disorder in their lifetime, the global economic

burden of schizophrenia ranges from 0.02% to 1.65% of the gross domestic product,

depending on the country (Chong et al., 2016; McGrath et al., 2008).

Schizophrenia was originally referred to as “dementia praecox”, or premature dementia, by

a German psychiatrist, Emil Kraepelin. In the 1890s, Kraepelin defined the disorder as a

chronic progressive illness with an early onset and poor prognosis, with the ultimate

cognitive and behavioural decline as the hallmark feature (Falkai et al., 2015; Jablensky,

2010; Kraepelin, 1896). The term “schizophrenia” was introduced in 1911 by Swiss

psychiatrist, Eugen Bleuler, who proposed that this disorder was a group of illnesses with

different clinical presentations, but with the “disturbances of associations” as the common,

fundamental feature (Bleuler, 1911; Heckers, 2011; Maatz et al., 2015). It is now accepted

that schizophrenia is a syndrome or spectrum due to the polygenic aetiology and the

influence of environmental and epigenetic factors, resulting in the heterogeneity in its

manifestation (Cariaga-Martinez et al., 2016; Owen et al., 2016; Tsuang, 2000; van Os and

Kapur, 2009). The symptoms of schizophrenia can be classified into three main domains:

positive symptoms (or psychotic symptoms, e.g. hallucinations, delusions, disorganised

thoughts), negative symptoms (e.g. lack of motivation, anhedonia, social withdrawal) and

cognitive impairment (e.g. deficits in attention, working memory, executive functions).

Chapter 1

12

The prognosis of schizophrenia is not as grave as it once was, particularly with the

development of antipsychotics, better understanding of the disorder and a decrease in

stigma towards this disorder (Carpenter and Koenig, 2008; Frese et al., 2009; van Os and

Kapur, 2009). Though prospective studies in schizophrenia showed diverse outcomes in

recovery (Cuyun Carter et al., 2011; Holla and Thirthalli, 2015; van Os and Kapur, 2009),

a meta-analysis of recovery in schizophrenia estimated that approximately 1 in 7

individuals diagnosed with schizophrenia recover, when recovery is described as “very

good outcome that considers both clinical and social/functional dimensions and includes a

duration criteria of at least 2 years for at least 1 of these measures” (Jaaskelainen et al.,

2013). However, there is a high mortality rate in schizophrenia, which is linked to high

suicide rate and death due to comorbid somatic disorders, such as cardiovascular diseases,

the prevalence of which is associated with atypical antipsychotic use (see 1.2.1.1) (Harris

and Barraclough, 1998; Laursen et al., 2014; Olfson et al., 2015; Saha et al., 2007).

Effective management of schizophrenia does not solely rely on pharmacotherapy, but

requires the addition of psychological intervention and social support (Owen et al., 2016).

However, the maintenance of medication is vital for the improvement of functional

outcomes; discontinuation of antipsychotic treatment is associated with increased risk of

relapse (Robinson et al., 1999). Nevertheless, around 30% of people with schizophrenia do

not respond to current antipsychotics (Kane et al., 1988; Meltzer, 1997). While these

treatment-refractory patients with schizophrenia can be prescribed clozapine (see below),

clozapine is only effective in treating the positive symptoms in approximately 60% of the

cases (Meltzer, 2013).

Chapter 1

13

1.2.1 GPCRs in Schizophrenia

1.2.1.1 Antipsychotics

The discovery of the antipsychotic effects of chlorpromazine in the 1950s initiated the

development of the first-generation, or typical, antipsychotics. Typical antipsychotics, such

as haloperidol, are all antagonists of the dopamine (DA) receptor of the D2 subtype (D2

DR). Their binding affinity for D2 DRs correlates with the therapeutic dosage prescribed to

treat positive symptoms of schizophrenia (Seeman et al., 1975). However, typical

antipsychotics can induce extrapyramidal side effects (EPS), which are movement

disorders such as acute dystonia, akathisia, Parkinsonism and tardive dyskinesia (Peluso et

al., 2012). Typical antipsychotics can also increase prolactin levels (Halbreich et al., 2003).

Furthermore, typical antipsychotics are not efficacious in ameliorating negative symptoms

and cognitive impairment, the improvement of which are just as important as positive

symptoms for re-insertion into the community and increasing the functional outcome and

quality of life of people with schizophrenia (Fervaha et al., 2014; Galuppi et al., 2010;

Norman et al., 2000; Savilla et al., 2008).

Second-generation or atypical antipsychotics, such as clozapine, differ from typical

antipsychotics by their mechanisms of action; the ratio of the affinity of atypical

antipsychotics at the 5-HT2 serotonin receptor over the D2 DR is generally higher than that

of typical antipsychotics (Meltzer et al., 1989b). Atypical antipsychotics are also thought

to have decreased occurrence of hyperprolactinemia and EPS liability, although some

publications suggest equivalent EPS occurrence with both typical and atypical

antipsychotic use (Leucht et al., 2009; Peluso et al., 2012). However, there is a strong

association between atypical antipsychotic use and the prevalence of metabolic syndrome

and cardiovascular risk, the occurrence of which contributes to the shortened average

Chapter 1

14

lifespan of people with schizophrenia (Correll et al., 2006; Laursen et al., 2014; McEvoy et

al., 2005; Orsolini et al., 2016).

Although initially thought to be more effective than typical antipsychotics, especially in

treating the negative symptoms, several studies have shown a lack of improved therapeutic

effects with atypical antipsychotics, with the exception of clozapine (Geddes et al., 2000;

Jones et al., 2006; Leucht et al., 2013; Leucht et al., 2009). Clozapine is often considered

the “gold-standard” treatment for schizophrenia due to its superior efficacy in treatment-

refractory schizophrenia, where patients do not respond to other antipsychotics (Kane et al.,

1988; Siskind et al., 2016). However, the utilization of clozapine is limited to treatment-

refractory schizophrenia, as it can induce agranulocytosis, as well as a number of other

adverse effects common with atypical antipsychotics (Fitzsimons et al., 2005; Ucok and

Gaebel, 2008).

With currently used antipsychotics demonstrating limited therapeutic effect, while being

associated with numerous side effects, there is still a great need to identify new therapeutic

targets and develop new drugs that can treat all symptom domains without eliciting

adverse effects.

1.2.1.2 GPCR targets for schizophrenia

The dysfunction of several neurotransmitters has been associated with the pathophysiology

of schizophrenia. The most prominent and well-studied hypothesis of schizophrenia is the

DA hypothesis, which postulates that positive symptoms are a result of elevated DA

transmission in the mesolimbic pathway in the brain of patients with schizophrenia,

whereas negative symptoms are thought to be a consequence of decreased DA signalling in

the prefrontal cortex (Figure 1.4A) (Boyd and Mailman, 2012; Jucaite and Nyberg, 2012).

Indeed, as mentioned in 1.2.1.1, all current antipsychotics act on the D2 DR as either

Chapter 1

15

Figure 1.4: Schematic representation of the dopaminergic and cholinergic systems in

the rodent brain. (A) Dopaminergic system (Bjorklund et al. 2007, used with permission) (B)

Cholinergic system (Woolf et al. 2011, used with permission). Abbreviations: bas, nucleus

basalis; BLA, basolateral amygdala; DR, dorsal raphe; EC, entorhinal cortex; hdb, horizontal

diagonal band nucleus; ICj, islands of Cajella; IPN, interpeduncular nucleus; LC; locus ceruleus;

ldt, laterodorsal tegmental nucleus; LH, lateral hypothalamus; ms, medial septal nucleus; ppt,

pedunculopontine nucleus; si, substantia innominata; SN, substantia nigra; vdb, vertical

diagonal band nucleus.

A

B

Chapter 1

16

antagonists or partial agonists, and are all very effective in treating positive symptoms,

though these drugs perform poorly when it comes to the other symptom domains.

Schizophrenia hypotheses of other neurotransmitter systems, including the aforementioned

serotonin (Selvaraj et al., 2014), glutamate (Coyle et al., 2012) and acetylcholine (ACh)

(McKinzie and Bymaster, 2012), have been proposed, and many drugs targeting the

GPCRs in these neurotransmitter systems are currently in clinical development for the

treatment of schizophrenia (Table 1.1) (Gray and Roth, 2007; Koster et al., 2014). The

focus of this thesis is the muscarinic acetylcholine hypothesis of schizophrenia, which will

be expanded upon in 1.2.2.

1.2.2 Muscarinic Acetylcholine Hypothesis of Schizophrenia

The cholinergic system in the brain is an extensive network that provides cholinergic input

in virtually all brain regions and interacts with range of neurotransmitter systems,

including dopaminergic projections in the midbrain (ventral tegmental area and substantia

nigra) (Figure 1.4) (Woolf and Butcher, 2011). The cholinergic hypothesis of

schizophrenia was first proposed by Neubauer et al. (1975), after observations that high

doses of cholinergic antagonists induced psychotomimetic effects, including auditory and

visual hallucinations (Neubauer et al., 1975; Perry and Perry, 1995). Early clinical studies

found that cholinergic agonists, such as physostigmine, a reversible cholinesterase

Table 1.1: GPCR targets of key neurotransmitter systems indicated in the pathophysiology

of schizophrenia

Neurotransmitter GPCR Targets

Dopamine D1/2/3/4

Serotonin 5-HT1A/1B/2A/2C/3/4/6/7

Glutamate mGlu1/2/3/5

Acetylcholine M1/4

References (Gray and Roth, 2007; Koster et al., 2014; Miyamoto et al., 2012)

Chapter 1

17

inhibitor, modestly improved symptoms in patients with schizophrenia (Janowsky et al.,

1973; Pfeiffer and Jenney, 1957). Now, cholinesterase inhibitors, such as donepezil, are an

approved treatment for cognitive decline in Alzheimer’s disease, and have been shown to

be promising in treating neuropsychiatric symptoms in Alzheimer’s disease (Wynn and

Cummings, 2004).

Recent radioligand binding studies in post-mortem brain tissues of subjects with

schizophrenia showed decreased mAChR expression in the cortical and subcortical regions,

and these findings were supported by in vivo binding experiments using single photon

emission computed tomography (SPECT), confirming the role of mAChRs in the

pathophysiology of the disorder (for review, see McKinzie and Bymaster (2012)).

Specifically, expressions of M1 mAChR in the cortex (Dean et al., 2002; Zavitsanou et al.,

2004), M2/M4 mAChR in the striatum (Crook et al., 1999) and M4 mAChR in the

hippocampus (Scarr et al., 2007) were all found to be decreased in subjects with

schizophrenia.

The proof-of-concept for the use of mAChR agonist in the treatment of schizophrenia

stems from research involving the M1/M4 mAChR-preferring orthosteric agonist,

xanomeline (Andersen et al., 2015; Andersen et al., 2003; Bodick et al., 1997; Dencker et

al., 2011; Shannon et al., 1994; Shekhar et al., 2008). Xanomeline was originally

developed for the treatment of Alzheimer’s disease as a receptor-targeted approach, a

departure from the inhibition of ACh breakdown, which is the mainstay of current

Alzheimer’s disease treatment (Bodick et al., 1997). It was found that xanomeline not only

improved cognitive performance in subjects with Alzheimer’s disease, but also decreased

the number of neuropsychiatric symptoms, including delusions and hallucinations (Bodick

et al., 1997). In a pilot study with subjects with schizophrenia, treatment of xanomeline

improved symptoms from all symptom domains, with significant reduction in positive

Chapter 1

18

symptoms by the end of the first week of treatment (Shekhar et al., 2008). However,

xanomeline failed clinical trials for Alzheimer’s disease due to adverse gastrointestinal

side effects induced by the off-target activity of xanomeline on the mAChRs in the

periphery (Bodick et al., 1997). Nonetheless, the success of xanomeline in treating positive

and negative symptoms, as well as cognitive impairment in subjects with schizophrenia

confirms the hypothesis of enhancing mAChR activity to treat this disorder. Due to the

relative locations of the receptor expression in the brain, it is postulated that the pro-

cognitive effects and the antipsychotic effects of xanomeline were mediated through the

M1 and the M4 mAChRs, respectively, which will be expanded on in 1.3 and 4.1 (Foster et

al., 2014; Langmead et al., 2008; Nickols and Conn, 2014).

Chapter 1

19

1.3 Muscarinic Acetylcholine Receptors

Muscarinic acetylcholine receptors (mAChRs) belong to the Rhodopsin family (Family A)

of GPCRs (Caulfield and Birdsall, 1998). Along with ionotropic nicotinic acetylcholine

receptors (nAChRs), mAChRs mediate the functions of the neurotransmitter acetylcholine

(ACh) in the central and peripheral nervous systems, and in non-neuronal tissues

(Caulfield and Birdsall, 1998; Eglen, 2006; Eglen, 2012). The genes encoding these

receptors are intronless, and exhibit high sequence homology across receptor subtypes and

species (Hulme et al., 1990; Kubo et al., 1986).

The mAChR family consists of five receptor subtypes (M1, M2, M3, M4 and M5), which are

grouped into two classes based on their G protein-coupling preferences and signal

transduction. M1-like mAChRs (M1, M3 and M5 subtypes) preferentially couple to Gαq/11

proteins, activating PLCβ and mobilising intracellular calcium, whereas M2-like mAChRs

(M2 and M4 subtypes) preferentially couple to Gαi/o proteins, inhibiting AC production of

cAMP (Felder, 1995).

The mAChR subtypes have distinct distribution patterns both in the periphery and the

brain, though most tissues and cell types express more than one mAChR subtype (Wess et

al., 2007). In this section, the distributions and functions of each mAChR subtype will be

discussed, with more emphasis on the M4 mAChR, which is the focus of this thesis.

1.3.1 M1 mAChR

The M1 mAChR subtype is the highest expressing mAChR subtype in the central nervous

system (CNS), with the majority of the receptor found in the hippocampus, cortex,

olfactory tubercule and the striatum (Hersch et al., 1994; Levey et al., 1995; Levey et al.,

1991; Wall et al., 1991a). M1 mAChRs are also expressed to a lesser extent in the

Chapter 1

20

periphery, mainly in the salivary glands and vas deferens (Dorje et al., 1991). Expressed

on postsynaptic neurons in the brain, M1 mAChRs are important for learning and memory,

sensory perception and attention (Foster et al., 2014; Langmead et al., 2008; Wess et al.,

2007). Indeed, there has been a tremendous effort in developing PAMs selective for M1

mAChRs for the treatment of Alzheimer’s disease and the cognitive deficits in

schizophrenia (for review, see Davie et al. (2013) and Melancon et al. (2013)).

Additionally, M1 mAChRs have been implicated in substance abuse (Dencker et al.,

2012a).

1.3.2 M2 mAChR

The M2 mAChR subtype is expressed predominantly in the heart, smooth muscle and lung

in the periphery, but is also found centrally in the hindbrain, thalamus, cerebral cortex and

the striatum (Dorje et al., 1991; Hersch et al., 1994; Levey et al., 1991; Li et al., 1991).

Centrally, the M2 mAChR is the predominant presynaptic autoreceptor regulating ACh

release in the hippocampus and cerebral cortex (Zhang et al., 2002a). M2 mAChRs are the

predominant autoreceptors in the diaphragm, and are involved in the autoinhibition of ACh

in the heart (Slutsky et al., 2003; Zhou et al., 2002). Knockout studies demonstrated that

the M2 mAChR subtype is involved in thermoregulation, regulation of heart rate, smooth

muscle contractility, learning and memory, hippocampal synaptic plasticity and

nociception (Bymaster et al., 2001; Gomeza et al., 2001; Seeger et al., 2004). Due to the

wide distribution of M2 mAChRs and their role in many different processes, there is

potential for M2 mAChR-selective ligands to treat a number of conditions, such as

overactive bladder syndrome, asthma, chronic obstructive pulmonary disease and pain

(Dale et al., 2014; Meurs et al., 2013; Wess et al., 2007). However, feasibility of targeting

M2 mAChR for the treatment of these conditions remains to be seen due to the lack of M2

mAChR subtype-selective orthosteric or allosteric compounds. Recent identification of

Chapter 1

21

PAMs and NAMs selective for the M2 mAChR subtype will help the advancement of this

field (Miao et al., 2016).

1.3.3 M3 mAChR

While there is a very low expression of M3 mAChRs in the cerebral cortex and

hippocampus, this receptor subtype is found mostly in the glandular tissues and smooth

muscle in the periphery (Caulfield, 1993; Dorje et al., 1991; Levey et al., 1991; Wall et al.,

1991b). The M3 mAChR is the predominant mAChR subtype involved in smooth muscle

contractility, and plays a role in the regulation of appetite, glucose homeostasis and

salivation (Gautam et al., 2006; Guo et al., 2006; Matsui et al., 2000; Wess et al., 2007;

Yamada et al., 2001b). Hence, M3 mAChRs can be targeted to treat overactive bladder

syndrome, asthma, chronic obstructive pulmonary disease, cancer, xerostomia, type II

diabetes and obesity (Dale et al., 2014; Meurs et al., 2013; Russo et al., 2014; Wess et al.,

2007; Weston-Green et al., 2013). However, M3 mAChR-selective compounds have not

been developed, and therefore, the effectiveness of selectively targeting M3 mAChR to

treat these conditions remains to be elucidated.

1.3.4 M4 mAChR

The M4 mAChR subtype is found most predominantly in the striatum, but is also

expressed in the cerebral cortex and hippocampus in the CNS, as well as the lung and

smooth muscle in the periphery (Dorje et al., 1991; Hersch et al., 1994; Levey et al., 1995;

Levey et al., 1991; Yasuda et al., 1993). In the periphery, M4 mAChRs are involved in the

autoinhibition of ACh release in the heart, and are the predominant autoreceptors in the

bladder (Zhou et al., 2002).

Chapter 1

22

M4 mAChRs are closely associated with DRs in the brain. In the striatum, M4 mAChRs are

co-localised with M2 mAChRs and D2 DRs on cholinergic interneuron (Dawson et al.,

1988; Hersch et al., 1994; Yan and Surmeier, 1996), where the M4 mAChR is the main

mAChR subtype involved in the autoinhibition of ACh release in this brain region (Zhang

et al., 2002a). M4 mAChRs are also co-expressed with D1 DRs on the striatonigral

GABAergic medium spiny output neurons of the direct pathway (Hersch et al., 1994; Ince

et al., 1997; Levey et al., 1991), and have been shown to regulate D1 DR-mediated

signalling, such as stimulation of AC-mediated production of cAMP and phosphorylation

of ERK1/2 (DeLapp et al., 1996; Kelly and Nahorski, 1986; Olianas and Onali, 1996;

Olianas et al., 1983; Xue et al., 2015). Additionally, studies using whole-body M4 mAChR

knockout mice and mice with conditional M4 mAChR knockout only in D1 DR-expressing

neurons showed that DA release in the striatum is regulated by M4 mAChRs (Jeon et al.,

2010; Threlfell et al., 2010; Tzavara et al., 2004).

Due to the role of M4 mAChRs in DA signalling and neurotransmission in the brain,

knockout studies have demonstrated that the M4 mAChR subtype is involved in DA driven

functions, such as striatal motor control and regulation of reward circuitry (Gomeza et al.,

1999b; Schmidt et al., 2011). Indeed, M4 mAChRs have been implicated as potential

targets for the treatment for schizophrenia, Parkinson’s disease and substance abuse

(Dencker et al., 2012a; Eglen, 2012; Foster et al., 2014; Langmead et al., 2008; Levran et

al., 2016; Schmidt et al., 2011). In particular, the development of M4 mAChR selective

compounds for the treatment of schizophrenia has been quite active following publication

of research showing the antipsychotic ability of xanomeline (Langmead et al., 2008). As

mentioned in 1.2.2, xanomeline demonstrated antipsychotic-like effects in animal models

of aspects of psychosis through the attenuation of amphetamine-induced increases in

locomotor activity (LMA) and reversal of scopolamine-induced disruption of prepulse

Chapter 1

23

inhibition (PPI) in wildtype mice (see 1.5) (Dencker et al., 2011; Woolley et al., 2009).

These effects were abolished in M4 mAChR knockout mice, suggesting that M4 mAChRs

are important in mediating these effects (Dencker et al., 2011; Woolley et al., 2009).

Several PAMs selective for M4 mAChRs have been developed, with LY2033298 being the

first to show antipsychotic-like effects in rodent models of aspects of psychosis (Brady et

al., 2008; Bubser et al., 2014; Chan et al., 2008; Le et al., 2013; Salovich et al., 2012;

Shirey et al., 2008; Wood et al., 2016a; Wood et al., 2016b). Table 1.2 lists some of the

M4 mAChR PAMs that have shown efficacy in in vivo.

Due to its close association with DRs, which have been the main treatment targets for

schizophrenia, studies on the M4 mAChR have demonstrated that it is a promising target

for the treatment of this disorder. Therefore, the focus of this thesis is to better understand

of the relationship between the M4 mAChR and the DRs with the use of PAMs selective

for M4 mAChRs.

1.3.5 M5 mAChR

M5 mAChRs have the lowest CNS expression of all the mAChRs, with discrete expression

in the ventral tegmentum area and substantia nigra (Vilaro et al., 1990; Weiner et al., 1990).

M5 mAChRs play a role in regulation of cerebrovascular blood flow, striatal DA release

and salivary secretion (Forster et al., 2002; Takeuchi et al., 2002; Yamada et al., 2003;

Yamada et al., 2001a). Due to their expression and function, M5 mAChRs are implicated

in substance abuse (Basile et al., 2002; Dencker et al., 2012a; Fink-Jensen et al., 2003;

Langmead et al., 2008; Thomsen et al., 2005; Wess et al., 2007). Recent development of

M5 mAChR NAMs will aid the understanding of the role of M5 mAChRs in this disorder

(Berizzi et al., 2016; Gentry et al., 2014; Gentry et al., 2013).

Chapter 1

24

Table 1.2: In vivo efficacy of some CNS-penetrant M4 mAChR PAMs

Name (activity at M4

mAChR) Chemical structure In vivo efficacy References

LY2033298

(PAM-agonist)

When co-administered with a sub-effective

dose of oxotremorine:

Reversed apomorphine-induced

disruption of PPI

Reversed amphetamine-induced increase

in LMA

Decreased CAR

Chan et al.

(2008); Leach et

al. (2010);

Suratman et al.

(2011)

ML253

(PAM-agonist)

Reduced amphetamine-induced increase

in LMA Le et al. (2013)

VU0152099

(PAM)

Reduced amphetamine-induced increase

in LMA

Brady et al.

(2008)

VU0152100

(PAM)

Improved NOR in poorly performing

rats

Reversed amphetamine-induced learning

deficit in contextual fear learning

Reversed amphetamine-induced

disruption of PPI

Reduced cocaine or amphetamine-

induced increase in LMA

Decreased cocaine self-administration

Brady et al.

(2008); Byun et

al. (2014);

Dencker et al.

(2012b);

Galloway et al.

(2014)

VU0467154

(PAM)

Reversed amphetamine or MK-801-

induced increase in LMA

Reversed MK-801-induced learning

deficit in touchscreen pairwise

discrimination task

Reversed MK-801-induced learning

deficit in contextual fear learning

Enhanced acquisition of contextual and

cue dependent fear learning

Bubser et al.

(2014)

CAR, conditioned avoidance response; LMA, locomotor activity; MK-801, non-competitive NMDA receptor

antagonist; NOR, novel object recognition; oxotremorine, non-specific mAChR orthosteric agonist; PAM,

positive allosteric modulator; PPI, prepulse inhibition

Chapter 1

25

1.4 Challenges in Translational Research

Despite the development of the first neuropsychiatric drug being over 60 years ago, drug

discovery for CNS disorders has suffered high attrition rates, largely due to insufficient

efficacy (Kola and Landis, 2004; Millan et al., 2015). Often, drug candidates for CNS

disorders that show promising efficacy in vitro do not show efficacy in animal models, and

of those that do proceed to clinical trials, the majority fail due to lack of efficacy

(Arrowsmith and Miller, 2013; Kola and Landis, 2004). There are many factors

contributing to the impediment in the research and development of psychiatric therapeutics,

and these have been discussed in several reviews (Chandler, 2013; Dean et al., 2014;

Millan et al., 2015; Pankevich et al., 2014; Sams-Dodd, 2013). One of these factors is the

use of animal models to predict therapeutic efficacy of drug candidates (Markou et al.,

2009; McGonigle and Ruggeri, 2014).

The use of animal models has been essential for the understanding of the underlying

pathophysiology of disorders and the prediction of therapeutic efficacy, as well as toxicity,

of drug candidates and therapeutic interventions (McGonigle, 2014; van der Staay et al.,

2009). However, a major limitation and the inherent challenge of modelling

neuropsychiatric disorders in animals is that they are complex human disorders, polygenic

in their aetiology and heterogeneous in their manifestation, where the diagnosis is often

reliant on the patient’s descriptive account of their symptoms (Fernando and Robbins,

2011; McGonigle, 2014). This is particularly the case for schizophrenia, where the precise

aetiology and pathophysiology of the disorder are still unknown (Fernando and Robbins,

2011). Therefore, it is impractical to expect animal models of neuropsychiatric disorders to

represent a disorder in its entirety; rather, ones that model aspects or symptoms of a

disorder are said to have increased validity and utility (Dean et al., 2014; Fernando and

Chapter 1

26

Robbins, 2011; Pankevich et al., 2014). To assess the utility of an animal model and its

representation of the human disorder, a key criterion to consider is validity (Homberg,

2013; McGonigle, 2014). The three common types of validity are face, predictive and

construct validity, first proposed by Willner (1984). Face validity describes the degree of

similarity between the behaviour displayed in the animal model and symptom of the

human disorder. Predictive validity describes the ability of the animal model to respond to

clinically effective therapeutics and to identify compounds with potential therapeutic

efficacy. Construct validity describes the similarity between underlying mechanisms of the

behaviour displayed in the animal model and the aetiology of the human disorder

(Homberg, 2013; Jones et al., 2011; McGonigle, 2014; van der Staay et al., 2009).

One of the main problems facing CNS drug discovery programs is the failure of animal

models to predict therapeutic efficacy in clinical trials (Dean et al., 2014; Markou et al.,

2009; McGonigle and Ruggeri, 2014; Millan et al., 2015; Pankevich et al., 2014). Due to

the lack of understanding of the mechanisms involved in the pathophysiology of the

disorders, many animal models of neuropsychiatric disorders were created using

established therapeutics as reference drug, or “gold standard” (Markou et al., 2009;

McGonigle, 2014; Pankevich et al., 2014). While these assays have predictive validity in

identifying new compounds for the same target, they may not be able to identify

compounds with novel activity, which presented a problem, as drug discovery programs

have been shifting away from developing “me-too” drugs and towards drugs with distinct

mechanism of action (Pankevich et al., 2014). A prime example is the Forced Swim Test

(FST). It was established by Porsolt et al. (1977) to detect the activity of first generation

tricyclic antidepressants, but the test had to be revised to detect the activity of a new class

of antidepressants, the selective serotonin reuptake inhibitors (SSRIs) (Lucki, 1997;

McGonigle, 2014). However, with better understanding of the underlying mechanisms of

Chapter 1

27

the disorders, such as identification of endophenotypes, disease biomarkers or genetic

mutations, animal models can be established to have better construct validity, which can in

turn improve the predictive validity (Markou et al., 2009).

Promising drug compounds may also fall through the cracks with false-negative results

due to species variability of activity. For instance, LY2033298, a M4 mAChR-selective

PAM in the presence of ACh, exhibited high cooperativity with ACh at the human M4

mAChR in both radioligand binding and cell signalling assays (Chan et al., 2008; Leach et

al., 2010). However, LY2033298 alone was unable to elicit an effect in animal models

predictive of antipsychotic activity (Chan et al., 2008; Leach et al., 2010; Suratman et al.,

2011). This is due to the species variability of LY2033298. At the rodent M4 mAChR, the

allosteric potentiation of ACh-induced signalling by LY2033298 was reduced compared to

that at the human variant; therefore, the cooperativity was not high enough to translate to

an effect in vivo (Chan et al., 2008; Leach et al., 2010; Suratman et al., 2011). As drug

candidates are usually only characterised at the human receptor in vitro before proceeding

to in vivo validation, there is the potential that, due to species variability, some drug

candidates in past drug discovery programs were perceived to lack therapeutic efficacy in

animal models, when they do potentially have efficacy in humans (Suratman et al., 2011).

In this case, LY2033298 was able to elicit antipsychotic-like effect in rodent models when

co-administered with a sub-effect dose of a non-specific mAChR orthosteric agonist,

oxotremorine, highlighting probe dependence as another property that should be

considered in drug discovery, especially for allosteric modulators (see 1.1.3.) (Chan et al.,

2008; Leach et al., 2010; Suratman et al., 2011).

Reproducibility is another major issue in preclinical research (Prinz et al., 2011). There are

many factors that can affect animal behaviour and physiology, ranging from inherent

factors, such as species, strain, age and sex of the animals, through to environmental

Chapter 1

28

factors, such as housing, handling and noise exposure of the animals (see Everitt (2015)

and Toth (2015) for reviews). In particular, for neuropsychiatric disorders, where both

genetic and environmental factors are implicated in their aetiology, variations in the

environment may confound results of animal studies. Even within the same strain of inbred

mice, where the mice are genetically identical, the phenotype of each individual mouse

(their “individuality”) is a result of the interaction between genetic and environmental

factors (Claassen, 1994; Loos et al., 2015). Additionally, some environmental factors can

lead to physiological changes. For instance, exposure to noise in laboratory animals can

lead to non-auditory changes in these animals, including cardiovascular, hormonal and

reproductive changes (Turner et al., 2005). Variations in the experimental protocol or data

handling can also lead to different results, which can attribute to reproducibility problems.

Therefore, to improve reproducibility, researchers should be mindful about the internal and

environmental factors in their experiments that can potentially confound the results, and be

thorough when reporting animal experiments to allow for replication studies (Kilkenny et

al., 2010).

1.5 Prepulse Inhibition and Locomotor Activity

In this thesis, animal models and tests were used as a means to measure D1 DR agonist-

induced behaviours associated with the striatum, rather than to model aspects of

schizophrenia (see Chapter 4). The following sections will expand on the two behaviour

tests used in this thesis: prepulse inhibition (PPI) and locomotor activity (LMA). The

rationale of why these tests were chosen is further explained in 4.1.

1.5.1 Prepulse Inhibition of the Startle Reflex

Prepulse inhibition (PPI) is a measure of sensorimotor gating, a cross-species phenomenon

in which a weak sensory input (prepulse) inhibits the motor startle reflex to an subsequent

Chapter 1

29

intense sensory stimulus (pulse) (Swerdlow et al., 2001). The sensorimotor gating is a

protective mechanism designed to filter excess stimuli from the environment out of

awareness, to allow for more important information to be the focus of attention for an

individual (Braff and Geyer, 1990; Braff et al., 2001).

While the exact mechanism by which the prepulse stimulus attenuates the startle response

to a subsequent pulse stimulus is still unknown, key brain areas have been identified

through invasive methods and genetic approaches in rodents (for reviews see Fendt et al.

(2001) and Swerdlow et al. (2001)). Furthermore, a recent study using non-invasive

[18F]fluoro-2-deoxyglucose positron emission tomography (FDG-PET) in rats confirmed

that these previously proposed brain regions for startle and PPI mediation and modulation

were activated during PPI sessions (Figure 1.5) (Rohleder et al., 2014). The neural circuits

involved in PPI of acoustic startle reflex are divided into three pathways: startle, PPI

mediation and PPI modulation pathways (Figure 1.5) (Fendt et al., 2001; Rohleder et al.,

2014; Swerdlow et al., 2001). Both startle and PPI mediation pathways are located in the

brainstem, where the caudal pontine reticular nucleus and the ventrolateral tegmental

nucleus are important areas for mediating startle response and the pedunculopontine

tegmental nucleus and the cuneiform nucleus are important for the mediation of PPI

(Rohleder et al., 2014). The PPI mediation pathway is activated in a phasic manner by the

prepulse, which results in attenuation of the startle response induced by the subsequent

pulse stimulus (Swerdlow et al., 2001). The PPI modulation network comprises of brain

regions associated with the limbic system, including nucleus accumbens, hippocampus,

basolateral amygdala and ventral tegmental area (Figure 1.5). It is hypothesised that the

PPI modulation network acts as a “thermostat” on the PPI mediation pathway by

evaluating the potential danger of the prepulse and/or pulse stimuli, which can be

Chapter 1

30

influenced by affective or attentional states, administration of drugs or neuropathological

changes (Cromwell and Atchley, 2015; Rohleder et al., 2014; Swerdlow et al., 2001).

When PPI is disrupted, the prepulse stimulus has limited ability to attenuate the startle

response induced by the pulse stimulus, which is a reflection of a deficit in sensorimotor

gating (Swerdlow et al., 2000). Deficits in PPI are implicated in several CNS disorders,

such as schizophrenia, obsessive compulsive disorder and Tourette’s syndrome (Braff et

al., 2001; Kohl et al., 2013). Additionally, healthy individuals treated with indirect or

direct DR agonists, such as amphetamine or apomorphine, showed disruption of PPI,

which can be replicated in rodents (Geyer et al., 2001). As such, PPI testing in rodents has

been reported to detect the antipsychotic activity of current antipsychotics, through the

ability of antipsychotics to reduce the psychostimulant-induced disruption of PPI (Dean et

al., 2014; Geyer et al., 2001).

Figure 1.5: Neural network of startle reflex and PPI (Rohleder et al. 2014, used with

permission).

Chapter 1

31

1.5.2 Locomotor Activity

Historically, direct or indirect DR agonists-induced increase in locomotor activity (LMA)

in rodents is commonly used to model positive symptoms of schizophrenia (van den Buuse,

2010). This is in line with the DA hypothesis of schizophrenia, where it is posited that the

positive symptoms are a result of hyperdopaminergic transmission in the mesolimbic

pathway in the brain of patients with schizophrenia (Boyd and Mailman, 2012; Jucaite and

Nyberg, 2012). As all current antipsychotics are either antagonists or partial agonists at D2

DRs, direct or indirect DR agonists-induced hyperlocomotor activity has predictive

validity in identifying potential antipsychotics (Meltzer et al., 1989a; Seeman et al., 1975;

van den Buuse, 2010). Additionally, it has been suggested that hyperlocomotor activity in

rodents may be a model for psychomotor agitation that is found in some patients with

schizophrenia (Kilts, 2001)

The control of movement involves the basal ganglia, which are a collection of nuclei

consisting of the nucleus accumbens (or ventral striatum), the caudate nucleus and the

putamen (or dorsal striatum), the internal segment of the globus pallidus (or medial globus

pallidus in rodents), the external segment of the globus pallidus (or globus pallidus in

rodents), the subthalamic nucleus, the substantia nigra pars reticulata and the substantia

nigra pars compacta (Figure 1.6) (Graybiel, 2005; Lanciego et al., 2012). The striatum is

the primary input nucleus of the basal ganglia, where it receives glutamatergic cortical and

thalamic afferents, as well as dopaminergic projections from the midbrain (Kreitzer and

Malenka, 2008). The majority of the neurons in the striatum are GABAergic medium spiny

neurons (MSNs), which are classified into two subtypes: the striatonigral MSNs, which

highly express D1 DRs, and the striatopallidal MSNs, which highly express D2 DRs

(Cepeda et al., 2008; Ince et al., 1997; Kreitzer and Malenka, 2008). The striatonigral

MSNs make up the direct pathway of the basal ganglia and projects directly to the internal

Chapter 1

32

segment of the globus pallidus and the substantia nigra pars reticulata, which are the output

nuclei of the basal ganglia. Conversely, the striatopallidal MSNs make up the indirect

pathway, which projects to the external segment of the globus pallidus (Figure 1.6)

(Kreitzer and Malenka, 2008). Activation of the direct pathway have been shown to

increase LMA, whereas activation of the indirect pathway suppresses movement initiation,

and together, these pathways play an important role in the bidirectional regulation of motor

behaviour by the basal ganglia (Freeze et al., 2013; Kravitz et al., 2010; Kreitzer and

Malenka, 2008).

Figure 1.6: Schematic representation of the direct and indirect pathways of the basal

ganglia circuit in the rodent brain (Gerfen et al. 2006, used with permission). Abbreviations:

GP, globus pallidus; GPm, medial globus pallidus; SNr, substantia nigra pars reticulata; STN,

subthalamic nucleus.

Chapter 1

33

1.6 Scope of Thesis

As highlighted, there is a great need to identify novel targets for the treatment of

schizophrenia, and M4 mAChRs have been shown to be promising targets. Preclinical

studies have suggested that the modulation of DR functions, specifically that of D1 DRs,

by M4 mAChRs could be a mechanism by which the antipsychotic-like effects of non-

selective muscarinic agonists and PAMs of M4 mAChRs are produced. Most studies

investigating the pharmacology of PAMs have been performed on cell lines over-

expressing the receptor of interest. While such models are useful in amplifying cellular

signalling responses to be detected, such manipulation can also produce responses that do

not represent what occurs in the native environment. Therefore, this thesis aims to better

understand the cross-talk between M4 mAChRs and DRs using a cell line endogenously

expressing the receptors, as well as mice, with the view to aid the development of positive

allosteric modulators of M4 mAChR as treatments for schizophrenia.

In Chapter 2, experiments are described that investigated the potential of an impedance-

based label-free technology to detect and quantify positive allosteric modulation of M4

mAChRs endogenously expressed in a rodent neuronal cell line. The findings of this

chapter revealed that despite the low expression of native M4 mAChRs, positive allosteric

modulation can be detected at the level of whole cell changes, and the parameters

estimated from the data are comparable to those estimated from end-point based assays.

Therefore, these results show that label-free technologies can be used to screen for

allosteric modulators, even for GPCRs with no known G protein coupling preferences.

Chapter 3 describes studies that investigated the potential functional cross-talk between

M4 mAChRs and DRs endogenously expressed in a rodent neuronal cell line. The DR

subtype expressed in this cell line was identified to be the D2 subtype, and the cross-talk

Chapter 1

34

with M4 mAChRs was explored in end-point based assays using combinations of agonists,

antagonists and an M4 mAChR PAM. However, the results revealed that the model used in

these experiments was not suitable for identifying functional cross-talk between these two

receptors. Additionally, the majority of the literature points to a functional cross-talk

between D1 DRs and M4 mAChRs, and with the absence of cell lines that endogenously

express these two subtypes, Chapters 4 and 5 explored the functional cross-talk in vivo.

Chapter 4 describes experiments where a D1 DR-selective orthosteric agonist was used to

induce behavioural changes in mice, which were measured using two behavioural tests

known to involve DRs and the striatum: PPI and LMA. The findings of this chapter

showed that selective activation of M4 mAChRs by a PAM could modulate some D1 DR-

induced behaviours. Experiments described in Chapter 5 sought to confirm the role of M4

mAChRs in the modulation of D1 DR-induced behaviours in whole-body M4 mAChR

knockout mice, but, unfortunately, the results were inconclusive.

Chapter 2:

Detection and Quantification of

Allosteric Modulation of Endogenous

M4 Muscarinic Acetylcholine

Receptor Using Impedance-Based

Label-Free Technology in a Neuronal

Cell Line

Amy N. Y. Chen, Daniel T. Malone, Kavita Pabreja, Patrick M.

Sexton, Arthur Christopoulos, and Meritxell Canals

Journal of Biomolecular Screening 2015, 20(5):646–654

Chapter 2

36

Chapter 2

37

Chapter 2

38

Chapter 2

39

Chapter 2

40

Chapter 2

41

Chapter 2

42

Chapter 2

43

Chapter 2

44

Chapter 3:

Determination of Signalling Cross-

Talk between M4 Muscarinic

Acetylcholine and Dopamine

Receptors Endogenously Expressed

in a Neuronal Cell Line

Chapter 3

46

3.1 Introduction

Belonging to the Rhodopsin family (Family A) of GPCRs, mAChRs are expressed in both

the periphery and the CNS. In the CNS, mAChRs play important roles in regulation of

processes such as cognition, sensory processing and motor control (Felder et al., 2000;

Wess et al., 2007). The M4 mAChR subtype, in particular, is found most abundantly in the

striatum, and dysregulation of this receptor subtype has been associated with disorders

such as schizophrenia (for review, see Carruthers et al. (2015)). Previous work using

whole body M4 mAChR knockout mice has shown that M4 mAChRs play a role in

regulating DA activity in the CNS (Gomeza et al., 1999b; Schmidt et al., 2011; Tzavara et

al., 2004; Zhang et al., 2002b). Furthermore, M4 mAChRs are co-expressed with D1 DRs

in striatonigral MSNs (that form the direct striatal output pathway) in the striatum, and

conditional knockout studies have shown that this subpopulation of M4 mAChRs are

critically involved in the modulation of DA-dependent behaviours (Hersch et al., 1994;

Ince et al., 1997; Jeon et al., 2010; Levey, 1993; Levey et al., 1991; Yan et al., 2001).

DRs, another member of the Rhodopsin family of the GPCRs, are also expressed

throughout the periphery and the CNS. Central DRs are involved in functions such as

locomotor activity, reward and reinforcement, and learning and memory (Beaulieu and

Gainetdinov, 2011). Furthermore, disruption of DA signalling is implicated in a number of

CNS disorders such as schizophrenia and Parkinson’s disease (Boyd and Mailman, 2012).

The main DRs expressed in the striatum are the D1 and D2 subtypes, expressed in MSNs

that make up over 90% of the striatal neuronal population (Kreitzer and Malenka, 2008).

While D1 DRs are predominantly expressed in striatonigral (direct) MSNs, D2 DRs are

found on striatopallidal MSNs that form the indirect striatal output pathway (Shuen et al.,

2008). Additionally, D2 DRs are co-expressed with M4 mAChRs on cholinergic

Chapter 3

47

interneurons in the striatum (Dawson et al., 1988; Kreitzer and Malenka, 2008; Yan and

Surmeier, 1996). D2 DRs are highly implicated in schizophrenia, with all clinical

antipsychotics possessing antagonist or partial agonist activity at the D2 DR (Boyd and

Mailman, 2012). On the other hand, D1 DRs have only recently re-emerged as a

therapeutic target for psychotic disorders (Boyd and Mailman, 2012). Previous studies

using D1 DR-selective antagonists have shown that they can worsen extrapyramidal side

effects in patients with schizophrenia (Den Boer et al., 1995; Karle et al., 1995; Karlsson et

al., 1995), but a recent study demonstrated that selective activation of the D1 DR has the

potential to improve working memory impairment in patients with schizotypal personality

disorder (Rosell et al., 2015).

The interest in the regulation of DA activity by M4 mAChRs, and the therapeutic potential

in the manipulation of this system, was increased recently by studies involving xanomeline

(see 1.2.2), a M1/M4 mAChR-preferring agonist that has been demonstrated to improve

cognitive impairments and ameliorate psychotic effects in patients with schizophrenia

(Shekhar et al., 2008). While xanomeline lacks affinity for DRs, studies in whole-body M4

mAChR knockout mice and in mice with conditional M4 mAChR knockout in D1 DR-

expressing striatal neurons demonstrated that the antipsychotic effects of xanomeline are

mediated predominantly through the M4 mAChR (Dencker et al., 2011; Woolley et al.,

2009).

Prior to the development of allosteric ligands of GPCRs, the ex vivo and in vitro

exploration of signalling cross-talk between the M4 mAChR and the DRs was hampered

due to the high sequence conservation at the orthosteric binding site of mAChRs, resulting

in limited subtype selectivity of the orthosteric ligands targeting this receptor. While it has

been shown that activation of M4 mAChRs reduces DA-induced increases in cAMP

accumulation in rat striatal membranes, these studies were performed with mAChR

Chapter 3

48

orthosteric antagonists and agonists with limited subtype selectivity (Olianas et al., 1996;

Sanchez-Lemus and Arias-Montano, 2006). As mentioned in 1.1.3, allosteric ligands bind

to a topographically distinct binding site to that of endogenous ligands, which is less

conserved across receptor subtypes of the same family, and therefore allosteric ligands can

afford receptor subtype selectivity not seen with orthosteric ligands (Christopoulos, 2014;

Gregory et al., 2010). Additionally, allosteric ligands can enhance or diminish the affinity

and/or efficacy of a co-bound orthosteric ligand, and can also exhibit agonistic activity in

the absence of orthosteric ligands. The development of a number of M4 mAChR subtype-

selective PAMs in recent years (see Table 1.2), made it possible to further explore the

potential signalling cross-talk between the M4 mAChR and DRs (Brady et al., 2008;

Bubser et al., 2014; Chan et al., 2008; Le et al., 2013; Salovich et al., 2012; Shirey et al.,

2008).

In this chapter, we investigated the endogenous expression of DRs and the potential cross-

talk between DRs and M4 mAChRs in a mouse neuroblastoma x rat glioma hybrid cell line,

NG108-15. The allosteric potentiation of ACh-mediated inhibition of cAMP accumulation

and phosphorylation of ERK1/2 by an M4 mAChR PAM were demonstrated in this cell

line in Chapter 2 (Chen et al., 2015). In the present study, we demonstrated that D2-like

DRs, but not D1-like DRs, are endogenously expressed in this cell line. We then performed

interaction studies using M4 mAChR and D2 DR ligands to determine the functional cross-

talk between these two receptors. Our results show a lack of signalling cross-talk between

M4 mAChRs and D2 DRs when tested in cAMP BRET biosensor and ERK1/2

phosphorylation assays. This suggests that the cell line and the assays used in this chapter

are not suitable for the investigation of the cross-talk between M4 mAChRs and D2 DRs.

Chapter 3

49

3.2 Materials and Methods

3.2.1 Materials

High glucose DMEM without sodium pyruvate, high glucose DMEM, HBSS, HAT

supplement, Flp-In CHO cells and PTX were obtained from Life Technologies (Mulgrave,

VIC, Australia). HygroGold was purchased from InvivoGen (San Diego, CA, USA). FBS

was purchased from In Vitro Technologies (Noble Park, VIC, Australia). PEI was

purchased from Polysciences (Warrington, PA, USA). CAMYEL construct and NG108-15

cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Coelenterazine h was purchased from Promega (Alexandria, NSW, Australia). LY2033298

was a generous gift from Christian Felder (Eli Lilly & Co.). All other chemicals were

purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

3.2.2 Cell Culture

NG108-15 cells were grown and maintained in high glucose DMEM without sodium

pyruvate supplemented with 10% FBS and HAT supplement. To differentiate NG108-15

cells, when cells were 70% confluent, full growth medium was replaced with

differentiation medium (high glucose DMEM without sodium pyruvate supplemented with

0.5% FBS, penicillin, streptomycin, HAT and 1% DMSO) and cells were allowed to

differentiate for 3 days. Flp-In CHO stable cell lines were generated as previously

described for human D2L DR (hD2L-FlpIn-CHO; Shonberg et al. (2013)), and human M4

mAChR (hM4-FlpIn-CHO; Nawaratne et al. (2008)). These cells were grown and

maintained in high glucose DMEM supplemented with 10% FBS and 700 µg/mL

HygroGold. All cells were maintained at 37 °C in a humidified incubator containing 5%

CO2.

Chapter 3

50

3.2.3 cAMP Bioluminescence Resonance Energy Transfer Biosensor Assay

A bioluminescence resonance energy transfer (BRET) biosensor, CAMYEL, was used to

measure cAMP levels in live cells (Jiang et al., 2007; Xu et al., 2003). The CAMYEL

sensor is an Epac1 protein with YFP (acceptor) and Rluc (donor) on either termini.

Binding of cAMP to Epac1 induces a conformational change that results in an increase in

the distance between the YFP and Rluc proteins, and thus a decrease in the BRET signal.

Both undifferentiated and differentiated NG108-15 cells were seeded at 2,000,000 per 10

cm culture dish in their respective culture medium and grown overnight. The cells were

transiently transfected with 2 µg of CAMYEL using PEI. Cells were seeded into poly-ᴅ-

lysine coated 96-well Culturplates (PerkinElmer; Waltham, MA) 24 h post-transfection

and assayed at 48 h post-transfection. For PTX experiments, cells were treated with PTX

25 ng/mL for 24 h before assaying. Prior to the start of the assay, cells were allowed to

equilibrate in HBSS at 37°C. Under low light conditions, coelenterazine h was added at a

final concentration of 5 μM 15 min prior to BRET detection. Ligands were added either

alone (for concentration-response curves) or simultaneously (for interaction studies) 5 min

after coelenterazine h. Forskolin was added at a final concentration of 0.1 μM after a

further 5 min. All ligands were dissolved in HBSS and DA was dissolved immediately

prior to use to prevent ligand oxidation. BRET readings were captured with LUMIstar

Omega instrument (BMG LabTech, Offenburg, Germany) that allows for sequential

integration of the signals detected at 475 ± 30 and 535 ± 30 nm, using filters with the

appropriate band pass.

3.2.4 ERK1/2 Phosphorylation Assay

ERK1/2 phosphorylation was measured using the ALPHAScreen SureFire phospho-ERK

kit (TGR Biosciences; Adelaide, SA, Australia). NG108-15 cells were seeded at 30,000

Chapter 3

51

cells per well into a transparent 96-well plate coated with poly-ᴅ-lysine and grown

overnight in culture medium with 2.5% FBS. The next day, culture medium was aspirated,

the cells rinsed with PBS and incubated for 4 h in culture medium with 0.5% FBS before

assaying. For PTX experiments, cells were treated with PTX 25 ng/mL for 20 h before

assaying and during the serum starving incubation. ERK1/2 phosphorylation time-course

experiments were initially performed at least twice to determine the time at which the

ligands were able to elicit the maximum pERK1/2 response (7.5 min for ACh, DA and

LY2033298). Concentration-response curve and functional interaction experiments were

performed at 37°C with single addition and simultaneous addition of the ligands,

respectively. ACh 10 μM was used as a positive control. DA was dissolved in serum-free

DMEM with 0.1%w/v ascorbic acid to prevent ligand oxidation. All other ligands were

dissolved in serum-free DMEM. After 7.5 min incubation, ligand stimulation was

terminated by removal of medium and cells were lysed by addition of cold 100 μL

SureFire lysis buffer (PerkinElmer; Waltham, MA) to each well. Lysates were shaken in

plates for 5 min at RT prior to transferring 5 μL lysate to a white 384-well Proxiplate

(PerkinElmer; Waltham, MA). Under low light conditions, 8 μL of a 240:1440:7:7 mixture

of Surefire activation buffer:Surefire reaction buffer:Alphascreen acceptor

beads:Alphascreen donor beads was added to each well. Plates were incubated in the dark

at 37 °C for 1 h and read with a Fusion-α plate reader (PerkinElmer; Waltham, MA) using

standard AlphaScreen settings. For hM4-FlpIn-CHO and hD2L-FlpIn-CHO cells, the time-

course experiments were performed as described above with some modifications. The cells

were seeded at 50,000 cells per well into a transparent 96-well plate. After 6 h, cells were

washed with PBS and incubated with serum-free DMEM overnight before assaying. FBS

was used as positive control.

Chapter 3

52

3.2.5 Data Analysis

Data were analysed with GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA).

Agonist concentration-response curves were fitted empirically to a three-parameter logistic

equation, where bottom (baseline) and top (Emax) are the lower and upper plateaus of the

concentration-response curve, respectively; [A] is the molar concentration of the agonist;

EC50 is the molar concentration of the agonist required to generate a 50% of the full

response:

Equation 3.1:

𝒀 = 𝒃𝒐𝒕𝒕𝒐𝒎 +𝒕𝒐𝒑 − 𝒃𝒐𝒕𝒕𝒐𝒎

𝟏 + 𝟏𝟎(𝒍𝒐𝒈 𝑬𝑪𝟓𝟎−𝒍𝒐𝒈[𝑨])

Statistical comparisons between Emax, EC50 and baseline values were by one-way analysis

of variance (ANOVA) with a Tukey’s multiple comparisons post-test.

Chapter 3

53

3.3 Results

3.3.1 NG108-15 Cells Endogenously Express D2-like, but Not D1-like, Dopamine

Receptors

A cell line that endogenously expresses M4 mAChR and DRs was used in order to study

the potential functional cross-talk between M4 mAChRs and D1-like or D2-like DRs. The

mouse neuroblastoma x rat glioma hybrid cell line, NG108-15, has been previously

described by our laboratory to endogenously express rodent M4 mAChRs (see Chapter 2)

(Chen et al., 2015; Leach et al., 2010). NG108-15 cells have also been shown to contain

rodent D1 DR mRNA in undifferentiated cells, as well as in cells differentiated in culture

medium containing low FBS and 1% DMSO, though the expression of the receptor protein

at the cell membrane has yet to be determined (Chan et al., 1994; Kaushal et al., 2012).

Therefore, the expression of DRs was explored first in undifferentiated NG108-15 cells by

characterising the effect of the endogenous DR agonist, DA. D1-like and D2-like DRs have

opposing effects on the production of cAMP by AC: D1-like DRs couple to Gαs/olf, which

activates AC and stimulates cAMP production, whereas D2-like DRs preferentially couple

to Gαi/o, which inhibits AC and therefore reduces cAMP levels. Hence, we detected

changes in cAMP levels using the BRET biosensor, CAMYEL, transfected 48 h prior to

assaying. Forskolin was used to induce AC production of cAMP to detect ligand-induced

decreases in cAMP levels. In undifferentiated cells, DA was able to inhibit cAMP

accumulation induced by forskolin. However, when added in the absence of forskolin, DA

did not induce cAMP accumulation (Figure 3.1A). To further delineate the DR subtype

involved, as well as the contribution of Gαi proteins to the observed effect on cAMP

accumulation, we treated the cells with quinpirole, a selective D2 DR-like agonist, and

PTX, which inactivates Gαi proteins, preventing its inhibition of cAMP production by AC

Chapter 3

54

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

L o g [l ig a n d ] (M )

% F

ors

ko

lin

in

du

ce

d c

AM

P D A

Q u in p iro le

Fo

rsko

lin

0.1

uM

D A (w /o fo rs k o lin )

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4


ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

D A

Q u in p iro le

Vehic

le

A B

Figure 3.1: D2-like DRs are endogenously expressed in undifferentiated NG108-15 cells.

(A) DA was unable to induce cAMP accumulation in the absence of forskolin in NG1081-5 cells;

n=1. DA and quinpirole, a D2-like DR partial agonist, both inhibited forskolin 0.1 µM-induced

cAMP accumulation; n=2. (B) In cells treated with PTX, the ability of quinpirole to inhibit

forskolin-induced cAMP accumulation was reduced; n=1. All ligand concentrations are 100 µM.

(C) DA and quinpirole also induced ERK1/2 phosphorylation; n=2-3. (D) DA-induced ERK1/2

phosphorylation was abolished in cells treated with PTX; n=1. All ligands concentrations are

100 µM. Data are presented as mean + SEM, with exception of DA (w/o forskolin), forskolin +

PTX, quinpirole + forskolin + PTX and DA + PTX, which are presented as mean of triplicates.

% F

ors

ko

lin

in

du

ce

d c

AM

P

-4 0

0

4 0

8 0

1 2 0

P T X

F o rs k o lin

D A

Q u in p iro le

- - - - + ++++++-

++ - -- -- +

--

-+

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

-4 0

0

4 0

8 0

1 2 0

P T X

D A

Q u in p iro le

+- -+ +--+ -

C D

Chapter 3

55

(Mangmool and Kurose, 2011). Quinpirole inhibited forskolin-induced cAMP

accumulation with lower potency and efficacy compared to DA (Figure 3.1A). In cells

treated with PTX, this inhibition was reduced, demonstrating the involvement of Gαi

proteins in mediating this effect (Figure 3.1B). These results indicate that undifferentiated

NG108-15 cells endogenously express the D2-like DR subtype, which couples to Gαi/o, and

not Gαs/olf, proteins, and inhibits forskolin-induced cAMP accumulation when activated.

Since differentiation has been suggested to increase D1 DR mRNA, NG108-15 cells that

were allowed to differentiate were then used to examine the expression of D1 DRs by

characterising the effect of the endogenous DR ligand, DA, as well as the D1 DR-selective

agonist, SKF83822, on cAMP production by AC (Kaushal et al., 2012). As it has been

demonstrated that M4 mAChRs are endogenously expressed in undifferentiated NG108-15

cells, it was anticipated that their expression would not change upon NG108-15 cell

differentiation. Therefore, the endogenous agonist, ACh, was used to confirm the

expression of M4 mAChRs by its ability to inhibit forskolin-induced cAMP accumulation

in these cells (see Chapter 2 Figure 1) (Chen et al., 2015; Leach et al., 2010). NG108-15

cells were differentiated by incubation in differentiation medium for 3 days, with

CAMYEL plasmid transfected on day 2 (i.e. 48 h prior to assaying). The differentiation of

NG108-15 cells can be observed as morphological changes after day 2, with cells showing

increased numbers and length of neurites. However, the transfection efficiency of the

CAMYEL plasmid under these conditions was very low, as evidenced by low raw

luminescence and fluorescence counts and weak, non-consistent BRET signals following

ligand addition (Figure 3.2A).

Chapter 3

56

Figure 3.2: DR ligands and ACh inhibit cAMP accumulation in NG108-15 cells

differentiated via different methods. (A) NG108-15 cells were incubated in differentiation

medium for 3 days. DA and SKF83822 did not increase cAMP accumulation in these cells.

Inconsistent data was due to low BRET signals. DA, but not quinpirole and ACh, was able to

inhibit cAMP accumulation induced by forskolin; n=1. (B) NG108-15 cells were incubated in

differentiation medium for 3 days, only substituted for full growth media for 4 h post CAMYEL

transfection on day 2. DA and SKF83822 both showed slight inhibition of baseline cAMP

accumulation. DA, quinpirole and ACh all inhibited forskolin-induced cAMP accumulation; n=1.

(C) NG108-15 cells were incubated in differentiation medium only after 4 h post CAMYEL

transfection and sustained for 44 h until assaying. DA, SKF83822 and ACh showed inhibition of

baseline cAMP accumulation. DA inhibited forskolin-induced cAMP accumulation; n=1. Data are

presented as mean of duplicates.


% F

ors

ko

lin

in

du

ce

d c

AM

P

-8 0

0

8 0

1 6 0

-1 2 -1 0 -8 -6 -4 -2

D A

S K F 8 3 8 2 2 Q u in p iro le + fo rs k o lin

D A + fo rs k o lin

A C h + fo rs k o lin

Fo

rsko

lin

L o g [l ig a n d ] (M )%

Fo

rs

ko

lin

in

du

ce

d c

AM

P

-8 0

0

8 0

1 6 0

-1 2 -1 0 -8 -6 -4 -2

D o p a m in e

S K F 8 3 8 2 2 Q u in p iro le + fo rs k o lin

D A + fo rs k o lin

A C h + fo rs k o lin

Fo

rsko

lin

A B


% F

ors

ko

lin

in

du

ce

d c

AM

P

-8 0

0

8 0

1 6 0

-1 4 -1 2 -1 0 -8 -6 -4 -2 0

A c e ty lc h o lin eD o p a m in e

D A + fo rs k o linS K F 8 3 8 2 2

Fo

rsko

lin

C

Chapter 3

57

As the differentiation medium may be unfavourable for transfection to occur, two other

differentiation methods were applied:

1. Incubate the cells in differentiation medium for 3 days, with medium substituted

with full growth medium for only 4 h post CAMYEL transfection on day 2; or

2. Change the full growth medium to differentiation medium only after 4 h post

CAMYEL transfection, and sustained for 44 h until assaying.

Both methods resulted in higher transfection efficiency, and therefore, better BRET signals,

but as the cells spent less time in differentiation medium, their extent of differentiation was

decreased. While ACh and DA were both able to inhibit forskolin-induced accumulation of

cAMP in cells subject to both treatments, DA in the absence of forskolin was still did not

stimulate cAMP accumulation. Rather, it inhibited basal cAMP accumulation, indicating

the presence of Gαi/o protein-coupled DRs expressed in differentiated NG108-15 cells

(Figures 3.2B, C). Additionally, SKF83822, a D1 DR-selective agonist, did not induce

cAMP accumulation, suggesting that the DR subtype endogenously expressed in

differentiated NG108-15 cells is not the D1 subtype and does not couple to Gαs/olf proteins.

Therefore, these results suggested that in both undifferentiated and differentiated NG108-

15 cells, the endogenously expressed DR is the D2-like DR.

We then investigated the ability of DA and quinpirole to phosphorylate ERK1/2, a

downstream signalling end-point with a higher level of stimulus-response amplification

than that of cAMP, by using the AlphaScreen SureFire Phospho-ERK1/2 assay. Similar to

the results obtained using the cAMP biosensor assay, both DA and quinpirole induced

phosphorylation of ERK1/2, with quinpirole exhibiting lower efficacy compared to DA

(Figure 3.1C). PTX treatment completely abolished the DA-induced phosphorylation of

ERK1/2, indicating that the phosphorylation of ERK1/2 induced by DA was mediated

Chapter 3

58

through activation of Gαi proteins (Figure 3.1D). Collectively, these data suggest that only

D2-like DRs are endogenously expressed in undifferentiated NG108-15 cells.

3.3.2 Interaction Studies Reveal a Lack of Signalling Cross-Talk between M4

Muscarinic Acetylcholine and D2-like Dopamine Receptor Ligands

Following the confirmation that NG108-15 cells endogenously express rodent M4

mAChRs and D2-like DRs, the potential signalling cross-talk between these two receptors

was investigated. As M4 mAChRs and D2 DRs both preferentially couple to Gαi/o proteins

and, therefore, may have similar canonical signalling profiles, combinations of M4

mAChR- and D2 DR-selective agonists, antagonists and an M4 mAChR PAM were used to

study the potential signalling cross-talk between these two receptors (Figure 3.3).

However, it was important to first establish that these ligands were selective for their

respective receptors and would not elicit a response via the other receptor and potentially

confound the results.

Figure 3.3: Chemical structures of M4 mAChR and D2 DR ligands used in interaction

studies.

Chapter 3

59

To confirm the ligand-receptor selectivity, Flp-In CHO cells heterologously expressing the

human M4 mAChR or D2L DR (hM4-FlpIn-CHO and hD2L-FlpIn-CHO, respectively)

were tested in ERK1/2 phosphorylation time-course assays. The heterologous system was

chosen as overexpression of the receptor may amplify any receptor-mediated response that

may occur. ACh, the endogenous mAChR orthosteric agonist, was able to induce robust

ERK1/2 phosphorylation in hM4-FlpIn-CHO cells, reaching a maximum effect after 7.5

min (Figure 3.4A). However, the endogenous DR orthosteric agonist, DA, and D2 DR-like

antagonist, haloperidol, both did not elicit an effect over the 30 min experiment.

Conversely, in hD2L-FlpIn-CHO cells, DA caused phosphorylation of ERK1/2 that

reached a maximum response at 2.5 min (Figure 3.4B). Atropine, a non-selective mAChR

antagonist, LY2033298, a PAM at the M4 mAChR in the presence of ACh, and ACh did

not induce ERK1/2 phosphorylation in these cells. These results confirm that the M4

mAChR and D2 DR ligands are selective for their respective receptors.

h M 4 -F lp In -C H O

T im e (m in )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% F

BS

)

0 1 0 2 0 3 0

0

5 0

1 0 0 D A

H a lo p e r id o l

A C h

h D 2 L -F lp In -C H O

T im e (m in )

0 1 0 2 0 3 0

0

5 0

1 0 0A C h

A tro p in e

L Y 2 0 3 3 2 9 8

D A

A B

Figure 3.4: M4 mAChR and D2 DR ligands are selective for their respective receptors. (A)

In hM4-FlpIn-CHO cells, ACh induced ERK1/2 phosphorylation, but D2 DR ligands, DA and

haloperidol both did not; n=1. (B) In hD2L-FlpIn-CHO cells, DA induced ERK1/2

phosphorylation, but M4 mAChR ligands, ACh, atropine and LY2033298 did not; n=1. All ligand

concentrations are 1 µM. Data are expressed as mean of duplicates.

Chapter 3

60

3.3.2.1 Agonist – antagonist interactions

The potential signalling cross-talk between M4 mAChRs and D2 DRs was then

investigated by first studying the effect of an antagonist of one receptor on the ability of

the orthosteric agonist of the other receptor to inhibit cAMP accumulation and

phosphorylate ERK1/2 in NG108-15 cells. ACh caused a concentration-dependent

decrease of forskolin-induced cAMP accumulation and increase in ERK1/2

phosphorylation (Figures 3.5A, B). Increasing concentrations of the D2 DR-like antagonist,

haloperidol, did not change the maximum response (Emax) or the potency (EC50) of ACh in

cAMP biosensor assay (Figure 3.5A; Appendix 1.1 Table 1). In the ERK1/2

phosphorylation assay, the highest concentration of haloperidol (10 nM) significantly

decreased the potency of ACh; however, this change was not concentration-dependent

(Figure 3.5B; Appendix 1.1 Table 2). Similarly, DA inhibited forskolin-induced cAMP

accumulation and stimulated ERK1/2 phosphorylation in a concentration-dependent

manner, and addition of increasing concentrations of the non-selective mAChR antagonist,

atropine, did not change the maximum response or potency of DA in these assays (Figures

3.5C, D; Appendix 1.1 Tables 3, 4). These results suggest that inhibition of mAChRs or

DRs does not have any impact on the full agonist signalling of the other receptor.

Chapter 3

61

L o g [A C h ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Veh

icle

+

halo

peri

dol

[H a lo p e r id o l]

0 n M

0 .0 0 1 n M

0 .0 1 n M

0 .1 n M

1 n M

1 0 n M

L o g [A C h ] (M )

% F

ors

ko

lin

in

du

ce

d c

AM

P

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Fo

rsko

lin

+

halo

peri

dol

L o g [D A ] (M )

% F

ors

ko

lin

in

du

ce

d c

AM

P

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Fo

rsko

lin

+

atr

op

ine

A

C

B

D

cAMP

L o g [D A ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

-4 0

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Veh

icle

+

atr

op

ine

[A tro p in e ]

0 n M

0 .0 0 1 n M

0 .0 1 n M

0 .1 n M

1 n M

1 0 n M

pERK1/2

Figure 3.5: Interaction study between M4 mAChR and D2 DR agonists and antagonists in

NG108-15 cells did not reveal functional interaction between these two receptors in

cAMP accumulation and ERK1/2 phosphorylation assays. (A-B) Concentration-response

curves to ACh with increasing concentrations of haloperidol in cAMP accumulation and ERK1/2

phosphorylation assays; n=3. (C-D) Concentration-response curves to DA with increasing

concentrations of atropine in cAMP accumulation and ERK1/2 phosphorylation assays; n=3-4.

Data are presented as mean + SEM.

Chapter 3

62

3.3.2.2 Full agonist – full agonist interaction

Interaction studies between the two full agonists, ACh and DA, showed that while

increasing concentrations of DA treatment alone induced a concentration-dependent

increased ERK1/2 phosphorylation, when added with ACh, DA did not affect the potency

nor the maximum response of ACh to phosphorylate ERK1/2 (Figure 3.6A). The same

was seen vice versa, where ACh alone treatment demonstrated a concentration-dependent

increase in ERK1/2 phosphorylation, though co-addition with DA resulted in no change in

both potency and maximum response (Figure 3.6B).

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

0 µ M

0 .1 µ M

0 .3 µ M

1 µ M

[D A ]

L o g [A C h ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

Veh

icle

+

DA

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4 -2

0 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

[A C h ]

L o g [D A ] (M )

Veh

icle

+

AC

h

A B

Figure 3.6: Interaction study between DA and ACh in NG108-15 cells showed additive

agonist effects in ERK1/2 phosphorylation assay. (A) Concentration-response curves to

ACh with increasing concentrations of DA in ERK1/2 phosphorylation assay; n=1. (B)

Concentration-response curves to DA with increasing concentrations of ACh in ERK1/2

phosphorylation assay; n=1. Data are presented as mean of duplicates.

Chapter 3

63

3.3.2.3 Agonist – PAM interactions

The effect of the M4 mAChR PAM, LY2033298, on the signalling responses of DA was

investigated. Similar to what was observed previously, when administered alone,

LY2033298 showed slight agonistic activity in both cAMP accumulation and pERK1/2

assays, as demonstrated by the shift in baselines (Appendix 1.1 Tables 5, 6; also see

Chapter 2 Figures 1, 2) (Chen et al., 2015). DA treatment alone inhibited forskolin-

induced cAMP accumulation and increased ERK1/2 phosphorylation in a concentration-

dependent manner, as demonstrated previously (Figures 3.7A, B; Appendix 1.1 Tables 5,

6). However, increasing concentrations of LY2033298 did not significantly change the

maximum response or potency of DA-mediated signalling.

L o g [D A ] (M )

% F

ors

ko

lin

in

du

ce

d c

AM

P

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Fo

rsko

lin

+

LY

2033298

A BcAMP ERK1/2

L o g [D A ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

0

4 0

8 0

1 2 0

-1 0 -8 -6 -4

Veh

icle

+

LY

2033298

[L Y 2 0 3 3 2 9 8 ]

0

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 M

3 µ M

Figure 3.7: Interaction study between M4 mAChR PAM, LY2033298, and DA in NG108-15

cells did not reveal functional interaction between these two receptors in cAMP

accumulation and ERK1/2 phosphorylation assays. (A-B) Concentration-response curves to

DA with increasing concentrations of LY2033298 in cAMP accumulation and ERK1/2

phosphorylation assays; n=3-6. Data are presented as mean + SEM.

Chapter 3

64

Interestingly, when the DA and LY2033298 interaction study was performed in the

presence of a low ACh concentration (10 nM), the concentration-response curves of DA

changed dramatically (Figure 3.8A). As a M4 mAChR-selective PAM, in addition to

exhibiting agonistic activity at the M4 mAChR, co-addition of LY2033298 can also

positively modulate the affinity and efficacy of ACh (Leach et al., 2010). The effect of

LY2033298 on ACh potency was demonstrated in Figure 3.8A, where increasing

concentrations of LY2033298 robustly potentiated the response to a low concentration of

ACh (10 nM; Appendix 1.1 Table 7). Despite this, the interaction study between DA and

LY2033298 in the presence of a low concentration of ACh revealed that this allosteric

potentiation of ACh at M4 mAChR by LY2033298 did not affect the potency of DA to

phosphorylate ERK1/2 (Figure 3.8A; Appendix 1.1 Table 7). There was a slight increase

in maximal DA response, though this was likely due to the additive effect of 10 nM ACh

maximum response. When the interaction between ACh and LY2033298 was investigated

in the presence of 10 nM DA, increasing concentrations of LY2033298 potentiated both

the maximal response and the potency of combined ACh and 10 nM DA ERK1/2

phosphorylation (Figure 3.8B; Appendix 1.1 Table 8). The 2-fold leftward shift in

potency was likely via potentiation of ACh phosphorylation of ERK1/2 by LY2033298.

DA exerted a limited effect on the change in potency of ACh, as this shift was similar to

that seen in ACh and LY2033298 interaction (see Chapter 2 Figure 2) (Chen et al., 2015).

The robust potentiation of maximal response was likely due to the potentiation of ACh

maximal response by LY2033298, though 10 nM DA may have contributed to the further

increase in maximal response (see Chapter 2 Figure 2) (Chen et al., 2015).

Chapter 3

65

L o g [A C h ] (M )

0

1 0 0

2 0 0

3 0 0

4 0 0

-1 0 -8 -6 -4

0 µ M (w /o D A )

0 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

Veh

icle

+

LY

2033298

[L Y 2 0 3 3 2 9 8 ]

+ D A 1 0 n M

L o g [D A ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% A

Ch

10

M)

0

5 0

1 0 0

1 5 0

-1 0 -8 -6 -4

0 µ M (w /o A C h )

0 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

Veh

icle

+

LY

2033298

[L Y 2 0 3 3 2 9 8 ]

+ A C h 1 0 n M

A B

Figure 3.8: Interaction study between DA or ACh and LY2033298 in the presence of low

concentration of the other orthosteric agonist in NG108-15 cells. (A) Concentration-

response curves to DA with increasing concentrations of LY2033298, with and without 10 nM

ACh in ERK1/2 phosphorylation assay; n=3. Data are presented as mean + SEM. (B)

Concentration-response curves to ACh with increasing concentrations of LY2033298, with and

without 10 nM DA in ERK1/2 phosphorylation assay; n=2. Data are presented as mean + SD.

Chapter 3

66

3.4 Discussion

In this chapter, the potential cross-talk between M4 mAChR and DRs endogenously

expressed in NG108-15, a neuroblastoma x glioma hybrid cell line was investigated. In

undifferentiated NG108-15 cells, D2-like DRs, but not D1-like DRs, were endogenously

expressed as DA inhibited cAMP accumulation induced by forskolin in a PTX-sensitive

manner. This was in contrast to an early study that showed that undifferentiated NG108-15

cells expressed murine D1 DR mRNA (Chan et al., 1994). However, the presence of the

mRNA encoding for the D1 DR does not necessarily mean that this receptor is expressed as

a functional protein at the cell membrane, where the receptor may be activated to stimulate

cAMP production. When we attempted to differentiate NG108-15 cells to induce

expression of D1 DR mRNA as described by Kaushal et al. (2012), we were unable to

produce reliable data with the cAMP biosensor assay to determine the DR expression in

these cells (Figure 3.2A). The advantage of the cAMP biosensor assay is its ability to

detect changes in cAMP in live cells with high sensitivity in real time, and therefore, it is

equally suitable for both Gs and Gi protein-coupled receptors (Salahpour et al., 2012).

However, as this assay requires the transfection of the biosensor CAMYEL, low BRET

signals from this experiment suggests that CAMYEL transfection efficiency was limited

by the differentiated state of NG108-15 cells. To circumvent the need for transient

transfection of a biosensor in differentiated cells, an alternative is to use a non-biosensor

assay to detect cAMP accumulation, such as those that utilise the ALPHAScreen

(amplified luminescent proximity homogeneous assay; Perkin Elmer) or the enzyme

complementation (DiscoveRx) technologies (for review of cAMP detection methods, see

Williams (2004)). However, the major limitations for these alternative technologies are

that cAMP accumulation is detected in cell lysates and this detection cannot be performed

in real-time (Sprenger and Nikolaev, 2013).

Chapter 3

67

While both M4 mAChRs and D2 DRs are co-expressed by cholinergic interneurons in the

striatum, studies exploring the cross-talk between these two receptors are very limited.

Generally, functional cross-talk can be presented as the increase or decrease of signalling

intensity of one GPCR by another, or even altogether a different signal type, whereas

physical cross-talk is presented by the formation of GPCR homo- or heteromers (Guixa-

Gonzalez et al., 2012). Indeed, the D2 DR has been shown to not only exhibit functional,

but also physical cross-talk with a number of GPCRs that are co-localised with D2 DRs in

the striatum, including the D1 DR, D3 DR and the adenosine A2A receptor (for review, see

Guixa-Gonzalez et al. (2012)). Therefore, given the co-localisation of D2 DR and M4

mAChR in striatal cholinergic interneurons, there is the potential that these receptors may

also form heteromers (Dawson et al., 1988; Kreitzer and Malenka, 2008; Yan and

Surmeier, 1996).

The most common techniques used to investigate GPCR homo- and heteromerisation are

FRET and BRET, often complemented by microscopy techniques or biochemical

techniques (Kaczor and Selent, 2011). Both FRET and BRET technologies are based on

the energy transfer of a donor protein to the acceptor protein upon excitation if the

acceptor protein is in close proximity to the donor protein. Major caveats with these two

technologies are the requirements of the attachment of fluorescent proteins or enzymes

onto the receptors of interest and overexpression of these tagged receptors, which may

interfere with natural protein-protein interactions and thus alter the physiological relevance

of the cellular environment (Hebert et al., 2006). Additionally, these assays measure

protein proximity, and therefore, cannot definitively determine the physical cross-talk

between protomers (Gomes et al., 2016). Hence, co-immunoprecipitation assays are often

performed in addition to FRET or BRET assays to confirm that the proteins of interest are

in complex (Gomes et al., 2016). Further investigations into the presence of physical cross-

Chapter 3

68

talk between M4 mAChRs and D2 DRs should be conducted to explore this potential.

However, the present interaction study between M4 mAChR and D2 DR ligands in both

cAMP biosensor and phosphorylation of ERK1/2 assays did not reveal any alterations in

functional signalling between these two receptors. These data suggest that the existence of

functional, and also physical, cross-talk between these two receptors seems highly unlikely.

As with studies investigating M4 mAChR and D2 DR cross-talk, the study of the physical

cross-talk between M4 mAChRs and D1 DRs is limited, despite the co-localisation of M4

mAChRs and D1 DRs in striatonigral MSNs (Hersch et al., 1994; Ince et al., 1997; Levey,

1993; Levey et al., 1991; Yan et al., 2001). The functional cross-talk between M4 mAChRs

and D1 DRs, on the other hand, is much more studied. Whole-body M4 mAChR knockout

mice have been shown to possess increased sensitivity to hyperlocomotor activity induced

by the D1 DR-like selective agonist SKF28292 (Gomeza et al., 1999b). Additionally, mice

with conditional M4 mAChR knockout only in D1 DR-expressing neurons also maintained

this sensitivity, suggesting that this subpopulation of M4 mAChRs are involved in

regulating D1 DR-mediated effects (Jeon et al., 2010). In fact, it has been long suggested

that M4 mAChRs and D1 DRs are functionally coupled (DeLapp et al., 1996). Early studies

using rat striatal tissues showed that DA or the D1 DR-like selective agonist, SKF38393,

stimulated the production of cAMP by AC, and this stimulation was inhibited by addition

of non-selective mAChR agonists that were suggested to be acting through the M4 mAChR

(DeLapp et al., 1996; Kelly and Nahorski, 1986; Olianas et al., 1996; Olianas et al., 1983).

More recently, it has been shown that systematic administration of the M4 mAChR PAM

VU0152100 into rats can regulate ERK1/2 phosphorylation induced by the D1 DR agonist

SKF81297 in the striatum (Xue et al., 2015). Overall, there is a substantial amount of

evidence of both in vitro and in vivo experiments in the literature to suggest a functional

cross-talk between M4 mAChRs and D1 DRs.

Chapter 3

69

The establishment of the role of M4 mAChRs in the regulation of D1 DR functional

activity has catalysed the development of M4 mAChR-selective PAMs for the treatment of

CNS disorders where striatal DA dysfunction is indicated in the pathophysiology (see

table 1.2) (Dencker et al., 2012a; Foster et al., 2014). However, to our knowledge, there

are no immortalised cell lines of neuronal origin available that endogenously express these

two receptors to perform a thorough pharmacological evaluation of the signalling cross-

talk between M4 mAChRs and D1 DRs. Hence, investigating the functional cross-talk

between these two receptors was changed to in vivo methods, which utilised animal

models of psychosis-like behaviours associated with D1 DRs. While many recently

developed M4 mAChR PAMs have been shown to reverse non-selective or indirect DR-

mediated psychosis-like behaviours in rodents, the ability of a M4 mAChR PAM to reverse

behaviours mediated by selective D1 DR activation has yet to be studied (see Table 1.2)

(Brady et al., 2008; Byun et al., 2014; Dencker et al., 2012b; Le et al., 2013; Suratman et

al., 2011). Therefore, attempts to investigate this will be the focus of the rest of the thesis.

Chapter 4:

Studying the Effect of Positive

Allosteric Modulation of M4

Muscarinic Acetylcholine Receptors

on Psychosis-like Behaviours Induced

by a D1 Dopamine Receptor-selective

Agonist in Mice

Chapter 4

71

4.1 Introduction

As discussed in 1.2, schizophrenia is a debilitating disorder of the CNS, with around seven

in 1000 individuals affected by it in their lifetime (McGrath et al., 2008). The symptoms of

schizophrenia are classified into three domains: positive symptoms (or psychosis,

including hallucinations, delusions and disorganised thoughts), negative symptoms

(including avolition and social withdrawal) and cognitive impairment (including deficits in

memory, attention and executive functions) (van Os and Kapur, 2009). While there have

been major milestones in the development of treatments for the positive symptoms – from

the discovery of first-generation (typical) antipsychotics in the 1950s to the development

of second-generation (atypical) antipsychotics in the 1990s – current treatments still

perform poorly in terms of alleviating negative symptoms and improving cognitive deficits

(Hartling et al., 2012; Lewis and Lieberman, 2000). Additionally, antipsychotics are

associated with side effects, including extrapyramidal side effects, cardiovascular

complications, weight gain and other metabolic syndromes (Baptista et al., 2002; Gerlach

et al., 1975; Idanpaan-Heikkila et al., 1975; Parsons et al., 2009). Therefore, there is a

great need for novel therapeutic targets that can treat all three symptom domains without

eliciting unwanted side effects.

The prevalent hypothesis of schizophrenia is the dysregulation of DA transmission in a

number of brain regions, proposed after the discovery that the D2 DR is a common target

for antipsychotics (Davis et al., 1991; Howes and Kapur, 2009; Seeman et al., 1975;

Snyder, 1976). However, the involvement of mAChR dysregulation in the pathology and

the potential of mAChRs as therapeutic targets for schizophrenia have been increasingly

recognised in recent years (Foster et al., 2012; Langmead et al., 2008; Scarr and Dean,

2009). Importantly, as mentioned in 1.2.2, the development of xanomeline, a M1/M4

Chapter 4

72

mAChR subtype-preferring orthosteric agonist, provided a proof-of-concept for the

muscarinic cholinergic hypothesis of schizophrenia (Bodick et al., 1997; Shekhar et al.,

2008). In a pilot study, xanomeline alleviated positive symptoms and ameliorated

cognitive deficits in patients with schizophrenia (Shekhar et al., 2008). Unfortunately,

xanomeline failed in Alzheimer’s disease clinical trials due to an unacceptable proportion

of participants experiencing severe peripheral side effects as a result of the drug acting on

peripheral M2 and M3 mAChRs (Bodick et al., 1997; Shannon et al., 1994; Wess, 2004).

Despite this, the mechanism by which xanomeline mediates its antipsychotic effects

continues to be of great interest. Unlike other antipsychotics, xanomeline has little affinity

for DRs (Shannon et al., 1994). Instead, the antipsychotic effects of xanomeline were

found to be mediated predominately through M4 mAChRs, specifically the M4 mAChRs

co-expressed with D1 DRs in the striatonigral MSNs of the striatum (Dencker et al., 2011;

Woolley et al., 2009). Furthermore, it has been shown that M4 mAChRs are involved in the

regulation of D1 DR-mediated functions, as whole-body M4 mAChR knockout mice, as

well as mice with conditional M4 mAChR knockout only in D1 DR-expressing neurons,

possess increased sensitivity to hyperlocomotor activity induced by D1 DR-like selective

agonists (Gomeza et al., 1999b; Jeon et al., 2010). The positive symptoms of schizophrenia

have been hypothesised to be the result of increased DA transmission in the striatum;

hence, the activation of M4 mAChRs to regulate D1 DR-mediated functions may be a

potential mechanism to obtain antipsychotic activity (Davis et al., 1991; Woolley et al.,

2009).

To further study the therapeutic potential of selective activation of M4 mAChRs, allosteric

modulators were developed (Brady et al., 2008; Bubser et al., 2014; Chan et al., 2008; Le

et al., 2013; Salovich et al., 2012; Shirey et al., 2008). As mentioned in 1.1.3, allosteric

modulators of GPCRs bind to a site (termed the allosteric binding site) that is

Chapter 4

73

topographically distinct from the orthosteric binding site where the endogenous ligand

binds. Allosteric binding sites are less conserved across receptor subtypes, and therefore,

allosteric modulators can be more subtype-selective compared to orthosteric ligands

(Christopoulos, 2014). One of the first reported M4 mAChR PAMs is LY2033298 (Chan et

al., 2008; Leach et al., 2010). LY2033298 is functionally selective for M4 mAChRs in the

presence of ACh, but it can also bind to M2 mAChRs and positively modulate the activity

of oxotremorine at this receptor subtype (Valant et al., 2012). This characteristic is termed

probe dependence (see 1.1.3) (Kenakin, 2005). Additionally, although in vitro experiments

revealed that LY2033298 also showed species variability in that it exhibited greater

potentiation of ACh responses at the human M4 mAChR compared to the rodent receptor,

LY2033298 was the first M4 mAChR PAM to show efficacy in vivo, in rodent models of

psychosis-like behaviour (Chan et al., 2008; Leach et al., 2010; Suratman et al., 2011).

When co-administered with a sub-effective dose of a non-selective mAChR orthosteric

agonist, oxotremorine, LY2033298 reversed PPI deficits in rats induced by non-selective

DR agonist, apomorphine (Chan et al., 2008). PPI is a measure of sensorimotor gating of

the startle reflex, which is a cross-species mechanism where excess stimuli from the

environment are filtered out of awareness in order for more important information to be

the focus of attention for an individual (see 1.5.1) (Braff et al., 2001; Swerdlow et al.,

2008). Deficits in PPI are implicated in a number of CNS disorders, including

schizophrenia (Braff et al., 2001; Kohl et al., 2013). Additionally, the co-treatment of

LY2033298 and oxotremorine was shown to reverse the hyperlocomotor activity induced

by the indirect DA agonist, amphetamine (Suratman et al., 2011). M4 mAChR PAMs,

VU0152099 and VU0152100, which are structurally distinct from LY2033298, also

showed the ability to reverse indirect DA agonist-induced hyperlocomotor activity and

disruption of PPI, though without the requirement of co-treatment of an exogenous

Chapter 4

74

orthosteric agonist, reinforcing the role of M4 mAChR in modulating striatal DA functions

(Brady et al., 2008; Byun et al., 2014; Dencker et al., 2012b).

However, the ability of selective activation of M4 mAChRs by a PAM to regulate specific

D1 DR-induced behaviours has yet to be studied, and therefore, is the main focus of this

chapter. In this chapter, the species variability of LY2033298 and a structural analogue,

ML253 was first compared, and it was found that LY2033298 exhibited less species

variability in its allosteric modulation of ACh. LY2033298 also had better aqueous

solubility compared to ML253 in the formulation used in this study. Therefore,

LY2033298 was selected for these behavioural experiments. The ability of LY2033298 to

reverse the disruption of PPI and increase in LMA induced by a D1 DR-selective

orthosteric agonist, R(+)-6-Br-APB, in mice was then investigated. Previous studies have

co-treated LY2033298 with a sub-effective dose of oxotremorine to enhance the allosteric

potentiation mediated by LY2033298 in rodents (Chan et al., 2008; Gannon and Millan,

2012; Suratman et al., 2011). However, due to the probe dependence nature of LY2033298,

the potentiation of oxotremorine by LY2033298 can be via M2 mAChRs, in addition to M4

mAChRs (Valant et al., 2012). Therefore, to avoid confounding off-target effects on M2

mAChRs, LY2033298 was co-administered with a sub-effective dose of donepezil, an

acetylcholinesterase inhibitor, to increase endogenous ACh tone. We demonstrated that the

combined treatment of LY2033298 and donepezil reversed the disruption of PPI and

increase in LMA induced by R(+)-6-Br-APB in C57Bl/6J mice.

Chapter 4

75

4.2 Material and Methods

4.2.1 Materials

High glucose DMEM, Flp-In CHO cells, geneticin (G418) and PTX were obtained from

Life Technologies (Mulgrave, VIC, Australia). CHO-K1 cells were purchased from

American Type Culture Collection (Manassas, VA, USA). HygroGold was purchased from

InvivoGen (San Diego, CA, USA). FBS was purchased from ThermoTrace (Melbourne,

VIC, Australia). Cis-(Z)-flupentixol dihydrochloride was purchased from Santa Cruz

(Dallas, TX, USA). [3H]NMS, [3H]SCH23390 and [3H]spiperone were purchased from

PerkinElmer (Waltham, MA, USA). Donepezil was purchased from Sapphire Bioscience

(Redfern, NSW, Australia). LY2033298 was a gift from Dr Christian Felder (Eli Lilly &

Co., USA) and ML253 was synthesised according to the general synthetic procedure

previously reported for ML253 (Le et al., 2013) by Monika Szabo and Tracey Huynh

(Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Australia). All other

chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

4.2.2 Cell Culture

Flp-In CHO stable cell lines were generated as previously described for human D2L DR

(hD2L-FlpIn-CHO; Shonberg et al. (2013)), and human M4 mAChR (hM4-FlpIn-CHO;

Nawaratne et al. (2008)). Flp-In CHO cells stably expressing endogenous levels of mouse

D1 DR (mD1-FlpIn-CHO) were generated by Dr Ann Stewart using the mouse D1 DR

cDNA purchased from OriGene (Rockville, MD, USA), subcloning it into pcDEST using

the Gateway (Invitrogen; Carlsbad, CA, USA) strategy and then transfecting this construct

and pOG44 in Flp-In CHO cells (Invitrogen) using a 1:9 ratio. Flp-In CHO cell lines were

grown and maintained in high glucose DMEM supplemented with 10% FBS and 700

µg/mL HygroGold. CHO-K1 cells stably expressing mouse M4 mAChR (mM4-CHO-K1)

Chapter 4

76

were generated as previously described (Suratman et al., 2011), and were grown and

maintained in high glucose DMEM supplemented with 10% FBS and 200 µg/mL G418.

Cells were maintained at 37 °C in a humidified incubator containing 5% CO2.

4.2.3 Preparation of Cell Membranes

When cells were approximately 90% confluent, they were detached using 2 mM EDTA in

PBS (150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4) and centrifuged (300 g, 4°C, 10

min). The resulting pellets were resuspended in 30 ml of ice-cold buffer containing 20mM

HEPES and 10mM EDTA at pH 7.4. The cell suspension was homogenized using a

Polytron homogenizer (PT 1200 CL; Kinematica; Basel, Switzerland), with three 10 s

bursts and 30 s periods of cooling on ice between each burst. The cell homogenate was

centrifuged (300 g, 4°C, 10 min), and the supernatant was transferred to new tubes and re-

centrifuged (30,000 g, 4°C, 1 h) in a Sorvall Evolution RC ultracentrifuge with a SA-600

rotor (Thermo Scientific; Scoresby, VIC, Australia). The pellet was resuspended in 5 ml of

assay buffer (20 mM HEPES and 0.1 mM EDTA, pH 7.4) and briefly homogenized to

ensure uniform consistency. The cell homogenate was then separated into 250 µL aliquots

and stored at -80°C. The protein concentration was determined by the method of Bradford

(Bradford, 1976).

4.2.4 Radioligand Binding Assays in Membrane Preparations

Saturation binding assays in mM4-CHO-K1 cell membranes were performed by

incubating 5 µg membranes with increasing concentrations of [3H]NMS (85.5 Ci/mmol) in

HEPES binding buffer (20 mM HEPES, 100 mM NaCl, and 10 mM MgCl2, pH 7.4) for 1

h at 37°C. Nonspecific binding was defined in the presence of 10 µM atropine and binding

was terminated by fast-flow filtration onto GF/B grade filter paper (Whatman; Maidstone,

UK) using a Brandel harvester, followed by three washes with ice-cold 0.9% NaCl. Bound

Chapter 4

77

radioactivity was measured in a Tri-Carb 2900TR liquid scintillation counter (PerkinElmer;

Waltham, MA, USA). Saturation binding assay in mD1-FlpIn-CHO and hD2L-FlpIn-CHO

cell membranes followed the same procedures, incubating 10 µg mD1-FlpIn-CHO

membranes with increasing concentrations of [3H]SCH23390 (81.9 Ci/mmol), and 5 µg

hD2L-FlpIn-CHO membranes with increasing concentrations of [3H]spiperone (73.4

Ci/mmol), for 3 h at 37°C. Nonspecific binding was defined in the presence of 1 µM cis-

(Z)-flupentixol dihydrochloride for mD1-FlpIn-CHO cell membranes and 10 µM

haloperidol for hD2L-FlpIn-CHO cell membranes.

For competition binding assays, cell membranes (mD1-FlpIn-CHO, 10 μg; hD2L-FlpIn-

CHO, 5 μg) were incubated with increasing concentrations of DA or R(+)-6-Br-APB in

binding buffer (20 mM HEPES, 100 mM NaCl, and 10 mM MgCl2, pH 7.4) containing

100 μM GppNHp and 0.4 nM of [3H]SCH23390 and 0.1 nM [3H]spiperone, for mD1-

FlpIn-CHO and hD2L-FlpIn-CHO membranes, respectively, to a final volume of 0.5 mL.

The cell membranes were incubated at 37 °C for 3 h, and binding was terminated by fast-

flow filtration onto GF/B grade filter paper using a Brandel harvester, followed by three

washes with ice-cold 0.9% NaCl. Nonspecific binding was defined as above and bound

radioactivity was measured in a Tri-Carb 2900TR liquid scintillation counter (PerkinElmer;

Waltham, USA).

4.2.5 ERK1/2 Phosphorylation Assays

ERK1/2 phosphorylation was measured using the AlphaScreen SureFire phospho-ERK kit

(PerkinElmer; Waltham, MA, USA). FlpIn CHO and CHO-K1 cells were seeded at 40,000

cells per well into a transparent 96-well plate. After 6 h, cells were washed with PBS and

incubated in serum-free DMEM overnight before assaying. Time-course experiments were

performed three times to determine the times at which the ligands were able to elicit the

Chapter 4

78

maximum ERK1/2 phosphorylation response (hM4-FlpIn-CHO cells: FBS, ACh,

LY2033298 and ML253 7.5 min; mM4-FlpIn-CHO cells: FBS and ACh 5 min,

LY2033298 and ML253 10 min; mD1-FlpIn-CHO and hD2-FlpIn-CHO cells: FBS, DA

and R(+)-6-Br-APB 5 min).

Functional interaction experiments of ACh and the PAMs (LY2033298 and ML253) were

performed at 37 °C. Increasing concentrations of ACh were simultaneously added in the

absence or presence of increasing concentrations of the PAMs in hM4-FlpIn-CHO cells

and stimulated for 7.5 min. In mM4-CHO-K1 cells, vehicle or increasing concentrations of

the PAMs were added for 5 min before the cells were incubated with increasing

concentrations of ACh for a further 5 min. Concentration-response experiments of DA and

R(+)-6-Br-APB were performed at 37 °C in the presence of 0.1% ascorbic acid. Cells were

stimulated with the ligands for 5 min. 10% FBS was used as positive control for both

experiments.

Ligand stimulation was terminated by removal of medium and cells were lysed by addition

of cold 100 μL SureFire lysis buffer to each well. Lysates were shaken in plates for 5 min

at RT prior to transferring 5 μL lysate to a white 384-well Proxiplate (PerkinElmer;

Waltham, MA, USA). Under low light conditions, 8 μL of a 240:1440:7:7 mixture of

Surefire activation buffer:Surefire reaction buffer:Alphascreen acceptor beads:Alphascreen

donor beads was added to each sample well. Plates were incubated in the dark at 37 °C for

1 h and read with Fusion-α plate reader (PerkinElmer; Waltham, MA, USA) using

standard AlphaScreen settings.

4.2.6 Animals

The Monash Institute of Pharmaceutical Sciences Animal Ethics Committee approved all

procedures on experimental animals. All in vivo studies were conducted using male

Chapter 4

79

C57Bl/6J mice (8 weeks old at the commencement of habituation), obtained from Monash

Animal Research Platform (Clayton, VIC, Australia), and habituated in the Murine

Disease Model Facility holding room for at least a week before experiments. Mice were

acclimatised to being handled at least three times over the 5 days prior to being tested.

Mice were group housed and kept in a holding room with an ambient temperature of 22°C,

humidity 30–40% and a reverse-phase lighting cycle (lights on 7:00 PM, off 07:00 AM).

All test sessions were conducted between 8:00 AM and 6:00 PM, during the most active

phase of the mice. Food and water were available ad libitum. All mice were tested for both

PPI and LMA, with a two-week washout period between tests, each time treated with

different drug combinations (Figure 4.1A). A separate cohort of male, 8 week old

C57Bl/6J mice were used for a brain and plasma exposure pharmacokinetic (PK) study.

A sample size of 10 for both PPI and LMA experiments was calculated using a power

calculation (unpaired t-test) based on the magnitude of effect observed for the drug

treatments versus vehicle in a preliminary study.

4.2.7 Drugs

All drugs were administered i.p., LY2033298 in a 13.33 mL/kg volume, and donepezil and

R(+)-6-Br-APB in 3.33 mL/kg volume. LY2033298 was dissolved in V1: 10% DMSO/5%

Tween 80 in Tris buffer pH 8.9. Donepezil was dissolved in V2: 2% Tween 80 in saline.

R(+)-6-Br-APB was dissolved in V3: water for injection. Mice were treated with the drug

combinations in a pseudorandomised order, and no mouse received the same drug

combination twice.

Chapter 4

80

4.2.8 Prepulse Inhibition of the Acoustic Startle Response (PPI)

PPI was measured using SR-LAB startle chambers (San Diego Instruments, San Diego,

CA, USA). A pilot study was performed to determine the optimum number of pulse-alone

trials conducted at the start of the PPI test session to sufficiently stabilise the startle

amplitude. From this pilot, it was determined that the optimal number of pulse alone trials

was 24 (i.e. 8 of each pulse intensity). These 24 pulse-alone trials were presented at the

beginning and the end of the PPI session to stabilise the startle amplitude and to allow for

observation of acoustic startle habituation, but were not included in PPI calculations

(Figure 4.1C). All mice were acclimatised to the startle chambers during a 30 min session

the day before testing. As shown in Figure 4.1B, on the test day, 40 min prior to testing,

mice were injected with LY2033298 (10 mg/kg) or V1 and donepezil (1 mg/kg) or V2.

After 20 min, mice were injected with R(+)-6-Br-APB (0.1, 0.3 or 0.1 mg/kg) or V3.

Twenty min after the third injection, mice were placed in startle chambers for an initial 5

min acclimatisation period where mice were exposed to a background noise of 65 dB

(Figure 4.1C). This was followed by the PPI session, which was composed of pulse trials

of 100, 110 or 120 dB intensity (denoted as PX, where X = pulse intensity) for 40 ms each,

either alone or preceded by a prepulse (6, 12 or 18 dB above background; denoted as ppY,

where Y = prepulse intensity above background) with an interstimulus interval of 100 ms,

prepulse alone trials and no stimulus trials (background noise alone; Figure 4.1C). The

prepulse plus pulse trials are denoted as PXppY, where X = pulse intensity and Y =

prepulse intensity above background; e.g. P120pp6 = pulse 120 dB, prepulse 6 dB above

background. The PPI session was divided into 10 blocks, where the 16 different trials were

presented once within a block in a pseudorandomised order. Trials were presented with

variable inter-trial intervals (7-23 s) with an average of 15 s. An accelerometer detected the

whole-body flinch of the mouse elicited by the presentation of each trial. The startle

Chapter 4

81

Arrive, 8

weeks old

2 weeks washout period

PPI test

1 week

habituation

LMA test

Cull mice

Behavioural experiment timeline

0 20 80 Time

(min)

Vehicle or

LY or Don

Vehicle or

Br-APB

LMA experiment protocol

Habituation

Placed

in arena

-30 55

Quantified LMA

End of experiment

0 20 40 100 Time

(min)

Vehicle or

LY or Don

Vehicle or

Br-APB Placed in startle chamber

PPI test

PPI experiment protocol

End of experiment

PPI test timeline

Background

habituation

5 min 65 dB

PPI trials:

No stimulus (65 dB)

Pulse alone (100, 110, 120 dB)

Prepulse alone* (pp6, pp12, pp18)

Prepulse + pulse

(P100pp6 P110pp6 P120pp6

P100pp12 P110pp12 P120pp12

P100pp18 P110pp18 P120pp18)

Randomised X 10 blocks

Startle

habituation

8 x (100,

110, 120 dB)

Startle

habituation

8 x (100,

110, 120 dB)

A

B

Figure 4.1: Timeline of behavioural experiments. Coloured arrows along the timeline

indicate the time points of the PK study. Blue arrow: LY2033298 (30, 50, 95 min post i.p.

administration); purple arrow: donepezil (30, 45, 95 min post i.p. administration); green arrow:

R(+)-6-Br-APB (30, 50 min post i.p. administration). PPI: prepulse inhibition; LMA: locomotor

activity; LY: LY2033298; Don: donepezil; Br-APB: R(+)-6-Br-APB; P: pulse; pp: prepulse.

*Denotes dB above 65 dB background (e.g. pp6 = 71 dB).

C

D

Chapter 4

82

magnitude was defined as the average of 100 one-millisecond samples of accelerometer

startle amplitudes immediately following the onset of the pulse. The raw startle amplitudes

from each of the 16 different trials were subjected to an outlier test where values greater or

less than two standard deviations from the mean were excluded (limited to one exclusion

per trial). PPI was calculated as a percentage of startle amplitude using equation:

Equation 4.1:

%PPI = 100 x (pulse alone) − (prepulse plus pulse)

pulse alone

4.2.9 Locomotor Activity (LMA)

LMA was conducted in a well-ventilated dark room with infrared lights in open-field

arenas (40 x 40 x 40 cm). As shown in Figure 4.1D, mice were habituated in the arenas for

30 min, then injected with LY2033298 (10 mg/kg) or V1 and donepezil (0.6 or 1 mg/kg)

or V2, and immediately placed back in the arenas. After 20 min, mice were injected with

R(+)-6-Br-APB (0.3, 1 or 3 mg/kg) or V3, and their LMA was recorded for a further 60

min. LMA was recorded for a total of 110 min with an infra-red camera and the video was

analysed in real time with video-tracking analysis system Viewer® software (BiObserve

GmbH, Bonn, Germany), with distance travelled in cm recorded in 5 min blocks.

4.2.10 Assessment of Compound Exposure in Brain and Plasma

Sample collection was performed with the help of Kwok Ho Christopher Choy (Drug

Discovery Biology, Monash Institute of Pharmaceutical Sciences, Australia) and members

of the Biopharmaceutics Section of the Centre for Drug Candidate Optimisation (Monash

Institute of Pharmaceutical Sciences, Australia). LY2033298 (10 mg/kg), donepezil (0.3

Chapter 4

83

and 1 mg/kg) and R(+)-6-Br-APB (0.6 and 1 mg/kg) were formulated in their respective

vehicles as above and dosed to mice. Blood and brain were collected at 30, 50, 95 min

post-injection for LY2033298 treated mice, at 30, 45, 95 min post-injection for donepezil

treated mice, and at 30 and 50 min post-injection for R(+)-6-Br-APB treated mice. The

time points for LY2033298 and donepezil were chosen to reflect the start and end of the

behavioural tests (Figures 4.1B, D). The 30 min time point for R(+)-6-Br-APB reflects the

time point with the highest hyperlocomotor activity and the 50 min time point is the end of

the LMA test (Figure 4.1D). Three mice were used for each drug dose and time point.

Mice were deeply anesthetised using isoflurane and blood was collected via terminal

cardiac puncture using a 1 mL syringe fitted with a 21 G needle. Following cervical

dislocation, the brain was removed, weighed (0.41 – 0.48 g) and frozen on dry ice.

Collected blood was transferred to an Eppendorf tube containing 10 µL of heparin +

stabilising cocktail solution (1 tablet of Complete® EDTA-free dissolved in 1 mL of 4

mg/kg KF and 0.1 M EDTA solution, and 200 µL of 1000 U/mL heparin) and kept on ice.

The Eppendorf tubes were inverted to mix, followed by centrifugation in a microcentrifuge

to separate plasma at 10,000 rpm for 5 min, and the supernatant was stored at -80°C until

analysis.

Sample analyses were performed by members of the Biopharmaceutics Section of the

Centre for Drug Candidate Optimisation (Monash Institute of Pharmaceutical Sciences,

Australia). Plasma standards were prepared with addition of 10 µL of solution standards

(known concentrations of the LY2033298, donepezil or R(+)-6-Br-APB diluted with 50%

acetonitrile in water) and 10 µL of internal standard (diazepam, 6 µg/mL) to 50 µL of

plasma collected from treatment-naïve mice. Plasma samples (50 µL) were similarly

prepared, with addition of 10 µL 50% acetonitrile instead of solution standards. Protein

precipitation was performed with addition of 130 µL acetonitrile, followed by vortexing

Chapter 4

84

for 20 s and centrifugation at 10,000 rpm for 3 min in a microcentrifuge. Subsequently, the

supernatant was isolated and injected into LC-MS.

Whole brains collected from treatment-naïve mice were homogenised in 3 volume/weight

of water with a glass rod. Brain homogenate standards were prepared by adding 10 µL of

solution standards and 10 µL of internal standard (diazepam, 6 µg/mL) to 200 µL brain

homogenate containing 50 mg tissue. Brain samples were similarly prepared, with addition

of 10 µL 50% acetonitrile instead of solution standards. Protein precipitation was

performed with addition of 600 µL acetonitrile, followed by vortexing for 20 s and

centrifugation at 10,000 rpm for 3 min in a microcentrifuge. Finally, the supernatant was

isolated and injected into LC-MS.

Both plasma and brain homogenates were analysed by LC-MS. LC separation was

performed using an Acquity UPLC (Waters; Rydalmere, NSW, Australia) with an Ascentis

Express RP-Amide column (2.7 µm, 50 x 2.1 mm; Sigma-Aldrich; Castle Hill, NSW,

Australia) fitted with a Security Guard column and Synergi polar packing material

(Phenomenex; Lane Cove, NSW, Australia) at a flow rate of 0.4 mL/min at 40°C. HPLC

analysis was implemented with the use of acetonitrile-water gradients containing 0.05%

formic acid. MS was performed using a Xevo TQ triple quadruple Micromass (Waters;

Rydalmere, NSW, Australia) mass spectrometer in positive ion mode, controlled by

system software, QuanLynx. Multiple-reaction monitoring was employed to confirm the

elution of analytes and internal standard, using the transitions from m/z 312.04 to 255.03 at

12 eV for LY2033298, m/z 380.32 to 90.83 at 30 eV for donepezil, m/z 373.76 to 214.49 at

45 eV for R(+)-6-Br-APB and m/z 285.08 to 154.05 at 25 eV for the internal standard

(diazepam).

Chapter 4

85

Calibration curves were constructed using the plasma and brain homogenate standards,

extracted as described above. Calibration curves for all drugs were defined by a quadratic

function. The concentrations of plasma and brain homogenate samples were determined

relative to calibration curves, and the concentrations were given as non-salt equivalent.

4.2.11 Data and Statistical Analysis

4.2.11.1 Cell-based Assays

For cell-based assays, data were analysed with GraphPad Prism 6.01 (GraphPad Software,

La Jolla, CA). Concentration-response data from ERK1/2 phosphorylation studies were

normalised to maximum response induced by 10% FBS. Allosteric modulation was

quantified by applying the operational model of allosterism to the concentration-response

curves of the interaction between ACh and the PAMs (Leach et al., 2007):

Equation 4.2:

𝐸 =𝐸𝑚

1 + [([𝐴]𝐾𝐵 + 𝐾𝐴𝐾𝐵 + 𝐾𝐴[𝐵] + 𝛼[𝐴][𝐵])

(𝜏𝐴[𝐴](𝐾𝐵 + 𝛼𝛽[𝐵]) + 𝜏𝐵[𝐵]𝐾𝐴)]

𝑛

where Em is the maximal effect of the pathway; [A] and [B] are concentrations of the

orthosteric agonist and the allosteric modulator, respectively; KA and KB are the

equilibrium dissociation constant of the orthosteric agonist and allosteric modulator,

respectively; τA and τB are operational measures of the respective signalling efficacies of

orthosteric agonist and allosteric modulator that incorporate receptor expression levels and

efficiency of stimulus-response coupling; α is the cooperativity factor of the allosteric

effect of the modulator on orthosteric agonist binding affinity, whereas β is that of the

signalling efficacy; and n is the transducer slope factor linking occupancy to response. All

affinity, potency and cooperativity values were estimated as logarithms (Christopoulos,

Chapter 4

86

1998). Statistical comparisons between values were by one-way analysis of variance

(ANOVA) using a Sidak’s multiple comparisons post hoc analysis.

Agonist concentration-response curves were fitted empirically to a three-parameter logistic

equation:

Equation 4.3:

𝑌 = 𝑏𝑜𝑡𝑡𝑜𝑚 +𝑡𝑜𝑝 − 𝑏𝑜𝑡𝑡𝑜𝑚

1 + 10(𝑙𝑜𝑔 𝐸𝐶50−𝑙𝑜𝑔[𝐴])

where bottom (baseline) and top (Emax) are the lower and upper plateaus of the

concentration-response curve, respectively; [A] is the molar concentration of the agonist;

EC50 is the molar concentration of the agonist required to generate a 50% of the full

response.

4.2.11.2 Behavioural Experiments

For animal experiments, statistical analysis was performed using IBM® SPSS® Statistics

Version 23 for Windows (IBM® Corp., NY, USA). Although three pulse intensities were

tested, disruption of PPI induced by R(+)-6-Br-APB was most prominent at 120 dB,

therefore only the data from 120 dB pulse intensity are presented in this chapter (see

Appendix 2 for results from 100 and 110 dB pulse intensities). PPI data were analysed

using a repeated-measures ANOVA to compare startle amplitude and PPI values between

all treatment groups, with prepulse intensity (6, 12 or 18 dB above background) as within-

subjects factor and drug treatment as between-subjects factor. For the R(+)-6-Br-APB

dose-response PPI experiments, where there was a main effect of drug treatment on PPI or

significant drug treatment and prepulse intensity interaction, a one-way ANOVA with

Dunnett t-test post hoc analysis was performed with vehicle treatment group as control to

Chapter 4

87

determine the level of significance at each prepulse intensity for PPI. For the LY2033298

and donepezil combined treatment PPI experiments, where there was a main effect of drug

treatment on PPI or significant drug treatment and prepulse intensity interaction, a one-

way ANOVA with Tukey HSD post hoc analysis was performed to determine the level of

significance at each prepulse intensity for PPI. Effect of drug treatment on startle

amplitude induced by 120 dB was analysed with a one-way ANOVA with Tukey HSD

post hoc analysis. Significant differences between treatment groups were indicated with *

p < 0.05, ** p < 0.01 and *** p < 0.001.

LMA over time from 25 min after the first two injections onwards were analysed using a

repeated-measures ANOVA, with time as a within-subjects factor and treatment groups as

a between-subjects factor. When there was a main effect of drug treatment or a significant

drug treatment and time interaction on LMA, a one-way ANOVA with drug treatment as

fixed factor and each time point as dependent variables were used. For R(+)-6-Br-APB

dose-response experiments, Dunnett t-test post hoc analysis was applied, with vehicle

treatment group as control. For LY2033298 and donepezil combined treatment

experiments, Tukey HSD post hoc analysis was performed to determine the level of

significance at each time point.

Distances travelled every 5 min from 25 to 55 min after the first two injections were

summed to calculate cumulated distance travelled. This timeframe was used to capture the

maximum increase in LMA induced by R(+)-6-Br-APB in C57Bl/6J mice. Cumulated

distance travelled was analysed using a one-way ANOVA, with distance travelled as fixed

factor and treatment groups as between-subjects factors. For both the R(+)-6-Br-APB

dose-response experiments and the LY2033298 and donepezil combination treatment

experiments, when there was a main effect of drug treatment on LMA, Tukey HSD post

Chapter 4

88

hoc analysis was applied. Significant differences between treatment groups were indicated

with * p < 0.05, ** p < 0.01 and *** p < 0.001.

4.2.11.3 Assessment of Compound Exposure in Brain and Plasma

The determined brain homogenate concentrations of LY2033298, donepezil and R(+)-6-

Br-APB were the total concentrations of the drugs in the brain, including those found in

the microvasculature of the brain. To determine the drug concentration in the brain

parenchyma (Cparenchyma), the contribution of drug within the brain microvasculature was

subtracted from the total brain concentration (Cbrain) for each mouse:

Equation 4.4:

𝐶𝑝𝑎𝑟𝑒𝑛𝑐ℎ𝑦𝑚𝑎 = 𝐶𝑏𝑟𝑎𝑖𝑛 − (𝐶𝑝𝑙𝑎𝑠𝑚𝑎×𝑉𝑣𝑎𝑠𝑐𝑢𝑙𝑎𝑡𝑢𝑟𝑒)

where Cplasma is the concentration of the drug that was determined from the plasma samples

and Vvasculature is the plasma volume of the brain vasculature, which was found to be 0.017

mL/g for C57Bl/6 mice (Nicolazzo et al., 2010).

Compound exposure data were analysed with GraphPad Prism 6.01 (GraphPad Software,

La Jolla, CA). Compound exposure experiments of donepezil were conducted with 0.3 and

1 mg/kg doses; however, the exposure data for donepezil 0.6 mg/kg was also required, as

this was the dose used in LMA experiments. As the values for donepezil 1 mg/kg at each

time-point were approximately 3 times that of the corresponding values for donepezil 0.3

mg/kg, it can be assumed that the brain homogenate concentration of donepezil after 0.3,

0.6 and 1 mg/kg i.p. administration at each time point follows a linear correlation.

Therefore, to minimise animal use, the exposure data of donepezil 0.6 mg/kg were

estimated from the interpolation of the linear regression of donepezil 0.3 and 1 mg/kg at

Chapter 4

89

each time point (30 min equation: Y = 260.2*X - 15.52; 45 min equation: Y = 142.3*X +

4.8; 95 min equation: Y = 94.86*X + 0.14).

Drug concentrations in the brain (expressed as ng/g) and plasma (expressed as ng/mL)

were converted to molar concentrations by taking consideration of a brain density of 1

g/mL (Barber et al., 1970).

Chapter 4

90

4.3 Results

4.3.1 Potentiation of ACh Function at M4 mAChRs by a Next Generation M4

Muscarinic Receptor Positive Allosteric Modulator, ML253, is Subject to Species

Variability

ML253 was developed recently as a next generation M4 mAChR PAM, with improved

aqueous solubility and reduced allosteric modulation species variability compared to

previously reported M4 mAChR PAMs, such as LY2033298 (Figure 4.2) (Le et al., 2013).

These purported properties make ML253 an ideal alternative to LY2033298 for in vivo

testing, as it potentially overcomes previous issues of differential PAM effects between

rodent and human receptors, and may achieve increased brain penetration (Le et al., 2013;

Suratman et al., 2011). However, in the Le et al. (2013) study, ML253 was characterised in

CHO cells co-transfected with human or rat M4 mAChR and a chimeric Gαqi5 protein,

allowing the M4 mAChR, which predominantly couples to Gαi/o proteins, to signal through

Gαq proteins and mobilise intracellular calcium (Conklin et al., 1993; Salovich et al., 2012).

While this method is beneficial for high-throughput screening of test compounds for

allosteric modulators, further characterisation of ML253 in cells with wildtype G proteins

is needed to determine its functional allosteric parameters (i.e. potency (KB), efficacy (τB)

and ability to potentiate ACh function (composite cooperativity; αβ)) in more “native”

systems. Additionally, a thorough comparison of the functional allosteric parameters

between ML253 and LY2033298 at the human and mouse M4 mAChR is needed to

determine the true extent of the difference in species variability of these two PAMs.

Therefore, we performed ERK1/2 phosphorylation interaction experiments on the PAMs

and ACh to address these issues.

Chapter 4

91

Figure 4.2: Chemical structures of LY2033298 and ML253 (M4 mAChR PAMs), donepezil

(acetylcholinesterase inhibitor) and R(+)-6-Br-APB (D1 DR-selective agonist).

H u m a n M 4 m A C h R

L o g [A C h ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% F

BS

)

0

5 0

1 0 0

1 5 0

-1 0 -8 -6 -4

0 µ M

0 .0 1 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

[L Y 2 0 3 3 2 9 8 ]

Veh

icle

+

LY

2033298

H u m a n M 4 m A C h R

L o g [A C h ] (M )

0

5 0

1 0 0

1 5 0

-1 0 -8 -6 -4

0 µ M

0 .0 1 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

[M L 2 5 3 ]

Veh

icle

+

ML253

M o u s e M 4 m A C h R

L o g [A C h ] (M )

ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% F

BS

)

0

5 0

1 0 0

-1 0 -8 -6 -4

0 M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

[L Y 2 0 3 3 2 9 8 ]

3 µ M

Veh

icle

+

LY

2033298

M o u s e M 4 m A C h R

L o g [A C h ] (M )

0

5 0

1 0 0

-1 0 -8 -6 -4

0 µ M

0 .0 3 µ M

0 .1 µ M

0 .3 µ M

1 µ M

[M L 2 5 3 ]

Veh

icle

+

ML253

Figure 4.3: LY2033298 and ML253 potentiation of ACh-induced ERK1/2 phosphorylation

in CHO cells stably expressing human or mouse M4 mAChRs are subject to species

variability. Concentration-response curves of ACh-induced ERK1/2 phosphorylation with

increasing concentrations of either PAMs in CHO cells stably expressing the human [(A)

LY2033298; (B) ML253] or the mouse M4 mAChRs [(C) LY2033298; (D) ML253]. Curves shown

represent the best fit of the operational model of allosterism, Equation 4.2. Data are presented

as mean + SEM; n=3.

A B

C D

Chapter 4

92

In both hM4-FlpIn-CHO and mM4-CHO-K1 cells, ACh induced phosphorylation of

ERK1/2 in a concentration-dependent manner (Figures 4.3A-D, open circles). Both

LY2033298 and ML253 induced ERK1/2 phosphorylation in a concentration-dependent

manner at the human M4 mAChR on their own, demonstrating their allosteric agonist

properties (Figures 4.3A, B). These properties were retained at the mouse M4 mAChR,

although they were markedly reduced (Figures 4.3C, D). Addition of increasing

concentrations of LY2033298 robustly potentiated the potency of the ACh response at the

human M4 mAChR, as evident by the LY2033298 concentration-dependent left-ward shift

of the ACh curves (Figure 4.3A). This potentiation of ACh response was reduced at the

mouse receptor, which is in agreement with previous observations (Figure 4.3C)

(Suratman et al., 2011). Similarly, while ML253 strongly potentiated the potency of ACh

response at the human receptor, this potentiation was also reduced at the mouse receptor,

confirming that the allosteric interaction between both PAMs and ACh are subject to

species variability (Figures 4.3B, D).

To quantify and compare the allosteric effect of LY2033298 and ML253 across species,

the operational model of allosterism (Equation 4.2) was applied to the concentration-

response curves to yield allosteric parameters (Table 4.1). To aid analysis, the equilibrium

dissociation constants of ACh and LY2033298 (denoted as KA and KB, respectively) at the

human and the mouse M4 mAChR were fixed to those determined from radioligand

binding assays (Suratman et al., 2011). Efficacy values of the agonist and PAMs (denoted

as τA and τB, respectively) at the human and mouse M4 mAChR were adjusted for the

different M4 mAChR receptor expression levels for each cell line obtained by [3H]NMS in

membrane saturation binding experiments (hM4-FlpIn-CHO Bmax = 1.1 ± 0.2 pmol/mg

(Nawaratne et al., 2008) and mM4-CHO-K1 Bmax = 0.48 ± 0.05 pmol/mg of membrane

protein (n=3)).

Chapter 4

93

The affinity of ML253 (denoted as the equilibrium dissociation constant, KB) at the human

M4 mAChR was estimated to be 1.20 µM (pKB = 5.92), which was approximately 8 times

lower than at the mouse receptor (0.15 µM; pKB = 6.82). In contrast, the affinities of

LY2033298 at the human and the mouse M4 mAChR were very similar (KB = 4.07 µM,

pKB = 5.39 for the human receptor vs KB = 3.24 µM, pKB = 5.49 for the mouse receptor)

(Suratman et al., 2011). This suggests that, in contrast to LY2033298, the affinity of

ML253 at the M4 mAChR is subject to species differences and exhibited higher affinity for

the mouse than for the human receptor. The efficacy of ACh (denoted as τA) was

significantly higher at the mouse M4 mAChR than the human receptor (τA, 57 vs 9,

respectively, in ACh and LY2033298 interaction experiments; τA, 63 vs 14, respectively,

Table 4.1: Operational Model Parameters for Functional Interaction between ACh and

LY2033298 or ML253 at Human and Mouse M4 mAChR

Parameter ACh + LY2033298 ACh + ML253

Human M4 Mouse M4 Human M4 Mouse M4

pEC50a 7.11 ± 0.13 7.10 ± 0.10 7.32 ± 0.14 7.19 ± 0.19

pKAb = 5.84 = 5.74 = 5.84 = 5.74

pKBc = 5.39 = 5.49 5.92 ± 0.70 6.82 ± 0.33

LogτAd (τA) 0.96 ± 0.10 (9) *1.75 ± 0.15 (57) 1.16 ± 0.13 (14) *1.80 ± 0.16 (63)

LogτBe (τB) 1.35 ± 0.09 (22) 0.65 ± 0.12 (4) 1.11 ± 0.63 (13) -0.15 ± 0.18 (1)

Logαβf (αβ) 4.10 ± 0.14 (12531) *1.86 ± 0.24 (72) 3.17 ± 0.72 (1476) *1.24 ± 0.25 (17)

Allosteric parameters were estimated from the operational model of allosterism, Equation 4.2, and are

presented as mean ± SEM; n=3. Asterisk indicates value significantly different from corresponding value

at the human receptor (*p < 0.05; one-way ANOVA with Sidak’s multiple comparisons test). a Negative logarithm of the concentration of ACh that produces half the maximal agonist response. b Negative logarithm of the equilibrium dissociation constant of ACh; value was fixed to that determined

from radioligand binding assays at the human or mouse mAChR expressed in CHO cells (Suratman et

al., 2011). c

Negative logarithm of the equilibrium dissociation constant of the PAM; value for LY2033298 was

fixed to that determined from radioligand binding assays at the human or mouse M4 mAChR expressed

in CHO cells (Suratman et al., 2011). d Logarithm of the operational efficacy parameter of ACh as an orthosteric agonist. Antilogarithm shown

in parentheses. e Logarithm of the operational efficacy parameter of the PAM as an allosteric agonist. Antilogarithm

shown in parentheses. f Logarithm of the product of the binding (α) and efficacy (β) cooperativity factors between ACh and the

PAM. Antilogarithm shown in parentheses.

Chapter 4

94

in ACh and ML253 interaction experiments; both p<0.05, one-way ANOVA with Sidak’s

multiple comparisons test). The allosteric agonism, or efficacy, of LY2033298 (denoted as

τB, and corrected for receptor expression levels) was markedly reduced at the mouse M4

mAChR compared to the human receptor (τB, 4 vs 22, respectively), which was in

agreement with previous observations (Suratman et al., 2011). This was also the case for

ML253 (τB, 1 vs 13, respectively), though the difference between the efficacy of ML253 at

the mouse and the human receptor was greater compared to LY2033298.

The composite cooperativity (αβ) is a global measure of the ability of the PAMs to

potentiate ACh affinity and efficacy at the M4 mAChR. The αβ for LY2033298 was

estimated to be 12531 at the human receptor (Logαβ = 4.10), which was higher compared

to a previously reported value of 372 (Leach et al., 2010). The magnitude of positive

cooperativity between ACh and LY2033298 was significantly reduced at the mouse

receptor (αβ = 72; Logαβ = 1.86; p < 0.05, one-way ANOVA with Sidak’s multiple

comparisons test). The positive cooperativity between ACh and ML253 was also

significantly decreased at the mouse receptor compared to the human (αβ = 17; Logαβ =

1.24 for the mouse receptor vs αβ = 1476; Logαβ = 3.17 for the human receptor; p < 0.05,

one-way ANOVA with Sidak’s multiple comparisons test), though the difference was less

compared to LY2033298.

Overall, while ML253 demonstrated less species variability in functional cooperativity

compared to LY2033298, it has higher affinity for the rodent receptor compared to the

human, and its allosteric agonism was impacted by species differences to a greater extent

than LY2033298. However, in our hands, ML253 had poorer aqueous solubility compared

to LY2033298, as at 3 µM concentration, ML253 fell out of solution, while LY2033298

stayed soluble. As the animal experiments for the present study will require more

Chapter 4

95

concentrated drug solutions, LY2033298 was chosen for the subsequent animal

behavioural studies.

4.3.2 In Vitro and In Vivo Characterisation of R(+)-6-Br-APB, a Selective D1

Dopamine Receptor Agonist

To investigate the ability of selective M4 mAChR positive allosteric modulation to alter

specific D1 DR-mediated behavioural effects in mice, we chose to study two mouse

models of aspects of psychosis-like behaviour that involve the striatum where the receptors

are co-localised, namely drug-induced disruption of PPI and hyperlocomotor activity.

(R)-3-Allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepinium

(R(+)-6-Br-APB; Figure 4.2), a D1 DR-selective agonist, has been shown to disrupt PPI

and induce hyperlocomotor activity in C57BL/6J mice (Ralph and Caine, 2005; Thomsen

et al., 2011). To confirm the selectivity of R(+)-6-Br-APB, we tested the compound in a

competition radioligand binding assay to determine its affinity, as well as in a functional

assay to determine its potency and efficacy, at the D1 and D2 DRs. We also assessed the

brain (and plasma) exposure in vivo in mice following i.p. administration to determine the

concentration of R(+)-6-Br-APB in an attempt to correlate with the in vitro data.

4.3.2.1 R(+)-6-Br-APB displays higher affinity for, and has higher potency at D1

compared to D2 Dopamine Receptors

R(+)-6-Br-APB was developed from a stereoisomeric study of SKF38393, a D1/D5 DR-

selective partial agonist of the benzazepine class (Neumeyer et al., 1992). Of the

compounds described in this study, R(+)-6-Br-APB displayed the highest D1 DR affinity

of 4.3 nM and a D1 DR selectivity of 119-fold over D2 DR in rat forebrain tissues. We

tested the affinity of this ligand in our hands using FlpIn CHO cells overexpressing the

mouse D1 or the human D2L DRs (mD1-FlpIn-CHO and hD2L-FlpIn-CHO, respectively).

Chapter 4

96

Membrane radioligand competition binding studies estimated that the affinity of R(+)-6-

Br-APB at the mouse D1 DR was 8.91 nM (pKi = 8.05; n=1), whereas its affinity at the

human D2L DR was 977 nM (pKi = 6.01, SD = 0.27; n=2). These results showed that R(+)-

6-Br-APB had a D1 DR selectivity of 110-fold over D2 DR, which were in accordance with

the values from literature (Neumeyer et al., 1992).

We then tested the ability of R(+)-6-Br-APB to induce ERK1/2 phosphorylation in mD1-

FlpIn-CHO and hD2L-FlpIn-CHO cells. R(+)-6-Br-APB induced phosphorylation of

ERK1/2 at the mouse D1 DR in a concentration-dependent manner, with similar efficacy as

the full agonist, DA, and exhibited picomolar potency, with an EC50 of 310 pM (pEC50 =

9.51; Figure 4.4A). In contrast, the potency of R(+)-6-Br-APB to induce ERK1/2

phosphorylation at the D2L DR was 80 times lower, with an EC50 of 24 nM (pEC50 = 7.61;

Figure 4.4B). Furthermore, the higher potency of R(+)-6-Br-APB at the mouse D1 DR was

not a result of a higher receptor expression in these cell lines, as membrane radioligand

saturation binding studies on the two cell lines showed that the human D2L DRs were

A B

Figure 4.4: R(+)-6-Br-APB has higher potency at the mouse D1 DR than human D2 DR in

ERK1/2 phosphorylation. DA and R(+)-6-Br-APB induced ERK1/2 phosphorylation in Flp-In

CHO cells heterologously expressing the (A) mouse D1 DRs and (B) human D2 DRs. Data are

presented as mean + SEM; n=3. Br-APB: R(+)-6-Br-APB.

M o u s e D 1 D R


ER

K1

/2 p

ho

sp

ho

ry

lati

on

(% F

BS

)

-2

0

2

4

6

8

1 0

-1 4 -1 2 -1 0 -8 -6 -4

D A

B r-A P B

Vehic

le

H u m a n D 2 D R


-1 0

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1 0

2 0

3 0

4 0

-1 4 -1 2 -1 0 -8 -6 -4

D A

B r-A P B

Vehic

le

Chapter 4

97

expressed at a higher level than the mouse D1 DRs (Bmax, 2219 ± 157 vs 758 ± 86 pmol/mg,

respectively; n=3).

Whilst R(+)-6-Br-APB demonstrated high selectivity for D1 DR over D2 DR in vitro, when

administered in mice, it is still possible for the drug to bind to D2 DRs and exert an effect

if R(+)-6-Br-APB is present in high enough concentrations in the brain. Therefore, brain

exposure was assessed in mice after i.p. administration of 0.3 and 1 mg/kg R(+)-6-Br-APB.

At 30 min after administration of 1 mg/kg of R(+)-6-Br-APB, total brain concentration

was 169 nM (63 ng/g), which was reduced to 74 nM (28 ng/g) at 50 min post

administration (Figure 4.5). Total brain concentration of R(+)-6-Br-APB after

administration of 0.3 mg/kg was substantially lower at both time-points, only reaching 41

and 36 nM at 30 and 50 min post-administration, respectively (15 and 14 ng/g,

respectively; Figure 4.5). The plasma concentrations of R(+)-6-Br-APB could not be

detected at either doses, suggesting that the brain:plasma ratio for this compound is very

high and, therefore, vascular correction was not needed for the total brain concentrations

(see 4.2.11.3). However, the total brain concentration value includes both the bound and

unbound drug concentrations, and it is the free, unbound drug that is available to act on

targeted receptors and induce physiological effects (Reichel, 2009). Due to time

constraints, an in silico prediction of drug-brain tissue binding was applied in lieu of

performing further experiments to determine the unbound fraction of R(+)-6-Br-APB in

the brain (Wan et al., 2007). R(+)-6-Br-APB has a calculated partition-coefficient (logP)

value of 4.3, and according to the data compiled by Wan et al. (2007), compounds with

logP values of 4 or greater have a 88.6% chance of having an unbound fraction of less than

1%. Therefore, at 30 min post 1 mg/kg i.p. administration, the unbound concentration of

R(+)-6-Br-APB was estimated to be, at most, 1.69 nM in the brain, which lies close to the

affinity (8.91 nM) and above the potency (0.31 nM) of this drug at the D1 DR. In contrast,

Chapter 4

98

the estimated unbound concentration was well below both the affinity and potency of

R(+)-6-Br-APB at the D2L DR (Ki = 977 nM; EC50 = 25 nM). Administration of 0.3 mg/kg

R(+)-6-Br-APB gave an even lower unbound concentration estimate of 0.41 nM in the

brain after 30 min, though this value was above potency at the D1 DR, and this low dose

was still able to induce disruption of PPI in mice (see 4.3.2.2).

4.3.2.2 R(+)-6-Br-APB induced disruption of prepulse inhibition and increase in

locomotor activity in mice

To determine the optimum dose to disrupt PPI in C57Bl/6J mice, three R(+)-6-Br-APB

doses (0.1, 0.3 and 1 mg/kg) were investigated in a PPI experiment. R(+)-6-Br-APB

treatments had no effect on the startle amplitude (Figure 4.6A). There was a significant

within-subjects main effect of prepulse intensity on PPI (F2,66 = 36.01, p < 0.001),

indicating that higher prepulse intensities produced greater PPI values (Figures 4.6B).

There was a significant between-subjects main effect of R(+)-6-Br-APB treatment on PPI

(F3,33 = 3.24, p = 0.035). A one-way ANOVA with Dunnett t-test post hoc analysis showed

significant disruptions of PPI by R(+)-6-Br-APB 0.3 mg/kg at P120pp6 (p < 0.05) and

T im e (m in )

Co

nc

en

tra

tio

n (

nM

)

0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 4 0 5 0 6 0

0 .3 m g /k g

1 m g /k g

R (+ )-6 -B r-A P B

Figure 4.5: Brain exposure of R(+)-6-Br-APB in C57Bl/6J mice post i.p. administration.

Concentration of R(+)-6-Br-APB in the brain at 30 and 50 min after 0.3 and 1 mg/kg i.p.

administration. Data were presented as mean SEM; n=3.

Chapter 4

99

P120pp18 (p < 0.05) when compared to vehicle, whereas the PPI values of either lower or

higher R(+)-6-Br-APB doses were not significantly different from vehicle (Figure 4.6B).

Post hoc analysis of average PPI values of all prepulse intensities confirmed that R(+)-6-

Br-APB 0.3 mg/kg significantly disrupted PPI (p < 0.05), whereas the PPI values of either

lower or higher R(+)-6-Br-APB doses were not significantly different from vehicle

(Figure 4.6C).

Sta

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e a

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(arb

itu

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un

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)

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

V 1 + V 2 + B r -A P B 0 .3 (n = 1 1 )

V 1 + V 2 + V 3 (n = 1 1 )

V 1 + V 2 + B r-A P B 0 .1 (n = 8 )

V 1 + V 2 + B r-A P B 1 (n = 7 )

P r e p u ls e In te n s ity

(d B a b o v e b a c k g r o u n d )

% P

PI

6 1 2 1 8

-4 0

-2 0

0

2 0

4 0

6 0

*

*

Av

era

ge

% P

PI

0

1 0

2 0

3 0

4 0 *

A

B

Figure 4.6: R(+)-6-Br-APB at 0.3 mg/kg dose was optimum in disrupting PPI. (A) Startle

amplitude of mice at 120 dB pulse intensity. (B) Percent inhibition of acoustic startle in mice

when presented with prepulses of 6, 12 or 18 dB above background prior to pulse intensity of

120 dB. (C) Average % PPI of all prepulse intensities. Data are presented as mean + SEM;

n=7-11. * p < 0.05. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in

saline. V3: water for injection. Br-APB: R(+)-6-Br-APB.

C

Chapter 4

100

Next, the optimal R(+)-6-Br-APB dose to induce hyperlocomotor activity in mice was

determined. There was a significant effect of time and R(+)-6-Br-APB on LMA (F11,264 =

3.14, p = 0.001 and F3,24 = 15.14, p < 0.001, respectively; Figure 4.7A; Appendix 2.2),

though there was no significant interaction between time and R(+)-6-Br-APB treatment.

The LMA profile of R(+)-6-Br-APB 1 mg/kg over time revealed that 55 min after the first

two injections, the ability of R(+)-6-Br-APB to induce hyperlocomotor activity decreased.

This was potentially due to a decrease in R(+)-6-Br-APB brain concentration after this

time, which went from 169 nM at 30 min post R(+)-6-Br-APB administration (or 50 min

after the first two injections) and dropped 50% to 74 nM at 50 min post R(+)-6-Br-APB

administration (or 70 min after the first two injections; Figures 4.1D, 4.5). Therefore, to

quantify the effect of the drug, the cumulated distance travelled by mice treated with

vehicle or R(+)-6-Br-APB from 25 to 55 min after the first two injections was used as an

assessment of LMA (Figure 4.7B). Treatment with R(+)-6-Br-APB had a significant

effect on distance travelled (F3,24 = 19.65, p < 0.001). Post hoc analysis revealed that

although all three doses significantly increased LMA, R(+)-6-Br-APB presented a bell-

shaped response profile, with 1 mg/kg inducing the most robust hyperlocomotor activity

(Figure 4.7B). From these data, it was determined that R(+)-6-Br-APB 1 mg/kg is the

optimum dose for inducing hyperlocomotor activity in C57Bl/6J mice

Chapter 4

101

T im e (m in )

Dis

tan

ce

tra

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d (

cm

)

-2 0 0 2 0 4 0 6 0 8 0

0

1 0 0 0

2 0 0 0

3 0 0 0

V 1 + V 2 + B r -A P B 0 .3

V 1 + V 2 + V 3 V 1 + V 2 + B r-A P B 1

V 1 + V 2 + B r -A P B 3

3 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1

2 n d - V 2

D is ta n c e T ra v e lle d

2 5 to 5 5 m in

Dis

tan

ce

Tra

ve

lle

d (

cm

)

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

V 1 + V 2 + V 3 (n = 8 )

V 1 + V 2 + B r-A P B 0 .3 (n = 4 )

V 1 + V 2 + B r-A P B 1 (n = 8 )

V 1 + V 2 + B r-A P B 3 (n = 8 )

* * *

* * *

* *

*

Figure 4.7: R(+)-6-Br-APB displays a bell-shaped dose-response profile in LMA, with 1

mg/kg dose the most efficient in increasing LMA. (A) Distance travelled every 5 min by mice

treated with different doses of R(+)-6-Br-APB recorded 30 min before and 80 min after

treatment of V1+V2. Vertical dotted lines and arrows indicate the time at which the drugs were

administered. (B) Cumulated distance travelled between 25 and 55 min after treatment of

V1+V2. Data are presented as mean + SEM; n=4-8. ** p < 0.01 and *** p < 0.001. V1: 10%

DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection.

Br-APB: R-6-Br-APB.

A B

Chapter 4

102

4.3.3 Drug Vehicles do not Affect Prepulse Inhibition and Locomotor Activity

compared to Saline and Water for Injection in Mice

Three different vehicles were used to dissolve the drugs administered in mice for

behavioural experiments: V1 – 10% DMSO/5% Tween 80 in Tris buffer pH 8.9 for

LY2033298; V2 – 2% Tween 80 in saline for donepezil; and V3 – water for injection for

R(+)-6-Br-APB. Combinations of these vehicles were tested against saline + saline + V3

treatments in PPI and LMA to investigate whether the vehicles themselves had any effect

on mouse behaviour.

In the PPI experiments, mice treated with V1 + V2 + V3 did not display any differences in

acoustic startle when exposed to 120 dB pulse intensity compared to saline + saline + V3

treated mice (Figure 4.8A inset). Additionally, these treatments did not alter PPI in mice

compared to saline + saline + V3 treated group (Figures 4.8A). Similarly, mice treated

with V1 + V2 + V3 did not display any differences in LMA compared to saline + saline +

V3 treated mice (Figure 4.8B; Appendix 2.4). These results indicate that the vehicles used

in this study do not have effects on their own in PPI and LMA experiments.

Chapter 4

103

P r e p u ls e In te n s ity

(d B a b o v e b a c k g r o u n d )

% P

PI

6 1 2 1 8

0

2 0

4 0

6 0

8 0

S a lin e + s a lin e + V 3 (n = 8 )

V 1 + V 2 + V 3 (n = 8 )

Sta

rtl

e a

mp

litu

de

(arb

itu

ra

ry

un

its

)

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

Figure 4.8: Drug vehicles do not affect PPI or LMA compared to saline+saline+V3

treatment. (A) Percent inhibition of acoustic startle in mice when presented with prepulses of 6,

12 or 18 dB above background prior pulse intensity of 120 dB. (Inset) Startle amplitude of mice

at 120 dB pulse intensity. (B) Cumulated distance travelled between 25 and 55 min after

treatment of 1st and 2nd injections. Data are presented as mean + SEM; n=4-8. V1: 10%


A B

D is ta n c e tra v e lle d

2 5 to 5 5 m in

Dis

tan

ce

tra

ve

lle

d (

cm

)

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

S a lin e + s a lin e + V 3 (n = 4 )

V 1 + V 2 + V 3 (n = 4 )

Chapter 4

104

4.3.4 Assessment of Compound Exposure in Plasma and Brain

Plasma and brain exposure of LY2033298 and donepezil post i.p. administration were

measured to assess the drug concentrations in the brain at various time-points,

corresponding to different stages of the PPI and LMA experiments (Figures 4.1B, D).

Thirty min after 10 mg/kg i.p. administration of LY2033298, there was a total brain

parenchyma concentration of approximately 7 µM (2132 ng/kg), which was decreased to 2

µM (745 ng/g) and lower in later time points (Figure 4.9A). AUC brain parenchyma:AUC

plasma ratio of LY2033298 over the 30 to 170 min period post administration was 6.95,

confirming the high brain penetrance of the drug (Table 4.2). Following donepezil 1

mg/kg administration, there was a total brain concentration of 645 µM (245 ng/g) at 30

min post administration, which dropped to 388 µM (147 ng/g) at 45 min, and 250 µM (95

ng/g) at 95 min post administration (Figure 4.9B). As expected, donepezil 0.3 mg/kg

exhibited lower brain concentrations, reaching a maximum of 165 µM (63 ng/g) at 30 min,

decreasing to 125 (48 ng/g) and 75 µM (29 ng/g) at 45 and 95 min post administration,

respectively (Figure 4.9B). The plasma concentrations of donepezil were not measured, as

donepezil has a high brain:plasma ratio, which was reported to be 1.7 – 2.24, therefore,

vascular correction was not needed for the total brain concentrations (see 4.2.11.3) (Matsui

et al., 1999). As the values for donepezil 1 mg/kg at each time-point were approximately 3

times those of the corresponding values for donepezil 0.3 mg/kg, we estimated the

concentrations for donepezil 0.6 mg/kg administration to be 370, 238 and 150 µM at 30,

45 and 95 min post administration, respectively (141, 90 and 57 ng/g, respectively; see

4.2.11.3; Figure 4.9B).

Chapter 4

105

L Y 2 0 3 3 2 9 8 1 0 m g /k g

T im e (m in )

Co

nc

en

tra

tio

n (

M)

0

0

3

6

9

1 2

3 0 6 0 9 0 1 2 0 1 5 0 1 8 0

P a re n c h y m a (n g /g )

P la s m a (n g /m L )

D o n e p e z il

T im e (m in )

Co

nc

en

tra

tio

n (

M)

0

0

2 0 0

4 0 0

6 0 0

8 0 0

3 0 6 0 9 0

0 .3 m g /k g

1 m g /k g

0 .6 m g /k g (e s t im a te d )

A B

Figure 4.9: Plasma and brain exposure of LY2033298 and donepezil in C57Bl/6J mice

post i.p. administration. (A) Concentration of LY2033298 in the brain parenchyma (see

4.2.11.3) and plasma at 30, 50, 95 and 170 min after 10 mg/kg i.p. administration. Data were

presented as mean SEM; n=3. (B) Concentration of donepezil in the brain at 30, 45 and 95

min after 0.3 and 1 mg/kg i.p. administration. Dotted line represents the estimated brain

concentration of donepezil 0.6 mg/kg administration, calculated from data from donepezil 0.3

and 1 mg/kg (see 4.2.11.3). Data were presented as mean SEM; n=3.

Table 4.2: Pharmacokinetic analysis of LY2033298

Parameter LY2033298

Mean AUC(30→170) brain parenchyma (ng ∙ min/g) 81791.00 ± 14054.92

Mean AUC(30→170) plasma (ng ∙ min/mL) 12288.00 ± 3220.35

AUC brain parenchyma:AUC plasma ratio 6.95 ± 0.63 AUC from 30 to 170 min and brain/plasma ratio values of LY2033298 in plasma and brain exposure

studies in male C57Bl/6J mice post i.p. administration of 10 mg/kg, presented as mean ± SEM; n = 3.

Chapter 4

106

4.3.5 Treatments of LY2033298 alone or with Donepezil, an Acetylcholinesterase

Inhibitor, Showed a Trend to Reverse Disruption of Prepulse Inhibition Induced by

R(+)-6-Br-APB

The effect of LY2033298 and donepezil, either alone or in combination, on disruption of

PPI induced by R(+)-6-Br-APB was investigated in mice. There was no significant effect

of drug treatments on the startle amplitude (Figure 4.10A). There was a significant within-

subjects main effect of prepulse on PPI (F2,118 = 64.76, p < 0.001). Additionally, there was

a significant prepulse x R(+)-6-Br-APB interaction (F2,118 = 3.58, p = 0.031). There were

significant between-subjects main effects of donepezil (F1,59 = 5.63, p = 0.021) and R(+)-

6-Br-APB (F1,59 = 23.38, p < 0.001) on PPI, and a significant LY2033298 x donepezil x

R(+)-6-Br-APB interaction (F1,59 = 7.35, p = 0.009).

One-way ANOVA analysis with Tukey HSD post hoc analysis revealed that treatment of

R(+)-6-Br-APB significantly disrupted PPI at P120pp12 (p < 0.001) and P120pp18 (p <

0.01; Figures 4.10B). Both LY2033298 alone and combined LY2033298 + donepezil

treatments significantly reversed the disruption of PPI induced by R(+)-6-Br-APB at

P120pp12 (both p < 0.05; Figure 4.10B). However, despite showing a trend to reverse,

this significance was not seen at other prepulse intensities (Figure 4.10B). Post hoc

analysis of average PPI of all prepulse intensities also showed that treatment of R(+)-6-Br-

APB significantly disrupted PPI (p < 0.01), and while treatments of LY2033298 and/or

donepezil showed trend to reverse this disruption, the effects were not significant (Figure

4.10C).

Chapter 4

107

Sta

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litu

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(arb

itu

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un

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)

0

5 0

1 0 0

1 5 0

2 0 0V 1 + V 2 + V 3 (n = 1 0 )

L Y 1 0 + V 2 + V 3 (n = 7 )

V 1 + D o n 1 + V 3 (n = 8 )

L Y 1 0 + D o n 1 + V 3 (n = 9 )

V 1 + V 2 + B r-A P B 0 .3 (n = 8 )

L Y 1 0 + V 2 + B r -A P B 0 .3 (n = 9 )

V 1 + D o n 1 + B r -A P B 0 .3 (n = 8 )

L Y 1 0 + D o n 1 + B r -A P B 0 .3 (n = 8 )

P re p u ls e In te n s ity (d B a b o v e b a c k g ro u n d )

% P

PI

6 1 2 1 8

-4 0

-2 0

0

2 0

4 0

6 0

8 0

* * ** *

*

*A

ve

ra

ge

% P

PI

-2 0

0

2 0

4 0

6 0

* *

A

B

Figure 4.10: Treatment of LY2033298 alone and in combination with donepezil reverse

disruption of PPI induced by R(+)-6-Br-APB, reaching significance at P120pp12. (A)

Startle amplitude of mice at 120 dB pulse intensity. (B) Percent inhibition of acoustic startle in

mice when presented with prepulses of 6, 12 or 18 dB above background prior to pulse intensity

of 120 dB. (C) Average % PPI of all prepulse intensities. Data are presented as mean + SEM;

n=7-10. * p < 0.05, ** p < 0.01 and *** p < 0.001. V1: 10% DMSO/5% Tween 80 in Tris buffer

pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection. LY: LY2033298; Don: donepezil; Br-

APB: R(+)-6-Br-APB.

C

Chapter 4

108

4.3.6 Combined Treatment of LY2033298 and Donepezil Reversed Hyperlocomotor

Activity Induced by R(+)-6-Br-APB

Next, the ability of LY2033298 and donepezil to reverse the increase of LMA induced by

R(+)-6-Br-APB was investigated in mice. Figures 4.11A-D show the distance travelled

every 5 min over time of each treatment group (Appendix 2.5). There was a significant

within-subjects main effect of time on LMA (F11,957 = 26.11, p < 0.001), as well as

significant time x R(+)-6-Br-APB (F11,957 = 11.43, p < 0.001), time x LY2033298 (F11,957 =

3.78, p < 0.001), time x donepezil (F11,957 = 3.08, p < 0.001) and time x R(+)-6-Br-APB x

donepezil (F11,957 = 3.10, p < 0.001) interactions. R(+)-6-Br-APB (F1,87 = 134.51, p <

0.001), LY2033298 (F1,87 = 4.19, p = 0.044) and donepezil (F1,87 = 7.26, p = 0.001) all had

significant between-subjects main effects on LMA. There was also a significant R(+)-6-

Br-APB x donepezil interaction (F1,87 = 4.53, p = 0.013).

Post hoc analysis revealed that R(+)-6-Br-APB 1 mg/kg significantly increased distance

travelled from 25 to 55 min after the first two injections (Figure 4.11A-D, salmon filled

circles; Appendix 2.5). Treatments of LY2033298 (10 mg/kg) or donepezil (0.6 mg/kg or

1 mg/kg) alone did not affect LMA (Figure 4.11A; Appendix 2.5). However, when

combined, LY2033298 + donepezil 1 mg/kg decreased distance travelled throughout the

duration of the test, though this decrease was not significant (Figure 4.11B, blue diamond-

crosses; Appendix 2.5). This reduction in distance travelled was less pronounced in mice

treated with LY2033298 + donepezil 0.6 mg/kg (Figure 4.11B, blue diamonds).

LY2033298 + R(+)-6-Br-APB treatment did not significantly affect the increase in LMA

induced by R(+)-6-Br-APB (Figure 4.11C, purple triangles; Appendix 2.5). Treatment of

donepezil 0.6 mg/kg + R(+)-6-Br-APB significantly reduced distance travelled at 25, 30

and 35 min after the first two injections (p < 0.001, p < 0.001 and p < 0.05, respectively)

when compared to mice treated with R(+)-6-Br-APB alone (Figure 4.11C, purple squares;

Chapter 4

109

Appendix 2.5). Interestingly, the higher dose of donepezil 1 mg/kg + R(+)-6-Br-APB was

less effective at decreasing hyperlocomotor activity induced by R(+)-6-Br-APB, reaching

significance only at 25 and 30 min after the first two injections (both p < 0.05; Figure

4.11C, purple square-x’s; Appendix 2.5). The combined treatments of LY2033298 and

either doses of donepezil were both effective in significantly reducing the increase in LMA

induced by R(+)-6-Br-APB at 25 to 40 min after the first two injections (all p < 0.001;

Figure 4.11D; Appendix 2.5). However, this effect was transient for both treatments, as

the distance travelled became significantly increased compared to vehicle treatment after

55 to 65 min after the first two injections (Figure 4.11D; Appendix 2.5). This is

potentially due to the decrease of LY2033298 and donepezil total brain concentrations

after this time, which led to a reduction of M4 mAChR activity (Figures 4.9).

Figure 4.11E shows the cumulated distance travelled over the 25 to 55 min period after

the first two injections. There were significant between-subjects main effects of R(+)-6-Br-

APB (F1,87 = 68.49, p < 0.001), LY2033298 (F1,87 = 9.41, p = 0.003) and donepezil (F2,87 =

10.76, p < 0.001), as well as a significant R(+)-6-Br-APB x donepezil interaction (F2,87 =

6.09, p = 0.003). Post hoc analysis revealed that R(+)-6-Br-APB, LY2033298 + R(+)-6-

Br-APB, and donepezil 1 mg/kg + R(+)-6-Br-APB treatments caused a significant increase

in cumulated distance travelled compared to vehicle (p < 0.001, p < 0.001 and p < 0.05,

respectively; Figure 4.11E). Both treatments of LY2033298 + donepezil 0.6 mg/kg +

R(+)-6-Br-APB and LY2033298 + donepezil 1 mg/kg + R(+)-6-Br-APB significantly

reversed hyperlocomotor activity induced by R(+)-6-Br-APB (p < 0.001 and p < 0.01,

respectively; Figure 4.11E). Donepezil 0.6 mg/kg + R(+)-6-Br-APB treatment also

reduced the increased LMA induced by R(+)-6-Br-APB, though this effect was not

significant (Figure 4.11E).

Chapter 4

110

Dis

tan

ce

tra

ve

lle

d (

cm

)

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

V 1 + V 2 + V 3 (n = 1 3 )

L Y 1 0 + V 2 + V 3 (n = 6 )

V 1 + D o n 0 .6 + V 3 (n = 6 )

V 1 + D o n 1 + V 3 (n = 6 ) V 1 + V 2 + B r -A P B 1 (n = 9 )

L Y 1 0 + D o n 1 + B r-A P B 1 (n = 1 0 )

L Y 1 0 + D o n 0 .6 + B r-A P B 1 (n = 1 0 )

V 1 + D o n 1 + B r -A P B 1 (n = 7 )

V 1 + D o n 0 .6 + B r-A P B 1 (n = 8 )

L Y 1 0 + V 2 + B r-A P B 1 (n = 8 )

L Y 1 0 + D o n 1 + V 3 (n = 7 )

L Y 1 0 + D o n 0 .6 + V 3 (n = 9 )

3 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1 /L Y 2 0 3 3 2 9 8

2 n d - V 2 /D o n e p e z il

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 03 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1 /L Y 2 0 3 3 2 9 8


T im e (m in )

Dis

tan

ce

tra

ve

lle

d (

cm

)

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T im e (m in )

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

A B

C D

Figure 4.11: Co-treatment of LY2033298 and donepezil reverses hyperlocomotor activity

induced by R(+)-6-Br-APB. (A-D) Distance travelled every 5 min by mice recorded 30 min

before and 80 min after treatment of 1st and 2nd injections. Vertical dotted lines and arrows

indicate the time at which the drugs were administered. (E) Cumulated distance travelled

between 25 and 55 min after treatment of 1st and 2nd injections. Data are presented as mean +

SEM; n=6-13. * p < 0.05, ** p < 0.01 and *** p < 0.001. V1: 10% DMSO/5% Tween 80 in Tris

buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection. LY: LY2033298; Don:

donepezil; Br-APB: R(+)-6-Br-APB.


2 5 to 5 5 m in

Dis

tan

ce

tra

ve

lle

d (

cm

)

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

V 1 + V 2 + V 3 (n = 1 3 )

L Y 1 0 + V 2 + V 3 (n = 6 )

V 1 + D o n 0 .6 + V 3 (n = 6 )

V 1 + D o n 1 + V 3 (n = 6 )

L Y 1 0 + D o n 0 .6 + V 3 (n = 9 )

L Y 1 0 + D o n 1 + V 3 (n = 7 )

V 1 + V 2 + B r-A P B 1 (n = 9 )

L Y 1 0 + V 2 + B r -A P B 1 (n = 8 )

V 1 + D o n 0 .6 + B r -A P B 1 (n = 8 )

V 1 + D o n 1 + B r -A P B 1 (n = 7 )

L Y 1 0 + D o n 0 .6 + B r -A P B 1 (n = 1 0 )

L Y 1 0 + D o n 1 + B r -A P B 1 (n = 1 0 )

* * *

* * *

*

* * *

* *

E

Chapter 4

111

4.4 Discussion

In this chapter, the allosteric parameters between two M4 mAChR PAM structural

analogues, LY2033298 and ML253, at the human and mouse M4 mAChRs were quantified

to compare the extent of species variability between the two ligands. The effect of the

difference in amino acid sequence of receptors between species on ligand pharmacology

has been documented for many receptors, including the serotonin receptors 5-HT1B and 5-

HT2A, D1 DRs, histamine receptors and M4 mAChRs (Canal et al., 2013; Chan et al., 2008;

Hamblin et al., 1992; Lewis et al., 2015; Strasser et al., 2013; Suratman et al., 2011). In

terms of M4 mAChRs, LY2033298 has been shown to exhibit species variability, whereby

its positive potentiation of ACh at the M4 mAChR was decreased at the rat and mouse

receptors compared to the human receptor (Chan et al., 2008; Suratman et al., 2011).

Additionally, due to probe dependence, at the mouse receptor, LY2033298 showed greater

cooperativity with oxotremorine compared to ACh (Suratman et al., 2011). Therefore,

these characteristics resulted in the requirement for LY2033298 to be co-administered with

a sub-effective dose of the non-selective mAChR orthosteric agonist, oxotremorine, in

order to exert antipsychotic-like effects in mice (Chan et al., 2008; Leach et al., 2010;

Suratman et al., 2011).

The source of species variability for LY2033298 was found to be primarily due to the

difference in positive cooperativity with ACh, but not in the affinity of the PAM for the

receptor, between the M4 mAChR of different species (Suratman et al., 2011). A

mutagenesis study revealed two divergent residues (D432 and T433) on the extracellular loop

3 (ECL3) and transmembrane helix 7 (TM7) junction that were critical for the activity of

LY2033298 at the human M4 mAChR, and when these residues were replaced with the

corresponding residues of the rodent receptor (E431 and R432), the activity of LY2033298

Chapter 4

112

was decreased (Chan et al., 2008; Nawaratne et al., 2010; van Koppen et al., 1993)

(Appendix 2.6). In the current study, it was found that, in contrast to LY2033298, the

estimate of ML253 affinity was subject to species variability, as it had higher affinity for

the mouse M4 mAChR than the human receptor (pKB, 6.82 vs 5.92, respectively).

However, despite the increased affinity at the mouse M4 mAChR, the functional

cooperativity between ACh and ML253 at the mouse receptor was still lower than that at

the human receptor, and also lower than the functional cooperativity between ACh and

LY2033298 at the mouse receptor.

Recently, the crystal structure of the human M4 mAChR in the inactive state bound to

tiotropium was solved, shedding light on key differences between the structures of the

mAChR subtypes, including differences in the orthosteric binding pocket (Thal et al.,

2016). Additionally, the authors performed mutagenesis and molecular homology studies

of human M4 mAChRs co-bound to ACh and LY2033298, based on the previously

published crystal structure of the M2 mAChR bound to the orthosteric agonist, iperoxo,

and PAM, LY2119620, to identify residues involved in the affinity and cooperativity of

LY2033298 (Kruse et al., 2013; Thal et al., 2016). This resulted in the identification of

residues along the interface between TM2, 3, 6 and 7, and ECL2, which are important for

the binding and cooperativity of LY2033298 (Thal et al., 2016). The mouse M4 mAChR

only contains three divergent residues from the human receptor in these regions, and those

include the aforementioned two residues on the ECL3 and TM7 junction, and one on TM2

(human V91; mouse L91; Appendix 2.6). The main difference between the structures of

LY2033298 and ML253 is the differential substitution of the amide moiety, cyclopropyl

for LY2033298 and pyridine-4-ylmethyl for ML253 (Figure 4.2). Although a thorough

comparison of the binding poses of LY2033298 and ML253 at the human and the mouse

receptor has not been explored, it can be hypothesised that the divergent residues on the

Chapter 4

113

top of TM7 of the mouse M4 mAChR created more favourable interactions with the

pyridine-4-ylmethyl moiety of ML253 than the reciprocal residues on the human receptor,

which led to an increased affinity of ML253 for the mouse receptor, whereas the difference

in these residues had minimal effect on the interactions with the cyclopropyl moiety of

LY2033298, thus leaving the affinities of LY2033298 for either species of receptor

unchanged.

The difference in positive cooperativity between ACh and the PAMs across the receptor

species, on the other hand, is more complex. Cooperativity between ACh and LY2033298

involved the allosteric network of residues along the interface between TM2, 3, 6 and 7,

and also ECL2, which link the allosteric and orthosteric site (Thal et al., 2016). As

mentioned above, the residues in these regions on the mouse M4 mAChR are essentially

identical to the human receptor, but for the three residues in TM2 and TM7, therefore it is

interesting that the positive cooperativity of these two ligands is still subject to species

variability. Further molecular modelling should be performed in the future in order to fully

understand the source of the species variability in positive cooperativity for these two

allosteric modulators.

The positive cooperativity (αβ) between LY2033298 and ACh at the human M4 mAChR in

the current study was higher than previously described values (Leach et al., 2010;

Suratman et al., 2011). Efficacy of the allosteric modulator (τB) can change with altered

receptor expression levels, which can in turn influence the activation cooperativity

parameter (β) (Conn et al., 2014; Langmead and Christopoulos, 2014; Leach et al., 2007).

Therefore, the increased positive cooperativity estimated in the current study may be due

to higher human M4 mAChR expression in the cells compared to previous studies.

Chapter 4

114

With the conclusion of LY2033298 as the more suitable M4 mAChR-selective PAM to

administer in vivo due to its better aqueous dissolution properties, the ability of

LY2033298 to modulate selective D1 DR-induced behavioural output in mice was

explored. The first behaviour that was tested was PPI, the deficit of which had been shown

in people with schizophrenia, as well as other disorders of the CNS (Braff et al., 1978;

Braff et al., 2001; Kohl et al., 2013; Swerdlow et al., 2008). Generally, most PPI tests in

rodents utilise just one pulse intensity paired with three or more prepulse intensities.

However, it has been shown that acoustic startle in response to pulse intensities may vary

across individual rats and mice (and especially between genetically modified and wildtype

mice), and this difference in acoustic startle can change PPI values (Brody et al., 2004;

Hince and Martin-Iverson, 2005; Stoddart et al., 2008; Yee et al., 2005). To address this

issue, it has been suggested that the use of different pulse intensities within a PPI session

can improve the interpretability of the data (Yee et al., 2005). Therefore, for this study,

three levels of pulse intensities (100, 110, and 120 dB) were used to provide a more

comprehensive characterisation of PPI. However, as the disruption of PPI induced by

R(+)-6-Br-APB was most prominent at 120 dB (see 4.2.11.2), only the data from 120 dB

were presented in this chapter.

The pharmacological activation of the dopaminergic system has long been shown to

induce deficits in PPI, though the relative involvement of D1 versus D2 DR is subject to

species and strains used (Geyer et al., 2001; Mansbach et al., 1988; Swerdlow et al., 2008;

van den Buuse, 2010). While disruption of PPI in rats is primarily mediated through

activation of D2 DRs, D1 DR activation is required to induce PPI deficits in several outbred

and inbred mice (Geyer et al., 2001; Peng et al., 1990; Ralph-Williams et al., 2003; Ralph

and Caine, 2005). R(+)-6-Br-APB has been shown to significantly disrupt PPI in C57Bl/6J

mice (Ralph and Caine, 2005), and this effect was reproduced in our hands.

Chapter 4

115

The second behaviour tested was LMA, due to the important role the striatum plays in

motor function, and that psychostimulants, including both direct and indirect DA agonists,

have been shown to induce hyperlocomotor activity in rodents (Durieux et al., 2012;

Graybiel, 1991; Groenewegen, 2003; Mink and Thach, 1993; Swerdlow et al., 1986; van

den Buuse, 2010). Similar to PPI, the role of D1 versus D2 DRs in the regulation of LMA

varies with species and strains, though it has been shown that D1 DRs expressed in the

striatonigral neurons are important for the stimulation of exploration in mice (Durieux et

al., 2012). Furthermore, R(+)-6-Br-APB has been shown to induce hyperlocomotor

activity in C57Bl/6J mice, which was also replicated in our hands (Ralph and Caine, 2005;

Ralph et al., 2001a; Thomsen et al., 2011).

The compound exposure study of R(+)-6-Br-APB estimated that at both the PPI disrupting

dose of 0.3 mg/kg and the hyperlocomotor activity inducing dose of 1 mg/kg, the unbound

concentration of the drug in the brain was much lower than the affinity and potency of the

drug at the D2 DR, but close to the affinity and above the potency of the drug at the D1 DR,

indicating that the disruption of PPI and hyperlocomotor activity induced by R(+)-6-Br-

APB in these mice were mediated through the activation of the D1 DR. However, as these

unbound concentrations in the brain were only in silico estimates, future experiments are

needed to determine the actual unbound fraction of R(+)-6-Br-APB in the brain of

C57Bl/6J mice.

Our results showed that treatment of LY2033298 in mice, with or without donepezil, show

a trend to reverse PPI deficits induced by R(+)-6-Br-APB, reaching significance only at

P120pp12 (Figure 4.10B). This was in line with previous findings that showed a

significant reversal of apomorphine-induced disruption of PPI by LY203398 + a sub-

effective oxotremorine dose in rats (Chan et al., 2008). However, the lack of significant

reversal of R(+)-6-Br-APB-induced disruption of PPI overall in this chapter may suggest

Chapter 4

116

that the adjunctive treatment of an exogenous mAChR orthosteric agonist is required for

LY2033298 to elicit a significant reversal of DR-induced PPI deficits, or even that the

reversal in the previous finding was driven by the potentiation of oxotremorine at both the

M2 and the M4 mAChRs by LY2033298, given the probe dependence nature of

LY2033298 (Suratman et al., 2011; Valant et al., 2012).

Alternatively, the modest effects of LY2033298 and donepezil treatments to reverse PPI

deficits induced by the D1 DR-selective agonist in this study could also be a result of

experimental design. Compound exposure studies that investigated brain and plasma

exposure of LY2033298 showed that the brain concentration of LY2033298 10 mg/kg was

7 µM at 30 min after i.p. administration, which was reduced to 2 µM at 50 min post

administration, just after the start of the PPI test (Figures 4.1B, 4.9A). With the

assumption that there is a direct correlation between the compound brain concentration and

behavioural outputs (i.e. between the pharmacokinetic and pharmacodynamics properties

of the drug compounds), the low brain concentration of LY2033298 during the PPI test

may not be sufficient to potentiate endogenous ACh function at the M4 mAChR. This is

further supported by the ability of the structurally distinct M4 mAChR PAM, VU0152100,

to reverse PPI disruption induced by amphetamine on its own, which was tested at the time

period correlating to high VU0152100 brain concentration in rats (Byun et al., 2014).

Future PPI studies with a shorter LY2033298 pre-treatment period should be conducted to

further determine the ability of LY2033298 to reverse D1 DR-selective agonist-induced

disruption of PPI. Additionally, a comprehensive pharmacokinetic and pharmacodynamics

characterisation of LY2033298 will be beneficial for the design of future behavioural

experiments.

The impact of experimental design was also demonstrated in the LMA data of the present

study. In this experiment, the combined treatments of LY2033298 + donepezil 0.6 or 1

Chapter 4

117

mg/kg both significantly reversed the hyperlocomotor activity induced by the D1 DR-

selective agonist (Figure 4.11). LMA data were quantified from 25 to 55 min post

LY2033298 and donepezil administrations, capturing the period of high drug

concentrations in the brain (Figure 4.9). The reversal effects of LY2033298 + donepezil

treatments diminished after 45 min post administration, reflecting the decrease in brain

concentrations of both drugs after this time.

While both doses of donepezil combined with LY2033298 significantly reversed the

hyperlocomotor activity induced by R(+)-6-Br-APB, LY2033298 + donepezil 1 mg/kg

treatment decreased the baseline LMA, confounding the reversal data. However, the lower

dose of donepezil 0.6 mg/kg with or without LY2033298 did not affect baseline LMA.

When treated on its own, donepezil 0.6 mg/kg showed a non-significant trend to reverse

R(+)-6-Br-APB induced hyperlocomotor activity, which became significant when

donepezil was co-administered with LY2033298 (Figure 4.11). This highlights one of the

advantages of using an M4 mAChR PAM as a therapeutic agent, as LY2033298 was able

to potentiate the function of the endogenous ACh, the concentration of which was elevated

by donepezil, without causing the sedative side effects seen with the higher dose of this

acetylcholinesterase inhibitor.

Therefore, the LMA data in this chapter demonstrated that selective activation of M4

mAChRs by a PAM could regulate D1 DR-induced behaviour in mice. Additionally,

LY2033298 and donepezil treatments showed a trend to reverse R(+)-6-Br-APB-induced

disruption of PPI. To determine the role of the M4 mAChR in the regulation of D1 DR-

induced behaviours, PPI and LMA tests were conducted on whole-body M4 mAChR

knockout mice, which is the focus of Chapter 5.

Chapter 5:

Studying the Role of M4 Muscarinic

Acetylcholine Receptors in the

Modulation of D1 Dopamine Receptor

Function Using Whole-body Knockout

Mice

Chapter 5

119

5.1 Introduction

In the previous chapter, it was demonstrated that while treatment with LY2033298 +

donepezil to activate M4 mAChRs showed a trend to reverse the disruption of PPI induced

by the D1 DR-selective agonist, R(+)-6-Br-APB, this combined treatment significantly

reversed the R(+)-6-Br-APB treatment-induced hyperlocomotor activity in C57Bl/6J mice.

While LY2033298 is functionally selective for the M4 mAChR in the presence of ACh

(Valant et al., 2012), donepezil, by inhibiting the breakdown of ACh by

acetylcholinesterase and thereby increasing endogenous levels of ACh, does not display

any subtype selectivity for mAChRs. Therefore, it is possible that the reversal effects

found in the previous chapter are, at least in part, produced by the off-target activation of

other mAChR subtypes.

To determine the role of M4 mAChRs in the modulation of D1 DR-selective agonist-

induced disruption of PPI and increases in LMA, experiments presented in this chapter

were performed in M4 mAChR knockout (M4-/-) mice on a C57Bl/6NTac background.

Both C57Bl/6J and C57Bl/6NTac mice were developed from the same C57Bl/6 ancestral

line (developed by C. C. Little), with C57Bl/6J established when the C57Bl/6 mice were

sent to The Jackson Laboratory (Bar Harbor, ME, USA) in 1948. A subset of these mice

were sent to the National Institute of Health (Bethesda, MD, USA) in 1951 to form the

C57Bl/6N substrain, and then to Taconic Farm (Hudson, NY, USA) in 1991 to form the

C57Bl/6NTac substrain (Mekada et al., 2009). While both C57Bl/6J and C57Bl/6N mice

are of the same strain, phenotypic and genetic differences have been observed between

these two substrains (Ashworth et al., 2015; Garcia-Menendez et al., 2013; Heiker et al.,

2014; Kendall and Schacht, 2014; Matsuo et al., 2010; Mekada et al., 2009; Radulovic et

al., 1998; Rendina-Ruedy et al., 2015; Stiedl et al., 1999; Zurita et al., 2011).

Chapter 5

120

Therefore, all experiments used the C57Bl/6NTac wildtype (M4+/+) mice as controls. In

this chapter, vehicle-treated M4+/+ mice displayed higher PPI values and lower LMA

compared to vehicle-treated C57Bl/6J mice seen in the previous chapter, similar to what is

reported in the literature (Matsuo et al., 2010). Due to insufficient breeding resulting in a

relatively small sample size, data obtained in M4-/- mice were inconclusive in the

determination of the role of M4 mAChRs in the modulation of R(+)-6-Br-APB treatment-

induced disruption of PPI. In terms of LMA, the treatments of LY2033298 + R(+)-6-Br-

APB and donepezil + R(+)-6-Br-APB attenuated the hyperlocomotor activity induced by

R(+)-6-Br-APB in M4-/- mice, which were not seen in M4

+/+ mice, and LY2033298 +

donepezil + R(+)-6-Br-APB treatment also reduced D1 DR agonist-induced increases in

LMA in M4-/- mice.

Chapter 5

121

5.2 Material and Methods

5.2.1 Animals

Monash Institute of Pharmaceutical Sciences Animal Ethics Committee approved all

procedures on experimental animals. C57Bl/6NTac mice heterozygous for the M4 mAChR

mutation (M4+/-) were a generous gift from Dr Jürgen Wess (National Institute of Diabetes

and Digestive and Kidney Disorders, Bethesda, MD), from Taconic Farms (Hudson, NY).

The M4 mAChR knockout (M4-/-) were generated on a mixed genetic background

(129S6/SvEv + CF1) and the founder mice were subsequently backcrossed to the

C57Bl/6NTac strain for more than 10 generations (Gomeza et al., 1999b; Schmidt et al.,

2011). At the Animal House at Monash Institute of Pharmaceutical Sciences (Parkville,

VIC, Australia), the M4+/- mice were interbred to produce wildtype (M4

+/+) and M4 mAChR

knockout (M4-/-) mice. Due to the slow breeding rate of M4

-/- mice, pure M4+/+ and M4

-/-

breeding colonies were established to produce more mice of both genotypes, but these

mice were backcrossed with M4+/- mice every 2 to 3 generations. Male M4

+/+ and M4-/-

mice were transported from the Animal House to the holding room in the Murine Disease

Model Facility (8 weeks at the commencement of habituation), and were habituated in the

holding room for at least a week before experiments. Mice were acclimatised to being

handled at least three times over the 5 days prior to being tested. Mice were group-housed

and kept in a holding room with an ambient temperature of 22°C, humidity 30–40% and a

reverse-phase lighting cycle (lights on 7:00 PM, off 07:00 AM). All test sessions were

conducted between 8:00 AM and 6:00 PM, during the most active phase of mice. Food and

water were available ad libitum. All mice were tested for both PPI and LMA, with a two-

week washout period between tests. Due to the slow breeding rate of M4-/- mice, initial

studies on V1+V2+V3-treated M4-/- mice were performed to compare the effect of re-

Chapter 5

122

testing of PPI and LMA, with a mind to use the same mice for multiple drug treatment

groups for both behavioural tests. A PPI re-test was conducted 7 days after the first LMA

test, and LMA re-test was conducted 7 days after PPI re-test (Figure 5.1A). Re-testing

affected the acoustic startle and PPI in M4-/- mice, but not LMA, therefore following the

first LMA test, M4-/- mice were re-tested only in LMA, with a one-week washout period

between tests (Figure 5.1B). All mice were treated with different drug treatment groups

for each test.

A sample size of 10 for both PPI and LMA experiments was calculated using a power

calculation (unpaired t-test) based on the magnitude of effect observed for the drug

treatments versus vehicle in a preliminary study.

Figure 5.1: Timeline of behavioural experiments. (A) Timeline of initial study on the effect of

repeated testing on PPI and LMA results. Repeated PPI testing was performed 7 days after first

LMA test, and repeated LMA testing was performed 7 days after repeated PPI testing. (B)

Timeline of final behavioural experiment protocol. Timeline drawn in increments of 1 week. PPI:

prepulse inhibition; LMA: locomotor activity.

Arrive, 8

weeks old


PPI test

1 week

habituation

LMA test

Cull mice

Repeated PPI and LMA testing timeline

LMA

re-test

PPI

re-test

Arrive, 8

weeks old


PPI test

1 week

habituation

LMA test

Cull mice

Behavioural experiment timeline

1 week

washout

LMA

re-test

A

B

Chapter 5

123

5.2.2 Drugs

Drugs were sourced and prepared as previously described in sections 4.2.1 and 4.2.7.

5.2.3 Prepulse Inhibition of the Acoustic Startle Response (PPI)

PPI was conducted as previously described in section 4.2.8.

5.2.4 Locomotor Activity (LMA)

LMA was conducted as previously described in section 4.2.9.

5.2.5 Data and Statistical Analysis

Statistical analysis was performed using IBM® SPSS® Statistics Version 23 for Windows

(IBM® Corp., NY, USA). For the experiment comparing PPI of V1 + V2 + V3 treated

M4+/+ and M4

-/- mice (see 5.3.2.1), PPI data were analysed using a repeated-measures

ANOVA to compare startle amplitude and PPI values between genotype, with pulse (100,

110 or 120 dB) and prepulse intensity (6, 12 or 18 dB above background) as within-

subjects factors and genotype as between-subjects factor. Where there was a significant

main effect of genotype, a one-way ANOVA was performed to determine the level of

significance at each pulse and prepulse intensity for PPI.

For the LY2033298 and donepezil combined treatment PPI experiments, although three

pulse intensities were tested, disruption of PPI induced by R(+)-6-Br-APB was most

prominent at 100 and 110 dB pulse intensities, therefore only the data from these two pulse

intensities were presented in this chapter (see Appendix 3.4 for results from 120 dB pulse

intensity). PPI data were analysed using a repeated-measures ANOVA to compare startle

amplitude and PPI values between all treatment and genotype groups, with pulse (100 or

110 dB) and prepulse intensity (6, 12 or 18 dB above background) as within-subjects

Chapter 5

124

factors and treatment and genotype as between-subjects factors. Where there was a

significant main effect of treatment or genotype, a one-way ANOVA with Tukey HSD

post hoc analysis was performed to determine the level of significance at each pulse and

prepulse intensity for PPI. Greenhouse-Geisser correction was applied per Mauchley’s

Test of Sphericity. Significant differences between treatment groups were indicated with *

p < 0.05, ** p < 0.01 and *** p < 0.001.

Statistical analysis of spontaneous LMA of vehicle-treated M4+/+ and M4

-/- mice (measured

over the 30 min habituation period before administration of the first two injections) and

LMA during the initial and final test phases (measured over the 60 min after

administration of the third injection) were performed using a repeated-measures ANOVA,

with time as a within-subjects factor and genotype as a between-subjects factor. When

there was a main effect of genotype, a one-way ANOVA with genotype as a fixed factor

and each time point as dependent variables were used. Distance travelled for each phase

(habituation, testing – initial and testing – final) were summed to calculate cumulated

distance travelled. Cumulated distance travelled was analysed using a one-way ANOVA,

with distance travelled for each phase as dependent variables and genotype as a fixed

factor. Significant differences between genotypes were indicated with * p < 0.05, ** p <

0.01 and *** p < 0.001.

To compare LMA between the first and re-test periods, a repeated-measures ANOVA was

performed with time as a within-subjects factor and re-testing as a between-subjects factor.

A one-way ANOVA was performed on cumulated distance travelled data, with distance

travelled as a fixed factor and re-testing as a between-subjects factor. LMA over time from

25 min after first two injections onwards was analysed using a repeated-measures ANOVA,

as previously described in section 4.2.11.2 with the addition of genotype as a between-

subjects factor. Distance travelled every 5 min from 25 to 55 min after first two injections

Chapter 5

125

was summed to calculate cumulated distance travelled. This timeframe was used to capture

the maximum increase in LMA induced by R(+)-6-Br-APB in C57Bl/6NTac M4+/+ mice.

Cumulated distance travelled data was analysed as previously described in section 4.2.11.2

with the addition of genotype as a between-subjects factor. Significant differences between

groups were indicated with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Chapter 5

126

5.3 Results

5.3.1 When Re-tested, M4-/- Mice Exhibited Reduced Startle Amplitude and

Improved Prepulse Inhibition, but Exhibited No Change in Locomotor Activity

As shown in Figure 5.1A, M4-/- mice were tested in PPI and LMA first, using the same

experimental timeline as the behavioural experiments in the previous chapter. Then, these

mice were re-tested in PPI 7 days after the first LMA test, and re-tested in LMA 7 days

after PPI re-test (Figure 5.1B). The results showed that in vehicle-treated M4-/- mice, the

startle amplitude to pulse intensities 110 and 120 dB was decreased in the PPI re-test

compared to the first test (Figure 5.2A). Additionally, these mice demonstrated improved

PPI in the re-test, with higher PPI values in all pulse and prepulse intensities, particularly

for pulse intensities 110 and 120 dB (Figures 5.2B-D). While the PPI data from the first

test showed incremental increases of PPI values as a function of prepulse intensities, when

the mice were re-tested, their PPI value seemed to reach a maximum of 80% at prepulse

intensities 12 and 18 dB above background (Figures 5.2B-D). This was further

demonstrated in averaged PPI values of all pulse intensities (Figure 5.2E). Averaged PPI

values for all pulses and prepulses showed that M4-/- mice showed improved PPI values

when re-tested in PPI, with an increase of approximately 1.7-fold (Figure 5.2F).

Chapter 5

127

1 0 0 1 1 0 1 2 0

0

5 0

1 0 0

1 5 0

A c o u s tic S ta r t le

P u ls e In te n s ity (d B )

Sta

rtl

e a

mp

litu

de

(arb

itu

ra

ry

un

its

)

p p 6 p p 1 2 p p 1 8

0

2 0

4 0

6 0

8 0

1 0 0

P P I s 1 0 0


% P

PI

p p 6 p p 1 2 p p 1 8

0

2 0

4 0

6 0

8 0

1 0 0

P P I s 1 1 0


% P

PI

p p 6 p p 1 2 p p 1 8

0

2 0

4 0

6 0

8 0

1 0 0

P P I s 1 2 0


% P

PI

p p 6 p p 1 2 p p 1 8

0

2 0

4 0

6 0

8 0

1 0 0

A v e ra g e A ll P u ls e s


% P

PI

0

2 0

4 0

6 0

8 0

1 0 0

A v e ra g e A ll P u ls e s + P re p u ls e s

% P

PI

V 1 + V 2 + V 3 M 4- / -

(n = 8 )

V 1 + V 2 + V 3 M 4- / -

re -te s t (n = 2 )

A B

Figure 5.2: Re-testing PPI in M4-/- mice decreased startle amplitude and increased PPI

values. (A) Startle amplitude of mice at three pulse intensities 100, 110 and 120 dB. (B-D)

Percent inhibition of acoustic startle in mice when presented with prepulses of 6, 12 or 18 dB

above background prior to 100, 110 or 120 dB pulse intensities. (E) Averaged %PPI values of

all pulse intensities at each prepulse intensities. (F) Averaged %PPI values of all pulse and

prepulse intensities. Data presented as mean + SEM; n=2-8. V1: 10% DMSO/5% Tween 80 in

Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection.

C D

E F

Chapter 5

128

Next, the effect of re-testing on LMA was investigated. Repeated-measures ANOVA with

time as a within-subjects factor and re-testing as a between-subjects factor was used to

analyse the effect of time and re-testing on LMA in vehicle treated M4-/- mice. While time

did not have a significant effect on LMA, re-testing of LMA did (F1,9 = 9.34, p = 0.014;

Figure 5.3A; Appendix 3.1). A one-way ANOVA at each time point was performed to

reveal that distance travelled in the re-test was significantly different from the first LMA

test at 25 (p < 0.05), 30 (p < 0.05), 60 (p < 0.05), 75 (p < 0.01) and 80 (p < 0.01) min after

the first two injections (Figure 5.3A; Appendix 3.1). However, when distance travelled

was quantified as cumulated distance travelled between 25 and 55 min after the first two

injections, there was no significant difference in LMA between first test and re-test

(Figure 5.3B).

Therefore, to increase the sample size of the treatment groups, M4-/- mice were tested for

PPI and LMA, and then re-tested for LMA, but not PPI, 7 days after first LMA test

(Figure 5.1B).

T im e (m in )

Dis

tan

ce

tra

ve

lle

d (

cm

)

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0 V 1 + V 2 + V 3 M 4

- / -(n = 8 )

V 1 + V 2 + V 3 M 4

- / - re - te s t (n = 3 )

3 rd - V e h

1 s t - V e h

2 n d - V e h

**

* * ** *

D is ta n c e T r a v e lle d

2 5 to 5 5 m in

Dis

tan

ce

tra

ve

lle

d (

cm

)

Fir

st

Re-t

est

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0 n .s .

A B

Figure 5.3: Re-testing LMA in M4-/- mice did not significantly change LMA overall. (A)

Distance travelled every 5 min by mice recorded 30 min before and 80 min after treatment of 1st

and 2nd injections. Vertical dotted lines and arrows indicate the time at which the drugs were

administered. (B) Cumulated distance travelled between 25 and 55 min after treatment of 1st

and 2nd injections of first and repeated LMA tests. Data are presented as mean + SEM; n=3-8. *

p<0.05 and ** p<0.01 vs V1+V2+V3 M4KO group. V1: 10% DMSO/5% Tween 80 in Tris buffer

pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection.

Chapter 5

129

5.3.2 M4-/- Mice Exhibit Phenotypic Differences in PPI and LMA Compared to

M4+/+ Mice

5.3.2.1 M4-/- mice presented decreased PPI compared to M4

+/+ mice

A previous study has shown that M4-/- mice on a 129S6/SvEv background have

significantly reduced PPI compared to M4+/+ mice (Koshimizu et al., 2012). However,

another study using female M4-/- mice on the same C57Bl/6NTac background as the

present study showed that there was no difference in PPI, though a significant increase in

startle amplitude was seen, but male mice were not tested (Thomsen et al., 2010). The

divergent findings of the previous studies may be due to strain or sex differences; therefore,

to determine if male M4-/- mice on a C57Bl/6NTac background have lower PPI compared

to M4+/+ mice, PPI data from vehicle-treated mice of both genotype were analysed (Figure

5.4). There was a significant within-subjects main effect of pulse on startle amplitude,

indicating that higher pulse intensity induce greater startle amplitude (F2,38 = 77.281, p =

0.001). There was no significant pulse x genotype interaction, and there was no significant

between-subjects main effect of genotype on startle amplitude (Figure 5.4A).

There were significant within-subjects main effects of pulse (F2,38 = 8.94, p = 0.001) and

prepulse (F2,38 = 207.89, p < 0.001) on PPI. There was a significant between-subjects main

effect of genotype on PPI (F1,19 = 42.03, p < 0.001). As there were no significant

interactions between pulse or prepulse with genotype, the PPI data was averaged across all

pulse and prepulse intensities (Figure 5.4B; see Appendix 3.2 for PPI values from

individual pulse and prepulse intensities). One-way ANOVA analysis of the average PPI

data revealed that M4-/- mice have significantly reduced PPI compared to M4

+/+ mice (p <

0.001; Figure 5.4B).

Chapter 5

130

5.3.2.2 M4-/- mice displayed increased spontaneous LMA compared to M4

+/+ mice

Previous studies have shown discrepancies in the occurrence of increased baseline LMA in

M4-/- mice compared to M4

+/+ mice (Fink-Jensen et al., 2011; Gomeza et al., 1999b;

Koshimizu et al., 2012; Woolley et al., 2009). These discrepancies may have been a result

of different experimental methods (e.g. without or with habituation prior to measurement,

spontaneous LMA vs LMA following saline treatment). Therefore, to compare the results

of the present study with previous studies, both spontaneous LMA (measured during the

30 min habituation period prior V1 + V2 administration) and habituated LMA following

vehicle treatment (measured for 60 min following administration of V3) were analysed

(see Figure 4.1D). Figure 5.5A shows the distance travelled every 5 min for 30 min

before V1 + V2 injections (habituation phase), for 30 min immediately after V1 + V2

injections (testing – initial phase) and for the last 30 min of the testing phase (testing –

final phase). There was a significant main effect of time on LMA (F17,306 = 59.17, p <

0.001) and a significant time x genotype interaction (F17,306 = 3.33, p < 0.001). There was

also a significant main effect of genotype (F1,18 = 29.29, p < 0.001). One-way ANOVA

analyses of distance travelled per time point showed that M4-/- mice displayed significantly

1 0 0 1 1 0 1 2 0

0

5 0

1 0 0

1 5 0

2 0 0


Sta

rtl

e a

mp

litu

de

(arb

itu

ra

ry

un

its

)

0

2 0

4 0

6 0

8 0

Av

era

ge

% P

PI

V 1 + V 2 + V 3 M 4+ /+

(n = 1 3 )

V 1 + V 2 + V 3 M 4- / -

(n = 8 )* * *

A B

Figure 5.4: M4-/- mice have significantly reduced PPI compared to M4

+/+ mice. (A) Startle

amplitude of mice at three startle intensities 100, 110 and 120 dB. (B) Averaged %PPI values of

all startle pulses and prepulses. Data presented as mean + SEM; n=8-13. *** p<0.001 vs

V1+V2+V3 M4+/+ group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80

in saline. V3: water for injection. PPI: prepulse inhibition; pp: prepulse.

Chapter 5

131

higher spontaneous LMA compared to M4+/+ mice at all time points during the habituation

phase (all p < 0.001, except -5 min time point, which was p < 0.05; Figure 5.5A;

Appendix 3.3). However, M4-/- mice only showed significantly higher LMA at 35 to 45

min after first two injections (all p < 0.01) during the testing – initial phase, and only at 55

min after first two injections (p < 0.05) during the testing – final phase (Figure 5.5A;

Appendix 3.3).

Figure 5.5B shows the cumulated distance travelled during each of the three phases for

M4+/+ and M4

-/- mice. M4-/- mice displayed significantly greater spontaneous LMA during

the habituation phase compared to M4+/+ mice (p < 0.001; Figure 5.5B). This increased

LMA of M4-/- mice was also seen during the initial phase of the testing period (p < 0.001),

which was lost in the second half of the testing period (Figure 5.5B).

T e s tin g - f in a l

T e s tin g - in it ia l

T im e (m )

Dis

tan

ce

Tra

ve

lle

d (

cm

)

-3 0 -2 0 -1 0 0

0

1 0 0 0

2 0 0 0

3 0 0 0

2 0 3 0 4 0 5 0 6 0 7 0 8 0

V 1 + V 2 + V 3 M 4+ /+

(n = 9 )

V 1 + V 2 + V 3 M 4- / -

(n = 1 1 )

H a b itu a tio n

Hab

itu

at i

on

Test i

ng

- In

itia

l

Test i

ng

- F

inal

0

2 5 0 0

5 0 0 0

7 5 0 0

1 0 0 0 0

1 2 5 0 0

1 5 0 0 0

V 1 + V 2 + V 3 M 4+ /+

(n = 9 )

V 1 + V 2 + V 3 M 4- / -

(n = 1 1 )

* * *

* * *

A B

Figure 5.5: Spontaneous LMA of M4-/- mice was significantly increased compared to M4

+/+

mice, though this difference was reduced in the testing phase. (A) Distance travelled every

5 min by mice recorded for 30 min before the treatment of V1+V2 and for 60 min after the

treatment of V3. (B) Cumulated distance travelled for the three 30 min phases: habituation,

testing – initial and testing - final. Data are presented as mean + SEM; n=9-11. *** p < 0.001 vs

V1+V2+V3 M4+/+ mice. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in

saline. V3: water for injection.

Chapter 5

132

5.3.3 Determination of the Role of M4 mAChRs in the Modest Reversal of R(+)-6-

Br-APB Treatment-Induced Disruption of Prepulse Inhibition by LY2033298 and

Donepezil Treatments Using M4-/- Mice was Inconclusive

In the previous chapter, it was shown that in C57Bl/6J wildtype mice, treatments of

LY2033298 and/or donepezil displayed a trend to reverse R(+)-6-Br-APB-induced

disruption of PPI (see 4.3.5; Figure 4.9). Though modest, these effects were hypothesised

to be mediated through the selective activation of M4 mAChRs by LY2033298 and

endogenous ACh, the level of which was increased by the treatment of donepezil. To

confirm the hypothesis, the effect of LY2033298 and donepezil, treated either alone or in

combination, on disruption of PPI induced by R(+)-6-Br-APB in C57Bl/6NTac M4+/+ and

M4-/- mice was investigated.

There was a significant within-subjects main effect of pulse intensity on startle amplitude

(F1,127 = 334.17, p < 0.001), with greater pulse intensities giving greater startle amplitude

(Figure 5.6A). There was also significant pulse x R(+)-6-Br-APB (F1,127 = 3.96, p = 0.049)

and pulse x LY2033298 x R(+)-6-Br-APB (F1,127 = 5.24, p = 0.024) interactions. There

was a significant between-subjects main effect of R(+)-6-Br-APB on startle amplitude

(F1,127 = 8.68, p = 0.004), and significant LY2033298 x R(+)-6-Br-APB (F1,127 = 6.48, p =

0.012), donepezil x genotype (F1,127 = 5.81, p = 0.017), LY2033298 x donepezil x

genotype (F1,127 = 3.94, p = 0.049) and LY2033298 x donepezil x R(+)-6-Br-APB x

genotype (F1,127 = 5.19, p = 0.024) interactions. However, post hoc analysis revealed that

none of the drug treatment groups had significant effects on startle amplitude at any pulse

intensity (Figure 5.6A).

Chapter 5

133

1 0 0 1 1 0

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0


Sta

rtl

e a

mp

litu

de

(arb

itu

ra

ry

un

its

)

V 1 + V 2 + V 3 M 4+ /+

(n = 1 3 )

L Y 1 0 + V 2 + V 3 M 4+ /+

(n = 1 0 )

V 1 + D o n 1 + V 3 M 4+ /+

(n = 1 0 )

L Y 1 0 + D o n 1 + V 3 M 4+ /+

(n = 1 1 )

V 1 + V 2 + B r-A P B 0 .3 M 4+ /+

(n = 1 5 )

L Y 1 0 + V 2 + B r -A P B 0 .3 M 4+ /+

(n = 1 2 )

V 1 + D o n 1 + B r -A P B 0 .3 M 4+ /+

(n = 1 1 )

L Y 1 0 + D o n 1 + B r -A P B 0 .3 M 4+ /+

(n = 1 2 )

V 1 + V 2 + V 3 M 4- / -

(n = 8 )

L Y 1 0 + V 2 + V 3 M 4- / -

(n = 3 )

V 1 + D o n 1 + V 3 M 4- / -

(n = 3 )

L Y 1 0 + D o n 1 + V 3 M 4- / -

(n = 5 )

V 1 + V 2 + B r-A P B 0 .3 M 4- / -

(n = 1 2 )

L Y 1 0 + V 2 + B r -A P B 0 .3 M 4- / -

(n = 4 )

V 1 + D o n 1 + B r -A P B 0 .3 M 4- / -

(n = 4 )

L Y 1 0 + D o n 1 + B r -A P B 0 .3 M 4- / -

(n = 1 0 )

p p 6 p p 1 2 p p 1 8

-5 0

0

5 0

1 0 0

P P I s 1 0 0


% P

PI

*

* * * ** *

* * ** * *

p p 6 p p 1 2 p p 1 8

-5 0

0

5 0

1 0 0

P P I s 1 1 0


% P

PI

** * * * *

* *

0

2 0

4 0

6 0

8 0

A v e ra g e A ll P u ls e s + P re p u ls e s

% P

PI

* * ** * *

A

B

Figure 5.6: M4+/+ mice showed similar PPI data to the C57Bl/6J mice (Chapter 4), though

effect of the drugs were less clear in M4-/- mice, likely due to floor effect. (A) Startle

amplitude of mice at 100 and 110 dB startle intensities. (B, C) Percent inhibition of acoustic

startle in mice when presented with prepulses of 6, 12 or 18 dB above background prior to

pulse intensities of 100 or 110 dB. (D) Averaged %PPI values of all startle pulses and

prepulses. Data presented as mean + SEM; n=3-13. * p<0.05, ** p<0.01 and *** p<0.001. V1:

10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for

injection. LY: LY2033298; Don: donepezil; Br-APB: R(+)-6-Br-APB; PPI: prepulse inhibition; pp:

prepulse.

C

D

Chapter 5

134

There was a significant within-subjects main effect of prepulse intensity (F2,254 = 459.28, p

< 0.001) on PPI, indicating that higher prepulse intensities were efficient in producing

greater PPI, as well as significant prepulse x LY2033298 x donepezil (F2,254 = 4.30, p =

0.019) interaction. There was a significant within-subjects main effect of pulse intensity

(F1,127 = 6.35, p = 0.013), as well as significant pulse x LY2033298 x R(+)-6-Br-APB

(F1,127 = 5.88, p = 0.017) and pulse x LY2033298 x donepezil x R(+)-6-Br-APB x

genotype (F1,127 = 4.43, p = 0.037) interactions. There were significant between-subjects

main effects of R(+)-6-Br-APB (F1,127 = 36.17, p < 0.001) and genotype (F1,127 = 8.12, p =

0.005), as well as significant donepezil x R(+)-6-Br-APB x genotype interaction (F1,127 =

4.09, p = 0.045).

One-way ANOVA analysis with Tukey HSD post hoc analysis revealed that in M4+/+ mice,

treatment of R(+)-6-Br-APB significantly disrupted PPI at all pulse and prepulse

conditions (P100pp6, p < 0.05; P100pp12, p < 0.05; P100pp18, p < 0.01; P110pp6, p <

0.05; P110pp12, p < 0.001; P110pp18, p < 0.01; Figures 5.6B, C). LY2033298 + R(+)-6-

Br-APB treatment also significantly disrupted PPI in these mice at the 100 dB pulse

intensity (P100pp6, p < 0.001; P100pp12, p < 0.01; P100pp18, p <0.001; Figure 5.6B).

Post hoc analysis of average PPI values of all pulse and prepulse intensities also showed

that R(+)-6-Br-APB and LY2033298 + R(+)-6-Br-APB treatments significantly disrupted

PPI (both p < 0.001; Figure 5.6D). However, post hoc analyses showed that although there

is a trend for treatment of donepezil or combined treatment of LY2033298 + donepezil to

reverse the disruption of PPI induced by R(+)-6-Br-APB, these effects were not significant

(Figures 5.6B-D).

In M4-/- mice, R(+)-6-Br-APB treatment significantly disrupted PPI only at P100pp18 (p <

0.05; Figures 5.6B, C). Interestingly, at P110pp12, M4-/- mice treated with LY2033209 +

donepezil + R(+)-6-Br-APB had significantly different PPI compared to R(+)-6-Br-APB

Chapter 5

135

treated M4-/- mice (p < 0.01), though R(+)-6-Br-APB treatment did not significantly disrupt

PPI in these mice (Figure 5.6C). However, this was not seen at other pulse and prepulse

conditions. Post hoc analysis of averaged PPI values of all pulse and prepulse intensities

showed that none of the treatments were significantly different from vehicle-treated group

in M4-/- mice (Figure 5.6D).

5.3.4 R(+)-6-Br-APB 1 mg/kg Dose Induces Stereotypic Behaviour in C57Bl/6NTac

Wildtype Mice Not Seen in C57Bl/6J Mice

A pilot experiment tested the effect of R(+)-6-Br-APB on LMA in C57Bl/6NTac M4+/+

mice using the 1 mg/kg dose as used in the previous chapter to induce hyperlocomotor

activity. Results from this pilot experiment showed a reduction in LMA in mice from 40 to

55 min after the first two injections, which was not seen previously in C57Bl/6J mice

(Figure 5.7A; Appendix 3.5). Video recordings of these mice showed that during this

period, the mice displayed stereotypic behaviour, such as repeated sniffing around the

mouth and grooming of the snout, resulting in a decrease in LMA. Lower doses of R(+)-6-

Br-APB (0.3 and 0.6 mg/kg) were also tested to determine the optimum dose of inducing

hyperlocomotor activity without stereotypy. There was a significant between-subjects

main effect of R(+)-6-Br-APB (F3,16 = 18.75, p < 0.001), but not within-subjects effect of

time, and there was no significant time x R(+)-6-Br-APB interaction.

Analysis of cumulated LMA from 25 to 55 min after first two injections (see 5.2.5)

revealed the robust increase in LMA induced by all three doses of R(+)-6-Br-APB (Figure

5.7B). There was a significant between-subjects main effect of R(+)-6-Br-APB on

cumulated distance travelled (F3,16 = 23.35, p < 0.001). Post hoc analysis further confirmed

that all three doses significantly increased LMA, as seen in C57Bl/6J mice (see 4.3.2.2)

with the 0.6 mg/kg dose being the optimum dose for inducing hyperlocomotor activity in

Chapter 5

136

these mice without inducing stereotypy behaviour (Figure 5.7B, Appendix 3.5). Therefore,

R(+)-6-Br-APB 0.6 mg/kg was used to induce hyperlocomotor activity in M4+/+ and M4

-/-

mice for the rest of the study.

A B

Figure 5.7: Treatment of R(+)-6-Br-APB 1 mg/kg caused a decrease in LMA between 40

and 55 min post first two injections. R(+)-6-Br-APB 0.6 mg/kg dose was the most

effective in increasing LMA in M4+/+ mice. (A) Distance travelled every 5 min by mice treated

with different doses of R(+)-6-Br-APB recorded 30 min before and 80 min after treatment of

V1+V2. Vertical dotted lines and arrows indicate the time at which the drugs were administered.

(B) Cumulated distance travelled between 25 and 55 min after treatment of V1+V2. Data are

presented as mean + SEM; n=3-6. ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 group. V1: 10%


Br-APB: R-6-Br-APB.

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

T im e (m in )

Dis

tan

ce

Tra

ve

lle

d (

cm

)

V 1 + V 2 + V 3

V 1 + V 2 + B r-A P B 1V 1 + V 2 + B r -A P B 0 .3

V 1 + V 2 + B r -A P B 0 .6

3 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1

2 n d - V 2

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0


2 5 to 5 5 m in

Dis

tan

ce

tra

ve

lle

d (

cm

)

V 1 + V 2 + V 3 (n = 5 )

V 1 + V 2 + B r-A P B 1 (n = 6 )

V 1 + V 2 + B r-A P B 0 .3 (n = 3 )

V 1 + V 2 + B r-A P B 0 .6 (n = 6 )

* *

* * ** * *

Chapter 5

137

5.3.5 LY2033298, Donepezil or LY2033298 and Donepezil Combined Treatment

Decreased Hyperlocomotor Activity Induced by R(+)-6-Br-APB in M4-/- Mice

In the previous chapter, only the combined treatment of LY2033298 + donepezil + R(+)-6-

Br-APB reversed the D1 DR-selective agonist-induced hyperlocomotor activity (see 4.3.6).

To determine the role of M4 mAChRs in this modulation of effect, the ability of

LY2033298 or donepezil, or combined treatment, to modulate R(+)-6-Br-APB-induced

hyperlocomotor activity was investigated in M4+/+ and M4

-/- mice.

Figures 5.8A-D and Appendices 3.6, 3.7 show the distance travelled every 5 min over

time of each treatment group and genotype. There were significant within-subjects main

effects of time on LMA (F11,1364 = 11.01, p < 0.001). There were also significant time x

R(+)-6-Br-APB (F11,1364 = 9.83, p < 0.001), time x genotype (F11,1364 = 2.40, p = 0.029)

and time x R(+)-6-Br-APB x genotype (F11,1364 = 4.81, p < 0.001) interactions. There were

significant between-subjects main effects of R(+)-6-Br-APB (F1,124 = 267.12, p < 0.001),

LY2033298 (F1,124 = 20.80, p < 0.001), donepezil (F1,124 = 31.85, p < 0.001) and genotype

(F1,124 = 4.19, p = 0.043) on LMA. Additionally, there were significant LY2033298 x

R(+)-6-Br-APB (F1,124 = 4.82, p = 0.030), donepezil x R(+)-6-Br-APB (F1,124 = 7.18, p =

0.008), LY2033298 x donepezil x R(+)-6-Br-APB (F1,124 = 4.36, p = 0.039), donepezil x

R(+)-6-Br-APB x genotype (F1,124 = 4.73, p = 0.032) and LY2033298 x donepezil x R(+)-

6-Br-APB x genotype (F1,124 = 12.53, p = 0.001) interactions. See Appendices 3.6 and 3.7

for post hoc analysis of LMA at each time point. It should be noted that the distance

travelled over time profile for LY2033298 + donepezil + R(+)-6-Br-APB in M4+/+ mice

displayed a long-lasting reduction of hyperlocomotor activity induced by R(+)-6-Br-APB

(Figure 5.8C, Appendix 3.6), which is in contrast with what was observed in C57Bl/6J

mice, where the reduction was transient (see 4.3.6, Figure 4.10D). Additionally, in M4-/-

mice, treatments of LY2033298 + R(+)-6-Br-APB, donepezil + R(+)-6-Br-APB and

Chapter 5

138

V 1 + D o n 0 .6 + B r-A P B 0 .6 M 4

- / -(n = 6 )

V 1 + D o n 0 .6 + V 3 M 4

- / -(n = 7 )

V 1 + D o n 0 .6 + V 3 M 4

+ / +(n = 8 )

L Y 1 0 + D o n 0 .6 + A P B 0 .6 M 4

- / - (n = 8 )

L Y 1 0 + D o n 0 .6 + V 3 M 4

- / -(n = 6 )

0

2 5 0 0

5 0 0 0

7 5 0 0

1 0 0 0 0

1 2 5 0 0

1 5 0 0 0

D is ta n c e T ra v e lle d 2 5 to 5 5 m in

Dis

tan

ce

tra

ve

lle

d (

cm

)

V 1 + V 2 + V 3 M 4

+ / + (n = 9 )

L Y 1 0 + D o n 0 .6 + V 3 M 4

+ / +(n = 1 0 )

V 1 + V 2 + B r-A P B 0 .6 M 4

+ / +(n = 1 3 )

L Y 1 0 + D o n 0 .6 + A P B 0 .6 M 4

+ / +(n = 1 0 )

V 1 + V 2 + V 3 M 4

- / -(n = 1 1 )

V 1 + V 2 + B r-A P B 0 .6 M 4

- / -(n = 1 2 )

L Y 1 0 + V 2 + V 3 M 4

- / -(n = 7 )

L Y 1 0 + V 2 + B r-A P B 0 .6 M 4

- / -(n = 6 )

L Y 1 0 + V 2 + B r-A P B 0 .6 M 4

+ / +(n = 1 0 )

V 1 + D o n 0 .6 + B r-A P B 0 .6 M 4

+ / + (n = 1 0 )

L Y 1 0 + V 2 + V 3 M 4

+ / +(n = 7 )

* * ** * *

* * ** * *

*

* * *** ** * *

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

M 4+ /+

M ic e

T im e (m in )

Dis

tan

ce

Tra

ve

lle

d (

cm

)

V 1 + V 2 + V 3 (n = 9 )

3 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1 /L Y 2 0 3 3 2 9 8


L Y 1 0 + D o n 0 .6 + V 3 (n = 1 0 )

V 1 + V 2 + B r-A P B 0 .6 (n = 1 3 )

L Y 1 0 + V 2 + V 3 (n = 7 )

V 1 + D o n 0 .6 + V 3 (n = 8 )

L Y 1 0 + D o n 0 .6 + B r-A P B 0 .6 (n = 1 0 )

V 1 + D o n 0 .6 + B r-A P B 0 .6 (n = 1 0 )

L Y 1 0 + V 2 + B r-A P B 0 .6 (n = 1 0 )

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

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M 4-/-

M ic e

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V 1 + V 2 + V 3 (n = 1 1 ) V 1 + V 2 + B r-A P B 0 .6 (n = 1 2 )

L Y 1 0 + D o n 0 .6 + V 3 (n = 6 )

L Y 1 0 + V 2 + V 3 (n = 7 )

V 1 + D o n 0 .6 + V 3 (n = 7 )

3 rd - V 3 /R (+ ) -6 -B r -A P B

1 s t - V 1 /L Y 2 0 3 3 2 9 8


L Y 1 0 + D o n 0 .6 + B r-A P B 0 .6 (n = 8 )

V 1 + D o n 0 .6 + B r-A P B 0 .6 (n = 6 )

L Y 1 0 + V 2 + B r-A P B 0 .6 (n = 6 )

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T im e (m in )

Dis

tan

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-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T im e (m in )

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A B

Figure 5.8: Co-treatment of LY2033298 and donepezil reverses hyperlocomotor activity

induced by R(+)-6-Br-APB in M4+/+ mice. Treatments of LY2033298 and donepezil either

alone or in combination reduced the baseline and R(+)-6-Br-APB-induced

hyperlocomotor activity in M4-/- mice. Distance travelled every 5 min by (A, C) M4

+/+ and (B,

D) M4-/- mice recorded 30 min before and 80 min after treatment of 1st and 2nd injections.

Vertical dotted lines and arrows indicate the time at which the drugs were administered. (E)

Cumulated distance travelled between 25 and 55 min after treatment of 1st and 2nd injections.

Data presented as mean + SEM; n=6-13. * p<0.05, ** p<0.01 and *** p<0.001. V1: 10%


LY: LY2033298; Don: donepezil; Br-APB: R-6-Br-APB.

C D

E

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LY2033298 + donepezil + R(+)-6-Br-APB all decreased the hyperlocomotor activity

induced by R(+)-6-Br-APB in M4-/- mice, although the effects were transient compared to

that of LY2033298 + donepezil + R(+)-6-Br-APB treatment in M4+/+ mice (Figures 5.8C,

D, Appendix 3.7).

Figure 5.8E shows the cumulated distance travelled of M4+/+ and M4

-/- mice treated with

the drug treatments from 25 to 55 min after first two injections. There were significant

main effects of LY2033298 (F1,124 = 20.05, p < 0.001), donepezil (F1,124 = 35.48, p < 0.001)

and R(+)-6-Br-APB (F1,124 = 165.85, p < 0.001) treatments on cumulated distance

travelled. There were also significant donepezil x R(+)-6-Br-APB (F1,124 = 7.21, p = 0.008),

R(+)-6-Br-APB x genotype (F1,124 = 4.37, p = 0.039), LY2933298 x donepezil x genotype

(F1,124 = 4.74, p = 0.031), donepezil x R(+)-6-Br-APB x genotype (F1,124 = 4.79, p = 0.031)

and LY2033298 x donepezil x R(+)-6-Br-APB x genotype (F1,124 = 12.04, p = 0.001)

interactions. Post hoc analysis revealed that in M4+/+ mice, R(+)-6-Br-APB, LY2033298 +

R(+)-6-Br-APB and donepezil + R(+)-6-Br-APB treatments significantly increased LMA

(all p < 0.001; Figure 5.8E). LY2033298 + donepezil + R(+)-6-Br-APB treatment

significantly reversed hyperlocomotor activity induced by R(+)-6-Br-APB (p < 0.001;

Figure 5.8E).

In M4-/- mice, R(+)-6-Br-APB also significantly increased LMA (p < 0.001; Figure 5.8E).

However, treatments of LY2033298 + R(+)-6-Br-APB and donepezil + R(+)-6-Br-APB

both significantly reversed the hyperlocomotor activity induced by R(+)-6-Br-APB (p <

0.05 and p < 0.01, respectively; Figure 5.8E). Additionally, LY2033298 + donepezil +

R(+)-6-Br-APB treatment also reduced the hyperlocomotor activity (p < 0.001), though

this effect was confounded by the significantly reduced LMA induced by combined

LY2033298 + donepezil treatment (p < 0.05; Figure 5.8E).

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5.4 Discussion

In this chapter, the role of M4 mAChR in the ability of LY2033298 and donepezil

treatments to reverse disruption of PPI and hyperlocomotor activity induced by R(+)-6-Br-

APB (as reported in Chapter 4) was investigated in whole-body M4 mAChR knockout

mice. Due to the lack of receptor subtype selectivity of muscarinic pharmacological tools,

whole-body knockout mice of each mAChR subtype were created in the late 1990s and

early 2000s, with the objective to delineate the specific physiological functions of each

mAChR subtype (Gomeza et al., 1999a; Gomeza et al., 1999b; Hamilton et al., 1997;

Matsui et al., 2000; Yamada et al., 2001a). Specifically, M4-/- mice on a mixed

129S6/SvEv + C57Bl/6 + CF-1 genetic background were created by Gomeza et al. (1999b).

Mice used in this chapter were generated from these founder mice backcrossed to the

C57Bl/6NTac strain for over 10 generations.

In the present study, vehicle-treated M4-/- mice had lower PPI compared to vehicle-treated

M4+/+ mice (5.3.2.1; Figure 5.4), potentially due to the increased basal DA efflux in the

nucleus accumbens, which was reported for both whole-body M4-/- mice and mice with

conditional M4 mAChR knockout only in D1 DR-expressing neurons (Jeon et al., 2010;

Tzavara et al., 2004). While this is in agreement with one study, where M4-/- mice on a

129S6/SvEv background have been shown to have significantly reduced PPI compared to

M4+/+ mice (Koshimizu et al., 2012), another study using M4

-/- mice on a CF1 x 129/SvEv

background did not detect any difference in PPI compared to M4+/+ mice (Felder et al.,

2001). Additionally, in female M4-/- mice on the same C57Bl/6NTac background as the

present study, there was no difference in PPI, though a significant increase in startle

amplitude was seen (Thomsen et al., 2010). However, the Thomsen et al. (2010) study did

not investigate PPI in male M4-/- mice, and while it is possible that these effects, or lack

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thereof, are sex specific, without data from male M4-/- mice, a direct comparison could not

be made. It should be noted that the literature on the investigation of PPI in M4-/- mice is

limited, and further studies on this topic should be performed to determine the role of M4

mAChRs in PPI.

The combined treatments of LY2033298 and donepezil or treatment of donepezil alone in

M4+/+ mice showed a trend to reverse R(+)-6-Br-APB treatment-induced disruption of PPI,

though these effects were not significant (Figures 5.6), similar to effects observed in

C57Bl/6J mice in Chapter 4. In M4-/- mice, though R(+)-6-Br-APB treatment reduced PPI,

this effect was not significant, possibly due to a floor effect (Figures 5.6) (Swerdlow et al.,

2000). Interestingly, combined treatment of LY2033298 and donepezil significantly

reversed the R(+)-6-Br-APB-induced decrease in PPI at the P110pp12 condition in these

Figure 5.9: Simplified schematic representation of reported localisation of DRs, mAChRs

and nAChRs in the striatum. Adapted from Quik et al. 2011; Bonsi et al. 2011; Calabresi et al.

2014. M1: M1 mAChR; M2: M2 mAChR; M3: M3 mAChR; M4: M4 mAChR; D1: D1 DR; D2: D2

DR; MSN: medium spiny neuron.

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mice lacking M4 mAChRs, and also showed a trend to reverse at some pulse and prepulse

conditions (Figure 5.6). These effects could be mediated indirectly by donepezil, through

its inhibition of acetylcholinesterase, resulting in an increase in ACh, which can bind to

other mAChR subtypes or nAChRs to exert its effect. Indeed, activation of α7 or α4β2

nAChRs, which are expressed in striatal neurons, has been shown to reverse disruption of

PPI (Kaiser and Wonnacott, 2000; Kohnomi et al., 2010; Kucinski et al., 2012; Marchi et

al., 2002; Wallace et al., 2011; Wildeboer and Stevens, 2008) (Figure 5.9). In an attempt

to distinguish the relative contributions of LY2033298 and donepezil to this reversal effect,

LY2033298 + R(+)-6-Br-APB and donepezil + R(+)-6-Br-APB treatments were tested in

M4-/- mice (Figures 5.6). However, due to the slow breeding and the inability of re-testing

mice for PPI experiments mentioned above, unfortunately, there were not enough M4-/-

mice to obtain a large enough sample size of these treatment groups. Future studies with

M4-/- mice to increase the sample size of the treatment groups will address this question.

In the present study, M4-/- mice backcrossed to the C57Bl/6NTac strain were found to

exhibit increased spontaneous LMA (habituation phase) and increased LMA following

vehicle injections (testing – initial phase) compared to M4+/+ mice (Figure 5.5, Appendix

3.3). Similar to the reduced PPI seen in these mice, the significantly increased spontaneous

LMA seen in M4-/- mice in this chapter are potentially due to the increased basal DA efflux

in the nucleus accumbens reported for mice lacking M4 mAChRs (Jeon et al., 2010;

Tzavara et al., 2004). However, previous studies have reported conflicting results

regarding LMA in M4-/- mice compared to wildtype. Gomeza et al. (1999b) reported that

M4-/- mice displayed increased spontaneous LMA and enhanced hyperlocomotor activity

induced by D1 DR activation compared to M4+/+ mice. Additionally, a recent study using

M4-/- mice on a pure 129S6/SvEv background showed increased spontaneous LMA, but

only in the initial 10 min of novelty-induced LMA (Koshimizu et al., 2012). Conversely,

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in other studies using congenic M4-/- mice that were backcrossed to the C57Bl/6J or

C57Bl/6NTac strains for over 10 generations, the mice did not display increased

spontaneous and DA agonist-induced LMA compared to wildtype mice (Fink-Jensen et al.,

2011; Woolley et al., 2009).

Phenotypic variations between inbred mouse strains are a result of their genotypic

differences, and behaviours, such as spontaneous LMA, have been shown to vary

(Claassen, 1994; Crabbe et al., 1999; Crawley et al., 1997; Crusio et al., 1991; Fink and

Reis, 1981; Ralph et al., 2001b). The differences may suggest that the contributions of

neurotransmitters DA and ACh and their corresponding receptors, DRs and mAChRs (and

nAChRs), to the regulation of LMA may also differ between mouse strains. This could

explain the discrepancies in the literature regarding the presence of the increased LMA

phenotype of M4-/- mice.

Additionally, it should be noted that in the present study, spontaneous LMA was measured

for only 30 min (habituation phase), which was followed by administration of vehicles V1,

V2 and V3 during the 20 min before testing phase, where LMA was measured for 60 min.

In the Fink-Jensen et al. (2011) study, while there were no significant differences in

spontaneous LMA measured for 120 min between the two genotypes overall, M4-/- mice

did show higher, though non-significant, activity in the first 30 min of the test compared to

M4+/+ mice. There is the potential that if the habituation phase of the present study was

extended beyond 30 min, the increased LMA phenotype of M4-/- mice may have reduced

over time. This hypothesis is supported by the loss of a significant difference in LMA in

the testing – final phase (second 30 min of the testing phase) of vehicle treated mice in this

study (Figure 5.5; Appendix 3.3).

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In M4+/+ mice, combined LY2033298 and donepezil treatment caused a complete reversal

of R(+)-6-Br-APB-induced hyperlocomotor activity (Figure 5.8), which was also seen in

C57Bl/6J mice in Chapter 4. However, as seen in PPI, combined LY2033298 and

donepezil treatment also produced a reversal of R(+)-6-Br-APB-induced hyperlocomotor

activity in M4-/- mice. Furthermore, both LY2033298 + R(+)-6-Br-APB and donepezil +

R(+)-6-Br-APB treatment groups also had significantly lower LMA compared with the

R(+)-6-Br-APB treatment group in M4-/- mice, which were effects that were not seen in

M4+/+ mice (Figure 5.8). It is possible that these changes seen in M4

-/- mice could be due to

the drugs producing effects on other receptors that were upregulated due to the potential

compensatory effects from a lack of M4 mAChRs (Holschneider and Shih, 2000).

Although previous studies have not seen changes in M2 mAChR expression in key brain

regions in M4-/- mice (Gomeza et al., 1999b), further experiments to determine changes in

receptor expression of all mAChR subtypes are needed to confirm the lack of such

compensatory changes.

Other receptors may also be involved in the reversal effects of the combined LY2033298

and donepezil treatment on R(+)-6-Br-APB treatment-induced hyperlocomotor activity. In

the striatum, M4 mAChRs are expressed presynaptically on cholinergic interneurons,

regulating ACh release, and postsynaptically on striatonigral MSNs, the activation of

which leads to the activation of movement and locomotion (direct pathway; see 1.5.2;

Figures 1.6, 5.9) (Calabresi et al., 2014; Hersch et al., 1994; Santiago and Potter, 2001;

Yan et al., 2001; Zhang et al., 2002a). M1 mAChRs are also expressed postsynaptically,

not only on the striatonigral MSNs, but also on the striatopallidal MSNs (Levey et al.,

1991; Weiner et al., 1990; Yan et al., 2001). Activation of M1 mAChRs have been shown

to increase MSN excitability, particularly for striatopallidal MSNs (Akins et al., 1990;

Kreitzer, 2009; Shen et al., 2005; Shen et al., 2007). It has been shown that activation of

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M1 mAChRs inhibits the inwardly rectifying K+ channel, Kir2.3, which is highly expressed

in the striatopallidal MSNs, leading to an increase in the state transitions of these neurons

(Shen et al., 2007). Activation of striatopallidal MSNs lead to a reduction in LMA and

movement (indirect pathway; see 1.5.2) (Calabresi et al., 2014), which may explain the

significant reduction of the D1 DR agonist-induced hyperlocomotor activity by donepezil

treatment in M4-/- mice.

M2 mAChRs are also expressed presynaptically on cholinergic and GABAergic

interneurons, glutamatergic afferent neurons and dopaminergic afferent neurons, where

they act as auto- or heteroreceptors, regulating the release of ACh, DA, glutamate and

GABA (Bernard et al., 1992; Hersch et al., 1994; Hersch and Levey, 1995; Yan and

Surmeier, 1996) (Figure 5.9). LY2033298 has been shown to potentiate the effect of

choline, a metabolite of ACh, at the M2 mAChR (Wootten et al., 2012). Activation of M2

mAChRs, by either ACh or choline, leads to a reduction of the release of these

neurotransmitters, which may lead to a reduction of LMA seen in M4-/- mice with

LY2033298 and/or donepezil treatment.

Furthermore, nAChRs are expressed on nigrostriatal dopaminergic afferent neurons,

corticostriatal glutamatergic afferent neurons and GABAergic interneurons, and are

involved in the cholinergic control of DA, glutamate and GABA release in the striatum

(Quik and Wonnacott, 2011) (Figure 5.9). These nAChRs, as well as those expressed in

the ventral tegmental area, have been shown to be involved in the nAChR modulation of

dopaminergic activity, including locomotion (Faure et al., 2014), and may also be involved

in the reduction of R(+)-6-Br-APB treatment-induced hyperlocomotor activity by

treatments of LY2033298 and donepezil seen in M4-/- mice.

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Outside of the mAChR family, Chan et al. (2008) found that LY2033298 did not bind to

the orthosteric site for other GPCRs, ion channels, signalling proteins and enzymes.

However, there is still the possibility that LY2033298 can bind to the allosteric site for

other GPCRs, producing effects that only became apparent when M4 mAChRs were not

present in the system. A previous study has shown that the combined LY2033298 and sub-

effective oxotremorine treatment could significantly reduce the conditioned avoidance

response, which is a measure of potential antipsychotic activity, and this effect was not

completely abolished in M4-/- mice (Leach et al., 2010; Wadenberg, 2010). Due to the

probe dependence nature of LY2033298, the residual effects of LY2033298 and

oxotremorine in M4-/- mice may be due to the drugs acting on M2 mAChRs (Valant et al.,

2012).

Due to the added complications with the potential of compensatory effects in whole-body

knockout mice, which can be difficult to account for (Holschneider and Shih, 2000), other

systems may be more suitable for the investigation the role of M4 mAChR in the

modulation of D1 DR function using an M4 mAChR PAM. For example, mice with

conditional M4 mAChR knockout in D1 DR-expression neurons using the Cre/loxP

technology were generated recently, where the authors demonstrated that this

subpopulation of M4 mAChR is critically involved in the modulation of DA-dependent

behaviours (Jeon et al., 2010). These mice were reported to have no changes in M1 and M2

mAChR and D1 DR expressions in the striatum, and showed normal expression of M4

mAChRs in areas other than the nucleus accumbens and caudate putamen, which makes

these mice a suitable alternative for future studies (Dencker et al., 2012b; Jeon et al., 2010).

Chapter 6:

General Discussion

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Due to the expression of M4 mAChRs and the close association of these receptors with the

DA system in the CNS, the M4 mAChR is an emerging novel target for the treatment of

schizophrenia, particularly for treating the positive symptoms (Dencker et al., 2012a;

Foster et al., 2012; Langmead et al., 2008). However, owing to the high sequence

homology of the orthosteric binding site within the mAChR family, orthosteric ligands for

this receptor family have limited subtype selectivity, which often leads to the occurrence

of undesirable side effects when administered in humans (Christopoulos, 2014). As

highlighted in 1.1.3, allosteric ligands offer greater receptor subtype selectivity, due to the

less conserved allosteric binding site. In the absence of allosteric agonism, allosteric

ligands can also maintain the spatiotemporal control of the receptor activity, as they can

only exert their modulatory effect in the presence of an orthosteric ligand. These properties

make allosteric modulators attractive therapeutic agents. In fact, many allosteric

modulators are currently in development or being marketed for CNS disorders (such as

schizophrenia, Fragile X syndrome, Parkinson’s disease and Alzheimer’s disease) and

other disease states, such as hyperparathyroidism and HIV infection (for reviews, see Conn

et al. (2009) and Conn et al. (2014)).

Novel allosteric modulators are generally identified through high-throughput screening

using cell lines overexpressing the human receptor of interest, often with an intracellular

Ca2+ mobilisation assay as a readout (Bertekap et al., 2015). As Ca2+ mobilisation is a

signalling end-point in the Gαq protein pathway, for GPCRs that preferentially couple to

Gαs or Gαi/o proteins, Gα15/16 or the chimeric Gαqi5 proteins are used to artificially allow

the activation of these receptors to mobilise intracellular Ca2+ (Bertekap et al., 2015;

Conklin et al., 1993; Offermanns and Simon, 1995). However, by changing the expression

of proteins in these cells, the physiological relevance of such cellular environment is

decreased.

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Additionally, it has been recognised that GPCRs can adopt different active conformations

that recruit or disrupt different signalling effectors, and endpoint-based approaches are

unable to capture such diverse signalling profile in one assay (Galandrin et al., 2007;

Kenakin and Miller, 2010). Label-free technologies are proposed to address these

limitations as they monitor whole-cell changes, such as cell morphology, adhesion and

cytoskeleton reorganisation, in real-time (Halai and Cooper, 2012; Scott and Peters, 2010).

While label-free technologies have been used to study allosteric modulators in

recombinant cell lines (Klein et al., 2013; Peters et al., 2007), we were the first to

demonstrate in an endogenous neuronal cell line that the allosteric modulation of an

orthosteric ligand by a PAM can be detected and quantified with this technology (Chapter

2). More importantly, the allosteric parameters estimated from the label-free approach are

comparable to those estimated from endpoint-based assays. Future studies can utilise this

approach to screen for allosteric modulators for GPCRs with unknown G protein-coupling

preferences.

The main aim of this thesis was to investigate the potential functional cross-talk between

endogenous M4 mAChRs and the D1 or D2 DRs. The dysregulation of mAChRs is

implicated in schizophrenia, and mAChRs are increasingly being recognised as potential

novel targets for the treatment of this disorder (Carruthers et al., 2015; McKinzie and

Bymaster, 2012). M4 mAChRs, in particular, have been shown to be involved in the

regulation of DA activity in the CNS (Gomeza et al., 1999b; Tzavara et al., 2004; Zhang et

al., 2002b). Furthermore, the modulation of DR functions by M4 mAChRs expressed in the

striatum is suggested to be the mechanism through which the non-selective mAChR

orthosteric agonist, xanomeline, mediates its antipsychotic effects (Dencker et al., 2011;

Woolley et al., 2009). Therefore, in Chapters 3 and 4, we sought to better understand the

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relationship between M4 mAChRs and the DRs in a neuronal cell line endogenously

expressing these receptors and in mice.

M4 mAChRs and DRs are co-localised in the striatum: M4 mAChRs and D2 DRs are on

tonically active cholinergic interneurons that provide cholinergic tone to the striatum, and

M4 mAChRs and D1 DRs are on striatonigral (direct) MSNs that are involved in the

regulation of motor behaviour (Dawson et al., 1988; Hersch et al., 1994; Ince et al., 1997;

Kreitzer and Malenka, 2008; Yan and Surmeier, 1996). In Chapter 3, the investigation of

the functional cross-talk between endogenous M4 mAChRs and D2 DRs in a neuronal cell

line using cAMP BRET biosensor and ERK1/2 phosphorylation assays did not reveal a

functional cross-talk between these two receptors. However, these results do not

necessarily indicate that functional cross-talk does not exist between these two receptor,

but that the cell line and assays used may not have been suitable for this investigation. As

there are limited studies in the literature on the potential cross-talk between M4 mAChRs

and D2 DRs, further experiments using a label-free approach may be useful in determining

the potential of this relationship (Fang, 2011). Furthermore, future studies can also explore

the possibility of a physical cross-talk between M4 mAChRs and D2 DRs using FRET or

BRET techniques, coupled with co-immunoprecipitation assays.

In the literature, there is more evidence of a functional cross-talk between M4 mAChRs

and D1 DRs. These receptors preferentially couple to G proteins of opposing actions: M4

mAChRs couple to Gαi/o proteins to inhibit AC production of cAMP, and D1 DRs couple

to Gαs proteins to activate AC production of cAMP (Beaulieu and Gainetdinov, 2011;

Felder, 1995). Indeed, activation of M4 mAChRs have been shown to inhibit cAMP

production and regulate ERK1/2 phosphorylation induced by D1 DR-like selective agonists

in the striatum (DeLapp et al., 1996; Kelly and Nahorski, 1986; Olianas and Onali, 1996;

Olianas et al., 1983; Xue et al., 2015). Furthermore, mice with either whole-body M4

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mAChR knockout or conditional M4 mAChR knockout in D1 DR-expressing neurons both

show heightened sensitivity to D1 DR-like selective agonist-induced hyperlocomotor

activity, indicating that M4 mAChRs play a role in regulating D1 DR-mediated functions

(Gomeza et al., 1999b; Jeon et al., 2010). Following studies reporting the antipsychotic

effects of xanomeline in patients with schizophrenia and that these effects were mediated

mainly by the subpopulation of M4 mAChRs found on D1 DR-expressing neurons

(Dencker et al., 2011; Shekhar et al., 2008), M4 mAChR PAMs were developed as

potential antipsychotics with greater receptor subtype selectivity (see Table 1.2 and

references therein). These M4 mAChR PAMs reversed disruption of PPI or

hyperlocomotor activity induced by either direct (apomorphine) or indirect (amphetamine,

cocaine) non-selective DR agonists. However, none of the studies explored the ability for

M4 mAChR PAMs to reverse specific D1 DR-induced behaviours.

In Chapter 4, the functional cross-talk between M4 mAChRs and D1 DRs was explored in

mice by investigating the ability of an M4 mAChR PAM, LY2033298, to modulate D1 DR-

selective ligand induced disruption of PPI and increase in LMA. In both behavioural tests,

LY2033298 required the co-administration of donepezil, an acetylcholinesterase inhibitor,

to elicit an effect. This is in line with previous studies, where co-treatment of a sub-

effective dose of non-selective mAChR orthosteric agonist, oxotremorine, was required to

show antipsychotic-like effects in rodents (Chan et al., 2008; Leach et al., 2010; Suratman

et al., 2011). This is due to the species variability of LY2033298, in that its potentiation of

the ACh response is greater at the human M4 mAChRs than at the rodent variant (Leach et

al., 2010; Suratman et al., 2011). Additionally, this is due to probe dependence, whereby,

at the mouse M4 mAChR, LY2033298 shows higher cooperativity with oxotremorine than

with ACh (Suratman et al., 2011). However, in the present study, donepezil was used in

place of oxotremorine as a co-treatment, because despite LY2033298 being functionally

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selective for M4 mAChRs in the presence of ACh, it can also bind to the M2 mAChR

subtype in the presence of oxotremorine and potentiate its response, again, due to probe

dependence (Valant et al., 2012).

The treatments of LY2033298 and/or donepezil showed a trend to reverse the disruption of

PPI induced by a D1 DR-selective agonist, R(+)-6-Br-APB, reaching significance at only

one prepulse intensity in C57Bl/6J mice. In Chapter 5, PPI experiments using M4+/+ mice

on a C57Bl/6NTac background also showed similar results, where the treatments showed

trend to reverse but do not reach significance. These modest effects could be due to the

experimental design, as the pharmacokinetics study of the drugs revealed that the brain

concentrations of LY2033298 and donepezil were already reduced by the start of the PPI

test. This is supported by the study using a structurally distinct M4 mAChR PAM,

VU0152100, where the PPI test was performed at the time corresponding to high

VU0152100 brain concentration, and the PAM treatment alone significantly reversed the

disruption of PPI induced by amphetamine (Byun et al., 2014). Future experiments with a

shorter LY2033298 pre-treatment period may increase the ability of LY2033298 to

significantly reverse R(+)-6-Br-APB-induced disruption of PPI.

In Chapter 5, M4-/- mice on a C57Bl/6NTac background were tested to determine the role

of M4 mAChRs in the modest effects of LY2033298 and donepezil treatments in reversing

the disruption of PPI induced by R(+)-6-Br-APB. The data was inconclusive, due to the

inability of R(+)-6-Br-APB to disrupt PPI in these mice, on account of a floor effect, as

M4-/- mice exhibited significantly decreased baseline PPI compared to M4

+/+ mice

(Swerdlow et al., 2000). As a result, we were unable to explore the role of M4 mAChR in

mediating the reversal effects of LY2033298 and donepezil using these mice.

Chapter 6

153

On the other hand, the combined treatment of LY2033298 and donepezil significantly

reversed the hyperlocomotor activity induced by R(+)-6-Br-APB in C57Bl/6J mice

(Chapter 4). This significance was maintained in M4+/+ mice on a C57Bl/6NTac

background (Chapter 5). Treatments of either LY2033298 or donepezil alone in these

mice did not induce a reversal of R(+)-6-Br-APB induced increase of LMA. However,

when administered in M4-/- mice on a C57Bl/6NTac background, all three treatments

(LY2033298, donepezil, and LY2033298 and donepezil combined) significantly reversed

the hyperlocomotor activity induced by the D1 DR-selective agonist (Chapter 5).

Additionally, the combined treatment of LY2033298 and donepezil significantly decreased

baseline LMA, which was not seen in M4+/+ mice. This supports the possibility that, in the

absence of M4 mAChRs, the effects of LY2033298 and endogenous ACh at other

receptors becomes apparent, potentially due to compensatory effects (Holschneider and

Shih, 2000).

As discussed in 5.4, donepezil prevents the breakdown of ACh, which can activate

mAChRs, as well as nAChRs. In the striatum, M1 mAChRs are found postsynaptically on

both the striatonigral and the striatopallidal MSNs (Levey et al., 1991; Weiner et al., 1990;

Yan et al., 2001), and activation of this receptor leads to increased neuronal excitation

(Kreitzer, 2009). In particular, it has been shown that activation of M1 mAChRs on the

striatopallidal MSNs increases its state transitions and excitability (Shen et al., 2007).

Activation of striatopallidal MSNs of the indirect pathway lead to a reduction in LMA and

movement initiation (Calabresi et al., 2014). Additionally, ACh can activate M2 mAChRs,

which are expressed presynaptically on cholinergic, GABAergic, glutamatergic and

dopaminergic neurons in the striatum, inhibiting the release of these neurotransmitters,

which may lead to a reduction of LMA (Bernard et al., 1992; Hersch et al., 1994; Hersch

and Levey, 1995; Yan and Surmeier, 1996). Alternatively, M2 mAChRs can also be

Chapter 6

154

activated by LY2033298 through the potentiation of an ACh metabolite, choline (Wootten

et al., 2012). Furthermore, nAChRs found presynaptically on dopaminergic, glutamatergic

and GABAergic neurons in the striatum, as well as those expressed in the ventral

tegmental area, can also be activated by ACh (Quik and Wonnacott, 2011). These nAChRs

have been shown to modulate dopaminergic activity (Faure et al., 2014), and can

potentially contribute to the reversal of R(+)-6-Br-APB-induced increase of LMA

mediated by LY2033298 and donepezil in M4-/- mice.

Taken together, this study demonstrated that the combined treatment of an M4 mAChR

PAM, LY2033298 and donepezil could reverse the disruption of PPI and increase in LMA

induced by a D1 DR-selective agonist. However, the specific role of M4 mAChRs in these

reversal effects could not be established, due to the possible compensatory effects

exhibited in M4-/- mice. Future experiments using mice with Cre/loxP-mediated conditional

M4 mAChR knockout only in D1 DR-expressing neurons can explore the role of this

subpopulation of M4 mAChR in mediating the reversal effects of LY2033298 and

donepezil (Dencker et al., 2011; Jeon et al., 2010). Improved understanding of the

mechanisms through which non-selective mAChR orthosteric agonists, such as

xanomeline, and M4 mAChR-selective PAMs mediate their antipsychotic-like effects, can

add to the current knowledge of the pathophysiology of schizophrenia and aid the

development of better therapeutics for this disorder.

Importantly, the results from this study highlighted several factors that should be taken

into account for future translational research using GPCR allosteric modulators. Allosteric

modulators should be profiled for their probe dependence and species variability properties

to avoid loss of efficacy in animals. Additionally, the potential modulation of endogenous

ligand metabolites by the allosteric modulators should also be explored. Prior to

administration of allosteric modulators into animals, the pharmacokinetic and

Chapter 6

155

pharmacodynamics properties of the ligands are needed to determine the optimum

timeframe for behavioural studies. Lastly, allosteric modulators should be quantified for

their allosteric parameters using the operational model of allosterism, as these parameters

may help predict in vivo effect of allosteric modulators using in vitro data. Future studies

can explore the potential relationship between affinity or efficacy cooperativities with

effects seen in an in vivo setting.

Appendix 1:

Chapter 3 Supporting Information

Appendix 1

157

Appendix 1.1: Parameters for functional interaction between M4 mAChR and

D2 DR ligands

Values were estimated from the three-parameter logistic equation (Chapter 3, Equation 1),

presented as mean ± S.E.M. n=3-5, except for ACh + LY2033298 (w/ DA 10 nM) interaction in

pERK1/2, where n=2. Baseline and Emax are the lower and upper plateaus of the concentration-

response curve, respectively, and were expressed as % forskolin-induced response (cAMP) or %

ACh 10 µM response (pERK1/2). EC50 is the molar concentration of the agonist required to

generate a 50% of the full response, and was expressed as µM. Statistical comparisons between

Emax, baseline and EC50 values were by one-way analysis of variance using a Tukey’s multiple

comparisons post-test. Statistical analysis for ACh + LY2033298 (w/ DA 10 nM) interaction in

pERK1/2 (n=2) was not performed.

Table 1: ACh + haloperidol interaction – cAMP

Parameter [Haloperidol] (M)

0 1e-11 1e-10 1e-9 1e-8

Emax 9.92 ± 6.60 12.87 ± 4.23 6.71 ± 8.10 7.99 ± 12.31 7.08 ± 9.71

Baseline 99.40 ± 2.56 95.52 ± 4.93 95.29 ± 7.70 96.00 ± 2.28 102.0 ± 7.6

EC50 0.80 ± 0.18 0.81 ± 0.24 0.70 ± 0.16 0.70 ± 0.25 0.62 ± 0.28

Table 2: ACh + haloperidol interaction – pERK1/2

Parameter [Haloperidol] (M)

0 1e-11 1e-10 1e-9 1e-8

Emax 102.8 ± 4.0 75.86 ± 5.85 78.42 ± 7.85 82.94 ± 10.20 75.11 ± 11.77

Baseline -2.88 ± 2.10 -6.57 ± 3.42 -4.72 ± 2.90 -5.13 ± 4.23 -3.83 ± 3.42

EC50 0.073 ± 0.007 0.096 ± 0.007 0.076 ± 0.007 0.095 ± 0.020 0.127 ± 0.008*

Asterisk indicates significant difference compared to haloperidol 0 M (one-way analysis of variance using a Tukey’s

multiple comparisons post-test, * p<0.05)

Table 3: DA + atropine interaction – cAMP

Parameter [Atropine] (M)

0 1e-12 1e-11 1e-10 1e-9 1e-8

Emax 11.11 ± 2.98 14.50 ± 2.06 13.74 ± 4.10 3.07 ± 5.57 15.91 ± 2.30 6.28 ± 7.82

Baseline 105.6 ± 2.4 110.0 ± 2.8 100.7 ± 4.2 106.0 ± 4.9 104.5 ± 5.8 104.8 ± 4.5

EC50 6.49 ± 2.65 7.38 ± 3.58 8.36 ± 3.39 8.03 ± 3.24 6.99 ± 4.03 6.99 ± 2.00

Appendix 1

158

Table 4: DA + atropine interaction – pERK1/2

Parameter [Atropine] (M)

0 1e-12 1e-11 1e-10 1e-9 1e-8

Emax 92.55 ± 11.71 79.32 ± 25.48 76.03 ± 7.77 88.04 ± 13.86 51.29 ± 14.92 60.76 ± 10.40

Baseline -4.62 ± 2.46 -11.29 ± 2.01 -11.93 ± 6.91 4.59 ± 11.85 -15.33 ± 7.26 -14.87 ± 14.80

EC50 0.76 ± 0.52 0.27 ± 0.11 0.18 ± 0.06 0.18 ± 0.08 0.18 ± 0.10 0.26 ± 0.10

Table 5: DA + LY2033298 interaction – cAMP

Parameter [LY2033298] (M)

0 3e-8 1e-7 3e-7 1e-6 3e-6

Emax 17.24 ± 3.19 10.11 ± 6.36 5.59 ± 10.69 13.96 ± 6.35 18.92 ± 6.02 23.12 ± 3.00

Baseline 102.7 ± 2.2 101.6 ± 3.0 99.03 ± 4.78 95.08 ± 7.82 98.01 ± 13.02 91.38 ± 14.75

EC50 2.50 ± 0.99 3.43 ± 1.52 4.22 ± 2.10 3.62 ± 1.93 3.06 ± 1.64 5.08 ± 2.20

Table 6: DA + LY2033298 interaction – pERK1/2


0 3e-8 1e-7 3e-7 1e-6 3e-6

Emax 67.60 ± 9.32 66.09 ± 8.35 66.45 ± 9.65 72.78 ± 10.37 71.78 ± 6.07 61.03 ± 4.21

Baseline -2.03 ± 2.83 -5.66 ± 3.00 -3.82 ± 3.75 -1.08 ± 4.14 5.88 ± 5.06 12.96 ± 5.93

EC50 0.24 ± 0.05 0.17 ± 0.05 0.16 ± 0.06 0.16 ± 0.02 0.08 ± 0.04 0.14 ± 0.07

Table 7: DA + LY2033298 (w/ ACh 10 nM) interaction – pERK1/2


0 (w/o ACh) 0 3e-8 1e-7 3e-7 1e-6

Emax 76.39 ± 4.84 95.65 ± 3.37 99.90 ± 9.11 91.53 ± 5.32 107.9 ± 13.4 105.5 ± 12.8

Baseline -0.94 ± 0.68 26.36 ± 12.26 34.34 ± 16.54 46.42 ± 19.27 74.96 ± 11.84† 87.51 ± 9.03††

EC50 0.17 ± 0.02 0.14 ± 0.05 0.07 ± 0.02 0.45 ± 0.37 0.04 ± 0.01 0.06 ± 0.05

Daggers indicate significant difference compared to LY2033298 0 M without ACh (one-way analysis of variance using a

Tukey’s multiple comparisons post-test, † p<0.05, †† p<0.01)

Table 8: ACh + LY2033298 (w/ DA 10 nM) interaction – pERK1/2


0 (w/o DA) 0 3e-8 1e-7 3e-7 1e-6

Emax 145.3 ± 12.1 179.7 ± 36.9 213.4 ± 1.0 180.0 ± 21.3 210.2 ± 29.5 257.0 ± 66.0

Baseline 0.88 ± 1.43 5.11 ± 4.57 10.81 ± 0.35 15.70 ± 9.02 33.36 ± 10.22 52.75 ± 15.89

EC50 0.48 ± 0.26 1.01 ±0.74 0.16 ± 0.05 0.20 ± 0.10 1.82 ± 1.79 0.02 ± 0.00

Appendix 2:


Appendix 2

160

Appendix 2.1: Effect of V1 + V2 + R(+)-6-Br-APB 0.1 – 1 mg/kg on acoustic

startle and PPI at 100 and 110 dB pulse intensities in C57Bl/6J mice

100 dB pulse intensity Startle amplitude

(arbitrary unit)

PPI (%)

pp6 pp12 pp18

V1+V2+V3 60.1 ± 13.1 14.3 ± 6.4 30.2 ± 6.9 42.0 ± 8.7

+Br-APB 0.1 40.8 ± 7.3 7.7 ± 13.3 31.5 ± 7.5 42.8 ± 7.8

+Br-APB 0.3 37.8 ± 6.3 -6.8 ± 9.7 5.0 ± 9.1 33.9 ± 4.8

+Br-APB 1 27.5 ± 5.6 10.3 ± 16.5 26.0 ± 9.4 35.4 ± 9.4


(arbitrary unit)

PPI (%)

pp6 pp12 pp18

V1+V2+V3 144.5 ± 25.8 16.5 ± 3.4 33.0 ± 5.7 44.5 ± 4.4

+Br-APB 0.1 101.4 ± 19.3 -0.8 ± 16.6 13.3 ± 12.5 38.0 ± 9.9

+Br-APB 0.3 128.4 ± 22.1 3.2 ± 7.6 20.6 ± 9.2 32.2 ± 7.4

+Br-APB 1 94.1 ± 17.8 7.4 ± 10.6 16.5 ± 5.6 51.5 ± 3.6

Data are presented as mean ± SEM; n=7-11. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9.

V2: 2% Tween 80 in saline. V3: water for injection. Br-APB: R(+)-6-Br-APB, dosage in mg/kg.

Appendix 2

161

Appendix 2.2: LMA after V1 + V2 + R(+)-6-Br-APB administration in

C57Bl/6J mice

Time

(min)

R(+)-6-Br-APB dosage (mg/kg)

0 (V3) 0.3 1 3

25 879 ± 119 1368 ± 122 **1652 ± 172 1313 ± 177

30 773 ± 138 **1992 ± 145 ***1858 ± 189 1070 ± 203

35 924 ± 181 *1802 ± 141 **2081 ± 76 1182 ± 272

40 785 ± 139 **2403 ± 309 ***2410 ± 214 *1718 ± 301

45 951 ± 115 *2127 ± 205 ***2454 ± 273 *1967 ± 286

50 1037 ± 152 *2074 ± 386 ***2504 ± 250 *2025 ± 202

55 937 ± 139 *2165 ± 589 **2158 ± 318 1779 ± 189

60 1049 ± 132 1687 ± 242 *2279 ± 411 1812 ± 226

65 953 ± 122 1568 ± 129 *2132 ± 381 1729 ± 302

70 1070 ± 154 1186 ± 98 *2161 ± 269 1781 ± 310

75 1239 ± 197 1322 ± 110 1975 ± 326 2011 ± 244

80 965 ± 151 1124 ± 183 *1756 ± 309 *1829 ± 144

Distance travelled every 5 minutes after R-6-Br-APB i.p. administration, presented as mean ± SEM;

n=4-8. Time refers to min after the V1 + V2 injections. Refer to 4.2.11.2 for statistical analysis. **

p < 0.01 and *** p < 0.001 vs V1+V2+V3 group (R(+)-6-Br-APB 0 mg/kg). V1: 10% DMSO/5%

Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection.

Appendix 2

162

Appendix 2.3: Comparison between saline + saline + V3 and V1 + V2 + V3

treatments on acoustic startle and PPI at 100 and 110 dB pulse intensities in

C57Bl/6J mice


(arbitrary unit)

PPI (%)

pp6 pp12 pp18

Saline+saline+V3 68.3 ± 15.7 22.7 ± 7.1 33.2 ± 5.8 51.1 ± 3.0

V1+V2+V3 68.8 ± 17.3 18.5 ± 7.2 34.9 ± 8.7 43.7 ± 11.3


(arbitrary unit)

PPI (%)

pp6 pp12 pp18

Saline+saline+V3 149.2 ± 21.5 27.9 ± 4.9 34.9 ± 6.9 50.4 ± 2.6

V1+V2+V3 160.7 ± 34.2 19.3 ± 2.4 37.5 ± 5.8 47.0 ± 5.9

Data are presented as mean ± SEM; n=8. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2:

2% Tween 80 in saline. V3: water for injection.

Appendix 2

163

Appendix 2.4: Comparison between saline + saline + V3 and V1 + V2 + V3

treatments on LMA over time in C57Bl/6J mice

Time (min) Saline+saline+V3 V1+V2+V3

25 740 ± 90 925 ± 206

30 838 ± 46 936 ± 149

35 799 ± 194 983 ± 292

40 1051 ± 203 899 ± 187

45 902 ± 213 1019 ± 171

50 1068 ± 251 1088 ± 199

55 1212 ± 193 983 ± 187

60 1265 ± 167 1145 ± 88

65 995 ± 244 1064 ± 178

70 1203 ± 92 1347 ± 180

75 1220 ± 281 1561 ± 268

80 1247 ± 298 1151 ± 234

Distance travelled every 5 minutes after vehicle i.p. administration, presented as mean ± SEM; n=4.

Time refers to min after the first and second injections. V1: 10% DMSO/5% Tween 80 in Tris

buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection.

Appendix 2

164

Appendix 2.5: Effect of LY2033298 treatment, with or without donepezil, on hyperlocomotor activity induced by R(+)-6-Br-

APB in C57Bl/6J mice

Drug treatments (1st, 2nd, 3rd injections)

1st V1 LY10 V1 V1 LY10 LY10 V1 LY10 V1 V1 LY10 LY10

2nd V2 V2 Don0.6 Don1 Don0.6 Don1 V2 V2 Don0.6 Don1 Don0.6 Don1

3rd V3 V3 V3 V3 V3 V3 Br-APB1 Br-APB1 Br-APB1 Br-APB1 Br-APB1 Br-APB1

Tim

e (m

in)

25 953

± 165

636

± 106

664

± 185

724

± 103

560

± 82

261

± 63

***1969

± 119

1550

± 156

†††717

± 171

†1136

± 190

†††465

± 68

†††743

± 221

30 1045

± 73

660

± 84

825

± 296

825

± 204

628

± 98

296

± 66

***2095

± 133

1726

± 142

†††934

± 253

†1234

± 302

†††533

± 128

†††773

± 186

35 1025

± 105

739

± 104

984

± 299

1003

± 257

794

± 144

427

± 113

**2134

± 200

1747

± 143

†1139

± 259

1337

± 350

†††720

± 212

†††887

± 180

40 949

± 130

917

± 113

928

± 229

991

± 264

811

± 151

392

± 81

***2324

± 244

***2360

± 141

1499

± 240

1705

± 357

†††960

± 269

†††992

± 182

45 1139

± 67

939

± 58

932

± 331

930

± 191

807

± 132

505

± 77

*2279

± 293

**2458

± 154

1782

± 190

2213

± 379

1290

± 369

1454

± 287

50 1075

± 62

846

± 123

1041

± 346

1009

± 190

808

± 110

504

± 125

**2348

± 369

***2962

± 290

1881

± 190

**2466

± 306

1495

± 352

1806

± 331

55 996

± 113

1146

± 77

989

± 260

1022

± 127

931

± 159

617

± 135

***2427

± 276

***2666

± 197

***2508

± 194

***2766

± 263

1616

± 297

**2171

± 314

60 1117

± 126

1074

± 131

1154

± 281

818

±187

975

± 126

634

± 145

2038

± 315

**2536

± 298

2136

± 101

**2597

± 292

1943

± 278

***2578

± 340

65 1056

± 95

1310

± 118

1013

± 231

946

± 223

1009

± 115

741

± 161

1668

± 188

**2407

± 247

**2403

± 289

**2379

± 290

*2007

± 214

***2603

± 351

70 1050

± 100

1102

± 180

1161

± 294

945

± 131

975

± 167

851

± 148

1651

± 233

1897

± 262

1925

± 327

*2058

± 194

*1937

± 187

***2352

± 229

75 1062

± 113

1174

± 112

1104

± 243

1082

± 214

945

± 131

678

± 142

1588

± 139

*2032

± 250

1592

± 252

1802

± 317

1714

± 223

*1966

± 204

80 1111

± 91

1079

± 89

1256

± 280

921

± 203

985

± 119

673

± 151

1526

± 258

1859

± 291

1169

± 227

1452

± 264

1769

± 233

1785

± 140

Appendix 2

165

Appendix 2.5 (continue)

Distance travelled every 5 minutes post 3rd i.p. injection, presented as mean ± SEM; n=6-13. Time

refers to min after the 1st and 2nd injections. Refer to 4.2.11.2 for statistical analysis. * p < 0.05,

** p < 0.01 and *** p < 0.001 vs V1+V2+V3 group. † p < 0.05 and ††† p < 0.001 vs V1+V2+Br-

APB 1 group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline.

V3: water for injection. LY10: LY2033298 10 mg/kg; Don1: donepezil 1 mg/kg; Br-APB1: R(+)-

6-Br-APB 1mg/kg.

Appendix 2

166

Appendix 2.6: Snake plot of the mouse M4 mAChR, with residues different

from the human receptor highlighted in red

Obtained from GPCR database (http://gpcrdb.org/).

Appendix 3:


Appendix 3

168

Appendix 3.1: Effect of re-testing on LMA in C57Bl/6NTac M4-/- mice

Time (min) V1+V2+V3 first test V1+V2+V3 re-test

25 862 ± 110 *1364 ± 180

30 683 ± 72 *1065 ± 205

35 1216 ± 169 1283 ± 98

40 1305 ± 125 1080 ± 59

45 967 ± 113 1169 ± 75

50 1023 ± 112 1048 ± 237

55 876 ± 113 1122 ± 287

60 744 ± 100 *1349 ± 188

65 864 ± 144 1211 ± 56

70 890 ± 81 877 ± 144

75 757 ± 86 **1338 ± 84

80 764 ± 80 **1291 ± 140

Distance travelled every 5 min post V3 i.p. administration, presented as mean ± SEM; n=3-8. Time

refers to min post 1st and 2nd injections. Refer to 5.2.5 for statistical analysis. * p < 0.05 and ** p <

0.01 vs V1+V2+V3 first test group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2%

Tween 80 in saline. V3: water for injection.

Appendix 3

169

Appendix 3.2: Comparison of PPI at 100, 110 and 120 dB pulse intensities

between V1 + V2 + V3 treated M4+/+ and M4

-/- mice on a C57Bl/6NTac

background

100 dB pulse intensity PPI (%)

pp6 pp12 pp18

V1+V2+V3 M4+/+ mice 45.14 ± 4.05 68.61 ± 2.97 83.20 ± 2.21

V1+V2+V3 M4-/- mice **24.42 ± 6.00 **44.55 ± 6.63 **69.00 ± 4.52


pp6 pp12 pp18

V1+V2+V3 M4+/+ mice 41.99 ± 4.45 60.49 ± 2.29 73.72 ± 2.23

V1+V2+V3 M4-/- mice **10.01 ± 7.98 ***30.84 ± 5.01 ***56.20 ± 2.34


pp6 pp12 pp18

V1+V2+V3 M4+/+ mice 32.58 ± 4.07 49.77 ± 3.66 68.18 ± 3.52

V1+V2+V3 M4-/- mice *12.25 ± 7.39 *35.40 ± 5.67 *51.40 ± 5.19


V2: 2% Tween 80 in saline. V3: water for injection. Refer to 5.2.5 for statistical analysis. * p <

0.05, ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 M4+/+ group

Appendix 3

170

Appendix 3.3: Comparison of baseline LMA between V1+V2+V3 treated M4+/+

and M4-/- mice

Time (min) V1+V2+V3 M4+/+ V1+V2+V3 M4

-/-

-25 1710 ± 152 ***2562 ± 72

-20 1547 ± 86 ***2309 ± 108

-15 1468 ± 127 ***2193 ± 87

-10 1445 ± 73 ***2049 ± 109

-5 1448 ± 105 *1865 ± 146

0 1309 ± 100 ***2023 ± 115

25 713 ± 66 999 ± 114

30 633 ± 72 787 ± 89

35 712 ± 68 **1234 ± 123

40 779 ± 72 **1244 ± 96

45 682 ± 61 **1022 ± 87

50 774 ± 70 1030 ± 97

55 593 ± 85 *943 ± 110

60 646 ± 94 909 ± 120

65 726 ± 118 959 ± 115

70 807 ± 140 887 ± 67

75 757 ± 119 915 ± 104

80 642 ± 82 908 ± 99

Distance travelled every 5 min for 30 min pre V1+V2 i.p. administration, presented as mean ±

SEM; n=9-11. Time refers to min relative to the time V1+V2 injections were administered. Refer

to 5.2.5 for statistical analysis. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 M4+/+

group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water

for injection.

Appendix 3

171


disruption of PPI induced by R(+)-6-Br-APB in M4+/+ and M4

-/- mice on a

C57Bl/6NTac background at 120 dB pulse intensity

M4+/+ mice

Drug treatments Startle amplitude

(arbitrary unit)

PPI (%)

1st 2nd 3rd pp6 pp12 pp18

V1 V2 V3 155.08 ± 13.19 32.58 ± 4.07 49.77 ± 3.66 68.18 ± 3.52

LY10 V2 V3 139.70 ± 21.02 27.81 ± 7.17 58.97 ± 4.04 66.18 ± 5.55

V1 Don1 V3 102.96 ± 12.98 39.71 ± 6.58 59.83 ± 3.72 71.20 ± 2.04

LY10 Don1 V3 176.80 ± 17.74 22.03 ± 4.87 48.47 ± 4.50 65.27 ± 4.08

V1 V2 Br-APB0.3 144.58 ± 24.97 9.61 ± 8.82 24.83 ± 6.34 52.72 ± 4.62

LY10 V2 Br-APB0.3 117.33 ± 16.62 17.88 ± 7.17 41.66 ± 6.12 51.13 ± 5.67

V1 Don1 Br-APB0.3 161.16 ± 19.06 15.38 ± 4.03 42.42 ± 4.03 55.08 ± 4.66

LY10 Don1 Br-APB0.3 162.38 ± 19.05 17.11 ± 7.09 35.26 ± 7.05 57.92 ± 4.29

M4-/- mice

Drug treatments Startle amplitude

(arbitrary unit)

PPI (%)

1st 2nd 3rd pp6 pp12 pp18

V1 V2 V3 130.14 ± 12.96 12.25 ± 7.39 35.40 ± 5.67 51.40 ± 5.19

LY10 V2 V3 229.17 ± 48.05 28.90 ± 14.84 52.67 ± 3.62 67.43 ± 10.16

V1 Don1 V3 175.57 ± 30.57 28.87 ± 5.89 56.57 ± 7.97 60.67 ± 8.33

LY10 Don1 V3 145.28 ± 32.60 9.16 ± 22.10 34.40 ± 7.10 52.98 ± 6.52

V1 V2 Br-APB0.3 124.94 ± 11.34 0.94 ± 4.82 14.52 ± 7.44 33.01 ± 6.99

LY10 V2 Br-APB0.3 124.55 ± 26.06 -28.55 ± 24.22 21.08 ± 10.29 53.48 ± 13.59

V1 Don1 Br-APB0.3 146.45 ± 19.72 0.80 ± 13.56 29.32 ± 11.87 46.10 ± 8.63

LY10 Don1 Br-APB0.3 97.58 ± 17.12 3.69 ± 15.99 18.25 ± 12.74 47.94 ± 5.39


V2: 2% Tween 80 in saline. V3: water for injection. LY10: LY2033298 10 mg/kg; Don1:

donepezil 1 mg/kg; Br-APB0.3: R(+)-6-Br-APB 0.3 mg/kg.

Appendix 3

172

Appendix 3.5: LMA post R(+)-6-Br-APB administration in C57Bl/6NTac M4+/+

mice

Time

(min)

R(+)-6-Br-APB dosage (mg/kg)

0 0.3 0.6 1

25 692 ± 119 802 ± 69 *1318 ± 178 **1578 ± 181

30 601 ± 105 1106 ± 132 **1510 ± 60 ***1683 ± 230

35 764 ± 96 1107 ± 123 **1467 ± 94 **1629 ± 207

40 773 ± 130 1287 ± 205 *1541 ± 82 1346 ± 234

45 662 ± 82 1189 ± 288 **1775 ± 81 1041 ± 245

50 667 ± 84 1180 ± 253 **1753 ± 128 1207 ± 234

55 530 ± 138 *1379 ± 95 ***1723 ± 102 **1425 ± 231

60 628 ± 157 *1518 ± 47 *1451 ± 208 **1833 ± 207

65 670 ± 155 1041 ± 304 1546 ± 275 *1712 ± 312

70 851 ± 248 913 ± 441 1383 ± 207 1798 ± 295

75 763 ± 196 1150 ± 216 *1759 ± 267 *1764 ± 230

80 654 ± 146 *1404 ± 163 1158 ± 153 ***1661 ± 125

Distance travelled every 5 min post R-6-Br-APB i.p. administration, presented as mean ± SEM;

n=3-6. Time refers to min post 1st and 2nd injections. Refer to 5.2.5 for statistical analysis. * p <

0.05, ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 group (R(+)-6-Br-APB 0 mg/kg).

Appendix 3

173


APB in C57Bl/6NTac M4+/+ mice


1st V1 LY10 V1 LY10 V1 LY10 V1 LY10

2nd V2 V2 Don0.6 Don0.6 V2 V2 Don0.6 Don0.6

3rd V3 V3 V3 V3 Br-APB0.6 Br-APB0.6 Br-APB0.6 Br-APB0.6

Tim

e (m

in)

25 713 ± 66 408 ± 68 477 ± 103 518 ± 65 1201 ± 103 *1306 ± 155 *1328 ± 140 ††466 ± 63

30 633 ± 72 569 ± 117 508 ± 104 565 ± 68 ***1496 ± 47 ***1454 ± 113 ***1448 ± 140 †††663 ± 81

35 712 ± 68 565 ± 100 519 ± 90 525 ± 87 **1402 ± 60 ***1522 ± 128 **1458 ± 136 †††703 ± 81

40 779 ± 72 622 ± 84 722 ± 92 763 ± 71 ***1601 ± 91 ***1633 ± 154 1303 ± 152 †††760 ± 90

45 682 ± 61 648 ± 130 636 ± 91 817 ± 86 ***1707 ± 112 ***1561 ± 102 **1392 ± 159 †††883 ± 109

50 774 ± 70 513 ± 111 531 ± 98 901 ± 77 ***1698 ± 104 ***1715 ± 120 1362 ± 145 ††977 ± 123

55 593 ± 85 486 ± 103 522 ± 85 728 ± 93 ***1750 ± 73 ***1813 ± 155 ***1707 ± 210 ††977 ± 120

60 646 ± 94 587 ± 115 727 ± 91 670 ± 78 ***1671 ± 119 ***1937 ± 153 ***1686 ± 185 1033 ± 117

65 726 ± 118 509 ± 111 581 ± 111 754 ± 101 ***1727 ± 168 **1642 ± 182 **1643 ± 194 †1010 ± 120

70 807 ± 139 475 ± 99 575 ± 102 906 ± 79 *1551 ± 124 *1584 ± 200 *1582 ± 213 1050 ± 106

75 757 ± 119 582 ± 115 483 ± 84 828 ± 89 **1566 ± 145 1419 ± 116 1480 ± 227 †844 ± 129

80 642 ± 82 485 ± 49 701 ± 80 792 ± 88 1251 ± 115 *1388 ± 150 **1565 ± 217 898 ± 149

Distance travelled every 5 min post 3rd i.p. injection, presented as mean ± SEM; n=7-13. Time refers to min post 1st and 2nd injections. Refer to 5.2.5 for

statistical analysis. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 M4+/+ group. † p < 0.05, †† p < 0.01 and ††† p < 0.001 vs V1+V2+Br-APB 0.6

M4+/+ group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection. LY10: LY2033298 10 mg/kg; Don0.6:

donepezil 0.6 mg/kg; Br-APB0.6: R(+)-6-Br-APB 0.6 mg/kg.

Appendix 3

174


APB in C57Bl/6NTac M4-/- mice


1st V1 LY10 V1 LY10 V1 LY10 V1 LY10

2nd V2 V2 Don0.6 Don0.6 V2 V2 Don0.6 Don0.6

3rd V3 V3 V3 V3 Br-APB0.6 Br-APB0.6 Br-APB0.6 Br-APB0.6

Tim

e (m

in)

25 999 ± 114 784 ± 103 853 ± 130 393 ± 52 1257 ± 152 1052 ± 280 1085 ± 114 673 ± 120

30 787 ± 89 714 ± 170 665 ± 105 373 ± 41 ***1711 ± 124 1284 ± 215 †1088 ± 149 †††945 ± 163

35 1234 ± 123 865 ± 108 795 ± 122 **448 ± 76 1673 ± 131 1120 ± 209 ††946 ± 111 ††949 ± 145

40 1244 ± 96 866 ± 76 *644 ± 88 *560 ± 113 1706 ± 135 †1073 ± 203 †1026 ± 143 ††984 ± 158

45 1022 ± 87 881 ± 77 690 ± 87 569 ± 83 ***1823 ± 161 1281 ± 188 †1137 ± 181 †1139 ± 199

50 1030 ± 97 678 ± 163 680 ± 70 570 ± 114 ***1797 ± 142 1171 ± 174 1262 ± 207 1208 ± 169

55 943 ± 110 748 ± 191 613 ± 79 568 ± 66 ***1892 ± 185 1441 ± 265 1411 ± 241 1393 ± 156

60 909 ± 120 887 ± 102 577 ± 113 444 ± 84 ***2030 ± 206 1528 ± 240 1418 ± 205 1433 ± 172

65 959 ± 115 900 ± 98 772 ± 99 545 ± 112 ***1866 ± 171 1695 ± 217 *1793 ± 149 1382 ± 148

70 887 ± 67 764 ± 88 710 ± 92 601 ± 83 ***1915 ± 143 1582 ± 242 *1785 ± 227 1461 ± 240

75 915 ± 104 783 ± 132 666 ± 104 407 ± 66 ***2116 ± 134 **1845 ± 272 1563 ± 300 1428 ± 195

80 908 ± 99 694 ± 137 735 ± 90 450 ± 55 ***2021 ± 165 **1870 ± 214 **1797 ± 191 †1306 ± 238

Distance travelled every 5 minutes post 3rd i.p. injection, presented as mean ± SEM; n=6-12. Time refers to min post 1st and 2nd injections. Refer to 5.2.5 for

statistical analysis. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs V1+V2+V3 M4-/- group. † p < 0.05, †† p < 0.01 and ††† p < 0.001 vs V1+V2+Br-APB 0.6 M4

-/-

group. V1: 10% DMSO/5% Tween 80 in Tris buffer pH 8.9. V2: 2% Tween 80 in saline. V3: water for injection. LY10: LY2033298 10 mg/kg; Don0.6:

donepezil 0.6 mg/kg; Br-APB0.6: R(+)-6-Br-APB 0.6 mg/kg.

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