Discovery and Development of Selective M1 Agonists that Protect Against Alzheimer's Pathology and Bias Hippocampal Circuitry Dynamics in Rodents Open Access
Lebois, Evan P. (2014)
Abstract
M1 muscarinic receptors are heavily expressed in brain areas important for memory (e.g. the hippocampus) and are known to both modulate hippocampal memory circuits and regulate amyloid pathology in AD mice. M1-/- mice develop memory impairments and high Aβ levels in their brain, highlighting a potentially disease-modifying role of M1 receptors in AD. Although acute improvements in memory and pathology have been shown in AD mice with M1 activators, it remains unclear whether chronically activating M1 can lead to persistent disease-modifying effects that prevent memory impairments and neuropathology from forming. Thus, the present work used chronic dosing of the selective M1 agonist, VU0364572, to 5X FAD mice from an age prior to appreciable amyloid pathology (2 months) to an age where these mice display memory impairments (6 months). M1 activation was found to both prevent mice from becoming memory impaired as well as significantly reduce levels of soluble and insoluble Aβ40,42 in the cortex and hippocampus of these animals.
Although M1 modulates hippocampal circuitry, how M1 biases circuits in vivo to improve memory remains unclear. Pharmacological work indicates M1 activation improves spatial memory, and in vitro studies with M1-/- mice have indicated M1 activation suppresses CA3-CA1 transmission. However, the effects of M1 activation on spatial representations and CA3-CA1 functional connectivity remains unclear. In vivo tetrode recording from dCA3 and dCA1 was used to obtain spiking and local field potential data in adult rats during open field exploration in a morph box. The M1 agonist, VU0364572, was used to examine the impact of M1 activation on hippocampal place fields and CA3-CA1 synchrony. Consistent with prior findings, the present results indicated that M1 activation decreased CA3-CA1 functional connectivity yet benefitted spatial representations. Specifically, M1 activation decreased CA3-CA1 spike-field synchrony in the theta range and the temporal organization of CA3 spikes to CA1. However, M1 activation also increased responsiveness of hippocampal place fields to changes in the shape of the recording enclosure. One possible interpretation is that less coherent CA3-CA1 synaptic transmission might act to prioritize current information coming from entorhinal cortex.
Table of Contents
TABLE OF CONTENTS
Chapter I: INTRODUCTION............................................................................................................1
Alzheimer's Disease: Overview and
Significance..................................................................................1
The Pathophysiology of Alzheimer's
Disease........................................................................................4
APP Processing and the Amyloid Cascade Hypothesis............................................................................9
Aβ-Induction of Synaptic Dysfunction.................................................................................................11
Cholinergic Neurotransmission and its Role in Alzheimer's
Disease..........................................................12
Neuroanatomy of the Cholinergic
System...........................................................................................12
Muscarinic Acetylcholine Receptor Expression and
Function....................................................................14
Alzheimer's Disease
Treatment.........................................................................................................21
Currently Approved Therapeutics and Limitations..................................................................................21
Therapeutic Strategies Under Investigation..........................................................................................23
Pro-muscarinic strategies under therapeutic development for AD.............................................................31
M1 Allosteric Agonist Development for Alzheimer's Disease....................................................................35
Animal Models of Alzheimer's
Disease................................................................................................44
The Hippocampal Memory
System.....................................................................................................49
Functional Neuroanatomy and Overview..............................................................................................49
Hippocampal Place Cells: Properties and Relevance to Memory...............................................................56
Place Field Properties.......................................................................................................................56
Place Cell Remapping.......................................................................................................................59
Local Field Potential Oscillations and Relevance to
Memory.....................................................................63
Proposed
Research..........................................................................................................................76
Chapter II: MATERIALS AND METHODS............................................................................................79
Subjects for Transgenic AD Mouse Studies...........................................................................................79
Animal Perfusion.............................................................................................................................79
Biochemical Tissue Fractionation........................................................................................................80
Behavioral Testing...........................................................................................................................82
Morris Water Maze...........................................................................................................................82
Cued and Contextual Fear Conditioning................................................................................................83
Immunohistochemistry.....................................................................................................................84Immunohistochemical Quantification...................................................................................................85
Aβ40 and Aβ42 ELISA........................................................................................................................85
Drugs for Transgenic AD Mouse Studies...............................................................................................86
Determination of CNS VU572 Concentrations for Chronic Dosing...............................................................86
Primary Neuron Culture....................................................................................................................87
Statistical Analyses..........................................................................................................................88 Drugs for In Vivo Electrophysiology Studies..........................................................................................89 Subjects for In Vivo Electrophysiology Studies.......................................................................................90Morph Box Open Field Exploration Task................................................................................................90
Surgeries and Positioning of Recording Electrodes..................................................................................91
In Vivo Electrophysiology Data Acquisition............................................................................................95
In Vivo Electrophysiology Data Analyses...............................................................................................95
Place Field Analyses and Spatial Discrimination Scores............................................................................95
Spatial Information Analyses..............................................................................................................96
Relation of Local Field Potentials to Behavior.........................................................................................97
Analyses of Hippocampal Synchrony....................................................................................................97
Statistical Analyses...........................................................................................................................99
Electrolytic Lesioning.........................................................................................................................99
Chapter III: IN VITRO AND IN VIVO EFFECTS OF SELECTIVE M1 AGONISTS ON NEUROPATHOLOGY
AND BEHAVIORAL DEFICITS IN A CHRONIC, PREVENTATIVE TREATMENT TRIAL OF TRANSGENIC
AD MICE………...............................................................................................................................101
Introduction…………………………………………………………………….........................................................................101 Results…………………………………………………………………………...........................................................................103M1 Activation by VU572 Lowers Aβ40 and Aβ42 Levels In Vitro in Mouse Primary Cortical and Hippocampal
Neurons.......................................................................................................................................103
M1 Activation by VU572 Preserves Hippocampal Memory When Dosed Chronically in 5X FAD Transgenic
Alzheimer's Mice............................................................................................................................105
VU572 Decreases Soluble and Insoluble Aβ40 and Aβ42 Levels in Cortex and Hippocampus of 5X FAD Mice.....113
M1 Activation by VU572 Treatment Abolishes Soluble Aβ42 Correlation with Memory Impairment..................114
M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Hippocampus and cortex of 5X FAD Mice..........119
Effect of M1 Activation by VU572 on Contextual and Cued Fear Conditioning..............................................122
Discussion.....................................................................................................................................135Chapter IV: EFFECTS OF SELECTIVE M1 ACTIVATION ON HIPPOCAMPAL CIRCUITRY DYNAMICS...........141
Introduction...................................................................................................................................141 Results..........................................................................................................................................143Place Cells Code Contextual Change in the Proposed Morph Box Test Paradigm..........................................143
M1 Activation by VU572 Increases Spatial Discrimination of Hippocampal Place Cells..................................147
M1 Activation by VU572 Does Not Impair Spatial Information of CA3 or CA1 Place Cells, but Activation by the
M1 PAM BQCA Does.........................................................................................................................153
M1 Activation by VU572 Suppresses CA3-CA1 Field-Field Coherence.........................................................156
M1 Activation by VU572 Suppresses CA3 Spike-CA1 Field Coherence........................................................156
M1 Activation by VU572 Renders CA3 Spikes to CA1 Less Coherent..........................................................162
Discussion.....................................................................................................................................165Chapter V: SUMMARY AND FUTURE DIRECTIONS..............................................................................173
DECLARATION OF CONFLICTS OF INTEREST.....................................................................................182
REFERENCES.................................................................................................................................183
LIST OF FIGURES
1.1 Alzheimer's Disease Pathology........................................................................................................31.2 Pathological Cascade of Alzheimer's Disease......................................................................................5
1.3 Amyloid Cascade Hypothesis of AD: Amyloid Buildup is Responsible for AD.............................................7
1.4 Cholinergic Hypothesis of AD: Decreased Acetylcholine is Responsible for AD.........................................14
1.5 Muscarinic Receptor Function and Expression in the CNS.....................................................................16
1.6 Current Therapy for AD Patients.....................................................................................................22 1.7 Novel Therapeutic Strategies for AD Patients....................................................................................251.8 M1 Muscarinic Receptors Bias APP Toward Non-Amyloidogenic Processing..............................................29
1.9 M1 Agonist Discovery and Characterization.......................................................................................38
1.10 M1 Agonist Optimization for In Vivo Studies....................................................................................39
1.11 M1 Activation by VU572 Robustly Impacts Hippocampal Synaptic Plasticity............................................42
1.12 M1 Allosteric Agonists Do Not Robustly Desensitize M1 Receptor Signaling............................................43
1.13 Animal Models of AD: The 5X FAD Mouse is a Robust Animal Model of AD.............................................47
1.14 The Hippocampus Combines Spatial and Nonspatial Inputs in Order to Form Episodic Memories................51
1.15 Hippocampal Place Cells Form Place-Specific Firing Fields..................................................................57
1.16 Place Cells Form Context-Specific Representations............................................................................61
1.17 Oscillations Lay the Foundation for Spike-Timing Dependent Plasticity: They Serve to Turn Many Small
Signals Into a Big Signal that Can Elicit Target Neuron Discharge ..............................................................65
1.18 The Mammalian CNS Has Many Different Neural Oscillators Whose Frequencies Subserve Different Memory
Processes.........................................................................................................................................66
1.19 Intra- Versus Extra-Hippocampal Microcircuits Contribute to Different Aspects of Information Encoding,
Where Intrahippocampal Connectivity is Modulated by Muscarinic Recepors..................................................69
1.20 CA3 and CA1 Hippocampal Subfields Show Theta-Modulated Gamma Activity That Serves to Organize
Pyramidal Cell Spiking During Behavior..................................................................................................70
1.21 Coherence is a Measure of Functional Connectivity............................................................................72
1.22 Coherence Reflects How Much Two LFPs Co-Vary in Time....................................................................73
1.23 Coherograms are a Way of Simultaneously Depicting Coherence Across Many Frequency Bands of Interest..74
2.1 Terminal Pathology Processing.........................................................................................................812.2 32-Tetrode Microdrive Permits High-Density Neuronal Recording From the Rodent Hippocampus..................92
2.3 Tetrode Recordings Allow for Optimum Single Unit Isolation..................................................................93
3.1 M1 Activation by VU572 Lowers Aβ40/42 Levels in Mouse Primary Cortical and Hippocampal Neurons...........104
3.2 M1 Agonist VU572 Preventative Treatment Trial in 5X FAD Mice.............................................................107
3.3 M1 Activation by VU572 Preserves Hippocampal Memory When Dosed Chronically in 5X FAD Transgenic
Alzheimer's Mice................................................................................................................................111
3.4 M1 Activation by VU572 Does Not Influence Morris Water Maze Performance During Training......................112
3.5 M1 Activation by VU572 Decreases Soluble Aβ40 and Aβ42 Levels in Cortex and Hippocampus of 5X FAD
Mice.................................................................................................................................................115
3.6 M1 Activation by VU572 Decreases Insoluble Aβ40 and Aβ42 Levels in Cortex and Hippocampus of 5X FAD
Mice.................................................................................................................................................116
3.7 M1 Activation by VU572 Treatment Abolishes Soluble Aβ42 Correlation with Memory Impairment.................118
3.8 Insoluble Aβ42 Levels Are Not Correlated with Memory.........................................................................120
3.9 M1 Activation by VU572 Does Not Impact Soluble or Insoluble Aβ40/42 Ratios in the Cortex or Hippocampus
of 5X FAD Mice...................................................................................................................................121
3.10 M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Hippocampus of 5X FAD Mice.......................123
3.11 M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Cortex of 5X FAD Mice................................125
3.12 M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Brains of 5X FAD Mice.................................126
3.13 M1 Activation by VU572 Reduces Aβ42 Neuropathology in the Brains of 5X FAD Mice.................................128
3.14 M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Brains of 5X FAD Mice.................................130
3.15 M1 Activation by VU572 Reduces Aβ40 Neuropathology in the Brains of 5X FAD Mice.................................131
3.16 M1 Activation by VU572 Does not Rescue Deficits in Contextual Fear Conditioning in 5X FAD Mice................133
3.17 Cued Fear Conditioning is Not Impacted by 5X FAD Genotype or M1 Activation by VU572 Treatment in 5X
FAD Mice............................................................................................................................................134
4.1 Morph Box Test Paradigm to Manipulate Hippocampal Place Field Activity.................................................144
4.2 Place Cells Code Contextual Change in the Proposed Morph Box Test Paradigm..........................................145
4.3 Representative Place Cell Examples From Experimental Drug Sessions.....................................................149
4.4 M1 Activation by VU572 Increases Spatial Discrimination of Hippocampal Place Cells..................................151
4.5 M1 Activation by VU572 Does Not Impair Spatial Information of CA3 or CA1 Place Cells, but Activation by
the M1 PAM BQCA Does.........................................................................................................................154
4.6 M1 Activation by VU572 Suppresses CA3-CA1 Field-Field Coherence.........................................................157
4.7 M1 Activation by VU572 Suppresses CA3 Spike-CA1 Field Coherence........................................................160
4.8 M1 Activation by VU572 Renders CA3 Spikes to CA1 Less Coherent..........................................................163
4.9 Under Normal Circumstances CA1 Listens Equally to Either CA3 or EC......................................................168
4.10 The Suppression of CA3-CA1 Circuitry by M1 Agonists May Bias CA1 Towards Synchronizing More with
Other Partners Such as EC to Drive Novel Information Encoding...................................................................169
5.1 Cholinergic Dysfunction is Central to Age-Associated Alterations in Hippocampal Function and May Be a Key
Contributor to Age-Related Memory Impairment........................................................................................176
5.2 Aging Detrimentally Impacts the Ability of Place Cells to Represent Space and Biases the Hippocampus
Towards Pattern Completion...................................................................................................................177
5.3 Mouse Models of AD Show Disrupted Place Fields and Decreased Spatial Information Content of Place Cells....179
5.4 M1 Agonists For the Treatment of Memory Dysfunction...........................................................................181
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