Synaptic and Cellular Evaluation of NMDA Receptors in Health and Disease Restricted; Files Only
Camp, Chad (Spring 2023)
Published
Abstract
N-methyl-D-aspartate receptors are glutamate-gated, calcium-permeable ion channels involved in a host of normal brain functions, including neuronal development. To non-specialists, NMDARs are important neurotransmitter receptors involved in synaptic plasticity, mainly long-term potentiation. In this dissertation, however, expanded roles of NMDARs are described that have come to light thanks to recent technological advances in genetic sequencing, transgenic mouse model production, and small molecule drug design. The overarching goals of the work presented in this thesis are two-fold: 1) to provide cellular and synaptic evaluation of mouse models based on NMDAR human patient-derived variants and 2) show efficacy of subunit-selective NMDAR modulation in altering circuit function for potential therapeutic gain. In Chapter 1, various roles of NMDARs are reviewed and intended to highlight the true omnipresent nature of these receptors within the central nervous system. In Chapters 2 and 3, mouse models of human patient-derived NMDAR variants are used to explore the pathophysiology of disease at the cellular and circuit levels using patch-clamp electrophysiology and immunohistochemical techniques. Chapter 3 also highlights the importance of viewing animal model age as a potent experimental variable while providing novel supporting data for GluN2A’s role in neurodevelopment and circuit maturation. In Chapter 4, a novel positive allosteric modulator is used to provide functional data on synaptic NMDAR subunit expression in groups of relatively understudied GABAergic interneurons. Overall, the work presented in this thesis is aimed at pushing the field of precision medicine forward via detailed cellular and synaptic analyses of mouse models of human disease, combined with novel subunit-selective NMDAR modulators.
Table of Contents
TABLE OF CONTENTS
CHAPTER 1: NMDA Receptors 101 – Structure, Function, Kinetics, Pharmacology, Expression, Roles in Normal Brain Function, and Genetic Variation. 1
1.1 Glutamatergic Neurotransmitter Receptors. 2
1.2 NMDAR Subunit Stoichiometry and Structure. 3
1.3 NMDAR Activation and Deactivation in Heterologous Expression Systems. 7
1.4 Synaptic NMDARs: Activation and Deactivation. 8
1.5 Synaptic NMDARs: Spatiotemporal Expression Patterns. 14
1.6 Synaptic NMDARs: Roles in Neurogenesis and Neuronal Migration. 18
1.7 Synaptic NMDARs: Roles in Neurite Development 19
1.8 Synaptic NMDARs: Roles in Synapse Formation. 20
1.9 Roles of NMDARs in Presynaptic Signaling. 21
1.10 Roles of NMDARs in Postsynaptic Signaling. 22
1.11 Roles of NMDARs in Extrasynaptic Signaling. 23
1.12 Roles of NMDARs in Glial Cells. 26
1.13 Roles of NMDARs in Synaptic Plasticity. 27
1.14 NMDARs, Genetic Variation, and Disease. 35
1.15 NMDAR Variants and Models of Disease. 43
1.16 Conclusions. 45
1.17 References for Chapter 1. 47
CHAPTER 2: Mouse Models of Gain-of-Function NMDAR Variants. 77
2.1 Introduction. 78
2.2 Methods. 80
2.2.1 Mouse Model Generation. 80
2.2.2 Electroconvulsive Threshold. 81
2.2.3 Acute Hippocampal Slice Preparation and Electrophysiological Recordings. 81
2.2.4 Statistical Analysis and Figure Preparation. 83
2.3 Results. 84
2.4 Discussion. 101
2.5 References for Chapter 2. 107
CHAPTER 3: GRIN2A Null Variants and Parvalbumin-Positive Interneuron Maturation. 110
3.1 Introduction. 111
3.2 Methods. 113
3.2.1 Animals and Breeding. 113
3.2.2 Human Patient Data. 115
3.2.3 Acute Hippocampal Slice Preparation and Electrophysiological Recordings. 115
3.2.4 Interneuron Anatomical Reconstructions. 118
3.2.5 Immunohistochemistry for GABAergic Interneuron Markers. 119
3.2.6 Image Acquisition and Analysis. 120
3.2.7 Statistical Analysis and Figure Preparation. 120
3.3 Results. 121
3.3.1 Null GRIN2A Variants May Have a Transient Seizure Burden. 121
3.3.2 Developing Hippocampus Shows Hyperexcitability in Grin2a+/- and Grin2a-/- Mice. 121
3.3.3 Alterations in Hippocampal PV Cell Density. 125
3.3.4 Age-Dependent Changes in Passive and Action-Potential Firing Properties of CA1 PV Cells. 126
3.3.5 Electrophysiological Maturation of PV Cells is Delayed in Grin2a+/- and Grin2a-/- Mice. 132
3.4 Discussion. 139
3.5 References for Chapter 3. 143
CHAPTER 4: Subunit-Specific Modulation of NMDARs for Therapeutic Gain. 163
4.1 Introduction. 164
4.2 Methods. 167
4.2.1 Animals and Breeding. 167
4.2.2 Acute Hippocampal Slice Preparation and Electrophysiological Recordings. 167
4.2.3 Cell Selection and Post-Hoc Slice Processing. 169
4.2.4 Statistical Analysis and Figure Preparation. 171
4.3 Results. 171
4.4 Discussion. 191
4.5 References for Chapter 4. 194
CHAPTER 5: Conclusions, Discussion, and Future Directions. 230
5.1 Initial Behavioral Characterization of Gain-of-Function Mouse Models. 231
5.2 Differences between heterologous and synaptic data. 233
5.3 GRIN2A null variants, seizure susceptibility, and parvalbumin-positive interneurons. 239
5.4 GluN2C/GluN2D Subunit-Selective Modulation of Synaptic NMDARs. 247
5.5 Conclusions. 251
5.6 References for Chapter 5. 252
LIST OF FIGURES
Figure 1.1. Ionotropic glutamate receptor functional classes and domain structure……………….4
Figure 1.2. NMDA receptor subunit stoichiometry……………………………………………….6
Figure 1.3. Time course of synaptic AMPA, kainate, and NMDA receptors…………………….11
Figure 1.4. NMDAR subunit spatiotemporal expression patterns in developing rat brain ………15
Figure 1.5. Organization of a typical glutamatergic synapse…………………………………….29
Figure 2.1. Topographical location of variants used in mouse model experiments………………85
Figure 2.2. Seizure phenotypes of mouse models of human NMDAR variants………………….87
Figure 2.3. Comparison of GluN2A-P552R kinetic properties in HEK cells and at native synapses……………………………………………………………………………………….…90
Figure 2.4. Comparison of GluN2A-S644G kinetic properties in HEK cells and native synapses………………………………………………………………………………………….95
Figure 2.5. Comparison of GluN2D-V667I kinetic properties in HEK cells and native synapses………………………………………………………………………………………….98
Figure 2.6. Spontaneous and miniature inhibitory postsynaptic currents (IPSCs) onto CA1 pyramidal cells…….……………………………………………………………………………102
Figure 2.7. The decay time of mIPSCs onto CA1 pyramidal cells is unchanged in GluN2D-V667I/+ mice…………………………………...………………………………………………104
Figure 3.1. Null GRIN2A null patients display a transient seizure burden not seen in missense GRIN2A patients……………………………………………………………………...…………122
Figure 3.2. Juvenile CA1 circuit shows hyperexcitability in Grin2a+/- and Grin2a-/- mice……124
Figure 3.3. Loss of Grin2a causes an increase in parvalbumin (PV) cell density in CA1…...…..127
Figure 3.4. CA1 PV cells undergo electrophysiological maturation of passive and action-potential firing properties………………………………………………………………………………....130
Figure 3.5. The loss of Grin2a causes a transient change in passive electrophysiological properties in CA1 PV cells…………………………………………………………………………………133
Figure 3.6. The loss of Grin2a causes a transient change in action-potential waveform properties of CA1 PV cells…………………………………………………………………………………137
Figure 3.7. The loss of Grin2a causes a transient change in action-potential firing properties of CA1 PV cells……………………………………………………………………………………140
Supplemental Figure S3.1. Juvenile CA1 circuit hyperexcitability is not due to changes in CA1 pyramidal cell intrinsic or action-potential firing properties……………………………………148
Supplemental Figure S3.2. Loss of Grin2a does not alter cholecystokinin (CCK) cell density in CA1……………………………………………………………………………………………..149
Supplemental Figure S3.3. CA1 PV cells undergo electrophysiological maturation of passive and action-potential firing properties………………………………………………………………..150
Figure 4.1. Distinctions between the exemplar GluN2C/GluN2D positive allosteric modulator, CIQ, and its predecessor, EU1180-465………………………………………………...………..172
Figure 4.2. Grin2d mRNA is found in GABAergic interneurons in mouse hippocampus……..174
Figure 4.3. Application of EU1180-465 increases inhibitory tone in CA1…………………..….175
Figure 4.4. Application of EU1180-465 increases amplitude, but not decay time, of sIPSCs onto CA1 pyramidal cells………………………………………………………………..…………...177
Figure 4.5. EU1180-465 potentiates CA1 stratum radiatum GABAergic interneurons………..179
Figure 4.6. EU1180-465 response on stratum radiatum interneurons can be stratified into responders and non-responders…………………………………….…………………………...182
Figure 4.7. NPY-expressing neurogliaform cells are potentiated by EU1180-465……………183
Figure 4.8. VIP-expressing interneuron-selective interneurons are not potentiated by EU1180-465……………………………………………………………………………………………....186
Figure 4.9. VIP/CCK-expressing basket cells are potentiated by EU1180-465…………..…….188
Figure 4.10. Validation of CCK antibody and post-hoc staining technique…………...………..190
LIST OF TABLES
Table 1.1 Pharmacological and kinetic properties of diheteromeric and triheteromeric NMDARs expressed in heterologous systems……………………………………….…………………...…...9
Table 1.2. NMDAR variants and intolerance scores.………………………………………..…...37
Table 1.3. NMDAR variants and associated disease………………………………………....…..38
Table 1.4. NMDAR subunit null mouse models and prevalence of null mutations in affected individuals………………………………………………..………………………………….…...46
Table 2.1. Human patient data of gain-of-function missense NMDAR variants…………………86
Table 2.2. Kinetic properties of NMDAR-mediated EPSCs onto CA1 pyramidal cells from GoF GluN2A missense mouse models……………………………...…………………………………92
Table 2.3. Kinetic properties of NMDAR-mediated EPSCs onto CA1 pyramidal cells and CA1 stratum radiatum from GluN2D-V667I mice…………………………………………………..100
Table 2.4. Properties of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) onto CA1 pyramidal cells from GluN2D-V667I mice………………………………………………………………………………………..……103
Supplemental Table S3.1. Null GRIN2A variant patient data and seizures burden………….…152
Supplemental Table S3.2. Missense GRIN2A variant patient and seizure burden……………..154
Supplemental Table S3.3. Action-potential spiking probability in Grin2a+/+, Grin2a+/-, and Grin2a-/- juvenile (P14-16) mice………………………………………………………………..156
Supplemental Table S3.4. Passive and action-potential firing properties of CA1 pyramidal cells in Grin2a+/+, Grin2a+/-, and Grin2a-/- juvenile (P14-16) mice…………………………………..157
Supplemental Table 3.5. Parvalbumin-positive CA1 density and cellular lamination in Grin2a+/+, Grin2a+/-, and Grin2a-/- preadolescent (P21-26) mice…………………………………………..158
Supplemental Table S3.6. Cholecystokinin-positive CA1 density and cellular lamination in Grin2a+/+, Grin2a+/-, and Grin2a-/- preadolescent (P21-26) mice………………………………159
Supplemental Table S3.7. Electrophysiological properties of wildtype CA1 PV cells during development………………………………………………………………………………….…160
Supplemental Table S3.8. Passive electrophysiological properties during development in Grin2a+/+, Grin2a+/-, and Grin2a-/- CA1 PV cells………………………………………………161
Supplemental Table S3.9. Action-potential waveform and firing properties during development in Grin2a+/+, Grin2a+/-, and Grin2a-/- CA1 PV cells……………………………………………162
Table 4.1. Summary of sIPSCs onto hippocampal CA1 pyramidal cells with EU1180-465….176
Table 4.2. Summary data from all evoked NMDAR-mediated EPSCs onto stratum radiatum interneurons……………………………………………………………..………………………180
Table 4.3. Summary data from all evoked NMDAR-mediated EPSCs onto NPY-expressing neurogliaform cells……………………………………………...……………………………....184
Table 4.4. Summary data from all evoked NMDAR-mediated EPSCs onto VIP-expressing interneuron-selective interneurons……………………………………………..……………….187
Table 4.5. Summary data from all evoked NMDAR-mediated EPSCs onto VIP/CCK co-expressing basket cells……………………………………………………….………………....189
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