Neural ensemble and subcellular dynamics of seizures and spreading depolarizations Restricted; Files Only

Abstract

Epilepsy is marked by recurrent, unprovoked, aberrant neural firing. This often hypersynchronous and widespread activity “seizes” the brain and man, frequently resulting in paroxysms and loss of consciousness. As our tools for investigation have evolved from astute and vigilant clinical observation to highly sensitive and discriminating electrophysiologic measurement, so too has our understanding this debilitating disease. Yet, despite the decades of resulting pharmacologic and surgical treatment advances, there still remain millions of patients suffering from poorly controlled seizures. While the mechanism underlying seizures is often framed as an imbalance between excitatory and inhibitory signaling, we are coming to realize that seizure manifestation is not a simple tipping of the scales. Rather, seizure pathology is rooted in complex interactions of neural ensembles within highly recurrent and intricate microcircuits of the brain. Deepening our comprehension of these dynamics is of paramount importance to close the epilepsy treatment gap.

In this dissertation, I detail my investigation of generalized seizures using in vivo two-color two-photon calcium imaging, with simultaneous multimodal electrophysiology, to record the spatiotemporal activity patterns of hundreds of cells across recurrent seizures. Within this, I leveraged two sophisticated genetic approaches to measure the firing of both glutamatergic and GABAergic neurons simultaneously and to concurrently capture calcium changes in the cytosol and endoplasmic reticulum (ER). I also present my algorithm developed for automated detection of neuron recruitment to epileptiform activity in calcium data. In brief, I discovered dynamics consistent with an increasing afferent drive into unrecruited cortex ahead of seizure onset and a subsequent breakdown of a feedforward inhibitory surround upon ictal invasion of a variable glutamatergic wavefront. I further reveal post-ictal slow calcium waves are definitively cortical spreading depolarizations (CSDs), and simultaneously recruit GABAergic and glutamatergic neurons. I also elucidate that CSDs are marked by a depletion of ER calcium stores consistent with a calcium-induced calcium release, contribute to a post-ictal suppression of epileptiform activity and have mechanistic implications for therapeutic electrical stimulation for epilepsy. Collectively, this work provides insight into the biological underpinnings of and interplay between generalized seizures and CSD, informing development of neuromodulatory approaches to control these intractable diseases.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1 Of Epilepsy and Spreading Depolarization: an introduction 1

1.1 Seizures and Epilepsy Overview 2

1.2 Historical Perspective 2

1.3 Modern Approaches for the Study of Epilepsy 5

1.3.1 Electrophysiology 5

1.3.2 Functional optical imaging 7

1.4 Seizure Physiology 11

1.4.1 Interictal activity 11

1.4.2 Pre-ictal to ictal transition 15

1.4.3 Seizure propagation and ictal dynamics 18

1.4.4 Seizure termination and post-ictal activity 22

1.5 Spreading Depolarization 24

1.6 Dissertation Overview 26

CHAPTER 2 Of Waves and Currents: optical and electrophysiological methods to study in vivo neural dynamics during seizures 30

2.1 Introduction 31

2.2 GECI Paradigms 31

2.2.1 Molecular Biology 32

2.2.1.1 DDO-jYCaMP1s and DIO-jRGECO1a 32

2.2.1.2 XCaMP-Y-P2A-RCatchER 33

2.2.2 Production of Viral Vector 33

2.3 In Vivo Experiments 34

2.3.1 Fabrication of cranial windows 34

2.3.2 Stereotaxic surgery 37

2.3.3 Two-photon imaging and electrophysiology 38

2.3.4 Acute seizure model 39

2.3.5 Electrical stimulation 40

2.3.6 Histology 41

2.3.7 Two-photon excitation spectra collection 42

2.4 Image Processing and Seizure Event Detection 43

2.4.1 Description of event detection algorithm 44

2.4.1.1 Data pre-processing 46

2.4.1.2 Movie and representative frame processing 47

2.4.1.3 Pre-ictal spike recruitment detection 47

2.4.1.4 Wavefront recruitment detection 49

2.4.2 Algorithm evaluation approach 50

2.4.3. Algorithm evaluation results 53

2.4.3.1. Pre-ictal spike detection 53

2.4.3.2. Seizure invasion and activity at termination detection 55

2.4.3.3. Seizure wavefront modeling 57

2.4.3.4 ER imaging modifications 58

2.4.4. Discussion of automated seizure event detection approach 59

2.5 Data Analysis 64

2.5.1 Calcium change magnitude 64

2.5.2 PIS recruitment dynamics 65

2.5.3 Calcium wavefront properties 66

2.5.3 Electrophysiology Analysis 66

CHAPTER 3 Of Excitation and Inhibition: a spatiotemporal study of neural ensemble activity during seizures and spreading depolarization 68

3.1 Introduction 69

3.2 Results 71

3.2.1 Intravital imaging of excitatory and inhibitory neurons in awake mice during generalized seizures 71

3.2.2 Neurons are progressively recruited in a heterogenous fashion and synchronize within and across subtypes pre-ictally 74

3.2.3 GABAergic neurons are recruited ahead of a variable glutamatergic ictal wavefront 79

3.2.4 Glutamatergic and GABAergic neurons are simultaneously recruited to post-ictal CSD 82

3.3 Discussion 84

3.3.1 Two-color mutually exclusive subtype imaging 85

3.3.2 Pre-ictal dynamics 87

3.3.3 Ictal invasion 89

3.3.4 Seizure propagation and stereotypy 91

3.3.5 Seizure termination 91

3.3.6 Limitations 93

3.3.7 Conclusion 94

CHAPTER 4 Of Crests and Troughs: a spatiotemporal study of intracellular calcium dynamics during seizures and spreading depolarization 95

4.1 Introduction 96

4.2 Results 98

4.2.1 Intravital imaging of cytosol and ER calcium stores in awake mice 98

4.2.2 CSD observed at generalized seizure termination is marked by a unique depletion of ER calcium 100

4.2.3 ER calcium depletion during seizure CSD is consistent with a CICR 103

4.2.4 ER calcium depletion is a conserved feature across multiple types of CSD 109

4.3 Discussion 114

4.3.1 Fast in vivo ER imaging through RCatchER 115

4.3.2 Implications of CSD in seizure physiology 115

4.3.3 CSD, CICR and ER calcium flux 117

4.3.4 CSD and additional intracellular dynamics 119

4.3.5 Calcium changes of TSD 120

4.3.6 Translational implications 121

CHAPTER 5 Of Reflections and Projections: concluding remarks 123

5.1 Dissertation Summary and Contextualization 124

5.2 Future Directions 130

Afterword 136

References 139

Appendix 174

Figure Legends for Movies 174

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	Calcium Imaging Two-Photon Epilepsy Seizure Intravital Microscopy Spreading Depolarization
Committee Chair / Thesis Advisor	Robert E. Gross, Emory University
Committee Members	Jon T. Willie, Washington University Raymond J. Dingledine, Emory University Garrett B. Stanley, Georgia Institute of Technology Nigel P. Pedersen, University of California Davis

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