Lights, Gamma, Action: Gamma Visual Flicker Activates Neuroimmune Signaling Restricted; Files Only
Garza, Kristie (Spring 2021)
Published
Abstract
Many neurodegenerative and neurological diseases are rooted in dysfunction of the neuroimmune system. Therefore, the ability to manipulate this system has strong therapeutic potential. Prior work has shown that exposing mice to flickering lights at 40Hz drives gamma frequency (∼40Hz) neural activity and recruits microglia, the primary immune cells of the brain. This stimulation, termed gamma visual flicker or 40Hz flicker, provides a novel method to manipulate the neuroimmune system. However, the biochemical signaling mechanisms between 40Hz neural activity and immune recruitment remain unknown. This gap in the literature presents a barrier to develop this stimulation to its full therapeutic potential. In this thesis, we exposed wild-type mice to gamma visual flicker or control flicker stimulations for durations ranging from five to sixty minutes. Following flicker exposure, we assessed cytokine and phosphoprotein networks known to play roles in immune functioning. Using these methods, we found, for the first time, that gamma visual flicker leads to increases in the expression of cytokines known to be involved in microglial recruitment. To identify possible mechanisms underlying cytokine expression, we quantified the effect of the flicker on intracellular signaling pathways known to regulate cytokine levels. We found 40Hz flicker upregulates phospho-signaling within the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) pathways. We next used inhibitors of these pathways to prove that activity of these pathways is necessary for the increased cytokine profile seen following gamma visual flicker. Last, we use an established microglia depletion paradigm to show these cytokine signals are not fully dependent on microglia. We then provide evidence to suggest a neuronal origin for our observed neuroimmune signaling changes. The results presented in this thesis are the first to address a possible mechanism underlying the neuroimmune impacts of gamma visual flicker. These results provide a major contribution to the field and will be essential for the use of flicker as a therapeutic for brain disease.
Table of Contents
CHAPTER 1: INTRODUCTION 1
1.1 Scope and Organization 2
1.2 Gamma Oscillations 3
1.2.1 Gamma Deficits and Alzheimer’s Disease 5
1.2.2 Gamma Entrainment 6
1.3 The Neuroimmune System 8
1.3.1 Cells of the Neuroimmune System 8
1.4 Neuroimmune Signaling Mechanisms 10
1.4.1 Phosphoprotein Pathways 11
1.4.2 Cytokines 15
1.4.3 Neuroinflammation 16
1.5 Gamma Entrainment and the Neuroimmune system 17
1.6 Thesis Objectives and Hypothesis 18
CHAPTER 2: MATERIALS AND METHODS 21
2.1 Animals 21
2.2 Visual Stimulation Exposure 22
2.3 Lipopolysaccharides (LPS) Stimulation 22
2.4 Phosphoprotein Inhibitors 23
2.5 Microglia Depletion 23
2.6 Immunohistochemistry (IHC) and Microscopy 24
2.7 Cytokine and phospho-protein assays 25
2.8 Partial least squares discriminant analysis 27
2.9 Animal behavior assays 28
2.10 Experimental design and statistical analysis 29
CHAPTER 3: GAMMA VISUAL FLICKER INDUCES CYTOKINE EXPRESSION IN THE BRAIN 31
3.1 Introduction 31
3.2 Results 33
3.2.1 40Hz Visual Flicker Induces Increases in Cytokine Expression Profile 33
3.2.2 Flicker Frequencies Each Induce Unique Cytokine Profiles 38
3.2.3 Animal Behavior is similar across flicker stimulations 39
3.2.4 40Hz Flicker induces neuroimmune profile distinct from acute and chronic pathological inflammation 41
3.3 Discussion 46
3.3.1 40Hz flicker-induces a unique cytokine profile in the visual cortex 46
3.3.2 40Hz flicker-induced cytokines have neuroprotective functions 47
3.3.3 Behavior is similar across different visual stimulation conditions 49
3.3.4 40Hz flicker cytokine response differs from acute pathological inflammation 50
3.3.5 40Hz flicker cytokine response differs from chronic, pathological inflammation 52
CHAPTER 4: GAMMA VISUAL FLICKER ACTIVATES PHOSPHOPROTEIN PATHWAYS NECESSARY FOR CYTOKINE EXPRESSION 54
4.1 Introduction 54
4.2 Results 56
4.2.1 40Hz Flicker Induces NFB and MAPK Signaling 56
4.2.2 Phosphoprotein Network Correlations 62
4.2.3 Cytokine signaling after 40Hz Flicker is dependent on both NFB and MAPK pathways 63
4.3 Discussion 70
CHAPTER 5: GAMMA VISUAL FLICKER INDUCES IMMUNE SIGNALS IN NON-MICROGLIAL CELLS 75
5.1 Introduction 75
5.2 Results 77
5.2.1 Confirmation of Microglial Depletion 77
5.2.2 Microglia are not necessary for cytokine expression 80
5.2.3 Microglia are not necessary for all 40Hz gamma flicker-induced cytokines 82
5.2.4 pNFB Expression After Gamma Flicker Occurs in Neurons 86
5.2.4 M-CSF Expressed after Gamma Flicker is Expressed Neurons 88
5.3 Discussion 90
5.3.1 Confirmation of Microglial Depletion 90
5.3.2 Cytokines Persist in the Brain in the Absence of Microglia 90
5.3.3 Microglia are not necessary for all 40Hz flicker-induced cytokines 92
5.3.4 Phosphorylated NFB Gamma Flicker Localizes to Neurons 94
5.3.5 M-CSF Induced after 40Hz Flicker Colocalizes with NeuN 95
CHAPTER 6: GENERAL DISCUSSION 98
6.1 Summary 98
6.2 Major Contributions 98
6.3 Flicker’s Dynamic Effect on the Neuro-Immune System 99
6.4 Proposed Mechanism 101
6.4.1 The Calcium Hypothesis 101
6.4.2 Calcium and the NFB Pathway 102
6.4.3 Calcium and the MAPK Pathway 104
6.4.4 Microglia and Amyloid Beta 104
6.5 Flicker as a Therapeutic 105
6.5.1 Alzheimer’s Disease 105
6.5.2 Implications for other disease types 107
6.6 Future Directions 108
6.6.1 In Vivo Experiments 108
6.6.2 The Role of Akt phospho-signaling in Gamma Flicker 111
6.6.3 Brain Cells and Gamma Sensory Flicker 112
6.7 Concluding Remarks 114
APPENDIX 116
Appendix 1: Gamma visual flicker does not change cytokine levels in the periphery 116
Appendix 2: Gamma visual flicker and Monoamines 117
REFERENCES 119
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