Lights, Gamma, Action: Gamma Visual Flicker Activates Neuroimmune Signaling Restricted; Files Only

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 NFB and MAPK Signaling          56

4.2.2 Phosphoprotein Network Correlations 62

4.2.3 Cytokine signaling after 40Hz Flicker is dependent on both NFB 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 pNFB 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 NFB 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 NFB 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

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	Gamma Immune
Committee Chair / Thesis Advisor	Annabelle Singer, Emory University
Committee Members	James Lah, Emory University Levi Wood, the georgia institute of technology Machelle Pardue, Emory University Malu Tansey, Emory University

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