An exploration into the link between brain rhythms and synaptic plasticity in health and infectious disease Iain James Hartnell PhD University of York Biology September 2018 Abstract During wakefulness, synapses are strengthened to enable memory formation. Whereas, during sleep, weaker connections are ‘pruned’ to help consolidate memories. These synaptic alterations are related to cortical oscillations, which are generally faster during wakefulness (30- 80Hz, gamma), and slower during deep sleep (1-4Hz, delta). Synaptic strength is thought to decrease during delta rhythms (compared to gamma rhythms). Neuroinflammation can disturb these brain rhythms and lead to a decline in cognitive function, which may result from aberrations in synaptic plasticity. To test the laminar and cellular changes in synaptic plasticity during sleep- and wake-related oscillations, in vitro electrophysiology and immunofluorescence were employed using acute rat neocortical slices. To examine the effect of neuroinflammation on these brain states, systemic infection was induced using synthetic analogues of pathogenic bacterial and viral material, and a biological parasitic disease model. The expression of an immediate early gene (IEG) marker of neuronal plasticity (Arc) was higher during delta oscillations compared to gamma oscillations and was concentrated to mid-apical dendrite bundles from layer V intrinsically bursting cells. These bundles represented cortical microcolumns which are known to exhibit synchronous activity, allowing parallel processing of information. Increased Arc expression in these columns during delta oscillations may promote synaptic rescaling and highlights the role of cortical microcolumns in memory consolidation. A balance of pro- and anti-inflammatory cytokines was found after short term systemic infection which gave way to a predominately pro-inflammatory state when the infection was longer term. The oscillatory activity also changed, with a continued decline in gamma power. However, delta power increased short term but decreased with a longer infection. The systemic infection had no effect on cortical plasticity. These results were corroborated in a mouse model of Leishmaniasis and show that systemic infection alters neuronal communication by changes to oscillatory activity, but does not change synaptic plasticity levels. 2 Declaration I declare that this thesis is a presentation of original work and I am the sole author except where otherwise indicated in the text. This work has not previously been presented for an award at this, or any other, University. All sources are acknowledged as References. 3 Contents ABSTRACT ............................................................................................................................................. 2 DECLARATION ....................................................................................................................................... 3 ACKNOWLEDGEMENTS. ........................................................................................................................... 8 ABBREVIATIONS ..................................................................................................................................... 9 LIST OF FIGURES .................................................................................................................................. 10 AIMS AND OBJECTIVES .......................................................................................................................... 13 CHAPTER ONE – GENERAL INTRODUCTION ............................................................................................... 14 1.1 THE NEOCORTEX ............................................................................................................. 15 1.1.1 Intracortical transfer of information through the neocortex .................................... 16 1.1.2 Oscillatory activity in neocortical columns ................................................................ 17 1.1.3 Intercolumnar communication in the neocortex ....................................................... 19 1.1.4 Primary somatosensory cortex .................................................................................. 21 1.1.5 Secondary somatosensory cortex .............................................................................. 21 1.1.6 Primary auditory cortex ............................................................................................. 22 1.2 NEURAL OSCILLATIONS .................................................................................................... 22 1.2.1 Recording the electrical activity of the human brain ................................................. 22 1.2.2 Recording oscillatory activity in vivo .......................................................................... 25 1.2.3 Recording oscillations in vitro .................................................................................... 25 1.2.4 Gamma Oscillations ................................................................................................... 26 1.2.5 Delta Oscillations ....................................................................................................... 34 1.3 IMMEDIATE EARLY GENES (IEGS) ........................................................................................ 40 1.3.1 C-fos - Finkel–Biskis–Jinkins osteogenic sarcoma homologue ................................... 41 1.3.2 Arc - Activity-related cytoskeleton-associated protein ............................................. 44 1.3.3 General IEG changes during different brain states .................................................... 45 1.3.4 Regional IEG changes across brains states................................................................. 46 1.4 NEUROINFLAMMATION .................................................................................................... 47 1.4.1 The blood-brain barrier .............................................................................................. 48 1.4.2 Microglia .................................................................................................................... 49 1.4.3 Cytokines and chemokines ........................................................................................ 50 1.4.4 Neuroinflammation in disease ................................................................................... 53 1.5 LEISHMANIASIS............................................................................................................... 56 1.5.1 Visceral Leishmaniasis ................................................................................................ 57 1.5.2 Leishmaniasis and peripheral neuropathy ................................................................. 58 1.5.3 Leishmaniasis in the CNS ............................................................................................ 58 1.5.4 Leishmaniasis and neuroinflammation ...................................................................... 59 1.6 AIMS AND OBJECTIVES ..................................................................................................... 61 4 CHAPTER TWO – MATERIALS AND METHODS ............................................................................................ 62 2.1 MATERIALS ................................................................................................................... 63 2.1.1 List of drugs and chemicals used ............................................................................... 63 2.1.2 ACSF Formulations ..................................................................................................... 63 2.1.3 Antibodies used ......................................................................................................... 64 2.2 ELECTROPHYSIOLOGY ....................................................................................................... 65 2.2.1 Animal provision ........................................................................................................ 65 2.2.2 Brain slice preparation ............................................................................................... 65 2.2.3 Slice maintenance ...................................................................................................... 67 2.2.4 Oscillation induction .................................................................................................. 70 2.2.5 Extracellular (LFP) Recordings .................................................................................... 70 2.2.6 Analysis of field potential recordings ......................................................................... 73 2.2.7 Intracellular Recordings ............................................................................................. 73 2.2.8 Biocytin labelling and imaging ................................................................................... 74 2.3. INFECTION AND INFLAMMATION MODELS ............................................................................ 75 2.3.1 Acute inflammation ................................................................................................... 75 2.3.2 Chronic inflammation ................................................................................................ 75 2.3.3. Leishmaniasis Infection ............................................................................................. 76 2.4. IMMUNOFLUORESCENCE .................................................................................................