Neurophysiological characterisation of neurons in the rostral nucleus reuniens in health and disease. Submitted by Darren Walsh, to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Medical Studies, September 2017. This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. (Signature) ……………………………………………………………………………… Word Count = 44,836 1 Abstract Evidence is mounting for a role of the nucleus reuniens (Re) in higher cognitive function. Despite growing interest, very little is known about the intrinsic neurophysiological properties of Re neurons and, to date, no studies have examined if alterations to Re neurons may contribute to cognitive deficits associated with normal aging or dementia. Work presented chapter 3 provides the first detailed description of the intrinsic electrophysiological properties of rostral Re neurons in young adult (~5 months) C57- Bl/6J mice. This includes a number of findings which are highly atypical for thalamic relay neurons including tonic firing in the theta frequency at rest, a paucity of hyperpolarisation-activated cyclic nucleotide–gated (HCN) mediated currents, and a diversity of responses observed in response to depolarising current injections. Additionally this chapter includes a description of a novel form of intrinsic plasticity which alters the functional output of Re neurons. Chapter 4 investigates whether the intrinsic properties of Re neurons are altered in aged (~15 month) C57-Bl/6J mice as compared to a younger control group (~5 months). The intrinsic properties were remarkably similar across age ranges suggesting that alterations to the intrinsic properties of Re neurons do not contribute to age-related cognitive deficits. Chapter 5 investigates whether alterations to the intrinsic properties of Re neurons occur in the J20 model of amyloidopathy. Alterations to the resting membrane potential (RMP), propensity to rebound fire, and a reduction in action potential (AP) width were observed. This suggests that alterations to the intrinsic properties of Re neurons may contribute to cognitive deficits observed in Alzheimer’s disease (AD). Chapter 6 investigates whether alterations to the intrinsic properties of Re neurons occur in a mouse model (CHMP2Bintron5) of frontotemporal dementia (FTD). Only subtle changes were observed suggesting that alterations to the intrinsic properties of Re neurons does not contribute to cognitive deficits observed in FTD linked to chromosome 3 (FTD-3). 2 Acknowledgements First and foremost, I would like to thank my supervisors Prof. Andy Randall and Dr. Jon Brown for all their help and support over the last 4 years. Needless to say without their input none of this would have been possible. I would also like to thank all of the Hatherly crew, past and present, for their warmth, friendship and making this Irish country bumpkin feel at home in the metropolis that is Devon. Particular thanks go to Hannah Smithers (who prepared some of the slices used in chapter 3) and Tom Ridler, who have been there all the way. To quote Drizzy Drake, “Started from the bottom now we’re here”. Next a special thanks goes to the Fabreeze Brothers (Ben H, Myles J, Tom H, and Jeff L) for the stimulating quasi-intellectual pub talk, both scientific and otherwise, and for not losing faith when I wandered. To Soraya, for believing in me when I didn’t and your unwavering ability to make me smile, I am more grateful than you could ever know. Finally to my family at home. I did this for you guys. I hope ye are proud. This work is dedicated to the memory of my late grandfather Tommy Kelly. A victim of Alzheimer’s disease; I couldn’t have done it without him. 3 Table of Contents Abstract ..................................................................................................................... 2 Acknowledgements .................................................................................................. 3 List of figures ........................................................................................................... 7 List of tables ............................................................................................................. 9 Abbreviations ........................................................................................................... 9 1 Introduction ....................................................................................................... 12 1.1 General electrophysiological properties of neurons. ................................... 12 1.1.1 Resting membrane potential. ................................................................ 12 1.1.2 Passive membrane properties .............................................................. 14 1.1.3 Voltage-gated ion channels. ................................................................. 15 1.1.4 The neuronal action potential. .............................................................. 16 1.1.5 Synaptic transmission. .......................................................................... 18 1.1.6 Neuronal oscillations ............................................................................ 20 1.2 The thalamus .............................................................................................. 21 1.2.1 Neuroanatomy of the thalamus. ............................................................ 21 1.2.2 First and higher order relays influence on cortical function. .................. 22 1.2.3 Intrinsic electrophysiological properties of thalamic relay neurons ....... 24 1.3 The nucleus reuniens. ................................................................................. 27 1.3.1 Connectivity of the nucleus reuniens. ................................................... 28 1.3.2 The role of the nucleus reuniens in hippocampal prefrontal interactions 29 1.3.3 Cellular diversity in the nucleus reuniens.............................................. 32 1.3.4 The effect of experimental manipulation of the nucleus reuniens on cognitive function. ............................................................................................. 32 1.3.5 The role of the nucleus reuniens in disease. ........................................ 35 1.4 The effects of physiological aging on the brain. .......................................... 37 1.4.1 Regional decline in brain volume with physiological aging ................... 38 1.4.2 Changes in intrinsic excitability with physiological aging ...................... 38 1.5 Major Neurocognitive Disorders. ................................................................. 39 1.5.1 Alzheimer’s Disease. ............................................................................ 39 1.5.2 Frontotemporal Dementia. .................................................................... 43 1.5.3 Mouse models of dementia. .................................................................. 44 1.6 Aims ............................................................................................................ 46 2 Materials and Methods ...................................................................................... 52 4 2.1 Ethical approval and animals ...................................................................... 52 2.2 Slice preparation ......................................................................................... 52 2.3 Visual identification of neurons in the rostral nucleus reuniens ................... 53 2.4 Electrophysiological recordings ................................................................... 54 2.4.1 Whole cell patch clamp ......................................................................... 54 2.5 Data analysis ............................................................................................... 60 2.6 Staining for amyloid plaques ....................................................................... 64 3 Intrinsic electrophysiological properties of neurons of the rostral nucleus reuniens ................................................................................................................... 60 3.1 Introduction ................................................................................................. 60 3.2 Methods ...................................................................................................... 61 3.3 Results ........................................................................................................ 62 3.3.1 Cellular properties at in the absence of injected current ....................... 62 3.3.2 Spontaneous synaptic input .................................................................. 68 3.3.3 Passive and active membrane properties ............................................. 72 3.3.4 Apparent absence of HCN like current ................................................. 76 3.3.5 Afterpotential following elicitation of a single spike. .............................. 83 3.3.6 Action potential production in response to αEPSC current injections. .. 84 3.3.7 Firing behaviour in response to square current potentials are voltage dependant ......................................................................................................... 88 3.3.8 Electrophysiological and