This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Functional Characterisation of Spontaneously Active GABAA Receptors in Rat Dentate Gyrus Granule Cells Nathanael O’Neill B.Medsc. (Hons) Doctor of Philosophy The University of Edinburgh 2020 ii Abstract GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the adult mammalian central nervous system. GABAARs mediate two forms of inhibition: fast, phasic conductance; and slow, tonic conductance. Tonic conductance arises due to the persistent activation of GABAARs. This persistent activation can occur by GABA-dependent or GABA- independent mechanisms. Low concentrations of ambient GABA activate high affinity GABAARs located outside the synapse – at peri-/extra-synaptic sites – to generate GABA-dependent tonic conductance. In contrast, GABA-independent tonic conductance is generated by GABAARs that activate spontaneously, in the absence of GABA, due to constitutive receptor gating. Because spontaneously active GABAARs (s-GABAARs) do not require GABA to activate, they are resistant competitive antagonists, e.g. SR-95531, but can be inhibited by the channel-blockers, e.g. picrotoxin. s-GABAARs have been shown to produce GABA-independent tonic conductances in the hippocampus and the amygdala. However, despite the good evidence for the presence of s- GABAARs, their function and pharmacology remain largely unknown. Here we show, for the first time, using both current- and voltage-clamp recording techniques, that the s-GABAAR-mediated tonic conductance exerts a powerful inhibitory effect in rat dentate gyrus granule cells. We find that at resting membrane potential, s-GABAARs generate a shunting conductance that decreases both the membrane resistance and the membrane time constant of the neuron. When the membrane potential is depolarised, s-GABAARs conduct hyperpolarising currents that exhibit outward-rectification; this means that their net inhibitory effect is greater when the neuron is close to firing threshold than when it is at rest. Consistent with this, we find that block of s-GABAARs shifts the neuron into a more excitable state, as evidenced by the increase in the gain of the input-output relationship and the decrease in the rheobase current and the hyperpolarisation of the action potential threshold. At the network level, s-GABAARs regulate the precision of signal transmission in the dentate gyrus: blocking s- GABAARs widens the temporal window over which multiple excitatory inputs can be successfully summated to generate an action potential. Finally, we report that s-GABAAR tonic currents are resistant to pharmacological compounds that target extrasynaptic GABAARs (L-655,708 and DS2), but are augmented by the clinically used benzodiazepine site modulators, zolpidem and midazolam, and partially inhibited by the inverse agonist, DMCM. The sensitivity of s-GABAARs to these compounds suggests the involvement of the γ2-subunit. iv Lay Summary Neurons communicate with each other using a group of proteins called receptors. Receptors are found on the surface of neurons and when they are activated (switched ON) they cause either excitation or inhibition. The appropriate balance between excitation and inhibition is critical for proper brain function. When this balance is upset, brain dysfunction quickly follows; as happens in a variety of diseases such as epilepsy. The main inhibitory receptor in the brain – and the topic of my PhD – is called the GABAA receptor. GABAA receptors can be thought of as switches. A simple view states that GABAA receptors are normally switched OFF, and are only switched ON in the presence of their neurotransmitter, which is called GABA (-aminobutyric acid). According to this view, all GABAA receptor effects therefore require GABA. However, is this an oversimplification? Are we seeing these receptors and, in turn, the brain, for what it really is? Prior to my PhD it was shown that, contrary to the simplistic view expressed above, GABAA receptors can actually switch ON in the absence of GABA. However, at this point in time, nobody knew if these GABA-independent GABAA receptors did anything important in the brain. During my PhD I recorded the electrical activity from thin slices of rat brains in order to examine the role of GABA-independent GABAA receptors. I was able to show, for the first time, that these receptors play an important role in maintaining the proper balance between excitation and inhibition in the brain. When I blocked these receptors from turning ON, neurons and their networks became hyper-excitable and were no longer able to filter incoming signals as effectively. This meant neurons that had previously been silent were now active. The hyper- excitability that I observed in the brain was similar to what has been found in some cases of epilepsy. This led me to test if anti-convulsants could modulate GABA-independent GABAA receptors. I was able to show that midazolam, a clinically used anti-convulsant, makes these receptors turn ON more. And, as a result, increases the inhibitory signal that they provide. In a narrow sense, my PhD shows that GABA-independent GABAA receptors play an important role in maintaining proper brain function. It also shows that these receptors can be modulated by anti-epileptic drugs, and thus can potentially be considered as new drug targets. But more broadly, it forces us to wonder: How many other receptors, in other brain regions, mediate some of their effects in the absence of their neurotransmitter? And what might this tell us about the brain? v Acknowledgements I would first like to thank Dr Sergiy Sylantyev for his supervision throughout my PhD. Without Sergiy, I would not have been able to enjoy three excellent years in the wonderful city of Edinburgh. I am thankful for the trust he put in me: I was able to take the project in the direction that I wanted to and was given the freedom to make mistakes (and hopefully learn from them) without judgement. This was especially generous of him, given that most of the equipment was brand new (and expensive!). I am also indebted to him for his inclusiveness on projects outside of my PhD. The Sylantyev Lab became a much less lonely place when Molly Hickey, a gifted undergraduate student, arrived to undertake a summer project with us. Molly generated some of the data in Chapter 4 and adapted to life in the lab exceedingly well. She was able to learn new and difficult skills quickly and had a knack for asking interesting questions. None of the lab work would have been possible without the lovely Sprague Dawley rats. And thanks to Will and Ami for taking such good care of them. I would also like to thank Professor Seth Grant, who co-supervised this project. It was a privilege to attend the Grant Lab’s weekly meetings. Seth broadened my scientific horizons and was a constant source of good, solid advice – especially on presentations. I am also thankful to Seth, as it was through in his lab that I met some of my closest friends in Edinburgh. Vlad Anton, forever a legend: discretion never was your middle name, but this, coupled with the daily compost-coffee, livened up Little France tremendously. And the regular dose of absurdist humour (and fluorescent PSD-95) that you provided got me through some of the toughest bits of my PhD. Thanks also to George, for being such a passionate advocate of the good life: good friends, good food, good drink, and, of course, Greece. And to Sarah, for lovely chats, good humour and thoughtful advice; and to Cathy for always keeping cheerful and for help with the cell culture. And to Malik, Max, Jamie, Dimitra, Edita, Noboru, Erik, Ann and Colin. I also want to express my deepest gratitude to Dr Matt Livesey. Matt helped tremendously with putting this PhD together and was a constant source of support in the last few months of writing. I cannot express how much I appreciate the time that Matt gave up, and the fact he went so out of his way to help me out. Throughout the PhD, Matt also instilled in me a greater sense of confidence in my work and was always on hand to provide expert experimental advice. He is also incredibly kind and will make an excellent Lab PI. I would also like to thank my previous supervisors, Dr Jonathan Wolf Mueller, Dr Andrew Powell, Dr Gillian Grafton, Professor Nick Barnes, and Professor Richard Barrett-Jolley. And, finally, to thank the people, without whom, I could not have got through the PhD: Phoebe, Mum, Dad, and Lillie. To Phoebe, thank you for all the love and support. The best times in Edinburgh were spent with you, be it at the Fringe, dog-spotting on the meadows, or eating ice-cream at Mary’s. Words don’t do justice to how amazing you have been. And to my family, whose selflessness and