Increased activity of GABAA receptors contributes to postanesthetic memory deficits by Agnieszka A. Zurek A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Agnieszka A. Zurek, 2015 Increased activity of GABAA receptors contributes to postanesthetic memory deficits Agnieszka A. Zurek Doctor of Philosophy Department of Physiology University of Toronto 2015 Abstract General anesthetics are widely used to allow patients to tolerate surgery and to sedate patients in intensive care units. Anesthesia causes long-term memory deficits in laboratory animals, suggesting it could contribute to memory loss in patients. However, the mechanisms underlying postanesthetic memory deficits are unknown. Most anesthetics cause neurodepression by allosterically increasing the activity of γ- aminobutyric acid type A (GABAA) receptors. In particular, positive allosteric modulation of α5 subunit-containing GABAA (α5GABAA) receptors contributes to the acute, amnestic effects of the anesthetic etomidate. Once the anesthetic has been eliminated, allosteric modulation of GABAA receptors is rapidly reversed and it is assumed that GABAA receptors do not contribute to memory deficits that persist after anesthesia. However, previous work from our lab suggests that α5GABAA receptors may play a role in postanesthetic memory loss, as treatment with a drug that inhibits these receptors before anesthesia prevents memory deficits. I hypothesized that exposure to anesthetics results in a sustained increase in α5GABAA receptor activity, which causes memory deficits. ii First, my data showed that α5GABAA receptors are required for postanesthetic memory deficits as wild-type, but not Gabra5 null-mutant mice (Gabra5-/-), exhibit impaired memory performance on the object recognition memory task. These deficits are evident for 24 hours after isoflurane anesthesia and persist for up to 1 week after exposure to the injectable anesthetic etomidate. The mechanism underlying these memory deficits is likely a sustained increase in the function of α5GABAA receptors as a single, in vivo exposure to the anesthetic etomidate (8 mg/kg i.p.) or isoflurane (1.3%, 1 h) increases a tonic inhibitory current that is mediated by α5GABAA receptors and increases cell-surface expression of α5GABAA receptors for least 1 week with full recovery by 2 weeks. Memory deficits in wild-type mice are reversed after anesthesia by inhibiting α5GABAA receptor activity with the inverse agonist L-655,708. Collectively, these results refute the widely-held belief that the function of GABAA receptors returns to baseline after the anesthetic agent has been eliminated. Furthermore, the data present a mechanism and a plausible treatment strategy for postanesthetic memory deficits. iii Acknowledgments I would like to thank my supervisor and mentor Dr. Beverley Orser, without you none of this would be possible. Thank you for your guidance, support, unwavering patience, and all the opportunities that allowed me to develop as a scientist and as a person. I admire your adventurous spirit in science and life. I thank my Supervisory Committee Drs. Evelyn Lambe and Paul Frankland for your advice, support and kindness over the years. Thank you also to Drs. Melanie Woodin, Mike Salter and Milton Charlton for your mentorship and counsel on my project. Thank you to lab alumni, Drs. Loren Martin and Robert Bonin, for your friendship and for always sharing your wisdom with me, especially in times of need. I am very grateful for the contributions of the entire Orser lab. Thank you Dr. Dianshi Wang for being the lab sage, the crusader for perfection, and the patient teacher. Thank you Ella Czerwinska for all your jokes, hugs, and zucchinis, you made me feel at home from day one. Thank you Anine (Jieying) Yu for your immense generosity and help on my project. Thank you to all the summer students I’ve had the pleasure to work with over the years; Burç Aydin, Mohammed Haijha, Erica Bridgwater, Zeenia Aga and Eric Salter – you all taught me so much. Also thank you to everyone who has been a part of the lab family, Dr. Paul Whissell, Irene Lecker, William To, Dr. Sinziana Avramescu, Dr. Antonello Penna, Dr. Gang Li, Dr. Stephen Kemp, Dr. Junhui Wang, Sean Haffey, and Fariya Mostafa. You have taught me the power of teamwork and have made the lab feel like a second home. Most importantly, I would like to thank my family. I thank my parents, Beata and Zdzislaw Zurek, for your love, constant encouragement, and interest in my work, especially over the last decade of university. I will be forever grateful. Lastly, thank you to my partner-in-crime, Peter Dziak, for your love, understanding, and always reassuring me that “yes, I can”. iv List of Contributions Agnieszka Zurek produced all of the results with the exception of those listed below. The behavioural data presented in Chapter 4 were collected with the assistance of Ms. Erica Bridgwater, who performed the experiment with sevoflurane. In Chapter 5, the behavioural studies of mice treated with a sedative dose of etomidate were performed with the assistance of Ms. Erica Bridgwater. Studies of cell-surface expression were performed by Ms. Jieying Yu, Dr. Gang Li, and Mr. Tom Chang. Recordings from cultured neurons were performed with the assistance of Dr. Antonello Penna and Dr. Dianshi Wang. The recordings in astrocyte-neuron and microglia-neuron cocultures were performed by Mr. Sean Haffey and Ms. Irene Lecker. The experiments presented in Appendix 1 were performed with the help of Ms. Zeenia Aga. Dr. Dianshi Wang and Dr. Beverley Orser helped with the writing of the articles published in Anesthesia and Analgesia and in The Journal of Clinical Investigation, modified versions of these articles are presented in Chapters 4 and 5. v Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations .....................................................................................................................xv List of Appendices ....................................................................................................................... xix Chapter 1. Thesis Structure .......................................................................................................1 1.1 General Overview ................................................................................................................1 1.2 Hypothesis and Specific Aims .............................................................................................2 1.2.1 Hypothesis................................................................................................................2 1.2.2 Specific Aims ...........................................................................................................2 1.3 Thesis Structure ...................................................................................................................3 Chapter 2. General Introduction ................................................................................................5 2.1 Postoperative Cognitive Dysfunction ..................................................................................5 2.1.1 Incidence ..................................................................................................................5 2.1.2 Cognitive Domains Affected by POCD ...................................................................6 2.1.3 Effects on patient outcome.......................................................................................7 2.1.4 Risk factors and potential causes of POCD in patients............................................8 2.2 Memory deficits after surgery and anesthesia in animal models .......................................12 2.2.1 Memory deficits after surgery in animal models ...................................................14 2.2.2 Memory deficits after anesthesia in animal models ...............................................18 2.3 Proposed mechanisms of postanesthetic memory deficits in animals ...............................18 2.3.1 Inflammation and activation of apoptotic pathways ..............................................19 vi 2.3.2 Impaired neurogenesis ...........................................................................................21 2.3.3 Alzheimer Disease-related mechanisms ................................................................21 2.3.4 Potential GABAA receptor-dependent mechanisms ..............................................24 2.4 GABA and GABAA receptor-mediated inhibition ............................................................24 2.4.1 GABA Synthesis and release .................................................................................24 2.4.2 GABA transport and metabolism...........................................................................26 2.4.3 GABA receptors.....................................................................................................27