Investigating the Central Role of Astrocytes in Mediating Postanesthetic Memory Deficits

Kirusanthy Kaneshwaran

A thesis submitted in conformity with the requirements for the degree of Master in Science

Department of Physiology University of Toronto

Investigating the Central Role of Astrocytes in Mediating Postanesthetic Memory Deficits Kirusanthy Kaneshwaran Master of Science Department of Physiology University of Toronto 2017 Abstract

Anesthetics cause postanesthetic memory deficits in animal models, and similar deficits may contribute to postoperative delirium and cognitive dysfunction in patients. We previously showed using a mouse model that etomidate-induced persistent memory deficits result from increased tonic GABAA receptor-mediated inhibitory current in the hippocampus, through an astrocyte-mediated mechanism.

However, the underlying mechanisms are uncertain.

The aims of this study were to: 1) determine whether widely used anesthetic and sedative agents persistently increase tonic GABAA current; 2) determine whether human astrocytes mediate the increase, and 3) identify the underlying mechanisms. The results show that several GABAergic drugs persistently increase tonic current and human astrocytes release soluble factors that mediate etomidate’s effect on tonic current. Furthermore, etomidate and sevoflurane act through astrocytic

GABAA receptors to trigger a pro-inflammatory signaling pathway, which underlies the persistent increase in tonic current. These results increase the mechanistic understanding of postanesthetic memory deficits and delineate treatment targets.

Acknowledgements

During the past two years I have undergone immense growth as a researcher and more importantly, as an individual. I have been privileged enough to be surrounded by wonderful people who have supported me and encouraged me in all my endeavors, research-related and otherwise.

I would like to thank my supervisor, Dr. Beverley Orser for your immense support and guidance. You have offered me pearls of wisdom pertaining to not only research, but also life. I strive to be as strong and inspiring a woman as you are in my future. Until then “I will fake it, till I become it”.

Thank you for giving me this opportunity to work with amazing individuals and hone my research skills.

I also thank the members of my Advisory Committee, Dr. Rob Bonin and Dr. Richard Horner for your time, support, excellent advice (and reference letters upon short notice!). Your advice has really helped me develop my ideas and realize that there is still so much to learn. You have helped me develop a love for learning through example. I would also like to thank Dr. Richard Horner for providing me with an important learning experience as a TA.

I would like to convey my sincerest gratitude to the entire Orser Lab. Thank you Dr. Sinziana

Avramescu for being my “laboratory mom”: giving me hugs when I needed them most, listening to my worries, sharing my happiness, supporting me even when I was at my worst, and so much more. My gratitude cannot be put in to words. You were everything I needed: a caring friend, an incredible mentor, an inspiring researcher, etc.. Thank you Dr. Dian-Shi Wang, for being one of the best teachers I have had in my life! Your guidance has really helped me improve my critical thinking, organizational, and even technical and math skills! Thank you Dr. Irene Lecker for giving me some of the best advice I have ever received and for supporting me throughout this phase of my life. Thank you Fariya Mostafa for being an incredible friend. I have become a better person through our friendship, and I hope to one day be as kind and caring an individual as you are. I am extremely grateful that you were with me

iii through the majority of these past two years: as a fellow laboratory mate to discuss experiments and results, as a friend to share both sad and happy things alike, and as an older sister who gave me incredible advice and support. Thank you Dr. Shelly Au, for being a great friend, listening to my worries and teaching me about life. Thank you Shahin for being my rock, inspiring me to have a love for science and helping me out whenever I needed it. Thank you Nathan Chan for being my ray of sunshine! Thank you Agnes Crnic for being you! Your great humor, Microsoft Word and PowerPoint skills, and friendship have made my life significantly more enjoyable! Also, thank you to everyone who has been a part of the lab: Michael, Alejandro, Dr. Gang Lei, Dr. Junhui Wang, Dr. Yu Feng Xia, Sean Haffey and

Dr. Agnes Zurek. I am extremely privileged to have worked with you in the last two years. I would also like to thank all my fellow Physiology graduate students and GASP (Melanie, Ankur, Hanna, Susmita,

Farwah, Vivian, Feiya…), who have given me valuable experiences and tolerated my craziness.

Last but not least, I would like to thank my family, who has provided me with immense support during the past two years. Thank you for tolerating me when I was at my worst, keeping me humble when I was at my best, and supporting and encouraging me whenever I needed it. Thank you Appa for listening to me talk about science and life during our car rides and giving me your opinion and advice.

Thank you Amma for listening to me ramble and vent and supporting me in all that I do. I can never repay you for all that you do for me, and I sincerely value every little thing that you do to make my life better. Thank you Guhaverl and Lokeesan for letting me burrow your stuff and take up your time.

These two years have been the best years of my life thanks to you, and I will cherish my relationship with all of you and the experiences we have had together as precious memories for the rest of my life!

List of Contributions

Kirusanthy Kaneshwaran produced all of the results and content of this thesis except those listed below.

The electrophysiological data presented in Figure 4.1 were collected with the help of Ms. Fariya

Mostafa, Dr. Sinziana Avramescu, Dr. Irene Lecker, and Dr. Dianshi Wang. They contributed to the recording of tonic current in neurons using different anesthetics. Also, in Chapter 5, Dr. Sinziana

Avramescua and Dr. Irene Lecker contributed recordings from neurons for data presented in Figure 5.1C, and Mr. Sean Haffey provided data for Figures 5.3B and 5.4A.

This work was completed with the financial support from a Kirk Weber Graduate Award provided by the

Department of Anesthesia, Sunnybrook Health Sciences Centre and an Ontario Graduate Scholarship.

Table of Contents

Abstract ...... ii Acknowledgements ...... iii List of Contributions ...... v Table of Contents ...... vi List of Figures ...... viii List of Abbreviations ...... ix Chapter 1. Thesis overview ...... 1 1.1. Rationale ...... 1 1.2. Hypothesis and specific aims ...... 6 1.3. Thesis structure ...... 9 Chapter 2. General Introduction ...... 11 2.1. Postoperative delirium and cognitive dysfunction ...... 11 2.1.1. Defining postoperative delirium and POCD ...... 11 2.1.2. Incidence ...... 12 2.1.3. Effects on patient outcomes ...... 14 2.1.4. Risk factors and potential causes of postoperative delirium and POCD in patients ...... 15 2.2. Cognitive deficits after anesthesia in animal models ...... 19 2.2.1. Evidence in the literature ...... 19 2.2.2. Proposed mechanisms of postanesthetic cognitive deficits in animals ...... 20 2.3. GABA and GABAA receptors-mediated inhibition ...... 24 2.3.1. GABA: synthesis, release, transport and metabolism ...... 24 2.3.2. Overview of GABA receptors ...... 26 2.3.3. GABAA receptors mediated inhibition ...... 27 2.3.4. GABAA receptors antagonists (bicuculline) ...... 31 2.3.5. Subunit composition of GABAA receptors ...... 31 2.3.6. Synaptic GABAA receptors ...... 33 2.3.7. Extrasynaptic GABAA receptors ...... 34 2.3.8. α5 subunit containing GABAA receptors ...... 36 2.4. General anesthetics, benzodiazepines and GABAA receptors ...... 42 2.4.1. General anesthetics ...... 42 2.4.2. Types of anesthetics and their use ...... 43 2.4.3. Mechanism(s) of action...... 49 2.4.4. Benzodiazepines ...... 49 2.4.5. Mechanism(s) of action...... 52 2.5. Astrocytes ...... 53 2.5.1. Role in cognitive behaviours (memory) ...... 56 2.5.2. Anesthetic action on astrocytes ...... 57 2.5.3. Role in postanesthetic cognitive deficits ...... 60 2.5.4. GABA receptors in astrocytes ...... 63 2.5.5. α5GABAA receptors in astrocytes ...... 65 2.5.6. Astrocyte-neuron interactions ...... 65 vi

2.6. Inflammation and anesthesia ...... 66 2.6.1. Anesthetic-induced inflammation ...... 66 2.6.2. Inflammation and cognition (memory) ...... 68 2.6.3. IL-1 secretion and signaling ...... 69 2.6.4. IL-1β and cognition (memory) ...... 70 2.7. Summary ...... 71 Chapter 3. General Materials and Methods ...... 72 3.1. Study approval ...... 72 3.2. Electrophysiology in cell culture ...... 72 3.1.1. Preparation of primary cell cultures ...... 72 3.1.2. Human optic nerve head astrocyte cultures ...... 74 3.1.3. Whole-cell voltage-clamp recordings in cell culture ...... 74 3.2. Preparation of pharmacological agents used in vitro ...... 76 3.3. Statistical analyses ...... 81 Chapter 4. Multiple GABAergic anesthetics trigger a persistent increase in tonic current in hippocampal neurons ...... 82 4.1. Introduction ...... 82 4.2.Methods ...... 84 4.1.1. Preparation of primary cell cultures ...... 84 4.1.2. Whole-cell voltage-clamp recordings in cell culture ...... 84 4.1.3. Statistical analyses ...... 85 4.3. Results ...... 86 4.4. Discussion ...... 90 Chapter 5. Mechanism(s) mediating anesthetic-induced persistent increase in tonic current ...... 93 5.1. Introduction ...... 93 5.2. Methods ...... 96 5.1.1. Preparation of primary cell cultures ...... 96 5.1.2. Whole-cell voltage-clamp recordings in cell culture ...... 96 5.1.3. Statistical analyses ...... 97 5.3. Results ...... 98 5.3.1. Activation of astrocytic α5 GABAA receptors ...... 98 5.3.2. Inflammatory pathway mediating postanesthetic increase in tonic current ...... 101 5.4. Discussion ...... 107 Chapter 6. General Discussion ...... 110 6.1. Summary ...... 110 6.1.1. A central role for astrocytes in postanesthetic memory deficits ...... 111 6.1.2. A role for α5GABAA receptors in cognition ...... 113 6.1.4. Common mechanisms and targets: Implications for treating memory disorders ...... 114 6.2. Future directions ...... 116 6.3. Conclusion ...... 119 Chapter 7. References ...... 121

vii

List of Figures

Figure 1.1. Hypothesized mechanism of anesthetic-induced increase in tonic current ...... 7

Figure 4.1. Multiple general anesthetics and a benzodiazepine trigger a persistent increase in tonic current in neurons ...... 87

Figure 4.2. Human astrocytes can mediate the etomidate-induced increase in tonic current in neurons ...... 89

Figure 5.1. Astrocytic α5GABAA receptors mediate persistent increase in tonic current in neurons induced by etomidate and sevoflurane ...... 100

Figure 5.2. Etomidate-induced persistent increase in tonic current may be mediated by an inflammatory pathway ...... 102

Figure 5.3. Etomidate- and sevoflurane- induced persistent increase in tonic current in neurons is mediated by IL-1 receptors activity ...... 104

Figure 5.4. Etomidate- and sevoflurane- induced persistent increase in tonic current in neurons is mediated by p38-MAP kinase phosphorylation ...... 106

viii

List of Abbreviations

α5GABAA α5 Subunit-containing γ-aminobutyric acid subtype A

(receptors) ACM Astrocyte conditioned medium

AMPA α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate

ANOVA Analysis of variance

APV (2R)-Amino-5-phosphonovaleric acid

ATP Adenosine triphosphate

BIC Bicuculline

CA1 Cornu Ammonis area 1

CA3 Cornu Ammonis area 3 cAMP Cyclic adenosine monophosphate

CNQX 6-Cyano-7-nitroquinoxaline-2,3-dione

D-AP5 D(-)Aminophosphopentanoic acid

ECF Extracellular fluid

EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid

GABA γ-Aminobutyric acid

GABAA γ-Aminobutyric acid subtype A (receptors)

GABAB γ-Aminobutyric acid subtype B (receptors)

GABA-T γ-Aminobutyric acid transaminase

Gabrα5-/- α5 Subunit-containing γ-aminobutyric acid subtype A gene deletion

GAD Glutamate decarboxylase

GAT γ-Aminobutyric acid transporter

GFAP Glial fibrillary acidic protein

GIRK G protein-coupled inwardly-rectifying potassium channels

GTP Guanosine triphosphate

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid i.p. Intraperitoneal i.t. Intrathecal

ICF Intracellular fluid

IL-1α Interleukin-1α

IL-1β Interleukin-1β

IL1-ra Interleukin-1 receptors antagonist

IL-6 Interleukin-6

IPSC Inhibitory postsynaptic current

KCC2 K+ - Cl- co-transporter

LTD Long-term depression

LTP Long-term potentiation

MAC Minimum alveolar concentration mIPSC Miniature inhibitory postsynaptic current

NKCC1 Na+-K+-Cl- co-transporter

NMDA N-methyl-D-aspartic acid

RONRs Reactive oxygen & nitrogen radicals

TEA Triethylammonium

TNFα Tumor necrosis factor α

TTX Tetrodotoxin

Vm Membrane potential

WT Wild type

Chapter 1. Thesis overview

1.1. Rationale

General anesthetics that are used during surgery may contribute to cognitive deficits that persist after the anesthetics have been eliminated from the body (Moller, et al. 1998). Such prolonged cognitive deficits typically involve deficits in executive function and memory (Price et al., 2008; Silverstein et al.,

2007), and are an underlying feature of postoperative delirium and cognitive dysfunction. Postoperative delirium and post-operative cognitive dysfunction (POCD) result in billions of dollars in increased health care costs per year (Moller et al., 1998, Djaiani et al., 2016) and are associated with poor long-term outcomes, including loss of independence, premature retirement and increased mortality (Steinmetz et al., 2009).

A systematic review revealed that postoperative delirium occurs in 11-50% of patients undergoing non-cardiac surgery, where the prevalence increases with age and varies with the type of surgery (Inouye et al., 2014). POCD occurs in around 1 in 3 patients at 1 week after surgery and 1 in 10 patients at 3 months after surgery (Weiser et al., 2008). Furthermore, approximately 14% of patients experience memory decline 3 months after surgery and 9% of patients show declines in both memory and executive function (Price et al., 2008). Thus, it becomes very important to develop both preventative and restorative treatments for these disorders.

Although the specific mechanisms underlying these postoperative cognitive disorders are unknown and are believed to be multifactorial, many preclinical studies indicate that general anesthetics are a major contributing factor (Inouye et al, 2014; Mo & Zimmermann et al., 2013; Sanders et al., 2011;

Rappaport et al., 2015). Nevertheless, the use of general anesthetics is unavoidable during surgery and, but no adequate strategies are available for treating postoperative delirium and POCD (Inouye et al.,

1 2014). The aim of my project was to determine how general anesthetics cause persistent long- term cognitive deficits that last long after anesthetics have been eliminated.

Given that over 312.9 million anesthetics are administered each year (Weiser et al., 2016), new prevention strategies and treatments identified in my studies will have broad implications for improving patient care. Identifying strategies that prevent the harmful effects of anesthetics will help reduce the incidence of postoperative delirium and POCD. My studies will also provide insight into cellular mechanisms of memory and behaviour, and particularly the role of astrocytes. In this thesis, I explore the cellular and molecular mechanisms underlying postanesthetic cognitive deficits using an in vitro cell culture model and electrophysiological recording methods.

Most anesthetics achieve their desired effects of amnesia, sedation, unconsciousness and immobility by increasing the potency of γ-aminobutyric acid (GABA) at GABA type ‘A’ receptors (GABAA receptors), which in turn increases chloride influx and reduces neuronal excitability in the central nervous system (Orser et al., 2008; Rudolph & Antkowiak, 2004). GABAA receptors that are expressed in neurons have a well-defined role in learning and memory. Our laboratory has shown that the effects of anesthetics do not cease once the drugs are eliminated, as previously believed (Zurek et al., 2014).

Specifically, we have shown that the anesthetic etomidate triggers a persistent increase in extrasynaptic

GABAA receptors function in hippocampal neurons that persists long after the anesthetics have been eliminated and this increase in GABAA receptors function may underlie persistent postanesthetic memory deficits (Zurek et al., 2014). Specifically, etomidate increases extrasynaptic GABAA receptors cell-surface expression, resulting in enhanced tonic GABAA receptor-mediated inhibitory current in the hippocampus. Inhibiting GABAA receptors reverses memory deficits after etomidate exposure.

Our previous studies were conducted using the prototypic anesthetic etomidate, which is not commonly used in clinical practice. Thus, further studies that use more commonly used anesthetic drugs

2 are required to increase the physiological and clinical relevance of our previous findings. Furthermore, these initial studies were conducted using murine cell cultures. Experiments using human cell cultures will support the notion that the mechanisms delineated from our studies underlie postanesthetic cognitive deficits in humans. Several clinical studies suggest that more commonly used general anesthetics and sedatives, such as the injectable anesthetic propofol, the inhalational anesthetics isoflurane and sevoflurane, and the GABAergic benzodiazepine midazolam can also contribute to postoperative cognitive deficits (Royse et al., 2011; Zhang et al., 2012; Rortgen et al., 2009; Rasmussen et al., 2009). It is currently unknown whether the persistent cognitive deficits caused by these drugs are mediated by a sustained increase in extrasynaptic GABAA receptors function in hippocampal neurons.

Also, ketamine, which can be used to initiate and maintain anesthesia, has been associated with attenuation of postoperative cognitive deficits following surgery (Deiner et al., 2009; Hudetz et al.,

2009). The effect of ketamine on tonic GABA current has not been previously investigated.

Findings from our laboratory implicate glial cells in the development of these postanesthetic cognitive deficits. Glia play a central role in network plasticity and cognitive behaviors. Glia, and particularly astrocytes, communicate bi-directionally with neurons (Perea et al., 2009; Araque et al.,

2008) and contribute to the learning and memory processes. For example, astrocytes release soluble signaling factors that induce and regulate long-term potentiation (LTP) of synaptic transmission, a cellular model of memory (Ota et al., 2013; Henneberger et al., 2010). However, despite significant advances, we do not fully understand the function of astrocytes and their contribution to cognitive behavior. Previous findings from our laboratory suggest that astrocytes play a central role in mediating anesthetic-induced persistent increase in GABAA receptors function, underlying memory loss. Etomidate exposure failed to cause a sustained increase in tonic current when neurons were treated alone, or when co-cultured with microglia. In contrast, etomidate consistently triggered an increase in the tonic current when neurons were co-cultured with astrocytes. When astrocytes alone were treated with

3 etomidate and conditioned medium from these astrocytes was applied to neurons, the tonic current was increased for at least 24 h after etomidate treatment. Heating the conditioned medium from astrocytes exposed to anesthetics prevented the increase in tonic current in neurons, suggesting that a heat-sensitive solute triggers the increased tonic current in neurons. Collectively, these results suggest astrocyte–neuron coupling mediates the anesthetic-induced increase tonic current that underlies postanesthetic cognitive deficits and specifically that anesthetics act through astrocytes to cause the release of soluble factor(s) that trigger a persistent increase in tonic GABA current. Given these results, there are two main questions that need to be addressed: 1) how are astrocytes stimulated to release soluble factors by anesthetics; and 2) what is the mechanistic pathway mediating anesthetic-induced persistent increase in GABAA receptors function.

Our laboratory and others have shown that one particular subtype of GABAA receptors, α5 subunit- containing GABAA receptors (α5GABAA receptors) critically regulate acquisition and extinction of memory (Collinson et al., 2002; Yee et al., 2004). These receptors play a significant role in hippocampal physiology and the formation of hippocampus-dependent memory. Specifically, these receptors regulate the excitability of hippocampal pyramidal neurons, the stimulatory threshold that is required to induce long-term potentiation (LTP), and hippocampus-dependent memory (Bonin et al., 2007; Martin et al.,

2010). Our laboratory has shown that pharmacological blockade or genetic deletion of α5GABAA receptors in mice after exposure to anesthetics prevents impairment of hippocampus-dependent memory (Saab et al., 2010; Zurek et al., 2012). Specifically, inhibition of α5GABAA receptors improves memory performance and reverses memory deficits after anesthetic exposure, whereas activation of

α5GABAA receptors causes memory deficits (Rudolph et al., 2006; Martin et al., 2010; Wang et al., 2012).

These effects have been solely attributed to α5GABAA receptors that are expressed in neurons.

However, α5GABAA receptors are also expressed in astrocytes (Bovolin et al., 1992).

4 Several results support the idea that astrocytes express GABAA receptors (Fraser et al., 2005;

MacVicar et al., 1989). Preliminary data from our laboratory show that astrocytes contain functional

α5GABAA receptors and that activation of α5GABAA receptors triggers the release of soluble factors that may underlie anesthetic-induced memory deficits. Specifically, Western Blot showed that the α5 subunit is expressed in astrocytes. Furthermore, GABA current recorded from cultured astrocytes or astrocytes in slices can be inhibited by an inverse agonist specific for α5GABAA receptors, L-655,708, and this effect is abolished in slices from Gabra5-/- mice. α5GABAA receptors on astrocytes may be the major subtype of GABAA receptors mediating the increase in the neuronal tonic current. The following evidence supports this idea: 1) preliminary results show that α5GABAA receptors are expressed in astrocytes (as mentioned before); 2) astrocytic GABAA receptors exhibit a high affinity for GABA and benzodiazepine- sensitivity, which are signature characteristics of α5GABAA receptors; 3) previous published results from our laboratory show that pre-treatment with an inverse agonist (L-655,708) which preferentially inhibits

α5GABAA receptors, prevents post-anesthetic memory deficits in mice; and 4) when cocultures of astrocytes and neurons are treated with L-655,708 and etomidate, the tonic current in neurons is not increased. These findings lead to the question of whether astrocytic GABAA receptors, and in particular

α5GABAA receptors, are key regulators of memory processes and mediate anesthetic-induced memory deficits.

The identity of the factor(s) and the signaling pathway that mediate the increase in tonic GABA current in neurons is currently unclear. Our laboratory has characterized a mechanism underlying inflammation-induced persistent memory deficits. We reported that an increase in cell-surface expression of α5GABAA receptors may underlie hippocampus-dependent memory deficits caused by inflammation (Wang et al., 2012). Specifically, we showed that inflammation (induced through lipopolysaccharide treatment), which triggers the release of the pro-inflammatory cytokine interleukin-

1β (IL-1β) (Hu et al., 2010; Vacas et al., 2013), causes a persistent increase in α5GABAA receptors surface 5 expression in cultured hippocampal neurons. Also, an IL-1β-induced increase in tonic GABA current and memory deficits are mediated by a signaling pathway involving the IL-1 receptors and phosphorylation of a downstream factor, p38 mitogen-activated protein kinase (p38-MAP kinase) (Wang et al., 2012).

Furthermore, elevated levels of IL-1β are associated with inflammation-induced memory deficits

(Serrantes et al., 2006; Cibelli et al., 2010). Another paper from our laboratory also shows that inflammation, and more specifically pre-treatment of mice with IL-1β, increases neuronal sensitivity to general anesthetics by increasing the function of extrasynaptic GABAA receptors (Avramescu et al.,

2016). These findings suggest that anesthetic-induced memory deficits may be mediated by an inflammatory pathway.

In conclusion, previous findings from our laboratory and others provide the basis for a hypothesis regarding a mechanism that underlies anesthetic-induced cognitive deficits.

1.2. Hypothesis and specific aims

I hypothesized that anesthetics activate α5GABAA receptors expressed in astrocytes to trigger the release of soluble factors. Furthermore, I postulate that these soluble factors then act on neurons through an inflammatory pathway to cause a persistent increase in tonic current (Figure 1.1).

Specific Aims

1. To test whether commonly used general anesthetics (i.e. isoflurane, sevoflurane, and propofol) and a benzodiazepine (i.e., midazolam) can trigger a persistent increase in tonic current.

2. To determine whether anesthetics act on human astrocytes to release soluble factors that increase the tonic current.

3. To determine whether anesthetics activate astrocytic α5GABAA receptors to trigger the downstream increase in neuronal tonic current.

6 4. To determine whether an inflammatory pathway mediates anesthetic-induced persistent increase in tonic current.

Soluble factor(s)

α5GABAAR

Figure 1.1 Hypothesized mechanism of anesthetic-induced increase in tonic current.

The anesthetic binds to α5GABAA receptors on astrocytes, which triggers the release of a soluble factor that acts on IL-1 receptors on neurons. This then leads to phosphorylation of p38-MAP kinase, which leads to a persistent increase in tonic current in neurons.

8 1.3. Thesis structure

In Chapter 2, I present an overview of the relevant literature concerning postoperative cognitive deficits and the proposed mechanisms for these deficits derived through animal models. I also review

GABAA receptor-mediated inhibition, anesthetics, astrocytes, and inflammation.

In Chapter 3, the methods used in my experiments are described in detail to ensure the reproducibility of my findings.

In Chapter 4, I present the results that address Aims 1 and 2. I use the in vitro model developed in our laboratory to model postanesthetic cognitive deficits. Previously, we showed using murine cultures that a prototypical anesthetic etomidate causes a persistent increase in tonic current in hippocampal neurons. Here, I show that more commonly used general GABAergic anesthetics, including the inhalational anesthetics isoflurane and sevoflurane, the injectable anesthetic propofol, and a benzodiazepine midazolam, cause a persistent increase in tonic current, similar to etomidate. Ketamine, an anesthetic that inhibits NMDA receptors, does not cause a persistent increase in tonic current.

Furthermore, I show that conditioned medium from human astrocytes treated with anesthetics also mediate the anesthetic-induced increase in tonic current in neurons. Collectively, these results enhance the clinical and physiological relevance of our previous findings and further suggest that the persistent increase in tonic current may underlie cognitive deficits in humans induced by commonly used general anesthetics and benzodiazepines.

In Chapter 5, I present the results that address Aims 3 and 4, which together seek to determine the signaling pathway mediating anesthetic-induced increase in tonic current in hippocampal neurons. I use the previously developed in vitro model of postanesthetic cognitive deficits to elucidate the cellular and molecular components of this mechanism. I show that co-application of etomidate or sevoflurane with L-655,708 prevented the increase in tonic current in astrocyte-conditioned medium experiments.

9 Next, based on our results from a previous study that investigates memory deficits caused by inflammation (Wang et al., 2012), I hypothesized that a similar mechanism may underlie postanesthetic cognitive deficits. Thus, I examined the roles of the IL-1 receptors and the p38-MAP kinase signaling pathway in the anesthetic-induced increase in tonic current. I show that the anesthetic-induced increase in tonic current is prevented when neurons are treated with an IL-1 receptors antagonist (IL-1ra) and a specific p38 MAPK inhibitor (SB-203,580). Collectively, these results demonstrate that activation of astrocytic α5GABAA receptors is necessary for anesthetics to cause a persistent increase in tonic current and that the neuronal signaling involves IL-1 receptors and p38 MAP kinase.

Finally, in Chapter 6, I summarize my main findings and discuss how these results compare to relevant current literature. I also offer some insight into the future directions, which may stem from my findings.

10 Chapter 2. General Introduction

2.1. Postoperative delirium and cognitive dysfunction

2.1.1. Defining postoperative delirium and POCD

A subset of patients who undergo surgery and anesthesia may experience postoperative

cognitive disorders, which manifest as impairments in learning, memory, attention, and executive

cognitive function. Two major categories of postoperative cognitive disorders that are of particular

interest in relation to our studies are postoperative delirium and postoperative cognitive dysfunction

(POCD).

Postoperative delirium and POCD can be loosely distinguished based on the time course and the

clinical features for each respective disorder. Postoperative delirium is diagnosed 24 – 72 h after surgery

and may emerge in the operating theatre or postanesthesia care unit. In contrast, POCD is usually

diagnosed weeks to months after surgery (Silverstein, 2007). Furthermore, there are differences in the

symptomatic presentation of these postoperative disorders. Postoperative delirium is characterized by

an acute state of confusion with disruptions in attentiveness, alterations in psychomotor functions, and

reduced awareness of the environment (Inouye et al., 1990). A key component of postoperative delirium

is the fluctuating levels of disorientation experienced by patients throughout the day, and can often

include the development of hallucinations or inappropriate behaviors (Lipowski, 1987; Inouye et al.,

1990). Contrastingly, patients with POCD are typically oriented but display a substantial decline from

their baseline cognitive performance, as assessed by tests of learning, memory, attention, and executive

function (Hanning, 2005; Wu et al., 2004; Makensen et al., 2004; Morandi et al., 2012; Deiener &

Silverstein, 2009).

Postoperative delirium and POCD are also likely related to each other. Postoperative delirium is

often a precursor to POCD (Tsai et al., 2010), and in some patients the two disorders can often be

difficult to distinguish (Lewis et al., 2004). Further issues with categorizing these two disorders occurs 11 due to the inconsistent definitions of POCD used in the literature. Investigators often define POCD to suit their methodology (Lewis et al., 2004). For example, reviews of POCD after cardiac and noncardiac surgery reveal that the two most commonly assessed cognitive domains assessed were 1) learning and memory and 2) attention and concentration. 97% of studies including patients undergoing cardiac surgery and 70% of studies including patients undergoing noncardiac surgery included a memory and learning test; 94% of studies including patients undergoing cardiac surgery and 57% of studies including patients undergoing noncardiac surgery included an attention and concentration test. About one-third of studies included tests of verbal and language skills and tests of visual and spatial skills and few studies included tests of numerical reasoning (6% for both types of subjects) or executive function (14% for cardiac surgery and 6% for noncardiac surgery subjects). Consequently, it is difficult to determine whether patterns of deficits in specific domains emerge across studies. One review concluded that declines in memory and attention and psychomotor function were consistently found after coronary artery bypass graft, but stated that the domains of POCD detected are likely to be a function of the tests used to assess POCD.

Therefore, both postoperative delirium and POCD result in long-term consequences and may be related, and although they can be defined and certain criteria and tests can be used for assessment, there are still some limitations in understanding these disorders.

2.1.2. Incidence

While an alarming number of patients who undergo surgery and general anesthesia incur cognitive impairments, the incidence and severity of postoperative delirium and POCD remains debated due to the variability reported in literature. This variability is the result of several factors believed to influence the reported incidence. Sources of this variability include the type of surgical procedure (Monk et al., 2008; Rasmussen, 2006), the age of the patient (Price et al., 2008; Rasmussen, 2006), variations in the methods used to perform the neuropsychological assessment and the definitions of these disorders

12 that are used for assessment (Lewis et al., 2004; Rasmussen, 2006). Predisposing factors including medication or substance abuse also contribute to variability in the reported incidence of these disorders

(Monk & Price, 2011; Whitlock et al., 2011; Krenk et al., 2011; Rudolph & Marcantonio, 2011).

The variability in the incidence of postoperative delirium and POCD stems in part from the nature of the surgery. The reported incidence of postoperative delirium varies considerably (9 – 87%).

For instance, hip fracture patients have a high incidence of postoperative delirium (35 – 65%) (Weed et al., 1995; Alcover et al., 2013), which may be partly due to the urgency of surgery and high comorbidity of this population. Abdominal surgery (34 – 54%) and coronary artery bypass graft surgery (37 – 52%)

(Rudolph & Marcantonio, 2011; Roach et al., 1996; Maldonado et al., 2003; Alcover et al., 2013) are also associated with a high incidence of postoperative delirium.

Cardiac surgery appears to carry the highest risk of POCD, with the incidence ranging from 30-

80% of patients three weeks after surgery, and 10- 60% three to six months after surgery (Rasmussen,

2006). However, many types of surgical patients are at risk of persistent deficits. One study reported that after major non-cardiac surgery, 56% of patients incurred cognitive impairments at discharge, while

24.9% exhibited symptoms three months after major non-cardiac surgery (Price et al., 2008). Another study reported that POCD occurs in 10-49% of patients aged 18 years or older one week after noncardiac surgery (Coburn et al., 2010).

Another variable that contributes to discrepancies in the reported incidences of postoperative delirium and POCD is age. Although postoperative delirium and POCD can affect patients of any age, the incidence is greater in elderly patients who have undergone major surgeries (Bedford, 1995). Elderly patients present with POCD symptoms at higher incidences and with greater severity than younger patients (Monk et al., 2008; Rasmussen, 2006). The International Study of Postoperative Cognitive

Dysfunction (ISPOCD I) found that around 25% of elderly patients undergoing major non-cardiac surgery presented symptoms of POCD 1 week after surgery and around 10% had symptoms three months after surgery (Moller et al., 1998). In another study, around 45% of patients with an average age of 60 years 13 experienced POCD 6-12 weeks after thoracic or vascular surgery (Rasmussen et al., 2001). Only a few studies have reported the incidence of POCD in younger patients. One such study, a prospective longitudinal study, reported that the incidence of POCD at the time of discharge in young (18-39 years), middle aged (40-59 years) and elderly (60+ years) patients was 36.6%, 30.4% and 41.4%, respectively, and 5.7%, 5.6% and 12.7%, respectively, at three months after surgery (Monk et al., 2008). Thus, even younger patients are at risk. Notably, this study also showed that only elderly patients had an incidence of POCD at significantly higher rates than non-surgical age-matched controls (Monk et al., 2008).

Collectively, these studies suggest that POCD occurs at all age groups, but incidence increases with age, likely due to the multitude of medical co-morbidities in elderly patients.

Finally, the reported incidences of postoperative delirium and POCD vary depending on the diagnostic criteria and statistical methodologies used for the analyses (Lewis et al., 2004; Rasmussen,

2006). Some of the variation in the reported incidence of postoperative delirium and POCD can be attributed to a lack of standardized testing across studies and challenges in assessment. Previous studies did not utilize standardized tests to assess the occurrence of postoperative delirium. Although recent clinical studies use validated testing such as the Confusion Assessment Method for the ICU (CAM-ICU) and the Intensive Care Delirium Screening Checklist (ICDSC) (Mo & Zimmermann, 2013; O’Keeffe et al.,

1997; Ely et al., 2001; Gaudreau et al., 2005; Alcover et al., 2013), further work needs to be done to truly capture the rates of postoperative cognitive disorders in surgical patients. Furthermore, as mentioned, it is difficult to distinguish POCD from other states of confusion such as postoperative delirium or pre- existing signs of dementia (Lewis et al., 2004), which also lead to variations in reported incidences.

Despite these discrepancies it is clear that these disorders invariably continue to affect a staggering number of patients (Rasmussen & Siersma, 2004; Steinmetz et al., 2009).

2.1.3. Effects on patient outcomes

Postoperative delirium is associated with increased morbidity and healthcare costs, as well as difficulties with tasks that are necessary for self-preservation and overall well-being (Rudolph & 14 Marcantonio, 2011, Monk & Price, 2011). Patients with postoperative delirium have even been shown to have higher mortality rates than their counterparts (Rudolph et al., 2007; Marcantanio et al., 1994;

Norkiene et al., 2007; Robinson et al., 2009; Koster et al., 2009). It has also been reported that patients with postoperative delirium take a significantly longer time to be discharged from the hospital, and elderly patients may require prolonged nursing (Norkiene et al., 2007), leading to increased healthcare costs of millions of dollars every year (Witlox et al., 2010).

Patients with POCD experience a noticeable decline in cognitive function, including memory loss and an inability to focus and concentrate. This cognitive decline may lead them to seek premature retirement, experience loss of independence, and demonstrate an increased need for social assistance

(Steinmetz et al., 2009; Alcover et al., 2013). One observational study which studied outcomes for a median of 8.5 years after surgery reports increased mortality at three months after surgery (Steinmetz et al., 2009). Furthermore, patients with POCD at one week postoperatively were at a higher risk of prematurely leaving the labour force due to disability or voluntary early retirement, and showed a greater dependency on social transfer payments, from 2 years following the operation (Steinmetz et al.,

2009). Another study showed that patients who displayed combined deficits in memory and executive function three months following surgery experienced a higher degree of functional impairments, which they described as difficulties with “instrumental activities in daily life” (Price et al., 2008).

Given that those afflicted with postoperative delirium and POCD experience profound cognitive deficits that impact their well-being, it is crucial that we identify the risk factors associated with these disorders and develop prevention and treatment strategies.

2.1.4. Risk factors and potential causes of postoperative delirium and POCD in patients

There are many risk factors associated with postoperative delirium and POCD, which are complex in nature. Postoperative delirium and POCD share many overlapping and inter-related risk factors, which can be broadly categorized as being either patient or surgery-based (Rundshagen, 2014,

15 Krenk et al., 2010; Alcover et al., 2013).

The most predictable patient-based risk factor for developing postoperative delirium and POCD is age (Bekker & Weeks, 2003; Rundshagen, 2014; Saleh et al., 2015; Sauer et al., 2009; Wang et al.,

2014). Aging is associated with impairments in the functionality of many physiological regulatory processes (Wang et al., 2014) and changes in pharmacokinetic and pharmacodynamic capacities, which may affect the inability to fully recover from anesthesia. For example, aged patients may have compromised renal and hepatic clearance, which can lead to prolongation of the elimination half-life of the drug and cause changes to the patients’ drug sensitivities (Mclean & Le Couteur, 2004). Elderly patients also have an increased likelihood of suffering from multiple concurrent medical conditions, which lead to increases in the likelihood of perioperative complications (Wang et al., 2014). However, there may be more literature supporting the notion that age is a risk factor for postoperative cognitive deficits because it is easier to detect cognitive disorders in adults, due to the availability of multiple test metrics (Cole et al., 2004; Inouye et al., 2005; Lewis et al., 2006; Silverstein et al., 2007).

Children may also be vulnerable to long-term impairments in executive function and neurodevelopment after surgery; however, it is difficult to assess their cognitive performance using standardized tests (Wilder et al., 2009; Millar et al., 2014; Miller et al., 2006). It is also difficult to evaluate the psychometric performance of pediatric patients due to rapid developmental changes and the medical conditions that created the need for the surgery in the first place (Janssen et al., 2011;

Millar et al., 2006; Todd et al., 2002). Finally, most clinical studies focus on the elderly population because they are more likely to undergo surgery than their pediatric counterparts.

Several other patient-related factors can increase the risk for postoperative cognitive disorders, including use of certain medications. Anticholinergic medications, such as atropine and scopolamine

(Bekker & Weeks, 2003; Rundshagen, 2014; Wang et al., 2014), benzodiazepines, dopamine antagonists,

α2 agonists, α1 antagonists, phenobarbital and opiate pain medications (Bekker & Weeks, 2003; Wang et al., 2014) increase the risk of developing POCD. Other risk factors include toxicity and abnormalities in 16 neurotransmitter levels resulting from multiple drug use, a history of alcohol addiction and abuse, severe illness such as organ failure, previous physical or cognitive damage, preexisting health conditions

(especially cerebral, cardiac or vascular disease), and even a lower level of education and lower socioeconomic status (Rasmussen et al., 2001; Whitlock et al., 2011; Guenter et al., 2011; Krenk &

Rasmussen, 2011; Vasilevskis, et al., 2012; Alcover et al., 2013; Bekker & Weeks, 2003; Rundshagen,

2014; Sauer et al., 2009; Wang et al., 2014; Schoen et al., 2011; Alcover et al., 2013). Lastly, some patients may naturally have low brain reserve which makes them more vulnerable to insults from surgery and anesthesia (Staz, 1993; Jankowski et al., 2011).

The most independent patient-based risk factor for the development of postoperative cognitive disorders is pre-existing cognitive impairment (Jones et al., 2010; Alcover et al., 2013). These patients may have an underlying cognitive insult that can be further potentiated with the stress of surgery and the effects of anesthestics. Other cognitive processes, such as a history of psychiatric conditions, especially depression, or deficits in visual or auditory function may also exacerbate the development of postoperative cognitive disorders (Greene et al., 2009; Smith et al, 2009; Kazmierski et al., 2006;

Kazmierski et al., 2010; Rudolph & Marcantanio, 2011).

There are some surgery-based risk factors for developing postoperative cognitive deficits; one such major risk factor is the type of surgery. There is a higher risk of developing POCD following extensive and highly invasive surgical procedures, such as hip-replacement, thoracic, abdominal and cardiac surgery, compared to minor surgical procedures (Rundshagen, 2014; Sauer et al., 2009; Wang et al., 2014). Furthermore, intraoperative and perioperative complications such as hypothermia, hypotension and hypoperfusion are associated with increased incidence and severity of POCD

(Rundshagen, 2014; Sauer et al., 2009).

Many clinical studies suggest that general anesthetics and sedatives, such as benzodiazepines, contribute to the development of postoperative delirium and POCD, especially in the elderly and children (Deiner & Silverstein, 2009; Newman et al., 2001; Caza et al., 2008; Marcantanio et al., 1994; 17 Alldred, 2011; Sanders & Maze, 2011; Alcover et al., 2013). Specifically, there are strong correlations between the use of different general anesthetics and benzodiazepines and the incidence of these postoperative cognitive disorders (Moller et al., 1998; Monk et al., 2008; Sanders et al., 2011; Seiber et al., 2011; Fong et al., 2006; Guenter et al., 2011; Schoen et al., 2011). For example, a sedative dose of propofol exacerbates the incidence of POCD (Rasmussen et al., 2003; Weber et al., 2009; Papaioannaau et al., 2005). Furthermore, the duration of anesthesia and depth of sedation exacerbate the prognosis of these disorders (Mandal et al., 2009; Steinmetz et al., 2010; Guenter et al., 2011; Alcover et al., 2013).

For example, studies have shown that the incidence of delirium is proportional to the quantities of propofol or opioid use (Alcover et al., 2013) and deeper sedation with propofol itself is associated with a higher incidence of postoperative delirium in hip fracture patients (Sieber et al., 2010; Sanders, 2011).

Lastly, studies suggest that the duration of POCD symptoms is proportional to the duration of action of the anesthetic agent (Rundshagen, 2014). Taken together, these studies strongly implicate general anesthetics and GABAergic sedatives in the pathogenesis of postoperative cognitive deficits.

One potential risk factor that remains controversial is the type of anesthetic that is administered. Although some studies suggest that the incidence of POCD following regional anesthesia is less than that of general anesthesia (Sauer et al., 2009; Wu et al., 2004), there is no conclusive evidence indicating regional anesthesia can mitigate the likelihood of developing POCD (Rundshagen,

2014). There is no definitive clinical data to suggest that the type of anesthetic drug influences the risk of developing POCD (Rundshagen, 2014; Sauer et al., 2009); however, there are some interesting findings that may support this notion. For example, one study shows that patients undergoing pulmonary surgery with isoflurane have a higher incidence of POCD than patients treated with desflurane or sevoflurane anesthesia (Tsai et al., 1992). Furthermore, recovery from cognitive deficits also appear to be slower for isoflurane, compared to other inhalational anesthetics (Dupont et al., 1999;

Mandal et al., 2009). More research is needed to investigate whether the type of anesthetic affects incidence of postoperative cognitive disorders. 18 Amongst the common risk factors for postoperative cognitive disorders outlined here, anesthesia is of particular interest. Given that millions of patients are exposed to prolonged sedation and anesthesia during surgery (Weiser et al. 2016), it is important to understand the mechanism of anesthetic-induced cognitive impairment to enable the development of treatment strategies. Clinical studies cannot be used to investigate the mechanism(s) underlying postanesthetic cognitive deficits and animal models are needed for this purpose.

2.2. Cognitive deficits after anesthesia in animal models

2.2.1. Evidence in the literature

The mechanism(s) underlying postoperative cognitive disorders are unknown but appear to be multifactorial as sedation regimen, sleep deprivation, and invasiveness of surgery have all been cited as contributing factors (Monk & Price, 2011; Alcover et al., 2013). Anesthesia, as described in the previous chapter, is likely an important factor contributing to postoperative cognitive disorders, and thus it is important to understand the role of anesthesia and surgery in causing cognitive deficits.

Since it is difficult to conduct clinical studies to understand the role of anesthetics in postoperative cognitive disorders, animal models, in which cognitive performance is studied following exposure to surgery with general anesthetics or anesthesia alone, are used instead.

Studies in aged mice show that anesthetics cause persistent effects on cognition and memory.

Anesthesia alone causes impairment in performance in a Y-maze task, used to assess cognitive performance, in mice aged 20-22 months (Qian et al., 2015). These impairments appear to be long term, as adult and elderly mice exposed to general anesthesia show deficits in memory tasks that persist for weeks after exposure (Culley et al., 2003; Culley et al., 2004). Furthermore, even brief anesthetic administration can have an effect, as a single exposure to isoflurane anesthesia leads to deficits in learning and spatial memory that persists for three weeks afterwards (Si et al., 2016).

Studies in neonatal and juvenile animals also demonstrate significant effects of anesthesia in

19 learning and memory. Fetal exposure to isoflurane anesthesia causes impairments in learning and memory, assessed using the Morris Water Maze task (Wang et al., 2016). The effects have also been seen in young rodents, as postnatal exposure to anesthetics leads to significant learning and memory deficits that last for up to three months into adulthood (Brambrink et al., 2010; Slikker et al., 2007;

Murphy et al., 2013). The effects are comparable to those often seen in aged animals, as studies performed on neonatal and aged rats demonstrate that anesthesia can cause deficits in various retrograde memory tasks that persist even two weeks after exposure for both groups of animals (Crosby et al., 2005; Culley et al., 2004).

Taken together, these studies suggest that anesthetics alone can cause significant cognitive impairments that can be assessed using various assays, and implicate animal models as an effective way to study the nature of postanesthetic cognitive impairments and their underlying mechanism(s).

2.2.2. Proposed mechanisms of postanesthetic cognitive deficits in animals

Impairments in neurogenesis, synaptogenesis, increased apoptosis and neuroinflammation have been implicated as key mechanisms that may underlie the pathogenesis of postoperative cognitive disorders and specifically postanesthetic cognitive deficits. In this section, I will be focusing on studies using animal models, as animal studies enable us to investigate the mechanism(s) of postanesthetic cognitive deficits.

Defeats in neurogenesis and increased apoptosis have garnered considerable attention as potential mechanisms underlying POCD. Neurogenesis is the coordinated proliferation, differentiation, migration, and integration of neuronal progenitor stem cells into cellular networks (Hudson &

Hemmings, 2011). Animal studies show that anesthetics disrupt neurogenesis at particular life stages.

For example, isoflurane decreases differentiation of neural progenitor stem cells in the dentate gyrus in postnatal day (P)7 rats, which may underlie anesthetic-induced deficits in hippocampal memory

(Stratmann et al., 2009). Exposure to a single dose of inhalational anesthetics can reduce neurogenesis

20 in the dentate gyrus of neonatal rodents that last for several weeks (Zhu et al., 2010; Stratmann et al.,

2010). This prolonged reduction in neurogenesis may contribute to impairments in hippocampal- dependent learning and memory during adulthood. Isoflurane exposure to young mice and rats causes a decrease in hippocampal stem cells and persistent reductions in neurogenesis, which underlie impairments in hippocampus-dependent memory (Zhu et al., 2010). In contrast, no effect is seen with exposure to older mice and rats (Zhu et al., 2010). Rats older than P60 do not display any deficits in neurogenesis or memory performance (Stratmann et al., 2009). However, anesthetics can decrease the number of new, maturing, and differentiating neurons in the adult rat brain (Lin & Zuo et al., 2011).

Thus, anesthetics significantly disrupt neurogenesis and memory, if rodents are exposed at a young age, but have limited effects when exposure occurs at an older age. Furthermore, despite the presence of studies that implicate impairment of neurogenesis as an underlying mechanism of postanesthetic memory deficits, no clinical evidence exists to support this notion.

Anesthetic-induced impairments in synaptogenesis in the developing rodent brain have also been implicated as a putative mechanism for postanesthetic memory deficits. Exposure to anesthesia in

P7 rats reduces synapse formation in the hippocampus and loss of neuroglia later in life (Lunardi et al.,

2010). Exposure to inhalational anesthetics in P16 rats reduces the diameter of apical and dendritic spines in cortical pyramidal neurons (Briner et al., 2010). Collectively, these studies provide a putative mechanism for how anesthetic-induced neurotoxicity in the developing brain can predispose patients to postanesthetic cognitive deficits at later life stages.

Apoptosis is another candidate mechanism for postanesthetic cognitive deficits. Apoptosis is the process of programmed cell-death, and has differential effects in young and aged brains (Sinner et al.,

2014). Apoptotic pruning of brain cells is required for normal cortical architecture and function in the developing brain (Hudson & Hemmings, 2011). Approximately 50-70% of developing neurons undergo apoptosis under normal conditions (Oppenheim, 1991). Similarly, in the elderly brain, age-related factors and underlying conditions cause neurons to undergo apoptosis. Thus, it is difficult to determine the 21 extent to which anesthetics cause apoptosis in and of themselves, if so, whether this process underlies postanesthetic cognitive deficits.

Studies in rodents and non-human primates of all ages suggest that anesthetics have proapoptotic properties, which correlate with anesthetic-induced memory deficits, with neonates being especially vulnerable (Sanders et al., 2009; Valentim et al., 2010; Brambrink et al., 2010). Isoflurane and propofol cause increased apoptosis and cognitive deficits in the developing brain that persist even during later stages in life (Sanders et al., 2013). Anesthetics exposure increases the expression of the apoptosis marker caspase-3 protein (Bekker et al., 2009; Slikker et al., 2007, Brambrink et al., 2010), and cyclin D1 and B-cell lymphoma 2 (Bcl-2) cell death proteins, which cause subsequent caspase activation and apoptosis in neonatal rodents (Liang et al., 2010; Walker et al, 2010; Bekker et al., 2009). Caspase activity also leads to neuronal death in adult and senile rodents (Fang et al., 2012; Komita et al., 2013;

Istanphanous et al., 2011; Mawhinney et al., 2012; Lin et al., 2011).

Several studies have investigated the pathway underlying anesthetic-induced neuroapoptosis.

Some of these studies implicate anesthetic-induced elevations in calcium as a mechanism underlying increased neuroapoptosis following anesthetic exposure. Isoflurane causes abnormal increases in cytosolic calcium by triggering the inositol-triphosphate (IP3) pathway, which exacerbates caspase activation and subsequent neuroapoptosis. The following evidence supports this notion: (1) chelation of calcium ions attenuates neuroapoptosis in cultured neurons (Zen et al., 2009; Stover et al., 2004); and

(2) pharmacologic inhibition of IP3 reduces intracellular calcium and caspase activation, thereby reducing neuroapoptosis (Wei et al., 2008; Inan et al., 2010).

Although the same mechanisms of apoptosis are seen in adult rodent models, the degree of anesthetic-induced neuroapoptosis is less severe than in younger rodent models (Lin & Zuo et al., 2011;

Pan et al., 2011; Hofecer et al., 2013). Isoflurane increases the expression of the apoptosis-related factor capsase-3 in the hippocampus of aged mice, which correlates with impairments in hippocampus- dependent memory (Qian et al., 2015). Taken together, these studies suggest that anesthetic-induced 22 neuroapoptosis may be a mechanism underlying postanesthetic cognitive deficits, especially in young animals.

Collectively, these studies suggest that an anesthetic-induced decrease in neurogenesis and increase in apoptosis may underlie postanesthetic memory deficits, mostly in young animals. Therefore, a more comprehensive pathophysiological mechanism is required to explain the acquisition of cognitive deficits following exposure to anesthetics. One potential mechanism that links anesthetic-induced deficits with neurogenesis and apoptosis is inflammation, as inflammation regulates neurogenesis and apoptosis. A pro-inflammatory cranial radiation therapy model in adult rats causes chronic inflammation, which reduces neurogenesis and causes cognitive decline (Monje et al., 2003). Treatment with a non-steroidal anti-inflammatory drug (indomethacin) restores neurogenesis and improves memory. Thus, neuroinflammation inhibits neurogenesis (Fan et al., 2014; Kubera et al., 2011).

Furthermore, proinflammatory cytokines, including TNFα and IL-1β, are also well-known for their ability to trigger apoptosis (Janes et al., 2005).

There is much evidence to support the notion that postoperative cognitive deficits can be caused by neuroinflammation during and following surgery. Surgery-induced infection or trauma can trigger a massive systemic inflammatory response that impairs hippocampus-dependent memory (Cibelli et al., 2010; Fidaldo et al., 2011; Terrando et al., 2010; Wan et al., 2007). According to the neuroinflammatory hypothesis, surgery and anesthesia can induce a “cytokine storm” (Osterholm,

2005), which is a widespread up-regulation of transcription, translation and secretion of proinflammatory cytokines that can cause significant cellular and tissue damage (Osterholm, 2005). This

“cytokine storm” may underlie postoperative cognitive deficits. The cytokines trigger neuroinflammatory processes through activating glia, such as microglia and astrocytes (Vacas et al., 2013), which produce additional cytokines (Cibelli et al., 2010; Santos-Galindo et al., 2011; Wan et al., 2007), including IL-1β.

IL-1β has significant effects on hippocampal function and memory, and is thus a major candidate as a mediator of postoperative cognitive deficits. Anesthetic exposure can also stimulate activation of 23 reactive astrocytes and thereby trigger the release of proinflammatory cytokines which result in neuroinflammation (Erasso et al., 2013). Collectively, these results suggest that neuroinflammation likely contributes to postanesthetic cognitive deficits.

Taken together, anesthetic-induced disruptions in neurogenesis, apoptosis, and neuroinflammation impair brain regions involved in cognitive function and thereby contribute to anesthetic-induced cognitive deficits, as demonstrated using animal models.

Studies have also implicated anesthetic-potentiation of GABAA receptors activity as a mechanism underlying postanesthetic cognitive, and specifically memory, deficits in rodent models (Zurek et al.

2014). Thus, the next chapter will provide a brief review of the GABA neurotransmitter system, before outlining the aforementioned mechanism for anesthetic-induced memory deficits.

2.3. GABA and GABAA receptors-mediated inhibition

2.3.1. GABA: synthesis, release, transport and metabolism

GABA, is a small amino acid, which acts as the primary inhibitory neurotransmitter in the mammalian central nervous system (Roth, et al. 2003). It exists as a zwitterion with a neutral charge at physiological pH (7.4) (Rowley, et al. 2012). The enzyme glutamic acid decarboxylase (GAD) synthesizes

GABA by catalyzing the removal of two CO2 groups from the amino acid glutamate. GAD exists as two isoforms (GAD67 and GAD65), which are encoded by separate genes that differ in their amino acid sequence, molecular weight, and distribution within neurons (Kaufman et al., 1991; Rowley et al., 2012).

GAD67, (also known as GAD1) is a 67 kDa protein (from which it derives its name). It is mostly predominantly found to be distributed broadly in the cytoplasm of GABAergic neurons and saturated with its activating cofactor, pyridoxal phosphate (PLP). PLP converts GAD67 into its activated holoenzyme form, so that it can produce GABA for metabolic purposes (Kaufman et al., 1991). GAD65 is a membrane-bound isoform, which is highly concentrated at axon terminals. Approximately 50% of

GAD65 exists in its active form, which produces GABA that is released via vesicular neurotransmission at

24 inhibitory synapses (Kaufman, et al. 1991; Walls, et al. 2010).

The vesicular GABA transporter (VGAT) is spatially and functionally coupled with GAD65. This coupling allows it to quickly and efficiently pack GABA into synaptic vesicles after it has been synthesized for neurotransmission in the presynaptic terminals of GABAergic interneurons (Chaudhry et al., 1998; Jin et al., 2003). The vesicles are docked at the plasma membrane and fuse with the plasma membrane in a

Ca2+-dependent manner when an action potential induces depolarization of the nerve terminal and activates voltage-gated calcium channels (Jin et al., 2003). The contents of the vesicles are then released into the synaptic cleft and GABA concentrations reach 1.5 – 1.8 mM (Barberis et al., 2004). Some of the spillover GABA is also found in the extracellular space (Lerma et al., 1986).

GABA is also released through non-vesicular methods of release (i.e. reverse GABA transporter

(GAT) activity in neurons and astrocytic GABA release through bestrophin-1 channels) (Heja et al., 2012;

Lee et al., 2011; Yang et al., 2014). Transport through the channels primarily contributes to ambient

GABA levels in the extracellular space, similar to spillover of GABA from the synaptic cleft (Song et al.,

2013). Extracellular GABA concentrations in the hippocampus range from 0.2 – 0.8 μM (Lerma et al.,

1986; Tossman et al., 1986; Avramescu et al., 2016).

Enzymatic breakdown of GABA does not occur in the extracellular space. Thus, it must be cleared from the synaptic cleft through passive diffusion and active reuptake. Four GAT isoforms (GAT1-

4) function as electrogenic co-transporters to allow both neurons and astrocytes to take part in GABA recycling, in addition to VGAT in neurons (Awad et al., 2009; Scimemi, 2014). These transporters use the inward driving forces of Na+ and Cl- to transport GABA molecules into the cell (Scimemi, 2014; Zhou &

Danbolt, 2013). GAT1 and 3 are mostly expressed in the brain (Zhou & Danbolt, 2013), and specifically in the cerebral cortex, while GAT2 and GAT4 are predominantly expressed in the liver, kidneys and leptomeninges (Conti, 2004; Zhou & Danbolt, 2013). GAT1 is predominantly expressed perisynaptically in neuronal axon terminals expressing GAD67 (Conti et al., 2004), but it can also be found in astrocytic processes (Melone et al., 2014). It regulates presynaptic homeostasis and phasic and tonic GABAA 25 receptor- mediated inhibition (Melone et al., 2014). GAT3 is predominantly expressed at distal astrocytic processes in extrasynaptic regions (Melone et al., 2014), and modulates GABA levels in the extracellular space (Conti et al., 2004; Melone et al., 2014). Recycled GABA is catabolized by another PLP-dependent enzyme, GABA transaminase (GABA-T) (Rowley et al., 2012) into succinic semialdehyde using either pyruvate or α-ketoglutarate as an amino-group acceptor (Shelp et al., 1999). Succinic semialdehyde is an intermediate in the tricyclic acid cycle (TCA), and therefore GABA in neurons and astrocytes is either used as a source of metabolic energy or is cycled back into the glutamine cycle for glutamate synthesis and resynthesis of GABA (Rowley et al., 2012; Shelp et al., 1999).

2.3.2. Overview of GABA receptors

In the central nervous system, GABA mediates inhibitory neurotransmission by targeting specific receptors in the postsynaptic neuronal membrane (Botzolakis, 2009). GABA receptors are categorized into two main groups: (1) GABAB receptors which are metabotropic and generate a slow, long-lasting response to GABA; and (2) GABAA receptors which are ionotropic and generate fast responses to GABA

(Olsen & Sieghart, 2008).

2.3.2.1. GABAB receptors

GABAB receptors are heterodimeric proteins consisting of two subunits, GABAB1 and GABAB2

(Bettler et al., 2004). As metabotropic receptors, they are coupled to G-proteins, especially the Gi/o family subtype. GABAB receptor signaling modulates ion channel and enzyme activity. When GABA or other ligands bind to the N-terminal of Gi/o-coupled GABAB receptor, the Gi/o heterotrimer exchanges a

GDP for a GTP and dissociates into Gαi/o and Gβi/o subunits (Pinard et al., 2010). The signaling pathways of Gαi/o are not well-studied but some studies suggest that Gαi/o inhibits adenylyl cyclase and lowers the levels of cyclic adenylyl monophosphate (cAMP) in the cell (Urwyler et al., 2001). Signaling pathways of

Gβi/o are well known to regulate the activities of ion channels close to the stimulated GABABR.

2+ Particularly, Gβi/o inhibits N, P, and Q-type voltage-gated Ca channels and activates G-protein gated inward rectifying K+ (GIRK) channels, which stimulate K+ efflux and prevent Ca2+ influx, resulting in 26 hyperpolarization (Mintz & Bean, 1993).

2.3.2.2. GABAA receptors

GABAA receptors are ligand-gated ion channels in the cysteine-loop superfamily of receptors that are structurally analogous to nicotinic acetylcholine receptors, glycine receptors, 5- hydroxytryptamine type 3 (5HT3) receptors and zinc-activated receptors (Sigel & Steinmann, 2012). GABAA receptors can be either homo- or heteropentamers, composed of five subunits forming a central pore permeable to Cl-

- and HCO3 (Sigel & Steinmann, 2012). Each subunit consists of a hydrophilic extracellular N-terminal domain coupled to a Cys-loop that plays an important role in ligand binding, four transmembrane α- helical regions (M1-4), and an extracellular C-terminal domain (Sigel & Steinmann, 2012). The N-terminal domain plays an important role in the assembly of subunits into a pentameric receptors, provides binding sites for allosteric modulators and forms five extracellular binding sites at the interface between subunits, two of which bind GABA molecules at the junctions between α and β subunits (Olsen &

Sieghart, 2009; Sigel & Steinmann, 2012). The M2 region lines the channel pore, while a large intracellular loop between M3 and M4 allows modulation of subunits by phosphorylation (Sigel &

Steinmann, 2012). All GABAA subunits share these commonalities, but each possesses distinct pharmacological properties and have distinct expression patterns.

2.3.3. GABAA receptors mediated inhibition

Ligand-gated ion channels, such as GABAA receptors, generate electrical currents by allowing ions to pass through their channel pore and across the cell membrane. These currents can be measured using whole-cell electrophysiology, which can be used to study the properties and functions of GABAA receptor-mediated currents in different brain regions (Hille, 2001). Ion channels move cations in or anions out of the cell to produce an inward current that increases the membrane potential to become more “positive” and depolarize the cell. Alternatively, they move cations out or anions into the cell to produce an outward current that decreases the membrane potential to become more “negative” and hyperpolarize the cell. The flow of ions, or amount of current, that flows through a channel is 27 determined by the channel’s conductance and membrane potential, and can be described by Ohm’s law.

Ohm’s law states that the current (I) through an ion channel is directly proportional to the membrane potential or voltage (V). So for a given membrane potential (V), an ion channel with a high conductance

(g) will have a larger current than one with low conductance (Hille, 2001). The equation for Ohm’s law is given below, where I is the current in amperes, g is the conductance, and V is the voltage in watts:

I = g V

The direction of current flow (i.e. into or out of the cell membrane) is determined by the electrochemical gradient of the ion in question, which gives rise to a driving force, which is determined by the difference between the cell membrane potential (Vm) and the equilibrium potential or reversal potential for that ion E (Hille, 2001). Therefore, the equation above can be revised so that driving force represents the voltage, as follows:

I = g (Vm – E)

The equilibrium or reversal potential of an ion depends on the concentrations of that ion on either side of the cell membrane, and is equal to the membrane potential at which its net current across the membrane is zero (Kandel et al., 2013). At the equilibrium potential of an ion, Vm = E, and so there is no driving force for the ion (Hille, 2001), and per Ohm’s Law, it follows that I = 0. Reversal potential for an ion, such as chloride, can be calculated using the Nernst equation, which accounts for the concentration of the ion on either side of the membrane:

RT [x]outside E = ln X zF [x]inside

Here, Ex is the reversal potential for ion X, R represents the thermodynamic gas constant, T represents the temperature in Kelvin, z represents the valence of the ion (e.g. +1 for Na and K, -1 for Cl ion), F represents Faraday’s constant (i.e., the amount of charge in coulombs per mole of ion), and X

- represents the ion. Note that if the valence is negative, as in the case of Cl , [X]outside and [X]inside are

28 written inversely due to the logarithmic equation.

The cell membrane is permeable to many ions, and so the relative permeability to these ions must be considered to calculate the or reversal potential. The mammalian nervous system is most permeable to K+, Na+, and Cl-, and so the equilibrium potential for a mammalian neuronal membrane is given by the Goldman-Hodgkin-Katz (GHK) equation, which is an extension of the Nernst equation:

+ + - Pion is the relative permeability of each ion, K , Na and Cl (Hille, 2001), which are 1.0, 0.04 and

0.45, respectively, at the resting membrane potential. Permeability of Na+ increases to 20 during an action potential (Kandel et al., 2013).

- - GABAA receptors are permeable to two anions, Cl and HCO3, which contribute to the

- - equilibrium potential for GABAergic neurotransmission (EGABA). At baseline, GABAA receptors are 5 times

- - more permeable for Cl ions compared to HCO3 ions, and consequently EGABA is closer to the equilibrium potential for Cl than HCO3, which is determined by intracellular chloride concentrations. Thus, EGABA, like

- - ECl-, is dependent on factors that regulate intracellular Cl concentrations ([Cl ]i )(Kandel et al., 2013).

- Historically, [Cl ]i has been thought to be determined by the activity of cation-chloride cotransporters (CCCs). In neurons, the Na+-K+-Cl- cotransporter (NKCC1) and the K+-Cl- cotransporter

(KCC2) are the primary CCCs (Glykys et al., 2014). NKCC1 uptakes Cl- by transporting 1 Na+, 1 K+, and 2 Cl-

- ions into the cell, resulting in a [Cl ]i equilibrium of around 60 mM (Glykys et al., 2014; Hartmann &

Nothwang, 2015; Kaila et al., 2014); KCC2 extrudes Cl- by transporting 1 K+ and 1 Cl- out of the cell,

- resulting in a [Cl ]i equilibrium of around 3 mM (Glykys et al., 2014; Hartmann & Nothwang, 2015; Kaila et al., 2014). Since these CCCs have different properties, imbalance in their expression can have significant effects on GABAergic transmission in development, plasticity and disease. During development, immature

29 neurons lack functional KCC2. Unopposed NKCC1 activity keeps [Cl-]i close to 60 mM, higher than in

- mature cells (Ben-Ari et al., 2007). Thus, the driving force on Cl is directed outward and GABAA receptors

- activation results in the depolarizing efflux of Cl and GABAA receptor-mediated excitatory signaling, which triggers intracellular Ca2+ transients that activate downstream signaling and have neurotrophic effects on the developing neuron (Ben-Ari, 2007; Owens & Kriegstein, 2002). As the neuron develops, it expresses- more functional KCC2 than NKCC1. Depending on the expression and activity levels of KCC2, GABAA receptors activation triggers either shunting or hyperpolarizing inhibition. If EGABA is equal to the Vm (-65 to

-70 mV in hippocampal neurons), GABAA receptors opening will increase conductance, which will “short- circuit” temporal and spatial summation of depolarizing excitatory postsynaptic currents (EPSCs) (Kaila et

- al., 2014). If EGABA is more negative than the Vm, GABAA receptors activation will trigger Cl influx leading to hyperpolarizing inhibitory postsynaptic current (IPSC) (Kaila et al., 2014).

- When GABAA receptors are over-stimulated, Cl influx can exceed removal, which may lead to temporary accumulation of Cl- in the cell, and consequently disruptions in the Cl- gradient and increased flux of HCO3 ions through GABAA receptors Consequently, EGABA shifts towards the more positive EHCO3-, and GABAA receptors activation leads to an initial hyperpolarization, followed by a depolarization

(Isomuara et al, 2003). GABAA receptors activation in mature neurons is only depolarizing under pathological conditions where the function or expression of KCC2 is downregulated (Kaila et al., 2014). An aberrant expression of KCC2 is implicated in many neuropathologies such as epilepsy and chronic pain

(Kaila et al., 2014). Thus, CCCs play an important role in regulating GABAergic transmission.

GABAA-mediated inhibition is also determined by the cellular localization of the GABAA receptors. GABAA receptors are either localized within the synapse, as synaptic GABAA receptors, which mediate phasic, synaptic inhibitory current, or far away from the synapse, as extrasynaptic GABAA receptors, which mediate tonic inhibitory current (Khale et al., 2008), depending on the subunit composition and brain area.

30 For the purposes of this thesis, I will only focus on GABAA receptors activation in mature hippocampal and cortical neurons where regular Cl- transport occurs through KCC2 and therefore GABAA receptors activation results in inhibitory current.

2.3.4. GABAA receptors antagonists (bicuculline)

Bicuculline (BIC) is a phthalide isoquinoline derivative (Curtis et al., 1971). It is both a competitive and allosteric GABAA receptor antagonist with strong proconvulsant and epileptic properties

(Johnston, 2013). Its initial interaction with the GABAA receptors occurs at the orthosteric GABA-binding site, where it reduces times and frequencies of opening. It also binds allosteric sites, where it stabilizes the closed state of the ion channel (Johnston, 2013). BIC can bind outside of the orthosteric pocket on an additional allosteric site because of its large molecular weight (i.e. approximately 3X that of

GABA) (Johnston, 2013). BIC is largely a specific and potent anti-GABAergic drug, other than minor inhibitory effects on Ca2+-activated K+ channels (Khawaled et al., 1999) and mammalian acetylcholinesterase (Olsen et al., 1976), which makes it very useful to study the physiology and pharmacology of GABAA receptors. BIC binds to specific residues of GABAA receptors. Substituting phenylalanine (Phe)64 with leucine (Leu) in the extracellular- N-terminus of the α subunit, which allows competitive blockers to interact with the orthosteric site of the receptors, decreases affinity by 60- to

200-fold (Ueno et al., 1997; Johnston, 2013). Mutating the tyrosine (Tyr)157 to Ser in the β2 subunit of

α1β2γ2 receptors results in BIC acting as a weak GABAA agonist (Ueno et al., 1997), supporting the notion that BIC binds competitively and allosterically to the GABAA receptors.

We chose to use BIC over other competitive inhibitors for this study, because: (1) it causes a near-complete block of GABAA conductance, and (2) its crystalline salt derivatives are highly water soluble.

2.3.5. Subunit composition of GABAA receptors

GABAA receptors are heteropentamers with variable subunit compositions. Although nineteen

31 subunits have been cloned from the mammalian CNS (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3), only some of the many possible combinations of these subunits exist (Farrant & Nusser, 2005; Mody & Pearce, 2004;

Olsen & Sieghart, 2009). There are basic rules of assembly, and differential expression patterns of subunit types across brain regions and neuronal populations (Farrant, 2005). Some of these subunits

(α1, β1, β2, β3, and γ2) are found throughout the brain, while others (α2, α3, α4, α5, α6, γ1, and δ) have region-specific expression patterns (i.e. α5 is predominantly expressed in the hippocampus) (Olsen &

Sieghart, 2009).

Studies using immunocytochemistry and electron microscopy provide insight into the colocalization patterns of most receptors subtypes. Most GABAA receptors consist of two α subunits, two β subunits, and one γ subunit arranged “pseudo-symmetrically” around the ion channel in the sequence γ-β-α-β-α (Farrant & Nusser, 2005), to create GABA-binding pockets at each of the β-α junctions (Sigel & Steinmann, 2012). α1, β2 and γ2 subunits constitute the most abundantly expressed receptors subtype in the brain, while other common assemblies include α2β3γ2, α3β3γ2, α4βxγ2,

α5β3γ2 and α6βxγ2 (Farrant & Nusser, 2005). Receptors subtypes with γ1, γ3 or δ subunits in place of the γ2 subunit are not common (Olsen & Sieghart, 2009). Other more complex subunit combinations are rare, but can be found. For instance, some pentamers may contain two different α or β isoforms, or the

γ subunit can be replaced with a δ, ε, π or θ (Farrant & Nusser, 2005). There is also a subclass of ionotropic GABA receptors composed entirely of ρ subunits, which may be classified as “type C” (GABAC) receptors, (Olsen & Sieghart, 2009). However, since ρ subunits can also form receptors with γ2 or with

α1 and γ2 (Farrant & Nusser, 2005), these receptors can be considered as “GABAA-ρ” receptors rather than being considered a distinct subclass (Olsen & Sieghart, 2009).

The molecular heterogeneity of GABAA receptors determines their pharmacological properties, cell surface distribution and dynamic regulation (Farrant & Nusser, 2005), which results in functional diversity of GABAA receptor-mediated inhibition.

32 2.3.6. Synaptic GABAA receptors

Synaptic GABAA receptors mediate phasic inhibition. The release of GABA from presynaptic terminals causes high GABA concentrations (0.3 – 1.0 mM) in the synaptic cleft that last for less than 1 ms (Mody & Pearce, 2004). This increase in GABA causes them to conduct large, transient inhibitory postsynaptic currents (IPSCs) (Farrant & Nusser, 2005; Mody & Pearce, 2004; Olsen & Sieghart, 2009).

The magnitude and duration of IPSCs can vary between synapses depending on the quantity and properties of the GABAA receptors that are activated (Mody & Pearce, 2004).

Some receptors subtypes can be characterized as “synaptic” GABAA receptors, although none of them exist exclusively on the postsynaptic membrane. Subunits that are predominantly found in postsynaptic membranes of GABAergic synapses include α1, α2, α3, α6, β2/3 and γ2 (Farrant & Nusser,

2005). GABAA receptors subtypes that mediate phasic synaptic inhibition predominantly contain a γ2 subunit associated with α1, α2 or α3 subunits (Farrant & Nusser, 2005).

The kinetics of IPSCs can vary between synapses. IPSC amplitudes can vary between synapses as there may be tens to hundreds of GABAA receptors in a given synapse (Farrant & Nusser, 2005; Mody &

Pearce, 2004). Presynaptic mechanisms such as multivesicular release can increase the peak GABA concentrations in the synaptic cleft and thereby affect the kinetics of IPSCs (i.e. increase amplitude and slow decay of IPSCs) through partially dictating the gating of ion channels (Mody & Pearce, 2004).

Specifically, GABAA receptors gating depends on the conformational state of its three-dimensional protein structure, which transitions between closed, open (ion-conducting) and desensitized (relatively long- lived, agonist-bound closed) (Farrant & Nusser, 2005), based on the temporal profile of GABA transmission, and the intracellular phosphorylation of different subunits (Mody & Pearce, 2004).

Synaptic GABAA receptors are characterized by their intrinsic biophysical properties in addition to their cellular localization and subunit composition. Measurements of two key electrophysiological parameters: (1) the concentration of GABA that elicits a half-maximal response (EC50), and (2) the rate and duration of current desensitization in the continued presence of GABA have helped characterize 33 that synaptic GABAA receptors exhibit a low affinity for GABA and rapidly desensitize (Farrant & Nusser,

2005). They may desensitize quickly to prevent excessive channel activation.

The α subunit is the primary determinant of GABA sensitivity in GABAA receptors containing α, β and γ subunits; α3 subunits confer the highest and α6 subunits the lowest EC50 values, corresponding to the lowest and highest affinities, respectively (Farrant & Nusser, 2005). Specifically, the order of affinities conferred by α subunits is: α6<α1<α2<α4<α5<<α3 (Bohme et al., 2004). The EC50 values range from 1 to 50 µm (Bohme et al., 2004), which lead to typically low GABA affinities in synaptic GABAA receptors. It makes sense that synaptic GABAA receptors would not have high sensitivity to GABA since

GABA concentrations can reach >1 mM in the synaptic cleft. Similar to affinity, desensitization rates of

αβγ and αβδ subtypes are also conferred primarily by the α isoform (Farrant & Nusser, 2005). Receptors with a α1βxγx composition desensitize more rapidly than those containing an α5 or α6 subunit, whereas the opposite trend is observed in αβδ receptors (Farrant & Nusser, 2005).

In summary, synaptic receptors that mediate phasic inhibition can be distinguished from extrasynaptic receptors that mediate tonic inhibition, based on their low affinity for GABA and rapid, prolonged desensitization (Farrant & Nusser, 2005).

2.3.7. Extrasynaptic GABAA receptors

Extrasynaptic GABAA receptors mediate slow, tonic inhibitory currents (Farrant & Nusser, 2005;

(Glykys & Mody, 2007; Mody & Pearce, 2004), which means that there is always a baseline current from these receptors (Glykys & Mody, 2007). Tonic current is quite small (0.5 pA to 40 pA) (Lee & Maguire,

2014), and so it can usually only be measured when the neuron is clamped at a specific membrane potential and a GABAA receptors antagonist is applied. GABAA receptors- mediated tonic current is found in multiple brain areas, including the hippocampus, cortex (layers I-V), and medulla (Lee et al., 2014).

Extrasynaptic GABAA receptors have a high affinity for GABA and are activated by low ambient extracellular concentrations (10 nM – 1 μM) (Farrant & Nusser, 2005), which may released from spontaneous or action potential induced synaptic release, reverse activity of GABA transporters, and 34 non-vesicular release from astrocytes (Lee et al., 2010; Song et al., 2013). The degree to which ambient

GABA levels are due to spillover of synaptic transmission or non-neuronal cells, such as astrocytes, is still being investigated.

As GABAA receptors are activated by low ambient extracellular GABA concentrations, and their affinity values are much higher than the affinity of synaptic GABAA receptors. The EC50 values for extrasynaptic GABAA receptors range from nanomolar (δGABAA receptors) to 30 μM (α5GABAA receptors) concentrations (Karim et al., 2013). Extrasynaptic GABAA receptors also have much longer activation times than synaptic GABAA receptors since they desensitize more slowly (Bianchi et al., 2002).

Similar to synaptic GABAA receptors, the subunit composition and expression patterns in the brain dictate inhibitory function of extrasynaptic GABAA receptors, among other properties (Lee &

Maguire, 2014). However, unlike synaptic GABAA receptors, the number of possible combinations of extrasynaptic GABAA receptors is limited. Extrasynaptic GABAA receptors contain the δ, α5 or ε subunits, or αβ subunits, exclusively (Glykys & Mody, 2007). δ-subunit-containing GABAA (δGABAA) receptors predominantly generate tonic conductance in most brain regions, including granule cells in the cerebellum and dentate gyrus, thalamic neurons, layer 2/3 pyramidal neurons, and interneurons of the dentate molecular layer (Farrant & Nusser, 2005; Glykys & Mody, 2007). They typically combine only with α4/6 and β2/3 subunits, and are diffusely scattered across the plasma membrane, specifically at the soma or perisynaptic regions (Belelli et al., 2009; Farrant & Nusser, 2005). Much of tonic inhibition in the brain is mediated by α4δ-containing GABAA receptors, apart from the cerebellum (which mostly contains α6δGABAA receptors) and the dentate gyrus (which mostly contains α1δGABAA receptors)

(Chandra et al., 2006; Glykys & Mody, 2007).

Another major subtype of extrasynaptic GABAA receptors is the α5 subunit-containing GABAA

(α5GABAA) receptors, which are critical in mediating tonic inhibition in CA1 and CA3 pyramidal cells, cortical layer 5 pyramidal cells and olfactory cortical neurons (Farrant & Nusser, 2005). Approximately

75% of these receptors are located extrasynaptically and most possess the α5β3γ2 configuration

35 (Loebrich et al., 2006). α5GABAA receptors-mediated tonic inhibition in the CA1 and CA3 regions of the hippocampus modulate hippocampal memory function (Caraiscos et al., 2004), which is the focus of this thesis.

2.3.8. α5 subunit containing GABAA receptors

α5-subunit containing GABAA receptors (α5GABAA receptors) generate most of the tonic inhibition that regulates the excitability of pyramidal neurons in CA1 and CA3 regions of the hippocampus (Caraiscos et al., 2004; Glykys and Mody, 2006, 2007; Pavlov et al., 2009; Prenosil et al.,

2006; Semyanov et al., 2004) and layer 5 cortical neurons (Yamada et al., 2007), even though they comprise less than 5% of all GABAA receptors in the brain (Piker et al, 2010).

To effectively mediate tonic current, extrasynaptic α5GABAA receptors must possess two crucial properties: (1) high affinity for GABA, enabling activation by near-nanomolar concentrations of the extracellular space, and (2) little or no channel desensitization (Farrant & Nusser, 2005; Glykys &

Mody, 2007; Mody & Pearce, 2004). α5GABAA receptors have a higher affinity for GABA than most GABAA receptors and are activated by low, ambient concentrations of GABA (Martin et al., 2009). α5 subunits possess a low EC50 of approximately 30 µm (Karim et al., 2013). The α5 subunit also confers slower desensitization, causing α5GABAA receptors to generate slowly desensitizing tonic inhibitory currents

(Karim et al., 2013). Although the α5 subunit can associate with multiple α, β and γ subunits, it is mostly found with the β3 subunit (Ju et al., 2009; Martin et al., 2009), and electrophysiological evidence shows that it can also be found as part of the α – γ2 combination (Martin et al., 2009; Savic et al., 2008).

Recombinant studies suggest that α5β3γ2 is the predominant configuration of GABAA receptors in CA1 hippocampal pyramidal neurons (Caraiscos et al., 2004; Martin et al., 2009).

Recently, researchers have focused on α5GABAA receptors as a target for cognition-enhancing compounds such as α5-selective inverse agonists, L-655,708 (L6), α5IA and RO4938583 (D'Hulst et al.,

2009), due to their unique pharmacological properties and expression profile in the hippocampus, which confer a central role for memory. However, the neuropharmacological properties of α5GABAA receptors 36 in the human brain must be investigated before such treatments can become available.

2.3.8.1. Physiological role in memory

Researchers have only recently begun to understand the physiological role of α5GABAA receptors in the mammalian central nervous system, in particular, as a key memory regulating receptors. Genetic studies in mutant mice lacking the α5 subunit (Gabrα5-/- mice), which demonstrated improved spatial learning in the Morris Water Maze (MWM), with an otherwise normal behavioural phenotype (Collinson et al., 2002), were the first to confer a role for α5GABAA receptors in memory processes. Following this, numerous genetic studies in mice have shown that α5GABAA receptor-mediated tonic inhibition is involved in hippocampus-dependent learning and memory (Martin et al., 2010). Gabrα5-/- mice show enhanced performance in various hippocampus-dependent learning and memory tasks (i.e. Morris water maze and trace fear conditioning) compared to wild-type controls (Collinson et al., 2002; Yee et al., 2004; Martin et al., 2010). Pharmacological studies show that inhibiting α5GABAA receptors before learning or during recall using L6, improves learning and memory in wild-type mice, tested using various behavioural assays (i.e. radial arm maze, Morris water maze, and fear conditioning) (Koh et al., 2013;

Martin et al., 2010), suggesting that α5GABAA receptors are involved in acquisition and recall of hippocampus-dependent memory (Collinson et al., 2006).

Electrophysiological studies have been used to characterize the cellular correlates of cognitive enhancement following reduced α5GABAA function. α5GABAA receptors have “non-synaptic” properties that make them ideal for facilitating tonic inhibition; namely high affinity for GABA, ability to detect ambient GABA concentrations, and slow desensitization kinetics (Caraiscos et al., 2004). α5 subunit expression is found predominantly in hippocampal CA1 pyramidal neurons (Pirker et al., 2000; Sur et al.,

1999). Furthermore, there is an inverse relationship between α5GABAA receptors function and hippocampus-dependent memory. These findings suggest that α5GABAA receptors play a critical role in restricting learning and memory by regulating tonic inhibition in the hippocampus (Caraiscos et al.,

2004). Bonin and colleagues (2007) demonstrated that α5GABAA receptors are instrumental in regulating 37 the excitability of hippocampal CA1 pyramidal cells. Specifically, fully functioning α5GABAA receptors typically hyperpolarize the resting membrane potential by ~3 mV (Bonin et al., 2007), leading to an increase in the amount of depolarizing current required to trigger an action potential (Bonin et al.,

2007). Martin and colleagues (2010) showed that α5GABAA receptors activity is directly proportional to the threshold stimulation required to induce LTP in CA1 neurons (Martin et al., 2010), thus leading to a

“LTP suppressing” effect and consequent constrains on hippocampus-dependent memory that is entirely independent of synaptic inhibition. Taken together, these results implicate α5GABAA receptors as key

“memory-blocking” receptors in the hippocampus and provide insight into the underlying mechanism involved.

2.3.8.2. Pathophysiological role

α5GABAA receptors are implicated in the pathophysiology of cognitive deficits associatd with developmental disorders such as Down’s syndrome and autism and injury processes such as inflammation and stroke (Martinez-Cue et al., 2013; Clarkson et al., 2010; Wang et al., 2012). Following its physiological role, aberrant α5GABAA receptors function has been implicated in neuropathologies affecting hippocampus-dependent memory (Wang et al., 2012; Zurek et al., 2012).

Our group investigated the relationship between α5GABAA receptor-mediated tonic inhibition and inflammation-induced memory deficits. We showed that inflammation-induced impairment of LTP in hippocampal CA1 neurons and hippocampus-dependent contextual fear memory can be reversed with pharmacological blockade (with L6) or genetic deletion of α5GABAA receptors (Wang et al., 2012).

The impairments in LTP are mediated by an increase in α5GABAA receptors expression (Wang et al.,

2012). Microglia secrete the inflammatory cytokine interleukin-1β (IL-1β), which acts on the IL-1 receptors in neurons, thereby activating the p38 MAP Kinase signaling cascade. This leads to an increase in the surface expression of α5GABAA receptors (Wang et al., 2012). Thus, α5GABAA receptors link inflammation and memory deficits.

Our laboratory has demonstrated that α5GABAA receptors play a role in anesthetic-induced 38 cognitive deficits (Zurek et al., 2014). Since anesthetics cause their clinical actions through acting on

GABAA receptors, we hypothesized that anesthetics also cause postanesthetic cognitive impairment through actions on GABAA receptors.

Our first studies investigated the effect of anesthetics on cognitive performance in rodents. Adult mice were administered a single dose of the anesthetic isoflurane and trained on the fear-conditioning task 1 or 24 h after exposure (Saab et al., 2010), and short- and long- term memory were assessed 30 minutes and 48 h after training, respectively. Mice showed short- and long-term memory impairments 1 h after isoflurane treatment, and only short-term memory impairments 24 h later. In other studies, adult mice were exposed to a single sedative dose of the anesthetic etomidate and trained in the novel object recognition task either 24 h, 72 h, or 1 week after exposure (Zurek et al., 2014) and memory was tested

24 h after training. Memory loss persisted for up to a week after exposure, suggesting that anesthetics cause long-term cognitive impairments.

We next investigated the mechanisms underlying the anesthetic-induced memory deficits that were observed. We first investigated whether anesthetics disrupt synaptic plasticity, which is a cellular correlate of memory in the hippocampus, long after the anesthetics are eliminated from the body. A sedative dose of etomidate (8 mg/kg) in vivo significantly reduces synaptic plasticity and causes a persistent increase in extrasynaptic tonic current, which last up to a week (Zurek et al., 2014), with no changes in postsynaptic GABAA receptor-mediated current. Thus, anesthetics cause persistent changes in tonic current in the hippocampus (Zurek et al., 2014).

We first became interested in α5GABAA receptors when postanesthetic memory impairments in the fear-conditioning task were abolished when mice were pretreated with L-655,708, a specific inhibitor of α5GABAA receptors (Saab et al., 2010). Memory deficits in the novel object recognition task caused by GABAergic anesthetics (i.e. etomidate, isoflurane, sevoflurane) result from a persistent increase in α5GABAA receptors function (Zurek et al., 2014), strongly implicating a role for α5GABAA receptors in postanesthetic cognitive deficits. α5GABAA receptors are mainly responsible for tonic 39 current in hippocampal regions involved in synaptic plasticity, so given our results where anesthetics cause persistent changes in synaptic plasticity, it makes sense that α5GABAA receptors play a role in anesthetic-induced persistent increase in tonic current. We conducted experiments to test this hypothesis. Specifically, null mutant Gabrα5-/- mice, lacking α5GABAA receptors and control mice were injected with the sedative dose of etomidate and hippocampal tonic current was measured from slices

24 h later (Zurek et al., 2014). Etomidate enhanced tonic current in wild-type mice, but failed to do so in mice lacking α5GABAA receptors (Zurek et al., 2014), suggesting that etomidate acts via α5GABAA receptors to cause a persistent increase in tonic current in the hippocampus.

We then investigated the mechanism that underlies etomidate-induced persistent increase in tonic current in the hippocampus. The fact that a single etomidate treatment causes a persistent increase in α5GABAA receptor-mediated tonic current suggests that etomidate increases the expression of α5GABAA receptors. This was verified using cell-surface biotinylation in mice hippocampal slices

(Zurek et al., 2014), which showed that etomidate and isoflurane induced increase in α5GABAA receptor surface expression lasts up to a week after treatment, with no effect on total protein expression in the hippocampus (Zurek et al., 2014). Collectively, these results suggest that anesthetics cause a persistent increase in hippocampal tonic current by increasing α5GABAA receptors cell surface expression.

As mentioned previously, anesthetic-induced increase in α5GABAA receptors surface expression lasts for a week, which is the same time-frame for which anesthetic-induced memory deficits are observed, suggesting that α5GABAA receptors may play a critical role in anesthetic-induced memory deficits.

Collectively, these studies suggest that a single anesthetic exposure causes memory deficits, which can be prevented by pharmacologic blockade of α5GABAA receptors and that anesthetic-induced increase in α5GABAA receptor-mediated hippocampal tonic current is central to the pathogenesis of postanesthetic memory deficits. The fact that there are similarities between inflammation and anesthetic induced memory impairment, as they are both mediated by an increase in α5GABAA

40 receptors function, suggests that these cognitive disorders share common underlying pathways.

2.3.8.3. Inverse agonists (L-655, 708)

L-655,708 (L6) is an imidazobenzodiazepine that is 50- to 100-fold more selective for α5GABAA receptors than GABAA receptors containing α1, α2 or α3 (Atack et al., 2006; Quirk et al., 1996). It is a potent partial inverse agonist, which means that beyond simply blocking the effect of GABA (as an antagonist does), it causes the reverse pharmacological effect of the agonist. The highest binding density of L6 takes place in the hippocampus, as α5β2γ2 receptor expression is highest in this region in rats and humans (Sur et al., 1998, 1999). Thus, L6 is a useful tool for studying hippocampus-dependent memory and LTP. L6 enhances LTP in mouse hippocampal slices and improves performance in the Morris water maze task, without the convulsant or proconvulsant properties of non-selective GABAA inverse agonists

(Atack et al., 2006; Chambers et al., 2004). Some evidence suggests that it has anxiogenic properties in animal models (Chambers et al., 2004; Navarro et al., 2002).

The α5 residues threonine (Thr)208 and isoleucine (Ile)215 each confer a 10-fold binding selectivity factor for L6 when compared to α1. Collectively, these residues confer 100-fold selectivity to

L6 (Casula et al., 2001). This is further validated as point mutations in the α1 subunit, where Ser205 is replaced with Thr or Val212 is replaced with Ile confer L6 selectivity similar to α5 subunits (Casula et al.,

2001). These residues form the benzodiazepine binding site with residues on the γ subunit of GABAA, which is where L6 binds the receptors (Atack et al., 2006; Casula et al., 2001; Chambers et al., 2004).

L6 offers exciting new possibilities in the development of cognition-enhancing treatment for humans and for studying the role of α5GABAA receptors in learning and memory. It displays a high level of α5- selectivity and is orally bioavailable (Chambers et al., 2004). L6 is suitable for in vitro studies in targeting α5GABAA receptors in vitro, but is not an ideal drug as it still possesses a low affinity for other α subtypes.

41 2.4. General anesthetics, benzodiazepines and GABAA receptors

2.4.1. General anesthetics

General anesthetics have made complex invasive surgeries tolerable, by enabling a complex sedative state characterized by immobility, amnesia, unconsciousness, and analgesia (Hemmings Jr. et al., 2005; Rudolph & Antkowiak, 2004). Anesthetics affect multiple neurotransmitter systems, brain regions and cognitive faculties. This chapter will focus on the pharmacological nature of GABAergic and non-GABAergic anesthetics.

General anesthetics have many different molecular structures and target different molecular and cellular substrates. There is no universal molecular mechanism underlying the actions of different anesthetics. Historically, there has been an erroneous theory that general anesthetics have a general, non-selective effect on consciousness via interactions and disruptions of the cellular membranes in the brain (Hemmings Jr. et al., 2005; Thompson & Wafford, 2001). However, decades of research have gradually led to the modern hypothesis that anesthetics target specific proteins, especially ligand-gated ion channels, and either reduce or enhance the function of that protein (Thompson & Wafford, 2001).

Anesthetics can be classified based on target specificity and routes of administration.

General anesthetics are broadly classified based on the route of administration. Inhalational anesthetics include the halogenated ethers and alkanes (i.e. isoflurane, sevoflurane, and diethyl ether), and other gaseous anesthetics (i.e. nitrous oxide and xenon) (Thompson & Wafford, 2001). Sometimes alcohols are included in this group as they may share similar mechanistic sites of action (Thompson &

Wafford, 2001). These anesthetics are largely GABAergic in nature. Intravenous anesthetics include barbiturates, propofol, etomidate and ketamine (Thompson & Wafford, 2001). Most of these compounds are also GABAergic, apart from ketamine, although their site of action differs from volatile anesthetics (Thompson & Wafford, 2001).

42 2.4.2. Types of anesthetics and their use

2.4.2.1. Etomidate

Etomidate [or (R)-(+)-ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate] is a prototypic intravenous anesthetic that acts primarily by potentiating GABAA receptors in the central nervous system

(Belelli et al., 1997). It also weakly potentiates glycine receptors, and has a weak inhibitory effect on nicotinic acetylcholine receptors (neuronal and muscular) (Rudolph & Antkowiak, 2004). Nevertheless, compared to other anesthetics, etomidate is a relatively GABA-selective drug (Garcia et al, 2010).

Etomidate’s pharmacokinetic properties make it well-suited for rapid intravenous use. It is short- acting, with a rapid onset that leads to its properties of having clinically insignificant hemodynamic changes and few side effects (Erdoes et al., 2014; Vinson & Bradbury, 2002), which make it useful in emergency procedures, especially rapid sequence intubation (Vinson & Bradbury, 2002). After intravenous induction, typically at a dose of 0.2 – 0.3 mg/kg (Erdoes et al., 2014), its onset of action occurs rapidly within 30 to 60 seconds with a peak effect at 1 minute (Bergen & Smith, 1997). Effects generally last three to five minutes (Bergen & Smith, 1997).

One of the few adverse effects of etomidate is adrenocortical suppression, as it disrupts cortisol and corticosterone synthesis. These are steroid hormones that regulate blood glucose levels in response to stress (Erdoes et al., 2014; Forman, 2011). Specifically, etomidate blocks the activity of 11β- hydroxylase, a cytochrome enzyme in the adrenal glands that converts 11-deoxycortisol and 11- deoxycorticosterone to cortisol and corticosterone, respectively (Erdoes et al., 2014; Forman, 2011).

Etomidate’s half-life values for distribution, redistribution and elimination are 2.7 minutes, 29 minutes and 2.9 – 5.3 hours, respectively (Bergen & Smith, 1997).

The molecular structure of etomidate contains one chiral carbon, and thus it can exist as either the R(+)-enantiomer and S(-)-enantiomer isoforms. The R(+)-enantiomer is the only isomer that is active as a hypnotic (Bergen & Smith, 1997), and is 100x more potent than the S(–)-enantiomer (Forman, 2011;

43 Proctor et al., 1986).

Etomidate’s ability to modulate and activate GABAA receptors is dependent on the β subunit present in the receptors. Namely, the GABA-modulatory and mimetic effects of etomidate, at clinical and supraclinical doses, respectively, can be seen in the presence of either β2 or β3 subunits over β1

(Belelli et al., 1997; Forman, 2011). Etomidate’s preference for β2/3 is conferred by a single amino acid in the M2 domain. A point mutation replacing asparagine (Asn)-289 with serine (Ser; the homologous residue in β1) markedly reduces etomidate’s GABA-modulatory and GABA-mimetic effects effects on

GABAA receptors (Belelli et al., 1997), while replacing with methionine confers insensitivity (Belelli et al.,

1997). Collectively, these pharmacodynamic properties make etomidate a unique anesthetic agent.

Etomidate has been used as our prototypic drug in previous studies in our laboratory for the following reasons. First, as a non-volatile anesthetic, etomidate is highly practical for the treatment of cell culture dishes. Volatile agents can dissipate and skew the effective dose of the treatment. Second, as mentioned earlier, etomidate is comparatively more selective for targeting GABAA receptors, which are the primary focus of the proposed mechanism in this thesis. Third, etomidate causes minimal cardiovascular or respiratory effects that would confound behavioral studies. Fourth, it has a rapid onset and is rapidly eliminated with no active metabolites. Fifth, etomidate analogues represent the next generation of anesthetics (Cotten et al., 2009). Therefore, although experiments must also be performed with other anesthetic agents, etomidate was best suited for our purposes in initial studies.

2.4.2.2. Isoflurane and Sevoflurane

Isoflurane and sevoflurane are general inhalational anesthetics used for induction or maintenance of general anesthesia. Isoflurane is not as widely used as other inhalational anesthetics as it may cause respiratory depression, low blood pressure and airway irritation due to its pungency

(Alagesan et al., 1987; Saraswat, 2015). More serious side effects include malignant hyperthermia and high blood potassium (McGuire & Easy, 1990; Visoiu et al., 2014; Wedel et al, 1993; Heard et al., 1998).

On the other hand, sevoflurane is one of the most commonly used inhalational anesthetic agents, as it 44 causes less irritation to mucous membranes and is a relatively safe drug (Delgado-Herrera et al., 2001).

Adverse side effects of sevoflurane use include respiratory depression and increase in intracranial pressure (Doi et al., 1987; Brown et al., 1998; Bundgaard et al., 1998; Goren et al., 2001). An increasing number of studies continue to investigate sevoflurane’s potential neurotoxic effect, especially when used for infants and children (Lu et al., 2010; Zhou et al., 2014; Jevtovic-Todorovic et al., 2013).

The exact mechanism(s) of action of isoflurane and sevoflurane have not been clearly delineated. Isoflurane has been shown to bind to GABA, glycine and glutamate receptors with different effects (Lugli et al., 2009; Garcia et al., 2010; Henschel et al., 2008; Mihic et al., 1997; Campagna et al.,

2003). Isoflurane potentiates glycine receptors activity to cause a decrease in motor function and inhibits receptors activity in the NMDA glutamate receptorsubtypes (Lugli et al. 2009). Sevoflurane is a positive allosteric modulator of the GABAA receptors, acts as an NMDA receptors antagonist, potentiates glycine receptor currents, and inhibits nicotinic acetylcholine and serotonin 5-HT3 receptors (Lugli et al.,

2009; Garcia et al., 2010; Henschel et al., 2008; Mihic et al., 1997; Campagna et al., 2003). Since the effect of these anesthetics on GABAA receptors is the primary focus of our studies, I will elaborate further on the literature concerning this topic.

Understanding of the molecular pharmacology of isoflurane and sevoflurane has been enhanced by studies implicating GABAA receptors as a critical target site for these anesthetic drugs. Isoflurane and sevoflurane enhance the amplitude of responses to low concentrations of GABA and prolong the duration of GABA-mediated synaptic inhibition (Harrison et al., 1993; Garcia et al., 2010). At supraclinical concentrations, isoflurane and sevoflurane can open GABAA receptors anion channels in the absence of

GABA in a process known as “direct activation” (Garcia et al., 2010) There is a limited understanding of which receptors subunits are required for the actions of isoflurane and sevoflurane, despite many studies using the appropriate concentrations of anesthetics for in vitro studies (Franks et al., 1996;

Harrison et al., 1993). EC50 for general anesthesia translate to the following micromolar concentrations that should be used for in vitro experiments: 280 µM isoflurane, 330 µM, sevoflurane (Franks et al., 45 1996). It is known however, that all GABAA receptors containing an α subunit are sensitive to at least one inhalational general anesthetic. Co- transfection of HEK293 cells with β1 subunit cDNAs with either an

α1 or an α2 subunit cDNA is sufficient to confer sensitivity to isoflurane. Thus, the binding site(s) for isoflurane and sevoflurane are likely located within α and β subunits (Harrison et al., 1993).

Research on the effects of isoflurane and sevoflurane on extrasynaptic GABAA receptors in hippocampal brain slice preparations suggest that extrasynaptic GABAA receptors exhibit higher affinities for GABA and decreased desensitization after exposure to these anesthetics (Bai et al., 2001). The

α1β2γ2s, α5β2γ2s, or α6β2γ2s subunit combinations exhibit enhanced GABA sensitivity with isoflurane in a dose-dependent fashion (Carasicos et al., 2004). Furthermore, low, sub-anesthetic concentrations of

- isoflurane potentiate the Cl current at α5β3γ2L GABAA receptors but not at α1β3γ2L GABAA receptors in hippocampal neurons (Caraiscos et al., 2004). There is still a lack of information on the effect of isoflurane and sevoflurane on different GABAA receptors populations in the central nervous system. In conclusion, sevoflurane and isoflurane increase the affinity of GABAA receptors for GABA, while little is known about their relative effects on different GABAA receptors subunits.

2.4.2.3. Propofol

Propofol (2,6, diisopropylphenol) is one of the most extensively used intravenous anesthetic agents. It is commonly used to start and maintain general anesthesia, sedate mechanically ventilated adults who are not undergoing surgery (such as patients in the intensive care unit) and for procedural sedation (Miner et al. 2007). Propofol has been widely used for sedation and induction of anesthesia due to its fast induction, short-acting nature, and rapid and clear recovery. It has its maximal effect at two minutes after administration and its effect lasts typically five to ten minutes (Miner et al. 2007;

Larijani et al. 1989).

Propofol has more pronounced hemodynamic effects than many other intravenous anesthetic agents (Weisenberg et al., 2010). Other side effects include low blood pressure related to vasodilation, transient apnea following induction doses, and cerebrovascular effects (Weisenberg et al., 2010). 46 Recreational use of propofol via self-administration is relatively rare due to its potency and the level of monitoring required for safe use, but has been reported nevertheless due to its desired short term effects of mild euphoria, hallucinations, and disinhibition (Tezcan et al., 2014). The steep dose- response curve of propofol makes potential misuse very dangerous without proper monitoring, and deaths from self-administration continue to be reported (Tezcan et al., 2014). Attention to the risks of off-label use of propofol increased after the death of Michael Jackson following use of a mixture of propofol and the benzodiazepine drugs lorazepam, midazolam and diazepam.

Propofol has been proposed to have several mechanisms of action, but has been shown to primarily act through potentiation of GABAA receptors activity and by acting as a sodium channel blocker

(Yamanoue et al., 1994; Alkire et al., 2001). Propofol’s actions on GABAA receptors are of interest to our studies, and therefore are further delineated here. Propofol potentiates GABA responses and directly activates GABAA receptor function (Alkire et al., 2001). α, β and γ subunits all confer GABAA receptor sensitivity to propofol (Hales et al., 1991; Jones et al., 1995; Lam et al., 1998). Propofol is less efficacious at β1 containing receptors than at those containing β2 or β3 subunits (Sanna et al., 1997).

The potency and efficacy of propofol at GABAA receptors is dependent on key moieties, namely the phenolic hydroxyl group and the number and arrangement of methyl groups at the 2- and 6- positions that flank it (Krasowski et al., 2001). The discovery of the essential pharmacophore for propofol’s action, has led to the development of FOS-propofol, a new anesthetic that releases a propofol molecule after liver metabolism. FOS-propofol is a water-soluble anesthetic that has slower induction kinetics than its active metabolite, which may be desirable in some surgical settings (Moore et al., 2001).

Interestingly, propofol may be actively involved in the recruitment of new subunits to the surface of the neuron. Namely, quantitative PCR revealed that during deep anesthesia, propofol increases α4 subunit mRNA (Sekine et al., 2006).

2.4.2.4. Ketamine

Ketamine is a phencyclidine derivative widely used as a short-acting “dissociative” general 47 anesthetic (Bergman et al., 1999). Ketamine causes limited cardiovascular and respiratory depression compared to most other general anesthetics, and so it is frequently administered for the induction of anesthesia in hemodynamically unstable patients, brief surgical procedures, treatment of mass casualties in disaster relief efforts, and combat-related injuries (Bergman et al., 1999; Mulvey et al.,

2006; Morris et al., 2009).

Ketamine acts as a noncompetitive and uncompetitive antagonist for excitatory glutamatergic N- methyl-D-aspartate (NMDA) receptors, which primarily mediate their anesthetic effects (Bergman et al.,

1999; Orser et al., 1997). Ketamine also modifies the function of other neurotransmitter receptors and ion channels, including acetylcholine receptors, hyperpolarization- activated cyclic nucleotide-gated channel, voltage gated ion channels, and dopamine and opioid receptors (Chen et al., 2009; Duan et al.,

2003; Hirota et al., 1996; Hustveit et al., 1995; Sanacora et al., 2015; Schnoebel et al., 2005).

Studies examining the effects of ketamine on GABAA receptors have produced inconsistent results. Some studies suggest that ketamine does not act on GABAA receptors. Namely, one study shows that ketamine does not affect GABAA receptor-mediated currents in the dorsal root neurons of the spinal cord in vitro or the inhibitory effects of GABA on excitatory firing in vivo (Anis et al., 1983;).

Contrastingly, other studies suggest that ketamine has actions on GABAA receptors (Gage et al., 1985;

Little et al., 1984; Scholfield et al., 1980; Lin et al., 1992; Wakasugi et al., 1999). In vitro studies show that ketamine enhances GABAA receptor-mediated inhibition, behavioral studies in mice show that bicuculline attenuates ketamine-induced loss of righting reflex (Ueno et al., 1997; Irifune et al., 2000), and human brain imaging studies suggest that S-ketamine increases the function of GABAA receptors in the prefrontal cortex (Heinzel et al., 2008). Results from a study published from our laboratory shows that ketamine acutely increases the function of extrasynaptic GABAA receptors at high concentrations in cortical and hippocampal neurons, but has no effect on synaptic currents (Wang et al., 2017). Given these findings, it is of interest to investigate the persistent effects of ketamine on extrasynaptic GABAA receptors activity, at clinically relevant concentrations. 48 2.4.3. Mechanism(s) of action

Most inhaled and intravenous anesthetics function by enhancing GABAA receptors function through allosterically binding to GABAA receptors and increasing affinity for GABA (Rudolph &

Antkowiak, 2004; Urban et al., 2006). This leads to increased opening time of the channel pore when

GABA directly activates the receptors (Rudolph & Antkowiak, 2004), and consequently increased Cl- flux across the plasma membrane and reduced neuronal excitability. Most general anesthetics require the presence of GABA to affect GABAA receptors function at clinical concentrations, but many of the intravenous drugs can also directly activate the receptors in the absence of GABA at higher concentrations (Moody et al., 1998; Urban et al., 2006).

Anesthetics have different effects on neuronal excitability, and even consciousness, depending on the subunit composition and cellular localization of the activated GABAA receptors. Synaptic and extrasynaptic GABAA receptors interact with anesthetics differently due to differences in their kinetic and pharmacodynamic properties. Isoflurane prolongs inhibitory postsynaptic currents when it binds to synaptic subtypes (Hemmings Jr. et al., 2005; Jones & Harrison, 1993), while anesthetics enhance overall tonic inhibition when they bind to extrasynaptic GABAA receptors (Bai et al., 2001). α5GABAA receptors, which are primarily found in the hippocampus, are especially sensitive to low concentrations of propofol and isoflurane (Hemmings Jr. et al., 2005), so it is highly plausible that they may be a neural substrate for the amnestic properties of anesthetics.

2.4.4. Benzodiazepines

Benzodiazepines are a class of psychoactive drugs whose core chemical structure is the fusion of a benzene ring and a diazepine ring (Richter et al. 2011; Sieghart et al. 1994). Benzodiazepines enhance the effect of GABA at the GABAA receptors, resulting in sedative, hypnotic, anxiolytic, anticonvulsant, and muscle relaxant properties (Fox et al. 2011; Sieghart et al. 1994). Benzodiazepines also have a dose- dependent ventilatory depressant effect, reduce arterial blood pressure and increase heart rate

49 because of a decrease in systemic vascular resistance (Fox et al. 2011). Benzodiazepines are categorized as either short-, intermediate-, or long-acting, by its elimination half-life (Fox et al. 2002).

Short-acting compounds, such as midazolam, have a median half-life of 1-12 hours; intermediate-acting compounds have a median half-life of 12-40 hours; long-acting compounds, such as diazepam, have a half-life of 40-250 hours (Fox et al. 2002). Short- and intermediate-acting benzodiazepines are preferred for the treatment of insomnia; longer-acting benzodiazepines are recommended for the treatment of anxiety. High doses of shorter-acting benzodiazepines may also cause anterograde amnesia and dissociation.

Benzodiazepines are generally safe and effective for short-term use, although they can cause cognitive impairment and paradoxical effects such as aggression, behavioral disinhibition and worsened agitation or panic. Benzodiazepines are also associated with increased risk of suicide. Long-term use can result in adverse psychological and physical effects, decreasing effectiveness, and physical dependence and withdrawal. Benzodiazepines can be overdosed and can cause dangerous deep unconsciousness, although they do not result in death when taken alone. Benzodiazepines are commonly misused when taken in combination with other drugs of abuse or other central nervous system depressants such as alcoholic drinks and opioids, which increases the potential for toxicity and overdose.

Benzodiazepines share a similar chemical structure. The term “benzodiazepine” is the chemical name for the heterocyclic ring system, which is a fusion between the benzene and diazepine ring systems (Fox et al. 2011; Kelly et al. 2002; Sieghart et al. 1994). Benzodiazepine drugs are substituted

1,4-benzodiazepines, and different benzodiazepine drugs have different side groups attached to the central structure. Benzodiazepines predominantly have their effects in humans through allosteric modification of the GABAA receptors , which increases the overall conductance and results in the various therapeutic and adverse effects of benzodiazepines (Kelly et al. 2002; Sieghart et al. 1994). The different side groups affect the binding of the benzodiazepine molecule to the 50 GABAA receptors and consequently modulate pharmacological properties. Many of the pharmacologically active, classical benzodiazepine drugs contain the 5-phenyl-1H-benzo[e] [1,4] diazepin-2(3H)-one substructure (Kelly et al. 2002; Sieghart et al. 1994).

The four widely used benzodiazepines are midazolam, diazepam and lorazepam and the antagonist flumazenil. Midazolam, diazepam and flumazenil are metabolized by cytochrome P450 (CYP) enzymes and glucuronide conjugation, whereas lorazepam directly undergoes glucuronide conjugation. The duration of action of all benzodiazepines is strongly dependent on the duration of their administration.

Midazolam has the shortest recovery profile. Midazolam, diazepam and lorazepam are widely used for sedation and induction and maintenance of anaesthesia. Flumazenil can be used to reverse benzodiazepine-induced sedation and diagnose benzodiazepine overdose.

2.4.4.1. Midazolam

Midazolam is a benzodiazepine used during anesthesia, procedural sedation and severe agitation

(Kissin, 1993; Reves et al., 1985). Midazolam can be delivered through various methods, namely orally, intravenously, by injection into a muscle, sprayed into the nose, or in the cheek (Reves et al., 1985).

Midazolam is short-acting with an elimination half-life of 1.5-2.5 hours (Reves et al., 1985). Midazolam is metabolised by cytochrome P450 (CYP) enzymes and by glucuronide conjugation into an active metabolite α1-hydroxymidazolam, which contributes to its biological activity (Seo et al., 2010). The therapeutic as well as adverse effects of midazolam and almost all its pharmacological properties (i.e. sedation, induction of sleep, reduction in anxiety, anterograde amnesia, muscle relaxation and anticonvulsant effects) are due to its effects on GABAA receptors, as a benzodiazepine (Edwards et al.,

1990). Midazolam does not activate GABAA receptors directly but, as with other benzodiazepines, it enhances the effect of GABA on the GABAA receptors, resulting in neural inhibition (Edwards et al.,

1990).

Midazolam induces sleepiness, decreasing anxiety, and a loss of ability to create new memories.

51 Midazolam typically begins to have its effects within five minutes when given intravenously, and after fifteen minutes when injected into a muscle (Kissin, 1993; Reves et al., 1985). Effects last for between one and six hours. Intravenous midazolam is indicated for procedural sedation, preoperative sedation, induction of general anesthesia, and sedation of people who are ventilated in critical care units. It is the most popular benzodiazepine for use in the intensive care unit because of its short elimination half-life, water solubility and suitability for continuous infusion (Kissin, 1993; Reves et al., 1985).

Midazolam can have adverse side effects, including respiratory depression and hypotension, due to a reduction in systematic vascular resistance, and an increase in heart rate. Long-term use can lead to tolerance to its effects, withdrawal syndrome and long-lasting memory deficits. A “midazolam infusion syndrome” may result from high doses, and is characterized by delayed arousal hours to days after discontinuation of midazolam, and may lead to an increase in the length of ventilatory support needed

(Mets et al., 1991). In susceptible individuals, midazolam can cause an altered state of consciousness or disinhibition that leads to a paradoxical reaction, wherein individuals experience anxiety, involuntary movements, aggressive or violent behavior, uncontrollable crying or verbalization, and other similar effects. Paradoxical behavior is often not recalled by the patient due to the amnesic effects of the drug

(Mets et al., 1991). Flumazenil can be used to inhibit or reverse the effects of midazolam (Ghouri et al.,

1994).

2.4.5. Mechanism(s) of action

Benzodiazepines cause their effects through increasing the efficiency of GABA in decreasing the excitability of neurons. A subset of GABAA receptors complexes, referred to as benzodiazepine receptors, contain a single binding site for benzodiazepines (Sieghart et al. 1994). Benzodiazepines bind at the interface of the α and γ subunits on the GABAA receptors, but only at GABAA receptors that contain α subunits containing a histidine amino acid residue, (i.e., α1, α2, α3, and α5 containing GABAA receptors) (Rudolph et al. 2000). Benzodiazepines have no affinity for GABAA receptors that

52 contain α4 and α6 subunits. These subtypes contain an arginine instead of a histidine residue (Rudolph et

al. 2000; Kaufmann et al. 2003). Once bound to the benzodiazepine receptor, the benzodiazepine

ligand locks the benzodiazepine receptors into a conformation which has a greater affinity for

GABA (Sieghart et al. 1994). Unlike other positive allosteric modulators that increase ligand binding,

benzodiazepines increase the total conduction of chloride ions across the neuronal cell membrane by

increasing the frequency of the opening of the chloride ion channel when GABA is already bound to the

GABAA receptors (Sieghart et al. 1994; Kelly et al. 2002). This results in hyperpolarization of the neuron's

membrane potential, and consequently decreased probability of firing. By this mechanism, the inhibitory

effect of available GABA is potentiated, leading to sedatory and anxiolytic effects (Sieghart et al. 1994;

Kelly et al. 2002). Different GABAA receptors subtypes have varying distributions within different regions

of the brain and, therefore, control distinct neuronal circuits (Rudolph et al. 2000). Thus, benzodiazepine

activation of different GABAA receptors subtypes results in distinct pharmacological actions. For

instance, benzodiazepines with high affinity for GABAA receptors containing α1 subunits are associated

with stronger hypnotic effects, whereas those with higher affinity for GABAA receptors containing α2

and/or α3 subunits have good anti-anxiety activity (Rudolph et al. 2000).

2.5. Astrocytes

Astrocytes are specialized glial cells that outnumber neurons by over fivefold (Sofroniew et al.,

2010). They contiguously tile the central nervous system and play an essential role in complex functions in

the healthy central nervous system and central nervous system disorders and pathologies (Sofroniew et

al., 2010; Barres, 2008; De Keyser et al., 2008; Seifert et al., 2006). Understanding the morphological,

physiological and functional properties of astrocytes can provide insight into their function in the central

nervous system in healthy and diseased states.

Astrocytes can be divided into two main subtypes, protoplasmic or fibrous, based on differences

in cellular morphologies and anatomical locations (Cajal, 1909). Protoplasmic astrocytes are found

53 throughout gray matter (including the cortex) and consist of several stem branches that give rise to many finely branching processes in a uniform globoid distribution. Fibrous astrocytes are found in white matter

(including the optic nerve head) and consist of many long fiber-like processes (Cajal, 1909). The processes of protoplasmic astrocytes envelop synapses, while the processes of fibrous astrocytes contact nodes of

Ranvier, and both types of astrocytes form gap junctions between distal processes of neighboring astrocytes (Peters et al., 1991). In addition, there are other astroglial cells that share similarities, but also exhibit differences to, protoplasmic and fibrous astrocytes, such as Muller glia in the retina, Bergmann glia in the cerebellum, and cribrosocytes at the optic nerve head (Cajal, 1909; Sofroniew et al., 2010).

Astrocytes are the major cell type in the optic nerve head, and thus are easily accessible in this tissue. Astrocytes in the optic nerve head differ from gray matter astrocytes due to their distinct appearance and morphology. They are spatially aligned to ensheathe axons in bundles in a unique arrangement that provides essential structural support for the axons, and secrete and extracellular matrix referred to as the lamina cribrosa (Hernandez et al., 1988). Astrocytes in the optic nerve head express astrocyte-related molecules such as GFAP and exert functions similar to astrocytes in the central nervous system in manners specialized to their locations. They also become reactive in response to central nervous system insults and can play important roles in pathological changes (Sofroniew et al., 2010).

Astrocytes form tiles in a contiguous, non-overlapping and organized manner. Protoplasmic astrocytes can extend from five to ten main stem branches, each giving rise to many evenly distributed finely branching processes (Bushong et al., 2002; Halassa et al., 2007; Ogata et al., 2002). In the hippocampus or cortex, these finely branching processes contact several hundred dendrites from multiple neurons and envelope 100,000 or more synapses (Bushong et al., 2002; Halassa et al., 2007; Ogata et al.,

2002).

Astrocytes express potassium and sodium channels and exhibit evoked inward currents, but do not propagate action potentials along their processes (Nedergaard et al., 2003; Seifert et al., 2006). They exhibit regulated increases in intracellular calcium concentration which correlates with excitability, and 54 are of functional importance in astrocyte-astrocyte and astrocyte-neuron communication (Charles et al.,

1991; Cornell et al., 1990). Intracellular calcium increase in astrocytes can result from calcium released from intracellular stores leading to intrinsic oscillations, or be triggered by transmitters (i.e. glutamate and purines) released during neuronal activity. The increase in calcium causes the release of transmitters, such as glutamate, from astrocytes, which triggers receptors mediated currents in neurons, and can propagate to neighboring astrocytes (Nedergaard et al., 2003; Seifert et al., 2006). Calcium signaling enables astrocytes to play a direct role in synaptic transmission, which will be discussed further in the next chapter.

Rodent and human astrocytes share many similarities; however, there are also some key differences in their morphological and functional properties. Namely, human astrocytes are structurally more complex, larger, and propagate calcium signals significantly faster than rodent astrocytes

(Oberheim et al., 2012). Specifically, astrocytes in the human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP+ primary processes than their rodent counterparts, and propagate Ca2+ waves at approximately 4-fold faster than rodent. The human neocortex also contains several anatomically-defined subclasses of astrocytes that are not represented in rodents. Thus, human cortical astrocytes are larger and structurally more complex and diverse than those of rodents (Oberheim et al.,

2012). These differences are of particular interest, because astrocytes coordinate and modulate neural signal transmission (Rusakov et al., 2011; Verkhratsky et al., 1998).

Current concepts of the role of astrocytes in neural network performance are based almost entirely on studies of astrocytic physiology in the rodent brain (Oberheim et al., 2006). The increase in astrocytic complexity may permit the increased functional competence of the adult human brain, and provide reasoning to conduct studies in more relevant and representative models. For example, engrafting human astrocytes into mice improves memory performance, providing direct evidence for the critical role of astrocytes in learning and memory process and suggesting that human astrocytes have properties that impart different functional consequences (Han, et al. 2013). These results suggest that 55 further studies using human astrocytes are needed to fully understand and model astrocytic function in the human brain.

2.5.1. Role in cognitive behaviours (memory)

Astrocytes play an integral role in network plasticity as part of the “tripartite synapse”

(presynpatic neuron + glia + postsynaptic neuron), which posits that astrocytes play a direct and interactive role with neurons during synaptic activity in a manner that is essential for information processing by neural circuits. With an increasing body of evidence supporting a central role for astrocytes in regulating synaptic plasticity, it follows that these cells may play a significant role in cognition and behaviour. One area of particular interest concerns the functional role of astrocytes in learning and memory. Astrocytes communicate bi-directionally with neurons (Perea, et al. 2009; Araque, et al. 2008) and contribute to learning and memory processes. For example, astrocytes release soluble signaling factors that induce and regulate long-term potentiation (LTP) of synaptic transmission, a cellular model of memory (Ota, et al. 2013; Henneberger, et al. 2010). However, despite significant advances, we do not fully understand the function of astrocytes and their contribution to cognitive behavior.

Astrocytes have been shown to affect memory through several distinct physiological pathways that enable a high degree of intercellular communication between neurons and astrocytes. They can up- and downregulate LTP and affect synaptic plasticity, by releasing gliotransmitters like glutamate, GABA,

ATP, adenosine, Dserine, and glycine into the synaptic cleft (Halassa et al., 2009; Moraga-Amaro et al.,

2014; Orr et al., 2015; Perea et al., 2014; Velez-Fort et al., 2012), and receiving and processing reciprocal signals carried by these neurotransmitters (Anchour et al., 2012; Araque et al., 2014; Yoon et al., 2012).

Lactate released from astrocytes is necessary for long-term memory consolidation in the hippocampus (Gibbs et al., 2008; Moraga-Amaro et al., 2014), and several hormones and neurotransmitters (i.e. epinephrine, norepinephrine and acetylcholine) enhance memory by increasing

56 astrocytic lactate via glycogenolysis (Gibbs et al., 2008; Gold, 2014). Astrocytes can also affect memory by releasing cytokines during neuroinflammatory responses. Astrocytes may contribute to memory deficits associated with acute stress, traumatic brain injury and malignant glioma (Floyd & Lyeth, 2007;

Sugama et al., 2011; Tarassishin et al., 2014) by contributing to pathological levels of interleukin-1β (IL-

1β). Aberrant IL-1β signaling has detrimental effects on hippocampal neurogenesis and memory consolidation (Ben Menachem-Zidon et al., 2011; Goshen et al., 2007; Moraga-Amaro et al., 2014). Thus, astrocytes can affect learning and memory processes in the hippocampus.

Astrocytes may also affect memory as per the “Astrocentric Hypothesis” of memory, which states that astrocytes may act as the primary substrate of memory and consciousness (Robertson, 2002).

Astrocytes are able to communicate with neurons at the synaptic level, and so are well positioned to influence the information stored in neurons (Caudle, 2006; Robertson, 2002). Astrocytes can support electrical potentials in the form of Ca2+ fluxes for several seconds, which allows them to retain, store and process information on a longer time scale than neurons, thus serving as a potential substrate for consciousness and memory (Caudle, 2006).

Despite discrepancies in theories used to explain the role of astrocytes in learning and memory, it remains clear that astrocytes play an integral role in these cognitive processes.

2.5.2. Anesthetic action on astrocytes

Studies report many different effects of general anesthetics on the central nervous system, nearly all of which involve neurons. Although anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity, growing evidence suggests that anesthetics may also act on astrocytes independently of neuronal activity and have glial effects that constitute non-neuronal mechanisms for actions of anesthetic drugs. However, only a few studies have investigated the effects of general anesthetics on astrocyte physiology, and especially on astrocytic GABAA signaling, and these effects are not well understood.

57 Anesthetics have direct effects on astrocytes, which cause changes in synaptic transmission, and provide insight into how anesthetics cause sedation. Calcium signaling is the principal mechanism by which astrocytes respond to neuronal activity and modulate synaptic activity. The role of Ca2+ signaling in astrocyte physiology and its relation to various neuropathologies is an area of growing interest.

Specifically, intracellular Ca2+ transients are implicated in astrocytic modulation of synaptic plasticity, neurovascular coupling and extracellular ion homeostasis (Dallerac et al., 2013; Pirttimaki & Parri, 2013).

2+ Given that in astrocytes, GABAA-mediated depolarizing currents rapidly activate voltage-gated Ca channels (Meier et al., 2008), it is likely that anesthetics disrupt intracellular Ca2+ signaling by targeting

GABAA receptors. There are studies which support this notion. For example, one study shows that three general anesthetics, including isoflurane, disrupt widespread synchronized Ca2+ transients in neocortical astrocytes in awake, head-restrained mice (Thrane et al., 2012) and disrupts the frequency, kinetics and pattern of astrocyte calcium signaling. General anesthesia causes direct effects on astrocytes, including direct impairment of calcium mobilization, decrease in sensory-evoked calcium responses, and an almost complete rapid suppression of synchronous spontaneous calcium activity that may represent a non-neuronal hallmark of wakeful cortical activity (Thrane et al., 2012). These effects occur at anesthetic doses insufficient to affect neuronal responses and are largely independent of neuronal effects, local synaptic and neuronal activity, and purinergic signaling (Thrane et al., 2012). Taken together, the study suggests that general anesthetics may exert sedative effects by directly suppressing astrocyte calcium signaling. Anesthetic-induced disruptions in astrocytic calcium could also be attributed to anesthetic- induced closure of astrocytic gap junctions known as the “uncoupling effect” (Mantz et al., 1993). The effect of anesthetics on astrocytes via activation of GABAA receptors constitutes a gap in our knowledge concerning the action of anesthetics in the brain.

Anesthetics can also affect astrocytes by altering “leaky” K+ currents that are especially prominent in hippocampal astrocytes (Chu et al., 2010; Seifer et al., 2009) and largely contribute to the resting membrane potential of astrocytes (Chu et al., 2010). These leaky, outwardly rectifying, 58 hyperpolarizing currents may be mediated by TWIK-related acid-sensitive K+ (TASK) channels (Patel et al., 1999; Chu et al., 2010). Most anesthetics activate most subtypes of tandem two-pore K+ channels

(Steinberg et al., 2014). Thus, the anesthetic-induced potentiation of leaky K+ channels may have effects on Ca2+ transients in astrocytes, which may contribute to effects on memory, but this remains to be explored.

Glutamate is a major excitatory neurotransmitter in the brain and has been established as the most important fast-acting neurotransmitter in the central nervous system. Glutamate released from presynaptic terminals is scavenged quickly to terminate synaptic transmission. Most of the glutamate is removed by astrocytes, which have fine processes that surround each synapse and are the only cells in the brain that possess glutamine synthase. Commonly used volatile anesthetics potentiate glutamate uptake in astrocytes in a dose-dependent manner, at clinically relevant concentrations. This may cause a rapid decrease in glutamate concentration in the cleft and decrease postsynaptic glutamate receptors action, contributing to anesthesia.

Astrocytes are coupled by gap junctions into networks, which facilitate cellular communication via ionic and metabolic exchange between adjacent astrocytes. Gap junction permeability in astrocytes can be blocked by anesthetics. Notably, halothane causes a significant and reversible reduction of astrocytic coupling and conductance (Dermietzel, et al. 1991), the intravenous anesthetics etomidate and propofol induce a significant reduction in gap junction permeability, and inhalational anesthetics halothane and isoflurane induce a dose-dependent closure of gap junctions, while ketamine has no effect on gap junction permeability in astrocytes (Mantz, et al. 1993). These studies suggest that general anesthetics affect gap junction permeability, and may consequently alter astrocyte communication and underlie anesthetic actions at the level of the central nervous system.

Erythropoietin, which is mainly produced in astrocytes, is induced under hypoxic conditions and plays a prominent role in neuroprotection and neurogenesis. General anesthetics significantly suppress hypoxia-induced up-regulation of erythropoietin mRNA expression in astrocytes in the mouse brain 59 through a hypoxia-inducible-factor-dependent pathway. These results suggest that general anesthetics have some direct effect on astrocytes and a major impact on the hypoxic response of the central nervous system.

There is growing concern that pediatric exposure to anesthetic agents may cause long-lasting deficits in learning by impairing brain development. Most studies have focused on the direct effects of anesthetics on neurons. However, astrocytes play an important role in supporting neuronal development and therefore may be a target for anesthetics and a mediator of these effects. When astrocytes were exposed to isoflurane then co-cultured with unexposed neurons, the neurons showed a reduction in neuronal growth, which may be attributable to reduced levels of brain derived neurotrophic factor in the coculture media. Overall, these results suggest that isoflurane interferes with the ability of cultured astrocytes to support neuronal growth, which may lead to reduced brain development.

These studies suggest that anesthetics may disrupt learning and memory via several putative astrocyte-dependent mechanisms, but there is a lack of a definitive mechanism linking them together.

Thus, more studies are needed to investigate the role of astrocytes in anesthetic-induced cognitive deficits. These studies, however, do provide sufficient evidence to shows that anesthetics impact astrocyte physiology. Investigating the effect of anesthetics on astrocyte physiology will have significant implications for the field of anesthesiology.

2.5.3. Role in postanesthetic cognitive deficits

Data from our laboratory suggests that astrocytes play a central role in mediating anesthetic- induced persistent cognitive deficits. We have shown that treatment with etomidate causes a persistent increase in tonic current in hippocampal slices. Treating pure hippocampal neurons with etomidate (1 h treatment) failed to increase tonic current in neurons 24 h after treatment (Zurek et al., 2014), suggesting that etomidate acts on non-neuronal (i.e. glial) cells in the hippocampus to increase neuronal tonic current.

60 There are two major types of non-neuronal or glial cells: (1) microglia, which provide immunity to surrounding neurons (Aloisi, 2001); and (2) astrocytes, which nourish and regulate functions of nearby neurons (Sofroniew, 2005). To study whether anesthetics act on microglia or astrocytes, microglia- hippocampal neuron cococultures and astrocyte-hippocampal neuron cocultures were treated with etomidate for 1 h (Zurek et al., 2014), and tonic current in neurons was measured 24 h later. Etomidate failed to increase neuronal tonic current in microglia-neuron cocultures, but contrastingly triggered a persistent increase in tonic current in neurons in astrocyte-neuron cocultures. Thus, astrocytes are necessary for anesthetics to persistently increase tonic current in neurons. These results suggest that astrocyte-neuron (and not microglia-neuron) coupling mediates the persistent increase in tonic current that underlies postanesthetic memory deficits (Zurek et al., 2014).

We then investigated whether anesthetic action on astrocytes alone is sufficient to increase neuronal tonic current in the hippocampus. We treated pure astrocyte cultures with etomidate for 1 hour, and 2 h after incubation, etomidate-treated astrocyte conditioned media (ACM) was transferred to pure hippocampal neuron cultures. Tonic current from these neuron cultures was recorded 24 h later

(Zurek et al., 2014). Neurons treated with conditioned media from etomidate-treated astrocytes had persistent increase in tonic current, just like neurons in astrocyte-neuron cocultures (Zurek et al., 2014), suggesting that astrocytes are necessary and sufficient for etomidate to persistently increase tonic current in hippocampal neurons.

Since astrocytes are central to anesthetic-induced persistent increase in hippocampal tonic current, anesthetic action on astrocytes likely plays a key role in postanesthetic memory deficits.

Therefore, our next objective was to investigate the mechanism of anesthetic action on astrocytes.

Astrocytes are known to express functional GABAA receptors (Araque et al., 2014). Furthermore, our unpublished studies show that application of the GABAA receptors antagonist, bicuculline, inhibits anesthetic-induced increase in GABAA receptor-current in astrocyte cultures and slices. Thus, GABAergic anesthetics (i.e. most general anesthetics) may target GABAA receptors in astrocytes to mediate their 61 actions. Particularly, activation of GABAA receptors in astrocytes depolarizes the astrocyte membrane, resulting in intracellular calcium influx (Angulo et al., 2008), which triggers vesicular release of signaling molecules such as neurotransmitters, peptides, and proinflammatory cytokines (Araque et al., 2014).

Astrocytic release of signaling proteins such as proinflammatory cytokines have been shown to stimulate an increase in neuronal cell-surface expression of GABAA receptors (Perea et al., 2009). These studies, in addition to previous studies in our lab, suggest that anesthetic activation of astrocytic GABAA receptors may stimulate the release of signaling proteins which target neurons and lead to the persistent increase in neuronal tonic current.

To study the role of soluble factors released by astrocytes, pure astrocyte cultures were treated with etomidate for 1 h. 2 h later etomidate-treated astrocyte-conditioned medium was heated for 5 minutes before transferring to pure neuron cultures. The heat treatment was expected to denature signaling proteins suspected to increase the tonic current in neurons. Tonic current was recorded from neurons 24 h later. As predicted, heating the etomidate-treated astrocyte-conditioned medium prevented etomidate-induced increase in neuronal tonic current. Collectively, these results suggest that etomidate triggers astrocytes to release heat-sensitive soluble factors, likely signaling proteins, which increase the tonic current in neurons.

Overall, literature suggests that anesthetic action on astrocytic GABAA receptors triggers the release of soluble factors, which cause a persistent increase in tonic current in hippocampal neurons that may underlie postanesthetic memory deficits. Thus, astrocytes may play a key role in GABAergic anesthetic- mediated cognitive deficits.

Most commonly used general anesthetics are GABAergic agents, i.e. they target GABAA receptors to mediate their actions (Ben-Ari, 2012; Mo & Zimmermann, 2013). The use of common

GABAergic general anesthetics is associated with increased incidences of postoperative cognitive disorders. Thus, astrocytes may likely be activated via GABAA receptors to mediate their effects on postoperative cognition. 62 2.5.4. GABA receptors in astrocytes

GABA receptors are widely distributed throughout the central nervous system on various cell types, including astrocytes. Astrocytes help terminate inhibitory synaptic transmission via GABA uptake mechanisms (Martin, 1976), and GABA receptors are also present on astrocytes, suggesting that these cells may respond and contribute to inhibitory synaptic transmission in a manner that was previously not considered.

Autoradiographic binding experiments show GABAB receptors expression on cultured astrocytes from the cerebellum, spinal cord and brain stem; albeit a lower density than in neurons. A specific

GABAB receptors agonist, Baclofen, causes membrane hyperpolarization, which is depressed by GABAB receptors antagonists. Some studies with Baclofen suggest that GABAB receptorstimulation may be negatively coupled to an inositol phosphate second-messenger cascade and subsequent Ca2+ gating mechanisms. Other studies in mixed neuronal/astrocyte cultures suggest that Baclofen induces a transient increase in cytosolic Ca2+ dependent on influx from the external media and partially blocked by

GABAB antagonist phaclofen. However, application of GABA itself results in membrane depolarization that can be accounted for by GABAA receptors activity, and which are completely insensitive to GABABR antagonist blockade. So, the physiological relevance of GABAB associated hyperpolarization remains obscure.

However, these experiments were performed using the whole-cell patch technique, which may mask a GABAB-mediated response by dialyzing the cytoplasmic constituents. Another study demonstrates that γ-hydroxybutyric acid, which is a precursor to GABA, glutamate and glycine, elicits

2+ robust [Ca ]i transients in astrocytes of the ventral tegmental area (VTA) and ventrobasal thalamus, brain areas involved in reward properties. This effect is mediated by actions on astrocytic GABAB receptors, as γ-hydroxybutyric acid is a weak agonist for GABAB receptors, and is comparable to the effect evoked by baclofen. Furthermore, prolonged γ-hydroxybutyric acid and baclofen exposure in the

63 VTA causes a reduction in spontaneous glutamate release from astrocytes (Gould, et al. 2014). Thus, there may be pathophysiological roles for GABAB receptors on astrocytes.

Astrocytes express many of the ligand-gated ion channels found in neurons. Of these, the GABAA receptors remains one of the least characterized. The expression of GABAA receptors on glial cells is controversial. Although autoradiographic and biochemical studies have failed to demonstrate the presence of GABAA receptors on cultured astrocytes, electrophysiological studies have shown that astrocytes are depolarized by GABA, independent of electrogenic uptake mechanism(s), and this effect is mimicked by GABAA receptors agonists (i.e. muscimol) and blocked by antagonists (i.e. bicuculline).

GABAA receptors on astrocytes have properties similar to their neuronal counterparts, although there are a few pharmacological differences (Fraser et al., 1994, 1995; Kettenmann et al., 1987; Liu et al.,

1996).

Researchers have only recently begun to study the physiological function of GABAA receptors expressed on astrocytes. Some studies suggest that they may be involved in extracellular ion homeostasis and pH regulation. Astrocytes do not elicit or propagate action potentials, so GABA does not affect astrocytes in the same way as in neurons. Activation of astrocytic GABAA receptors triggers an outward Cl- current, due to relatively high intracellular Cl- concentrations (Velez-Fort et al., 2012; Yoon et al., 2012), resulting in GABA-induced membrane depolarization that leads to an increase in intracellular

Ca2+ via voltage-gated Ca2+ channel activation and release of sequestered ER Ca2+ stores. Consequently, this triggers the secretion of various gliotransmitters including GABA and glutamate (Velez-Fort et al.,

2012; Yoon et al., 2012).

Thus, astrocytes are “GABAergic and GABAceptive” cells, as they have the ability to secrete and detect GABA (Yoon et al., 2012), which allows them to respond to and produce GABAergic signaling.

Although microglia – a cell type predominantly involved in CNS inflammatory processes – produce and secrete GABA, there is no evidence to suggest they express GABAA receptors (Velez-Fort et al., 2012). In fact, one of the main functions of astrocytic GABA release is to modulate microglia activity and 64 interleukin release via GABAB signaling (Kuhn et al., 2004; Lee et al., 2011).

Therefore, neurons and astrocytes are sources and targets of GABA in the brain, and can thus communicate with each other via GABAergic signaling (Yoon et al., 2012). This ability for bidirectional

GABA signaling between neurons and astrocytes may have a regulatory role in neurotransmission.

Astrocytes may prevent “hyper-inhibition” by responding to excessive GABA release and suppressing neurotransmission in GABAergic cells. However, the precise intracellular signaling events that follow

GABAA receptors activation in astrocytes remain poorly understood.

2.5.5. α5GABAA receptors in astrocytes

Preliminary data from our laboratory show that astrocytes contain functional α5GABAA receptors and that activation of α5GABAA receptors receptors in astrocytes causes the release of soluble factors that may underlie anesthetic-induced memory deficits. α5GABAA receptors on astrocytes may be the major subtype of GABAA receptors mediating the anesthetic-induced increase in neuronal tonic current. The following evidence supports this notion: 1) preliminary results show that the α5 subunit is expressed in astrocytes; 2) astrocytic GABAA receptors exhibit a high affinity for GABA and benzodiazepine-sensitivity, which are signature characteristics of α5GABAA receptors ; 3) our published results show that pre-treatment with an inverse agonist (L-655,708) that inhibits α5GABAA receptors , prevents postanesthetic memory deficits in mice and 4) when cocultures of astrocytes and neurons are treated with L-655,708 and etomidate, the tonic current in the neurons is not increased.

Furthermore, a study using the competitive polymerase chain reaction assay shows that astrocytes express mRNA for α5GABAA receptors, albeit at lower levels than in neurons (Bovolin et al. 1992).

2.5.6. Astrocyte-neuron interactions

Much evidence suggests that astrocytes have an important role in regulating the structure and function of the synaptic cleft (Anchour & Pascual, 2012; Perea et al., 2009), as per a “tripartite” model, which explains traditional synaptic transmission and bidirectional communication between astrocytes

65 and neurons (Anchour & Pascual, 2012).

Astrocytes envelope synapses in the central nervous system. While earlier findings suggested

that the role of these cells in synaptic transmission was limited to potassium and glutamate clearance

(Anchour & Pascual, 2012; Walz, 2000), recent electrophysiology studies have provided more thorough

insight into how astrocytes impact synaptic plasticity and neurotransmission. Increased synaptic activity

causes dynamic morphological responses in astrocytes, which are temporally coupled with increased

intracellular Ca2+ levels and gliotransmitter release (Dallerac et al., 2013; Newman, 2003). These dynamic

responses modulate synaptic activity by stabilizing aberrant hyperexcitability at synapses, thereby

facilitating long-term potentiation, or regulating the activity of larger nearby neuronal networks

(Dallerac et al., 2013; Newman, 2003; Pirttimaki & Parri, 2013).

Since astrocyte gliotransmission plays a central role in regulating synaptic transmission, much

effort has been put into delineating the specific functions of different gliotransmitters, such as

glutamate, ATP, adenosine and D-serine. Together, these transmitters synchronize neuronal activity

within and across brain regions, in addition to regulating sleep, arousal and other complex behaviors

(Angulo et al., 2004; Araque et al., 2014; Halassa et al., 2009). Understanding of how GABA functions as

a gliotransmitter lags in comparison, although some evidence suggests that it regulates neuronal

excitability like glutamate (Angulo et al., 2008; Koch & Magnusson, 2009). Behavioral implications of

bidirectional GABA signaling between neurons and astrocytes remain poorly understood.

2.6. Inflammation and anesthesia

2.6.1. Anesthetic-induced inflammation

General anesthesia may influence inflammatory responses that are essential in maintaining the

homeostatic state during the postoperative period (Schneemilch et al., 2004). Severe dysregulation of

these inflammatory responses may provoke or aggravate postoperative complications, such as increased

susceptibility to infections, inadequate stress reactions and hypercatabolism (Kelbel et al. 2001).

66 Anesthetics have been associated with impairing various immune system functions either directly, via disturbing the functions of immune-competent cells, or indirectly, via modulating the stress response

(Kurosawa et al. 2008). Suppression of cellular and humoral immunity following surgery and general anesthesia may have profound implications for postoperative patient health, especially for patients with pre-existing immune disorders and high-risk, immunocompromised patients (Scheemilch et al. 2004;

Kelbel et al. 2001).

Animal studies demonstrate that exposure to general anesthetics induces neuroinflammation in the developing and aging brain (Shen et al., 2013; Zhang et al., 2014). Repeated exposure to sevoflurane in neonatal rats causes a significant increase in interleukin-6 (IL-6) and tumor necrosis factor (TNF) protein levels in the brain and decreased activity of the AKT-glycogen synthase kinase 3β (GSK3β) signaling pathway. Isoflurane exposure leads to increased levels of the pro-inflammatory cytokines TNF,

IL-6 and IL-1β in the central nervous system and this effect is reported to be aggravated in the brains of transgenic mice used to model Alzheimer’s disease (Wu et al., 2012). Anesthesia-induced activation of canonical nuclear factor-kB (NF-kB) signaling underlies general anesthetic-linked increase in IL-1β expression in aged rats.

Clinical studies indicate that anesthetics can induce increases in cytokine production. Namely, one study in elective patients receiving long-term sedation for more than 2 days shows that propofol causes a significant increase in IL-1β, IL-6 and TNF-α levels after 48 hours of exposure (Helmy, et al.

2001). Surgery also causes an increase in the levels of proinflammatory cytokines such as interleukin 6

(IL-6) and tumour necrosis factor α (TNFα), which can alter the integrity of the blood-brain barrier and increase systemic inflammation which leads to neuronal death and abnormal synaptic function (Cottrell et al., 2012; Rudolph et al., 2008; Alcover et al., 2013). Plasma levels of inflammatory cytokines are also significantly higher in patients with postoperative delirium and POCD (Cunningham, 2011).

Some studies have provided contradictory results and suggest that general anesthetics impact the active process of resolution of inflammation. Namely, one study shows that lipopolysaccharide- 67 induced glial cell IL-1β mRNA expression in the brain is significantly reduced with co-treatment of general anesthetics including isoflurane, midazolam, ketamine, and propofol, whereas IL-6 and TNF-α levels remain largely unaffected. The anesthetics did not activate NF-kB. Taken together, the results suggest that general anesthetics inhibit LPS-induced IL-1β upregulation in glial cells (Tanaka et al., 2013).

One study shows that isoflurane promotes resolution of inflammation by down-regulating many proinflammatory chemokines and cytokines and proteins known to be active in cell migration and chemotaxis (Chiang et al., 2008).

Despite reports of controversial effects, clinical and animal studies support the notion that anesthetics have significant effects on inflammation, and specifically levels of inflammatory cytokines.

2.6.2. Inflammation and cognition (memory)

Acute systemic inflammation caused by a variety of disorders, including autoimmune diseases, infection, traumatic brain injury, and stroke, can lead to memory loss (Dantzer et al., 2008; Di Filippo et al., 2008; Kipnis et al., 2008; Yirmiya and Goshen, 2011), which manifests as impaired explicit recall in humans, and deficiencies of fear-associated memory and reduced performance for object-recognition tasks in animal models (Yirmiya and Goshen, 2011). Inflammation also contributes to chronic neurodegenerative diseases characterized by memory loss, including Alzheimer disease, Parkinson disease, multiple sclerosis, and even AIDS-related dementia (Di Filippo et al., 2008; Kipnis et al., 2008;

Yirmiya and Goshen, 2011).

Systemic inflammation increases the production of multiple cytokines in the brain, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 (Dantzer et al., 2008; Yirmiya and

Goshen, 2011). In particular, increases in IL-1β levels have been shown to contribute to inflammation- induced memory deficits. Increased plasma levels of IL-1β in patients with sepsis-associated encephalopathy, correlate with cognitive deficits (Serantes et al., 2006). Decreased levels of IL-1β caused by a genetic variant of the IL-1β-converting enzyme leads to better cognitive performance (Trompet et

68 al., 2008). Elevated levels of IL-1β in the hippocampus of laboratory animals that have undergone orthopedic surgery correlate with memory deficits (Cibelli et al., 2010). Memory deficits associated with elevated levels of IL-1β are typically hippocampus dependent, while hippocampus- independent memory is unaffected (Yirmiya and Goshen, 2011).

The mechanisms underlying inflammation-induced memory deficits are not well understood.

There are studies to support various underlying mechanisms for inflammation-induced memory deficits, including changes in multiple neurotransmitter receptors, such as reduction in N-methyl-D-aspartate

(NMDA) receptors activity, alterations in the trafficking and phosphorylation of 2-amino-3-(5-methyl-3- oxo-1,2-oxazol-4-yl) propanoic acid receptors, and activation of the α7 subtype of nicotinic acetylcholine receptors (Stellwagen et al., 2005; Terrando et al., 2011; Viviani et al., 2003). However, treatments based on these mechanisms have not been effective in reversing or preventing memory deficits associated with inflammation. Furthermore, general inhibition of the inflammatory response or specific blocking of IL-1β activity by inhibiting the membrane-bound type 1 IL-1 receptors are impractical treatment strategies as they cause risks of infection and delayed wound healing (Fleischmann et al.,

2006), and low basal levels of IL-1β in the hippocampus are necessary to play a physiological role in maintaining normal memory performance (Yirmiya and Goshen, 2011). Thus, the downstream mediators of inflammation-induced memory deficits should be investigated, to facilitate the development of effective treatment strategies.

2.6.3. IL-1 secretion and signaling

IL-1 plays a central role in mediating inflammatory processes (Goshen et al., 2007; Huang &

Sheng, 2010). IL-1 exists in either an α or β isoform (Allan et al., 2005; Huang & Sheng, 2010). The mRNA of both precursor proteins is transcribed (i.e. pro-IL-1α/β) and, depending on the cleaving enzyme present in the cell, plasma-membrane-bound enzyme calpain or intracellular enzyme capsase-1, either

IL-1α or β is secreted, respecitvely (Huang & Sheng, 2010). Both isoforms share similar structures,

69 activate the same receptors (IL-1 receptors), and exert similar physiological effects (Huang & Sheng,

2010; Sims et al., 1993). However, IL-1α remains largely bound to the plasma membrane to activate immune cells via direct cell-to-cell contact, while IL-1β is secreted into the extracellular environment

(Pugh et al., 2001). Microglia express activated capsase-1 upon stimulation by lipopolysaccharide (LPS) or TNFα to produce IL-1β, as the predominant source of IL-1β in the central nervous system (Cibelli et al., 2010; Wang et al., 2012). Astrocytes express capsase-1 and release IL-1β, but only in certain pathological inflammatory conditions (Santos-Galindo et al., 2011; Sugama et al., 2011; Tarassishin et al.,

2014). IL-1β is the major secreted form of IL-1 (Pugh et al., 2001).

2.6.4. IL-1β and cognition (memory)

IL-1β plays an important role in regulating several cognitive processes and particularly memory

(Huang & Sheng, 2010). IL-1β restricts LTP in the hippocampus and impairs hippocampus-dependent memory, but not hippocampus-independent memory (Huang & Sheng, 2010; Pugh et al., 2001; Vereker et al., 2000). Elevated IL-1β levels in the hippocampus completely block LTP in vitro and in vivo, and administration of the IL-1 receptors antagonist IL-1ra reverses these deficits (Barrientos et al., 2015;

Chapman et al., 2010; Coogan et al., 1999; Kelly et al., 2003; Vereker et al., 2000). LTP is also blocked by treatment with an inhibitor of p38 mitogen-activated protein (MAP) kinase, a key protein kinase that acts downstream of the IL-1 receptors (Barrientos et al., 2015; Chapman et al., 2010; Kelly et al., 2003).

Mice treated with IL-1β are impaired in several hippocampal-dependent memory tasks (e.g. contextual fear conditioning, Morris water maze, radial arm maze) (Pugh et al., 2001). Neuroinflammatory processes that involve IL-1β play an important role in surgery-induced impairment of LTP and cognitive decline, but the underlying mechanisms are still unclear. A study from our laboratory provides insight into the mechanism underlying inflammation-induced impairment in LTP and cognition. Treatment with

IL-1β increases the cell-surface expression of extrasynaptic α5GABAA receptors in cultured hippocampal neurons via an IL-1 receptors- and p38 MAP kinase-dependent mechanism (Wang et al., 2012),

70 suggesting that inflammation-induced increase in α5GABAA receptors expression underlies

inflammation-induced effects on cognition.

2.7. Summary

Patients with postoperative delirium or POCD are critically affected by memory deficits that negagively impact their health and wellbeing (Price et al., 2008; Steinmetz et al., 2009). The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is a powerful regulator of learning, memory, and synaptic plasticity (Collinson et al., 2002). GABA type A (GABAA) receptors generate two distinct forms of inhibition: phasic, fast inhibitory postsynaptic currents and a tonic form of inhibition that is primarily mediated by extrasynaptic GABAA receptors (Brickley and Mody, 2012; Glykys and Mody, 2007; Luscher et al., 2011).

Our laboratory has identified a relationship between sustained tonic GABA current in hippocampal neurons and postanesthetic memory deficits (Martin et al., 2010; Zurek et al., 2014). However, the cellular and molecular mechanisms underlying this anesthetic-induced increase in GABAA receptor function remain unknown.

In this thesis, I will utilize a reduced cell culture model to explore the role of astrocytic GABAA receptor activation in postanesthetic memory deficits, as well as the molecular factors secreted from astrocytes following exposure to these drugs. My goal is to enhance understanding of the molecular mechanisms underlying postoperative delirium and POCD to enable development of better preventative and restorative treatment options.

71 Chapter 3. General Materials and Methods

3.1. Study approval

All experimental procedures were approved by the Animal Care Committee of the University of Toronto and performed in accordance with guidelines from the Canadian Council on Animal Care. The methods have been described in great detail to ensure the reproducibility of the results reported in this thesis.

3.2. Electrophysiology in cell culture

3.1.1. Preparation of primary cell cultures

Primary neuron cultures

Primary cell cultures of hippocampal neurons were prepared from CD1 mice (Charles River, Montreal,

Canada), as described previously (MacDonald et al. 1989; Zurek et al. 2014) with minor modifications.

Briefly, fetal pups (embryonic day 18) were removed from time-pregnant mice euthanized by cervical dislocation. The hippocampi were dissected from each fetal pup and placed in an ice-cooled culture dish.

Next, the hippocampi were digested with 0.05% trypsin (Life Technologies, Grand Island, New York) for

20 min at 37°C and were then dissociated by mechanical titration using two Pasteur pipettes (tip diameter, 150-200 μm). The dissociated neurons were then plated on 35-mm culture dishes, coated with collagen or poly-D-lysine (Sigma-Aldrich Co., St. Louis, Missouri), at a density of approximately 1 x

105 cells/ml. The cell cultures were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Life

Technologies, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS). Two hours later, the cell culture media were changed to neurobasal medium supplemented with 2% B27 and 0.5 mM L-glutamate (Invitrogen Corporation, Carlsbad, California). The neurons were cultured in a Napco incubator (Precision Scientific Inc., Buffalo, New York) set at 37°C with 95% humidified air and 5% CO2.

72 For the first 5 days in vitro, cells were maintained in minimal essential media (MEM) supplemented with

10% fetal bovine serum and 10% horse serum (Life Technologies, Grand Island, New York). After the cells had grown to confluence, 0.1 ml of a mixture of 4 mg 5-fluorodeoxyyuridine and 10 mg uridine in 20 ml minimal essential media was added to the extracellular solution to reduce the number of dividing cells.

Subsequently, neurobasal media in the cell cultures was supplemented with 10% horse serum and replaced every 2 to 3 days. The hippocampal neurons were maintained under these culture conditions for 14 to 21 days prior to recording.

Primary astrocyte cultures

Cerebral cortical astrocytes were isolated from CD1 embryonic day 18 mouse embryos as described previously (Albuquerque et al. 2009; Wang et al. 2014; Qiao et al. 2016), with minor modifications.

Briefly, meninges-free cortices were cut into small cubes (<1 mm3), placed in modified Dulbecco’s

Modified Eagle’s Medium (DMEM; Invitrogen Corporation, Carlsbad, California), and mechanically dissociated by vortex for 60 s. The resulting cell suspension was sieved through 40 μm filters (Millipore,

Etobicoke, Ontario). The filtrated cells were plated in a flask consisting of 5 x 105 cells/ml in DMEM supplemented with 10% FBS (Life Technologies, Grand Island, New York). The astrocyte cultures were incubated in a Napco incubator (Precision Scientific Inc., Buffalo, New York) set at 37°C with 95% humidified air and 5% CO2. The astrocyte cultures were maintained in DMEM supplemented with 10%

FBS (Life Technologies, Grand Island, New York) and were allowed to grow to confluence for 14 days.

They were then maintained in DMEM supplemented in 7% FBS for the following 14 days. Cells were then enzymatically dissociated with trypsin-EDTA (0.05%; Life Technologies, Grand Island, New York), and passaged three times to obtain a nearly pure astrocytic culture. Next, the astrocytes were plated on 35 mm culture dishes at a density of 25,000 cells per dish.

73 Primary astrocyte-neuron cocultures

For astrocyte-neuron cocultures, 28 day-old cortical astrocyte cell suspension was placed over

hippocampal neurons cultured at 14 days in neurobasal media. Astrocytes were monitored visually to

ensure survival and confluence for the duration of the experiment.

3.1.2. Human optic nerve head astrocyte cultures

The laminar cribosa from 4 healthy deceased human donors (Eye Bank of Canada, Ontario Division) were

dissected into explants and grown in Dulbecco Modified Eagle Medium (DMEM) Nutrient Mixture F12 (4

mM L- glutamine;1g/L glucose; 1.5 g/L sodium bicarbonate; 10% FBS; penicillin/streptomycin) until

confluent. The astrocytes were isolated from other cell types using a technique previously described (Yu

et al. 2008). Briefly, explants were split into serum-free astrocyte growth media (AGM -serum, Lonza,

Switzerland) for 2 weeks, when the media was replaced with Dulbecco Modified Eagle Medium /F12.

The cells were grown to confluence and split in this media until there were enough viable cells to

conduct an experiment. Cultures were maintained in sterile incubators at 37 °C and 5% CO2, and media

was changed twice a week. Morphologically, the astrocytes were cultured in a monolayer and were

similar to those previously reported (Yang et al. 2003; Hernandez et al. 1988; Lambert et al. 2001). All

cultures are of at least 95% purity based on morphology and staining.

For all reported studies in this thesis, data were acquired from cells that were prepared from at least

three different dissections of brain tissue.

3.1.3. Whole-cell voltage-clamp recordings in cell culture

For whole-cell voltage-clamp recordings, the recording electrode (2-3 MΩ) was filled with intracellular

solution containing (in mM): 140 CsCl, 10 HEPES, 11 EGTA, 2 TEA, 1 CaCl2, 2 MgCl2, 4 Mg2ATP (pH

adjusted to 7.3 with CsOH and osmolarity adjusted to 290-2955 mOsm). The pipette offset was

74 compensated when the recording electrode was in the bath which consisted of the extracellular solution containing (in mM): 140 NaCl, 25 HEPES, 2 KCl, 1.3 CaCl2, 1 MgCl2, 28 glucose (pH adjusted to 7.4 with

NaOH and osmolarity adjusted to 322-328 mOsm).

A tight gigaohm seal (>1 GΩ) was formed on the cell body of the neuron. Pipette capacitance was compensated before obtaining whole cell configuration, which was obtained by applying negative pressure to the recording electrode and rupturing the cell membrane. Obtaining whole cell configuration allows electrical access to the entire cell and control of the intracellular environment by resulting in the replacement of the intracellular contents with the artificial intracellular solution in the patch pipette. The series resistance was monitored using a 10 mV hyperpolarizing voltage step in

Multiclamp software (Molecular Devices, Sunnyvale, California). Currents were sampled at 5 kHz. All cells were recorded at a holding potential of -60 mV and the Multiclamp software applied automatic capacitance compensation.

To measure the amplitude of the tonic extrasynaptic GABAA receptors-mediated current, exogenous

GABA (0.5 μM) was added to the extracellular solution and the change in holding current was revealed during application of a GABAA receptors-selective antagonist, bicuculline (BIC, 20 μM). GABA (0.5 μM) is similar to the physiological levels of extracellular GABA that occur in vivo (Bright & Smart, 2013) and is in the range of low ambient extracellular concentrations (10 nM – 1 µM) that activate extrasynaptic receptors (Farrant & Nusser, 2005).

75 3.2. Preparation of pharmacological agents used in vitro

All drugs were stored in the refrigerator after use in order to minimize degradation and contamination.

Etomidate

Etomidate (Sigma-Aldrich, Canada) was first dissolved in 35% propylene glycol at 2 mg/mL. Next, the etomidate in propylene glycol was diluted with phosphate buffered saline (PBS; Gibco, Thermofisher

Scientific, Canada) to a stock concentration of 200 μM. Cell cultures were treated with this stock solution of etomidate to achieve a final concentration of 1 μM, which corresponds to an anesthetic dose in vitro.

Sevoflurane

Sevoflurane (AbbVie Corporation, Saint Laurent, Quebec) was dissolved in freshly prepared extracellular solution to obtain a stock concentration of 11.8 mM. Specifically, one part of sevoflurane was added to two parts of extracellular solution and left to stand overnight in order to saturate the solution with sevoflurane. Cell cultures were treated with this stock solution of sevoflurane to achieve a final concentration of 266 μM, which corresponds to an anesthetic dose in vitro.

Isoflurane

Isoflurane (AbbVie Corporation, Saint Laurent, Quebec) was dissolved in freshly prepared extracellular solution to obtain a stock concentration of 2500 μM. Specifically, one part of isoflurane was added to two parts of extracellular solution and left to stand overnight in order to saturate the solution with isoflurane. Cell cultures were treated with this stock solution of isoflurane to achieve a final concentration of 250 μM, which corresponds to an anesthetic dose in vitro.

76 Propofol

Propofol (Sigma-Aldrich, Canada) was dissolved in phosphate buffered saline (PBS; Gibco, Thermofisher

Scientific, Canada) to a stock concentration of 200 μM. Cell cultures were treated with this stock solution of etomidate to achieve a final concentration of 3 μM, which corresponds to an anesthetic dose in vitro.

Midazolam

Midazolam (Sandoz, Canada) 1 mg/mL stock was dissolved in phosphate buffered saline (PBS; Gibco,

Thermofisher Scientific, Canada) to obtain a stock concentration of 40 μM. Cell cultures were treated with this stock solution of midazolam to achieve a final concentration of 200 nM.

Ketamine

Ketamine (50 mg/mL; Wyeth, Canada) was dissolved in phosphate buffered saline (PBS; Gibco,

Thermofisher Scientific, Canada) to obtain stock concentrations of 2mM and 100 mM. Cell cultures were treated with these stock solutions of ketamine to achieve a final concentration of 10 and 300 μM.

Bicuculline

Bicuculline (Abcam, Canada) was dissolved in phosphate buffered saline (PBS; Gibco, Thermofisher

Scientific, Canada) to obtain a stock concentration of 10 mM. Cell cultures were treated with this stock solution of bicuculline to achieve a final concentration of 20 μM.

L-655,708

L-655,708 (Sigma-Aldrich, Canada) was dissolved in phosphate buffered saline (PBS; Gibco, Thermofisher

Scientific, Canada) to obtain a stock concentration of 5 mM. Cell cultures were treated with this stock solution of L-655,708 to achieve a final concentration of 50 nM.

77 Minocycline

Minocycline (Sigma-Aldrich, Canada) was dissolved in deionized water to obtain a stock concentration of

20 mM. Cell cultures were treated with this stock solution of minocycline to achieve a final concentration of 100 μM.

IL-1β

IL-1β (R&D Systems, Minneapolis, USA) was dissolved in 0.1 % Bovine Serum Albumin (BSA; Sigma-

Aldrich, Canada) in phosphate buffered saline (PBS; Gibco, Thermofisher Scientific, Canada) to obtain a stock concentration of 10 μg/mL. Cell cultures were treated with this stock solution of IL-1β to achieve a final concentration of 60 ng/mL.

IL-1 receptors antagonist

IL-1 receptors antagonist (Sigma-Aldrich, Canada) was dissolved in 0.1 % Bovine Serum Albumin (BSA;

Sigma-Aldrich, Canada) in phosphate buffered saline (PBS; Gibco, Thermofisher Scientific, Canada) to obtain a stock concentration of 0.05 μg/mL. Cell cultures were treated with this stock solution of IL-1 receptors antagonist to achieve a final concentration of 100 ng/mL.

SB 203,580

SB 203,580 (R&D Systems, Minneapolis, USA) was dissolved in phosphate buffered saline (PBS; Gibco,

Thermofisher Scientific, Canada) to obtain a stock concentration of 4 mM. Cell cultures were treated with this stock of solution of SB 203,580 to achieve a final concentration of 20 μM.

80 SB 202,474

SB 202,474 (Millipore, Massachusetts, USA) was dissolved in phosphate buffered saline (PBS; Gibco,

Thermofisher Scientific, Canada) to obtain a stock concentration of 4 mM. Cell cultures were treated with this stock of solution of SB 202,474 to achieve a final concentration of 20 μM.

3.3. Statistical analyses

All results are presented as mean ± standard error of the mean (SEM). An unpaired Student’s t-test was used to compare groups where appropriate. When comparing three or more groups, a one-way analysis of variance followed by Tukey’s Honestly Significant Difference was applied. The Kolmogorov-Smirnov test and Shapiro-Wilk test were used to validate the assumption of normality. In cases where the assumption of normality was not met for one or more groups, the Mann-Whitney U test was employed.

Statistical testing was performed with GraphPad Prism software version 6.0 (GraphPad software, San

Diego, California). A P value less than 0.05 was considered statistically significant.

81 Chapter 4. Multiple GABAergic anesthetics trigger a persistent increase in tonic current in hippocampal neurons

4.1. Introduction

In the mammalian central nervous system GABA functions as the principal inhibitory neurotransmitter in the brain (Farrant & Nusser, et al. 2005). GABA reduces the excitability of neurons

- by binding to Cl channel-coupled GABA type A receptors (GABAA receptors). GABAA receptors that are expressed in neurons have a well-defined role in learning and memory. GABAA receptors are broadly divided into synaptic and extrasynaptic categories. Synaptic GABAA receptors mediate phasic inhibition by conducting large, transsient inhibitory postsynaptic currents (IPSCs) in response to presynaptic GABA release. Extrasynaptic GABAA receptors mediate slow, tonic inhibitory currents that are relatively constant in neurons (Farrant & Nusser, et al. 2005; Mody, et al. 2004). Our laboratory has shown that the anesthetic etomidate acts on astrocytes to trigger a sustained increase in extrasynaptic GABAA receptors function in hippocampal neurons. The etomidate-induced persistent increase in extrasynaptic

GABAA receptors function persists long after the anesthetics have been eliminated and may underlie persistent postanesthetic memory deficits (Zurek, et al. 2014). Specifically, etomidate markedly increases extrasynaptic GABAA receptors cell-surface expression, resulting in enhanced tonic GABAA receptor-mediated inhibitory current in the hippocampus. Inhibiting GABAA receptors reverses memory deficits observed after etomidate exposure. These findings have garnered considerable scientific and media attention because they refute the widely held assumption that brain function rapidly returns to baseline after anesthesia.

Although etomidate, our prototypic intravenous anesthetic for previous studies, is frequently used as an induction agent for emergency anesthesia, its use for long-term sedation in the intensive care has been prohibited due to its effects on the adrenal cortex associated with increase in mortality (van

82 den Heuvel, et al. 2013). Several clinical studies suggest that the use of the injectable anesthetic propofol, the inhalational anesthetics isoflurane and sevoflurane, and the GABAergic benzodiazepine midazolam, which are more widely used in clinical practice than etomidate, can also contribute to postoperative cognitive deficits (Royse, et al. 2011; Zhang, et al. 2012; Rortgen, et al. 2009; Rasmussen, et al. 1999). These drugs cause their anesthetic and sedative actions through GABAA receptor- dependent pathways, similar to etomidate (Garcia, et al. 2010; Olkkola, et al. 2008). The mechanism underlying postoperative cognitive deficits induced by these drugs is currently unknown. Given our results with etomidate, it seems likely that the persistent memory deficits caused by these drugs may be mediated by a sustained increase in extrasynaptic GABAA receptors function in hippocampal neurons.

Ketamine, which can be used to initiate and maintain anesthesia, has been associated with attenuation of postoperative cognitive deficits following surgery (Deiner, et al. 2009; Hudetz, et al. 2009). Thus, it can be used a control for our studies. Furthermore, the effect of ketamine on tonic GABA current has not been previously investigated.

Our previous studies were conducted in murine cultures. Although murine cultures are a good model, there are some differences between human and murine cells (Zhang, et al. 2016). In order to increase the physiological relevance of our findings, we decided to perform experiments using human cell cultures. Specifically, we performed experiments using human optic nerve head astrocyte cultures provided to us by Dr. Jeremy Sivak (Toronto Western Hospital).

Here, I conducted experiments to demonstrate that anesthetic-induced postoperative cognitive deficits may be mediated by a persistent increase in tonic extrasynaptic GABAA receptor-mediated inhibitory current. I tested the hypothesis that treatment with GABAergic anesthetics and sedative agents other than etomidate and treatment of human cell cultures can cause a persistent increase in tonic current. My primary aims were to (1) determine whether widely used anesthetic and sedative agents associated with postoperative cognitive deficits also cause a sustained increase in tonic GABAA receptor-mediated inhibitory current in hippocampal neurons, and (2) determine whether human 83 astrocyte cultures can also mediate anesthetic-induced persistent increase in tonic current. I developed an in vitro model to explore how various anesthetic and sedative agents affect tonic inhibition in hippocampal neurons cultured with astrocytes. I collected electrophysiological recordings to measure the tonic GABA-evoked currents in neurons 24 h after 1 h exposure to the anesthetic and sedative agents. I also used an astrocyte-conditioned medium method, wherein I transferred the supernatant collected from anesthetic-treated human astrocyte cultures onto murine hippocampal neurons.

4.2.Methods

4.1.1. Preparation of primary cell cultures

Primary cell cocultures of hippocampal neurons and cortical astrocytes, and human optic nerve head astrocytes were prepared as previously described in Chapters 3.2.1 and 3.2.2, respectively.

4.1.2. Whole-cell voltage-clamp recordings in cell culture

For whole-cell voltage-clamp recordings, the recording electrode (2-3 MΩ) was filled with intracellular solution containing (in mM): 140 CsCl, 10 HEPES, 11 EGTA, 2 TEA, 1 CaCl2, 2 MgCl2, 4

Mg2ATP (pH adjusted to 7.3 with CsOH and osmolarity adjusted to 290-2955 mOsm). The pipette offset was compensated when the recording electrode was in the bath which consisted of the extracellular solution containing (in mM): 140 NaCl, 25 HEPES, 2 KCl, 1.3 CaCl2, 1 MgCl2, 28 glucose (pH adjusted to

7.4 with NaOH and osmolarity adjusted to 322-328 mOsm). Cells were treated with drugs for 1 hour. The media was then removed and replaced with fresh culture media. Recordings were performed 24 hours later; all cells were recorded at a holding potential of -60 mV and automatic capacitance compensation from the Multiclamp software was applied. Cells were treated with the sevoflurane (SEVO; 266 μM) or isoflurane (ISO; 250 μM) or vehicle solution (extracellular solution); propofol (PROP; 3 μM) or vehicle solution (phosphate buffered saline); midazolam (MDZ; 200 nM) or vehicle solution (phosphate buffered saline); ketamine (KET; 10 μM) or vehicle solution (benzethonium chloride dissolved in phosphate 84 buffered saline). These concentrations were selected because they correspond to anesthetizing doses in humans. To prepare conditioned supernatant media, human astrocyte cultures were treated with etomidate (ETOM; 1 μM) for 1 hour. The media was then removed and replaced with fresh culture media. The astrocytes were kept in fresh media for 2 hours before the conditioned media was collected and applied to neuronal culture for 24 hours. The concentration of 1 μM for etomidate was chosen because it corresponds to an anesthetizing dose in humans (Alkire & Gorski, 2004; Benkwitz et al. 2007).

To measure the amplitude to tonic current, exogenous GABA (0.5 μM) was added to the extracellular solution and the change in holding current was measured during application of bicuculline (BIC; 20 μM).

4.1.3. Statistical analyses

Statistical analyses were performed as previously described in Chapter 3.3.

85 4.3. Results

First, we asked whether anesthetics other than etomidate can also cause a persistent increase in tonic GABA current when applied to cocultures of murine astrocytes and neurons. Isoflurane (ISO; 250

µM) and sevoflurane (SEVO; 266 µM) caused a significant increase in tonic GABA current in cultured hippocampal neurons 24 hours after treatment. Similarly, intravenous anesthetic propofol (PROP; 3 µM) caused a persistent increase in tonic current. Interestingly, the benzodiazepine midazolam (MDZ; 200 nM) also caused an increase in tonic current that persisted 24 hours after the treatment period.

However, ketamine (KET), an NMDA receptors antagonist that was used as a control, did not cause a persistent increase in tonic current at sedative (10 µM) concentration (Figure 4.1). Altogether, these results show that various widely used GABAergic anesthetics and sedative agents trigger a persistent increase in tonic current in hippocampal neurons when applied at clinically relevant concentrations, while the NMDA receptors antagonist and sedative agent ketamine does not.

86 A Drug 1 h 24 h wash out astrocytes + neurons

B C

D E

9 7

Figure 4.1 Multiple general anesthetics and a benzodiazepine trigger a persistent increase in tonic current in neurons. (A) Cocultures of neurons and astrocytes were treated with anesthetics for 1 h. The medium was then replaced with fresh culture medium. 24 h later, bicuculline (BIC, 20 µM) was used to measure the tonic current. Dashed lines indicate the difference in holding current during application of 0.5 μM GABA alone and during application of 0.5 μM GABA with 20 μM BIC. Summarized data show that the (B) inhalational anesthetics isoflurane (ISO, 250 µM) and sevoflurane (SEVO, 266 µM), (C) intravenous anesthetics etomidate (ETOM, 1μM) and propofol (PROP, 3 µM), and (D) GABAergic benzodiazepine midazolam (MDZ, 200 nM) cause a persistent increase in GABA-evoked tonic current in astrocyte-neuron cocultures compared to vehicle controls, while (E) NMDAergic sedative agent ketamine (KET, 1 mM and 10 µM) does not. n = 7-16 cells, * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t-test of one-way ANOVA. CTRL/ Veh: control.

Our previous findings showed that etomidate triggers a persistent increase in tonic current in

hippocampal neurons when conditioned medium from etomidate-treated astrocytes is applied to

neurons. Based on these findings, we next asked whether we could observe the same effect when the

experiment was performed with primary astrocyte cultures from humans. Murine hippocampal neurons

treated with conditioned medium from etomidate (ETOM; 1 µM) -treated human optic nerve head

astrocytes showed a significant increase in tonic current that persisted 24 hours after treatment (Figure

4.2). These results suggest that etomidate can stimulate the release of soluble factors from human-

derived astrocytes, which increase tonic current in hippocampal neurons, thus increasing the

physiological relevance of our findings.

A ETOM

1 h wash 2 h 24 h out Human astrocytes neurons

2.0 ***

) pF

A 1.5

p (

t i

s 1.0

n e

n 0.5

e 12 12 r

u C 0.0 CTRL ETOM 1 μM

Figure 4.2 Human astrocytes release factors that mediate the etomidate-induced increase in tonic current in neurons. (A) Cultured human astrocytes were treated with etomidate (ETOM, 1 μM) for 1 h. The medium was then replaced with fresh culture medium for 2 h before the medium was collected to treat cultured neurons. Tonic current in neurons was recorded 24 h later. (B) Summarized data show that etomidate (ETOM, 1 μM) causes a significant persistent increase in neuronal tonic current. n = 12 cells *** P < 0.001, Student’s t-test. CTRL: control.

4.4. Discussion

Our findings help increase the clinical and physiological relevance of our previous findings that demonstrate that anesthetics cause a persistent increase in tonic current in neurons, which may underlie postanesthetic cognitive deficits. Specifically, our findings present the first evidence that various widely used anesthetics cause a persistent increase in tonic GABA current and that this anesthetic-induced tonic current increase can be mediated by soluble factors released by human astrocytes. A clinically relevant anesthetic dose of the anesthetics isoflurane (250 µM), sevoflurane (266

µM) and propofol (3 µM), and a benzodiazepine midazolam (200 nM) increased tonic current 24 hours after removal of the drug in neurons that were cocultured with astrocytes. This provides a novel mechanism by which these general anesthetics and benzodiazepines may contribute to postoperative delirium and POCD, as reported in clinical studies. Conditioned media collected from etomidate (1 µM) - treated human astrocytes also increased tonic current in neurons 24 hours later. Collectively, these results increase the physiological and clinical relevance of our lab’s previous findings and suggest that the mechanistic understanding gained through studies using etomidate in murine cultures may be relevant and applicable to humans and various other commonly used GABAergic anesthetics and sedative drugs.

These results suggest that GABAergic general anesthetics and benzodiazepines cause an increase in GABAA receptors surface expression, which results in an increase in tonic current. It is noteworthy that other labs have produced results that support this finding. Namely, Li et al., 2015 have shown that propofol causes an increase in neuronal GABAA receptors surface expression in the hippocampus of mice. Previous findings from our laboratory show that the etomidate-induced increase in tonic current is mediated by an increase in specifically α5GABAA receptors surface expression. It is likely that an increase in α5GABAA receptors surface expression on neurons may also underlie the increase in tonic current induced by the general anesthetics and benzodiazepine tested here. Further 90 biochemical studies are needed to investigate this question. Interestingly, in humans the GABAA receptors α5 subunit gene has also been identified as a susceptibility locus for schizophrenia

(Maldonado-Aviles et al., 2009) and depression (Kato, 2007). Autopsy studies from individuals who have suffered from major depression exhibit marked alterations in the expression of α5GABAA receptors

(Choudary et al., 2005; Sequeira et al., 2009). The idea that changes in extrasynaptic GABAA receptors expression are associated with stress-related disorders, and specifically α5GABAA receptors are heavily regulated by stress hormones is a recent emerging theme in literature.

Our previous studies had been conducted using murine cell cultures, and although this may be a good initial model for our studies, further studies using human cell cultures were needed to enhance the physiological relevance of our findings. Rodent and human astrocytes share many similarities; however, there are also some key differences that may affect the results of our experiments. Namely, human astrocytes are structurally more complex, larger, and propagate calcium signals significantly faster than rodent astrocytes (Openheimer et al. 2006). Specifically, astrocytes in human neocortex are 2.6 fold larger in diameter and extend 10-fold more GFAP+ primary processes than their rodent counterparts, and propagate Ca2+ waves at approximately 4-fold faster than rodent. The human neocortex also contains several anatomically-defined subclasses of astrocytes that are not represented in rodents.

Thus, human cortical astrocytes are larger and structurally more complex and diverse than those of rodents (Openheimer et al. 2010). The increase in astrocytic complexity may permit the increased functional competence of the adult human brain, and provide reasoning to conduct studies in more relevant and representative cell culture models.

One question that arises from these studies is whether GABAA receptors activation alone in astrocytes is sufficient to cause the increase in tonic current in neurons. It is interesting to note that these results demonstrate that several GABAergic drugs cause an increase in the tonic current, while the

NMDAergic drug ketamine fails to do so. Multiple studies have reported activation of astrocytic GABAA

91 currents by high levels of exogenous GABA or GABAergic compounds (i.e. anesthetics, benzodiazepine, barbiturates, etc.) (Felisberti et al., 1997; Fraser et al., 1994, 1995; Rosewater & Sontheimer, 1994;

Thrane et al., 2012). Thus, it seems likely that activation of astrocytes with high levels of GABA or

GABAergic compounds may be sufficient to induce changes in tonic current. However, further studies are required to test this notion.

92 Chapter 5. Mechanism(s) mediating anesthetic-induced persistent increase in tonic current

5.1. Introduction

Some patients undergoing surgery and general anesthesia incur postanesthetic memory deficits, which compromise their ability to learn and recall information and makes it difficult to complete common daily tasks necessary for self-preservation and well being (Monk & Price, 2011). However, despite the severity of these symptoms and their prevalence roughly one quarter of patients 3 months after surgery (Price et al., 2008), no effective treatments exist to prevent or reverse postanesthetic memory deficits.

In the mammalian central nervous system, GABA functions as the predominant inhibitory neurotransmitter. GABA achieves reduced the excitability of neurons by binding the Cl- channel-coupled

GABA type A (GABAA) receptors, which are broadly divided into synaptic and extrasynaptic subcategories. Synaptic GABAA receptors mediate phasic inhibition by conducting large, transient inhibitory postsynaptic currents (IPSCs) in response to presynaptic GABA release, while extrasynaptic

GABAA receptors mediate slow, tonic inhibitory currents that are relatively constant in the neuron

(Farrant & Nusser, 2005; Mody & Pearce, 2004). In particular, one subtype of extrasynaptic GABAA receptors that contain α5 subunits (α5GABAA) are highly expressed in hippocampal CA1 pyramidal neurons (Pirker et al., 2000; Sur et al., 1999) where they play a critical role in restricting hippocampal- dependent learning and memory by mediating tonic inhibition (Caraiscos et al., 2004). Therefore, given these physiological properties of α5GABAA receptors, they could play an important role in the pathogenesis of postanesthetic memory deficits.

93 Our group has helped to characterize the neurobiological underpinnings of postanesthetic memory deficits. Specifically, we found that anesthetics alone (i.e. without surgery) caused persistent deficits in hippocampal-dependent memory tasks in rodents by increasing the expression of the same

α5GABAA receptors in hippocampal neurons (Saab et al., 2010; Zurek et al., 2012, 2014). However, the cellular and molecular mechanisms that lead to this anesthetic induced enhancement of α5GABAA receptors function remained unclear. Taking into consideration how astrocytes express functional

GABAA receptors (Velez-Fort et al., 2012) in addition to playing a central role in modulating tonic inhibition (Yoon et al., 2014; Yoon & Lee, 2014; Yoon et al., 2012), we postulated that these glial cells might be integral to the development of memory deficits following exposure to anesthetics.

Numerous studies show that major surgery triggers a massive systemic inflammatory response that can impair hippocampal function and, ultimately, memory (Cibelli et al., 2010; Fidaldo et al., 2011;

Terrando et al., 2010; Wan et al., 2007). Mechanistically speaking, many researchers believe that one of the primary functions of IL-1β is the restriction of LTP in the hippocampus, and thus the impairment of hippocampus-dependent memory (Huang & Sheng, 2010; Pugh et al., 2001; Vereker et al., 2000).

Additionally, elevated IL-1β levels in the hippocampus can completely block LTP both in vitro and in vivo, while central administration of the IL-1 receptors antagonist (IL-1ra) reverses these deficits (Barrientos et al., 2015; Chapman et al., 2010; Coogan et al., 1999; Kelly et al., 2003; Vereker et al., 2000). Similar results have also been achieved by inhibiting one of the key protein kinases downstream of the IL-1 receptors known as the p38 mitogen-activated protein (MAP) kinase (Barrientos et al., 2015; Chapman et al., 2010; Kelly et al., 2003), thereby reaffirming the role of this signaling cascade in inflammation- induced cognitive decline. Since we previously demonstrated the involvement of p38 MAP kinase in memory deficits caused by microglia-facilitated inflammation (Wang et al., 2012), we surmised that a similar mechanism may be contributing to postanesthetic memory deficits. Although microglia serve as the primary immune cell in the CNS, astrocytes also facilitate a number of neuroinflammatory processes.

94 For instance, astrocytes contribute to pathological levels of IL-1β in acute stress, traumatic brain injury and malignant glioma (Floyd et al., 2007; Sugama et al., 2011; Tarassishin et al., 2014). Given that aberrant IL1β signaling has detrimental effects on hippocampal neurogenesis and hippocampus- dependent memory consolidation (Ben Menachem-Zidon et al., 2011; Goshen et al., 2007; Moraga-

Amaro et al., 2014), it is no surprise that these conditions are associated with memory deficits. With this in mind, along with our previously presented finding that etomidate triggers a sustained increase in

GABAA receptors activity in neurons by activating GABAA receptors in astrocytes, we hypothesized that etomidate achieves this physiological endpoint by activating an inflammatory-like response in the form of astrocytic IL-1β secretion.

Here, I tested our hypothesis that the activation of α5GABAA receptors in astrocytes is necessary to trigger a sustained increase in tonic current in neurons following anesthetic exposure and that the activation of GABAA receptors in astrocytes triggers the release of IL-1β, which subsequently increases neuronal GABAA receptors activity via an IL-1 receptors- and p38 MAP kinase-dependent pathway. My primary aims were to (1) determine the molecular mechanisms by which astrocytes are activated by anesthetics and (2) identify a candidate factor and signaling pathway by which astrocytes may alter neuronal excitability through changes in tonic current. To do so, I used an in vitro cell culture model established by our laboratory wherein neurons and astrocytes are cultured either together or separately. In the co-culture model, I treated cocultures of astrocytes and neurons. In the astrocyte- conditioned medium model, I transferred the supernatant collected from treated astrocytes onto neurons. I collected electrophysiological recordings to measure the tonic GABA-evoked currents in neurons 24 after a 1 h exposure to etomidate, a prototypic intravenous anesthetic, or sevoflurane, a commonly used inhalational anesthetic, co-applied with a GABAA-blocking compound to test whether a specific subtype of GABAA receptors is necessary to trigger a sustained increase in neuronal tonic inhibition, and various blocking compounds to identify the molecular components underlying etomidate

95 and sevolfurane-induced enhancement of neuronal tonic current.

5.2. Methods

5.1.1. Preparation of primary cell cultures

Primary cell cocultures of hippocampal neurons and cortical astrocytes, and human optic nerve head astrocytes were prepared as previously described in Chapters 3.2.1 and 3.2.2, respectively.

5.1.2. Whole-cell voltage-clamp recordings in cell culture

For whole-cell voltage-clamp recordings, the recording electrode (2-3 MΩ) was filled with intracellular solution containing (in mM): 140 CsCl, 10 HEPES, 11 EGTA, 2 TEA, 1 CaCl2, 2 MgCl2, 4 Mg2ATP (pH adjusted to 7.3 with CsOH and osmolarity adjusted to 290-2955 mOsm). The pipette offset was compensated when the recording electrode was in the bath which consisted of the extracellular solution containing (in mM): 140 NaCl, 25 HEPES, 2 KCl, 1.3 CaCl2, 1 MgCl2, 28 glucose (pH adjusted to 7.4 with

NaOH and osmolarity adjusted to 322-328 mOsm). Cocultures of astrocytes and neurons were treated with drugs for 1 hour. The media was then removed and replaced with fresh culture media. Recordings were performed 24 hours later; all cells were recorded at a holding potential of -60 mV and automatic capacitance compensation from the Multiclamp software was applied. To prepare conditioned supernatant media, astrocyte cultures were treated with drugs for 1 hour. The media was then removed and replaced with fresh culture media. The astrocytes were kept in fresh media for 2 hours before the conditioned media was collected and applied to neuronal culture for 24 hours. For some coculture experiments, L-655, 708 (L6; 50 nM) was co-applied with etomidate (1 μM) or sevoflurane (266 μM). The concentrations of 1 μM for etomidate and 266 µM for sevoflurane were chosen because they correspond to an anesthetizing dose in humans (Alkire & Gorski, 2004; Benkwitz et al. 2007; Garcia et al.

96 2010). For ACM experiments, L5 was co-applied with etomidate or sevoflurane to astrocytes, since the

action of α5GABAA receptors was being tested only on astrocytes. In other coculture experiments, IL-1β

(60 ng/mL) or minocycline (100 µM) were co-applied with etomidate (1 μM). In other coculture

experiments, interleukin 1 receptors antagonist (IL1ra; 100 ng/mL) or SB 203,580 (SB; 20 μM) were co-

applied with etomidate (1 μM) or sevoflurane (266 μM), and a second application was made after the

media was replaced with fresh culture media. This was done to prevent the actions of IL1β and p38-MAP

kinase both in the presence and absence of etomidate and sevoflurane. However, for ACM experiments,

IL1ra or SB was applied only when the ACM was transferred to neuron cultures, since the action of IL1β

and p38-MAP kinase were only being tested in neurons. In all cases, the amplitude of tonic current by

measured by adding exogenous GABA (0.5 μM) to the extracellular solution and the change in holding

current was measured during application of BIC (20 μM).

5.1.3. Statistical analyses

Statistical analyses were performed as described previously in Chapter 3.3.

97 5.3. Results

5.3.1. Activation of astrocytic α5 GABAA receptors

Considering our previous findings that the α5GABAA inverse agonist L-655,708 (L6) reverses

postanesthetic memory deficits in mice and prevents any increase in neuronal tonic current in ex vivo

slice recordings (Zurek, 2014), we next asked if L6 could prevent etomidate and sevoflurane from

increasing tonic current in vitro. Since the presence of astrocytes is necessary for this effect, etomidate

and sevoflurane are known as relatively GABAA-selective drug (Rudolph & Antkowiak, 2004), and the

anesthetic-induced persistent increase in tonic current is mediated by actions on α5GABAA receptors, we

postulated that α5GABAA receptors in astrocytes contribute to postanesthetic memory deficits.

Specifically, we asked whether the effect is indeed the result of the action of etomidate and sevoflurane

on α5GABAA receptors in astrocytes.

To test this hypothesis, we co-treated astrocyte cultures with etomidate (1 μM) or sevoflurane

(266 μM) and L-655, 708 (L6, 50 nM) for 1 hour to test whether L-655,708 (L6) could block the increase

in tonic current induced by etomidate or sevoflurane. Indeed, L6 prevented etomidate and sevoflurane

from triggering a sustained increase in tonic current in neurons exposed to medium from astrocytes

conditioned with both etomidate or sevoflurane (Figure 5.1). These results demonstrate that the

activation of astrocytic α5GABAA receptors on astrocytes is necessary for etomidate and sevoflurane to

trigger the release of soluble factors from astrocytes, which in turn cause an increase in tonic current in

neurons. Not only do these results reaffirm our previous findings, but they also offer exciting new

evidence implicating α5GABAA receptors in the etiology of postanesthetic memory deficits.

However, as I will discuss later in greater detail, the absence of any effect following the

coapplication of etomidate or sevoflurane and L6 could simply mean that the anesthetics are unable to

98 activate a sufficient number of GABAA receptors on the surface of the astrocytes in order to trigger the downstream release of soluble factors. Therefore, the effect may not be specifically α5- dependent, but rather it may rely on a minimum (or threshold) amount of depolarization.

99 A ETOM/ SEVO + L6

1 h wash 2 h 24 h out astrocytes neurons B

Figure 5.1 Astrocytic α5GABAA receptors mediate persistent increase in tonic current in neurons induced by etomidate and sevoflurane. (A) Cultured astrocytes were treated with etomidate (Etom, 1 μM) or sevoflurane (SEVO, 266 µM) and an inverse agonist, L-655,708 (L6, 50 nM); for 1 h. The medium was then replaced with fresh culture medium for 2 h before the medium was collected to treat cultured neurons. Tonic current in neurons was recorded 24 h later. Summarized data show that (B) etomidate (ETOM, 1μM) and (C) sevoflurane (SEVO) induce a persistent increase in GABA-evoked tonic current that can be blocked by application of L6 (50 nM) to astrocytes. n = 5 - 9 cells, * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t-test of one-way ANOVA. Ctrl/ Veh: control.

5.3.2. Inflammatory pathway mediating postanesthetic increase in tonic current

Given the pathological similarities between cognitive deficits induced by inflammation and

anesthetics, it seems plausible that there are common physiological mechanisms underlying both

anesthetic and inflammation-induced cognitive deficits. Furthermore, nonspecific attenuation of innate

immunity with an anti-inflammatory agent, minocycline, has been shown to mitigate surgery-induced

elevations in IL-1β and memory impairment (Serantes et al. 2006). When minocycline (MINO; 100 µM)

was co-applied with etomidate, there was a reversal of etomidate-induced increase in tonic GABA

current (Figure 5.2B), suggesting that etomidate causes a sustained increase in tonic current through a

pro-inflammatory pathway.

We next asked whether IL-1β, a proinflammatory cytokine linked to α5GABAA receptors and

memory, contributes to etomidate induced enhancement of tonic current. IL- 1β activates neuronal IL-1

receptors, which increases α5GABAA surface expression via a p38 MAP Kinase-dependentpathway (Wang

et al., 2012). Given the tendency of astrocytes to secrete IL-1β in various inflammatory conditions (Floyd

et al., 2007; Sugama et al., 2011; Tarassishin et al., 2014), we tested whether IL-1 receptorsignaling

contributes to etomidate-induced enhancement of tonic current. IL-1β (60 ng/mL) caused a persistent

increase in tonic current, similar to etomidate. Co-treatment of IL-1β and etomidate did not cause a

further enhancement in persistently increased tonic current (Figure 5.2C), suggesting that both IL-1β and

etomidate may cause the persistent increase in tonic current through the same signaling pathway.

101

A Drug(s) 1 h 24 h wash out astrocytes + neurons

Figure 5.2 Etomidate-induced persistent increase in tonic current in neurons may be mediated by an inflammatory pathway. (A) Cocultures of neurons and astrocytes were treated with etomidate and various drugs for 1 h. The medium was then replaced with fresh culture medium. 24 h later, bicuculline (BIC, 20 µM) was used to measure the tonic current. Summarized data show that (B) an anti-inflammatory agent, minocycline (MINO, 100 µM) reverses and (C) pro-inflammatory cytokine, IL- 1β, mimics etomidate-induced increase in GABA-evoked tonic current. n = 6 - 13 cells, * P < 0.05, ** P < 0.01, Student’s t-test of one-way ANOVA. Veh: control.

When IL-1 receptors antagonist (IL-1ra, 20 ng/mL) was co-applied to cocultures of astrocytes

and neurons treated with etomidate or sevoflurane, no increase in neuronal tonic current was observed

24 hours later. When IL-1 receptors antagonist (IL-1ra, 20 ng/mL) was applied to the conditioned

medium collected from etomidate or sevoflurane-treated astrocytes, no increase in neuronal tonic

current was observed 24 hours later (Figure 5.3). These results confirm our hypothesis that, like

inflammation- induced enhancement of tonic current, etomidate increases α5GABAA receptors activity

via an IL-1 receptors-dependent pathway. Importantly, these results do not confirm that astrocytes

release IL- 1β in response to etomidate. Nevertheless, the data provide a convenient framework for

future studies.

103

A ETOM + IL-1ra 1 h 24 h wash out astrocytes + neurons

ETOM/ SEVO IL-1ra

1 h wash 2 h 24 h out astrocytes neurons

C D

Figure 5.3 Etomidate- and sevoflurane-induced persistent increase in tonic current in neurons is mediated by IL-1 receptors activity. (A) Cultures were treated with etomidate or sevoflurane and an IL-1 receptors antagonist, IL-1ra, in the coculture and conditioned medium treatment paradigms.(B) Summarized data show that an IL-1 receptors antagonist (IL-1ra, 100 ng/mL) reverses etomidate (ETOM, 1 µM) -induced increase in tonic current in cocultures, and (C) reverses etomidate (ETOM, 1 μM)- induced increase in tonic current and (D) has a trend toward reversing sevoflurane (SEVO, 266 µM)- induced persistent increase in tonic current when IL-1ra is added to the conditioned medium from anesthetic-treated astrocytes used to treat neurons. n = 5 - 8 cells, * P < 0.05, ** P < 0.01, Student’s t- test of one-way ANOVA. Veh: control.

We next asked whether p38 mitogen-activated protein kinase (MAPK), a signaling enzyme that

acts downstream of the IL-1 receptors, plays a role in increasing neuronal tonic current following

exposure to etomidate or sevoflurane. To test our hypothesis, we co-treated neuron-astrocyte

cocultures with SB-203,580 (SB, 20 μM), a specific p38 MAPK antagonist, and measured tonic current 24

hours later. As predicted, SB prevented etomidate and sevoflurane from triggering the increase in tonic

current in neurons cocultured with astrocytes and medium from neurons treated with anesthetic-

treated astrocytes (Figure 5.4). These results further implicate the “IL-1 receptors-p38” signaling

pathway in postanesthetic memory deficits pathophysiology, as well as reaffirm results presented earlier

in this thesis and in previous publications.

105

B C

Figure 5.4 Etomidate- and sevoflurane-induced persistent increase in tonic current in neurons is mediated by p38 MAPK phosphorylation. (A) Summarized data show that a p38-MAP kinase inhibitor, SB203580 (20 µM) reverses etomidate (ETOM, 1 µM) -induced persistent increase in tonic current in cocultures, whiles its inactive analog (SB202,474, 20 µM) does not, and (B) reverses etomidate (ETOM, 1 μM)-induced and (C) sevoflurane (SEVO, 266 µM)- induced persistent increase in tonic current when SB203,580 is added to the conditioned medium from anesthetic-treated astrocytes used to treat neurons. n = 6 - 12 cells, * P < 0.05, ** P < 0.01, *8* P < 0.001, Student’s t-test of one-way ANOVA. Veh: control.

5.4. Discussion

These findings present the first evidence that the activation of astrocytic α5GABAA receptors by anesthetics is necessary to trigger a sustained increase in tonic current in cultured hippocampal neurons. Specifically, co- application of L6 with etomidate and sevoflurane prevented any increase in tonic current 24 hours later. Our findings also suggest that an inflammatory pathway and particularly IL-

1 signaling plays an important role in the mechanism underlying etomidate and sevoflurane-induced increase in tonic current. Specifically, co-application of IL-1 receptors antagonist and p38 MAP kinase inhibitor on neurons with medium from etomidate or sevoflurane treated astrocytes prevented the persistent increase in tonic current.

These results lead to two possible conclusions, but in either case, the activation of astrocytic

GABAA receptors is necessary for etomidate and sevoflurane to persistently increase neuronal tonic current. In the first possible case, the effect may be subunit-dependent, and etomidate and sevoflurane may activate α5GABAA receptors on astrocytes and trigger the release of soluble factors. This proposed mechanism, although possible, is problematic due to the scarcity of evidence of α5GABAA expression in astrocytes. An alternative mechanism is that the effect is not dependent on the subunit composition of the astrocytic GABAA receptors, but rather the absolute number of receptors that are activated. In other words, L6 may prevent the increase in tonic current because it precludes a sufficient etomidate- triggered excitatory GABAA current from taking place in the astrocytes. These possibilities will be explored in greater detail in the general discussion of this thesis.

We have not yet identified the soluble factor released from astrocytes. However, we can infer from these results and the pharmacology of IL-1 that the molecule secreted in response to etomidate and sevoflurane treatment is likely IL-1β. Specifically, considering how IL-1α often remains membrane- bound following the cleavage of pro-IL-1α by calpain (Pugh et al., 2001; Schobitz et al., 1994), while IL-1β

107 acts as the main secreted form of the cytokine (Pugh et al., 2001; Rothwell & Luheshi, 1994), it seems unlikely that IL-1α could account for the level of IL-1 receptors activation that is necessary to enhance tonic current in neurons. Nevertheless, more precise methods such as western blotting or ELISA will be needed to confirm this assumption. Preliminary results from our laboratory show that IL-1β and phosphorylated p38-MAP kinase levels are increased in ex vivo mouse tissue 24 hours after injection with a sedative dose (8 mg/kg) of etomidate, further suggesting that IL-1β and p38-MAP kinase mediate the anesthetic-induced increase in tonic current.

In addition to this novel finding, the results suggest that a similar mechanism may underlie the pathogenesis of memory deficits triggered by anesthesia and inflammation. Specifically, the findings reported here demonstrate that the IL-1--p38 MAP kinase pathway contributes to etomidate- and sevoflurane-induced enhancement of tonic current in the same manner that it contributes to inflammation-induced enhancement of tonic current (Wang et al., 2012). Furthermore, studies have demonstrated that the IL-1β–p38 MAP kinase pathway plays a central role in regulating LTP and memory

(Coburn et al., 2010; Huang & Sheng, 2010; Kelly et al., 2003; Vereker et al., 2000), and also that its hyperactivity contributes to memory deficits that are incurred following severe inflammatory injuries

(i.e. surgery) (Cibelli et al., 2010; Coogan et al., 1999; Pugh et al., 2001). The larger implications of multiple memory disorder that share a common cellular pathway will be explored in greater detail in the general discussion of this thesis.

These studies suggest that IL-1β may be the factor that mediates the anesthetic-induced increase in tonic current in neurons. However, the source of IL-1β remains to be investigated. Our studies suggest that astrocytes may be the source of IL-1β. Literature suggests that anesthetic exposure can also cause the activation of reactive astrocytes and thereby trigger the release of proinflammatory cytokines which result in neuroinflammation (Erasso et al., 2013). Furthermore, astrocytes have been shown to contribute to memory deficits associated with acute stress, traumatic brain injury and

108 malignant glioma (Floyd & Lyeth, 2007; Sugama et al., 2011; Tarassishin et al., 2014) by contributing to pathological levels of interleukin-1β (IL-1β). However, whether IL-1β is released from astrocytes, and if so, the mechanism underlying this release remains controversial (Verkhatsky et al., 2016; Moynagh et al., 2005). The study from our laboratory which investigated inflammation-induced memory deficits suggested that microglia may be the source of IL-1β (Wang et al. 2012). Our reductionist model consists of astrocytes and neurons, albeit some possible contamination with microglia. Furthermore, published studies demonstrate that microglia are unable to mediate anesthetic-induced increase in tonic current

(Zurek et al. 2014). These lines of evidence suggest that either astrocytes release IL-1β, or induce the release of IL-1β from neurons or the small number of microglia, which may be sufficient to cause effects on tonic inhibition in neurons.

In summary, we used a reduced cell culture model to show that etomidate and sevoflurane trigger a sustained increase in neuronal tonic current by activating astrocytic α5GABAA receptors. Our findings shed light on the intercellular communication that takes place between astrocytes and neurons following exposure to anesthetics. Specifically, we have shown that a neuronal signaling cascade involving the IL-1 receptors and p38 MAP kinase contributes to this effect, as specific antagonists for each protein prevented any increase in tonic current.

109 Chapter 6. General Discussion

6.1. Summary

The objective of these studies was to: (1) increase the physiological and clinical relevance of our lab’s previous findings that provide insight into the molecular underpinning of postanesthetic memory deficits and (2) further characterize the cellular and molecular pathways underlying anesthetic-induced enhancement of neuronal tonic current. Together, the results show that several general anesthetics and a benzodiazepine increase a tonic current in neurons that may underlie postanesthetic memory deficits.

The results also demonstrate that human astrocytes release soluble factors that mediate the anesthetic- induced increase in tonic current. In addition, the activation of GABAA receptors in astrocytes is required for etomidate and sevoflurane to trigger a pro-inflammatory pathway in neurons involving IL-1 receptors and p38 MAP kinase to cause a sustained increase in this neuronal tonic current.

The results expand the relevance of the previously identified mechanism that underlies postanesthetic memory deficits. Specifically, they suggest that multiple GABAergic drugs, such as commonly used anesthetics and benzodiazepines, cause persist memory deficits through increasing a tonic GABAA receptors-mediated inhibitory current in hippocampal neurons. Furthermore, human astrocytes can also mediate this increase in tonic current through the release of soluble factors, suggesting the relevance of the pathway in humans. Given that human and rodent astrocytes have different morphological and physiological characteristics that affect their functional properties, it was important to conduct these studies to confirm the physiological relevance of our previous findings to humans.

The data present two novel and significant findings. First, they elucidate a possible mechanism by which anesthetics interact with astrocytes, namely via activation of GABAA receptors. Second, they suggest that a proinflammatory pathway mediates postanesthetic memory deficits. These findings 110 provide insight into the physiological nature of postoperative delirium and POCD. Specifically, not only do my results describe the cellular and molecular components of this phenomenon in greater detail, but they also present novel targets for the treatment and prevention of postanesthetic memory deficits.

These results align with previous studies demonstrating that α5GABAA receptors play a critical role in the development of postanesthetic memory deficits (Saab et al., 2010; Zurek et al., 2012, 2014).

These results support the notion that astrocytes are an important cellular intermediate in the mechanism underlying increase in neuronal tonic current. However, the intracellular events that occur following activation of GABAA receptors in astrocytes that lead to the induction of a pro-inflammatory pathway in neurons cannot be delineated based on these results, and are thus open to speculation.

Furthermore, this thesis proposes that anesthetics may impair memory via the same IL-1β – p38 MAP kinase signaling cascade in hippocampal neurons that has been previously shown to mediate inflammation-induced memory deficits (Wang et al., 2012).

6.1.1. A central role for astrocytes in postanesthetic memory deficits

The results show that activation of astrocytic GABAA receptors plays a central role in etomidate- induced enhancement of neuronal tonic current. However, exactly what takes place on a subcellular level during and after this initial GABAergic activation remains uncertain. Astrocytes express slow-

- desensitizing, low affinity GABAA receptors that, when activated, generate an outward depolarizing Cl current that triggers Ca2+ influx via voltage-gated Ca2+ channels (Velez-Fort et al., 2012; Yoon et al.,

+ 2012). GABAA-mediated depolarization also triggers a long- term blockade of voltage gated K channels, which conduct outward hyperpolarizing currents (Bekar et al., 1999; Velez-Fort et al., 2012), thus positively feeding back onto the depolarization-induced increase in intracellular Ca2+ levels. There is a limited understanding of the mechanistic events that occur following this increase in astrocytic Ca2+ levels. Thus we can only speculate upon the down-stream components of the mechanism.

111 Multiple studies have reported activation of astrocytic GABAA currents by high levels of exogenous GABA or GABAergic compounds (i.e. anesthetics, benzodiazepine, barbiturates, etc.)

(Felisberti et al., 1997; Fraser et al., 1994, 1995; Rosewater & Sontheimer, 1994; Thrane et al., 2012).

These studies support the idea that deviations from physiological states, such as injury and exposure to

GABAergic drugs, can lead to perturbations in astrocytic Ca2+ levels. Increased intracellular Ca2+ can lead to Ca2+-dependent vesicular release of gliotransmitters or cytokines (Haydon & Carmignoto, 2006; Perea et al., 2009; Velez-Fort et al., 2012; Yoon et al., 2012). Anesthetic-induced Ca2+ changes might also initiate downstream intracellular cascades that activate transcription and expression of new peptides, consequently resulting in a reactive, inflammatory-like cellular phenotype (Corps et al., 2015; Jo et al.,

2014; Narayan et al., 2014). Altogether, it seems likely that anesthetics trigger an upregulation of neuronal tonic current via a combination of astrocytic mechanisms, namely the Ca2+-dependent release of soluble factors and activation of inflammatory-like genetic programs, although we will need further studies to confirm this idea.

In addition to considering how the anesthetic-induced activation of GABAA receptors affects a single astrocyte during postanesthetic memory loss, we must also consider the broader, network-wide changes that result from anesthetic exposure. Intercellular connections formed by gap junctions lead to complex astrocyte networks (Mantz et al., 1993), which allow for cellular and cytoplasmic continuity.

The increase in Ca2+ occurring in a single cell can propagate as a wave across vast cellular networks, ultimately affecting synaptic transmission through “astrocyte plasticity” (Dallerac et al., 2013; Mantz et al., 1993; Pirttimaki & Parri, 2013). These changes can lead to especially rapid and robust morphological and electrophysiological changes in the hippocampus (Haber & Murai, 2006). As discussed previously anesthetics also disrupt widespread Ca2+ waves by differentially affecting gap junction permeability in astrocytes (Mantz et al., 1993; Thrane et al., 2012). Thus, anesthetics may cause the cognitive symptoms of POCD by changing Ca2+ wave signaling throughout the hippocampus and in other brain regions (e.g.

112 impairing PFC dependent executive function). However, it is still unclear how anesthetics affect memory by perturbing network-wide astrocyte signaling.

6.1.2. A role for α5GABAA receptors in cognition

The findings from this study, strongly implicate a role for α5GABAA receptors in cognition, which aligns with current literature. Specifically, these studies demonstrate that blocking anesthetic-induced

α5GABAA receptors on astrocytes reverses anesthetic-induced increase in tonic inhibition, which may underlie postanesthetic memory deficits. Other studies have shown that specific blockers of α5GABAA receptor-mediated tonic inhibition and α5GABAA receptors knockout mice have provided insights into how these receptors, and the tonic inhibition they mediate, impede learning and cognition (Atack, 2010;

Martin et al., 2009).

Mice with a partial or full deficit of α5GABAA receptors show improved performance in associative learning and memory tasks (Collinson et al., 2002; Crestani et al., 2002; Yee et al., 2004), and negative allosteric modulators and inverse agonists selective for α5GABAA receptors, such as α5IA, L-

655,708 or RO- 493851, enhance learning and cognitive performance (Ballard et al., 2009; Chambers et al., 2004; Dawson et al., 2006; Navarro et al., 2002). In humans, ethanol-induced amnesia is reduced by administering α5IA (Nutt et al., 2007). There is also another clinical study underway, testing the efficiency of basmisanil, a derivative of RO-493851 (another inverse agonist for α5GABAA receptors) in treating cognitive deficits in patients with Down’s Syndrome. Thus, α5GABAA receptors may mediate tonic inhibition that underlies not only postanesthetic cognitive deficits, but also cognitive deficits in various other neurological and psychiatric disorders characterized by deficits in learning, memory or cognition. The continuing development and refinement of negative allosteric modulators specific for

α5GABAA receptors (Knust et al., 2009), and other drugs that modulate tonic inhibition, holds promise as novel treatments for such disorders.

113 Interestingly, some studies indicate that mechanisms involving an enhanced tonic inhibition that impede the functional plasticity of the adult brain in learning and memory, such as those found in mice lacking α5GABAA receptors or animals treated with a negative allosteric modulator of α5GABAA receptors, might also be operational during post stroke recovery (Clarkson et al., 2010). Therefore,

α5GABAA receptors inverse agonists developed for treating cognitive disorders may equally be useful as the first clinical treatment to enhance functional recovery after stroke or possibly other devastating brain injuries.

6.1.4. Common mechanisms and targets: Implications for treating memory disorders

One of the most important findings presented in this thesis is that anesthetic- and inflammation- induced memory loss may have similar underlying mechanism(s). The fact that the mechanisms underlying both disorders have similarities, such as roles for glial intermediate(s) and the activation of a neuronal signaling cascade involving the IL-1 receptors and p38 MAP kinase, provide us with similar therapeutic targets for future studies. The p38 MAP kinase inhibitor, SB203580, improves clinical scores and reduces mRNA expression of inflammatory cytokines in a rodent model of colitis (Hollenbach et al.,

2004) and clinical trials with a p38 MAP kinase inhibitor, Semapimod, suggest that it is promising in

Crohn’s disease (Hommes et al., 2002). Thus, p38 MAP kinase inhibitors have a therapeutic effect on inflammatory processes, suggesting that they may be used to treat inflammation-induced pathologies.

Furthermore, literature suggests that there is interplay between anesthetic sensitivity and inflammation, further suggesting that a common signaling pathway is involved. Namely, volatile anesthetics have been shown to induce oxidative stress and inflammation (Lee et al. 2015), inflammation has been shown to induced increased anesthetic sensitivity (Avramescu et al., 2016), and there are interesting effects associated with the use of local anesthesia (Ueno et al. 2008), leading to therapeutic indications (Hollmann et al. 2000). Furthermore, inflammation has been shown to induce 114 changes in GABAergic activity. Namely, chronic exposure to LPS induces changes in intrinsic membrane properties and causes a sustained IL-1β dependent increase in GABAergic inhibition in hippocampal CA1 pyramidal neurons (Hellstrom et al. 2005).

As previously discussed, IL-1β plays an essential role in regulating LTP and hippocampus- dependent memory. Specifically, IL-1β suppresses LTP in the CA1 and CA3 regions of the hippocampus

(Bellinger et al., 1993; Katsuki et al., 1999), thereby regulating memory consolidation (Pugh et al., 2001).

IL-1β plays a central role in the pathogenesis of inflammation-induced memory deficits by increasing the expression of α5GABAA receptors in hippocampal neurons (Wang et al., 2012), and our results here implicate that a similar mechanism underlies postanesthetic memory deficits. Specifically, in both cases, antagonists for the IL-1 receptors or p38 MAP kinase abolish the increase in tonic GABA inhibition.

However, neither of these is a viable option for the treatment of anesthetic- or inflammation-induced memory loss since this intracellular pathway is necessary for regular learning and memory and other functions throughout the body (Huang & Sheng, 2010; Pugh et al., 2001). Therefore, we must find alternative treatment targets for these disorders.

The possibility that inflammation- and anesthetic-induced memory loss share a common underlying mechanism also has profound implications for the fields of anesthesia, surgery and neurology. These results imply that severe cognitive deficits incurred following surgery may be the result of neuroinflammatory responses to anesthetics and traumatic tissue injury incurred during surgery

(Krenk et al., 2010; Terrando et al., 2011; Vacas et al., 2013). Therefore, if we can attenuate the activation of astrocytes and microglia, which appears to be the converging point of these two mechanisms, we may prevent the long-term cytokine-triggered suppression of LTP in the hippocampus, thereby improving patients’ cognitive outcomes. Furthermore, characterizing a common mechanism that underlies multiple memory disorders, such as the one outlined here, enables us to have a better understanding of the nature of memory and consciousness in general.

115 6.2. Future directions

The results presented in this thesis demonstrate that multiple GABAergic anesthetics and a benzodiazepine increase the tonic inhibition, which has previously been shown to underlie postanesthetic memory deficits. The results also demonstrate that human astrocytes can mediate the anesthetic-induced increase in tonic current, through the release of soluble factors. Collectively, these results suggest that the mechanisms that we have previously identified and proposed to underlie postanesthetic memory deficits are relevant to humans and the use of multiple GABAergic drugs used in clinical practice. The results also show that astrocytic GABAA receptors activation and the participation of the IL-1 receptor-p38 MAP kinase signaling pathway underlie anesthetic-induced increase in the tonic inhibition, as demonstrated using the anesthetics etomidate and sevoflurane. However, although these findings provide deeper insight into the mechanisms underlying postanesthetic memory deficits, several questions remain unanswered and must be addressed in future studies.

Firstly, the pharmacodynamic and subcellular events that take place in the astrocytes following exposure to anesthetics remain largely unknown. The subunit composition and biophysical properties of astrocytic GABAA receptors must be further characterized to answer one key outstanding question: is the anesthetic-induced increase in neuronal tonic current dependent on the specific subunits comprising the receptors (i.e. α5), or is it simply a matter of activating a sufficient number of GABAA receptors so as to generate a sufficient level of depolarization? L6 prevents etomidate and sevoflurane from triggering an increase in tonic current in both the coculture and astrocyte-conditioned medium paradigms. However, this could simply be the result of L6 blocking a large enough proportion of the total GABAA receptors population; the effect may not be subunit-dependent. One way to address this issue would be to culture wild-type (WT) hippocampal neurons with either WT or α5GABAA-null (Gabrα5-/-) astrocytes and test whether the anesthetics can still trigger an increase in tonic current in the absence of α5. Furthermore, creating a conditional astrocyte-specific α5-null mouse line would allow us to test whether

116 postanesthetic memory impairments are dependent on astrocytic α5GABAA receptors. These experiments are already underway, and we hope to get the results from these experiments soon.

Several important studies must be done to characterize the subcellular events taking place in the astrocytes following anesthetic exposure. For instance, Ca2+ imaging can be used to observe how anesthetics alter Ca2+ signaling on a single-cell level. Also, Western Blot analysis would help determine which protein kinases and phosphatases are activated in response to anesthetics, reverse transcription polymerase chain reaction (RT-PCR) will identify which genes are activated, and immunocytochemistry would reveal whether any morphological changes take place in the astrocytes. Together, these studies will help to further elucidate the cellular mechanisms underlying postanesthetic memory deficits.

Experiments are also needed to determine the nature of intercellular communication between astrocytes and neurons.

Although this study strongly suggests a role for IL-1β in the pathogenesis of postanesthetic memory deficits, electrophysiology is not an ideal means for screening soluble factors secreted from astrocytes. Additionally, since cytokines circulate in the brain at concentrations less than 10 pg/mL under physiological conditions (Pollmacher et al., 2002), Western Blot would also be an inefficient screening approach. Therefore, studies using much more sensitive techniques such as mass spectrometry and enzyme-linked immunosorbent assay (ELISA) will be essential in identifying additional candidate soluble factors and, more importantly, confirming the release of IL-1β. Furthermore, while we show here that the action of IL-1ra on neurons prevents etomidate from increasing tonic current, the source of IL-1β remains unclear. Since neurons are also capable of synthesizing and secreting IL-1β in the brain (Allan et al., 2005; Freidin et al., 1992), it is possible that the cytokine may not simply be released by astrocytes in response to etomidate, but rather an additional unknown astrocytic factor could be triggering its release from the neurons and subsequently activating an autocrine signaling pathway.

The mechanism that takes place in the hippocampal neurons also requires further investigation.

117 We must confirm the participation of p38 MAP kinase using Western Blot analyses. Specifically, an increase in the neuronal levels of p38 MAP kinase phosphorylated at the Thr180 and Tyr182 residues, the activated form of the enzyme (Cuadrado & Nebreda, 2010), with no change in the total expression of p38 MAP kinase would confirm its role in the mechanism.

Future studies should also use in vivo mouse models or even human studies to fully elucidate the nature of postanesthetic memory deficits. One interesting experiment would be to intraventricularly inject supernatant collected from anesthetic-treated cultured astrocytes and compare their memory performance to that of saline injected mice. Results from these experiments would confirm that astrocytes impair memory by releasing soluble factors in response to anesthetics. Furthermore, applying astrocyte conditioned medium directly onto the hippocampus using microdialysis techniques would provide more precise results with a less invasive procedure, while conferring a higher level of brain region specificity.

Although the human astrocytes from human optic nerve head were useful as an initial screening tool, a better and more easily available cell culture model is needed for future experiments. Astrocytes are the major cell type in the optic nerve head, and so they are easily accessible in this tissue (Morgan et al. 2000) making them an attractive cell type for our initial studies. Although astrocytes from the optic nerve head and cortex share similarities in form and response to injury (Sun et al., 2010), there are differences due to their regional specificity and functions in those regions (Sun et al., 2010). Thus, it is important to conduct studies using cortical astrocytes.

The use of induced pluripotent stem cells derived into astrocytes holds promise for future studies, as cultures can be regularly made and the phenotype of astrocytes may be more similar to astrocytes from the cortex than astrocytes from the optic nerve head. It is also important that we push for clinical trials of novel preventative treatments, such as L6, and further our translational studies.

There is much compelling evidence from animal models to support the use of L6 in the prevention of 118 postanesthetic memory deficits in humans (Saab et al., 2010; Xie et al., 2015), therefore we must make human trials a priority if we are to eventually bring our findings from bench to bedside. Our laboratory has already received a US patent for the use of L6 as a treatment for postoperative delirium and POCD, and so we are well-positioned to drive this movement from bench to bedside.

Interestingly, ketamine, an NMDA receptor antagonist that is used as a control in our studies presented here, has been shown to attenuate postoperative cognitive deficits in some clinical studies

(Hudetz et al., 2009). Specifically, ketamine has been shown to attenuate postoperative delirium and

POCD in patients undergoing cardiac surgery. These results suggest that ketamine may be able to reverse anesthetic-induced increase in tonic inhibition in neurons, and thereby attenuate postoperative cognitive deficits. Future electrophysiological studies may be used to test this hypothesis. Interestingly, there is some debate surrounding this topic, as a recent clinical trial suggests that ketamine administration has no effect on the incidence of delirium (Avidan et al., 2017) while others suggest that the effect of ketamine on delirium may be due to the fact that it reduces the amount of anesthetic used and that it has no effect in and of itself (Sharma et al., 2011). Electrophysiological studies in an in vitro model, where a constant concentration of anesthetic is applied, may help eliminate these discrepancies.

There are still several questions regarding the mechanism of postanesthetic memory deficits that must be addressed. Each step of the putative pathway, from initial interactions between anesthetics and astrocytes to eventual enhancement of tonic GABA current in neurons, demands its own set of future experiments. Our group, as well as others, must rely on a combination of repeated, traditional techniques in conjunction with more ambitious, creative ones to fully understand the mechanisms underlying postoperative delirium and POCD and, ultimately, treat it.

6.3. Conclusion

In summary, several GABAergic anesthetics and a benzodiazepine cause a persistent increase in

119 tonic GABAA conductance in hippocampal neurons, which may underlie postanesthetic memory deficits.

The activation of α5GABAA receptors in astrocytes is necessary for anesthetics to trigger the release of soluble factors from astrocytes, which subsequently enhance neuronal GABAA receptors function via a signaling pathway involving the IL-1 receptors and p38 MAP kinase.

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