A Novel Mechanism for Gabapentin Actions

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A NOVEL MECHANISM FOR GABAPENTIN ACTIONS:

UP-REGULATION OF δGABAA RECEPTORS

Jieying Yu

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto

A Novel Mechanism for Gabapentin Actions: Up-regulation of

δGABAA Receptors

Jieying Yu

Master of Science

Department of Physiology University of Toronto

2014

Abstract

Gabapentin is a widely used anticonvulsant and analgesic drug that also causes anxiolysis, sedation and ataxia. The mechanisms underlying these effects are poorly understood. Previously, we showed that gabapentin increases a tonic inhibitory conductance in hippocampal neurons that is generated by α5 subunit- and δ subunit-containing GABAA receptors. Several pharmacological and physiological factors that activate δGABAA receptors cause behavioural effects similar to gabapentin.

This thesis explores the hypotheses that: (1) δGABAA receptors are necessary for the antinociceptive, anxiolytic and ataxic properties of gabapentin and (2) gabapentin up-regulates

δGABAA receptor expression in the brain. The results show that δGABAA receptors contribute to the anxiolytic and ataxic but not anti-nociceptive properties of gabapentin. Also, gabapentin increases the expression of δGABAA receptors in several brain regions. These results identify a highly novel and plausible mechanism to account for many of the behavioural properties of gabapentin.

Acknowledgments

I would sincerely like to thank my wonderful supervisor, Dr. Beverley A. Orser for her mentorship, guidance and care over the past 2.5 years that we have worked together. Dr. Orser‟s daily actions and journey as a clinician-scientist have inspired me to aim high and pursue my dream of becoming a clinician-scientist myself. She has been a pillar of encouragement, positivity and strength during the tough times in my degree, and has always been supportive of all my career decisions. Bev as the lab warmly calls her is always willing to give expert advice on academic and career subjects. I know I would not have received better training and support anywhere else.

I also want to thank my great supervisory committee members Dr. Sergio Grinstein and Dr. Michael (Mike) Salter, who have been very kind to me. They both provided invaluable guidance and suggestions on many occasions, and I thank them sincerely for their time and patience.

I would also like to thank all the members of the Orser lab that I have worked with; everyone has been a great supporter in my Masters journey. Thanks to Dr. Robert Bonin for his patience and his continous aid in my degree. Rob spent a lot of time explaining things to me when I first started my project and was the one who made the initial observations that seeded this thesis. Thanks to Agnieszka Zurek, Irene Lecker, William To, Paul Whissell, Sinziana Avramescu, Antonello Penna Silva, Dianshi Wang, Sean Haffey, and Ella Czerwinska for all the personal and academic discussions and advice. It has been a great honor to participate in your respective projects, and you guys and gals have all been great collaborators, friends and colleagues.

I reserve a special thank you for my family members who have been supportive of my ambitions. I would like to thank my parents Kenny and Iellene who instilled the value of hard work in me, my in-laws, Jacinta and Rabindra, as well as my sister Fiona, who have all provided love, prayers and emotional support. I would especially like to thank my husband, Justin who has provided support in every possible way. He has always loved me and has been my number one supporter throughout my career.

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List of Contributions

Several investigators contributed to the work presented in this thesis. In Chapter 4, Robert P. Bonin executed the tail-flick assay and elevated plus maze experiments. In Chapter 6, Robert P Bonin along with Agnieszka Zurek injected all the mice and extracted their brains for the high- performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS) work. Also in Chapter 6, Dr. Glen B. Baker and Gail Rauw performed all the HPLC, GC and MS experiments.

This work was completed with the financial support from an Alexander Graham Bell Canada Graduate Scholarship provided by the Natural Sciences and Engineering Research Council of Canada, a University of Toronto Fellowship provided by the Department of Physiology, a James F. Crothers Family Fellowship in Peripheral Nerve Damage provided by the Faculty of Medicine and travel grants provided by the School of Graduate Studies at the University of Toronto.

Table of Contents

Abstract ...... ii

Acknowledgments...... iii

List of Contributions ...... iv

Table of Contents ...... v

List of Figures ...... xi

List of Tables ...... xiii

List of Abbreviations ...... xiv

Chapter 1 : Introduction ...... 1

1.0 Overview ...... 1

1.1 GABA ...... 3

1.1.1 GABA synthesis...... 3

1.1.2 GABA release and metabolism ...... 4

1.2 GABA receptors...... 7

1.2.1 GABAB receptors ...... 7

1.2.2 GABAA receptors ...... 7

1.2.3 Synthesis, formation and trafficking of GABAA receptors ...... 10

1.3 Synaptic GABAA receptors – Phasic inhibition ...... 16

1.4 Extrasynaptic GABAA receptors – Tonic inhibition ...... 16

1.4.1 α5GABAA receptors ...... 20

1.4.2 δGABAA receptors ...... 20

1.5 δGABAA receptor: pharmacological and physiological outcomes ...... 28

1.6 Gabapentin ...... 31

1.6.1 Pharmacokinetic and pharmacodynamic properties of gabapentin ...... 35

1.7 α2δ subunit of VDCC and gabapentin ...... 36

1.8 GABA receptors and gabapentin...... 39

1.8.1 Tonic GABAA receptors and gabapentin ...... 40

Chapter 2 : Hypotheses and Aims ...... 41

2.0 Overview ...... 41

2.1 General Hypotheses ...... 42

2.2 Specific Aims ...... 42

Chapter 3 : Materials and Methods ...... 44

3.0 Overview ...... 44

3.1 Animal models ...... 44

3.2 Behavioural assays ...... 45

3.2.1 Materials for behaviour ...... 45

3.2.2 Drug administration ...... 46

3.2.3 Tail flick assay ...... 46

3.2.4 Formalin injection test ...... 47

3.2.5 Rotarod assay ...... 48

3.2.6 Elevated plus assay ...... 49

3.3 Total protein, surface protein and mRNA methods ...... 51

3.3.1 Materials for total protein, surface protein and mRNA ...... 51

3.3.2 Tissue preparation ...... 54

3.3.3 Acute brain slice generation...... 54

3.3.4 Treatment in vitro ...... 55

3.3.5 Treatment ex vivo ...... 55

3.3.6 Total protein and surface protein isolation ...... 56

3.3.7 Biotinylation assay in vitro and ex vivo ...... 58

3.3.8 Western blot ...... 61

3.3.9 Western blot quantification and equipment ...... 63

3.3.10 Real time quantitative PCR ...... 63

3.3.11 RNA extraction ...... 63

3.3.12 RT-qPCR...... 64

3.4 High-performance liquid chromatography, Gas Chromatography and Mass Spectrometry .. 65

3.4.1 Materials ...... 65

3.4.2 Animal subjects ...... 65

3.4.3 Analysis of amino acids ...... 66

3.4.4 Analysis of neuroactive steroids ...... 67

Chapter 4 : δGABAA receptors contribute to some of gabapentin‟s acute effects ...... 69

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4.0 Overview ...... 69

4.1 Introduction ...... 69

4.2 Materials and Methods ...... 70

4.3 Results ...... 71

4.3.1 δGABAA receptors do not contribute to the analgesic effects of gabapentin ...... 71

4.3.2 δGABAA receptors contribute to the anxiolytic-like effects of gabapentin ...... 74

4.3.3 δGABAA receptors contribute to the ataxic effects of gabapentin ...... 76

4.4 Discussion ...... 78

Chapter 5 : Gabapentin increases δGABAA receptor expression in vitro and ex vivo ...... 81

5.0 Overview ...... 81

5.1 Introduction ...... 81

5.2 Materials and Methods ...... 83

5.3 Results ...... 85

5.3.1 Gabapentin exposure in vitro increases δGABAA receptor expression ...... 85

5.3.2 Acute gabapentin exposure ex vivo increases δGABAA receptor surface expression ...... 92

5.3.3 Chronic gabapentin exposure ex vivo increases δGABAA receptor expression ...... 99

5.4 Discussion ...... 101

Chapter 6 : Gabapentin does not alter ligand or neurosteroid levels ...... 103

6.0 Overview ...... 103

6.1 Introduction ...... 103

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6.2 Materials and Methods ...... 104

6.3 Results ...... 105

6.3.1 Gabapentin does not increase GABAA receptor activating amino acids in murine brain tissue ...... 105

6.3.2 Gabapentin does not increase neurosteroid levels in murine brain tissue ...... 107

6.4 Discussion ...... 109

Chapter 7 : PKA and PKC are implicated in gabapentin-induced δGABAA receptors expression increase ...... 110

7.0 Overview ...... 110

7.1 Introduction ...... 110

7.2 Materials and Methods ...... 112

7.3 Results ...... 112

7.3.1 Inhibition of PKC does not prevent gabapentin induced δGABAA receptor total expression increase, but attenuates surface expression increase ...... 112

7.3.2 Inhibition of PKA attenuates gabapentin induced δGABAA receptor total and surface expression increase ...... 113

7.4 Discussion ...... 116

Chapter 8 : Discussion ...... 119

8.0 Overview ...... 119

8.1 The roles and implications of δGABAA receptors in behaviour ...... 119

8.2 Tonic inhibition and neuronal excitability ...... 120

8.3 Phosphorylation states and tonic inhibition ...... 122 ix

8.3.1 Kinases and gabapentin...... 122

8.3.2 Kinases and δGABAA receptor expression ...... 123

8.4 Future directions ...... 124

Appendix ...... 126

References ...... 127

List of Figures

Fig 1.0: GABA Synthesis and Metabolism...... 5

Fig 1.1: GABA Synthesis, Reuptake and Metabolism in cells ...... 6

Fig 1.2 GABA Receptors ...... 9

Fig 1.3 GABA sources for Synaptic and Extrasynaptic GABAA receptors...... 19

Fig 1.4 GABA and Gabapentin Structures ...... 33

Fig 1.5 Gabapentin binding in rat brain ...... 34

Fig 3.0 Elevated Plus Maze ...... 50

Fig 3.1 Sulfo-NHS-SS-Biotin Structure ...... 57

Fig 3.2 Surface protein biotinylation – in vitro experiments ...... 60

Fig 3.3 Surface protein biotinylation – ex vivo experiments ...... 60

Fig 4.0: Gabapentin does not alleviate acute pain – tail flick assay ...... 72

Fig 4.1: The antinociceptive effects of gabapentin are not δGABAA receptor dependent – formalin assay ...... 73

Fig 4.2 δGABAA receptors contribute to the anxiolytic effects of gabapentin – elevated plus maze ...... 75

Fig 4.3 δGABAA receptors contribute to the ataxic effects of gabapentin – rotarod assay ...77

Fig 5.0 In vitro application of 300 µM gabapentin upregulates surface δGABAA receptor expression in the hippocampus ...... 87

Fig 5.1 In vitro application of 300 µM gabapentin upregulates total and surface δGABAA receptor expression in the thalamus ...... 89 xi

Fig 5.2 In vitro application of 300 µM gabapentin upregulates total and surface δGABAA receptor expression in the cerebellum...... 90

Fig 5.3 Acute in vivo application of 100 mg/kg gabapentin upregulates surface δGABAA receptor expression in the hippocampus ...... 93

Fig 5.4 Acute in vivo application of 100 mg/kg gabapentin upregulates surface δGABAA receptor expression in the cerebellum ...... 95

Fig 5.5 Regional differences in α5 and δ GABAA subunit mRNA expression ...... 97

Fig 5.6 No changes in mRNA expression with acute in vivo gabapentin treatment ...... 98

Fig 5.7 Chronic in vivo application of 100 mg/kg gabapentin upregulates total and surface

δGABAA receptor expression in the cerebellum ...... 100

Fig 6.0 Chronic in vivo administration of gabapentin does not change neurotransmitter levels ...... 106

Fig 6.1 Acute and chronic in vivo administration of gabapentin does not change GABAergic neurosteroid levels ...... 108

Fig 7.0 PKC and PKA may be implicated in gabapentin induced upregulation of surface

δGABAA receptors ...... 114

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List of Tables

Table 1 Kinase/phosphatase action on GABAA receptor expression and function ...... 12

Table 2 Function of different GABAA receptor interacting proteins ...... 14

Table 3 Potency of GABA on different human GABAA receptors ...... 22

Table 4 Regional and subcellular localization of neuronal GABAA receptor subtypes ...... 24

Table 5 Primers used in RT-qPCR...... 84

Table 6 Total protein data: 300 µM Gabapentin treatment in vitro ...... 91

Table 7 Surface protein data: 300 µM Gabapentin treatment in vitro ...... 91

Table 8 Total protein data: 100 mg/kg Gabapentin treatment in vivo ...... 96

Table 9 Surface protein data: 100 mg/kg Gabapentin treatment in vivo ...... 96

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List of Abbreviations

aCSF = Artificial cerebral spinal fluid

α2/δ-1 = α2/δ-1 subunit of voltage-dependent calcium channel

AMPA = α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate

ANOVA = Analysis of variance

ATP = Adenosine triphosphate

α5GABAA = α5 subunit-containing γ-aminobutyric acid subtype A (receptor)

BIG2 = brefeldin A-inhibited GDP/GTP exchange factor 2

CA1 = Cornu Ammonis area 1

CA3 = Cornu Ammonis area 3

CaMKII = Ca2+ / calmodulin-dependent protein kinase II

CNS = Central nervous system

δGABAA = δ subunit-containing γ-aminobutyric acid subtype A (receptor)

DHEA = Dehydroepiandrosterone

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

GABA = γ-Aminobutyric acid

GABAA = γ-Aminobutyric acid subtype A (receptor)

GABAA-ρ = γ-Aminobutyric acid subtype A-ρ (receptor)

GABAB = γ-Aminobutyric acid subtype B (receptor)

GABAc = γ-Aminobutyric acid subtype C (receptor)

GABA-T = GABA transaminase

GABARAP = GABAA receptor-associated protein

Gabra5–/– = α5 subunit-containing γ-aminobutyric acid subtype A gene deletion xiv

Gabrd–/– = δ subunit-containing γ-aminobutyric acid subtype A gene deletion

GBP = Gabapentin

GAD = Glutamic acid decarboxylase

GAT = GABA transporter

GRIF1 = GABAA receptor interacting factor-1

GTP = Guanosine triphosphate

HCN = Hyperpolarization-activated, cyclic nucleotide-gated channel

HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Ihold = Holding current i.p. = Intraperitoneal

IPSC = Inhibitory postsynaptic current

IPSP = Inhibitory postsynaptic potential

KCC2 = K+ - Cl- co-transporter

L6 = L-655,708

LAT1 = L-type amino acid transporter 1

LTD = Long-term depression

LTP = Long-term potentiation mIPSC = Miniature inhibitory postsynaptic current

NMDA = N-methyl-D-aspartic acid

PKA = Protein kinase A

PKC = Protein kinase C

PLIC1 = Protein linking IAP with cytoskeleton 1

PRIP = Phospholipase C-related inactive protein

Pregnen = Pregnenolone

PTK = Protein tyrosine kinase

RMS = Root mean square

RT-PCR = Reverse transcription polymerase chain reaction

THIP = 4,5,6,7-Tetrahydroisoxazolo[4,5c]pyridine-3-ol

THDOC = 3α,21-dihydroxy-5α-pregnan-20-one; Tetrahydrodeoxycorticosterone

3α,5α-THPROG = 3α-hydroxy-5α-pregnane-20-one

VDCC = Voltage-dependent calcium channel

VGCC = Voltage-gated calcium channel

WT = Wild type

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Chapter 1 : Introduction

1.0 Overview

Gabapentin is a ɣ-aminobutyric acid (GABA) analogue that is primarily used to treat neuropathic pain and epilepsy (Kukkar et al., 2013). Gabapentin also causes many off-target effects that may be therapeutic or adverse such as anxiolysis, sedation and ataxia in humans and laboratory animals (Clarke et al., 2013; Mack, 2003; Peterson, 2009; Somerville and Michell, 2009). The mechanisms underlying these effects of gabapentin remain poorly understood.

Surprisingly, gabapentin does not produce its pharmacological effects from the direct binding of

GABAA or GABAB receptors despite being a GABA analog (Taylor et al., 1998). Presently, the α2δ subunits of voltage-dependent calcium channels (VDCC) have been identified as primary receptor targets of gabapentin (Dolphin, 2012b; Eroglu et al., 2009; Gee et al., 1996). However, the interactions at these subunits do not fully account for the multiple behavioural properties of gabapentin as described in section 1.7.

We previously reported that gabapentin increased a tonic inhibitory conductance, which is mediated by extrasynaptic GABAA receptors (Cheng et al., 2006). GABAA receptors that contain the α5 subunit (α5GABAA) or the δ subunit (δGABAA) tend to be extrasynaptic and mediate tonic GABAergic conductances (Brickley and Mody, 2012). Interestingly, the activation of

δGABAA receptors results in many similar physiological effects to gabapentin treatment. Some physiological outcomes that can result from gabapentin treatment or δGABAA receptor activation are: analgesia (Bonin et al., 2011; Kukkar et al., 2013), anticonvulsion (Goa and Sorkin, 1993; Petersen et al., 1983), anxiolysis (Clarke et al., 2013; Hoehn-Saric, 1983), ataxia (Gerlach et al., 1984; Somerville and Michell, 2009), hypnosis through increased non-REM sleep (Foldvary-Schaefer et al., 2002; Lancel and Faulhaber, 1996), and impairment of short term memory (Lindner et al., 2006; Whissell et al., 2013a).

There are 16 different GABAA receptor subunits that form different pentameric subtypes of

GABAA receptors. GABAA receptor subunit composition can determine the receptor‟s pharmacological and functional properties, as well as its localization in the synaptic or extrasynaptic area (Farrant and Nusser, 2005; Nusser et al., 1996a; Nusser et al., 1998). GABAA 1 receptors have been explored as targets for gabapentin actions in the past, but many of the past studies focused on only synaptic GABAA receptors (Gotz et al., 1993; Taylor et al., 1998). These studies investigated the binding affinity of gabapentin to the synaptic GABAA receptors since gabapentin is a GABA analog. The effect(s) of gabapentin on extrasynaptic GABAA receptors are unknown.

Extrasynaptic GABAA receptors are located outside the synaptic area and are activated even in ambient levels of GABA (Bonin et al., 2007; Farrant and Nusser, 2005; Martin et al., 2010). They tend to be temporally dispersed throughout the neuron and are continuously present where they persistently generate „tonic‟ inhibitory currents (Karayannis et al., 2010). The role(s) of GABAergic tonic inhibition are currently being explored for a wide range of physiological outcomes from learning and memory to stress regulation (Biggio et al., 2007; Martin et al., 2010).

δGABAA receptors are widely expressed in the CNS (Wisden et al., 1992), and the activation of these receptors result in similar physiological effects to gabapentin action. Due to these reasons, this thesis primarily investigates δGABAA receptors. In contrast, α5GABAA receptors show relatively restricted expression (Wisden et al., 1992), and do not elicit similar effects to gabapentin action. However, α5GABAA receptor surface and total expression were also investigated and reported in this thesis since they contribute to tonic GABAergic inhibition in the CNS.

This thesis investigates the effects of gabapentin on δGABAA receptors at the behavioural, cellular and molecular level. Using behaviour and a null mutant mouse model, δGABAA receptors‟ contribution to the anti-nociceptive, anxiolytic and ataxic effects of gabapentin were explored. Additionally, using biochemical and molecular tools, we explored whether gabapentin can upregulate δGABAA receptor surface, total and mRNA expression. Furthermore, the levels of ligands and endogenous neurosteroids, which activate GABAA receptors were measured after gabapentin treatment. Finally, we investigated the contribution of protein kinase A (PKA) and protein kinase C (PKC) to the effects of gabapentin on the GABAA receptor system. This chapter provides an introduction to the topics and concepts that are central to this thesis.

1.1 GABA

Dr. Eugene Roberts and Dr. Jorge Awapara independently discovered γ-aminobutyric acid (GABA) in the brain in 1950 (Awapara et al., 1950; Roberts and Frankel, 1950). Subsequently, GABA was characterized as an inhibitory neurotransmitter in the mammalian central nervous system (CNS) (Krnjevic and Schwartz, 1966). Presently, GABA is considered the major inhibitory neurotransmitter in the CNS (Botzolakis, 2009). GABA released from presynaptic vesicles can exert a fast and slow inhibitory effect through the activation of GABA receptors (Botzolakis, 2009). GABA and its receptors have profound behavioural and developmental effects on all organisms; hence they have been the subject of numerous studies and target sites for drug development (Rudolph et al., 2001; Whiting, 2003).

1.1.1 GABA synthesis

Two pools of GABA exist in the CNS: the transmitter and the metabolic pool. These pools of GABA are present throughout neurons (Martin and Barke, 1998). The synthesis of GABA from glutamate is facilitated by the enzyme glutamic acid decarboxylase (GAD) (Erlander and Tobin, 1991) (Fig 1.0). Two different genes encode the two major isoforms of GAD - GAD65 and GAD67 (Erlander et al., 1991).

GAD65 and GAD67 exhibit some different characteristics from each other such as molecular size, amino acid sequence, and intracellular distribution (Erlander et al., 1991). These differences help facilitate the maintenance of different pools of GABA, which vary by location and function. GAD65 is enriched near the neuronal plasma membrane and catalyzes the synthesis of GABA near synaptic terminals where it supplies GABA to the extracellular space via vesicular release (Martin and Rimvall, 1993). Additionally, an abundant amount of apoGAD (GAD without cofactors) allows GAD65 at synapses to respond to transient changes in GABA demand (Martin and Rimvall, 1993). Due to its synaptic localization and enrichment, GAD65 is usually associated with synthesizing GABA for rapid synaptic transmission (Soghomonian and Martin, 1998).

In contrast, GAD67 is enriched in the cell soma, where it catalyzes the synthesis of cytoplasmic GABA (Martin and Rimvall, 1993). Cytoplasmic GABA output usually occurs via non-vesicular mechanisms such as GABA co-transporters (Soghomonian and Martin, 1998). Null mutant mice 3 and pharmacological studies indicate that GAD67 is responsible for the majority of GABA synthesis in the brain (Soghomonian and Martin, 1998). This pool of GABA can contribute to synaptic transmission, but is usually associated with diffused signaling mechanisms (Kaufman et al., 1991; Soghomonian and Martin, 1998).

1.1.2 GABA release and metabolism

Activation of GABAergic synapses induces the release of high concentrations of GABA from the post-synaptic membrane into the synaptic cleft. GABA then diffuses to the postsynaptic membrane where it acts upon GABA receptors. GABA is then removed from the synaptic cleft via several distinct mechanisms (Cavelier et al., 2005; Conti et al., 2004). However, the complete removal of GABA from the synaptic cleft does not usually occur. As a result, there is residual GABA in the extracellular space, which constitutes “ambient” GABA concentration that ranges from 0.1 – 0.4 μM (Cavelier et al., 2005).

Additionally, GABA may diffuse from the synaptic cleft and activate receptors located on the extrasynaptic membrane; this is often referred to as “spillover” (Semyanov et al., 2004). The spillover of neurotransmitter can also affect neighbouring presynaptic and postsynaptic terminals. There is evidence to show that GABA can be released by mechanisms that are independent of action potentials, and from cells besides neurons via a process referred to as non- synaptic release. GABA can be released by Golgi cells or astrocytes in this manner (Rossi et al., 2003). These pools of GABA can contribute to the ambient GABA concentration.

GABA can be recycled or cleared via passive diffusion or via reuptake through GABA transporters (GATs) into presynaptic terminals or surrounding astrocytes (Cavelier et al., 2005; Conti et al., 2004) (Fig 1.1). There are four known subtypes of GAT (GAT 1-4). In neurons, GAT1 is the primary transporter (Fig 1.1). GAT3 and GAT4 are the major GATs in astrocytes (Conti et al., 2004; Song et al., 2013) (Fig 1.1). Thus, GAT1 is usually associated with maintaining ambient GABA levels from vesicular release, whereas GAT3/4 is from non- vesicular sources. Although GABA reuptake is the primary mechanism of extracellular GABA clearance, GABA outside of cells can also be metabolized by GABA transaminase (GABA-T), which catalyzes the conversion of GABA to succinic semialdehyde and glutamate (Madsen et al., 2008) (Fig 1.0).

Fig 1.0: GABA Synthesis and Metabolism. GAD catalyzes the formation of GABA from glutamic acid. GABA can be anabolized via two pathways: (1) α-ketoglutarate plus GABA catalyzed by GABA transaminase to form glutamic acid and succinate, which is metabolized through the Kreb cycle, (2) GABA alone catalyzed by GABA transaminase to form succinic semialdehyde, which is converted to succinate by succinic semialdehyde dehydrogenase.

Fig 1.1: GABA Synthesis, Reuptake and Metabolism in cells. The precursor to GABA, glutamate, comes from two different sources: (1) Kreb's cycle in astrocytes and other glial cells and (2) glutamine in nerve terminals. Once released into the synapse, GABA can be recycled or cleared via passive diffusion or via reuptake by GABA transporters (GATs). GAT1 generally transports GABA into the presynaptic terminals. GAT3/4 transports GABA into the surrounding astrocytes and other glial cells. Modified from (Brambilla et al., 2003).

1.2 GABA receptors

There are two types of GABA receptors: GABAA and GABAB receptors. GABAA receptors are ionotropic whereas GABAB receptors are considered metabotropic due to differences in function and structure (Olsen and Sieghart, 2008). Ionotropic receptors form ion channel pores and are able to directly regulate ion passage, while metabotropic receptors usually act through secondary messenger systems and indirectly regulate ion channels on the plasma membrane (Fig 1.2). GABA receptors are found both presynaptically, where they can regulate neurotransmitter release, and postsynaptically, where they can regulate neuronal firing (Botzolakis, 2009).

1.2.1 GABAB receptors

GABAB receptors are heteromeric proteins and in the vertebrate brain, that are form via a combination of GABAB(1) and GABAB(2) genes (Bettler et al., 2004). Unlike GABAA receptors which form ion channels, GABAB receptors are G protein-coupled receptors that act through a secondary messenger system. Giα and Goα are the main types of G proteins that are coupled with

GABAB receptors (Bettler et al., 2004). GABAB receptors work by affecting other channels downstream. They can exert slow inhibitory effects via the activation of outwardly rectifying voltage-gated potassium channels or by the inhibition of voltage-gated calcium channels (Mintz and Bean, 1993; Wagner and Dekin, 1993). Presynaptic GABAB receptors can control GABA release (autoreceptors) or inhibit neurotransmitter release (heteroreceptors). There is some literature to suggest that GABAB receptors may contribute to some of the effects of gabapentin (Bertrand et al., 2001; Ng et al., 2001). However, there is also a body of literature that directly counteracts those findings and concludes that GABAB receptors are not involved in mediating the effects of gabapentin (Lanneau et al., 2001; Patel et al., 2001; Shimizu et al., 2004). As a result, GABAB receptors will not be explored in this thesis.

1.2.2 GABAA receptors

- GABAA receptors are ionotropic Cl channels from the Cys-loop family of ligand-gated ion channels (Macdonald and Olsen, 1994). Although GABAA receptors are generally known to - - be selectively permeable to Cl , they can also be permeable to bicarbonate anions (HCO3 )

(Kaila, 1994). GABAA receptors are considered the dominant inhibitory receptor channels in the 7

CNS (Sarto-Jackson and Sieghart, 2008), and are targets for many clinical and recreational drugs

(Burt and Kamatchi, 1991). Depending on their subunit composition, GABAA receptors can generate either a phasic or tonic conductance (Stell and Mody, 2002).

Generally, under homeostatic conditions, a relatively low concentration of Cl- is found intracellularly in most adult CNS neurons. Accordingly, GABAA receptors activation usually - hyperpolarizes the membrane through Cl influx (Botzolakis, 2009). However, GABAA receptors can also have a depolarizing effect in both immature and mature CNS neurons. This occurs when intracellular Cl- concentration is above the electrochemical equilibrium; hence the activation of - GABAA receptors can cause a Cl efflux resulting in depolarization. Hence, the activation of

GABAA receptors can result in neuronal inhibition or excitation (Botzolakis, 2009).

There are 8 known GABAA receptor subunit families (α, β, δ, γ, θ, ε, π, ρ) encoded by different genes, many of these subunits have several subtypes (α1-6, β1-3, γ1-3, ρ1-3) and splice variants (e.g. γ2L and γ2S, β3-v1and β3-v2) (Botzolakis, 2009; Mertens et al., 1993; Olsen and Sieghart,

2009). Native GABAA receptors mainly exist as a pentamers consisting of two α, two β, and one

γ/ δ/ θ/ ε/ π subunit. However, there is also evidence of functional homo-oligomeric GABAA receptors consisting of only two α and two β subunits (Sieghart and Sperk, 2002). The existence of a large variety of GABAA receptor subunits and subtypes allows GABAA receptors to exhibit a wide range of pharmacological, geographical, biophysical and functional variability and specialization.

A variant of GABAA receptors exist which is composed solely of ρ subunits and is insensitive to typical allosteric modulators of GABAA receptors (Olsen and Sieghart, 2008). These receptors were formerly known as GABAC receptors, but their official designation is GABAA-ρ receptors.

GABAA-ρ receptors are typically found in retinal bipolar or horizontal cells (Wegelius et al.,

1998). This thesis will mainly focus on typical GABAA receptors since GABAA-ρ receptors are not likely to be involved in mediating the effects of gabapentin.

Fig 1.2: GABA Receptors. There are two types of GABA receptor: GABAA and GABAB receptors. GABAA receptors are ionotropic and hence can form ion channel pores and are able to - directly regulate ion passage (mainly Cl ). GABAB receptors are metabotropic and hence usually act through secondary messenger systems and indirectly regulate ion channels on the plasma membrane. Presynaptic GABA receptors can regulate the likelihood of neurotransmitter release, and postsynaptic GABA receptors can regulate the likelihood of neuronal firing.

1.2.3 Synthesis, formation and trafficking of GABAA receptors

GABAA receptors are dynamic proteins that are constantly being synthesized, degraded, recycled, and trafficked to and from the cell surface. The proteins and factors that control these mechanisms can regulate the strength and type of GABAergic current generated (phasic/tonic) and the temporal and spatial distribution of the receptors. GABAA receptors that participate in the generation of current are usually located on the cell surface. GABAA receptor surface expression is regulated by protein synthesis, phosphorylation/dephosphorylation activity, and trafficking, clustering and stabilizing proteins (Table 1 and 2).

Many, but not all GABAA receptor subunit genes are found in tight clusters on the same chromosomes. Accordingly, it is hypothesized that subunits that are close together on the same chromosome tend to express and form functional receptors (Darlison et al., 2005; Russek and Farb, 1994). An example of subunits that are on the same chromosome that tend to partner are α1, β2 and γ2; the genes of these three subunits are all in close proximity on chromosome number 5 (Botzolakis, 2009; Simon et al., 2004). The α1 subunit frequently partners with β2 and

γ2 to form functional pentameric α1β2γ2 GABAA receptors. In fact, this pentamer is one of the most common synaptic GABAA receptors found in the CNS (Sieghart and Sperk, 2002).

The significance of gene clustering for functional GABAA receptor formation is debatable, since individual neurons are more than capable of expressing multiple genes from multiple chromosomes simultaneously. In fact, not all GABAA receptor subunits that endogenously partner are found on the same chromosomes. For example the δ subunit is encoded by genes on chromosome 1, while its binding partners the α4 or α6 subunit are encoded by genes on chromosome 4 and 5, respectively (Botzolakis, 2009; Darlison et al., 2005; Simon et al., 2004).

GABAA receptors possess many phosphorylation sites and their function and expression is regulated by different kinases and phosphatases (Stelzer et al., 1988). Kinases such as protein kinase A (PKA), protein kinase C (PKC), P38 mitogen-activated protein kinases (p38-MAPK) and protein tyrosine kinase (PTK) affect the surface expression of different subtypes of GABAA receptors as well as their activity (Botzolakis, 2009; Kittler and Moss, 2003; Michels and Moss, 2007; Wang et al., 2012). Differential kinase activity can up- or down-regulate the expression of different subtypes of receptors separately or simultaneously. For example, the activation of p38-

MAPK has been found to up-regulate α5GABAA receptor expression while simultaneously down-regulating α1GABAA receptor expression (Wang et al., 2012). Alternatively, phosphatases such as protein phosphatase 1α (PP1α), calcineurin, and protein phosphatase 2A (PP2A) can dephosphorylate GABAA receptors (Table 1). The dephosphorylation of GABAA receptors has mostly been linked to endocytosis of the receptors (Jacob et al., 2008). Table 1 provides a summary of the action of different kinases and phosphatases on GABAA receptor function and expression.

In addition to kinase and phosphatase control, different subtypes of GABAA receptor subunits contain different protein binding domains. These domains influence GABAA receptor clustering, sorting, trafficking and spatial location on the membrane surface (Chen et al., 2007; Kittler and

Moss, 2003; Michels and Moss, 2007). Well known interacting proteins that affect GABAA receptor expression and/or localization are brefeldin A-inhibited GDP/GTP exchange factor 2

(BIG2), GABAA receptor interacting factor-1 (GRIF1), GABAA receptor-associated protein (GABARAP), Golgi-specific DHHC zinc finger protein (GODZ), phospholipase C-related inactive protein (PRIP), gephyrin, radixin, a ubiquitin-like protein - protein linking IAP with cytoskeleton 1 (PLIC1), and adaptin complex AP2. Table 2 provides a summary of the function of different GABAA receptor interacting proteins.

GABAA receptors are mostly inserted into the extrasynaptic membrane (Arancibia-Carcamo and

Kittler, 2009; Jacob et al., 2005; Thomas et al., 2005; Triller and Choquet, 2005). GABAA receptors laterally diffuse into the synaptic area where they are stabilized by scaffolding proteins such as gephyrin (Arancibia-Carcamo and Kittler, 2009; Jacob et al., 2005; Renner et al., 2008).

The mobility of the GABAA receptor and its ability to be stabilized by synaptic scaffolding proteins depends on the receptor‟s subunit composition. Synaptic receptors also diffuse back into the extrasynaptic area to be internalized (Leidenheimer, 2008).

Almost all of the mechanistic trafficking work has been done on synaptic GABAA receptors. It is postulated that the trafficking of extrasynaptic GABAA receptors is similar to that of their synaptic counterparts. Currently, it is hypothesized that extrasynaptic GABAA receptors trafficking and stabilization are mainly dependent on their α and β subunits.

Table 1: Kinase/ phosphatase action on GABAA receptor expression and function

Phosphorylation or Kinase/ Dephosphorylation Phosphatase site Effect Reference Enhances β2 containing GABAA receptor activity through reduced endocytosis from reduced interaction with AP2 resulting in increased Wang, Liu et al. Akt β2GABAAR(S410) surface expression, 2003b

Increases insertion of Brandon, GABAA receptors to Jovanovic et al. membrane surface when 2002, Saliba, β3GABAAR(S383), phosphorylated at Kretschmannova CaMKII γ2GABAAR(S327/343) GABRβ3(S383). et al. 2012 Enhances β3 containing GABAA receptor activity through reduced endocytosis from reduced interaction with AP2 resulting in increased surface expression. Reduces β1 containing GABAA receptor activity. Brandon, Phosphorylates PRIP1, Jovanovic et al. phospho-PRIP1 dissociates 2002; Kittler, from PP1α, therefore can not Chen et al. 2005; inactivate PP1α resulting in Terunuma, Jang β1GABAAR(S409), increase endocytosis and et al. 2004; β3GABAAR(S408/409), decrease surface expression Kumar, Ren et PKA PRIP1(T94) of GABAA receptors. al. 2012

Brandon, Jovanovic et al. Enhances α4, β2 or β3 2002; Brandon, containing GABAA receptor Jovanovic et al. activity through reduced 2003; Kittler, endocytosis from reduced Chen et al. 2005; interaction with AP2 Abramian, resulting in increased surface Comenencia- expression. Ortiz et al. 2010; α4GABAAR(S443), Reduces β1 or γ2 containing Bright and Smart β1GABAAR(S409), GABAA receptor activity. In, 2013; Peirs, Patil β2GABAAR(S410), cortical neurons - decreased et al. 2014, β3GABAAR(S408/409), GABAA receptor function by Wang, Liu et al. PKC γ2GABAAR(S327/343) modifying receptor gating. 2003a

Table 1: Kinase/ phosphatase action on GABAA receptor expression and function continue

Phosphorylation or Kinase/ Dephosphorylation Phosphatase site Effect Reference Moss, Gorrie et Enhances γ2 containing al. 1995; GABAA receptor activity Brandon, through reduced endocytosis Jovanovic et al. from reduced interaction with 2002, Smith, AP2 resulting in increased McAinsh et al. PTK γ2GABAAR(Y365/367) surface expression. 2008 Increases α5 containing GABAA receptor activity through increased surface expression. Decreases α1 containing Wang, Zurek et p38-MAPK unknown GABAA receptor expression. al. 2012

Dephosphorylates β1, β2 or β3 resulting in enhanced GABAA receptor function for β1containing receptors and β1GABAAR(S409), reduced GABAA receptor β2GABAAR(S410), function for β2 or β3 Terunuma, Jang PP1α β3GABAAR(S408/409) containing receptors. et al. 2004

Dephosphorylates β3 and induces endocytosis of GABAA receptors, decreases Luscher and PP2A β3GABAAR(S408/409) surface expression. Keller 2004 Dephosphorylates γ2 and enhances γ2 containing Wang, Liu et al. Calcineurin γ2GABAAR(S327/343) GABAA receptor function. 2003a

Table 2: Function of different GABAA receptor interacting proteins

Interacting GABAA Interacting receptor protein subunit(s) Effect Reference

Reduces GABAA receptors surface expression through promoting Kittler, Chen et al. endocytosis of receptors. Binds to 2005; Kittler, Chen dephosphorylated β and γ2 subunits to et al. 2008; Smith, All β subunits, induce endocytosis via clathrin and McAinsh et al. AP2 γ2 subunit dynamin dependent mechanisms. 2008 Promotes the translocation of GABAA receptors from the Golgi apparatus to the cell surface, thereby increasing the surface expression of Charych, Yu et al. GABAA receptors through increased 2004; Luscher, BIG2 All β subunits insertion. Fuchs et al. 2011

Promotes the translocation of GABAA Marsden, Beattie et receptors to the cell surface in an al. 2007; Luscher, GABARAP All γ subunits activity dependent manner. Fuchs et al. 2011 Tretter, Jacob et al. Scaffold protein which stabilizes 2008; Fritschy, GABAA receptor surface expression. Harvey et al. 2008; α2 and α3 Clusters GABAA receptors at synaptic Luscher, Fuchs et Gephyrin subunits sites. al. 2011

Facilitates the palmitoylation of synaptic GABAA receptors enhancing GABAA receptor translocation through the Golgi apparatus. Palmitoylation stabilizes cell surface receptors resulting Keller, Yuan et al. in increased synaptic GABAA receptor 2004; Rathenberg, GODZ γ2 subunit surface expression. Kittler et al. 2004

The exact function is unclear, but it is postulated to regulate the exocytosis of Beck, Brickley et β2 containing GABAA receptors to the al. 2002; Luscher, GRIF1 β2 subunit plasma membrane. Fuchs et al. 2011

Table 2: Function of different GABAA receptor interacting proteins continued

Interacting GABAA Interacting receptor protein subunit(s) Effect Reference Inhibits ubiquitin-mediated proteolysis of receptors intracellularly. Regulates GABAA receptor surface and total expression through increasing the number of receptors available for plasma membrane insertion. Overexpression of PLIC1 results in Bedford, Kittler et PLIC1 and All α and β increased surface expression of al. 2001; Kleijnen, 2 subunits GABAA receptors Shih et al. 2000

Regulates GABAA receptor surface expression by regulating phosphatase activity. PRIP binds to PP1α and PP2A to inactive those phophatases. However, Brandon, Jovanovic when PRIP is phosphorylated at T94, it et al. 2002; dissociates from PP1α/PP2A resulting Terunuma, Jang et in the activation of the phosphatases. al. 2004; The phosphatases dephosphorylate the β Kanematsu, subunits resulting in AP2 binding which Yasunaga et al. initiates endocytosis resulting in 2006; Yanagihori, decreased surface expression of GABAA Terunuma et al. PRIP receptors. 2006 Christie and de Blas Clusters α5GABAA receptors at 2002; Loebrich, Radixin α5 subunit extrasynaptic sites. Bahring et al. 2006

1.3 Synaptic GABAA receptors – Phasic inhibition

Depending on their subunit composition, different GABAA receptors are preferentially localized to either synaptic or extrasynaptic sites (Farrant and Nusser, 2005; Nusser et al., 1996a; Nusser et al., 1998). Receptor combinations without the α5 or δ subunits are most likely synaptic

(Farrant and Nusser, 2005; Nusser et al., 1998). Synaptic GABAA receptors are typically expressed at postsynaptic terminals and mediate phasic inhibition. This type of inhibition is fast

(10-100 ms) and is spatially and temporally discrete, most likely because synaptic GABAA receptors tend to only briefly bind to GABA (Maconochie et al., 1994). Hence GABAA receptors that mediate “phasic” or “fast” inhibitory signaling are time-locked to presynaptic action potentials.

GABAA receptors involved in phasic inhibition require a high concentration of GABA (1-5 mM) to be released from presynaptic terminals, since they usually have a low affinity for GABA as shown in Table 3 (Farrant and Nusser, 2005). Phasic inhibition is important for many neuronal functions; it is essential for neuronal information transfer, the synchronization of neuronal rhythms and precision of action potential generation (Cobb et al., 1995; Farrant and Nusser, 2005).

1.4 Extrasynaptic GABAA receptors – Tonic inhibition

Unlike synaptic GABAA receptors, which mediate phasic or fast inhibitory transmission, extrasynaptic GABAA receptors can mediate a tonic inhibitory current that is not time-locked to presynaptic action potentials. Extrasynaptic GABAA receptors are activated at ambient GABA levels or even when no GABA is present (Wlodarczyk et al., 2013).

Receptor combinations that include the α5 or δ or ε subunits are most likely extrasynaptic (Farrant and Nusser, 2005; Nusser et al., 1998; Wagner et al., 2005). Tonic GABAergic inhibition is mediated by extrasynaptic GABAA receptors on postsynaptic neurons. In contrast to phasic inhibition, this type of inhibition is slow to desensitize, and involves the persistent activation of random, temporally dispersed extrasynaptic receptors (Karayannis et al., 2010).

Although tonic GABAergic inhibition is present throughout the CNS, extrasynaptic GABAA receptors and hence tonic current are highly enriched in certain cells within brain regions. These tonically enhanced cells include cerebellar granule cells (Brickley et al., 1996; Nusser et al., 16

1998), thalamocortical relay neurons of the ventral basal complex (Belelli et al., 2005; Porcello et al., 2003), hippocampal pyramidal cells (Martin et al., 2010; Whissell et al., 2013a), dentate granule cells (Nusser and Mody, 2002; Whissell et al., 2013b), and neocortical neurons (Drasbek et al., 2007) amongst others.

Extrasynaptic GABAA receptors exhibit high affinities for GABA (Table 3) and hence may be activated by the low concentrations of GABA present in the extrasynaptic space following action potential-dependent release of the transmitter. Ambient GABA levels in the brain are also sufficient for the activation of many extrasynaptic GABAA receptors in both brain slice and neuronal cell models (Bonin et al., 2007; Farrant and Nusser, 2005; Martin et al., 2010).

Although ambient GABA concentrations can activate extrasynaptic GABAA receptors, it has been shown that tonic current can be augmented through increased GABA concentrations through the application of exogenous GABA (McCartney et al., 2007) or by blocking GABA metabolism (Caraiscos et al., 2004).

Tonic inhibition is important in regulating neuronal excitability and gain which can affect firing rates. Tonic inhibition has also been implicated in mediating network oscillatory behaviour, action potential conduction and neurotransmitter release (Farrant and Nusser, 2005). Due to its broad network activity effects, imbalances in GABAergic tonic inhibition have been implicated in several physiological and pathological conditions such as motor impairments (Frye and Breese, 1982), anxiety (Maguire et al., 2005), learning and memory deficits (Saab et al., 2010; Wang et al., 2012; Whissell et al., 2013a; Whissell et al., 2013b; Zurek et al., 2012), epilepsy (Cope et al., 2009; Walker and Kullmann, 2012), mood disorders (Stell et al., 2003), stress (Biggio et al., 1990; Biggio et al., 2007; Serra et al., 2008; Serra et al., 2007), and pain (Bonin et al., 2011).

Factors that regulate tonic signaling can be cell-type specific. For example, in the cerebellar granule cells, which possess an abundance of δGABAA receptors, the tonic current is positively modulated by endogenous neurosteroids. δGABAA receptors are highly sensitive to neurosteroid changes (Belelli and Lambert, 2005). Neurosteroids synthesis and release can fluctuate with changes related to hormonal status and stress (Walker and Kullmann, 2012). Alternatively, the pyramidal cells in the hippocampus possess an abundance of α5GABAA receptors. The tonic current in these cells can be modulated through changes in inflammatory states and anesthetic 17 action, which has been shown to alter α5GABAA receptor expression and activity (Wang et al., 2012; Zurek et al., 2012).

It must be noted that in some instances GABAergic tonic current can also have an excitatory effect. This can occur either via neuron depolarization or by a network effect due to a dominant effect on interneurons (Walker and Kullmann, 2012). However, in most instances the tonic current mediated by extrasynaptic GABAA receptors is inhibitory. Generally, tonic inhibition is crucial for creating a persistent increase in cell input conductance, which in turn affects the duration and magnitude of a voltage response and ultimately action potential generation (Farrant and Nusser, 2005).

Fig 1.3: GABA sources for Synaptic and Extrasynaptic GABAA receptors. Depolarization of the presynaptic neurons releases GABA into the synaptic cleft (vesicular release); this high concentration of GABA binds to the synaptic GABAA receptors. The GABA from vesicular release can also diffuse to the extrasynaptic region (GABA spillover) and activate extrasynaptic

GABAA receptors. Non-vesicular GABA release from astrocytes and other glial cells can contribute to ambient GABA levels. Although the release of GABA from these sources is usually low, it can contribute to the activation of extrasynaptic GABAA receptors.

1.4.1 α5GABAA receptors

α5GABAA receptors are major contributors of GABAergic tonic inhibition in certain regions of the CNS. α5GABAA receptors are relatively restricted in where they are expressed compared to δGABAA receptors (Wisden et al., 1992). α5GABAA receptors are highly enriched in the hippocampus (Rudolph et al., 2001) but are absent in the cerebellum. They can be found in other brain regions such as deep cortical layers and olfactory bulbs as well (Wisden et al., 1992).

Additional regions where α5GABAA receptors are expressed are listed in table 4. The most common α5GABAA receptor isoform is α5β3γ2, which consist of a pentameric receptor formed from two α5 subunits, two β3 subunits and one γ2 subunit (Luddens and Korpi, 1995; Sur et al., 1998).

α5GABAA receptors are primarily known for mediating learning and memory. The importance of α5GABAA receptors in learning and memory has been demonstrated in α5GABAA receptor suppression and enhancement studies. Suppression of α5GABAA receptor function using knockout mouse models or the pharmacologic α5GABAA receptor-selective inverse agonist L-655, 708 results in increased rates of associative memory acquisition and slower rates of extinction in hippocampal-dependent learning tasks (Collinson et al., 2002; Wang et al., 2012; Yee et al., 2004; Zurek et al., 2012). Conversely, conditions such as anesthesia and inflammation which increase the activation of α5GABAA receptors result in increased memory deficits (Wang et al.,

2012; Zurek et al., 2012). Thus, the selective activation or suppression of α5GABAA receptors can result in profound learning and memory changes.

1.4.2 δGABAA receptors

δGABAA receptors are major contributors of GABAergic tonic inhibition in the CNS. They are expressed throughout the CNS, but they are generally enriched in granule cells and interneurons (Olsen and Sieghart, 2009; Wisden et al., 1992). Hence brain regions rich in granule cells such as the dentate gyrus and cerebellum are also rich in δGABAA receptors (Pirker et al.,

2000). Additional regions where δGABAA receptors are highly expressed are listed in table 4.

The major isoforms of δGABAA receptors are α4β2/3δ in most brains regions, and α6β2/3δ in the cerebellum. The α1β2/3δ isoform can also be found in the interneurons of the hippocampus in the molecular layer and the parvalbumin-positive interneurons of the dentate gyrus (Glykys et 20 al., 2007; Milenkovic et al., 2013). The α4β2/3δ isoform is highly enriched in the thalamus and the α6βxδ isoform is restricted to the cerebellar granule cells (Brickley et al., 1996; Laurie et al., 1992a; Nusser et al., 1998; Nusser et al., 1996b; Pirker et al., 2000). In fact, the deletion of the α4 subunit or α6 subunit in knock-out mouse models reduces the tonic current recorded in the thalamus and cerebellum, respectively (Fritschy and Panzanelli, 2006; Liang et al., 2008).

Furthermore, the enhanced activation of δGABAA receptors using the GABAA receptor agonist

4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, gaboxadol) at δGABAA receptor-selective concentrations can yield antinociception in rodent models of pain (Enna and McCarson, 2006), but THIP‟s antinociceptive effects were attenuated in α4 null mutant mice (Chandra et al., 2006).

The pharmacological and physiological functions of δGABAA receptors will be explored in the next section.

Table 3: Potency of GABA on different human GABAA receptors Receptor - composition GABA EC50 (µM) (95% Cl ) nH α1β1 141 (51.9–381) 0.7 ± 0.3 α2β1 45.4 (16.6–49.2) 1.5 ± 0.7 α3β1 268 (146–491) 0.6 ± 0.3 α4β1 0.72 (0.60–0.80) 1.0 ± 0.2 α5β1 41.8 (29.8–58.5) 0.9 ± 0.2 α6β1 7.7 (5.0–11.6) 1.2 ± 0.2 α1β1γ2L 259 (179–375) 0.7 ± 0.3 α2β1γ2L 51.3 (32.6–80.7) 0.8 ± 0.5 α3β1γ2L 249 (64.2–193) 0.6 ± 0.5 α4β1γ2L 80 (51.5–127) 0.9 ± 0.2 α5β1γ2L 31.1 (23.4–41.2) 0.7 ± 0.3 α6β1γ2L 18 (13.2–25.1) 0.8 ± 0.4 α4β1δ 0.024 (0.019–0.030) 1.1 ± 0.1 α6β1δ 0.35 (0.24–0.52) 0.9 ± 0.3 α1β2 22 (14.4–32.5) 0.7 ± 0.3 α2β2 56 (26.9–119) 0.6 ± 0.3 α3β2 56.2 (34.8–90.7) 0.7 ± 0.5 α4β2 2.29 (1.54–3.40) 0.9 ± 0.3 α5β2 23.6 (14.6–38.0) 0.6 ± 0.3 α6β2 2 (1.4–2.8) 1.3 ± 0.3 α1β2γ2L 107 (74.2–156) 1.0 ± 0.2 α2β2γ2L 131 (93.8–182) 0.8 ± 0.3 α3β2γ2L 133 (88–201) 1.1 ± 0.2 α4β2γ2L 103 (61.9–173) 0.8 ± 0.3 α5β2γ2L 41.1 (34.2–49.2) 1.0 ± 0.2 α6β2γ2L 25 (18.8–33.2) 0.9 ± 0.3 α4β2δ 1.00 (0.89–1.31) 1.3 ± 0.2 nH (1) = 0.9 ± EC50 (1) = 0.05 (0.02–0.15), 0.3, nH (2) = 1.7 α6β2δ EC50 (2) = 8.7 (3.7–20) ± 1.0

Table 3: Potency of GABA on different human GABAA receptors continued

Receptor - composition GABA EC50 (µM) (95% Cl ) nH α1β3 34.5 (23.4–50.8) 0.8 ± 0.3 α2β3 89.7 (44.3–182) 0.7 ± 0.4 α3β3 90.7 (44.3–183) 0.7 ± 0.5 α4β3 0.41 (0.25–0.69) 0.6 ± 0.1 α5β3 14.6 (10.4–20.4) 0.8 ± 0.3 α6β3 1.7 (0.8–3.5) 0.5 ± 0.2 α1β3γ2L 156 (96.0–252) 0.9 ± 0.2 α2β3γ2L 157 (94.9–259) 0.8 ± 0.4 α3β3γ2L 127 (81.7–206) 0.9 ± 0.2 α4β3γ2L 127 (68.2–236) 1.0 ± 0.2 α5β3γ2L 33.3 (22.4–49.3) 0.6 ± 0.5 α6β3γ2L 23.3 (15.5–34.9) 0.9 ± 0.3 α1β3δ 8.7 (6.4–11.7) 0.8 ± 0.3

EC50 (1) = 0.012 (0.006– nH (1) = 1.2 ± α4β3δ biphasic 5:1:5 0.025), EC50 (2) = 1.3 (0.7– 0.5, nH (2) = 1.1 injection ratio 2.5) ± 0.3 α4β3δ monophasic 0.016 (0.014–0.018) 0.8 ± 0.1 α6β3δ 0.44 (0.33–0.53) 0.9 ± 0.4

Table 3: Potency of GABA on different human GABAA receptors. This table is reproduced from data in (Karim et al., 2013). The higher the EC50, the lower the affinity for GABA the

GABAA receptor has, since a higher concentration of GABA is needed to produce a response in the receptor. The δGABAA receptors are of particular interest in this thesis. The table shows that

δGABAA receptors typically have a very low EC50 (ranging from 0.012 - 1 µM GABA) compared to other subtypes of GABAA receptors. δGABAA receptors can be activated at ambient concentrations of GABA due to their high GABA affinity. Biphasic concentration responses are observed in some receptor combinations which contain GABA binding sites of unequal affinities, hence they have two EC50 values.

Table 4: Regional and subcellular localization of neuronal GABAA receptor subtypes

Isoforms (Major Expression as a Main isoform in whole, and Subcellular MW Subunit bold) expression sites localization (kDa) References Pirker et al., Mainly synaptic 2000; Michels (soma, dendrites), and Moss Low expression can be extrasynaptic 2007; Sieghart during in some neurons. and Sperk development. α1β2/3δ 2002; Fritschy Highly and widely extrasynaptic in and Brunig α1β2/3γ2, expressed in adult cortical/hippocampal 2003; Glykys α1 α1β2/3δ CNS. interneurons 50-51 et al, 2008 Highly expressed during development. More restricted expression in adult brains. Cerebral cortex (layers I- IV), brainstem, spinal cord, Pirker et al., hippocampus, 2000; Michels amygdala, Mainly synaptic, and Moss striatum, enriched in axon 2007; Sieghart hypothalamus, initial segment of and Sperk α2β2/3γ2, superior colliculus, cortical and 2002; Fritschy α2β1γ1, inferior olive, hippocampal and Brunig α2 α2β1θ motor nuclei pyramidal neurons 52-53 2003 Highly expressed during development. Low and restricted expression in adult Mainly synaptic, in Pirker et al., CNS. Cerebral axon initial segment. 2000; Michels cortex (layers V- Extrasynaptic in and Moss VI), brainstem, inferior olivary 2007; Sieghart α3β2/3γ2, thalamic reticular neurons. Found in and Sperk α3β1γ2, and intralaminar both noradrenergic 2002; Fritschy α3α1β1/2/3γ2, nuclei, spinal cord, and serotonergic and Brunig α3 α3γ2, α3θ medial septum neurons. 53-55 2003

Table 4: Regional and subcellular localization of neuronal GABAA receptor subtypes cont’

Isoforms (Major Expression as a Main isoform in whole, and Subcellular MW Subunit bold) expression sites localization (kDa) References

Low expression during development. Pirker et al., Relatively widely 2000; Michels expressed in mature Synaptic or and Moss 2007; neurons. Thalamus, extrasynaptic, Sieghart and dentate gyrus, highly enriched in Sperk 2002; α4β2/3δ, striatum, cerebral the thalamus and Fritschy and α4 α4β2/3γ2 cortex, cerebellum dentate gyrus 60-66 Brunig 2003 Mainly perisynaptic or Low expression in extrasynaptic, general. Highly highly enriched in restricted expression. the hippocampus, Pirker et al., Hippocampus, deep can be 2000; Michels cortical layers, extrasynaptic or and Moss 2007; olfactory bulb, synaptic in spinal Sieghart and hypothalamus, spinal trigeminal nucleus Sperk 2002; α5β3γ2, trigeminal nucleus, and superior Fritschy and α5 α5β3γ3 spinal cord olivary nucleus 53-56 Brunig 2003 Highly enriched in granule cells of the cerebellum. Highly restricted α6βxδ - Pirker et al., expression. Low Extrasynaptic, 2000; Michels expression in the only in and Moss 2007; CNS except for the cerebellum. Sieghart and α6β2/3δ, cerebellum. α6βxγ2 -synaptic, Sperk 2002; α6β2/3γ2, Cerebellum, dorsal only in Fritschy and α6 α6α1β2/3γ2 cochlear nucleus cerebellum. 53-56 Brunig 2003

Pirker et al., 2000; Michels and Moss 2007; Partners Sieghart and with mainly Low expression. Mainly associated Sperk 2002; synaptic Hippocampus, with synaptic Fritschy and β1 subunits. cerebral cortex receptors 54-56 Brunig 2003

Table 4: Regional and subcellular localization of neuronal GABAA receptor subtypes cont’

Isoforms (Major Expression as a Main isoform in whole, and Subcellular MW Subunit bold) expression sites localization (kDa) References Pirker et al., Partners with Widely expressed, 2000; Michels almost all found in almost all and Moss subunit brain regions but 2007; Sieghart combinations, particularly Can be synaptic and Sperk both synaptic enriched in the or extrasynaptic, 2002; Fritschy and cerebral cortex and found in many and Brunig β2 extrasynaptic cerebellum regions. 55-57 2003 Highly expressed during development. Lower and more Can be synaptic Pirker et al., restricted or extrasynaptic, 2000; Michels expression than β2 but has been and Moss in adult CNS. associated more 2007; Sieghart Partners with Cerebellum, with and Sperk mainly hippocampus, extrasynaptic 2002; Fritschy extrasynaptic cerebral cortex, receptors except and Brunig β3 subunits striatum in the thalamus. 54-56 2003 Pirker et al., 2000; Michels and Moss Low expression. 2007; Sieghart Pallidum, central and Sperk α1β2γ1, and medial 2002; Fritschy α2β2γ1,α1β1γ1, amygdaloid nuclei, and Brunig γ1 α2β1γ1 substantia nigra Mainly synaptic 45-51 2003 Pirker et al., 2000; Michels and Moss Widely expressed 2007; Sieghart Partners with throughout the Mainly synaptic, and Sperk almost all CNS, found in mainly found in 2002; Fritschy subunit almost all brain soma and and Brunig γ2 combinations regions dendrites 45-51 2003

Table 4: Regional and subcellular localization of neuronal GABAA receptor subtypes cont’

Isoforms (Major Expression as a Main isoform in whole, and Subcellular MW Subunit bold) expression sites localization (kDa) References

Pirker et al., 2000; Michels and Moss 2007; Sieghart and Very low expression. Sperk 2002; Hippocampus, Fritschy and γ3 α1β2γ3 cerebral cortex Mainly synaptic 43-46 Brunig 2003 Low expression during development. Well expressed in adult CNS with enrichments in Extrasynaptic, Pirker et al., certain brain regions. highly enriched in 2000; Michels Cerebellum, the granule cells in and Moss 2007; thalamus, dentate the cerebellum Sieghart and gyrus, lower layers and dentate gyrus, Sperk 2002; of the cortex, as well as the Fritschy and α6β2/3δ, hippocampus, interneurons in the Brunig 2003; α4β2/3δ, striatum, olfactory thalamus and Glykys et al, δ α1β2/3δ bulb, amydala hippocampus 45-54 2008

1.5 δGABAA receptor: pharmacological and physiological outcomes

Through recombinant studies it was discovered that δGABAA receptors have an extremely high affinity/sensitivity to GABA. Studies showed that replacing the γ subunit with the δ subunit decreases the EC50 of GABA from the millimolar range to the low micromolar range, increase channel opening time and decrease the rate of desensitization (Brown et al.,

2002; Saxena and Macdonald, 1994). However, δGABAA receptors generate low amplitude currents that persist with the continued presence of agonist (Saxena and Macdonald, 1994). Due to their high affinity for GABA, δGABAA receptors can respond to low concentrations of GABA (from GABA spillover or ambient GABA levels) and mediate a persistent inhibitory conductance.

Despite their insensitivity to benzodiazepines (Nusser and Mody, 2002), enhanced activation of

δGABAA receptors using the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4- c]pyridin-3-ol (THIP, gaboxadol) at concentrations selective for δGABAA receptors can produce sedation and hypnosis (Drasbek et al., 2007; Drasbek and Jensen, 2006). Activation of δGABAA receptors using THIP has also been shown to decrease anxiety, be antinociceptive, lower seizure frequency, enhance discrimination memory, increase neurogenesis, induce ataxia and impair short term memory (Bonin et al., 2011; Gerlach et al., 1984; Hoehn-Saric, 1983; Petersen et al., 1983; Vaught et al., 1985; Whissell et al., 2013a; Whissell et al., 2013b)

Acute application of THIP evokes a larger maximal current than GABA, but only by δGABAA receptors (Drasbek et al., 2007; Drasbek and Jensen, 2006). The potency of THIP is much higher for δGABAA receptors than non-δGABAA receptors (Krogsgaard-Larsen et al., 2004). Hence,

THIP is considered selective for δGABAA receptors at low concentrations. Currently, there is no specific δGABAA receptor inhibitor available; hence THIP is being used as the main pharmacological tool to probe the effects of δGABAA receptors in vitro and in vivo (Brown et al., 2002; Storustovu and Ebert, 2006).

δGABAA receptors have also been highly implicated in the actions of ethanol. Many studies have linked δGABAA receptors to the neurodepressive consequences of ethanol use (Follesa et al., 2004; Follesa et al., 2006; Santhakumar et al., 2013; Talani et al., 2013). Chronic ethanol administration is generally associated with increased δGABAA receptor activity and expression

(Follesa et al., 2006; Follesa et al., 2005; Talani et al., 2011), conversely during ethanol withdrawal δGABAA receptor activity and expression generally decreases, which can contribute to hyperexcitability (Follesa et al., 2006; Kumar et al., 2009).

The activity of δGABAA receptors is also modified by neurosteroids. Endogenous neurosteroids are synthesized in the brain and spinal cord, and can rapidly modulate neuronal excitability through membrane receptor and ion channel interactions (Reddy, 2003). These compounds can positively modulate all GABAA receptors, but the δGABAA receptors are considered highly sensitive to neurosteroids compared to other GABAA receptors (Belelli and Lambert, 2005; Bianchi and Macdonald, 2003; Hosie et al., 2006; Maguire and Mody, 2009). As a result,

δGABAA receptors are greatly implicated in physiological and pathological conditions where endogenous neurosteroid levels change such as pregnancy (Maguire and Mody, 2008), the ovarian cycle (Maguire et al., 2005), stress (Biggio et al., 2007; Dazzi et al., 1996; Serra et al., 2008; Serra et al., 2007), mood disorders (Smith, 2013) and depression (Maguire and Mody, 2008).

Neurosteroids act as partial agonists to all GABAA receptors (Bianchi and Macdonald, 2003).

However, δGABAA receptors are the most functionally responsive and sensitive to neurosteroid changes (Wohlfarth et al., 2002). This notion has been confirmed repeatedly in both in vitro and in vivo studies using many methods including using null mutant mice that do not express

δGABAA receptors (Gabrd−/−). Gabrd−/− mice demonstrate vastly attenuated responses to neurosteroids compared to their wild type (WT) counter parts (Maguire and Mody, 2008; Maguire et al., 2005; Sarkar et al., 2011; Wu et al., 2013).

There are endogenous and synthetic neurosteroids. The most commonly studied endogenous neurosteroids that greatly potentiate δGABAA receptor-mediated tonic current are 5α-pregnan- 3α,21-diol-20-one (THDOC), progesterone and its metabolites such as 5α-pregnan-3α-ol-20-one (3α,5α-THPROG or allopregnanolone). Neurosteroids enhances GABA activation by binding to the α subunit of GABAA receptors (Hosie et al., 2006). These compounds confer a higher gating efficacy to δGABAA receptors which usually experiences low gating efficacy patterns (Bianchi and Macdonald, 2003). In addition to allosterically modulating GABAA receptor gating, neurosteroids can selectively increase δ subunit surface expression through its binding partner

29 the α4 subunit (Abramian et al., 2014; Smith et al., 2007). Neurosteroids increases the vesicular insertion of α4βδ GABAA receptors to the cell membrane (Abramian et al., 2014).

Many physiological and pathophysiological conditions are linked to changes in δGABAA receptor function and expression (Brickley and Mody, 2012; Maguire and Mody, 2008; Maguire et al., 2005; Smith, 2013). Hence δGABAA receptor-preferring synthetic steroids such as ganaxalone may be possible treatments for conditions where δGABAA receptor activity is downregulated such as post-partum depression (Maguire and Mody, 2008). Additionally, neurosteroids may be explored as possible treatments for conditions where neurogenic and cognitive deficits occur such as in Alzheimer‟s disease, since δGABAA receptor activity has been shown to promote neurogenesis (Whissell et al., 2013b). Indeed neurosteroids have been shown to be effective in reversing neurogenic and cognitive impairments in a mouse model of Alzheimer‟s disease (Wang et al., 2010). Whether this treatment is applicable to humans is currently unknown.

To date, non-pharmacological behavioural experiments performed on Gabrd -/- mice yield very few differences from their WT counterparts. The exceptions being Gabrd -/- mice exhibiting abnormal maternal behaviours, and elevated baseline pain scores (Bonin et al., 2011; Maguire et al., 2009; Mihalek et al., 1999).

Gabrd -/- mice exhibit relatively normal baseline behaviours, possibily because α5GABAA receptor activity is upregulated in some brain regions such as the CA 1 and 3 of the hippocampus to compensate for the loss of δGABAA receptors (Glykys et al., 2008). Additionally, the loss of

δGABAA receptor mediated tonic inhibition may be compensated by increases in other forms of inhibition, such as elevated synaptic inhibition through increased γ2 subunit expression in the forebrain (Peng et al., 2002), and increased TASK-1 „leak‟ potassium channel activity in the cerebellum, which results in elevated neuronal inhibition (Brickley et al., 2001).

1.6 Gabapentin

Gabapentin is a drug marketed by Pfizer and is commercially known as Neurontin™. Gabapentin is a structural analog of GABA (Fig 1.4) and was first synthesized to be used as an anticonvulsant drug that crossed the blood brain barrier (BBB) (Bockbrader et al., 2010; Somerville and Michell, 2009). Indeed, gabapentin was efficacious in the treatment of focal epilepsy, but surprisingly it did not produce its pharmacological effects from the direct binding of GABAA or GABAB receptors (Taylor et al., 1998). Additionally, gabapentin is eliminated renally in an unchanged form, therefore it is not metabolized to any GABA receptor binding metabolites (Goa and Sorkin, 1993).

Gabapentin is shown in autoradiograph studies to bind to all regions of the CNS with stronger binding in some regions than others (Fig 1.5). Gabapentin can be injected or orally taken with a oral bioavailability of approximately 65%. This value decreases with increasing dose (Somerville and Michell, 2009). Gabapentin absorption usually peaks at 2 to 3 hours post-intake with an elimination half-life of 5 to 9 hours. As a result, treatment typically involves 2 to 3 doses of gabapentin administration throughout the day (Somerville and Michell, 2009). Gabapentin exist as a zwitterion at physiological pH in an aqueous environment, a chemical characteristic that enables gut absorption via the L-type amino acid transporter system (Stewart et al., 1993).

As of 1994, gabapentin was approved for epilepsy management in North America, and as of 2004 was also approved in the United States and Europe for neuropathic pain, especially neuropathy associated with diabetes and post herpetic neuralgia (Kukkar et al., 2013). An analog of gabapentin is pregabalin. This compound is widely prescribed for the same uses as gabapentin as both drugs act similarly (Bockbrader et al., 2010; Sills, 2006). As a result, many researchers consider studies performed using gabapentin to be applicable to pregabalin, and vice-versa.

Gabapentin and pregabalin are also widely prescribed for a large number of off-label uses such as attention deficit disorder (ADD), bipolar disorder, restless leg syndrome, periodic limb movement disorder of sleep, migraine headaches, alcohol withdrawal syndrome, diabetic neuropathy (before 2004 approval), peripheral neuropathy (before 2004 approval), complex regional pain syndrome, and trigeminal neuralgia (Mack, 2003; Radley et al., 2006), both drugs are also noted to have anxiolytic properties (Clarke et al., 2013; Strawn and Geracioti, 2007;

Taylor et al., 1998). Common adverse effects of gabapentin are somnolence, ataxia, asthenia, weight gain, nausea, headaches, dizziness and vertigo (Somerville and Michell, 2009).

A primary reason for the wide off-label use of gabapentin was due to Pfizer illegally promoting off-label use for many years (Steinman et al., 2006). This practice resulted in a multimillion dollar lawsuit in 2004. Despite all the controversy over gabapentin‟s and pregabalin‟s off-label use, gabapentin and pregabalin are found to be effective clinically for several off-label uses such as anxiety (Clarke et al., 2013; Spiegel and Webb, 2012; Strawn and Geracioti, 2007), assorted pain syndromes (Bohlega et al., 2010; Ebell and Kripke, 2006; Mack, 2003), and restless leg syndrome (Mack, 2003).

Fig 1.4: GABA and Gabapentin Structures. Gabapentin is a structural analog of GABA.

Fig 1.5: Gabapentin binding in rat brain. This image is taken from (Hill et al., 1993). This autoradiographic image shows [3H]-gabapentin binding in rat brain (horizontal section). The highest levels of [3H]-gabapentin binding occurs in the cortex, hippocampus and cerebellum. Moderate binding levels are observed in the thalamus and striatum. Legend: Layers I and II of the cerebral cortex (1,2), fields Cal, Ca2 and Ca3 of Ammon's horn of the hippocampus, dentate gyrus (dg), central gray (cg), granule (gr) and molecular (mol) layers of the cerebellum, lateral septum (Is), white matter (w).

1.6.1 Pharmacokinetic and pharmacodynamic properties of gabapentin

Gabapentin is a white crystalline solid with a molecular weight of 171.24 g/mol (Bockbrader et al., 2010). Gabapentin is highly water soluble and has an acid dissociation constant (pKa) of 3.7 and a base dissociation constant (pKb) of 10.7, and exists as a zwitterion at neutral pH (Bockbrader et al., 2010; Somerville and Michell, 2009). Orally administered gabapentin is absorbed in the gut and transported across the epithelia by L-type amino acid transporter 1 (LAT1) (Bockbrader et al., 2010). LAT1 belongs to a family of transporters that facilitates the absorption of large and neutral amino acids (Stewart et al., 1993). The maximum plasma concentrations of gabapentin occurs at 2 to 3 hours post-ingestion (Somerville and Michell, 2009). Oral administration of gabapentin exhibits nonlinear kinetics, in the case of gabapentin as the dose increases, the bioavailability decreases (Bockbrader et al., 2010). The bioavailability of orally administered gabapentin in humans is approximately 60%, 47%, 34%, 33%, and 27% following 900, 1200, 2400, 3600, and 4800 mg/day given in 3 divided doses, respectively (Pfizer, 2012). The increasing popularity of using the gabapentin analog pregabalin over gabapentin is due to the fact that pregabalin has a better oral bioavailability (Bockbrader et al., 2010).

Gabapentin does not bind to any plasma proteins (Bockbrader et al., 2010) and is excreted renally unmetabolised in rodents and primates (Bockbrader et al., 2010). The elimination half- life of gabapentin in humans with healthy renal function is 5 to 7 hours and is unaltered by dose or dose frequency (Blum et al., 1994; Pfizer, 2012). Of course, individuals with impaired renal functions will experience reduced plasma and renal clearance rates of gabapentin (Blum et al., 1994; Boyd et al., 1999).

Gabapentin can cross the blood brain barrier (BBB) through the L-amino acid transport system (Luer et al., 1999). Interestingly, gabapentin accumulation in the CNS can persist beyond the attainment of maximal plasma concentrations (Bockbrader et al., 2010). Additionally, the ratio of gabapentin in the cerebrospinal fluid (CSF) to plasma increases with time after multiple dosages (Bockbrader et al., 2010; Muscas et al., 2000). This may explain why some of the effects of gabapentin manifest only after multiple dosings, in fact the dosing schedule is usually 3 times a day for epilepsy and pain management (Bockbrader et al., 2010; Pfizer, 2012; Somerville and Michell, 2009). 35

Gabapentin demonstrates a dose-response relationship in the treatment of postherpetic neuralgia and partial seizures (Anhut et al., 1994; Chadwick et al., 1998; Pfizer, 2012; Rice et al., 2001; Rowbotham et al., 1998). The lowest clinical dose used for individuals over 12 is typically 300 mg/day, however, dosages used clinically and in studies are generally 900, 1800, 2400 and 3600 mg/day (some times more) divided into 3 doses daily (Bockbrader et al., 2010; Pfizer, 2012; Somerville and Michell, 2009). Since gabapentin is a drug that manages disorders but does not cure diseases, the dosing schedule continues for extended periods of time.

1.7 α2δ subunit of VDCC and gabapentin

Initially, when gabapentin was discovered to be efficacious as an anticonvulsant and analgesic, little was known about its mechanism(s) of action. Continued research lead to the discovery of a likely target; the auxiliary α2δ subunit of voltage-dependent Ca2+ channels (VDCC). This protein contains a high affinity binding site for gabapentin (Gee et al., 1996; Suman-Chauhan et al., 1993) and pregabalin (Li et al., 2011). Two isoforms of the α2δ subunit - α2δ-1 and α2δ-2, have been identified to contribute to some of the therapeutic and adverse effects of gabapentin and pregabalin (Dolphin, 2012a, b; Dooley et al., 2007). The binding affinity of the α2δ-1 and α2δ-2 subunit to gabapentin is 59 nM and 153 nM respectively (Marais et al., 2001), and to pregabalin is 6.0 and 7.2 nM respectively (Li et al., 2011).

The α2δ-1 and α2δ-2 subunits are widely expressed in the CNS and the peripheral nervous system, and can modulate excitability in neurons (Dolphin, 2012b). Functionally, when the α2δ subunit binds to VDCCs, it increases both the expression and the conductance of the channels which can result in modified neurotransmitter release (Dooley et al., 2007; Walker and De Waard, 1998). Additionally, the α2δ subunit can regulate synaptogenesis through thrombospondin interactions which is a VDCC-independent mechanism (Eroglu et al., 2009).

Gabapentin is efficacious in the management of neuropathic pain and epilepsy, and presently the inhibition of the α2δ subunit is proposed to be the mechanism underlying these effects (Dolphin, 2012a; Maneuf et al., 2006), as it is postulated that the α2δ subunits contribute to pain and epilepsy. Some supporting evidence include: in experimental models of tactile allodynic pain (Li et al., 2006), peripheral nerve injury (Bauer et al., 2009; Newton et al., 2001), and hyperalgesia (Li et al., 2006), upregulated expression of the α2δ subunits occur. However, the exact link

36 between the α2δ subunit to epilepsy is unclear since no known human α2δ subunit mutations have been linked to epileptic phenotypes. However, in several strains of α2δ-2 mutant mice such as the entla, ducky and ducky2J mice exhibited an absence of generalized epilepsy (Brill et al., 2004; Dolphin, 2012b). Hence, the modulation of the α2δ subunit by gabapentin may contribute to the anticonvulsant effects of gabapentin.

Surprisingly, despite gabapentin‟s high affinity for the α2δ subunit, it actually exerts minimal inhibitory effects on VDCC mediated currents when applied acutely in multiple experimental models (Davies et al., 2006; Hendrich et al., 2008; Martin et al., 2002; Schumacher et al., 1998; Stefani et al., 1998; Sutton et al., 2002). However, it has been demonstrated that the binding of gabapentin and pregabalin to the α2δ-1 subunit is essential for the analgesic effect of these compounds in experimental models of neuropathic pain (Field et al., 2006). In animal studies, gabapentin and pregabalin binding is attenuated in their respective α2δ subunits in mutant mice which possess a point mutation in either the α2δ-1 subunit (R217A), or in the α2δ-2 subunit (R279A) (Lotarski et al., 2011). Additionally, although gabapentin produces no inhibitory effects on VDCC mediated currents in dorsal root ganglion (DRG) neurons from wild type (WT) mice, the VDCC mediated currents in DRG neurons from α2δ-1 overexpressing mutant mice experienced inhibition by gabapentin (Li et al., 2006). This suggests that the α2δ-1 up-regulation during neuropathic pain may be necessary for gabapentin‟s effects. Unlike acute applications, chronic applications of gabapentin in cell lines and in DRG neurons decreases the cell surface expression of α2δ subunits which results in reduced calcium channel currents (Hendrich et al., 2008).

The inhibition of the α2δ-1 or α2δ-2 subunit accounts for some of the physiological and behavioural effects of gabapentin, but there is evidence to suggest that multiple molecular targets may be involved in producing the effects of gabapentin. Clues that indicate that the modulation of the α2δ-1 subunit of VDCC does not account for all the effects of gabapentin include:

1) In ex vivo autoradiographic studies in mice, the point mutation R217A in the α2δ-1 subunit does not prevent gabapentin or pregabalin binding in all brain regions. In fact, both genotypes of mice (WT and point mutant mice) exhibited equally and extremely strong binding of gabapentin and pregabalin in the cerebellum (Bian et al., 2006). Hence, cerebellum-mediated behavioural functions, such as motor coordination, may be affected by gabapentin and pregabalin through 37

α2δ-independent mechanisms. The results from the study (Bian et al., 2006) also imply that gabapentin and pregabalin may exhibit a high affinity to other molecular target(s) yet to be identified.

2) The change in VDCC current can only be detected in conditions that increased the α2δ subunit expression (Dolphin, 2012b). However the analgesic properties of gabapentin are observed in models of pain where the α2δ subunit expression does not change (Luo et al., 2001).

3) The effects of gabapentin are observed with acute, short term applications in humans and animals (Bazil et al., 2005; Meymandi and Sepehri, 2008; Zhang et al., 2011). However, acute, short term application of gabapentin does not change Ca2+ channel mediated synaptic transmission (Brown and Randall, 2005) or α2δ expression (Hendrich et al., 2008).

4) Gabapentin has a high affinity (Kd = 59nM) to the α2δ-1 subunit of VDCC, yet clinically large doses ranging from 300 – 3600 mg per day are necessary for its therapeutic effects (Chadwick, 1994; Chen, 2013; Somerville and Michell, 2009). Therefore, another target which possesses a lower affinity to gabapentin than the α2δ subunit may be responsible for mediating some of the effects of gabapentinoids.

5) Stereoisomeric analogues of gabapentin with a high affinity for the α2δ subunit on the same binding site do not exhibit antinociceptive properties (Urban et al., 2005).

6) The disruption of synaptogenesis though gabapentin binding on the α2δ subunit may also contribute to the physiological and behavioural effects of gabapentin. However, gabapentin only disrupts synapse formation in approximately 50% of animals tested; hence not all subjects are similarly affected (Eroglu et al., 2009). Also, in vivo studies showed that chronic gabapentin exposure resulted in no significant synaptogenesis defects on infants exposed to the drug in utero (Molgaard-Nielsen and Hviid, 2011; Morrow et al., 2006).

Due to the reasons outlined above, it is hypothesized that other unknown molecular targets of gabapentin exist. Understanding the other modes of action underlying gabapentin‟s effects can improve patient outcome, and novel therapies using these drugs may be discovered.

1.8 GABA receptors and gabapentin

Gabapentin can exert a wide range of influence on a range of molecular systems. In vitro studies showed that gabapentin can increase N-methyl-D-aspartate (NMDA) receptor activity, inhibit glutamate release, inhibit voltage-gated sodium channel activity, and increase voltage- gated potassium channel activity (Sills, 2006; Taylor et al., 1998). However as of date, none of these mechanisms have been shown to contribute the behavioural effects of gabapentin.

Although gabapentin is a GABA-mimetic, gabapentin is not a substrate or direct modulator of

GABAA or GABAB receptors. However, the off-target and therapeutic effects of gabapentin are very similar to the effects produced by GABAergic drugs such as THIP. More specifically, the effects of gabapentin mirror the effects of δGABAA receptor activation. Some physiological outcomes that can result from either gabapentin treatment or δGABAA receptor activation are analgesia (Bonin et al., 2011; Kukkar et al., 2013), anticonvulsion (Goa and Sorkin, 1993; Petersen et al., 1983), anxiolysis (Clarke et al., 2013; Hoehn-Saric, 1983), ataxia (Gerlach et al., 1984; Somerville and Michell, 2009), hypnosis through increased non-REM sleep (Foldvary- Schaefer et al., 2002; Lancel and Faulhaber, 1996), and impairment of short term memory (Lindner et al., 2006; Whissell et al., 2013a).

Some studies showed that GABAB receptor function is altered by gabapentin (Bertrand et al., 2001; Ng et al., 2001). However, there is also a body of literature that directly contradicts those findings and concludes that GABAB receptors are not involved in mediating the effects of gabapentin (Lanneau et al., 2001; Patel et al., 2001; Shimizu et al., 2004). As a result, the contribution of GABAB receptors to gabapentin action is controversial.

In many studies gabapentin is found to increase GABA concentrations in the brain in both rodents and humans (Cai et al., 2012; Errante et al., 2002; Kocsis and Honmou, 1994; Kuzniecky et al., 2002; Loscher et al., 1991; Petroff et al., 1996). This increase in GABA may account for some of the GABAergic-like effects observed behaviourally.

Some studies indicate that gabapentin can increase GABA levels, more specifically ambient GABA levels through several mechanisms. In vitro studies revealed that gabapentin can activate GAD at drug concentrations of 1.0 to 2.5 mM (Silverman et al., 1991; Taylor et al., 1992), resulting in increased GABA synthesis. Also very high concentrations of gabapentin (25mM) 39 can inhibit GABA-T, a GABA-catabolizing enzyme (Taylor et al., 1992), resulting in decreased GABA metabolism. It must be noted that despite many supporting studies demonstrating increases in GABA levels with gabapentin treatment, several studies have not found this effect (Errante and Petroff, 2003; Errante et al., 2002). One possible reason that might account for the differences may be that gabapentin only increases GABA levels in certain experimental conditions and brain regions. Indeed, studies have shown that gabapentin increased GABA turnover in some brain regions tested only, and the temporal effect of the drug varied from region to region (Loscher et al., 1991).

In addition to possible GABA level changes, gabapentin and its closely related compound pregabalin have been reported to affect a wide range of neurotransmitters including: acetylcholine, glutamate, noradrenaline, substance P and calcitonin gene-related peptide (Taylor et al., 2007). The inhibition of neuronal calcium influx through the inhibition of VDCC via gabapentin binding to the α2δ subunits is postulated to be responsible for these changes in neurotransmitter release (Somerville and Michell, 2009).

Despite the fact that gabapentin does not show direct binding to GABA receptors, gabapentin exerts an increase in GABAA receptor-mediated membrane conductance in hippocampal pyramidal neurons, with no observable effects on GABAB receptor-mediated current (Lanneau et al., 2001). The mechanism(s) by which this increase in membrane conductance occurs and the corresponding behavioural outcome(s) are unknown.

1.8.1 Tonic GABAA receptors and gabapentin

In neuronal culture studies, it has been demonstrated that gabapentin exposure increases tonic GABAergic conductance (Cheng et al., 2006); however the exact mechanism causing this increase in conductance is not known. This change in tonic inhibitory conductance may account for some of the effects of gabapentin which appear GABAergic in nature such as hypnosis, sedation, anxiolysis, ataxia, and somnolence (Davis, 2003; Mack, 2003; Somerville and Michell, 2009; Strawn and Geracioti, 2007). This thesis aims to discover the mechanism(s) which results in the increase in GABAergic tonic current after gabapentin treatment.

Chapter 2 : Hypotheses and Aims

2.0 Overview

Gabapentin is an antiepileptic and antinociceptive drug (Kukkar et al., 2013), with many off-target effects (Clarke et al., 2013; Mack, 2003; Peterson, 2009; Somerville and Michell, 2009). The mechanisms underlying the many therapeutic and adverse effects of gabapentin remain poorly understood. Pharmacological enhancement of δGABAA receptor mediated tonic inhibition can produce similar effects to gabapentin such as antinociception, anticonvulsion, hypnosis, anxiolysis, sedation, ataxia and amnesia (Belelli et al., 2009; Bonin et al., 2011; Drasbek et al., 2007; Drasbek and Jensen, 2006; Hoehn-Saric, 1983; Whissell et al., 2013a).

Many of the past studies on gabapentin and GABAA receptors focused on only synaptic GABAA receptors. Synaptic receptors do not appear to be involved in the effects of gabapentin, as gabapentin does not enhance synaptic GABAergic activity (Cheng et al., 2006), and does not directly bind to GABAA receptors (Taylor et al., 1998). However, the effect(s) of gabapentin on extrasynaptic GABAA receptors are largely unknown. Previously, our lab showed that gabapentin increases a tonic GABAergic inhibitory conductance while the synaptic GABAergic conductance was unchanged (Cheng et al., 2006). Determining how gabapentin enhances tonic inhibition would shed light on the mechanisms of gabapentin action which may account for some of the behavioural effects of gabapentin.

The α5GABAA and δGABAA receptors are the main contributors to tonic GABAergic inhibition in the CNS. However, the α5GABAA receptors tend to be more restricted in expression compared to the δGABAA receptors. Furthermore, total α5GABAA receptor expression is unchanged after gabapentin treatment (Cheng et al., 2006). Accordingly, this thesis focuses mainly on δGABAA receptors, although α5GABAA receptors were also studied in some experiments. A combination of behavioural, biochemical and molecular techniques were utilized to determine whether δGABAA receptors contributed to some of the physiological effects of gabapentin. How gabapentin increases tonic GABAergic inhibition was also investigated via probing into the total and surface expressions of different GABAA receptor subunits using different models. Some intracellular mechanisms which may contribute to the increase in tonic GABAergic inhibition observed after gabapentin treatment were also investigated.

2.1 General Hypotheses

This thesis will address the following hypotheses:

(1) δGABAA receptors are necessary for some of the acute effects of gabapentin.

(2) δGABAA receptor expression is up-regulated by gabapentin.

(3) Neuroactive steroid and/or GABAA receptor ligand levels are altered by gabapentin.

(4) PKC and PKA are necessary for the δGABAA receptor expression change associated with gabapentin treatment.

2.2 Specific Aims

Aim 1: To determine whether δGABAA receptors are necessary for some of the acute effects of gabapentin. This aim was addressed by using wild type (WT) and null mutant mice that do not express δGABAA receptors (Gabrd−/−). Increased δGABAA receptor activity induces anxiolysis, analgesia and ataxia, outcomes which are all effects of gabapentin. To determine if

δGABAA receptors were involved in the effects of gabapentin anxiety-like behaviour, nociception and changes in motor coordination were tested on WT and Gabrd-/- mice after a single treatment with gabapentin (acute). The results of this study are described in Chapter 4.

Aim 2: To test whether δGABAA receptor expression is up-regulated by gabapentin. This aim was addressed by using an in vitro and ex vivo model. In the in vitro model, brain slices from WT mice were treated with 300 µM gabapentin or control solution. The samples were then biotinylated to isolate surface proteins. Different brain regions were probed to see if there are regional differences in expression. Total and surface proteins were subjected to western blot, and the relative changes were quantified. In the ex vivo model, WT mice were treated with gabapentin (100mg/kg) or control solution acutely or chronically, surface and total proteins were obtained and subjected to western blot analysis. Using the ex vivo model, mRNA was also measured using RT-qPCR.

Relative quantification of total and surface proteins will elucidate the mechanism(s) of gabapentin action. An increase in total protein and mRNA may not result in an increase in

42 functional surface receptors if the trafficking of the receptors to the membrane surface is unchanged, or endocytosis rates increase. The results of this study are described in Chapter 5.

Aim 3: To determine whether alterations in neuroactive steroid and/or GABAA receptor ligand levels by gabapentin account for changes in δGABAA receptor activity. This aim was addressed by using an in vitro and ex vivo model. In the in vitro model, murine neuronal cultures were treated with 300 µM gabapentin or control solution; the media were sent for mass spectrometry analysis. In the ex vivo model, WT mice were treated with 30 mg/kg gabapentin or control solution chronically, the brains were harvested and sent for mass spectrometry analysis. The results of this study are described in Chapter 6.

Aim 4: To test whether PKC and PKA are necessary for δGABAA receptor expression change associated with gabapentin treatment. This aim was addressed by using an in vitro model. Brain slices from WT mice were treated with one of the following conditions (1) 300 µM gabapentin, (2) control solution, (3) 300 µM gabapentin + PKC inhibitor 5 µM chelerythrine chloride, (4) control solution + PKC inhibitor 5 µM chelerythrine chloride, (5) 300 µM gabapentin + PKA inhibitor 20 µM H89, (6) control solution + PKA inhibitor 20 µM H89. The samples were then biotinylated to isolate surface proteins. Total and surface proteins were subjected to western blot, and relative changes were quantified. The results of this study are described in Chapter 7.

Chapter 3 : Materials and Methods

3.0 Overview

This section provides an overview of the experimental animals, drugs, materials, solutions, behavioural protocols, biochemical protocols, and molecular protocols used in this thesis. Specific details regarding drug application and dosages, timing and specific protocols used are described in chapters 4 through 7.

3.1 Animal models

All experiments were approved by the Animal Care Committee of the University of Toronto. Adult (2.5 – 4.5 months of age) male mice were used for all experiments. Adult mice were used since adolescent mice exhibit high variability in hormonal changes, which can affect

δGABAA receptor expression and activity (Shen et al., 2007; Smith et al., 2007). Also, female mice were not used in this study since female mice exhibit altered δGABAA receptor activity and expression profiles which are linked to their estrous cycle and other hormonal changes (Maguire et al., 2009; Maguire et al., 2005). These confounding factors could result in a high variability in data and may produce different responses to gabapentin. It would be of interest in the future to explore the effect of gender on behavioural responses to gabapentin treatment.

In all experiments, the experimenter was blinded to the genotype and the pharmacological intervention. WT and Gabrd −/− mice were bred from stock obtained from Dr. Gregg Homanics (Department of Anesthesiology, University of Pittsburgh, USA); they were bred on a C57BL/6J x 129 SvJ genetic background. The generation of the Gabrd −/− mice lines were bred as previously described (Mihalek et al., 1999). The mice were housed between 2 - 4 mice per cage. All animals had food and water ad libitum and were subjected to a 12 hour light/dark schedule. The mice were bred and maintained under the supervision of the Department of Comparative Medicine in the Medical Sciences Building in the University of Toronto. The mice used in all the experiments were derived from a mix of heterozygotic and homozygotic mating pairs. Homozygotic pairing was used to maximize animal breeding, since the generation of Gabrd +/+ and Gabrd −/− male mice from heterozygotic mating provided a very low yield of male mice of the desired genotypes.

The weaning and genotyping of mice were performed at approximately 3 weeks of age. Tissue for genotyping was obtained by clipping the tip of the tails of mice. The DNA from the tail tissue was extracted and amplified using Extract-N-Amp Tissue PCR Kit (Sigma Aldrich, ON, Canada).

The Gabrd −/− mice do not exhibit vastly different phenotypes from their WT counterparts; the main phenotypic differences are somewhat subtle. The exceptions are Gabrd −/− mice exhibit abnormal maternal behaviours, and slightly elevated baseline pain scores for inflammatory pain but not acute pain (Bonin et al., 2011; Maguire et al., 2009; Mihalek et al., 1999). Gabrd −/− mice also demonstrate attenuated sensitivity to exogenous and endogenous neuroactive steroids (Mihalek et al., 1999; Spigelman et al., 2002). Gabrd −/− mice exhibit impaired memory extinction (Whissell et al., 2013b), but enhanced acquisition of fear memory (Whissell et al., 2013b; Wiltgen et al., 2005). Gabrd -/- mice also exhibit impaired recognition memory and contextual discrimination relative to WT mice (Whissell et al., 2013b). Despite Gabrd −/− mice exhibiting relatively normal baseline behaviours, no compensatory changes have been identified which replaces the loss of δGABAA receptor mediated tonic inhibition.

3.2 Behavioural assays

All individuals involved in the behavioural experiments were certified by the Department of Comparative Medicine at the University of Toronto, and all experiments were performed in accordance with standard operating procedures.

3.2.1 Materials for behaviour

The gabapentin used were purchased either from Abcam (Cambridge, MA, USA ) or was gifted materials from Dr. Yves De Koninck at Centre de recherche de l'Institut universitaire en santé mentale de Québec. The saline was purchased from Baxter (Mississauga, ON, Canada). The gabapentin was prepared in saline, the vehicle injections were saline. The gabapentin treatment was made freshly everyday to prevent possible degradation. Adult (2.5 – 4.5 months of age) male WT and Gabrd −/− mice were used for all experiments; more detailed animal information was given in chapter 3.1. Mice were administered drugs or vehicle via intraperitoneal injections using sterile disposable 30.5 gauge needles attached to sterile disposable 1 ml tuberculin slip tip syringes (BD, Franklin Lakes, NJ, USA). The formalin was 45 purchased from Sigma Aldrich (St. Louis, MO, USA) and diluted in saline to a 5% formalin concentration. The formalin solution was made fresh daily. A 50 μl Hamilton syringe with a 30.5 gauge needle was used to administer the formalin into the hindpaw for the formalin assay.

3.2.2 Drug administration

In the behavioural experiments, gabapentin was dissolved in sterile saline, while the vehicle (control solution) was saline. Fresh drug solutions were made daily. Mice were administered drugs or vehicle via intraperitoneal injections. Sterile disposable 30.5 gauge needles attached to sterile disposable 1 ml tuberculin slip tip syringes were used to inject mice. Gabapentin dosages were measured in mg/kg and ranged from 30 mg/kg, 60 mg/kg to 100 mg/kg depending on the experiment. The mice were injected with gabapentin or vehicle 2 hours before behavioural testing.

The 2-hour time point was chosen because the behavioural effects of gabapentin were observed 2 hours post-injection in previous studies (De Sarro et al., 1998; Welty et al., 1993). Also, the brain and serum levels of gabapentin peaked at 1 hour post-treatment (i.p) (Kusunose et al., 2010; Vollmer et al., 1986), but gabapentin accumulation in the CNS persist beyond the time point of maximal serum concentrations, hence gabapentin accumulation in the CNS increases with time (Bockbrader et al., 2010). The lag time in observing the effects of gabapentin after peak serum levels has been documented (Welty et al., 1993), but the reason why this occurs is unclear. Perhaps, the proteins or systems affected need time to be trafficked, synthesized or degraded for an observable behavioural effect.

3.2.3 Tail flick assay

The tail flick assay is a common measure of immediate pain-related behaviours and is commonly used to test the analgesic effect of treatments (Bannon and Malmberg, 2007). In this study, the tail flick assay was used to assess whether WT and Gabrd -/- mice exhibit different acute nociceptive responses to gabapentin.

Prior to the experiment, different light intensities were tested to determine which intensity level produced a tail flick response in mice within 3 - 4 seconds. The light intensity which produced the tail flick in the desired time frame was set for all experiments. Treatments of gabapentin (60

46 mg/kg) or vehicle were injected intraperitoneally 2 hours before testing. Acute thermal nociception was assessed using a radiant heat tail flick meter. The mouse was restrained and an intense light beam was directed on the mid-point of the mouse‟s tail until the mouse spontaneously moved its tail from the light path. The light intensity was set to a level which was empirically determined to produce a tail flick response in mice within 3 - 4 seconds. The latency to flick the tail away from the activated light source (latency to tail flick) was automatically measured to the nearest 1/100th of a second using an integrated light beam detector. A maximal cutoff time of 10 seconds to respond was set to prevent tissue damage. Analysis was conducted on the latency to tail flick.

3.2.4 Formalin injection test

The formalin test is a standard and popular method of nociception assessment since subjects can be assessed unrestrained. This test also assesses the distinct phases of nociception, including the nociception associated with the direct activation of nociceptors (acute phase) and the nociception associated with the subsequent inflammation (late or tonic phase) (Bannon and Malmberg, 2007; Tjolsen et al., 1992).

Treatments of gabapentin (60 mg/kg) (Abcam, Cambridge, MA, USA) or vehicle were injected intraperitoneally 2 hours before testing. The mice were allowed to acclimate to the environment by being placed in the observation chamber for 20 minutes prior to the start of the experiment. The observation chamber consisted of a round acrylic cylinder (15 cm diameter and 30 cm tall) suspended above an angled mirror to facilitate observation from all vantage points. The mice were restrained using a modified decapicone and a glass 50 μl Hamilton syringe with a 30.5 gauge needle was used for the formalin injection. 20 μl of 5% formalin (diluted in saline) was injected in the dorsal side of the right hindpaw.

Two animals were tested simultaneously. The experimenter alternated between observing each mouse at a 2 minute time interval. The formalin injection of each mouse was staggered by 2 minutes apart to maintain temporally consistent measurement intervals after the formalin injection. The first two minutes immediately after the formalin injection was not scored. Subsequently, the mice were observed for the next 44 minutes after the formalin injection.

Induced nociception-related behaviours exemplified by licking or biting of the injected hindpaw were recorded. The time spent licking or biting the injected hindpaw follows a strongly biphasic response in mice. The phase 1 response (2 – 4 min) mainly results from the direct activation of primary afferent nociceptors, whereas the phase 2 response (6 – 44 min) results from the combined effects of primary afferent activity and central sensitization in the dorsal horn (Coderre et al., 1993; Tjolsen et al., 1992).

The total time spent licking and biting the injected hindpaw in phases 1 and 2 of the formalin response were calculated by adding the time spent licking and biting in each 2 minute time bin that existed in phase 1 (2 to 4 min) and phase 2 (6 to 46 min). Results are presented as mean +/- S.E.M. Differences between groups are considered significant for P < 0.05, using a 2 way ANOVA.

3.2.5 Rotarod assay

The rotarod assay is a standard assay to assess changes in motor coordination in rodents (Carter et al., 2001). The rotarod apparatus consists of an elevated rotating rod on which mice must actively move in a coordinated manner or fall. Mice will try to stay on the rotating rod to prevent the unpleasant experience of falling. Prior to testing, all groups of mice underwent a training period which ensured homogenous performance of the task. During training, mice were trained to stay on the rotating rod with a fixed rotation speed for a fixed minimum amount of time. Mice that experience impaired motor coordination during testing will not be able to stay on the rotating rod as long as they did during the pre-testing period.

The experimenter was blinded to genotype only. Mice were placed in the experimental room 1 hour prior to training or testing in order to acclimatize to the environment. Prior to testing, the mice were trained on the rotarod (Economex, Columbus Instruments) for 4 times each day with 15 minutes rest between intervals, until they can stay on the rod consistently for 300 seconds minimum. The rotation speed of the rod was set to 12 rotations per minute (rpm). Training took approximately 2 weeks.

Treatments were injected intraperitoneally 2 hours before testing. On day 1 of testing, all the mice were injected with vehicle (saline). On day 3 (two days after saline injection), the mice were injected with gabapentin (30 mg/kg). On day 5, the mice were injected with gabapentin (60 48 mg/kg) and on day 7, the mice were injected with gabapentin (100 mg/kg). The testing protocol consisted of mice being placed on the rotarod rotating at 12 rpm and the time spent on the rotating rod from 4 same day trials were recorded, the mice were given 15 minutes rest between each same day trial. The same mice were used due to a shortage of mice available for testing. Gabapentin is renally excreted unmetabolised, gabapentin is usually completely cleared from the body 12 hours post-injection in mice (Radulovic et al., 1995). Since the mice were dosed with new concentrations of gabapentin 2 days apart, on each testing day, all the drugs from the prior treatments should be eliminated. The mice were trained during the non-testing days to ensure their ability to stay on the rotarod was maintained at 300 seconds.

The times spent on the rotarod from all 4 testing intervals were averaged for each mouse for each condition. Results are presented as mean +/- S.E.M. Differences between groups were considered significant at the P < 0.05 level, using a 2 way ANOVA analysis.

3.2.6 Elevated plus assay

The elevated plus assay is a standard assay to test anxiety-like behaviour in rodents (Hogg, 1996; Lister, 1987; Pellow et al., 1985). An elevated plus maze contains 2 enclosed and 2 open arms as shown in Fig 3.0. This assay is useful for predicting a drug‟s anxiolytic effect in humans. Mice generally consider dark and enclosed spaces as “safe” and would tend to stay in these areas when anxious. On the other hand, exploration of open areas is deemed to reflect low levels of anxiety.

Treatments of gabapentin (30 mg/kg) (Abcam, Cambridge, MA, USA) or vehicle were injected intraperitoneally 2 hours before testing. At 2 hours post-injection, the treated mouse was removed from his home cage and was placed at the junction of the open and closed arms of the elevated plus maze, facing the open arm opposite to where the experimenter was located. The number of entries made by the mouse into the open and closed arms and the time spent in the open arms and closed arms were recorded for 5 minutes beginning immediately after the mouse‟s placement into the maze.

The total amount of time spent in the open or closed arms were analyzed respectively. Results are presented as mean +/- S.E.M. Differences between groups were considered significant at the P < 0.05 level, using a 2 way ANOVA analysis. 49

Fig 3.0: Elevated Plus Maze. This maze is a good predictor of anxiety-like effects in rodents. Generally, when mice are anxious they prefer to stay in enclosed areas (closed arms). However, mice are also exploratory creatures; hence in less anxious states they would most likely explore the open arms. An increased amount of time spent in the open arms of the maze is therefore an indicator of reduced anxiety.

3.3 Total protein, surface protein and mRNA methods

All samples were acquired from C57BL/6J x 129 SvJ mice. Surface label biotinylation was used to isolate surface proteins. Western blot was used to measure relative changes in total and surface proteins of interests. RT-qPCR was used to measure mRNA levels.

3.3.1 Materials for total protein, surface protein and mRNA

The gabapentin used were purchased either from Abcam or were gifted materials from Dr. Yves De Koninck at Centre de recherche de l'Institut universitaire en santé mentale de Québec. For the in vitro experiments, the gabapentin was prepared in oxygenated artificial cerebrospinal fluid (ACSF) (95% O2, 5% CO2) containing (in mM): 124 NaCl, 3 KCl, 26

NaHCO3, 1.3 MgCl2, 2.6 CaCl2, 1.25 NaH2PO4, and 10 glucose, pH 7.4, 300 – 310 mOsm; control is oxygenated ACSF. For the ex vivo experiments gabapentin was prepared in saline (Baxter, Mississauga, ON, Canada), the vehicle injections were saline.

Adult (2 – 3 months of age) male WT mice were used for all experiments; more detailed animal information was given in chapter 3.1. Mice for ex vivo experiments were administered drugs or vehicle via intraperitoneal injections using sterile disposable 30.5 gauge needles attached to sterile disposable 1 ml tuberculin slip tip syringes (BD, Franklin Lakes, NJ, USA). Acute brain slices were made using a vibrotome (Leica VT1200S, Deerfield, IL, USA).

The phosphate buffered saline (PBS) used for washing in biotinylation experiments contained (in mM): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.47 KH2PO4, pH 7.4. The EZ-Link Sulfo-NHS-SS- Biotin was purchased from Pierce (Rockford, IL, USA). The Dulbecco PBS (DPBS) was purchased from Gibco (Grand Island, NY, USA). The modified Tris buffered saline (TBS) used for the quenching step of biotinylation contained the following (in mM); 25 Tris-Cl, 137 NaCl, 5

KCl, 2.3 CaCl2, 0.5 MgCl2, pH 7.4. The lysis buffer contained (in mM): 20 HEPES, 150 NaCl, 2 EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium dodecyl sulfate (SDS) and cOmplete Protease Inhibitor Cocktail (Roche, Laval, QC, Canada), pH 7.4. The samples were homogenized by using a probe sonicator (Cole Parmer, Montreal, QC, Canada).

All centrifugation steps were performed on the Microfuge 22R Centrifuge (Beckman Coulter, Mississauga, ON, Canada). The total protein concentration was determined using the

51 bicinchoninic acid assay (BCA) (Bio-Rad, Hercules, CA, USA ), and the standard curve was made using bovine serum albumin (BSA). The BCA was performed on 96 well plates and the results were read on a spectrophotometer (BioTek, Winooski, VT, USA). The High Capacity Neutravidin agarose resin used was purchased from Pierce (Rockford, IL, USA). The spin columns used to wash the agarose were purchased from Qiagen (Toronto, ON, Canada). The agarose wash buffer contained (in mM): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.47 KH2PO4, 0.05% (w/v) SDS, pH 7.4. The elution buffer contained (in mM): 50 Tris-Cl, 2% (w/v) SDS, 2 DTT, pH 7.4, and surface protein concentration was determined using DC™ Protein Assay (Bio-Rad, Hercules, CA, USA).

SDS polyacrylamide gels were cast using the Mini-PROTEAN casting molds using 0.75 mm spacer plates (Bio-rad, Hercules, CA, USA). The Mini-PROTEAN® Tetra Cell with Mini Trans- Blot® Module and PowerPac (Bio-Rad, Hercules, CA, USA) was used for SDS page and transfer.

For SDS Page, samples were prepared in 5X sample buffer with 5% (v/v) betamercaptoethanol (BME), the final sample buffer concentration in the sample contained (in mM): 10% (w/v) sucrose, 10% (w/v) SDS, 62.5 Tris-Cl, trace bromophenol blue (for color), pH 6.8. The samples were loaded into a 10% SDS polyacrylamide gel with a stacking layer containing (in mM): 125 Tris-Cl, 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.001% (v/v) TEMED, 4% polyacrylamide/bis (37.5:1), pH 6.8, and a resolving layer containing (in mM): 375 Tris-Cl, 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.0005% (v/v) TEMED, 10% polyacrylamide/bis (37.5:1), pH 8.8. The gels were run in running buffer containing (in mM): 25 Tris-Base, 192 glycine, 0.1% (w/v) SDS in a mini electrophoresis system (Bio-Rad, Hercules, CA, USA).

For the transfer step, transfer buffer containing (in mM): 25 Tris-Base, 192 glycine, 20 % (v/v) methanol was used. Proteins from the SDS Page gel were transferred onto a nitrocellulose blotting membrane (Pall, Pensacola, FL, USA). All the electrophoresis transfer equipment (transfer electrode, cassette, power pack) were all from Bio Rad (Hercules, CA, USA).

For blotting, the membrane was blocked using a solution containing 5% milk dissolved in TBS- Tween containing (in mM): 50 Tris-Cl, 150 NaCl, 0.1% (v/v) Tween-20, pH 7.4. The primary

52 antibodies were diluted to various concentrations depending on the antibody in either 3% (w/v) BSA in TBS-Tween or 3% (w/v) milk in TBS-Tween. The primary antibodies used in different experiments included anti-GABAA receptor α1 antibody 1:1000 (Millipore, Billerica, MA, USA), α5 antibody 1:1000 (PhosphoSolutions, Aurora, CO, USA), β3 1:1000 (Thermo Scientific, Rockford, IL, USA), or δ 1:1000 (Millipore, Billerica, MA, USA), anti-β-actin antibody 1:2000 (Millipore, Billerica, MA, USA ), anti-Na+/K+ ATPase antibody 1:5000 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The membrane washed in between steps with TBS-Tween. A horseradish peroxidase- conjugated (HRP-conjugated) secondary antibody matching the host of the primary antibody was applied. The anti-rabbit and anti-mouse secondary antibodies were diluted 1:1000 in either 3% (w/v) BSA in TBS-Tween or 3% (w/v) milk in TBS-Tween, both secondary antibodies were from Cell Signaling (Danvers, MA, USA).

The membranes were exposed by applying enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) to the membranes. The chemiluminescence signal from the membrane was captured with either the Image Station 2000R (Kodak, USA) or the Chemidoc XRS+ system (Bio-Rad, Hercules, CA, USA). Blot images were quantified using Image Station software (Kodak, USA) or Image Lab software (Bio-Rad, Hercules, CA, USA).

For the RNA extraction, tissues were homogenized in 500 µl TRIZOL (Invitrogen, Burlington, ON, Canada) per 50 mg of tissue. Chloroform (Sigma Aldrich, ON, Canada) was also added to the samples. All centrifugation steps were performed on the Microfuge 22R Centrifuge (Beckman Coulter, Mississauga, ON, Canada). The isopropanol and ethanol used were obtained from Sigma Aldrich (ON, Canada), the ethanol was diluted in DEPC H2O (Invitrogen, Burlington, ON, Canada). The RNA concentration and purity was found using the Nanodrop (NanoDrop, DE, USA).

For the PCR steps, the Dnase 1, and 10X Dnase 1 buffer were obtained from New England BioLabs (Whitby, ON, Canada). The EDTA and DTT were obtained from Sigma Aldrich (ON, Canada). All the primers were designed according to primers found in literature, the exact primer sequences used are listed in Table 5; the primers were obtained from Sigma Aldrich (ON,

Canada). The DEPC H2O, 5X First-strand buffer, RNase OUT, dNTP mix, MMLV and Power SYBR Green were obtained from Invitrogen (Burlington, ON, Canada). A Chromo 4 real-time 53

PCR detector (Bio-Rad, Hercules, CA, USA) was used for RT-qPCR. Data were analyzed using Opticon software (Bio-Rad, Hercules, CA, USA).

3.3.2 Tissue preparation

Precautions in handling the mice were exercised to reduce anxiety, since stress can affect

δGABAA receptor activity and expression (Maguire and Mody, 2007; Shen et al., 2007). Precautions included homogenizing the scents from the guillotine and the decapitation area using 70% ethanol and allowing the mice to acclimatize to the decapitation room for a minimum for 2 hours prior to live decapitation. Mice were live decapitated since anesthetics can affect GABAA receptor expression or activity (Zurek et al., 2012). This procedure is approved by the Department of Comparative Medicine at the University of Toronto. The brains were immediately harvested and placed in an ice cold oxygenated ACSF bath before slicing.

3.3.3 Acute brain slice generation

Coronal brain slices (350 µm thick) were prepared from 10 – 14 week old WT mice using a vibrotome (Leica VT1200S, Deerfield, IL, USA) in ice cold oxygenated ACSF. The hippocamus, thalamus and cerebellum were micro-dissected out of the brain slices in an ice cold oxygenated ACSF bath.

For in vitro experiments, the regional slices were transferred individually into room temperature oxygenated ACSF, and equilibrated at room temperature for 1 hour before treatment.

For acute ex vivo samples, the mice were treated with either gabapentin (100 mg/kg) or saline (control) 2 hours prior to live-decapitation. The regional brain slices were obtained as described above. The slices were transferred individually into ice cold oxygenated ACSF and equilibrated for 30 minutes to 1 hour on ice to prevent receptor trafficking. The slices were biotinylated without further treatment.

For chronic ex vivo samples, the mice were treated via intraperitoneal injections with gabapentin (100 mg/kg) or saline (control) for 6 days; on the last day mice were treated 2 hours prior to live- decapitation. The slicing protocol is the same as the acute ex vivo slice protocol.

3.3.4 Treatment in vitro

Treatment methods differ between in vitro and ex vivo experiments. The concentrations of drug used also differ but are consistent with the amount use in literature for the respective methods. For in vitro experiments,10 hippocampal slices or 5 thalamic slices or 4 cerebellar slices were treated with either 300 µM gabapentin dissolved in oxygenated ACSF or oxygenated ACSF alone (control). The treatment was applied for 6 hours at 35 to 37 oC to enable receptor trafficking. 300 µM of gabapentin was used in in vitro treatments since that was the concentration used to elicit an increase in tonic current in neuronal cell cultures (Cheng et al., 2006). A higher concentration of gabapentin and longer treatment time may be needed for in vitro studies versus in vivo studies for an effect since the network and transporter systems may be disrupted in in vitro models.

For the PKA and PKC inhibition experiments all the ACSF used was oxygenated, gabapentin was dissolved in ACSF and the stock solutions for the PKA inhibitor H-89 (20 mM) and the PKC inhibitor chelerythrine chloride (5 mM) were dissolved in DMSO. For the PKA and PKC experiments, cerebellar slices were treated with one of the following conditions for 6 hours at 35 to 37 oC: (1) ACSF + 0.1% (v/v) DMSO, (2) 300 µM gabapentin dissolved in ACSF + 0.1% (v/v) DMSO, (3) 20 µM H-89 in ACSF, (4) 20 µM H-89 + 300 µM gabapentin dissolved in ACSF, (5) 5 µM chelerythrine chloride in ACSF, (6) 5 µM chelerythrine chloride + 300 µM gabapentin dissolved in ACSF. The 0.1% (v/v) DMSO added in some of the conditions is to ensure homogenous vehicle conditions since the H-89 and chelerythrine chloride stocks were made in DMSO. 5 µM of chelerythrine chloride was used since this was a comparable amount used in previous studies (Takasu et al., 2008). 20 µM of H-89 was used since this was a comparable amount used in previous studies (Liu et al., 2014; Williams et al., 2000; Zhu et al., 2003).

3.3.5 Treatment ex vivo

The mice were treated with gabapentin (100 mg/kg) or saline (control) via intraperitoneal injections 2 hours prior to live-decapitation. This treatment protocol is similar to the one used in the behavioural studies described in sections 3.1 and 3.2.

3.3.6 Total protein and surface protein isolation

Total protein and surface protein fractions were obtained. Surface protein was isolated from samples using cell surface protein biotinylation. The exact methods will be further described in chapter 3.3.6. Biotin was used to label surface proteins for isolation. Biotin is a small protein with a molecular weight of 244.31 g/mol. Biotin binds to the amine groups of compounds, and has an extremely high affinity to streptavidin and avidin (Weber et al., 1989). As a result, biotinylated products can be pulled down using streptavidin or avidin coated beads.

A modified biotin - Sulfo-NHS-SS-biotin (Fig 3.1) was used since this biotin contains a disulfide bond in a spacer arm which allows the biotin to be removed from the bound protein through thiol cleavage using reducing agents such as DTT. Biotin is usually not water soluble, hence it needs to be dissolved in organic solvents such as DMSO; however this solvent is shown to block

GABAA receptor activity (Nakahiro et al., 1992), we were interested in isolating surface GABAA receptors, hence DMSO was avoided in our preparation. Additionally, organic solvents can penetrate the cell membrane, hence intracellular protein contamination will occur. The Sulfo- NHS-SS-biotin possess a sulfonate group giving it a polar negative charge, this allows the compound to become water-soluble (Elia, 2012); hence the biotinylation experiments were performed in the absence of organic solvents. The negative charge of the Sulfo-NHS-SS-biotin is also useful in preventing intracellular protein contamination, since the negative charge prevents the modified biotin from passing the intact cell membrane.

Fig 3.1: Sulfo-NHS-SS-Biotin Structure. Due to its structure Sulfo-NHS-SS-Biotin is water soluble and does not cross the cell membrane.

3.3.7 Biotinylation assay in vitro and ex vivo

The slices treated in vitro were washed 5 times with ice cold PBS to ensure no treatment contamination, as some of the treatments contain amine groups which can affect the biotinylation process. For the ex vivo slices, they were immediately subjected to biotinylation after equilibration.

After washing (in vitro slices) or equilibration (ex vivo slices), the slices were incubated for 30 minutes at 4 oC with 0.75 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) dissolved in ice cold DPBS (Gibco, Grand Island, NY, USA). The biotin was dissolved right before use to minimize hydrolysis. The biotin solution was then aspirated and the slices were incubated with new 0.75 mg/ml EZ-Link Sulfo-NHS-SS-Biotin for another 30 minutes at 4 oC with light agitation.

The biotin solution was aspirated and excess biotin was quenched and removed by gently washing the slices 6 times with ice cold modified TBS. The slices were then placed in an eppendorf tube with lysis buffer containing protease and phosphatase inhibitors. The samples were homogenized on ice by using a probe sonicator (Cole Parmer, Montreal, QC, Canada) at 65% output using twelve 1 second pulses with 5 seconds intervals between each pulse to prevent the samples from getting warm. The homogenized samples were rotated at 4 oC for 30 minutes.

Insoluble material was removed by centrifugation at 18,000g for 20 minutes at 4 oC. The supernatant was isolated and bicinchoninic acid assay (BCA) (Bio-Rad, Hercules, CA, USA) was performed on the samples to determine protein concentration. A standard curve made through the serial dilution of a standard BSA sample was run with each assay so that the concentration and yield of protein in the unknown samples can be calculated. All the standard and unknown protein samples were run in triplicates on a 96 well plate. The colorimetric results were detected using a spectrophotometer (BioTek, Winooski, VT, USA) at 562 nm. An aliquot of the supernatant was retained and this fraction was known as “total protein”. This sample was stored at -20 oC until SDS page.

A fixed amount (0.8 mg) of the supernatant was incubated overnight with 200 µl of 50% slurry High Capacity Neutravidin agarose resin (Pierce, Rockford, IL, USA) at 4 oC with constant rotation. The agarose and protein slurry for each sample was completely transferred into 58 individual spin columns (Qiagen, Toronto, ON, Canada) for washing. Unbound protein was then removed through centrifugation at 2000 g for 1 minute, the flow through was considered unbound protein and was discarded. The bound protein was in the stationary agarose fraction. The agarose were washed by applying washing buffer to the spin column, followed by centrifugation at 2000 g for 1 minute, the flow through is was discard. The agarose was washed 10 times.

The agarose samples were completely transferred into an eppendorf tube, and bound materials were eluted with equal volumes of elution buffer containing DTT. The agarose were incubated with the elution buffer at room temperature with constant rotation for 1 hour, with 10 minute incubation on a heat block at 37 oC at the 30 minute time point. The samples were returned to their respective spin columns, with a clean eppendorf tube attached. The samples were centrifuged at 10, 000 g for 5 minutes. The flow through was kept since it is the eluted protein, which are the isolated surface proteins.

The protein concentrations of the “surface protein” samples were determined using the DC™ Protein Assay (Bio-Rad, Hercules, CA, USA). All the standard and unknown surface protein samples were run in triplicates on a 96 well plate. The colorimetric results were detected using a spectrophotometer (BioTek, Winooski, VT, USA) at 670 nm. The total and surface proteins were subjected to SDS polyacrylamide gel electrophoresis (SDS page) immediately or stored at -20 oC.

Fig 3.2: Surface protein biotinylation - In vitro experiments. Diagramatic description of the protocol described previously.

Fig 3.3: Surface protein biotinylation - Ex vivo experiments. Diagramatic description of the protocol described previously.

3.3.8 Western blot

Equal amounts of each protein fraction for each condition were loaded onto a 10% SDS polyacrylamide gel for comparison studies. A 10% polyacrylamide gel was used since the proteins of interest were between 25 to 100 kDa. Typically, 8 or 45 µg of total hippocampal protein, 4 or 15 µg of total thalamic protein and 3 or 8 µg of cerebellar total protein were loaded and 8 µg of surface hippocampal protein, 4 µg of surface thalamic protein and 3 µg of cerebellar surface protein were loaded. Significantly more protein were loaded for the hippocampal samples since δGABAA receptors are very lowly expressed in the hippocampus compared to the cerebellum and thalamus (Wisden et al., 1992). Hence, a higher protein load was needed to detect a signal for hippocampal samples. Conversely, a lot less protein was loaded for cerebellar slice samples since δGABAA receptors are highly expressed in the cerebellum, loading more protein would result in signal saturation and non-analyzable data.

Equal amounts of each sample were used for same gel comparisons. Since not all samples are of the same concentration, samples were prepared and diluted in distilled water so that their concentrations were homogenous. For every 4 µl of homogenous sample, 1 µl of 5X sample buffer with 5% (v/v) betamercaptoethanol (BME) was used in the sample preparation, the final sample buffer concentration in the sample contains (in mM): 10% (w/v) sucrose, 10% (w/v) SDS, 62.5 Tris-Cl, trace bromophenol blue (for color), pH 6.8. The final prepared samples were incubated at 37 oC for 15 minutes. 15 µl of each sample were loaded onto a 10% SDS polyacrylamide gel with a stacking layer containing (in mM): 125 Tris-Cl, 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.001% (v/v) TEMED, 4% polyacrylamide/bis (37.5:1), pH 6.8, and a resolving layer containing (in mM): 375 Tris-Cl, 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.0005% (v/v) TEMED, 10% polyacrylamide/bis (37.5:1), pH 8.8.

The gels were run in running buffer containing (in mM): 25 Tris-Base, 192 glycine, 0.1% (w/v) SDS in a mini electrophoresis system (Bio-Rad, Hercules, CA, USA). Gels were run at 35 V through the stacking layer and 100 V through the resolving layer. The electrophoresis was stopped when the bromophenol blue dye has just run out of the gel.

The gels were lightly washed with distilled water to remove the SDS from the running buffer and were left to set in 4 oC transfer buffer containing (in mM): 25 Tris-Base, 192 glycine, 20 % (v/v)

61 methanol. The transfer sandwich was made with two sponges presoaked with transfer buffer, followed by blotting paper presoaked with transfer buffer, followed by the gel with a nitrocellulose blotting membrane (Pall, Pensacola, FL, USA) presoaked in transfer buffer on top. All these layers were held together in a transfer cassette. The assembled cassette was placed in the electrophoresis transfer box containing transfer buffer. The transfer apparatus was placed in the cold room to maintain buffer temperatures of approximately 4 oC. The gels were allowed to transfer for 12 hours at 0.12 mA at 4 oC.

To block unspecific binding, the nitrocellulose membrane which contains the transferred proteins was incubated at room temperature for 1.5 hours or overnight at 4 oC with 5% milk dissolved in TBS-Tween containing (in mM): 50 Tris-Cl, 150 NaCl, 0.1% (v/v) Tween-20, pH 7.4. Next, a primary antibody which probes for the protein of interest was applied to the membrane for 2 hours at room temperature or overnight at 4 oC. The primary antibody was diluted to various concentrations depending on the antibody in either 3% (w/v) BSA in TBS-Tween or 3% (w/v) milk in TBS-Tween. The primary antibodies used in different experiments included anti-GABAA receptor α1 antibody 1:1000 (Millipore, Billerica, MA, USA), α5 antibody 1:1000 (PhosphoSolutions, Aurora, CO, USA), β3 1:1000 (Thermo Scientific, Rockford, IL, USA), or δ 1:1000 (Millipore, Billerica, MA, USA), anti-β-actin antibody 1:2000 (Millipore, Billerica, MA, USA ), anti-Na+/K+ ATPase antibody 1:5000 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The membrane was then washed for 15 minutes with TBS-Tween for 3 times. A horseradish peroxidase- conjugated (HRP-conjugated) secondary antibody matching the host of the primary antibody was applied for 1.5 hours at room temperature or overnight at 4 oC. The membrane was then washed for 15 minutes with TBS-Tween for 3 times.

Enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) was applied to the membrane for 3 to 5 minutes, and the chemiluminescence signal from the membrane was captured with either the Image Station 2000R (Kodak, USA) or the Chemidoc XRS+ system (Bio-Rad, Hercules, CA, USA).

3.3.9 Western blot quantification and equipment

Gels were cast using the Mini-PROTEAN casting molds using 0.75 mm spacer plates (Bio-rad, Hercules, CA, USA). The Mini-PROTEAN® Tetra Cell with Mini Trans-Blot® Module and PowerPac (Bio-Rad, Hercules, CA, USA) was used for SDS page and transfer. Blots were imaged using the Image Station 2000R (Kodak, USA) or Chemidoc XRS+ system (Bio- Rad, Hercules, CA, USA). Blot images were quantified using Image Station software (Kodak, USA) or Image Lab software (Bio-Rad, Hercules, CA, USA).

3.3.10 Real time quantitative PCR

The messenger RNA (mRNA) levels were measured in WT mice treated with gabapentin (100 mg/kg) or saline (control) via intraperitoneal injections 2 hours prior to live-decapitation. The hippocampus, thalamus and cerebellum were isolated from the harvested brains for real time quantitative polymerase chain reaction (RT-qPCR) assay and analysis.

3.3.11 RNA extraction

The regional tissues were homogenized in 500 µl TRIZOL per 50 mg of tissue in an RNase-free environment. Chloroform was added to the samples in a ratio of 0.2 ml chloroform per 1 ml of TRIZOL. The mixture was homogenized by light trituration and vortex and incubated at room temperature for 2 to 3 minutes. The homogenate was centrifuged 12,000 g for 15 minutes at 4 oC. The supernatant was retained and 0.5 ml of isopropanol per 1 ml of TRIZOL was added to the supernatant. The mixture was incubated for 10 minutes at room temperature followed by centrifugation at 12,000 g for 10 minutes at 4 oC. The supernatant was discarded and the RNA pellet was washed 3 times with 500 µl of 70% ethanol. The ethanol was removed and after the final wash, the RNA pellet was air-dried for 10 minutes under a sterile hood. The RNA pellet was then dissolved in 30 µl of DEPC H2O. The RNA concentration and purity was assessed using the Nanodrop (NanoDrop, DE, USA).

3.3.12 RT-qPCR

Each sample was prepared as follows: 10 µg of RNA, 1 µl of Dnase 1 (2units), 1 µl 10X

Dnase 1 buffer and various volumes of DEPC H2O was mixed to give a final total volume of 10 µl. The samples should all have a final RNA concentration of 1 µg/1 µl. The samples were thoroughly mixed and incubated at 37 oC for 10 minutes. 1 µl of 50 mM EDTA was added to the samples followed by heat inactivation at 75 oC for 10 minutes.

Since RNA is unstable, it must be converted to cDNA. 1 µl of 1 µg/1 µl RNA, 1 µl of primer

(50-500 ng), 1 µl of 10 mM dNTP mix and 9 µl of DEPC H2O was combined to give a final volume of 12 µl. This mixture was incubated at 65 oC for 5 minutes, followed by an 1 minute incubation on ice. 4 µl of 5X First-strand buffer, 2 µl of 0.1 M DTT, 1 µl RNase OUT (40units/ µl) was added to the sample. The sample was lightly mixed and incubated at 37 oC for 2 minutes. 200 µl of MMLV is added to sample. The samples were placed in a Chromo 4 real-time PCR detector (Bio-Rad, Hercules, CA, USA) and programmed to execute the following cycle conditions: 25 oC for 10 minutes, 37 oC for 50 minutes and 70 oC for 15 minutes. A total of 35 cycles were then performed. All the samples were run in triplicates. The Nanodrop was used to measure DNA concentration and purity.

To quantify the RNA in samples, equal amounts of the cDNA from the previous step, equal amounts of the reverse and forward primers of interest, sterile RNAse free water and equal amounts of Power SYBR Green (Invitrogen, Burlington, ON, Canada) were lightly mixed together. The samples were placed in a Chromo 4 real-time PCR detector (Bio-Rad, Hercules, CA, USA) and programmed to execute the following cycle conditions: 95 oC for 10 minutes, 95 oC for 15 seconds and 60 oC for 30 seconds. A total of 40 cycles were then performed. All the samples were run in triplicates. Data were analyzed using Opticon software (Bio-Rad, Hercules, CA, USA). Quantified data were normalized to β-Actin as the endogenous control mRNA.

3.4 High-performance liquid chromatography, Gas Chromatography and Mass Spectrometry

3.4.1 Materials

Gabapentin for all experiments were from Abcam Biochemicals (Toronto, ON, Canada). The isofluane used to anesthetize mice before decapitation were from Abbott Laboratories (Abbott Park, IL, USA).

High-performance liquid chromatography (HPLC) was used for amino acid analysis. For the amino acid analysis, amino acid standards, methanol, isopentane, 2-mercaptoethanol and o- phthaldialdehyde are from Sigma Aldrich (St. Louis, MO, USA). HPLC was performed using an Alliance 2690XE (Waters, Mississauga, ON, Canada) with an autosampler and a Waters 474 programmable fluorescence detector attached (Waters, Mississauga, ON, Canada). Data were acquired and processed using Empower Pro software (Waters, Mississauga, ON, Canada). A Symmetry C18 column (4.5 × 100 mm, 5 μm) coupled with a guard column (Waters, Mississauga, ON, Canada) was used for separation.

Gas Chromatography and mass spectrometry were used for neuroactive steroid analysis. Solid- phase extractions on the supernatants were performed using an Oasis HLB 30 mg extraction plate (Waters, Mississauga, ON, Canada). All chemical reagents: methanol, methylene dichloride, heptafluorobutyrylimidazole (HFBI), ethyl acetate, and toluene were purchased from Sigma Aldrich (St. Louis, MO, USA). An Agilent 6890 gas chromatograph (Mississauga, ON, Canada) with a HP-MS5 5% phenylmethylsiloxane column (30 cm × 250 μm, 0.25 μm film thickness) was used for gas chromatography. The gas chromatograph was coupled to an Agilent 5973 mass spectrometer (Mississauga, ON, Canada).

3.4.2 Animal subjects

Robert Bonin treated all the mice for this series of experiments. Robert Bonin and Agnes Zurek harvested all tissues for this series of experiments. Tissues were collected from 3 – 4 month old male Swiss Webster mice (Charles River, Montreal, QC, Canada). Mice were administered 30 mg/kg gabapentin dissolved in saline or saline (vehicle) via intraperitoneal injections using sterile disposable 30.5 gauge needles attached to sterile disposable 1 ml

65 tuberculin slip tip syringes (BD, Franklin Lakes, NJ, USA). The brains of mice were harvested 2 hours post-treatment in the acute model. In the chronic model, the mice were treated for 4 consecutive days every 12 hours; the mice brains were harvested 2 hours after the last treatment.

3.4.3 Analysis of amino acids

Dr. Glen B. Baker at the University of Alberta performed all the amino acid measurements. High-performance liquid chromatography (HPLC) was used for amino acid analysis.

The whole brains of mice were collected for amino acids and neuroactive steroids analysis as described previously by Dr. Baker‟s lab (Ahboucha et al., 2008; Rauw et al., 2010). The mice were anesthetized with isoflurane using a bell jar apparatus and decapitated. The whole brain was flash frozen in isopentane cooled with a slurry of dry ice and ethanol. The flash frozen brains were shipped on dry ice to Dr. Baker at the University of Alberta.

The frozen brain tissues were homogenized in 80 % (v/v) methanol at 0 °C, and a 50 μl sample of each homogenate was removed and further diluted into 200 μl of methanol at 0°C. The samples were incubated on ice for ten minutes prior to centrifugation at 12,000 g for 4 minutes at 4 °C. The supernatant was kept, and the pellet discarded. The supernatant was diluted ten-fold with deionized water; all the samples were kept ice cold.

The free amino acids from the standards and samples were converted to highly fluorescent thioalkyl-substituted isoindoles by mixing 5 μl of the sample or the amino acid standards with 2- mercaptoethanol and o-phthaldialdehyde for 1.5 minutes. The prepared samples and standards were injected into an Alliance 2690XE HPLC system (Waters, Mississauga, ON, Canada) equipped with an autosampler and a Waters 474 programmable fluorescence detector (Waters, Mississauga, ON, Canada). All the data were acquired and processed using Empower Pro software (Waters, Mississauga, ON, Canada).

A Symmetry C18 column (4.5 × 100 mm, 5 μm) coupled with a guard column was used for the separation step. A mobile phase gradient consisting of varying amounts of sodium phosphate buffer and methanol was used to separate the amino acids; a flow rate of 0.5 ml/minute was used. The samples were maintained at 4 °C and the column compartments were maintained at 30 °C.

The excitation wavelength used was 260 nm and the emission wavelength used was 455 nm. Empower software was used to control the instrument, acquire the data and perform the analysis. Graph Pad Prism software (La Jolla, CA, USA) was used to perform a student‟s t-test for each amino acid concentration measure, the amino acid levels in the brain tissues of the gabapentin treated mice were compared to their respective vehicle treated counterparts.

3.4.4 Analysis of neuroactive steroids

The laboratory of Dr. Glen B. Baker at the University of Alberta performed all measurements of neuroactive steroids. Gas Chromatography and mass spectrometry were used for neuroactive steroid analysis.

The frozen brain tissues were homogenized in 80 % (v/v) methanol at 0 °C. Alfaxalone was used as an internal standard. Alfaxalone was added to each homogenate and then vortexed. The samples were incubated on ice for ten minutes prior to centrifugation at 12,000 g for 5 minutes at 4 °C. The supernatants were kept, and the pellets discarded. Solid-phase extractions on the supernatants were performed using an Oasis HLB 30 mg extraction plate (Waters, Mississauga, ON, Canada). The wells were washed with 1 ml of 5 % (v/v) methanol, and the neuroactive steroids were eluted using 1.5 ml of 9:1 (v/v) methylene dichloride:methanol solution. The eluents were dried under a vacuum and the analytes were then derivatized for 1 hour at 45 °C with 10 μl of heptafluorobutyrylimidazole (HFBI) in 50 μl of ethyl acetate, this step was crucial to convert the analytes to more volatile and less reactive compounds for gas.

The sample were then dried and reconstitution in 50 μl of toluene. They were then each washed with 50 μl of deionized water to remove all water soluble compounds, and centrifuged at 12,000 g for 2 minutes. The toluene layer was then analyzed by gas chromatography using an Agilent 6890 gas chromatograph (Mississauga, ON, Canada) with a HP-MS5 5% phenylmethylsiloxane column (30 cm × 250 μm, 0.25 μm film thickness) coupled to an Agilent 5973 mass spectrometer (Mississauga, ON, Canada) in the negative ion-chemical ionization mode. The inlet temperature was 250 °C, and the oven temperature was initialized at 100 °C, this temperature was increased to 295 °C at a rate of 8 °C/minute, and the temperature was maintained at 295 °C for 5 minutes.

The mass spectrometer was operated in the single ion monitoring mode and standard curves (0 - 2000 pg) in 80 % (v/v) methanol were run in parallel with each assay. Data capture and analysis were performed using the Agilent MSD Chemstation software (Mississauga, ON, Canada). Tissue concentrations of neuroactive steroids in a sample that were below the limit of detection were assigned a value of 1 pg steroid /g tissue in the analysis. Neuroactive steroid levels were compared between vehicle and gabapentin treated groups using the non-parametric Mann- Whitney test using Graph Pad Prism software (La Jolla, CA, USA).

Chapter 4 : δGABAA receptors contribute to some of gabapentin’s acute effects

4.0 Overview

Gabapentin is a GABA analog that does not bind to GABA receptors despite exhibiting GABAergic-like effects such as neurodepression (Sills, 2006). Gabapentin is widely prescribed for its anticonvulsant, antinociceptive and anxiolytic effects, however many individuals also report adverse side effects such as somnolence and ataxia (Somerville and Michell, 2009).

Increased δGABAA receptor activity and expression can result in similar outcomes to gabapentin treatment such as anticonvulsion (Peng et al., 2004), antinociception (Bonin et al., 2011), anxiolysis (Lagrange, 2006), and ataxia (Herd et al., 2009). In this chapter, Gabrd -/- and WT mice were used to explore whether δGABAA receptors contribute to the acute effects of gabapentin. The results indicate that δGABAA receptors contribute to the anxiolytic and ataxic, but not antinociceptive effects of gabapentin.

4.1 Introduction

Gabapentin is widely prescribed for epilepsy management and neuropathic pain (Kukkar et al., 2013). It is also frequently prescribed for many off-label uses such as attention deficit disorder (ADD), bipolar disorder, restless leg syndrome, periodic limb movement disorder of sleep, migraine headaches, alcohol withdrawal syndrome, complex regional pain syndrome, trigeminal neuralgia (Mack, 2003; Radley et al., 2006), and generalized and pre-operative anxiety (Clarke et al., 2013; Strawn and Geracioti, 2007; Taylor et al., 1998). Common adverse effects of gabapentin are somnolence, ataxia, asthenia, weight gain, nausea, headaches, dizziness and vertigo (Somerville and Michell, 2009). The mechanism(s) underlying all these therapeutic and adverse effects of gabapentin are poorly understood.

The inhibition of the α2δ subunit of VDCC accounts for some of the effects of gabapentin when it is chronically administered (Dolphin, 2012b). However, the modulation of this subunit does not fully account for all the effects of gabapentin as decribed in section 1.7. Furthermore, the mechanisms associated with acute gabapentin administration are unknown.

Gabapentin is structurally related to GABA, but does not bind to either GABAA or GABAB receptors (Taylor et al., 1998). It can cross the blood brain barrier due to the addition of a lipophilic cyclohexyl group (Sills, 2006). We showed that gabapentin treatment increased a tonic inhibitory current by nearly 3 folds, while no change was observed in phasic inhibitory current

(Cheng et al., 2006). Tonic inhibitory currents are primarily mediated by extrasynaptic GABAA receptors that contain either the δ or α5 subunit (Semyanov et al., 2004). Enhanced δGABAA receptor activity and expression produced similar behavioural effects to gabapentin treatment (Bonin et al., 2011; Herd et al., 2009; Lagrange, 2006; Somerville and Michell, 2009). This chapter explores whether δGABAA receptors contribute to the antinociceptive, anxiolytic and ataxic effects of gabapentin.

4.2 Materials and Methods

The methods used are briefly described in this section. All the behavioural methods and materials used in this section were described in greater detail in section 3.2.

To determine whether δGABAA receptors contribute to the antinociceptive effects of gabapentin; WT and Gabrd −/− mice were treated with 60 mg/kg gabapentin or saline (control). The tail flick assay was used to assess acute pain associated with thermal nociception. The formalin assay was used to assess acute and inflammatory pain. The formalin assay has a biphasic response. Phase 1 corresponds to the nociceptive response associated with the direct activation of nociceptors (acute pain), while phase 2 corresponds to the nociception associated with inflammation (inflammatory pain).

To determine whether δGABAA receptors contributed to the acute anxiolytic effects of gabapentin, WT and Gabrd −/− mice were treated with 30 mg/kg gabapentin or saline (control). The mice were subjected to the elevated plus assay. The maze consists of two open arms and two closed arms; less anxious mice spend more time in the open arms.

To determine whether δGABAA receptors contribute to the acute ataxic effects of gabapentin; WT and Gabrd −/− mice were treated with saline (control) and then 30, 60 or 100 mg/kg gabapentin. To assay for ataxia, the rotarod test was used. Initially, the mice were trained to stay on a rotating rod for a fixed amount of time (300 seconds) before testing. Subsequently, the mice received an injection of vehicle or gabapentin during testing. The mice that were unable to 70 remain on the rod consistently during testing were deemed to exhibit impaired motor coordination which is a sign of ataxia.

4.3 Results

4.3.1 δGABAA receptors do not contribute to the analgesic effects of gabapentin

In the tail flick assay, the response latency was similar between genotypes (Fig 4.0). Gabapentin (60 mg/kg) treatment did not significantly change acute thermal nociceptive behaviours in both genotypes (WT vehicle: 3.525 ± 0.124 s, n = 13; WT GBP: 3.441 ± 0.135 s, n = 14, P > 0.05), (Gabrd -/- vehicle: 3.562 ± 0.089 s, n = 13; Gabrd -/- GBP: 3.388 ± 0.089 s, n = 12, P > 0.05).

In the formalin assay (Fig 4.1), gabapentin (60 mg/kg) treatment produced a non-significant downwards trend in nociceptive behaviours in phase 1 in both genotypes compared to vehicle (WT vehicle: 18.8 ± 2.8 s, n = 10; WT GBP: 12.8 ± 2.5 s, n = 10, P > 0.05), (Gabrd -/- vehicle: 27.9 ± 5.1 s, n = 8; Gabrd -/- GBP: 19.4 ± 4.1 s, n = 8, P > 0.05). However, gabapentin treatment significantly reduced nociceptive behaviours in phase 2 in both genotypes (WT vehicle: 99.0 ± 9.4 s, n = 10; WT GBP: 32.2 ± 5.0 s, n = 10, P < 0.0001), (Gabrd -/- vehicle: 135.1 ± 5.4 s, n = 8; Gabrd -/- GBP: 47.8 ± 7.2 s, n = 8, P < 0.0001). Interestingly at baseline conditions (vehicle groups) in phase 2, WT mice displayed lower nociceptive behaviours than Gabrd -/- mice (WT vehicle: 99.0 ± 9.4 s, n = 10; Gabrd -/- vehicle: 135.1 ± 5.4 s, n = 8, P = 0.0069). However, both genotypes of mice displayed similar levels of nociceptive behaviours after gabapentin (60 mg/kg) treatment (WT GBP: 32.2 ± 5.0 s, n = 10; Gabrd -/- GBP: 47.8 ± 7.2 s, n = 8, P = 0.0856).

These results demonstrate that gabapentin attenuates inflammatory pain but not acute nociception. The results also show that endogenous δGABAA receptors do not contribute to the antinociceptive properties of gabapentin, since Gabrd -/- and WT mice responded similarly to gabapentin in all the assays

Fig 4.0: Gabapentin does not alleviate acute pain – tail flick assay. 60 mg/kg gabapentin treatment did not significantly change nociceptive behaviours associated with acute thermal pain in both genotypes of mice (WT vehicle: 3.525 ± 0.124 s, n = 13; WT GBP: 3.441 ± 0.135 s, n = 14, P > 0.05; two-way ANOVA), (Gabrd -/- vehicle: 3.562 ± 0.089 s, n = 13; Gabrd -/- GBP: 3.388 ± 0.089 s, n = 12, P > 0.05; two-way ANOVA). Data are represented as mean ± SEM.

Fig 4.1: The antinociceptive effects of gabapentin are not δGABAA receptor dependent – formalin assay. 60 mg/kg gabapentin did not produce significant differences in nociceptive behaviour in phase 1 in both genotypes compared to vehicle (WT vehicle: 18.8 ± 2.8 s, n = 10; WT GBP: 12.8 ± 2.5 s, n = 10, P > 0.05; two-way ANOVA), (Gabrd -/- vehicle: 27.9 ± 5.1 s, n = 8; Gabrd -/- GBP: 19.4 ± 4.1 s, n = 8, P > 0.05; two-way ANOVA). Gabapentin significantly reduced nociceptive behaviours in phase 2 in both genotypes (WT vehicle: 99.0 ± 9.4 s, n = 10; WT GBP: 32.2 ± 5.0 s, n = 10, P < 0.0001; two-way ANOVA), (Gabrd -/- vehicle: 135.1 ± 5.4 s, n = 8; Gabrd -/- GBP: 47.8 ± 7.2 s, n = 8, P < 0.0001; two-way ANOVA). Data are represented as mean ± SEM.

4.3.2 δGABAA receptors contribute to the anxiolytic-like effects of gabapentin

The baseline levels of anxiety-like behaviour in the elevated plus maze were not significantly different between genotypes (WT vehicle: 0.97 ± 0.53 %, n = 9; Gabrd -/- vehicle: 3.74 ± 2.04 %, n = 11, P = 0.2483) (Fig 4.2). The results (Fig 4.2) further show that WT mice spent a larger percentage of time in the open arms when treated with gabapentin compared to their vehicle treated counterparts (WT vehicle: 0.97 ± 0.53 %, n = 9; WT GBP: 11.30 ± 3.21 %, n = 10, P < 0.01). This effect was absent in Gabrd -/- mice, as Gabrd -/- mice treated with either gabapentin or vehicle spent comparable amounts of time in the open arms (Gabrd -/- vehicle: 3.74 ± 2.04 %, n = 11; Gabrd -/- GBP: 3.23 ± 1.06 %, n = 11, P > 0.05).

These results suggest that endogenous δGABAA receptors contribute to the anxiolytic properties of gabapentin, since WT mice exhibited increased anxiolytic-like responses to gabapentin treatment but Gabrd -/- mice did not.

Fig 4.2: δGABAA receptors contribute to the anxiolytic effects of gabapentin – elevated plus maze. 30 mg/kg gabapentin treatment significantly increased anxiolytic-like behaviour in WT mice, but not in Gabrd -/- mice (WT vehicle: 0.97 ± 0.53 %, n = 9; WT GBP: 11.30 ± 3.21 %, n = 10, P < 0.01; two-way ANOVA), (Gabrd -/- vehicle: 3.74 ± 2.04 %, n = 11; Gabrd -/- GBP: 3.23 ± 1.06 %, n = 11, P > 0.05; two-way ANOVA). Data are represented as mean ± SEM.

4.3.3 δGABAA receptors contribute to the ataxic effects of gabapentin

A 30 mg/kg dose of gabapentin did not significantly change motor coordination in WT and Gabrd -/- mice (WT vehicle: 298.4 ± 1.0 s, n = 6; WT 30 mg/kg GBP: 251.5 ± 22.2 s, n = 6, P > 0.05), (Gabrd -/- vehicle: 292.7 ± 3.2 s, n = 5; Gabrd -/- 30 mg/kg GBP: 291.3 ± 3.8 s, n = 5, P > 0.05). However, 60 mg/kg and 100 mg/kg doses of gabapentin significantly impaired motor coordination in WT mice, but not in Gabrd -/- mice (WT vehicle: 298.4 ± 1.0 s, n = 6; WT 60 mg/kg GBP: 139.3 ± 13.8 s, n = 6, WT 100 mg/kg GBP: 251.5 ± 16.6 s, n = 6 P < 0.0001), (Gabrd -/- vehicle: 292.7 ± 3.2 s, n = 5; Gabrd -/- 60 mg/kg GBP: 255.4 ± 18.0 s, n = 5, Gabrd - /- 100 mg/kg GBP: 229.8 ± 34.86 s, n = 5 P > 0.05).

These results suggest that endogenous δGABAA receptors contribute to the ataxic properties of gabapentin, since WT mice exhibited impaired motor coordination following gabapentin treatment in a dose-dependent manner, but Gabrd -/- mice did not.

Fig 4.3: δGABAA receptors contribute to the ataxic effects of gabapentin – rotarod assay. A 30 mg/kg dose of gabapentin did not significantly change motor coordination in WT and Gabrd -/- mice (WT vehicle: 298.4 ± 1.0 s, n = 6; WT 30 mg/kg GBP: 251.5 ± 22.2 s, n = 6, P > 0.05; two- way ANOVA), (Gabrd -/- vehicle: 292.7 ± 3.2 s, n = 5; Gabrd -/- 30 mg/kg GBP: 291.3 ± 3.8 s, n = 5, P > 0.05; two-way ANOVA). However, 60 mg/kg and 100 mg/kg doses of gabapentin significantly reduced motor coordination in WT mice but not Gabrd -/- mice (WT vehicle: 298.4 ± 1.0 s, n = 6; WT 60 mg/kg GBP: 139.3 ± 13.8 s, n = 6, WT 100 mg/kg GBP: 251.5 ± 16.6 s, n = 6 P < 0.0001; two-way ANOVA), (Gabrd -/- vehicle: 292.7 ± 3.2 s, n = 5; Gabrd -/- 60 mg/kg GBP: 255.4 ± 18.0 s, n = 5, Gabrd -/- 100 mg/kg GBP: 229.8 ± 34.86 s, n = 5 P > 0.05; two-way ANOVA). Data are represented as mean ± SEM.

4.4 Discussion

These results revealed that δGABAA receptors contribute to the anxiolytic and ataxic effects of gabapentin. However, δGABAA receptors do not appear to contribute to the antinociceptive effects of gabapentin, but may contribute to the sensitization of inflammatory pain as shown by the difference in baseline pain scores in phase 2 of the formalin assay. A low dose (30 mg/kg) of gabapentin decreased anxiety-like effects in WT mice, but not in Gabrd -/- mice. WT mice treated with gabapentin spent significantly more time in the open arms of the elevated plus maze which is an indicator of anxiolysis in rodents (Lister, 1987; Pellow et al.,

1985). These data indicate that δGABAA receptors may be necessary for gabapentin‟s acute anxiolytic effects.

Similarly, there was evidence to support that δGABAA receptors were necessary for the ataxic effects of gabapentin. Gabapentin significantly decreased motor coordination in WT mice at dosages of 60 mg/kg and above in a dose dependent manner, but not in Gabrd -/- mice. WT mice treated with 30 mg/kg gabapentin did not show impaired motor skills, while WT mice treated with 60 mg/kg and 100 mg/kg exhibited severe motor coordination impairments. Conversely, gabapentin-treated and vehicle-treated Gabrd -/- mice did not show significant differences in motor coordination skills even at the highest dose tested in this study (100 mg/kg). The baseline performance between genotypes on the rotarod was not significantly different since all the mice were trained to be equally skilled at this task before testing. This set of data indicates that

δGABAA receptors may be necessary for gabapentin‟s acute ataxic effects.

It is important to note that for these behavioural studies in mice, the highest dose of 100 mg/kg per day gabapentin used is considered a mid range dosage in mice. In contrast, 30 mg/kg per day is considered a low dose. Many other mice studies typically use dosages ranging from 30 mg/kg per day up to 3000 mg/kg per day gabapentin (Pfizer, 2012). In human studies and clinically, patients are prescribed daily total doses ranging from 300 mg up to 3600 mg (some times more), these total doses are usually divided into 3 treatments daily (Bockbrader et al., 2010; Pfizer, 2012; Somerville and Michell, 2009). Whether higher daily doses of gabapentin would yield more pronounced drug effects were not investigated in this study, but would be of future interest.

The tail flick assay was used to assess acute thermal nociception. The response latency values were similar between genotypes with gabapentin and vehicle treatment. The formalin assay produces a biphasic response. Phase 1 is associated with nociceptive behaviours resulting from the direct activation of primary afferent sensory neurons, and phase 2 is associated with nociception resulting from inflammation (Tjolsen et al., 1992). Gabapentin treatment resulted in no significant changes in nociception in phase 1 in both genotypes. In phase 2, gabapentin treatment significantly reduced nociception in both genotypes to a similar degree. These results suggest that δGABAA receptors do not contribute to the antinociceptive and analgesic effects of gabapentin.

The phase 2 formalin assay results show that gabapentin is effective for attenuating inflammatory pain. These results are consistent with previous findings, since almost all studies in different species conclude that gabapentin is effective in attenuating chronic inflammatory pain (Bockbrader et al., 2010; Dworkin et al., 2003; Pfizer, 2012; Somerville and Michell, 2009). The reduction in inflammatory pain experienced by both genotypes of mice after gabapentin treatment may be dependent on the high affinity binding of gabapentin to the α2δ subunit of VDCCs (Field et al., 2000). Gabapentin can impair excitatory CNS synaptogenesis through disrupting the interaction between the α2δ subunit with thrombospondin, which can result in pain reduction (Eroglu et al., 2009). Also, gabapentin binding can inhibit the trafficking of the α2δ subunit in DRG (Hendrich et al., 2008). This can result in attenuated glutamate and substance P release resulting in reduced pain sensitization (Hendrich et al., 2008; Kukkar et al., 2013). However, all the α2δ subunit mechanisms described result from chronic or long term gabapentin treatment, how gabapentin attenuates inflammatory pain in an acute model of treatment as shown in this thesis study is unclear.

The tail flick assay and the phase 1 formalin assay results both show that gabapentin is ineffective for reducing acute pain. It is still unclear whether gabapentin attenuates acute pain since results vary in each study (Berry and Petersen, 2005; Dworkin et al., 2009; Fassoulaki et al., 2006; Pfizer, 2012; Werner et al., 2001).

Caution must be exercised when interpreting these results. A global δGABAA receptor knock out mouse was used, which exhibits several known compensatory changes in the expression of ion channels that maintain neuronal inhibition. Gabrd -/- mice exhibit increased expression of 79

γ2GABAA and α5GABAA receptor subunits (Glykys et al., 2008; Korpi et al., 2002). Increased activity of γ2GABAA receptors through benzodiazepine treatment can result in anxiolysis (Sieghart and Sperk, 2002), however the baseline anxiety-like behaviour in WT and Gabrd -/- were not significantly different. Also, WT mice displayed lower baseline nociception than Gabrd -/- mice in phase 2 of the formalin assay, this difference is abolished with gabapentin treatment. This may be due to: (1) an unknown compensatory mechanism in the Gabrd -/- mice or, (2)

δGABAA receptor expression contributes to reduce pain sensitization associated with inflammation. A ceiling effect could account for the fact that the difference is no longer observed after gabapentin treatment. Finally, the rotarod data indicate that at higher dosages (greater than

100 mg/kg), δGABAA receptors as well as another receptor may contribute to the ataxic effects of gabapentin since Gabrd -/- mice trends toward impaired locomotor skills at 100 mg/kg.

All together, the results suggest that δGABAA receptors contribute to the acute anxiolytic and ataxic effects but not the antinociceptive effects of gabapentin. The relative regional differences in δGABAA receptor expression may account for this. δGABAA receptors are highly expressed in the cerebellar granule cells, increased activity of δGABAA receptors in this brain region has been associated with impaired motor coordination (Hanchar et al., 2005). In contrasts, δGABAA receptors are lowly expressed in the DRG and spinal cord (Laurie et al., 1992b), which may account for the lack of antinociceptive differences between genotypes with gabapentin treatment. How gabapentin produces the anxiolytic and ataxic effects in WT mice will be explored in the following chapters. Finally, pathological states such as inflammation, alcohol abuse, depression and chronic stress are associated with altered δGABAA receptor expression (Diaz et al., 2013; Maguire et al., 2005; Serra et al., 2008; Serra et al., 2007) which may explain the variability in the efficacy of gabapentin between individuals in different states (Fehrenbacher et al., 2003; Lal et al., 2013; Takasu et al., 2008).

Chapter 5 : Gabapentin increases δGABAA receptor

expression in vitro and ex vivo

5.0 Overview

The δGABAA receptors contribute to the anxiolytic and ataxic effects of gabapentin

(Chapter 4). Pharmacological enhancement of δGABAA receptor activity increases GABAergic tonic currents (Drasbek and Jensen, 2006; Jia et al., 2005), which may result in anxiolysis (Maguire et al., 2005) and ataxia (Hanchar et al., 2005). Previously, we showed that gabapentin increased a GABAergic tonic current (Cheng et al., 2006). Hence, some of the effects of gabapentin may be due to an increase in tonic current mediated by δGABAA receptors. How gabapentin increases this current is unknown.

The increase in total and/or surface expression of δ or α5 GABAA receptors can account for the increase in GABAergic tonic current observed. Other factors that can account for the increase in tonic current are: (1) the alteration of receptor kinetics, (2) an increase in GABAA receptor ligands such as GABA, β-alanine (Horikoshi et al., 1988) and taurine (Jia et al., 2008), and (3) an increase in neurosteroid levels as described in section 1.5. The changes in ligands and neurosteroid levels are explored in Chapter 6. This chapter explores the total and surface GABAA receptor subunit expression profiles under different experimental conditions. The results indicate that δGABAA receptor surface expression is increased after gabapentin treatment in vitro and in vivo, while α5- and α1- GABAA receptor surface expression was unchanged.

5.1 Introduction

Increased cell surface expression of δ or α5 GABAA receptors can lead to increased GABAergic tonic current (Michels and Moss, 2007). Enhanced total expression of these receptors usually leads to increased cell surface expression. However, increased surface expression can also occur even when total expression does not change. This result may occur from changes in trafficking mechanisms. For example, the receptor may undergo reduced endocytosis from the cell membrane or increased insertion into the cell membrane from a readily available pool of receptors. Factors which can affect GABAA receptor trafficking and cell surface stabilization are summarized in Table 1 and 2.

In this chapter, the total and surface expression of δ, α5, and α1 subunits of GABAA receptors were probed. The expression of δ and α5 subunits of GABAA receptors were investigated since they are the primary contributors of tonic current in the CNS. However, overall δGABAA receptors are much more widely expressed than α5GABAA receptors in the CNS (Wisden et al.,

1992). The α1 subunit of GABAA receptors was probed since it is a widely expressed synaptic

GABAA receptor, and contributes the most to synaptic inhibitory conductance in most brain regions (Nusser et al., 1996a; Wisden et al., 1992). Since synaptic inhibition does not change with gabapentin treatment (Cheng et al., 2006), synaptic GABAA receptor expression is expected to be unaltered.

Anxiety involves a complex neuronal circuit which is not well understood. The brain regions that are often implicated in the anxiety circuitry are the amygdala, prefrontal cortex, thalamus and hippocampus (Bannerman et al., 2004; Bannerman et al., 2014; Li et al., 2010; McHugh et al., 2004; Rotge et al., 2012). Motor coordination is highly regulated by the cerebellum (Colucci- Guyon et al., 1999; Gowen and Miall, 2007; Kashiwabuchi et al., 1995; Miall, 1998; Miall et al.,

2001). δGABAA receptors are highly expressed in the cerebellar granule cells (Laurie et al., 1992a), dentate granule cells (Brooks-Kayal et al., 1999; Wei et al., 2003), hippocampal interneurons (Glykys et al., 2007), and thalamic interneurons (Bright et al., 2007). Since

δGABAA receptors contribute to the anxiolytic and ataxic effects of gabapentin (Chapter 4), and are highly expressed in the cerebellum, thalamus and hippocampus, these regions were selectively probed for changes in δGABAA receptor expression. The α5GABAA receptors are highly expressed in the hippocampus, and are key contributors to tonic inhibition (Rudolph et al.,

2001). Hence α5GABAA receptor expression was probed for in hippocampal preparations. The

α1GABAA receptors are highly expressed in all brain regions probed (Wisden et al., 1992), and are the key contributors to synaptic inhibition in the CNS. As a result, α1GABAA receptor expression was probed for in all three brain regions investigated.

5.2 Materials and Methods

To determine whether gabapentin increases the total number of extrasynaptic or synaptic

GABAA receptors, we performed western blot analyses using subunit–selective antibodies. To determine whether gabapentin increases the cell surface number of extrasynaptic or synaptic

GABAA receptors, we isolated all the surface proteins via surface label biotinylation followed by western blot analyses using subunit–selective antibodies.

All the methods and materials used in this section were described in great detail in section 3.3. The primers used for RT-qPCR are listed in Table 5.

Table 5: Primers used in RT-qPCR

Primer Forward strand Revers strand Tm Tm Size sequence sequence Forward Reverse (bp) (oC) (oC)

α2 GABAA CTTGGGACGGGAA GGAAAGATTCGGGG 60.1 60.3 110 subunit GAGTGTA CATAGT

α5 GABAA CCCTCCTTGTCTTC TGATGTTGTCATTG 62.9 60.9 99 subunit TGTATTTCC GTCTCGTCT

δ GABAA ACCAGTTACCGCTT GGAGGACAGAGGG 60.5 60.5 133 subunit CACCAC CATGTAA

β-actin AGG CCA ACC GTG ACC AGA GGC ATA 58.4 61.2 101 AAA AGA TG CAG GGA CAA

5.3 Results

5.3.1 Gabapentin exposure in vitro increases δGABAA receptor expression

Gabapentin does not change the total expression (Fig 5.0 A), but increased the surface expression (Fig 5.0 B) of δGABAA receptors in hippocampal slices. Gabapentin increased both the total and the surface δGABAA receptor expression in the thalamus and cerebellum but to different degrees. (Fig 5.1 A and B, Fig 5.2 A and B). In the hippocampus, gabapentin does not change δGABAA receptor total expression in vitro (Control: 100.0 ± 2.8 %, n = 4; GBP: 106.9 ±

6.7 %, n = 4, P > 0.05), but increased δGABAA receptor surface expression by 32.6 % (Control: 100.0 ± 2.4 %, n = 4; GBP: 132.6 ± 3.3 %, n = 4, P < 0.001). In the thalamus, gabapentin increased δGABAA receptor total expression in vitro by 54.2 % (Control: 100.0 ± 8.8 %, n = 3;

GBP: 154.2 ± 10.3 %, n = 3, P < 0.05) and increased δGABAA receptor surface expression by 31.4 % (Control: 100.0 ± 0.4 %, n = 3; GBP: 131.4 ± 7.7 %, n = 3, P < 0.05). In the cerebellum, gabapentin increased δGABAA receptor total expression in vitro by 79.2 % (Control: 100.0 ± 1.0

%, n = 3; GBP: 179.2 ± 5.9 %, n = 3, P < 0.001) and increased δGABAA receptor surface expression by 67.7 % (Control: 100.0 ± 4.6 %, n = 3; GBP: 167.7 ± 3.6 %, n = 3, P < 0.001).

The α5GABAA receptor total and surface expression was unchanged in the hippocampus by gabapentin treatment (Fig 5.0 E and F): total expression (Control: 100.0 ± 0.6 %, n = 3; GBP: 103.6 ± 3.3 %, n = 3, P > 0.05), surface expression (Control: 100.0 ± 6.1 %, n = 3; GBP: 96.0 ±

6.5 %, n = 3, P > 0.05). The α1GABAA receptor total and surface expression was unchanged in all 3 brain regions tested (Fig 5.0 C and D, Fig 5.1 C and D, Fig 5.2 C and D) (P > 0.05 for all groups tested), refer to Table 6 and 7 for data and statistics.

The results showed that in vitro treatment with gabapentin increased the surface expression of

δGABAA receptors in all brain regions, but to varying degrees. In some brain regions an increase in the total δGABAA receptor expression was also observed. No changes in total or surface expression were observed for α5GABAA receptors. Hence the increase in tonic current observed previously is most likely attributed to a change in δGABAA receptor expression. α1GABAA receptor total and surface expression was also unchanged for all brain regions. This suggests that synaptic inhibition is unchanged with gabapentin treatment. This set of data and the behavioural data in chapter 4 suggest that δGABAA receptors contribute to some of the effects of gabapentin.

Furthermore, the results in this section suggest that the upregulation of surface δGABAA receptor expression may be an influencing factor in gabapentin action.

Fig 5.0: In vitro application of 300 µM gabapentin upregulates surface δGABAA receptor expression in the hippocampus. In the hippocampus, gabapentin does not change δGABAA receptor total expression in vitro (A) (Control: 100.0 ± 2.8 %, n = 4; GBP: 106.9 ± 6.7 %, n = 4,

P > 0.05), but increased δGABAA receptor surface expression by 32.6 % (B) (Control: 100.0 ±

2.4 %, n = 4; GBP: 132.6 ± 3.3 %, n = 4, P < 0.001). The total and surface expressions of α1 (C,

D) and α5 (E, F) GABAA receptor subunits are unchanged with gabapentin treatment (P > 0.05 for all groups, exact values found on Table 6 and 7). Con stands for control. GBP for gabapentin. Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Fig 5.1: In vitro application of 300 µM gabapentin upregulates total and surface δGABAA receptor expression in the thalamus. In the thalamus, gabapentin increases δGABAA receptor total expression in vitro by 54.2 % (A) (Control: 100.0 ± 8.8 %, n = 3; GBP: 154.2 ± 10.3 %, n =

3, P < 0.05) and increases δGABAA receptor surface expression by 31.4 % (B) (Control: 100.0 ± 0.4 %, n = 3; GBP: 131.4 ± 7.7 %, n = 3, P < 0.05). The total and surface expressions of

α1GABAA receptor subunits are unchanged with gabapentin treatment (C, D) (P > 0.05 for all groups, exact values found on Table 6 and 7). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Fig 5.2: In vitro application of 300 µM gabapentin upregulates total and surface δGABAA receptor expression in the cerebellum. In the cerebellum, gabapentin increases δGABAA receptor total expression in vitro by 79.2 % (A) (Control: 100.0 ± 1.0 %, n = 3; GBP: 179.2 ± 5.9

%, n = 3, P < 0.001) and increases δGABAA receptor surface expression by 67.7 % (B) (Control: 100.0 ± 4.6 %, n = 3; GBP: 167.7 ± 3.6 %, n = 3, P < 0.001). The total and surface expressions of α1GABAA receptor subunits are unchanged with gabapentin treatment (C, D) (P > 0.05 for all groups, exact values found on Table 6 and 7). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Table 6: Total protein data: 300 µM Gabapentin treatment in vitro

Brain region Cerebellum Cerebellum Thalamus Thalamus Hippocampus Hippocampus Hippocampus Subunit analyzed δ α1 δ α1 δ α1 α5 P value 0.0002 0.5056 0.0161 0.8058 0.3815 0.9936 0.349 P value summary *** ns * ns ns ns ns Control 100.0 ± 100.0 ± Mean ± 100.0 ± 100.0 ± 8.753 4.325 100.0 ± 2.834 100.0 ± 8.121 100.0 ± SEM 1.037 N=3 5.060 N=3 N=3 N=3 N=4 N=3 0.5880 N=3 154.2 ± 97.67 ± GBP Mean 179.2 ± 108.6 ± 10.34 7.747 106.9 ± 6.696 100.1 ± 10.21 103.6 ± 3.318 ± SEM 5.946 N=3 10.63 N=3 N=3 N=3 N=4 N=3 N=3 Difference between -79.24 ± -8.598 ± -54.19 ± 2.330 ± -6.865 ± -0.1115 ± -3.570 ± means 6.036 11.77 13.55 8.872 7.271 13.04 3.369

Table 7: Surface protein data: 300 µM Gabapentin treatment in vitro

Brain region Cerebellum Cerebellum Thalamus Thalamus Hippocampus Hippocampus Hippocampus Subunit analyzed δ α1 δ α1 δ α1 α5 P value 0.0003 0.6544 0.0148 0.9431 0.0002 0.5246 0.6774 P value summary *** ns * ns *** ns ns Control 100.0 ± 100.0 ± Mean ± 100.0 ± 100.0 ± 0.3539 3.605 100.0 ± 2.424 100.0 ± 3.332 100.0 ± 6.112 SEM 4.259 N=3 5.117 N=3 N=3 N=3 N=4 N=3 N=3 GBP 131.4 ± 99.63 ± Mean ± 167.7 ± 104.5 ± 7.659 3.202 132.6 ± 3.277 94.76 ± 6.750 96.00 ± 6.508 SEM 3.624 N=3 7.715 N=3 N=3 N=3 N=4 N=3 N=3 Difference between -67.71 ± -4.470 ± -31.45 ± 0.3660 ± -32.56 ± means 5.592 9.258 7.667 4.822 4.076 5.242 ± 7.528 3.999 ± 8.928

5.3.2 Acute gabapentin exposure ex vivo increases δGABAA receptor surface expression

In order to investigate if δGABAA receptor expression is upregulated in the behavioural conditions in Chapter 4, gabapentin treatment in an in vivo model was used. In the hippocampus after in vivo gabapentin treatment, δGABAA receptor total expression was unchanged (Fig 5.3A) (Control: 100.0 ± 4.2 %, n = 6; GBP: 101.5 ± 0.6 %, n = 6, P > 0.05), but the surface expression was increased by 22.4% (Fig 5.3B) (Control: 100.0 ± 3.3 %, n = 6; GBP: 122.4 ± 3.8 %, n = 6, P

= 0.0013). The α5 and α1GABAA subunits total expression and surface expression were unchanged (Fig 5.3 C, D, E, F; Table 8, 9). These in vivo experimental results in the hippocampus are similar to the results observed in the in vitro experiments in section 5.3.1.

Unlike the in vitro results in section 5.3.1, gabapentin was found to only increase the surface (Fig 5.4B) (Control: 100.0 ± 4.8 %, n = 6; GBP: 136.3 ± 5.4 %, n = 6, P = 0.0005), but not the total

(Fig 5.4A) (Control: 100.0 ± 4.0 %, n = 6; GBP: 109.1 ± 4.4 %, n = 6, P > 0.05) δGABAA receptor expression in the cerebellum. However, similarly to the in vitro results, the α1GABAA subunit total and surface expression were unchanged in the cerebellum (Fig 5.4 C and D; Table 8, 9).

The expression profile and mRNA levels of δ, α5, and α2 GABAA subunits differ in each brain region (Fig 5.5). The mRNA levels for δ, α5, and α2 GABAA subunits in the hippocampus and thalamus were unchanged after in vivo gabapentin treatment (Fig 5.6 A and B) (P > 0.05 for each comparison). The δ and α2 GABAA subunits in the cerebellum were also unchanged after in vivo gabapentin treatment (Fig 5.6C) (P > 0.05 for each comparison).

The results showed that one dose of gabapentin in vivo can increase the surface expression of

δGABAA receptors in the hippocampus and cerebellum. The increase in δGABAA receptor surface expression observed is higher in the cerebellum than in the hippocampus. The mRNA levels and the total expression of extrasynaptic GABAA subunits: δ and α5, and synaptic GABAA subunits: α1 or α2 were unchanged in the hippocampus, thalamus and cerebellum after in vivo gabapentin treatment. This set of data and the behavioural data in chapter 4 suggest that the upregulation of cell surface δGABAA receptors may be an influencing factor in the acute actions of gabapentin.

Fig 5.3: Acute in vivo application of 100 mg/kg gabapentin upregulates surface δGABAA receptor expression in the hippocampus. In the hippocampus after gabapentin treatment,

δGABAA receptor surface expression increased by 22.4% (B) (Control: 100.0 ± 3.3 %, n = 6; GBP: 122.4 ± 3.8 %, n = 6, P = 0.0013), but the total expression was unchanged (A) (Control: 93

100.0 ± 4.2 %, n = 6; GBP: 101.5 ± 0.6 %, n = 6, P > 0.05). The total and surface expression of

α5 (E, F) and α1 (C, D) GABAA receptor subunits are unchanged with gabapentin treatment (P > 0.05 for all groups, exact values found on Table 8 and 9). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Fig 5.4: Acute in vivo application of 100 mg/kg gabapentin upregulates surface δGABAA receptor expression in the cerebellum. In the cerebellum, the δGABAA surface expression increased by 36.3 % (B) (Control: 100.0 ± 4.8 %, n = 6; GBP: 136.3 ± 5.4 %, n = 6, P = 0.0005), but not the δGABAA total expression (A) (Control: 100.0 ± 4.0 %, n = 6; GBP: 109.1 ± 4.4 %, n

= 6, P > 0.05). The total and surface expressions of α1GABAA receptor subunits are unchanged with gabapentin treatment (C, D) (P > 0.05 for all groups, exact values found on Table 8 and 9). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Table 8: Total protein data: 100 mg/kg Gabapentin treatment in vivo

Brain region Cerebellum Cerebellum Hippocampus Hippocampus Hippocampus Subunit analyzed δ α1 δ α1 α5 P value 0.158 0.4619 0.737 0.2459 0.2211 P value summary ns ns ns ns ns Control Mean 100.0 ± 100.0 ± 3.610 100.0 ± 4.201 100.0 ± 4.474 100.0 ± 2.589 ± SEM 4.003 N=6 N=5 N=6 N=5 N=4 GBP Mean ± 109.1 ± 96.96 ± 1.554 101.5 ± 107.6 ± 4.087 95.17 ± 2.412 SEM 4.387 N=6 N=5 0.5547 N=6 N=5 N=4 Difference between -9.062 ± -1.464 ± -7.586 ± means 5.938 3.037 ± 3.930 4.238 6.059 4.831 ± 3.538

Table 9: Surface protein data: 100 mg/kg Gabapentin treatment in vivo

Brain region Cerebellum Cerebellum Hippocampus Hippocampus Hippocampus Subunit analyzed δ α1 δ α1 α5 P value 0.0005 0.1276 0.0013 0.341 0.7679 P value summary *** ns ** ns ns Control Mean 100.0 ± 100.0 ± 4.307 100.0 ± 3.344 100.0 ± 1.562 100.0 ± 3.002 ± SEM 4.819 N=6 N=6 N=6 N=3 N=6 GBP Mean ± 136.3 ± 91.36 ± 2.908 122.4 ± 3.849 95.65 ± 3.714 98.48 ± 4.015 SEM 5.357 N=6 N=6 N=6 N=3 N=6 Difference between -36.34 ± -22.44 ± means 7.205 8.636 ± 5.197 5.098 4.352 ± 4.030 1.521 ± 5.013

Fig 5.5: Regional differences in α5 and δ GABAA subunit mRNA expression. All data is normalized to hippocampal expression, so expression is relative to hippocampus. The hippocampus expresses significantly more α5GABAA subunit mRNA than the thalamus and cerebellum (Hippocampus: 100 %, n = 3; Thalamus: 31.6 ± 5.6 %, n = 3, P < 0.001; Cerebellum:

1.1 ± 0.5, n = 3, P < 0.0001). α5GABAA mRNA and protein is not typically expressed in the cerebellum. Conversely, the cerebellum expresses significantly more δGABAA subunit mRNA than the thalamus and hippocampus (Hippocampus: 100 %, n = 3; Thalamus: 744.3 ± 52.55 %, n = 3, P < 0.001; Cerebellum: 1824 ± 459.8, n = 3, P < 0.05). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

Fig 5.6: No changes in mRNA expression with acute in vivo gabapentin treatment. (P > 0.05 for each comparison). Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

5.3.3 Chronic gabapentin exposure ex vivo increases δGABAA receptor expression

Gabapentin is often taken 3 times per day for an extended period of time (Bockbrader et al., 2010; Pfizer, 2012; Somerville and Michell, 2009). Hence the effect on δGABAA receptor expression after chronic administration of gabapentin in vivo was investigated. The cerebellum was used because it is the region with the greatest δGABAA receptor expression (Sieghart and Sperk, 2002). Additionally, the cerebellum is involved in motor coordination and can contribute to the ataxic effects of gabapentin (Chapter 4). The results shown in this section are preliminary data.

Preliminary data indicate that total and surface expression of δGABAA receptors increase after chronic gabapentin administration (Fig 5.7). The preliminary results in this section are similar to the cerebellar slice in vitro results in section 5.3.1.

Fig 5.7: Chronic in vivo application of 100 mg/kg gabapentin upregulates total and surface

δGABAA receptor expression in the cerebellum. N = 2 for all groups, preliminary data indicate that total and surface expression of δGABAA receptors increase after chronic gabapentin administration. More data points are needed for statistical analysis.

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5.4 Discussion

The cell surface δGABAA receptor expression is upregulated in an in vitro and an ex vivo model of gabapentin administration. Interestingly, the in vitro administration model, and the chronic administration model of gabapentin shows an increase in total δGABAA receptors in brain regions rich in endogenous δGABAA receptor expression such as the thalamus and cerebellum (Wisden et al., 1992). Additionally, in all the models used, the total and surface expression of the α5GABAA receptor, which is a major extrasynaptic GABAA receptor in the hippocampus, was not altered. This result suggest that the increased tonic GABAergic current observed after gabapentin treatment (Cheng et al., 2006) is most likely attributed to an increase in δGABAA receptor expression. Furthermore, in all the models used, the total and surface expression of the α1GABAA receptor which is the predominant synaptic GABAA receptor in the CNS did not change. This result supports the previous finding which observed a lack of change in GABAergic synaptic currents following gabapentin treatment (Cheng et al., 2006).

Regional differences in how gabapentin affect δGABAA receptor expression were also observed. The degree of total and surface receptor change varied from region to region, and not all regions showed an increase in total protein in the in vitro study. In the in vitro model, hippocampal

δGABAA receptor surface expression increased but not the total expression. In contrast, the thalamus and cerebellum showed enhanced δGABAA receptor surface and total expression. The cerebellum showed a greater increase in both total and surface expression of δGABAA receptors compared to both the hippocampus and thalamus. In the in vitro study, δGABAA receptor total expression increased by 79.2 ± 5.9 % in the cerebellum and 54.2 ± 10.3 % in the thalamus and was unchanged in the hippocampus after 6 hours of treatment. In the same study, δGABAA receptor surface expression increased by 67.7 ± 3.624 % in the cerebellum, 31.4 ± 7.6 % in the thalamus and 32.6 ± 3.3 % in the hippocampus. The regional differences in the degree and type of δGABAA receptor expression upregulation observed within the same time frame may be attributed to the receptor expression profile being different in each brain region.

Gabapentin may elicit different physiological responses depending on whether the user is a first time user or chronic user of the drug (Cilio et al., 2001). Some of the effects may be dependent on the α2δ subunit of VDCC but some may be δGABAA receptor dependent. Gabapentin can inhibit calcium currents through a reduction in the surface expression of the α2δ subunit only 101 when applied chronically but not acutely (Hendrich et al., 2008). Hence the δGABAA receptor expression profile may differ in acute and chronic applications of gabapentin as well. The ex vivo data shows that with an acute injection of gabapentin the cell surface δGABAA receptor expression is upregulated, while total expression remains unchanged. Preliminary data indicates that with chronic gabapentin treatment ex vivo, both cell surface and total δGABAA receptor expression is upregulated. Further experiments to increase the sample size are needed to confirm this effect. If confirmed, it will indicate that the effects of gabapentin can vary between acute and chronic treatments. This would be consistent with the effects of other drugs which affect GABAA receptors, such as ethanol (Grobin et al., 1998).

Collectively, the results show that gabapentin administration increases δGABAA receptor cell surface expression (in all regions and instances) and total expression (in some brain regions and instances). An increase in δGABAA receptor expression can lead to an increase in tonic inhibitory conductance (Farrant and Nusser, 2005). This increase in tonic conductance may result in anxiolysis (Maguire et al., 2005; Shen et al., 2005) and ataxia (Hanchar et al., 2005). All together, gabapentin can increase δGABAA receptor expression, which can result in increased

GABAergic tonic current (Cheng et al., 2006; Jacob et al., 2008). The δGABAA receptor- mediated increase in tonic current may contribute to the acute anxiolytic and ataxic effects of gabapentin (Chapter 4) (Hanchar et al., 2005; Shen et al., 2005). In addition to the effects studied, δGABAA receptor upregulation may be implicated in other gabapentin physiological outcomes such as hypnosis, sedation and anticonvulsion, since enhanced δGABAA receptor activity has been shown to elicit those effects described (Drasbek et al., 2007; Drasbek and Jensen, 2006; Walker and Kullmann, 2012).

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Chapter 6 : Gabapentin does not alter ligand or neurosteroid

levels

6.0 Overview

Chapter 5 demonstrated that gabapentin can increase δGABAA receptor surface expression in vitro and ex vivo. The intracellular mechanism by which gabapentin facilitates this increase in expression is unknown. The increase in the surface expression of δGABAA receptors can account for the increase in GABAergic tonic current (Cheng et al., 2006). However, other factors can also contribute to the increase in tonic current such as: (1) the alteration of receptor kinetics, (2) an increase in GABAA receptor ligands such as GABA, β-alanine (Horikoshi et al., 1988) and taurine (Jia et al., 2008), and (3) an increase in neurosteroid levels (Belelli and

Lambert, 2005). Additionally, neurosteroids can alter δGABAA receptor expression (Abramian et al., 2014; Smith et al., 2007). The changes in ligands and neurosteroids levels are explored in this chapter. The results showed that GABAergic ligands and endogenous neurosteroids levels were unchanged after gabapentin treatment. Hence, the increased tonic current after gabapentin treatment is not a result of changes in ligands or neurosteroids levels.

6.1 Introduction

The increase in channel opening probability of extrasynaptic GABAA receptors can result in increased tonic current. Enhancement of channel opening probability can result from an increase in either ambient concentrations of ligands that activate GABAA receptors or neuroactive steroids which can increase gating efficacy (Bianchi and Macdonald, 2003).

Increased levels of GABA and/or other amino acids that can activate GABAA receptors can account for the increase in tonic inhibitory current observed and some of the effects of gabapentin. Therefore, in this chapter the level of amino acids of different neurotransmitters and ligands were investigated. In addition to GABA levels, taurine and β-alanine were investigated since they can both activate GABAA receptors (Horikoshi et al., 1988), taurine especially is a potent activator of extrasynaptic GABAA receptors (Jia et al., 2008). Other inhibitory amino acids such as glycine were also investigated. Excitatory amino acids such as glutamate and

103 aspartate, and amino acids that can increase glutamate levels such as asparagines were also measured.

Some studies have shown that gabapentin can increase GABA levels in the brain (Cai et al., 2012; Errante et al., 2002), however, this notion is controversial. It is controversial because, other studies observed no increase in GABA levels after gabapentin treatment (Errante and Petroff, 2003; Errante et al., 2002). Additionally, gabapentin increased GABA turnover in some brain regions tested only, and the temporal effect of the drug varied from region to region (Loscher et al., 1991).

Alternatively, the effects of gabapentin may be explained by alterations in the level of neurosteroids. δGABAA receptors are highly sensitive to neurosteroids compared to other

GABAA receptors, hence they are highly implicated in physiological and pathological conditions where endogenous neurosteroid levels are altered (Belelli and Lambert, 2005; Bianchi and Macdonald, 2003; Hosie et al., 2006; Maguire and Mody, 2009). Neurosteroids can alter

δGABAA receptor expression and activity (Belelli and Lambert, 2005; Sarkar et al., 2011; Smith et al., 2007; Wu et al., 2013). It is therefore possible that changes in neurosteroid levels may account for the changes in receptor expression and tonic current after gabapentin treatment.

Common endogenous neurosteroids that greatly potentiate δGABAA receptor activity were investigated. These neurosteroids were dehydroepiandrosterone (DHEA), progesterone metabolites 3α-hydroxy-5α-pregnane-20-one (3α,5α-THPROG), 3α-hydroxy-5β-pregnane-20- one (3α,5β-THPROG) and 3β-hydroxy-5α-pregnane-20-one (3β,5α-THPROG), pregnenolone (Pregnen), and tetrahydrodeoxycorticosterone (THDOC) (Belelli and Lambert, 2005).

6.2 Materials and Methods

All materials and methods were decribed in chapter 3.4. The experiments performed in this chapter were conducted by Dr. Robert Bonin in collaboration with Dr. Glen B Baker of the University of Calgary. The analysis of neurosteroid levels and amino acid levels were performed by Dr. Baker‟s research technologist, Gail A. Rauw.

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6.3 Results

6.3.1 Gabapentin does not increase GABAA receptor activating amino acids in murine brain tissue

Contrary to some published data, the concentration of GABA was comparable in gabapentin-treated and vehicle-treated mice (Fig 6.0). Additionally, the levels of all other amino acids measured were similar in both gabapentin-treated and vehicle-treated mice (Fig 6.0) (P >

0.05 for each comparison). This data suggests that the increase in tonic current and δGABAA receptor expression observed with gabapentin treatment is not due to changes in ligand or other amino acid neurotransmitter levels.

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Fig 6.0: Chronic in vivo administration of gabapentin does not change neurotransmitter levels. All neurotransmitter and amino acid levels measured were similar in gabapentin-treated and vehicle-treated mice (P > 0.05 for each comparison). ASP = aspartic acid, GLU = glutamic acid, ASN = asparagine, GLY = glycine, TAUR = taurine, ALA = β-Alanine. Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

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6.3.2 Gabapentin does not increase neurosteroid levels in murine brain tissue

The results showed that the levels of neurosteroids did not change after either a single dose (acute) or chronic administrations of gabapentin (Fig 6.1) (P > 0.05 for each comparison).

This data suggest that the increase in tonic current and δGABAA receptor expression observed with gabapentin treatment is not due to changes in neurosteroid levels.

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Fig 6.1: Acute and chronic in vivo administration of gabapentin does not change GABAergic neurosteroid levels. The levels of neurosteroids did not change after acute or chronic gabapentin treatment compared to vehicle-treated mice (P > 0.05 for each comparison). DHEA = 5- dehydroepiandrosterone, 3α,5 α = 5α-pregnan-3α-ol-20-one (3α,5 α-THPROG), 3α,5β = 3α,5β- THPROG, Pregnen = pregnenolone, 3β,5α = 3β,5α-THPROG, THDOC = 3α,21-dihydroxy-5α- pregnan-20-one. Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

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6.4 Discussion

Some studies showed that gabapentin can increase GABA levels in the brain (Cai et al., 2012; Errante et al., 2002), which can contribute to the enhancement in tonic current observed following gabapentin treatment (Cheng et al., 2006). However, our results showed that GABAA receptor ligand levels (including GABA) were unchanged after chronic gabapentin administration. This is not completely unexpected since the notion that gabapentin increased GABA levels is controversial. Similar to our findings, several other studies have also found no significant changes in neurotransmitter levels after gabapentin treatment (Errante and Petroff, 2003; Errante et al., 2002). The variability in results across studies may be explained by differences in strain and/or species of animals used. Additionally, gabapentin may enhance GABA turnover in some brain regions and not others, and the temporal effect of the drug varied from region to region (Loscher et al., 1991). Since we measured neurotransmitter levels in whole brain tissue, regional changes in neurotransmitter levels may not be detected. Future studies should measure neurotransmitter levels in specific brain regions.

Neurosteroids can alter δGABAA receptor expression and activity (Belelli and Lambert, 2005; Sarkar et al., 2011; Smith et al., 2007; Wu et al., 2013). In this study we showed that neurosteroids levels were unchanged after acute and chronic gabapentin treatment. This suggests that the enhancement in δGABAA receptor expression and increase in tonic current observed after gabapentin treatment is not a result of neurosteroid upregulation.

As a whole, these data suggest that increased δGABAA receptor expression and tonic current following gabapentin treatment is not a result of changes in GABAA receptor ligand levels, or neurosteroid levels. As a result, the link between gabapentin treatment and the resulting rise in

δGABAA receptor expression and enhancement in tonic current remains unknown.

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Chapter 7 : PKA and PKC are implicated in gabapentin-

induced δGABAA receptors expression increase

7.0 Overview

So far, our results showed that δGABAA receptors contribute to some of the anxiolytic and ataxic effects of gabapentin (Chapter 4). Furthermore, gabapentin treatment increased

δGABAA receptor total and surface expression in vitro and in vivo (Chapter 5), which may contribute to the increased tonic current observed following gabapentin treatment (Cheng et al., 2006). Finally, we showed that the concentration of GABAergic ligands and endogenous neurosteroids which can modulate δGABAA receptor activity and expression were unchanged after gabapentin treatment (Chapter 6). In this chapter, we investigated whether gabapentin- induced upregulation of δGABAA receptors was mediated by intracellular factors known to phosphorylate GABAA receptors such as protein kinase C (PKC) and protein kinase A (PKA) (Abramian et al., 2010; Brandon et al., 2002a; Bright and Smart, 2013; Kittler et al., 2005; Song and Messing, 2005). The results showed that PKC or PKA inhibition can attenuate gabapentin- induced upregulation of cell surface δGABAA receptors. These findings suggest that PKC and PKA may be necessary for some of the GABAergic effects of gabapentin.

7.1 Introduction

Gabapentin increased δGABAA receptor expression (Chapter 5). The expression of

GABAA receptors can be regulated by kinase and phosphatase action (Table 1). GABAA receptors possess several known phosphorylation sites which can affect receptor insertion and cell surface stability. Kinases known to affect GABAA receptor surface expression are protein kinase A (PKA), protein kinase C (PKC), P38 mitogen-activated protein kinases (p38-MAPK) and protein tyrosine kinase (PTK) (Botzolakis, 2009; Kittler and Moss, 2003; Michels and Moss, 2007; Wang et al., 2012). Different kinases can up- or down-regulate the expression of different subtypes of receptors (Brandon et al., 2002a; Kittler et al., 2005; Saliba et al., 2012; Song and

Messing, 2005). Besides directly phosphorylating GABAA receptors, PKC and PKA may phosphorylate proteins that are associated with GABAA receptor trafficking (Terunuma et al., 2004). Table 1 provides a summary of the action of different kinases and phosphatases on

GABAA receptor function and expression. 110

Currently the molecular mechanisms of δGABAA receptor insertion, stabilization, endocytosis and recycling are unclear. Also, how gabapentin treatment leads to the increase in δGABAA receptor total and surface expression is unknown. However, there are a few clues in literature that suggest that PKC and PKA may regulate δGABAA receptor trafficking (Abramian et al., 2010; Bright and Smart, 2013; Kumar et al., 2012; Saliba et al., 2012; Tang et al., 2010).

PKC activation can increase the cell surface expression of δGABAA receptors (Abramian et al., 2010; Joshi and Kapur, 2009). Interestingly, PKC activity can enhance the cell surface stability and insertion of α4 subunit-containing GABAA (α4GABAA) receptors by phosphorylating serine 443 (S443) (Abramian et al., 2014; Abramian et al., 2010). Since the α4 subunit partners with the δ subunit (Jia et al., 2005; Smith, 2013; Sun et al., 2004), the phosphorylation of the α4 subunit may result in increased expression of α4β2/3δ GABAA receptors. Similarly, the β2 and the β3 subunits partner with the δ subunit to form functional receptors. PKC enhances the surface expression of β2/β3 containing GABAA receptors through phosphorylating the β2 and β3 subunits (Brandon et al., 2002a). These mechanisms may contribute to the gabapentin-induced upregulation of δGABAA receptors. Specifically, PKC phosphorylates β2 at serine 410 (S410) and β3 at serine 408/409 (S408/409) (Brandon et al., 2002a; Brandon et al., 2003; Kittler et al., 2005; McDonald and Moss, 1997). The phosphorylation of these two subunits prevents the

GABAA receptor(s) from interacting with AP2, and hence disrupts endocytic signaling, resulting in increased surface expression (Brandon et al., 2003; Brandon et al., 2002b).

Similarly, PKA can phosphorylate the β3 subunit of GABAA receptors at serine 408/409

(S408/409) to promote increase GABAA receptor surface expression (Kittler et al., 2005).

Additionally, PKA inhibition can attenuate induced δGABAA receptor total protein expression (Uusi-Oukari et al., 2010). Furthermore, there is evidence to show that PKA is necessary for the effects of gabapentin on other channels (Lee et al., 2008; Martin et al., 2002; Takasu et al.,

2008). Hence, PKA may be involved in the gabapentin-induced upregulation of δGABAA receptors.

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7.2 Materials and Methods

A cerebellar slice in vitro model was used since the cerebellum had the biggest δGABAA subunit total protein and surface protein increase in response to gabapentin treatment (Chapter 5).

The stock solutions for the PKA inhibitor H-89 (20 mM) and the PKC inhibitor chelerythrine chloride (5 mM) were dissolved in DMSO. For the PKA and PKC experiments, cerebellar slices were treated with one of the following conditions: (1) ACSF + 0.1% (v/v) DMSO, (2) 300 µM gabapentin + 0.1% (v/v) DMSO, (3) 20 µM H-89, (4) 20 µM H-89 + 300 µM gabapentin, (5) 5 µM chelerythrine chloride, (6) 5 µM chelerythrine chloride + 300 µM gabapentin. The 0.1% (v/v) DMSO added in some of the conditions is to ensure homogenous vehicle conditions since the H-89 and chelerythrine chloride stocks were made in DMSO. All the treatments applied in vitro were applied for 6 hours at 35 to 37 oC to enable receptor trafficking.

Materials and methods were described in greater detail in section 3.3.

7.3 Results

7.3.1 Inhibition of PKC does not prevent gabapentin induced δGABAA receptor total expression increase, but attenuates surface expression increase

Total δ subunit expression was increased in the gabapentin (GBP) treated, the chelerythrine chloride (CCl) treated, and the CCl + GBP treated groups relative to control (Fig 7.0A) (Control: 100.0 ± 3.3 %, n = 3; GBP: 192.3 ± 9.3 %, n = 3, P < 0.001; CCl: 140.3 ± 5.5 %, n = 3, P < 0.01; CCl + GBP: 188.0 ± 8.1 %, n = 3, P < 0.001). Interestingly, PKC inhibition alone increased total δ subunit expression compared to control (Control: 100.0 ± 3.3 %, n = 3; CCl: 140.3 ± 5.5 %, n = 3, P < 0.01). The total δ subunit expression in GBP treated and CCl + GBP treated groups were not significantly different (GBP: 192.3 ± 9.3 %, n = 3; CCl + GBP: 188.0 ± 8.1 %, n = 3, P > 0.05). Hence, PKC inhibition does not attenuate GBP-induced upregulation of total δGABAA receptor subunit expression.

However, PKC attenuated GBP-induced upregulation of cell surface δGABAA receptor expression (Fig 7.0B). Cell surface δGABAA receptor expression increased in the GBP treated groups, but not in the CCl + GBP treated groups relative to control (Fig 7.0B) (Control: 100.0 ± 112

2.4 %, n = 3; GBP: 160.9 ± 9.3 %, n = 3, P < 0.01; CCl: 106.7 ± 6.3 %, n = 3, P > 0.05; CCl +

GBP: 100.7 ± 11.32 %, n = 3, P >0.05). The change in surface δGABAA receptor expression was significantly different in the GBP treated versus CCl + GBP treated groups (GBP: 160.9 ± 9.3 %, n = 3; CCl + GBP: 100.7 ± 11.32 %, n = 3, P < 0.05). Hence, PKC inhibition can attenuate GBP- induced upregulation of surface δGABAA receptors.

7.3.2 Inhibition of PKA attenuates gabapentin induced δGABAA receptor total and surface expression increase

Total δ subunit expression was not significantly different in the H-89 treated and the H- 89 + GBP treated groups relative to control (Fig 7.0A) (Control: 100.0 ± 3.3 %, n = 3; GBP: 192.3 ± 9.3 %, n = 3, P < 0.001; H-89: 112 ± 5.2 %, n = 3, P > 0.05; H-89 + GBP: 107.9 ± 4.4 %, n = 3, P > 0.05). The total δ subunit expression was significantly different in the GBP treated and the H-89 + GBP treated groups (Fig 7.0A) (GBP: 192.3 ± 9.3 %, n = 3; H-89 + GBP: 107.9 ± 4.4 %, n = 3, P < 0.01). Hence, PKA inhibition attenuates GBP-induced upregulation of total

δGABAA receptor subunit expression.

The cell surface δGABAA receptor expression was significantly increased in the GBP treated group, but not in the H-89 treated or the H-89 + GBP treated groups relative to control (Fig 7.0B) (Control: 100.0 ± 2.4 %, n = 3; GBP: 160.9 ± 9.3 %, n = 3, P < 0.01; H-89: 94.8 ± 6.8 %, n

= 3, P > 0.05; H-89 + GBP: 92.3 ± 3.3 %, n = 3, P > 0.05). The change in surface δGABAA receptor expression was significantly different in the GBP treated versus H-89 + GBP treated groups (Fig 7.0B) (GBP: 160.9 ± 9.3 %, n = 3; H-89 + GBP: 92.3 ± 3.3 %, n = 3, P < 0.01).

Hence, PKA inhibition attenuates GBP-induced upregulation of surface δGABAA receptors.

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Fig 7.0: PKC and PKA may be implicated in gabapentin induced upregulation of surface

δGABAA receptors. (A) PKC inhibition does not attenuate GBP-induced increase of δGABAA receptor total expression (Control: 100.0 ± 3.3 %, n = 3; GBP: 192.3 ± 9.3 %, n = 3, P < 0.001; CCl: 140.3 ± 5.5 %, n = 3, P < 0.01; CCl + GBP: 188.0 ± 8.1 %, n = 3, P < 0.001). PKA inhibition attenuates GBP-induced increase of δGABAA receptor total expression (Control: 100.0 ± 3.3 %, n = 3; GBP: 192.3 ± 9.3 %, n = 3, P < 0.001; H-89: 112 ± 5.2 %, n = 3, P > 0.05; H-89

114

+ GBP: 107.9 ± 4.4 %, n = 3, P > 0.05). GBP stands for gabapentin, CCl stands for chelerythrine chloride. Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

(B) PKC inhibition attenuates GBP-induced increase of δGABAA receptor surface expression (Control: 100.0 ± 2.4 %, n = 3; GBP: 160.9 ± 9.3 %, n = 3, P < 0.01; CCl: 106.7 ± 6.3 %, n = 3, P > 0.05; CCl + GBP: 100.7 ± 11.32 %, n = 3, P >0.05). PKA inhibition attenuates GBP-induced increase of δGABAA receptor surface expression (Control: 100.0 ± 2.4 %, n = 3; GBP: 160.9 ± 9.3 %, n = 3, P < 0.01; H-89: 94.8 ± 6.8 %, n = 3, P > 0.05; H-89 + GBP: 92.3 ± 3.3 %, n = 3, P > 0.05). GBP stands for gabapentin, CCl stands for chelerythrine chloride. Student‟s t-test used for all analysis. Data are represented as mean ± SEM.

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7.4 Discussion

The results showed that in vitro treatment with PKC inhibitor (chelerythrine chloride) or

PKA inhibitor (H-89) can significantly reduce gabapentin-induced surface δGABAA receptor upregulation. However, PKC inhibition does not prevent gabapentin-induced total δGABAA receptor upregulation, while PKA inhibition does. These data suggest that PKC and PKA activity may contribute to gabapentin-induced surface δGABAA receptor upregulation. These kinases may be involved through the modulation of trafficking of δGABAA receptor subunit(s) such as: α6, α4, β2, β3 or δ. Future investigations are necessary to isolate the exact subunit(s) altered by PKC and PKA after gabapentin treatment.

PKC activity can enhance the cell surface stability and insertion of δGABAA receptors by phosphorylating its binding partners such as: S443 on the α4 subunit (Abramian et al., 2014; Abramian et al., 2010), S410 on the β2 subunit, and S408/409 on the β3 subunit (Brandon et al., 2003; Brandon et al., 2002b). The phosphorylation of the α4 subunit increases the insertion of

α4βδ GABAA receptors to the cell surface (Abramian et al., 2010), while the phosphorylation of the β2/3 subunits disrupts endocytic signaling (Brandon et al., 2002a). All of these mechanisms may result in increased δGABAA receptor surface expression. The data in this chapter suggest that PKC activation is involved in the gabapentin-induced enhancement of surface δGABAA receptor expression.

PKA activation can promote the upregulation of surface δGABAA receptor expression through the phosphorylation of the β3 subunit on S408/409 (Kittler et al., 2005). Furthermore, PKA inhibition has been shown to attenuate AMPA receptor-induced total δGABAA receptor protein expression (Uusi-Oukari et al., 2010). Hence, PKA inhibition may also prevent gabapentin- induced upregulation of total δGABAA receptor expression. This notion was supported by the results in this chapter. Additionally, there is evidence in literature to show that PKA is necessary for the effects of gabapentin on other channels (Lee et al., 2008; Martin et al., 2002; Takasu et al., 2008). However, how gabapentin changes PKA activity is currently unknown.

In interpreting the data, there are several limitations that must be noted. For one, the experiments were all performed on cerebellar slices where the predominant form of δGABAA receptors are α6βxδ (Sieghart and Sperk, 2002). In contrast, the α4βxδ form is predominant in other brain

116 regions such as the thalamus and dentate gyrus, but is also present in the cerebellum (Sieghart and Sperk, 2002). PKC is known to phosphorylate the α4 subunit (Abramian et al., 2010), but its effect(s) on the α6 subunit is currently unknown.

Additionally, the β2 or β3 subunits can partner with other subunits besides the δ subunit to form either synaptic GABAA receptors such as α1β2/3γ, or other extrasynaptic GABAA receptors such as α5β3γ (Sieghart and Sperk, 2002). Hence, PKA or PKC activation may not selectively upregulate δGABAA receptors. Interestingly, the results in chapter 5 indicate that the surface expression of both α1 and α5 GABAA receptors were unchanged with gabapentin treatment.

How gabapentin induces PKA and PKC activity to be selective for δGABAA receptor upregulation is unknown.

A possible mechanism by which PKA and PKC activity may be selective for δGABAA receptors may be through the activation of a specific isoform of PKA or PKC. There are many different isoforms of PKA and PKC in cells (Singh et al., 1998; Way et al., 2000), and previously it has been shown that different isoforms of PKC can exert a variety of effects on specific GABAA receptors (Kumar et al., 2002; Song and Messing, 2005; Werner et al., 2011). The PKA inhibitor (H-89) and PKC inhibitor (chelerythrine chloride) used in our experiments were not isoform specific. Perhaps gabapentin directly or indirectly activates a specific isoform of PKA and PKC which selectively targets δGABAA receptors. This hypothesis would be of interest for future explorations. Finally, although H-89 is promoted as a selective and potent inhibitor of PKA and is commonly used in studies for PKA inhibition, there is literature to show that H-89 can also inhibit other kinases such as S6K1, MSK1, ROCKII, PKBα and MAPKAP-K1b (Lochner and Moolman, 2006). This fact may be a confounding factor in data interpretation.

Together, the data in this section suggest that gabapentin may directly or indirectly alter PKC and PKA activity, which may contribute to the gabapentin-induced upregulation of total and surface δGABAA receptors. The current experiments do not directly show that gabapentin alter PKC and PKA activity, the notion that it does is an inference based on the data. Future experiments may be performed to see if gabapentin changes PKC and PKA activity using mutant mice which contain PKA-sensitive luciferase or PKC-sensitive luciferase. These compounds can change luminescent signals when they are in their active form (Moskaug et al., 2008). Also, how gabapentin changes PKC and PKA activity (directly or indirectly), and the specific isoform(s) of 117

PKC and PKA responsible for δGABAA receptor modulation would be of interest in future studies.

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Chapter 8 : Discussion

8.0 Overview

This thesis describes a novel mechanism to account for some of gabapentin‟s actions. The results showed that: (1) δGABAA receptors contribute to the anxiolytic and ataxic effects of gabapentin, (2) gabapentin upregulates δGABAA receptor surface expression and in some instances total expression, and (3) PKA and PKC inhibition can attenuate the gabapentin-induced upregulation of δGABAA receptor expression. This study provides a foundation for many further studies. Future studies should investigate if δGABAA receptors contribute to other effects of gabapentin such as anticonvulsion, sedation and hypnosis. Also, studies should be performed to map out the exact link between gabapentin and changes in PKA and PKC activity. Finally, how

(directly or indirectly) gabapentin treatment can lead to δGABAA receptor upregulation should be investigated.

8.1 The roles and implications of δGABAA receptors in behaviour

δGABAA receptors are a major contributor of GABAergic tonic inhibition in the CNS, where they are widely expressed (Farrant and Nusser, 2005; Wisden et al., 1992). The activation of δGABAA receptors using THIP results in: decreased anxiety (Hoehn-Saric, 1983), decreased nociception (Bonin et al., 2011; Vaught et al., 1985), reduced seizure frequency (Petersen et al., 1983), enhanced discrimination memory likely through increased neurogenesis (Whissell et al., 2013b), impaired motor coordination (Gerlach et al., 1984), increased non-REM sleep (Lancel and Faulhaber, 1996) and impaired short term memory (Whissell et al., 2013a). Some of these THIP effects are consistent with those observed after gabapentin treatment in this study.

We showed that gabapentin treatment decreased anxiety and impaired motor coordination in WT mice, but not in Gabrd -/- mice. THIP increases δGABAA receptor activity through the generation of a greater peak response than GABA on δGABAA receptors, hence it acts as a

“super” agonist on δGABAA receptors (Brown et al., 2002). Meanwhile, the results from this thesis suggest that gabapentin increases δGABAA receptor activity through increasing surface receptor numbers. Also, currently gabapentin is presumed to not bind to GABAA receptors (Sills, 2006; Somerville and Michell, 2009; Taylor et al., 1998). Accordingly, THIP allosterically

119 modulates δGABAA receptor activity, while gabapentin does not. Interestingly, gabapentin and its analog pregabalin elicit many behavioural effects similar to those caused by THIP treatment in humans and laboratory animals. These effects include: decreased anxiety (Clarke et al., 2013; Strawn and Geracioti, 2007), decreased nociception (Pfizer, 2012; Somerville and Michell, 2009), reduced seizure frequency (Goa and Sorkin, 1993), impaired motor coordination (Somerville and Michell, 2009), increased non-REM sleep (Foldvary-Schaefer et al., 2002), and impaired short term memory (Lindner et al., 2006).

There are several implications associated with the finding that gabapentin can upregulate

δGABAA receptor surface expression and activity. A reduction in δGABAA receptor activity or expression has been implicated in ethanol withdrawal (Follesa et al., 2006; Kumar et al., 2009), mood disorders (Smith, 2013) and depression (Maguire and Mody, 2008). Since we found that gabapentin upregulates δGABAA receptor expression; gabapentin may be useful in treating conditions where δGABAA receptor activity or expression is downregulated.

8.2 Tonic inhibition and neuronal excitability

Tonic GABAergic inhibition is usually mediated by extrasynaptic GABAA receptors on postsynaptic neurons (Farrant and Nusser, 2005). Receptor combinations with either α5 or δ or ε subunits are most likely extrasynaptic and contribute to tonic conductance (Farrant and Nusser,

2005; Nusser et al., 1998; Wagner et al., 2005). Extrasynaptic GABAA receptors are activated at ambient levels of GABA, or even under conditions with no GABA present (Wlodarczyk et al., 2013), hence once expressed at the cell surface they tend to be constitutively active. Tonic inhibition is important in regulating neuronal excitability and gain which can affect firing rates (Lee and Maguire, 2014; Mitchell and Silver, 2003). Additionally, tonic inhibition has also been implicated in modulating network oscillatory behaviour, action potential conductance and neurotransmitter release (Farrant and Nusser, 2005).

Generally, the tonic current mediated by extrasynaptic GABAA receptors is inhibitory. However, GABAergic tonic currents can also cause an excitatory effect either via neuron depolarization or a network effect due to a dominant effect on interneurons (Walker and Kullmann, 2012). The effects of increased tonic inhibition may account for some of the behavioural effects of

120 gabapentin. For example, increased tonic inhibition may lead to reduced neuronal excitability, which may have an anxiolytic and antiepileptic effect (Maguire et al., 2005).

Previously, we discovered that gabapentin increased GABAergic tonic inhibition (Cheng et al., 2006). The results from this thesis demonstrated that this enhancement may be attributed to an increase in δGABAA receptor expression. Also, gabapentin is found to inhibit the α2δ subunits of VDCC, which can lead to reduced neuronal and network excitability (Dolphin, 2012b; Eroglu et al., 2009). These effects of gabapentin may be additive to synergistically reduce neuronal and network excitability.

Although tonic GABAergic inhibition is present on all neuron types, extrasynaptic GABAA receptors and accordingly tonic current are highly enriched in certain cells such as the cerebellar granule cells (Nusser et al., 1996b; Payne et al., 2007). Also, the subtype of GABAA receptors that are enriched in each brain region and type of cell differ (Olsen and Sieghart, 2009), this may facilitate different physiological and pharmacological effects. The results showed that gabapentin increased δGABAA receptor expression, which are highly enriched in the cerebellar granule cells (Brickley et al., 1996; Nusser et al., 1998), the thalamocortical relay neurons (Belelli et al., 2005; Porcello et al., 2003), and the dentate granule cells (Mtchedlishvili and Kapur, 2006).

Some of the effects of gabapentin tested in this thesis were mediated by brain regions rich in

δGABAA receptor expression. For example, the ataxic phenotype observed after gabapentin treatment by WT but not Gabrd -/- mice is most likely mediated by the cerebellum, a brain region rich in δGABAA receptor expression (Wisden et al., 1992), and is important for fine motor control. A possible neuronal pathway that may account for the ataxic behaviour observed after gabapentin treatment is through increased tonic inhibition in the cerebellar granule cells which is rich in δGABAA receptor.

Cerebellar granule cells are generally considered excitatory interneurons which can relay mossy fiber inputs via parallel fiber projections to the Purkinje cells. The Purkinje cells can then project to the deep cerebellar nuclear cells. The cerebellar nuclear cells project signals to the upper motor neurons in the brainstem and cortex. The firing pattern and firing frequency of the Purkinje cells can modulate the timing of motion and is important for real-time error corrections during ongoing movement (Purves D, 2004). The gabapentin induced upregulation of cell

121 surface δGABAA receptor expression would result in increased tonic GABAergic inhibition in the cerebellar granule cells. This can potentially interfere with the signal relay frequency and amplitude of current from the granule cells to the Purkinje cells, which can impair the timing and real-time error corrections during movement, resulting in ataxia.

8.3 Phosphorylation states and tonic inhibition

GABAA receptors possess many phosphorylation sites and their function and expression can be regulated by different kinases and phosphatases (Table 1). Kinases such as PKA, PKC,

PTK, and p38-MAPK affect the surface expression of different types of GABAA receptors as well as their activity through phosphorylating target residue(s) (Botzolakis, 2009; Kittler and Moss, 2003; Michels and Moss, 2007; Wang et al., 2012). Alternatively, phosphatases such as

PP1α, calcineurin, and PP2A can dephosphorylate GABAA receptors, which also results in altered expression and activity (Table 1). The dephosphorylation of GABAA receptors has mostly been linked to endocytosis of the receptors (Jacob et al., 2008).

The results in this thesis show that gabapentin-induced upregulation of surface δGABAA receptors are attenuated by PKA inhibition and PKC inhibition. It is speculated from the results that PKA and PKC activation can increase tonic GABAergic inhibition through facilitating the increase of δGABAA receptor expression. Indeed, PKA and PKC activation has been linked to increased cell surface stability of δGABAA receptor‟s binding partners, such as the β2, β3 (Brandon et al., 2002a; Saliba et al., 2012) and α4 (Abramian et al., 2010) subunits.

Furthermore, PKA and PKC activation has been linked to increased tonic inhibition through increased surface expression and activity of extrasynaptic GABAA receptors (Abramian et al., 2010; Bright and Smart, 2013; Saliba et al., 2012; Tang et al., 2010). There are many isoforms of PKA and PKC, future studies should investigate which isoform(s) of PKA and PKC are responsible for the specific upregulation of δGABAA receptors.

8.3.1 Kinases and gabapentin

It is speculated from inhibition experiments that gabapentin may alter PKA and PKC activity, however, this has never been definitively shown. How gabapentin alters PKA and PKC activity is also unknown. There is some literature to show that some of the effects of gabapentin

122 are eliminated, or reduced with PKA inhibition (Lee et al., 2008; Takasu et al., 2008) or PKC inhibition (Gu and Huang, 2001). In this thesis, we demonstrated that PKA inhibition or PKC inhibition attenuated the gabapentin-induced upregulation of surface δGABAA receptor expression. Future studies should investigate how gabapentin can alter PKA and PKC activity. Structurally gabapentin does not resemble a direct modulator of PKA or PKC activity; hence the mechanism is most likely through an indirect pathway.

8.3.2 Kinases and δGABAA receptor expression

Currently, there are no direct or specific mechanisms to account for δGABAA receptor synthesis or trafficking. Most of the mechanistic trafficking work have been done on synaptic

GABAA receptors, however it is speculated that the endocytosis and exocytosis of extrasynaptic

GABAA receptors are similar to their synaptic counterparts (Leidenheimer, 2008; Luscher et al.,

2011; Michels and Moss, 2007). Presently, it is postulated that extrasynaptic GABAA receptor trafficking and stabilization are mainly dependent on their α and β subunits. How different kinases and phosphatases influence GABAA receptor trafficking is summarized in Table 1. We demonstrated that PKA inhibition attenuated gabapentin-induced upregulation of total and surface δGABAA receptor expression. In contrast, PKC inhibition did not prevent gabapentin- induced upregulation of total δGABAA receptor expression. However, PKC inhibition attenuated the gabapentin-induced upregulation of surface δGABAA receptors.

PKA activation can promote the enhancement of surface δGABAA receptor expression through the phosphorylation of the β3 subunit at S408/409 (Kittler et al., 2005). Also, PKA inhibition can prevent AMPA receptor-induced upregulation of total δGABAA receptor expression (Uusi-

Oukari et al., 2010). PKC activity can enhance the cell surface stability of the δGABAA receptors by phosphorylating its binding partners, S443 on the α4 subunit (Abramian et al., 2010), S410 on β2 and S408/409 on β3 (Brandon et al., 2003; Brandon et al., 2002b). The phosphorylation of the

α4 subunit increases the insertion of α4βxδ GABAA receptors to the cell surface (Abramian et al., 2010), and the phosphorylation of the β2/3 subunits disrupts endocytic signaling (Brandon et al., 2002a). All of these mechanisms result in increased δGABAA receptor surface expression.

Although, the β2 and β3 subunits can partner with δ to form functional δGABAA receptors, they can also partner with other subunits to form other GABAA receptors. For example, the β2 and β3 123 subunits can form synaptic GABAA receptors such as α1β2/3γ or other extrasynaptic GABAA receptors such as α5β3γ (Olsen and Sieghart, 2009). Interestingly, the results in chapter 5 indicate that the surface expression of both α1 and α5 GABAA receptors are unchanged with gabapentin treatment. How gabapentin induces PKA and PKC to selectively upregulate δGABAA receptor expression is unknown. Also presently, no kinases have been identified to phosphorylate or modulate the δ subunit of GABAA receptors.

Cells possess many different isoforms of PKA and PKC (Singh et al., 1998; Way et al., 2000). Previously, it has been shown that different isoforms of PKC can exert a variety of effects on specific GABAA receptors (Kumar et al., 2002; Song and Messing, 2005; Werner et al., 2011). The PKA inhibitor (H-89) and PKC inhibitor (chelerythrine chloride) used in the thesis experiments were not isoform specific. Perhaps gabapentin directly or indirectly activates a specific isoform of PKA and PKC, which selectively targets δGABAA receptors. This speculation would be of interest for future explorations.

Collectively, the data shows that gabapentin upregulates δGABAA receptor surface expression, which contribute to the anxiolytic and ataxic effects of gabapentin. Also, PKA and PKC may be involved in the gabapentin-induced upregulation of δGABAA receptors. This study provides a novel mechanism to account for some of the physiological effects of gabapentin.

8.4 Future directions

Gabapentin and its analog pregabalin have been shown to be an anticonvulsant and analgesic (Kukkar et al., 2013). Additionally gabapentinoids have been shown to elicit a range of therapeutic and adverse effects such as anxiolysis (Clarke et al., 2013; Strawn and Geracioti, 2007; Taylor et al., 1998), sedation, hypnosis through increased non-REM sleep (Foldvary- Schaefer et al., 2002), short term memory loss (Lindner et al., 2006), somnolence and ataxia

(Somerville and Michell, 2009). Interestingly, the activation of δGABAA receptors can also result in analgesia (Bonin et al., 2011; Vaught et al., 1985), anticonvulsion (Petersen et al., 1983), anxiolysis (Hoehn-Saric, 1983), ataxia (Gerlach et al., 1984), hypnosis also through increased non-REM sleep (Lancel and Faulhaber, 1996) and impairment of short term memory

(Whissell et al., 2013a). We found that δGABAA receptors contribute to the anxiolytic and ataxic

124 effects of gabapentin. Future studies should investigate if all the other gabapentinoid effects listed above are implicated by δGABAA receptors as well.

The results in chapter 5 showed that the surface expression of δGABAA receptors is selectively upregulated by gabapentin, and the upregulation may involve PKA and PKC activity (Chapter 7). Future studies should investigate:

(1) Whether δ subunit binding partners such as the α4 and α6 subunits are also upregulated after gabapentin treatment.

(2) Whether GABAergic tonic current increases with gabapentin treatment in vivo.

(3) Whether gabapentin changes PKA or PKC activity using a direct method of measurement (described in section 7.4)

(4) The PKA and PKC isoform(s) which can elicit a selective upregulation of δGABAA receptors by gabapentin. This can be done by using isoform-specific PKA and PKC inhibitors.

(5) Whether Gabrd -/- mice exhibit an altered α2δ subunit expression profile. Additionally, experiments on the α2δ point mutant mice which prevent gabapentin binding should be performed to see if δGABAA receptors are still upregulated after gabapentin treatment. This will address whether the gabapentin-induced upregulation of surface δGABAA receptors is dependent or independent of the α2δ-gabapentin binding mechanism.

This study provides a novel mechanism to account for the anxiolytic and ataxic effects of gabapentin. The gabapentin field has long assumed that GABAA receptors play no or a minimal role in gabapentin‟s actions. However, this study shows that δGABAA receptors contribute to some of gabapentin‟s effects through the upregulation of surface δGABAA receptors. Knowing the GABAergic effects of gabapentin would help improve patient outcomes and can open up new avenues for gabapentinoid use.

125

Appendix Publications and papers in submission resulting from my graduate studies

1) Penna A, Wang DS*, Yu J, Lecker I, Brown PM, Bowie D, Orser BA. Hydrogen peroxide increases GABAA receptor-mediated tonic current in hippocampal neurons. 2014 (In press) J Neurosci. [JY contributed to 10% of experiments and analyses, and 5% of writing]

2) Diaz MR, Vollmer CC, Zamudio-Bulcock PA, Vollmer W, Blomquist SL, Morton RA, Everett JC, Zurek AA, Yu J, Orser BA, Valenzuela CF. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats. 2014. Neuropharmacology 79:262-74. [JY contributed to western blot experiments and figures]

3) Bonin RP, Zurek AA, Yu J, Bayliss DA, Orser BA. Hyperpolarization-activated current (Ih) is reduced in hippocampal neurons from Gabra5-/- mice. 2013. PLoS One 8(3):e58679. [JY contributed to 10% of experiments and analyses, and 5% of writing]

4) Wang DS, Zurek AA, Lecker I, Yu J, Abramian AM, Avramescu S, Davies PA, Moss SJ, Lu WY, Orser BA. Memory deficits induced by inflammation are regulated by α5-subunit- containing GABAA receptors. 2012. Cell Rep. 2(3):488-96. [JY contributed to 20% of experiments and analyses, and 5% of writing]

5) Zurek AA, Yu J, Wang DS, Haffey SC, Bridgwater EM, Penna A, Lecker I, Lei G, Salter EWR, Orser BA. Sustained increase in GABA(A) receptor function impairs memory after anesthesia. (Under review) J. Clin. Invest. [JY contributed to 35% of experiments and analyses, and 10% of writing]

6) Whissell PD, Lecker I, Wang DS, Yu J, Orser BA. Altered Expression of δGABAA Receptors in Health and Disease. (Under review) Neuropharmacology [JY contributed to 50% of figures and 5% writing]

126

References

Abramian, A.M., Comenencia-Ortiz, E., Modgil, A., Vien, T.N., Nakamura, Y., Moore, Y.E., Maguire, J.L., Terunuma, M., Davies, P.A., and Moss, S.J. (2014). Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors. Proceedings of the National Academy of Sciences of the United States of America.

Abramian, A.M., Comenencia-Ortiz, E., Vithlani, M., Tretter, E.V., Sieghart, W., Davies, P.A., and Moss, S.J. (2010). Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition. Journal of Biological Chemistry 285, 41795-41805.

Ahboucha, S., Butterworth, R.F., Pomier-Layrargues, G., Vincent, C., Hassoun, Z., and Baker, G.B. (2008). Neuroactive steroids and fatigue severity in patients with primary biliary cirrhosis and hepatitis C. Neurogastroenterology and Motility 20, 671-679.

Anhut, H., Ashman, P., Feuerstein, T.J., Sauermann, W., Saunders, M., and Schmidt, B. (1994). Gabapentin (Neurontin) as add-on therapy in patients with partial seizures: a double-blind, placebo-controlled study. Epilepsia 35, 795-801.

Arancibia-Carcamo, I.L., and Kittler, J.T. (2009). Regulation of GABA(A) receptor membrane trafficking and synaptic localization. Pharmacology & Therapeutics 123, 17-31.

Awapara, J., Landua, A.J., Fuerst, R., and Seale, B. (1950). Free gamma-aminobutyric acid in brain. Journal of Biological Chemistry 187, 35-39.

Bannerman, D.M., Rawlins, J.N., McHugh, S.B., Deacon, R.M., Yee, B.K., Bast, T., Zhang, W.N., Pothuizen, H.H., and Feldon, J. (2004). Regional dissociations within the hippocampus-- memory and anxiety. Neuroscience and Biobehavioral Reviews 28, 273-283.

Bannerman, D.M., Sprengel, R., Sanderson, D.J., McHugh, S.B., Rawlins, J.N., Monyer, H., and Seeburg, P.H. (2014). Hippocampal synaptic plasticity, spatial memory and anxiety. Nature Reviews Neuroscience 15, 181-192.

127

Bannon, A.W., and Malmberg, A.B. (2007). Models of nociception: hot-plate, tail-flick, and formalin tests in rodents. Current Protocols in Neuroscience Chapter 8, Unit 8 9.

Bauer, C.S., Nieto-Rostro, M., Rahman, W., Tran-Van-Minh, A., Ferron, L., Douglas, L., Kadurin, I., Sri Ranjan, Y., Fernandez-Alacid, L., Millar, N.S., et al. (2009). The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. Journal of Neuroscience 29, 4076-4088.

Bazil, C.W., Battista, J., and Basner, R.C. (2005). Gabapentin improves sleep in the presence of alcohol. Journal of Clinical Sleep Medicine 1, 284-287.

Belelli, D., Harrison, N.L., Maguire, J., Macdonald, R.L., Walker, M.C., and Cope, D.W. (2009).

Extrasynaptic GABAA receptors: form, pharmacology, and function. Journal of Neuroscience 29, 12757-12763.

Belelli, D., and Lambert, J.J. (2005). Neurosteroids: endogenous regulators of the GABA(A) receptor. Nature Reviews Neuroscience 6, 565-575.

Belelli, D., Peden, D.R., Rosahl, T.W., Wafford, K.A., and Lambert, J.J. (2005). Extrasynaptic

GABAA receptors of thalamocortical neurons: a molecular target for hypnotics. Journal of Neuroscience 25, 11513-11520.

Berry, J.D., and Petersen, K.L. (2005). A single dose of gabapentin reduces acute pain and allodynia in patients with herpes zoster. Neurology 65, 444-447.

Bertrand, S., Ng, G.Y., Purisai, M.G., Wolfe, S.E., Severidt, M.W., Nouel, D., Robitaille, R., Low, M.J., O'Neill, G.P., Metters, K., et al. (2001). The anticonvulsant, antihyperalgesic agent gabapentin is an agonist at brain gamma-aminobutyric acid type B receptors negatively coupled to voltage-dependent calcium channels. Journal of Pharmacology and Experimental Therapeutics 298, 15-24.

Bettler, B., Kaupmann, K., Mosbacher, J., and Gassmann, M. (2004). Molecular structure and physiological functions of GABA(B) receptors. Physiological Reviews 84, 835-867.

128

Bian, F., Li, Z., Offord, J., Davis, M.D., McCormick, J., Taylor, C.P., and Walker, L.C. (2006). Calcium channel alpha2-delta type 1 subunit is the major binding protein for pregabalin in neocortex, hippocampus, amygdala, and spinal cord: an ex vivo autoradiographic study in alpha2-delta type 1 genetically modified mice. Brain Research 1075, 68-80.

Bianchi, M.T., and Macdonald, R.L. (2003). Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns. Journal of Neuroscience 23, 10934-10943.

Biggio, G., Concas, A., Corda, M.G., Giorgi, O., Sanna, E., and Serra, M. (1990). GABAergic and dopaminergic transmission in the rat cerebral cortex: effect of stress, anxiolytic and anxiogenic drugs. Pharmacology & Therapeutics 48, 121-142.

Biggio, G., Concas, A., Follesa, P., Sanna, E., and Serra, M. (2007). Stress, ethanol, and neuroactive steroids. Pharmacology & Therapeutics 116, 140-171.

Blum, R.A., Comstock, T.J., Sica, D.A., Schultz, R.W., Keller, E., Reetze, P., Bockbrader, H., Tuerck, D., Busch, J.A., Reece, P.A., et al. (1994). Pharmacokinetics of gabapentin in subjects with various degrees of renal function. Clinical Pharmacology and Therapeutics 56, 154-159.

Bockbrader, H.N., Wesche, D., Miller, R., Chapel, S., Janiczek, N., and Burger, P. (2010). A comparison of the pharmacokinetics and pharmacodynamics of pregabalin and gabapentin. Clinical Pharmacokinetics 49, 661-669.

Bohlega, S., Alsaadi, T., Amir, A., Hosny, H., Karawagh, A.M., Moulin, D., Riachi, N., Salti, A., and Shelbaya, S. (2010). Guidelines for the pharmacological treatment of peripheral neuropathic pain: expert panel recommendations for the middle East region. Journal of International Medical Research 38, 295-317.

Bonin, R.P., Labrakakis, C., Eng, D.G., Whissell, P.D., De Koninck, Y., and Orser, B.A. (2011). Pharmacological enhancement of delta-subunit-containing GABA(A) receptors that generate a tonic inhibitory conductance in spinal neurons attenuates acute nociception in mice. Pain 152, 1317-1326.

129

Bonin, R.P., Martin, L.J., MacDonald, J.F., and Orser, B.A. (2007). Alpha5GABAA receptors regulate the intrinsic excitability of mouse hippocampal pyramidal neurons. Journal of Neurophysiology 98, 2244-2254.

Botzolakis, R.L.M.a.E.J. (2009). GABAA Receptor Channels, Vol 1 (Boston: Elsevier/Academic Press).

Boyd, R.A., Turck, D., Abel, R.B., Sedman, A.J., and Bockbrader, H.N. (1999). Effects of age and gender on single-dose pharmacokinetics of gabapentin. Epilepsia 40, 474-479.

Brambilla, P., Perez, J., Barale, F., Schettini, G., and Soares, J.C. (2003). GABAergic dysfunction in mood disorders. Molecular Psychiatry 8, 721-737, 715.

Brandon, N., Jovanovic, J., and Moss, S. (2002a). Multiple roles of protein kinases in the modulation of gamma-aminobutyric acid(A) receptor function and cell surface expression. Pharmacology & Therapeutics 94, 113-122.

Brandon, N.J., Jovanovic, J.N., Colledge, M., Kittler, J.T., Brandon, J.M., Scott, J.D., and Moss, S.J. (2003). A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta subunits. Molecular and Cellular Neurosciences 22, 87-97.

Brandon, N.J., Jovanovic, J.N., Smart, T.G., and Moss, S.J. (2002b). Receptor for activated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABA(A) receptors with the activation of G-protein-coupled receptors. Journal of Neuroscience 22, 6353-6361.

Brickley, S.G., Cull-Candy, S.G., and Farrant, M. (1996). Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. Journal of Physiology 497 ( Pt 3), 753-759.

Brickley, S.G., and Mody, I. (2012). Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73, 23-34.

130

Brickley, S.G., Revilla, V., Cull-Candy, S.G., Wisden, W., and Farrant, M. (2001). Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409, 88-92.

Bright, D.P., Aller, M.I., and Brickley, S.G. (2007). Synaptic release generates a tonic GABA(A) receptor-mediated conductance that modulates burst precision in thalamic relay neurons. Journal of Neuroscience 27, 2560-2569.

Bright, D.P., and Smart, T.G. (2013). Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus. European Journal of Neuroscience 38, 3408-3423.

Brill, J., Klocke, R., Paul, D., Boison, D., Gouder, N., Klugbauer, N., Hofmann, F., Becker, C.M., and Becker, K. (2004). entla, a novel epileptic and ataxic Cacna2d2 mutant of the mouse. Journal of Biological Chemistry 279, 7322-7330.

Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Lin, D.D., Rikhter, T.Y., Holloway, K.L., and Coulter, D.A. (1999). Human neuronal gamma-aminobutyric acid(A) receptors: coordinated subunit mRNA expression and functional correlates in individual dentate granule cells. Journal of Neuroscience 19, 8312-8318.

Brown, J.T., and Randall, A. (2005). Gabapentin fails to alter P/Q-type Ca2+ channel-mediated synaptic transmission in the hippocampus in vitro. Synapse 55, 262-269.

Brown, N., Kerby, J., Bonnert, T.P., Whiting, P.J., and Wafford, K.A. (2002). Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors. British Journal of Pharmacology 136, 965-974.

Burt, D.R., and Kamatchi, G.L. (1991). GABAA receptor subtypes: from pharmacology to molecular biology. FASEB Journal 5, 2916-2923.

Cai, K., Nanga, R.P., Lamprou, L., Schinstine, C., Elliott, M., Hariharan, H., Reddy, R., and Epperson, C.N. (2012). The impact of gabapentin administration on brain GABA and glutamate concentrations: a 7T (1)H-MRS study. Neuropsychopharmacology 37, 2764-2771.

131

Caraiscos, V.B., Elliott, E.M., You-Ten, K.E., Cheng, V.Y., Belelli, D., Newell, J.G., Jackson, M.F., Lambert, J.J., Rosahl, T.W., Wafford, K.A., et al. (2004). Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma- aminobutyric acid type A receptors. Proceedings of the National Academy of Sciences of the United States of America 101, 3662-3667.

Carter, R.J., Morton, J., and Dunnett, S.B. (2001). Motor coordination and balance in rodents. Current Protocols in Neuroscience Chapter 8, Unit 8 12.

Cavelier, P., Hamann, M., Rossi, D., Mobbs, P., and Attwell, D. (2005). Tonic excitation and inhibition of neurons: ambient transmitter sources and computational consequences. Progress in Biophysics and Molecular Biology 87, 3-16.

Chadwick, D. (1994). Gabapentin. Lancet 343, 89-91.

Chadwick, D.W., Anhut, H., Greiner, M.J., Alexander, J., Murray, G.H., Garofalo, E.A., and Pierce, M.W. (1998). A double-blind trial of gabapentin monotherapy for newly diagnosed partial seizures. International Gabapentin Monotherapy Study Group 945-77. Neurology 51, 1282-1288.

Chandra, D., Jia, F., Liang, J., Peng, Z., Suryanarayanan, A., Werner, D.F., Spigelman, I., Houser, C.R., Olsen, R.W., Harrison, N.L., et al. (2006). GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proceedings of the National Academy of Sciences of the United States of America 103, 15230- 15235.

Chen, C. (2013). Meta-analyses of dose-exposure relationships for gabapentin following oral administration of gabapentin and gabapentin enacarbil. European Journal of Clinical Pharmacology 69, 1809-1817.

Chen, Z.W., Chang, C.S., Leil, T.A., and Olsen, R.W. (2007). C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking. Journal of Neuroscience 27, 6655-6663.

132

Cheng, V.Y., Bonin, R.P., Chiu, M.W., Newell, J.G., MacDonald, J.F., and Orser, B.A. (2006). Gabapentin increases a tonic inhibitory conductance in hippocampal pyramidal neurons. Anesthesiology 105, 325-333.

Cilio, M.R., Bolanos, A.R., Liu, Z., Schmid, R., Yang, Y., Stafstrom, C.E., Mikati, M.A., and Holmes, G.L. (2001). Anticonvulsant action and long-term effects of gabapentin in the immature brain. Neuropharmacology 40, 139-147.

Clarke, H., Kirkham, K.R., Orser, B.A., Katznelson, R., Mitsakakis, N., Ko, R., Snyman, A., Ma, M., and Katz, J. (2013). Gabapentin reduces preoperative anxiety and pain catastrophizing in highly anxious patients prior to major surgery: a blinded randomized placebo-controlled trial. Canadian Journal of Anaesthesia 60, 432-443.

Cobb, S.R., Buhl, E.H., Halasy, K., Paulsen, O., and Somogyi, P. (1995). Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons. Nature 378, 75-78.

Coderre, T.J., Katz, J., Vaccarino, A.L., and Melzack, R. (1993). Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52, 259- 285.

Collinson, N., Kuenzi, F.M., Jarolimek, W., Maubach, K.A., Cothliff, R., Sur, C., Smith, A., Otu, F.M., Howell, O., Atack, J.R., et al. (2002). Enhanced learning and memory and altered

GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor. Journal of Neuroscience 22, 5572-5580.

Colucci-Guyon, E., Gimenez, Y.R.M., Maurice, T., Babinet, C., and Privat, A. (1999). Cerebellar defect and impaired motor coordination in mice lacking vimentin. Glia 25, 33-43.

Conti, F., Minelli, A., and Melone, M. (2004). GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Research Reviews 45, 196-212.

Cope, D.W., Di Giovanni, G., Fyson, S.J., Orban, G., Errington, A.C., Lorincz, M.L., Gould,

T.M., Carter, D.A., and Crunelli, V. (2009). Enhanced tonic GABAA inhibition in typical absence epilepsy. Nature Medicine 15, 1392-1398.

133

Darlison, M.G., Pahal, I., and Thode, C. (2005). Consequences of the evolution of the GABA(A) receptor gene family. Cellular and Molecular Neurobiology 25, 607-624.

Davies, A., Douglas, L., Hendrich, J., Wratten, J., Tran Van Minh, A., Foucault, I., Koch, D., Pratt, W.S., Saibil, H.R., and Dolphin, A.C. (2006). The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function. Journal of Neuroscience 26, 8748-8757.

Davis, S. (2003). Gabapentin may be appropriate for off-label uses. Journal of Managed Care Pharmacy 9, 569-570.

Dazzi, L., Sanna, A., Cagetti, E., Concas, A., and Biggio, G. (1996). Inhibition by the neurosteroid allopregnanolone of basal and stress-induced acetylcholine release in the brain of freely moving rats. Brain Research 710, 275-280.

De Sarro, G., Spagnolo, C., Gareri, P., Gallelli, L., and De Sarro, A. (1998). Gabapentin potentiates the antiseizure activity of certain anticonvulsants in DBA/2 mice. European Journal of Pharmacology 349, 179-185.

Diaz, M.R., Vollmer, C.C., Zamudio-Bulcock, P.A., Vollmer, W., Blomquist, S.L., Morton, R.A., Everett, J.C., Zurek, A.A., Yu, J., Orser, B.A., et al. (2013). Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA receptor delta subunit in cerebellar granule neurons and delays motor development in rats. Neuropharmacology 79C, 262-274.

Dolphin, A.C. (2012a). Calcium channel alpha2delta subunits in epilepsy and as targets for antiepileptic drugs. In Jasper's Basic Mechanisms of the Epilepsies, J.L. Noebels, M. Avoli, M.A. Rogawski, R.W. Olsen, and A.V. Delgado-Escueta, eds. (Bethesda (MD)).

Dolphin, A.C. (2012b). Calcium channel auxiliary alpha2delta and beta subunits: trafficking and one step beyond. Nature Reviews Neuroscience 13, 542-555.

Dooley, D.J., Taylor, C.P., Donevan, S., and Feltner, D. (2007). Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission. Trends in Pharmacological Sciences 28, 75-82.

134

Drasbek, K.R., Hoestgaard-Jensen, K., and Jensen, K. (2007). Modulation of extrasynaptic THIP conductances by GABAA-receptor modulators in mouse neocortex. Journal of Neurophysiology 97, 2293-2300.

Drasbek, K.R., and Jensen, K. (2006). THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb Cortex 16, 1134-1141.

Dworkin, R.H., Backonja, M., Rowbotham, M.C., Allen, R.R., Argoff, C.R., Bennett, G.J., Bushnell, M.C., Farrar, J.T., Galer, B.S., Haythornthwaite, J.A., et al. (2003). Advances in neuropathic pain: diagnosis, mechanisms, and treatment recommendations. Archives of Neurology 60, 1524-1534.

Dworkin, R.H., Barbano, R.L., Tyring, S.K., Betts, R.F., McDermott, M.P., Pennella-Vaughan, J., Bennett, G.J., Berber, E., Gnann, J.W., Irvine, C., et al. (2009). A randomized, placebo- controlled trial of oxycodone and of gabapentin for acute pain in herpes zoster. Pain 142, 209- 217.

Ebell, M., and Kripke, C. (2006). Gabapentin for pain: balancing benefit and harm. American Family Physician 73, 435.

Elia, G. (2012). Cell surface protein biotinylation for SDS-PAGE analysis. Methods in Molecular Biology 869, 361-372.

Enna, S.J., and McCarson, K.E. (2006). The role of GABA in the mediation and perception of pain. Advances in Pharmacology 54, 1-27.

Erlander, M.G., Tillakaratne, N.J., Feldblum, S., Patel, N., and Tobin, A.J. (1991). Two genes encode distinct glutamate decarboxylases. Neuron 7, 91-100.

Erlander, M.G., and Tobin, A.J. (1991). The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochemical Research 16, 215-226.

Eroglu, C., Allen, N.J., Susman, M.W., O'Rourke, N.A., Park, C.Y., Ozkan, E., Chakraborty, C., Mulinyawe, S.B., Annis, D.S., Huberman, A.D., et al. (2009). Gabapentin receptor alpha2delta-1

135 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139, 380-392.

Errante, L.D., and Petroff, O.A. (2003). Acute effects of gabapentin and pregabalin on rat forebrain cellular GABA, glutamate, and glutamine concentrations. Seizure 12, 300-306.

Errante, L.D., Williamson, A., Spencer, D.D., and Petroff, O.A. (2002). Gabapentin and vigabatrin increase GABA in the human neocortical slice. Epilepsy Research 49, 203-210.

Farrant, M., and Nusser, Z. (2005). Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nature Reviews Neuroscience 6, 215-229.

Fassoulaki, A., Stamatakis, E., Petropoulos, G., Siafaka, I., Hassiakos, D., and Sarantopoulos, C. (2006). Gabapentin attenuates late but not acute pain after abdominal hysterectomy. European Journal of Anaesthesiology 23, 136-141.

Fehrenbacher, J.C., Taylor, C.P., and Vasko, M.R. (2003). Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C. Pain 105, 133-141.

Field, M.J., Cox, P.J., Stott, E., Melrose, H., Offord, J., Su, T.Z., Bramwell, S., Corradini, L., England, S., Winks, J., et al. (2006). Identification of the alpha2-delta-1 subunit of voltage- dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. Proceedings of the National Academy of Sciences of the United States of America 103, 17537-17542.

Field, M.J., Hughes, J., and Singh, L. (2000). Further evidence for the role of the alpha(2)delta subunit of voltage dependent calcium channels in models of neuropathic pain. British Journal of Pharmacology 131, 282-286.

Foldvary-Schaefer, N., De Leon Sanchez, I., Karafa, M., Mascha, E., Dinner, D., and Morris, H.H. (2002). Gabapentin increases slow-wave sleep in normal adults. Epilepsia 43, 1493-1497.

136

Follesa, P., Biggio, F., Caria, S., Gorini, G., and Biggio, G. (2004). Modulation of GABA(A) receptor gene expression by allopregnanolone and ethanol. European Journal of Pharmacology 500, 413-425.

Follesa, P., Biggio, F., Talani, G., Murru, L., Serra, M., Sanna, E., and Biggio, G. (2006). Neurosteroids, GABAA receptors, and ethanol dependence. Psychopharmacology 186, 267-280.

Follesa, P., Mostallino, M.C., Biggio, F., Gorini, G., Caria, S., Busonero, F., Murru, L., Mura, M.L., Sanna, E., and Biggio, G. (2005). Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons. Journal of Neurochemistry 94, 659-671.

Fritschy, J.M., and Panzanelli, P. (2006). Molecular and synaptic organization of GABAA receptors in the cerebellum: Effects of targeted subunit gene deletions. Cerebellum 5, 275-285.

Frye, G.D., and Breese, G.R. (1982). GABAergic modulation of ethanol-induced motor impairment. Journal of Pharmacology and Experimental Therapeutics 223, 750-756.

Gee, N.S., Brown, J.P., Dissanayake, V.U., Offord, J., Thurlow, R., and Woodruff, G.N. (1996). The novel anticonvulsant drug, gabapentin (Neurontin), binds to the alpha2delta subunit of a calcium channel. Journal of Biological Chemistry 271, 5768-5776.

Gerlach, J., Bjorndal, N., and Christensson, E. (1984). Methylphenidate, apomorphine, THIP, and diazepam in monkeys: dopamine-GABA behavior related to psychoses and tardive dyskinesia. Psychopharmacology 82, 131-134.

Glykys, J., Mann, E.O., and Mody, I. (2008). Which GABA(A) receptor subunits are necessary for tonic inhibition in the hippocampus? Journal of Neuroscience 28, 1421-1426.

Glykys, J., Peng, Z., Chandra, D., Homanics, G.E., Houser, C.R., and Mody, I. (2007). A new naturally occurring GABA(A) receptor subunit partnership with high sensitivity to ethanol. Nature Neuroscience 10, 40-48.

Goa, K.L., and Sorkin, E.M. (1993). Gabapentin. A review of its pharmacological properties and clinical potential in epilepsy. Drugs 46, 409-427.

137

Gotz, E., Feuerstein, T.J., Lais, A., and Meyer, D.K. (1993). Effects of gabapentin on release of gamma-aminobutyric acid from slices of rat neostriatum. Arzneimittel-Forschung 43, 636-638.

Gowen, E., and Miall, R.C. (2007). The cerebellum and motor dysfunction in neuropsychiatric disorders. Cerebellum 6, 268-279.

Grobin, A.C., Matthews, D.B., Devaud, L.L., and Morrow, A.L. (1998). The role of GABA(A) receptors in the acute and chronic effects of ethanol. Psychopharmacology 139, 2-19.

Gu, Y., and Huang, L.Y. (2001). Gabapentin actions on N-methyl-D-aspartate receptor channels are protein kinase C-dependent. Pain 93, 85-92.

Hanchar, H.J., Dodson, P.D., Olsen, R.W., Otis, T.S., and Wallner, M. (2005). Alcohol-induced motor impairment caused by increased extrasynaptic GABA(A) receptor activity. Nature Neuroscience 8, 339-345.

Hendrich, J., Van Minh, A.T., Heblich, F., Nieto-Rostro, M., Watschinger, K., Striessnig, J., Wratten, J., Davies, A., and Dolphin, A.C. (2008). Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin. Proceedings of the National Academy of Sciences of the United States of America 105, 3628-3633.

Herd, M.B., Foister, N., Chandra, D., Peden, D.R., Homanics, G.E., Brown, V.J., Balfour, D.J., Lambert, J.J., and Belelli, D. (2009). Inhibition of thalamic excitability by 4,5,6,7- tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for delta-GABA(A) receptors. European Journal of Neuroscience 29, 1177-1187.

Hill, D.R., Suman-Chauhan, N., and Woodruff, G.N. (1993). Localization of [3H]gabapentin to a novel site in rat brain: autoradiographic studies. European Journal of Pharmacology 244, 303- 309.

Hoehn-Saric, R. (1983). Effects of THIP on chronic anxiety. Psychopharmacology 80, 338-341.

Hogg, S. (1996). A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacology, Biochemistry, and Behavior 54, 21-30.

138

Horikoshi, T., Asanuma, A., Yanagisawa, K., Anzai, K., and Goto, S. (1988). Taurine and beta- alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA. Brain Research 464, 97-105.

Hosie, A.M., Wilkins, M.E., da Silva, H.M., and Smart, T.G. (2006). Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 444, 486-489.

Jacob, T.C., Bogdanov, Y.D., Magnus, C., Saliba, R.S., Kittler, J.T., Haydon, P.G., and Moss,

S.J. (2005). Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. Journal of Neuroscience 25, 10469-10478.

Jacob, T.C., Moss, S.J., and Jurd, R. (2008). GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nature Reviews Neuroscience 9, 331-343.

Jia, F., Pignataro, L., Schofield, C.M., Yue, M., Harrison, N.L., and Goldstein, P.A. (2005). An extrasynaptic GABAA receptor mediates tonic inhibition in thalamic VB neurons. Journal of Neurophysiology 94, 4491-4501.

Jia, F., Yue, M., Chandra, D., Keramidas, A., Goldstein, P.A., Homanics, G.E., and Harrison, N.L. (2008). Taurine is a potent activator of extrasynaptic GABA(A) receptors in the thalamus. Journal of Neuroscience 28, 106-115.

Joshi, S., and Kapur, J. (2009). Slow intracellular accumulation of GABA(A) receptor delta subunit is modulated by brain-derived neurotrophic factor. Neuroscience 164, 507-519.

Kaila, K. (1994). Ionic basis of GABAA receptor channel function in the nervous system. Progress in Neurobiology 42, 489-537.

Karayannis, T., Elfant, D., Huerta-Ocampo, I., Teki, S., Scott, R.S., Rusakov, D.A., Jones, M.V., and Capogna, M. (2010). Slow GABA transient and receptor desensitization shape synaptic responses evoked by hippocampal neurogliaform cells. Journal of Neuroscience 30, 9898-9909.

Karim, N., Wellendorph, P., Absalom, N., Johnston, G.A., Hanrahan, J.R., and Chebib, M. (2013). Potency of GABA at human recombinant GABA(A) receptors expressed in Xenopus oocytes: a mini review. Amino Acids 44, 1139-1149.

139

Kashiwabuchi, N., Ikeda, K., Araki, K., Hirano, T., Shibuki, K., Takayama, C., Inoue, Y., Kutsuwada, T., Yagi, T., Kang, Y., et al. (1995). Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant mice. Cell 81, 245-252.

Kaufman, D.L., Houser, C.R., and Tobin, A.J. (1991). Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. Journal of Neurochemistry 56, 720-723.

Kittler, J.T., Chen, G., Honing, S., Bogdanov, Y., McAinsh, K., Arancibia-Carcamo, I.L., Jovanovic, J.N., Pangalos, M.N., Haucke, V., Yan, Z., et al. (2005). Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission. Proceedings of the National Academy of Sciences of the United States of America 102, 14871-14876.

Kittler, J.T., and Moss, S.J. (2003). Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition. Current Opinion in Neurobiology 13, 341-347.

Kocsis, J.D., and Honmou, O. (1994). Gabapentin increases GABA-induced depolarization in rat neonatal optic nerve. Neuroscience Letters 169, 181-184.

Korpi, E.R., Mihalek, R.M., Sinkkonen, S.T., Hauer, B., Hevers, W., Homanics, G.E., Sieghart, W., and Luddens, H. (2002). Altered receptor subtypes in the forebrain of GABA(A) receptor delta subunit-deficient mice: recruitment of gamma 2 subunits. Neuroscience 109, 733-743.

Krnjevic, K., and Schwartz, S. (1966). Is gamma-aminobutyric acid an inhibitory transmitter? Nature 211, 1372-1374.

Krogsgaard-Larsen, P., Frolund, B., Liljefors, T., and Ebert, B. (2004). GABA(A) agonists and partial agonists: THIP (Gaboxadol) as a non-opioid analgesic and a novel type of hypnotic. Biochemical Pharmacology 68, 1573-1580.

Kukkar, A., Bali, A., Singh, N., and Jaggi, A.S. (2013). Implications and mechanism of action of gabapentin in neuropathic pain. Archives of Pharmacal Research 36, 237-251.

140

Kumar, S., Porcu, P., Werner, D.F., Matthews, D.B., Diaz-Granados, J.L., Helfand, R.S., and Morrow, A.L. (2009). The role of GABA(A) receptors in the acute and chronic effects of ethanol: a decade of progress. Psychopharmacology 205, 529-564.

Kumar, S., Ren, Q., Beckley, J.H., O'Buckley, T.K., Gigante, E.D., Santerre, J.L., Werner, D.F., and Morrow, A.L. (2012). Ethanol Activation of Protein Kinase A Regulates GABA(A) Receptor Subunit Expression in the Cerebral Cortex and Contributes to Ethanol-Induced Hypnosis. Frontiers in Neuroscience 6, 44.

Kumar, S., Sieghart, W., and Morrow, A.L. (2002). Association of protein kinase C with GABA(A) receptors containing alpha1 and alpha4 subunits in the cerebral cortex: selective effects of chronic ethanol consumption. Journal of Neurochemistry 82, 110-117.

Kusunose, N., Koyanagi, S., Hamamura, K., Matsunaga, N., Yoshida, M., Uchida, T., Tsuda, M., Inoue, K., and Ohdo, S. (2010). Molecular basis for the dosing time-dependency of anti- allodynic effects of gabapentin in a mouse model of neuropathic pain. Molecular Pain 6, 83.

Kuzniecky, R., Ho, S., Pan, J., Martin, R., Gilliam, F., Faught, E., and Hetherington, H. (2002). Modulation of cerebral GABA by topiramate, lamotrigine, and gabapentin in healthy adults. Neurology 58, 368-372.

Lagrange, A. (2006). Dancing the delta shuffle: neurosteroids regulate GABAA receptor expression. Epilepsy Currents 6, 14-17.

Lal, R., Sukbuntherng, J., Luo, W., Tovera, J., Lassauzet, M.L., and Cundy, K.C. (2013). Population pharmacokinetics and pharmacodynamics of gabapentin after administration of gabapentin enacarbil. Journal of Clinical Pharmacology 53, 29-40.

Lancel, M., and Faulhaber, J. (1996). The GABAA agonist THIP (gaboxadol) increases non- REM sleep and enhances delta activity in the rat. Neuroreport 7, 2241-2245.

Lanneau, C., Green, A., Hirst, W.D., Wise, A., Brown, J.T., Donnier, E., Charles, K.J., Wood,

M., Davies, C.H., and Pangalos, M.N. (2001). Gabapentin is not a GABAB receptor agonist. Neuropharmacology 41, 965-975.

141

Laurie, D.J., Seeburg, P.H., and Wisden, W. (1992a). The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. Journal of Neuroscience 12, 1063-1076.

Laurie, D.J., Wisden, W., and Seeburg, P.H. (1992b). The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. Journal of Neuroscience 12, 4151-4172.

Lee, C.H., Tsai, T.S., and Liou, H.H. (2008). Gabapentin activates ROMK1 channels by a protein kinase A (PKA)-dependent mechanism. British Journal of Pharmacology 154, 216-225.

Lee, V., and Maguire, J. (2014). The impact of tonic GABAA receptor-mediated inhibition on neuronal excitability varies across brain region and cell type. Frontiers in Neural Circuits 8, 3.

Leidenheimer, N.J. (2008). Regulation of excitation by GABA(A) receptor internalization. Results and Problems in Cell Differentiation 44, 1-28.

Li, C.Y., Zhang, X.L., Matthews, E.A., Li, K.W., Kurwa, A., Boroujerdi, A., Gross, J., Gold, M.S., Dickenson, A.H., Feng, G., et al. (2006). Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation. Pain 125, 20-34.

Li, Y., Li, S., Wei, C., Wang, H., Sui, N., and Kirouac, G.J. (2010). Orexins in the paraventricular nucleus of the thalamus mediate anxiety-like responses in rats. Psychopharmacology 212, 251-265.

Li, Z., Taylor, C.P., Weber, M., Piechan, J., Prior, F., Bian, F., Cui, M., Hoffman, D., and Donevan, S. (2011). Pregabalin is a potent and selective ligand for alpha(2)delta-1 and alpha(2)delta-2 calcium channel subunits. European Journal of Pharmacology 667, 80-90.

Liang, J., Suryanarayanan, A., Chandra, D., Homanics, G.E., Olsen, R.W., and Spigelman, I.

(2008). Functional consequences of GABAA receptor alpha 4 subunit deletion on synaptic and extrasynaptic currents in mouse dentate granule cells. Alcoholism, Clinical and Experimental Research 32, 19-26.

142

Lindner, M.D., Bourin, C., Chen, P., McElroy, J.F., Leet, J.E., Hogan, J.B., Stock, D.A., and Machet, F. (2006). Adverse effects of gabapentin and lack of anti-allodynic efficacy of amitriptyline in the streptozotocin model of painful diabetic neuropathy. Experimental and Clinical Psychopharmacology 14, 42-51.

Lister, R.G. (1987). The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 92, 180-185.

Liu, S., Lai, L., Zuo, Q., Dai, F., Wu, L., Wang, Y., Zhou, Q., Liu, J., Liu, J., Li, L., et al. (2014). PKA turnover by the REGgamma-proteasome modulates FoxO1 cellular activity and VEGF- induced angiogenesis. Journal of Molecular and Cellular Cardiology 72C, 28-38.

Lochner, A., and Moolman, J.A. (2006). The many faces of H89: a review. Cardiovascular Drug Reviews 24, 261-274.

Loscher, W., Honack, D., and Taylor, C.P. (1991). Gabapentin increases aminooxyacetic acid- induced GABA accumulation in several regions of rat brain. Neuroscience Letters 128, 150-154.

Lotarski, S.M., Donevan, S., El-Kattan, A., Osgood, S., Poe, J., Taylor, C.P., and Offord, J. (2011). Anxiolytic-like activity of pregabalin in the Vogel conflict test in alpha2delta-1 (R217A) and alpha2delta-2 (R279A) mouse mutants. Journal of Pharmacology and Experimental Therapeutics 338, 615-621.

Luddens, H., and Korpi, E.R. (1995). GABA antagonists differentiate between recombinant GABAA/benzodiazepine receptor subtypes. Journal of Neuroscience 15, 6957-6962.

Luer, M.S., Hamani, C., Dujovny, M., Gidal, B., Cwik, M., Deyo, K., and Fischer, J.H. (1999). Saturable transport of gabapentin at the blood-brain barrier. Neurological Research 21, 559-562.

Luo, Z.D., Chaplan, S.R., Higuera, E.S., Sorkin, L.S., Stauderman, K.A., Williams, M.E., and Yaksh, T.L. (2001). Upregulation of dorsal root ganglion (alpha)2(delta) calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats. Journal of Neuroscience 21, 1868-1875.

143

Luscher, B., Fuchs, T., and Kilpatrick, C.L. (2011). GABAA receptor trafficking-mediated plasticity of inhibitory synapses. Neuron 70, 385-409.

Macdonald, R.L., and Olsen, R.W. (1994). GABAA receptor channels. Annual Review of Neuroscience 17, 569-602.

Mack, A. (2003). Examination of the evidence for off-label use of gabapentin. Journal of Managed Care Pharmacy 9, 559-568.

Maconochie, D.J., Zempel, J.M., and Steinbach, J.H. (1994). How quickly can GABAA receptors open? Neuron 12, 61-71.

Madsen, K.K., Larsson, O.M., and Schousboe, A. (2008). Regulation of excitation by GABA neurotransmission: focus on metabolism and transport. Results and Problems in Cell Differentiation 44, 201-221.

Maguire, J., Ferando, I., Simonsen, C., and Mody, I. (2009). Excitability changes related to GABAA receptor plasticity during pregnancy. Journal of Neuroscience 29, 9592-9601.

Maguire, J., and Mody, I. (2007). Neurosteroid synthesis-mediated regulation of GABA(A) receptors: relevance to the ovarian cycle and stress. Journal of Neuroscience 27, 2155-2162.

Maguire, J., and Mody, I. (2008). GABA(A)R plasticity during pregnancy: relevance to postpartum depression. Neuron 59, 207-213.

Maguire, J., and Mody, I. (2009). Steroid hormone fluctuations and GABA(A)R plasticity. Psychoneuroendocrinology 34 Suppl 1, S84-90.

Maguire, J.L., Stell, B.M., Rafizadeh, M., and Mody, I. (2005). Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nature Neuroscience 8, 797-804.

Maneuf, Y.P., Luo, Z.D., and Lee, K. (2006). alpha2delta and the mechanism of action of gabapentin in the treatment of pain. Seminars in Cell & Developmental Biology 17, 565-570.

144

Marais, E., Klugbauer, N., and Hofmann, F. (2001). Calcium channel alpha(2)delta subunits- structure and Gabapentin binding. Molecular Pharmacology 59, 1243-1248.

Martin, D.J., McClelland, D., Herd, M.B., Sutton, K.G., Hall, M.D., Lee, K., Pinnock, R.D., and Scott, R.H. (2002). Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression. Neuropharmacology 42, 353-366.

Martin, D.L., and Barke, K.E. (1998). Are GAD65 and GAD67 associated with specific pools of GABA in brain? Perspectives on Developmental Neurobiology 5, 119-129.

Martin, D.L., and Rimvall, K. (1993). Regulation of gamma-aminobutyric acid synthesis in the brain. Journal of Neurochemistry 60, 395-407.

Martin, L.J., Zurek, A.A., MacDonald, J.F., Roder, J.C., Jackson, M.F., and Orser, B.A. (2010). Alpha5GABAA receptor activity sets the threshold for long-term potentiation and constrains hippocampus-dependent memory. Journal of Neuroscience 30, 5269-5282.

McCartney, M.R., Deeb, T.Z., Henderson, T.N., and Hales, T.G. (2007). Tonically active GABAA receptors in hippocampal pyramidal neurons exhibit constitutive GABA-independent gating. Molecular Pharmacology 71, 539-548.

McDonald, B.J., and Moss, S.J. (1997). Conserved phosphorylation of the intracellular domains of GABA(A) receptor beta2 and beta3 subunits by cAMP-dependent protein kinase, cGMP- dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase. Neuropharmacology 36, 1377-1385.

McHugh, S.B., Deacon, R.M., Rawlins, J.N., and Bannerman, D.M. (2004). Amygdala and ventral hippocampus contribute differentially to mechanisms of fear and anxiety. Behavioral Neuroscience 118, 63-78.

Mertens, S., Benke, D., and Mohler, H. (1993). GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by alpha 5- and delta-subunit-specific immunopurification. Journal of Biological Chemistry 268, 5965-5973.

145

Meymandi, M.S., and Sepehri, G. (2008). Gabapentin action and interaction on the antinociceptive effect of morphine on visceral pain in mice. European Journal of Anaesthesiology 25, 129-134.

Miall, R.C. (1998). The cerebellum, predictive control and motor coordination. Novartis Foundation Symposium 218, 272-284; discussion 284-290.

Miall, R.C., Reckess, G.Z., and Imamizu, H. (2001). The cerebellum coordinates eye and hand tracking movements. Nature Neuroscience 4, 638-644.

Michels, G., and Moss, S.J. (2007). GABAA receptors: properties and trafficking. Critical Reviews in Biochemistry and Molecular Biology 42, 3-14.

Mihalek, R.M., Banerjee, P.K., Korpi, E.R., Quinlan, J.J., Firestone, L.L., Mi, Z.P., Lagenaur, C., Tretter, V., Sieghart, W., Anagnostaras, S.G., et al. (1999). Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice. Proceedings of the National Academy of Sciences of the United States of America 96, 12905- 12910.

Milenkovic, I., Vasiljevic, M., Maurer, D., Hoger, H., Klausberger, T., and Sieghart, W. (2013). The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits alpha1, beta2, and delta along their extrasynaptic cell membrane. Neuroscience 254, 80- 96.

Mintz, I.M., and Bean, B.P. (1993). GABAB receptor inhibition of P-type Ca2+ channels in central neurons. Neuron 10, 889-898.

Mitchell, S.J., and Silver, R.A. (2003). Shunting inhibition modulates neuronal gain during synaptic excitation. Neuron 38, 433-445.

Molgaard-Nielsen, D., and Hviid, A. (2011). Newer-generation antiepileptic drugs and the risk of major birth defects. Journal of the American Medical Association 305, 1996-2002.

Morrow, J., Russell, A., Guthrie, E., Parsons, L., Robertson, I., Waddell, R., Irwin, B., McGivern, R.C., Morrison, P.J., and Craig, J. (2006). Malformation risks of antiepileptic drugs

146 in pregnancy: a prospective study from the UK Epilepsy and Pregnancy Register. Journal of Neurology, Neurosurgery, and Psychiatry 77, 193-198.

Moskaug, J.O., Carlsen, H., and Blomhoff, R. (2008). Noninvasive in vivo imaging of protein kinase A activity. Molecular Imaging 7, 35-41.

Mtchedlishvili, Z., and Kapur, J. (2006). High-affinity, slowly desensitizing GABAA receptors mediate tonic inhibition in hippocampal dentate granule cells. Molecular Pharmacology 69, 564- 575.

Muscas, G.C., Chiroli, S., Luceri, F., Mastio, M.D., Balestrieri, F., and Arnetoli, G. (2000). Conversion from thrice daily to twice daily administration of gabapentin (GBP) in partial epilepsy: analysis of clinical efficacy and plasma levels. Seizure 9, 47-50.

Nakahiro, M., Arakawa, O., Narahashi, T., Ukai, S., Kato, Y., Nishinuma, K., and Nishimura, T. (1992). Dimethyl sulfoxide (DMSO) blocks GABA-induced current in rat dorsal root ganglion neurons. Neuroscience Letters 138, 5-8.

Newton, R.A., Bingham, S., Case, P.C., Sanger, G.J., and Lawson, S.N. (2001). Dorsal root ganglion neurons show increased expression of the calcium channel alpha2delta-1 subunit following partial sciatic nerve injury. Brain Research Molecular Brain Research 95, 1-8.

Ng, G.Y., Bertrand, S., Sullivan, R., Ethier, N., Wang, J., Yergey, J., Belley, M., Trimble, L., Bateman, K., Alder, L., et al. (2001). Gamma-aminobutyric acid type B receptors with specific heterodimer composition and postsynaptic actions in hippocampal neurons are targets of anticonvulsant gabapentin action. Molecular Pharmacology 59, 144-152.

Nusser, Z., and Mody, I. (2002). Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. Journal of Neurophysiology 87, 2624-2628.

Nusser, Z., Sieghart, W., Benke, D., Fritschy, J.M., and Somogyi, P. (1996a). Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells. Proceedings of the National Academy of Sciences of the United States of America 93, 11939-11944.

147

Nusser, Z., Sieghart, W., and Somogyi, P. (1998). Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. Journal of Neuroscience 18, 1693-1703.

Nusser, Z., Sieghart, W., Stephenson, F.A., and Somogyi, P. (1996b). The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. Journal of Neuroscience 16, 103-114.

Olsen, R.W., and Sieghart, W. (2008). International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacological Reviews 60, 243-260.

Olsen, R.W., and Sieghart, W. (2009). GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology 56, 141-148.

Patel, S., Naeem, S., Kesingland, A., Froestl, W., Capogna, M., Urban, L., and Fox, A. (2001). The effects of GABA(B) agonists and gabapentin on mechanical hyperalgesia in models of neuropathic and inflammatory pain in the rat. Pain 90, 217-226.

Payne, H.L., Connelly, W.M., Ives, J.H., Lehner, R., Furtmuller, B., Sieghart, W., Tiwari, P., Lucocq, J.M., Lees, G., and Thompson, C.L. (2007). GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer. Journal of Biological Chemistry 282, 29130-29143.

Pellow, S., Chopin, P., File, S.E., and Briley, M. (1985). Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 14, 149-167.

Peng, Z., Hauer, B., Mihalek, R.M., Homanics, G.E., Sieghart, W., Olsen, R.W., and Houser, C.R. (2002). GABA(A) receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain. Journal of Comparative Neurology 446, 179-197.

Peng, Z., Huang, C.S., Stell, B.M., Mody, I., and Houser, C.R. (2004). Altered expression of the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. Journal of Neuroscience 24, 8629-8639.

148

Petersen, H.R., Jensen, I., and Dam, M. (1983). THIP: a single-blind controlled trial in patients with epilepsy. Acta Neurologica Scandinavica 67, 114-117.

Peterson, B.L. (2009). Prevalence of gabapentin in impaired driving cases in Washington State in 2003-2007. Journal of Analytical Toxicology 33, 545-549.

Petroff, O.A., Rothman, D.L., Behar, K.L., Lamoureux, D., and Mattson, R.H. (1996). The effect of gabapentin on brain gamma-aminobutyric acid in patients with epilepsy. Annals of Neurology 39, 95-99.

Pfizer (2012). Neurontin (Gabapentin). In USPI_Med Guide - Neurontin - gabapentin - Cap,Tab,OS-PDF, Pfizer, ed. (http://labeling.pfizer.com/ShowLabeling.aspx?id=630).

Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., and Sperk, G. (2000). GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815-850.

Porcello, D.M., Huntsman, M.M., Mihalek, R.M., Homanics, G.E., and Huguenard, J.R. (2003). Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking delta subunit. Journal of Neurophysiology 89, 1378-1386.

Purves D, A.G., Fitzpatrick D, et al. (2004). Neuroscience, 3rd edn (Sinauer Associates).

Radley, D.C., Finkelstein, S.N., and Stafford, R.S. (2006). Off-label prescribing among office- based physicians. Archives of Internal Medicine 166, 1021-1026.

Radulovic, L.L., Turck, D., von Hodenberg, A., Vollmer, K.O., McNally, W.P., DeHart, P.D., Hanson, B.J., Bockbrader, H.N., and Chang, T. (1995). Disposition of gabapentin (neurontin) in mice, rats, dogs, and monkeys. Drug Metabolism and Disposition 23, 441-448.

Rauw, G.A., Grant, S.L., Labrie, V., Roder, J.C., Antflick, J.E., Hampson, D.R., and Baker, G.B. (2010). Determination of L-serine-O-phosphate in rat and mouse brain tissue using high- performance liquid chromatography and fluorimetric detection. Analytical Biochemistry 405, 260-262.

149

Reddy, D.S. (2003). Pharmacology of endogenous neuroactive steroids. Critical Reviews in Neurobiology 15, 197-234.

Renner, M., Specht, C.G., and Triller, A. (2008). Molecular dynamics of postsynaptic receptors and scaffold proteins. Current Opinion in Neurobiology 18, 532-540.

Rice, A.S., Maton, S., and Postherpetic Neuralgia Study, G. (2001). Gabapentin in postherpetic neuralgia: a randomised, double blind, placebo controlled study. Pain 94, 215-224.

Roberts, E., and Frankel, S. (1950). gamma-Aminobutyric acid in brain: its formation from glutamic acid. Journal of Biological Chemistry 187, 55-63.

Rossi, D.J., Hamann, M., and Attwell, D. (2003). Multiple modes of GABAergic inhibition of rat cerebellar granule cells. Journal of Physiology 548, 97-110.

Rotge, J.Y., Aouizerate, B., Amestoy, V., Lambrecq, V., Langbour, N., Nguyen, T.H., Dovero, S., Cardoit, L., Tignol, J., Bioulac, B., et al. (2012). The associative and limbic thalamus in the pathophysiology of obsessive-compulsive disorder: an experimental study in the monkey. Translational Psychiatry 2, e161.

Rowbotham, M., Harden, N., Stacey, B., Bernstein, P., and Magnus-Miller, L. (1998). Gabapentin for the treatment of postherpetic neuralgia: a randomized controlled trial. Journal of the American Medical Association 280, 1837-1842.

Rudolph, U., Crestani, F., and Mohler, H. (2001). GABA(A) receptor subtypes: dissecting their pharmacological functions. Trends in Pharmacological Sciences 22, 188-194.

Russek, S.J., and Farb, D.H. (1994). Mapping of the beta 2 subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant

GABAA receptor isoform. Genomics 23, 528-533.

Saab, B.J., Maclean, A.J., Kanisek, M., Zurek, A.A., Martin, L.J., Roder, J.C., and Orser, B.A. (2010). Short-term memory impairment after isoflurane in mice is prevented by the alpha5 gamma-aminobutyric acid type A receptor inverse agonist L-655,708. Anesthesiology 113, 1061-1071.

150

Saliba, R.S., Kretschmannova, K., and Moss, S.J. (2012). Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current. EMBO 31, 2937-2951.

Santhakumar, V., Meera, P., Karakossian, M.H., and Otis, T.S. (2013). A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition. PloS One 8, e72976.

Sarkar, J., Wakefield, S., MacKenzie, G., Moss, S.J., and Maguire, J. (2011). Neurosteroidogenesis is required for the physiological response to stress: role of neurosteroid- sensitive GABAA receptors. Journal of Neuroscience 31, 18198-18210.

Sarto-Jackson, I., and Sieghart, W. (2008). Assembly of GABA(A) receptors (Review). Molecular Membrane Biology 25, 302-310.

Saxena, N.C., and Macdonald, R.L. (1994). Assembly of GABAA receptor subunits: role of the delta subunit. Journal of Neuroscience 14, 7077-7086.

Schumacher, T.B., Beck, H., Steinhauser, C., Schramm, J., and Elger, C.E. (1998). Effects of phenytoin, carbamazepine, and gabapentin on calcium channels in hippocampal granule cells from patients with temporal lobe epilepsy. Epilepsia 39, 355-363.

Semyanov, A., Walker, M.C., Kullmann, D.M., and Silver, R.A. (2004). Tonically active

GABAA receptors: modulating gain and maintaining the tone. Trends in Neurosciences 27, 262- 269.

Serra, M., Pisu, M.G., Mostallino, M.C., Sanna, E., and Biggio, G. (2008). Changes in neuroactive steroid content during social isolation stress modulate GABAA receptor plasticity and function. Brain Research Reviews 57, 520-530.

Serra, M., Sanna, E., Mostallino, M.C., and Biggio, G. (2007). Social isolation stress and neuroactive steroids. European Neuropsychopharmacology 17, 1-11.

Shen, H., Gong, Q.H., Aoki, C., Yuan, M., Ruderman, Y., Dattilo, M., Williams, K., and Smith,

S.S. (2007). Reversal of neurosteroid effects at alpha4beta2delta GABAA receptors triggers anxiety at puberty. Nature Neuroscience 10, 469-477.

151

Shen, H., Gong, Q.H., Yuan, M., and Smith, S.S. (2005). Short-term steroid treatment increases delta GABAA receptor subunit expression in rat CA1 hippocampus: pharmacological and behavioral effects. Neuropharmacology 49, 573-586.

Shimizu, S., Honda, M., Tanabe, M., and Ono, H. (2004). GABAB receptors do not mediate the inhibitory actions of gabapentin on the spinal reflex in rats. Journal of Pharmacological Sciences 96, 444-449.

Sieghart, W., and Sperk, G. (2002). Subunit composition, distribution and function of GABA(A) receptor subtypes. Current Topics in Medicinal Chemistry 2, 795-816.

Sills, G.J. (2006). The mechanisms of action of gabapentin and pregabalin. Current Opinion in Pharmacology 6, 108-113.

Silverman, R.B., Andruszkiewicz, R., Nanavati, S.M., Taylor, C.P., and Vartanian, M.G. (1991). 3-Alkyl-4-aminobutyric acids: the first class of anticonvulsant agents that activates L-glutamic acid decarboxylase. Journal of Medicinal Chemistry 34, 2295-2298.

Simon, J., Wakimoto, H., Fujita, N., Lalande, M., and Barnard, E.A. (2004). Analysis of the set of GABA(A) receptor genes in the human genome. Journal of Biological Chemistry 279, 41422- 41435.

Singh, A.K., Tasken, K., Walker, W., Frizzell, R.A., Watkins, S.C., Bridges, R.J., and Bradbury, N.A. (1998). Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells. American Journal of Physiology 275, C562-570.

Smith, S.S. (2013). alpha4betadelta GABAA receptors and tonic inhibitory current during adolescence: effects on mood and synaptic plasticity. Frontiers in Neural Circuits 7, 135.

Smith, S.S., Shen, H., Gong, Q.H., and Zhou, X. (2007). Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits. Pharmacology & Therapeutics 116, 58-76.

Soghomonian, J.J., and Martin, D.L. (1998). Two isoforms of glutamate decarboxylase: why? Trends in Pharmacological Sciences 19, 500-505.

152

Somerville, E.R., and Michell, A.W. (2009). Gabapentin. In The Treatment of Epilepsy (Wiley- Blackwell), pp. 519-526.

Song, I., Volynski, K., Brenner, T., Ushkaryov, Y., Walker, M., and Semyanov, A. (2013). Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources. Frontiers in Cellular Neuroscience 7, 23.

Song, M., and Messing, R.O. (2005). Protein kinase C regulation of GABAA receptors. Cellular and Molecular Life Sciences 62, 119-127.

Spiegel, D.R., and Webb, K. (2012). A case of treatment refractory hyperemesis gravidarum in a patient with comorbid anxiety, treated successfully with adjunctive gabapentin: a review and the potential role of neurogastroentereology in understanding its pathogenesis and treatment. Innovations in Clinical Neuroscience 9, 31-38.

Spigelman, I., Li, Z., Banerjee, P.K., Mihalek, R.M., Homanics, G.E., and Olsen, R.W. (2002).

Behavior and physiology of mice lacking the GABAA-receptor delta subunit. Epilepsia 43 Suppl 5, 3-8.

Stefani, A., Spadoni, F., and Bernardi, G. (1998). Gabapentin inhibits calcium currents in isolated rat brain neurons. Neuropharmacology 37, 83-91.

Steinman, M.A., Bero, L.A., Chren, M.M., and Landefeld, C.S. (2006). Narrative review: the promotion of gabapentin: an analysis of internal industry documents. Annals of Internal Medicine 145, 284-293.

Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M., and Mody, I. (2003). Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit- containing GABAA receptors. Proceedings of the National Academy of Sciences of the United States of America 100, 14439-14444.

Stell, B.M., and Mody, I. (2002). Receptors with different affinities mediate phasic and tonic GABA(A) conductances in hippocampal neurons. Journal of Neuroscience 22, RC223.

153

Stelzer, A., Kay, A.R., and Wong, R.K. (1988). GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science 241, 339-341.

Stewart, B.H., Kugler, A.R., Thompson, P.R., and Bockbrader, H.N. (1993). A saturable transport mechanism in the intestinal absorption of gabapentin is the underlying cause of the lack of proportionality between increasing dose and drug levels in plasma. Pharmaceutical Research 10, 276-281.

Storustovu, S.I., and Ebert, B. (2006). Pharmacological characterization of agonists at delta- containing GABAA receptors: Functional selectivity for extrasynaptic receptors is dependent on the absence of gamma2. Journal of Pharmacology and Experimental Therapeutics 316, 1351- 1359.

Strawn, J.R., and Geracioti, T.D., Jr. (2007). The treatment of generalized anxiety disorder with pregabalin, an atypical anxiolytic. Neuropsychiatric Disease and Treatment 3, 237-243.

Suman-Chauhan, N., Webdale, L., Hill, D.R., and Woodruff, G.N. (1993). Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies. European Journal of Pharmacology 244, 293-301.

Sun, C., Sieghart, W., and Kapur, J. (2004). Distribution of alpha1, alpha4, gamma2, and delta subunits of GABAA receptors in hippocampal granule cells. Brain Research 1029, 207-216.

Sur, C., Quirk, K., Dewar, D., Atack, J., and McKernan, R. (1998). Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics. Molecular Pharmacology 54, 928-933.

Sutton, K.G., Martin, D.J., Pinnock, R.D., Lee, K., and Scott, R.H. (2002). Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones. British Journal of Pharmacology 135, 257-265.

Takasu, K., Ono, H., and Tanabe, M. (2008). Gabapentin produces PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice. Journal of Neurochemistry 105, 933-942.

154

Talani, G., Biggio, G., and Sanna, E. (2011). Enhanced Sensitivity to Ethanol-Induced Inhibition of LTP in CA1 Pyramidal Neurons of Socially Isolated C57BL/6J Mice: Role of Neurosteroids. Frontiers in Endocrinology 2, 56.

Talani, G., Licheri, V., Masala, N., Follesa, P., Mostallino, M.C., Biggio, G., and Sanna, E. (2013). Increased Voluntary Ethanol Consumption and Changes in Hippocampal Synaptic Plasticity in Isolated C57BL/6J Mice. Neurochemical Research.

Tang, X., Hernandez, C.C., and Macdonald, R.L. (2010). Modulation of spontaneous and

GABA-evoked tonic alpha4beta3delta and alpha4beta3gamma2L GABAA receptor currents by protein kinase A. Journal of Neurophysiology 103, 1007-1019.

Taylor, C.P., Angelotti, T., and Fauman, E. (2007). Pharmacology and mechanism of action of pregabalin: the calcium channel alpha2-delta (alpha2-delta) subunit as a target for antiepileptic drug discovery. Epilepsy Research 73, 137-150.

Taylor, C.P., Gee, N.S., Su, T.Z., Kocsis, J.D., Welty, D.F., Brown, J.P., Dooley, D.J., Boden, P., and Singh, L. (1998). A summary of mechanistic hypotheses of gabapentin pharmacology. Epilepsy Research 29, 233-249.

Taylor, C.P., Vartanian, M.G., Andruszkiewicz, R., and Silverman, R.B. (1992). 3-alkyl GABA and 3-alkylglutamic acid analogues: two new classes of anticonvulsant agents. Epilepsy Research 11, 103-110.

Terunuma, M., Jang, I.S., Ha, S.H., Kittler, J.T., Kanematsu, T., Jovanovic, J.N., Nakayama,

K.I., Akaike, N., Ryu, S.H., Moss, S.J., et al. (2004). GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein. Journal of Neuroscience 24, 7074-7084.

Thomas, P., Mortensen, M., Hosie, A.M., and Smart, T.G. (2005). Dynamic mobility of functional GABAA receptors at inhibitory synapses. Nature Neuroscience 8, 889-897.

Tjolsen, A., Berge, O.G., Hunskaar, S., Rosland, J.H., and Hole, K. (1992). The formalin test: an evaluation of the method. Pain 51, 5-17.

155

Triller, A., and Choquet, D. (2005). Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move! Trends in Neurosciences 28, 133-139.

Urban, M.O., Ren, K., Park, K.T., Campbell, B., Anker, N., Stearns, B., Aiyar, J., Belley, M., Cohen, C., and Bristow, L. (2005). Comparison of the antinociceptive profiles of gabapentin and 3-methylgabapentin in rat models of acute and persistent pain: implications for mechanism of action. Journal of Pharmacology and Experimental Therapeutics 313, 1209-1216.

Uusi-Oukari, M., Kontturi, L.S., Coffey, E.T., and Kallinen, S.A. (2010). AMPAR signaling mediating GABA(A)R delta subunit up-regulation in cultured mouse cerebellar granule cells. Neurochemistry International 57, 136-142.

Vaught, J.L., Pelley, K., Costa, L.G., Setler, P., and Enna, S.J. (1985). A comparison of the antinociceptive responses to the GABA-receptor agonists THIP and baclofen. Neuropharmacology 24, 211-216.

Vollmer, K.O., von Hodenberg, A., and Kolle, E.U. (1986). Pharmacokinetics and metabolism of gabapentin in rat, dog and man. Arzneimittel-Forschung 36, 830-839.

Wagner, D.A., Goldschen-Ohm, M.P., Hales, T.G., and Jones, M.V. (2005). Kinetics and spontaneous open probability conferred by the epsilon subunit of the GABAA receptor. Journal of Neuroscience 25, 10462-10468.

Wagner, P.G., and Dekin, M.S. (1993). GABAb receptors are coupled to a barium-insensitive outward rectifying potassium conductance in premotor respiratory neurons. Journal of Neurophysiology 69, 286-289.

Walker, D., and De Waard, M. (1998). Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function. Trends in Neurosciences 21, 148-154.

Walker, M.C., and Kullmann, D.M. (2012). Tonic GABAA Receptor-Mediated Signaling in Epilepsy. In Jasper's Basic Mechanisms of the Epilepsies, J.L. Noebels, M. Avoli, M.A. Rogawski, R.W. Olsen, and A.V. Delgado-Escueta, eds. (Bethesda (MD)).

156

Wang, D.S., Zurek, A.A., Lecker, I., Yu, J., Abramian, A.M., Avramescu, S., Davies, P.A., Moss, S.J., Lu, W.Y., and Orser, B.A. (2012). Memory deficits induced by inflammation are regulated by alpha5-subunit-containing GABAA receptors. Cell Reports 2, 488-496.

Wang, J.M., Singh, C., Liu, L., Irwin, R.W., Chen, S., Chung, E.J., Thompson, R.F., and Brinton, R.D. (2010). Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer's disease. Proceedings of the National Academy of Sciences of the United States of America 107, 6498-6503.

Way, K.J., Chou, E., and King, G.L. (2000). Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends in Pharmacological Sciences 21, 181-187.

Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J., and Salemme, F.R. (1989). Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85-88.

Wegelius, K., Pasternack, M., Hiltunen, J.O., Rivera, C., Kaila, K., Saarma, M., and Reeben, M. (1998). Distribution of GABA receptor rho subunit transcripts in the rat brain. European Journal of Neuroscience 10, 350-357.

Wei, W., Zhang, N., Peng, Z., Houser, C.R., and Mody, I. (2003). Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. Journal of Neuroscience 23, 10650-10661.

Welty, D.F., Schielke, G.P., Vartanian, M.G., and Taylor, C.P. (1993). Gabapentin anticonvulsant action in rats: disequilibrium with peak drug concentrations in plasma and brain microdialysate. Epilepsy Research 16, 175-181.

Werner, D.F., Kumar, S., Criswell, H.E., Suryanarayanan, A., Fetzer, J.A., Comerford, C.E., and Morrow, A.L. (2011). PKCgamma is required for ethanol-induced increases in GABA(A) receptor alpha4 subunit expression in cultured cerebral cortical neurons. Journal of Neurochemistry 116, 554-563.

Werner, M.U., Perkins, F.M., Holte, K., Pedersen, J.L., and Kehlet, H. (2001). Effects of gabapentin in acute inflammatory pain in humans. Regional Anesthesia and Pain Medicine 26, 322-328.

157

Whissell, P.D., Eng, D., Lecker, I., Martin, L.J., Wang, D.S., and Orser, B.A. (2013a). Acutely increasing deltaGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus. Frontiers in Neural Circuits 7, 146.

Whissell, P.D., Rosenzweig, S., Lecker, I., Wang, D.S., Wojtowicz, J.M., and Orser, B.A. (2013b). gamma-aminobutyric acid type A receptors that contain the delta subunit promote memory and neurogenesis in the dentate gyrus. Annals of Neurology 74, 611-621.

Whiting, P.J. (2003). The GABAA receptor gene family: new opportunities for drug development. Current Opinion in Drug Discovery & Development 6, 648-657.

Williams, D.P., Pirmohamed, M., Naisbitt, D.J., Uetrecht, J.P., and Park, B.K. (2000). Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine. Molecular Pharmacology 58, 207-216.

Wiltgen, B.J., Sanders, M.J., Ferguson, C., Homanics, G.E., and Fanselow, M.S. (2005). Trace fear conditioning is enhanced in mice lacking the delta subunit of the GABAA receptor. Learning & Memory 12, 327-333.

Wisden, W., Laurie, D.J., Monyer, H., and Seeburg, P.H. (1992). The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. Journal of Neuroscience 12, 1040-1062.

Wlodarczyk, A.I., Sylantyev, S., Herd, M.B., Kersante, F., Lambert, J.J., Rusakov, D.A.,

Linthorst, A.C., Semyanov, A., Belelli, D., Pavlov, I., et al. (2013). GABA-independent GABAA receptor openings maintain tonic currents. Journal of Neuroscience 33, 3905-3914.

Wohlfarth, K.M., Bianchi, M.T., and Macdonald, R.L. (2002). Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. Journal of Neuroscience 22, 1541-1549.

Wu, X., Gangisetty, O., Carver, C.M., and Reddy, D.S. (2013). Estrous cycle regulation of extrasynaptic delta-containing GABA(A) receptor-mediated tonic inhibition and limbic epileptogenesis. Journal of Pharmacology and Experimental Therapeutics 346, 146-160.

158

Yee, B.K., Hauser, J., Dolgov, V.V., Keist, R., Mohler, H., Rudolph, U., and Feldon, J. (2004). GABA receptors containing the alpha5 subunit mediate the trace effect in aversive and appetitive conditioning and extinction of conditioned fear. European Journal of Neuroscience 20, 1928- 1936.

Zhang, J., Ho, K.Y., and Wang, Y. (2011). Efficacy of pregabalin in acute postoperative pain: a meta-analysis. British Journal of Anaesthesia 106, 454-462.

Zhu, W.Z., Wang, S.Q., Chakir, K., Yang, D., Zhang, T., Brown, J.H., Devic, E., Kobilka, B.K., Cheng, H., and Xiao, R.P. (2003). Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. Journal of Clinical Investigation 111, 617-625.

Zurek, A.A., Bridgwater, E.M., and Orser, B.A. (2012). Inhibition of alpha5 gamma- Aminobutyric acid type A receptors restores recognition memory after general anesthesia. Anesthesia and Analgesia 114, 845-855.

159