The Role of Δ Subunit-Containing Γ-Aminobutyric Acid Type a Receptors

THE ROLE OF δ SUBUNIT-CONTAINING γ-AMINOBUTYRIC ACID TYPE A RECEPTORS

IN MEMORY AND SYNAPTIC PLASTICITY

Paul David Whissell

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Institute of Medical Science

University of Toronto

Paul Whissell

The role of δ subunit-containing γ-aminobutyric acid type A receptors in memory and

synaptic plasticity

Doctor of Philosophy, Institute of Medical Science, University of Toronto, 2014

Abstract

Background: Extrasynaptic γ-aminobutyric acid type A receptors that contain the δ subunit

(δGABAA receptors) are highly expressed in the dentate gyrus (DG) of the hippocampus, where they generate a tonic conductance that regulates activity. GABAA receptor-dependent signaling regulates memory and neurogenesis in the adult DG; however, the role of δGABAA receptors in these processes is unclear. Accordingly, it was postulated that δGABAA receptors regulate memory and neurogenesis in the DG.

Methods: A combination of genetic and pharmacologic techniques was employed. Memory in wild-type (WT) and δ subunit null (Gabrd–/–) mice was assessed using object-place recognition, novel object recognition, contextual discrimination, fear conditioning, fear extinction and water maze tasks. Long-term potentiation, a molecular correlate of memory, was examined using the in vitro hippocampal slice preparation. To ascertain the effects of enhanced δGABAA receptor activity, the receptor-preferring agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; 4 mg/kg) was applied either as a pre-treatment

(2 weeks prior to testing) or an acute treatment (30 min prior to testing).

Results: Gabrd–/– mice exhibited impaired object-place recognition, novel object recognition and contextual discrimination relative to WT mice. Further, Gabrd–/– mice exhibited impaired fear extinction, although fear acquisition was enhanced. Pre-treatment with THIP improved memory in WT but not Gabrd–/– mice. Consistent with these behavioural findings,

ii neurogenesis was impaired in Gabrd–/– mice and enhanced in WT mice by pre-treatment with THIP. In contrast to the beneficial effects of pre-treatment with THIP, acute THIP impaired memory and long-term potentiation in WT mice.

Conclusions: These results indicate that δGABAA receptors promote memory and neurogenesis under baseline conditions. These processes may also be enhanced by long- term activation of δGABAA receptors with selective drugs, provided that these drug are absent during testing. Further, these findings show that acute activation of δGABAA receptors impairs memory and long-term potentiation.

Implications: δGABAA receptors may be a therapeutic target for the long-term treatment of memory dysfunction during aging, injury and disease. These findings also have clinical implications, as δGABAA receptors are molecular targets for therapeutic and recreational drugs. The acute amnestic effects of these compounds may be partially explained by δGABAA receptor activity.

iii

Acknowledgements

First and foremost, I would like to thank my supervisor and mentor, Dr. Beverley Orser, for her inspiration, dedication, integrity and patience throughout this auspicious journey. I am grateful to her for setting up an environment where success was not only possible, but also tremendously enjoyable.

To all post-doctoral researchers and research associates who came through the lab

(including Dian-Shi, Xuanamao, Hongbin, Michael, Sinziana, and Antonello) I would like to extend my thanks for their expertise, advice and uncompromising standards. A special thanks goes to Dian-Shi for doing it all with a smile. I am also very grateful to all Orser lab students

(brothers- and sisters-in-arms). I would like to thank Loren, Rob, Agnes, Irene, Anine, Will and

Dave for making research fun, thought-provoking and productive. I would like to personally thank Irene for her patience in enduring a painful saga of experiments that never seemed to end. To the Physiology student community, I'm grateful for the support, lunch room timbits and all the journal club cookies. To the summer students (Erica, Zeenia, Eric, Michael, Dave,

Bonnie and Jane) I would like to say thanks for reminding me why I got into research. I'd like to thank the technical staff (Ella and Yao-Fang) as well as my committee members (Dr.

Wojtowicz, Dr. Zhang, Dr. Feng, Dr. Yeomans, Dr. Frankland, Dr. Osborne and Dr. Smith) for their support and input. I'm particularly indebted to Dr. Wojtowicz and Shira Rosenzweig for the opportunity to be involved in such an exciting collaboration.

Finally, I'd like to thank my girlfriend Heather, my family (4 brothers, 1 sister, 2 nieces and 1 nephew) and my mother, Dr. Cynthia Whissell, for being awesome.

List of Contributors

The majority of Chapter 4 was derived from the article, "δGABAA receptors promote memory and neurogenesis" in the journal Annals of Neurology (Whissell et al. 2013), which was the work of several investigators. I served as co-first author of this paper along with Dr. Shira

Rosenzweig. Other authors include Irene Lecker (Ph.D. candidate), Dr. Dian-Shi Wang, Dr.

Beverley Orser and Dr. J. Martin Wojtowicz. Irene Lecker also contributed to several behavioural experiments (Figure 4.6, Figure 4.7) by handling animals and performing drug injections. Dr. Rosenzweig collected and analyzed all data relating to neurogenesis (Figure

4.8, Figure 4.9) with the assistance of technician Yao-Fang Tan. Portions of this data have been presented previously (Rosenzweig 2011) but have not been published. I contributed to the presentation and interpretation of this data for the paper. Finally, Dr. Wang, Dr. Orser and

Dr. Wojtowicz contributed to the writing of the paper. Technician Ella Czerwinska (M. Sc.) managed the animal population used in this study.

The majority of Chapter 5 was derived from the article, "Acute activation of δGABAA receptors impairs memory and synaptic plasticity in the hippocampus" that is currently under review for publication in Frontiers in Neural Circuits. I served as first author of this paper.

Other authors include Dave Eng (M.Sc.), Irene Lecker, Dr. Loren Martin, Dr. Wang and Dr.

Orser. Dave Eng performed several behavioural experiments (Figure 5.1C, Figure 5.2B and

Figure 5.2C) which are partly documented in his thesis (Eng 2008) but have not been published. I contributed to the collection, analysis and presentation of this data. Irene Lecker contributed to behavioural experiments by handling animals and performing drug injections

(Figure 5.3) while the remaining authors contributed to writing. Technician Ella Czerwinska again managed the animal population used.

Table of Contents

Abstract ...... ii

Acknowledgements ...... iv

List of Contributors ...... v

Table of Contents ...... vi

List of Figures ...... ix

List of Tables ...... xii

List of Abbreviations...... xiii

Chapter 1. Thesis Structure ...... 1

1.1. Chapter Structure ...... 1

1.2. Overview ...... 1

1.3. Hypothesis ...... 4

1.4. Specific aims...... 4

1.5. Results ...... 4

1.6. Conclusions ...... 5

1.7. Implications ...... 5

Chapter 2. Introduction ...... 7

2.1. Overview ...... 7

2.2. GABA ...... 7

2.3. GABAA receptors ...... 11

2.4. GABA activation of GABAA receptor channels ...... 34

2.5. The hippocampal formation and memory ...... 43

2.6. Anatomy of the hippocampal formation ...... 46

2.7. Postnatal neurogenesis and the DG ...... 60

2.8. Measuring hippocampal function with memory assays ...... 65

2.9. Synaptic plasticity ...... 77

Chapter 3. Methods ...... 87

3.1. Animals ...... 87

3.2. Behavioural methods ...... 87

3.3. Electrophysiological methods ...... 91

3.4. Drugs ...... 93

3.5. Statistics and analysis ...... 95

Chapter 4. δGABAA receptors promote memory performance regulated by the DG and adult- born neurons ...... 98

4.1. Introduction ...... 98

4.2. Specific Methods ...... 100

4.3. Results ...... 103

4.4. Discussion ...... 127

Chapter 5. Acute activation of δGABAA receptors impairs memory and synaptic plasticity in the hippocampus ...... 131

5.1. Introduction ...... 131

5.2. Specific methods ...... 133

vii

5.3. Results ...... 138

5.4. Discussion ...... 162

Chapter 6. General Discussion ...... 167

6.1. Summary ...... 167

6.2. Baseline δGABAA receptor activity promotes memory performance ...... 167

6.3. Acute activation δGABAA receptors during a memory task impairs performance ... 174

6.4. Acute activation of δGABAA receptors impairs long-term potentiation ...... 174

6.5. Limitations...... 178

6.6. Future Directions ...... 180

6.7. Implications ...... 183

References ...... 186

viii

List of Figures

Figure 2.1. Synthesis of GABA...... 9

Figure 2.2. GABA flux depends upon electrochemical chloride gradient...... 15

Figure 2.3. Structure of the GABAA receptor...... 19

Figure 2.4. Synaptic and extrasynaptic GABAA receptors...... 22

Figure 2.5. Phasic inhibition versus tonic inhibition in a whole cell recording...... 23

Figure 2.6. Synaptic pathways in the hippocampus...... 45

Figure 2.7. Schematic of the hippocampus and DG showing laminar structure...... 48

Figure 2.8. Perforant pathway termination in the molecular layer...... 54

Figure 2.9. Inhibitory circuitry in the DG...... 58

Figure 2.10. Common assays of hippocampus-dependent memory...... 69

Figure 2.11. Standard protocol for contextual discrimination...... 73

Figure 2.12. Assays of recognition memory in rodents...... 75

Figure 2.13. Recording long-term potentiation in the hippocampal slice preparation...... 82

Figure 3.1. Acute THIP treatment protocol...... 96

Figure 3.2. Long-term THIP treatment protocol...... 97

Figure 4.1. Recognition memory was impaired in Gabrd–/– mice...... 105

Figure 4.2. Difficult contextual discrimination is impaired in Gabrd–/– mice...... 109

Figure 4.3. Elevated plus maze performance is not regulated by δGABAA receptors...... 111

Figure 4.4. Electroshock sensitivity is not regulated by δGABAA receptors...... 112

Figure 4.5. Acquisition of conditioned fear is enhanced but extinction of fear is impaired in

Gabrd–/– mice...... 114

Figure 4.6. Long-term but not acute treatment with THIP promotes memory...... 118

Figure 4.7. Long-term THIP treatment does not affect anxiety...... 120

Figure 4.8. δGABAA receptors promote neurogenesis in the dentate gyrus...... 124

Figure 4.9. Long-term THIP treatment accelerates maturation of adult-born neurons...... 126

Figure 5.1. Acute pharmacologic activation of δGABAA receptors impairs spatial navigation in the water maze...... 141

Figure 5.2. Acute pharmacologic activation of δGABAA receptors impairs contextual but not cued fear memory...... 145

Figure 5.3. Acute pharmacologic activation of δGABAA receptors impairs novel object recognition...... 147

Figure 5.4. THIP increases tonic inhibitory conductance in cells from WT but not Gabrd–/– mice...... 150

Figure 5.5. THIP inhibits DG-LTP in slices from WT but not Gabrd–/– mice...... 151

THIP depressed LTP in the DG in slices from WT ...... 151

Figure 5.6. THIP inhibits CA1-LTP in slices from WT but not Gabrd–/– mice...... 152

THIP depressed LTP in CA1 in slices from WT ...... 152

Figure 5.7. Co-application of SR-95531 does not occlude THIP-mediated impairment of DG-

LTP...... 155

THIP depressed LTP in the DG in slices from WT ...... 155

Figure 5.8. BIC occludes THIP-mediated impairment of DG-LTP...... 156

THIP does not impair LTP in the DG in BIC-treated slices. BIC (100 μM) was perfused throughout the recordings...... 156

Upper panels: ...... 156

Figure 5.9. THIP has no effects on basal synaptic transmission or presynaptic function in slices from WT or Gabrd–/– mice...... 161

Figure 6.1. Theoretical model explaining the effects of long-term THIP on DG-dependent memory...... 173

Figure 6.2. Theoretical model explaining the effects of acute THIP on memory...... 177

List of Tables

Table 2.1. δGABAA receptor expression is affected by pathophysiological and pharmacological states...... 85

Table 4.1. Interaction time is recognition memory assays is not affected by expression of

δGABAA receptors or α5GABAA receptors...... 106

Table 4.2. Absolute freezing (%) in shock, safe and novel chambers for contextual discrimination during a 9-day protocol ...... 110

Table 4.3. Absolute freezing (%) in the shock, safe and novel chambers for extended contextual discrimination ...... 119

xii

List of Abbreviations

ASCF = artificial cerebrospinal fluid

BIC = Bicuculline

CA = Cornu Ammonis

DG = Dentate gyrus fPSP = field postsynaptic potential

GABA = gamma aminobutyric acid

Gabra5–/– = Transgenic mice lacking the α5 subunit

Gabrd–/– = Transgenic mice lacking the δ subunit

Gabrd+/– = Transgenic mice with one copy of the δ subunit

GAD = glutamic acid decarboxylase

GAT = GABA transporter

Ihold = Holding current

IP3 = inositol triphosphate

KCC2 = Potassium-Chloride ion co-transporter

LPP = Lateral perforant pathway

LTD = long-term depression

xiii

LTP = long-term potentiation mIPSC = miniature inhibitory postsynaptic current

NMDA = N-methyl D-aspartate receptor

NR2A = N-methyl D-aspartate receptor type 2A subunit

NR2B = N-methyl D-aspartate receptor type 2B subunit

THIP = 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol

WT = Wild-type mice

α5GABAA receptor = GABA type A receptor containing the α5 subunit

δGABAA receptor = GABA type A receptor containing the δ subunit

xiv

Chapter 1. Thesis Structure

1.1. Chapter Structure

This thesis consists of six Chapters, the first of which serves as an overview. In Chapter 2, a review of the literature that motivated this thesis is presented. Chapter 3 describes the methods used to investigate the hypothesis. The main findings of this thesis are presented in

Chapters 4 and 5 and are discussed in further detail in Chapter 6.

1.2. Overview

Memory, defined as 'the capacity to retain information', is thought to be supported by persistent changes in the structure and function of neuronal networks throughout the brain.

The hippocampus, a small horn-shaped structure located within the medial temporal lobe, is a critical regulator of memory processes (Eichenbaum 2004). In humans and laboratory animals, injury or pharmacologic inactivation of the hippocampus profoundly impairs most forms of memory, particularly those that require the processing of declarative, spatial or contextual information (Eichenbaum 2004, Lee et al. 2005, Lee et al. 2004, Makkar et al.

2010, Scoville et al. 1957). Identifying the properties of the hippocampus which support memory is critical for understanding the mechanisms underlying the cognitive impairment that occurs during aging, injury and diseases and for developing treatments.

The function of the hippocampus is governed by signaling through a variety of neurotransmitters, including γ-aminobutyric acid (GABA) (Andersen et al. 1980, Wigstrom et al. 1983a, 1985). The activity of GABA at its receptors significantly affects multiple memory processes (Makkar et al. 2010, Myhrer 2003). GABA type A (GABAA) receptors, which are ionotropic chloride-permeable channels, mediate much of the cognitive effects attributed to

GABA. GABAA receptors are composed from a diverse array of subunits (α1-6, β1-3, γ1-3, δ,

π, θ, ε, ρ1-3) that form a wide variety of GABAA receptor subtypes. Multiple subtypes of the

GABAA receptor regulate memory (Collinson et al. 2002, Cushman et al. 2011, Martin et al.

2010, Moore et al. 2010) at least partially through the control of hippocampal activity (Chang et al. 2012, Mello-Carpes et al. 2013, Wang et al. 2012b, Yousefi et al. 2013). The role of individual GABAA receptor subtypes in memory remains to be determined.

This thesis specifically examines the role of δ subunit-containing GABAA (δGABAA) receptors in memory. These receptors are expressed at extrasynaptic regions of neurons and mediate a persistent, low amplitude conductance termed 'tonic inhibition' (Farrant et al. 2005).

For several reasons, δGABAA receptors may regulate memory. First, these receptors are highly expressed in brain regions which contribute to memory, including the dentate gyrus

(DG) subfield of the hippocampus (Pirker et al. 2000). Second, δGABAA receptors mediate a tonic conductance which regulates neuronal and network excitability (Glykys et al. 2008, Herd et al. 2009, Maguire et al. 2009a), which are molecular correlates of memory. Third, δGABAA receptors may facilitate postnatal neurogenesis in the DG, a process which involves the generation of new neurons in the adult brain (Altman et al. 1965) and is important for memory performance (Marin-Burgin et al. 2012b). Neurogenesis in the DG is promoted by a tonic

GABAA receptor-mediated current (Ge et al. 2006) which is likely mediated by δGABAA receptors, as these receptors are the predominant source of tonic current in this region

(Glykys et al. 2008). Currently, the role of δGABAA receptors in memory is unclear.

By promoting neurogenesis, the δGABAA receptor may facilitate forms of memory that are regulated by the DG or adult-born neurons, such as place recognition (Lee et al. 2005), contextual discrimination (Sahay et al. 2011) and extinction (Deng et al. 2009) (Aim 1).

Furthermore, long-term pharmacologic activation of δGABAA receptors prior to a memory task might facilitate subsequent performance of that task (Aim 2).

While long-term activation of δGABAA receptors prior to a memory task may promote performance, several lines of evidence suggests that acute activation of these receptors during a memory task may have the opposite effect and impair performance (Aim 3). First, acute increases in δGABAA receptor activity depress excitability in mature neurons (Herd et al. 2009, Maguire et al. 2009a, Shen et al. 2010). Furthermore, others have observed that pharmacologic increases in GABAA receptor activity during a memory task typically impair performance (Cushman et al. 2011, Martin et al. 2010) while pharmacologic reductions in

GABAA receptor activity improve performance (Atack et al. 2006). Finally, reduced expression of GABAA receptor subtypes is generally associated with impaired performance, at least in fear-associated memory tasks (Cushman et al. 2012, Moore et al. 2010). The effects of acute administration of δGABAA receptor agonists on memory is currently unknown.

Acute pharmacologic activation of δGABAA receptors may also depress synaptic plasticity in the hippocampus, a correlate of memory formation (Aim 4). Long-term potentiation (LTP), a persistent increase in synaptic efficiency, is a prominent form of synaptic plasticity that is observed in the hippocampus following learning (Whitlock et al. 2006) and is correlated with memory performance (Saab et al. 2009, Sahay et al. 2011, Saxe et al. 2006).

LTP is tightly constrained by GABAA receptors at baseline (Wigstrom et al. 1983a) and is attenuated by increases in ambient GABA (Arima-Yoshida et al. 2011) and other enhancers of GABAA receptor activity (Shen et al. 2010). Further, transgenic deletion or pharmacologic blockade of GABAA receptor subtypes generally enhances LTP and memory (Collinson et al.

2006, Martin et al. 2010, Wigstrom et al. 1985). These findings suggest that δGABAA

3 receptors, which mediate an inhibitory conductance in mature neurons (Glykys et al. 2008), constrain LTP at baseline and depress LTP when supra-activated by pharmacologic agonists.

1.3. Hypothesis

Based on the above evidence, it was hypothesized that δGABAA receptors upregulate and downregulate distinct memory processes.

1.4. Specific aims

1. To determine whether baseline activity of δGABAA receptors promotes forms of

memory performance regulated by the DG and adult-born neurons.

2. To determine whether long-term activation of δGABAA receptors prior to a memory task

promotes performance.

3. To determine whether acute activation of δGABAA receptors during a memory task

impairs performance.

4. To determine whether acute activation of δGABAA receptors depresses synaptic

plasticity in the hippocampus (DG and CA1 subfields).

1.5. Results

In Chapter 4, the results show that δGABAA receptors promote forms of memory that are regulated by the DG and adult-born neurons. Specifically, I demonstrate that mutant mice null for the δ subunit (Gabrd–/–) are impaired at recognition memory, contextual discrimination and extinction tasks relative to wild-type (WT) mice. Further, I illustrate that long-term pharmacologic activation δGABAA receptors prior to a memory task improves performance. A one week pre-treatment period with δGABAA receptor-preferring agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances memory performance that was

4 measured two weeks later, even though the drug is likely to be eliminated at this time. These results identify a novel and important role of the δGABAA receptor in promoting memory, and illustrate how a controlled increase in GABAergic transmission can improve memory performance.

In Chapter 5, I demonstrate that acute activation of δGABAA receptors impairs memory and attenuates synaptic plasticity in the DG and CA1 subfields of the hippocampus.

Specifically, I show that the δGABAA receptor-preferring agonist THIP impairs memory that is measured 30 minutes after it is injected. I further find that THIP attenuates LTP in the DG and

CA1 subfields. These findings are the first to demonstrate that acute administration of a

δGABAA receptor agonist impairs memory and plasticity in male mice, and are also the first to compare synaptic plasticity in male WT and Gabrd–/– mice.

1.6. Conclusions

This thesis illustrates that long-term δGABAA receptor activation prior to a memory task promotes performance, but also shows that acute supra-physiological increases in δGABAA receptor activation impair memory and synaptic plasticity.

1.7. Implications

The current findings predict that reduced δGABAA receptor expression and activity, which occurs is observed in animal models of post-partum depression (Maguire et al. 2008), epilepsy (Peng et al. 2004) and Fragile X syndrome (D'Hulst et al. 2006), will be associated with specific forms of memory impairment. Further, these results demonstrate that δGABAA receptor-preferring agonists can enhance memory function, a finding which suggests these compounds may be useful in the treatment of memory dysfunction that occurs during aging, injury and disease. 5

Additionally, these findings have important clinical implications, as δGABAA receptors are molecular targets for a number of drugs which affect memory function, including anesthetics (Lees et al. 1998), sedative-hypnotics (Wafford et al. 2006), neuroactive steroids

(Belelli et al. 2005) and ethanol (Olsen et al. 2007). The results presented in this thesis suggest that activation of the δGABAA receptor contributes to the potent amnestic effects of these compounds. Further implications and caveats are discussed in Chapter 6.

Chapter 2. Introduction

2.1. Overview

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. It is estimated that over 20% of synapses use GABA as their primary neurotransmitter (Somogyi et al. 1998), and GABA receptors are expressed in virtually every type of neuron and brain region (Brickley et al. 2012). GABA signaling regulates neuronal activity and network plasticity (Staubli et al. 1999, Wigstrom et al. 1983a), learning and memory processes (Collinson et al. 2002, Martin et al. 2010), and a host of behavioural functions including pain, anxiety, sleep and awareness (Enna et al. 2006, Rudolph et al. 2011,

Wang et al. 2011). Many subtypes of GABA receptors exist and there is considerable diversity in the structural and signaling properties of these receptor subtypes. The focus of this dissertation will be the role of GABA type A receptors that contain the δ subunit (δGABAA receptors) in memory and synaptic plasticity.

2.2. GABA

2.2.1. GABA synthesis

GABA is synthesized from the amino acid L-glutamate in a reaction that is catalyzed by the enzyme glutamic acid decarboxylase (GAD) (Roberts et al. 1951) (Figure 2.1). Two isoforms of GAD exist, GAD65 and GAD67 (Erlander et al. 1991). These isoforms differ in intracellular distribution, weight and amino acid sequencing, which suggests that they mediate distinct functions (Esclapez et al. 1994, Kaufman et al. 1991). GAD65 is closely associated with synaptic vesicles and may assist in the generation and packaging of GABA for vesicular release (Jin et al. 2003). In this manner, GAD65 may facilitate rapid, local GABAergic

7 signaling. In contrast to GAD65, GAD67 is found throughout the cell and is not specifically localized to vesicular regions (Kaufman et al. 1991). GAD67 expression predominates during development of and following injury to the nervous system, which suggests it may generate

GABA for trophic use (Pinal et al. 1998). Alternatively, another function of GAD67 may be the production of GABA for paracrine signaling (Kaufman et al. 1991).

Figure 2.1. Synthesis of GABA.

GABA is produced from L-glutamate by the enzyme glutamic acid decarboxylase. GABA is metabolized to succinic semialdehyde by the enzyme GABA transaminase. Succinic semialdehyde is metabolized to succinate, which can then enter the Kreb's cycle and be used in the generation of energy. 9

2.2.2. GABA release

GABA loading into synaptic vesicles is facilitated by the vesicular GABA transporter (VGAT)

(Buddhala et al. 2009). Evidence suggests that GABA is released into the extracellular space following calcium-mediated exocytosis of a GABA-loaded vesicle from the synaptic terminal

(Moss et al. 2001). The concentration of GABA in the synaptic cleft is rapidly increased following vesicular release to ~1.5 - 3 mM (Mozrzymas et al. 2003). Non-vesicular forms of

GABA release are also observed, some of which are mediated by the reverse function of other non-vesicular GABA transporters (GATs, described below) (Borden 1996, Richerson et al. 2003).

Importantly, the ability to release GABA is not restricted to neurons. Notably, astrocytes (a subtype of glial cell) also release GABA (Le Meur et al. 2012). GABA released from astrocytes can activate microglia (another subtype of glial cell) thereby modulating immune responses (Lee et al. 2011).

2.2.3. GABA transport and metabolism

Clearance of GABA from the synapse is important for the termination of GABA signaling.

Following GABA release, the concentration of GABA in the synaptic cleft is rapidly decreased by two principle processes: 1) passive diffusion of the transmitter from the synapse and 2) reuptake of the transmitter by either the presynaptic terminal of the neuron or by nearby cells

(such as astrocytes) (Conti et al. 2004).

GABA re-uptake is thought to be the predominant mechanism (Glykys et al. 2007a) and is mediated by GATs, which are high-affinity, Na+/Cl--dependent transporters (Conti et al.

2004). Four separate GATs have been identified in the mouse brain (GAT1-4) and these have homologs in rats and humans (Madsen et al. 2008). GAT1 and GAT4 are brain-specific, but 10 are localized to different brain regions and cell types (Jursky et al. 1994). GAT1 is found in neurons and glia while GAT4 is largely specific to neurons (Jursky et al. 1994). GAT1 has a wider distribution pattern that strongly overlaps with that of GABA. Thus, it has been proposed that this transporter mediates the majority of GABA transport from the synaptic cleft (Jursky et al. 1994). Drugs that modulate GAT function may prove therapeutic in disorders that are characterized by irregularities in GABA signaling, such as epilepsy (Madsen et al. 2010).

While the uptake of GABA by neurons has been estimated to be three- to six-fold greater than the uptake of GABA by astrocytes (Madsen et al. 2008), astrocytic regulation of the concentration of GABA in the synapse may be critical in preventing pathologies of disinhibition such as epilepsy (Pirttimaki et al. 2012) and ischemia (Conti et al. 2004).

Importantly, the clearance of GABA from the extracellular space by GATs is incomplete and a low concentration of GABA remains (~0.01-0.4 µM) (Attwell et al. 1993).

Extracellular GABA is ultimately metabolized to succinic semialdehyde by GABA transaminase (GABA-T) (Madsen et al. 2008) (Figure 2.1). Drugs which inhibit GABA-T function increase the concentration of extracellular GABA and, similar to GAT inhibitors, may be of therapeutic benefit (Sarup et al. 2003).

2.2.4. The GABA receptor family

There are two principle classes of GABA receptors: type A (GABAA receptors) and type B

(GABAB) receptors (Chebib et al. 1999). GABAA receptors are the predominant mediators of inhibition in the central nervous system and are the principle focus of this dissertation; GABAB receptors will not be discussed further.

2.3. GABAA receptors

The GABAA receptor is a member of the ligand-gated ion channel superfamily which includes the nicotinic acetylcholine receptor, glycine receptor and 5-hydroxytryptamine type 3 receptor

(Connolly et al. 2004). Integral to the structure of the GABA receptor is the receptor channel, which is highly permeable to chloride ions (Cl-) but is also permeable to bicarbonate ions

- - (HCO3 ). As the permeability for Cl is roughly five-fold higher (Bormann et al. 1987), GABAA receptors are often described as 'chloride-selective.'

2.3.1. Regulation of chloride flux through GABAA receptors

The direction of Cl- flux through the GABA channel is determined by the electrochemical gradient for Cl-, which is affected by several factors including the intracellular Cl- concentration. In neurons, intracellular Cl- concentration is regulated by a number of different proteins, most notably cation chloride co-transporters (CCCs) (Payne et al. 2003). Within the central nervous system, members of the CCC family likely to be involved in the regulation of intracellular Cl- concentration include the Na+-Cl- ion co-transporter (NCC), Na+-K+-2Cl- ion co-transporter 1 (NKCC1) and several K+-Cl- ion co-transporters (KCC1-4) (Payne et al.

2003). NKCC1 and KCC2 are thought to play a predominant role in regulating neuronal Cl- concentration and therefore Cl- flux through the GABA receptor channel.

In most mature neurons, the intracellular concentration of Cl- is significantly lower than the extracellular concentration due to high expression of KCC2, which extrudes Cl- and K+ from the neuron (Ben-Ari 2002, Rivera et al. 1999) (Figure 2.2). In these neurons, the reversal potential for Cl- is also typically negative to the resting membrane potential (2.3.2) (Raimondo et al. 2012). This electrochemical gradient encourages a strong inward driving force for the Cl- anion. When the GABA receptor is activated, there is inward Cl- flux through the channel and the cell is hyperpolarized below the action potential threshold, making an action potential less

12 likely. In addition to hyperpolarizing inhibition, GABA receptors may regulate a shunting form of inhibition in mature neurons which is also described in 2.3.2. As a result of these two prominent forms of inhibition, GABA receptors are considered predominantly inhibitory in mature neurons.

In contrast to their generally inhibitory effects on mature neurons, GABA receptors are generally excitatory in immature neurons (Ben-Ari 2002). The reason for this discrepancy in

GABA action is the relatively greater intracellular Cl- concentration in immature neurons due to low expression of KCC2 (Payne et al. 2003) and high expression of NKCC1, which gates

Na+, K+ and Cl- into the cell (Ben-Ari 2002, Yamada et al. 2004) (Figure 2.2). In immature cells, the reversal potential for Cl- is positive to the membrane potential, rather than negative.

Accordingly, when the GABA channel is opens, Cl- flows outward and the neuron is depolarized (Ben-Ari 2002). GABA-mediated depolarization may be important for the development of neuronal networks in the perinatal brain (Ben-Ari 2002) and neurogenesis within the postnatal brain (Ge et al. 2006) (2.7). The existence of GABA-mediated depolarization in immature cells is not without controversy (Bregestovski et al. 2012), but is generally accepted to be an important developmental phenomenon (Ben-Ari et al. 2012).

Though a full review of all the mechanisms regulating Cl- concentration is beyond the scope of this thesis, it is important to note that many other mechanisms exist for the regulation of intracellular Cl- concentration besides ion co-transport by members of the CCC family. Notably, anion exchangers such as anion exchanger 3 (AE3) may regulate neuronal

Cl- concentration (Casey et al. 2009) as can chloride channels such as voltage-dependent chloride channel 2 (ClC-2) (Staley et al. 1996). Extensive GABAA receptor activation can also significantly alter the intracellular Cl- concentration, changing the reversal potential for Cl-

13 therefore the direction of Cl- flux (Raimondo et al. 2012). This phenomenon, termed 'ionic plasticity', is observed at many GABAergic synapses (Raimondo et al. 2012).

Figure 2.2. GABA flux depends upon electrochemical chloride gradient.

A. In mature cells, a low intracellular concentration of chloride is present due to high expression levels of the ion co-transporter KCC2. In these cells, the reversal potential of chloride is typically negative relative to the membrane potential. When the GABAA receptor is activated in these cells, chloride flux is inward and the cell is hyperpolarized. B. In immature cells, there is high intracellular chloride due to high expression of ion co-transporter NKCC1 and comparatively lower expression of KCC2. In these cells, the reversal potential for chloride is typically positive to the membrane potential. When the GABAA receptor is activated in immature cells, chloride flux is outward.

2.3.2. Mechanisms of GABAergic inhibition

GABAergic inhibition can occur through two principle mechanisms: either hyperpolarization or shunting (Andersen et al. 1980, Staley et al. 1992). In the case of hyperpolarizing inhibition,

- the reversal potential for Cl (VCl-) is negative to the membrane potential (VM) and threshold for action potential generation (VAP) (e.g.: VCl- = -70 mV, VM = -85 mV, VAP = -55 mV). In this scenario, the driving force for Cl- is inward and opening of the GABA channel significantly hyperpolarizes the neuron, moving it much further away from the threshold for action potential generation.

In the case of shunting inhibition, the reversal potential for Cl- need not be substantially negative - and in fact can be slightly positive - to the membrane potential, provided it is still sufficiently below the threshold for action potential generation (e.g.: VCl- = = -65 mV, VM = -70 mV, VAP = -55 mV). While there is little net change in membrane potential when the channel opens in this situation, there is still current flow across the membrane. This increase in membrane conductance decreases membrane resistance (RM). As RM is reduced, Ohm`s law predicts that the depolarization (VE) produced by local excitatory currents (IE) is attenuated

(as VE = IE RM). Shunting inhibition has therefore been explained colloquially as a 'short- circuit` of nearby excitatory currents. Shunting inhibition may play a key role in regulating the excitability of the hippocampus (Riekki et al. 2008) and is argued to be the primary mechanism by which interneurons regulate signaling in the dentate gyrus (Vida et al. 2006).

2.3.3. GABAA receptor composition

The native GABAA receptor is typically a heteropentamer assembled from 5 subunits arranged to form a central pore for permeant ions (Macdonald et al. 1994) (Figure 2.3A).

Each subunit consists of a large extracellular N-terminus domain, four transmembrane

16 domains (TM1-4) and an intracellular loop between TM3 and TM4 (Figure 2.3B). The extracellular N-terminus domain includes the binding site for GABA (Olsen et al. 2009). The

TM3-TM4 intracellular loop contains interaction sites for putative proteins that are involved in receptor trafficking and many potential interaction sites for serine/threonine and tyrosine kinases (Davis et al. 2001, Luscher et al. 2004, Swope et al. 1999, Wang et al. 1999). The

TM2 region of each subunit is thought to line the pore and is important in determining ion permeability in GABAA receptors and other members of the Cys Loop family (Wotring et al.

2008).

The 5 subunits that comprise the GABAA receptor are drawn from a family of at least

19 members: α(1-6), β(1-3), γ(1-3), δ, θ, π, ε and ρ(1-3) (Whiting et al. 1999). If all these subunits were allowed to freely combine to generate functional receptors, an enormous array of GABAA receptor subtypes would exist. However, as only a relatively limited number of subtypes is observed in the brain (McKernan et al. 1996), it is likely that GABAA receptor assembly is governed by a number of constraints. Most GABAA receptor subunits typically combine in a 2α:2β:γ or 2α:2β:δ stoichiometry (Sieghart et al. 1999) (Figure 2.3C) and this preferred stoichiometry is further constrained by preferences in subunit combination (Olsen et al. 2009). For instance, the δ subunit most commonly pairs with the α4 and/or α6 subunit, but not with the α1, α2 or α3 subunits (Olsen et al. 2009).

Certain GABAA receptor subtypes that are particularly prevalent include the α1β2γ2 receptor (~43% of all GABAA receptors), α2β2γ2/α2β3γ2 receptors (~18%) and α3βγ2/α3βγ3 subtypes (~17%) (McKernan et al. 1996). α1β2γ2 receptors are widely expressed, particularly within interneurons of the brain (Olsen et al. 2009). Other receptor subtypes are less common, including α5β3γ2 receptors and α4βδ receptors (<5% each) (Olsen et al. 2009), and are localized to specific cell types in specific areas (Pirker et al. 2000). 17

Different GABAA receptor subtypes demonstrate considerable heterogeneity in intracellular localization, pharmacologic properties and distribution patterns (Olsen et al.

2009). Based on their composition, localization and function, GABAA receptors are here classified into two broad categories: synaptic GABAA receptors (2.3.4) and extrasynaptic

GABAA receptors (2.3.5).

Figure 2.3. Structure of the GABAA receptor.

A. The GABAA receptor consists of 5 subunits arranged around a central pore. B. Each subunit is composed of 4 transmembrane (TM) domains. C. Stoichiometry of the GABAA receptor showing subunit families.

2.3.4. Synaptic GABAA receptors and phasic inhibition

The subunit composition of the GABAA receptor determines its intracellular localization.

GABAA receptors containing the γ2 subunit are mainly expressed at the postsynaptic regions of neurons. Synaptic targeting of most γ2 subunit-containing GABAA receptors is facilitated by the membrane protein gephyrin (Moss et al. 2001). Inhibiting gephyrin expression reduces clustering of synaptic GABAA receptors while global deletion of the γ2 subunit reduces gephyrin clustering at synapses (Essrich et al. 1998).

Knockdown of gephyrin does not affect the localization of several GABAA receptor subunits, such as the α5 subunit (Kneussel et al. 2001, Marchionni et al. 2009). This result implies that alternative, gephyrin-independent mechanisms exist for receptor trafficking of α5 subunit-containing GABAA receptors (α5GABAA receptors). Trafficking of the α5GABAA receptor may instead be regulated by the protein radixin (Loebrich et al. 2006). While

α5GABAA receptors typically contain the γ2 subunit, these receptors are not specifically targeted to synapses and are predominantly found in extrasynaptic regions (Brunig et al.

2002) (Figure 2.4).

Synaptic GABAA receptors in the postsynaptic membrane are typically activated by action potential-dependent release of vesicular GABA from the presynaptic neuron (Figure

2.4). These receptors typically generate a high amplitude postsynaptic current (IPSC) with fast rise and decay kinetics (Figure 2.5). The characteristics of the IPSC are shaped by the properties and number of receptors and the magnitude and duration of the GABA transient at the synapse. As a result of their distinctive temporal properties, IPSCs have been termed

'phasic inhibition'. Alternatively, as IPSCs are typically mediated by synaptic receptors, these

20 currents are also called 'synaptic inhibition.' Phasic inhibition is thought to be particularly important in the generation of network oscillations (Farrant et al. 2005).

Figure 2.4. Synaptic and extrasynaptic GABAA receptors.

Just below the presynaptic terminal are synaptic GABAA receptors (left, orange). These receptors are exposed to large concentrations of GABA following action potential-dependent release of vesicular GABA (filled blue circles). Lateral to the synaptic cleft are extrasynaptic

GABAA receptors (right, purple). These receptors are exposed to low, ambient concentrations of GABA. These receptors typically contain α5 or δ subunits. Figure is adapted from Bonin et al. (2008).

Figure 2.5. Phasic inhibition versus tonic inhibition in a whole cell recording.

Holding current (IHold) is represented by the dashed grey line. Left. Phasic inhibition is primarily mediated by synaptic GABAA receptors, which generate high-amplitude, fast inhibitory postsynaptic currents (IPSCs). Middle. Tonic inhibition is mediated by extrasynaptic

GABAA receptors, and manifests as a decrease in holding current following the application of a GABAA receptor antagonist such as bicuculline or picrotoxin. Right. Agonists of extrasynaptic GABAA receptors, such as THIP, produce an increase in tonic inhibition which manifests as an increase in holding current. Abbreviations: BIC = bicuculline, PTX = picrotoxin.

2.3.5. Extrasynaptic GABAA receptors and tonic inhibition

As stated earlier, clustering of GABAA receptors occurs at extrasynaptic locations (Mody et al.

2004). Two major subtypes of GABAA receptors are found predominantly in the extrasynaptic regions: δ subunit-containing GABAA receptors (2.3.5.1), which are the focus of this dissertation, and the aforementioned α5GABAA receptors (2.3.5.2) (Farrant et al. 2005). At least in the hippocampus, α5GABAA receptors and δGABAA receptors are the primary extrasynaptic receptor subtypes (Glykys et al. 2008). Other extrasynaptic GABAA receptors may exist, including those comprised of αβ subunits (Mortensen et al. 2006), but these have not been well characterized.

Since extrasynaptic receptors are located outside of the synapse, they are not exposed to the high concentrations of GABA that occur following vesicular release (Mozrzymas et al.

2003). However, these receptors have an exquisite sensitivity to GABA and can detect low concentrations of the transmitter present in the extracellular space (ambient GABA; 0.01-0.4

µM) (Houston et al. 2012). The effective concentration of GABA required for a half-maximal response for α5GABAA receptors and δGABAA receptors is within the nM range (Bohme et al.

2004, Brown et al. 2002) with δGABAA receptors showing a higher affinity (Bohme et al. 2004,

Brown et al. 2002). A major source for ambient GABA is thought to be spill-over of the transmitter from neighbouring synapses following action potential-dependent GABA release from neurons (Glykys et al. 2007b). However, other possible sources have been identified

(Attwell et al. 1993, Farrant et al. 2005, Koch et al. 2009). Alterations in GAT function, for example, can result in significant changes in ambient GABA and tonic current (Richerson et al. 2003).

Extrasynaptic GABAA receptors generate a low-amplitude, persistently-active or 'tonic' current that contrasts with the fast phasic current generated by synaptic receptors. In most mature neurons, tonic current is inhibitory and constrains excitability and plasticity (Martin et al. 2010, Mitchell et al. 2003, Shen et al. 2010). Tonic current can be revealed in whole-cell, voltage-clamp recordings through application of GABAA receptor competitive antagonists such as bicuculline. In cells with tonic inhibitory currents, pharmacologic blockade of extrasynaptic GABAA receptors decreases holding current (Figure 2.5). Conversely, activation of extrasynaptic GABAA receptors with a selective agonist increases holding current.

Extrasynaptic receptors are generally thought to be relatively non-desensitizing.

Accordingly, the properties of tonic current are not thought to change despite prolonged exposure to ambient GABA. However, over a short timeframe, profound desensitization of extrasynaptic receptors and corresponding changes in tonic current have been observed

(Bright et al. 2011). Rapid regulation of extrasynaptic receptor activity and tonic current can occur depending upon the intracellular Cl- concentration within the neuron, the post- depolarization potentiation of the receptor and the degree of nonvesicular GABA release

(Ransom et al. 2012). Further, the intracellular Cl- concentration can affect the expression of extrasynaptic GABAA receptors (Succol et al. 2012) which in turn affects tonic current.

2.3.5.1. δGABAA receptors: Expression and function

δGABAA receptors can be found within the DG, hippocampus, thalamus, cerebellum and many other brain regions (Pirker et al. 2000). Outside of the cerebellum and thalamus, expression of δGABAA receptors is perhaps most significant in the DG (Pirker et al. 2000). In the DG, δGABAA receptors are strongly expressed in the granular cell layer and molecular layer, where the cell bodies and dendritic processes of granule cells reside, respectively

(Maguire et al. 2009a, Pirker et al. 2000). Importantly, δGABAA receptors are not just expressed in granule cells, but in DG interneurons (Glykys et al. 2008, Pirker et al. 2000)

δGABAA receptors are localized to perisynaptic or extrasynaptic regions in the DG (Wei et al.

2003) and are the predominant source of tonic conductance (Glykys et al. 2008). Accordingly,

δGABAA receptors are thought to shape neuronal activity within the DG and regulate behavioural functions of the DG. The possibility that δGABAA receptors constrain memory

(i.e.: like α5GABAA receptors) has long been speculated but has not been shown in male mice (Mihalek et al. 1999, Wiltgen et al. 2005) and is only evident in female mice under special circumstances, such as puberty (Shen et al. 2010).

Importantly, δGABAA receptors may not be strictly inhibitory. δGABAA receptors may generate depolarizing or 'excitatory' currents in immature neurons as the reversal potential for

Cl- in these cells is significantly positive to the resting membrane potential (2.3, Figure 2.2).

Interestingly, young adult-born neurons generate depolarizing tonic GABAergic currents which are important for proper synapse formation and dendritic development (Ge et al. 2006).

These currents are likely generated by δGABAA receptors, as these receptors are the predominant source of tonic current in the DG (Glykys et al. 2008). An investigation into the role of δGABAA receptors in the development of adult-born neurons, and the forms of memory which are regulated by adult-born neurons, is presented in Chapter 4.

δGABAA receptors are also expressed in hippocampal subfields other than the DG. In the CA1 subfield, δGABAA receptors are typically expressed at low levels and mediate a low amplitude conductance under baseline conditions (Glykys et al. 2008, Pirker et al. 2000) which suggests they do not significantly regulate neuronal activity in this region at baseline.

However, during certain physiological conditions such as puberty, δGABAA receptor expression in the CA1 region is greatly increased and impairments in hippocampus- 26 dependent memory and plasticity are observed (Shen et al. 2010), as well as increases in anxiety (Shen et al. 2007).

In the CA3 region of the hippocampus, there is also expression of δGABAA receptors

(Pirker et al. 2000). These receptors are likely found in presynaptic mossy fiber terminals

(Ruiz et al. 2010) and interneurons (Mann et al. 2010) rather than simply being located in the dendritic processes of CA3 pyramidal neurons. Interestingly, presynaptic δGABAA receptors are thought to be depolarizing and facilitate CA3 neurotransmission (Ruiz et al. 2010).

δGABAA receptors are thought to be expressed outside of the brain and involved in functions besides memory. Spinal δGABAA receptors are thought to regulate nociception, as pharmacologic activation of δGABAA receptors reduces nociception in acute and persistent pain models (Bonin et al. 2011). δGABAA receptors are also thought to regulate mood and affect, as maternal behaviour and the physiologic response to stress are distrupted in transgenic mice lacking the δ subunit (Maguire et al. 2008, Sarkar et al. 2011).

2.3.5.1.1. Plasticity of δGABAA receptor expression

The δ subunit demonstrates remarkable plasticity in expression (Table 2.1). Changes in δ subunit expression are often accompanied by significant changes in tonic current (Maguire et al. 2007, 2008, Maguire et al. 2005, Serra et al. 2006, Sundstrom-Poromaa et al. 2002), sensitivity to pharmacologic compounds with high activity at the δGABAA receptor (Follesa et al. 2005, Liang et al. 2004, Liang et al. 2007, Mtchedlishvili et al. 2010, Peng et al. 2004, Qi et al. 2006, Shen et al. 2005, Zhan et al. 2009), neuronal and network excitability (Maguire et al.

2009a, Maguire et al. 2007, Maguire et al. 2005, Peng et al. 2004, Qi et al. 2006, Sanna et al.

2011, Shen et al. 2010) and behaviour (Maguire et al. 2008, Maguire et al. 2005, Nin et al.

2012, Shen et al. 2005, Shen et al. 2010, Sundstrom-Poromaa et al. 2002). While most

27 studies specifically examine δ subunit expression in the hippocampus, plasticity of the subunit has also been recognized in the thalamus (Maguire et al. 2009a, Pisu et al. 2008), cerebellum

(Lin et al. 1996), cortex (D'Hulst et al. 2006, Gantois et al. 2006, Peng et al. 2004), periaqueductal grey (Griffiths et al. 2005, Lovick et al. 2009) and elsewhere (Maguire et al.

2009a). However, as the focus of this thesis is the role of δGABAA receptors in regulating memory, this section will primarily discuss receptor expression in the hippocampus and DG.

2.3.5.1.2. Developmental regulation of δGABAA receptor expression

Cumulative evidence suggests substantial developmental regulation of δGABAA receptor expression and function. Importantly, the expression of δ subunit mRNA in the brain is usually undetectable during the embryonic period and is typically first observed between postnatal day 6-12 (Laurie et al. 1992). In contrast, other GABAA receptor subunit mRNAs (e.g. α1, α2,

α3, α4 and α5 subunits) are detectable even in the embryonic period (Laurie et al. 1992). The expression of δGABAA receptors is assumed to increase from the early postnatal period at least until the early adult period, as evidenced by age-dependent increases in signal strength for δ subunit mRNA (Laurie et al. 1992), δ subunit protein (Shen et al. 2011) and binding of

[H3]THIP (Friemel et al. 2007) in the brain during this time. The presence of developmentally- regulated receptor expression is further corroborated by the observation that δGABAA receptor-mediated tonic current does not appear until the juvenile period in rats, at least in cerebellar granule cells (Brickley et al. 1996). During puberty, particularly dramatic increases in δ subunit expression and δGABAA receptor-mediated tonic current are observed in female mice (Shen et al. 2010). However the persistence of these changes in receptor expression and/or function during puberty have not been well-characterized.

The plasticity of δGABAA receptor expression in late adulthood and advanced aging is more controversial. Interestingly, some rat strains show further progressive increases in δ 28 subunit expression within the thalamus from 2 to 6 months of age (Pisu et al. 2008), a time point which roughly corresponds to middle age in humans. Given the general decline in neurosteroid levels with aging (Ritsner et al. 2008), a compensatory increase in δGABAA receptor expression in some areas seems intuitive and is consistent with these results.

However, this prediction of increased receptor expression would be opposed to the normally pervasive age-dependent reduction in GABAA receptor subunit expression (Hoekzema et al.

2012) and down-regulation of the δGABAA receptor gene that is observed in the frontal cortex

(Lu et al. 2004). These studies suggest that δGABAA receptor plasticity during advanced aging is complex; δGABAA receptor expression may be both upregulated and downregulated in different brain regions.

2.3.5.1.3. Regulation of δGABAA receptor expression by neurosteroids

Neurosteroids are steroid compounds which significantly affect neural function and have high activity at the δGABAA receptor (2.4.1.2). Significant fluctuations in neurosteroid levels occur during a multitude of physiological states, including stress, aging and pregnancy, and are also observed following use of psychiatric and recreational drugs (Belelli et al. 2005). These fluctuations are often accompanied by changes in the expression of the δ subunit and its partner, the α4 subunit (Smith et al. 2007).

Fluctuations in the levels of neurosteroids are often tied to fluctuations in peripherally- derived steroid hormones, as neurosteroids are metabolites of these compounds (Belelli et al.

2005). In rodents, increased levels of the steroid hormone progesterone and decreased levels of δ subunit expression are observed during pregnancy and late diestrus in female mice

(Maguire et al. 2008, Maguire et al. 2005). At least in late diestrus, this reduction in δ subunit expression can be blocked by the administration of finasteride, a drug that inhibits the synthesis of neurosteroids from progesterone by inhibiting the enzyme 5α-reductase (Maguire 29 et al. 2007). This suggests that neurosteroids specifically are responsible for progesterone- mediated downregulation of δ subunit expression.

Importantly, the effects of neurosteroids on δ subunit expression are determined by the temporal dynamics (e.g. duration) of exposure. While prolonged exposure to neurosteroids decreases δ subunit expression, acute exposure to these compounds has a different effect.

Surprisingly, acute neurosteroid exposure may increase δ subunit expression, as might acute progesterone exposure (Maguire et al. 2007, Nin et al. 2012, Shen et al. 2005). Further, withdrawal from exposure to these compounds may also increase δ subunit expression

(Maguire et al. 2007, Nin et al. 2012, Shen et al. 2005).

The mechanism by which neurosteroids alter δGABAA receptor expression is poorly understood. As neurosteroids activate protein kinase C, which regulates surface expression of GABAA receptors, it has been proposed that this kinase is involved (Maguire et al. 2007).

However, this possibility remains to be explored and little is known about δGABAA receptor trafficking in general.

2.3.5.1.4. δGABAA receptor expression and pathological conditions

Notably, δGABAA receptor expression varies significantly in pathological conditions (Table

2.1). Following traumatic brain injury, δ subunit expression and the activity of δGABAA receptor-preferring agonists in the hippocampus is increased (Kharlamov et al. 2011b,

Mtchedlishvili et al. 2010). These changes are thought to be related to the memory impairment that occurs following brain injury; however it is noteworthy that these effects are not observed in all studies (Pavlov et al. 2011).

In animal models of epilepsy, δ subunit receptor expression and the activity of δGABAA receptor-preferring compounds is reduced in the hippocampus and DG (Nishimura et al.

2005, Peng et al. 2004, Qi et al. 2006). Interestingly, an increase in δ subunit receptor expression was also observed in interneurons in these models (Peng et al. 2004).

Theoretically, both these changes in subunit expression may result in a disinhibition of neural activity which could facilitate the generation of seizures. However, it is worth noting that net tonic inhibition is generally maintained in animal models of epilepsy, so this reduction in

δGABAA receptor expression is likely not without compensation (Houser et al. 2012).

In animal models of Fragile X syndrome, researchers have observed reduced δ subunit expression, impaired learning, abnormal behaviour and hyperactivity (D'Hulst et al. 2006,

Gantois et al. 2006). Interestingly, the δGABAA receptor-preferring agonist THIP (2.4.1.1) can ameliorate some behavioural deficits in these models (Olmos-Serrano et al. 2011).

2.3.5.2. α5GABAA receptors: Expression and function

α5GABAA receptors are expressed in many brain regions, including the dentate gyrus, hippocampus (CA1/CA3), olfactory system and cortex (Pirker et al. 2000). Expression of the receptor is most significant in the olfactory cortex and hippocampus. Approximately 20% of all

α5GABAA receptors are found in the hippocampus (Sur et al. 1999), a brain region critically involved in memory processes (2.5). In the CA1 subfield of the hippocampus, α5GABAA receptors are the predominant source of tonic inhibitory current (Glykys et al. 2008).

Evidence strongly suggests that α5GABAA receptors constrain hippocampus- dependent memory. Transgenic mice null for the α5 subunit (Gabra5–/–) show improved performance on the Morris Water maze and trace fear conditioning tasks (Collinson et al.

2002, Martin et al. 2010). Similarly, transgenic mice with reduced α5 subunit expression and activity show improved performance in some memory tasks (Crestani et al. 2002).

Conversely, increased expression of the α5GABAA receptor, which occurs in pathologies such

31 as stroke and inflammation, is associated with impaired memory (Clarkson et al. 2010, Wang et al. 2012a).

While studies in Gabra5–/– mice suggest that the α5GABAA receptor constrains memory, other mouse models have showed different results. One transgenic model of reducing α5GABAA receptor function, which involves introducing a point mutation in position

105 of the α5 subunit, is associated with impaired memory and abnormal behaviour on some tasks. Mice with this mutation exhibit impaired spatial memory (Prut et al. 2010), pre-pulse inhibition (Hauser et al. 2005), latent inhibition (Gerdjikov et al. 2008) and fear extinction (Yee et al. 2004) relative to wild-type mice. These findings illustrate that the α5GABAA receptors may play a broader role in memory than originally speculated and suggest care must be taken in comparing studies utilizing different mouse models.

Pharmacologic studies also suggest that α5GABAA receptor activity inhibits memory.

Pharmacologic enhancement of α5GABAA receptor activity, with a drug such as an anesthetic, impairs performance of the Morris water maze (Cheng et al. 2006), fear conditioning (Cheng et al. 2006) and novel object recognition tasks (Saab et al. 2010, Zurek et al. 2012). Surprisingly, the memory impairments produced by anesthetics persist even after these compounds have been eliminated from the body (Zurek et al. 2012). This suggests that acute α5GABAA receptor activation by anesthetics leads to long-term neurophysiologic changes which inhibit subsequent memory formation. In contrast to these amnestic effects of pharmacologic α5GABAA receptor activation, pharmacologic blockade of α5GABAA receptors enhances memory (Atack 2011, Atack et al. 2006, Collinson et al. 2006).

How might α5GABAA receptors constrain memory? A commonly-held assumption is that α5GABAA receptors constrain memory by attenuating synaptic plasticity (Martin et al.

2010) and excitability (Bonin et al. 2007) in neuronal networks, properties on which memory is thought to depend (Whitlock et al. 2006). A review of synaptic plasticity, and its relationship to memory, can be found 2.9.

2.4. GABA activation of GABAA receptor channels

Efficient gating of the GABA receptor by GABA requires the binding of two GABA molecules

(Olsen et al. 2009). The binding pocket is found at the interface between the α and β subunits. At most synapses, GABAA receptors are not fully occupied following action potential-dependent release of vesicular GABA. This is likely due to the comparatively long duration of GABA binding relative to the short GABA transient (Barberis et al. 2011).

GABA sensitivity varies considerably among receptor subtypes. In comparing GABA sensitivity, the effective concentration that produces the half-maximal response (the EC50) is useful. In studies of αβγ2 subunit-containing receptors, the rank order of EC50 values, as determined by the α subunit present, is α6 < α1 < α2 < α4 < α5 <<< α3 (Bohme et al. 2004,

Brickley et al. 1996). When the γ2 subunit in the αβγ2 is replaced with the δ subunit, the sensitivity to GABA of the resulting receptor (e.g. α4βδ) increases considerably (Brown et al.

2002). Most synaptic GABAA receptors demonstrate a lower sensitivity (EC50 = 6-14 µM) than do extrasynaptic δGABAA receptors (EC50 = 0.3-0.7 µM) (Brown et al. 2002). It has been argued that GABA sensitivity may not be relevant for some channel subtypes as the opening of some channels may in fact be GABA-independent (Farrant et al. 2005, Walker et al. 2008) particularly within the dentate gyrus (Wlodarczyk et al. 2013).

Desensitization varies considerably among GABAA receptor subtypes. It is commonly believed that extrasynaptic receptors demonstrate less desensitization (Brown et al. 2002), though this argument has been challenged recently (Bright et al. 2011). The potentially low desensitization and high sensitivity of extrasynaptic GABAA receptors have been argued to be ideal properties for the generation of tonic current in response to persistent exposure to low concentrations of ambient GABA (Mody et al. 2004).

Most GABAA receptors have a single channel conductance of ~25-28 pS (Farrant et al.

2005). Importantly, the conductance of a GABAA receptor channel is only one of several factors that determines the response to GABA. The response of a receptor channel to GABA is also modified by the kinetic properties of that channel's open states. Notably, δGABAA receptor channels have a low open probability and mean open time in the presence of GABA

(Farrant et al. 2005). Accordingly, δGABAA receptors are said to be of low efficacy.

Furthermore, GABA has been described as being only a partial agonist for δGABAA receptors.

2.4.1. Pharmacologic modulation of GABAA receptors

GABAA receptor activity can be affected by a wide variety of compounds besides GABA.

Some of these compounds can activate the receptor directly, functioning as full or partial agonists at the GABA binding site (2.4.1.1). Other compounds do not activate the receptor directly, but instead modulate receptor activity through activity at allosteric sites (2.4.1.2,

2.4.1.3). Finally, other compounds also function as antagonists at the GABAA receptor (such as picrotoxin and bicuculline, 2.4.1.4). This section will review some of the major compounds with activity at the GABAA receptor, with a focus on those compounds that affect δGABAA receptors.

2.4.1.1. Agonists

2.4.1.1.1. THIP

4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, also called Gaboxadol) is a synthetic muscimol derivative. Molecular modeling suggests that THIP acts at the GABA binding site

(Bergmann et al. 2013). Electrophysiological evidence suggests that THIP competes with

GABA for this binding site, as high concentrations of GABA occlude THIP-mediated enhancement in tonic current (Houston et al. 2012).

THIP has a number of therapeutic properties, including anxiolytic (Chandra et al.

2006), analgesic (Bonin et al. 2011) and sedative-hypnotic activity (Wafford et al. 2006).

Originally developed with a view towards treating epilepsy (Wafford et al. 2006), THIP was subsequently tested as a potential drug for the treatment of insomnia, but clinical trials were abandoned following evidence of a poor risk-benefit ratio (Saul 2007). The drug was shown to have unusual side effects - including disorientation and hallucination - and also failed an efficacy trial (Saul 2007).

In spite of the current lack of a defined therapeutic use, THIP is highly useful as a pharmacologic tool due to its relative selectivity. Recombinant electrophysiological studies in cell culture suggest THIP selectively activates α4β3δ receptors at low concentrations (~1 µM)

(Brown et al. 2002). The EC50 for THIP at α4β3δ GABAA receptors (6.3 μM) is much lower than that at α4β3γ2 GABAA receptors (101.6 μM) (Brown et al. 2002) or other synaptic receptors. Consistent with the results in culture models, low concentrations of THIP (1-5 μM) significantly increase δGABAA receptor-mediated tonic inhibition while minimally affecting synaptic inhibition in hippocampal slice (Maguire et al. 2005).

THIP generates a greater maximal response than GABA at recombinant α4β3δ GABAA receptors, a phenomenon termed 'super agonist behaviour' (Mortensen et al. 2010). This effect of THIP has been attributed to increased channel open probability and the duration of channel open time relative to GABA (Mortensen et al. 2010). However, the status of THIP as a super agonist at δGABAA receptors has been challenged recently, as the steady-state response to THIP and GABA is comparable (Houston et al. 2012).

THIP demonstrates some interesting pharmacokinetic properties. The drug shows only limited protein binding in the plasma (<15%) in rats and humans (Cremers et al. 2007, Lund et al. 2006). The drug readily penetrates the blood-brain barrier, likely via passive diffusion, and as yet has no identified active transport mechanism (Cremers et al. 2007). Following subcutaneous injection of a low dose of THIP (2.5 mg/kg), levels of the drug in the brain peak within approximately 30 minutes (Cremers et al. 2007). Intramuscular admnistration or oral ingestion of the drug in humans also leads to peak drug levels within 30 minutes (Madsen et al. 1983, Schultz et al. 1981). The half-life of THIP, at least rodents, is roughly 28 minutes

(Cremers et al. 2007). Studies in humans using radio-ligated THIP suggest effectively complete excretion of the drug within 24 hours (Schultz et al. 1981).

It is likely that some species differences in the pharmacokinetics of THIP exist.

Notably, humans show lower plasma THIP concentrations than rats (Cremers et al. 2007,

Lund et al. 2006). Further, THIP sensitivity may differ even within rodent species.

Interestingly, low doses of THIP (4 mg/kg) have hypnotic effects in rats, but not mice (Lancel

1997, Winsky-Sommerer et al. 2007).

In humans, a therapeutic oral dose of THIP (15 mg) is correlated with low μM range in plasma and likely in the central nervous system (Cremers et al. 2007). Notably, this concentration range corresponds to that which is thought to selective δGABAA receptors in recombinant systems (1-10 μM) (Brown et al. 2002). Consistent with this suggestion, most of the salient behavioural effects of low doses of THIP are mediated by δGABAA receptors.

Gabrd–/– mice and Gabra4–/– mice, which both lack α4βδ receptors, are largely insensitive to the electrophysiological and sedative-hypnotic effects of THIP (Boehm et al. 2006, Chandra et al. 2006, Herd et al. 2009). However, the mechanism of THIP's actions are controversial

(Houston et al. 2012) and some therapeutic effects of the drug may not require activation of 37

δGABAA receptors. Notably, THIP still has analgesic effects in Gabrd–/– mice in persistent pain models (Bonin et al. 2011). These findings suggest that THIP is not as selective as originally expected and have lead to the compound being termed 'δGABAA receptor- preferring' rather than 'δGABAA receptor-selective.'

2.4.1.1.2. Muscimol

Muscimol, a compound derived from mushrooms of the Amanita genus (Michelot et al. 2003), selectively activates multiple subtypes of GABAA receptors and has sedative-hypnotic and psychoactive effects (Mortensen et al. 2010). Muscimol utilizes the same binding site as

GABA, and like THIP has been described to have moderate 'super agonist behaviour'

(Mortensen et al. 2010). Muscimol is useful in binding assays (Sotiriou et al. 2005) and for reversible inactivation of the hippocampus during the study of memory (Majchrzak et al. 2000,

Oliveira et al. 2010).

2.4.1.2. Positive allosteric modulators

2.4.1.2.1. Barbiturates

Barbiturates are used to induce anesthesia and treat epilepsy (Smith et al. 1991) but also have hypnotic, analgesic and anxiolytic effects. These compounds enhance GABAA receptor- mediated current likely by increasing mean channel open time in the presence of GABA

(Olsen et al. 2009). At lower concentrations, these compounds interact with a binding site that is separate from that of GABA, and are hence described as positive allosteric modulators

(Olsen et al. 2009). At high concentrations, some barbiturates can activate the receptor directly (Rho et al. 1996).

Barbiturates show only limited selectively for the GABAA receptor and also affect the glutamate receptor channels and other members of the ion channel superfamily to which the

GABA receptor belongs (Olsen et al. 2009). While clinically useful, barbiturates have a high potential for addiction, tolerance, and overdose (Ito et al. 1996), which has lead to a reduction in their popularity in recent years.

2.4.1.2.2. Benzodiazepine-type compounds

Benzodiazepines are popular clinically-relevant drugs with anxiolytic and sedative-hypnotic effects. Most benzodiazepines significantly enhance GABAA receptor activity and are hence described as positive allosteric modulators (Rudolph et al. 2011). The likely interaction site for benzodiazepines is distinct from that of GABA and barbiturates, and is found is between the α and γ subunits (Olsen et al. 2009). Classical benzodiazepines, such as diazepam and flunizatrepam, significantly modulate GABA-mediated current at α1βγ2, α2βγ2, α3β2γ2 and

α5βγ2 receptors (Olsen et al. 2009). In general, benzodiazepines do not demonstrate any activity at α4βδ and α6βδ receptors (i.e., δGABAA receptors) (Olsen et al. 2009) as these receptors lack the γ subunit component of the benzodiazepine binding site. To a limited extent, the individual therapeutic effects of benzodiazepines can be attributed to their activity at distinct receptor subtypes (Rudolph et al. 2004). While benzodiazepines are highly useful as therapeutic compounds, their use is constrained by a high potential for addiction and tolerance (Lancel et al. 2000, Tan et al. 2011).

Imidazobenzodiazepines are a structurally distinct subtype of benzodiazepines. Most of these compounds are positive allosteric modulators of the GABAA receptor (e.g. midazolam). However, some imidazobenzodiazepines are exceptions to this rule, such as flumazenil and Ro15-4513. Flumazenil is an antagonist of the benzodiazepine site while

Ro15-4513 is a inverse agonist (Meera et al. 2010, Olsen et al. 2009). As these two compounds are not positive allosteric modulators of the GABAA receptor, they are covered separately in 2.4.1.3. 39

2.4.1.2.3. Other compounds with activity at the benzodiazepine site

A variety of compounds have activity at the benzodiazepine site but have a structure that is distinct from benzodiazepines. Non-benzodiazepines, for example, are a class of compounds which interact with the benzodiazepine site and generally act as positive allosteric modulators. An example of a non-benzodiazepine is the compound zolpidem, which has a very high affinity for α1β2γ2 receptors and a much lower affinity for other GABAA receptor subtypes, particularly α5βγ2 receptors (Farrant et al. 2005).

The compound L-655,708 also interacts with the benzodiazepine site. L-655,708 shows high selectivity for the α5 subunit and acts as an inverse agonist (Atack 2011).

2.4.1.2.4. Neurosteroids

Neurosteroids are steroid compounds that can significantly modulate neural function (Belelli et al. 2005). The compounds enhance GABAA receptor-mediated current, presumably by enhancing the gating efficiency of the receptor (Belelli et al. 2005). Endogenous neurosteroids are either generated de novo in the brain or produced via metabolism of peripherally-derived stress hormones (Belelli et al. 2005) (2.3.5.1). Typical endogenous neurosteroids include 5α- pregnan-3α-ol-20-one (3α,5α-THPROG, also called allopregnanolone) and 5-pregnan-3α,21- diol-20-one (3α,5α-THDOC). Synthetic neurosteroids have been also been produced which are structurally and functionally similar to endogenous neurosteroids, such as the compound alphaxalone (Lambert et al. 2001). As both endogenous and synthetic neurosteroids modulate neural function, these compounds are often referred to as 'neuroactive steroids'

(Belelli et al. 2005). Here, only endogenous neurosteroids will be discussed.

δGABAA receptors are perhaps most affected by low concentrations of neurosteroids as these receptors have a comparatively lower efficacy for GABA (2.3.5.2). Indeed,

40 physiologic concentrations of neurosteroids (~10-100 nM) selectively enhance δGABAA receptor-mediated tonic inhibition (Stell et al. 2003). Based on this evidence, it has been proposed that that δGABAA receptors are the preferred, if not sole, target of neurosteroids in the central nervous system (Belelli et al. 2005). In support of this claim, Gabrd–/– mice show reduced sensitivity to the electrophysiological and behavioural effects of neurosteroids

(Mihalek et al. 1999, Stell et al. 2003).

At low concentrations, neurosteroids are thought to exert their effects by binding to sites on the α and β subunits (Hosie et al. 2007), that are distinct from the GABA binding site.

At concentrations modestly above physiological levels, neurosteroids can interact with the

GABA binding site, acting as GABA mimetics (Belelli et al. 2005). In certain cases, neurosteroids can also prolong the decay of the mIPSC, which suggests they may also significantly affect postsynaptic GABAA receptors (Belelli et al. 2005).

2.4.1.2.5. Ethanol

GABAA receptors have long been viewed a potential target of ethanol. Ethanol has similar physiological and behavioural effects to barbiturates and benzodiazepines, and interacts with members of both classes of these drugs (Olsen et al. 2009). However, recombinant studies have shown that postsynaptic GABAA receptors are typically insensitive to ethanol, as high concentrations of the drug are required to elicit an effect (Wallner et al. 2006). In contrast, some reports show that δGABAA receptors are sensitive to low concentrations of ethanol

(~mM) (Wallner et al. 2006) though there is some controversy in this regard (Borghese et al.

2007). These concentrations represent those in humans following recreational drinking or mild intoxication (Olsen et al. 2009). The compound Ro15-4513 antagonizes many behavioural actions of alcohol in mammals, and may share a binding site with ethanol at the δGABAA receptor (Olsen et al. 2009). 41

2.4.1.2.6. Anesthetics

Anesthetics are drugs which induce anesthesia, or the loss of sensation. These drugs are administered to allow patients to tolerate surgery (Wang et al. 2011). Anesthetics can be classified into two general categories: 1) general anesthetics, which cause a reversible loss of consciousness, and 2) local anesthetics, which cause a loss sensation in a particular region of the body. General anesthetics can be further defined by their route of administration

(injected or inhaled). Prototypic general anesthetics thoroughly investigated in the experimental setting include etomidate (injected), isoflurane (inhaled), sevoflurane (inhaled) and propofol (injected). Local anesthetics include compounds such as lidocaine.

GABAA receptors are important molecular targets for anesthetics (Franks 2006). Many of the actions of anesthetics are explained by the activation of specific GABAA receptor subtypes (Cheng et al. 2006), though anesthetics such as dexmedetomidine have different mechanisms (Maze et al. 1991). Most anesthetics enhance GABAA receptor-mediated current and act as positive allosteric modulators (Wang et al. 2011). Some of the individual effects of anesthetics can be attributed to activity at particular GABAA receptor subunits (Cheng et al.

2006). Many of the amnestic effects of anesthetics are mediated by α5GABAA receptors

(Wang et al. 2011). While δGABAA receptors are activated by anesthetics (Meera et al. 2009), these receptors are not likely involved in post-anesthetic memory impairment. Gabrd–/– mice, which lack δGABAA receptors, still show memory deficits following exposure to anesthetics (Eng 2008).

2.4.1.3. Negative allosteric modulators

Negative allosteric modulators of the GABAA receptor include flumazenil (Weinbroum et al.

1997), Ro15-4513 (Olsen et al. 2007), and the ionic form of Zinc (Brown et al. 2002,

Mortensen et al. 2006, Smart et al. 2004). Flumazenil competes for the benzodiazepine binding site, and is useful in reducing the undesired symptoms of benzodiazepine use, such as excessive drowsiness (Weinbroum et al. 1997). Ro15-4513 is an inverse agonist of the benzodiazepine binding site on the GABAA receptor, and can antagonize the behavioural effects of ethanol (Olsen et al. 2007). Both compounds are useful in labelling and imaging of

GABAA receptors. Ionic Zinc suppresses tonic GABAergic inhibition but has relatively modest effects on synaptic inhibition (Smart et al. 2004).

2.4.1.4. Antagonists and channel blockers

A number of antagonists exist for the GABAA receptor (Siegel 1999). Three notable antagonists used in experimental settings are bicuculline, picrotoxin and SR-95531 (also called gabazine). Bicuculline and SR-95531 are competitive antagonists of the GABA binding site while picrotoxin is a non-competitive antagonist, effectively equivalent to a channel blocker (Ueno et al. 1997).

High concentrations of bicuculline, picrotoxin and SR-95531 block both synaptic and tonic inhibition, and are used to reveal tonic current (Figure 2.5) (Stell et al. 2002). However, low concentrations of SR-95531 (~200 nM) selectively block synaptic inhibition while having a minimal effect on tonic inhibition, making the drug a useful experimental tool (Stell et al.

2002). There is some discrepancy in the concentration of SR-95531 that affects tonic inhibition. One report suggests blockade of tonic inhibition with a concentration of 10 μM (Stell et al. 2002) while another report suggest that concentrations as high as 20 μM have no effect on tonic inhibition (Bai et al. 2001).

2.5. The hippocampal formation and memory

Neuroscience has long been interested in determining the locus of memory within the brain.

While early experimental evidence demonstrated that memories could be stored throughout the brain (Lashley 1950), the mechanism of memory storage was unclear. Later clinical observations specifically implicated the medial temporal lobe in memory storage (Dickerson et al. 2010). The hippocampal formation, contained with the medial temporal lobe, was found to play an important role in the process (Rempel-Clower et al. 1996, Scoville et al. 1957). In humans, damage to the hippocampal formation produced a profound impairment in the acquisition of new memories (termed anterograde amnesia) and a graded impairment in the recall of previously acquired memories (retrograde amnesia) (Rempel-Clower et al. 1996,

Scoville et al. 1957, Squire et al. 2001). Convergent evidence from animal models reinforced the importance of the hippocampal formation for memory storage (Sutherland et al. 2010).

Due to the obligatory requirement of the hippocampal formation in multiple memory processes, the structure is often affectionately dubbed "the gateway to memory" and is a primary focus of neuroscience research.

In the literature, the term 'hippocampal formation' usually refers to a collection of structures which includes the hippocampus proper, dentate gyrus (DG), subicular complex and entorhinal cortex (Figure 2.6). The term 'hippocampus proper' refers only to the CA subfields and does not include the other structures (Andersen et al. 2007). In this dissertation, the terms 'hippocampus proper' and 'hippocampus' will be considered synonymous and discussed in one section. The DG will be discussed separately.

Figure 2.6. Synaptic pathways in the hippocampus.

Top. General schematic of the hippocampus showing main excitatory pathways. Bottom. The trisynaptic circuit. Trisynaptic pathways are highlighted in green while other pathways are highlighted in red. Figure adapted from Nakashiba et al. (2008). Abbreviations: PP = perforant pathway, DG = dentate gyrus, MFP = mossy fiber pathway, RCP = recurrent collateral pathway, SCP = schaffer collateral pathway, EC = entorhinal cortex, MC = mossy cells, S = subiculum.

2.6. Anatomy of the hippocampal formation

2.6.1. The hippocampus

The hippocampus is a horn-shaped structure that is contained within the medial temporal lobe (Andersen et al. 2007). The dorsal and ventral portions of the hippocampus are thought to have distinct connections (Moser et al. 1998) and mediate distinct functions

(Fanselow et al. 2010). The dorsal hippocampus has been argued to be more involved in cognition while the ventral hippocampus has been argued to be more involved in emotion and affect (Fanselow et al. 2010). Accordingly, most investigations into the role of the hippocampus in memory focus upon the dorsal hippocampus.

The hippocampus is divided into multiple subregions, termed CA subfields (Figure 2.7).

According to Andersen and colleagues (2007), there are three distinct CA subfields (CA1,

CA2 and CA3). These subfields have largely distinct anatomical, electrophysiological and functional properties. As differentiation of the CA2 subfield is difficult in many species, the existence of the CA2 subfield is often considered controversial and use of the term is discouraged (Andersen et al. 2007).

The hippocampus has a well-defined laminar structure (Andersen et al. 2007) (Figure

2.7). In the CA1 and CA3 subfields, the bodies of pyramidal cells are packed into the stratum pyramidale (or pyramidal cell layer). The basal dendritic processes of pyramidal cells extend into the stratum oriens, which is also occupied by interneurons (Ganter et al. 2004). The apical dendritic processes of pyramidal cells pass through another layer, termed the stratum radiatum, all the way to the hippocampal fissure. In the CA1 subfield, the stratum radiatum is the site where the dendritic processes of CA1 pyramidal cells synapse with Schaffer collateral fibers from CA3 pyramidal neurons. In the CA3 subfield, the stratum radiatum is instead

46 occupied by auto-associational connections (from CA3 to CA3) and outgoing Schaffer collateral fibers. At each of these synapses, significant and distinct forms of synaptic plasticity can be observed (2.9) that are associated with memory.

In the CA3 subfield only is the stratum lucidum, where incoming mossy fibers from the

DG synapse with CA3 pyramidal cells. Finally, internal to the stratum lucidum is a thin, superficial layer of the hippocampus termed the stratum lacunosum-moleculare, where afferent fibers from the entorhinal cortex, perirhinal cortex and other brain regions terminate.

Figure 2.7. Schematic of the hippocampus and DG showing laminar structure.

The dentate gyrus is divided into three layers: the stratum granulosum (sg), stratum moleculare (sm) and polymorphic layer (pl). In the CA3 subregion are the stratum lacunosum- moleculare (slm), stratum radiatum (sr), stratum lucidum (sl), stratum pyramidale (sp) and the stratum oriens (so). The CA1 subregion has a similar laminar structure to CA3, but lacks the stratum lucidum. Other abbreviations: hf = hippocampal fissure, spb = suprapyramidal blade, and the ipb = infrapyramidal blade.

2.6.1.1. Intrinsic hippocampal connections

A remarkable feature of the hippocampus is its circuitry, which emphasizes unidirectional inputs and lacks the type of significant reciprocal backprojections which characterize the layers of the neocortex (Felleman et al. 1991). Collectively, the hippocampus and DG contain a series of three synapses that form a single, unified circuit termed 'the trisynaptic loop' or

'trisynaptic circuit' (Figure 2.6) (Anderson et al. 1971).

The first stage of the trisynaptic circuit is the entorhinal cortex, which sends inputs to the DG via the perforant pathway (synapse 1). In the second stage of the circuit, the DG sends inputs to the CA3 subfield via the mossy fiber pathway (synapse 2). In the third stage of the circuit, the CA3 subfield then sends inputs to the CA1 subfield via the Schaffer collateral pathway (synapse 3). The CA1 subfield then sends inputs to the subicular complex, which in turn sends inputs to the entorhinal cortex, completing the circuit. The different stages of the trisynaptic circuit may mediate distinct behavioural functions. In an elegant series of experiments, the respective contribution of each of the hippocampal synapses to memory was examined by Nakashiba and colleagues (Nakashiba et al. 2009, Nakashiba et al. 2012,

Nakashiba et al. 2008, Suh et al. 2011).

Adding a layer of complexity to the trisynaptic circuit are other intrinsic connections.

Importantly, the entorhinal cortex does not simply send inputs to the DG, but directly to CA3 and CA1 subfields (Andersen et al. 2007). Further, the CA3 subfield has a backprojection to the DG which is thought to regulate the behavioural functions of this region (Myers et al.

2011) and neurogenesis (Vivar et al. 2012). Also in the CA3 subfield is an extensive recurrent collateral network wherein CA3 pyramidal cells are connected to other CA3 pyramidal cells

(Andersen et al. 2007). These recurrent collateral connections outnumber other afferent connections in the region (Rolls 2010).

Finally, the activity of each subfield of the hippocampus, and of the DG, is modulated by a diverse population of local inhibitory interneurons (Wojtowicz 2012) (2.6.2.2).

2.6.1.2. Extrinsic hippocampal connections

Both the CA1 and CA3 subfield of the hippocampus are connected with structures outside of the hippocampal formation (Andersen et al. 2007). The CA1 subfield receives additional inputs from the amygdala, perirhinal cortex, nucleus reuniens, locus coeruleus and raphe nuclei and sends inputs to the amygdala. The CA3 subfield receives inputs from the septum, amygdala, locus coeruleus and raphe nuclei and also sends inputs to the lateral septal nucleus.

2.6.2. The dentate gyrus

The DG differs from the hippocampus in several major ways. Firstly, the DG exhibits lower overall baseline activity (Barnes et al. 1990, Jung et al. 1993, O'Reilly et al. 1994). This tightly constrained or 'sparse' activity is likely the result of comparatively stronger GABAergic inhibition in the region (Coulter et al. 2007) some of which may be mediated by extrasynaptic

GABAA receptors (Arima-Yoshida et al. 2011). This level of sparse activity is believed to facilitate the processing of contextual information and pattern separation (O'Reilly et al. 1994).

When GABAA receptor-mediated inhibition is pharmacologically blocked, sparse activity is lost and cells are easily activated by stimulation (Marin-Burgin et al. 2012a). Interestingly, no one has tested the effects of reduced GABAA receptor-mediated inhibition on contextual discrimination, a form of pattern separation.

Secondly, the DG exhibits limited synaptic plasticity relative to the hippocampus (2.9), at least in the in vitro slice preparation (Snyder et al. 2001). In the mature DG, two prominent forms of synaptic plasticity are observed which are correlated with memory (long-term potentiation and long-term depression) (Dong et al. 2012, Whitlock et al. 2006). These forms of plasticity are notoriously difficult to induce in the DG (Derrick 2007, Snyder et al. 2001).

This reluctant plasticity, at least in the case of long-term potentiation, is attributed to GABAA receptor-mediated inhibition. When GABAergic inhibition is pharmacologically blocked with a selective GABAA receptor antagonist, long-term potentiation is robust and easy to induce

(Wigstrom et al. 1983b). Conversely, when GABAergic inhibition is increased by the addition of GABA or compounds which enhance GABAA receptor activity, long-term potentiation is impaired (Arima-Yoshida et al. 2011, Shen et al. 2010).

Thirdly, and perhaps most remarkably, the DG is one of the few recognized sites of postnatal neurogenesis in the central nervous system (Gould 2007) (2.7). Postnatal neurogenesis, the process wherein new, adult-born neurons are generated in the adult brain, regulates memory (Marin-Burgin et al. 2012b), emotion and affect (Snyder et al. 2011b).

Importantly, neurogenesis is also regulated by GABAA receptor-mediated signaling (Ge et al.

2006).

Much like the hippocampus, the dentate gyrus (DG) is perceived as an early, integral structure in the formation of episodic memories. In rodents, lesion of the DG is associated with significant behavioural impairments in many forms of spatial and contextual learning

(Gilbert et al. 2001, Lee et al. 2005, Lee et al. 2004). The DG is involved in a diverse array of learning tasks; a brief review of these is presented in 2.8.

2.6.2.1. General anatomy of the DG

Like the hippocampus, the DG is a horn-shaped structure that runs from the septal nuclei

(rostral) to the temporal cortex (caudal) in the brain (Amaral et al. 2007, Andersen et al.

2007). The anatomical and physiological properties of the DG can differ significantly along the rostral-caudal axis (Snyder et al. 2011a). When viewed through a coronal section, the DG is

U-shaped or V-shaped throughout its extent. The two arms of the U-shape or V-shape are separated by the insertion of CA3 into the polymorphic layer of the DG (Figure 2.7) and are considered anatomically separate. The arm dorsal to the CA3 insertion is termed the suprapyramidal blade while the arm ventral to the CA3 insertion is termed the infrapyramidal blade (Figure 2.7).

The suprapyramidal and infrapyramidal blades differ substantially in structural and physiological properties. Most remarkably, granule cells in the suprapyramidal blade appear to be much more readily activated by spatial experiences (Chawla et al. 2005, Satvat et al.

2011) and have longer and more complex dendritic processes (Desmond et al. 1985).

Neurogenesis also differs between the blades (Snyder et al. 2012). The relative quiescence of the infrapyramidal blade and its lack of an identified function have been puzzling to neuroscientists.

The DG is a laminar structure with three distinct layers: the stratum granulosum

(granule cell layer), stratum moleculare (the molecular layer) and polymorphic layer (including the hilus) (Amaral et al. 2007) (Figure 2.7). In the granule cell layer are the densely-packed cell bodies of granule cells, the main projection cells of the DG. The molecular layer is where the apical dendritic processes of granule cells synapse with the afferent fibers of the perforant pathway from the entorhinal cortex and is also where the commissural/associational fiber

52 system can be found (Figure 2.7, Figure 2.8). In the polymorphic layer is a diverse variety of cells which can act locally to modulate granule cell activity. Within these cell layers is a wide variety of other cell types (see below).

Figure 2.8. Perforant pathway termination in the molecular layer.

A typical granule cell (shown in blue) extends processes to the hippocampal fissure in the

DG. The lateral perforant pathway (LPP, orange) terminates in the outer third of the molecular layer closest to the hippocampal fissure. In contrast, the medial perforant pathway (MPP, green) terminates in the middle of the molecular layer. In the inner third of the molecular layer are fibers of the commissural/associational pathway (CAP, not labelled here). Other abbreviations: ML = molecular layer, GCL = granule cell layer, PL = polymorphic layer.

2.6.2.2. Principle cell types in the DG

2.6.2.2.1. Granule cells

Granule cells are the main projection cells of the DG. These cells are small (with cell bodies between 8-10 µM), numerous (~1 x 106 in rodents, 15 x 106 in humans), and densely packed into the granule cell layer (Amaral et al. 2007). The apical dendritic processes of granule cells extend into the molecular layer, where they synapse with the fibers of the perforant pathway from the entorhinal cortex. The most prominent output of granule cells is the CA3 subfield via the mossy fiber pathway (Figure 2.6).

2.6.2.2.2. Mossy cells

Mossy cells are located in the polymorphic layer (Amaral et al. 2007) and use glutamate as their primary transmitter (Soriano et al. 1994). The dendritic processes of these cells are largely confined to the polymorphic layer, where they are contacted by mossy fiber collaterals from granule cells. Mossy cell axons form the majority of the associational/commissural pathway. Via this pathway, mossy cells contact granule cells and other inhibitory cells

(Scharfman 1995).

2.6.2.2.3. Inhibitory interneurons

Within the DG there exists a diverse array of inhibitory interneurons. In addition to their role in regulating local cell activity (Houser 2007), interneurons may also regulate neurogenesis

(Masiulis et al. 2011, Waterhouse et al. 2012). While several classification systems exist for these interneurons (Houser 2007) this dissertation will employ the system outlined by Halasy et al. (1993) which categorizes cells according to the location of their cell bodies and distribution of their axon terminals (Figure 2.9).

According to this classification system (Halasy et al. 1993), at least 5 types of interneurons exist in the DG (Wojtowicz 2012). Within the hilus reside two distinct types of interneurons. The first of these extends fibers into the perforant path region (termed a hilar to perforant pathway-associated cell or HIPP cell) (Halasy et al. 1993). HIPP cells extend processes which intersect with the distal dendrites of granule cells. Also located in the hilus are the cell bodies of interneurons that extend axonal processes into the commissural/associational pathway region of the molecular layer (these are termed HICAP cells) (Halasy et al. 1993). Both HIPP and HICAP cells are thought to mediate important feedback inhibition in the DG (Houser 2007).

In the molecular layer reside two other distinct types of interneurons. The first of these interneurons extends axons which are contained within the perforant path region (molecular layer/perforant pathway-associated or MOPP cell) while the other extends axons downward to the granule cell layer (molecular layer/granule cell layer-associated or MOGCL cell).

Together, MOPP and MOGCL cells are thought to mediate important feedforward inhibition

(Houser 2007).

In the inner granule cell layer are basket cells, which are perhaps the most extensively studied type of inhibitory cell in the DG (Amaral et al. 2007). These cells have apical dendrites that extend into the molecular layer and basal dendrites which extend into the polymorphic layer. Basket cells extend axonal processes that form a dense plexus around the cell bodies of granule cells and the proximal regions of their apical dendrites (Amaral et al. 2007, Houser

2007). A single basket cell can potentially influence a large proportion of the granule cell population (as much as %1) due to its numerous connections (Amaral et al. 2007).

Importantly, though these interneurons are largely GABAergic, they are likely capable of releasing other peptide co-transmitters, such as enkephalins (Drake et al. 2007).

Figure 2.9. Inhibitory circuitry in the DG.

In the hilus are hilar-perforant pathway-associated cells (HIPP cells, red), hilar commissural/associational pathway-associated cells (HICAP cells, green) and basket cells

(blue). In the molecular layer are molecular perforant pathway-associated cells (MOPP cells, purple) and molecular layer-granule cell layer-associated cells (MOGCL cells, orange). Other abbreviations: ML = molecular layer, GCL = granule cell layer, PL = polymorphic layer. Figure adapted from Wojtowicz (2012) and Amaral et al. (2007).

2.6.2.3. Inputs to the DG: The perforant pathway

The principle input to the DG is from layer II of the entorhinal cortex via the perforant pathway

(Figure 2.6, Figure 2.8). Fibers of the perforant pathway synapse with the dendritic processes of granule cells and with interneurons in the molecular layer of the DG. The perforant pathway is divided into two distinct fiber systems: the medial perforant pathway (MPP) and the lateral perforant pathway (LPP). The MPP is formed by fibers originating from the medial entorhinal cortex that contact more distal dendritic processes. The LPP is formed by fibers originating from the lateral entorhinal cortex that contact proximal dendritic processes (Figure 2.8).

In addition to their anatomical differences, the MPP and LPP may mediate distinct functions: the MPP might be involved in the transmission of spatial information while the LPP might be involved in the transmission of multimodal sensory information (Kesner 2007).

Further, both pathways differ in their electrophysiological properties: MPP synapses exhibit paired pulse depression of synaptic responses while LPP synapses exhibit paired pulse facilitation (Christie et al. 1994). In this dissertation, only MPP function was studied.

Other important inputs to the DG arise from the presubiculum, parasubiculum, medial septal nucleus, nucleus of the diagonal band of broca, supramammillary nucleus, locus coeruleus, median raphe and dorsal raphe (Andersen et al. 2007). These diverse inputs can target granule cells, basket cells and other cell types. There is also a notable back-projection from the CA3 subfield to the DG (Myers et al. 2011), as mentioned previously.

2.6.2.4. Output of the DG: The mossy fiber pathway

The main output of DG is the CA3 subfield via the mossy fiber pathway (Blaabjerg et al. 2007)

(Figure 2.6). This pathway is composed of the thin, unmyelinated axons which synapse mainly on the proximal regions of the apical processes of CA3 pyramidal cells. Mossy fiber 59 input is highly divergent; the odds that any two DG granule cells contact the same two CA3 pyramidal cell are low (Rolls 2010). However, while these synaptic inputs are dispersed, they are highly effective at exciting the neuron, perhaps because they are located close to the soma. Burst activity in a single granule cell can drive its target CA3 pyramidal cell to fire

(Henze et al. 2002). Owing to this strong input, mossy fiber-CA3 synapses are often referred to as 'conditional detonator synapses.' The exceptional structural and electrophysiological characteristics of the mossy fiber pathway have lead to intense speculation about its function.

Many computational models emphasize the role of the mossy fiber in computational processes such as pattern separation (O'Reilly et al. 1994, Rolls 2010) which may be relevant for stimulus discrimination (Aimone et al. 2011).

Importantly, the mossy fiber pathway sends collaterals that synapse on to mossy cells and basket cells in the DG polymorphic layer and other inhibitory neurons (Figure 2.9).

Remarkably, the mossy fiber pathway actually contacts more CA3 interneurons than CA3 pyramidal cells (Acsady et al. 1998).

2.7. Postnatal neurogenesis and the DG

Most neurons in the central nervous system do not have the capacity to divide. However, there is continuous production of new neurons in at least two discrete regions of the brain: the olfactory bulb and DG (Gould 2007). In the DG, adult-born neurons integrate into functional brain circuitry (Ge et al. 2008, Stone et al. 2011) and demonstrate significant synaptic plasticity (Snyder et al. 2001) and excitability (Marin-Burgin et al. 2012a, Mongiat et al. 2009) relative to older, developmentally-generated neurons. Neurogenesis is significant in juvenile animals and contributes significantly to the structure of the mature DG (Cushman et al. 2012).

At any given time, young adult-born neurons constitute only a small portion of the total

60 population in the DG (>5-10%) (Imayoshi et al. 2008, Nakashiba et al. 2012) but can regulate memory (Marin-Burgin et al. 2012b), mood and affect (Snyder et al. 2011b). Neurogenesis is also implicated in the cognitive and emotional response to stress (Snyder et al. 2011b), exercise (Creer et al. 2009) and anti-depressant drugs (Eisch et al. 2012). Others have speculated that neurogenesis is also related to the cognitive impairments that occur in neurodegenerative diseases (Kempermann 2012, Ming et al. 2011).

Significant neurogenesis has been observed in many species, including birds, rodents and primates (Amrein et al. 2009, Kempermann 2012). The extent and temporal dynamics of neurogenesis vary considerably between species (Amrein et al. 2009, Kempermann 2012).

Rats, for example, not only produce more new neurons than mice, but also produce faster- maturing neurons (Snyder et al. 2009). It is important to consider that while neurogenesis is presumed to be significant in humans, most conclusions about neurogenesis have been drawn from animal models (Ming et al. 2011). However, several studies have suggested that there is significant neurogenesis in adult humans (Eriksson et al. 1998, Spalding et al. 2013).

Remarkably, as much as a third of neurons in the human hippocampus may be subject to exchange through neurogenesis throughout the life of the individual (Spalding et al. 2013).

The extent of neurogenesis can be affected by a diverse array of factors, particularly aging (Ming et al. 2011). Neurogenesis declines steadily with age (McDonald et al. 2005), and this may be related to age-related cognitive decline (Creer et al. 2009, Lee et al. 2012).

Experimental manipulations which enhance neurogenesis, such as exercise, may be useful in reversing age-related cognitive impairment (Creer et al. 2009). Neurogenesis is also enhanced by a number of other factors besides exercise, including learning and environmental enrichment (Ming et al. 2011).

Importantly, neurogenesis also varies by sex (Pawluski et al. 2009). These effects may be due to sex-dependent differences in the levels of the steroid hormones estradiol, progesterone and corticosterone. In female animals, levels of these hormones can vary during aging, stress, estrous and reproductive states (including pregnancy and lactation)

(Pawluski et al. 2009). Some reports also suggest that neurogenesis is higher in females

(Roughton et al. 2012). In males, the gonadal hormone testosterone can also have significant and complex influences on neurogenesis, including potential effects on the survival of adult- born cells (Galea 2008).

A number of neurotransmitter receptors regulate neurogenesis (Zhao et al. 2008), including N-methyl-D-aspartate (NMDA) receptors, GABAA receptors and neurotrophin receptors (Catts et al. 2008, Ge et al. 2008, Nacher et al. 2006, Shimazu et al. 2006). The following section focuses on the contribution of GABAA receptors to neurogenesis in the DG.

2.7.1. Stages of neurogenesis

Neurogenesis actually encompasses several distinct and temporally sequential cellular processes (Ming et al. 2011). The first stage of neurogenesis is proliferation, which typically begins with the neuronal stem cell, termed a Type-1 cell (or radial glial-like stem cell). In the

DG, these cells are located within the subgranular zone of the granule cell layer. Type-1 cells divide asymmetrically to produce one Type-1 cell and one daughter cell, called a Type-2 cell.

Type-2 cells can then divide symmetrically multiple times before differentiating. Differentiated

Type-2 cells can go on to become Type-3 cells (an intermediate type of cell). These cells demonstrate proliferative capacity, but can also differentiate and eventually become adult- born neurons.

Prior to synapse formation, both Type-2 and Type-3 cells generate a GABA receptor- mediated tonic current, and this current is important for the development of these cells (Ge et al. 2006). Evidence suggests that GABAA receptors mediate some of this current. However, as the effects of GABAA receptors on development can be occluded by blockade of NMDA receptors, it is quite possible that GABAA receptors regulate development by modifying the activity of NMDA receptors (Rosenzweig 2011). Besides playing a role in neuronal development, GABA contributes to activity-dependent neuronal differentiation (Tozuka et al.

2005) and may contribute to proliferation (Duveau et al. 2011, Young et al. 2012).

Survival of newly-generated cells following proliferation is not guaranteed. The 1-2 week timeframe following division marks a critical window for survival, as over 50% of newly- generated cells do not survive this period (McDonald et al. 2005). Survival of adult-born neurons is regulated by a number of factors, including NMDA receptor-mediated excitation and engagement in learning tasks (Shors et al. 2012, Tashiro et al. 2006).

2.7.2. Maturation of adult-born neurons

As adult-born neurons mature, they begin to extend processes (mossy fibers) towards the

CA3 region and collaterals towards the hilus. Fiber extension to the CA3 region can be observed as early as 5-10 days after cell division (Hastings et al. 1999), with synapses being formed at 17 days and continuing to develop for many weeks thereafter (Toni et al. 2008).

Dendritic processes also develop significantly during maturation, with the peak growth period being observed between 21-28 days (Shapiro et al. 2007, Zhao et al. 2006).

This peak growth roughly coincides with the development of enhanced excitability in adult-born neurons. By 24-29 days of age, adult-born neurons can fire action potentials as well as mature neurons (Mongiat et al. 2009). This comparable excitability is surprising given

63 that young adult-born neurons have formed so few synapses (i.e.: they receive less excitatory input). Adult-born neurons are perhaps better at integrating excitatory input because they demonstrate lower magnitude inward-rectifying K+ currents (Mongiat et al. 2009), which are hyperpolarizing and inhibit the generation of action potentials (Takigawa et al. 2002). Another possible explanation for the enhanced excitability of adult-born neurons is their lack of inhibition by GABAA receptors. Perisomatic inhibition, which is typically mediated by α1 subunit-containing GABAA receptors, temporally overlaps with excitatory input in older neurons but not young adult-born neurons (Marin-Burgin et al. 2012a). Blockers of GABAA receptors (such as picrotoxin) elevate excitability in older neurons significantly, making them functionally similar to young adult-born neurons (Marin-Burgin et al. 2012a).

The maturity of adult-born neurons also affects their capacity to demonstrate long-term potentiation (LTP), a form of synaptic plasticity. It has been repeatedly demonstrated that young (3-8 week old) adult-born neurons demonstrate significantly greater LTP than do older, developmentally-generated neurons (Ge et al. 2007), though this property is lost with age

(Mongiat et al. 2011). The modest baseline DG-LTP that is observed in the slice preparation is attributed to young adult-born neurons (Snyder et al. 2001). Irradiation, which selectively ablates the adult-born neuron progenitor population while sparing the older, developmentally- generated neuronal population, prevents the induction of DG-LTP, at least for several weeks

(Singer et al. 2011, Snyder et al. 2001). The failure of older neurons to demonstrate significant DG-LTP may be due to the fact they receive significantly more GABAA receptor- mediated inhibition (Coulter et al. 2007), as competitive antagonists of GABAA receptors greatly enhance LTP in the DG (Snyder et al. 2001).

Interestingly, following irradiation, a recovery of LTP in the DG is observed after several weeks (Singer et al. 2011). This restoration of plasticity may be due to compensatory 64 disinhibition of mature neurons. In support of this hypothesis, there is a reduction in GABA synthesizing enzymes following irradiation (Singer et al. 2011).

Importantly, synaptic plasticity in adult-born neurons is regulated by the expression of

NMDA receptors containing the NR2B subunit (Kheirbek et al. 2012, Snyder et al. 2001). The expression of the NR2B subunit is significant in young adult-born neurons but declines with age. Transgenic deletion or pharmacologic blockade of the NR2B subunit with ifenprodil attenuates DG-LTP (Kheirbek et al. 2012, Snyder et al. 2001). NR2B-mediated synaptic plasticity in adult-born neurons may be important for memory processes as transgenic deletion of the NR2B subunit impairs forms of memory which depend upon the DG (Kheirbek et al. 2012).

2.8. Measuring hippocampal function with memory assays

In humans, the hippocampal formation is thought to mediate declarative memory processes (Eichenbaum 1999). However, declarative memory is difficult to observe in experimental settings as laboratory animals lack the capacity for language. As a result, researchers have employed spatial and contextual learning assays (also termed 'place' learning assays) (Silva et al. 1998) as surrogate measures of hippocampal function. Place learning and declarative memory may share common mechanisms (Eichenbaum 1999). In place learning, the animal leans to associate a place with a salient experience (McDonald et al. 2004, Silva et al. 1998). While a number of place learning assays exist (Sharma et al.

2010), the two most popular are the Morris water maze and contextual fear conditioning task

(Figure 2.10). The hippocampal formation regulates many other forms of learning; those most relevant to this thesis are described in 2.8.3-2.8.6.

While many of these tasks are routinely described as 'hippocampus-dependent', many other brain regions besides the hippocampal formation are involved in successful performance. Performance in the Morris water maze, for example, is not just impaired by lesions of the hippocampus, but is also impaired by selective lesions of the DG, basal forebrain, cerebellum and even cerebral cortex (D'Hooge et al. 2001). Similarly, contextual fear conditioning is profoundly impaired by lesions of the amygdala, DG, perirhinal cortex and other cortical regions (Bucci et al. 2002, Burwell et al. 2004, Lee et al. 2004, Phillips et al.

1992). It also important to consider that some forms of fear conditioning may not even require the hippocampus at all, such as cued fear conditioning (Phillips et al. 1992). These data confirm that the hippocampus is not the sole facilitator of memory processes, but is a participant in a complex neural circuit that includes multiple brain regions.

2.8.1. Morris water maze

The Morris water maze is perhaps the 'gold standard' test of hippocampal function

(D'Hooge et al. 2001). In this test, an animal is placed in a pool of water which is surrounded by external extramaze cues. To escape the pool, the animal must successfully navigate towards a submerged platform. The animal acquires the location of the platform over many trials (acquisition) (Figure 2.10). As the start position in the maze changes every trial and the platform is not visible, the animal must utilize the extramaze cues to determine the platform's location. Following extensive training, recall of platform location is tested (probe trial). In this trial, the animal is returned to the maze with the platform removed, and the time it spends in the area formerly containing the platform is measured and used as an index of spatial memory.

2.8.2. Fear conditioning

Another prominent test of hippocampal function is the contextual fear conditioning assay. Contextual fear conditioning is favoured because it is a simple form of Pavlovian conditioning (Fanselow 2000) that is robust and easily trained. In the contextual fear conditioning task, the animal learns to associate a particular context (a chamber) with an aversive experience (an electric footshock). As a result of this association, the animal selectively expresses fear whenever it is returned to that chamber (Figure 2.10).

In Pavlovian terms, the footshock may be considered an unconditioned stimulus (US) as it reliably elicits a stereotyped unconditioned response (UR) in the animal: a profound burst in locomotor activity. Locomotor activity is eventually followed by freezing behaviour (the lack of movement except that related to respiration), which is indicative of fear (a conditioned response, or CR). During training, an animal is exposed to repeated shocks while in the context. As a result, the context becomes a conditioned stimulus (CS) which signals the US.

Eventually, the animal exhibits the CR whenever it is placed in the context (CSContext →

CRFreezing). This is termed contextual fear conditioning. Animals that learn the CS + US association should demonstrate high levels of freezing when re-exposed to the CSContext.

When freezing to the CSContext is lower than normally expected in a treatment group, memory is said to be impaired in that group.

In some fear conditioning protocols, the shock is also accompanied by an auditory tone

(CSTone + USShock pairing). This leads to freezing behaviour whenever the tone is heard

(CSTone → CRFreezing). This is termed cued fear conditioning, and is measured by re-exposure to the CSTone rather than the CSContext.

Fear conditioning is mediated by a complex circuit which includes the amygdala

(Fanselow et al. 2003). Depending on the protocol, the hippocampus and other brain regions

67 are also involved to varying degrees (Fanselow 2000, Lee et al. 2004, Phillips et al. 1992).

Contextual fear conditioning is thought to require the hippocampus because the task entails the processing and integration of contextual information (Fanselow 2000). According to this assumption, damage to the hippocampus impairs the ability to form associations between the context and aversive experience because it is impairs the ability to 'gather' information about the context.

Cued fear conditioning does not normally require the hippocampus (Phillips et al.

1992). However, in protocols in which there is a time delay between the delivery of the footshock and the delivery of the auditory tone, the hippocampus contributes to performance.

This form of conditioning, termed trace fear conditioning, might require the hippocampus to encode the temporal relationship between the tone and shock (Misane et al. 2005).

While a role of the DG in contextual fear conditioning appears clear (Lee et al. 2004), the role of neurogenesis is heavily debated. Some studies have shown that neurogenesis contributes to fear conditioning (Drew et al. 2010, Saxe et al. 2006) while others have not

(Clark et al. 2008, Nakashiba et al. 2012, Shors et al. 2002, Zhang et al. 2008a). Differences in testing methodology may accommodate these discrepancies. Recently, Drew et al. (2010) proposed that neurogenesis contributes to learning in single-trial fear conditioning protocols but not other protocols. While these findings collectively indicate that fear conditioning can be modulated by neurogenesis in certain situations, they also illustrate that neurogenesis is not essential for the behaviour. Accordingly, fear conditioning is not considered a form of

'neurogenesis-dependent' behaviour. In this regard, context discrimination (2.8.4) is strongly preferred.

Figure 2.10. Common assays of hippocampus-dependent memory.

A. In the Morris water maze, animals learn to navigate towards a submerged platform in a water pool using extramaze cues. Originally, the path length to the platform and the escape latency from the maze is long (A, left). With training, path length and escape latency decreases (A, middle). Later, recall of the platform location is tested by removing the platform in a probe trial (A, right). B. In the fear conditioning task, animals acquire a fear to a particular conditioned stimulus. In contextual fear conditioning, this stimulus is the context (B, top). In cued fear conditioning, the conditioned stimulus is the tone (B, bottom).

2.8.3. Fear extinction

Fear conditioning leads to a robust freezing response following re-exposure to the CS that is highly resistant to decay over time (Wang et al. 2009). However, with repeated and/or prolonged exposure to a CS without a shock being delivered, there is a progressive reduction in the freezing response to that CS. This phenomenon is termed fear extinction. Fear extinction is often applied to the study of psychological conditions such as post-traumatic stress disorder (Milad et al. 2012) that are characterized by an inability to extinguish acquired fear or anxiety to a particular stimulus.

The mechanism of fear extinction is frequently debated. Despite the fact that fear extinction manifests as a gradual decline in a learned fear response, extinction does not necessarily involve the 'forgetting' or 'erasure' of fear memory. While fear extinction may involve erasure of memory in some cases (Quirk et al. 2010), other mechanisms of extinction have also been identified. A compelling body of evidence supports that fear memory is often intact following extinction, and is actively being suppressed rather than being erased. Even after extinction of the fear response in a given context, the original fear response can be: 1) renewed following a time delay (a phenomenon referred to as renewal), 2) reinstated with a single re-pairing of the CS and US (reinstatement) and 3) exhibited to other similar CS

(generalization) (Quirk et al. 2010). In light of these findings, fear extinction has also been described as an active learning process wherein a new response is learned and an old response (fear) is suppressed.

Like fear conditioning, fear extinction is regulated by a complex circuit which includes the amygdala, hippocampus, and medial prefrontal cortex (Milad et al. 2012). The hippocampus appears to contribute to the extinction of contextual fear in particular (Fischer et

70 al. 2007, Mamiya et al. 2009, Radulovic et al. 2010). In the hippocampus, expression of a cellular transcription factor, cyclic adenosine monophosphate response element binding protein (CREB), is increased following extinction of contextual fear and is necessary for extinction to occur (Radulovic et al. 2010). Other forms of molecular signaling in the hippocampus are also critical for the extinction of conditioned fear (Agis-Balboa et al. 2011,

Fischer et al. 2007). Importantly, neurogenesis promotes the extinction of contextual fear conditioning (Ko et al. 2009, Pan et al. 2012).

2.8.4. Contextual discrimination

Contextual discrimination refers to the successful discrimination of contexts (places, environments, etcetera; Figure 2.11). While the dorsal hippocampus is required for contextual discrimination (Frankland et al. 1998), many other brain regions play key supporting roles, including the postrhinal and perirhinal cortices (Bucci et al. 2002, Burwell et al. 2004).

Depending on the difficulty of the discrimination task, the DG can play a major role.

Specifically, the DG is thought to be significantly involved when the discrimination task is difficult (i.e.: the contexts to be differentiated are highly similar) (McHugh et al. 2007,

Nakashiba et al. 2012, Sahay et al. 2011, Scobie et al. 2009) but not when the task is easy

(Scobie et al. 2009).

Contextual discrimination is typically measured using a modified fear conditioning protocol and is inferred from the presence of context-specific fear responses in the animal

(Figure 2.11). In the standard contextual discrimination paradigm, the animal is taught to discriminate only between two contexts. In one context, the animal receives a mild footshock

(shock chamber) while in the other context they do not (safe chamber). The safe and shock contexts are highly similar and differ in only a few features (such as lighting, wallpaper or

71 odour). Successful discrimination is deemed to have occurred if the animal freezes more in the shock chamber than in the safe chamber. A long training period is normally required before contextual discrimination is exhibited (≥9 days in most protocols) (Nakashiba et al.

2012, Sahay et al. 2011).

Contextual discrimination is considered a sensitive assay of DG function and is promoted by adult-born neurons (McHugh et al. 2007, Nakashiba et al. 2012, Sahay et al.

2011, Scobie et al. 2009), particularly those within 4-6 weeks of age (Nakashiba et al. 2012).

With a delay in the maturation of adult-born neurons, a corresponding impairment in contextual discrimination is observed (Scobie et al. 2009). Conversely, when neurogenesis is enhanced, contextual discrimination is enhanced (Sahay et al. 2011). Further, synaptic plasticity within the DG network, which is primarily mediated by adult-born neurons (Snyder et al. 2001), is strongly correlated with contextual discrimination performance (Kheirbek et al.

2012, McHugh et al. 2007). In cases in which synaptic plasticity is reduced or absent, corresponding impairments in contextual discrimination are observed (Kheirbek et al. 2012,

McHugh et al. 2007).

Figure 2.11. Standard protocol for contextual discrimination.

2.8.5. Object-place recognition

The object-place recognition assay is a test of spatial working memory (Dix et al. 1999,

Ennaceur et al. 1992, Lee et al. 2005). In this task, the animal must discriminate between two sequential arrays of objects that differ in spatial arrangement (Figure 2.12). In the training phase, the animal is exposed to an array of objects and is allowed to explore them freely.

During the subsequent testing phase, the animal is exposed to a new arrangement of the objects in which one has been displaced. As animals are attracted towards novelty, they will interact more with the displaced object if they recognize that it has been moved to a new location (Dix et al. 1999). An interaction with the displaced object that is significantly above chance levels indicates the animal has successfully recognized the displacement. This assay is also referred to as the 'object location' or 'memory for object location' test (Dix et al. 1999,

Goodman et al. 2010, Prut et al. 2010).

Performance on the object-place recognition task is facilitated by the DG (Lee et al.

2005, Saab et al. 2009). Object-place recognition induces the expression of plasticity-related immediate early genes in the DG, as well as proteins related to long-term memory (Soule et al. 2008). Consistent with this observation, synaptic plasticity within the DG is strongly correlated with performance on the task (Saab et al. 2009). The important role of the DG in object-place recognition is further reinforced by the observation that performance on the test is impaired by DG lesion (Lee et al. 2005) and by ablation of postnatal neurogenesis

(Goodman et al. 2010). However, other brain regions within the hippocampus may contribute to this behaviour (Oliveira et al. 2010).

Figure 2.12. Assays of recognition memory in rodents.

In the initial training period, animals are exposed to an environment in which three identical objects reside. The environment is surrounded by visual cues. Top. In object-place recognition, one object (designated D) is displaced. The animal is deemed to have recognized this spatial novelty and succeeded at the task if it interacts with the displaced object at above chance levels. Bottom. In novel object recognition, one of the objects has been replaced by a novel object (designated N). An animal is deemed to have recognized this object novelty and succeeded at the task if it interacts with the novel object at above chance levels.

2.8.6. Novel object recognition

Novel object recognition is a test of non-spatial working memory (Ennaceur et al. 1992) that is useful in studying the amnestic effects of drugs (Zurek et al. 2012) and amnesia following brain injury (Winters et al. 2010). In this test, the animal must recognize a novel object amidst a set of familiar objects (Figure 2.12). Due to the fact that novel object recognition is mediated primarily by extrahippocampal regions such as the perirhinal cortex

(Winters et al. 2010), the behaviour is sometimes described as hippocampus-independent.

However, this term is misleading, as the hippocampus can play a role in novel object recognition under certain conditions (Barker et al. 2011, Broadbent et al. 2004, Oliveira et al.

2010, Sannino et al. 2012).

The role of the DG in novel object recognition is controversial. Some studies have shown that novel object recognition is not mediated by the DG or by adult-born neurons

(Goodman et al. 2010, Madsen et al. 2003), while other studies have shown that the DG and adult-born neurons can indeed regulate the behaviour (Denny et al. 2012, Hunsaker et al.

2007, Jessberger et al. 2009, Kheirbek et al. 2012). More puzzling is the seemingly contradictory roles of neurogenesis in novel object recognition. Some studies have demonstrated that a disruption or ablation of neurogenesis impairs novel object recognition

(Goodman et al. 2010, Jessberger et al. 2009), while others have shown that ablation of neurogenesis improves the behaviour (Denny et al. 2012). This disparity in results may be accommodated by differences in methodology. Depending on the experimental treatment used to impede neurogenesis and the time course of the experiment, a distinct adult-born neuron population may contribute to behaviour in each of these studies, making comparing these findings difficult.

2.9. Synaptic plasticity

Determining the neural substrate of learning is a longstanding goal of neuroscience research.

One of the most influential theories proposed to explain learning was put forward by psychologist Donald Hebb (Hebb 1949). Hebb postulated the particular set of neurons activated by a learning task became more closely associated with one another following learning. His theory also proposed that the repetitive activation of these neurons would lead to long-term changes in their structure or physiology that would increase the efficiency of their intercommunication. Hebb, and many scientists thereafter, would argue that these long-term changes were a substrate of learning.

Compelling evidence to support Hebb's postulate came with the discovery of a remarkable form of synaptic plasticity by Bliss and Lomo (Bliss et al. 1970), who observed that synaptic responses within the DG were profoundly enhanced by brief high-frequency electrical stimulation (Bliss et al. 1970). This phenomenon, originally christened long-lasting potentiation, was later renamed long-term potentiation (LTP). LTP was an attractive model for learning because it fit the criteria outlined by Hebb's postulate: a profound and prolonged increase in synaptic strength that was dependent upon experience (or in this case, electrical stimulation). Others would later observe that long-term reductions in synaptic response could occur following certain stimulation protocols, and this phenomenon was correspondingly termed long-term depression (LTD) (Andersen et al. 2007).

Together, LTP and LTD are the two most prominently studied forms of synaptic plasticity and have been found to have a strong, if correlative, relationship with learning

(Martin et al. 2002, Neves et al. 2008). These forms of plasticity can persist in vivo for hours, days, weeks or even months (Abraham 2003). Both forms of synaptic plasticity have been

77 studied extensively and have been observed in many brain regions besides the hippocampus

(Martin et al. 2002, Neves et al. 2008). As this dissertation is concerned primarily with measuring synaptic plasticity in the hippocampus, synaptic plasticity within other brain regions will not be discussed further.

2.9.1. LTP

2.9.1.1. Properties and Domains of LTP

LTP is a heterogeneous phenomenon; distinct forms of LTP can be observed between different brain regions and sometimes even within the same synaptic region (Andersen et al.

2007). Nevertheless, most forms of LTP, particularly those in the hippocampal formation, share several properties in common.

One important property of LTP is that it is input-specific. That is to say that LTP occurs only at the tetanized input and not others (Bliss et al. 1973). The second important property of

LTP is cooperativity. To induce some forms of LTP, a certain 'threshold' amount of afferent fibers must be activated (McNaughton et al. 1978). A third and final general property of LTP is associativity (McNaughton et al. 1978). When the activation of a weak input coincides temporally with activation of the strong input, the weak input is potentiated.

LTP has been described as occurring in two distinct stages. The first stage of LTP lasts from 1-6 hours, and is characterized by protein kinase activity. This stage is termed early LTP and is the form typically observed in the in vitro slice preparation. The second stage of LTP is termed late LTP, takes places over many hours and is dependent upon protein synthesis

(Frey et al. 1988).

2.9.1.2. NMDA receptor-dependent LTP

One commonly studied form of LTP that is readily observable in the hippocampal formation and strongly correlated with learning depends upon the activation of NMDA receptors, which are ionotropic receptors for glutamate. NMDA receptor-dependent LTP can be observed at

Schaffer collateral synapses, perforant pathway synapses and at many other locations, typically following high frequency (or tetanic) stimulation of afferent fibers (Neves et al. 2008)

(Figure 2.13).

The activation of NMDA receptors during tetanic stimulation is critical to induction of these forms of LTP (Collingridge et al. 1983). Under normal conditions (when the membrane is near resting potential), NMDA receptors are blocked by Magnesium (Mg2+) ions (Nowak et al. 1984). However, after tetanic stimulation, presynaptic fibers are activated and the postsynaptic membrane is sufficiently depolarized such that the Mg2+ blockade is relieved

(Coan et al. 1985). Following relief from Mg2+ ion blockade, the NDMA receptor channel then opens, permitting the entry of Ca2+ ions into the neuron.

Pharmacologic blockade of NMDA receptors during tetanic stimulation with antagonists

D(-)aminophosphopentanoic acid (Collingridge et al. 1983) or MK-801 (Coan et al. 1987) impairs the induction of this form of LTP. However, while these experiments identified a requirement of NMDA receptors for some forms of LTP, they did not implicate any specific

NMDA receptor subtypes as the antagonists used were not very specific. Later experiments suggested an important role of NR2A subunit-containing receptors in CA1-LTP, as NR2A subunit antagonists blocked LTP in this region (Andersen et al. 2007). In contrast, an important role of NR2B subunit-containing NMDA receptors was observed in DG-LTP, as

NR2B subunit antagonist ifenprodil blocked LTP in this region (Snyder et al. 2001).

These pharmacologic studies are congruent with transgenic studies. Deletion of the

NR1 subunit of the NMDA receptor, which is obligatory for NMDA receptor formation, impairs

CA1-LTP (Tsien et al. 1996b) and DG-LTP (McHugh et al. 2007). More specifically, deletion of the NR2A subunit of the NMDA receptor impairs CA1-LTP (Sakimura et al. 1995) while deletion of the NR2B subunit impairs LTP in the DG (Kheirbek et al. 2012). However, the role of the NMDA receptor in the induction of LTP may depend upon the stimulation protocol employed (Zhang et al. 2008b).

It is worth noting that significant learning impairments are observed with pharmacological blockade of NMDA receptors (Morris et al. 1986) and with genetic deletion of the NR1, NR2A and NR2B subunits (Kheirbek et al. 2012, McHugh et al. 2007, Sakimura et al. 1995, Tsien et al. 1996b). Thus, under conditions in which synaptic plasticity is impaired, learning is often impaired. This relationship, while correlational, supports the notion that alterations in synaptic plasticity support learning.

The influx of Ca2+ through the NMDA receptor has a number of important consequences. Several intracellular enzymes, including cyclic AMP-dependent protein kinase

A (PKA) and calcium/calmodulin dependent kinase 2 (CaMKII) (Lisman et al. 2012), are sensitive to Ca2+ levels and become active following Ca2+ influx (Bliss et al. 2011). These kinases are central to the induction of NMDA receptor-dependent LTP. CaMKII phosphorylates AMPA receptors, which are important in the expression of LTP (Lee et al.

2003). Notably, CaMKII knockouts exhibit impaired synaptic plasticity in the CA1 subfield

(Hinds et al. 1998) and abnormal emotional behaviour (Chen et al. 1994), which supports the importance of CaMKII in regulating behaviour through LTP. The importance of this Ca2+ influx in LTP is supported by the observation that LTP is inhibited by calcium chelators such as

80 ethylene glycol tetraacetic acid (EGTA) (Lynch et al. 1983, Malenka et al. 1988) and can be mimicked in the postsynaptic neuron by increasing intracellular calcium (Malenka et al. 1988).

Ca2+ influx through the NMDA receptor channel can also facilitate release of Ca2+ from internal stores. This Ca2+-induced Ca2+ release from internal stores is important for LTP. The compound thapsigargin, which blocks the refilling of internal Ca2+ stores, prohibits LTP

(Harvey et al. 1992). Ca2+-induced Ca2+ release occurs via activation of inositol triphosphate

(IP3) and ryanodine receptors. Blockade of ryanodine receptors with dantrolene, which attenuates Ca2+-induced Ca2+ release, can occlude LTP (Obenaus et al. 1989). Genetic deletion of ryanodine type 3 receptors also impairs many forms of LTP and learning

(Balschun et al. 1999). Similarly, genetic deletion of IP3 receptors can have significant effects on LTP and LTD (Taufiq et al. 2005, Yoshioka et al. 2010).

While NMDA receptor-dependent LTP is thought to be largely mediated by changes in the postsynaptic neuron, there is sufficient evidence to support that changes in the presynaptic neuron may occur as well (Kullmann 2012, Martin et al. 2002).

A B

Figure 2.13. Recording long-term potentiation in the hippocampal slice preparation.

A. Schematic showing arrangement of electrodes in the DG. The stimulating electrode (black) stimulates the perforant path fibers (purple). The recording electrode (white) detects the resulting field postsynaptic potential (fPSP) in the middle third of the molecular layer, where the dendritic processes reside. Right. Example of long-term potentiation. The initial response fPSP (left, shown in blue) is increased significantly (middle and right, shown in red) following tetanic stimulation through the stimulating electrode.

2.9.2. LTD

Long-term depression (LTD) is another prominent form of synaptic plasticity. While LTD has been studied extensively, it is not favoured as a correlate of learning and is more commonly associated with the erasure or decay of memory (Tsumoto 1993). Considering that LTP is correlated with enhanced learning, that LTD is associated with impairing learning seems intuitive. However, LTD is not simply the mirror image of LTP, and a strong body of evidence supports that LTD occurs with learning in many behaviours (Collingridge et al. 2010). In particular, LTD may play a critical role in forming spatial cognitive maps (Kemp et al. 2007), motor learning mediated by the cerebellum (Ito 1986), novelty-evoked exploration and other behaviours (Collingridge et al. 2010). Further, besides its role in learning, LTD may also play a role in developing networks within the brain (Lisman 2011).

2.9.2.1. Types of LTD

LTD is a heterogeneous phenomenon and multiple forms have been discovered (Collingridge et al. 2010). Two principle forms of LTD exist and are mediated by ionotropic and metabotropic glutamate receptors (NMDA and mGlu receptors, respectively). This dissertation is primarily concerned with NMDA receptor-dependent LTD as this form of LTD can be regulated by extrasynaptic receptors. Other forms of LTD will not be discussed further.

NMDA receptor-dependent LTD can be elicited in the CA1 and DG in the in vitro slice preparation. Interestingly, experimental induction of LTD in the DG is considerably more difficult than induction of LTP, in both the in vitro and in vivo preparations (Collingridge et al.

2010).

2.9.2.2. NMDA receptor-dependent LTD

LTD is normally elicited following weak, low-frequency stimulation. While some forms of LTD appear to involve NMDA receptors, the exact role of the NMDA receptor is not straightforward. It is unclear, for example, which NMDA receptor subunits facilitate LTD. For example, while the nonspecific NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid impairs LTD, other antagonists targeting specific NMDA receptor subtypes have conflicting effects. Antagonists of specific NMDA receptor subtypes (such as ifenprodil) can occlude LTD, but this result is not always observed (Andersen et al. 2007).

Ca2+ influx through the NMDA receptor is important to the induction of NMDA receptor- dependent LTD. The observation that a reduction in Ca2+ influx could convert LTP into LTD not only verified that Ca2+ influx was important in generating LTD, but also suggested that a low magnitude Ca2+ transient was specifically required for LTD induction (Cummings et al.

1996). It has been speculated that this low Ca2+ transient would activate a distinct population of high affinity protein phosphatases, such as protein phosphatase 2B (PP2B). PP2B is thought dephosphorylate phosphoprotein called inhibitor 1 (I1), rendering it inactive. As a result of this deactivation of I1, protein phosphatase 1 (PP1) is no longer inhibited. PP1 then desphorylates and deactivates CaMKII. More importantly, this phosphatase cascade may lead to a dephosphorylation of the AMPA receptor and a subsequent reduction in receptor expression at the synapse. The end result of this would be a reduction in synaptic efficacy.

Table 2.1. δGABAA receptor expression is affected by pathophysiological and pharmacological states.

Condition Species/Sex Brain region/Cell type Effect Reference

Alcohol exposure/withdrawal Rat - Cerebellar granule cells ↓ δ subunit mRNA (Biggio et al. 2007)

Rat - Cerebellar granule cells ↓ THIP-sensitive current (Biggio et al. 2007)

Rat M Hippocampus ↓ δ subunit protein (Cagetti et al. 2003)

Rat M Hippocampus (DG, CA1) ↓ Surface δ subunit protein (Gonzalez et al. 2012)

Rat M Hippocampus (CA1) ↓ THIP-sensitive current (Liang et al. 2004)

Rat M Hippocampus (CA1) ↓ Surface δ subunit protein (Liang et al. 2007)

Rat M Hippocampus ↓ δ subunit protein (Marutha Ravindran et al. 2007)

Mouse - Hippocampus ↓ Surface δ protein (Shen et al. 2011)

Mouse M Hippocampus ↑ Internalized δ subunit (Suryanarayanan et al. 2011)

Monkey M Dorsolateral prefrontal cortex ↓ δ subunit mRNA (Hemby et al. 2006)

Human - Orbitofrontal cortex ↓ δ subunit mRNA (Jin et al. 2011)

Rat M Hippocampus (DG, CA1,3) ↑ δ subunit mRNA (Pisu et al. 2011)

Rat - Cerebellum ↑ δ subunit mRNA (Follesa et al. 2005)

Rat M Perifornical hypothalamus ↑ δ subunit mRNA (Volgin 2008)

Rat M Perifornical hypothalamus ↑ Behavioural effects of THIP (Volgin 2008)

Brain injury Rat M Hippocampus ↑ δ subunit protein (Kharlamov et al. 2011a)

Rat M DG granule cells ↑ THIP-sensitive current (Mtchedlishvili et al. 2010)

Depression/Depressive-like Human - Frontopolar brain ↓ δ subunit mRNA (Merali et al. 2004)

Human M Ventral frontal cortex ↑ δ subunit gene (Klempan et al. 2009)

Rat M Ventral Pallidum ↑ δ subunit mRNA (Skirzewski et al. 2011)

Rat M DG granule cells ↑ THIP-sensitive current (Holm et al. 2011)

Epilepsy models Rat M DG ↓ δ subunit mRNA (Nishimura et al. 2005)

Mice M DG ↓ δ subunit mRNA (Zhang et al. 2007) 85

Mice M DG ↓ Neurosteroid sensitivity (Zhang et al. 2007)

Noda rat M DG ↓ δ subunit protein (Pandit et al. 2013)

Noda rat M DG ↓ THIP-sensitive current (Pandit et al. 2013)

Mouse M DG molecular layer ↓ δ subunit labelling (Peng et al. 2004)

Mouse M DG interneurons ↑ δ subunit labelling (Peng et al. 2004)

Rat - DG granule cells ↑ δ subunit mRNA (Brooks-Kayal et al. 1998)

Rat M Hippocampus ↑ δ subunit labelling (at 1-2d) (Schwarzer et al. 1997)

Rat M Hippocampus ↑ δ subunit labelling (at 30d) (Schwarzer et al. 1997)

Fragile X models FMR1 M Cortex ↓ δ subunit (D'Hulst et al. 2006)

FMR1 M Hippocampus, cortex ↓ δ subunit (Gantois et al. 2006)

FMR1 M Subiculum ↓ δ subunit mRNA (Curia et al. 2009)

FMR1 M Forebrain ↓ δ subunit mRNA (Adusei et al. 2010)

Schizophrenia Human - Dorsolateral prefrontal cortex ↓ δ subunit mRNA (Hashimoto et al. 2008a)

Human - Dorsolateral prefrontal cortex ↓ δ subunit mRNA (Hashimoto et al. 2008b)

Human - Dorsolateral prefrontal cortex ↓ δ subunit mRNA (Maldonado-Aviles et al. 2009)

Pentobarbital Mouse M Cerebellum ↓ δ subunit mRNA (Lin et al. 1996)

Stress Rat M Parvocellular hypothalamus ↑ δ subunit mRNA (Verkuyl et al. 2004)

Mouse M Hippocampus ↑ δ subunit mRNA (Maguire et al. 2007)

Mouse M DG ↑ δ subunit labelling (Sanna et al. 2011)

Mouse M Hippocampus ↑ δ subunit mRNA (Sanna et al. 2011)

Mouse M DG granule cells ↑ THIP-sensitive current (Sanna et al. 2011)

Rat M CA1, CA3, DG ↑ δ subunit labelling (Serra et al. 2006)

Rat M DG granule cells ↑ Neurosteroid sensitivity (Serra et al. 2006)

Rat M DG granule cells ↑ THIP-sensitive current (Holm et al. 2011)

Chapter 3. Methods

In this Chapter is a general description of the methods employed in this thesis. More specific and detailed descriptions are included in Chapters 4 and 5.

3.1. Animals

All procedures were approved by the local Animal Care Committee and employed 2- to 6- month-old male mice. This age range in mice corresponds to the age range of early adulthood in humans (Flurkey et al. 2007). Wild-type (WT) mice (C57BL/6 × SvJ129) and transgenic mice either heterozygous (Gabrd+/–) or null for the δ subunit gene (Gabrd–/–) were used

(Mihalek et al. 1999). Gabrd–/– mice exhibit no overt phenotype abnormalities (bodyweight, brain size, etcetra) but do have a slightly reduced birth rate (Mihalek et al. 1999). Some studies also used mice lacking the α5 subunit gene (Gabra5–/–) and their WT controls

(C57BL/6 × SvEv129) (Martin et al. 2010). These mice are also phenotypically normal

(Collinson et al. 2002). The C57BL/6 background was preferred as mice of this background exhibit robust fear memory and the capability for fear extinction (Balogh et al. 2003,

Macpherson et al. 2013). Experimenters were blinded to genotypes and treatment conditions.

Prior to all behavioural experiments, animals were handled in 5 min epochs for between 3-5 days.

3.2. Behavioural methods

3.2.1. Memory assays

3.2.1.1. Object-place recognition

In the object-place recognition assay, animals were required to discriminate between two arrays of objects that differ in spatial arrangement (Saab et al. 2009). This assay was performed in an exposure chamber with opaque walls (20 cm × 20 cm × 20 cm) on which different visual cues were mounted (Figure 2.12). The day before testing, each animal was habituated to the chamber for 15 min. The next day, during the training phase, the animal was placed in the chamber for 2 min with an array of three identical objects (e.g., toy frogs, cars or blocks, designated O1, O2, and O3). The animal was then removed for 2 min, during which time the chamber was cleaned with 70% ethanol and one of the objects (O3) was placed in a new location. The short length of time between training and testing periods was used to facilitate accurate measurement of working memory (Ennaceur 2010). During the testing phase, the animal was exposed to this new environment and the trial was recorded on videotape. Afterwards, the trial was viewed by an experimenter blind to condition and the interaction time of the mice with all objects (in seconds) was measured. An interaction was defined as an active investigation of the object when the mouse was within 1 cm of the object and oriented towards it. As an a priori exclusion criterion, animals with a total interaction time

(O1 + O2 + O3) less than 3 s were omitted from analysis. Preference for the displaced object

(%) was calculated as O3/(O1 + O2 + O3) × 100. Successful performance was deemed to have occurred if object preference exceeded chance (33%).

3.2.1.2. Novel object recognition

The novel object recognition assay followed a similar protocol to the object-place recognition assay (Saab et al. 2009), only instead of an object being displaced, an object was replaced

88 with a novel one (Figure 2.12). In most experiments, novel object recognition was measured on a separate day than object-place recognition.

3.2.1.3. Contextual discrimination

To assess contextual discrimination, an exposure chamber (25 cm × 25 cm × 30 cm) with a shock grid floor consisting of stainless steel bars (2 cm apart, diameter = 2 mm) was used

(Med Associates Inc., St. Albans, VT, USA). Within this chamber, two distinct contexts were generated through the manipulation of various cues (either visual, textural, spatial or olfactory).

In the difficult discrimination task, each mouse was exposed to two similar chambers that differed only in visual cues (lighting/wallpaper). The mouse received a footshock in the

“shock” chamber but not the “safe” chamber. Each mouse was pre-exposed to the shock chamber for 1-3 days and then exposed to both chambers for 9 consecutive days (Figure

2.11) (Sahay et al. 2011). Each exposure began with a habituation period of 180 s followed by a 2 s, 0.70-mA footshock; the mouse was removed 15 s later. The percentage of time spent

“freezing” (absence of movement except respiration) was scored for each chamber exposure.

All scoring was done by computerized FreezeFrame software. Chambers were cleaned with

70% ethanol between exposures. Successful difficult discrimination was defined by two criteria: (1) FreezingShock > FreezingSafe or (2) normalized discrimination ratio > 0.5. The normalized discrimination ratio was calculated as follows: (FreezingShock)/(FreezingShock +

FreezingSafe).

In the easy discrimination task, freezing in the shock chamber was compared to freezing in a highly dissimilar "novel" chamber with a new floor, a modified shape, and a

89 different scent. Easy discrimination was measured once on the final day of discrimination testing.

3.2.1.4. Fear extinction

Each animal was exposed to the chamber and given a footshock as described above for 3 days to acquire contextual fear conditioning (termed the acquisition period, Fig 5.3). The animal was then re-exposed to the same chamber for 180 s once per day for a further 10 days without being shocked (termed the extinction period, Days 0–9). The percentage time spent freezing during each exposure period was calculated. Summarized data for every 3 days of fear extinction (D1–3, D4–6, D7–9) were analyzed.

3.2.2. Other assays

3.2.2.1. Elevated plus maze

The elevated plus maze is an assay of anxiety (Sahay et al. 2011). In this test, animals are placed in a plus-shaped maze that is elevated significantly above the ground. All trials were 5 min and scored by an experimenter blind to condition. The time spent in open and closed arms of the maze was recorded. A high proportion of time spent in the closed arms is deemed to reflect high anxiety, while time spent in the open arms reflects low anxiety.

3.2.2.2. Electroshock sensitivity

Electroshock sensitivity was measured using methods defined previously (Tronel et al. 2010).

In this test, the animal was placed in a standard fear conditioning chamber. It then received seven shocks of incrementally increasing intensity (from 0.1-0.7 mA). Each shock was separated by 30 seconds. The flinching, running/jumping and vocalization responses to each

90 shock were scored using a binary system ('0' or '1') by an observer blind to condition. The probability of each response was then calculated for each group.

3.3. Electrophysiological methods

3.3.1. Slice preparation

Mice were anaesthetized with isoflurane and their brains were harvested and placed in oxygenated artificial cerebrospinal fluid (aCSF) (composition in mM: 124 NaCl, 3 KCl, 1.3

MgCl2, 2.6 CaCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 D-glucose) with the osmolarity adjusted to 300–310 mOsm. Coronal hippocampal slices (350-400 µm thick) were then cut with a vibratome (VT1000E tissue slicer; Leica, Deerfield, IL). This method is ideal for the examination of electrophysiological function within the CA1 and DG subfields of the dorsal hippocampus, which was the primary focus of this thesis. Other methods are better suited for the examination of electrophysiological function in the CA3 subfield, such as the thick slice preparation (Wong et al. 2005). Slices were allowed to recover for at least 1h before being transferred to the recording chamber. In the recording chamber, slices were perfused with aCSF (~3mL/min).

3.3.2. Extracellular field recordings of long-term potentiation

In all experiments, extracellular field postsynaptic potentials (fPSPs) were recorded using an aCSF-filled borosilicate glass electrode (World Precision Instruments, Sarasota, FL, USA) following stimulation of the relevant excitatory pathway with a bipolar tungsten electrode

(Rhodes Medical Instruments, Summerland, CA, USA). The distance separating the recording and stimulating electrodes was kept relatively constant throughout experiments (~300-400

µM), as this distance can affect the characteristics of the field response.

In experiments examining DG plasticity, the recording and stimulating electrodes were placed in the stratum moleculare and medial perforant pathway, respectively (Fig 2.13).

Placement of the electrodes was verified by the presence of paired pulse depression at an interval of 50 ms (Christie et al. 1994). Baseline fPSPs were then measured at 0.05 Hz using a stimulation intensity that produced a half-maximal response. Following a stable baseline period of 10 min, an LTP-inducing stimulation protocol was applied (Snyder et al. 2001). This protocol has been shown to elicit LTP without pharmacologic blockade of GABAA receptor- mediated inhibition (Snyder et al. 2001). This protocol consisted of 4 stimulus trains of 50 pulses at 100 Hz with an inter-train interval of 20 s.

In experiments examining CA1 plasticity, the recording and stimulating electrodes were placed in the stratum radiatum and Schaffer collateral pathway, respectively. Baseline responses were obtained as normal but an alternate protocol was used to induce LTP. This protocol consisted of 10 stimulus trains of 4 pulses at 100 Hz with an inter-train interval of 500 ms (Martin et al. 2009).

All drugs (3.4) were allowed to perfuse the slice for at least 15 min before recording.

3.3.3. Whole cell recordings

Hippocampal slices were obtained from 14- to 21-day-old male mice. Mice of this age range were utilized as their brains exhibit are more resistant to the dissection process (Moyer et al.

1998) and offer a larger population of healthy cells for easier patching. These cells show significant δGABAA receptor expression and δGABAA receptor-mediated currents (Laurie et al. 1992, Shen et al. 2011). All recordings were obtained from cells located in the granule cell layer of the DG that were visually identified with a Olympus BX51WI microscope (Center

Valley, PA, USA). Recording pipettes (3–5 MΩ) were filled with the intracellular solution

92 containing (in mM) 140 CsCl, 11 ethylene glycol tetra-acetic acid, 10 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid, 2 K2-ATP, 1 CaCl2, 2 MgCl2 and 2 tetraethylammonium with osmolarity adjusted to 290–295 mOsm and pH adjusted to 7.3. To block glutaminergic neurotransmission and voltage-dependent sodium channels, 6-Cyano-7-nitroquinoxaline-2,3- dione (10 µM), (2R)-amino-5-phosphonovaleric acid (40 µM), and tetrodotoxin (0.5 µM) were added to the ACSF. All recordings were performed at a holding potential of –70 mV, sampled at 10 kHz and filtered at 2 kHz by a low-pass Bessel filter. Cells were included in analysis only if they had an access resistance of ≤ 20 MΩ and this resistance did not vary by more than

20% during the recording period. We restricted our experiments to the measurement of inhibitory currents and did not measure membrane potential or other intrinsic membrane properties extensively, as previous investigations have found no differences in membrane properties between WT and Gabrd–/– mice (Spigelman et al. 2002).

3.4. Drugs

3.4.1. THIP

The δGABAA receptor–preferring agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol

(THIP; Tocris Bioscience, Bristol, UK) was used to preferentially activate δGABAA receptors

(Brown et al. 2002) in some behavioural and electrophysiologic experiments. THIP is described in more detail in 2.4.1.1.

3.4.1.1. THIP - Electrophysiologic experiments

THIP (1 µM) was employed in electrophysiologic experiments as recombinant models have shown that this concentration selectively activates human α4βδ GABAA receptors expressed in mouse L(-tk) cells (Brown et al. 2002). Further, in vitro studies have shown that concentrations of THIP in the 1 - 5 µM significantly increase tonic inhibitory conductance 93 without appreciably affecting synaptic events (Herd et al. 2009, Maguire et al. 2005).

Importantly, Gabrd–/– mice and Gabra4-/- mice, which lack δGABAA receptors, are insensitive to concentrations of THIP in the µM range (Chandra et al. 2006, Maguire et al.

2005, Winsky-Sommerer et al. 2007).

3.4.1.2. THIP - Behavioural experiments

In behavioural experiments, two drug treatment protocols were used to assess the effects of acute and long-term enhancement of δGABAA receptor function. In the acute protocol, a single dose of THIP (4 mg/kg i.p.) was administered and behaviour was tested 30 min later as

THIP levels peak at this time (Cremers et al. 2007) (Figure 3.1). At this dose, THIP is mildly analgesic in WT but not Gabrd–/– mice and has no significant hypnotic or motor-impairing effects (Bonin et al. 2011, Winsky-Sommerer et al. 2007). The long-term THIP protocol was designed to chronically influence the development of adult-born neurons, which require several weeks to mature (Snyder et al. 2009), and to ensure that THIP was eliminated at the time of behavioural testing. The long-term treatment protocol involved a daily injection of THIP

(4 mg/kg i.p.) for seven consecutive days. Two weeks later, behavioural or neurogenesis experiments were conducted (Figure 3.2). In both protocols, saline (vehicle) served as the control solution.

3.4.2. BIC

To block both phasic and tonic inhibition, competitive GABAA receptor agonist bicuculline methiodide (BIC, obtained from Tocris Bioscience) was used. At concentrations at or greater than 10 µM, BIC blocks the majority of both phasic and tonic inhibition in hippocampal slice (Bai et al. 2001, Nusser et al. 2002) (2.4.1.4). In slice experiments,

94 concentrations of 10, 20 and 100 µM were used. Unless otherwise stated, the concentration employed is 100 µM.

3.4.3. SR-95531

The competitive GABAA receptor agonist SR-95531 (obtained from Tocris Bioscience) was used in some experiments. Low concentrations of SR-95531 selectively block phasic inhibition while minimally affect tonic inhibition (Bai et al. 2001, Stell et al. 2002) (2.4.1.4).

3.5. Statistics and analysis

All results are reported as mean ± standard error of the mean (SEM). For analysis of acquisition data (during Morris water maze, fear conditioning, contextual fear discrimination and extinction experiments), a MANOVA with the appropriate within subject and between subject factors was used and followed by the appropriate post-hoc test. In all other cases, statistical significance was assessed using the student’s t-test, one-way ANOVA (followed by

Tukey’s post-hoc test) or two-way ANOVA (followed by Bonferroni post-hoc test), as appropriate. Differences between groups were considered significant at p < .05. Analysis was conducted using Graphpad Prism 5.0 and SPSS17 for Windows.

Figure 3.1. Acute THIP treatment protocol.

In the acute treatment protocol, the drug is admnistered and memory is measured 30 minutes later, when the levels of the drug in the brain are expected to peak (Cremers et al. 2007). This protocol therefore measures memory under the influence of the drug.

Figure 3.2. Long-term THIP treatment protocol.

In the long-term treatment protocol, the drug is administered daily for one week. Memory and neurogenesis are measured two weeks later, when the drug is likely to be eliminated. This protocol is therefore a pretreatment protocol which examines memory and neurogenesis when the drug is absent.

Chapter 4. δGABAA receptors promote memory performance regulated by the DG and adult-born neurons

This chapter is derived from the article "δGABAA receptors promote memory and neurogenesis" in the journal Annals of Neurology (Whissell et al. 2013). Behavioural data were collected by myself with the assistance of Irene Lecker, who performed all drug injections. All neurogenesis data (Figure 4.8, Figure 4.9) was collected by Dr. Shira

Rosenzweig.

4.1. Introduction

δGABAA receptors are expressed in several specific brain regions, including the cortex, cerebellum and thalamus (Farrant et al. 2005). Notably, these receptors are highly expressed in the dentate gyrus (DG) subfield of the hippocampus (Pirker et al. 2000), where they generate a tonic inhibitory conductance that constrains network excitability (Farrant et al.

2005, Maguire et al. 2009b). As excitability in the DG and hippocampus is correlated with memory performance, it has been widely assumed that increased δGABAA receptor activity impairs memory (Shen et al. 2010, Wiltgen et al. 2005). Surprisingly, there is little evidence to support this specific hypothesis, and the conditions under which δGABAA receptors regulate memory are highly specific. For example, studies of δGABAA receptor null mutant (Gabrd–/–) mice show that these receptors constrain fear-associated memory, but only in female mice when receptor expression or activity is greatly increased, such as during puberty (Shen et al.

2010, Wiltgen et al. 2005). Further, the effects of δGABAA receptor on memory performance in male mice are unclear (Mihalek et al. 1999, Wiltgen et al. 2005).

A regulatory role for δGABAA receptors in memory processes may have been overlooked due to the limited focus of previous behavioural investigations, which have probed only fear-associated memory (Mihalek et al. 1999, Shen et al. 2010, Wiltgen et al. 2005). The

DG, where δGABAA receptors are expressed at high levels, regulates a number of other cognitive processes, most notably pattern separation. Pattern separation refers to the discrimination or disambiguation of similar stimuli (e.g. objects or contexts) (Sahay et al.

2011) and may facilitate the discrimination of similar memories in humans (Aimone et al.

2011). In animals, the recognition of spatial novelty (Lee et al. 2005, Saab et al. 2009) and the discrimination of similar contexts (Sahay et al. 2011, Scobie et al. 2009) are forms of pattern separation, yet the role of δGABAA receptors in these behaviours has not been investigated.

Understanding the molecular substrates underlying pattern separation is of interest because the ability to distinguish between similar stimuli can be impaired in aging and neuropathologies such as Fragile X syndrome (Eadie et al. 2012, Yassa et al. 2011).

Many forms of memory regulated by the DG are facilitated by postnatal neurogenesis, a process whereby new neurons are generated in the adult brain (Marin-Burgin et al. 2012b).

These adult-born neurons constitute only a small percentage of the total population of granule cells in the DG (5-10%) (Nakashiba et al. 2012). However, adult-born neurons demonstrate enhanced synaptic plasticity and excitability (Marin-Burgin et al. 2012b) and play a critical role in DG-dependent behavioural tasks (Sahay et al. 2011). Enhanced neurogenesis secondary to exercise, genetic or pharmacologic interventions improves memory, whereas reduced neurogenesis secondary to lesions, radiation or neurotoxicity, impairs memory (Marin-Burgin et al. 2012b). Neurogenesis declines during aging and in pathologies such as ischemia and depression (Marin-Burgin et al. 2012b), conditions where memory is often impaired.

Neurogenesis in the DG is facilitated by tonic GABAergic signalling, as loss of a tonic depolarizing conductance impairs the development of synapses and dendrites in adult-born neurons (Ge et al. 2006). Because extrasynaptic δGABAA receptors are the major mediators of the tonic conductance in the DG, a potential physiological function of these receptors might be to promote neurogenesis.

Based on the above evidence, we postulated that δGABAA receptors enhance certain forms of DG-dependent memory and promote neurogenesis.

4.2. Specific Methods

4.2.1. Animals

All studies were approved by the local Animal Care Committee and employed 2- to 5-month- old male mice. Wild-type (WT) mice (C57BL/6 × SvJ129) and transgenic mice either heterozygous (Gabrd+/–) or homozygous null for the δ subunit gene (Gabrd–/–) were used

(Mihalek et al. 1999). Some studies used mice that lacked the α5 subunit gene (Gabra5–/–) and their WT controls (C57BL/6 × SvEv129) (Martin et al. 2010). Experimenters were blinded to genotypes and treatment conditions. Generally, an animal was utilized in only a single experiment and was not re-tested. However, in some experiments involving long-term THIP injection (Figures 4.3D-G), a small population of animals was tested on multiple behavioural assays. These assays were admnistered to the animal in the following order: object-place recognition, elevated plus maze and contextual discrimination.

4.2.2. Behavioural Experiments

100

4.2.2.1. Object-place recognition

One day before behavioural testing, each animal was habituated for 15 min in a chamber (20 cm × 20 cm × 20 cm) that was marked with visual cues (Saab et al. 2009). On the test day, each mouse was placed in the chamber for 2 min with three identical objects (O1, O2 and

O3), then removed for 2 min, during which time the chamber was cleaned with 70% ethanol and one of the objects (O3) was moved to a new location (Figure 4.1A, top). The mouse was then placed back in the chamber, and the time that it spent interacting with each object (sec) was measured. Preference for the displaced object (%) was calculated as follows: O3/(O1 +

O2 + O3) (Saab et al. 2009).

4.2.2.2. Novel object recognition

A protocol similar to that described above was used; however, during the 2-min removal period, one of the familiar objects was replaced with a novel object and preference for the novel object was measured (Fig 4.1B, bottom) (Saab et al. 2009).

4.2.2.3. Contextual discrimination

An exposure chamber (25 cm × 25 cm × 30 cm) with a shock grid floor consisting of stainless steel bars (2 cm apart, diameter = 2 mm) was used for this task (Med Associates Inc., St.

Albans, VT, USA). In the “difficult” discrimination task, each mouse was exposed to two similar chambers that differed only in visual cues (lighting/wallpaper). The mouse received a footshock in the “shock” chamber but not in the “safe” chamber. Each mouse was pre- exposed to the shock chamber and then exposed to both the safe and shock chambers for 9 consecutive days (Figs 4.2A, 4.4G) (Sahay et al. 2011). Each exposure session began with a habituation period of 180 s that was followed by a 2 s, 0.70-mA footshock. The mouse was

101 removed from the chamber 15 sec later. The percentage of time spent “freezing” (absence of movement except respiration) was scored each time the mouse was exposed to the chamber.

Chambers were cleaned with 70% ethanol between exposures. Successful difficult discrimination was defined by two criteria: (1) FreezingShock > FreezingSafe and (2) normalized discrimination ratio > 0.5. The normalized discrimination ratio was calculated as follows:

(FreezingShock)/(FreezingShock + FreezingSafe). In the easy discrimination task, freezing was compared in the shock chamber and a highly dissimilar "novel" chamber which had a novel floor, shape and scent. Easy discrimination was measured once, on the final day of discrimination testing.

4.2.2.4. Fear extinction

Each animal received a footshock at either 0.45 mA or 0.70 mA, as described above for 3 consecutive days (acquisition, Figs 4.1A,D). The animal was then re-exposed to the same chamber for 180 s once per day for a further 10 days without being shocked (extinction, Days

1–9). Data for every 3 days of fear extinction (D1–3, D4–6, D7–9) were summated.

4.2.2.5. Elevated Plus Maze

This assay was employed to study anxiety (Sahay et al. 2011). Time spent within open and closed arms was recorded. A high percentage of time in the closed arms of the maze is deemed to reflect high anxiety.

4.2.2.6. Electroshock sensitivity

To study nociception, mice received a footshock (0.7 mA) as described above, and the responses (flinching, running/jumping, and vocalization) were scored (Tronel et al. 2010).

4.2.3. Histological experiments 102

Adult-born neurons were labeled with 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, St.

Louis, MO, USA) by administering a dose of 100 mg/kg by intraperitoneal (i.p.) injection.

Hippocampal coronal sections (40 µM) were prepared from mice injected with BrdU

(Rosenzweig et al. 2011). Sections were immunostained for BrdU and additional markers, including Ki-67, a marker of proliferating cells, doublecortin (DCX), a marker of immature cells and calbindin (CaBP), a marker of mature cells, as described previously (Rosenzweig et al.

2011, Wojtowicz et al. 2006). Quantification of labelled cells and dendrites was performed with a fluorescence microscope (Optiphot-2, Nikon Inc., Melville, NY, USA) and ImageJ software (National Institute of Health) (Rosenzweig et al. 2011).

4.3. Results

4.3.1. δGABAA receptors promote recognition memory

The role of δGABAA receptors in recognition memory was first studied using the object-place recognition assay, which tests spatial memory (Dix et al. 1999, Lee et al. 2005, Saab et al.

2009). In this assay, mice must discriminate between two sequential arrays of three objects that only differ in their spatial arrangement (Figure 4.1A, top). During training, the animals were allowed to explore the objects freely. During the testing phase, mice were exposed to a new arrangement of the objects in which one had been displaced. Because animals are attracted to novelty, they tend to interact more with the displaced object if they recognize that its location has been changed (Dix et al. 1999). As such, increased preference for the displaced object is deemed to indicate recognition of spatial novelty. Gabrd–/– mice exhibited a reduced preference for the displaced object relative to WT mice (p < 0.05; Figure 4.1B, left).

This difference between genotypes could not be attributed to any differences in exploratory drive as the total object interaction time did not differ between groups (p = 0.65; Table 4.1).

103

To determine whether non-spatial recognition memory was also impaired, mice were studied using a novel object recognition task (Denny et al. 2012, Dix et al. 1999, Saab et al.

2009). In this test, the mice must recognize a novel object within a set of familiar objects to which they were previously exposed (Figure 4.1A, bottom). Again, because animals are driven to investigate novelty, mice that recall the familiar objects will preferentially interact with the novel object (Dix et al. 1999). Gabrd–/– mice were impaired relative to WT mice, as evidenced by their lower object preference scores (p < 0.05; Figure 4.1B, right). Interaction times did not differ between genotypes, as observed in the previous test (p = 0.98; Table 4.1).

Since tonic conductance in the DG is generated by both α5GABAA receptors and

δGABAA receptors (Glykys et al. 2008), we also investigated whether the regulation of recognition memory could be generalized to another source of tonic GABAergic conductance.

Accordingly, the performance of WT and Gabra5–/– mice was compared for the object-place recognition and novel object recognition tasks. No differences were observed between these genotypes on either task (all ps > 0.05; Figure 4.1C). Also, WT and Gabra5–/– mice did not differ in total interaction times on either test (all ps > 0.05; Table 4.1). Collectively, these results show that δGABAA receptors, but not α5GABAA receptors, promote recognition memory.

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Figure 4.1. Recognition memory was impaired in Gabrd–/– mice.

A. Schematic diagram showing the protocol. B. Gabrd–/– mice demonstrated a reduced preference for both the displaced and novel objects compared with WT mice (n = 13-18). C.

Gabra5–/– mice did not differ from WT mice in preference for the displaced or novel object (n

= 15-18).

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Table 4.1. Interaction time is recognition memory assays is not affected by expression of

δGABAA receptors or α5GABAA receptors.

Interaction time (s) Genotype Object-place Novel object Gabrd+/+ 9.1 ± 1.1 8.7 ± 2.0 Gabrd–/– 8.5 ± 1.0 8.8 ± 1.0

Gabra5+/+ 8.0 ± 0.8 9.4 ± 1.3 Gabra5-/- 9.5 ± 0.8 9.5 ± 1.4

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4.3.2. δGABAA receptors promote difficult contextual discrimination

The object-place and novel object recognition tasks probe short-term working memory because they test memory immediately following a short, one-trial training period (Dix et al.

1999). To study long-term memory, a contextual discrimination task that required extended training was used. In this task, mice learn to discriminate between a chamber in which they receive a mild electric shock ("shock" chamber) and a highly similar chamber in which they are not shocked (“safe” chamber) after many days of training. If discrimination is successful, mice freeze more in the shock chamber than in the safe chamber. The two chambers differ only in terms of visual cues, which makes discrimination extremely difficult. Contextual discrimination is considered a sensitive assay of DG function and is promoted by neurogenesis (Sahay et al. 2011, Scobie et al. 2009).

A 9-day protocol that produces discrimination by the final day of testing was adopted

(Figure 4.2A) (Sahay et al. 2011). Freezing scores and normalized discrimination ratios were compared between genotypes on Day 9. WT mice but not Gabrd–/– mice froze more in the shock chamber than the safe chamber (p < 0.05; Figure 4.2B, left; Table 4.2). An analysis of discrimination ratios further illustrated that WT mice learned to distinguish between the shock and safe chambers better than Gabrd–/– mice (p < 0.001; Fig 4.2B, right).

Next, to determine whether δGABAA receptors played a role in an easy discrimination task, the ability to discriminate a highly dissimilar “novel” chamber from the shock chamber was examined (Figure 4.2C). The two chambers differed considerably in terms of visual, tactile, spatial and olfactory cues. As the DG is not required for easy discrimination tasks (Lee et al. 2010), it was anticipated that δGABAA receptors would not contribute to performance on this assay. As predicted, the two genotypes exhibited similar discrimination ratios (p = 0.78;

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Figure 4.2C) and similar freezing scores in the novel chamber (Table 4.2). Collectively, these results are consistent with the notion that δGABAA receptors promote difficult but not easy contextual discrimination. This difference in contextual discrimination was not due to differences in the level of anxiety or nociception, as performance in the elevated plus maze

(Figure 4.3) and sensitivity to the shock did not differ between groups (Figure 4.4).

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Figure 4.2. Difficult contextual discrimination is impaired in Gabrd–/– mice.

A. Schematic diagram showing the protocol. Discrimination was compared on day 9. B. WT mice but not Gabrd–/– mice showed more freezing in the shock chamber than in the safe chamber. WT mice also demonstrated a higher discrimination ratio (n = 24-31). C. Both WT and Gabrd–/– mice showed comparable discrimination ratios on the easy discrimination task

(n = 7-15).

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Table 4.2. Absolute freezing (%) in shock, safe and novel chambers for contextual discrimination during a 9-day protocol (Fig 4.2A to 4.2C).

Days 1-8 Day 9 Genotype Shock Safe Shock Safe Novel 73.9 ± 2.0 71.7 ± 2.3 77.8 ± 2.1 63.1 ± 3.1 40.3 ± 3.8 WT (n = 31) (n = 31) (n = 31) (n = 31) (n = 15) 67.3 ± 2.3 69.0 ± 2.4 75.5 ± 2.2 75.8 ± 2.2 40.8 ± 5.5 Gabrd–/– (n = 24) (n = 24) (n = 24) (n = 24) (n = 7)

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Figure 4.3. Elevated plus maze performance is not regulated by δGABAA receptors.

WT and Gabrd–/– mice spent similar amount of time in the closed or open arms. All n values

≥ 10.

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Figure 4.4. Electroshock sensitivity is not regulated by δGABAA receptors.

Flinching, running/jumping and vocalization responses are comparable in WT and Gabrd–/– mice. All n values ≥ 10.

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4.3.3. δGABAA receptors regulate the acquisition and extinction of conditioned fear

We next studied whether δGABAA receptors regulate the acquisition and extinction of fear memory, as GABAA receptors are involved in these forms of learning (Makkar et al. 2010).

Mice were first fear conditioned using a shock intensity of 0.70 mA and a 3-day acquisition protocol. Extinction was tested by returning the animal to the context without delivering the footshock over nine days (Figure 4.5A). On day 2 of acquisition, Gabrd–/– mice froze slightly more in the shock chamber than the WT mice (p < 0.05; Figure 4.5B), suggesting enhanced acquisition of the conditioned fear response in Gabrd–/– mice. However, by day 3 of acquisition, WT and Gabrd–/– mice exhibited comparable levels of fear memory. Notably, after 3 days of fear extinction (D1–3), Gabrd–/– mice exhibited higher levels of freezing than the WT mice (p < 0.05; Figure 4.5C). After 3 additional days (D4–6), greater freezing was still evident in Gabrd–/– mice (p < 0.05) whereas freezing was similar between genotypes after 9 days (D7–9, p > 0.05).

The above results could indicate that δGABAA receptors promote fear extinction.

Alternatively, the apparent impaired extinction in Gabrd–/– mice could result from higher initial fear levels during acquisition. Higher levels of fear may not have been evident due to an asymptote in freezing behaviour associated with the high intensity shock stimulus (0.70 mA).

Therefore, the experiment was repeated using a low intensity shock stimulus (0.45 mA,

Figure 4.5D). Freezing responses were greater in Gabrd–/– mice on day 2 and day 3 of training (all ps < 0.05). On day 4, freezing also tended to be greater (p = 0.16, Figure 4.5E).

On all days of extinction, Gabrd–/– mice showed higher freezing scores than WT mice (Figure

4.5F). Together, these results suggest that δGABAA receptors constrain fear acquisition but may also promote fear extinction. Additionally, or alternatively, differences in acquisition of fear memory may contribute to impaired extinction in Gabrd–/– mice. 113

Figure 4.5. Acquisition of conditioned fear is enhanced but extinction of fear is impaired in

Gabrd–/– mice.

A. Schematic diagram showing the protocol for the high intensity shock (0.70 mA) experiment.

B. At a high shock intensity, Gabrd–/– mice showed more freezing than WT mice on day 2 of acquisition. However, freezing was comparable in both genotypes on days 3 and 4 of acquisition (n = 14-15). C. Gabrd–/– mice showed more freezing after three (D1-3) and six days (D4-6) of extinction. After nine days (D7-9), freezing was comparable in both genotypes.

D. Schematic diagram showing the protocol for the low intensity shock (0.45 mA) experiment.

E. At this shock intensity, Gabrd–/– mice showed more freezing than WT mice on day 2 and day 3 of the acquisition period (n = 10-17). F. Gabrd–/– mice showed more freezing for all nine days of extinction (D1-9).

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4.3.4. Acute treatment with THIP does not enhance memory

Since reduced expression of δGABAA receptors generally impairs DG-dependent memory, then pharmacologically increasing receptor activity may enhance memory performance. To test this hypothesis, WT and Gabrd–/– mice were treated with a single acute dose of THIP (4 mg/kg i.p.), a drug that preferentially activates δGABAA receptors (Brown et al. 2002), 30 min before behavioural testing (Figure 4.6A) (Bonin et al. 2011). The object-place recognition assay was selected because it revealed a distinct difference between genotypes and is relatively high throughput compared to the contextual discrimination assay. Vehicle-treated

WT mice performed better than Gabrd–/– mice, as described above (main effect of genotype, p < 0.01; Figure 4.6B, left). However, acute treatment with THIP had no effect on memory performance in either genotype.

It is possible that THIP may have failed to enhance performance in WT mice because of a “performance ceiling” in the object-place recognition assay. Thus, we next studied heterozygous mice (Gabrd+/–) which might exhibit lower scores at baseline. THIP had no effect on performance of Gabrd+/– mice (p = 0.34, Figure 4.6B, right).

4.3.5. Long-term treatment with THIP enhances memory

As long-term enhancement of δGABAA receptors facilitates neurogenesis, it is possible that long-term activation of these receptors also facilitates memory. To study the effects of a sustained increase in δGABAA receptor activity, WT and Gabrd–/– mice were treated with a

“long-term” THIP regimen. Mice received a daily injection of THIP (4 mg/kg) for 7 days (Figure

4.6C). Object-place recognition was studied 2 weeks later. THIP did not improve object-place recognition in WT or Gabrd–/– mice (main effect of drug, p = 0.80; drug × genotype

115 interaction, p = 0.28; Figure 4.6D, left) but improved performance in Gabrd+/– mice (p < 0.05,

Figure 4.6D, right).

Next, to determine whether long-term THIP treatment improved long-term memory, a rigorous training protocol that allowed examination of extended contextual discrimination was employed (Figure 4.6E) (Tronel et al. 2010). THIP had no effect on the acquisition of the conditioned fear response (p > 0.50 for all drug effects and interactions; Figure 4.6F). During training on the difficult discrimination task (Days 1–8), WT mice performed better than Gabrd–

/– mice, as previously observed (main effect of genotype, p < 0.05; Figure 4.6G, left).

Performance also tended to be enhanced in THIP-treated WT and Gabrd+/– mice, although this difference was not significant (ps > 0.05 for the main effect of drug and drug × genotype interaction). On Day 9, the WT mice again performed better than Gabrd–/– mice (main effect of genotype, p < 0.05). Furthermore, THIP-treated WT and Gabrd+/– mice performed better than vehicle-treated controls (main effect of genotype, p < 0.05; p < 0.05 for all post-hoc comparisons; Figure 4.6G, right). THIP had no effect in Gabrd–/– mice. Freezing scores for this task are summarized in Table 4.3.

In an easy discrimination task, WT mice treated with THIP performed slightly better than vehicle controls (p < 0.05; Figure 4.6H), whereas THIP had no effect in Gabrd+/– or

Gabrd–/– mice. Also, the differences in contextual discrimination were not due to different levels of anxiety, as performance on the elevated plus maze was similar in all genotypes (p >

0.5 for all comparisons; Figure 4.7). Taken together, these data show that long-term treatment with THIP enhances memory performance for both object-place recognition and contextual discrimination tasks.

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Figure 4.6. Long-term but not acute treatment with THIP promotes memory.

A-B. Acute injection of THIP did not improve memory. A. Schematic diagram showing the acute injection protocol. B. Summarized data for object-place recognition (n = 16-21). C-H.

Long-term injection of THIP improved memory. C. Schematic diagram showing the long-term injection protocol. D. Long-term THIP increased preference for the displaced object in

Gabrd+/- mice but not in WT or Gabrd–/– mice (n = 12-21). E. Schematic diagram showing the protocol. F. Long-term THIP did not affect the acquisition of the conditioned fear response in WT or Gabrd–/– mice (n = 24-25). G. Long-term THIP enhanced difficult discrimination ratios in WT and Gabrd+/- mice, but not in Gabrd–/– mice (n = 14-25). H. Long-term THIP enhanced the easy discrimination ratio only in WT mice (n = 14-25).

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Table 4.3. Absolute freezing (%) in the shock, safe and novel chambers for extended contextual discrimination (Fig 4.4G,H).

Days 1-8 Day 9 Group Shock Safe Shock Safe Novel 68.5 ± 2.6 63.9 ± 2.4 72.5 ± 2.7 60.8 ± 3.1 30.4 ± 2.6 WT + Veh (n = 25) (n = 25) (n = 25) (n = 25) (n = 25) 68.3 ± 2.6 61.6 ± 3.1 78.3 ± 2.8 58.6 ± 3.3 24.1 ± 2.5 WT + THIP (n = 24) (n = 24) (n = 24) (n = 24) (n = 24)

67.9 ± 2.8 64.9 ± 2.1 68.3 ± 3.2 63.3 ± 3.4 37.7 ± 4.9 Gabrd+/- + Veh (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) 70.6 ± 3.6 65.3 ± 4.0 78.2 ± 3.6 61.2 ± 3.7 38.2 ± 3.9 Gabrd+/- + THIP (n = 14) (n = 14) (n = 14) (n = 14) (n = 14)

70.5 ± 3.5 68.6 ± 3.4 72.0 ± 3.9 63.9 ± 4.0 37.9 ± 2.7 Gabrd–/– + Veh (n = 24) (n = 24) (n = 24) (n = 24) (n = 24) 71.2 ± 3.2 69.5 ± 3.2 77.0 ± 3.5 68.1 ± 3.7 39.4 ± 3.4 Gabrd–/– + THIP (n = 21) (n = 21) (n = 21) (n = 21) (n = 21)

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Figure 4.7. Long-term THIP treatment does not affect anxiety.

Long-term treatment with THIP does not affect the time spent in the closed or open arms in

WT or Gabrd–/– mice. All n values ≥ 10.

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4.3.6. δGABAA receptors promote postnatal neurogenesis in the DG

Neurogenesis is facilitated by a tonic GABAergic conductance (Ge et al. 2006) and δGABAA receptors are the predominant source of a tonic current in the DG (Glykys et al. 2008). Thus, it was postulated that long-term activation of δGABAA receptors facilitates neurogenesis whereas genetic deletion of these receptors impairs this process. Neurogenesis within the DG was first compared in WT mice and Gabrd–/– mice. Proliferation of adult-born cells was studied by labelling proliferating cells with Ki-67. No baseline differences in Ki-67 expression were observed between genotypes (p = 0.38; Figure 4.8A). Survival of adult-born neurons was studied by injecting the mice with BrdU, which labels newly generated cells. The number of BrdU-positive (BrdU+) cells was comparable in WT mice and Gabrd–/– mice, at both 2 weeks and 4 weeks after injection of BrdU (all ps > 0.05; Figure 4.8B). Collectively, these results show that proliferation and survival of adult-born cells did not differ between genotypes.

During migration, adult-born granule cells move from the inner subgranular zone (SGZ) to the granule cell layer (GCL), and this process is regulated by GABA (Duveau et al. 2011).

Thus, we compared migration between WT and Gabrd–/– mice. The percentage of BrdU+ neurons in the SGZ was higher in Gabrd–/– mice at 2 weeks and 4 weeks after injection of

BrdU (all ps < 0.05; Figure 4.8C) suggesting that a lower proportion of adult-born neurons migrated to the GCL.

The maturation of adult-born neurons was also studied, as this property determines the electrophysiological properties of neurons and their ability to regulate memory behaviour

(Scobie et al. 2009). The expression of DCX, a reliable marker of immature neurons (Snyder et al. 2009), was compared in the DG of WT and Gabrd–/– mice. The density of immature,

DCX+ neurons was greater in Gabrd–/– mice compared to WT mice (p < 0.01; Figure 4.8D). 121

This increase in density of DCX+ neurons was attributed to a larger percentage of immature neurons because the proliferation and overall survival of adult-born neurons were similar in

Gabrd–/– and WT mice. Thus, the expression of δGABAA receptors promotes the maturation of adult-born neurons in the DG.

Next, since learning tasks induce significant changes in dendritic structures (O'Malley et al. 2000), including the complexity and dendritic branching of neurons, we studied branching of dendritic processes in immature neurons. We utilized a novel method that permits the simultaneous visualization of a large population of immature neurons

(Rosenzweig et al. 2011). In Gabrd–/– mice, the ratio of secondary and tertiary dendrites was lower than that in WT mice (all ps < 0.05; Figure 4.8G). These results suggest a reduction in dendritic branching of neurons in Gabrd–/– mice.

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Figure 4.8. δGABAA receptors promote neurogenesis in the dentate gyrus.

A-B. No significant difference in the number of immunoreactive cells for Ki-67 (A) and BrdU

(B) was observed between WT and Gabrd–/– mice. C. Migration of adult-born neurons was impaired in Gabrd–/– mice, as a significantly greater percentage of adult-born neurons was found in the subgranular zone (SGZ) in Gabrd–/– mice. Scale bar = 20 µM. D-F. Maturation of adult-born neurons was impaired in Gabrd–/– mice. D. A significant increase in the number of

DCX immunoreactive cells was detected in Gabrd–/– mice. Scale bar = 100 µM. E. There was a significant increase in the percentage of BrdU immunoreactive cells colabelled for DCX in

Gabrd–/– mice two weeks following BrdU injection. Scale bars = 15 µM. F. A significant decrease in the percentage of BrdU immunoreactive cells colabelled for CaBP in Gabrd–/– mice four weeks following BrdU injection was observed. Scale bars = 15 µM. G. The branching ratio of secondary and tertiary dendrites of the DCX immunoreactive cells was reduced in Gabrd–/– mice. Scale bar = 100 µM. All n values = 5-6.

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4.3.7. Long-term THIP treatment promotes the maturation of adult-born neurons

We next sought to determine whether long-term pharmacologic activation of δGABAA receptors with THIP (Figure 4.6C) enhances neurogenesis. THIP did not affect the survival of adult-born neurons, as the number of BrdU+ cells was similar in WT and Gabrd–/– mice (all ps > 0.05; Figure 4.9A). In contrast, a greater percentage of BrdU+ neurons was co-labelled for CaBP in THIP-treated WT mice but not THIP-treated Gabrd–/– mice (p < 0.05; Figure

4.9B). Interestingly, the baseline difference in CaBP+ neurons exhibited by WT and Gabrd–/– mice was no longer detected in the mice that received repeated injections. Collectively, these results suggest that the maturation of adult-born neurons is promoted by long-term treatment with THIP and this effect requires the expression of δGABAA receptors.

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Figure 4.9. Long-term THIP treatment accelerates maturation of adult-born neurons.

A. The number of immunoreactive cells for BrdU in WT and Gabrd–/– mice was similar in vehicle- and THIP-treated mice. n = 5-6. B. A significant increase in the percentage of BrdU immunoreactive cells colabelled for CaBP for THIP injection group was observed only in WT mice. All n values = 5-6. Scale bar = 15 µM.

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4.4. Discussion

These results identify a novel function of δGABAA receptors in promoting performance for certain memory behaviours. Reduced expression of δGABAA receptors impaired recognition memory, contextual discrimination and fear extinction but enhanced fear acquisition. In addition, pharmacological activation of δGABAA receptors with THIP facilitated object-place recognition and contextual discrimination. The impaired memory performance of Gabrd–/– mice was unexpected given that enhanced δGABAA receptor activity generally inhibits the excitability of neuronal networks (Maguire et al. 2009b, Shen et al. 2010) and increased

δGABAA receptor expression impairs fear-associated memory (Shen et al. 2010).

Furthermore, treatment with drugs that non-selectively activate GABAA receptors, including anesthetics, barbiturates and benzodiazepines, usually impairs memory performance

(Chavant et al. 2011, Makkar et al. 2010, Wang et al. 2011).

The impaired recognition memory and contextual discrimination exhibited by Gabrd–/– mice may have been due to disrupted neurogenesis in the DG. In Gabrd–/– mice, the percentage of immature, DCX+ adult-born neurons is greater than in WT mice. This suggests that comparatively more neurons in Gabrd–/– mice exhibit structural and electrophysiological properties that are similar to neurons 3 weeks of age or younger, as this corresponds to the timeframe of DCX expression (Snyder et al. 2009). At this age, adult-born neurons do not demonstrate significant synaptic plasticity or excitability and are unlikely to have formed functional connections to circuits within the hippocampus (Ge et al. 2007, Mongiat et al.

2009). These properties do not become apparent until 4–6 weeks of age, when adult-born neurons begin to play a critical role in learning (Denny et al. 2012, Gu et al. 2012, Nakashiba et al. 2012). Therefore, the majority of neurons in Gabrd–/– mice may not be sufficiently mature to participate in learning tasks. In support of this interpretation, a delay in the 127 maturation of adult-born neurons has previously been correlated with reduced performance for contextual discrimination (Scobie et al. 2009). Also, when neurogenesis is arrested or attenuated, animals exhibit deficits in contextual discrimination (Nakashiba et al. 2012), novel object recognition (Jessberger et al. 2009, Madsen et al. 2003) and extinction (Deng et al.

2009) in a manner similar to Gabrd–/– mice.

Consistent with our findings, genetic deletion of the α4 subunit, which typically partners with the δ subunit to form α4βxδ receptors, also impaired neurogenesis (Duveau et al. 2011).

The migration and dendritic development of adult-born neurons were disrupted in Gabra4–/– mice (Duveau et al. 2011). Interestingly, no change in neurogenesis in Gabrd–/– mice was observed in a previous study (Duveau et al. 2011). The discrepancy between these findings and our own may have been due to differences in experimental procedures. Specifically, the handling and injection of animals with BrdU was much more frequent in the previous study

(eight injections over 3 days) (Duveau et al. 2011). Experimental factors including handling and stress influence neurogenesis in laboratory animals (Lemaire et al. 2006). These factors are also a consideration in our long-term THIP experiment; extensive handling and injections and may account for the lack of differences in maturation between vehicle-treated WT and

Gabrd–/– mice.

Several lines of evidence suggest that the activity of δGABAA receptors in the DG accounts for the behavioural impairments exhibited in Gabrd–/– mice. Contextual discrimination depends on neurogenesis and is correlated with synaptic plasticity in the DG

(Kheirbek et al. 2012, Nakashiba et al. 2012, Sahay et al. 2011). Also, object-place recognition induces the expression of immediate early genes in the DG (Soule et al. 2008).

Performance of the object-place recognition task is correlated with synaptic plasticity in the

DG and is impaired by lesion of the DG (Lee et al. 2005, Saab et al. 2009). Novel object 128 recognition, although primarily mediated by the perirhinal cortex (Winters et al. 2010), may also be promoted by neurogenesis in the DG (Jessberger et al. 2009, Kheirbek et al. 2012,

Madsen et al. 2003).

Interestingly, acquisition of contextual fear conditioning was enhanced in Gabrd–/– mice. This finding is consistent with previous results which showed that α4βxγ2 receptors restrict this form of learning (Cushman et al. 2011, Moore et al. 2010, Wiltgen et al. 2005).

The enhanced acquisition in Gabrd–/– mice might be caused by the absence of δGABAA receptors on mature neurons in regions other than the DG, as this receptor population is predominantly inhibitory and typically constrains memory (Shen et al. 2010). Further supporting evidence shows that the DG (Nakashiba et al. 2012) and adult-born neurons may not play a major role in contextual fear conditioning (Deng et al. 2009, Shors et al. 2002,

Zhang et al. 2008a), although this depends upon the testing protocol (Drew et al. 2010). In comparison with enhanced acquisition, fear extinction was impaired in Gabrd–/– mice. A reduction in neurogenesis in DG was shown previously to disrupt extinction (Deng et al.

2009). However, in our study, enhanced acquisition might contribute to impaired fear extinction in Gabrd–/– mice, as stronger fear memories are typically resistant to extinction.

A role of δGABAA receptors in pattern separation is suggested by the impaired discrimination of similar contexts observed in Gabrd–/– mice. Pattern separation is a putative function of the DG and is thought to critically depend upon adult-born neurons (Aimone et al.

2011). Aimone and colleagues recently proposed a “memory resolution” hypothesis whereby adult-born neurons facilitate pattern separation by maximizing the information stored in the hippocampus and producing ‘high resolution' memories (Aimone et al. 2011). When neurogenesis is disrupted and the functional population of adult-born neurons is reduced, 'low resolution' memories are formed. These memories, akin to blurry and pixilated photographs, 129 are difficult to distinguish from each another. According to this perspective, one intriguing interpretation of this data is that Gabrd–/– mice acquire a stronger, but lower resolution fear memory, which is harder to extinguish and distinguish.

An outstanding question is whether there are sex-dependent differences in the role of

δGABAA receptors in DG-dependent memory and neurogenesis. Only male mice were employed in the current study as females show marked changes in δGABAA receptor expression during the estrous cycle (Maguire et al. 2009b). A previous study shows enhanced trace fear conditioning in female Gabrd–/– mice, but not males (Wiltgen et al. 2005). The effect of δGABAA receptors on learning in females may be due to changes in the expression of these receptors caused by fluctuating levels of steroid hormones during the estrous cycle

(Maguire et al. 2009b). It is of future interest to determine whether δGABAA receptors influence DG-dependent memory and neurogenesis in females. Also, it remains unknown whether δGABAA receptors directly regulate memory and neurogenesis, or act indirectly by modifying the activity of other receptor populations, such as N-methyl-D-aspartate or neurotrophin receptors (Kheirbek et al. 2012, Shimazu et al. 2006).

Finally, the current results may have therapeutic implications because the expression of δGABAA receptors is altered in various physiological and pathophysiological conditions

(Brickley et al. 2012). Long term-administration of neuroactive steroids, which are positive allosteric modulators of the δGABAA receptor, has been proposed as a treatment for the cognitive decline associated with aging and neurodegenerative diseases (Wang et al. 2007), although such compounds may impair memory in healthy controls (Matthews et al. 2002).

Similarly, our results suggest that long-term treatment with THIP enhances memory and neurogenesis.

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Chapter 5. Acute activation of δGABAA receptors impairs memory and synaptic plasticity in the hippocampus

This Chapter is based on the article, "Acute activation of δGABAA receptors impairs memory and synaptic plasticity in the hippocampus" which is currently under review by the journal

Frontiers in Neural Circuits. Figure 5.1C, Figure 5.2B and Figure 5.2C have appeared previously in Dave Eng's thesis document (Eng 2008) but have not been published. These figures are included here for completeness of story. For the experiment in Figure 5.3, Irene

Lecker assisted by performing drug injections. All remaining figures include novel data collected, analyzed and interpreted by myself.

5.1. Introduction

γ-Aminobutyric acid type A (GABAA) receptors are the primary mediators of inhibitory neurotransmission in the mammalian nervous system and are molecular targets for a variety of drugs that are used to treat anxiety, pain, seizures and insomnia (Sieghart 2006). Recently,

δGABAA receptors have attracted considerable attention as therapeutic targets because these receptors robustly regulate neuronal excitability in vitro (Maguire et al. 2009a, Stell et al.

2003) and modulate memory (Shen et al. 2010, Wiltgen et al. 2005), nociception (Bonin et al.

2011), maternal behaviour (Maguire et al. 2008) and the physiologic responses to stress in vivo (Holm et al. 2011, Sarkar et al. 2011).

Drugs that directly activate the δGABAA receptor, or act as positive allosteric modulators, have been suggested as potential therapeutic agents for the treatment of a wide variety of disorders including insomnia (Wafford et al. 2006), pain (Bonin et al. 2011), cognitive dysfunction (Wang et al. 2007) and mood disorders (Christensen et al. 2012,

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Maguire et al. 2008). The most widely studied of these compounds is THIP, a δGABAA receptor-preferring agonist (Brown et al. 2002). Hypnotic properties of THIP have been demonstrated in humans and laboratory animals (Faulhaber et al. 1997, Wafford et al. 2006) and analgesic properties of the drug have been confirmed in models of acute and persistent pain (Bonin et al. 2011). THIP and related δGABAA receptor-preferring compounds are promising candidates for long-term use because they may have a reduced risk for tolerance and addiction relative to other less selective enhancers of GABAA receptor activity, such as benzodiazepines (Ebert et al. 2008, Tan et al. 2011). THIP may also have nootropic effects, as it was recently demonstrated in a rodent model that a one week pre-treatment with the drug improved memory performance studied two weeks after drug treatment (Chapter 4) and also enhanced neurogenesis (Rosenzweig 2011).

While long-term treatment with THIP may be useful in a therapeutic context, several lines of evidence suggest that acutely increasing δGABAA receptor activity impairs memory.

First, activation of δGABAA receptors typically constrains neuronal firing (Bonin et al. 2011) and network excitability (Maguire et al. 2009a), which are molecular correlates of memory processes. Second, genetically modified mice deficient in δGABAA receptors exhibit improved fear-associated memory (Cushman et al. 2011, Moore et al. 2010, Wiltgen et al. 2005) which suggests that δGABAA receptors constrain at least this form of memory. Transgenic deletion of δ subunit, or its partner the α4 subunit, reduces expression of δGABAA receptors and enhances contextual fear memory in female and male mice, respectively (Cushman et al.

2011, Moore et al. 2010, Wiltgen et al. 2005).

This study tests the hypothesis that acutely increasing δGABAA receptor activity impairs memory. To identify the molecular basis for memory impairment caused by enhancement of δGABAA receptor activity, long-term potentiation (LTP) was measured. LTP 132 is a putative molecular substrate of memory formation and is correlated with memory performance on a variety of memory tasks (Saab et al. 2009, Sahay et al. 2011, Saxe et al.

2006). Importantly, LTP is tightly constrained by GABAA receptor activity. Pharmacologic blockade of GABAA receptors greatly increases LTP (Snyder et al. 2001, Wigstrom et al.

1985) while pharmacologic enhancement of GABAA receptor activity substantially depresses

LTP (Arima-Yoshida et al. 2011, Shen et al. 2010). As δGABAA receptors are the predominant source of tonic inhibitory conductance in the DG (Glykys et al. 2008), these receptors may constrain DG-LTP at baseline and depress DG-LTP when activated by THIP or other δGABAA receptor-selective drugs. Similarly, as δGABAA receptors are expressed in the

CA1 subfield of the hippocampus (Pirker et al. 2000), these receptors might also constrain baseline synaptic plasticity in CA1-LTP and depress CA1-LTP when activated by agonists such as THIP.

In the present study, it is demonstrated that increasing δGABAA receptor activity impairs memory and LTP in WT but not δGABAA receptor null mutant (Gabrd–/–) mice.

5.2. Specific methods

5.2.1. Animals

All procedures were approved by the local Animal Care Committee. Gabrd–/– mice and WT mice were generously provided by Dr. Gregg Homanics (Mihalek et al. 1999). Only male mice 3-6 months old were used for behavioural experiments. Researchers were blinded to genotype and drug conditions.

5.2.2. Drugs

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The δGABAA receptor-preferring agonist THIP was used to increase receptor activity (Brown et al. 2002). THIP was obtained from Tocris Bioscience (Bristol, UK). In behavioural experiments, the dose of THIP was 4 mg/kg administered by intraperitoneal (i.p.) injection.

This dose was selected as it has no sedative effects but does has a mild analgesic effect

(Bonin et al. 2011). In electrophysiological experiments, slices were treated with THIP (1 µM) as this concentration is expected to preferentially activate δGABAA receptors (Brown et al.

2002). The competitive GABAA receptor antagonists SR-95531 (Bai et al. 2001, Nusser et al.

2002) and bicuculline methiodide (BIC) and were obtained from Tocris Bioscience (Bristol,

UK).

5.2.3. Morris water maze

Memory performance was first assessed with the Morris water maze. The water maze was a circular pool (ø = 1.2 m) that was surrounded by visual cues, filled with opaque white nontoxic paint and kept at 25 ± 2°C. The escape platform was a 10 cm x 10 cm square of plexiglass that was positioned 0.5 cm below the pool surface so that it was not visible during the experiment. On the training day (acquisition), the platform was placed in a random quadrant of the pool and mice were given 4 trials to learn its location. The time (s) taken to find and remain upon the platform (escape latency) was recorded during each trial. If a mouse did not find the platform after 60 s, a maximum time of 60 s was recorded and the mouse was guided to the platform. 24 h after acquisition, long-term recall of the platform location was tested in a

60 s probe trial. During this trial, the platform was removed and the percentage of time mice spent in the quadrant formerly containing the platform was calculated. Data was recorded using SMART video tracking software (San Diego Instruments, San Diego, CA, USA).

5.2.4. Fear conditioning

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An exposure chamber (20 cm x 20 cm x 30 cm) with a shock grid floor consisting of stainless steel bars (2 cm apart, ø = 2 mm) was used for this task (Med Associates Inc., St. Albans, VT,

USA) (Wang et al. 2012a). During acquisition, each mouse was allowed to explore the chamber for 180 s. A 4000 Hz tone, created by a frequency generator, amplified to 100 dB and lasting 20 s was then presented. The last 2 seconds of the auditory tone were paired with an electric footshock (2 s, 0.7 mA). This tone-shock pairing was presented three times (S1-

S3), separated by 60 s. On day 2, 24 h after acquisition, contextual fear memory was assessed by returning the mouse to the context for 8 min and measuring the percentage of time that the mouse spent freezing. On day 3, the conditioning chamber was modified to measure the freezing response to the auditory tone (cued fear memory). Mice were monitored for 180 s for freezing to the modified context, to rule out contextual influences. After this monitoring period, the auditory tone was presented for 5 min and percentage freezing was scored. The percentage of time that each mouse spent freezing was determined using

FreezeView software (Version 2.26, Actimetrics Inc, Wilmette, IL, USA).

5.2.5. Novel object recognition

24 h prior to testing, mice were habituated for 15 min in a chamber (20 cm x 20 cm x 20 cm) marked with visual cues (Whissell et al. 2013). During testing, mice were exposed to a set of three identical objects in the chamber for 2 min (Figure 2.12). The mice were then removed from the chamber for 2 min while the entire setup was cleaned with 70% ethanol and one of the objects was replaced with a novel object. Mice were then returned to the chamber and the interaction time (s) with the two familiar objects (O1 + O2) and the novel object (NO) was recorded. Total interaction time was the sum of these totals (O1 + O2 + NO). Novel object preference (%) was defined as NO/(O1+O2+NO). An interaction was defined as an active

135 investigation of the object while the mouse was within 1 cm of the object and orientated towards it. Mice with a total interaction time of less than 3 s were excluded.

5.2.6. Electrophysiology

Male mice were anaesthetized deeply with isoflurane and decapitated, and then the brains were removed. Coronal hippocampal slices (350-400 µm thick) were cut with a vibratome

(VT1000E; Leica, Deerfield, IL, USA) while immersed in ice-cold artificial cerebrospinal fluid

(ACSF) containing (in mM): 124 NaCl, 3 KCl, 1.3 MgCl2, 2.6 CaCl2, 1.25 NaH2PO4, 26

NaHCO3 and 10 D-glucose. The ACSF was saturated with 95% O2 and 5% CO2 with the osmolarity adjusted to 300–310 mOsm. The slices were allowed to recover for at least 1 h at room temperature (23-25 oC) before being transferred to the recording chamber, where they were perfused with ACSF at 3-4 ml/min. All recordings were performed at room temperature using a MultiClamp 700A amplifier (Molecular Devices, Sunnyvale, CA, USA) controlled with pClamp 9.0 software via a Digidata 1322A interface (Molecular Devices, Sunnyvale, CA,

USA).

5.2.6.1. Field recordings of LTP

Hippocampal slices obtained from 3– 6 month-old mice were used. In recordings of DG-LTP, extracellular field postsynaptic potentials (fPSPs) were recorded from the stratum moleculare of the DG using an ACSF-filled borosilicate pipette (World Precision Instruments, Sarasota,

FL, USA) upon stimulation of the medial perforant pathway (MPP) with a bipolar tungsten electrode (Rhodes Medical Instruments, Summerland, CA, USA). To measure presynaptic plasticity and to determine the placement of the stimulating electrode in the MPP, a pair of stimuli was applied at various intervals (50, 100, 150, 200 or 300 ms) to generate a pair of responses. The paired pulse ratio was defined as (slope of response 2)/(slope of response 1).

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The presence of paired pulse depression (ratio < 1), which is one of the criteria for detecting

MPP inputs (Colino et al. 1993), was used to verify successful stimulation of the MPP. To record LTP, baseline fPSPs were measured for at least 10 min at 0.05 Hz using a stimulation intensity that produced a half-maximal response. LTP was then induced with theta burst stimulation (TBS), which consisted of 4 stimulus trains of 50 pulses delivered at 100 Hz with an inter-train of 20 s. fPSPs were monitored for 60 min after TBS, and the average of the last

5 min of recording was compared with the average of the baseline fPSPs. All drugs were allowed to perfuse the slices for 15 min before recording.

In recordings of CA1-LTP, the stimulating and recording electrodes were placed in the

Schaffer collateral pathway and stratum radiatum, respectively. Baseline responses were obtained as normal but an alternate protocol was used to induce LTP. This protocol consisted of 10 stimulus trains of 4 pulses at 100 Hz with an inter-train interval of 500 ms (Martin et al.

2009).

5.2.6.2. Whole-cell voltage-clamp recordings

Hippocampal slices obtained from 14–21 day-old mice were used. To maximize the yield of whole cell recordings, slices from younger mice were preferred as cell survival is comparatively poorer in slices from older mice (Ogden 1994). All recordings were obtained from putative granule cells in the DG identified with an Olympus BX51WI microscope (Center

Valley, PA, USA). Recording pipettes (3-5 MΩ) were filled with the intracellular solution containing (in mM): 140 CsCl, 11 ethylene glycol tetraacetic acid (EGTA), 10 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 K2-ATP, 1 CaCl2, 2 MgCl2 and 2 tetraethylammonium (TEA) with osmolarity adjusted to 290-295 mOsm and pH at 7.3. 6-

Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), (2R)-amino-5-phosphonovaleric acid

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(APV, 40 µM), and tetrodotoxin (0.5 µM) were added to the ACSF to block glutaminergic neurotransmission and voltage-dependent sodium channels. All recordings were performed at a holding potential of -70 mV.

5.2.7. Statistical Analysis

Analysis was conducted using Graphpad Prism 5.0 and SPSS17 for Windows. Acquisition data for Morris water maze and fear conditioning were analyzed using repeated measures analysis of variance (ANOVA). In other cases, either student’s t-test, standard one-way or two-way ANOVA followed by Bonferroni post hoc test was used. All the values are expressed as the mean ± SEM, and the level of p < 0.05 was considered statistically significant. Outliers with performance scores of more than 2 standard deviations from the mean were excluded from analysis.

5.3. Results

5.3.1. Acute THIP impairs spatial learning in the Morris water maze

The effects of acute THIP on spatial memory in the Morris water maze were examined as this form of memory is hippocampus-dependent and regulated by GABAA receptors (Cheng et al.

2006, Collinson et al. 2002, D'Hooge et al. 2001, Myhrer 2003). WT and Gabrd–/– mice were administered either THIP (4 mg/kg, i.p.) or physiological saline (vehicle) 30 min before being trained to locate a hidden platform over 4 trials. All groups were able to acquire the platform location successfully, as evidenced by reduced latency to locate the platform over subsequent trials (Figure 5.1A). There were no differences in acquisition between vehicle-treated WT and

Gabrd–/– mice (Figure 5.1A; genotype × trial, p = 0.28, n = 16-19). However, THIP impaired acquisition in WT mice (genotype × drug × trial, p < 0.05), as shown by the slower escape

138 latencies of the 3rd and 4th trials in THIP-treated WT mice compared with vehicle-treated mice

(Figure 5.1A, left). THIP had no effects on acquisition in Gabrd–/– mice (Figure 5.1A, right).

To investigate whether THIP also impairs long-term memory, recall of the platform location was tested in a probe trial 24 h after the last acquisition trial. THIP-treated WT mice spent less time in the quadrant formerly containing the platform than did vehicle-treated controls (Figure 5.1B, left; genotype × drug, p < 0.05, n = 14-17). In contrast, THIP-treated

Gabrd–/– mice performed similarly to vehicle-treated mice (Figure 5.1B, right). Collectively, these findings suggested that THIP impaired spatial memory in WT but not Gabrd–/– mice.

This result was not likely due to an effects of THIP on motor activity, as swim speed was unchanged by the drug (Figure 5.1C, drug and drug × genotype, ps > 0.05).

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Figure 5.1. Acute pharmacologic activation of δGABAA receptors impairs spatial navigation in the water maze.

A. THIP impairs acquisition of spatial memory in WT but not Gabrd–/– mice. THIP-treated WT mice have slower escape latencies than vehicle-treated controls on the 3rd and 4th trials of the training session. B. THIP impairs recall of spatial memory in WT but not Gabrd–/– mice. In a probe trial, THIP-treated WT mice show less preference for the goal quadrant formerly containing the escape platform than vehicle-treated controls. Stippled line indicates chance performance. C. THIP does not affect swim speed in WT or Gabrd–/– mice. All n values = 16-

19.

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5.3.2. Acute THIP impairs contextual but not cued fear memory

To determine whether THIP impaired other forms of hippocampus-dependent memory, aversive contextual fear conditioning was examined in WT and Gabrd–/– mice following acute injection with THIP or vehicle. 30 min after injection, mice were trained to associate an electric footshock (an unconditioned stimulus, US) with a training context and auditory cue

(conditioned stimuli, CS). A one day acquisition protocol with three footshocks (S1-3) was employed (Mihalek et al. 1999). During acquisition, all groups showed successive increases in freezing after each shock (Figure 5.2A), indicating that they had learned the task successfully. There was no difference in acquisition between vehicle-treated WT and Gabrd–

/– mice (genotype × shock, p > 0.5, n = 25-30), which is consistent with previous results using this protocol (Mihalek et al. 1999). However, THIP-treated WT mice showed reduced freezing following the third shock relative to vehicle-treated controls (Figure 5.2A, left); genotype × drug × shock, p < 0.01, n = 25-30). In contrast, THIP did not affect acquisition in Gabrd–/– mice (Figure 5.2A, right). These results indicated that THIP impaired the acquisition of fear memory.

To study the effects of THIP on contextual fear memory, which is hippocampus- dependent (Phillips et al. 1992), mice were returned to the same training context 24 h after acquisition. THIP-treated WT mice showed reduced freezing scores compared with vehicle- treated controls, suggesting impaired contextual fear memory (Figure 5.2B, left; genotype × drug, p < 0.01, n = 25-30). In contrast, Gabrd–/– mice treated with THIP exhibited no memory deficits (Figure 5.2B, right).

To examine the effects of THIP on cued fear memory, which is hippocampus- independent (Phillips et al. 1992), mice were re-exposed to the tone in a novel context.

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Interestingly, THIP did not affect cued fear memory in either WT or Gabrd–/– mice (Figure

5.2C; ps > 0.2 for main effects and interaction, n = 25-30). These results show that THIP impairs contextual but not cued fear memory.

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Figure 5.2. Acute pharmacologic activation of δGABAA receptors impairs contextual but not cued fear memory.

A. THIP impairs acquisition of fear memory in WT but not Gabrd–/– mice. THIP-treated WT mice show lower freezing scores following the presentation of the 3rd tone-shock pairing than vehicle-treated controls. B. THIP-treated WT mice show reduced freezing relative to vehicle- treated mice in a recall trial for contextual fear memory. C. THIP had no effects on freezing in a recall trial for cued fear memory in either WT or Gabrd–/– mice. All n values = 25-30.

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5.3.3. Acute THIP impairs novel object recognition

The Morris water maze and contextual fear conditioning tasks probe hippocampus- dependent, long-term memory (D'Hooge et al. 2001, Phillips et al. 1992), whereas cued fear conditioning probes hippocampus-independent long-term memory (Phillips et al. 1992). To determine whether THIP could impair short-term working memory, the novel object recognition task was used. In this assay, mice must recognize a novel object within a set of familiar objects to which they have been previously exposed (Figure 5.3A). As animals are driven to investigate novelty, mice that recall the familiar objects will preferentially interact with the novel object (Ennaceur et al. 1992). Novel object recognition is a non-aversive task that depends primarily upon the perirhinal cortex (Barker et al. 2011), and is regulated by GABAA receptors (Zurek et al. 2012).

THIP-treated WT mice showed impaired novel object preference relative to vehicle- treated controls (Figure 5.3B, left; drug × genotype, p < 0.05, n = 11-14). THIP did not have effects in Gabrd–/– mice (Figure 5.3B, right). These results demonstrate that acute THIP impairs recognition memory in WT but not Gabrd–/– mice. This result could not be attributed to an effect of THIP on exploratory drive in WT mice as total object interaction time was unchanged by the drug (Figure 5.3C). Consistent with past results, novel object recognition performance was reduced in Gabrd–/– mice relative to WT mice (p < 0.05).

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Figure 5.3. Acute pharmacologic activation of δGABAA receptors impairs novel object recognition.

A. Schematic showing protocol. B. THIP impairs novel object recognition in WT but not

Gabrd–/– mice. THIP-treated WT mice show lower preference for the novel object than vehicle-treated mice. C. THIP had no effect on total interaction times in WT or Gabrd–/– mice.

All n values = 12-21.

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5.3.4. THIP depresses LTP in the DG and CA1 region of the hippocampus

As THIP impaired memory, we next examined the effects of the drug on long-term potentiation (LTP), a molecular correlate of learning and memory, using the in vitro hippocampal slice preparation. We specifically examined LTP in the DG (DG-LTP) as

δGABAA receptors are densely expressed in this region (Pirker et al. 2000).

Prior to DG-LTP recordings, we first confirmed that THIP (1 μM) preferentially increased δGABAA receptor-mediated tonic conductance using whole-cell voltage-clamp recordings in DG granule cells. Perfusion of THIP activated a significant inward current in cells from WT mice but not in cells from Gabrd–/– mice (Figure 5.4A; p < 0.01, n = 7-8), indicating the drug preferentially acted upon δGABAA receptors. This was further verified by the observation that competitive GABAA receptor antagonist bicuculline (BIC, 20 µM) completely blocked the effects of THIP. Further, BIC revealed a tonic current that was greater in cells from WT mice than in Gabrd–/– mice (Figure 5.4A; p < 0.05, n = 6-7).

Next, the effects of THIP on DG-LTP were examined in hippocampal slices from WT and Gabrd–/– mice. Theta burst stimulation of the perforant pathway was used to induce LTP in the stratum moleculare of the DG, and field postsynaptic potentials (fPSPs) were recorded before and after stimulation. The slope of the fPSPs increased to 112.2% ± 6.7% and 110.0%

± 6.5% of baseline for WT and Gabrd–/– mice, respectively, after stimulation (Figure 5.5).

There was no effect of genotype (p > 0.4). Interestingly, THIP treatment completely blocked

DG-LTP in slices from WT mice (Figure 5.5A; genotype × drug, p > 0.05, n = 9-10) without affecting DG-LTP in slices from Gabrd–/– mice (Figure 5.5B). These results indicate that

THIP depresses DG-LTP by acting upon δGABAA receptors.

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In a separate series of experiments, the effects of THIP on CA1-LTP were examined, as δGABAA receptors mediate a tonic inhibitory conductance in this region (Glykys et al.

2008, Pirker et al. 2000). Theta burst stimulation of the Schaffer collateral pathway elicited robust CA1-LTP in both WT and Gabrd–/– mice, but there were no differences between the genotypes. Application of THIP depressed CA1-LTP in WT but not Gabrd–/– mice (Figure 5.6; genotype × drug, p > 0.05, n = 8-9).

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Figure 5.4. THIP increases tonic inhibitory conductance in cells from WT but not Gabrd–/– mice.

A, Left. Representative recording showing the effects of THIP on tonic current in cells from

WT mice. THIP increases tonic current while BIC blocks tonic current. A, Right. Quantified data showing absolute change in baseline holding current amplitude in WT mice. B, Left.

Representative recording showing the effects of THIP on tonic current in cells from Gabrd–/– mice. B, Right. Quantified data for Gabrd–/– mice. All n values = 5-7.

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Figure 5.5. THIP inhibits DG-LTP in slices from WT but not Gabrd–/– mice.

THIP depressed LTP in the DG in slices from WT but not Gabrd–/– mice. Upper panels:

Representative traces before and after tetanic stimulation. Middle panels: Normalized slope of fPSPs following tetanic stimulation. Bottom panels: Summarized data showing the last 5 min of recording. Note that THIP depressed LTP in DG only in WT mice. n = 8-13, *p < 0.05.

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Figure 5.6. THIP inhibits CA1-LTP in slices from WT but not Gabrd–/– mice.

THIP depressed LTP in CA1 in slices from WT but not Gabrd–/– mice. Upper panels:

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5.3.5. Extrasynaptic GABAA receptors likely mediate the effects of THIP on DG-LTP

To further verify that the effects of THIP were mediated by extrasynaptic GABAA receptors,

DG-LTP was re-measured when synaptic GABAA receptors were blocked by a low concentration of the competitive GABAA receptor antagonist, SR-95531 (1 µM). If synaptic

GABAA receptors mediated the effects of THIP, then antagonism of these receptors by SR-

95531 should prevent THIP-mediated depression of DG-LTP. While application of SR-95531 tended to increase DG-LTP relative to control conditions (WT = 123.3% ± 5.2% and Gabrd–

/– = 125.7% ± 5.4%, n = 10-12; Figure 5.7), it failed to reverse THIP-mediated impairment of

DG-LTP in WT mice (Figure 5.7).

Next, DG-LTP was measured in the presence of bicuculline (BIC, 100 µM), a blocker of both synaptic and extrasynaptic GABAA receptors. Application of BIC dramatically increased

DG-LTP to 136.7% ± 8.4% and 142.3% ± 11.7% for WT and Gabrd–/– mice, respectively

(Figure 5.8, n = 9-10). Additional administration of THIP (1 µM) did not impair DG-LTP in the presence of BIC (Figure 5.8; drug and drug × genotype, p > 0.40, n = 9-10).

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Figure 5.7. Co-application of SR-95531 does not occlude THIP-mediated impairment of DG-

LTP.

THIP depressed LTP in the DG in slices from WT but not Gabrd–/– mice co-treated with SR-

95531. Upper panels: Representative traces before and after tetanic stimulation. Middle panels: Normalized slope of fPSPs following tetanic stimulation. Bottom panels: Summarized data showing the last 5 min of recording. Note that THIP depressed LTP in WT mice. n = 8-9,

*p < 0.05.

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Figure 5.8. BIC occludes THIP-mediated impairment of DG-LTP.

THIP does not impair LTP in the DG in BIC-treated slices. BIC (100 μM) was perfused throughout the recordings.

Upper panels: Representative traces before and after tetanic stimulation. Middle panels:

Normalized slope of fPSPs following tetanic stimulation. Bottom panels: Summarized data

156 showing the last 5 min of recording. Note that THIP did not depress LTP in the DG in both WT and Gabrd–/– mice. n = 9-10.

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5.3.6. THIP does not affect basal excitatory transmission or presynaptic function in the

To confirm that THIP acts upon δGABAA receptors without any other secondary effects, we examined the effects of THIP on basal excitatory neurotransmission using input-output relationship in both WT and Gabrd–/– mice. To generate these relationships, stimulus intensity was increased incrementally to generate fPSPs that increase in strength. The amplitude of the presynaptic fiber volley versus the slope of each fPSP was graphed as a scatter plot. The presynaptic fiber volley and the slope of each fPSP are indicative of presynaptic fiber activation (input) and postsynaptic activation (output), respectively. A 'best fit line' representing the input-output relationship was then computed using linear regression.

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Figure 5.9A). There was no difference between the slopes of the input-output relationship for either genotypes or THIP treatment.

We next investigated the effects of THIP on the paired pulse ratio, which represents a presynaptic form of short-term plasticity. To generate the paired pulse ratio, two fPSPs were elicited by applying two stimuli at varying time intervals (50-300 ms) to the perforant pathway.

A ratio of the resulting responses was then computed (response 2/response 1). As reported previously (Christie et al. 1994), paired pulse depression (ratio < 1) was observed in the DG with stimulation of the medial perforant path (Figure 5.9B). There was no difference between the paired pulse ratios for genotypes or THIP treatment (all ps > 0.05).

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Figure 5.9. THIP has no effects on basal synaptic transmission or presynaptic function in slices from WT or Gabrd–/– mice.

A. Input/output relationships, an indicator of basal synaptic transmission, are unaffected by

THIP. B. Paired pulse responses, an indicator of presynaptic function, are unaffected by

THIP. All n values = 8-9.

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5.4. Discussion

The above results show that acute THIP impairs multiple forms of memory and depresses

DG-LTP. These effects were dependent upon δGABAA receptor expression, as THIP did not impair memory or DG-LTP in Gabrd–/– mice.

Acute THIP impaired hippocampus-dependent memory in the Morris water maze and contextual fear conditioning tasks, but did not affect hippocampus-independent memory in the cued fear conditioning task. The greater vulnerability of hippocampus-dependent memory to the amnestic effects of THIP might be caused by the comparatively high expression of

GABAA receptors within the hippocampus relative to most other brain regions (Pirker et al.

2000) and the ability of these receptors to inhibit network excitability (Maguire et al. 2009a).

THIP-mediated impairments in long-term memory may have been due to impaired memory acquisition. These effects of THIP are consistent with those of neurosteroids, which are positive allosteric modulators of the δGABAA receptor that impair acquisition in the shock avoidance assay (Shen et al. 2010) and Y-maze recognition task (Mayo et al. 1993). Whether

THIP also impairs consolidation or retrieval of memory is an interesting topic for future study.

Interestingly, novel object recognition was also impaired by acute THIP, although this behaviour is primarily mediated by the perirhinal cortex (Winters et al. 2010). The hippocampus can regulate recognition memory when a novel and/or complex testing environment is used (Oliveira et al. 2010, Sannino et al. 2012) or when the delay between testing and training periods is short (Rose et al. 2012). In our experiments, we utilized a complex testing environment (multiple visual cues and three objects) and enforced a short delay between training and testing periods (2 min). These testing conditions may have facilitated the involvement of the hippocampus in novel object recognition. Accordingly, 162 pharmacologic activation of extrasynaptic GABAA receptors in the hippocampus may have also contributed to the memory impairment in this assay.

As activation of δGABAA receptors by THIP impaired memory, it might be expected that Gabrd–/– mice would show improved memory performance. However, we found no evidence of this in any behavioural test. Our data are consistent with previous results showing that deletion of the δ subunit is generally not beneficial for memory performance in male mice

(Mihalek et al. 1999, Wiltgen et al. 2005), although our results are in contrast with the enhanced memory in several varieties of GABAA receptor subunit knockout mice (Cushman et al. 2011, Martin et al. 2010, Moore et al. 2010). A potential explanation why memory is not enhanced in Gabrd–/– mice is that deletion of the δGABAA receptor impedes neurogenesis in the DG ((Rosenzweig 2011) , a process which contributes to memory performance (Marin-

Burgin et al. 2012b). Disruption of neurogenesis impairs Morris water maze, contextual fear conditioning and novel object recognition (Jessberger et al. 2009, Saxe et al. 2006, Snyder et al. 2005) and may counteract the benefits of reduced inhibition in Gabrd–/– mice, resulting in no net change in learning.

Another major extrasynaptic GABAA receptor that is similar in function to the δGABAA receptor is the α5 subunit-containing GABAA (α5GABAA) receptor, which also mediates tonic inhibition and constrains excitability (Bonin et al. 2007, Caraiscos et al. 2004). The effects of

THIP are highly similar to those of compounds which enhance α5GABAA receptor activity, such as anesthetics. These compounds impair memory performance in the Morris water maze, contextual fear conditioning and novel object recognition, but do not affect performance in the cued fear conditioning task (Cheng et al. 2006, Martin et al. 2009, Zurek et al. 2012), in a manner similar to THIP. Memory acquisition is also impaired by these drugs (Atack et al.

2006, Collinson et al. 2006). Collectively, these results indicate that α5GABAA and δGABAA 163 receptors can play similar roles in memory formation, and may even compensate for each other to some extent. This suggestion is supported by the observation that there is a compensatory up-regulation of α5GABAA receptor-mediated tonic inhibitory conductance in

Gabrd–/– mice (Glykys et al. 2008), which might be another contributing factor to the lack of enhanced memory in Gabrd–/– mice.

To identify a putative neurophysiologic substrate of THIP-induced memory deficits, we measured DG-LTP and CA1-LTP, which are molecular correlates of memory processes

(Bruel-Jungerman et al. 2007). DG-LTP is modest and difficult to induce in standard ACSF due to the presence of strong GABAA receptor-mediated inhibition (Arima-Yoshida et al. 2011,

Snyder et al. 2001, Wigstrom et al. 1983b). Consistent with these findings, we observed modest DG-LTP at baseline and significantly greater DG-LTP when GABAA receptors were blocked by BIC. As GABAA receptors constrain DG-LTP, it might be expected that Gabrd–/– mice would show enhanced DG-LTP. However, we saw comparable DG-LTP in both WT and

Gabrd–/– mice. Why was DG-LTP not enhanced in Gabrd–/– mice? Baseline DG-LTP is primarily mediated by adult-born neurons produced during neurogenesis (Snyder et al. 2001) which show increased excitability and plasticity relative to older neurons (Ming et al. 2011).

Development of adult-born neurons is facilitated by δGABAA receptors and is disrupted in

Gabrd–/– mice (Whissell et al. 2013). Impaired neurogenesis in Gabrd–/– mice might counteract the benefits of reduced GABAA receptor-mediated inhibition, resulting in no net change in DG-LTP. Importantly, the comparable levels of DG-LTP and CA1-LTP in both WT and Gabrd–/– mice is consistent with the fact these genotypes do not differ in memory performance.

In contrast to the minimal effects of δGABAA receptor deletion on CA1-LTP and DG-

LTP, δGABAA receptor activation with acute THIP depressed DG-LTP and CA1-LTP. Others 164 have observed impaired LTP when tonic inhibitory conductance is increased by the addition of neurosteroids or GABA to the ACSF (Arima-Yoshida et al. 2011, Shen et al. 2010). The potent effects of THIP on DG-LTP is consistent with the impaired memory performance exhibited in these animals.

The observation that acute THIP impairs memory in the current study contrasts with the finding that long-term pre-treatment of the drug promotes memory (Chapter 4). However, these two studies utilized very different protocols for dosing of the drug and testing of memory. In the current study, memory was examined 30 min after THIP injection, when the levels of the drug peak (Cremers et al. 2007). The current results suggest that this particular treatment protocol is likely to impair hippocampus-dependent memory and LTP. In the long- term pre-treatment study, memory was examined 2 weeks after the last dose of THIP, when the drug was unlikely to be present in the body. Long-term THIP pre-treatment enhanced recognition and discrimination memory, which is associated with enhanced neurogenesis

(Chapter 4). In contrast, an acute dose of THIP is unlikely to affect neurogenesis, as the process occurs over a period of many weeks (Zhao et al. 2008).

THIP and other drugs that enhance δGABAA receptor activity might be used for the treatment of insomnia (Wafford et al. 2006), pain (Bonin et al. 2011), cognitive dysfunction

(Wang et al. 2007) and mood disorders (Christensen et al. 2012, Maguire et al. 2008). Based on the current and previous findings (Whissell et al. 2013), these drugs are expected to have two distinct effects: acute memory impairment and long-term memory improvement. While the acute effects are highly desirable in certain contexts, such as the induction of anesthesia and the treatment of pain and insomnia (Bonin et al. 2011, Wafford et al. 2006), they are undesirable in other contexts, such as during demanding performance tasks (i.e. driving) or the treatment of cognitive dysfunction (Wang et al. 2007). Importantly, the long-term beneficial 165 effects must be weighed against the acute effects. Future studies will be required to investigate and optimize the therapeutic effects of the drugs in a given clinical context.

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Chapter 6. General Discussion

6.1. Summary

The current study tested the hypothesis that δGABAA receptors upregulate and downregulate distinct memory processes. In Chapter 4, I illustrated that baseline activity of δGABAA receptors promotes multiple forms of memory performance. Transgenic deletion of δGABAA receptors impaired memory on tasks regulated by the DG and/or adult-born neurons, such as object-place recognition and contextual discrimination. Furthermore, long-term treatment with the δGABAA receptor-preferring agonist THIP for 1 week improved memory performance that was measured 2 weeks later, a time point long after the drug was likely to be eliminated.

These findings, which verify that δGABAA receptors can upregulate certain memory processes, are further discussed in 6.2.

While these results suggested that long-term activation of δGABAA receptors prior to memory task could promote performance, I also hypothesized that acute supra-activation of these receptors would have the opposite effect and impair memory. In Chapter 5, I investigated this possibility. A single THIP injection impaired learning in the water maze, contextual fear conditioning and novel object recognition tasks that was measured 30 min later, when the levels of the drug were likely to have peaked (Cremers et al. 2007).

Furthermore, acute THIP treatment potently impaired synaptic plasticity in the hippocampus, a correlate of memory formation, in slices obtained from WT but not Gabrd–/– mice. These findings, which verify that δGABAA receptors can downregulate memory processes, are further discussed in 6.3.

6.2. Baseline δGABAA receptor activity promotes memory performance

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In Chapter 4, I investigated whether δGABAA receptors influenced memory behaviours regulated by the DG and/or adult-born neurons. I anticipated that δGABAA receptors would promote memory performance and neurogenesis. Consistent with this expectation, I observed that reduced expression of δGABAA receptors in Gabrd–/– mice was associated with impaired object-place recognition and contextual discrimination, two memory behaviours regulated by

DG function (Lee et al. 2005, Nakashiba et al. 2012). Further, I observed impaired novel object recognition and impaired fear extinction in Gabrd–/– mice, although the acquisition of fear memory was also enhanced. Neurogenesis was also disrupted in Gabrd–/– mice. In addition, I observed that pharmacological activation of δGABAA receptors prior to a memory task with THIP facilitated object-place recognition, contextual discrimination and neurogenesis.

The behavioural impairments exhibited by Gabrd–/– mice are consistent with disrupted neurogenesis in the DG. In Gabrd–/– mice, the percentage of immature, DCX+ adult-born neurons is greater than in WT mice. This suggests that comparatively more adult-born neurons in Gabrd–/– mice exhibit structural and electrophysiological properties that are similar to neurons 3 weeks of age or younger, as this corresponds to the timeframe of DCX expression (Snyder et al. 2009). At this age, adult-born neurons do not demonstrate significant synaptic plasticity or excitability and are unlikely to have formed functional connections (Ge et al. 2007, Mongiat et al. 2009). These properties do not become apparent until 4–6 weeks of age, when these neurons begin to play a critical role in learning (Denny et al. 2012, Gu et al. 2012, Nakashiba et al. 2012). Therefore, the majority of neurons in Gabrd–

/– mice may not be sufficiently mature to participate in learning tasks. In support of this interpretation, a delay in the maturation of adult-born neurons has previously been correlated with reduced performance for contextual discrimination (Scobie et al. 2009). Also, when

168 neurogenesis is arrested or attenuated, animals exhibit deficits in contextual discrimination

(Nakashiba et al. 2012), novel object recognition (Jessberger et al. 2009, Madsen et al. 2003) and extinction (Deng et al. 2009) in a manner similar to Gabrd–/– mice. A key function of

δGABAA receptors may therefore be to promote specific forms of memory, such as pattern separation, through neurogenesis.

An important outstanding question is how neurogenesis promotes pattern separation in general. Several theoretical models have been proposed to explain the role of adult-born neurons in pattern separation; two recent models - ideologically separate but potentially complimentary - have attracted considerable interest (Aimone et al. 2011, Piatti et al. 2013).

The first theoretical model proposes a “memory resolution” hypothesis to explain how adult- born neurons facilitate pattern separation (Aimone et al. 2011). According to this model, adult- born neurons are capable of densely sampling experiential information as they are highly excitable and likely to respond to multiple stimulus features. These neurons maximize the information stored following experience, thereby producing detailed memories which are akin to “high-resolution images". When neurogenesis is disrupted and the functional population of adult-born neurons is reduced, “low-resolution” memories are formed instead. These memories, akin to blurry and pixilated photographs, are difficult to distinguish from each another. Thus, adult-born neurons may play a direct role in pattern separation through enhancing the encoding of information.

A second influential model regarding the role of adult-born neurons in pattern separation was put forward more recently by Piatti et al (2013). This model is remarkable in that it proposes adult-born neurons promote pattern separation through connections on inhibitory interneurons rather than projection neurons. Importantly, adult-born neurons may have connectivity with inhibitory interneurons in the hilus and elsewhere (Piatti et al. 2013) 169 that mediate feedback inhibition on the DG network. Some potential support for the idea that adult-born neurons mediate feedback inhibition comes from the observation that GABAergic innervation in the DG is reduced following ablation of neurogenesis (Singer et al. 2011).

Thus, activation of adult-born neurons may shape the activity of the DG network through feedback inhibition. Possible consequences of this feedback inhibition include decreased overall excitability and increased sparse coding within projection neurons of the DG, conditions which may facilitate the generation of unique memory traces that are easily separated (Rolls 2010). According to the model of Piatti and colleagues (2013), adult-born neurons may play an indirect role in promoting pattern separation by modulating the activity of the DG network, akin to 'dictating the tone of the message' rather than delivering it.

Interestingly, δGABAA receptors did not uniformly promote memory, as the acquisition of contextual fear conditioning was enhanced in Gabrd–/– mice. This finding is consistent with experiments in Gabra4–/– mice which suggest that deletion of α4βδ receptors enhances this form of learning (Cushman et al. 2011, Moore et al. 2010, Wiltgen et al. 2005). It is noteworthy that contextual fear conditioning is not considered to be significantly regulated by the DG. Most studies of contextual fear conditioning do not show a major role of the DG

(Nakashiba et al. 2012) or adult-born neurons (Deng et al. 2009, Shors et al. 2002, Zhang et al. 2008a), although this depends upon the testing protocol (Drew et al. 2010). The enhanced fear acquisition in Gabrd–/– mice might thus be caused by the absence of δGABAA receptors on mature neurons in regions other than the DG, such as the CA1 subfield of the hippocampus. In the CA1 region, δGABAA receptors are predominantly inhibitory and constrain memory (Shen et al. 2010). Importantly, the enhanced fear acquisition in Gabrd–/– mice may have contributed to the delayed fear extinction in these animals, as stronger fear memories are typically resistant to extinction.

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The positive effects of THIP treatment on memory performance were observed 2 weeks after the drug treatment had terminated. At this time point, THIP was unlikely to be present even in trace amounts (Cremers et al. 2007). The effects of THIP on memory are therefore most likely explained by long-term neurophysiologic changes resulting from prolonged δGABAA receptor activation, such as enhanced neurogenesis (Figure 6.1). While the enhanced memory performance of THIP-treated mice is consistent with an enhancement of neurogenesis, there are other long-term changes which may be involved in this effect (6.6).

How might δGABAA receptor-mediated promotion of neurogenesis be explained? A significant possibility is that depolarizing δGABAA receptor-mediated currents in adult-born neurons modulate neurogenesis by regulating the currents mediated by other receptor channels (Figure 6.1). For example, depolarizing δGABAA receptor-mediated currents in adult-born neurons may amplify NMDA receptor-mediated currents or perhaps activate other voltage-gated calcium channels (Tozuka et al. 2005). Both these effects would likely lead to influx of Ca2+ and robust activation of intracellular Ca2+-dependent kinases and/or secondary messengers, such as cyclic adenosine monophosphoate (cAMP) (Sernagor et al. 2010).

These messengers would in turn activate cellular transcriptional factors, such as cAMP response element binding protein (CREB), which would influence gene expression and promote survival and maturation (Jagasia et al. 2009). Interestingly, there is some evidence to support that NMDA receptors may be necessary for δGABAA receptor-mediated promotion of neurogenesis (Rosenzweig 2011). Notably, the effects of THIP on promoting the maturation and survival of adult-born neurons are both occluded by antagonists of NMDA receptors

(Rosenzweig 2011). The fact that genetic deletion of the δGABAA receptor only modestly disrupts neurogenesis, rather than completely blocking the process, strongly suggests the

171

δGABAA receptor works in concert with several other receptor channels to regulate the development of adult-born neurons.

Taken together, the findings presented in Chapter 4 illustrate a dynamic, bidirectional role of δGABAA receptors in memory. Specific types of memory are promoted by the receptor, particularly forms of pattern separation such as object-place recognition and conextual discrimination. Other forms of memory, such as contextual fear conditioning, are constrained by the receptor.

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Figure 6.1. Theoretical model explaining the effects of long-term THIP on DG-dependent memory.

Repeated injections of THIP lead to a sustained increase in δGABAA receptor activity in immature adult-born neurons. In these neurons, δGABAA receptor-mediated currents are likely depolarizing and may amplify NMDA receptor-mediated currents or activate other voltage-gated channel currents which increase Ca2+ conductance. The resulting increase in intracellular Ca2+ eventually activates secondary messengers and transcription factors such as CREB, which promote neuronal maturation. This increase in maturation of adult-born neurons increases the size of the mature adult-born neuron population that is capable of engaging in memory behavior, and thereby promotes DG-dependent memory.

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6.3. Acute activation δGABAA receptors during a memory task impairs performance

In Chapter 5, I investigated the effects of acute THIP treatment on memory. In this study, I measured memory 30 min after THIP administration, at a time point at which THIP concentration in the serum was likely to have peaked (Cremers et al. 2007). I anticipated that acute THIP would impair memory significantly. Consistent with this expectation, I observed that acute THIP impaired memory performance in the Morris water maze, contextual fear conditioning and novel object recognition tasks.

The poor long-term memory performance in mice given acute THIP may have been specifically due to impaired memory acquisition during training. In the present study, acute

THIP impaired memory acquisition in both the Morris water maze and contextual fear conditioning tasks. Further, others have observed that acute δGABAA receptor activation inhibits memory acquisition. Neurosteroids, which are positive allosteric modulators of the

δGABAA receptor, impair memory acquisition in the shock avoidance assay (Shen et al. 2010) and Y-maze recognition task (Mayo et al. 1993). While an effect of THIP on acquisition appears likely, the drug may also have affected other memory processes, such as consolidation or retrieval. Evidence suggests that THIP may have significantly affected the consolidation of memory, as Makkar et al. (2010) have observed that post-training activation of GABAA receptors during consolidation impairs subsequent recall.

6.4. Acute activation of δGABAA receptors impairs long-term potentiation

To further examine the role of δGABAA receptors in memory and memory-related processes,

LTP was measured in the hippocampus (Bruel-Jungerman et al. 2007). These results are also presented in Chapter 5. It was anticipated that acute THIP treatment would impair long-term potentiation, as δGABAA receptors are predominantly inhibitory in mature neurons and

174

GABAA receptor-mediated inhibition constrains LTP (Arima-Yoshida et al. 2011, Snyder et al.

2001, Wigstrom et al. 1983b). Consistent with this expectation, acute THIP attenuated DG-

LTP and CA1-LTP.

If acute THIP treatment impaired LTP through activation of δGABAA receptors, why was DG-LTP not enhanced in Gabrd–/– mice? Importantly, δGABAA receptors also regulate neurogenesis in the DG, and neurogenesis is disrupted in Gabrd–/– mice (Whissell et al.

2013). Baseline DG-LTP is primarily mediated by adult-born neurons produced during neurogenesis (Snyder et al. 2001) which show increased excitability and plasticity relative to older neurons (Ming et al. 2011). Impaired neurogenesis in Gabrd–/– mice might thus counteract the benefits of reduced GABAA receptor-mediated inhibition, resulting in no net change in DG-LTP. The failure of δGABAA receptor deletion to affect CA1-LTP is less surprising, as δGABAA receptor activity is relatively low in CA1 compared to the DG under baseline conditions (Glykys et al. 2008).

The observation that acute THIP impairs memory contrasts with the finding that long- term pre-treatment of the drug promotes memory. However, these two studies utilized very different protocols for dosing of the drug and testing of memory (for comparison, see Figure

3.1 and Figure 3.2). In the acute injection study, memory was examined 30 min after THIP injection, when the levels of the drug peak (Cremers et al. 2007) (Figure 3.1). The current results suggest that this particular treatment protocol is likely to impair hippocampus- dependent memory and LTP (Figure 6.2). In the long-term pre-treatment study, memory was examined 2 weeks after the last dose of THIP, when the drug was unlikely to be present in the body (Figure 3.2). Long-term THIP pre-treatment enhanced recognition and discrimination memory, which is associated with enhanced neurogenesis (Chapter 4). In contrast, an acute

175 dose of THIP is unlikely to affect neurogenesis, as the process occurs over a period of many weeks (Zhao et al. 2008).

176

Figure 6.2. Theoretical model explaining the effects of acute THIP on memory.

Approximately 30 minutes after THIP administration, the levels of THIP peak in the hippocampus. In mature hippocampal neurons, the activation of δGABAA receptors by THIP leads to either hyperpolarizing inhibition or shunting inhibition of NMDA receptor-mediated currents. This decrease in NMDA receptor activity impairs synaptic plasticity, a potential substrate of memory acquisition. As a result, the acquisition of memory is impaired while

THIP is present in the brain.

177

6.5. Limitations

At the outset of this project, a global transgenic knockout model was employed as this was the only transgenic model available. Unfortunately, this global model does not permit temporal- or region-specific knockdown of the δ subunit, which it makes it difficult to conclusively identify the population of receptors within the brain that regulate memory (e.g.:

δGABAA receptors in the DG versus receptors in CA1). To provide this information, a transgenic model with region-specific knockout of δ subunit gene could be used. Such models already exist for other genes (Tsien et al. 1996a) and could be prepared for the δ subunit gene. To generate such a model, a Cre/LoxP system could be employed wherein the δ subunit gene is flanked by LoxP sites and the expression of Cre is driven by a promoter relatively specific to either CA1 pyramidal cells or DG granule cells (such as calcium/calmodulin-dependent protein kinase II alpha or proopiomelanocortin, respectively)

(McHugh et al. 2007, Tsien et al. 1996a). This model would elucidate the role of specific

δGABAA receptor populations in memory and neurogenesis.

To add another layer of specificity to the region-specific model, temporal control over δ subunit gene expression could also be implemented. Selective knockout of δ subunit gene expression in young adult-born granule cells, for example, can be accomplished using a

Cre/LoxP system wherein the expression of Cre is driven by a promoter specific to young adult-born granule cells (e.g. Nestin) (Kheirbek et al. 2012). This model would ascertain whether the effects of δGABAA receptors on memory are mediated by receptors on young adult-born granule cells or older, developmentally-generated granule cells. Alternatively, general temporal control of gene expression can be achieved by using a tetracycline transactivator system in combination with the Cre-LoxP system (Nakashiba et al. 2012).

178

While this thesis makes the argument that δGABAA receptors promote memory through neurogenesis, there is some controversy regarding the role of GABAA receptor- mediated tonic currents in neurogenesis which must be acknowledged (Ge et al. 2006). While one group has identified a role of δGABAA receptors in promoting neurogenesis (Chapter 4), another has not (Duveau et al. 2011). Furthermore, and most remarkably, the presence of a

δGABAA receptor-mediated tonic current in adult-born neurons has not yet been verified.

While it is known that α4 subunit-containing GABAA receptors mediate tonic currents in adult- born neurons (Duveau et al. 2011) it is still unclear whether these α4 subunit-containing receptors even contain δ subunits (i.e.: are α4βδ receptors). Indeed, it is not even clear whether δGABAA receptors are expressed in young adult-born cells or any type of young cell, as biochemical evidence suggests that the δ subunit is not expressed until at least postnatal day 12 (Duveau et al. 2011, Laurie et al. 1992). To resolve this controversy, future experiments should specifically examine the electrophysiologic properties of young, adult- born neurons in WT and Gabrd–/– mice (see below).

A central issue with all receptor knockdown models is compensation by other receptor types. Others have demonstrated that δ subunit deletion is associated with a compensatory increase in currents mediated by α5GABAA receptors and K+ channels (Brickley et al. 2001,

Glykys et al. 2008). An important question is whether these changes contribute to the behaviours studied here. At least in the case of α5GABAA receptors, this is unlikely. Baseline

α5GABAA receptor activity did not affect most of the memory behaviours studied in this thesis

(Figure 4.1) and does not affect neurogenesis (Duveau et al. 2011), which facilitates these same behaviours (Sahay et al. 2011). Together, these data suggest that compensatory changes in α5GABAA receptor activity is not relevant to the behaviours studied. However, other compensatory changes (such as changes in NMDA receptor expression) may exist and

179 future experiments will be required to determine whether these changes are relevant to memory processes.

Other compensatory changes may exist in the Gabrd–/– knockout model which do not involve receptor channels. Notably, it was recently observed that decreased δ subunit expression in immature (~6 day old) neurons is associated with increased KCC2 expression

(Succol et al. 2012). This finding would suggest that KCC2 expression is increased in Gabrd–

/– mice. This potential compensation may have implications for multiple cellular processes which depend upon chloride graident, and may explain the relatively modest changes in excitability and membrane properties observed in Gabrd–/– mice (Spigelman et al. 2002).

Further experiments will be required to determine if these compensatory increases occur and are functionally relevant in adult Gabrd–/– mice.

6.6. Future Directions

This thesis identifies a memory impairment in Gabrd–/– mice that is consistent with a disruption in the functioning of adult-born neurons. While others have shown that the migration, maturation and dendritic complexity of adult-born neurons is abnormal in Gabrd–/– mice (Chapter 4), it has not been demonstrated how these changes affect the activity of adult- born neurons. Future experiments using whole cell patch clamp could be performed to determine this information. To specifically identify adult-born neurons for patching in these experiments, retroviral labeling could be employed (Duveau et al. 2011). As adult-born neurons in Gabrd–/– mice mature slowly (Chapter 4), it is likely that these neurons may exhibit electrophysiological properties characteristic of relatively undeveloped neurons, namely a high threshold for activation and an inability to undergo burst firing (Mongiat et al.

180

2009). Conversely, it would be expected that these same properties would be enhanced in adult-born neurons studied in THIP-treated WT mice.

Another argument of this thesis is that adult-born neurons are active during the memory tasks studied and contribute to performance. While it is possible that adult-born neurons in Gabrd–/– mice are too immature to be involved in learning, activation of adult-born neurons during a memory task has yet to be quantified in these mice. Future research could determine whether the proportion of adult-born neurons activated by a memory task is reduced in Gabrd–/– mice relative to WT mice. This could be accomplished by measuring the expression fos, a protein correlated with neuronal activity, specifically in adult-born neurons

(Stone et al. 2011). If the activity of adult-born neurons contributes to memory performance on the behavioural assays employed in this thesis, then the proportion of active adult-born neurons should be lower in Gabrd–/– mice and greater in THIP-treated WT mice than in control animals.

An important question is whether long-term THIP treatment generally enhances memory performance or only influences memory behaviours besides those regulated by adult-born neurons or the DG, such as contextual discrimination (Nakashiba et al. 2012). For example, long-term THIP treatment could also affect memory performance on the Morris water maze or radial arm maze task. Similarly, another further question is whether long-term

THIP has effects on the expression of δGABAA receptors or other receptor systems in vivo, as prolonged THIP treatment has been shown to effect δGABAA receptors expression in vitro

(Shen et al. 2005), as has exposure to other compounds with activity at the δGABAA receptor

(Table 2.1).

181

While this thesis argues that acute THIP treatment impairs memory performance by impeding the acquisiton of memory, future experiments will be required to address the effects of the drug on other stages of memory. Virtually all components of the memory process are sensitive to GABAA receptor activity (including acquisition, consolidation and retrieval)

(Makkar et al. 2010) and could be affected by THIP treatment. To determine the effects of

THIP on other stages of memory, a separate experiment could be designed wherein mice were trained on a learning task without first receiving an injection of THIP or vehicle. This approach would exclude an effect of THIP on the acquisition of memory and allow the other stages of memory to be examined in isolation. To address the effects of THIP on memory consolidation, mice would receive an injection of the drug immediately after training. If THIP impaired memory by inhibiting memory consolidation, this post-training injection should impair performance in a subsequent recall trial. Similarly, if THIP impaired memory through affects on memory retrieval, an injection of THIP immediately prior to the recall trial should impair memory.

The ability of THIP to affect other electrophysiological parameters in the DG besides plasticity, such as excitability, is an important topic for future research. While it has been clearly demonstrated that THIP reduces excitability in spinal cord neurons (Bonin et al. 2011), the effect of the compound on granule cells of the DG has received comparatively little study.

It is possible, and very likely, that THIP inhibits the generation of action potentials through shunting inhibition (Staley et al. 1992). To better characterize the effects of THIP on excitability, several potential experiments could be performed. One such experiment could utilize whole-cell current clamp recordings to determine if THIP increases the minimal stimulation necessary to induce an action potential (referred to as the rheobase) in granule cells of the DG (Bonin et al. 2007). The effects of THIP on excitability could also be inferred

182 from field recordings of population responses in hippocampal slice. For this purpose, an examination of field responses in the dendritic and cell layer of the DG could be conducted using methods such as slope-spike coupling (Daoudal et al. 2002) and coastline analysis

(Martin et al. 2010), respectively.

Future experiments should also explore what neurophysiologic changes, besides neurogenesis in the DG, may contribute to the ability of δGABAA receptors to regulate memory. Notably, the CA3 subregion of the hippocampus is thought to facilitate memory in cooperation with the DG (Gilbert et al. 2006, Leutgeb et al. 2007, O'Reilly et al. 1994). The

CA3 subfield is a site where δGABAA receptors are expressed (Pirker et al. 2000) and may significantly modulate neurotransmission (Ruiz et al. 2010). Recently, Ruiz and colleagues demonstrated that neurotransmission at the CA3 pyramidal cell-DG mossy fiber synapse is facilitated by presynaptic, excitatory GABAA receptors which likely contain the δ subunit (Ruiz et al. 2010). This intriguing finding suggests that synaptic transmission in the CA3 subregion is reduced in Gabrd–/– mice, and is an alternate mechanism by which δGABAA receptors may regulate memory behaviours. However, synaptic plasticity in the CA3 region has not been explored in WT and Gabrd–/– mice.

6.7. Implications

This thesis demonstrates a complex, bidirectional role of δGABAA receptors in memory, neurogenesis and synaptic plasticity. These results have a number of clinical implications.

Importantly, the current results suggest that a prolonged reduction in δGABAA receptor expression and/or function may adversely affect memory and neurogenesis. Specifically, it is possible that impaired DG-dependent memory may be observed in physiologic and pathophysiologic conditions in which δGABAA receptor expression is reduced, such as post-

183 partum depression (Maguire et al. 2008), Fragile X syndrome (Curia et al. 2009, D'Hulst et al.

2006, Gantois et al. 2006) and epilepsy (Nishimura et al. 2005, Pandit et al. 2013, Zhang et al. 2007). Though DG-dependent memory has not been examined in all these situations, it is worth noting that contextual discrimination is impaired in animal models of Fragile X syndrome (Eadie et al. 2012).

Additionally, the current results also illustrate that δGABAA receptors are a novel therapeutic target for treating memory dysfunction. Notably, long-term pharmacologic enhancement of δGABAA receptor activity, with compounds such as THIP, may rescue cognitive function that is lost in aging, injury and disease.

These findings also have more immediate clinical implications, as δGABAA receptors are molecular targets for a wide variety of therapeutic drugs in use and in development, including anesthetics (Lees et al. 1998) and sedative-hypnotics (Wafford et al. 2006). The activity of δGABAA receptors is also regulated by endogenous hormone derivatives such as neurosteroids (Belelli et al. 2005), and recreational drugs such as ethanol (Olsen et al. 2007).

The current results suggest that the activation of δGABAA receptors may explain some of the recognized amnestic effects of these compounds.

An important consideration is that THIP induced a significant memory deficit in mice at a low, non-sedative dose (4 mg/kg) (Winsky-Sommerer et al. 2007). This dose would not be considered therapeutic in this species. Intuitively, it would be expected that higher, therapeutic doses of THIP would generate more significant memory deficits. Thus, it is possible that this thesis, which shows moderate memory deficits in mice with a sub- therapeutic dose of THIP, may underestimate the memory deficits in humans that would result from a therapeutic dose of the drug. The memory-impairing effects of THIP may also be

184 further exacerbated in situations in which δGABAA receptor expression is increased, such as during stress (Maguire et al. 2007, Pisu et al. 2011, Serra et al. 2006, Serra et al. 2007) and puberty (Shen et al. 2010). Memory loss may therefore be a significant side effect of pharmacologic agents which modify δGABAA receptor activity, particularly in certain clinical sub-populations.

In summary, this thesis illustrates that δGABAA receptor activity dynamically regulates distinct memory processes, and reaffirms the significant therapeutic potential of δGABAA receptor-preferring drugs. Depending upon the temporal parameters of pharmacologic

δGABAA receptor activation, both memory impairments and enhancements might be observed with the administration of drugs which enhance δGABAA receptor activity. These two distinct effects of δGABAA receptor-preferring drugs may be individually optimized within a given therapeutic context. Enhancement of memory by THIP, for example, may be useful in the treatment of cognitive dysfunction. On the other hand, impairment of memory by THIP may be desirable in a surgical context. For example, THIP may be useful as an adjunct to a principle anesthetic, preventing intra-operative awareness and the recall of the adverse events which occur during surgery.

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