CREB-Mediated Enhancement of Hippocampus-Dependent Memory Consolidation and Reconsolidation

CREB-mediated enhancement of hippocampus-dependent memory consolidation and reconsolidation

Melanie Jay Sekeres

A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy (PhD) Department of Physiology University of Toronto

Melanie Jay Sekeres

PhD

Department of Physiology University of Toronto

2012

Abstract

Memory stabilization following encoding (synaptic consolidation) or memory reactivation

(reconsolidation) requires gene expression and protein synthesis. Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms, disrupting the function of the transcription factor CREB (cAMP responsive element binding protein) impairs both processes. We use a gain-of-function approach to show that CREB (and CREB-coactivator CRTC1) can facilitate both synaptic and systems consolidation and reconsolidation.

We first examine whether acutely increasing CREB levels in the dorsal hippocampus is sufficient to enhance spatial memory formation in the watermaze. Locally and acutely increasing CREB in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions which do not normally support consolidation, weakly-trained wild-type (WT) mice and strongly-trained mutant mice with brain-wide disrupted CREB function.

CRTCs (CREB regulated transcription co-activators) are a powerful co-activator of

CREB, but their role in memory is virtually unexplored. We show, for the first time, that the novel CREB co-activator CRTC1 enhances memory consolidation. Locally increasing CRTC1 (or CREB) in the dorsal hippocampus of WT mice prior to weak context fear conditioning facilitates consolidation of precise context memory.

Last, we show that CREB or CRTC1 facilitates precise and enduring memory consolidation and reconsolidation. Acute enhancement of hippocampal CREB or

CRTC1 during initial synaptic consolidation can maintain precision of remote context memory, while increasing CREB or CRTC1 just prior to reactivation of a weak remote context memory enhances context memory reconsolidation. These gain-of-function manipulations indicate that increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as reactivated established, memories without compromising memory specificity.

Together with previous results, these findings indicate that CREB is both necessary and sufficient for hippocampal-dependent memory formation, and underline its pivotal role in the hippocampal molecular machinery underlying long-term memory consolidation and reconsolidation.

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Acknowledgments

I would like to acknowledge my PhD supervisor, Dr. Sheena Josselyn for her continuous mentorship throughout my graduate program. She is one of the toughest women scientists I have known, and she has been a tremendous example of dedication to science, and to pop culture. The contributions of Dr. Paul Frankland have also been an invaluable part of my graduate program, both as a collaborator and as a sports fan.

Life in the lab was always a fun and supportive environment thanks to the motley crew of graduate students and post-doctoral fellows in the Josselyn and Frankland labs. I would especially like to acknowledge Alonso Martinez-Cannabal, Anne Wheeler, Katherine Akers, Maithe Arruda-Carvalho, Christy Cole, Adelaide Yiu, Scellig Stone, and Catia Teixiera for years of collaboration, entertainment and enabling.

I also acknowledge the in-vitro work done by Valentina Mercaldo and Derya Sargin (Chapter 4).

Daily life in the lab would not have been nearly as smooth without the contributions of our outstanding lab manager Toni Decristofaro, and technicians Mika Yamamoto and Russell Braybon.

I thank Dr. Martin Wojtowicz and Dr. Zhengping Jia for their suggestions and guidance on my graduate supervisory committee.

I also acknowledge the contributions of Dr. Gordon Winocur for his ongoing advice and constructive criticism, and Jeff Winocur for his sharp eye and wit, and for his tolerance.

Funding of my graduate program was provided by CIHR Frederick Banting and Charles Best Canada Graduate scholarship - Master’s and Doctoral awards, Sick Kids Research Institute (Restracomp), the University of Toronto’s School of Graduate Studies (SGS), the University of Toronto’s Faculty of Medicine, and the University of Toronto Neuroscience Program (UTNP).

Table of Contents

Acknowledgments ...... iv

Table of Contents ...... v

List of Figures ...... xii

List of Abbreviations ...... xv

1 INTRODUCTION ...... 1

1.1 Consolidation ...... 2

1.1.1 Synaptic consolidation ...... 3

1.2 Transcription factors ...... 5

1.2.1 Transcriptional regulation by CREB...... 6

1.2.2 CREB co-activators ...... 11

1.3 CREB’s role in regulating behaviour ...... 15

1.3.1 Identification of CREB as a key mediator of memory consolidation ...... 16

1.3.2 Methods of genetically manipulating CREB function in the mammalian brain ...... 19

1.3.3 A role for CRTCs in memory consolidation? ...... 27

1.4 Introduction to Systems Consolidation ...... 29

1.4.1 The hippocampus ...... 31

1.4.2 Theories of systems consolidation of remote memory...... 41

1.4.3 Reconsolidation ...... 44

1.4.4 Time-dependent reorganization of remote memories ...... 45

1.5 Putting it all together: CREB is both necessary and sufficient for memory consolidation ...... 48

1.5.1 CREB is necessary for memory consolidation ...... 48

1.5.2 CREB is sufficient for memory consolidation ...... 54

1.5.3 CREB is necessary for memory reconsolidation ...... 58

1.6 The role of CREB co-activators in memory consolidation ...... 62

1.6.1 CBP/p300 disruption impairs memory consolidation ...... 62

1.6.2 What is the role of CRTCs in memory consolidation? ...... 63

1.7 Goals of this thesis: Hypotheses and predictions ...... 64

2 GENERAL METHODS...... 66

2.1 Mice ...... 66

2.1.1 Wild-type (WT) ...... 66

2.1.2 CREBαΔ−/− mice ...... 66

2.2 Preparation of HSV Vectors ...... 66

2.3 Surgery...... 68

2.4 General behavioural procedures ...... 68

2.4.1 Spatial watermaze experiments ...... 68

2.4.2 Context fear conditioning experiments ...... 72

2.5 Statistical analyses ...... 74

2.6 Histology ...... 74

2.7 Immunohistochemistry ...... 76

3 Dorsal hippocampal CREB is both necessary and sufficient for spatial memory ...... 77

3.1 Abstract ...... 77

3.2 Introduction ...... 77

3.2.1 Experiment 1: Effects of increasing or suppressing CREB on the consolidation of spatial memory that is only weakly acquired in control mice...... 79

3.2.2 Experiment 2: Effects of increasing or suppressing CREB on consolidation of a strong spatial memory...... 80

3.2.3 Experiment 3: Effects of increasing CREB on the consolidation of memory in the absence of spatial cues...... 81

3.2.4 Experiment 4: Effects of increasing CREB on retrieval/expression of a previously acquired spatial memory...... 82

3.2.5 Experiment 5: Effects of increasing CREB on consolidation of a spatial memory in CREB-deficient mice...... 82

3.3 Detailed Methods ...... 84

3.3.1 Mice ...... 84

3.3.2 Preparation of HSV Vectors ...... 84

3.3.3 Surgery ...... 84

3.3.4 Spatial watermaze training ...... 85

3.3.5 Probe test ...... 85

3.3.6 Experiment 1. Effects of increasing or suppressing CREB on the consolidation of spatial memory that is only weakly acquired in control mice...... 85

3.3.7 Experiment 2. Effects of increasing or suppressing CREB on consolidation of a strong spatial memory...... 85

3.3.8 Experiment 3. Effects of increasing CREB on the consolidation of memory in the absence of spatial cues...... 86

3.3.9 Experiment 4. Effects of increasing CREB on retrieval/expression of a previously acquired spatial memory...... 86

3.3.10 Experiment 5. Effects of increasing CREB on consolidation of a spatial memory in CREB-deficient mice...... 86

3.3.11 Statistical Analyses...... 86

3.3.12 Histology: Confirming transgene expression following behavioural testing...... 87

3.3.13 Immunohistochemistry: CREB staining...... 88

3.4 Results ...... 88

3.4.1 Microinjection of CREB vector increases CREB protein in the CA1 of dorsal hippocampus...... 88

3.4.2 Experiment 1: Increasing CREB in the hippocampus facilitates consolidation of spatial memory that is only weakly acquired in control mice ...... 90 vii

3.4.3 Experiment 2: Increasing CREB in the hippocampus further enhances consolidation of robust/strong spatial memory. Suppressing CREB in the hippocampus does not impair spatial memory...... 93

3.4.4 Experiment 3: Increasing CREB in the hippocampus does not enhance consolidation of memory in the absence of spatial cues ...... 97

3.4.5 Experiment 4: Increasing CREB in the hippocampus does not enhance retrieval/expression of a previously acquired spatial memory ...... 99

3.4.6 Experiment 5. Effects of increasing CREB in the hippocampus rescues the consolidation of spatial memory in CREB-deficient mice...... 102

3.5 Discussion ...... 109

3.6 Conclusion ...... 112

3.7 Contributions ...... 113

4 Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory precision . 114

4.1 Abstract ...... 114

4.2 Introduction ...... 115

4.2.1 Experiment 1: Effects of increasing CREB on the consolidation of context fear memory that is only weakly acquired in control mice ...... 117

4.2.2 Experiment 2&3: Effects of increasing CRTC1 or CREB on consolidation of a recent context fear memory ...... 118

4.2.3 Experiment 4: Effects of increasing CRTC1 or CREB on retrieval/expression of a weak context fear memory...... 119

4.2.4 Experiment 5: Effects of increasing CRTC1 or CREB on consolidation of a remote context fear memory ...... 120

4.2.5 Experiment 6: Effects of increasing CRTC1 or CREB on reconsolidation of a remote context fear memory ...... 121

4.3 Detailed Methods ...... 123

4.3.1 Mice ...... 123

4.3.2 Preparation of HSV vectors ...... 123

4.3.3 Transfection of primary hippocampal neurons and luciferase assay ...... 123

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4.3.4 Surgery ...... 125

4.3.5 Contextual fear conditioning ...... 125

4.3.6 Experiment 1. Effects of increasing CREB on the consolidation of context fear memory that is only weakly acquired in control mice...... 125

4.3.7 Experiment 2. Effects of increasing CRTC1 or CREB on consolidation of a weak context fear memory...... 126

4.3.8 Experiment 3. Effects of increasing CRTC1 or CREB on consolidation of a strong context fear memory...... 126

4.3.9 Experiment 4. Effects of increasing CRTC1 or CREB on retrieval of a weak context fear memory...... 126

4.3.10 Experiment 5. Examining the enduring effects of increasing CRTC1 or CREB on consolidation of a weak remote context fear memory...... 126

4.3.11 Experiment 6. Effects of increasing CRTC1 or CREB on reconsolidation of a weak remote context fear memory...... 127

4.3.12 Statistical analyses ...... 127

4.3.13 Histology: Confirming transgene expression following behavioural testing...... 128

4.3.14 Immunohistochemistry...... 128

4.4 Results ...... 130

4.4.1 Microinjection of CRTC1 or CREB vector increases CRTC1 and CREB function in the dentate gyrus of dorsal hippocampus...... 130

4.4.2 Experiment 1: Increasing CREB in the hippocampus facilitates consolidation of context fear memory that is only weakly acquired in control mice ...... 139

4.4.3 Experiment 2: Increasing CRTC1 or CREB in the hippocampus facilitates consolidation of context fear memory ...... 140

4.4.4 Experiment 3: Increasing CRTC1 or CREB in the hippocampus further enhances consolidation of robust /strong contextual fear memory ...... 143

4.4.5 Experiment 4: Increasing CRTC1 or CREB in the hippocampus does not enhance retrieval/expression of a previously acquired context fear memory ...... 144

4.4.6 Experiment 5: The context memory enhancement produced by increasing CRTC1 or CREB in the hippocampus at the time of conditioning is long-lasting ...... 146

4.4.7 Experiment 6: Increasing CRTC1 or CREB in the hippocampus facilitates reconsolidation of context fear memory ...... 147

4.5 Discussion ...... 150

4.6 Conclusion ...... 158

4.7 Contributions: ...... 158

5 GENERAL DISCUSSION ...... 159

5.1 CREB is sufficient to support consolidation of weak memory ...... 159

5.1.1 Candidate CREB-target genes facilitating memory consolidation ...... 161

5.2 CREB can further enhance strong memory formation ...... 162

5.3 Suppressing CREB in a limited population of hippocampal cells does not impair consolidation...... 163

5.3.1 Caveat ...... 164

5.4 CREB is necessary for memory consolidation ...... 165

5.5 CREB is both necessary and sufficient for memory consolidation ...... 166

5.6 CREB facilitates the consolidation, but not the retrieval of memory ...... 167

5.7 CRTC1 is sufficient to support memory consolidation in vivo ...... 169

5.8 CREB and CRTC1 promote consolidation of precise remote memory ...... 171

5.8.1 Caveats ...... 173

5.8.2 Are CREB or CRTC1-enhanced neurons required for the memory at a remote timepoint? ...... 174

5.8.3 Is this CREB-enhanced remote memory abnormally persistent? ...... 174

5.8.4 Time dependent structural reorganization of remote memory ...... 175

5.9 CREB or CRTC1 strengthen reconsolidation of remote memory ...... 176

5.9.1 The window for reconsolidation ...... 176

5.9.2 CREB or CRTC1 is necessary for reconsolidation ...... 176

5.9.3 CREB or CRTC1 is sufficient for reconsolidation ...... 177

5.9.4 How does CREB or CRTC1 change the representation of a strong remote context memory? ...... 178

5.9.5 Boundary conditions to reconsolidation ...... 180

5.10 General caveats ...... 181

5.11Practical applications of CREB enhancement in disease and aging ...... 182

5.11.1 CREB and aging ...... 182

5.11.2 CREB and disease ...... 183

6 CONCLUSION ...... 186

References ...... 187

List of Figures

Figure 1.1 Basic schematic of the major domains within the CRE site ...... 7

Figure 1.2 The upstream CREB signaling cascade...... 10

Figure 1.3 The CRTC1 signaling cascade...... 14

Figure 1.4 The traditional trisynaptic circuit of the hippocampus ...... 33

Figure 1.5 Basic schematic of the hippocampal-dependent memory consolidation process ...... 47

Figure 2.16 Watermaze apparatus...... 69

Figure 2.27 Timeline for weak and strong training spatial watermaze experiments ...... 70

Figure 2.38 Context fear conditioning apparatus and tracking ...... 72

Figure 2.49 Timeline for context fear conditioning and context generalization testing..73

Figure 3.110 Transgene expression in the CA1 of the dorsal hippocampus ...... 89

Figure 3.211 Microinjection of CREB vector in the CA1 increases levels of CREB protein...... 89

Figure 3.312 Acutely increasing CREB in the dorsal hippocampus enhances consolidation of weak spatial memory ...... 92

Figure 3.413 Acutely increasing CREB in the dorsal hippocampus further enhances consolidation of strong spatial memory ...... 96

Figure 3.514 CREB-enhanced performance in the watermaze is dependent on spatial memory...... 98

Figure 3.615 Figure 3.6 Acutely Increasing CREB does not affect expression of a previously acquired spatial memory ...... 101

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Figure 3.716 Microinjection of CREB vector increases levels of CREB protein in the CA1 region of CREB-deficient mice ...... 103

Figure 3.817 Acutely increasing CREB in dorsal hippocampus rescues the spatial memory deficit in CREB-deficient mice ...... 106

Figure 3.918 CREB-deficient mice fail to adopt a spatial search strategy ...... 108

Figure 4.119 Expression of CRTC1 in the dentate gyrus of the dorsal hippocampus . 131

Figure 4.220 Infection of dentate gyrus granule cells...... 132

Figure 4.321 Vector microinjection induces robust localized transgene expression in the dentate gyrus of dorsal hippocampus ...... 133

Figure 4.422 Estimations of the typical infection size for each vector ...... 135

Figure 4.523 Microinjection of CRTC1 vector in the dentate gyrus increases expression of CRTC1 protein ...... 136

Figure 4.624 Cellular localization of CRTC1 ...... 138

Figure 4.725 CRTC1 increases CRE-reporter activity ...... 138

Figure 4.826 Increasing wild-type CREB in the dentate gyrus induced strong context fear long-term memory ...... 140

Figure 4.927 Increasing CRTC1 or CREB levels in the dentate gyrus facilitates consolidation of weak contextual fear long-term memory; this enhancement is context- specific ...... 142

Figure 4.1028 Increasing CRTC1 or CREB levels in the dentate gyrus further enhances consolidation of strong contextual fear long-term memory ...... 144

Figure 4.1129 Increasing CRTC1 or CREB levels in the dentate gyrus does not facilitate retrieval of previously acquired context fear long-term memory ...... 145

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Figure 4.1230 Increasing CRTC1 or CREB levels in the dentate gyrus facilitates remote memory consolidation, which maintains context precision ...... 147

Figure 4.1331 Increasing CRTC1 or CREB levels in the dentate gyrus enhances reconsolidation of a remote contextual fear long-term memory ...... 149

Figure 5.132 Predicted enhanced spatial memory following retrieval in the presence of high hippocampal CREB...... 168

Figure 5.233 Predicted enhanced memory following new learning in the presence of high hippocampal CREB...... 169

Figure 5.334 Predicted freezing patterns for remote memory and remote reconsolidation of context memory following strong training ...... 179

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List of abbreviations

5-HT – serotonin

α-CaMKII - alpha-calcium/calmodulin-dependent protein kinase II

AAV – adeno-associated virus

AC – adenylyl cyclase

ACC – anterior cingulate cortex

ACh – acetylcholine

AMPA – 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid

AMPK – adenosine monophosphate activated protein kinase

ANOVA – analysis of variance

AP – anterior/posterior

AP-1 – activator protein 1

ApCREB1 - Aplysia CREB1 activator isoform

ApCREB2 – Aplysia CREB2 repressor isoform

Arc – activity-regulated cytoskeletal associated protein

ATF-1 – activating transcription factor-1

ATP – adenosine triphosphate

BDNF – brain-derived neurotropic factor

BLA – basolateral amygdala

BMAL-1 – brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 xv

BSA – bovine serum albumin bZIP – basic leucine zipper

C – carboxyl

(14C)2-DG – (14C)2-deoxyglucose

CA1 – Cornu Ammonis area 1

Ca2+ – calcium

CA3 – Cornu Ammonis area 3

CAD – constitutive active domain

CaM – calmodulin

CaMK – calcium/calmodulin-dependent protein kinase cAMP – cyclic adenosine monophosphate, cyclic AMP

CBP – CREB binding protein cDNA – complementary DNA

C/EBP – CCAAT-enhancer-binding proteins cm – centimeter

CRE – cAMP responsive element

CREM – CRE-modulating element

CREB – cAMP/Ca2+ responsive element binding protein

CRTC – CREB regulated transcription co-activators

CS – conditioned stimulus

xvi

CXT – context d – days

DAPI – 4',6-diamidino-2-phenylindole dCREB2-a – activator isoform CREB2-a dCREB2-b – inhibitory isoform CREB2-b

DG – dentate gyrus

DIV – days in-vitro dn – dominant negative

DNA – deoxyribonucleic dox – doxycycline

DREAM – downstream regulatory element antagonistic modulator

E18 – embryonic day 18

EC – entorhinal cortex eGFP – enhanced GFP

EPSC – excitatory post-synaptic current

EPSP – excitatory postsynaptic potential

ERK – extracellular-signal-regulated kinases fEPSP – field excitatory postsynaptic potential

FSK – forskolin

GABA – gamma-aminobutyric acid

xvii

GAD67 – glutamate decarboxylase 67

GFAP – glial fibrillary acidic protein

GFP – green fluorescence protein

GluR1 – glutamate receptor type 1

GPCR – G-protein coupled receptor h – hours

HAT – histone acetyltransferases

HEK – human embryonic kidney cells

HIV-1 – human immunodeficiency virus type 1 hs – heat shock

HSV – herpes simplex virus

Hz – hertz

ICER – inducible cAMP early repressor

IE – immediate early

IGF-1 – insulin-like growth factor iNOS – inducible nitric oxide synthase

IR – inducible repressor

ITI – inter-trial-interval

KCl – potassium chloride kg – kilograms

xviii

KID – kinase-inducible domain lacZ – β-galactosidase

L-LTP – late phase long-term potentiation

LSM – laser scanning microscope

LTD – long-term depression

LTF – long-term facilitation

LTP – long-term potentiation

LTM – long term memory mA – milliamperes

MAPK – mitogen-activated protein kinases

MARK – microtubule affinity regulating kinase mCREB – mutant CREB (also called CREBS133A )

MEF2 – myocyte enhancer factor-2 mg – milligrams ml – millilitres

M/L – medial/lateral mM – millimolar mRNA – messenger RNA

MSK – mitogen and stress activated protein kinase

MTT – Multiple Trace Theory

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MUT – mutant, also refers to CREB αδ-/- mutation

NF-1 – neurofibromin 1

NF-L – neurofilament light ng – nanogram

NGS – normal goat serum

NMDA – N-Methyl-D-aspartic acid

NMDAr – N-Methyl-D-aspartic acid receptor

NR – no reactivation group

Nr4a2 – nuclear receptor type 4a2

N-terminal – amino terminal

Nurr1 – nuclear receptor related 1 protein

ODN – oligodeoxynucleotides p300 – protein 300

PBS – phosphate-buffered saline pCREB – phosphorylated CREB

PDE4 – phosphodiesterase inhibitor 4

PFA – paraformaldehyde

PFC – prefrontal cortex

PKA – protein kinase A

PKM – protein kinase M

PP1 – protein phosphotase 1

PP2B – protein phosphotase 2B

PSI – protein synthesis inhibitor

PTZ – pentylenetetrazol

R – reactivation group

RA – retrograde amnesia

RAM – radial-arm maze

Ras – rat sarcoma

REM – rapid eye movement

RI – reactivity index

RNA – ribonucleic acid

RNA Pol lI – RNA polymerase 2 complex

RSC – retrosplenial cortex

RSK – ribosomal protein S6 kinase

RTS – Rubenstein Taybi Syndrome s – seconds

S133A – serine133 to alanine point mutation sc – subcutaneous

SCT – Standard Consolidation Theory

SEM – standard error of the mean

xxi

Ser – serine shRNAi – short hairpin RNA interference

SIK – salt-inducible kinase

Sirt – sirtuin

SRF – serum response factor

STFP – social transmission of food preference

STM – short term memory

TAF4 – TBD-associated factor 4

TBD – TATA-box binding domain tet – tetracycline

TFIID – transcription factor II D

TGF – transforming growth factor

TGRA – temporally graded retrograde amnesia

TORC – transducers of regulated CREB activity

TrKB – tyrosine kinase receptor type 2

TT – Transformation Theory tTa – tetracycline-controlled transactivator

US – unconditioned stimulus

V – ventral

VGCC – voltage-gated calcium channel

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VP16-CREB – constitutively active form of CREB

Q – glutamine

WM – watermaze

WT – wild-type

Zif268 – zinc finger transcription factor

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1 INTRODUCTION

Many reviews of the concept of ‘memory’ begin with a lofty description about the essence of the sentient being shaped by their experiences and memories. How one’s collective experiences, and the ability to recall these at will, give meaning to the person one becomes. How the loss of these memories is akin to the loss of one’s self identity. This, inevitably, will be accompanied by a Latin translation, and a quote by an obscure 200 year old poet pondering his existence. I hate that.

In reality, thinking about memory in these highly romanticized and abstract terms is much easier than understanding its biological basis. It is overwhelming to try and understand the complex molecular and cellular processes in the brain that have to occur in a precisely defined sequence in order to be able to recall a lifetime of events, a person, a place, at will. It is easiest to break down this process into discrete processes to best understand how this is possible. Here, the whole (memory) is greater than the sum of its parts (i.e. transcription, translation, synaptic consolidation, systems consolidation, reconsolidation). However, we need to understand the ‘parts’. It is not trivial that the inability to form or recall memories is devastating to an individual’s sense of personal identity and history, or at the most basic level, to the survival of an animal which requires the use of memory for information related to feeding, mating, and safety. To develop therapies aimed at remedying memory loss in conditions ranging from developmental disorders, to neurodegenerative diseases, aging, or accidental causes of traumatic brain injury in humans, it is critical that we understand the ‘parts’. We need a bottom-up approach to understand the complex process whereby a single experience of an event can produce a complex molecular signaling cascade that leads to lasting chemical, electrical, and structural changes in the brain. This is the essence of memory.

In this thesis, I present an overview of these ‘parts’ of memory, with emphasis on processes required to form and strengthen memory. We discuss the transcription factor CREB, identified as a critical protein required for the formation (consolidation) of many different types of memory across species. We show how CREB is not only required for normal memory formation, but how augmenting CREB activity in the hippocampus, a

1 key region implicated in memory consolidation, can actually allow for the formation of a strong, enduring memory under otherwise challenging learning conditions. While it is important to think of memory processing as a system, not as isolated and discrete processes, we investigate how manipulating one small part of this system can have dramatic effects on the whole.

1.1 Consolidation

This brings us to the first concept we need to understand in order to begin to comprehend how memories are physically represented in the brain. The term ‘consolidation’ is used to describe the process by which memories become progressively stabilized in the brain. Stemming from Latin word consolidaire, meaning ‘together’, and ‘make firm’, the term is attributed to Mueller and Pilzecker (1900), who realized that new memories require a certain length of time to fixate, and that interference with this fixation process immediately following the acquisition of new information leads to a disruption in the stabilization, or consolidation, of that new information. They termed this concept ‘retroactive interference’. After sufficient time has passed, they noted that the memory for the new information is no longer sensitive to disruption by interference (Dudai, 2004; Lechner et al., 1999). Consolidation is currently understood as a dual process, the first being a rapid consolidation (synaptic consolidation) occurring in synapses of neurons in the seconds and minutes following the new experience, and a second slower consolidation (systems consolidation) in which the memory becomes reorganized and distributed across multiple brain regions, a process occurring over the weeks, months, and even years following the event (for review see Dudai, 2004). To begin to understand the process required to ultimately result in the expression of a memory, we have to think small.

1.1.1 Synaptic consolidation

Synaptic plasticity is long-lasting changes in the strength of synaptic connections. Generally speaking, plasticity is a dynamic process by which something is modified or shaped by external force. Synapses, which are the communication junctions between neurons, exhibit plasticity, both structurally and functionally (reviewed in Holtmaat and Svoboda, 2009; Kasai et al., 2010). Here, the efficacy of communication between synapses can be strengthened or weakened depending on the external factors acting upon the synapse. A synapse that is activated following neuronal depolarization will become strengthened.

Synaptic transmission occurs in response to various signaling cascades. Depolarization of the cell via glutamate at an excitatory synapse triggers calcium (Ca2+) influx into the post-synaptic cell through either voltage-gated calcium channels (VGCCs) (Ghosh et al., 1994; Gallin and Greenberg, 1995; Greer and Greenberg, 2008)) or GluR1 subunit- containing AMPA receptors (Berridge, 1998). Binding of glutamate and depolarization triggers the release of the magnesium block in the NMDA receptor at the synapse, allowing calcium to rush into the post-synaptic cell through the NMDA ion channel (Morris et al., 1990a). The post-synaptic calcium influx and the release of intracellular calcium stores (Blochl and Thoenen, 1995) set off a cascade of molecular signaling in the cell, including catalyzing multiple protein kinases, which we will discuss in detail shortly.

In 1972, Bliss and Lømo realized that strong stimulation of input to a cell produces long- lasting changes in the strength or amplitude of the post-synaptic response, whereas weaker stimulation of inputs produces much smaller changes in the amplitude. Strong stimulation/depolarization can also induce long-lasting changes in synaptic strength, a process called long-term potentiation (LTP), which can last hours to days, and is characterized by two physiological stages: early and late-phase LTP (Bliss and Collingridge, 1993; Goelet et al., 1986; Bailey et al., 2000a; Bailey et al., 2000b); Kandel, 2001). The early phase (E-LTP) typically only last 1-2 h, and involves post- translational modifications of pre-existing proteins. It can also induce changes in receptor trafficking, such as the internalization of AMPA receptors. Protein-synthesis 3 independent E-LTP has been associated with short-term memory (Nguyen et al., 1994; Stork and Welzl, 1999; Soderling and Derkach, 2000; Kandel, 2001; Racaniello et al., 2009). The second phase, late-phase LTP (L-LTP) lasts between 6-24 h, and is dependent on the synthesis of new proteins. L-LTP is considered the cellular equivalent to long-term memory, where the generation of new proteins supports the consolidation, or stabilization of the change in strength between synapses. (Davis and Squire, 1984; Bailey and Chen, 1989; Frey et al., 1993; Goelet et al., 1986; Huang and Kandel, 1994; Kandel, 2001).

Early proposals of memory storage occurring by changes/growth of connections between cells originated from Santiago Ramón y Cajal in 1894. However his idea was not experimentally supported until the 1970s when Kandel and colleagues provided evidence that learning simple reflexive behaviour in the invertebrate Aplysia californica resulted in changes in the strength of synaptic connections between established, interconnected neurons (Kandel and Tauc, 1963; Castellucci et al., 1970; (Kupfermann et al., 1970). The modern understanding of memory storage is based on the observation that synapses are the site of neural transmission, and that synaptic plasticity is the mechanism for memory formation (Kandel, 2001). Synaptic plasticity is broadly defined as experience-dependent changes in the strength of synaptic connections leading to modifications of the neuronal circuitry via synaptic strengthening, generation, and remodeling. These structural and functional modifications to synapses in response to new learning and long-term memory formation underlie the concept of synaptic consolidation.

These are important distinctions between short-term memory (STM) and long-term memory (LTM) from the perspective of synaptic consolidation. At this stage, we will briefly note that synaptic consolidation (STM and LTM) differs from systems consolidation (Dudai 2004 for review), which distinguishes between many different types of memories (episodic, declarative, semantic, procedural; recent, remote). For the purpose of our review, we are primarily interested in episodic-like memories, which can be considered explicit memories for facts, places, and events (Scoville and Milner, 1957). We will later discuss recent and remote memories from the perspective of

4 systems consolidation. But first, we will explore the process of synaptic consolidation, which must first occur for a long-term memory to stabilize in the neuronal network.

1.2 Transcription factors

Long-term memories depend on gene expression and protein synthesis in the cell. Upon activation/depolarization of a cell, extracellular signaling initiates a molecular cascade of second messengers and kinases which ultimately result in the phosphorylation of transcription factors in the nucleus, leading to the transcription of genes involved in memory consolidation. Transcription factors are proteins that initiate or repress transcription of a particular gene by binding to the gene’s promoter region. Almost 30,000 genes have been identified within the human genome. Among them, almost 3000 transcription factors have been identified (Babu et al., 2004; Alberini, 2009).

All transcription factors share two characteristic domains: 1) A DNA-binding domain, and 2) and a transcriptional activator domain (Brivanlou and Darnell Jr., 2002; Alberini, 2009). The positive-acting transcription factors can be organized into two main categories. Constitutive transcription factors are always present and active in the nucleus, and regulatory transcription factors, which are present in the nucleus but remain inactive until initiated by intra or extracellular signaling. Among the class of signal-dependent transcription factors is a subcategory of resident nuclear factors that are activated by serine phosphorylation, which is the addition of a phosphate group to the amino acid sequence (Brivanlou and Darnell Jr., 2002). Among the class of regulatory nuclear factors is cyclic adenosine monophosphate (cAMP) response element (CRE)-binding protein (CREB), a well-characterized stimulus-induced transcription factor implicated in the initiation of gene transcription supporting memory consolidation.

1.2.1 Transcriptional regulation by CREB

The CREB family of regulatory transcription factors is part of the bZIP superfamily of transcription factors, which all bind to the CRE site of a gene. The CRE site was identified as an 8-bp sequence 5’-TGACGTCA-3’ (Comb et al., 1986; Montminy et al., 1986). Montminy and colleagues subsequently identified the cellular protein that binds to CRE, which they termed CRE binding protein (CREB) (Montminy and Bilezikjian, 1987). The CREB family is encoded by three genes: CREB, CREM (cAMP response element modulator) and ATF-1 (Activating Transcription Factor 1) (Gonzalez et al., 1989; Foulkes et al., 1991; Rehfuss et al., 1991). CREB encodes three alternatively spliced variants (CREB α, β and Δ) each of which stimulate transcription through promoters containing the CRE sequence in a gene’s promoter region, and are ubiquitously expressed in the body (Blendy et al., 1996). In contrast, CREM’s four major splice variants [CREM α, β, ϒ, and ICER (inducible cAMP early repressor)] can bind to the CRE site, but act to repress CRE-dependent transcription (Mellstrom et al., 1993).

1.2.1.1 CREB’s structure

The CREB family of proteins contain four highly conserved domains: the basic domain and leucine zipper domain (bZIP), two glutamine (Q)-rich domains (Q1, Q2/CAD, constitutive active domain), and the kinase inducible domain (KID). As part of the bZIP superfamily, CREB binds to DNA as dimers (Yamamoto et al., 1988). The carboxyl (C) - terminal basic domain mediates dimerization and the leucine zipper mediates DNA binding (Shaywitz and Greenberg, 1999; Lonze and Ginty, 2002). CREB family members can dimerize as homo or heterodimers, with CREB/CREB homodimers resulting in the strongest transcriptional activation (Hai et al., 1989; Foulkes et al., 1991; Laoide et al., 1993). Q1 and Q2/CAD induce low basal transcription activity, and interact with TAF4 (TBD associated factor 4) to recruit the basal transcription machinery to the promoter region (Quinn, 1993; Ferreri et al., 1994; Xing et al., 1995; Lonze and Ginty, 2002; Barco et al., 2003). Q1 and Q2/CAD are separated by the KID domain. The KID domain contains the critical residue Serine 133 (Ser133) required for

6 phosphorylation-dependent transcription of CREB target genes (Mayr and Montminy, 2001), and is the binding site for CREB co-activator CBP (CREB Binding Protein) and p300 (Chrivia et al., 1993; Kwok et al., 1994) (Fig 1.1). We will discuss the role of CBP/p300 in transcriptional activation shortly.

Figure 1.1 Basic schematic of the major domains within the CRE site

1.2.1.2 CREB signaling

Activation of CREB is accomplished through an upstream signaling cascade which ultimately results in the phosphorylation of CREB (pCREB) at Ser133. CREB phosphorylation can also occur on Ser129 and Ser142, but transcriptional activation of CREB-target genes requires phosphorylation at the key Ser133 residue within the KID (Sun et al., 1994, Giebler et al., 2000, Kornhauser et al., 2002) (Fig 1.1). This phosphorylation is dynamically regulated by kinases and phosphatases that respond to increased levels of intracellular second messengers calcium (Ca2+) and cAMP (West et al., 2002). In neurons, synaptic stimulation, depolarization, and behavioural training can all result in intracellular increases of calcium and cAMP (Flavell et al., 2008).

1.2.1.2.1 Calcium-induced signaling

Calcium entry to the post-synaptic cell triggers a signaling cascade beginning with an interaction with several calcium/calmodulin-dependent protein kinases (CaMKs). Multiple kinases can catalyze the phosphorylation of CREB at Ser133, including CaMK II and IV, mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinases (ERK), mitogen and stress activated protein kinase (MSK) 1-3, ribosomal protein S6 kinase (RSK) 1 and 2, and protein kinase A (PKA) genes (West et al., 2002; Brivanlou and Darnell, 2002; Cohen and Greenberg, 2008; Flavell and Greenberg, 2008).Calcium-induced phosphorylation of CREB is primarily mediated via two pathways. First, membrane depolarization allows calcium influx through VGCCs, or glutamatergic activation of NMDArs allows influx of ionic calcium, which interacts with the calcium-binding protein calmodulin (Dash et al., 1991; Sheng et al., 1991; Shaywitz and Greenberg, 1999; Wayman et al., 2008. The interaction of calcium with CaMKII and CaMKIV can directly phosphorylate CREB both in vitro and in vivo (Dash et al., 1991; Sheng et al., 1991; West et al., 1991; Kaang et al., 1993; Bito et al., 1996; Ho et al., 2000; Kang et al., 2001). Second, calcium entry can also trigger the activation of the Ras/ERK or the Ras/MAPK pathway activating several different kinases from the RSK and the MSK family of kinases which also phosphorylate CREB at Ser133 (Bading and Greenberg, 1991; Rosen et al., 1994; Xing and Quinn, 1994; Xing et al., 1996; De Cesare et al., 1998; Impey et al., 1998b; Roberson et al., 1999; Davis et al., 2000; Dolmetsch et al., 2001; Lonze and Ginty, 2002; Tian and Feig, 2006) (Fig 1.2).

Within minutes of neuronal activation, the molecular signaling of Ca2+ and cAMP second messengers and kinases results in CREB’s phosphorylation. Artificially inducing neuronal activation with PTZ-induced seizures results in sustained neuronal activation in the brain (Qian et al., 1993).The in vivo CREB phosphorylation pattern observed following seizure onset suggests that hippocampal and cortical CREB phosphorylation at Ser133 occurs within 3-8 minutes following neuronal activation (Moore et al., 1996), followed by a gradual decline in Ca2+/CaMKII and cAMP/PKA, and pCREB at 60 min. However, pCREB levels are still above control levels at this timepoint. In vitro, following strong tetanization of the Schaeffer’s collaterals which induced L-LTP, pCREB levels

8 peak at 45min, followed by a second wave 6hr following stimulation (Ahmed and Frey, 2005).

Calcium/calmodulin induced phosphorylation of CREB is mediated by a negative feedback mechanism to limit calcium-dependent activation of CREB in an activity- dependent manner. CaM-induced activation of the calcium-sensitive phosphatase calcineurin and protein phosphatase (PP) 1 and PP2A promotes dephosphorylation of Ser133, which inhibits gene transcription (Bito et al., 1996; Genoux et al., 2002). One way this is accomplished is by NR2B-containing NMDARs initiating intracellular signaling which promotes dephosphorylation of Ser133 by activating PP1 (Wadzinski et al., 1993; Alberts et al., 1994; Bito et al., 1996; Wu et al., 2001; Hardingham et al., 2002). Also, phosphorylation of Ser142 and Ser143 by CaMKII (Matthews et al., 1994) or CaMKIV (Ghosh and Greenberg, 1995) leads to a dissociation of CREB dimerization, which inhibits activity. Additionally, dephosphorylation can be induced by phosphodiesterase inhibitors, such as PDE4 (D'Sa et al., 2002). Together, these homeostatic mechanisms mediate CREB’s transcriptional activity.

Figure 1.2 The upstream CREB signaling cascade. Black arrows indicate signaling that results in the phosphorylation of CREB. Red arrows indicate negative regulation of phosphorylation. The AC/cAMP signaling cascade recruits PKA to phosphorylate CREB. Ca2+/CaMK signaling cascades recruit CaMKs and Ras/ERK/MAPK kinases to phosphorylate CREB. Ca2+/CaMK signaling also results in the mediation of phosphorylation of CREB via calcineurin and phosphatase signaling of PP1 and PP2A.

1.2.1.2.2 cAMP-induced signaling

The other main signaling cascade which triggers the phosphorylation of CREB begins with an increase in intracellular cAMP primarily via activation of G-protein coupled receptors (GPCRs) (Kandel, 2001), and stimulation of the enzyme adenylyl cyclase (AC) (Poser and Storm, 2001). AC stimulation converts adenosine triphosphate (ATP) to the second messenger cAMP, which triggers a release of PKA in the cytosol. PKA then translocates to the nucleus where it phosphorylates CREB (Fig 1.2). It should be

10 noted that increases in intracellular calcium can also result in an activation of Ca2+- sensitive isoform of AC, which also results in a calcium-induced increase in PKA (Xia and Storm, 1997). In 1986, Montminy and colleagues discovered that within the promoter region of the somatostatin gene, the CRE site was responsible for cAMP- induced transcription following stimulation with forskolin, a cAMP-agonist (Montminy et al., 1986). They went on to determine that cAMP activity regulated PKA (Yamamoto et al., 1988), and in turn, that PKA-mediated phosphorylation of Ser133 was critical for transcriptional activation of CRE-containing genes (Yamamoto et al., 1988; Gonzalez et al., 1989) (Fig 1.2). Thus, PKA was understood to be a critical kinase regulating the cAMP-induced activation of CREB target genes.

1.2.2 CREB co-activators

Above, we discussed the multiple signaling cascades that can result in the phosphorylation of CREB at Ser133. However, this phosphorylation event is not always sufficient to activate transcription (Impey et al., 1996; Mayr and Montminy, 2001) suggesting that additional mechanisms are involved in CREB mediated transcription. It is now known that interaction with different co-factors, including CBP/p300 and the recently identified family of CREB-regulated transcriptional co-activators (CRTCs) are also required (Goodman and Smolik, 2000; Mayr and Montminy, 2001; Vo and Goodman, 2001; Conkright et al., 2003a; Screaton et al., 2004).

1.2.2.1 CBP/p300

Phosphorylation of CREB stimulates the recruitment of the transcriptional co-activators CREB binding protein (CBP) and the closely related p300 [encoded by the EP300 gene (Chrivia et al., 1993; Parker et al., 1996)]. Following phosphorylation, CREB’s KID (kinase inducible domain) serves as a docking-site for CBP and p300, via their KIX (KID interaction) domains. CBP/p300 activate transcription by functioning as scaffolding proteins [recruiting additional transcriptional machinery components, including RNA

11 polymerase II (RNA Pol lI), to the binding complex] and histone acetyltransferases (HAT) (promoting unraveling of the chromatin structure to allow access to the transcription machinery), and counteracting the repressive effects of histone deacetylases (Bannister and Kouzarides, 1996; Nakajima et al., 1997b; Nakajima et al., 1997a; Vo and Goodman, 2001).

1.2.2.2 CRTCs

KID-mediated recruitment of CBP/p300 provides a mechanism for phosphorylation- dependent induction of CREB target genes. However, in 2003, a new family of CREB co-activators was identified, referred to as CRTCs (CREB regulated transcriptional co- activators, previously referred to as Transducers of Regulated CREB activity, or TORCs, but renamed to avoid confusion with the target of rapamycin complex) which can bind unphosphorylated CREB and promote its transcriptional activity (Conkright et al., 2003a; Iourgenko et al., 2003). Three isoforms of CRTC (CRTC1-3) have been identified in mammals (Bittinger et al., 2004), and were first recognized to be critical for mediating CREB-dependent transcription of glucogenic enzymes within the liver (Screaton et al., 2004; Poo et al., 2005). While all three isoforms are detectable in the brain, their expression patterns vary within different cortical and subcortical subregions. CRTC1 shows the highest brain expression, particularly with the hippocampus, prefrontal cortex, striatum, cerebellum, and hypothalamic regions (Conkright et al., 2003; Kovacs et al., 2007; Watts et al., 2011). CRTC2 shows moderate expression levels in these regions, while CRTC3 shows only trace amounts throughout the brain (Watts et al., 2011). Much of the in vitro research into the CRTC family has focused only on CRTC1, and 2, with little attention to CRTC3.

The N-terminal region of CRTC contains a coiled-coil structure that physically interacts with the bZIP domain of CREB to bind as a tetramer (Conkright et al., 2003a, 2003b). CRTC binding to CREB is thought to enhance transcription by facilitating the interaction of CREB with the TBD-associated factor 4 (TAF4) (previously called TAFII130/135 in mammals and TAFII110 in Drosophila (Tora, 2002;Conkright et al., 2003b) to its Q2

12 domain (Felinski and Quinn, 1999). TAF4 is a component of the transcription factor II D

(TFIID), one of the general transcription factors in the RNA polymerase II pre-initiation complex (Conkright et al., 2003a, 2003b; Xu et al., 2007; Altarejos and Montminy, 2011).

CRTC-mediated transcriptional activation requires CREB to be intact, but does not require phosphorylation of CREB at Ser133 (Conkright et al., 2003b). However, the subcellular location and activity of CRTC1 and CRTC2 themselves, are regulated by phosphorylation. Under basal conditions, CRTC1 is phosphorylated at Ser 151 and Ser 245 (Koo et al., 2005; Sasaki et al., 2011), and CRTC2 is phosphorylated at Ser171 and Ser275 (Screaton et al., 2004; Jansson et al., 2008) and sequestered in the cytosol by interactions with 14-3-3 scaffolding proteins (Screaton et al., 2004; Jansson et al., 2008; Mair et al. 2011) (Fig 1.3). Several kinases mediate the phosphorylation status of CRTC including salt-inducible kinases (SIK) 1-3, which are members of the 5’ adenosine monophosphate activated protein kinase (AMPK) family (Screaton et al., 2004; Koo et al., 2005). AMPK and its family member MARK2 (microtubule affinity regulating kinase 2) phosphorylate CRTC (Koo et al., 2005; Jansson et al., 2008; Sasaki et al., 2011), keeping it in its basal state in the cytosol. Dephosphorylation of CRTCs occurs in response to increases in intracellular calcium or cAMP/PKA and promotes nuclear localization, and subsequent interaction with CREB (Bittinger et al., 2004; Screaton et al., 2004; Dentin et al., 2007; Jansson et al., 2008; Li et al., 2009). CRTCs are maximally induced by coincident elevations of cAMP and calcium (Screaton et al., 2004). It was also recently demonstrated that Sirt1 (a member of the Sirtuin family) which is involved in lifespan regulations, and neuroprotection in various neurodegenerative disorders (Haigis and Guarente, 2006; Jiang et al., 2011) also contributes to the dephosphorylation of CRTC1 and promotes its nuclear translocation. Knockdown of Sirt1 in primary cortical neurons led to increased levels of phosphorylated CRTC1 even following forskolin stimulation (Jiang et al., 2011).

Interestingly, CRTC1-mediated gene transcription also has a negative feedback mechanism via SIK. SIK gene transcription is driven by CRE-mediated transcription. In cultured cortical neurons, following prolonged membrane depolarization, de novo SIK

13 will phosphorylate CRTC1 at Ser151, promoting its nuclear export and the return to its basal phosphorylated state in the cytosol (Li et al., 2009).

Figure 1.3 The CRTC1 signaling cascade Black arrows indicate signaling that results in the dephosphorylation of CRTC1. Red arrows indicate negative regulation of phosphorylation. Dephosphorylation releases CRTC1, and allows it to translocate the nucleus where CRTC1 binds as a tetramer to the bZIP domain of the CRE site.

Disrupting the interaction between CREB and CTRC blocks CRE-mediated transcription (Zhou et al., 2006). Although CRTC1 and CBP/p300 activate CRE-mediated transcription through different mechanisms, their effects can be synergistic (Ravnskjaer et al., 2007; Xu et al., 2007).

1.3 CREB’s role in regulating behaviour

Preliminary global analyses of CREB-DNA binding and transcriptional activation estimate that 4000-6000 of mammalian genes are regulated, at least in part, by CREB (Conkright et al., 2003b; Impey et al., 2004; Zhang et al., 2005). Recent chromatin immunoprecipitation assays of the mouse genome estimate that the CREB regulon is closer to 3400 genes (Benito et al., 2011). What is clear is that CREB-mediated transcription has the capacity to modulate a significant portion of the mammalian genome (Conkright et al., 2003a; Impey et al., 2004; Zhang et al., 2005), which suggests that different subsets of CREB-target genes are involved in regulating unique neural processes and behaviour.

CREB has been implicated in a wide range of neural and behavioural processes, including neuronal differentiation and development (Bonni et al., 1999; Riccio et al., 1999; Lonze et al., 2002; Jagasia et al., 2009), neuroprotection (Tan et al., 1996; Iordanov et al., 1997; Deak et al., 1998; Wiggin et al., 2002; Sasaki et al., 2011), addiction (Carlezon et al., 1998; McClung and Nestler, 2003; Carlezon et al., 2005; Briand and Blendy, 2010), depression (Duman et al., 1997; Chen et al., 2001), circadian rhythm (von Gall et al., 1998; Gau et al., 2002; Eckel-Mahan et al., 2008), synaptic plasticity (Bailey et al., 2000c; Kandel, 2001; Cohen and Greenberg, 2008) and learning and memory (Bourtchuladze et al., 1994; Yin et al., 1994; Ding et al., 1997; Silva et al., 1998; Lonze and Ginty, 2002; Won and Silva, 2008; Alberini 2009, Josselyn, 2010). Here, we are interested in CREB target genes involved in regulating synaptic plasticity and memory consolidation. These genes have multiple functions, and can be broadly classified in different categories, including transcription factors (c-fos, C/EBP, Jun-D, Nurr1, Egr-1), neurotransmission (acetylcholinesterase, β1-adrenergic receptor, β2- adrenergic receptor, corticotropin releasing hormone, norepinephrin transporter, somatostatin, vasopressin), growth factors (BDNF, IGF-1, TGF-β, TrKB); structural (fibronectin, NF-L), signal transduction (14-3-3, iNOS, NF-1, nNOS) (reviewed in Lonze and Ginty, 2002). Interestingly, these classes of CRE-mediated genes are similar to the proteins that Squire initially suggested are required to induce changes in synaptic efficacy along neural pathways or in populations of neuron following new learning:

1)enzymes regulating neurotransmitter levels, 2) post-synaptic receptor expression, 3) structural proteins (Barondes and Squire, 1972; reviewed in Davis and Squire, 1984).

1.3.1 Identification of CREB as a key mediator of memory consolidation

So far, we’ve discussed the complex molecular cascade which catalyzes the transcription of CREB-target genes regulating synaptic consolidation. Presently, the body of literature devoted to elucidating the role of CREB in memory formation and storage is overwhelming. But how did it first become apparent that CREB-mediated gene expression was even involved in memory? The first hint that cAMP signaling was implicated in memory formation came in the late 1960s, with several studies finding that depolarizing agents and application of neurotransmitters (serotonin) stimulate cAMP production in mammalian brain slices (Kakiucki et al., 1968a,b, 1969). Seminal studies of the molecular biology of sensitization in invertebrates revealed that cAMP signaling is essential for synaptic plasticity and long-term memory. In the mollusk Aplysia californica, synaptic stimulation of motor neurons results in a doubling of cAMP levels (Cedar et al., 1972), and addition of exogenous cAMP enhances synaptic transmission (Brunelli et al., 1976), while inhibition of cAMP-dependent protein phosphorylation in sensory neurons blocks synaptic strengthening (Castellucci et al., 1982). Interestingly, Kandel’s group also found that five bursts of serotonin result in an increase in nuclear translocation of both PKA and ERK/MAPK which, as we discussed, each are critical kinases regulating CREB phosphorylation, while weaker stimulation of only a single burst does not induce the same intracellular activity.

The first screening for genetic mutation which induced disruptions in learning and memory identified 2 genes encoding dunce (Dudai et al., 1976) and rutabaga (Aceves- Pina and Quinn, 1979) genes in the fruit fly Drosophila melanogaster. Further analyses revealed that these genes both encode key regulators of cAMP metabolism (Byers et al., 1981; Livingstone et al., 1984). Together, data from Aplysia and Drosophila clearly identified a potential role for cAMP in memory acquisition. Kandel’s work with Aplysia provided the framework to support all the subsequent work on CREB and memory

16 consolidation. Using a basic task in which a mild shock was delivered to the siphon of Aplysia, Kandel and colleagues studied a simplistic form of learning, sensitization in which an animal learns to enhance its defensive relex in response to any stimuli that is subsequently presented to the siphon area (Pinsker et al., 1970; Pinsker et al., 1973; Frost et al., 1985; Castellucci et al., 1989). Long-term sensitization, (defined by the withdrawl of the siphon/gill upon stimulation) for this form of learning was induced following multiple shocks (5) delivered at spaced intervals, and produced robust memory for the sensitization response lasting for weeks. This long-term sensitization (the proposed analog to long-term consolidation in mammals) was found to require new protein synthesis. Single shocks (as opposed to multiple shocks) failed to induce long- term sensitization (Castellucci et al., 1989). Kandel and colleagues went on to report that microinjection of oligodeoxynucleotides encoding consensus CREs into the nucleus of Aplysia sensory neuron blocks long-term facilitation (LTF), a strengthening of synaptic transmission thought to underlie learning in this system (Dash et al., 1990). They also found neuronal activation results in calcium and cAMP- dependent phosphorylation of CREB (Dash et al., 1991; Sheng et al., 1991), and that stimuli that produces long-term facilitation in Aplysia drives CREB-dependent transcription (Kaang et al., 1993). By 1993, CREB was believed to represent a key mediator of the transition between short-term and long-term memory.

The necessity of CREB in memory formation was first shown in 1994, using genetic inhibition of CREB activity during behavioural training paradigms in flies. Yin, Tully, and colleagues generated a transgenic inducible dominant-negative CREB line of Drosophila that conditionally-express an inhibitory isoform of CREB (dCREB2-b). To rule out the argument that disrupted CREB activity throughout the lifetime of the fly was responsible for its inability to form long-term memory, expression of the CREB repressor was placed under the control of a heat shock (hs) promoter (hs-dCREB2-b) so that CREB could be inducibly repressed just prior to odour discrimination training. Using a Pavlovian odour discrimination/avoidance task in which flies were trained to associate one of two odours with shock, they discovered that heat-shock induced flies expressing the CREB repressor gene were unable to form long-term memory (24 h) for the odour discrimination task. Importantly, un-induced flies (with normal CREB activity) developed 17 normal long term memory for the task. This was the first indication that, in flies, CREB is necessary for memory consolidation (Yin et al., 1994).

After showing that CREB-induced transcription is necessary for LTM in Drosophila, Yin, Tully, and colleagues performed an experiment in which they inducibly increased CREB function using a Drosophila CREB activator isoform (dCREB2-a, a PKA-responsive transcriptional activator of CREB). As before, the induction of the CREB transgene is controlled by a heat shock promoter, allowing temporal control over the activation of CREB. Similar to the previous study, they trained flies in an odour discrimination task (Yin et al., 1995). Like mammals, flies typically require “spaced-training” to form long- lasting memories in this task (i.e., 15 min rest intervals between the 10 training sessions); massed training (i.e., no intervals between training sessions) fails to produce long term memory. Upon conditional induction of CREB, however, flies were able to form long term memory following massed training on the odour discrimination task. While there was no increase in the strength of the memory in these flies, their results suggest that enhancing CREB alone does not produce stronger long-term memory relative to flies that received spaced training, but rather, induces equivalent memory after less training. These exciting data were the first to suggest that CREB may function to facilitate memory formation even under challenging learning conditions which do not typically support consolidation.

Based on both their loss and gain of function studies, they too suggested that CREB acts as a ‘molecular switch’ to convert short term memory into persistent long term memory (Yin and Tully, 1996). The long-term memory enhancing ability of the dCREB2a transgene has since been disputed (Perazzona et al., 2004). However, based on these Drosophila gain-of-function findings, back in Kandel’s lab, Bartsch and colleagues turned their focus to the facilitating effects of CREB in Aplysia. They found that blocking the activity of Aplysia CREB2 repressor (ApCREB2) with antibodies in cultured sensory neurons resulted in an increase in endogenous levels of Aplysia CREB1 activator (ApCREB1), and an increase of LTF at the sensory-motor neuron synapse following only a single application of serotonin (as opposed to five spaced applications typically required to induce LTF) (Bartsch et al., 1995). This confirmed that,

18 as in flies, CREB enhancement in Aplysia acts as a molecular switch for long-term memory consolidation at the synaptic level.

Following the seminal discovery that CREB is a potent mediator of protein-synthesis- dependent long term memory, multiple methods were rapidly developed to explore the effects of suppressing or enhancing CREB activity within the mammalian brain. Next we will discuss an overview of some of these methods, and their applications in rodent models of memory. Before we can thoroughly appreciate the effects of CREB manipulation in the mammalian brain on memory consolidation, we will move beyond the reductionist view of consolidation at the synaptic level, towards a more global view of consolidation within a network of interconnected brain regions. We will then return to the review of CREB manipulations in the rodent brain during memory consolidation and reconsolidation.

1.3.2 Methods of genetically manipulating CREB function in the mammalian brain

An extensive review of the methods for manipulating (increasing or decreasing) CREB activity levels has recently been published by Barco and Marie (2011). In it, they summarize the many methods of genetically manipulating neuronal CREB activity in vivo, including recombinant viral vectors, transgenic mouse models, and classical or conditional knock-in or knock-out models in which a gene of interest may be added or deleted. Each method has many variations, and while there are too many to describe each thoroughly, below we discuss several of the more well characterized methods for genetic manipulation of CREB in the brain which are of particular interest for this thesis.

1.3.2.1 Loss of function models

1.3.2.1.1 Point mutations mCREB: one popular method for disrupting CREB function is often referred to as mCREB (mutant CREB or CREBS133A) involves a point mutation in which the key phosphorylation residue serine 133 is mutated to alanine (S133A), an amino acid which cannot phosphorylate to recruit CBP and the RNA Pol lI complex to the promoter region (Gonzalez et al., 1989). Although mCREB cannot be phosphorylated, it can still bind to the CRE promoter region, competing with endogenous CREB and acting as a highly competitive antagonist. This model is used in Chapter 3. One limitation of this model is that mCREB can heterodimerize with CREB, and still allow for low levels of transcriptional activity by the intact CREB dimer.

K-CREB: This method has a point mutation at the K304 residue which heterodimerizes with CREB and prevents binding to the CRE site by disrupting the Mg2+ interaction required for DNA binding (Walton et al., 1992; Jean et al., 1998; Pittenger et al., 2002).

A-CREB: This method can heterodimerize with CREB family members and block binding to CRE site in the leucine zipper by the fusion of an acidic amphipathic extension onto the N-terminus of the CREB leucine zipper domain which prevents the basic region of CREB’s bZIP domain from binding to DNA (Ahn et al., 1998).

These popular methods of suppressing CREB with mCREB, K-CREB, or A-CREB in vivo have been combined with transgenic mouse models (Pittenger et al., 2002; Kida et al., 2002; Huang et al., 2004; Pittenger et al., 2006; Suzuki et al., 2007; Jancic et al., 2009; Lee et al., 2009; Wingate et al., 2009), and viral mediated gene transfer techniques (Glover et al., 2004; Zhu et al., 2004; Olson et al., 2005; Warburton et al., 2005; Dong et al., 2006; Han et al., 2006; Suzuki et al., 2007; Jagasia et al., 2009). In the proceeding experiments, we use viral vectors encoding mCREB to disrupt CREB function during memory consolidation.

1.3.2.1.2 Mouse models of CREB manipulation

As of 2011, over 20 CREB mutant strains of mice had been generated to enhance or reduce CREB activity in vivo (see Barco and Marie, 2011 for extensive review). Phenotypic analyses of behaviour in models disrupting CREB function report inconsistent findings in disruptions of long-term potentiation and long-term depression (LTD), memory impairment, addiction, and circadian rhythms. Due to the variety of models in which CREB may be regionally (global or local) or temporally (chronic or acute) regulated, there are mixed overall conclusions on the effects of CREB suppression on synaptic plasticity and behaviour.

1.3.2.1.2.1 CREB hypomorphic mice: CREBαΔ-/-

One model of particular interest to us was developed in 1994 by Shutz, in which two of the three CREB isoforms were ubiquitously deleted. CREB-deficient (CREBαΔ-/-) mice have a neomycin-resistance cassette (neo) inserted into exon 2 mice of the CREB gene that results in a deletion of the two main isoforms of CREB (α and Δ) (Hummler et al., 1994). The insertion of the neo gene into exon 2 does not disrupt the translation of another CREB isoform (CREBβ, which begins at exon 4). Deletion of all three isoforms is embryonically lethal (Rudolph et al., 1998), but CREBαΔ-/- mice retaining only the β isoform develop into adulthood. In fact, these mice have up-regulated levels of CREBβ as well as CREM activator (τ) and repressor (α and β) isoforms (Blendy et al., 1996). Despite these up-regulations, however, CRE-DNA binding is virtually abolished (by > 90%) (Pandey et al., 2000; Walters and Blendy, 2001) and the levels of CREB protein are dramatically reduced (roughly 80-90% reduction compared to controls) in the brains of CREB-deficient mice (Walters and Blendy, 2001; Walters et al., 2003).

Over a dozen papers have used this model to investigate the effects of continuous CREB disruption throughout development and adulthood. Bourtchuladze and colleagues (1994) performed the first experiments exploring the physiological and behavioural phenotype in these CREB-deficient mice. They recorded fEPSPs in the CA1 in hippocampal slice, and reported a deficit in LTP maintenance. Relative to 21 controls, LTP had decayed to baseline by 90 min following LTP induction by a train of 100 pulses at 100Hz. They, and others, went on to probe the behavioural phenotype of these mice. As we will soon discuss, studies investigating memory deficits in CREBαΔ-/- mice report divergent results. Early reports found that long-term memory deficits in these mice could be ameliorated through prolonged and spaced training (Kogan et al., 1997), suggesting that the training protocol may impact the rate and efficiency of synaptic consolidation in CREB-deficient mice.

Due to the chronic nature of disruption in classical knock-out models, genetic manipulations at this level have drawbacks, including the potential for developmental compensation for the deleted gene by up-regulation of related transcriptional regulators (reviewed in Josselyn and Cole, 2008). It has been argued that reports of intact memory in CREB hypomorphic mice is supported by the compensatory upregulation of the CREB-β isoform and CREM (Hummler et al., 1994; Blendy et al., 1996), which may be sufficient to support transcriptional activity required for the formation of new long term memories. The CREBαΔ-/- model has also been criticized for producing inconsistent learning and memory deficits, and for its sensitivity to the genetic background of different mouse strains (Graves et al., 2002). We will discuss the impact of CREB suppression on memory consolidation in these mice later in the chapter.

1.3.2.1.2.2 Transgenic models of CREB inhibition or expression

To more precisely dissect the role of CREB deletion in memory formation in the mouse, a second generation of transgenic has been developed which allows for inducible and regionally restricted disruption or expression of CREB protein. Tissue-specific expression under control of the α-CaMKII promoter restricts expression to excitatory neurons in the forebrain only (including the cortex, striatum, hippocampus, and amygdala) (Mayford et al., 1996). The ability to selectively turn expression of the CREB transgene ‘on’ or ‘off’ in the forebrain is accomplished using a tetracycline-controlled transactivator (tTa) (Gossen and Bujard, 1992) combined with the α-CaMKII forebrain promoter placed upstream of the gene of interest (Mayford et al., 1996). In this model,

CREB binding to DNA can be blocked or permitted in the presence or absence of tetracycline (tet) or the tetracycline analogue doxycline (dox) in the mouse diet, allowing for temporally precise control of gene expression (Mayford et al., 1996). This α-CaMKII- tTA/tetO double-transgenic system has also been used to induce expression of a constitutively active CREB transgene (VP16-CREB) (Barco et al., 2002) throughout the forebrain. Barco and colleagues first used model to study hippocampal LTP. While they found that VP16-CREB does not affect basal synaptic transmission levels in the Schaeffer’s collateral pathway of the hippocampus, they found that constitutively-active CREB expression lowers the threshold for inducing L-LTP (Barco et al., 2002; Alarcon et al., 2004; Barco et al., 2005) after only a single 100Hz tetanus train of stimulation, which is typically only sufficient to induce E-LTP. Interestingly, forebrain-wide expression of constitutively-active CREB has both facilitating and detrimental effects on memory, which we will discuss towards the end of the chapter (Viosca et al., 2009). Long-term expression of constitutively-active CREB produces enhanced excitatory effects and high plasticity that may actually interfere with normal memory processes, suggesting that the brain may require an optimal level of CREB activity to support normal transcriptional activity. In this case, bigger is not necessarily better.

At any rate, this model was revolutionary in the ability to examine synaptic plasticity and memory consolidation with regional control, and a new degree of temporal control. However, among its disadvantages to is that the tet/dox system requires several days to maximally activate or suppress transgene expression. To overcome this time lag and achieve higher temporal control of CREB activity, a more attractive inducible repressor (IR) mouse strain was developed in Silva’s lab (Kida et al., 2002) where the αCREB isoform with a S133A mutation (discussed above) was fused to the ligand binding domain of the estrogen receptor (Feil et al., 1996), under the control of the α-CaMKII promoter to restrict expression to excitatory forebrain neurons (α-CaMKII-CREBIR). In this model, the CREB fusion protein is sequestered in the cytosol in the absence of the estrogen receptor antagonist tamoxifen. Delivery of tamoxifen binds to the ligand binding domain and allows the CREB fusion protein to translocate to the nucleus where the mutant CREB protein competes with endogenous CREB protein for the CRE binding site. This model is interest to us, as its rapid mechanism of action upon delivery of 23 tamoxifen (several minutes) has allowed researchers to study discrete memory processes. Specifically, Kida and colleagues have used this clever model to study consolidation, reconsolidation, extinction processes with regional and temporal specificity not previously possible (Kida et al., 2002; Mamiya et al. 2009, Kim et al., 2011). We will review these exciting findings later in this chapter.

1.3.2.1.3 Viral vector-mediated gene transfer

While the application of tissue-specific promoters can provide some degree of control over the expression pattern of the transgene, virally-mediated gene transfer provides an even higher degree of regional and temporal control in the manipulation of a gene of interest. Currently, five types of replication-defective viruses are used to acutely manipulate CREB in the mouse brain in vivo (extensive review in Barco and Marie, 2011). Each method presents certain advantages (neurotropic expression, duration), and disadvantages (cytotoxicity). Below, we will review several of the better characterized viral methods.

1.3.2.1.3.1 Herpes Simplex Virus

In herpes simplex virus type1 (HSV) vector-mediated gene transfer, a plasmid carrying the CREB cDNA transgene is packaged into the HSV virus, causing infected neurons to express the gene of interest. Second generation viral vectors include the development of techniques to visualize cells infected by incorporating a green fluorescence protein (GFP) with the gene of interest. HSV is an ideal system for our purposes because, unlike many other viruses, HSV is naturally neurotropic, and infects non-dividing cells (Fink et al., 1996). In the hippocampus, HSV predominantly infects granule cells of the dentate gyrus, and large pyramidal cells of the CA1 and CA3 layers, without altering in vivo synaptic transmission (Dumas et al., 1999), or causing gliosis (Carlezon et al., 1998). Neve and colleagues designed the original HSV vectors used to study synaptic plasticity and memory consolidation (Carlezon et al., 1998; Neve et al., 2005), and it

24 continues to be the most frequently used viral vector system in neuroscience transgene research (Carlezon et al., 2005). However, one limitation to the HSV system is the transient transgene expression pattern.

As you will see in Chapters 3 and 4, CREB activity within the hippocampus can be enhanced through injections of exogenous activity-dependent CREB (CREBWT), or inhibited by over-expressing a dominant negative form of CREB (CREBS133A, mCREB). In 2001, Josselyn and colleagues provided the first demonstration that virus-mediated over-expression of CREB can actually facilitate memory formation in the rodent (Josselyn et al., 2001). Like Yin and Tully demonstrated in flies, rats require multiple space training sessions to form a long-term memory. Using HSV to deliver targeted microinjections of activity-dependent CREB into the basolateral amygdala (BLA) of rats, Josselyn and colleagues demonstrated that massed training sessions of a fear- potentiated startle task can support consolidation of a fear memory (Josselyn et al., 2001). This was accomplished by increasing the CREB available for transcriptional activation in the amygdala, a brain region known to be a critical site for plasticity in the encoding of these types of fear memories (Fanselow and LeDoux, 1999; LeDoux, 2003). This study was the first to use a gain-of-function approach to strengthening memory consolidation by increasing CREB function in mammals. Subsequent studies examining the role of CREB in memory formation have built on these initial findings. We will discuss this body of literature pertaining to CREB-mediated memory consolidation in rodents at the end of the chapter.

1.3.2.1.3.2 Sindbis Virus

Sindbis virus is also highly neurotropic, can co-express GFP to identify infected cells, and shows transgene expression within several days of infection. However, it is highly cytotoxic (Barco and Marie, 2011). Sindbis viral vectors carrying either the mCREB transgene or a constitutively active CREB (CREBY134F) transgene were used in the first electrophysiological studies both in cultured rat hippocampal neurons and in vivo. Relative to infection with activity-dependent CREB vector, infection with Sindbis vector

25 encoding constitutively-active CREBY134F drove high levels of immediate-early gene expression of c-fos in cultured neurons, a significant increase in NMDAr EPSCs, and enhanced maintenance of L-LTP (but normal LTD) following stimulation in hippocampal slice. They also found morphological changes indicative of enhanced synaptic plasticity, including an increase in dendritic spines within the CA1 region of the hippocampus. Specifically, they found an increase in the number of ‘silent synapses’ (Isaac et al., 1995; Liao et al., 1995), which contain NMDA receptors (but no AMPA receptors), potentiating NMDAr-mediated synaptic responses (Marie et al., 2005). This gain-of- function work with Sindbis viral vectors suggests that CREB activity is critically involved in strengthening synaptic plasticity in rodents. The use of Sindbis viral vectors encoding constitutively active CREB has been used extensively by Marie and colleagues to examine the behavioural effects of hippocampal CREB manipulation in memory consolidation (Restivo et al., 2009; Vetere et al., 2011a).

1.3.2.1.3.3 Adeno-Associated Virus

Adeno-associated virus (AAV) is a replication-defective parvovirus that infects neurons. Following infection with AAV, it takes several days to detect transgene expression. AAV has low in vivo toxicity, and is stably expressed periods up to a year (van den Pol et al., 2004; Barco and Marie, 2011). It has been used effectively to study the beneficial effects of long-term viral over-expression of CREB in aging rats (Mouravlev et al., 2006). We will discuss these findings later.

1.3.2.1.3.4 Lentivirus

Lentivirus is a retrovirus based on the HIV-1 genome, which infects both glia and neurons. One advantage to the use of Lentiviral vectors is its permanent transgene expression (Matrai et al., 2010; Barco and Marie, 2011). It has not been used to manipulate CREB in the brain, but it has been used to suppress CRTC1 in cultured primary cortical neurons. Infecting neurons with CRTC1 shRNAi prior to stimulation with

26 calcium and cAMP agonists potassium chloride (KCl) and forskolin (FSK) resulted in a decrease in CRE-luciferase reporter activity and a decrease in CRE-mediated expression of c-fos, BDNF, and Nr4a2 (Espana et al., 2010).

1.3.3 A role for CRTCs in memory consolidation?

CRTCs have been implicated in a range of physiological processes, including glucose regulation, energy homeostasis (Koo et al., 2005; Liu et al., 2008), neuroprotection in neurodegenerative disease (Jeong et al., 2012), cancer (Komiya et al., 2010), and lifespan regulation in aging (Mair et al., 2011). However, the role of CRTCs in memory has not yet been directly examined, though findings suggest that CRTCs are important in regulating forms of CREB-dependent synaptic plasticity. Four groups have examined the role of CRTCs in synaptic plasticity of cultured neurons (Zhou et al., 2006; Kovacs et al., 2007; Li et al., 2009; Espana et al., 2010; Ch’ng et al., 2012). Three of these groups find that CRTC1 (1) expresses in hippocampal and cortical pyramidal neurons, (2) translocates to the nucleus in response to neuronal activity, (3) is necessary for both activity-dependent expression of CREB target genes and L-LTP. The necessity of CRTC1 for CREB-dependent transcription can be shown using both dominant negative CRTC1 (dnCRTC1; truncation mutants that encode only the CREB-binding domain without the transcriptional activation or nuclear export domains), and small RNA- mediated knockdown of CRTC1, which decreases both basal- and induced-expression of CREB target genes. In response to 3-4 trains of high frequency stimulation, L-LTP (3 hour) is observed in control neurons; however, expression of dnCRTC1 results in failure to maintain potentiation. Interestingly, CRTC1 over-expression facilitates synaptic potentiation: a single high frequency train, which is unable to induce synaptic potentiation in control cells, promotes stable L-LTP in hippocampal slices transduced with a CRTC1-encoding viral vector (Zhou et al., 2006). Also, cultured hippocampal neurons transfected with plasmid containing wild-type CRTC1 show increased expression of CREB-target gene BDNF mRNA following stimulation KCl and FSK relative to stimulated controls. The converse effect was seen in stimulated neurons transfected with plasmid containing the dominant-negative CRTC1 which lacks the bZIP 27 binding domain (Kovacs et al., 2007). Enhancing CRTC1 in developing cortical neurons via transfection of CRTC1-containing plasmids have also resulted in morphological changes, with cortical neurons showing increased dendritic length and branching, both in vitro and in vivo (Li et al., 2009). Together, these results suggest that CRTC1- mediated CREB-target gene transcription might be a critical component of LTP, and perhaps memory formation. However, to date, no direct investigation in the role of CRTCs in an in vivo learning and memory paradigm exists.

Taken together, studies in Aplysia, Drosophila, and rodents clearly showed that CREB is a conserved protein necessary for synaptic consolidation of long-term memories in a variety of species. Above we have discussed the minutia of transcriptional mechanisms supporting synaptic consolidation of long-term memory, and how manipulations of CREB or CREB co-activators using genetic approaches can strengthen or weaken synaptic plasticity required for consolidation of a memory. But to understand how a memory becomes stably represented within the brain, you have to think bigger than the synapse.

1.4 Introduction to Systems Consolidation

So let’s think bigger. Think of the opening credits of any of those generic movies about the universe. The kind of movie scene that starts out as a close up shot of a child’s eye, then slowly zooms out to reveal the child standing in a house, within a city, on a continent, on Earth, within the solar system, in our galaxy, within… (those scene sequences always make me anxious). This is the kind of perspective that is needed to now imagine thinking about the biological basis of memory, from the level of neurotransmitter release, to kinases and phosphatase signaling, gene transcription, protein translation, at the individual synapse, within a neuron, within the neuronal network of a single subregion of the brain, within a broader interconnected cortical network. This scope of perspective is similarly anxiety provoking when trying to understand how all these small parts build upon one another to ultimately generate what we understand as ‘memory’. To better understand this, we will explore the concept of memory consolidation at the level of the ‘system’.

Systems Consolidation

Identifying the molecular mechanisms supporting long term memory allows us to understand how memories are initially consolidated (stably represented in the neuronal network). Once this initial rapid synaptic consolidation has occurred within the first few hours of memory encoding, a second, more prolonged form of consolidation occurs (Dudai, 2004). Systems consolidation is a process whereby the memory becomes represented within various regions in the brain. For these episodic-like memories (memory for places, events, the ‘what’ and ‘where’) (Tulving, 1972), the hippocampus is thought to be critical for the initial encoding. While the two forms of consolidation are often discussed independently, it is important to note that this is an artificial division between the two processes; both synaptic consolidation and systems consolidation are part of the same ongoing process of memory stabilization within a neuronal network. Although early work which examined neural processing in a very simple model [stimulation of motor neurons in Aplysia to examine the effects of long-term facilitation

29 and sensitization, from which they inferred the conditions required for long-term memory consolidation at the level of the single synapse (Pinsker et al., 1970; Pinsker et al., 1973; Frost et al., 1985; Castellucci et al., 1989; Dash et al., 1991; Sheng et al., 1991; Kaang et al., 1993)], the neuropsychological literature takes a more global, view of memory consolidation which takes into account the brain regions required for long-term memory consolidation.

The hippocampus first emerged as a candidate region for systems level memory consolidation following observations of the famous amnestic patient H.M., only recently identified as Henry Gustav Molaison following his death in 2008. H.M. received a resection of a large section of his medial temporal lobe (including the entire bilateral hippocampal complex) to treat a severe form of intractable epilepsy in 1957. While the surgery was effective in controlling the seizure activity, he developed a severe amnesia for events experienced recently prior to his surgery (retrograde amnesia, RA), and a complete inability to acquire any new memories (anterograde amnesia). In a landmark study by Scoville and Milner (1957), it was determined that H.M’s retrograde amnesia was not complete however – while most memories acquired more recently prior to removal of his medial temporal lobe were abolished, he displayed sparing of certain memories acquired many years prior to surgery (Scoville and Milner, 1957; Penfield and Milner, 1958). This temporal gradient of his memory loss for people, places, and events acquired prior to his surgery was termed ‘temporally graded retrograde amnesia’ (TGRA). It has since been debated that the pattern of H.M.’s amnesia shows both temporally graded and non-graded memory loss, depending of the ‘type’ of memory being probed. In the case of implicit or semantic memory (unconscious recall of general information ie. general facts, world knowledge, also called non-declarative memory) (Schacter, 1987), a temporal gradient is observed in which implicit memories acquired years before surgery were retained. However, when probed for explicit episodic memories (conscious recollection of ‘what’ and ‘where’, autobiographical information, also called declarative memory) (Cohen and Squire, 1980; Squire, 1982; Tulving, 1972, 1983, 2004; Schacter and Tulving, 1994) which are thought to always rely on the

30 hippocampus, memory loss is complete for memories acquired both recently and remotely before the surgery. Famous lesion studies in patients such as H.M., and other well-documented cases of K.C. (Rosenbaum et al., 2000; Rosenbaum et al., 2007), and E.P (Teng and Squire, 1999) have contributed greatly to the modern understanding of systems level memory consolidation, and storage in humans. Advantages to using human patients and healthy human participants to study memory processes is the ability to probe specific aspects of the memory with great details. Disadvantages, of course, include a lack of control over the case history, comorbid pathologies, type and extent of brain damage, and prior life experiences.

The use of animal models presents an attractive alternative to human lesion studies, with the greatest advantage being precise control over the experimental conditions (the use of naïve or explicitly trained animals, acute, controlled lesioning of desired tissue, such as the hippocampus). Animal models of memory consolidation, storage, and retrieval, however, are limited in the extent to which you can probe for highly complex and detailed memory processes. Unlike human participants who can verbalize their recollection, experimenters are limited to probing memory motivated by basic physiological and emotionally salient stimuli (LeDoux, 2012). Most frequently used are fear based paradigms, escape-motivated paradigms, such as the watermaze, and appetitive-based paradigms, such as the social-transmission of food preference task, and conditioned taste aversion. We will be focusing primarily on spatial memory for the watermaze, and contextual fear conditioning for our review, as these are the main behavioural tasks used in our studies.

1.4.1 The hippocampus

The medial-temporal lobe contains the hippocampal system, which is a highly interconnected system comprised of the hippocampus proper, the subiculum, and the entorhinal cortex, with direct and indirect projections to the thalamus, prefrontal cortex, amygdala, retrosplenial cortex, and projections via the entorhinal cortex to perirhinal and parahippocampal cortices and association areas in the temporal and parietal lobes

(Aggleton and Brown, 1999; Moscovitch et al., 2005). The hippocampus was identified over 400 years ago by the anatomist Giulio Cesare Arantius (1587), who gave it the Greek name ‘hippocampus’ based on its physical resemblance to the seahorse. The hippocampus proper is a laminar structure that is traditionally divided into 3 main subregions, first identified by Cajal, and later refined by Lorenté de No. He named the morphologically distinct regions cornu ammonis (meaning ‘ram’s horn’, named for its curved shape) areas 1-3 (CA1, CA2, CA3), and the dentate gyrus (DG) (Lorenté de No , 1934; Andersen et al., 1971), (Fig 1.4). The hippocampus and the discrete hippocampal subfields (with the exception of the small and often neglected CA2) has been the focus of much attention within the field of memory, as it is considered to be the site of initial memory consolidation.

The hippocampus is traditionally thought to communicate in a largely unidirectional manner through a pathway termed the trisynaptic circuit. Input from layer III of the entorhinal cortex projects via the perforant path to the molecular layer of the dentate gyrus. Granule cells of the dentate gyrus send direct projections via its mossy fibre axons to synapse onto the pyramidal cells in the stratum radiatum of the CA3. Axons of the CA3 pyramidal cells then project via the Schaeffer’s Collaterals to the apical dendrites in the stratum radiatum and basal dendrites in the stratum oriens of CA1 pyramidal cells (Schaffer, 1892; Blackstad, 1958; Storm-Mathisen and Fonnum, 1972; Hjorth-Simonsen, 1973). CA1 axons then send output signals directly to layer V of the entorhinal cortex or indirectly via the subiculum which projects to layer V of the entorhinal cortex (Nakashiba et al., 2009; Suh et al., 2011). In addition to the trisynaptic loop, there are also direct projections from layer III of the entorhinal cortex projecting directly to the CA1 via the temporoammonic pathway (Blackstad, 1958). The hippocampus also has many collateral and recurrent projections onto inhibitory cells which negatively regulate hippocampal activity and serve as internal feedback mechanisms (Fig 1.4).

Figure 1.4 The traditional trisynaptic circuit of the hippocampus (a) Coronal section of the dorsal hippocampus expressing GFP (green). Thick yellow line indicates the perforant path extending from the entorhinal cortex (EC) towards the molecular layer of the dentate gyrus (DG). The granule cells of the DG (red) receive inputs via the perforant path, and extend their axons towards the CA3 pyramidal cells (blue) via the mossy fiber pathway. CA3 extend its axons via the Schaeffer’s collateral towards the CA1 pyramidal cells (purple). CA1 axons, then project back towards layer V of the EC. CA1 also sends projections through the subiculum (not pictured) to the EC. CA1 also receives direct input from layer II or the EC, and CA3 received direct input from layer II of the EC. (b) Basic schematic of the major excitatory projections in the trisynaptic circuit.

The hippocampus in rodents is an anatomically large structure with heterogeneous inputs and outputs along the longitudinal axis between the dorsal (septal) and ventral (temporal) region (Moser and Moser, 1998b). Also, the density of hippocampal cells ranges from the septal to temporal poles, with the ratio of dentate gyrus to CA3 cells is approximately 12:1 at the septal, and 2:3 at the more temporal poles (Amaral and Witter, 1989). Though the hippocampus functions as a circuit, the major hippocampal subregions are thought to serve functionally discrete processes in memory encoding and consolidation (Moser and Moser, 1998a, b). Early studies into localization of function within the hippocampus used excitotoxic lesions of large portions of dorsal or

33 ventral hippocampal tissue along the longitudinal axis. It was first systematically demonstrated that the dorsal hippocampus is essential for the acquisition of spatial memory in a maze, but that ventral lesions had little effect on spatial memory (Hughes, 1965; Moser et al., 1993a, b; Moser et al., 1995; Bannerman et al., 2003). This pattern of spatial memory impairment is also seen following dorsal hippocampal loss due to transient ischemia (Volpe et al., 1992; Olsen et al., 1994a, b). However, it has been noted that even minimal (20-30%) of surviving dorsal hippocampal tissue is sufficient to support spatial memory formation (Moser et al., 1995). More anatomically-refined analyses of the hippocampal sub-fields have also suggested discrete characterizations of each region in memory processing.

1.4.1.1 The dentate gyrus

The dentate gyrus, a V-shaped region consisting of the upper and lower blades (termed the suprapyramidal and infrapyramidal blades, respectively), contains densely packed granule cells organized in the granule cell layer. It has been reported that the suprapyramidal blade contains larger, more complex neurons (longer dendrites, greater spine density) that those in the infrapyramidal blade (Desmond and Levy, 1982, 1985). Unlike pyramidal cells, most granule cells only extend apical dendrites (no basal dendrites), which form the molecular layer of the dentate gyrus. Dendritic spines in the molecular layer receive excitatory input from axons projecting from layer II of the entorhinal cortex, as well as inhibitory input from interneurons within the molecular layer (reviewed in Treves et al., 2008). Granule cells then project their axons along the mossy fiber pathway through the hilus where they form recurrent collateral connections within the molecular layer, or they form mossy fiber terminals that make excitatory synaptic contact with neurons in CA3 (Blackstad et al., 1970).

The adult mouse dentate gyrus contains an estimated 400,000 granule cells (O’Kusky et al., 2000), and is one of only two brain regions which continues to generate new cells into adulthood. The axon of each granule cell forms up to 50 synaptic contacts (Amaral et al., 1990) on up to 15 different neurons from the (Lavenex and Amaral, 2000). A

34 unique feature of the dentate gyrus is its ‘sparse coding’ activity pattern (Jung and McNaughton, 1993; Leutgeb et al., 2007). In response to new learning or exploration of a novel environment, for example, only approximately 2% of dentate gyrus neurons will be activated (Jung and McNaughton, 1993; Guzowski et al., 1999; Chawla et al., 2005; Ramirez-Amaya et al., 2006; Tashiro et al., 2007; Schmidt et al., 2012). Keeping in mind this small population of neurons activate many more cells within the CA3, it may be that this sparse activity is necessary to keep the overall level of activity in the brain from over-loading. Due to the sparse coding pattern in the dentate, it has been proposed to be involved in a process called ‘pattern separation’ (Leutgeb et al., 2007; McHugh et al., 2007; Nakashiba et al, 2012), which is a computational theory of brain processing for distinguishing between overlapping, but different, stimuli by orthogonalization of the synaptic input (Chadderton et al., 2004). The dentate gyrus is a candidate region for this process due to its very high number of cells, but sparse activation levels, making it an attractive candidate for encoding separate patterns.

1.4.1.1.1 The dentate gyrus and context memory

While the definition of ‘pattern separation’ was originally proposed as a computational theory, it has since evolved to encompass the encoding of basic discrimination tasks, such as contextual discrimination. Hippocampal lesion studies have demonstrated a critical role for both CA1 and dentate gyrus dorsal hippocampal regions in the consolidation of contextual fear memory in both mice and rats (Kim and Fanselow, 1992; Phillips and LeDoux, 1992; Frankland et al., 1998; Anagnostaras et al., 1999; Lee and Kesner, 2004; Biedenkapp and Rudy, 2007; Leutgeb et al., 2007; Wiltgen and Silva, 2007, Winocur et al., 2007; Hernandez-Rabaza et al., 2008; Winocur et al., 2009; Wang et al., 2012). It is important to keep in mind that, as part of the hippocampal circuit, contributions of the CA3 may be implicated in context memory formation as well.

Context memories, typically assessed by context fear conditioning in which an animal receives an aversive shock (unconditioned stimulus, US) in a particular context and learns to associate the context (conditioned stimulus, CS) with a fearful event, is

35 originally dependent on the hippocampus for memory consolidation (Sutherland and McDonald, 1990; Kim and Fanselow, 1992; Phillips and LeDoux, 1992). Frankland and colleagues demonstrated that pre-training lesions of the dorsal hippocampus block contextual discrimination in mice (Frankland et al., 1998). In this study, mice were trained to discriminate between the context in which they had received a footshock (Context-A), and a novel context (no shock, Context-B). Sham lesioned mice were able to discriminate between contexts, exhibiting high freezing only in Context-A only when tested 1d after conditioning, whereas hippocampal lesioned mice displayed equivalent freezing behaviour in both contexts, suggesting a lack of ability to discriminate the contextual detail associated with the fearful experience. Post-training lesions performed 1d after fear conditioning also result in impaired contextual discrimination in mice and in rats (Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009). As we will discuss, the continued role of the hippocampus in context memory after the initial acquisition/consolidation is often debated.

Over time (several weeks in rodents), the context memory undergoes systems consolidation, which is a time-dependent reorganization of the memory across neocortical areas (for review, see Frankland and Bontempi, 2005). At this time, lesions to the hippocampus do not impair expression of the context fear memory (Kim and Fanselow, 1992; Anagnostaras et al., 1999; Anagnostaras et al., 2001; Winocur et al., 2009; Wang et al., 2009; Wiltgen et al., 2010), as it is thought to be supported by the neocortical representation. There is evidence to suggest that the neocortical context memory is not an exact replication of the original context memory, but rather is a more general version which retains the general features of the context, but lacks the rich contextual detail (Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009). Whether a detailed hippocampal trace of the memory continues to exist in parallel following systems consolidation is a matter of great debate in the field of memory ( Zola- Morgan et al., 1986, Nadel and Moscovitch, 1997; Nadel et al., 2000; Teng and Squire, 2000; Squire et al., 2001; Squire et al., 2004; Frankland and Bontempi, 2005; Moscovitch et al., 2006; Squire and Bayley, 2007; Winocur et al., 2007; 2011; Winocur et al., 2010; Sutherland and Lehmann, 2011; Winocur and Moscovitch, 2011; Nadel et al., 2012). 36

While much can be gained from looking a loss-of-function models such as lesion and pharmacological inactivation studies of the hippocampus, modern gene imaging techniques have allowed us to see how the intact brain processes context memory consolidation. For example, context fear conditioning is associated with increases in phosphorylated CREB in the hippocampus (Stanciu et al., 2001), and expression of CREB-target gene Zif268 (Hall et al., 2001).

1.4.1.1.2 The dentate gyrus and neurogenesis

As briefly noted, one notable feature about the hippocampal dentate gyrus is its continued generation of newborn neurons into adulthood, a process called neurogenesis. While the field of neurogenesis has been the subject of much attention within the past two decades, the discovery that the mammalian dentate gyrus generates new neurons post-natally was originally discovered by Altman in the 1960s (Altman, 1962). It is one of only two regions in the mammalian brain which is capable of renewing cells (the other region being sub-ventricular zone, which sends adult-generated neurons to the olfactory bulb via the rostral migratory stream (Altman, 1969); however early evidence suggests that other brain regions may have ongoing neurogenesis as well (Altman, 1962). Neurons born in the subgranular zone of the dentate gyrus slowly migrate up towards the granule cell layer, where they extend dendritic processes into the molecular layer, and by four weeks, are capable of functionally integrating into the neuronal network (van Praag et al., 2002; Kee et al., 2007; Zhao et al., 2008 for review). The contribution of these adult-generated newborn neurons in memory has been widely investigated. There is ample evidence that, once mature, these new neurons are capable of functionally integrating into new memory networks for both context fear memories (Stone et al., 2011a), as well as spatial memory in the watermaze (Kee et al., 2007; Stone et al., 2011a,b), and that suppression of neurogenesis impairs hippocampal-dependent memory (Shors et al., 2002; Snyder et al., 2005; Winocur et al., 2006), although the results on this are mixed (Shors et al., 2002; Aimone et al., 2011). There is evidence that CREBαΔ-/- hypomorphic mice have increased levels of neurogenesis (Gur et al., 2007), which has been proposed as a systemic compensation 37 by the hippocampal network in CREB-deficient mice. CREB signaling has been shown to be involved in the survival, maturation, and integration of adult born granule cells (see Merz et al., 2011 for review), but it would be interesting to examine the effect of increasing CREB function in these already excitable new neurons to determine if they would be preferentially incorporated into memory networks; however, to date, no one has examined the role of CREB in adult-generated newborn neurons in a learning and memory paradigm.

1.4.1.2 CA3

CA3 gets comparably little attention within the study of the hippocampus. Between the highly popular CA1 and dentate gyrus, lowly CA3 has been referred to as a ‘bottleneck’ within the hippocampus. In the rat, the CA3 region consists of approximately 200,000 cells. It receives input from over 1,000,000 granule cells, then expands back out to the more densely packed CA1 of an estimated 400,000 cells (Rapp and Gallagher, 1996; Kubik et al., 2007). Those numbers are only slightly lower in the mouse hippocampus. As noted, CA3 excitatory pyramidal cells receive afferent fibers from the dentate gyrus via the mossy fiber pathway. CA3 is thought to play a role in ‘pattern completion (Treves and Rolls, 1994; Gold and Kesner, 2005; Leutgeb and Leutgeb, 2007), which involves forming familiar representations of a stimulus based on incomplete input from the sparse firing from the dentate gyrus. Unlike the dentate, CA3, and CA1 show high activity patterns.

1.4.1.3 CA1

Like CA3, CA1 is comprised on large excitatory pyramidal cells which receive input from the Schaeffer’s collaterals of CA3, or from direct input from layer III of the entorhinal cortex via the temporoammonic pathway (Fig 1.4). While localization of specific function to distinct sub-regions of the hippocampus is often debated (such as pattern separation and completion), there is evidence for a role of the CA1 region of the dorsal

38 hippocampus in consolidation of spatial memories (O'Keefe and Dostrovsky, 1971; O’Keefe and Nadel, 1978; Moser et al., 1995; Moser et al., 1995; Moser and Moser, 1998; Bannerman et al., 2003; Vazdarjanova and Guzowski, 2004; Porte et al., 2008a).

1.4.1.3.1 CA1 and spatial memory

Hippocampal place cells, which are neurons that fire with spatially selectivity, were first identified in CA1 by O’Keefe and Dostrovsky (1971). In what has become a seminal book on the role of the hippocampus, O’Keefe and Nadel suggest that place cells are the neural substrate of ‘cognitive maps’ in the hippocampus (O’Keefe and Nadel 1978).

Despite the ongoing argument on whether the hippocampus continues to be involved in memory once consolidated in the neocortex, successful performance on a spatial memory tasks always requires the hippocampus (Sutherland et al, 2001; Clark et al., 2005; Winocur et al, 2005). Spatial memories, which are richly detailed memories (akin to episodic memories in human) typically show non-graded retrograde amnesia, meaning that hippocampal lesioned animals show equal loss of the spatial memory if the lesion was performed just after memory acquisition or at a remote timepoint after acquisition, a timepoint when other contextual memories show resistance to disruption following hippocampal ablation. Notably, there is evidence that rats with hippocampal lesions can retain memory for spatial location acquired prior to hippocampal damage (Winocur et al., 2005; Winocur et al., 2010) but only under learning conditions that allow for prolonged and diverse experience with the environment prior to hippocampal lesioning. These studies showed that the spatial memories that survived hippocampal lesions were more schematic and relied less on precise spatial cues within the environment than those formed originally in the hippocampus as part of a detailed, hippocampus-dependent cognitive map.

Early work using hippocampal lesions prior to spatial learning found that hippocampal- lesioned rodents are severely impaired acquiring of memory for new spatial navigation tasks in the radial arm maze (Jarrard, 1978; Olton et al., 1978), and in the watermaze (Morris et al., 1982; Morris et al., 1990b). The standard spatial Morris Water Maze 39

(MWM) task is well-established as a hippocampal dependent task, where impaired hippocampal function (lesions, NMDAr antagonists) reliably produces deficits in spatial learning and retrieval (Morris, 1982; Morris et al., 1986). In this task, an animal is placed in a circular pool with an escape platform submerged just below the water. The animal must use distal spatial cues around the maze to learn and remember the location of the escape platform. However, what is less clear is if the hippocampus is required for successful navigation in the maze, or if it is critical to the storage of the spatial memory. Place cells within the CA1 region encode ‘place fields’, which will always fire in response to an animal’s precise position in a specific location in its environment (O’Keefe and Dostrovsky, 1971; McNaughton et al., 1983), a phenomenon which does not require learning. However, repeated exposure to the same environment will result in stable and reliable firing of the cell within the same location in the environment (its firing field). This repeated exposure to an environment results in the development of a cognitive map of an environment (O’Keefe and Nadel, 1978). Proponents of the cognitive map theory would argue that the hippocampus is required for successful navigation in a familiar environment.

Though earlier we discussed the critical role of the dentate gyrus in context memory, it is important to remember that the hippocampus functions as a circuit. Sparse coding input from the dentate gyrus travels towards CA3 and onto CA1, which is also critically involved in consolidation of context memory. Investigations to detect Arc (activity- regulated cytoskeletal associated protein) mRNA, an immediate early gene expressed in response to neuronal activation, following spatial exploration has identified that approximately 40% of CA1 neurons (and a similar population of CA3 neurons) are activated by exposure to any spatial environment (Guzowski et al., 1999). Using a technique to visualize neurons activated at two distinct time points within the same animal, investigators were able to determine that exposure to a distinct context, followed by re-exposure to that same context activated an over-lapping population of CA1 neuron. In contrast, subsequent exposure to a novel context activated a significant number of CA3 neurons and a new subset of CA1 neurons (Guzowski et al., 1999). This suggests that the CA1 region can actively recognize a familiar context, reactivating the same population of neurons within the neuronal network. In fact, repeated exposure to 40 the same environment leads to increasing reactivation of the same population of CA1 neurons (Guzowski et al., 2006), suggesting that CA1 Arc expression is similar to how place cells reliably fire in a place field within a familiar environment (Guzowski et al., 2004), whereas CA3 cells show activation in response to novel environments.

Along these lines, the CA1 region should be involved in consolidating spatial and contextual information in a spatial task such as the watermaze, where successful performance is dependent of forming a memory for stable spatial information following repeated exposure to a familiar set of spatial cues, and requires the constant updating of positional information relative to that set of distal cues.

While the literature often makes a distinction between spatial and contextual processing, it is important to understand that both spatial and contextual learning requires the subject to form relationships between elements within an environment. However, spatial memory may be considered a more complex form of context- dependent learning, requiring the use of detailed allocentric cues in the environment to successfully navigate. This means you must know your position and update it relative to major environmental cues, and decide upon a path based upon your relative position. Contextual learning is a more associative and egocentric form of learning, requiring the subject to form a representation of contextual cues, and to associate those cues with a fearful experience.

1.4.2 Theories of systems consolidation of remote memory

Competing theories exist to explain how the brain represents memories for events (episodic memory). Following synaptic consolidation in an ensemble of hippocampal- neocortical neurons, a memory undergoes slow re-organization into a distributed neocortical network (systems consolidation). Multiple theories have been put forth to explain how memories ultimately become stably represented in the brain. While leading theories agree that the hippocampus is required for the acquisition of a spatial memory, and that over time these memories become represented in distributed neuronal

41 networks, the continued involvement of the hippocampus over time is debated. The literature debating these theories is vast, so we will only touch on the main theories.

1.4.2.1 Standard Consolidation Theory

The standard view of consolidation posits that, over time, a memory will become represented in extensive neuronal networks, and eventually becomes completely independent of the hippocampus and represented in its identical form within the neocortex, where it remains immutable and insensitive to hippocampal disruption (Squire, 1992; Alvarez and Squire, 1994; Squire and Alvarez, 1995; Squire and Bayley, 2007). The standard model has often been challenged by studies which report both temporally graded and non-graded retrograde amnesia, as well as the recent emergence of reconsolidation, which argue against complete hippocampal disengagement and the immutability of the cortical memory.

1.4.2.2 Multiple Trace Theory / Transformation Theory

The multiple trace theory (MTT) originally proposed by Nadel and Moscovitch (1997) suggests that a memory does form extensive traces in extra-hippocampal networks, but recollection of detailed contextual information required for episodic memory always engages the hippocampus. It argues that repeated retrieval of a memory over time forms additional ‘traces’ of the memory in neocortical regions, with each subsequent trace adding new information which serves to strengthen the general features of a memory. Over time, the statistical regularities among the multiple traces of the memory in neocortex are sufficient to support the general schematic, or gist-like features of the original memory. However, it asserts that retrieval of richly detailed aspects of the original memory always relies on the hippocampus (Nadel and Moscovitch 1997, for review see Frankland and Bontempi, 2005; Winocur and Moscovitch, 2011, Nadel et al., 2012). The Transformation Theory (TT) of consolidation has recently been proposed as an elaboration of MTT, which further suggests that both the cortical memory containing

42 the general ‘schematic or gist-like’ version (which lacks perceptual detail features of the original memory), as well as the richly detailed hippocampal-dependent memory co- exist in the brain. TT proposes that there is a dynamic interplay between the general and the richly detailed memory, and situational demands upon retrieval will influence if one or the other trace dominates (Winocur et al, 2007, Winocur et al., 2010; Winocur and Moscovitch, 2011).

Taken together, MTT/TT offer a more parsimonious explanation for cases of both temporally graded and non-graded retrograde amnesia (RA) in cases of hippocampal damage, depending on the type of memory the subject is asked to recall, in which schematic/generalized remote memory can be supported by extra-hippocampal (neocortical) regions, and therefore survive hippocampal damage (temporally graded retrograde amnesia), whereas contextually-rich, detailed remote memories always require the hippocampus, and will be impaired following hippocampal damage (non- graded RA) (Rosenbaum et al., 2002; Moscovitch et al., 2005; Winocur et al., 2005, 2007, for review, see Winocur and Moscovitch, 2011; Nadel et al., 2012). This finding is not supported by the Standard Consolidation Theory, which asserts that once consolidated in extra-hippocampal regions, a memory remains in its identical form (does not generalize to other contexts, in the case of context fear memories). The MTT/TT theory is gaining popularity in acceptance, particularly in light of the recently emerged field of ‘reconsolidation’, which demonstrates that a well consolidated memory can be brought back to a sensitive hippocampal-dependent state through the process of reactivation. The ability to reactivate the hippocampal-dependent memory suggests that the hippocampus may not always be required, but can be reengaged. This too argues against the Standard Consolidation Theory, which asserts that once consolidated in neocortex, the memory no longer susceptible to hippocampal manipulation. Thus, reconsolidation offers an interesting way of examining the dynamic process of systems levels consolidation.

1.4.3 Reconsolidation

It would be easy to think of consolidation as a stable, one-way process whereby a memory is first ‘made firm’ in a local distribution of synapses in a small neuronal network, and if allowed to stabilize, persists in its original form. However, we now know that consolidation is a dynamic process, where the memory not only undergoes a process of reorganization at the systems level, but also may be returned to an unstable state where it is again susceptible to interference or disruption of the ‘reconsolidation’ process (Nader et al., 2000; Dudai, 2004; Nader and Hardt, 2009; Dudai, 2012). This idea is not new. It was originally proposed by Bartlett (1932) who suggested that memory stabilization is an ongoing reconstruction process in which past experiences are updated to incorporate new information from ongoing experiences. It is presently understood that memory ‘updating’ and ‘strengthening’ are among the main functions of reconsolidation (for review see Alberini, 2011).

Early work which led to the understanding that synaptic consolidation of long-term memory requires de novo protein synthesis used protein synthesis inhibitors (PSIs) at various delays to determine that disruption within the minutes following new memory acquisition results in a failure to consolidate long-term memory (Davis and Squire, 1984). Within the past two decades, the use of PSIs has been instrumental in discovering that reactivated established memories can be remade susceptible to disruption by protein synthesis inhibition. During reactivation (re-exposure to cues present during initial memory acquisition), a memory is recalled, and must once again restabilize in its neuronal network through a second consolidation phase called reconsolidation. This is also a protein synthesis dependent process, as delivering PSIs immediately following reactivation can impair subsequent recall of the memory (Nader el al., 2000; Sara et al., 2000). This was famously demonstrated in 2000 by Nader and colleagues using anisomycin infusions into the amygdala shortly after reactivation of a conditioned tone fear memory to interfere with the reconsolidation process (Nader et al., 2000). Interestingly, the proteins required for consolidation and reconsolidation may differ, suggesting a possible dissociation between consolidation (suggested to require

BDNF, but not Zif268) and reconsolidation (suggested to require Zif268, but not BDNF) processes (Lee et al., 2004).

Back in the late 1990’s, Sara and colleagues proposed the idea of reconsolidation, an idea that had not yet gained mainstream acceptance until Nader’s seminal study in 2000. They were originally investigating the role of CREB in reconsolidation of long-term memory using intracerbroventricular infusions of the β-noradrenergic antagonist Timolol or systemic injections of propranolol to interfere with metabotropic β-noradrenergic receptors activity regulating cAMP signaling (Roullet and Sara, 1998; Przybyslawski et al., 1999). Since then, the crucial role of CREB in both the consolidation and reconsolidation process has been demonstrated, where suppression of CREB activity in the hippocampus prior to reactivation of a contextual fear memory results in disruption of the second wave of CREB-mediated gene expression during the reconsolidation process that is required for the stabilization of a reactivated fear memory (Kida et al., 2002; Mamiya et al., 2009; Tronson et al., 2012). We will discuss these exciting findings soon.

1.4.4 Time-dependent reorganization of remote memories

Information we have gathered from human and animal lesion studies led to the idea that memories undergo a time-dependent qualitative change. Also, targeted pharmacological agents have permitted researchers to reversibly examine the transient effects of inhibiting neuronal activity within a particular brain region. In vivo imaging techniques, such as (14C)2-deoxyglucose [(14C)2-DG] radio imaging, and more recently immediate early gene activation has given us a window into the actual activation patterns with cellular and temporal resolution not previously possible.

Candidate regions for remote memory storage in systems consolidation are the anterior cingulate cortex (ACC), the prefrontal cortex (PFC), and the retrosplenial cortex (RSC). Imaging techniques using (14C)2-DG to visualize metabolic activity at recent or remote time points following spatial training of mice in the radial arm maze revealed that spatial memory does undergo a process of reorganization over time, with increased metabolic 45 activity in the ACC, PFC, and RSC when tested 25 d after training (considered a remote timepoint in the animal literature) (Bontempi et al., 1999). Bontempi’s group confirmed a similar pattern of remote spatial memory reorganization for the radial-arm maze at 30 d using immediate-early gene imaging of c-fos and Zif268 (Maviel et al., 2004). They also found a relative decrease in gene activation in the dorsal hippocampus at 30 d (compared to 1 d post training). This is an interesting finding in light of the fact that, despite undergoing systems reorganization, spatial memories like those in the radial arm maze, or in the watermaze, still require the hippocampus even at a remote timepoint. Immediate early gene imaging of c-fos and Zif268 at recent or remote time points following context fear conditioning also showed distributed activation patterns at remote time points, with increases in immediate early gene expression in the ACC, infralimbic and prelimbic cortices. At a recent time point following conditioning, higher c- fos and Zif268 expression was seen in CA1 of the dorsal hippocampus (Frankland et al., 2004; Wheeler and Frankland, unpublished observations). Additionally, inactivation of the ACC using infusions of the sodium-channel blocker lidocaine resulted in impaired retrieval of context fear memory at a remote (but not recent) timepoint (Frankland et al., 2004; Teixeira et al., 2006). Further work in the Frankland lab demonstrated that lesioning the dorsal hippocampus at either a recent (1d) or remote (30d) following spatial training in the watermaze resulted in impaired spatial memory for both time points, confirming the critical involvement of the hippocampus in spatial memory, regardless of the age of the memory (Teixeira et al., 2006). Finally, they showed that structural modification of dendritic excitatory spines in the ACC are necessary for systems consolidation of remote context fear memory. Blocking new excitatory dendritic spine growth in the ACC using MEF2, a transcription factor that negatively regulates synapse formation (Flavell et al., 2006), results in impaired consolidation of remote fear memory (Vetere et al., 2011). Together, these findings suggest that context memories do undergo a time-dependent reorganization to form a distributed cortical network, which allows these memories to survive following hippocampal lesioning or inactivation, but that richly detailed spatial memories always require an intact hippocampus. By examining the pattern of neuronal activation following memory testing at various time delay intervals following memory acquisition, we have a precise picture of the actual

46 neuronal activity pattern in the brain during the consolidation of recent and remote memories.

Figure 1.5 Basic schematic of the hippocampal-dependent memory consolidation process

We have taken a bottom-up approach in reviewing the ‘parts’ of long-term memory consolidation, from the level of the synapse, up to the level of the system. We have also discussed how the role of the hippocampus in consolidation is not a simple one-way process, but rather is a complex and conditional process whereby multiple representations of a memory can be gradually distributed throughout the brain, and can be returned to a hippocampal dependent state (Fig 1.5) [as the hippocampus is a highly interconnected brain region, it is important to note that other regions (ie. amygdala, prefrontal cortex) also play a key role in memory consolidation. However, for the purpose of our review, we must restrict most of our discussion of consolidation to the hippocampus]. Finally, we will now discuss how the transcription factor CREB has been used to investigate hippocampal-dependent memory consolidation in in vivo learning and memory paradigms.

1.5 Putting it all together: CREB is both necessary and sufficient for memory consolidation

There is an extensive body of literature surrounding the role of CREB in the hippocampus using many methods for in vivo manipulation of CREB on a range of hippocampal-dependent behavioural tasks. The overall finding is that CREB is both necessary and sufficient for memory consolidation.

1.5.1 CREB is necessary for memory consolidation

To infer that a molecule is ‘necessary’, a loss-of-function approach is typically used, where removal of the molecule produces a deficit. CREB has been demonstrated to be necessary for memory consolidation using a range of suppression models.

1.5.1.1 Memory deficits in the CREB hypomorphic mouse model

The behavioural phenotype of CREB αΔ-/- mutant mice (Hummler et al., 1994) has been investigated by several groups, with mixed findings, leaving the nature of memory deficits in this model of CREB deficiency unresolved. Bourtchuladze and colleagues were the first to investigate memory performance in CREB-hypomorphic mice across a range of hippocampal and amygdala-dependent tasks. They found that hippocampal- dependent context fear was impaired 1 h and 24 h following context fear conditioning, but that mice had high freezing levels equivalent to controls at 30 min following conditioning, suggesting intact short-term memory (which does not rely on protein synthesis), but impaired long-term memory for this task. During amygdala-dependent tone fear conditioning, CREB-hypomorphic mice froze at control levels when tested 30 min, or 1 h follow conditioning, but by 2 h or 24 h following conditioning, freezing to the tone dropped significantly, indicating poor long-term memory. They also trained and tested mice in a spatial version of the watermaze using a prolonged 15 d training protocol. While CREB-hypomorphic mice showed decreasing escape latencies across training days similar to controls, when probed for their spatial memory of the escape platform, they were significantly worse than controls, suggesting spatial memory impairments. Impairment was seen even when a more intensive training protocol was used (Bourtchuladze et al., 1994). Based on these early reports of memory impairment, impaired LTP maintenance in CREBαΔ-/- mice (discussed earlier), other labs have since further characterized the memory phenotype in this model of CREB deficiency.

Next, Kogan and colleagues explored variations in the learning protocols of several hippocampal-dependent tasks, including context fear conditioning, spatial memory in the watermaze, and the social transmission of food preference (STFP) task (Kogan et al., 1997). They confirmed earlier reports of spatial and context memory impairments, but found that these impairments could be overcome by providing mice with prolonged spaced training trials. For the spatial memory training, when mice were allowed a 10min inter-trial-interval (ITI) between each of the four trials within a daily training session (as opposed to 1 min ITI), CREB-hypomorphic mice were able to learn the spatial memory task across the 10 d of training days, and also displayed evidence of a spatial memory

49 when probed for the platform location in the absence of the platform. Similarly, if given an hour-long ITI between two daily context fear conditioning sessions (as opposed to 1 min ITI), CREB hypomorphic mice were able to form a robust context memory when tested 24 h after conditioning. But if given only 1 min ITI between five context fear conditioning sessions, CREB hypomorphic mice again displayed impaired context memory when tested 24 h later. Hippocampal place cell firing in CA1, discussed earlier to be critical for spatial memory formation and navigation, was measured in CREB αΔ-/- mice (Cho et al., 1998) using a radial-arm maze, which is a multi-armed circular maze with distal cues used for navigation and spatial memory formation. Recordings were obtained from CA1 place cells, and spatial firing patterns revealed a low spatial selectivity, and more scattered firing patterns in CREB αΔ-/- mice relative to wild-type controls while exploring the maze. Also, upon reconfiguring distal cues, CREB αΔ-/- place cells remapped their firing, whereas wild-type place cells remained stable, suggesting place cell instability in CREB αΔ-/- mice (Cho et al., 1998), which may account for some of their spatial memory deficits.

Hippocampal-dependent social transmission of food preference memory was investigated by Kogan and colleagues for the first time in a CREB-suppression model. In this task, the subject animal is allowed to interact with a demonstrator animal which has recently consumed a scented flavored food. When the subject is subsequently presented with a choice of the demonstrator-scented food, and a novel scented food, the subject should selectively consume the familiar food previously paired with the demonstrator (Galef and Wigmore, 1983). In their experiment, Kogan and colleagues gave two exposures to the demonstrator mouse, spaced either by 1 min or 1 h. Immediately following the second exposure, both CREB αΔ-/- and wild-type control mice selectively consumed the food paired with the demonstrator mouse, suggesting normal short-term memory for the task. However, when tested following a delay of 24 h after training, CREB αΔ-/- mice given only 1 min ITIs between training sessions exhibited significant impairment in long-term memory for the task, consuming equal amounts of both foods. However, lengthening the interval between exposure sessions to the demonstrator overcame this deficit when tested at 24 h (Kogan et al., 1997). They later investigated social recognition in CREB αΔ-/- mice. Consistent with their earlier findings, 50 they show that these CREB deficient mice show normal social recognition memory for a previously socialized juvenile mouse 30 min after training, but no recognition when tested after 24 h (Kogan et al., 2000), where CREB αΔ-/- mice spend equivalent time interacting with both a novel and a familiar, previously socialized mouse.

Taken together, though these CREB αΔ-/- mice exhibit impaired long-term memory across a range of hippocampal-dependent tasks, it appears that extensive spaced training over many days can overcome the deficits in spatial, context, and STFP memory induced by CREB suppression (Bourtchuladze, et al., 1994; Kogan et al., 1997; Balschun et al., 2003). This suggests that the stabilization of memory consolidation can be sensitive to the frequency and timing of training opportunities and potentially regulated by compensatory up-regulation of remaining CREB isoform (β) and CREM activator protein (Hummler et al., 1994, Blendy et al., 1996).

1.5.1.2 Inducibly disrupting CREB impairs memory consolidation

Using an α-CaMKII-tTA/tetO bitransgenic model for suppressing CREB function in the absence of dox, Pittenger and colleagues generated mice with K-CREB mutation, a dominant negative inhibitor of all 3 CREB isoforms, restricted to the dorsal CA1 region of the hippocampus (dCA1-KCREB). During periods of CA1 CREB disruption, they found deficits in L-LTP following forskolin stimulation, and impairments in spatial memory for the watermaze, which were both reversible when the mutant K-CREB transgene expression was suppressed. Surprisingly, when tested on a context conditioning task 24 h after conditioning, dCA1-KCREB mice were unimpaired in their ability to form a context memory, suggesting that not all hippocampal-dependent tasks were equally impaired (Pittenger et al., 2002).

Using a similar model for forebrain-wide CREB suppression, Josselyn and colleagues also investigated the memory for conditioned taste aversion, in which a novel taste is paired with transient nausea induced by lithium chloride. Animals should remember to avoid the illness-inducing taste, an amygdala-dependent process. In two models of CREB deficiency CREB αΔ-/- mice, and the inducible CREB repressor α-CaMKII-CREBIR 51 all show impaired long-term memory for conditioned taste aversion when tested 24 h after training (Kida et al., 2002; Josselyn et al., 2004).

Unlike forebrain-wide CREB suppression models, targeted injections of agents designed to disrupt CREB function have had mixed results. Microinjections of viral vector into discrete brain regions infects only a small population of cells within that region. Viral infection of vectors encoding mCREB into the hippocampus or lateral amygdala has been reported to be insufficient to significantly impair memory. Unlike more widespread CREB suppression models, impairing CREB function in only a limited number of cells allows for uninfected neurons with unimpaired CREB function to compensate for those with decreased CREB activity (Han et al., 2007). In mice microinjected with HSV-mCREB into the lateral amygdala, cellular activation patterns of the immediate early gene Arc immediately following tone fear conditioning showed that neurons with decreased CREB function are 11X less likely to be included in the active memory trace (Han et al., 2007). As with the hippocampus and spatial memory (Sekeres et al., 2010), infecting a subset of lateral amygdala neurons with mCREB did not impair memory for tone fear conditioning. While the selective exclusion of mCREB- expressing neurons has not yet been investigated in the hippocampus, it is likely that a similar pattern would be found, where there is a compensatory activation of uninfected neurons supporting normal spatial memory formation. There is at least one report of HSV-mCREB infection in the CA1 and dentate gyrus of the dorsal hippocampus resulting in long-term memory impairment for the social transmission of food preference task in rats when tested 10 d after training (Brightwell et al., 2005). However, when this same group performed a similar experiment using hippocampal mCREB vector injections, they failed to find an impairment in place response memory (Brightwell et al., 2008), suggesting that impairment following hippocampal disruption with mCREB may be selective and task-specific, and depend upon the extent of CREB disruption in the hippocampus.

Lastly, using intra-hippocampal infusions of antisense CREB oligodeoxynucleotides (ODN) in the hippocampus of wild-type rats, Guzowski and colleagues found that the pre-training CREB-ODN infusions blocked translation of RNA, and resulted in impaired

52 spatial memory in the watermaze (Guzowski and McGaugh, 1997). However, when CREB-ODN infusions were made 20 hours after spatial watermaze training (at a time when the initial window of synaptic consolidation has closed (Dudai, 2004), spatial memory was unimpaired when tested 2 d later. This suggests that, like in mice, CREB function in rats is necessary for spatial memory consolidation (Guzowski and MgGaugh, 1997).

Overall, CREB disruption throughout the entire brain, or restricted to the forebrain, or even individual subregions (hippocampus, lateral amygdala) shows an overall impairment across tasks, indicating that CREB is necessary for long-term memory consolidation. However, due to the varied methods, duration, and location of disruption, and the different tasks used to probe memory, the results of CREB’s necessity in long- term memory consolidation are mixed.

1.5.1.3 Upstream disruption of CREB impairs memory consolidation

As it became clear that CREB was a critical regulator of protein-synthesis dependent memory, several groups began looking upstream of CREB phosphorylation to see how disruption of the molecular signaling cascade upstream of the critical Ser133 residue can disrupt synaptic consolidation of long-term memory. Systemic (Atkins et al., 1998; Selcher et al., 1999), intracerebroventricular (Bourtchouladze et al., 1998; Schafe et al., 1999) or intra-amygdala (Schafe and LeDoux, 2000) administration of inhibitors of PKA or MAP kinase activity impairs long-term memory in rodents. Disruption of CREB phosphorylation also impairs learning and memory in humans. Coffin-Lowry Syndrome is an X-linked disorder caused by mutations in the gene encoding RSK2, a protein kinase that phosphorylates CREB at the key Ser133 residue (Trivier et al., 1996). The IQ of patients with Coffin-Lowry Syndrome is correlated with the capacity of their mutated RSK2 to phosphorylate CREB (Harum et al., 2001).

Genetically-engineered mice with mutations upstream of CREB phosphorylation provide additional support for the important role of CREB phosphorylation in memory formation. For instance, expression of a mutant form of the PKA transgene in the forebrain of mice 53 blocks hippocampus-dependent memory formation, and decreases PKA-dependent L- LTP (Abel et al., 1997). Deletion of the CREB kinases MSK1 (Chwang et al., 2007) or CaMKIV (Wei et al., 2002) not only decreases activity-dependent CREB phosphorylation but also impairs fear memory formation. RSK2-knockout mice also exhibit impaired spatial learning (Poirier et al., 2007). On the other hand, inhibition of the CREB phosphatase PP1 (which serves to negatively regulate serine phosphorylation) during training increases CREB phosphorylation and improves both novel object recognition and spatial learning (Genoux et al., 2002). Finally, double knockout animals missing the two calcium-dependent adenylyl cyclases (AC1 and AC8) fail to form long-term memory in the hippocampus-dependent passive avoidance task (Wong et al., 1999), although this defect is overcome by delivery of the adenylyl cyclase agonist forskolin to the CA1 region of the hippocampus. Together, these results suggest that CREB may act as a final central switch onto which various signaling pathways converge to regulate consolidation of long-term memory.

1.5.2 CREB is sufficient for memory consolidation

To infer that a molecule is ‘sufficient’, a gain-of-function approach is typically used, where addition of the molecule produces an enhancement. Earlier, we touched briefly upon the seminal finding in rodents which showed that virally over-expressing CREB in the basolateral amygdala can facilitate the formation of a long-term fear memory, even following massed-training sessions which do not typically support memory consolidation (Josselyn et al., 2001). This first gain-of-function study laid the groundwork for selectively enhancing CREB function. This idea that CREB is sufficient to induce consolidation under conditions in which long-term memory formation is not typically supported has since been demonstrated across a number of brain regions and cognitive tasks, including spatial memory tasks, and contextual and tone fear conditioning.

1.5.2.1 Virally enhanced CREB-mediated memory consolidation

We discussed earlier that microinjection of HSV-mCREB into the lateral amygdala did not impair fear memory (Han et al., 2007). However, the Josselyn lab found that increasing CREB function in the same proportion of amygdala neurons using HSV- CREB led to robust enhancements in memory formation (Han et al., 2007). Normal mice trained to associate a very weak shock with the tone failed to exhibit a freezing response when subsequently presented with the tone. However, micro-infusions of the HSV-CREB vector prior to this same weak conditioning produced robust freezing in these animals when tested for the fear memory after 2 d later, suggesting that, like in the hippocampus, increasing CREB function in the lateral amygdala is sufficient to induce a tone fear memory that is not supported in normal animals. Analysis of cellular activation patterns of Arc found that lateral amygdala neurons with enhanced CREB function are 3X more likely to be selected for inclusion in the memory trace than are neurons with normal CREB function (Han et al., 2007). The idea that CREB-enhanced neurons have a competitive advantage for incorporation into the active memory trace is gaining support. In addition to work out of the Josselyn lab, Silva’s lab performed whole cell recordings from lateral amygdala neurons infected with HSV-CREB, or non-infected neurons from the same slice. They find that CREB-enhanced neurons have enhanced synaptic transmission following tone-fear conditioning. Additionally, under non-training conditions, the resting membrane potential and synaptic amplitude of infected or non- infected neurons did not differ, although the threshold for firing action potentials was significantly lower in CREB-enhanced neurons, suggesting that these neurons have higher intrinsic excitability (Zhou et al., 2009; Benito and Barco, 2010).

As used in the lateral amygdala, HSV vectors have been used extensively to increase CREB function in the hippocampus. Work out of the Colombo lab has shown that microinjections of HSV-CREB vector into the dorsal hippocampus of rats facilitates place learning. Here, rats were given 30 trials to learn to find a hidden platform at the end of one arm of a plus (‘+’) maze. 5 d later, they were returned to the maze and probed for their memory of the platform location. While Colombo had previously established that this task results in an increase in pCREB and c-fos expression in the

55 dorsal hippocampus and striatum (Colombo et al., 2003), this was the first experiment to show that viral over-expression of CREB can enhance the formation of a spatial memory (Brightwell et al., 2007). Using another hippocampal-dependent task, this same groups found increased levels of c-fos expression in the dorsal hippocampus, and pCREB in the dorsal and ventral hippocampus following social transmission of food preference learning and recall (Countryman et al., 2005), but the potential for CREB- facilitated enhancements in STFP memory has not yet been investigated.

Acutely increasing CREB function in the dorsal hippocampus can also facilitate contextual fear memory consolidation Using a CRE-lacZ reporter mouse, Impey et al have shown an increase in CRE-dependent gene expression, and in pCREB in the dorsal CA1 and CA3 following context fear conditioning (Impey et al., 1998a). Restivo and colleagues (2009) showed that micro-injections of a Sindbis viral vector encoding a constitutively active form of CREB (CREBY134F) in the dorsal CA1 or dentate gyrus of wild-type mice prior to contextual fear conditioning enhanced memory for the fearful context when tested 24 h following conditioning. Importantly, by using a relatively low intensity shock during conditioning, they induced only weak freezing to the context in wild-type mice injected with control vector, but robust freezing in mice with constitutively active CREB in the CA1 or dentate gyrus. These findings suggest that increasing CREB function within the hippocampus is sufficient to induce a contextual fear memory following weak training. Using this same model, they next demonstrated that constitutively-active CREB in the dentate gyrus enhances context memory at 24 h, but that these mice still display normal extinction of fear over repeated and prolonged test sessions, suggesting that the strengthened memory consolidation is not abnormally persistent to new extinction learning (Vetere et al., 2011).

One drawback to using viral vectors to acutely increase CREB function is the limited penetrance of transgene expression. In the lateral amygdala, approximately 15% of neurons are infected (Han et al., 2007), and in the hippocampus, up to 20% of neurons in the dentate gyrus (Sekeres et al., 2012) are infected by viral microinjection. While we have evidence that the CREB-infected neurons are preferentially recruited for the memory trace, (Han et al., 2007), there are still many neurons within the target brain

56 region that we cannot manipulate using this method. Transgenic inducible-CREB models offer an attractive alternative for enhancing CREB in the hippocampus.

1.5.2.2 Transgenic inducible-CREB enhancement facilitates memory consolidation

Barco and colleagues used their α-CaMKII-/tTA/tetO-VP16 bitransgenic mouse model to turn on constitutively active CREB function throughout the forebrain prior to context fear conditioning. They found that constitutively active CREB activity could actually overcome the amnestic effects of the protein synthesis inhibitor anisomycin. They proposed that de novo protein synthesis may not have been necessary to support the consolidation of a context fear memory, as plasticity-related proteins within the synapses of the neurons may have been readily available due the high basal transcriptional activity of VP16-CREB neurons in these bitransgenic mice (Viosca et al., 2009b). They offer the synaptic tagging and capture hypothesis originally proposed by Morris and Frey (1997) as a potential explanation for the enhanced consolidation of new memory, even in the presence new protein synthesis inhibition (Kandel, 2001; Barco et al., 2002). VP16-CREB has 25-fold higher activation that endogenous CREB (Barco et al., 2002). As hippocampal CA1 neurons with chronically enhanced CREB function exhibit a lower threshold for L-LTP, and reduced after-hyperpolarization currents (Barco et al., 2002; Lopez de Armentia et al., 2007), it may be that this large population of hippocampal neurons with increased intrinsic excitability can actually disrupt the normal consolidation and retrieval of memory. Due to this high transcriptional activity of VP16- CREB throughout the entire forebrain, Barco’s lab later reported disruption of memory associated with such high CREB activation during the retrieval of a spatial memory (Viosca et al., 2009).

To address this, Kida’s lab designed a similar constitutively active CREB model (CREBY134F) with much lower basal activity (1.5-2.5 fold higher) throughout the forebrain. Their α-CaMKII-CREBY134F mice show enhanced long-term memory for context fear 2 h and 24 h after conditioning, but memory equivalent to controls after

57 only 30min. Relative to controls, CREBY134F mice also displayed enhanced social recognition memory when tested after 24 h, but equivalent memory at 30min, suggesting that more moderately constitutively active CREB enhances protein-synthesis dependent long-term memory, but has no effect of protein-synthesis independent short term memory (Suzuki et al., 2011). CREBY134F mice also showed enhanced spatial memory in the watermaze. Mice were trained for 10 d on the hidden platform version of the watermaze, and probed for their memory of the platform location on days 5 and 10 of training. While all mice successfully learned the platform location across days, CREBY134F mice displayed significantly better memory for the platform location during both probe tests relative to wild-type controls (Suzuki et al., 2011). Together, the transgenic induction of a more moderately active CREB throughout the forebrain most consistently produced enhancements of consolidation of multiple forms of memory.

1.5.3 CREB is necessary for memory reconsolidation

We have discussed many studies demonstrating that the transcription factor CREB is both necessary and sufficient for consolidation of memory. The necessity of CREB signaling in systems reconsolidation of memory was first identified in the late 1990’s by Sara and colleagues (Roullet and Sara, 1998; Przybyslawski et al., 1999; Sara, 2000). At the time, little attention was given to the findings.

They trained rats on a spatial task in the radial-arm maze (RAM) for seven days. Two days after training, rats were implanted with cannulas into the lateral ventricles, and ten days later were retrained in the RAM to criterion levels to ensure optimal spatial memory for the maze. 24 h following the final training session, rats were returned to the RAM for brief reactivation of the spatial memory. Rats then received intraventricular infusions of β-noradrenergic receptor antagonist Timolol or saline control at 5, 30, 60, or 300 min after reactivation. Rats were re-tested for spatial memory in the RAM 24 h later. Consistent with previous reports, protein-synthesis dependent long-term memory (but not short-term memory) is susceptible to disruption by amnestic agents. Only rats receiving Timolol 60 min after reactivation showed subsequent impairment in spatial

58 memory 24hrs later (Roullet and Sara, 1998). Their results suggest that β-noradrenergic antagonist disrupts cAMP-dependent reconsolidation of a well-consolidated spatial memory following reactivation within a discrete time window. They subsequently showed that post-reactivation disruption of cAMP using systemic injections of the β- noradrenergic receptor antagonist propranolol similarly disrupts reconsolidation of fear- based memory for the inhibitory avoidance task, conditioned taste aversion, and spatial memory in the RAM. Importantly, they showed here that in the absence of reactivation in the RAM, propranolol did not induce disruption of spatial memory. (Przybyslawski et al., 1999). Based on their findings, they suggested that interference with CREB’s upstream signaling cascade within the discrete timeframe for reconsolidation is sufficient to impair the restabilization of protein-synthesis memory in the hippocampus (Sara, 2000).

To more precisely investigate the effects of CREB suppression during the reconsolidation of a reactivated fear memory, Kida and colleagues used the CREB repressor mice (α-CaMKII-CREBIR, discussed earlier) to inducibly suppress CREB function throughout the forebrain (Kida et al., 2002). After first confirming in wild-type mice that delivery of the protein synthesis inhibitor anisomycin prior to re-exposure to the training context subsequently impairs fear memory testing 24hrs later (standard protocol for testing reconsolidation disruption,(Nader et al., 2000, Debiec et al., 2002; Suzuki et al., 2004; Wiltgen and Silva, 2007) they next specifically tested whether CRE- mediated transcription is necessary for the re-stabilization of a reactivated fear memory trace. Both wild-type and CREB repressor mice were fear conditioned (drug free) using a robust 3 shock protocol. The next day, mice were given either tamoxifen (to induce CREB suppression) or saline control injection prior to re-exposure to the conditioning context (no shock delivered during 90sec context re-exposure), and 24hrs later, mice were tested for their fear memory. Only mice with repressed CREB activity during the reconsolidation process displayed impaired fear memory when tested following context re-exposure. Importantly, CREB repressor mice receiving tamoxifen injections, but which did not receive the context re-exposure session, displayed intact fear memory during testing, supporting the idea that CREB-mediated gene expression during the reconsolidation process is required for the stabilization of a reactivated fear memory. 59

They quantified CREB activation in wild-type mice following brief re-exposure to the conditioning context, and found increased levels of pCREB in the CA1 and CA3 of the hippocampus 30min following context re-exposure relative to untrained control mice. Interestingly, no change in relative pCREB levels was reported in the dentate gyrus (Mamiya et al., 2009). Recently, the same group has reported similar findings in a spatial reconsolidation paradigm (Kim et al., 2011), building upon Sara’s initial proposals of the importance of CREB in reconsolidation of spatial memory (Roullet and Sara, 1998; Przybyslawski et al., 1999; Sara, 2000).

Kida’s lab followed up on their initial study of CREB suppression during reconsolidation (Kida et al., 2002) to show that CREB is necessary for reconsolidation following brief reactivation (3 min) of context memory, and also required for extinction following prolonged reactivation (30 min) of the context memory in CREBIR mice (Mamiya et al., 2009). An often debated subject is the difference between reconsolidation, and extinction. It is generally thought that brief ‘reminder’ serve to initiate a reconsolidation process, whereas longer reminders (30 min, hours), and particularly repeated reminders of the CS (presentation of the conditioning context alone, or in the case of cued fear memory, presentation of the tone) in the absence of the aversive US (shock) results in extinction (Monfils et al., 2009; Alberini, 2011). Extinction is a form of protein-dependent learning (not a form of forgetting), in which the animal learns that a stimuli previously thought to be aversive, is now safe or neutral.

A recent study examined the impact of regionally-specific CREB disruption in the basolateral amygdala in a reconsolidation paradigm. We will touch upon it here as it has relevance for one of our studies. Tronson and colleagues used microinjection of HSV- mCREB vector to suppress CREB activity of rats prior to reactivation of a tone-fear memory. They tested the hypothesis that CREB is necessary for reconsolidation, but not extinction of fear memory (Tronson et al., 2012). Rats were tone-fear conditioned using a robust conditioning which normally induces normal long-term memory. 24 h later, rats received microinjection of mCREB vector into the basolateral amygdala. 72 h later, when transgene expression was high (Barrot et al., 2002), subjects were placed in a novel context, and fear memory was reactivated with a 30 s tone. During the

60 reactivation session, both rats with mCREB vector and controls show normally high freezing to the tone, suggesting that the mCREB is not interfering with the retrieval of a previously acquired fear memory. However, when re-tested for the tone-fear memory 24 h after reactivation, rats with mCREB vector showed significantly lower freezing than controls, suggesting impaired CREB function in the basolateral amygdala during the post-reactivation reconsolidation process interfered with protein synthesis-dependent restabilization of the tone-fear memory (Tronson et al., 2012). Importantly, post-training microinjections of mCREB vector did not disrupt the fear memory when the tone reactivation session was omitted. This finding using region-specific disruption of CREB in the basolateral amygdala is consistent with finding that inducibly repressing CREB throughout the forebrain prior to reactivation of context memory disrupts reconsolidation (Kida et al., 2002).

To date, no one has directly investigated the ability of CREB to facilitate memory restabilization during the reconsolidation process. However, one study looked upstream of CREB to investigate PKA, a critical kinase required for the phosphorylation of CREB at Ser133 (Yamamoto et al., 1988; Gonzalez and Montminy, 1989). Tronson and colleagues infused the PKA-agonist 6-BNZ-cAMP into the basolateral amygdala of the rat prior to reactivation of a tone fear memory. Following 4 brief reactivation sessions in the presence of the PKA agonist, memory for the tone was significantly enhanced relative to vehicle injected controls. Post-training infusions of 6-BNZ-cAMP in the absence of the tone-fear reactivation had no effect on the memory, suggesting that enhancement of PKA is sufficient to facilitate reconsolidation of fear memory (Tronson et al., 2006). It is curious, however, that repeated exposure to the tone (in the absence of the reinforcing shock) would continue to enhance reconsolidation as opposed to inducing new learning of extinction in this case. This is a particularly interesting question in light of results by Vetere and colleagues which suggest, in the hippocampus, increasing CREB activity (via microinjection of Sindbis vector encoding constitutively active CREB into the dentate gyrus) enhances context fear memory consolidation, but does not interfere with normal extinction learning in the same animals (Vetere et al., 2011) Still, the enhancing effects of PKA in reconsolidation begs the question to be asked: can increasing CREB function facilitate the reconsolidation of memory? 61

1.6 The role of CREB co-activators in memory consolidation

CRE-dependent gene transcription has been found to be impaired not only by disruption of CREB activity, but also by disruption of CREB co-activators CBP/p300, and CRTCs. While the scope of review required to thoroughly characterize CBP/p300-dependent memory models is beyond this review, we will only briefly touch on the topic here.

1.6.1 CBP/p300 disruption impairs memory consolidation

A major cognitive and developmental disorder was identified in humans, Rubenstein- Taybi Syndrome (RTS; OMIM 180849) involves disruption of normal CBP function. RTS is an inherited disease with an incidence of 1 in 125,000 births, in which a mutation of the CBP gene causes severe developmental and mental retardation, including impairment in forming long-term memories (Petrij et al., 1995; Roelfsema and Peters, 2007) Approximately 45% of cases of RTS result from de novo loss mutations of CBP, with an additional ~5% resulting from mutations of p300. Many mouse-models have been generated to explore the effects of CBP or p300 mutations (see Josselyn, 2005 for extensive review). Of the most notable findings, heterozygous deletions (Alarcon et al., 2004) or truncations (Oike et al., 1999) of the CBP allele have demonstrated impaired long-term memory in tests for context and tone fear memory, passive avoidance (Alaracon et al., 2004; Oike et al., 1999) and novel object recognition (Bourtchouladze et al., 2003).

In humans, reported cases of EP300 (the gene encoding p300) mutations causing RTS are rare, with only 7 confirmed carriers (Roelfsema and Peters, 2007; Bartsch, et al., 2010; Viosca et al., 2010). Behavioural characterization of a heterozygous null mouse model of EP300, revealed only a mild cognitive phenotype, with minor impairment on spatial reversal learning task, but normal learning and memory in other tasks (Viosca et al., 2010). Brains from these animals also exhibit no changes in histone acetylation or gene expression. These data suggest that p300 activity may be less critical than CBP for memory formation. Together, the results described here show that the CREB co-

62 activator CBP, and to a lesser degree p300, are also essential for normal cognitive function.

1.6.2 What is the role of CRTCs in memory consolidation?

The role of CRTCs in memory has not yet been directly examined, though findings suggest that CRTCs are important in regulating forms of CREB-dependent synaptic plasticity, as previously discussed. To date, no published studies have investigated the role of CRTCs in an in vivo learning and memory paradigm. Recently, Espana and colleagues suggested a possible link between a mouse model of Alzheimer’s disease, and disruptions in CRTC1 activity in cultured primary cortical neurons harvested from transgenic mice with mutated β-amyloid precursor protein (APP). Relative to control, these APP mutant mice showed a deficit in CRE-luciferase activity following stimulation with KCl and forskolin, which was reversed in neurons transfected with plasmids containing CRTC1 or CRTC2 (Espana et al., 2011). While they also found that mutant APP mice also display deficits in spatial memory, the link to CRTC1 in memory formation was only inferred, not experimentally tested. Thus, to date, no direct investigation in the role of CRTCs in an in vivo learning and memory paradigm exists.

Together, all these data indicate that CREB and CREB co-activators play a conserved role in hippocampal memory consolidation and reconsolidation.

1.7 Goals of this thesis: Hypotheses and predictions

While the literature supporting a role for CREB in memory consolidation is vast, the majority of studies use a loss-of-function approach to determine CREB’s necessity for protein-synthesis dependent consolidation of long-term memory. We aim to take the opposite approach. Using gain-of-function techniques, we will demonstrate that CREB, and the CREB co-activator CRTC1 are sufficient to support consolidation and reconsolidation of hippocampal-dependent memory.

Based on the general hypothesis that enhancing CREB function or CRTC1 function in key hippocampal subregions prior to training could facilitate memory consolidation, we explored several hippocampal dependent tasks using limited training parameters that do not typically support spatial or contextual memory consolidation. We hypothesized that increasing hippocampal CREB function prior to training on these tasks would support consolidation under otherwise challenging conditions. We then aimed to rescue hippocampal-dependent memory deficits in CREB deficient mice (CREBαΔ-/- mice) using targeted enhancement of CREB. Finally, we hypothesized that enhancing CREB or CRTC1 function during the initial synaptic consolidation period would have enduring effects on the systems consolidation and reconsolidation of remote context memory.

There are three main goals for this thesis: 1) This is the first study to show that CREB is both necessary and sufficient for consolidation of spatial memory within the same model (addressed in Chapter 3). 2) This is the first study to show a role for CRTC1 in memory consolidation in an in vivo paradigm (addressed in Chapter 4) 3) It is the first investigation of the role of CREB (and CREB coactivators) in consolidation and reconsolidation of remote memory (addressed in Chapter 4).

To address these goals, first, we will combine classical gene knockdown mouse model (CREBαΔ-/- mice) with viral gene transfer techniques using HSV-CREB. We will build upon earlier findings in the CREBαΔ-/- mice to reveal hippocampal-dependent spatial memory impairments induced by a lifetime of CREB depletion. Next, we will rescue

64 spatial memory deficits in these CREB hypomorphic mice using target overexpression of CREB in the CA1 prior to training.

Second, we will use HSV vectors encoding the CREB co-activator CRTC1 in the dentate gyrus of wild-type mice to facilitate consolidation of context memory under limited training conditions which do not typically support memory formation.

Third, we will use viral gene transfer techniques to demonstrate how enhancing hippocampal CREB or CRTC1 during initial synaptic consolidation can maintain precision of context memory even at a remote timepoint. Also, we show that increasing CREB or CRTC1 prior to reactivation of a remote context memory can enhance reconsolidation of remote context memory.

2 General Methods 2.1 Mice

2.1.1 Wild-type (WT)

Adult F1 hybrid (C57Bl/6NTac x 129S6/SvEvTac) female mice were used for all experiments involving wild-type (WT) mice. Mice were bred in the colony at the Hospital for Sick Children and group housed (3-5 mice per cage) on a 12 h light/dark cycle with food and water available ad libitum. Behavioural experiments were conducted during the light-phase of the cycle. Mice were at least 8 weeks of age at the start of all experiments. All procedures were conducted in accordance with the policies of the Hospital for Sick Children Animal Care and Use Committee and conformed to both the Canadian Council on Animal Care (CCAC) and National Institutes of Health (NIH) Guidelines on the Care and Use of Laboratory Animals.

2.1.2 CREBαΔ−/− mice

The CREBαΔ mutation (Hummler et al., 1994) was backcrossed to inbred mouse strains C57Bl/6NTac (N10) and 129S6/SvEvTac (N11). Experimental mice (WT and mice homozygous for the CREB mutation) were the F1 offspring cross of mice heterozygote for the CREB mutation (CREBαΔ+/-). Experimental mice for the experiment depicted in Fig. 3.8, 3.9 (both male and female WT and homozygotes) were the F1 cross of mice heterozygote for the CREB mutation (CREBαΔ+/-). In all cases the genetic background of the experimental mice was 50% C57Bl/6NTac and 50% 129S6/SvEvTac. Mice that were homozygous for the CREB mutation (CREBαΔ−/−) and WT (CREBαΔ+/+) littermate controls were used in the experimental group for Experiment 5.

2.2 Preparation of HSV Vectors

We used replication-defective herpes simplex viral (HSV) vectors to locally and acutely increase transgene expression in the hippocampus. Genes of interest (CREB, CREBS133A or mCREB, CRTC1, GFP-LacZ) were cloned into the HSV with a Prpuc

66 backbone and packaged using a replication-defective helper virus as previously described (Han et al., 2007; Han et al., 2008; Han et al., 2009). To visualize transgene expression, eGFP was fused to the 5’ end of CREB WT, CREBS133A and CRTC1WT cDNA. Previous studies established that tagging CREB with eGFP does not interfere with its functional activity (Chao et al., 2002). Transgene expression is regulated by the constitutive promoter for the HSV immediate-early gene IE 4/5. As a control, we used an HSV expressing GFP alone.

Wild-type full-length CRTC1 or CREB cDNAs (kindly provided by Dr. Satoshi Kida, Tokyo University of Agriculture, Tokyo, Japan) were subcloned into the bi-cistronic HSV vectors that co-express GFP as a fluorescent reporter [HSV-p1005, (Russo et al., 2009)]. In this vector, GFP expression is driven by a CMV promoter whereas CRTC1 or CREB expression is driven by the constitutive promoter for the HSV immediate-early gene IE 4/5. Transgene expression using this viral system typically peaks 3 d, and dissipates within 10-14 d, following microinjection (Josselyn et al., 2001; Barrot et al., 2002; Vetere et al., 2011) (Fig. 4.2). HSV virus was packaged using a replication- defective helper virus, purified on a sucrose gradient, pelleted and resuspended in 10% sucrose, as previously described (Carlezon et al., 1998; Han et al., 2007; Han et al., 2008; Han et al., 2009). The average titer of the virus stocks is typically 4.0 x 107 infectious units/ml.

This method allows for neuronal and temporal specificity with significant intra- hippocampal expression in the infected neurons seen within 2-3 h of injection, and maximal expression at 24-72 h post infection (Barrot et al., 2002). We used HSV because, unlike many other viruses, HSV is naturally neurotropic (Fink et al., 1996). HSV predominantly infects granule cells of the dentate gyrus, and large pyramidal cells of the CA1 and CA3 layers, without altering in vivo synaptic transmission (Dumas et al., 1999), or causing gliosis (Carlezon et al., 1998).

2.3 Surgery

Mice were pre-treated with atropine sulfate (0.1 mg/kg, ip), anesthetized with chloral hydrate (400 mg/kg, ip) and placed in a stereotaxic frame. A small incision was made along the midline of the scalp, the skin was retracted, and holes drilled in the skull bilaterally above the dorsal hippocampus (AP = -2.3, ML = ±1.6, V = -1.6 mm from bregma for targeted CA1 infusion; AP = -2.3, ML = ±1.5, V = -1.8 mm from bregma for targeted dentate gyrus infusion) (Paxinos and Franklin, 1997). Bilateral microinjections of the vectors (2.0 l/side) were delivered though glass micropipettes connected via polyethylene tubing to Hamilton microsyringe (Hamilton, Reno, NV) in a microsyringe pump set to infuse at a rate of 0.1 l per minute. Following infusion, micropipettes were left in place an additional 5 min to ensure diffusion of the virus. Pipettes were then slowly retracted, and the incision site was closed using a surgical staple. Mice were post-operatively treated with Ketoprophen analgesic (5mg/kg, sub-cutaneous), and physiological saline (0.5-1ml, sub-cutaneous), and allowed to recover in a partially- heated cage for 24-48 h.

2.4 General behavioural procedures

2.4.1 Spatial watermaze experiments

2.4.1.1 Spatial watermaze apparatus

The circular water maze tank (120 cm diameter, 50 cm deep) was located in a dimly lit room (see Teixeira et al., 2006). The pool was filled to a depth of 40 cm with water made opaque by nontoxic white paint. Water temperature was maintained at 28±1°C using a heating pad under the base of the pool. A white circular escape platform (10 cm diameter) was submerged 0.5 cm below the water surface and located in a fixed position throughout training. The pool was divided into 4 quadrants based on the relative cardinal directions (Fig 2.1 a, b). The pool was surrounded by white curtains painted with large distinct cues, at a distance of 1m from the pool perimeter, except in the ‘no cues’ experiment (Experiment 3). In the ‘no cues’ experiment, solid black

68 curtains were hung over the original curtains to obscure all extra-maze spatial cues (Fig 2.1c).

Figure 2.16Watermaze apparatus (a) Schematic of distal spatial cues in the watermaze (black and red external cues). Arrows indicate the 20 cm radius circular ‘target’ zone (black dotted circle) centered on the former platform location, or the equivalent zones (grey dotted circles) in the other three quadrants of the pool. The grey circle indicates the 10cm diameter hidden platform within the ‘target’ zone. (b) Photograph of the spatial version of the watermaze. (c) Distal cues were obscured by a curtain (in probe 2 of the “no cues” condition, Chapter 3, Experiment 3).

2.4.1.2 Watermaze Behavioural Procedures

2.4.1.2.1 Strong training

Prior to beginning any experiment, mice were handled for 2 min/day for 1 week. For the strong training protocol, mice received 2 blocks of 3 consecutive trials per day, for 3 d (total of 6 blocks of training). This protocol reliably produced robust spatial memory in our hands. Within each training day, the inter-block interval was approximately 30min, and the inter-trial interval was approximately 30s. Each trial lasted a maximum of 60 s. To begin each trial, the mouse was placed in the pool, facing the wall in one of the 3 vacant quadrants (start location varied pseudo-randomly). The trial was complete once the mouse found the platform or 60 s had elapsed. If the mouse failed to find the platform on any trial, the experimenter guided the mouse onto the platform. After each training trial, the mouse was allowed 15 s to rest atop the platform prior to beginning the next trial (Fig 2.2).

2.4.1.2.2 Weak training

For the weak training protocol, mice received 1 block of 3 consecutive trials per d, for 3 d (total of 3 blocks of training). All other conditions were identical to the strong training conditions listed above. Previous pilot work in our lab established that this under- training protocol is not sufficient to induce spatial memory in WT mice (Fig 2.2).

Figure 2.27Timeline for weak and strong training spatial watermaze experiments. (a) Mice receive microinfusion of vector (CREB, mCREB, or GFP) into the CA1 of the dorsal hippocampus. 24 h later they begin 3 d training under either the weak protocol or

70 the strong protocol. One hour after the final training session, mice receive a single probe test.

2.4.1.2.3 Probe testing

Sixty min after the final training trial, spatial memory was assessed in a probe test in the watermaze during which the platform was removed from the pool and the mouse allowed to search for 60 s. The index of memory was the percentage of time spent searching in the target zone of the pool in which the escape platform was previously located was recorded. The target zone is a 20 cm radius centered on the training platform location. The percentage of time spent in the target zone was then compared to the percentage of time spent in the ‘other’ zones in the pool, which are comparable areas located at equivalent locations within the other 3 quadrants of the pool (Fig. 2.1a).

Behavioural data from the training and testing phases were acquired and analyzed using an automated tracking system (Actimetrics, Wilmette, IL). Using this software, we recorded a number of variables during training, including escape latency, swim speed, and path length. In probe tests we quantified spatial memory by measuring the amount of time mice spent searching in the target zone (20 cm radius, centered on the location of the platform during training; 11% of the pool surface) versus the average of the other equivalent zones in other areas of the pool (Moser et al., 1993; Moser and Moser, 1998; de Hoz et al., 2004). Thigmotaxic behaviour during training or the probe test was quantified by calculating the amount of time mice spent in the peripheral region of the pool (within an area of 5 cm from the wall) (Martin et al., 2005). Density plots (heat maps) for grouped data showing the probe test search patterns were generated using MATLAB (MATLAB 7.1, The MathWorks Inc.). During training, the platform was located in the lower right quadrant.

2.4.2 Context fear conditioning experiments

2.4.2.1 Context fear conditioning apparatus

Fear conditioning chambers (Context-A, CXT-A; 31 cm × 24 cm × 21 cm; MED Associates, St. Albans, VT), consisted of 2 stainless steel and 2 clear acrylic walls, with a stainless steel shock-grid floor (bars 3.2 mm diameter, spaced 7.9 mm apart). A stainless steel drop-pan containing a 70% ethanol solution was placed below the grid floor. A fan provided low-level white-noise during conditioning and testing in CXT-A. To examine the specificity of context fear memory, mice were also tested in a no-shock context, Context-B (CXT-B). CXT-B was the fear conditioning chamber but an opaque white acrylic triangular wall insert was placed inside the chamber and the floor bars were covered by an opaque white smooth acrylic floor. The door of the chamber was covered with an opaque sheet with horizontal black and white stripes. During testing in CXT-B, ethanol and the fan were not used (see Fig 2.3 and (Wang et al., 2009)).

Figure 2.38Context fear conditioning apparatus and tracking. (a) Conditioning and test Context-A (left) paired with shock during conditioning, and novel test Context-B (not paired with shock) (right). (b) Representative examples of frame-by-frame movement tracking within each context for mice microinfused with either CRTC1 vector (left) or GFP vector (right).

2.4.2.2 Context fear conditioning procedures

2.4.2.2.1 Weak training

Mice were placed in the conditioning chamber (CXT-A) and 2 min later, received an unsignalled shock (0.3 mA, 2 s). Mice remained in the chamber for an additional 60 s, before being returned to the homecage.

2.4.2.2.2 Strong training

Strong training was similar to above expect that mice received 3 unsignalled shocks (0.5 mA, 2 s) spaced 60 s apart. After the final shock, the mouse remained in the chamber for an additional 60s, before being returned to its homecage.

Figure 2.49Timeline for context fear conditioning and context generalization testing. (a) Mice received context fear conditioning using weak (one shock) or strong (three shocks) training. Following a delay, mice were then tested in Context-A (CXT-A) and Context-B (CXT-B).

Freezing behaviour (during context fear conditioning, reactivation and testing) was acquired by 4 overhead cameras, which digitized the video images at 4 Hz. An automated frame-by-frame analysis of movement using Freezeframe software (Actimetrics, Wilmette, IL) generated a freezing score. Reactivity to the shock was

73 assessed by comparing the distance travelled in the 2 s prior to the onset of shock (pre- US), to the distance traveled during the 2 s-shock (unconditioned stimulus, US). Reactivity Index (RI) was defined as the (US - pre-US) / (US + pre-US).

2.4.2.2.3 Context testing

Following delay (see individual experiment for precise delay), mice were replaced in CXT-A or CXT-B for 5 min. The percentage of time spent freezing during the 5 min test session was used as an index of memory. Freezing was defined as an immobilized, crouched position, with an absence of any movement except respiration (Blanchard and Blanchard, 1969; Bolles and Fanselow, 1982).

2.5 Statistical analyses

Data was analyzed using ANOVA. Significant interactions and main effects were further analyzed using Newman-Keuls (for Chapter 4), or Tukey’s HSD (for Chapter 5) post-hoc tests. Detailed descriptions of statistical analyses for each study can be found in the Methods section of Chapters 4 and 5.

2.6 Histology

Verifying vector microinjection and extent of viral infection. 90min following the final test session, mice received an overdose of chloral hydrate and were transcardially perfused with 0.1M PBS followed by 4% paraformaldehyde (PFA). Brains were fixed overnight at 4°C then transferred to a 30% sucrose solution. Brains were sliced coronally (40 m) across the entire anterior-posterior length of the hippocampus using a cryostat (Leica CM1850, Leica Microsystems), and every 2nd section was mounted on a gel-coated glass slide and coverslipped using Vectashield fluorescence mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA). Every other section was preserved in

PBS with 0.02% sodium azide for immunohistochemical analyses. Mice with extensive cortical or hippocampal damage were excluded from subsequent statistical analyses. Mice were perfused 4 d following vector microinjection for all behavioural experiments, except where noted.

Brain sections were imaged with a digital camera (Dxl 1200f, Nikon), affixed to a fluorescence microscope (Nikon Eclipse 801, Nikon) and GFP-fluorescence was analyzed in each brain using imaging software (ACT-1, Nikon). Consistent with previous reports from several labs (Carlezon et al., 1998; Josselyn et al., 2001; Wallace et al., 2004; Brightwell et al., 2005; Vetere et al., 2011a) microinjection of HSV vectors produces robust localized transgene expression and minimal tissue damage around the site of microinjection (Fig. 3.1, 4.3). GFP-fluorescence (which did not differ between vectors) was used to determine placement and extent of the viral infection for each mouse. Based on this, each mouse classified as a “hit” or “miss” by an examiner unaware of the treatment condition and behavioural results. Only mice determined to be a bilateral “hit” were included in subsequent data analysis. Mice were defined as “hit” if robust bilateral GFP expression was observed in the CA1 (Chapter 4) or dentate gyrus (Chapter 5) of dorsal hippocampus in at least 5 consecutive brain sections (across the anterior-posterior plane). All other mice were classified as “miss” (mice with weak transgene in the target region, mice with unilateral transgene expression in the target region, or mice with no expression in the target region).

Although our microinjections were aimed at the CA1 (for spatial memory experiments, Chapter 4) or the dentate gyrus (for contextual memory experiments, Chapter 4) region of the dorsal hippocampus, in some mice we observed GFP-positive neurons in other regions of the dorsal hippocampus. There was no difference in the performance of mice with robust bilateral GFP-expression restricted to the target region (CA1 or dentate gyrus) and mice with GFP-expression in other hippocampal sub-regions (CA1, CA3, or dentate gyrus) in any experiment. Therefore, we included all mice that showed robust bilateral expression of GFP minimally within the target region of the dorsal hippocampus (“hit”) in subsequent statistical analysis. See Methods in Chapters 3, 4 for detailed explanations

2.7 Immunohistochemistry

Expression of CREB or CRTC1 protein in infected neurons was examined (also looked at GFP) using immunohistochemistry. See Methods in Chapters 4 for CREB staining details, and Chapter 5 for CRTC1 staining details.

3 Dorsal hippocampal CREB is both necessary and sufficient for spatial memory

3.1 ABSTRACT

Although the CREB (cAMP/Ca2+ responsive element binding protein) family of transcription factors is critical for memory in many species from Aplysia and Drosophila to mice and rats (Bourtchuladze et al., 1994; Yin et al., 1994; Guzowski and McGaugh, 1997; Bartsch et al., 1998), whether it is sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals is unknown. Here we examine whether acutely increasing CREB levels in the dorsal hippocampus is sufficient to enhance the formation of a complex form of memory, spatial memory as measured by the watermaze. We found that locally and acutely increasing CREB levels in the dorsal hippocampus using viral vectors is sufficient to induce robust spatial memory in two conditions which do not normally support spatial memory, weakly-trained wild-type (WT) mice and strongly-trained mutant mice with a brain-wide disruption of CREB function. Together with previous results, these findings indicate that CREB is both necessary and sufficient for spatial memory formation, and underline its pivotal role in the hippocampal molecular machinery underlying the formation of spatial memory.

3.2 INTRODUCTION

A fundamental goal of neuroscience is to understand how memories are formed. One approach to study this question involves examining the molecular machinery underlying memory formation to determine the molecules that are necessary and sufficient for memory formation (Dudai, 2002). To investigate whether a molecule is necessary for memory formation, experiments typically interfere with the function of this molecule and determine whether memory is disrupted, thereby inferring the normal function of a molecule from dysfunction. Dozens of molecules satisfy this criterion (Grant, 2003). To examine sufficiency, experiments may enhance the function of a molecule and

77 determine whether memory can be artificially induced under conditions that do not normally support memory formation, thereby mimicking normal memory formation. Few, if any, molecules satisfy this criterion in mammals.

The CREB (cAMP/Ca2+ responsive element binding protein) family of transcription factors is critical for memory in many species from Aplysia and Drosophila to rats and mice (Bourtchuladze et al., 1994; Yin et al., 1994; Guzowski and McGaugh, 1997; Bartsch et al., 1998). As we discussed earlier, disrupting CREB function generally impairs the formation of many types of memory (Bourtchuladze et al., 1994; Yin et al., 1994; Guzowski and McGaugh, 1997; Bartsch et al., 1998; Kida et al., 2002; Pittenger et al., 2002; Frankland et al., 2004; Josselyn et al., 2004), while acutely increasing CREB function in rodents enhances various forms of simple memory such as one-trial social-defeat (Jasnow et al., 2005) and associative fear conditioning (Josselyn et al., 2001; Wallace et al., 2004; Han et al., 2007; Restivo et al., 2009). It is not known, however, if similarly increasing CREB function enhances the formation of more complex types of memory.

Unlike many one-trial or associative learning tasks, spatial memory formation, as assessed in the hidden-platform version of the Morris Watermaze (WM), requires multiple training sessions. In this complex cognitive task, subjects determine their present position and orientation (self-localization), desired position (goal determination), and the optimal route between the two (path integration) (Redish and Touretzky, 1998). The neural mechanisms mediating this type of visuospatial memory in mice likely contribute to general human cognitive processing as well. The formation of spatial memory critically relies on the hippocampus, including the dorsal CA1 region of the hippocampus (Morris et al., 1982). Disrupting CREB function in the dorsal hippocampus typically impairs watermaze performance [Bourtchuladze et al., 1994; Guzowski and McGaugh, 1997; Pittenger et al., 2002 (but see Balschun et al., 2003)], suggesting that hippocampal CREB is necessary for spatial memory formation. Whether CREB is sufficient for spatial memory formation, however, remains unknown.

Here we are interested in determining if acutely increasing CREB function specifically in the CA1 region of the dorsal hippocampus 1) strengthens the formation of spatial memory, and, 2) is sufficient to produce spatial memory under conditions that do not normally support spatial memory consolidation. At the time of our investigation, these questions had never before been investigated in vivo, and in freely behaving animals. We proposed investigating this by asking the following questions:

1. Can wild-type CREB over-expression in the hippocampus facilitate spatial memory that is only weakly acquired in control mice? 2. Can CREB over-expression in the hippocampus further strengthen a normal spatial memory? 3. Does suppression of CREB activity in the hippocampus impair the formation of spatial memory? 4. Can CREB over-expression in the hippocampus rescue spatial memory deficits in CREB-deficient mice?

3.2.1 Experiment 1: Effects of increasing or suppressing CREB on the consolidation of spatial memory that is only weakly acquired in control mice.

Rationale: Previous studies have used loss-of-function approaches to show that CREB is necessary to support spatial memory formation by knocking down CREB function either globally (Bourtchuladze et al., 1994; Kogan et al., 1997), or in a temporally, and regionally specific manner (Kida et al., 2002; Viosca et al., 2009). These findings all suggest that CREB is necessary for the formation of lasting spatial memory, although in some cases, the animal can overcome these deficits with intensive, prolonged training (Kogan et al., 1997). While these studies demonstrate the critical role of CREB in hippocampal-dependent spatial memory formation, they infer the role of CREB by interfering with CREB function, and looking at resulting impairments in learning and memory. To address the interesting question of CREB-mediated facilitation of hippocampal-dependent memory, we are interested in approaching this question from a gain-of-function perspective – that is, can we induce memory by increasing CREB 79 function? Specifically, is local and acute enhancement of CREB in the CA1 sufficient to induce spatial memory under conditions in which long-term memory formation is not typically supported? We extensively discussed the critical role of the dorsal hippocampal CA1 region in spatial memory consolidation in Chapter 1. While it has been shown that spatial learning and memory testing increases pCREB levels in the dorsal hippocampus (Colombo et al., 2003), it was only recently demonstrated that acutely increasing CREB function in the dorsal hippocampus can actually facilitate a more basic form of spatial memory for place learning in a plus-maze (Brightwell et al., 2007).

Here, we examined the ability of wild-type CREB to support formation of spatial memory, using a weak spatial training protocol which does not normally induce spatial memory under control conditions. This has never before been demonstrated. We also examined the impact of suppressing CREB function in a similar population of CA1 cells prior to spatial training to determine the effects of targeted CREB impairment on the formation of a weak memory.

Experimental Approach: We delivered microinjections of HSV viral vectors to acutely and locally increase or suppress CREB function in a portion of CA1 neurons in the dorsal hippocampus. We microinjected HSV encoding wild-type CREB (CREB vector), a dominant negative form of CREB (CREBS133A, mCREB vector) or a control GFP vector (LacZ fused with GFP, GFP vector) bilaterally into the dorsal CA1 of the dorsal hippocampus 1 d prior to weak watermaze training, then tested spatial memory with a single probe test.

3.2.2 Experiment 2: Effects of increasing or suppressing CREB on consolidation of a strong spatial memory.

Rationale: We were also interested in examining the potential enhancing abilities of CREB in a normally acquired memory to determine if any memory strengthening we see in the weak training experiments is simply crossing a threshold which permits synaptic consolidation of long-term memory following challenging learning conditions, or if we 80 can even further enhance a normal memory that is easily acquired by controls. To do so, we increased CREB prior to using a strong training protocol in the watermaze. Also, at the time of our study, no other labs had yet looked at the use of viral vectors to suppress CREB prior to a spatial memory task.

Previous research indicates that while a widespread disruption of CREB function throughout the brain or in the hippocampus impairs the acquisition of spatial memory (Bourtchuladze et al., 1994; Guzowski and McGaugh, 1997; Pittenger et al., 2002), a targeted deletion of CREB in as many as 70-80% of CA1 neurons in the dorsal hippocampus does not (Balschun et al., 2003). These findings are consistent with the notion that neurons with normal CREB function out-compete neighboring neurons with disrupted CREB function for integration into a memory trace (Han et al., 2007). Therefore we predicted that decreasing CREB function in a small portion of CA1 neurons in the dorsal hippocampus of wild-type mice would not disrupt spatial memory formation.

Experimental Approach: To test our ideas of normal memory strengthening, and of possible memory suppression, wild-type mice were microinjected with CREB vector, mCREB vector, or GFP vector into CA1 of the dorsal hippocampus 1 d prior to strong watermaze training, then tested spatial memory with a single probe test.

3.2.3 Experiment 3: Effects of increasing CREB on the consolidation of memory in the absence of spatial cues.

Rationale: To support the assertion that CREB enhancement at the time of training on a spatial task is sufficient to induce strong long-term memory consolidation, we realized that it would be important examine performance on the task in the absence of spatial cues. To rule out the possibility that CREB over-expression is facilitating the formation of a non-spatial based strategy, we next examined performance in the watermaze task both in the presence, and in the absence of spatial cues during training.

Experimental Approach: Wild-type mice were microinjected with CREB vector, and underwent weak training in the watermaze in the presence of distal spatial cues, then received a single probe test (with cues). All cues were then obscured, and mice received a second non-cued probe (no cues) test in the watermaze. This allowed us to examine whether the apparent enhancement in performance relied on these distal cues, reflecting spatial memory.

3.2.4 Experiment 4: Effects of increasing CREB on retrieval/expression of a previously acquired spatial memory.

Rationale: Our hypothesis that increasing CREB function in the key CA1 region is enhancing the synaptic consolidation of spatial long-term memory for the watermaze is dependent on CREB enhancement occurring during the memory acquisition phase (ie. during training in the watermaze). Previous studies using infusions of CREB oligodeoxynucleotides into the dorsal hippocampus of rats 20 h after the completion of spatial training did not impair the subsequent retrieval or expression of that spatial memory when tested 24 h later (Guzowski and McGaugh, 1997). To rule out the possibility that CREB over-expression is facilitating the retrieval, not the formation of the memory, we examined the effects of post-training CREB enhancement.

Experimental Approach: We trained wild-type mice in the watermaze using either the strong or weak training protocol, then gave a single probe test 24 h later. We next microinjected CREB or GFP vector, and gave a second probe test for the pre- operatively learned spatial location in the watermaze.

3.2.5 Experiment 5: Effects of increasing CREB on consolidation of a spatial memory in CREB-deficient mice.

Rationale: Discrepancies surrounding CREB’s necessity in long-term memory, as different models for suppressing CREB function have found different results. This is partially due to the varied regional (ie. global or regionally specific), and temporal

(chronic or acute/inducible suppression) techniques for interfering with CREB function. Additionally, different parameters used for learning and memory testing (brief or prolonged training opportunities) contribute to the inconsistencies in opinions on the necessity of CREB function in the literature. We aim to use the well characterized CREBαΔ-/- mouse model, which have a targeted disruption of the genes encoding the α and Δ CREB isoforms, deleting 80-90% of ubiquitous CREB activity throughout their lifetime (Pandey et al., 2000; Walters and Blendy, 2001). As discussed earlier, these mice have long-term memory deficits in hippocampal-dependent spatial and contextual memory tasks [although, as demonstrated by Kogan and colleagues (1997), these deficits can be laboriously overcome by prolonged, spaced training opportunities]. While this suggests that repeated learning opportunities may allow the residual CREB (the one remaining CREB β isoform, and CREM) to support sufficient CRE-transcriptional activity to allow for the laborious formation of memory, we are interested in expediting this process. If we exogenously supplement CREB levels with targeted microinjections of CREB vector into the key CA1 region of the dorsal hippocampus of CREBαΔ-/- mice, can they now form long-term memory?

Experimental Approach: We microinjected CREB or GFP vector into CREBαΔ-/- mice and wild-type littermate controls prior to strong training in the watermaze, then tested their spatial memory with a single probe test.

Together, these studies show that increasing CREB levels in dorsal CA1 hippocampal neurons is sufficient to induce robust spatial memory under weak training conditions that do not normally support consolidation of spatial long-term memory. We also show that increasing CA1 CREB enhances the already robust spatial memory produced by strong training in the watermaze. We confirm that the enhancement in spatial long-term memory is dependent on CREB-mediated facilitation of memory consolidation/formation (but not retrieval/expression), and is dependent upon spatial cues present during memory acquisition. Finally, we confirm a spatial long-term memory deficit in CREBαΔ-/- mice, and find that increasing CREB in the dorsal hippocampus is sufficient to rescue their spatial memory deficits. This is the first study to show, in the same model, that

CREB function in the dorsal hippocampus is both necessary and sufficient for spatial memory consolidation.

3.3 DETAILED METHODS

3.3.1 Mice

Adult C57Bl6x129 female mice were used for all experiments involving wild-type mice. Adult male and female CREBαΔ mutation (Hummler et al., 1994) were used for Experiment 5. Details on the rearing, housing conditions, and generation of CREBαΔ-/- mice can be found in the General Methods (Chapter 2).

3.3.2 Preparation of HSV Vectors

To locally and acutely increase CREB function we used replication-defective herpes simplex viral (HSV) vectors. To suppress CREB activity, we microinjected HSV encoding a mutant form of CREB that cannot be phosphorylated at the key Ser133 residue, and therefore, cannot activate transcription (CREBS133A, mCREB vector) (Gonzalez and Montminy, 1989). As a control, we microinjected HSV encoding LacZ fused with GFP (GFP vector). For details on the generation of HSV-vectors, see General Methods.

3.3.3 Surgery

All subjects underwent stereotaxic surgery to deliver bilateral microinjections of CREB, mCREB, or GFP vector (2.0 l/side) into the dorsal CA1. Mice were allowed at least 24 h to recover prior to further behavioural training or testing. See General Methods for surgical details.

3.3.4 Spatial watermaze training

Mice received weak (1 block of 3 consecutive trials per d, for 3 d) or strong (2 blocks of 3 consecutive trials per d, for 3 d) training sessions in the watermaze. The inter-trial interval was 30 s for both protocols and inter-block interval for the strong training protocol was 30 min. Each trial lasted a maximum of 60 s. After each training trial, the mouse was allowed 15 s on top of the platform (see Fig 2.1 in Methods for schematic of the watermaze).

3.3.5 Probe test

60 min after the final training trial, the platform was removed from the pool and the mouse was allowed to search for 60 s. Each mouse’s swim path was digitally recorded. See General Methods for details of watermaze training, testing and analyses.

3.3.6 Experiment 1. Effects of increasing or suppressing CREB on the consolidation of spatial memory that is only weakly acquired in control mice.

Wild-type mice were microinjected with vector (CREB vector, n=10; mCREB, n=8; GFP n=13) and 1 d later began weak spatial training in the watermaze. Mice received a single probe test 60 min following training (Fig 3.3a for timeline).

3.3.7 Experiment 2. Effects of increasing or suppressing CREB on consolidation of a strong spatial memory.

Wild-type mice were microinjected with vector (CREB n=10; GFP n=11; mCREB n=11; GFP n=10) and 1 d later began strong spatial training in the watermaze. Mice received a single probe test following training (Fig 3.4a for timeline).

3.3.8 Experiment 3. Effects of increasing CREB on the consolidation of memory in the absence of spatial cues.

Wild-type mice were microinjected with CREB vector (n=7) and 1 d later began weak training in the watermaze. Following training, mice received a first probe test in the presence of spatial cues (with cues). Cues were then obscured by a black curtain surrounding the watermaze, and 60 min later mice received a second non-cued probe test (no cues) (Fig 2.1 for schematic of un-cued watermaze and Fig 3.5a for timeline).

3.3.9 Experiment 4. Effects of increasing CREB on retrieval/expression of a previously acquired spatial memory.

Mice underwent weak or strong training in the watermaze, then received a single probe test (pre-vector) following training. 24 h later, mice were microinjected with vector (strong training: CREB n= 8; GFP n= 7; weak training: CREB n= 7; GFP n= 6). 4 d later, mice received a second probe test (post-vector) (Fig 3.6a for timeline).

3.3.10 Experiment 5. Effects of increasing CREB on consolidation of a spatial memory in CREB-deficient mice.

Wild-type (WT) or CREB αΔ-/- mutant (MUT) mice were microinjected with CREB or GFP vector (WT-CREB vector n= 10; WT-GFP vector n= 14; MUT-CREB n=10; MUT-GFP n=12) and 1 d later began strong spatial training in the watermaze. Mice received a single probe test following training (Fig 3.7a for timeline).

3.3.11 Statistical Analyses

We analyzed the time required to reach the platform (escape latency) using a 2-way Analysis of Variance (ANOVA) with between-group factor Vector (CREB, mCREB, GFP)

86 and within-group factor Block (3 levels for weak training, 6 levels for strong training). For the probe test, we first quantified spatial bias by comparing the amount of time mice spent in the target zone versus the average time spent in equivalent zones in the other thee quadrants of the pool using an ANOVA with a between-subjects factor Vector and within-subjects factor Zone (Target, Other). Next, we analyzed the time spent in the target zone between groups using a one-way ANOVA with between-group factor Vector. Significant effects from all analyses were further analyzed using Newman-Keuls post- hoc tests.

3.3.12 Histology: Confirming transgene expression following behavioural testing.

Following spatial memory testing, all subjects were perfused, and robust bilateral expression of GFP in the CA1 was confirmed for each subject included in the analyses by an examiner unaware of the behavioural data. Because transgene expression using this viral system peaks 3 d following surgery (Barrot et al., 2002), we trained mice 1 d following surgery for 3 d in all watermaze experiments except in the retrieval experiment, where mice were trained and given one probe test prior to surgery and an additional probe test 4 d following surgery. This delay was consistent with the delay between vector microinjection and probe testing in all other experiments, and ensures that the level of transgene expression during the final probe test was equivalent across all experiments.

The number of neurons in the CA1 region of the dorsal hippocampus expressing GFP was counted manually using a fluorescent microscope (Nikon) at 40X magnification. We observed that a portion of dorsal CA1 neurons around the site of microinjection (typically encompassing a circular region of 1.6 mm in diameter) were infected by HSV microinjection. Mice were classified as “hit” only if they had robust bilateral expression of GFP in over 2,000 neurons in the CA1 region of the dorsal hippocampus. All other mice were classified as “miss”, including mice with weak transgene expression (less than 2,000 GFP-positive neurons per side) in the dorsal hippocampus.

3.3.13 Immunohistochemistry: CREB staining.

To verify that microinjection of CREB vector increased expression of CREB protein, we performed immunohistochemistry using an antibody specific for CREB. 40 μm coronal sections from wild-type or CREB αδ-/- brains microinjected with GFP or CREB vector were incubated with blocking solution for 2 h at room temperature then incubated with anti-CREB primary mouse antibody (1:2500, Upstate Cell Signaling Solutions, NY). The CREB signal was visualized using an anti-mouse Alexa 568 secondary antibody (1:500, Invitrogen, Carlsbad, CA). Importantly, no staining was detected in the absence of the primary or secondary antibodies. Sections were washed with PBS 0.1M, counterstained with DAPI (Vectashield, Vector Labs, Burlingame, CA), mounted on gel coated slides and coverslipped using PermaFluor mounting medium (Thermo Scientific). Images were obtained using a fluorescence microscope (Nikon Eclipse 80i, Nikon).

3.4 RESULTS

3.4.1 Microinjection of CREB vector increases CREB protein in the CA1 of dorsal hippocampus.

Microinjection of vector produced robust, localized expression of GFP in the CA1 region (Fig 3.1). Brains microinjected with CREB vector show high expression of CREB protein restricted to the CA1 region, and a high co-localization of cells infected with CREB vector and cells expressing CREB protein (Fig 3.2). For all experiments, only mice with robust bilateral GFP expression in the CA1 region were included in subsequent analyses (see Methods for inclusion criteria). There was no difference in the performance of mice with robust bilateral GFP-expression restricted to the target region (CA1) and mice with bilateral GFP-expression in the target CA1 region and additional hippocampal sub-regions (CA3, or dentate gyrus) in any experiment. There was no correlation between the number of infected neurons, and performance in the probe test in any experiment.

Figure 3.110Transgene expression in the CA1 of the dorsal hippocampus. Robust, localized transgene expression (GFP, green) following vector microinjection into the CA1 region of the dorsal hippocampus. Scale bar = 100μm.

Figure 3.211Microinjection of CREB vector in the CA1 increases levels of CREB protein. Immunohistochemical staining for CREB protein (red) in the CA1 of the dorsal hippocampus 4d following microinjection of GFP vector (top) or CREB vector (bottom). Infected neurons (green) of mice microinjected with CREB vector show higher expression of CREB protein than those microinjected with GFP vector. Co-localization of infected neurons with CREB-expressing neurons is only seen in CA1 pyramidal neurons infected with CREB vector (yellow). Endogenous CREB expression can be seen co-localizing with DAPI labeled neurons (blue) in the CA1 infected with GFP vector, suggesting intact basal CREB expression in control animals. Scale bar = 50 μm.

3.4.2 EXPERIMENT 1: Increasing CREB in the hippocampus facilitates consolidation of spatial memory that is only weakly acquired in control mice In our first weak memory condition, we increased or suppressed CREB levels in the CA1 region of wild-type mice before sub-threshold (weak) training in the watermaze (one block of 3 trials per day for 3 d) that is not normally sufficient to produce spatial memory.

3.4.2.1 Increasing CREB enhances spatial memory produced by weak training in wild-type mice: the training data

To examine whether increasing CREB function in CA1 of the dorsal hippocampus is sufficient to produce spatial memory under challenging training, we microinjected wild- type mice with CREB, mCREB or GFP control vector in CA1 prior to weak training. Over the 3 training blocks, all groups required progressively less time to locate the platform, although this decrease was more pronounced in mice with CREB vector. The results of a ANOVA with between factor of Vector (CREB, mCREB, GFP) and within factor of Block (training block 1,2,3) supports this conclusion, showing a significant Vector x

Block interaction [F(4,56) = 3.56, p < .001] as well as a significant main effect of Block effect [F(2,56) = 7.55, p < .001], but no main effect of Vector [F(2,28) = 3.11, p > .05)] (Fig 3.3b). Post-hoc Newman-Keuls comparisons showed that the escape latencies on the first block of trials were similar in all groups, but that mice with CREB vector showed progressively shorter escape latencies over the blocks than mice with GFP or mCREB vector, which did not differ from each other. Importantly, all groups showed similar swim speeds (p > .05), suggesting that the difference in escape latency cannot be attributed to simple performance measures. Therefore, increasing CREB levels in the dorsal hippocampus of wild-type mice enhances their acquisition of a spatial search strategy over the course of weak training.

3.4.2.2 Increasing CREB enhances spatial memory produced by weak training in wild-type mice: the probe data

This is consistent with the finding that only mice with CREB vector acquired spatial memory, as assessed in a probe trial (in which the platform was removed) conducted after training. We first quantified this spatial bias by comparing the amount of time mice spent in the target (20 cm radius circular zone centered on the former platform location; 11% of the pool surface) versus the average time spent in equivalent zones in the other thee quadrants of the pool (see Fig 2.1a for schematic). Under the weak training conditions in the watermaze, mice microinjected with GFP or mCREB vectors showed no evidence of spatial memory during the probe test. Mice in these groups searched the target zone (that previously contained the platform) and the other equally-sized zones similarly. In contrast, mice microinjected with CREB vector showed robust spatial memory, searching selectively in the target zone (Fig 3.3c,d) These results are supported by an ANOVA, with between factor of Vector and within factor of Zone

(Target, Other) showing a significant Vector x Zone interaction (F(2,28) = 7.78, p < .001) and significant main effects of Vector (F(2,28) = 9.11, p < .001) and Zone (F(1,28) = 17.92, p < .001). Post-hoc analysis revealed that only mice with CREB vector searched preferentially in the target zone, indicating that this was the sole group to form a spatial memory. Furthermore, the time spent searching in the target zone was significantly higher in CREB vector mice than in mice with GFP or mCREB vector, which did not differ from each other (F(2,28) = 9.78, p < .001). Therefore, increasing CREB in the dorsal hippocampus is sufficient to produce a strong spatial memory under weak training conditions that do not normally support spatial long-term memory consolidation in wild- type mice.

3.4.2.3 Suppressing CREB does not further impair spatial memory produced by weak training in wild-type mice

The ability of CREB to induce a spatial memory depends on an intact Ser133 site, as microinjection of the mutated CREB vector (CREBS133A, mCREB) that lacks the capacity to phosphorylate at this site did not support consolidation of spatial long-term memory. 91

This finding is consistent with the key regulatory role of the Ser133 phosphorylation site in the transcriptional activity of CREB (Gonzalez et al., 1989).

Figure 3.312Acutely increasing CREB in the dorsal hippocampus enhances consolidation of weak spatial memory. (a) Schematic timeline for Experiment 1. (b) Wild-type mice microinjected with CREB, mCREB, or GFP vector were trained using a weak protocol. Mean escape latency decreased across training blocks in all groups. This decrease was more pronounced in mice with CREB vector, indicating enhanced spatial learning. (c) During probe testing, only mice with CREB vector showed robust

92 spatial memory after weak training, spending a greater amount of time in the target zone than in other zones. Error bars = ± SEM. (d) Density plots for grouped data showing the probe test search patterns of mice with CREB, mCREB or GFP vector. Platform position during training = lower right quadrant. Colour scale represents the number of visits per animal per 5 x 5 cm2 area of pool. Warm colours indicate more time spent in the area.

3.4.3 EXPERIMENT 2: Increasing CREB in the hippocampus further enhances consolidation of robust/strong spatial memory. Suppressing CREB in the hippocampus does not impair spatial memory. Does increasing CREB function to induce ‘normal’ memory under challenging learning conditions, or can it actually enhance normal memory formation? To address this interesting question, we used a ‘strong’ training protocol in which normal mice reliably form spatial memory in the watermaze.

3.4.3.1 Increasing CREB enhances spatial memory consolidation produced by strong training in wild-type mice: the training data

We next examined the effects of acutely increasing CREB in CA1 of the dorsal hippocampus in mice trained with a strong protocol (2 blocks of 3 trials per day, for 3 d; 6 blocks total). Mice were microinjected with CREB or GFP vector into the dorsal hippocampus prior to training. Over the 6 training blocks, both groups required progressively shorter times to locate the hidden platform, but again these escape latencies declined more rapidly in mice microinjected with CREB vector (Fig 3.4b). The results of a Vector x Block ANOVA supports this conclusion, showing a significant

Vector x Block interaction [F (5,95) = 3.44, p < .001], and a significant main effect of Block

[F (5,95) = 36.89, p < .001]. Post-hoc analyses revealed that on the first training block both groups required the same time to locate the platform, but that over training blocks 93 the mice with CREB vector located the platform progressively faster than mice with GFP vector. There was no difference in swim speed between the groups (p > .05). Therefore, although both groups showed decreased escape latencies over the course of training, mice with CREB vector showed a more pronounced decrease in escape latency. These data suggest that during training, all mice adopted a more focused, spatial search strategy, but that mice with CREB vector outperformed mice with GFP vector.

3.4.3.2 Increasing CREB enhances spatial memory produced by strong training in wild-type mice: the probe data

We next gave a probe test to examine if wild-type mice with enhanced CREB function in the CA1 maintained enhanced spatial memory following strong training. Relative to mice microinjected with GFP vector, mice with CREB vector showed enhanced spatial memory during the probe test, spending more time in the target zone of the pool where the platform was located during training (Fig 3.4c, d). Again, we quantified this spatial bias by comparing the amount of time mice spent in the target zone versus the average time spent in equivalent zones in the other thee quadrants of the pool. An ANOVA with a between-subjects factor Vector and within-subjects factor Zone revealed a significant

Vector x Zone interaction [F(1,19) = 7.42, p < .05] as well as a significant main effect of

Zone [F(1,19) = 68.21, p < .001]. Post-hoc comparisons on the significant interaction showed that both groups searched selectively in the target zone, with CREB vector mice showing a significantly stronger preference. The results of an ANOVA comparing the time spent in target zone between the two groups confirmed this [F(1,19) = 5.71, p < .05]. Therefore, similar to associative and one-trial memory, acutely increasing CREB further enhances the formation of a more complex form of memory, spatial memory in strongly-trained wild-type mice.

In Experiment 1 where we used a weak training protocol to induce weak memory in control animals, we did not expect that over-expression of the phosphorylation-deficient CREB vector (CREBSer133A, mCREB) would produce further deficits in spatial memory, given that the control’s baseline memory formation was predicted (and found) to be

94 minimal. Here, we were very interested in investigating whether similarly over- expressing the mutant CREB vector in CA1 of wild-type mice prior to strong training in the watermaze would produce impairments in spatial memory formation. Importantly, here we expected the strong training protocol to support consolidation of spatial long- term memory in mice receiving microinjections of the control GFP vector. To test this, wild-type mice were microinjected with vectors expressing a dominant negative form of CREB (mCREB vector) or GFP vector 1 d prior to beginning strong training in the watermaze.

3.4.3.3 Decreasing CREB does not impair spatial memory produced by strong training in wild-type mice: the training data

Over the 6 blocks of training, mice with mCREB or GFP vector showed similar decreasing escape latencies (Fig 3.4e). The results of a repeated measures ANOVA with between factor of Vector and within factor of Block supports this conclusion, showing a significant main effect of Block (F(5,95) = 10.71, p < .001, but no significant Vector X Block interaction, or main effect of Vector ). Importantly, swim speeds, distance travelled, or thigmotaxis (time spent near the periphery of the pool) between the two groups did not differ (p > .05). These data indicate that acutely decreasing CREB function in dorsal hippocampal CA1 neurons does not affect spatial learning.

3.4.3.4 Decreasing CREB does not impair spatial memory produced by strong training in wild-type mice: the probe data

During the probe test, all mice showed a robust preference for the target zone, regardless of vector (Fig 3.4f, g). An ANOVA with between factor of Vector and within factor of Zone revealed a significant main effect of Zone [F(1,19) = 28.14, p < .001], but no Vector X Zone interaction, or main effect of Vector. Furthermore, an ANOVA comparing the time spent in the target zone between the groups showed no difference

[F(1,19) = 0.13, p > .05]. Therefore, decreasing CREB function in a small population of

CA1 neurons in the dorsal hippocampus does not disrupt normal spatial memory consolidation.

Figure 3.413Acutely increasing CREB in the dorsal hippocampus further enhances consolidation of strong spatial memory. (a). Schematic timeline for Experiment 2. (b,e) Wild-type mice microinjected with CREB, mCREB, or GFP vector were trained in the watermaze using a strong protocol. Mean escape latency decreased across training blocks in all groups. This decrease was more pronounced in mice with CREB vector, indicating enhanced spatial learning. (c) During probe testing, both CREB and GFP groups show evidence of spatial memory, searching selectively in

96 the target zone. Mice microinjected with CREB vector show enhanced spatial memory for the target zone compared to GFP control (d) Density plots for grouped data showing the probe test search patterns of mice with CREB, or GFP vector. (f) During probe testing, both mCREB and GFP groups show evidence of spatial memory, spending comparable time searching in the target zone (g) Density plots for grouped data showing the probe test search patterns of mice with mCREB, or GFP vector.

3.4.4 EXPERIMENT 3: Increasing CREB in the hippocampus does not enhance consolidation of memory in the absence of spatial cues We next tested whether the enhancement in watermaze performance in mice microinjected with CREB vector was dependent on the use of distal spatial cues that surround the watermaze. Using wild-type mice all microinjected with CREB vector in CA1, we trained them in the watermaze using the weak training protocol, then assessed performance in two probe tests: first in the presence of distal spatial cues (with cues), and second in the absence of spatial cues (no cues) (see Fig 2.1 in Methods for schematic of cued and non-cued watermaze apparatus, and Fig 3.5a for schematic of timeline). During training, all mice showed the expected decrease in escape latencies across the 3 blocks of watermaze training (data not shown).

3.4.4.1 CREB-enhanced performance in the memory probe test depends on an accurate, allocentric representation of distal spatial cues: the probe data

As expected, all mice showed strong spatial memory in the first probe test in which the distal cues were present (Fig 3.5b). However, when these cues were subsequently obscured, mice searched randomly, no longer showing a preference for the target zone. This result was confirmed by an ANOVA [within-factors Test (With Cues or No Cues) and Zone] showing an interaction that approached significance [F (1,6) = 4.97, p = .06]. Planned comparisons revealed that mice searched selectively in the target zone when

97 the distal cues were present [F (1,6) = 9.99, p < .05] but not when the cues were obscured [F (1,6) = 0.03, p > .05]. Other performance parameters were equivalent between the two probe tests, including distance (Fig 3.5c), swim speed (Fig 3.5d), and thigmotaxis (Fig 3.5e) (all p > 0.05). Therefore, the enhancement in watermaze performance produced by increasing CREB in the dorsal hippocampus critically depends on spatial memory (an accurate, allocentric representation of distal cues surrounding the watermaze) and cannot be attributed to nonspecific changes in performance variables (anxiety, swim speed, motivation, etc.).

Figure 3.514CREB-enhanced performance in the watermaze is dependent on spatial memory. (a) Schematic timeline for Experiment 3. (b) Mice microinjected with CREB vector were trained with the weak protocol and given two probe tests in which the distal spatial cues were 1) present (with cues) or 2) obscured by a curtain (no cues). Mice spent more time in target, than in other, zones of pool when distal cues were present but spent an equal time when cues were obscured. (c) Distance travelled, (d) speed, and (e) thigmotaxic behaviour was equivalent between cued and non-cued probe tests, indicating that deficits in memory for the platform location were not due to non-specific performance effects.

3.4.5 EXPERIMENT 4: Increasing CREB in the hippocampus does not enhance retrieval/expression of a previously acquired spatial memory

So far, we have demonstrated that increasing CREB in CA1 neurons in the dorsal hippocampus enhances consolidation of spatial long-term memory assessed in the watermaze probe test. However, in these previous experiments, both training and testing were conducted during a period of high transgenic expression of CREB in the CA1. Therefore, it remains possible that the observed enhancement could be due to improved memory retrieval/expression, rather than to enhanced consolidation. To examine whether increasing CREB in the dorsal hippocampus alters retrieval/expression of a previously acquired spatial memory, we microinjected CREB or GFP vectors after, rather than before, strong training. In this way, training was conducted with normal (endogenous) CREB levels and memory retrieval/expression was assessed with high CREB levels.

3.4.5.1 Acutely enhancing CREB function in the dorsal hippocampus does not change the retrieval/expression of a previously acquired strong spatial memory

As expected, before vector microinjection, both groups showed equally robust spatial memory during the first probe test (pre-vector) following the strong watermaze training

[F(1,13) = 0.18, p > .05] (Fig 3.6b, probe 1 target). Subsequently increasing CREB levels in the dorsal hippocampus did not affect spatial memory retrieval/expression: In the second probe test (post-vector) both mice microinjected with CREB and GFP vectors after training showed similarly strong spatial memory [F(1,13) = 11.07, p > .05] (Fig 3.6b, probe 2 target). Therefore, acutely increasing CREB function before training enhances spatial memory whereas similarly increasing CREB function after training does not affect spatial memory retrieval/expression.

3.4.5.2 Acutely enhancing CREB function in CA1 neurons of the dorsal hippocampus does not facilitate the retrieval/expression of a previously acquired weak spatial memory

Having demonstrated that manipulation of CREB levels after training does not affect retrieval/expression of a normal spatial memory induced by strong training in the watermaze, we next wanted to investigate the effects on retrieval/expression of a previous spatial memory that had been only weakly acquired. Similar to the above experiment, we microinjected CREB or GFP vectors after, rather than before, weak training. In this way, weak training was conducted with normal (endogenous) CREB levels and memory retrieval/expression was assessed with high CREB levels.

Mice microinjected with CREB or GFP vector after (rather than before) weak training did not search selectively in the target zone during either probe test (both pre- and post- vector). During the first probe test (pre-vector), ANOVA revealed no significant Vector x

Zone interaction [F(1,11) = 0.0007, p>.05], and no main effects of Vector [F(1,11) = 0.259, p>.05], or Zone [F(1,11) = 2.705, p>.05]. Similarly, during the second probe test (post- vector), ANOVA revealed no significant Vector x Zone interaction [F(1,11) = 0.58278, p>.05], and no main effects of Vector [F(1,11) = 0.08387, p>.05], or Zone [F(1,11) = 2.705, p>.05], confirming that increasing CREB function after training does not facilitate the retrieval/expression of an earlier memory that is only weakly acquired. Strengthening of weak spatial memory consolidation requires high CREB levels at the time of memory acquisition.

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Figure 3.615Figure 3.6 Acutely Increasing CREB does not affect expression of a previously acquired spatial memory. (a) Schematic timeline for Experiment 4. (b) Mice microinjected with CREB or GFP vector after (rather than before) strong training showed similarly normal spatial memory during probe tests. (c) Mice microinjected with CREB or GFP vector after weak training show no preference for the target zone during probe testing. (d) Density plots for grouped data showing search patterns of probe 1 (pre-vector) and probe 2 (post-vector) following weak training shows no preference for the target zone in either test.

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3.4.6 EXPERIMENT 5. Effects of increasing CREB in the hippocampus rescues the consolidation of spatial memory in CREB-deficient mice.

Because one of the goals of our study was to determine if enhancement of CREB can induce normal memory formation under conditions in which memory consolidation is not typically supported, we used CREB-deficient (CREB αΔ−/−), mice that have reduced levels of CREB protein (> 90%) and CREB-DNA binding activity throughout the brain (Pandey et al., 2000; Walters and Blendy, 2001). These mice have been reported to have spatial memory impairment in the watermaze, although there have been conflicting reports on the extent of their hippocampal-dependent memory deficits (Bourtchuladze et al., 1994; Kogan et al., 1997; Hebda-Bauer et al., 2005).

In agreement with previous reports (Walters and Blendy, 2001; Walters et al., 2003), we found that CREB-deficient mice showed low levels of endogenous CREB protein in the dorsal hippocampus compared to wild-type littermate mice. However, microinjecting CREB vector into the dorsal hippocampus of CREB-deficient mice increased CREB levels in infected neurons only (Fig 3.7).

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Figure 3.716Microinjection of CREB vector increases levels of CREB protein in the CA1 region of CREB-deficient mice. Immunohistochemical staining for CREB protein (red) in the CA1 four d following microinjection of GFP or CREB vector. Wild- type (WT) or CREB-deficient (MUTANT) mice microinjected with CREB vector show higher levels of CREB protein levels than mice microinjected with GFP vector, in infected neurons (green). CREB-deficient mice microinjected with GFP vector do not express any detectable endogenous CREB protein (top). In both WT and MUTANT mice, co-localization of vector with CREB-expressing neurons (yellow) is only seen in CA1 pyramidal neurons infected with CREB vector. Endogenous CREB expression does not co-localize with neurons infected with GFP vector. Scale bar = 50 μm.

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3.4.6.1 Acutely increasing CREB rescues the spatial memory deficit in CREB-deficient mice: the training data

To examine whether acutely increasing CREB function in the dorsal hippocampus is sufficient to rescue the spatial memory deficit in CREB-deficient mice, we microinjected CREB or GFP vector into the CA1 region of CREBαΔ-/- mutant or wild-type littermate control mice before strong training (2 blocks of 3 trials a day for 3 d; 6 blocks total). Over the course of training, wild-type littermate mice (with either CREB or GFP vectors) showed the characteristic progressive decrease in the time required to locate the platform, while CREB-deficient mice with GFP vector showed a relatively flat escape latency curve (Fig 3.8b). However, microinjection of CREB vector into the CA1 region completely restored the escape-latency curve in CREB-deficient mice; CREB-deficient mice with CREB vector showed escape latencies similar to wild-type mice. The results of a Group (CREB-deficient mice/CREB vector, CREB-deficient mice/ GFP vector, wild- type mice/CREB vector, wild-type mice/ GFP vector) x Block (6 levels) ANOVA support this conclusion, showing a significant Group X Block interaction [F (15,210) = 1.84, p < .05] and significant main effects of Group [F (3,42) = 5.34, p < .05] and Block [F (5,210)= 22.80, p < .001]. Post-hoc analysis revealed that, in contrast to wild-type mice, CREB-deficient mice with GFP vector failed to show a progressive decline in escape latency over training blocks. There was no difference in the swim speeds between the groups (p > .05). Together, these data indicate that the CREB-deficient mice show impaired acquisition of a spatial learning strategy, but that this spatial learning deficit was rescued by acutely increasing CREB in dorsal hippocampal CA1 neurons.

3.4.6.2 Acutely increasing CREB rescues the spatial memory deficit in CREB-deficient mice: the probe data

Consistent with previous reports (Bourtchuladze et al., 1994; Kogan et al., 1997; Hebda- Bauer et al., 2005), we found that CREB-deficient mice with GFP control vector showed poor spatial memory as assessed in the probe test. However, the spatial memory deficit in CREB-deficient mice was completely rescued by microinjecting the CREB vector into the dorsal hippocampus. CREB-deficient mice with CREB (MUT-CREB) vector showed 104 comparably strong spatial memory to wild-type mice with CREB vector (WT-CREB) (Fig 3.8c). This interpretation is supported by the results of an ANOVA showing a significant

Group x Zone interaction [F (3,42) = 10.14, p < .001] as well as significant main effects of

Group (F (3,42) = 17.68, p < .001), and Zone (F (1,42) = 123.78, p < .001]. Post-hoc tests revealed that the CREB-deficient mice with GFP vector (MUT-GFP) did not search selectively in the target zone (spending equal time in the target and other zones) and spent less time searching in the target zone than wild-type mice with either GFP (WT- GFP) or CREB vector. In sharp contrast, CREB-deficient mice with CREB vector searched selectively in the target zone, and spent an equal amount of time in the target zone as wild-type mice with CREB vector [F (3,42) = 15.68, p < .001]. Therefore, the impairment in spatial memory in the CREBαΔ-/- mice suggests that functional CREB activity in necessary for consolidation of spatial long-term memory, but that acutely increasing CREB levels in the dorsal hippocampus of CREB-deficient mice with decreased CREB throughout the brain is sufficient to rescue the spatial memory deficit normally observed in these mice.

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Figure 3.817Acutely increasing CREB in dorsal hippocampus rescues the spatial memory deficit in CREB-deficient mice. (a) Schematic timeline for Experiment 5. (b) CREB-deficient (MUT) mice with CREB or GFP vector and wild-type (WT) mice with CREB or GFP vector were strongly trained in the watermaze. All groups except CREB- deficient mice with GFP vector showed a decrease in escape latency over training blocks, indicating spatial learning. (c) Increasing CREB in the dorsal hippocampus rescues the spatial memory deficit observed in CREB-deficient mice. (d) Density plots for grouped data showing the probe test search patterns of CREB-deficient and wild- type mice with CREB or GFP vector. Platform position during training = lower right quadrant.

3.4.6.3 CREB-deficient mice fail to adopt a spatial search strategy. Acutely increasing CREB in the dorsal hippocampus rescues this deficit: the thigmotaxis data

To more thoroughly characterize the spatial memory impairment observed in CREBαΔ-/- mice, we examined their swim patterns during training. These patterns are thought to reflect the search strategies developed by mice during training. Typically, during the

106 initial watermaze training trials, wild-type mice tend to swim near the wall of the pool, in an instinctive behaviour known as thigmotaxis [the tendency to search in the outer regions of the pool (Wolfer et al., 1998)]. After about two trials, most wild-type mice gradually begin to adopt more efficient search strategies, proceeding to scanning, chaining, focal searching and finally to a spatial search strategy (Wolfer and Lipp, 2000; Martin et al., 2005). This change in strategy is reflected by a decrease in thigmotaxic behaviour. Accordingly, we examined the levels of thigmotaxis (time spent within 5 cm of the pool wall) during training in CREB-deficient and wild-type mice microinjected with CREB or GFP vectors as an index of an inefficient search strategy. Consistent with the decreased escape latencies over the course of training, wild-type mice, regardless of vector, showed a dramatic decline in the time spent in the periphery of the pool over training blocks, suggesting the adoption of a more effective spatial search strategy (Martin et al., 2005; Stone et al., 2011) (Fig 3.9a). In contrast, CREB-deficient mice with GFP vector showed high levels of thigmotaxis throughout training. Disrupting CREB function seems to induce an increase in thigmotaxic behaviour, perhaps reflecting a failure to adopt a spatial search strategy.

However, it remained possible that this failure to naturally progress to a spatial search strategy was due to non-specific performance effects in the watermaze, such as an increase in anxiety in CREB-deficient mice. The thigmotaxic phenotype in CREBαΔ-/- mice has been previously reported (Gass et al., 1998; Balschun et al., 2003). To rule out this possible explanation, we observed that the high levels of thigmotaxis in CREB- deficient mice were rescued by microinjecting the CREB vector into the dorsal hippocampus of CREB-deficient mice [significant main effects of Group [F(3,42) = 7.58, p

< .001] and Block [F(5,210) = 40.22, p < .001] but no Group x Block interaction [F(15,210) = 0.96, p > .05]. Swim speeds between the four groups of mice were comparable (p > .05). Because thigmotaxic behaviour was rescued by increasing CREB in the dorsal hippocampus, it suggests that the high levels of thigmotaxis observed in CREB-deficient mice during training are due to the failure of CREB-deficient mice to adopt a spatial- based search strategy in the watermaze (to progress from the thigmotaxic strategy to a spatial strategy) and not a reflection of poor overall performance or increased anxiety in the CREBαΔ-/- mice. 107

We also examined the level of thigmotaxis during the probe test. Consistent with the training data, CREB-deficient mice with GFP vector spent more time searching in the periphery of the pool than wild-type mice with either CREB or GFP vectors [F(3,42) = 6.48, p < .001] (Fig 3.9b). Importantly, CREB-deficient mice with CREB vector showed similarly low levels of thigmotaxis as wild-type mice (post-hoc comparisons showed that thigmotaxis was greatest in CREB-deficient mice with GFP vector, while the other groups did not differ). Therefore, on two different measures (time spent in the target zone and level of thigmotaxis during the probe test), CREBαΔ-/- mice showed poor spatial memory, but acutely increasing CREB function just in the dorsal hippocampus rescues both of these measures of spatial memory. Because a brain-wide deficit in CREB function was rescued by locally increasing CREB levels, these results indicate that the formation of spatial memory requires intact CREB function minimally within in CA1 neurons of the dorsal hippocampus

Figure 3.918CREB-deficient mice fail to adopt a spatial search strategy. (a) High levels of thigmotaxis (time spent in periphery of the pool) decreased over training blocks in all groups, consistent with the adoption of a spatial search strategy. However, CREB- deficient mice with GFP vector (MUT-GFP) continued to show high levels of thigmotaxis throughout training. This effect was reversed by microinjecting the CREB vector in dorsal hippocampus of CREB-deficient mice (MUT-CREB). (b) CREB-deficient mice with GFP vector showed high levels of thigmotaxis during the probe test. This effect was abolished by microinjecting the CREB vector in CREB-deficient mice. (c). Representative swim paths for groups during the probe test. Only CREB-deficient mice

108 with GFP vector displayed high levels of thigmotaxis during the probe test. The position of platform during training is marked in the lower right quadrant.

In summary, CREB-deficient mice have a chronic disruption in CREB function. Although this disruption may produce changes in the developing brain [such as alterations in neurogenesis, cell migration and neuronal connectivity (Lonze and Ginty, 2002) that may account for the spatial memory deficits observed in adulthood, we found that acutely increasing CREB in CA1 neurons in the dorsal hippocampus in adult mice was sufficient to reverse their spatial memory deficit. This finding highlights the importance of acute CREB-mediated transcription in spatial memory consolidation and furthermore, shows the spatial memory deficit observed in these CREB-deficient mice cannot be attributed to developmental deficits.

3.5 DISCUSSION

Our results show that increasing CREB levels in CA1 neurons of the dorsal hippocampus 1) enhances the consolidation of a normal spatial memory, 2) is sufficient to induce spatial memory in wild-type mice under weak training conditions that do not normally support consolidation of spatial long-term memory, and, 3) rescues the spatial memory deficit in mice with a brain-wide disruption in CREB function. The enhancement produced by CREB was specific in terms of the molecular basis, behaviour and memory process. Specifically, we did not observe a similar enhancement following microinjection of the vector encoding the mutant form of CREB (mCREB) with a point mutation at the key Ser133 phosphorylation site (Gonzalez et al., 1989), indicating that the enhancement depended on intact and functional CREB protein. In addition, this enhancement was specific to spatial memory (as mice relied on distal cues to perform the task) and was not due to facilitation of retrieval or expression of a spatial memory. These findings are consistent with previous reports that CREB enhances other forms of 109 memory in rodents, such as one-trial social defeat (Jasnow et al., 2005) and associative fear conditioning (Josselyn et al., 2001; Wallace et al., 2004; Han et al., 2007; Restivo et al., 2009). Together with previous results showing that disrupting CREB function in the dorsal hippocampus impairs spatial memory formation (Guzowski and McGaugh, 1997; Pittenger et al., 2002), these results indicate that CREB function in the dorsal hippocampus is both necessary and sufficient for spatial memory formation.

We observed that under the strong training conditions used in the present experiments, CREB αΔ−/− mice with GFP vector showed increased escape latencies and higher levels of thigmotaxis during training compared to wild-type littermate control mice. This finding is in agreement with previous reports that several lines of mice with different genetic mutations that disrupt CREB function show increased escape latencies or longer path lengths (Pittenger et al., 2002; Balschun et al., 2003; Hebda-Bauer et al., 2005), and higher levels of thigmotaxis (Gass et al., 1998; Balschun et al., 2003), during training than wild-type mice. Together, these findings suggest that disrupting CREB function impairs the acquisition of spatial learning. We also observed that CREB-deficient mice showed poor spatial memory in the probe test. It is interesting to note that the deficits in water maze behaviour exhibited by CREB αΔ−/− mice with GFP vector (increase in thigmotaxis and escape latency in training and impaired spatial memory in the probe test) are similar to those observed in rodents with hippocampal lesions (Morris et al., 1982; Logue et al., 1997; Wang et al., 2009). This similarity suggests that a deficit in hippocampal function may be at the core of the spatial memory deficit in CREB-deficient mice.

Previous studies have emphasized that thigmotaxis during training and spatial memory in the probe test are independent variables (Wolfer and Lipp, 2000). Nevertheless, we found that thigmotaxis and escape latency increases during training as well as spatial memory during the probe test were rescued in CREB-deficient by acutely increasing CREB levels in the dorsal hippocampus. Because CREB levels were increased only in the dorsal hippocampus, these results also suggest that intact CREB function in the dorsal hippocampus alone is sufficient for spatial memory formation.

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As discussed in the introduction (Chapter 1), CREBαΔ-/- mice have an 80-90% reduction in CREB activity due to the mutation at exon 2 of the CREB gene, resulting in a ubiquitous deletion of the α and Δ CREB isoforms. The residual β and the CREM activator protein support the remaining 10-20% of CREB activity in the brain (and body) of CREBαΔ-/- mice (Hummler et al., 1994; Blendy et al., 1996). Consistent with this, we found that these adult mice have reduced CREB protein in the CA1 region of the dorsal hippocampus compared to wild-type littermate controls. It is important to note that the disruption in CREB function in these mice does not only occur in adulthood, but also during development. Although this disruption in CREB function may produce changes in the developing brain [such as alterations in neurogenesis, cell migration and neuronal connectivity (Lonze and Ginty, 2002)] that may account for the spatial memory deficits observed in adulthood, we found that acutely increasing CREB in CA1 neurons in the dorsal hippocampus in adult mice was sufficient to completely reverse the spatial memory deficit. Therefore, the spatial memory deficits observed in CREB-deficient mice cannot be solely attributed to developmental deficits.

The mechanism by which CREB enhances spatial memory formation is not known. However, increasing CREB function increases NMDA-receptor dependent LTP, spine density, the number of NMDA receptor-only containing ‘‘silent’’ synapses (Marie et al., 2005)(Marie et al., 2005) and neuronal excitability (Dong et al., 2006; Lopez de Armentia et al., 2007; Zhou et al., 2009). Any, or all, of these mechanisms may provide the conditions necessary for the formation of a spatial memory.

We found that acutely over-expressing a wild-type form of CREB in a portion of dorsal CA1 hippocampus neurons specifically enhanced spatial memory formation with no other change in behaviour or obvious detrimental effect. This is in contrast to recent findings showing that a more prolonged and widespread (ie. forebrain-wide) over- expression of a constitutively active form of CREB (VP16-CREB) in the hippocampus impairs the retrieval/ expression of spatial memory (Viosca et al., 2009) while chronic over-expression induces seizure and death (Lopez de Armentia et al., 2007). It may be that large-scale over-expression of a constitutively active form of CREB enhances general noise rather than a specific learning signal. Evidence for this is suggested by

111 results from flies with the dunce mutation, in which the generalized enhancement of cAMP signaling leads to impaired learning (Dudai et al., 1976).

The present finding that increasing CREB enhances spatial memory, a complex form of hippocampal-dependent memory suggests that CREB could be a viable therapeutic target for the treatment of memory disorders in humans (Tully et al., 2003). Increasing CREB function could be a relevant treatment goal for conditions in which hippocampal- dependent memory is impaired, such as Alzheimer’s disease (Yiu et al., 2011), or age- related dementia (Mouravlev et al., 2006). We will review the implications of this in the final discussion.

3.6 CONCLUSION

The formation of long-term memory is known to require transcription (Davis and Squire, 1984). The human genome contains as many as 3,000 transcription factors (Babu et al., 2004). Many of these transcription factors have been implicated in mammalian memory formation (e.g., CREB, Zif268, C/EBP, AP-1, SRF, BMAL-1, DREAM, MEF2 and Rel/nuclear factor B) (Alberini, 2009). However, no transcription factor (or other molecule, to the best of our knowledge) has been shown to be both necessary for spatial memory consolidation and sufficient to produce spatial memory under conditions that do not normally support memory formation in mammals. In these studies, we found that locally and acutely increasing CREB in the dorsal hippocampus was sufficient to induce robust spatial memory in two different conditions in which spatial memory is not normally observed. Together with previous findings, these results show that CREB in the dorsal hippocampus is both necessary and sufficient (with minimal training) for spatial memory formation, indicating that CREB-mediated transcription is a limiting step in the process of spatial memory formation.

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3.7 CONTRIBUTIONS:

Portions of this chapter are based on published findings from:

Sekeres, M.J., Neve, R.L., Frankland, P.W., and Josselyn, S.A. (2010). Dorsal hippocampal CREB is both necessary and sufficient for spatial memory. Learning & Memory, 17 (6): 180-3

MJS, PWF and SAJ conceived and designed the experiments and analyzed the data. MJS performed all experiments. RLN made the viral vectors. All authors contributed to writing the original manuscript.

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4 Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory precision

4.1 ABSTRACT

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011; Tronson et al., 2012). Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms (Lee et al., 2004), disrupting the function of the transcription factor CREB impairs both processes (Kida et al., 2002; Mamiya et al., 2009). Phosphorylation of CREB at Ser133 recruits CREB binding protein (CBP)/p300 co- activators to activate transcription (Chrivia et al., 1993; Parker et al., 1996). Besides this well-known mechanism, CRTCs (CREB regulated transcription co-activators stimulate CREB-mediated transcription, even in the absence of CREB phosphorylation. Despite being a powerful and specific co-activator of CREB, the role of CRTC in memory is virtually unexplored. To examine the effects of increasing CRTC levels, we used viral vectors to locally and acutely increase CRTC1 (the CRTC isoform with highest neural expression) in the dentate gyrus (DG) of the dorsal hippocampus of wild-type mice prior to training or memory reactivation in context fear conditioning. Overexpressing CRTC1 enhanced both memory consolidation and reconsolidation; the CRTC1-mediated facilitation of memory was context-specific and did not generalize to a non-trained context. Furthermore, this enhanced memory was long-lasting and observed even after virally-expressed CRTC1 dissipated. CREB overexpression produced strikingly similar effects on consolidation and reconsolidation. These gain-of-function manipulations indicate that increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as reactivated established, memories without compromising memory precision.

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4.2 INTRODUCTION

The stabilization of long-term memory following encoding (consolidation) or memory reactivation (reconsolidation) requires gene expression and de novo protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011; Tronson et al., 2012). Although the precise molecular mechanisms critically mediating these processes may differ (e.g. Lee et al., 2004; Alberini, 2005), we, and others, showed that disrupting CREB (cAMP/Ca2+ responsive element binding protein) function impairs both synaptic consolidation (Bourtchuladze et al., 1994; Yin et al., 1994; Guzowski and McGaugh, 1997; Bartsch et al., 1998; Kida et al., 2002; Pittenger et al., 2002; Frankland et al., 2004) and reconsolidation (Kida et al., 2002; Mamiya et al., 2009; Yang et al., 2011; Tronson et al., 2012). In contrast, increasing CREB function promotes memory consolidation (Josselyn et al., 2001; Wallace et al., 2004; Han et al., 2007; Restivo et al., 2009; Zhou et al., 2009; Sekeres et al., 2010). Whether increasing CREB function similarly promotes memory reconsolidation is unknown.

Phosphorylation of CREB at Ser133 modulates the transcription of genes with cAMP responsive elements (CRE) in the promoter regions (Shaywitz and Greenberg, 1999; De Cesare and Sassone-Corsi, 2000; Mayr and Montminy, 2001; Lonze and Ginty, 2002). Interestingly, phosphorylation of CREB is not always sufficient to stimulate transcription, suggesting that additional transcriptional modulators may be involved. Indeed, recent studies identified CRTCs (CREB regulated transcription co-activators) as potent modulators CREB-mediated transcription (Conkright et al., 2003b; Iourgenko et al., 2003). CRTCs may potentiate the ability of CREB to recruit CBP/p300 (Xu et al., 2007) to the promoter region, and to stimulate CRE-dependent transcription via a phosphorylation-independent interaction with the bZIP domain of CREB (Conkright et al., 2003b; Iourgenko et al., 2003). Therefore, CRTCs may provide a powerful mechanism for enhancing CREB function.

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We are interested in investigating CRTC1, the isoform showing the highest expression in the brain (particularly the hippocampus) of the 3 CRTC1 family members (Conkright et al., 2003b; Iourgenko et al., 2003; Wu et al., 2006; Zhou et al., 2006; Kovacs et al., 2007; Altarejos et al., 2008; Watts et al. 2011). To briefly review, dephosphorylation of CRTC1 by cAMP or calcium influx promotes nuclear translocation (Screaton et al., 2004). Intact CRTC1 function is necessary for CRE-mediated transcription; blocking the interaction between CRTC1 and CREB disrupts, while overexpressing CRTC1 increases, CRE-mediated transcription in culture (Zhou et al., 2006; Kovacs et al., 2007). The role of CRTC1 in memory is completely unexplored, although previous results show that blocking CRTC1 function disrupts, while increasing CRTC1 levels enhances, L-LTP in hippocampal slices (Zhou et al., 2006; Kovacs et al., 2007) suggesting that this novel CREB cofactor may be important in modulating forms of synaptic plasticity that are important for memory consolidation.

Here, we are interested in determining if increasing CRTC1 or CREB function in a regionally- and temporally-specific manner is sufficient to support complex forms of hippocampal-dependent contextual fear memory consolidation and reconsolidation. These questions have never before been investigated in vivo, and in freely behaving animals. We propose investigating this by asking the following questions:

1. Can wild-type CREB over-expression in the hippocampus facilitate consolidation of context fear memory that is only weakly acquired in control mice?

2. Can CRTC1 or CREB over-expression in the hippocampus facilitate recent context fear memory precision?

3. Can CRTC1 or CREB over-expression in the hippocampus facilitate remote context fear memory precision?

4. Can CRTC1 or CREB over-expression in the hippocampus facilitate reconsolidation of context fear memory?

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4.2.1 Experiment 1: Effects of increasing CREB on the consolidation of context fear memory that is only weakly acquired in control mice Rationale: Previous studies in our lab have shown that CREB is both necessary and sufficient to induce spatial memory formation (see Chapter 3, Sekeres et al., 2010). CREB-mediated facilitation of hippocampal-dependent memory extends to other well- established behavioural tasks, including contextual fear conditioning. Like with spatial memory, global and local inducible suppression of endogenous CREB function prior to contextual fear conditioning produces impairments in consolidation (Bourtchuladze et al., 1994; Kogan et al., 1997; Kida et al., 2002; but see Pittenger et al., 1994) and reconsolidation (Kida et al., 2002; Mamiya et al., 2009) of context fear memory. Contextual fear conditioning and reconsolidation increases CRE-dependent gene expression and levels of pCREB in the dorsal hippocampus (Impey et al., 1998; Mamiya et al., 2009). It was only recently demonstrated that acutely increasing CREB function in the CA1 or dentate gyrus of the dorsal hippocampus using viral vectors encoding constitutively-active CREB) can actually facilitate contextual fear memory formation in wild-type mice (Restivo et al., 2009). Importantly, by using a relatively low intensity shock during contextual fear conditioning, they induced only weak freezing to the fearful context in wild-type mice injected with control vector, but robust freezing in mice with constitutively active dorsal hippocampal CREB function, suggesting that, like with spatial memory, increasing CREB function with the hippocampus is sufficient to induce a contextual fear memory under conditions in which this memory is not typically supported. Lesion studies of the hippocampus have demonstrated a critical role for dorsal hippocampal regions including the dentate gyrus in the consolidation and context discrimination of contextual fear long-term memory (Phillips and LeDoux, 1992; Frankland et al., 1998; Lee and Kesner, 2004; Leutgeb et al., 2007; Hernandez-Rabaza, et al., 2008; Schmidt et al., 2012).

Here, we examined the ability of wild-type CREB to support contextual fear memory, using a weak fear stimulus which does not normally induce fear memory under control conditions. This has never before been demonstrated. Constitutively active CREB remains in its active, phosphorylated state, unlike wild-type CREB, which requires activity-dependent phosphorylation of CREB in order to initiate the process of 117 transcription. By over-expressing wild-type CREB in the dentate gyrus, we aimed to determine if CREB’s ability to facilitate long-term memory formation extends across multiple hippocampal-dependent cognitive tasks.

Experimental Approach: We investigated whether increasing wild-type CREB in a population of excitatory granule cells of the dentate gyrus in the dorsal hippocampus of wild-type mice is sufficient to support formation of contextual memory following weak context fear conditioning.

4.2.2 Experiment 2&3: Effects of increasing CRTC1 or CREB on consolidation of a recent context fear memory Rationale: The CREB co-activator CRTC1 is highly expressed across brain regions implicated in long-term memory, including the hippocampus (Conkright et al., 2003; Zhou et al., 2006; Watts et al., 2011). Recently, CRTC1 was found to increase hippocampal-dependent plasticity by lowering the threshold for LTP following only weak stimulation, prolonging the maintenance of L-LTP, as well as increasing BDNF mRNA synthesis in the CA1 region (Kovacs et al., 2007, Zhou et al., 2006), and promoting dendrite outgrowth in developing cortical neurons (Li et al., 2009). While this points to a key role of the CRTC family of proteins in regulating forms of plasticity associated with learning and memory, the role of CRTC1 in mediating memory formation has never before been investigated in vivo. We hypothesize that increasing CRTC1 levels in the hippocampus may support the formation of a robust long-term memory representation, similar to what we found when we directly increased CREB-expression within the dorsal hippocampus prior to learning.

As previously discussed, contextual memory and context discrimination have been shown to rely on the dorsal hippocampus (Kim and Fanselow, 1992; Phillips and LeDoux, 1992; McDonald et al., 1995; Frankland et al., 1998; Anagnostaras et al., 1999; Lee and Kesner, 2004; Hernandez-Rabaza, 2008). Post-training lesions performed 1d after fear conditioning also result in impaired contextual discrimination in mice and in rats (Wiltgen and Silva., 2007; Winocur et al., 2007; Wang et al., 2009). Understanding

118 the critical role of the dorsal hippocampus in contextual discrimination using a contextual fear paradigm, we were interested in determining if enhancing CRTC1 or CREB in the dorsal hippocampus is sufficient to induce a precise contextual fear memory. As in our spatial memory experiments, here we used both a weak and a strong fear conditioning protocol. First, we used a single low-intensity foot shock which fails to induce long-term memory for the conditioning context in control wild-type mice when tested after 24 h (Experiment 2: weak training). Second, we used multiple, higher intensity foot shocks to induce normal context memory in controls (Experiment 3: strong training).

Experimental Approach: We investigated whether increasing CRTC1 or CREB in the dentate gyrus of the dorsal hippocampus of wild-type mice is sufficient to support formation of precise contextual memory following weak or strong training conditions. We tested for the precision of this fear memory after 24 h by placing the mice in the conditioning context (Context-A, CXT-A), followed by a novel context (Context-B, CXT- B), and assessing freezing behaviour. This allowed us to determine (1) if mice over- expressing CRTC1 or CREB are able to form a robust memory for the context associated with the fearful experience, and (2) if CRTC1 or CREB enhancement is inducing a precise or a generalized fear memory.

4.2.3 Experiment 4: Effects of increasing CRTC1 or CREB on retrieval/expression of a weak context fear memory Rationale: De novo protein synthesis is required for the formation of a long-term memory. Manipulations of transcription factors CRTC1 or CREB outside of the time window of synaptic consolidation of new long-term memory (Guzowski and McGaugh, 1999; Dudai, 2004) should not have an effect on the stabilization or representation of that memory. As discussed, injections of protein synthesis inhibitors at a time after the wave of cellular consolidation has been complete have no effect on the subsequent expression of that fear memory (Davis and Squire, 1984). We previously demonstrated

119 that microinjection of CREB vector after spatial memory training in the watermaze had no effect on the retrieval/expression of either a weak or strongly acquired spatial memory [see Chapter 3, Fig 3.6c (WT-weak), 3.6b (WT-strong)]. Accordingly, we hypothesized here that microinjection of CRTC1 or CREB vector in the dentate gyrus after fear conditioning would not impact the retrieval/expression of a weakly acquired recent contextual memory.

Experimental Approach: We investigated whether increasing CRTC1 or CREB in the dentate gyrus 24 h after the completion of weak fear conditioning is sufficient to facilitate the retrieval of a previously acquired context memory. Mice underwent weak fear conditioning, received microinjection of viral vector (CRTC1, CREB, or GFP), then were tested in conditioning Context-A, and novel Context-B.

4.2.4 Experiment 5: Effects of increasing CRTC1 or CREB on consolidation of a remote context fear memory Rationale: Systems-level consolidation of remote memory results in a gradual reorganization of the memory in a distributed cortical network (see Frankland and Bontempi, 2005; Winocur et al., 2010; Moscovitch and Winocur, 2011 for review). While the hippocampus is important for the initial consolidation and storage of a fear memory, other cortical regions gradually become engaged in the storage of that memory, particularly medial prefrontal cortical regions, including the anterior cingulate cortex (Frankland et al., 2004; Texieria et al., 2006; Vetere et al., 2011). There is evidence that, over time, context memories generalize, becoming less reliant on the precise contextual cues present during encoding. Support for this idea comes from the findings that, after fear conditioning, rats or mice replaced in the conditioning context (Context-A) following a short delay will exhibit high freezing in that context, but not in a novel context (Context-B). Following a long delay (~30 d), the contextual fear memory generalizes, with mice exhibiting similarly high freezing in both Context -A and Context -B. On the other hand, mice which receive hippocampal lesions 24 h following fear conditioning exhibit abolished freezing in both Context-A and B. However, mice receiving lesions after a long delay following fear conditioning show spared memory like controls, 120 continuing to freeze at high levels in both Context -A and B (Wiltgen and Silva, 2007; Winocur et al., 2007). This suggests that the cortical representation is sufficient to support the remote fear memory, though the transformed representation of the fear memory is less contextually detailed as the original hippocampal trace (Winocur et al., 2010). While the continued role of the hippocampus in the expression of remote fear memories continues to be debated, a recent study looking at immediate-early gene expression of c-fos following recent and remote context fear memory testing suggests that if the hippocampus remains intact, it is likely still engaged during the retrieval of a remote fear memory (Wiltgen et al., 2010; Wheeler and Frankland, 2012, unpublished). Here we are manipulating CRTC1 and CREB levels in the dorsal hippocampus prior to weak fear conditioning to determine if enhancing CREB function during initial consolidation has persistent effects on the precision of a remote context fear memory.

Experimental Approach: Wild-type mice received microinjections of CRTC1 or CREB vector in the dentate gyrus prior to weak context fear conditioning. The precision of their remote fear memory was tested 30 d later in both Context-A and Context-B. .

4.2.5 Experiment 6: Effects of increasing CRTC1 or CREB on reconsolidation of a remote context fear memory Rationale: As discussed, reactivation of a memory by retrieval or re-exposure to the cues present during initial memory encoding returns the memory to a labile state in which can be sensitive to disruption during the re-stabilization of the second consolidation process, or ‘reconsolidation’ (Nader et al., 2000; Sara, 2000). The crucial role of CREB in both the consolidation and reconsolidation process has been demonstrated, where suppression of CREB activity in the hippocampus prior to reactivation of a contextual fear memory results in disruption of the second wave of CREB-mediated gene expression during the reconsolidation process that is required for the stabilization of a reactivated fear memory (Mamiya et al., 2009; Tronson et al., 2012).This disruption of CREB-mediated activity at the time of reactivation results in impaired fear memory when tested 24h later.

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There are reports that older memories are more resistant to disruption than recent memories (Milekic and Alberini, 2002; Suzuki et al., 2004). In our experiment, we will reactivate context memory at a remote time point. Unlike most reconsolidation studies which use a loss-of-function approach using inactivation of the hippocampus during this re-exposure period, we will take the opposite approach. By using a gain-of-function approach, we will increase CRTC1 or CREB levels of neurons in the dentate gyrus just prior to context re-exposure to determine if we can induce enhanced contextual memory following reconsolidation.

Experimental Approach: Wild-type mice underwent weak fear context fear conditioning, the received microinjection of vector (CRTC1, CREB, or GFP) 26 d after training. Mice then received a brief ‘reminder/reactivation’ in the conditioning Context-A. Mice were then tested 1 d later in both Context-A and Context-B.

Together, these studies provide a comprehensive overview of the role of transcription factors CRTC1 and CREB in multiple memory processes supporting contextual memory consolidation, reconsolidation, and retrieval. This is the first characterization of the role of the CREB co-activator CRTC1 in a learning and memory paradigm.

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4.3 DETAILED METHODS

4.3.1 Mice

Adult C57Bl6x129 female mice (wild-type) were used for all experiments. Details on the rearing and housing conditions of mice can be found in the Methods.

4.3.2 Preparation of HSV vectors

To locally and acutely increase CRTC1 or CREB function we used replication-defective herpes simplex viral (HSV) vectors. Wild-type full-length CRTC1 or CREB cDNAs (kindly provided by Dr. Satoshi Kida, Tokyo University of Agriculture, Tokyo, Japan) were subcloned into the bi-cistronic HSV vectors that co-express GFP as a fluorescent reporter [HSV-p1005, (Russo et al., 2009)]. In this vector, GFP expression is driven by a CMV promoter whereas CRTC1 or CREB expression is driven by the constitutive promoter for the HSV immediate-early gene IE 4/5. As a control, we used an HSV expressing GFP alone. For details on the generation of HSV-vectors, see Methods.

4.3.3 Transfection of primary hippocampal neurons and luciferase assay

To verify that infection with CRTC1 increases CRE-activity and promotes nuclear translocation in neurons, we conducted a CRE-luciferase assay, and stimulated primary hippocampal neurons with KCl/FSK.

4.3.3.1 Preparation of primary hippocampal neurons

Primary hippocampal neurons were prepared from E18-19 mice (see below). Briefly, hippocampi were collected in cold PBS and dissociated using trypsin (0.25%, 12 min at 37°C) and a glass Pasteur pipette. Neurons were plated onto poly-l-lysine-treated glass coverslips (immunostaining) or culture plates (luciferase assay) in MEM with 10% horse serum, 0.6% Glucose, 1 mM glutamax, 50 μg/ml streptomycin and 50 U/ml penicillin 123

(Gibco-Invitrogen). Media was replaced with Neurobasal Medium (Invitrogen) containing B27 supplement (2%; Invitrogen), penicillin-streptomycin (50 μg/ml penicillin, 50 U/ml streptomycin) and glutamine (1 mM; Sigma) 4-5 h later.

4.3.3.2 Immunostaining of primary hippocampal neurons

To visualize plasmid-induced CRTC1 protein expression and localization, DIV 5 hippocampal neurons were transfected with plasmids expressing CRTC1 or GFP alone. 24 h later, neurons were treated with KCl (50 mM)/FSK (20 µM) or vehicle for 4 h. Neurons were washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked [0.3% Triton X-100, 2% normal goat serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA), 0.5% bovine serum albumin (Bioshop Canada Inc., Burlington, ON) in PBS] then incubated at 4°C overnight with rabbit anti-CRTC1 polyclonal antibody (1:1000, Cell Signaling, Danvers, MA). After washing in PBS, neurons were incubated with goat anti-rabbit Alexa 568 antibody (1:500, Invitrogen, Molecular Probes, Eugene, Oregon, USA) for 1 h at room temperature, washed with PBS, counterstained with Hoechst and mounted with PermaFluor Mounting medium (Thermo Scientific, Fremont, CA). Images were obtained using a confocal laser scanning microscope (LSM 710, Zeiss).

4.3.3.3 Luciferase assays

DIV 5 neurons were transfected (using Lipofectamine 2000) with the CRE reporter plasmid [500 ng; generated by replacing d2eGFP transgene in pCRE-d2eGFP (Clontech) with the luciferase coding region from the MRE reporter plasmid, pGL3- TATA-DesMEF, with HindIII and XbaI (Vetere et al., 2011). TK-pRL vector expressing Renilla luciferase (250 ng, Promega) was used as the internal control. 24 h later, neurons were infected with GFP or CRTC1 vector. Medium was replaced 6 h after infection. 24 h later, neurons were treated with KCl (50 mM)/FSK (20 µM) or vehicle for 4 h. Neurons were lysed and luciferase assays conducted using a Dual Luciferase

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Assay kit (Promega). Firefly and Renilla luciferase activity levels were quantified by a luminometer (Berthold Microlumat LB 96V, Fisher Scientific) and CRE-luciferase activity was normalized to Renilla-luciferase activity. Data represent means from 4 independent experiments, with internal duplicates or triplicates for each condition.

4.3.4 Surgery

All subjects underwent stereotaxic surgery to deliver bilateral microinjections of wild- type CRTC1, CREB, or GFP vector (2.0 l/side) into the dorsal dentate gyrus. Mice were allowed at least 48 h to recover prior to further behavioural training or testing. See Methods for surgical details.

4.3.5 Contextual fear conditioning

Context fear conditioning was carried out as reported in Wang et al. (2009). Mice received a weak (1 x 0.3mA) or strong (3 x 0.5mA) unsignalled footshock(s) in the conditioning chamber. Following delay, mice were placed in the conditioning context (CXT-A) and in novel context (CXT-B) for 5 min each see { Fig 2.3 in Methods for schematic of test chambers, and Fig 2.4 for detailed schematic timeline of fear conditioning and test session). Freezing behaviour in each context was digitally recorded, and used as an index of memory for the fearful context. See Methods for details of fear conditioning and context discrimination testing and analyses.

4.3.6 Experiment 1. Effects of increasing CREB on the consolidation of context fear memory that is only weakly acquired in control mice. Wild-type mice were microinjected with vector (CREB vector n=12, GFP vector n=10) and 3 d later given weak fear conditioning. 24 h later, mice were placed in CXT-A (for 5 min) (Fig 4.8a for timeline).

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4.3.7 Experiment 2. Effects of increasing CRTC1 or CREB on consolidation of a weak context fear memory.

A within-subjects design was used to test context fear memory. Mice were microinjected with vector (CRTC1 vector n=29; CREB n=24; GFP n=27) and 3 d later given weak fear conditioning. 24 h later, mice were either placed in CXT-A or CXT-B (for 5 min) and 5 h later, placed in the alternate context. The order of context test was counterbalanced in this experiment only (Fig 4.9a for timeline).

4.3.8 Experiment 3. Effects of increasing CRTC1 or CREB on consolidation of a strong context fear memory.

Mice were microinjected with vector (CRTC1 n =10; CREB n=9; GFP n=8) and 3 d later given strong fear conditioning. 24 h later, mice were tested in CXT-A and 5 h later, tested in CXT-B (Fig 4.10a for timeline).

4.3.9 Experiment 4. Effects of increasing CRTC1 or CREB on retrieval of a weak context fear memory.

Mice received weak fear conditioning and 24 h later were microinjected with vector (CRTC1 n =12; CREB n=11; GFP n=11). 4 d later, mice were tested in CXT-A and 5 h later, tested in CXT-B (Fig 4.11a for timeline).

4.3.10 Experiment 5. Examining the enduring effects of increasing CRTC1 or CREB on consolidation of a weak remote context fear memory.

Mice were microinjected with vector (CRTC1 n = 12; CREB n= 16; GFP n= 9) and 3 d later given weak fear conditioning. 30 d later, at a time when transgene expression driven by the HSV vector had long dissipated, mice were tested in CXT-A and 5 h later, tested in CXT-B (Fig 4.12a for timeline).

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4.3.11 Experiment 6. Effects of increasing CRTC1 or CREB on reconsolidation of a weak remote context fear memory.

Mice were fear conditioned using the weak conditioning protocol and 26 d later, microinjected with vector (reactivation group (R): CRTC1 n=10; CREB n= 10; GFP n= 10). 3 d following microinjection, mice were replaced in CXT-A in the absence of the shock (for 45 s) to reactivate the context fear memory. Mice were removed from CXT-A and returned to the homecage. 24 h later, mice were tested as above in CXT-A then CXT-B. As a control, a ‘no memory reactivation’ condition was included in this experiment (no-reactivation groups (NR): CRTC1 n= 6; CREB n= 6; GFP n= 6). Mice were treated identically except that the reactivation procedure was omitted (mice remained in the homecage). The delay between conditioning, surgery, and context fear memory testing was identical between ‘reactivation’ and ‘no reactivation’ groups (Fig 4.13a, f for timelines).

4.3.12 Statistical analyses

To analyze CRE-luciferase activity, we used an ANOVA with between-group factor Plasmid (CRTC1-GFP, GFP) and within-group factor Stimulation [Stim (FSK/KCl), No Stim (unstimulated control)]. The infection rate between Vector (CRTC1, CREB, GFP) was analyzed using a 1-way ANOVA. For the contextual fear conditioning data, we used a 1-way ANOVA to analyze the shock reactivity during conditioning. Repeated measures ANOVAs were performed for fear conditioning and context generalization experiments. To analyze the fear condition session, we used an ANOVA with between- group factor Vector and within-group factor Shock [Pre-US (freezing prior to shock), Post-US (freezing after the shock)]. To analyze the fear generalization test sessions we used an ANOVA with between-group factor Vector and within-group factor Context [CXT-A (shock context), CXT-B (no-shock context)]. Significant interactions or main effects were further analyzed using Post-hoc Tukeys HSD tests. See Methods for statistical analyses details.

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4.3.13 Histology: Confirming transgene expression following behavioural testing.

Following context testing, all subjects were perfused, and expression of GFP was analyzed in each brain. Only mice determined to be bilateral “hit” in the DG were included in subsequent data analysis. Again, we observed a portion of GFP-expressing DG neurons around the site of microinjection (approximately 1.6mm in diameter). Mice are classified as “hit” only if they had robust bilateral expression of GFP across at least 5 consecutive coronal sections. All other mice were classified as “miss”, including mice with weak transgene expression. All brains were perfused 4 d following microinjection of vector except in Experiment 4 where brains were perfused 33 d following microinjection of vector. A subset of brains microinjected with CRTC1 (n=14), CREB (n=14), or GFP (n=12) vector were used to measure the area of infection in the dentate gyrus compared to a DAPI-labeled control brain (n=5). Using StereoInvestigator 8 software (MBF Bioscience, Vermont, USA), we traced the target region of the DG (-1.46 mm to -3.08 mm in the AP plane, corresponding to figures 43-56 in Paxinos & Franklin, 2001) over 15 serial sections (40 um thick) in random brains classified as “hit” to determine the percent target region infected. To estimate the number of cells infected by our microinjections, we then stereologically counted at 100X magnification to determine the mean number of infected DG cells per brain within the region of interest. All measurements and counts were performed on brains perfused 4 d post-infusion. The range of infection was calculated as a percentage of the total DAPI-labeled area of dentate gyrus within the sampled region (Fig 4.4). See Methods for histology inclusion criteria, and quantification of transgene expression.

4.3.14 Immunohistochemistry.

4.3.14.1 CRTC1 staining.

To verify that microinjection of CRTC1 vector increased expression of CRTC1 protein, we used an antibody specific for CRTC1. Coronal brain sections (50 μm) from mice 128 microinjected with GFP or CRTC1 vector were incubated with blocking solution (0.1% BSA, 2% NGS, 0.3% Triton X-100) for 2 h at room temperature, then incubated with rabbit anti-CRTC1 polyclonal antibody (1:500) at 4°C for 24 h. Sections were washed with PBS, then incubated with goat-anti-rabbit Alexa 568 (1:500) for 2 h at room temperature. Sections were washed with PBS, counterstained with Hoechst, mounted on gel coated slides and coverslipped using PermaFluor mounting medium. Images were obtained using a confocal laser scanning microscope (LSM 710, Zeiss).

4.3.14.1.1 Staining for endogenous CRTC1 protein.

To examine the cell type in which CRTC1 protein is endogenously expressed, we examined the overlap of antibodies specific for CRTC1 protein with cell markers specific for excitatory neurons (α-CaMKII, glial cells (GFAP) or interneurons (GAD67, Parvalbumin).

4.3.14.2 GFAP, GAD67 and Parvalbumin staining.

Brain sections (35 μm) from homecage wild-type mice were incubated with blocking solution (0.1% BSA, 2% NGS, 0.3% Triton-X) for 2 h at room temperature then incubated with rabbit anti-CRTC1 polyclonal (1:500) and one of the following primary antibodies: mouse monoclonal anti-GFAP (1:500, Cell Signaling), mouse monoclonal anti-GAD67 (1:500, Millipore, Billerica, MA), or mouse monoclonal anti-Parvalbumin (1:500, Sigma-Aldrich, St. Louise, MO) at 4°C for 24 h. Sections were washed with PBS 0.1M, then incubated with goat-anti-rabbit Alexa 568 (1:500, Invitrogen, Molecular Probes, Eugene, Oregon, USA) and goat-anti-mouse Alexa 633 (1:500, Invitrogen) for 2 h at room temperature. Sections were washed with PBS, counterstained with Hoechst, mounted on gel coated slides and coverslipped using PermaFluor mounting medium.

4.3.14.3 α-CaMKII staining.

Staining for α-CaMKII was similar except brain tissue was incubated with blocking solution (anti-mouse IgG blocking in 1% H2O2) for 1 h at room temperature then incubated with mouse monoclonal anti-α-CaMKII antibody (1:1000, Millipore, Billerica,

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MA) at 4°C for 24 h. Sections were washed with PBS, then incubated with donkey-anti- mouse HP (1:500) for 1 h at room temperature and signal amplified with TSA-FCM (30 min). Sections were washed with PBS, counterstained with Hoechst, mounted on gel coated slides and coverslipped using PermaFluor mounting medium. Images were obtained using a confocal laser scanning microscope (LSM 710, Zeiss).

4.4 RESULTS

4.4.1 Microinjection of CRTC1 or CREB vector increases CRTC1 and CREB function in the dentate gyrus of dorsal hippocampus.

We first examined endogenous expression of CRTC1 protein in the brain. Consistent with previous findings (Zhou et al., 2006; Watts et al., 2011), we observed high levels of CRTC1 in the hippocampus.

4.4.1.1 Endogenous CRTC1 expression is restricted to excitatory neurons in the hippocampus

Importantly, we found that in the dentate gyrus, CRTC1 is expressed exclusively in excitatory dentate granule cells, not in glia or interneurons (Fig 4.1). Microinjection of the neurotropic HSV-CRTC1 vector into the dentate gyrus selectively infects excitatory granule cells (Fig 4.2). Previous findings in our lab have shown that HSV-GFP co- localizes with α-CaMKII-expressing cells, but not GFAP, GAD67, or Parvalbumin expressing glia or interneurons (Mercaldo and Josselyn, unpublished results), confirming that our preparations of HSV vectors are neurotropic. In this way, our viral vectors increase CRTC1 levels only in neurons in which CRTC1 is endogenously expressed.

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Figure 4.119Expression of CRTC1 in the dentate gyrus of the dorsal hippocampus. In the dentate gyrus, CRTC1 is endogenously expressed exclusively in excitatory neurons (not interneurons or glia). Overlap of immunohistochemical staining for endogenous CRTC1 protein (red) and markers of different cell types [green, excitatory neurons (α-CaMKII), glia (GFAP) or interneurons (GAD67 or parvalbumin)]. Hoechst (blue) is used to identify dentate gyrus granule cell layer. Merged images shows that endogenous CRTC1 protein is co-localized in cells positive for α-CaMKII (yellow) (but not GFAP, GAD67 or parvalbumin). White arrows show lack of CRTC1 staining overlap in cells positive for GAD67 or parvalbumin cells. Scale bar = 50 μm.

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Figure 4.220Infection of dentate gyrus granule cells. In the dentate gyrus, HSV preferentially infects excitatory neurons (granule cells, labeled green) [assessed 4 d post-microinjection (top), counterstained with DAPI (blue)]. Transgene expression dissipates by 10 d post-microinjection (bottom). Scale bar = 50 μm.

4.4.1.2 Vector microinjection induces robust localized transgene expression in the dentate gyrus of dorsal hippocampus

To increase CRTC1 or CREB levels, we used HSV encoding wild-type CRTC1 (CRTC1 vector) or CREB (CREB vector); both vectors also expressed GFP, allowing visualization of infected neurons. As a control, we used a vector expressing GFP alone (GFP vector). Importantly, we observed no evidence of toxicity associated with these vectors either in cultured neurons or following microinjection in vivo. Bilateral

132 microinjections of CRTC1, CREB, or GFP vector into the dentate gyrus of dorsal hippocampus produced robust transgene expression in both the upper and lower dentate gyrus blades in a circular region (diameter of approx.1.6 mm) centered at the site of microinjection (target -2.3 mm posterior to bregma) (Fig 4.3).

Figure 4.321Vector microinjection induces robust localized transgene expression in the dentate gyrus of dorsal hippocampus. Left: Coronal brain images (adapted from Paxinos & Franklin, 2001) depicting the anterior-posterior extent of typical viral vector infections (-1.46 to -3.08 mm posterior to bregma). Right: Corresponding image showing transgene expression (GFP, green) following vector microinjection [assessed 4 d post-microinjection, counterstained with DAPI (blue)]. Scale bar = 200 μm. 133

We traced the target region of the dentate gyrus (-1.46 mm to -3.08 mm from bregma in the AP plane) across 15 serial sections in random brains classified as “hit” to determine the percent target region infected. We determined the infected area of the target region to be 63.00 ± 7.98 % for CRTC1 vector, 72.11 ± 4.67 % for CREB vector, and 62.54% ± 8.58 for GFP vector. Importantly, all vectors infected a similar percent area of dentate gyrus [F(2,36) = 0.59, p > 0.05] (Fig 4.4a). Previous studies using viral vectors with GFP to label infected neurons in the dentate gyrus have estimated the range of infection by simply tracing any area expressing GFP (Restivo et al., 2009). Accordingly, when we used this technique to estimate the size of our infections (as reported above), we found that the range of infection extends across 60-70% of the target area of the dorsal dentate gyrus. However, the penetrance of the infection within this target area was not complete, so that not all neurons across the infected range of the dentate gyrus express GFP. Therefore, to estimate the number of neurons infected by our microinjections, we stereologically counted infected neurons in a subset of brains (4-5 per group). The overall number of DAPI+ cells in the target region was 527027 ± 22108.43, and the number of cells in the target region infected by each vector was: CRTC1 vector: 120825 ± 19184.14; CREB vector: 114066 ± 11174.15; GFP vector: 138531 ± 47302.98 (Fig

4.4b). The mean number of cells infected did not vary between vectors [F(2,11) = 0.21, p > 0.05]. Finally, the number infected neurons in the “hit” groups did not correlate with any measure of memory in any experiment (p>0.05). We also first considered brains in which CA1 was highly infected with our CRTC1 or CREB vector, as CA1 is an area shown to have high pCREB activation in response to context fear conditioning (Impey et al., 1998; Mamiya et al., 2009). However, we chose to focus on the dentate gyrus, as the behavioural effects were most robust when brains expressed high GFP in this region. This does not necessarily exclude brains that had CA1 viral expression in additions to bilateral GFP expression in the dentate gyrus. Due to the relatively medial infusion site (+/- 1.6mm laterally from bregma), we rarely saw GFP expression in CA3 cells (with any of the 3 vectors). However, it is possible that input from the CRTC1 or CREB-infected granule cells in the dentate may be potentiating synaptic plasticity within the CA3 region.

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Figure 4.422Estimations of the typical infection size for each vector. (a) The area expressing GFP in the target regions was traced and measured to calculate the percent of area (range) of the dentate gyrus (DG) typically infected by each vector. (b) Stereological count of infected neurons in of a subset of brains microinjected with vector, or counterstained with DAPI to label nuclei in the dentate gyrus.

Previous findings indicate that the CREB vector increases CREB levels and CRE- mediated transcription both in vitro and in vivo (Barrot et al., 2002; Han et al., 2007; Han et al., 2009; Sekeres et al., 2010; Larson et al., 2011). To examine the effects of CRTC1 vector, we microinjected CRTC1 or GFP control vector into the dentate gyrus and observed CRTC1 protein levels. As expected, microinjection of CRTC1, but not GFP, vector robustly increased expression of CRTC1 protein in the dentate gyrus, well above endogenous levels (Fig 4.5).

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Figure 4.523Microinjection of CRTC1 vector in the dentate gyrus increases expression of CRTC1 protein. Immunohistochemical staining for CRTC1 protein (red) in the dentate gyrus 4 d following microinjection of GFP vector (top, green) or CRTC1 vector (bottom, green). Mice microinjected with CRTC1 vector show higher expression of CRTC1 protein than those microinjected with GFP vector. Co-localization of infected neurons with CRTC1 expressing neurons is only seen in dentate gyrus granule cells infected with CRTC1 vector (yellow). Dentate granule cell layer is labeled with Hoechst (blue). Endogenous CRTC1 expression can still be seen in the dentate gyrus infected with GFP vector, suggesting intact basal CRTC1 expression in control brains. Scale bars = 200 μm (top), 50 μm (bottom).

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4.4.1.3 Stimulation induced nuclear translocation of CRTC1, and an increase in CRE-reporter activity Similar to endogenous CRTC1 protein (Zhou et al., 2006; Li et al., 2009), transgenic CRTC1 protein is normally sequestered in the cytoplasm, but efficiently translocates to the nucleus following stimulation by cAMP and calcium (Fig 4.6).

Furthermore, we found that increasing CRTC1 levels in primary hippocampal neurons increased CRE-dependent transcription under unstimulated (basal) and stimulated [potassium chloride/forskolin (KCl/FSK) for 4 h] conditions (Fig 4.7). This observation was supported by the results of an with between factor Construct (CRTC1 plasmid, GFP plasmid) and within factor Stimulation [Stim (KCl/FSK), No Stim (unstimulated, vehicle control)] ANOVA which revealed a significant Construct X Stimulation interaction [F(1,12)

= 4.75; p < 0.05], as well as significant main effects of Construct [F(1,12) = 7.10, p < 0. 05] and Stimulation [F(1,12) = 9.10, p < 0.05]. Our finding that CRTC1 increases CRE- luciferase reporter activity in primary hippocampal neurons is consistent with previous results that overexpressing CRTC1 potentiates CRE-luciferase reporter activity and transcription of the CREB-target gene, BDNF, in stimulated primary hippocampal neurons and HEK cells (Zhou et al., 2006; Altarejos et al., 2008). Together, these data show that CRTC1 vector increases both CRTC1 levels and CRE-mediated transcription.

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Figure 4.624Cellular localization of CRTC1. Primary hippocampal neurons transfected with CRTC1 plasmid (green) show nuclear translocation of CRTC1 protein (red) following stimulation (KCl/FSK for 4 h). Scale bar = 20 μm

Figure 4.725CRTC1 increases CRE-reporter activity. Primary hippocampal neurons transfected with CRTC1 plasmid show increased CRE-dependent transcription following stimulation (KCl/FSK for 4 h).

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4.4.2 EXPERIMENT 1: Increasing CREB in the hippocampus facilitates consolidation of context fear memory that is only weakly acquired in control mice To examine the effects of increasing wild-type CREB function in dentate gyrus on contextual fear memory, we microinjected CREB or GFP vector 3 d prior to weak contextual fear conditioning which induces weak memory in wild-type mice (mice received a single 0.3 mA shock in Context A, CXT-A) (Fig 4.8a). During the fear conditioning session, all mice show similar activity levels, both prior to presentation of the shock [unconditioned stimulus, (US), (pre-US)], and during the 60 s post-shock (post-US). An ANOVA with between-group factor Vector (CREB, GFP) and within-group factor Shock (pre-US, post-US) showed no Vector X Shock interaction [F(1,20) = 0.49, p >

0.05], but a significant main effect of Shock [F(1,20) = 5.59, p < 0.05], with both groups showing higher freezing after the presentation of the mild shock during conditioning (Fig 4.8b). The day after conditioning, contextual fear memory was assessed by returning mice to the conditioning context (CXT-A). As expected, mice microinjected with GFP vector showed low levels of freezing when replaced in CXT-A; however, mice with CREB vector showed significantly higher levels of freezing. This was confirmed by t-test t(20) = 6.46, p<0.01.(Fig 4.8c). This finding confirmed, for the first time, that over- expression of activity-dependent CREB in the dentate gyrus prior to training is sufficient to support formation of hippocampal-dependent context memory that is not typically supported.

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Figure 4.826Increasing wild-type CREB in the dentate gyrus induced strong context fear long-term memory. (a) Schematic timeline for Experiment 1. (b) Freezing behaviour during the fear conditioning session. All groups show a slight increase in freezing following the presentation of the mild shock (post-US). (c) Freezing behaviour during the context memory test session. Microinjection of CREB vector in the dentate gyrus enhances long-term memory for contextual fear following weak conditioning.

4.4.3 EXPERIMENT 2: Increasing CRTC1 or CREB in the hippocampus facilitates consolidation of context fear memory Having demonstrated that CREB is sufficient to induce robust context fear memory following only weak conditioning in mice, we next investigated the novel effects of increasing CRTC1 or CREB function in dentate gyrus on the precision of contextual fear long-term memory. Here, we microinjected CRTC1, CREB, or GFP vector 3 d prior to weak contextual fear conditioning (mice received a single 0.3 mA shock in CXT-A) (Fig 4.9a), then tested their context long-term memory in both the conditioning Context-A, and novel Context -B.

During conditioning, all groups show similar activity levels, both prior to presentation of the shock, and during the 60 s post-shock. An ANOVA with between-group factor Vector (CRTC1, CREB, GFP) and within-group factor Shock (pre-US, post-US) showed

140 no Vector X Shock interaction [F(2,45) = 1.35, p > 0.05], but a significant main effect of

Shock [F(1,45) = 24.09, p < 0.01], with all groups showing higher freezing after the presentation of the mild shock during training (Fig 4.9b). Importantly, we observed no difference in shock reactivity between vectors. ANOVA did not reveal a significant main effect of Vector [F(2,45) = 0.33, p > 0.05].

The day after conditioning, contextual fear memory was assessed by returning mice to the conditioning context (CXT-A). As anticipated, mice microinjected with GFP vector showed low levels of freezing when replaced in CXT-A; however, mice with CRTC1 or CREB vector showed enhanced freezing (Fig 4.9c). To examine the specificity of this increased freezing, 5 h later we tested mice in a novel, alternate context not previously paired with shock (Context B, CXT-B). All groups, regardless of vector, showed similarly low freezing in CXT-B (Fig5.9d). An ANOVA with between-group factor Vector (CRTC1, CREB, GFP) and within-group factor Context (CXT-A, CXT-B) supported this observation and revealed a significant Vector X Context interaction [F(2,45) = 8.19, p <

0.001] as well as a significant main effect of Context [F(1,45) = 65.81, p < 0.001], but no main effect of Vector [F(2,45) = 3.02, p > 0.05]. Post-hoc analyses conducted on the significant interaction indicated that mice with either CRTC1 or CREB vector froze more in CXT-A than mice with GFP vector, but that all groups froze at equally low levels in CXT-B. Interestingly, we observed no difference between freezing levels in mice with CRTC1 or CREB vector. Testing mice first in CXT-B and then CXT-A produced similar results (significant Vector X Context interaction, [F(2,29) = 4.32, p < 0.05], significant main effect of Context [F(1,29) = 37.62, p < 0.001] and Vector [F(2,29) = 6.55, p < 0.001]) (Fig 4.9e). As shock reactivity and activity levels during conditioning was equivalent in CREB, CRTC1 and GFP control groups (Fig 4.9b,c), the increases in freezing behaviour and context discrimination seen in mice over-expressing CRTC1 and CREB cannot be attributed to differences in shock sensitivity during conditioning.

Therefore, increasing CRTC1 or CREB levels before training increased freezing in the context previously paired with shock, but did not enhance generalization of freezing to a

141 non-shock context. Over-expressing CRTC1 or CREB increased memory strength, without compromising memory specificity.

Figure 4.927Increasing CRTC1 or CREB levels in the dentate gyrus facilitates consolidation of weak contextual fear long-term memory; this enhancement is context-specific. (a) Schematic timeline for Experiment 2. (b) All groups show similar activity levels during the conditioning session (c), and similar reactivity to shock during conditioning. (d,e) Freezing behaviour during the context memory test session. Microinjection of CRTC1 or CREB vector in dentate gyrus before weak training (1 X 0.3 mA shock) enhances contextual fear memory. This memory enhancement is specific for the training context (CXT-A), and does not generalize to a similar, non-shocked context (CXT-B), regardless of context testing order [CXT-A then CXT-B (left) or CXT-B then CXT-A (right)]. Error bars = ± SEM.

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4.4.4 EXPERIMENT 3: Increasing CRTC1 or CREB in the hippocampus further enhances consolidation of robust /strong contextual fear memory We next examined the effects of increasing dentate gyrus CRTC1 or CREB function in context fear memory induced by a stronger training protocol (3 X 0.5 mA shocks in CXT-A) that produces strong contextual fear memory in wild-type mice (Fig 4.10a). During training, all groups show similar activity levels. An ANOVA showed no Vector X

Shock interaction [F(2,24) = 2.25, p > 0.05], but a significant main effect of Shock [F(1,24) = 266.41, p < 0.001], with all groups showing higher freezing after the presentation of the shock during training (Fig 4.10b). We observed no group differences in reactivity to the stronger shock stimulus [ANOVA, F(2,24) = 0.99, p > 0.05], Again, any increase in freezing cannot be attributed to group differences in shock sensitivity during stronger training (Fig 4.10c).

When tested in CTX-A, mice with CRTC1 or CREB vector showed higher levels of freezing than mice with GFP vector, but similar to Experiment 2, this memory enhancement was context-specific, as all groups showed low freezing in the no-shock context (Fig 10d) (significant Vector X Context interaction [F(2,24) = 4.90, p < 0.05], as well as significant main effects of Vector [F(2,24) = 3.77, p < 0.05] and Context [F(1,24) = 124.62, p < 0.001]. ]. Post-hoc analyses revealed that mice with CRTC1 or CREB vector froze more in CXT-A than mice with GFP vector, but that all groups showed equally low levels of freezing in CXT-B [p > 0.05]). Together, these results indicate that increasing CRTC1 or CREB function in the dentate gyrus prior to training enhances context fear memory induced by either weak or strong training. Furthermore, the enhanced memory produced by CRTC1 or CREB overexpression is specific and does not generalize to a novel context when tested after 24 h.

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Figure 4.1028Increasing CRTC1 or CREB levels in the dentate gyrus further enhances consolidation of strong contextual fear long-term memory. (a) Schematic timeline for Experiment 3. (b) All mice show similar activity levels during the training session, (c) and similar reactivity to shock during training. (d,e) Freezing behaviour during the context memory test session. Microinjection of CRTC1 or CREB vector in dentate gyrus before strong training (3 X 0.5 mA shocks) enhances memory for contextual fear; this memory is specific to the training context (CXT-A).

4.4.5 EXPERIMENT 4: Increasing CRTC1 or CREB in the hippocampus does not enhance retrieval/expression of a previously acquired context fear memory In the above experiments, we microinjected vectors prior to training such that mice were both trained and tested with high CRTC1 or CREB levels in the dentate gyrus. To examine whether the enhancement in freezing produced by CRTC1 or CREB vectors was due to facilitated memory retrieval/memory expression, we performed a similar experiment but microinjected CRTC1, CREB, or GFP vectors 24 h after weak training (Fig 4.11a timeline), at a time when synaptic consolidation is thought to be complete [see Dudai (2004) for review].

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During training, all groups show similar activity levels. An ANOVA showed no Vector X

Shock interaction [F(2,31) = 0.09, p > 0.05], but a significant main effect of Shock [F(1,31) = 11.94, p < 0.01], with all groups showing higher freezing after the presentation of the mild shock during training (Fig 4.11b). We observed no group differences in reactivity to the weak shock stimulus [ANOVA, F(2,31) = 0.40, p > 0.05], (Fig 4.11c).

When tested 4 d after microinjection, all groups froze at low levels in the shock-context (CXT-A) and still lower levels in the no-shock context (CXT-B) (no significant Vector X

Context interaction [F(2,31) = 0.69, p > 0.05] or main effect of Vector [F(2,31) = 1.45, p >

0.05] but a significant effect of Context [F(1,31) = 0.19, p < 0.001], in which all groups froze more in CXT-A than CXT-B) (Fig 2c). Therefore, increasing CRTC1 or CREB levels does not affect the expression of a previously acquired fear memory. Together, these results indicate that increasing CRTC1 or CREB function in the dentate gyrus before, but not after, context fear conditioning enhances long-term memory.

Figure 4.1129Increasing CRTC1 or CREB levels in the dentate gyrus does not facilitate retrieval of previously acquired context fear long-term memory. (a) Schematic timeline for Experiment 4. (b) All groups show similar activity levels during the training session, (c) and similar reactivity to shock during training (d) The enhancement of context memory by CRTC1 or CREB vector is not due to effects on memory retrieval/expression as microinjection of these vectors after training does not facilitate memory expression (in either context).

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4.4.6 EXPERIMENT 5: The context memory enhancement produced by increasing CRTC1 or CREB in the hippocampus at the time of conditioning is long-lasting

The above findings suggest that increasing CRTC1 or CREB at the time of conditioning enhances memory consolidation. Importantly, using this vector system, transgene expression peaks ~3 d following microinjection, and is absent by 10-14 d post- microinjection (Barrot et al. 2002; Vetere et al., 2011) (Fig 4.2). Therefore, to investigate whether the memory enhancement is enduring and persists beyond transgene expression, we trained mice as before (3 d following microinjection, at a time of high transgenic expression of CRTC1 or CREB) but tested mice long after transgene expression had dissipated (33 d following microinjection; see Fig 4.12a for timeline). During conditioning, all groups show similar activity levels. An ANOVA showed no

Vector X Shock interaction [F(2,34) = 0.39, p > 0.05], but a significant main effect of

Shock [F(1,34) = 5.32, p < 0.05], with all groups showing higher freezing after the presentation of the mild shock during training (Fig 4.12b). We observed no group differences in reactivity to the weak shock stimulus [ANOVA, F(2,34) = 0.53, p > 0.05], (Fig 4.12c).

When tested 30 d after conditioning, mice microinjected with CRTC1 or CREB vector froze higher in CXT-A than mice with GFP vector. This enhancement remained context- specific as all groups froze at equally low levels in CXT-B (significant Vector x Context interaction [F(2,34) = 5.44, p < 0.01], effect of Vector [F(2,34) = 5.90, p < 0.01] and Context

[F(1,34) = 37.35, p < 0.001] (Fig 4.12d). Therefore, increasing CRTC1 or CREB in the dentate gyrus prior to training facilitates the consolidation of remote context fear memory; this enhancement is both precise (observed only in the training context) and enduring (observed even after transgene expression had dissipated). Together, these results are consistent with the interpretation that increasing CRE-mediated transcription in the dentate gyrus around the time of training enhances memory consolidation. Once this memory has been consolidated, elevated CRE-mediated transcription is no longer necessary to maintain this context-specific memory.

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Figure 4.1230Increasing CRTC1 or CREB levels in the dentate gyrus facilitates remote memory consolidation, which maintains context precision. (a) Schematic timeline for Experiment 5. (b) All mice show similar activity levels during the training session, (c) and similar reactivity to shock during training. (d) Microinjection of CRTC1 or CREB vector before weak training enhances memory for contextual fear even when tested 30 d later (after transgene expression has dissipated). This memory enhancement maintains context specificity (only observed in CXT-A).

4.4.7 EXPERIMENT 6: Increasing CRTC1 or CREB in the hippocampus facilitates reconsolidation of context fear memory We found that increasing CRTC1 or CREB in the dentate gyrus prior to conditioning facilitates both recent (24 h) and remote (30 d) memory consolidation. Reactivation of a memory by re-exposure to the cues present during initial memory encoding may trigger a second wave of consolidation (referred to as reconsolidation). Similar to initial consolidation, reconsolidation also requires protein synthesis (Nader et al., 2000; Sara, 2000; Debiec et al., 2002) and intact CREB function (Kida et al., 2002; Mamiya et al., 2009; Tronson et al., 2012). Therefore, we next asked whether increasing CRTC1 or CREB in the dentate gyrus prior to remote memory reactivation would similarly enhance memory reconsolidation. We trained mice using weak training protocol but 2 d prior to

147 memory reactivation (placement in CXT-A for 45 s with no shock), we microinjected mice with CREB, CRTC1 or GFP vectors. The next day, mice were tested in CXT-A to examine the stability of the reactivated memory (memory reconsolidation). Again, memory specificity was assessed by testing mice in the no-shock context (CXT-B) (see timeline in Fig 4.13a).

During conditioning, all groups in either the memory reactivation (R) or no-reactivation (NR) groups show similar activity levels. For the reactivation group, ANOVA showed no

Vector X Shock interaction [F(2,27) = 0.59, p > 0.05], but a significant main effect of

Shock [F(1,27) = 15.03, p < 0.001], with all groups showing higher freezing after the presentation of the mild shock during training (Fig 4.13b). For the no-reactivation group, ANOVA showed no Vector X Shock interaction [F(2,15) = 1.34, p > 0.05], and no main effect of Shock [F(1,15) = 3.05, p > 0.05] (Fig 4.13g), suggesting no significant decrease in activity following the weak shock. Importantly we observed no group differences in reactivity to the weak shock stimulus for either set of groups (R group

[ANOVA, F(2,27) = 0.21, p > 0.05],Fig 4.13c); (NR group [ANOVA, F(2,15) = 0.17, p > 0.05], Fig 4.13h). As would be expected following weak fear conditioning, all groups froze at equally low levels during the memory reactivation session [F(2,19) = 0.72, p > 0.05] (Fig 4.13d). This finding is consistent with our previous data showing that increasing CRTC1 or CREB has no effect on memory retrieval or memory expression (Experiment 4, Fig 4.11d). However, when tested 24 h after memory reactivation, mice microinjected with CRTC1 and CREB vector showed significantly higher freezing than mice with GFP vector (Fig 4.13h). This enhancement in freezing was context-specific, as all groups showed equally low freezing when tested in CXT-B (significant Vector X Context interaction [F(2,

27) = 9.54, p < 0.001], as well as effects of Context [F(1, 27) = 39.06, p < 0.001] but not

Vector [F(2, 27) = 2.89, p > 0.05]). This finding is consistent with the interpretation that increasing CRTC1 or CREB prior to memory activation enhanced memory reconsolidation of remote memory.

To confirm that this memory enhancement was critically dependent on reactivation of the context fear memory at a time of high CRTC1 or CREB levels, we conducted a

148 control experiment where mice were not placed in CXT-A (no reactivation), but were maintained in the homecage 24 h prior to testing (see Fig 4.13f for timeline). When subsequently tested in both CXT-A and CXT-B, all mice showed equally low levels of freezing, regardless of vector or context (no significant Vector X Context interaction [F(2,

15) = 0.47, p > 0.05], main effect of Context [F(1, 15) = 4.32, p > 0.05] or Vector [F(2, 15) = 0.04, p > 0.05]) (Fig 4.13i). Therefore, the enhancement of memory reconsolidation by CRTC1 and CREB vector were critically dependent on memory reactivation.

Figure 4.1331Increasing CRTC1 or CREB levels in the dentate gyrus enhances reconsolidation of a remote contextual fear long-term memory. (a,f) Schematic

149 timelines for Experiment 6: (a) reactivation group, R, (f) no-reactivation group, NR. (b,g) All groups show similar activity levels during the training sessions, (c,h) and similar reactivity to shock during training. (d,e) Microinjection of CRTC1 or CREB vector before reactivation of an established weak contextual fear memory enhances subsequent memory expression in a context-specific manner. Naïve mice were fear conditioned with a weak protocol and 26 d later, were microinjected with vector. (d) 3 d following vector microinjection, all groups showed similar low levels of freezing when re-exposed to the training context (for 45 s). (e) In the subsequent test session (24 h later), mice with GFP vector showed low levels of freezing (in both contexts). However, mice with CRTC1 or CREB vector showed enhanced memory in the training context (CXT-A) but not in a non-shock context (CXT-B). (i) Memory reactivation is necessary for the enhancement of an established memory by CRTC1 or CREB vectors. Mice were trained as above, similarly microinjected with vectors, but were not re-exposed to the training context (no-reactivation, NR) after vector microinjection. During the subsequent test, all groups show equally low levels of freezing in both contexts.

4.5 DISCUSSION

Our studies are this first to identify a critical role for CRTC1 in both memory consolidation and reconsolidation. We show that increasing CRTC1 function enhances memory strength without compromising memory quality. To examine the effects of increasing CRTC1 levels on different memory phases, we used context fear conditioning. This task has often been used for investigating the molecular basis of consolidation and reconsolidation as infusions of the protein synthesis inhibitor anisomycin directly into the dorsal hippocampus around the time of training (synaptic consolidation) or memory reactivation (reconsolidation) disrupt subsequent memory expression (Quevedo et al., 1999; Taubenfeld et al., 2001; Debiec et al., 2002; Frankland et al., 2006; Suzuki et al., 2008; Mamiya et al., 2009). Moreover, by

150 examining fear in both a trained and a non-trained context, this task allowed us to assess the precision of the context memory.

We became interested in investigating the effects of increasing CRTC1 in context memory based on our preliminary findings using CREB vectors to increase context memory. Recall that one of our original questions for this study was: “Can wild-type CREB over-expression in the hippocampus facilitate context fear memory that is only weakly acquired in control mice?”

Wild-type CREB over-expression in the hippocampus facilitates context fear memory that is only weakly acquired in control mice. Consistent with the ‘CREB signature’ in which CREB facilitates long-term memory consolidation, our results confirm that CREB is sufficient to support contextual fear memory formation in normal mice under training conditions which do not typically support memory formation. Our data are in line with previous reports of enhanced contextual fear memory where infecting CA1 and dentate gyrus dorsal hippocampal neurons with constitutively active CREB vector has been shown to enhance consolidation of contextual fear memory (Restivo et al., 2009). However, unlike where they used constitutively active CREB, in which transcriptional activity is always turned on, our findings suggest that even low levels of transcriptional activity induced by activity-dependent CREB is sufficient to support this robust enhancement of contextual fear memory.

Based on this finding, we became interested in further exploring the precision of this contextual memory formed following enhanced CREB function. It remained possible that altered CREB function was not inducing a strong representation of the context memory, but rather inducing a generally fearful behaviour in new contexts. To address this point, we conducted the context discrimination experiments to examine the precision of the CREB-enhanced memory. We also wanted to test, for the first time, if increasing CREB co-activator CRTC1 in a regionally- and temporally-specific manner could also potentially have memory enhancing effects. We now address the findings from our

151 second question: “Can CRTC1 or CREB over-expression in the hippocampus facilitate recent context fear memory precision?”

Increasing CRTC1 or CREB in the hippocampus at the time of training facilitates consolidation of precise context fear memory. Next, we examined the precision of this contextual memory to determine if the memory for the fearful context is reliant on the precise contextual cues present during conditioning or if it generalizes to other similar contexts. If increasing CRTC1 levels simply enhanced non-specific aspects of this task (e.g., increased shock sensitivity or increased overall anxiety), then expression of fear would generalize to other similar contexts. We targeted our manipulation to the dentate gyrus , the primary relay station for inputs to the hippocampus (Treves et al., 2008), because previous studies show this region is particularly important in context fear acquisition and context discrimination (Lee and Kesner, 2004; Leutgeb et al., 2007; Hernandez-Rabaza et al., 2008; Nakashiba et al., 2012). We found that increasing CRTC1 or CREB levels in the dentate gyrus region of dorsal hippocampus prior to conditioning facilitates memory consolidation of a weak but context-specific fear memory. That is, we observed an increase in freezing only in the context previously paired with shock; this increase in freezing did not generalize to a similar no-shock context. This discrimination in mice with enhanced CRTC1 or CREB is also seen when the order of presentation of contexts is reversed so that mice are tested first in the novel context, followed by the shock-associated context. Similarly increasing CRTC1or CREB levels also enhanced precise contextual fear memory produced by even stronger training, in a context-specific manner.

Increasing CRTC1 or CREB in the hippocampus does not enhance expression of a previously acquired context fear memory. This CRTC1-induced enhancement in context memory consolidation is not due to an effect on memory retrieval/expression as similarly increasing CRTC1 levels after training did not affect the expression or specificity of a weak fear memory. Interestingly, increasing CREB levels produced 152 strikingly similar effects in all experiments. Therefore, increasing CRTC1 or CREB levels around the time of memory encoding enhances memory consolidation, while post-training manipulation of CRTC1 or CREB, outside of the window of synaptic consolidation (Guzowski and McGaugh, 1999; Dudai, 2004), has no facilitating effect on the retrieval/expression of a previously acquired memory. These findings are consistent with our similar manipulations of CREB following acquisition of a weak or strong spatial memory (Chapter 3, Experiment 4). Together, this supports the critical role of CREB or CREB co-activator activity during the formation of a hippocampal-dependent memory, but that the facilitating effects are limited to strengthening the consolidation/formation of the memory, and likely play a limited role in the retrieval process.

We have begun to understand the role of CRTC1 and CREB in different memory processes (ie. synaptic consolidation, retrieval). Our next question was about the duration of this CRTC1 or CREB-enhanced memory. We know that acutely increasing CRTC1 or CREB in the hippocampus facilitates the formation of a very precise context memory, but is this transient effect, or does it promote formation of an enduring, context-specific memory? So, we next asked: “Can CRTC1 or CREB over-expression in the hippocampus facilitate remote context fear memory precision?”

The memory facilitation produced by increasing CRTC1 or CREB in the hippocampus at the time of conditioning is long-lasting. As discussed, remote memories typically undergo a gradual reorganization across a cortical network. In lesion studies, post-training lesions of the hippocampus at a recent time point after training (24 h) abolish context fear memory and context discrimination. Lesions at a remote time point after training (~30 d) spare this fear memory, though both control and lesioned animals typically display reduced contextual discrimination at remote time points. This temporally-graded retrograde amnesia and generalization is commonly reported in rodents across hippocampal-dependent tasks (Winocur et al., 1990; Kim and Fanselow, 1992; Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009).

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In our experiment, the increase in memory produced by increasing CRTC1 or CREB levels at the time of training was long-lasting and was observed even after viral expression of CRTC1 or CREB dissipated. We took advantage of the relatively short time-course of transgene expression using HSV viral vectors (transgene expression dissipates after roughly 10 d) and conducted training at a time of high levels of CRTC1 or CREB but tested mice 30 d later, long after transgene over-expression. Mice with high CRTC1 or CREB at the time of training continued to show enhanced and context- specific memory when later tested, suggesting that once consolidated, this strong and precise context fear memory no longer required elevated CRTC1 or CREB function. While it may be that, in these mice, a distributed remote memory network exists, the hippocampal-dependent trace formed when mice had high hippocampal CRTC1 or CREB levels likely is sufficient to induce a robust, lasting contextually rich fear memory. It may be that testing the mice first in CXT-A, then later in CXT-B serves as a ‘reminder’ as in the reconsolidation experiments, and that this reminder in the training context is sufficient to reinstate context specificity of the fear memory. Alternatively, it may be increasing CRTC1 or CREB in hippocampal neurons during conditioning facilitated the synaptic consolidation of the context memory, and that these neurons continue to support the memory even at a remote time point. Unlike lesion or inactivation studies of remote memory, the neurons which had enhanced CRTC1 or CREB during conditioning are still intact in our study, and it is likely that these neurons are re-engaged during remote memory testing, although we did not test this in the present study. Previous studies in our lab have shown that lateral amygdala neurons with enhanced CREB during consolidation are selectively incorporated into the fear memory trace (Han et al., 2007). Targeted deletion of those key neurons prior to fear memory testing result in an erasure of the fear memory (Han et al., 2009). In addition to immediate-early gene imaging of active neurons, another way to determine if these neurons which had enhanced CRTC1 or CREB are still critically involved during remote context memory testing would be to lesion the hippocampus at a remote time point (ie. 30 d after context fear conditioning). It is predicted that, mice with enhanced CRTC1 or CREB during conditioning will no longer discriminate between contexts when tested following hippocampal lesioning at a remote time point. With the hippocampus no longer intact,

154 mice which initially retained highly specific contextual memory would rely on the less- contextually rich cortical trace supporting the remote memory, and it is predicted that they would exhibit freezing in both the conditioning context (CXT-A), and the novel context (CXT-B) (Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009; Wiltgen et al., 2010).

Now we know that CRTC1 or CREB enhancement at the time of training facilitates the formation of a precise, long-lasting context memory. But what happens if their levels are manipulated at a time long after the initial process of memory consolidation has passed? Based on our results in Experiment 4 in which we increased CRTC1 or CREB 24 h after context fear conditioning, we expect to see no effect of post-training manipulations of CRTC1 or CREB, especially at a much more remote time after context fear conditioning. But, what if we reactivated that remote context fear memory at a time when CRTC1 or CREB levels in the hippocampus were high? So our final question was: “Can CRTC1 or CREB over-expression in the hippocampus facilitate reconsolidation of context fear memory?”

Increasing CRTC1 or CREB in dentate gyrus facilitates reconsolidation of context fear memory. Memory retrieval is thought to be an active constructive process (Schacter et al., 1998) that functions to modify previously acquired memories (Sara, 2000; Dudai, 2006; Lee, 2009; Dudai 2012, Nadel et al., 2012). Here we showed that an established conditioned fear memory was strengthened (without further training) if simply reactivated at a time of high CRTC1 or CREB levels in the dentate gyrus. Specifically, we conditioned naïve mice using a weak protocol, and 26 d later, microinjected vector. Three days later, mice were re-exposed to the shock context (but did not receive a shock). It is important to note here that during the 45 s reminder session, both control mice and mice with CRTC1 or CREB vector show comparably low levels of freezing in the conditioning context, suggesting that CRTC1 or CREB manipulation at a remote time point following memory acquisition did not simply

155 facilitate the retrieval of a previously-acquired remote memory. When tested 24 h later, mice with GFP vector continued to show low conditioned fear memory (as expected). However, mice microinjected with CRTC1 or CREB vector prior to context re-exposure showed enhanced memory for the original conditioning context. This finding indicates that reconsolidation can strengthen an established memory can even relatively long after acquisition. Moreover, the quality of the memory was not affected, as conditioned fear did not generalize to the no-shock context. The enhanced memory was dependent on re-exposure to the training context, and not observed in similarly trained mice maintained in the homecage (no context re-exposure prior to testing). In this way, the memory enhancement produced by CRTC1 or CREB differs from that produced by overexpressing the atypical protein kinase C isoform, protein kinase M (PKM). For instance, Shema and colleagues (2011) found that virally increasing PKM expression in the insular cortex 6 d after conditioned taste aversion training (at a time when the memory trace had consolidated) enhanced subsequent memory even though rats were not re-exposed to the taste previously paired with illness. In the present experiments, memory enhancement was only observed when the memory was reactivated, showing that it is possible to increase the strength of an established memory by overexpressing CRTC1 or CREB during reconsolidation.

The majority of experiments investigating the molecular basis of memory consolidation, and especially reconsolidation, have examined whether a given molecule (or protein synthesis in general) is necessary for these processes by inferring normal function from loss-of-function studies. Although the results from these types of experiments have greatly increased our understanding of the mechanisms underlying memory, alternative interpretations to the observed behavioural deficits in memory reconsolidation experiments have been offered [i.e. the observed decrease in memory may be due to a temporary inability to access the memory trace (Lattal et al., 2004)]. Unlike previous reconsolidation studies using lesions or protein synthesis inhibitors to disrupt the second wave of gene expression during the re-stabilization of a reactivated fear memory, our gain-of-function approach allows for facilitation of the reconsolidation process by increasing the CRTC1 or CREB available for transcriptional activation

156 following context re-exposure. One advantage of this approach is that any enhancement in memory is unlikely to be easily attributable to memory retrieval effects.

As discussed in Chapter 1, the role of cAMP in reconsolidation of a reactivated memory was first introduced in 1998 by Roullet and Sara when they blocked the cAMP cascade using β noradrenergic receptor antagonist timolol following reactivation of well-learned spatial radial arm maze memory. When tested 24h following re-exposure to the training context, they found that blocking β noradrenergic activity 60 min (but not 30 min) following reactivation resulted in disruption of a well-consolidated memory, suggesting that there is a precise time window during which β noradrenergic receptors are critically involved in the re-activation of the cAMP pathway during reconsolidation of a memory (Roullet and Sara, 1998). Since then, it has become increasingly clear that CREB plays an important role in the reconsolidation of reactivated memories. Evidence from CREB repressor mice show that intact CREB function in required for the re-stabilization of both contextual and spatial memory, as well as tone fear memory (Kida et al., 2002; Suzuki et al., 2004; Mamiya et al., 2009).

So, while it has been demonstrated that disrupting CREB activity during this reconsolidation process is detrimental to, this is the first study to show a facilitation of the reconsolidation process by directly increasing CRTC1 or CREB levels in the hippocampus. Our findings are consistent with results from Tronson and colleagues (2006) who showed that infusions of a PKA agonist into the amygdala enhanced reconsolidation of a tone fear memory. As PKA may both phosphorylate CREB at Ser133 (Impey et al., 1998; West et al., 2002; Cohen and Greenberg, 2008) and promote nuclear translocation of CRTC1 (Bittinger et al., 2004), the present findings suggest a potential molecular mechanism for our result.

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4.6 CONCLUSION

Together, these studies have provided a novel and comprehensive view of the role of transcription factors CREB and the recently identified CREB co-activator CRTC1 in facilitating multiple memory processes supporting contextual memory consolidation, and reconsolidation (but not retrieval). These exciting new findings are the first step in characterizing the role of the CRTC family of genes in learning and memory. The overall implications of these findings will now be discussed in the general Discussion, Chapter 5.

4.7 Contributions:

Portions of this chapter are based on findings from:

Sekeres,M.J., Sargin, D., Mercaldo, V., Frankland, P.W., and Josselyn, S.A. (2012). Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory quality. Journal of Neuroscience (JN-RM-1419-12, under review)

MJS, PWF and SAJ conceived and designed the experiments and analyzed the data. MJS performed all surgery, behavioural experiments, and histology (excluding histology from immunohistochemistry and cell culture experiments). DS made the viral vectors. DS and VM performed immunohistochemistry and cell culture work. All authors contributed to writing the original manuscript.

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5 GENERAL DISCUSSION

We have shown the critical role of CREB and CREB co-activator CTRC1 in memory consolidation, and reconsolidation. While much of the reviewed literature focused on loss-of-function models to show CREB’s necessity in these processes, this is the first collection of studies to show that increasing CREB function in the hippocampus is sufficient to support both synaptic and systems consolidation.

Using limited training parameters that do not typically support spatial or contextual memory formation, we hypothesized that increasing hippocampal CREB function prior to training on these tasks would support consolidation under challenging conditions. We also hypothesized that enhancing CREB function during the initial synaptic consolidation period would have enduring effects on the systems consolidation and reconsolidation of remote context memory

The goals of this thesis were three-fold: 1. This is the first study to show that CREB is both necessary and sufficient to support spatial memory in the same model (addressed in Chapter 3). 2. This is the first study to show a role for CRTC1 in memory consolidation in an in vivo paradigm (addressed in Chapter 4) 3. It is the first investigation of the role of CREB (and CREB coactivators) in consolidation and reconsolidation of remote memory (addressed in Chapter 4).

5.1 CREB is sufficient to support consolidation of weak memory

Many behavioural studies in which CREB function is suppressed have suggested that CREB is necessary for long-term memory consolidation (Bourtchuladze et al., 1994; Kogan et al., 1997; Guzowski and McGaugh, 1997; Kogan et al., 2000; Kida et al., 2002; Pittenger et al., 2002; Balschun et al., 2003; Josselyn et al., 2004, Brightwell et al., 2005; Sekeres et al., 2010). Few have shown that increasing CREB is sufficient to 159 induce memory consolidation (Josselyn et al., 2001; Brightwell et al., 2007; Han et al., 2007; Restivo et al., 2009; Sekeres et al., 2010).

We established a spatial watermaze training protocol in which wild-type mice are given only a limited number of training trials across 3 days, and which reliably fails to induce spatial memory for the platform location in wild-type mice. By increasing CREB function via microinjections of CREB vector in this critical dorsal hippocampal CA1 region prior to training, we were able to induce formation of a strong spatial memory for the platform location in wild-type mice, while mice receiving control infusions failed to demonstrate any preference for the platform location. This suggests that CREB is sufficient to induce spatial memory under learning conditions which do not typically support consolidation in normal animals (Sekeres et al., 2010). Similarly, we found that acutely increasing CREB or the novel CREB co-activator, CRTC1, in the dentate gyrus prior to weak context fear conditioning induced consolidation of precise context fear long-term memory.

Spatial training induces changes in CA1 hippocampal levels of CREB phosphorylation, where there is a relationship between the duration and intensity of training, and the magnitude of increase in CREB phosphorylation (Porte et al., 2008). If limiting the intensity of training results in lower levels of CREB activation, this could potentially explain why control mice failed to develop a spatial memory for the platform location; however, by increasing the amount of CREB protein available for activation, we found that mice were able to form strong memory.

Our behavioural findings nicely compliment Barco’s findings using constitutively active CREB in the forebrain of bitransgenic mice (Barco et al., 2002). Constitutively active CREB remains in its active, phosphorylated state, unlike activity-dependent CREB, which requires phosphorylation of CREB in order to initiate the process of transcription. They found that weak stimulation of only a single tetanus of 100 Hz to the Schaeffer’s collaterals (a stimulus usually only capable of inducing transient E-LTP) is capable of inducing L-LTP. They argue that constitutively-active CREB expressing neurons have high levels of plasticity-related proteins available within the neuron, and that weak stimulation like a single train (or minimal training, in our case) can induce LTP (or LTM)

160 at the activated synapse by capturing the plasticity-related proteins available within the neuron and sending them to the activated (tagged) synapse. This model based on the ‘synaptic tagging and capture’ hypothesis proposed by Frey and Morris (1997) fits nicely with our findings. It suggests that even low levels of transcriptional activity induced by enhancing activity-dependent CREB prior to weak training is sufficient to support this robust enhancement of both spatial and contextual fear memory by increasing gene transcription and plasticity-related proteins available for the stabilization of memory that is on the cusp of consolidation.

5.1.1 Candidate CREB-target genes facilitating memory consolidation

CREB-regulated expression of candidate genes involved in regulating synaptic plasticity and enhanced memory consolidation include BDNF, NR4A2, and c-fos.

BDNF (brain-derived neurotrophic factor) is a CRE-mediated internal growth factor involved in modulating synaptogenesis, synaptic transmission, and spineogenesis (Poo et al., 2001; Gu et al., 2008; Hu et al., 2011). BDNF is commonly reported to be up- regulated in the hippocampus following tests of spatial memory, contextual, and cued fear memory (Kesslak et al., 1998; Hall et al., 2000; Mizuno et al., 2000; Rattinner et al., 2004). As BDNF mediates forms of plasticity required for consolidation within a neuronal network, suppression of BDNF results in impairment in spatial, cued fear memory (Mu et al., 1999; Rattinner et al., 2004). While there has yet to be any investigations of CRTC1- induced enhancement of BDNF in-vivo, treating hippocampal slices with CRTC1 resulted in an increase in BDNF mRNA synthesis in hippocampal neurons (Kovacs et al., 2007).

CREB-dependent transcription mediates the expression of other transcription factors, including c-fos, and NR4A2 (also known as Nurr1). NR4A2 is a nuclear receptor involved in regulating neuronal development. Suppression of NR4A2 has been associated with impaired spatial discrimination learning and long-term memory (Colon- Cesario et al., 2006). Following exposure to Ser/Thr kinase inhibitor staursporine, a promoter of CRTC dephosphorylation, mRNAlevels of NR4A2 was found to be 161 significantly elevated in culture (Ravnskjaer et al., 2007). CREB also regulates transcription of c-fos, an inducible transcription factor which, in turn, induces expression of other structural and growth-related proteins (Jeffrey et al., 1990; Worley et al., 1993; Herdenger and Leah, 1998). C-fos is known to be critical for memory formation c-fos knockout mouse models show impaired spatial memory (Paylor et al., 1994; Zhang et al., 2002), but like with the CREB hypomorphic mice, prolonged training in the watermaze was able to ameliorate the spatial memory deficits in c-fos deficient mice (Zhang et al., 2002). Similarly, targeted disruption of hippocampal c-fos using c-fos antisense ODN disrupted long-term spatial memory (Guzowski et al., 2002), and social- transmission of food preference (Countryman et al., 2005).

In cultured hippocampal neurons treated with CRTC1shRNA, there is a reported down- regulation of CREB-target genes c-fos, NR4A2, and BDNF in response to stimulation with forskolin and KCl, suggesting that functional CRTC1 is required for expression of these genes (Espana et al., 2010).

While these data suggest that CRTC1 is necessary for normal CREB-target gene expression, to date, no study has investigated the potential increase in CREB-target gene expression following CRTC1 enhancement. It would be interesting to examine levels of these candidate CREB-target genes following discrete contextual fear conditioning in mice with CREB or CRTC1 enhancement. We are currently collecting data examining c-fos expression in the gentate gyrus of mice which received microinjection of CRTC1 vector (or GFP control vector) prior to context fear conditioning (and in homecage control mice) to address this question.

5.2 CREB can further enhance strong memory formation

We also found that increasing CREB function in the hippocampus prior to strong training can even further strengthen memory consolidation. In their seminal study with Drosophila, where flies with an induced CREB activator isoform were able to form an 162 odour discrimination memory after only a single training session (as opposed to five sessions for control flies), Yin and Tully concluded that enhancing CREB function does not produce ‘stronger’ memory, but rather supports the formation of memory in fewer trials (Yin et al., 1995; Yin and Tully, 1996). We argue here that increasing CREB function can, in fact lead to stronger long-term spatial and context memory, at least when assessed at 24 h. Importantly, the strong fear memory induced by CREB enhancement is not abnormally persistent to extinction (Vetere et al., 2011), a point we will discuss in more detail shortly.

5.3 Suppressing CREB in a limited population of hippocampal cells does not impair consolidation.

While augmenting acute hippocampal CREB function with viral vectors leads to enhanced memory consolidation, similarly suppressing acute CREB function in a similar population of hippocampal neurons does not seem to have a negative impact on spatial memory formation. After injecting viral vectors encoding a mutant form of non- phosphorylable CREB (mCREB), we found that acute suppression of CREB function in a subset of dorsal hippocampal CA1 neuron does not impair spatial learning or memory in the watermaze (Sekeres et al., 2010) (Experiment 2, Chapter 3). Unlike more widespread CREB suppression models, impairing CREB function in only a limited number of hippocampal cells likely allows for uninfected neurons with normal CREB function to compensate for those with decreased CREB activity. This hypothesis is based on findings from the Josselyn lab using similar viral techniques to suppress CREB function in a limited population (~15%) of lateral amygdala neurons prior to tone fear conditioning. By visualizing immediate-early gene expression of Arc mRNA in lateral amygdala neurons immediately following fear conditioning, they determined that mCREB-infected neurons were selectively excluded from the active memory trace – in fact, mCREB-infected neurons were 11 X less likely to have been activated during fear conditioning than an uninfected neighboring neuron. Conversely, they found that lateral amygdala neurons infected with the CREB vector were preferentially incorporated into

163 the active memory trace – CREB-infected neurons were 3 X more likely to have been activated during fear conditioning than an uninfected neighboring neuron (Han et al., 2007). So, while it is estimated that between 30-40% of CA1 and approximately 2% of dentate gyrus neurons are active in a given spatial exploration task or learning event (Jung and McNaughton, 1993; Moore et al., 1996; Guzowski et al., 1999; Guzowski et al., 2000; Chawla et al., 2005; Vazdarjanova et al., 2006, Ramirez-Amaya et al., 2006, Tashiro et al., 2007; Schmidt et al., 2012), it is conceivable that neuronal selection would be biased towards CREB-infected (but not mCREB-infected) neurons in the hippocampus during spatial or context memory training.

5.3.1 Caveat

Infusion of a viral vector will not infect all cells within the targeted brain region. As a result, using mCREB vector is not an ideal method to infer CREB’s necessity in consolidation. Perhaps this explains why studies using mCREB vectors report mixed findings, depending on the brain region (hippocampus, striatum, or amygdala for example) and the task (social transmission of food preference impaired, but spatial memory, conditioned avoidance, and tone fear memory unimpaired) (Brightwell et al., 2005; Brightwell et al., 2008; Han et al., 2007; Sekeres et al., 2010). We found that suppression of CREB function in a limited population of hippocampal neurons with mCREB vector does not impair spatial memory consolidation (Experiment 2, Chapter 3). However, using this identical task and training protocol, we found that suppressing CREB function in an estimated 80-90% (Hummler et al., 1994; Blendy et al., 1996) of the brain does induce significant impairments in spatial memory consolidation in CREBαΔ-/- mice (Experiment 5, Chapter 3), suggesting that CREB in the hippocampus is necessary for spatial memory consolidation.

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5.4 CREB is necessary for memory consolidation

The above finding would suggest that normal spatial memory consolidation is impaired only when CREB function is disrupted in a very large population of hippocampal neurons. This is in agreement with previous research showing that more global suppression of CREB function results in impaired long-term memory consolidation (Bourtchuladze et al., 1994; Kogan et al., 1997; Guzowski and McGaugh, 1997; Kogan et al., 2000; Kida et al., 2002; Pittenger et al., 2002; Balschun et al., 2003).

The nature of memory deficits in CREBαΔ-/- mice has been shown to be sensitive to training intensities (Bourtchuladze et al., 1994; Kogan et al., 1997) and genetic background of the CREBαΔ-/- mice (Gass et al.,1998; Graves et al., 2002). Despite this, we found that CREBαΔ-/- mice reliably demonstrated long-term spatial memory impairment on our standard strong training protocol in the watermaze (Fig 3.8b,c,d). Notably, this is a training protocol with generous inter-trial intervals, and repeated daily session across 3 days, which wild-type control mice can learn with ease (Fig 3.4b,c,d).

Because the CREBαΔ-/- model involves a ubiquitous suppression of 80-90% of CREB throughout the lifetime of the mouse, it is conceivable that the CREB deficient mouse’s apparent spatial memory impairment may be due to other developmentally-related impairments, like motor dysfunction. Other labs have reported aberrant swimming behaviour in the watermaze, including high thigmotaxis (Gass et al.,1998; Balschun et al., 2003). We reported similar thigmotaxic behaviour in these CREB-deficient mice, but found that increasing CREB in the CA1 abolished this behaviour as mice adopted a more goal-directed, spatial search strategy in the watermaze. This suggests that the thigmotaxic phenotype in CREBαΔ-/- mice is due to the adoption of an inefficient search strategy, and not due to an anxiety-like behaviour or developmental impairment.

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5.5 CREB is both necessary and sufficient for memory consolidation

This brings us to the important conclusion that CREB is both necessary and sufficient in the same model. As discussed above, when mice were trained in the watermaze using a protocol that reliably induces robust spatial memory in normal wild-type mice, CREBαΔ-/- mice were severely impaired in both learning the platform location across days, as well as spatial memory during the probe test. We next demonstrated that it is possible to rescue this profound spatial memory deficit in these mice by acutely increasing CREB function in the dorsal hippocampal CA1 region. Following microinjection of CREB vector into CA1, CREB αΔ-/- mice subsequently formed robust spatial memory for the platform location in the watermaze at levels comparable to wild- type control animals. These findings suggest that CREB is, indeed, necessary for normal spatial memory consolidation, but that restoring CREB function to the critical CA1 region prior to learning is sufficient to rescue this deficit by supporting normal memory consolidation in these animals (Sekeres et al., 2010). While it can be argued that other brain regions also contribute to spatial memory consolidation, we can confidently say that the CA1 region of the dorsal hippocampus is critically involved in this type of memory consolidation, as replacing CREB in a limited population of CA1 neurons was sufficient to rescue this memory deficit (Experiment 5, Chapter 3).

It is not likely that we would not have seen the same rescue of context fear memory in these mice, due to the CREB dysfunction in the amygdala [known to be critical to for fear memory (Josselyn et al., 2001; Han et al., 2007; Han et al., 2009; reviewed in Rodrigues et al., 2004; Johansen et al., 2011). It might be interesting to determine if replacing CREB function in the hippocampus and the amygdala of CREB αΔ-/- mice is sufficient to rescue the contextual fear memory deficits exhibited by these animals (Bourtculadze et al., 1994; Kogan et al., 1997).

Taken together, our weak training experiments have confirmed the sufficiency of CREB to induce memory that is on the cusp of consolidation, and our memory rescue experiment in CREBαΔ-/- mice have shown, for the first time within the same model, that CREB is both necessary and sufficient to support long-term memory consolidation. 166

5.6 CREB facilitates the consolidation, but not the retrieval of memory

We next examined the memory retrieval process. Given that CREB is a transcription factor involved in mediating the transcription of genes critically involved in regulating synaptic plasticity (Bailey et al., 2000; Kandel, 2001; Cohen and Greenberg, 2008) and learning and memory (Bourtchuladze et al., 1994; Yin et al., 1994; Ding et al., 1997; Silva et al., 1998; Lonze and Ginty, 2002; Won and Silva, 2008; Alberini 2009, Josselyn, 2010) we next confirmed that enhancing CREB (or the CREB co-activator CRTC1) in the hippocampus after the wave of synaptic consolidation induced by a learning task did not have the same facilitation of memory expression upon retrieval. Estimates of the period of synaptic consolidation are typically under 24 h [(Dudai , 2004) but it has been argued that interference with the post-acquisition stabilization can interfere with consolidation for up to 102 h following memory acquisition, (Sutherland and Lehmann, 2011)].

Our post-training manipulations of CREB in spatial memory, or CREB and CRTC1 in context memory, did not facilitate the retrieval/expression of weak or strong spatial or contextual memory. This is in contrast to findings out of Barco’s lab, who found that inducing expression of constitutively-active CREB throughout the forebrain prior to the retrieval of a previously acquired spatial memory impaired the subsequent expression of that spatial memory in the watermaze (Viosca et al., 2009). The discrepancy in our findings may be attributable to a dose-dependent effect, where we only manipulated wild-type CREB function in a limited population of neurons the hippocampus, whereas their inducible transgenic model turns on constitutively-active CREB in all excitatory neurons in the hippocampus, and other regions including the amygdala and striatum.

We saw no enhancement in the expression of previously acquired spatial or context memory during retrieval. This is not surprising, as you would not expect the emergence of a non-specific enhancement of memory for events long preceding microinjection of vector. However, it would have been interesting to have retested the mice 24 h after the spatial memory retrieval test to see if we might see a subsequent enhancement of the reactivated spatial memory in the presence of high hippocampal CREB (or CRTC1) (Fig 167

5.1, probe 3). This experiment likely would not have as clean of an enhancing effect in a test of context memory following retrieval testing due to the confounding effect of retrieval testing in both conditioning Context-A and novel Context-B.

Figure 5.132Predicted enhanced spatial memory following retrieval in the presence of high hippocampal CREB. a) Proposed timeline to re-test a reactivated spatial memory following CREB enhancement. b) Results from probe 1 and 2 are adapted from Experiment 4, weak training, Chapter 3. It is possible that spatial memory retrieval (probe 2) would reactivate the hippocampal-dependent spatial memory in the presence of increased hippocampal CREB or CRTC1. When tested 24 h after reactivation (probe 3), we would expect an enhancement of the spatial memory following reconsolidation of the spatial memory.

Finally, in the case of the spatial memory retrieval experiment (Experiment 4, Chapter 3), mice that received weak training in the watermaze failed to form a robust spatial memory (Fig 3.6b). Post-training CREB enhancement did not facilitate spatial memory expressed during a second probe test in these mice. It would have been interesting to

168 then re-train the mice on a new platform location in the watermaze (now in the presence in high CA1 CREB) to determine if we subsequently see a facilitation of new learning and memory for the new platform location within these same animals (Fig 5.2).

Figure 5.233Predicted enhanced memory following new learning in the presence of high hippocampal CREB. a) Proposed timeline to train mice on a new platform location following CREB enhancement. b) Results from probe 1 and 2 are adapted from Experiment 4, weak training, Chapter 3). It is predicted that mice subsequently given weak spatial watermaze training in the presence of high hippocampal CREB or CRTC1 will form robust spatial memory for the new platform location.

5.7 CRTC1 is sufficient to support memory consolidation in vivo

The second major goal in of this thesis was to be the first to show a role for CRTC1 in in vivo memory consolidation. Early studies investigating CRTC1 found that it enhances several forms of synaptic plasticity. In developing cortical neurons (both in vitro, and in vivo), enhancing CRTC1 increased structural plasticity (Li et al., 2009). In hippocampal

169 slice, stimulation of Schaeffer’s collaterals of brains previously infected with CRTC1 vector showed a lower threshold for induction of LTP, and increased maintenance of L- LTP (Zhou et al., 2006).

Previous studies examining expression patterns of the CRTC family of proteins in the brain only reported expression levels across brain regions (Conkright et al., 2003; Kovacs et al., 2007; Watts et al., 2011). Our study was the first characterization of CRTC1 expression exclusively in excitatory neurons (but not in interneurons, or glia) (Fig 4.1). In hippocampal neurons transfected with CRTC1-containing plasmids, we confirmed that CTRC1 functionally translocates to the nucleus following stimulation with Ca2+ and cAMP agonists (Fig 4.6). We also showed that infection with our activity- dependent CRTC1 vector induced an increase in hippocampal expression of CRTC1 protein in vivo (Fig 4.5).

Finally, our studies were the first to show in vivo that increasing CRTC1 can facilitate recent memory (24 h), and remote memory (30 d) consolidation. Increasing CRTC1 induced remarkably similar facilitation effects to CREB. Given the rich literature on CREB’s role in memory consolidation, our findings that CRTC1 can similarly regulate memory opens the door to many additional studies of CRTC1 and behaviour. It will be important to investigate CRTC1 suppression models using dominant-negative CRTC1 viral constructs, or knock-out models to determine if CRTC1 function is also necessary for memory consolidation. Recently, a CRTC1 deletion model was generated in Cardinaux’s lab (Breuillaud et al., 2012). They have published a review of the behavioural phenotype, reporting that CRTC1-/- mice display impulsive aggression towards a novel mouse, social withdrawal, decreased sexual motivation, behavioural despair in a forced swim test, increased anxiety in an open-armed maze, and anhedonia in response to an appetitive sucrose solution. They suggest that CRTC1 is involved in regulating depressive mood disorders, but have yet to perform a thorough characterization of the cognitive phenotype of CRTC1 -/- mice.

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Taken together, our findings are the first characterization of CRTC1 in long-term memory consolidation.

5.8 CREB and CRTC1 promote consolidation of precise remote memory

Most investigations into CREB’s role in memory consolidation have focused on protein synthesis-dependent synaptic consolidation. In these cases, memory is typically tested 24 h after acquisition. As extensively discussed in the Introduction (Chapter 1), there have been many reported cases where CREB disruption immediately following memory acquisition disrupts memory when tested at 24 h (long-term memory), but not when tested at 30 min (short-term memory) (Roullet and Sara, 1999; Kogan et al., 2000; Bourtchuladze et al., 2004; Brightwell et al., 2005). This has been taken as evidence that CREB-dependent protein synthesis is required for long-term memory. While 24 h is considered long-term memory from the perspective of synaptic consolidation, from the perspective of systems consolidation 24 h is typically used as a measure of ‘recent’ memory.

To our knowledge, no other studies have looked at the role of CREB in the consolidation of remote memory. While the magnitude of the timescale of ‘remote memory’ differs between the human and the animal literature, a range between 28-50 d is typically used as a measure of remote fear memory in rodents (Kim and Fanselow, 1992; Anaganostaras et al., 1999; Debiec et al., 2002; Frankland et al., 2004; Teixeira et al., 2006; Wiltgen and Silva, 2007; Winocur et al., 2007, 2009; Wang et al., 2009).

In our experiment, mice received the weak fear conditioning protocol (a single low amplitude shock during one conditioning session). Using this protocol, GFP-injected control mice consistently failed to form a memory for the context when tested at a recent (24 h), or a remote (30 d) time point following training. There is evidence that stronger, and more prolonged training (repeated trials of multiple shock in the conditioning context, Context-A, and repeated exposure to the safe, non-shock associated context,

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Context -B) results in a strong memory that maintains context precision even at a remote timepoint (Wang et al. 2009), whereas single-trial context fear conditioning produces the predicted memory generalization when tested 30 d after training (Wang et al., 2009). It is remarkable that increasing CRTC1 or CREB in the hippocampus at the time of training induced strong, context-specific memory at a remote timepoint, similar to what Wang and colleagues found by using strong, repeated training. Again, this finding is reminiscent of the early findings in Drosophila, where Yin and Tully found that inducing a CREB-activator isoform in flies just prior to an odour discrimination task, could induce long-term memory after only 1 (as opposed to 5) conditioning sessions (Yin et al., 1995).

The maintained precision of context remote memory in mice that had enhanced CRTC1 or CREB during memory acquisition appears to be consistent with the Standard Consolidation Theory which asserts that a remote memory gradually becomes independent of the hippocampus, and represented in its identical form in neocortical structures (Squire, 1992; Squire and Alvarez, 1995; Squire and Zola, 1998). This theory argues against context generalization of remote memory.

An alternate interpretation is that by increasing hippocampal CRTC1 or CREB, we enhanced the synaptic consolidation within the hippocampus, which strengthened the hippocampal-dependent representation of memory for the contextual details. The Transformation Theory argues that, at a remote time point, both a context-general memory (thought to be represented in the neocortex), and the context-specific memory (represented in the hippocampus) trace can exist in parallel (Winocur et al., 2007; Winocur et al., 2011; Winocur and Moscovitch, 2011; Nadel et al., 2012). When we tested our mice long after CRTC1 or CREB transgene had dissipated, it is possible that by strengthening the hippocampal-dependent consolidation of the memory during acquisition, the hippocampal-dependent context-specific memory trace dominated over the cortical context-general trace upon testing, resulting in the emergence of the precise context memory.

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It would be interesting to follow-up on this hypothesis using immediate-early gene expression imaging at a remote timepoint to determine if we see an increase in hippocampal activation accompanying expression of precise context memory. Conversely, we might expect an increase in activation of cortical areas such as the anterior cingulate cortex (ACC), or prefrontal cortex (PFC) accompanying generalized context fear memory. It has been demonstrated that control rats trained with a robust context fear conditioning protocol in CXT-A can be separated into ‘generalizers’ (animals that show high freezing in both CXT-A and CXT-B) and ‘discriminators’ (animals that show high freezing in CXT-A but low freezing in CXT-B) when tested 14 d after conditioning (Wiltgen et al., 2010). Here, they found that discriminators exhibited high Arc and c-fos expression in the dorsal hippocampus, whereas generalizers showed relatively low hippocampal activation during testing, suggesting that the dorsal hippocampus continues to be important for the expression of precise contextual detail, even at a remote timepoint.

5.8.1 Caveats

It is also possible that the context-specific remote memory (where mice froze in Context-A, but not Context-B), was influenced by the test order. In the remote memory experiment (Experiment 5, Chapter 4), mice were always tested first in Context-A, (followed 5 h later by testing in Context-B). These test order effects may be a limitation to our interpretation of the memory precision. By first testing in the familiar Context-A, we may have reactivated the context-specific hippocampal-dependent memory. This may have served as a context ‘reminder’, which reinstated context specificity of the fear memory during the second test session. To address this, we could first test mice in the novel Context-B to see if, at a remote time point, we see the emergence of the context- general memory (which would be inferred by high freezing in Context-B). A previous study using a reconsolidation paradigm found that a Context-B ‘reminder’ is not effective in restoring context-specificity of a remote context memory (Winocur et al., 2009). As a result, subjects continued to exhibit high freezing in both contexts during the subsequent test session. However, if mice trained with high CRTC1 or CREB continued 173 to maintain low freezing first in Context-B, followed by high freezing in Context-A, we could be confident in saying that increasing CREB or CRTC1 during initial synaptic consolidation has persistent effects on systems consolidation of remote memory, which serves to strengthen the contextually-rich, hippocampal-dependent memory trace.

5.8.2 Are CREB or CRTC1-enhanced neurons required for the memory at a remote timepoint?

To test the continued role of CREB or CRTC1-enhanced neurons at a remote timepoint in weakly trained animals, we could lesion the hippocampus at 30 d post acquisition to see if freezing generalizes to both Context-A and Context-B. We know from published studies that most context fear memories undergo a time-dependent reorganization (Frankland and Bontempi, 2005; Moscovitch et al., 2006), and a transformation of the quality of memory from a richly detailed, hippocampal-dependent trace, to a more schematic cortical trace (Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009; Winocur et al., 2010). If mice over-expressing CREB or CRTC1 at the time of training only form hippocampal-dependent, persistent, context-specific memories, then you would expect that lesioning the hippocampus at a remote timepoint would result in an abolishment of freezing in both Context-A and in novel Context-B. If the mice trained with high CREB or CRTC1 have formed both a hippocampal-dependent context-specific memory, and a more generalized/schematic cortical representation of the fear memory, then you would expect that lesioning the hippocampus would result in spared freezing in both contexts.

5.8.3 Is this CREB-enhanced remote memory abnormally persistent?

Similar to our experiments, Vetere and colleagues found that acute over-expression of constitutively active CREB in the dentate gyrus enhances context fear memory at 24 h. They determined that this fear memory is not abnormally persistent, as it extinguishes following repeated and prolonged testing trials (extinction training) (Vetere et al., 2011).

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One could argue that our CREB-mediated enhancement of remote memory might suggest that we are inducing abnormally strong, persistent context fear memory in these mice. However, one important difference here is that our mice did not undergo typical extinction training to test this hypothesis. Extinction is not merely forgetting of a memory, but rather is a new type of learning, in which the mouse learns that a previously unsafe context is now safe. In our experiment, it is likely that if we had done a prolonged extinction trial in the conditioning context shortly after context fear memory acquisition, we would not have seen persistent memory at 30 d following extinction training. However, whether strengthening the consolidation of the fear memory increases the likelihood of incubation or spontaneous recovery of fear has not been investigated.

5.8.4 Time dependent structural reorganization of remote memory

Given that candidate regions for remote memory storage have been proposed (Bontempi et al., 1999; Frankland et al., 2004; Maviel et al., 2004; Frankland et al., 2006; Teixiera et al., 2006), it would be interesting to increase CREB or CRTC1 in neocortical regions such as the ACC or PFC at a remote timepoint to see if we can enhance the remote context memory, and specifically if we can enhance generalization of the context memory thought to be represented in the neocortex. This is the converse approach to that taken by Vetere et al. (2011) where they infused MEF2, a transcription factor involved in negatively regulating dendritic spine growth, into the ACC following context fear conditioning. They found that post-training microinjections of MEF2 in the ACC resulted in decreased spine density in the ACC at a remote timepoint, and impaired remote context fear memory. This suggests that time-dependent structural changes in the neocortical network are necessary to support systems consolidation, and that interfering with this process by restricting spine growth impairs remote memory consolidation. It is conceivable that taking the converse approach and increasing CREB levels in the ACC following context fear conditioning would produce an increase in dendritic spine density in CREB-infected neurons (Marie et al., 2005), at a remote timepoint, which might actually facilitate remote memory consolidation. 175

5.9 CREB or CRTC1 strengthen reconsolidation of remote memory

5.9.1 The window for reconsolidation

Early reconsolidation studies focused on disrupting protein synthesis within the amygdala following reactivation of a conditioned tone fear memory (Nader et al., 2000). Similar disruption of reconsolidation have been investigated in the hippocampus where delivery of protein synthesis inhibitors (PSI) block reconsolidation of context fear memory (Debiec et al., 2002) or inhibitory avoidance (Milekic and Alberini, 2002; Boccia et al., 2004; Boccia et al., 2007; Inda et al., 2011). Following reactivation, the time window for reconsolidation is estimated to begin within 3-10 min, and to last for at least 1 h (Monfils et al., 2009), during which time restabilization of the memory is susceptible to disruption with PSIs (Nader et al., 2000; Monfils et al., 2009). By 6 h following reactivation, the memory is no longer susceptible to disruption of protein synthesis (Nader et al., 2000; Duvarci and Nader, 2004).

5.9.2 CREB or CRTC1 is necessary for reconsolidation

The necessity of CREB in hippocampal-dependent reconsolidation was first hinted at in the late 1990’s (Roullet and Sara, 1998; Przybyslawski et al., 1999; Sara, 2000), then directly demonstrated by Kida’s lab over the next decade. They found that post- reactivation suppression of CREB in the forebrain resulted in impaired restabilization of the context memory 24 h later (Kida et al., 2002; Mamiya et al., 2009). Building upon Kida’s findings, most recently, Tronson and Taylor (2012) have demonstrated that targeted infusions of mCREB into the amygdala prior to memory reactivation also impairs the restabilization of a tone fear memory. These findings suggest that CREB is required for the protein-synthesis dependent restabilization of a reactivated memory; post-reactivation disruption of CREB function results in disruption of memory restabilization. In the hippocampus, Mamiya et al. (2009) report increased pCREB in CA1 and CA3 following context reactivation, but no increase in the dentate gyrus following a 3 min reactivation session in the conditioning context. This pattern of pCREB

176 activation following context fear conditioning has been reported elsewhere (Impey et al., 1998). The lack of detectable pCREB activation and CRE-reporter activity in the dentate gyrus is surprising, given our findings. However, it is very likely that the non-detectable increase in CRE-activity may be due to the sparse activity pattern of the dentate gyrus. An estimated 40% of neurons within CA1 and CA3 regions are activated following exposure to a novel context, as in context fear conditioning (Guzowski et al., 1999). As a result, the magnitude of CRE-activation in these regions is going to be much higher in CA1 and CA3 than in the sparsely coding neurons of the dentate, which likely obscured any relative increase in CRE-expression they might have seen following context memory reactivation.

5.9.3 CREB or CRTC1 is sufficient for reconsolidation

The sufficiency of CREB for strengthening memory reconsolidation was indirectly tested by Tronson and colleagues in 2006. They infused a cAMP agonist into the amygdala prior to reactivation of a tone-fear memory to strengthen memory over multiple reactivations. Our study is the first direct demonstration that CREB (and the novel CRTC1) strengthens memory reconsolidation. Even more novel is the fact that we found that CREB and CRTC1 are capable of supporting reconsolidation of a remote memory.

Our data argue that the hippocampus is actively engaged in reconsolidation of remote memory. By increasing the transcriptional capacity of neurons within the dentate gyrus prior to reactivation, we enhanced the protein-synthesis dependent restabilization of a reactivated weak fear memory. Upon restabilization, the increased CREB or CRTC1 actually strengthened the precision of the context memory when tested 24 h later. In the absence of reactivation however, increasing CREB or CRTC1 had no effect on the expression of the previously acquired remote memory (Experiment 6, Chapter 4), with mice expressing low, generalized freezing in both test contexts.

Though we do observe context-generalized freezing in controls at the remote time here and in Experiment 5 (Chapter 4), the memory is too weak to be able to say that what we are actually observing a context-general fear memory. Also, using the weak training 177 protocol, control mice always exhibit his pattern of weak, generalized freezing (even at recent time points following training). Training with a higher intensity protocol to induce strong, precise memory formation in controls could address the question of systems consolidation and reconsolidation of remote memory.

5.9.4 How does CREB or CRTC1 change the representation of a strong remote context memory?

Based on our Experiment 3 (Chapter 4), in which both GFP controls and mice over- expressing CREB or CRTC1 were trained using the strong shock protocol, we see robust, context-specific freezing (high freezing only in conditioning Context-A) in all groups when tested 24 h after memory acquisition. It would next be interesting to investigate the freezing behaviour of mice at 30 d (instead of 1 d) after strong training. Based on the literature, in GFP controls, we would expect to see high freezing in both Context-A and Context-B at this remote time point (Wiltgen and Silva, 2007; Winocur et al., 2007; Wang et al., 2009) (Fig 5.3f, grey). Similarly, if we were to perform our reconsolidation experiment (Experiment 6, Chapter 4) using the strong training protocol to induce normal long-term memory formation in controls, we would similarly predict a generalized context memory when mice were microinjected with GFP vector, then tested 30 d after training (4 d after GFP vector) in the ‘No Reactivation’ group (Fig 5.3g, grey). However, in the ‘Reactivation’ group, where GFP control mice are replaced briefly in the conditioning context (in the absence of shock), then tested 24 h later, we would expect to see a re-instatement of context specific memory, where mice should display high freezing only in Context-A during the post reactivation test (Fig 5.3h, grey).

What we do not know is how enhancing CREB or CRTC1 during acquisition of a strong context fear memory would affect the memory representation a remote timepoint, As depicted below, (Fig 5.3f, blue), if potentiating the hippocampal-dependent consolidation strengthens the hippocampal trace, it is possible that these mice will continue to express the context specific memory upon testing at a remote timepoint. In the reconsolidation paradigm, it is predicted that mice microinjected with CREB or

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CRTC1 vector at a remote timepoint following acquisition, then tested without reactivation 30 d after memory acquisition would display a generalized context memory (freezing in both contexts) (Fig 5.3g, blue). Here, we would not expect that increasing CREB or CRTC1 at a time long after the window for synaptic consolidation had closed to facilitate memory retrieval or expression. However as we demonstrated using the weak training protocol in Experiment 6 (Chapter 4), reactivating the remote fear memory with a brief context reminder in the presence of high hippocampal CREB or CRTC1 expression should re-instate context specificity of the reconsolidated memory (Fig 5.3h, blue)

Figure 5.334Predicted freezing patterns for remote memory and remote reconsolidation of context memory following strong training. The ‘weak training’ graphs (top row) are schematics of the general pattern of findings presented in Chapter

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4. Notably, due to the weak training protocol, controls (GFP) show low freezing in both the conditioning context (CXT-A) and the novel context (CXT-B).The ‘strong training graphs’ (bottom row) are predictions of freezing behaviour if we were to have tested mice in the same way as above, but using a strong training protocol to induce normal long-term memory formation in controls. Results from the ‘Strong-Recent’ graph (e) come from Experiment 3 (Chapter 4).

5.9.5 Boundary conditions to reconsolidation

In controls, we do not find evidence of reconsolidation using our weak context fear conditioning paradigm (Fig 4.13e). This may be due to several factors, including the weak strength of training, as well as the long delay between memory acquisition and testing. The literature has identified several boundary conditions which govern whether reconsolidation will be induced following reactivation of a memory (reviewed in Tronson and Taylor, 2007; Nader and Hardt, 2009; Nader and Einarsson, 2010; Finnie and Nader, 2012). Among the boundary conditions are the training strength (weak, single shock), reminder duration (brief, 45 s), and the age of the memory (remote, 29 d). Given these boundary conditions, it is not surprising that we do not see evidence of reconsolidation occurring in controls. What is even more remarkable is that we do see evidence of reconsolidation in our CREB and CRTC1 groups. Typically more strongly trained, and older memories require much longer reactivations in order to become labile and susceptible to interference during the reconsolidation process (Suzuki et al., 2004; Frankland et al., 2006; reviewed in Finnie and Nader, 2012). In our study, a brief reminder of a weak context memory was sufficient to induce strengthening on the remote context memory following reconsolidation in mice over-expressing hippocampal CREB or CRTC1. This suggests that perhaps it is more difficult to wipe out an established memory following reactivation than it is to strengthen it. Importantly in the absence of reactivation, there was no effect of increasing CREB or CRTC1 in the hippocampus just prior to remote memory testing.

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5.10 General caveats

Subjects were always tested during the ‘light phase’ of their 12 hr light-dark cycle. As mice are nocturnal, the ‘light phase’ during which the subjects were tested occurs during their sleep phase. While it would have been ideal to test the mice during their active dark phase, technical and logistical limitations of the behavioural testing facility did not permit reverse light cycle testing for these experiments. Hormonal expression patterns differ during the sleep and active cycles [ie. increases in hippocampal acetylcholine (ACh) and serotonin (5-HT) expression during REM sleep (Marrosu et al., 1995; Park et al., 1995; Diekelmann and Born, 2010) which may have affected memory consolidation.

Cholinergic activation of muscarinic receptors M1 and M4 have been shown to enhance cAMP signaling (Taussig et al., 1993; Dittman et al., 1994; Migeon and Nathanson, 1994), and to release intracellular Ca2+ stores, resulting in an increased expression of calcium-sensitive adenylyl cyclase (Choi et al., 1992). As adenylyl cyclase and cAMP both modulate PKA-induced phosphorylation of CREB at serine 133, it has been suggested that the increased hippocampal expression of ACh during post-learning REM sleep regulates CREB-regulated memory consolidation and hippocampal synaptic plasticity, through enhancement of PKA during REM sleep (Hasselmo, 1999; Graves et al., 2001).

Similarly, serotonin is thought to regulate theta oscillations during REM sleep (Graves et al., 2001; Sorman et al., 2011). However, different 5-HT receptors have been shown to either facilitate or impair memory consolidation (see Buhot, 1997 for review). However, several studies have shown that serotonin is critical for cAMP-regulated long-term synaptic facilitation (Kakiucki et al., 1968a,b, 1969; Bartsch et al., 1998; Liu et al., 2011). It is possible that high serotonin during sleep following the behavioural training and testing phase may have facilitated long-term memory consolidation in our subjects, although this was not measured.

Also, sleep is thought to play an important role in the stabilization of memory consolidation via hippocampal replay during slow wave sleep (Wilson and McNaughton, 1994). During exploration of an environment, ensembles of place cells show place-

181 specific firing within a spatial exploration task. Following spatial exploration, hippocampal place cells will exhibit a similar firing ensemble pattern with intermittent synchronized bursts of activity during slowwave sleep (Ranck et al., 1973; Pavlides and Winson, 1989; Wilson and McNaughton, 1994; Nadasdy et al., 1999; Ji and Wilson, 2007). In our case, it is possible that having the mice return to sleep immediately following spatial or contextual behavioural training also facilitated the stabilization of memory consolidation via hippocampal-replay of firing patterns which occurred during learning. These are interesting possibilities that could be further explored.

Although we cannot rule out any potential contributing effects of sleep (altered hormonal expression, hippocampal replay), we can assure that all subjects were trained at approximately the same time each day across experiments. Given this, any confounding factors related to changes in circadian arousal would be held constant for all subjects (and all groups) across experiments.

5.11 Practical applications of CREB enhancement in disease and aging

Finally, it is time to step outside the highly controlled experimental world, and consider the potential application of our findings to models of cognitive disorders involving CREB dysfunction, and normal aging.

5.11.1 CREB and aging

The decline in memory, particularly hippocampus-dependent memory, is a well-known consequence of aging (Craik, 1990; Albert, 1993). CREB protein and CREB activity levels in the brain also decline with age. For instance, Porte et al. find that phosphorylated CREB (pCREB) levels in CA1 and dentate gyrus of aged mice are lower than in young mice. Moreover, following training on a spatial memory task, they report

182 a positive correlation between CA1 pCREB levels and spatial memory performance in old mice, suggesting that the age-related decrease in hippocampal pCREB may be partially responsible for age-related memory impairment (Porte et al., 2008b). This decrease in pCREB of aged mice was accompanied by a decrease in CRE-dependent transcription of c-fos in old mice relative to young mice following spatial memory training (Porte et al., 2008b). Total CREB levels in the brain are also sensitive to age-related decline. Western blot analyses of total hippocampal CREB protein revealed reductions in aged rats, with a correlation between impaired spatial memory and reduced CREB protein (Brightwell et al., 2004). Interestingly, they found that aged rats with unimpaired spatial memory had hippocampal CREB protein levels comparable to young rats.

This finding leads to the appealing hypothesis that at least some of the memory deficits associated with aging may be overcome by enhancing CREB function. In fact, Mouravlev and colleagues found that microinjections of rAAV-CREB vector into the hippocampus (CA subfields and dentate gyrus) of young rats resulted in significant savings in spatial memory and passive avoidance performance when later tested at 15 months of age (Mouravlev et al., 2006). As rAAV is a viral method designed to stably and persistently express the transgene (Barco and Marie, 2011), the memory savings seen in CREB-overexpressing rats suggest that CREB enhancement throughout the adult life of the animal conferred a cumulative benefit of increased synaptic plasticity (Barco et al., 2002) and intrinsic neuronal excitability (Zhou et al., 2009) in old age. Alternately, it could be that sustained exogenous CREB in the aged rats was able to compensate for the typical loss of endogenous CREB accompanying normal aging (Mouravlev et al., 2006). It will be interesting to determine if acute over-expression of CREB in the hippocampus of aging mice can similarly rescue normal age-related impairments in hippocampal-dependent memory.

5.11.2 CREB and disease

Along these lines, CREB dysfunction is also a symptom reported in animal models of Alzheimer’s disease. Recently, using a transgenic mouse line expressing mutant

183 amyloid precursor protein (APP), the Josselyn lab examined the effects of enhancing CREB function using targeted delivery of HSV-CREB vector. These APP mutant mice typically develop a significant buildup of β-amyloid plaques throughout the prefrontal cortex and the hippocampus by 3 months of age, and display significant spatial memory impairments. Targeted over-expression of CREB in the CA1 region in these APP mutant mice rescued spatial memory deficits in the watermaze, suggesting that CREB dysfunction might account for, at least, some of the cognitive impairment associated with Alzheimer’s disease (Yiu et al., 2011).These findings should be interpreted cautiously though, as CREB over-expression did not correlate with decreases in amyloid plaque load in the brain, which is a major source of gross neurodegeneration in Alzheimer’s disease. Decreased CREB function has been linked to mutant APP in familial Alzheimer’s disease (Ikezu et al., 1996; Arvanitis et al., 2007), and post-mortem analysis report decrease CREB levels in the brains of Alzhemier’s patients (Yamamoto- Sasaki et al., 1999; Satoh et al., 2009). In human cases of Alzheimer’s disease, it is often reported that cognitive impairment precedes detectable morphological pathology, such as plaque deposition and neurofibrillary tangles (Braak and Braak, 1997). The results of Yiu and colleagues suggest the promising possibility that enhancing CREB function at the early stages of the disease may attenuate some of the memory impairment typically preceding neurodegeneration-induced memory impairment in Alzheimer’s disease.

A link between APP mutation and disrupted CRTC1 has been proposed by Espana and colleagues (2011). Using an APP mutant mouse, they report a reduction in CRE- reporter activity in cultured cortical neurons which was reversed in neurons transfected with plasmids containing CRTC1 or CRTC2. Like Yiu and colleagues, they also found a deficit in spatial memory consolidation in mutant APP mice. While they alluded to the potential for CRTCs to ameliorate the spatial memory deficit by increasing CRE-activity required to support normal memory consolidation, they did not test this hypothesis. Our data is the first to show that there could be a beneficial gain by increasing CRTC1 in the hippocampus of an Alzheimer’s mouse model, especially during the early stages of the disorder. Just as Yiu and colleagues (2011) showed that increasing CREB in the CA1 of the hippocampus can rescue spatial memory deficits in APP mice, it will be important to 184 test if the same rescue is possible using targeted delivery of CRTC1 in their hippocampus. While this method is of limited use for treatment of Alzheimer’s disease in human patients, it could provide insight into the molecular signaling disruption that is, at least in part, capable of improving synaptic consolidation in cases of Alzheimer’s disease.

As with Alzheimer’s disease, it would be valuable to investigate the use of CRTC1 vectors in models of CBP-dysfunction (see Josselyn, 2005 for review) to determine if increasing the availability of another CREB co-activator could compensate for some of the cognitive impairments associated with CBP haploinsufficiency commonly reported in cases of human Rubenstein-Taybi Syndrome (Petrij et al., 1995; Petrij et al., 2000; Wiley et al., 2003; Alarcon et al., 2004; Roelfsema and Peters, 2007).

Together, CREB and the CREB co-activator CRTC1 show a conserved role in consolidation across multiple aging and disease models, and suggest that CREB signaling could be an important treatment target for memory impairments in humans. This has profound implications for the development of learning and memory enhancing drugs aimed at enhancing CREB activity in challenging learning conditions, or in conditions of aging and disease.

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6 Conclusion

In the beginning of this thesis, we discussed the importance of seeing memory as a concrete, dynamic biological process comprised of many small ‘parts’. We took a bottom-up approach to putting those small parts together to build up to what we understand as ‘memory’. Specifically, we have seen how the highly conserved transcription factor CREB, and its transcriptional coactivators, are both necessary and sufficient to ultimately induce long-lasting, precise memory formation, but also to be capable of permitting memory formation under challenging learning conditions. These findings have wide-reaching application to the development of therapies aimed at remedying memory loss in conditions involving CREB dysfunction, or even targeting memory-enhancing compounds for healthy adults.

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Chapter 3: Portions of this chapter are based on published findings from Learning & Memory:

Sekeres, M.J1, 2., Neve, R.L. 3, Frankland, P.W1,2,4., and Josselyn, S.A1,2,4. (2010). Dorsal hippocampal CREB is both necessary and sufficient for spatial memory. Learning & Memory, 17 (6): 180-3

1Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, ON; 2Dept. of Physiology , University of Toronto, Toronto, ON; 3Dept. of Brain and Cognitive Sciences, MIT, Cambridge, MA; 4Institute of Medical Sciences, University of Toronto

Chapter 4: Portions of this chapter are based on a paper under review at the Journal of Neuroscience:

Sekeres,M.J. 1,2, Sargin, D. 2, Mercaldo, V. 2, Frankland, P.W1,2,3., and Josselyn, S.A. 1,2,3 (2012). Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory quality. Journal of Neuroscience (JN-RM-1419-12, under review)

1Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, ON; 2Dept. of Physiology , University of Toronto, Toronto, ON; 3Institute of Medical Sciences, University of Toronto

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CURRICULUM VITAE Updated: June 2012 MELANIE JAY SEKERES, PhD candidate

Address Program in Neurosciences & Mental Health, The Hospital for Sick Children Room 6020 McMaster 555 University Ave. Toronto, Ontario Canada M5G 1X8 Phone: 813-7654 x 6898 E-mail: [email protected]

1. EDUCATION

Summer 2008 - present PhD (in progress), Dept. of Physiology, University of Toronto, Toronto, ON, Canada Supervisor: Dr. Sheena A. Josselyn Thesis Title: Increasing CRTC (CREB regulated transcription co-activator) levels in the dorsal hippocampus is sufficient to induce contextual fear memory and discrimination

Fall 2006 – Summer 2008 MSc (transfer), Dept. of Physiology, University of Toronto, Toronto, ON, Canada Supervisor: Dr. Sheena A. Josselyn Thesis Title: Dorsal hippocampal CREB is both necessary and sufficient for spatial memory

Fall 1998 – Spring 2002 BSc, Dept. of Psychology, Trent University, Peterborough, ON, Canada

2. HONOURS AND AWARDS

2011 - 2012 Doctoral Completion Award University of Toronto $ 18,000

2011-2012 Dr. Joe A. Connolly Memorial Award Faculty of Medicine, University of Toronto $ 6000

2011-2012 Hayden Hantho Award Faculty of Medicine, University of Toronto $ 5000

2011-2012 Margaret & Howard Gamble Research Grant Faculty of Medicine, University of Toronto (declined) $ 5000

2011-2012 SCACE Graduate Fellowship in Alzheimer’s Research Faculty of Medicine, University of Toronto (declined) $ 5000 226

2011 University of Toronto Neuroscience Program Travel Award (Society for Neuroscience) University of Toronto $ 500

2011 Restracomp Travel Award, The Hospital for Sick Children $ 1,000

2011 School of Graduate Studies Conference Grant University of Toronto $ 150

2008 - 2011 CIHR Frederick Banting and Charles Best Canada Graduate Scholarships - Doctoral Award, CIHR $ 35,000/year

2010 Restracomp Travel Award, The Hospital for Sick Children $ 1,000

2010 Exceptional Trainee Award, Program in Neurosciences & Mental Health The Hospital for Sick Children $ 500

2010 University of Toronto Neuroscience Program Travel Award (Canadian Association for Neuroscience) University of Toronto $ 500

2009 Restracomp Travel Award, The Hospital for Sick Children $ 1,000

2009 BRAIN Research Symposium, winner (oral presentation) University of Toronto $ 200

2008 Restracomp Travel Award, The Hospital for Sick Children $ 1,000

2008 - 2009 Ontario Graduate Scholarship (declined) $ 15,000

2007 - 2008 Peterborough K.M. Hunter Graduate Studentship Award (declined) $ 20,000

2007 - 2008 University of Toronto Fellowship, University of Toronto $ 2,000

2006 - 2007 CIHR Frederick Banting and Charles Best Canada Graduate Scholarships - Master's Award, CIHR $ 17,500

2006 - 2007 University of Toronto Fellowship, University of Toronto $ 2,000

2006 - 2007 Restracomp Award, The Hospital for Sick Children $ 20,000

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2006 Research Training Competition Tuition Bursary The Hospital for Sick Children $ 2,000

2002 Trent University President’s Honour Roll, Trent University

1999 - 2001 Trent University Dean’s Honour Roll, Trent University

1999 - 2001 Trent University Scholarship, Trent University $ 1,000

1999 John Pettigrew Award (for English 100), Trent University $ 200

1998 Trent University Entrance Scholarship, Trent University $ 850

3. PEER REVIEWED PUBLICATIONS

Sekeres, M, Sargin, D, Mercaldo, V, Frankland, PW, Josselyn, SA. (2012). Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory quality. (under review, Journal of Neuroscience, JN-RM-1419-12).

Winocur, G, Sekeres, M, Moscovitch, M. (2012) Hippocampal lesions produce both non-graded and temporally-graded retrograde amnesia in the same rats. (under review, Journal of Neuroscience, JN-RM- 2968-12).

Cole, CJ, Restivo, L, Mercaldo, M, Yiu, A.P., Sekeres, M, Han, JH, Vetere, G, Pekar, T, Ross, PJ, Neve, RL, Frankland, PW, Josselyn, SA (2012) MEF2 negatively regulates learning-induced structural plasticity and memory formation. Nature Neuroscience (Epub ahead of print, doi: 10.1038/nn.3189).

Sekeres, M, Sargin, D, Ross, PJ, Josselyn, SA. (2012). The role of CREB and CREB co-activators in memory formation. In K.P. Giese (Ed.), The Memory Mechanisms in Health and Disease: Mechanistic Basis of Memory. World Scientific Publishing.

Rochon, P, Sekeres, M, Hoey, J, Lexchin, J, Ferris, LE, Moher, D, Wu, W, Kalkar, S, Van Laethem, M, Gruneir, A, Gold, J, Maskalyk, J, Streiner, DL, Tabak, N, Chan, AW. (2011). Investigator experiences with financial conflicts of interest in clinical trials. Trials, 12:9.

Sekeres, MJ, Neve, RL, Frankland, PW, Josselyn, SA. (2010). Dorsal hippocampal CREB is both necessary and sufficient for spatial memory. Learning & Memory, 17(6): 280-3.

Winocur, G, Moscovitch, M, Rosenbaum, RS, Sekeres, M (2010). Investigation of the effects of hippocampal lesions in rats on pre- and post-operatively acquired spatial memory in a complex environment. Hippocampus, 20 (12): 1350-65. 228

Rochon, P., Sekeres, M., Lexchin, J., Moher, D , Wu, W., Kalkar, S., Van Laethem, M., Hoey, J., Chan, AW., Gruneir, A., Gold, J., Maskalyk, J., Streiner, D.L., Tabak, N., Ferris, LE. (2010). Institutional financial conflicts of interest at Canadian academic health science centres: A national survey. Open Medicine, 4(3): 134-8.

Rochon, P., Hoey, J., Chan, AW., Ferris, LE., Lexchin, J., , Kalkar, S., Sekeres, M., Wu, W., Van Laethem, M., Gruneir, A., Maskalyk, J., Streiner, D.L., Gold, J., Moher, D (2010). Financial Conflicts of Interest Checklist 2010 for clinical research studies. Open Medicine, 4(1): 69-9.

Winocur, G, Moscovitch, M., Rosenbaum, R.S., Sekeres, M. (2010). A study of remote spatial memory in aged rats. Neurobiology of Aging (1):143-50.

Winocur, G, Frankland, P.W., Sekeres, M., Fogel, S., Moscovitch, M. (2009). Manipulating context- specificity in a memory reconsolidation paradigm: Selective effects of hippocampal lesions. Learning & Memory 16(11):722-9.

Sekeres, M., Gold, J., Chan, AW, Lexchin, J., Moher, D., Maskalyk, J., Ferris, L., Taback, N., Van Laethem, M., Rochon, P. (2008). Poor reporting of scientific leadership information in clinical trial registers. PLoS One, 3:2, e.1610.

Lexchin, J., Sekeres, M., Van Laethem, M., Ferris, L., Chan, AW, Moher, D., Gold, J., Maskalyk, J., Taback, N., Rochon, P. (2008). National evaluation of policies on individual financial conflicts of interest in Canadian academic health science centres. Journal of General Internal Medicine, 23(11):1896-903.

Winocur, G, Moscovitch, M., Sekeres, M. (2007). Memory consolidation or transformation: Context manipulation and hippocampal representations of memory. Nature Neuroscience, 10, 555-557.

Winocur, G., Wojtowicz, J.M., Sekeres, M., Snyder, J.S., Wang, S. (2006). Inhibition of neurogenesis interferes with hippocampus-dependent memory function. Hippocampus, 16: 296–304.

Winocur, G, Moscovitch, M., Fogel, S., Rosenbaum, R.S., Sekeres, M. (2005). Preserved spatial memory after hippocampal lesions: effects of extensive experience in a complex environment. Nature Neuroscience, 8, 273 – 275.

4. NON-PEER REVIEWED PUBLICATIONS AND PUBLISHED CONFERENCE PROCEEDINGS

Sekeres MJ, Neve RL, Josselyn SA (2009). Increasing CREB in the dorsal hippocampus is sufficient to induce spatial memory. Frontiers in Neuroscience. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.051

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5. RESEARCH, TEACHING AND EMPLOYMENT

September 2006 – Present Graduate student, Dept. of Physiology, University of Toronto; Program in Neurosciences & Mental Health, The Hospital for Sick Children Supervisor: Dr. Sheena Josselyn

August 2005 – August 2006 Research Assistant, Kunin Lunenfeld Applied Research Unit, Baycrest Supervisor: Dr. Paula Rochon

May 2002 – August 2005 Research Assistant, Psychology Department, Trent University. Supervisor: Dr. Gordon Winocur

2002 Teaching Assistant, Trent University PS379 Aging and Cognition and PS375 Neuropsychology Supervisor: Dr. Jocelyn Aubrey

6. PRESENTATIONS

Frontiers in Physiology Symposium (University of Toronto, Department of Physiology) April 13, 2012 (poster)

Society for Neuroscience Annual Conference (Washington, DC, USA) November 14, 2011 (poster)

Molecular and Cellular Cognition Society Meeting (Washington, DC, USA) November 11, 2011 (poster)

The Hospital for Sick Children Research Institute Research Retreat (Toronto, ON, Canada) November 1, 2011 (poster)

Canadian Association for Neuroscience (CAN) Annual Conference (Quebec City, QC, Canada) May 16, 2011 (poster)

Frontiers in Physiology Symposium (University of Toronto, Department of Physiology) April 8, 2011 (poster)

Brain Research and Integrated Neurophysiology Platform Research Day (University of Toronto, Department of Physiology) December 16, 2010 (poster)

Society for Neuroscience Annual Conference (San Diego, CA, USA) November 16, 2010 (poster)

Molecular and Cellular Cognition Society Meeting (San Diego, CA, USA) November 11, 2010 (poster)

Canadian Association for Neuroscience (CAN) Annual Conference (Ottawa, ON, Canada) May 16, 2010 (poster)

Frontiers in Physiology Symposium (University of Toronto, Department of Physiology) April 15, 2010 (poster)

University of Toronto Neuroscience Program (University of Toronto) April 14, 2010 (poster)

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Brain Research and Integrated Neurophysiology Platform Research Day (University of Toronto, Department of Physiology) December 16, 2009 (Winner of oral presentations)

Society for Neuroscience Annual Conference (Chicago, IL, USA) October, 2009 (poster)

Molecular and Cellular Cognition Society Meeting (Chicago, IL, USA) October, 2009 (poster)

Frontiers in Physiology Symposium (University of Toronto, Department of Physiology) April 22, 2009 (poster)

Brain Research and Integrated Neurophysiology Platform Research Day (University of Toronto, Department of Physiology) December 10, 2008 (poster)

Society for Neuroscience Annual Conference (Washington, DC, USA) November 16, 2008 (poster)

Molecular and Cellular Cognition Society Meeting (Washington, DC, USA) November 13, 2008 (poster)

Program in Neuroscience Research Day (University of Toronto) April 30, 2008 (poster)

Frontiers in Physiology Symposium (University of Toronto, Department of Physiology) April 24, 2008 (poster)

Brain Research and Integrated Neurophysiology Platform Research Day (University of Toronto, Department of Physiology) December 17, 2007 (poster)

7. COMMITTEES AND SUPERVISION

The Hospital for Sick Children Research Institute, Animal Care Committee (2009-2010)

Supervise 4th year undergraduate students in University of Toronto’s Human Biology Program, course HMB499Y: 2010-2011: Jane Wu (currently a 4th year honours student, University of Toronto) 2009-2010: Daniel Corrazolla (currently a 2nd year medical school student, Queens University) 2008-2009: Colin Kwok (currently a 3rd year medical school student, China)

Science Rendezvous volunteer 2009, 2011, 2012

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