fornix stimulation, effects on hippocampal oscillations

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Fornix stimulation, effects on hippocampal oscillations and memory in an AD mouse model Eva Vico Varela Integrated Program in Neuroscience McGill University Montreal, Quebec, Canada April 2020 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy, Ph.D. © Eva Vico Varela 2020

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Page 1: Fornix stimulation, effects on hippocampal oscillations

Fornix stimulation, effects on hippocampal oscillations and memory in an AD mouse model

Eva Vico Varela

Integrated Program in Neuroscience

McGill University

Montreal, Quebec, Canada

April 2020

A thesis submitted to McGill University in partial fulfillment of the requirements of the

degree of Doctor of Philosophy, Ph.D. © Eva Vico Varela 2020

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Abstract

Episodic memory depends on the integrity of the hippocampus and structures with

which it connects. Episodic memory is known to decline with age, but even more so in

progressive dementias such as Alzheimer’s disease (AD). Problems with episodic

memory generally follow the course of damage to the memory circuit in AD, with early

changes appearing in the hippocampus, a structure that is common across species.

Despite all efforts, there are currently no treatments that stop or reverse disease

progression in AD patients. Stimulation of the fornix, a major pathway connecting the

hippocampus with other essential memory areas, such as the medial septum, has the

potential of modulating hippocampal activity and enhance memory in disease. In AD

patients, the effect of fornix stimulation in cognitive measures has been variable, and

little is known about its mechanism of action. The work included in this thesis

investigates early oscillatory alterations in the hippocampus of freely-moving J20+ mice

(an amyloid precursor protein-overexpressing mouse model of AD), and how fornix

stimulation may modulate existing abnormal hippocampal activity and hippocampal-

dependent memory impairments. We focus our analysis on prominent hippocampal

oscillations, such as theta (4-10 Hz) and its coupling to gamma (30-120 Hz), which have

been linked to memory processes and occur during specific brain states.

In chapter 2, we examine early hippocampal rhythmic differences between non-

transgenic littermates and AD mice in wake and sleep periods. Following this, we

explore hippocampal hyperactivity during sleep in AD, and its correlation with memory

deficits. We demonstrate that during wake and rapid eye movement sleep, the peak of

theta frequency is lower in J20+, while theta- low gamma phase- amplitude coupling is

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reduced in wake periods only. Moreover, hyperactive events in the form of spike-wave

discharges are present only in AD mice, predominate during sleep and are particularly

prominent in rapid eye movement sleep. Lastly, the incidence of these aberrant

synchronous events correlates with memory performance in two hippocampal-

dependent tasks, novel place object recognition and passive avoidance. Thus, we

demonstrate that memory-associated early alterations can be detected in the

hippocampal activity of AD mice in a state-dependent manner. In chapter 3, we

investigate the effect of stimulating septo-hippocampal projections in these oscillatory

signatures using optogenetics. While activating septal fibers at the level of the fornix,

changes in hippocampal rhythms and hyperactivity are studied during wake and sleep,

together with the effect of stimulation on memory. The results reported here suggest

that GABAergic projections from the septum entrain hippocampal rhythms, lower

aberrant hyperactivity and improve memory performance in deficient mice. Finally, in

chapter 4, we show that electrical deep brain stimulation, approved for clinical use in AD

patients, is also able to enhance memory in J20+ when low, but not high, frequency

stimulation is delivered to the fornix. Our findings highlight differences between

stimulation paradigms for the modulation of memory.

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Résumé

La mémoire épisodique est dépendante de l'intégrité de l'hippocampe ainsi que de ses

structures connexes. Il est connu que ce type de mémoire se détériore avec l'âge, mais

d'autant plus dans le cas de démences progressives telles que la maladie d'Alzheimer

(MA). Dans la MA, les déficits de la mémoire épisodique sont généralement

temporellement corrélés aux dommages des circuits mnésique, avec les changements

les plus précoces apparaissant dans l'hippocampe, une structure commune à de

nombreuses espèces. Malgré de nombreux efforts, il n'existe à l'heure actuelle aucun

traitement pour arrêter ou améliorer la progression de la maladie chez les patients de la

MA. Le fornix est une voie majeure de connection entre l'hippocampe et d'autres

régions essentielles au traitement mnésique, telles que le septum médian, et la

stimulation de cette voie a le potentiel de pouvoir moduler l'activité de l'hippocampe et

d'améliorer la mémoire. Dans les patients atteints de la MA, les effets de la stimulation

du fornix sur les performances cognitives sont variables, et les mécanismes d'action

restent inconnus. Ce travail de thèse examine les altérations oscillatoires dans

l'hippocampe de souris J20+ (un modèle murin qui surexprime l'APP) libres de

mouvement, et la manière dont les stimulations du fornix pourraient moduler l'activité

hippocampique anormale ainsi que les troubles mnésiques hippocampes-dépendants.

Nous avons concentré notre analyse sur les principales oscillations de l'hippocampe,

telles que le thêta (4-10 Hz) et sont couplage au gamma (30-120 Hz), qui ont été liées

aux processus mnésiques et surviennent dans des états cérébraux spécifiques.

Dans le chapitre 2, nous examinons dans le sommeil et l'éveil les différences précoces

de rythmes hippocampiques entre souris MA et leurs contrôles non-transgéniques.

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Ensuite, nous explorons l'hyperactivité hippocampique pendant le sommeil dans la MA,

ainsi que sa corrélation avec les troubles de la mémoire. Nous démontrons que pendant

l'éveil et durant le sommeil paradoxal, la fréquence de pointe du thêta est diminuée

dans les souris J20+, alors que le couplage du gamma bas au thêta est seulement

réduit dans les périodes d'éveil. De plus, les événements d'hyperactivité qui prennent la

forme de décharges 'pointe-onde' sont seulement présents dans les souris MA, et

prédominent le sommeil, avec une prédominance pour le sommeil paradoxal. Enfin,

l'incidence de ces événements anormalement synchrones est corrélée avec la

performance mnésique dans deux tâches hippocampe-dépendantes: la reconnaissance

d'objet spatiale et la tâche d'évitement passif. En conséquence, nous démontrons que

les déficits précoces de la mémoire peuvent être détectés dans l'activité hippocampique

des souris MA en fonction de l'état cérébral. Dans le chapitre 3, nous avons examiné

l'effet des stimulations septo-hippocampiques sur ces événements oscillatoires en

utilisant l'optogénétique. Pendant l'activation des fibres septales au niveau du fornix,

des changements au niveau des rythmes hippocampiques et de l'hyperactivité sont

étudiés pendant l'éveil et le sommeil, ensemble avec les effets des stimulations sur la

mémoire. Les résultats rapportés ici suggèrent que les projections GABAergiques du

septum entraînent les rythmes hippocampiques, diminuent l'hyperactivité aberrante, et

améliorent la mémoire dans les souris déficientes. Enfin, dans le chapitre 4, nous

montrons que les stimulations cérébrales profondes électriques, qui ont été approuvées

pour usage clinique chez les patients atteints de la MA, sont également capables

d'améliorer la mémoire chez les souris J20+ seulement lorsque des motifs de

stimulation à basse fréquence, mais pas à haute fréquence, sont appliqués au fornix.

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Nos résultats soulignent l'importance du choix du motif de stimulation dans la

modulation de la mémoire.

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Acknowledgements

Many have accompanied me throughout the years and their help, support and

continued presence have allowed me to complete this thesis work. First and foremost is

my supervisor Sylvain Williams (PhD.) who gave me the opportunity to learn and grow

as a scientist in the work environment he created. In his laboratory I have enjoyed

scientific freedom and been exposed to a range of exciting techniques that have

considerably expanded my scientific horizons. I specially appreciate the opportunity to

take on new experimental and analytical challenges, which has been incredibly

stimulating and fulfilling. Sylvain has assembled a very talented group of people with

whom working with has been a privilege, and their perspective, invaluable. Thank you to

Richard Boyce (PhD.), Jennifer Robinson (PhD.), Ning Gu (PhD.), Bénédicte Amilhon

(PhD.), Guillaume Ducharme (PhD.), Frédéric Manseau (PhD.), Siddhartha Mondragon

(PhD.), Heather Nichol (MSc.), Jean-Bastien Bott (PhD.), Guillaume Etter (PhD.), Amy

Chee (PhD.), Jun Kang (PhD.), Lorène Penazzi (PhD.), Ingrid Inema (MSc.), Suzanne

van der Veldt (MSc.), Coralie-Ann Mosser (PhD.), Jisoo Choi (MSc.) and Ke Cui (BSc.).

A special thank you to Richard Boyce, Jennifer Robinson, Guillaume Etter, and Jean-

Bastien Bott for their mentorship and support during these years.

Thank you to my committee members, Mallar Chakravarty (PhD.) and Mark Brandon

(PhD.), and to my IPN mentor Erik Cook (PhD.) who have taken their time to offer me

guidance and counsel. Exchanging thoughts and ideas during my committee meetings

has been extremely useful and has contributed greatly to this work. Lastly thank you to

my proposal and thesis examiners for your time, and thoughtful comments and

questions.

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Contributions

Eva Vico Varela performed and analyzed all immunohistochemistry and in vivo

experiments described in this thesis, with the following exceptions: tetrode implants and

tetrode cluster cutting was performed by Johnson Ying, and the 16ch probe implant was

performed by Guillaume Etter. In vitro patch clamp data acquisition and analysis was

performed by Frederic Manseau. Writing of the thesis, including figure construction

(unless stated otherwise), was completed by Eva Vico Varela. GE and Sylvain Williams

contributed to and edited section 1.4 in chapter 1, which was published with some

modifications in Neurobiology of Disease (Vico Varela et al., 2019). GE translated the

thesis abstract to French. Amy Chee and SW provided many helpful revisions and

comments on the entirety of this work.

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Table of Contents

Abstract ............................................................................................................................ii

Résumé ...........................................................................................................................iv

Acknowledgements ........................................................................................................ vii

Contributions ................................................................................................................. viii

Table of Contents ............................................................................................................ix

List of figures .................................................................................................................. xii

Foreword: General overview of thesis ........................................................................... xiv

Chapter 1: Introduction .................................................................................................... 1

1.1. Hippocampal anatomy and circuit elements .......................................................... 1

1.1.2. Hippocampal projections................................................................................. 4

1.2. Major Hippocampal activity patterns ..................................................................... 6

1.2.1. Theta oscillations ............................................................................................ 7

1.2.2. Gamma oscillations ...................................................................................... 11

1.2.3 Sharp-wave ripple complexes ........................................................................ 12

1.3. Sleep and memory consolidation ........................................................................ 14

1.3.1 Differences between REM and NREM sleep ................................................. 15

1.3.2. REM sleep and memory ............................................................................... 17

1.4. Alterations in hippocampal activity and Alzheimer’s Disease* ............................ 19

1.4.1. Introduction ................................................................................................... 19

1.4.2. Abnormal rhythmic activity in AD .................................................................. 21

1.4.3. Abnormal network activity in AD promotes epileptiform activity .................... 23

1.4.4. Excitatory Disruption and Hyperactivity ........................................................ 27

1.4.5. Inhibitory Disruption ...................................................................................... 33

1.4.6. Targeting cognitive dysfunction by circuit modulation ................................... 38

1.4.7. Conclusions and Outlook .............................................................................. 43

1.5. Hypothesis and Aims .......................................................................................... 45

Chapter 2: Characterization of early hippocampal dysfunction and spatial memory in

Alzheimer’s Disease ...................................................................................................... 46

2.1. Introduction ......................................................................................................... 46

2.2. Methods .............................................................................................................. 48

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2.2.1. Animals ......................................................................................................... 48

2.2.2. Electrode implantation for in vivo experiments .............................................. 48

2.2.3. Post-surgery habituation and electrophysiological recordings ...................... 49

2.2.4. Novel object place recognition procedure and behavioral analysis ............... 50

2.2.5. Passive avoidance procedure and behavioral analysis ................................ 51

2.2.6. Vigilance state architecture analysis ............................................................. 52

2.2.7. Electrophysiological analysis of in vivo CA1 LFP and unit data .................... 53

2.2.8. SWD state-specific analysis of CA1 LFP ...................................................... 54

2.2.9. Ripple analysis .............................................................................................. 54

2.2.10. Phase-amplitude coupling........................................................................... 55

2.2.11. Statistical analysis ...................................................................................... 55

2.2.12. Histological confirmation of electrode/optic fiber placement ....................... 56

2.3. Results ................................................................................................................ 56

2.3.1. Time spent in REM, wake, and NREM does not differ in J20+ transgenic AD

mice compared to non-transgenic littermate control animals .................................. 56

2.3.2. Shift in hippocampal theta peak frequency in AD ......................................... 57

2.3.3. Hippocampal hyperactivity during sleep in AD .............................................. 64

2.3.4. Performance impairments during behavior in AD ......................................... 74

2.4. Discussion ........................................................................................................... 77

2.4.1. Deficits in theta frequency and theta-gamma coupling ................................. 78

2.4.2. Hyperactivity during sleep and its link to memory ......................................... 79

Chapter 3: Effects of optogenetic activation of septo-hippocampal fibers in AD. .......... 84

3.1. Introduction ......................................................................................................... 84

3.2. Additional Methods ............................................................................................. 85

3.2.1. Virus-mediated targeting of opsin and eYFP expression .............................. 85

3.3.2. Optic fiber implantation for in vivo experiments ............................................ 86

3.3.3. Light delivery to the fornix ............................................................................. 87

3.2.4. Immunohistochemistry and construct expression confirmation ..................... 87

3.3.5. In vitro patch-clamp electrophysiology. ......................................................... 88

3.3. Results ................................................................................................................ 90

3.3.1. Optogenetic targeting of medial septum neurons ......................................... 90

3.3.2 Characterizing the effect of theta stimulation of MS projections in hippocampal

rhythms ................................................................................................................... 93

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3.3.3. Modulation of SWDs by fornix optogenetic activation ................................... 99

3.3.4. Modulation of hippocampal memory by fornix optogenetic activation ........ 107

3.4. Discussion ......................................................................................................... 110

3.4.1. Stimulation of septo-hippocampal projections modulates hippocampal

rhythms ................................................................................................................. 110

3.4.2 Stimulation of septo-hippocampal projections decreases SWDs in a state-

dependent manner ................................................................................................ 112

3.4.3 Post-training stimulation improves spatial memory in AD ............................ 114

3.5 Supplementary Data .......................................................................................... 117

Chapter 4: Fornix deep brain theta-burst stimulation improves memory performance in

an Alzheimer’s disease mouse model ......................................................................... 122

4.1. Introduction ....................................................................................................... 122

4.2. Methods ............................................................................................................ 124

4.2.1. Surgical coordinates ................................................................................... 124

4.2.3 Post-training stimulation paradigm ............................................................... 124

4.2.4. Passive avoidance procedure. .................................................................... 124

4.2.5. Novel object place recognition procedure (NOPR) procedure. ................... 125

4.3. Results .............................................................................................................. 125

4.3.1. Passive avoidance memory is improved after fornix theta-burst stimulation in

J20+ mice. ............................................................................................................ 125

4.3.2. Novel object place recognition memory improves after fTBS in J20+ mice. 126

4.4. Discussion ......................................................................................................... 130

4.4.1. Possible therapeutic mechanisms of DBS in memory circuitry ................... 131

4.5. Supplementary Information ............................................................................... 135

Chapter 5: Concluding remarks ................................................................................... 138

References .................................................................................................................. 144

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List of figures

Chapter 1:

Fig. 1.1: The hippocampus.. ............................................................................................ 4

Fig. 1.2: The fornix. ......................................................................................................... 5

Fig. 1.3: Neural activity patterns associated with wake and sleep behavior in the human

and rodent brain. ........................................................................................................... 15

Fig. 1.4: Earliest recorded Spike Wave Discharges (SWDs) in AD models ................... 27

Fig. 1.5: Schematic of neural network in normal and AD conditions. ............................ 32

Chapter 2:

Fig. 2.1: Sleep Architecture in controls and J20+ mice. ................................................ 57

Fig. 2.2: Theta frequency decreased in J20+ during REM. ........................................... 61

Fig. 2 3: Theta frequency decreased in J20+ during wake and impaired theta-low

gamma coupling.. .......................................................................................................... 62

Fig. 2.4: Spike-wave discharges are specific to J20+ mice. .......................................... 66

Fig. 2.5: Spike-wave discharge characterization in sleep. ............................................. 68

Fig. 2.6: Unit activity in J20+ mice. ................................................................................ 70

Fig. 2.7: Example PSTHs.. ............................................................................................ 71

Fig. 2.8: PSTHs of all units classified by SWD response .............................................. 74

Fig. 2.9: Spatial memory impairments in J20+ mice ...................................................... 77

Table 2.1: Analysis of CA1 cell layer LFP ripple characteristics during NREM.. ........... 63

Supplemental Fig. 2.1: .................................................................................................. 83

Chapter 3:

Fig. 3.2: Optogenetics and MS neurons.. ...................................................................... 91

Fig. 3.1: In vitro characterization of AAV2-ChETA in medial septum neurons.. ............. 93

Fig. 3.3: Optogenetic driving of theta frequency in the hippocampus of J20+ during REM

sleep. ............................................................................................................................. 97

Fig. 3.4: Optogenetic driving of theta frequency in the hippocampus of J20+ mice during

wake.. ............................................................................................................................ 98

Fig. 3.5: Stimulation of fornix fibers does not alter sleep architecture in J20+.. ............. 99

Fig. 3.6: Effect of fornix stimulation in spike-wave discharge numbers ....................... 103

Fig. 3. 7: Effect of fornix stimulation in spike-wave discharge characteristics.. ........... 105

Fig. 3.8: Effect of fornix stimulation on hippocampal single unit activity. ..................... 107

Fig. 3. 9: Spatial memory impairments in J20+ mice ................................................... 109

Table 3. 1: Analysis of CA1 cell layer LFP ripple characteristics during NREM. ........... 97

Table 3.2: REM length -SWDs .................................................................................... 117

Table 3.3: REM segment -SWDs ................................................................................ 117

Table 3.4: NREM length -SWDs .................................................................................. 118

Table 3.5: NREM segment -SWDs .............................................................................. 119

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Table 3.6: Control behavioural measures .................................................................... 119

Table 3 7: Control behavioural measures .................................................................... 120

Table 3 8: Control behavioural measures .................................................................... 121

Chapter 4:

Fig. 4.1: Memory improves following fTBS in J20 mice ............................................... 129

Fig. 4.2: Novel object place recognition (NOPR) control measures. ........................... 130

Table 4.1: Passive Avoidance additional statistics. ..................................................... 135

Table 4. 2: Corresponds to data shown in Fig.4-1 F. .................................................. 136

Table 4. 3: Novel object place recognition (NOPR) additional statistics. ..................... 137

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Foreword: General overview of thesis

This thesis describes original research work carried out by the author which

investigate whether stimulation of the fornix modulates hippocampal activity, decreases

hippocampal hyperactivity, as well as restores memory performance in a rodent model

of Alzheimer’s disease. Accordingly, the first chapter contains an overview of

background literature integral to this thesis. First, a description of the basic

neuroanatomy of the hippocampus and its extensive connections through the fornix is

included. A more detailed presentation of major hippocampal electrophysiological

oscillations and their implication for memory is also provided, as these are a significant

focus of the current thesis. Next, since the majority of analyses performed in this thesis

are state-dependent, a general summary of wake and sleep behaviors and the latter’s

relevance for memory consolidation is included. The last sections of the introduction

review the role of aberrant hyperactivity in the cognitive deficits characteristic of

Alzheimer’s disease, and explores the mechanisms contributing to this altered

excitatory-inhibitory balance early in the disease progression. Although already

published (Vico Varela et al., 2019), these last sections have been modified here mainly

to include relevant literature pertaining to changes in rhythm dynamics in Alzheimer’s

disease. The chapter concludes with a statement of the rationale and specific

hypotheses of the thesis work.

In the second chapter, I describe early state-dependent alterations found in

hippocampal oscillatory patterns of freely-moving Alzheimer’s disease mice. First,

hippocampal rhythmic differences are assessed in Alzheimer’s disease and age-

matched controls, followed by a characterization of hippocampal hyperactivity in sleep,

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and a final assessment of spatial-memory consisting of the spatial novel object place

recognition and passive avoidance tasks. The results presented here serve as an

essential foundation in understanding how early existing deficits in our AD model

contribute to memory deficits.

In the third chapter, I present the effects of optogenetic stimulation of septo-

hippocampal forniceal projections on the altered hippocampal measures described in

our AD model in the previous chapter. The first section shows the specificity and

efficacy of optogenetic stimulation of neurons within the medial septum. The following

sections are laid out as in chapter 2, where the effects of optogenetic fornix stimulation

on hippocampal rhythms are assessed during different in vivo states. Finally, I present

data on how optogenetic fornix stimulation modifies network hyperactivity in the

hippocampus as well as performance in two spatial memory tasks.

Lastly, the fourth chapter explores the effect of non-specific fornix deep brain

stimulation, which is considered as a potential therapeutic treatment in AD patients. I

assess hippocampal-dependent memory after deep brain stimulation with two distinct

stimulation paradigms and find that stimulation patterns that more closely resemble

physiological frequencies have more significant enhancing effects on memory

performance. Finally, a general discussion is provided in chapter 5.

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Chapter 1: Introduction

1.1. Hippocampal anatomy and circuit elements

As one of the most studied areas in the brain, the hippocampus is anatomically

and functionally conserved across mammalian species and plays a critical role in

memory. Three distinct subregions can be distinguished: the dentate gyrus (DG), the

hippocampus proper (consisting of CA3, CA2 and CA1) and the subiculum (Fig. 1.1 A-

B). Within the CA regions, the cortex forming the hippocampus can be subdivided into

three main layers. The deep layer, or the stratum oriens, is comprised of the basal

dendrites of pyramidal cells and a mixture of afferent and efferent fibres. Superficial to

the stratum oriens is the cell layer, or stratum pyramidale, which is composed of the

somas of principal cells. The most superficial layer is subdivided into several sublayers,

mainly comprising the apical dendrites from the pyramidal layer. In CA3, three sublayers

are distinguished: the stratum lucidum, a thins stratum where Mossy fiber inputs from

the dentate gyrus terminate, the stratum radiatum and, most superficially, the stratum

lacunosum-moleculare. The lamination in CA2 and CA1 is similar, with the exception

that the stratum lucidum is missing (Andersen et al., 2007). Although pyramidal cell

somas are located solely in the stratum pyramidale, interneurons can be found in any of

the layers, wherein they finely modulate the output of principal neurons (Klausberger

and Somogyi, 2008).

The hippocampal circuit is most often described as a trisynaptic loop, where

information flows serially and unidirectionally (Fig. 1.1 C). In this classical scheme, the

neocortex projects to the parahippocampal region, consisting of the rhinal cortices,

which in turn provides the primary source of input to the hippocampus. One of these

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rhinal regions, the entorhinal cortex, is the source of the perforant pathway, which

projects to all hippocampal subregions. Specifically, layer II of the entorhinal cortex

projects to the DG and CA3, whereas layer III projects to CA1 and the subiculum,

synapsing at the level of the stratum lacunosum. The DG granule cells themselves give

rise to the mossy fiber pathway, which targets the stratum lucidum of CA3. Next, the

CA3 area gives rise to the Schaffer collaterals which project to CA1 across the stratum

radiatum and then CA1, in turn, projects to the subiculum. Finally, output from the

hippocampus arises in CA1 and the subiculum and is directed to the parahippocampal

region, in particular to the deep layers of the EC (Van Strien et al., 2009).

The trisynaptic circuit is an oversimplification of connectivity in the hippocampus,

and in contrast to what is depicted in the classical model, projections are neither serial

nor restricted to specific layers. In fact, several backprojections exist in the

hippocampus. For example, pyramidal cells in CA3 project back to the DG (Buckmaster

et al., 1993; Laurberg, 1979; Wittner et al., 2007, 2006), interneurons from CA1 oriens

and radiatum project to the same layers in CA3 (Cenquizca and Swanson, 2007;

Laurberg, 1979; Swanson et al., 1981) and the subiculum also backprojects to all layers

of CA1 and CA3 (Finch et al., 1983; Jackson et al., 2014; Köhler, 1985). Moreover,

recurrent collaterals which are generally viewed as a characteristic of CA3 (Buckmaster

et al., 1993; Laurberg, 1979; Sik et al., 1993) have also been described in other

hippocampal subregions. Although less extensive than in CA3, recurrent collaterals

have also been reported in the DG (Laurberg and Sørensen, 1981; van Groen and

Wyss, 1988), CA1 (Amaral et al., 1991; Cenquizca and Swanson, 2007; Van Groen and

Wyss, 1990). and the subiculum (Finch et al., 1983; Harris et al., 2001; Köhler, 1985).

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Finally, principal cells and interneurons also innervate other hippocampal areas

contralaterally through the hippocampal commissure (also called the commissure of the

fornix), and these projections have been described as originating from the dentate, CA3,

CA2 and CA1 (Eyre and Bartos, 2019; Shinohara et al., 2012; Sun et al., 2014; Zhou et

al., 2017).

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1.1.2. Hippocampal projections

The hippocampus not only sends and receives projections through the angular

bundle to the entorhinal cortex and other parahippocampal regions, but also to a myriad

of subcortical structures, primarily through the fornix (Andersen et al., 2007). Since the

work presented here is mostly concerned with projections passing though the fornix, the

following discussion will give an overview of fornix connectivity.

The fornix is the most extensive single pathway linking the hippocampus with

distal brain sites (Fig. 1.2). This fiber system is given different names at different points

in its trajectory from or towards anterior and posterior areas of the brain (Andersen et

al., 2007; Mark et al., 1993). Starting from the hippocampus, the alveus, which covers

the deep surface of the hippocampus, is a thin sheet of intrahippocampal as well as

afferent and efferent fibers covering the deep surface of the hippocampus. These

afferent and efferent projections collect laterally in a bundle named the fimbria, which

contains around 900.000 axons in the rodent (Wyss et al., 1980). The fimbria then

Fig. 1.1: The hippocampus. Nissl stained coronal views of A) mouse and B) human

brain sections from the Allen Brain Institute. Insets shows zoomed view of right

hippocampus. HPC: hippocampus; ff: fimbria-fornix; fim: fimbria alv: alveus; DG: dentate

gyrus; or: oriend; pyr: pyramidale; lu: lucidum; rad: radiatum; lm: lacunosum. C.

Schematic of the classical trysinaptic hippocampal circuit (entorhinal cortex (EC)–

dentate gyrus–CA3–CA1–EC) is depicted by solid arrows. MPP: medial perforin-path;

LPP: lateral performant path. Adapted from (Deng et al., 2010).

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proceeds to gives rise to the fornix. To emphasize the continuity of fibers in these

bundles and since the exact transition between fimbria and fornix is challenging to

define, the term fimbria-fornix is usually employed. The dorsal part of the fornix lies just

below the corpus callosum, very close to the midline. As the fibers of the fimbria leave

the hippocampus and rostrally descend into the forebrain at the level of the anterior

commissure, they are referred to as the columns of the fornix. At this level, it divides into

the precommissural and postcommisural branches to interconnect the hippocampus

with an array of subcortical sites. The more rostral precommisural fornix innervates the

Fig. 1.2: The fornix. Diagram of the fornix and the major structures it connects to the

hippocampus. The hippocampi illustrate the location of the fornix in the primate and

rodent brain. Pre. Com: pre-commisural fornix; Post. Com: post-commisural fornix; AC:

anterior commissure; N.ACC: nucleus accumbens; preoptic area; RE: nucleus reuniens;

ATN: anterior thalamic nuclei. MB: mamillary bodies.

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septal and preoptic nuclei, and nucleus accumbens (Kelley and Domesick, 1982;

Mathiasen et al., 2019; Saunders and Aggleton, 2007; Swanson and Cowan, 1979).

The caudally directed postcommisural fornix extend towards the diencephalon and

connects to the anterior nucleus of the thalamus, nucleus reuniens, locus coeruleus,

raphe nuclei, as well as to hypothalamic areas such as the mamillary nuclei (Bokor et

al., 2002; Bubb et al., 2017; Dillingham et al., 2015; Pasquier and Reinoso-Suarez,

1978; Saunders and Aggleton, 2007; Swanson and Cowan, 1975). Although many of

the projections mentioned above are bidirectional, those coming from the brain stem

(Mokler et al., 1998; Pasquier and Reinoso-Suarez, 1978) or going towards the nucleus

accumbens (Kelley and Domesick, 1982) appear unidirectional.

1.2. Major Hippocampal activity patterns

Brain rhythms are periodically fluctuating waves reflecting the electrical activity

produced by the synchronous fluctuations of large groups of neurons. These neural

oscillations can be measured using electroencephalogram (EEG) and local field

potential (LFP) recording techniques and can be used to detect general arousal patterns

in mammals, such as fundamental sleep and waking behaviors. In addition, local

rhythmic oscillations have been linked to many functions within the brain (Kahana,

2006; MacKay, 1997; Schnitzler and Gross, 2005). Of relevance to the present work,

these synchronous neural events are a critical component in current theories of learning

and memory which will be discussed in subsequent sections. One of the most

prominent features of the hippocampus is the presence of several patterns of oscillatory

activity related to cognitive functions. The hippocampal LFP shows three main rhythms

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which can be distinguished by their frequencies: theta (~4-10 Hz), gamma (~30-120 Hz)

and sharp-wave ripples (Buzsáki, 2002; Colgin, 2016).

1.2.1. Theta oscillations

Theta oscillations are relatively low frequency sinusoidal waves occurring during

periods of active wakefulness and rapid eye movement (REM) sleep (Winson, 1978).

These oscillations have been detected in several species ranging from rabbits to

humans (Aghajan et al., 2016; Jutras et al., 2013; Nokia et al., 2009), and can be

measured in cortical and subcortical areas (Holsheimer, 1982; Kocsis, 2006). They are

the most widely studied rhythm in the rodent hippocampus. This is due to the accessible

location of the rodent hippocampus, as it lies just below the dorsal cortical surface of the

brain, where large amplitude continuous theta can be clearly recorded.

1.2.1.1. The medial septum and hippocampal theta

As seen in section 1.1.2, the fornix connects many key memory regions to the

hippocampus, of which the medial septum is the most relevant for the present work. The

medial septum (MS) is a structure shaped like an inverted “Y” and is positioned centrally

across the midline, framed by the lateral ventricles and the anterior portion of the

anterior commisure. The MS receives input from several regions including the

hypothalamus, brain stem, and hippocampus (Swanson and Cowan, 1979), while its

major outputs are the entorhinal cortex and the hippocampus via the fornix (Gonzalez-

Sulser et al., 2014; Khakpai et al., 2013). The medial septum neural population consists

of three broad groups: GABAergic, cholinergic and glutamatergic neurons, and all three

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cell types project to all regions of the hippocampus, targeting both excitatory and

inhibitory neurons in different proportions (Amaral and Kurz, 1985; Colom et al., 2005;

Freund and Antal, 1988; Sotty et al., 2003). In CA1, principal cells receive a majority of

MS innervation from cholinergic (∼66%) and GABAergic (∼27%) cells (Sun et al., 2014).

In comparison, CA1 inhibitory cell types receive much less cholinergic MS innervation

(∼12%) and much stronger GABAergic innervation (∼67%). Finally, glutamaterigic MS

cells more strongly innervate CA1 inhibitory (∼21%) versus glutamatergic neurons

(~7%) (Freund and Antal, 1988; Gulyás et al., 1990; Sun et al., 2014).

GABAergic neurons within the MS are known to fire rhythmically at theta

frequencies and are phase-locked to hippocampal theta rhythms in vivo (Simon et al.,

2006). Although the reason why these neurons fire at theta frequency is not clearly

known, it may be due in part to the expression of specific conductances, such as HCN

channels, which confers them with an intrinsic ‘pacemaking’ rhythmicity (Varga et al.,

2008). Nevertheless, MS GABAergic neurons are critical for hippocampal theta, since

their inactivation significantly decreases theta power in the hippocampus, and

stimulation of these neurons entrains the network at the frequency of the stimulation

(Bender et al., 2015; Boyce et al., 2016; Etter et al., 2019; Tóth et al., 1997). In contrast,

MS cholinergic neurons show a slower firing pattern (Simon et al., 2006) and are

therefore unlikely to be directly involved in pacing the hippocampal theta rhythm. In

support of this, optogenetic manipulation of MS cholinergic neurons in vivo suggests

that these neurons may promote theta by suppressing peri-theta frequencies within the

hippocampus (Vandecasteele et al., 2014). Finally, optogenetic MS glutamatergic

stimulation can synchronize hippocampal theta oscillations in CA1, probably

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predominantly through local MS innervation rather than their direct projections to the

hippocampus (Fuhrmann et al., 2015; Robinson et al., 2016). Thus, based on the data

available to date, hippocampal theta rhythms in mice and rats likely arise from the

activity of GABAergic neurons, although the precise mechanism by which this occurs

remains to be uncovered.

1.2.1.2. Functional role of theta oscillations

Theta has been widely implicated in learning and memory(Winson, 1978). As

early as the 1970s, studies showed that higher amounts of theta was correlated with

future rate of learning (Berry and Thompson, 1978) or with the degree that rodents

remembered an event, such as a footshock (Landfield et al., 1972). Moreover,

disrupting theta rhythms in the hippocampus by inactivating the medial septum during

wakefulness or REM sleep consistently leads to memory deficits (Boyce et al., 2016;

Shirvalkar et al., 2010; Winson, 1978), while reinstating these rhythms restores memory

performance (McNaughton et al., 2006). Theta is also required to coordinate neural

assembly activity between distant brain structures (Sirota et al., 2008), as well as within.

For example, abolishing theta rhythms via medial septum inactivation disrupts

organized ensembles of place cells, called theta sequences, which are linked to

memory (Dragoi and Buzsáki, 2006; Skaggs et al., 1996; Wang et al., 2014). Finally,

this temporal code for neural populations may be enabled by theta through the

facilitation of synaptic plasticity (Hölscher et al., 1997; Huerta and Lisman, 1995). At the

neuronal level, memory formation may be due to changes in the strength of synaptic

connections in the networks underlying the memory. Exploration of a novel

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environment, in which theta frequency oscillations predominate in the hippocampus, is

associated with enhancement of persistent changes in synaptic strength, a

phenomenon termed long-term potentiation (LTP) (Davis et al., 2004; Orr et al., 2001).

Interestingly, applying electrical stimulation at theta frequencies can also efficiently

induce LTP-associated synaptic changes both in vitro and in vivo (Capocchi et al., 1992;

Larson et al., 1986; Larson and Munkácsy, 2015; Staubli and Lynch, 1987). The

association between theta activity and synaptic strength provides a possible mechanism

for the temporal coding of information. Theta activity can act as a temporal metric to

bind together or segregate neural activity, regulating synaptic connections important for

memory storage (Buzsáki, 2002).

1.2.1.3. Theta rhythm in humans

The hippocampus is located ventrally in humans and is therefore harder to

access, which may explain in part the relative scarcity of research on human

hippocampal theta. During REM sleep, studies using depth electrodes in epileptic

patients undergoing pre-surgical screening have described transient (average duration

of ~1.5s) and slower theta rhythms (~5 Hz) than in rodents (Cantero et al., 2003).

Furthermore, these hippocampal theta frequency oscillations were not found to couple

with gamma oscillations in sleep, a common feature in rodents (Bandarabadi et al.,

2019; Montgomery et al., 2008; Zhang et al., 2019)(see section 1.2.2).Given these

differences, it has been proposed that a continuous lower frequency oscillation (~1-3 Hz

equivalent to the rodent delta frequency range) could be the human analog to the rodent

hippocampal theta. Indeed, continuous 1-3 Hz oscillations have been recorded during

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REM sleep in epileptic patients (Bódizs et al., 2001) and gamma oscillations

significantly phase-lock to this frequency range (Clemens et al., 2009). Additionally,

similarly shifted low frequency rhythms have been reported within the hippocampus

during task acquisition and recall in a virtual environment (Clemens et al., 2013;

Watrous et al., 2013). However, this is at odds with a recent study by (Bohbot et al.,

2017), where clear hippocampal theta oscillations were reported in humans navigating

real rather than virtual environments. Bohbot et al’s results may reflect findings in

rodents where theta frequency, which usually increase with running speed, increased

less when running in virtual as opposed to real environments (Chen et al., 2018).

Adding to the difficulties in confirming a single theta band human analog during both

REM and wake behaviors, gamma oscillations have been shown to couple with ~2.5-

8hz theta in humans performing memory tasks and, mirroring evidence from rodent

studies, are also associated with memory performance (Heusser et al., 2016; Lega et

al., 2016; Mormann et al., 2005). Thus, although the proposed ~1-3 Hz human

hippocampal oscillations appear consistent with the basic features of theta rhythms

during REM sleep in rats and mice, confirming the human analog of the latter still

requires further research.

1.2.2. Gamma oscillations

It has been suggested that the transfer of information to hippocampal region CA1

from CA3 and the entorhinal cortex is organized in two separate gamma frequency

channels that are largest in amplitude during theta states (Colgin et al., 2009; Csicsvari

et al., 2003). Accordingly, these two gamma bands appear to be differentially prevalent

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in different layers of CA1 (Belluscio et al., 2012; Schomburg et al., 2014). The first type

of gamma, low gamma (~30- 60 Hz) is thought to be driven by CA3, since it has higher

amplitudes in stratum radiatum of CA1. The second, high gamma (~60-120 Hz), is

entrained by inputs from the medial entorhinal cortex and dominates in CA1 stratum

lacunosum-moleculare.

Functionally, since CA3 is associated with memory retrieval, low gamma is

proposed to be linked to these processes. Evidence supporting this idea comes from

behavioral studies in rats showing that, during periods of low gamma, place cell

ensembles preferentially represent upcoming locations rather than recent ones, when

navigating familiar environments (Bieri et al., 2014). Moreover, in an associative

memory task, the coupling between theta and low gamma amplitude at the time when

memory retrieval was expected to occur was correlated with task performance

(Shirvalkar et al., 2010; Tort et al., 2009). In contrast, the entorhinal cortex processes

sensory information and transmits this information to the hippocampus (Canto et al.,

2008). Consequently, high gamma has been linked to memory encoding and working

memory. For example, it has been shown that place cells tended to encode recent

locations and ongoing trajectories during high gamma (Bieri et al., 2014; Zheng et al.,

2016), and high gamma power increases when mice use cues in the external

environment to navigate towards the location of a reward (Cabral et al., 2014).

1.2.3 Sharp-wave ripple complexes

Sharp-wave ripple complexes (SWRs; labeled ripple in the thesis figures) consist

of 1-3 Hz sharp waves with superimposed ~110 -250 Hz fast oscillation ripples. These

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rhythms are thought to be generated within the hippocampus and are present in rodents

(Buzsáki, 1986), non-human primates (Hussin et al., 2020) and humans (Norman et al.,

2019). They are detected in periods of immobility, during consummatory behaviors, and

most frequently in non-REM (NREM) sleep (Buzsáki, 2015, 1998, 1986; Mölle et al.,

2006). Sharp-wave ripples have been hypothesized to carry out offline mnemonic

functions, such as memory consolidation (Clemens et al., 2007; Ramadan et al., 2009;

Siapas and Wilson, 1998), and their disruption impairs memory (Girardeau et al., 2009;

Talakoub et al., 2016). Moreover, SWRs contain trajectory information, since place cells

-pyramidal neurons that preferentially fire at given location in the environment- are

‘replayed’ in sequence during SWRs (Karlsson and Frank, 2009; Levenstein et al.,

2016; Nádasdy et al., 1999; Nakashiba, Buhl, McHugh, 2009; Wu et al., 2017). Although

sharp-waves and ripples often occur together, they are separate events with distinct

origins. Sharp-waves are excitatory events that are transmitted from CA3 to CA1

(Buzsáki, 1986; Sullivan et al., 2011). In contrast, ripples are generated locally. In CA1,

ripples result from strong inhibitory feedback on pyramidal cells via the activation of

reciprocally connected inhibitory interneurons (English et al., 2014; Klausberger T. et

al., 2003; Ylinen et al., 1995). This inhibition raises spiking thresholds and prevents

most cells from firing, which suggests that ripples may function to reinforce the most

strongly encoded memories for consolidation. In other words, only cells with synapses

that were recently potentiated during learning, which are likely more excitable, would fire

during this local inhibition and send their memory traces further downstream (Chrobak

and Buzsáki, 1994; Tingley and Buzsáki, 2020) for long-term storage in the neocortex.

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1.3. Sleep and memory consolidation

In mammals, sleep can be classified by combining analysis of brain and muscle

activity via EEG/LFP and electromyogram (EMG) recordings, respectively. Since state-

dependent analysis is presented in this thesis, a summary of the basic physiological

characteristics that distinguish this state is provided.

The first major distinction is observed between sleep and wake periods (Fig. 1.3).

Wakefulness can be characterized as a state of external awareness during which

subjects may engage in activities ranging from quiet rest to ambulation. A sustained

muscle tone is present within postural muscles throughout periods of wakefulness,

which is accompanied by large bursts of movement-associated muscle activity that can

be detected in EMG recordings without the need for video monitoring. During active

wake, theta and gamma frequencies can be easily detected from cortical and

hippocampal recordings. In contrast, quiet or drowsy wakefulness is accompanied by

prominent slow neural activity patterns in the delta (0.3-4 Hz) range, together with the

presence of discrete SWR events in the hippocampus (Brown et al., 2012).

During sleep, muscle tone decreases. Though no or barely any movement can

be detected during sleep, the brain is most definitely still active as sleep cycles through

electrophysiologically and neurochemically distinct sleep stages. These stages are

generally split into two main categories, based on the occurrence or absence of rapid

eye movement: REM and non-REM (NREM) sleep, respectively (Brown et al., 2012).

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1.3.1 Differences between REM and NREM sleep

REM and NREM sleep differ remarkably in several ways. NREM sleep is

characterized by high amplitude, low frequency (0.3–4 Hz) activity similar to quiet

awake which reflects synchronization across large cortical neuronal populations.

Furthermore, an increase in the number of SWRs occurs in NREM in comparison to

wake periods (Buzsáki, 2015). NREM is usually observed upon the initial transition from

Fig. 1.3: Neural activity patterns associated with wake and sleep behavior in the

human and rodent brain. Example EEG recordings from the human (left) and rat

(right) brain are for wakefulness, NREM, and REM sleep. Figure from Brown et al., 2012

with contribution from Purves et al., 1997

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wakefulness to sleep, although under certain sleep disorders, such as narcolepsy, REM

sleep may immediately follow wakefulness (Fujiki et al., 2009). Neuromodulator levels

also differ between these two brain states: during NREM sleep, acetylcholine levels in

the brain stem, forebrain, and hippocampus are at a physiological low (Hobson et al.,

1975; Marrosu et al., 1995), while serotonergic and noradrenergic neurons fire at

reduced rates compared to wake (Aston-Jones and Bloom, 1981).

In humans, NREM sleep is divided into light sleep and slow-wave (or deep) sleep

(Berry et al., 2015), while in rodents all NREM sleep stages are collectively referred to

as NREM or slow-wave sleep (Genzel et al., 2014; Oyanedel et al., 2014; Van Twyver,

1967). Only a few studies differentiate between light and deep NREM sleep in rodents

(Benedetto et al., 2013), or clarify if the spindle-rich transition to REM sleep is included

in the NREM analysis or excluded (Watts et al., 2012). This spindle-rich transition

appears to be unreported in the human literature and may not be a characteristic in

human sleep.

In normal conditions, REM always follows NREM periods, both in humans and

rodents (Genzel et al., 2014). Apart from the rapid darting eye movements, REM can

also be characterized by a complete absence of muscle tone. This atonia of the skeletal

musculature is a vital characteristic of REM sleep, the loss of which results in dream

enactment (McCarter et al., 2012). Cortical activity is reminiscent of active wake and

includes the presence of theta and gamma frequencies (Llinas and Ribary, 1993;

Steriade et al., 1996). Moreover, cholinergic modulation during REM sleep increases to

levels just below that of wake in the neocortex and even exceeds wake levels in the

hippocampus (Hasselmo, 1999). In contrast, serotonergic and noradrenergic neurons

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are almost completely silenced during REM sleep (Pace-schott et al., 2009). In human

nocturnal sleep, slow-wave sleep predominates during the first half of the night, with

REM sleep becoming increasingly prevalent toward the morning (Wurts and Edgar,

2000), while in rodents REM sleep does not appear to follow such circadian rhythm

(Yasenkov and Deboer, 2012).

1.3.2. REM sleep and memory

The hypothesis that sleep subserves memory consolidation is well established

(Rasch and Born, 2013). Current theories posit that memories are initially encoded into

a flexible learning store (i.e., the hippocampus) and then gradually transferred to the

neocortex for long-term stable storage. The hippocampus may ensure, even in one

attempt, the quick and efficient encoding of memories; yet, these representations are

unstable and vulnerable to interference by more recent experiences. Thus, information

must be gradually transferred to the neocortex to ensure that the gist of the memory is

preserved. It is presumed that the repeated reactivation of new memory traces during

off-line periods like sleep gradually strengthens and stabilizes this information.

Repeated reactivation in the form of SWRs during NREM periods has been amply

described in the hippocampus (see section 1.2.3). Notably, SWRs co-occur with cortical

spindles, highlighting a potential mechanism of cortico-hippocampal communication

during sleep (Gelinas et al., 2016; Siapas and Wilson, 1998). On the other hand, it is

thought that REM episodes following NREM periods may further stabilize memories, but

the mechanisms by which this process occurs is less clear.

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REM sleep has been clearly implicated in memory consolidation: in human and

rodent studies, REM sleep deprivation negatively affects memory performance

(Chernik, 1972; Karni et al., 1994; Pearlman and Becker, 1973, 1974; Smith and Butler,

1982; Walsh et al., 2011), while cue-induced re-exposure during REM enhances

subsequent memory performance (Guerrien et al., 1989; Hars et al., 1985; Hennevin et

al., 1989; Smith and Weeden, 1990).

Recent evidence indicates that synaptic downscaling may be one of the

mechanisms underlying memory stabilization in REM. This downscaling may enhance

the signal to noise ratio for more strongly encoded memory representations by nullifying

the strength of connections that were only weakly potentiated during wakefulness.

Indeed during REM sleep, new synapses are pruned, and the remaining synapses get

strengthened (Li et al., 2017). Network modeling suggests a strong link between the

strength of a neuron’s connectivity and its firing rate, indicating that firing rate

fluctuations may be a proxy for synaptic changes (Olcese et al., 2010; Tononi and

Cirelli, 2014). Supporting this notion, unit studies have shown that hippocampal

activation during REM sleep periods might contribute to an overall downscaling of

neuronal firing rates observed across sleep, as this decrease in firing rates during sleep

was correlated with REM theta power (Born and Feld, 2012; Grosmark et al., 2012;

Miyawaki et al., 2019; Miyawaki and Diba, 2016). Directly highlighting the importance of

theta oscillations in REM, inhibiting theta power in the hippocampus by optogenetically

inactivating the medial septum during post-training REM sleep results in memory

impairments (Boyce et al., 2016). Taken together, this data suggests that reactivation of

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sequences in SWRs during NREM, and subsequent theta power-associated synaptic

downscaling during REM may underlie memory consolidation.

1.4. Alterations in hippocampal activity and Alzheimer’s Disease*

The interplay between excitatory and inhibitory circuits underlies the brain’s

processes and their dysregulation has been linked to cognitive decline, psychiatric

disorders and epilepsy. In patients with Alzheimer’s disease (AD), an elevated

occurrence of seizures has been observed in both sporadic and familial forms of the

condition. Although seizure activity in AD has been mainly viewed as a result of

neuronal death and considered to occur in later stages, it is now increasingly clear that

aberrant neuronal activity may be more common in patients at earlier stages than

previously thought, and this aberrant activity may trigger and contribute significantly to

memory defects. In this section, we review abnormal oscillatory activity in the context of

AD, focusing on alterations of inhibitory and excitatory hippocampal and cortical circuits

that may lead to overexcitability and early dysregulation of neuronal networks, and the

therapeutic outcomes of restoring this excitatory-inhibitory balance.

1.4.1. Introduction

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by

declarative memory impairments and increasingly severe cognitive decline that

ultimately leads to dementia. To date, no successful treatment has been found. AD

pathology is predominantly characterized by the occurrence of amyloid beta (Ab)

plaques and neurofibrillary tangles of hyperphosphorylated tau in the brain, as well as

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neuronal loss. While Ab and tau may drive AD progression, the pathogenic cascade that

leads to AD appears to start decades before these clinical symptoms are present. In

particular, subjects at genetic risk for AD show hippocampal hyperactivity (as measured

with fMRI) during memory tasks (Bookheimer et al., 2000; Dickerson et al., 2005; Quiroz

et al., 2010) and impaired default network activity (Filippini et al., 2009), indicating the

presence of early network alterations. Importantly, although AD pathology is related to

global measures of cognition in unimpaired elderly subjects (Bennett et al., 2012), some

individuals display Ab accumulation without dementia (Bennett et al., 2012; Lue et al.,

1996; Roberts et al., 2018; SantaCruz et al., 2011). In fact, despite amyloid plaques

being a hallmark of the disease, the density of Ab deposits does not significantly

correlate with AD progression (Arriagada et al., 1992; Berg et al., 1993; Bierer et al.,

1995; Dickson et al., 1992; Guillozet et al., 2003; Schmitt et al., 2000), or does not

correlate as well as other measurements such as synaptic loss, which is regarded as

the best predictor (Bennett et al., 2004; Blennow et al., 1996; Giannakopoulos et al.,

2003; Lue et al., 1996; Scheff and Price, 1993; Terry et al., 1991). There is growing

evidence that alterations of the amyloid precursor protein (APP) and particular isoforms

of soluble Ab peptides can alter synaptic function and affect cognition before any

neurodegeneration or plaque deposition is observed in the brain (Balducci et al., 2010;

DʼHooge et al., 1996; Giacchino et al., 2000; Mucke et al., 2000, 1994; Oddo et al.,

2003; Shankar et al., 2008).

Beyond the main pathological hallmarks of AD, synaptic dysfunction is also of

major importance, and AD has consequently been referred to as a “disease of synaptic

failure” (Selkoe, 2002). Particularly, changes in excitatory and inhibitory synapses

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releasing glutamate and GABA, respectively, has gathered increasing interest as a

mechanism contributing to AD pathology. The fine balance between excitatory and

inhibitory transmission (E/I balance) is essential for brain oscillations (Amilhon et al.,

2015; Boyce et al., 2016; Huh et al., 2016) and normal cognitive function (Zhou and Yu,

2018). Perturbations in E/I balance probably contribute to cognitive changes in AD

(Busche and Konnerth, 2016; Selten et al., 2018), considering that abnormal oscillatory

rhythmic activity and network hypersynchrony are observed in AD mice models and AD

patients (de Waal et al., 2012; Goutagny et al., 2013; Irizarry et al., 2012; Lozsadi and

Larner, 2006; Osipova et al., 2005; Palop et al., 2007; Palop and Mucke, 2016; Vogt et

al., 2011).

1.4.2. Abnormal rhythmic activity in AD

Reflecting the progressive disruption in the fine balance between excitatory and

inhibitory transmission, EEG abnormalities are strongly related to disease severity and

disease stage in AD patients. Indeed, in the earliest stages of the disease, changes in

EEG tend to be mild or absent (Boerman et al., 1994; de Waal et al., 2012; Penttilä et

al., 1985). The most frequently observed spectral alteration is the “slowing” of the EEG

in resting state: a decrease in theta peak frequency, together with an increase of theta

power (~4-7 Hz) is expected in early AD patients, followed by a reduction of alpha

amplitude (~8-12 Hz) (Adler et al., 2003; Czigler et al., 2008; Huang et al., 2000; Jelic et

al., 2000; Moretti et al., 2010; van der Hiele et al., 2007). Although this is the most

common described characteristic, progressive EEG slowing could only be detected in a

proportion of early AD cases, with 50% showing no deterioration at 12 months follow-up

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(Rae-Grant et al., 1987; Soininen et al., 1989). In fact, theta power has been reported to

be unchanged at rest (Caravaglios et al., 2010; Wang et al., 2017) or even decreased

during visual and auditory stimulus processing (Başar et al., 2010; Caravaglios et al.,

2010; Yener et al., 2007). These discrepancies may in part be attributed to differences

in diagnostic criteria and methodological approaches, as well as to the fact that these

global EEG measures might not be quite sensitive enough (Jelic and Kowalski, 2009).

In AD rodent models, depth electrodes have been used to directly record

changes in oscillations from the hippocampus. In the early stages of the disease, before

plaque deposition is apparent, theta peak frequency has been shown to be decreased

in vivo (Cayzac et al., 2015; Siwek et al., 2015) and in vitro (Goutagny et al., 2013; Scott

et al., 2012). On the other hand, no changes in theta power have been found neither in

vivo (Rubio et al., 2012; Schneider et al., 2014) nor in vitro (Goutagny et al., 2013;

Mondragón-Rodríguez et al., 2018; Scott et al., 2012), with only one study detecting an

early decrease (Ittner et al., 2014). Once plaques are present, theta amplitude is

generally diminished (Mably et al., 2017; Rubio et al., 2012; Schneider et al., 2014;

Scott et al., 2012), although no changes have also been reported (Etter et al., 2019;

Siwek et al., 2015). All in all, the evidence here presented indicates that alterations in

theta amplitude, specially during rest, may not be a reliable early marker by itself for the

diagnosis of AD.

As seen in section 1.2.2, gamma amplitude and its coupling with theta are also

indicative of memory performance. Indeed, alteration in gamma and in its coupling with

slower frequencies have been observed in AD, although the studies in patients are few

and the results contradictory (Kim et al., 2012): both decreases (Goodman et al., 2018;

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Kurimoto et al., 2012) and increases (Van Deursen et al., 2008; Wang et al., 2017) in

gamma and theta-gamma coupling have been reported. The picture emerging from

hippocampal recordings in rodent models is clearer, with reductions in theta-gamma

coupling apparent early on (Goutagny et al., 2013; Mondragón-Rodríguez et al., 2018),

followed by decreases in gamma power (Etter et al., 2019; Gillespie et al., 2016; Mably

et al., 2017; Rubio et al., 2012; Schneider et al., 2014)(but see (Ittner et al., 2014)).

Interestingly, one early AD study has also reported decreased gamma power, but

specifically during sharp-wave ripples (Iaccarino et al., 2016). Although this SWR-

specific deficit may indicate that memory consolidation could be compromised in the

beginning stages of AD, it should be carefully interpreted: in hippocampal SWRs, the

presence of gamma appears to be spurious (Oliva et al., 2018), and may simply reflect

differences in inter-SWR separation between groups. Finally, gamma amplitude is not

only found to be lower in the hippocampus, but also in cortex (Verret et al., 2012). This

decrease in cortical gamma has been linked to aberrant GABAergic inhibition, which is

though to be reduced due to a loss of the voltage gated sodium channel subunit Nav 1.1

in interneurons (Martinez-Losa et al., 2018; Verret et al., 2012).

1.4.3. Abnormal network activity in AD promotes epileptiform activity

Significant E/I imbalance leading to epileptic-like activity probably contributes to

AD pathogenesis, since up to 22% of patients experience unprovoked seizures, with

rates increasing up to 58% in familial forms of AD (Amatniek et al., 2006; Friedman et

al., 2012; Larner, 2011; Mendez and Lim, 2003; Vossel et al., 2016, 2013). Seizures in

AD can be convulsive tonic-clonic, or more prevalently, non-convulsive with patients

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experiencing altered consciousness, amnestic periods, and confusion which can go

undiagnosed for years (Vossel et al., 2013). Furthermore, at least 20% of patients with

AD experience transient episodes of amnestic wandering and disorientation, common

elements of several dementias, which have been associated with epileptiform

discharges (Bradshaw et al., 2004; Lee et al., 2012; Rabinowicz et al., 2000). It is likely

that epileptiform activity in the early stages of AD is often underdiagnosed and

undetectable as intracranial electrodes positioned adjacent to the mesial temporal lobe

may be necessary to reveal abnormal electrical signals such as spike and wave activity

during sleep in patients with no history of epilepsy (Brown et al., 2018; Lam et al.,

2017).

Several studies suggest that increases in Ab, and particularly early soluble forms

of Ab such as oligomers, as well as Ab associated proteins, are key factors responsible

for altering E/I balance. Such rapid increases in soluble forms of Ab are central to

familial early onset forms of AD, which have mutations in the gene for APP or

presenilin-1 (PSEN1) (Brouwers et al., 2008; Potter et al., 2013; Shepherd et al., 2009;

Suzuki et al., 1994). In AD transgenic mouse models with similar APP mutations,

prominent neuronal hyperactivity and impaired E/I are also observed. For example,

amyloid oligomers in APP mice models were shown to acutely affect synaptic

transmission (Busche et al., 2012) and change the structure of the circuit (i.e. dendritic

morphology) that directly cause hyperexcitability (Šišková et al., 2014). Mice with

overexpression of mutated APP, or mutated PSEN1 or ApoE4 genes (a risk factor firmly

linked to sporadic AD in humans), display spontaneous seizures and spike-wave

discharges (SWDs), suggesting an early E/I imbalance linked to amyloid (Born, 2015;

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25

Born et al., 2014; Minkeviciene et al., 2009; Nuriel et al., 2017). SWDs are of particular

interest as an early feature of E/I imbalance in AD, as they occur even before the

presence of spontaneous seizures (Bezzina et al., 2015; Born et al., 2014; Kam et al.,

2016; Nygaard et al., 2015; Verret et al., 2012). SWDs reflect sudden, transient

synchronous hyperactivity, consisting of high voltage deflections with amplitudes

exceeding twice the baseline of recording, and resemble the waveform of interictal

spikes (Bezzina et al., 2015; Kam et al., 2016). Recent studies have focused on these

SWDs in AD mouse models as an important early factor (as illustrated in Fig.1.4)

underlying or contributing to cognitive defects, and are a relevant marker in the

diagnosis of epilepsy (Krendl et al., 2008; Rosati et al., 2003; Staley et al., 2011). SWDs

are notable in AD models carrying the APP Swedish mutation, which increases overall

Ab levels, in conjunction with other mutations, including the J20 (Palop et al., 2007),

Tg2576 (Bezzina et al., 2015), APP/TTA (Born et al., 2014), APP/PS1 (PSEN1)

(Minkeviciene et al., 2009; Reyes-Marin and Nuñez, 2017), and 3xTg (Nygaard et al.,

2015) models. To study the role of APP in the incidence of SWDs, models that enable

the expression of the human-mutated APP at different time points in a tetracycline-

responsive manner with the use of tetracycline analogs have been created (called

APP/TTA model, where TTA stands for tetracycline-controlled transactivator protein).

Once APP is expressed, SWDs are observed from the earliest time point that the

authors examined, and their number is higher during the light cycle when sleep is more

frequent (Born et al., 2014). The occurrence of SWDs was analyzed in more detail by

Kam et al. (2016) in Tg2576 mice, where they reported that SWDs appeared before

plaque deposition, and were more prominent in quiet wakefulness and sleep, and

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26

especially during rapid eye movement sleep (REM). A similar distribution of SWDs

during before widespread plaque deposition sleep has also been observed in the J20

model (Brown et al., 2018). On the other hand, Brown et al. also measured the

incidence of interictal spikes in a patient with amnesic mild cognitive impairment using

in-depth electrodes and found a higher incidence of events during non-REM rather than

REM sleep. This finding is in agreement with studies of temporal lobe epilepsy, where

the rate of interictal spikes is generally higher during non-REM sleep (Clemens et al.,

2003; Lieb et al., 1980; Rossi et al., 1984; Sammaritano et al., 1991). The discrepancy

with AD animal studies may be partially dependent on the recording area since, in

epileptic patients, the prevalence of interictal spikes is higher during REM sleep if

recording from the primary epileptogenic region (Lieb et al., 1980; Rossi et al., 1984;

Sammaritano et al., 1991). Nevertheless, the occurrence of SWDs during sleep and

quiet wakefulness may have significant implications for memory impairment since these

periods are known to be necessary for memory consolidation (Boyce et al., 2016;

Buzsáki, 2015; Girardeau et al., 2009; Karlsson and Frank, 2009).

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27

Fig. 1.4: Earliest recorded Spike-Wave Discharges (SWDs) in AD models. The

corresponding APP mutation is in parenthesis. References: J20 (Mucke et al., 2000;

Palop et al., 2007; Sanchez et al., 2012a; Verret et al., 2012). Tg2576 (Jacobsen et al.,

2006; Kam et al., 2016; Kawarabayashi et al., 2001). APP/PS1 (Minkeviciene et al.,

2009; Reyes-Marin and Nuñez, 2017; Schmid et al., 2016; Trinchese et al., 2004).

APP/TTA: (Born et al., 2014; Marin et al., 2016; Sri et al., 2019). 3xTg: 8 to 10 months is

the only time point were SWDs have been studied in this model to date (Billings et al.,

2005; Nygaard et al., 2015; Oddo et al., 2003).

Although not as much is known regarding the incidence of seizures in tau models

without APP mutations, tau aggregates have been reported in patient with epilepsy (Tai

et al., 2016) and reducing tau in experimental models of epilepsy ameliorates seizures

(Holth et al., 2013). FTDP-17 mice (a model of frontotemporal dementia with

parkinsonism linked to chromosome 17 which overexpresses human mutant tau) have

spontaneous seizures starting as early as 5 months of age, before tau aggregates are

present (García-Cabrero et al., 2013). Tau also appears to modulate hyperactivity and

seizure incidence in rodents, as knocking out tau completely, or reducing tau levels in

APP models, reduces SWDs and prevents increased susceptibility to induced seizures

(Roberson et al., 2011, 2007).

1.4.4. Excitatory Disruption and Hyperactivity

In AD, there has been a strong focus on excitatory dysfunction as the basis for

E/I disruption. Typically, changes in excitatory synaptic transmission have been

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28

suggested to play a key role in the aberrant hyperactivation and hypersynchrony of

circuits resulting in the generation and spreading of epileptic discharges. Similarly,

administration of soluble Ab in vivo (Busche et al., 2012), in vitro (Minkeviciene et al.,

2009) and in neuronal cultures (Cuevas et al., 2011) have also been shown to generate

neuronal hyperexcitability in hippocampal neurons and circuits. The Ab-mediated

increase in neuronal excitability could be due to several mechanisms associated with

glutamate synaptic transmission. Ab soluble oligomers are known to rapidly enhance

hippocampal NMDAR currents in vitro (Wu et al., 1995), in vivo (Molnár et al., 2004) and

in cell membranes of AD patients microtransplanted into oocytes (Texidó et al., 2011),

suggesting that these oligomers may directly cause increased neuronal firing.

Moreover, soluble Ab oligomers could cause hyperexcitability by disrupting

glutamate uptake as demonstrated by experiments employing cultured neurons and

astrocytes, wherein Ab directly alters glutamate transporter expression and/or function

(Fernández-Tomé et al., 2004; Harris et al., 1996; Matos et al., 2012). The disruption of

glutamate transporters, named excitatory amino acid transporters or EAATs, can cause

abnormal increases in extracellular glutamate concentrations, triggering elevated

spontaneous excitatory postsynaptic currents, population spike frequency, and

impairments in long-term-potentiation (Lei et al., 2016; S. Li et al., 2009). In addition,

reducing glutamate transporters can have significant effects on excitability because

these transporters can normally prevent the generation and spread of seizures

(Demarque et al., 2004). Glutamate is also a GABA precursor; consequently, disruption

of glutamate reuptake could indirectly reduce GABAergic synthesis, thereby intensifying

E/I imbalance and further promoting hyperexcitability (Sepkuty et al., 2012). Such

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29

reductions in glutamate transporter expression have been reported in AD patients and

may contribute to reductions in glutamate uptake, E/I imbalance and neurodegeneration

(Masliah et al., 1996; Scott et al., 2011).

Additionally, APP and soluble Ab can modulate synaptic plasticity and glutamate

receptor trafficking (see Hoe et al., 2012 for review). APP levels have been shown to

change early during development and peak in the second post-natal week, which

coincides with the NMDAR developmental receptor subtype switch from GluN2B to N2A

(Liu et al., 2004; Löffler and Huber, 1992). However, if APP is over-expressed, this can

cause an upregulation of NMDARs containing GluN2B subunits in the hippocampus due

to increased GluN2B mRNA levels (Cousins et al., 2009; Hoe et al., 2009). This

increase in GluN2B NMDA receptor subtype may, in turn, increase excitability and

directly contribute to seizures (Chen et al., 2016; Okuda et al., 2017). Although the

mediator behind this APP synaptic regulation may involve its intracellular domain

(Pousinha et al., 2017), the oligomeric form of Ab can alter surface levels of GluN2B

NMDARs, which is required for the presence of seizure phenotype in the APP/PS1 AD

mice model (Um et al., 2012). At the same time, Ab also disrupts the ability of other

mechanisms to limit excessive NMDAR activity, thus adding to network hyperexcitability

(You et al., 2012).

Apart from its role in modulating receptor trafficking, soluble Ab can directly

activate the GLuN2B NMDA receptor subunit, thereby disrupting intracellular calcium

homeostasis and synaptic plasticity (Ferreira et al., 2012; Li et al., 2011). Overactivation

of glutamate receptors has been shown to be highly toxic, an effect termed

excitotoxicity. Undesired increases of intracellular Na+ and Ca2+ that result in cell death

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30

will occur once the NMDAR Mg2+ block is released by persistent depolarization of the

cell (Arundine and Tymianski, 2003; Choi, 1985; Koh and Choi, 1991; Liu et al., 2007;

Rothman, 1985). A substantial body of evidence indicates that this neurotoxic

mechanism may contribute to the eventual neuronal loss seen in AD (Brorson et al.,

1995; Hynd et al., 2004; Mattson et al., 1992; Mattson and Goodman, 1995; Miguel-

Hidalgo et al., 2002; Tominaga-Yoshino et al., 2001; Yatin et al., 2001). Moreover,

activation of NMDARs is also known to regulate APP trafficking and processing, as well

as facilitating Ab production (Bordji et al. 2010; Lesne et al. 2005; Hoe et al. 2009),

potentially creating a feedback loop which eventually results in cell death.

Ab toxicity may also be potentiated by tau through increases in glutamatergic

signaling leading to excitotoxicity. Tau can stabilize NMDARs in synapses and increase

NMDA receptor-dependent currents through an aberrant association with the kinase

Fyn, ultimately strengthening glutamate neurotransmission (Ittner et al., 2010). Notably,

the reduction of tau suppresses NMDA dependent excitotoxicity in hippocampal slices

(Miyamoto et al., 2017) and decreases hyperexcitability in the form of SWDs and

seizure activity in animal models as noted previously (AD: Roberson et al. 2007;

Epilepsy: Holth et al. 2013). This enhanced glutamatergic signaling has been proposed

to account for the higher NMDAR binding found in post-mortem brain tissue from AD

patients (Ułas et al., 1994).

Finally, Ab can also interact with mGluRs, and its oligomeric form in particular, is

able to aberrantly cluster mGluR5, which in turn elevates intracellular calcium and could

ultimately cause synaptic deterioration (Renner et al., 2010). In agreement with this,

patients with AD have enhanced mGluR5 levels as measured by immunostaining in

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31

comparison to age-matched controls, at least in astrocytes (Casley et al., 2009) and

deletion of mGluR5 is known to improve cognitive decline in the APP/PS1 model of AD

(Hamilton et al., 2014).

Excitatory circuitry alterations may also directly dysregulate inhibitory

neurotransmission. There is evidence that glutamate may developmentally regulate

axons and dendrites of GABAergic interneurons (De Marco García et al., 2011), and

that glutamatergic signaling itself is capable of modulating post-synaptic GABAA

receptor expression and clustering (Bannai et al., 2015) as well as inhibitory circuit

plasticity (Mapelli et al., 2016; McLean et al., 1996; Moreau and Kullmann, 2013).

Although a great deal of data indicates that aberrant changes in excitatory synaptic

transmission are important for AD progression, significant evidence also exists

suggesting that disruption in GABAergic synaptic transmission is also important.

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32

Fig. 1.5: Schematic of neural network in normal and AD conditions, including

Glutamatergic/GABAergic neurons and connections. Presynaptic glutamate release

triggers postsynaptic activation through the binding of membrane receptors. In the

synaptic cleft, glutamate can be taken up by transporters located in astrocytes and

converted to glutamine and GABA. On the other hand, presynaptic GABA release

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33

triggers postsynaptic deactivation through GABAr, and can also be taken up by

astrocytic GABAergic transporters. In AD conditions, glutamatergic concentration in the

synaptic cleft is increased due to altered glutamatergic transport. This alteration leads to

overactivation of postsynaptic neurons through upregulated NMDA and mGluR

receptors. GABAergic transmission is impaired due to a loss of presynaptic sodium

currents and postsynaptic GABA receptors. To counteract hyperexcitability, astrocytes

may facilitate GABAergic synthesis, release and innervation. [1] Busche et al., 2012. [2]

Minkeviciene et al., 2009. [3] Cuevas et al., 2011. [4] Molnár et al., 2004. [5] Texidó et

al., 2011. [6] Wu et al., 1995. [7] Fernández-Tomé et al., 2004. [8] Harris et al., 1996. [9]

Matos et al., 2012. [10] Sepkuty et al., 2012. [11] Masliah et al., 1996. [12] Scott et al.,

2011. [13] Cousins et al., 2009. [14] Hoe et al., 2009. [15] Chen et al., 2016. [16] Okuda

et al., 2017. [17] Ferreira et al., 2012. [18] Li et al., 2011. [19] Ulrich, 2015. [20] Howell

et al., 2000. [21] Ikonomovic et al., 2003. [22] Limon et al., 2012. [23] Mizukami et al.,

1998. [24] Ren et al., 2018. [25] Mondragón-Rodríguez et al., 2018. [26] Verret et al.,

2012. [27] Martinez-Losa et al., 2018. [28] G. Li et al., 2009. [29] Bell et al., 2003. [30]

Born et al., 2014. [31] Hollnagel et al., 2019. [32] Palop et al., 2007.

1.4.5. Inhibitory Disruption

Although the predominant view has been that inhibitory synaptic transmission is

relatively more resilient in AD (Francis et al., 1993; Palmer and Gershon, 1990;

Reinikainen et al., 1988; Rissman and Mobley, 2011), it is now becoming clear that

inhibitory circuits are severely disrupted early in the disease process (Bell et al., 2006;

Rossor et al., 1982; Ulrich, 2015), as depicted in Fig.1.5. For example in the APP/TTA

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34

AD mice model, an E/I imbalance is apparent since administering 1.75mg/kg of the

GABAA antagonist picrotoxin leads to seizures within an hour in vivo, an effect not

observed with the same dosage in controls (Born et al., 2014). Another GABAA receptor

antagonist, Pentylenetrazol, has also been shown to induce seizures at a higher rate in

the Tg2576 (Westmark et al., 2008), J20 (Palop et al., 2007) and tgCRND8 (Del

Vecchio et al., 2004) AD models compared to controls. These effects may be explained

by factors related to reduced inhibitory function. For example, Ab application to cortical

slices in vitro results in the endocytosis of GABAA receptors (Ulrich, 2015), and

evidence from AD patients suggests that GABAergic receptor activation provides

smaller inhibitory currents, indicating a remodeling of GABAergic inhibition in human AD

(Limon et al., 2012). Other studies report that specific GABA receptor subunits are

downregulated in particular regions (Howell et al., 2000; Limon et al., 2012; Mizukami et

al., 1998; Rissman et al., 2003), while a preservation in the expression of other subunits

has also been observed (Limon et al., 2012; Mizukami et al., 1997; Rissman et al.,

2003), suggesting area-specific changes in receptor composition (see Rissman and

Mobley, 2011 for review).

Ab also produces a decrease in GABA release from fast-spiking interneurons

innervating principal cells in slice (Ren et al., 2018). Fast spiking firing patterns have

been associated with parvalbumin (PV) expressing GABAergic neurons since the 1980s

(Kawaguchi et al., 1987; see Hu et al., 2014 for review), have been linked to memory

consolidation (Ognjanovski et al., 2017; Xia et al., 2017a) and are essential for spatial

working memory (Murray et al., 2011). Stimulating PV interneurons, but not other cell

types, can enhance gamma oscillatory activity (20-80 Hz) (Cardin et al., 2009), and

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35

increases in this frequency band during memory encoding have been linked with

memory performance (Sederberg et al., 2006; Yamamoto et al., 2014), see section

1.2.2). Early in the J20 AD model, a reduction of PV interneuron firing has been

observed in areas such as the hippocampus (Mondragón-Rodríguez et al., 2018) and

has been linked to the loss of the voltage-gated sodium channel subunit Nav 1.1,

leading to a decrease of GABAergic inhibition and possibly gamma power (Verret et al.,

2012). Importantly, interneuron transplants overexpressing Nav1.1 in cortex and

hippocampus were able to reduce hypersynchrony, enhance gamma oscillations and

improve memory in the same transgenic mouse line (Martinez-Losa et al., 2018). Along

the same line, stimulating PV interneurons at gamma frequency appears to decrease

Ab-40 and 42 isoforms (Iaccarino et al., 2016). In findings linked to interneuron

hypoactivity, neuronal pentraxin-2, a protein secreted by excitatory neurons which

specifically mediates activity-dependent strengthening of excitatory synapses onto PV

interneurons (Chang et al., 2010), is down-regulated in the brains of AD patients (Xiao

et al., 2017).

Several lines of research also show that APP itself may modulate GABAergic

inhibition and GABAA receptors (Chen et al., 2017; Seabrook et al., 1999; Yang et al.,

2009). Most recently, Rice et al. (2019) demonstrated that secreted APP is able to bind

to GABAB receptors to suppress synaptic vesicle release, thus modulating hippocampal

synaptic plasticity. In addition to APP, the AD risk factor ApoE4 is known to decrease

both the number of interneurons as well as GABAergic innervation in ApoE4 knockin

mice (G. Li et al., 2009), whereas conditionally deleting ApoE4 in neurons rescues

GABAergic neuronal loss and memory deficits (Knoferle et al., 2014). Other studies

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36

point to important changes in synaptic inhibition and the subsequent potential

compensatory responses of the GABAergic circuitry that are associated with

hyperactivity and cognitive decline in AD models. For instance, in transgenic models

where early epileptic discharges have been observed, an increase in the number of

GABAergic presynaptic boutons and GABAergic terminals in cortex (Bell et al., 2003;

Born et al., 2014), CA1-3 hippocampal regions (Hollnagel et al., 2019) and dentate

gyrus (Palop et al., 2007) have been noted.

As the disease progresses and once amyloid deposits are present, the number of

GABAergic presynaptic boutons is severely decreased near plaques in the cortex of

APP/PS1 mice at 18 months of age (Bell et al., 2006). A diminishing number of

GABAergic cortical terminals at the perisomatic level immediately adjacent to Ab

plaques has also been observed in AD patients (Garcia-Marin et al., 2009; Hardy et al.,

1987), as well as general lower levels of GABA neurotransmitters in temporal cortices

(Gueli and Taibi, 2013; Seidl et al., 2001). In particular, somatostatin-expressing

GABAergic hippocampal interneurons, which are essential for learning and memory

(Davies et al., 1980; Siwani et al., 2018), appear to be preferentially affected. In a

complete longitudinal study spanning the age of 4 to 11 months, Schmid et al. (2016)

imaged this population in APP/PS1 mice in vivo, and found, dependent on plaque

proximity, progressive axonal loss starting at 5 months of age and additional plasticity

deficits after a learning task. Related findings have been described in temporal lobe

epilepsy models and patients (Dinocourt et al., 2003; Robbins et al., 1991), as well as in

AD patients, where somatostatin-like immunoreactivity is reduced in cortex and

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37

hippocampus (Davies et al., 1980; Davis et al., 1999; Grouselle et al., 1998; Rossor et

al., 1980).

Consistent with a reduction in GABAergic inhibition, a decreased number of

GABAergic neurons in the hippocampus has also been found in the tgCRND8 (Krantic

et al., 2012), APoE4 knockin (G. Li et al., 2009) and 3xTg AD models (Zallo et al.,

2018). Specially neuronal loss (50-60%) is markedly pronounced in the stratum oriens

of CA1-3 and dentate regions of APP/PS1 mice, before any other neural loss is

observed in these regions of the hippocampus (Baglietto-Vargas et al., 2010; Ramos et

al., 2006). In AD patients, a reduction of GABAergic neurons measured using GAD65

immunostaining has been noted in the dentate gyrus (Schwab et al., 2013), and region-

specific hippocampal GABAergic morphological changes and neural loss has been

described in PV (Brady and Mufson, 1997) and somatostatin/NPY (Chan-Palay, 1987;

Chan-Palay et al., 1986) interneuron populations. Other cell type changes were not

compared in these hippocampal measurements, but in the entorhinal cortex, GABAergic

interneuron degeneration precedes changes to principal cells, at least in the early

stages of the disease (Mikkonen et al., 1999; Solodkin et al., 1996).

In addition to neurons, astrocytes modulate the brain’s excitatory/inhibition

balance and play an important role in brain homeostasis (McKenna et al., 2002; Pekny

et al., 2016; Rose and Ransom, 1996; Rothstein et al., 1996; Schousboe and

Waagepetersen, 2003; White et al., 2002). Changes in their function appear early on in

AD patients (Carter et al., 2012) and AD animal models (Heneka et al., 2005). For

example, as a response to glutamatergic stimulation, astrocytes can reverse the

function of GABAergic GAT2/3 receptors in slice, increasing GABA release (Héja et al.,

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38

2009). This reversal has been shown to lead to an abnormal tonic inhibition in the

5xFAD mouse model, which can be countered by the application of GABA transporter

inhibitors, supporting a dysregulation of GABAergic mechanisms (Wu et al., 2014).

Indeed in this same model, the enzymes GAD65 and MaoB are upregulated and GABA

synthesis is therefore enhanced in areas such as the dentate gyrus (Jo et al., 2014; Wu

et al., 2014). Results from patients further corroborate the involvement of astrocytes in

the early remodelling of the GABAergic system, as the largest signal of MaoB is

detected in activated astrocytes in prodromal AD (Saura et al., 1994). The expression of

astrocytic transporters in patients is clearly altered as well, but these changes are

complex and region-specific (Fuhrer et al. 2017). While GAT3, the human/rat equivalent

of GAT4 in mice, has been found to be upregulated in the dentate gyrus (Wu et al.,

2014), it is downregulated in other hippocampal and cortical areas (Fuhrer et al., 2017).

In Fuhrer et al.’s study, GAT3 downregulation was accompanied by an increase of the

astrocytic transporter BGT1, and a similar co-regulation between GAT3 and BGT1 has

been described after excitatory injury of the hippocampus (Zhu and Ong, 2004) which

the authors propose is a protective mechanism.

1.4.6. Targeting cognitive dysfunction by circuit modulation

Identifying the mechanisms that promote E/I dysfunction in early AD can guide the

development of future therapies. To date, only four drugs have been approved to treat

the symptoms of AD, and they can be separated into two categories:

acetylcholinesterase inhibitors (AChEI) comprising Donepezil, Galantamine and

Rivastigmine, and the NMDAR GluN2B antagonist Memantine. AChEI drugs increase

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39

cholinergic transmission and are recommended for the treatment of mild to moderate

AD (Arce et al., 2009; Ballard, 2002; NICE, 2011). Memantine is the only NMDAR

antagonist used by AD patients to prevent the decline in cognition (although the effects

are modest) and is recommended for the treatment of moderate to severe AD

(Limapichat et al., 2013; Matsunaga et al., 2015; NICE, 2011). These drugs only

temporarily reduce the rate of decline, but do not stop its progression.

Presently, treatments focusing on amyloid reduction and clearance have

dominated the therapeutic landscape even though amyloid only weakly correlates with

cognitive decline in the symptomatic phase of the disease. These drugs aim to clear

pre-existing plaques or inhibit the formation of new ones by using monoclonal

antibodies against Ab or by inhibiting the secretases that cleave APP and produce the

plaque-forming Ab isoforms. However, it remains to be determined if eliminating or

reducing Ab will have a significant effect on the cognitive decline in AD patients. A

recent phase III clinical trial with the monoclonal antibody Solanezumab, reduced free

Ab levels by at least 90%, but was unable to clear existing plaques or slow-down

cognitive decline (Honig et al., 2018). Likewise, the beta-secretase inhibitor

Verubecestat was shown to reduce Ab cerebrospinal fluid levels by 75%, but had no

effect in decreasing the rate of cognitive decline (Egan et al., 2018). Similar results have

been found in clinical trials with gamma-secretase inhibitors (Doody et al., 2013) and

although these and other anti-amyloid drugs have been proven ineffective (Biogen,

2019; Sevigny et al., 2016), clinical trials targeting different Ab molecules are still

ongoing and may show promise (Salloway et al., 2014; see Folch et al., 2017 for

review).

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The overall lack of success of amyloid therapies suggest that once dementia is

present, disease progression is likely independent of Ab production and could be

irreversible. It is reasonable to assume that treatments targeting amyloid should be

implemented early in the disease and not simply focus on plaque clearance, as the

pathological cascade is thought to begin 10 to 20 years before the onset of clinical

symptoms (Bateman et al., 2012; Villemagne et al., 2013). This highlights the need for

developing useful biomarkers which may aid clinicians in the detection of the disease at

earlier stages, and ease the recruiting of patients for clinical trials before the dementia

stage has been reached (Blennow and Zetterberg, 2018).

Interventions targeting mechanisms reducing the E/I imbalance discussed in this

review may provide new therapeutic opportunities to improve cognition and quality of life

in patients. For example, memantine is one such drug that targets NMDAR GluN2B and

can taper the rate of cognitive decline by directly regulating hyperexcitability. Other

glutamate receptor antagonists such as Ifenprodil, a GluN2B selective antagonist, can

prevent Ab induced Ca2+ rise and synaptic plasticity impairments in vitro (Ferreira et al.,

2012; Hu et al., 2009; Rönicke et al., 2011). mGluR5 antagonists show some promise

when tested in models of AD since they can reduce Ab production, seizures, and help

restore LTP (Kazim et al., 2017; Rammes et al., 2011). Overall, these results suggest

the validity of developing drugs targeting the glutamatergic system as they may

potentially provide beneficial effects to help restore E/I balance. Using a combination of

glutamate receptor antagonists with other drugs in the treatment of AD is now being

explored. One such combination is Memantine and AChEIs to potentially achieve

additive positive effects in patients (Parsons et al., 2013; Tariot et al., 2004).

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41

Anti-epileptic drugs such as the closely related Brivaracetam and levetiracetam

have been shown to successfully normalize the excitation/inhibition balance observed in

AD mice models. Specifically, when J20 mice are treated with levetiracetam, they show

a reduction in epileptiform activity (such as SWDs) and an improvement in memory

(Sanchez et al., 2012a). Brivaracetam has also been successful at decreasing SWD

hyperactivity in the APP/PS1 and 3xTg AD models while ameliorating spatial memory

(Nygaard et al., 2015). Although their mode of action remains to be clearly established,

brivaracetam and levetiracetam are known to interact with the synaptic vesicle protein

2A (SV2A)(Lynch et al., 2004) and may regulate its expression. Since SV2A knockout

animals or mice with missense mutations of SV2A have severe seizures, these drugs

may regulate the expression of the Ca2+ sensor protein Syt1, thereby changing the

sensitivity of synaptic vesicles to Ca2+, and specifically affecting the modulation of

GABA release in the hippocampus (Tokudome et al., 2016). In humans, these drugs

have shown promising results as levetiracetam administered to patients suffering from

mild cognitive impairment (which often progress to AD), results in reduced hippocampal

hyperactivation as measured by BOLD levels, and improve performance in a memory

task (Bakker et al., 2012).

Medications that restore E/I balance which are already clinically approved could

be potentially repurposed as valuable disease modifying treatments for AD and should

be taken into consideration. One such example is Acamprosate, a synthetic GABA

analog that is thought to interact with NMDARs (Qatari et al., 1998). Acamprosate is

often prescribed as an anti-craving medication to prevent alcohol relapse and can

decrease glutamate levels and reduce hyperexcitability (Dahchour et al., 1998; Kalk and

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42

Lingford-Hughes, 2014; Umhau et al., 2010). Other examples include Baclofen, a

GABAB receptor agonist, which in conjunction with Acamprosate is currently being

tested in AD patients (Chumakov et al., 2015).

Beyond pharmacology, novel therapies such as deep brain stimulation (DBS), a

neurosurgical procedure able to focally modulate circuit activity, are being developed

(Lozano et al., 2016; Smith et al., 2012). In rodents, there is strong evidence that

memory can be enhanced by indirectly stimulating the hippocampus through electrical

activation of associated regions or the white matter tracts that connect them. Stimulation

of the entorhinal cortex (Stone et al., 2011), fornix (Hescham et al., 2017, 2016),

hypothalamus (Soriano-Mas et al., 2005) and anterior thalamic nuclei (Hamani et al.,

2011) have been associated with significant improvement in spatial memory in healthy

animals. Memory deficits are also abolished on spatial learning tasks, particularly after

stimulation of the fornix (McNaughton et al. 2006- tetracaine; Zhang et al. 2015- Aβ 1-42

injection; Hescham et al. 2013- scopolamine injection; Hao et al. 2015- RTT mouse

model; Gallino et al., 2019; Mann et al., 2017; Xia et al., 2017b; - AD transgenic

models).

In humans, evidence from drug-resistant epileptic populations and in one case

of morbid obesity appears to reinforce the idea that stimulation of hippocampal

associated areas may improve memory (Fell et al., 2013; Hamani et al., 2008;

Koubeissi et al., 2013; Miller et al., 2015; Oh et al., 2012; Suthana et al., 2012). In

patients with AD, a handful of studies particularly on the effect of fornix DBS have been

conducted. In an initial Phase 1 clinical trial, six patients with AD who were treated with

fornix DBS for a year showed that the implant procedure and stimulation were well-

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tolerated, together with improvement in a subset of symptoms. A possible slowing of

cognitive decline and increased glucose metabolism in the temporal lobe as measured

with PET were observed, together with slower rates of hippocampal atrophy (Laxton et

al., 2010; Sankar et al., 2015; Smith et al., 2012). Similar results were found in a case

study by Fontaine et al. (2013). In a subsequent phase 2 clinical trial, no significant

differences in cognitive scores were observed in patients who had received fornix DBS

treatment vs sham stimulation, although if patients were segregated by age, a positive

cognitive change was noted if 65 or older (Holroyd et al., 2015; Lozano et al., 2016;

Ponce et al., 2015). Finally, fornix stimulation has been recently reported, in a case

study, to elicit memory flashback in a portion of AD patients (Deeb et al., 2019).

Taken together, the evidence presented here suggests that fornix DBS may have

the potential to modulate the memory network and reduce cognitive decline at least in a

subset of patients. Up to now, these studies have only employed high frequency

parameters, while stimulating chronically well in advance of any cognitive/memory

testing, and the effectivity of other stimulation paradigms remains unknown. A

systematic characterization of how these and other stimulation paradigms affect E/I

balance and memory mechanisms in the context of disease will be critical for the

efficient development of optimal stimulation protocols for AD.

1.4.7. Conclusions and Outlook

Although it is becoming increasingly clear that an E/I imbalance and epileptic

activity may be an early stage dysfunction in the brains of AD patients, non-convulsive

network anomalies may have gone unnoticed and undiagnosed in the patient

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population, and it is not apparent when and how these anomalies start or progress.

Despite an increased number of studies pointing to multiple alterations to excitatory and

inhibitory mechanisms, the interrelation between these systems and how their

interaction progressively changes should be considered from the context of AD. Before

plaques are present in AD brains, APP and amyloid beta are able to disrupt both

glutamatergic and GABAergic signaling, thus upsetting the networks’ fine

excitatory/inhibitory balance. Glutamate uptake and enhanced glutamatergic signaling

via changes in NMDARs and mGluRs are involved in synaptic dysfunction in the early

stages of AD, and pharmacologically blocking these receptors shows promising results.

Recent research has also highlighted the changes in GABAergic innervation and

impaired firing of fast-spiking GABAergic neurons, and how antiepileptic drugs may be

able to abolish hyperexcitability and ameliorate cognitive decline. Finally, the

importance of astrocytes in balancing the system by reversing GABAergic transport

function and increasing GABA synthesis should also be considered, as astrocytic

alterations are detected early on in patients. These results suggest that determining the

effect and causes leading to neuronal network imbalance in neurodegenerative

diseases is an important question that spans beyond neuronal death and can lead to

the development of potential therapeutic targets.

Based on results from recent anti-amyloid clinical trials, it has become apparent that

reducing Ab levels in the later stages of AD is unlikely to result in better patient

outcome, highlighting the need to develop useful preclinical biomarkers. The use of

additional treatment approaches to modulate early E/I imbalance in AD may offer

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promising disease-modifying strategies. Further studies on the effect of antiepileptic

drugs and other inhibitory modulating drugs in AD should be considered.

1.5. Hypothesis and Aims

Hypothesis: The modulation of hippocampal activity will improve memory

performance in an Alzheimer’s Disease mouse model.

To test this central hypothesis, the current work will address the following aims:

• Our first aim is to describe the early hippocampal oscillatory alterations in AD and

assess if a corresponding memory deficit is present. In chapter 2, we use

electrophysiological recordings of area CA1 in freely-moving mice, to evaluate

state-dependent hippocampal alterations in the J20+ APP-overexpressing AD

mouse model.

• The second aim of this project is to determine if rhythmic activation of septo-

hippocampal projections modulates oscillatory hippocampal alterations in J20+.

Using recordings from freely-moving animals, the effect of fornix optogenetic

stimulation is assessed on hippocampal rhythms, hyperactivity, and memory.

• Our final aim is to compare two stimulation paradigms on memory rescue by

employing electrical fornix stimulation, a potential therapeutic treatment tested in

AD patients. We used a similar physiological pattern to that employed in chapter

3 and contrasted its effects to the high-frequency paradigm applied in clinical

settings.

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Chapter 2: Characterization of early hippocampal dysfunction and

spatial memory in Alzheimer’s Disease

2.1. Introduction

Currently, one of the critical efforts in Alzheimer’s disease research is the

identification of early markers to aid with diagnosis and guide the development of

effective therapies. The advanced stage at which the disease is typically diagnosed is

partly why existing disease-modifying therapies may have only a limited symptomatic

benefit. Indeed timing may be crucial for therapeutic intervention to be effective (Wolfe,

2016). Notably, before neural loss becomes widespread, it is thought that an initial

synaptic failure leading to hippocampal dysfunction gives rise to the memory deficits

associated with AD (Selkoe, 2002; Small et al., 2001). Even at the disease’s end stage,

as indicated by postmortem studies, synaptic loss is more robustly correlated with

premortem cognitive deficits than the number of amyloid plaques or tau tangles,

neuronal loss, or cortical gliosis (Terry et al., 1991). In accordance, compelling evidence

from the rodent literature indicates that synaptic and behavioral alterations arise in AD

before plaque deposition is apparent (DʼHooge et al., 1996; Giacchino et al., 2000;

Mucke et al., 2000; Oddo et al., 2003).

Neurophysiological tools are able to detect alterations in neural network activity

in patients (Babiloni et al., 2009; Rossini et al., 2007) and animal models (Palop et al.,

2007). Although network alterations are a reflection of these early excitatory-inhibitory

imbalances (Hollnagel et al., 2019; Martinez-Losa et al., 2018; Verret et al., 2012;

Volman et al., 2011; Wulff et al., 2009; Zarnadze et al., 2016), apparent rhythmic

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alterations such as decreases in theta-gamma coupling or in theta and gamma power

have mainly been described once plaques are already present in vivo (Etter et al., 2019;

Iaccarino et al., 2016; Palop et al., 2007; Rubio et al., 2012; Verret et al., 2012), earlier

in the disease if under anesthesia (Hamm et al., 2017; Scott et al., 2012) or in vitro

(Goutagny et al., 2013; Mondragón-Rodríguez et al., 2018). Whether these same

network deficits can be detected in freely-behaving mice is unclear, with studies either

showing no (J20+, (Rubio et al., 2012)), slight (5xFAD (Siwek et al., 2015)) or overt

(APP23 (Ittner et al., 2014)) changes in power. Electrophysiological techniques have the

potential of providing a sensitive marker for aiding in early AD diagnosis, but the

predictive utility of currently employed measures, such as theta and gamma power,

appears to be lacking.

This chapter explores possible markers of early dysfunction in J20+ mice, an

APP-overexpressing mouse model of Alzheimer’s disease. To characterize early

alterations that may be relevant to spatial memory, we recorded from the hippocampus

of freely moving mice, before plaque deposition was apparent. Recordings were

analyzed in a state-dependent manner by segregating sleep and wake periods. First,

we compare the CA1 LFP of age matched J20+ and control littermates during 4 hours of

homecage recordings, assessing frequency bands which have been widely linked to

memory processes (see section 1.2). Then, we provide a detailed characterization of

spike-wave discharges (SWD), hypersynchronous aberrant events which clearly reflect

existing excitatory-inhibitory network imbalances. Lastly, we evaluate J20+ memory

performance in two hippocampal-dependent tasks and correlate the existing

impairments to SWD rate.

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2.2. Methods

2.2.1. Animals

Hemizygous (J20+) male and female mice expressing a mutant form of the human

amyloid protein precursor bearing both the Swedish (K670N/M671L) and the Indiana

(V717F) mutations (APPSwInd) under the control of the PDGFB promoter [J20 (PDGF-

APPSw,Ind) (Mucke et al., 2000), (Jackson laboratory)] and their non-transgenic

littermates were used in all experiments. For the collection of in vivo data, 3 to 5 months

old mice were used. After surgeries, mice were housed individually in polycarbonate

cages at constant temperature (22° C) and humidity (30-50%) and were kept on a

regular circadian cycle (12 h:12 h light: dark cycle, lights on at 8 a.m.). Mice were

provided with food and water ad libitum. Animals were treated according to protocols

and guidelines approved by McGill University and the Canadian Council of Animal Care.

Animals used in this chapter were injected, and an optic fiber was implanted as they

were also used in the optogenetic experiments of chapter 3. Therefore, the viral

injection and optic fiber implant procedures are further detailed in sections 3.2.2 and

3.2.3 respectively.

2.2.2. Electrode implantation for in vivo experiments

At ~12 weeks mice were anesthetized with isoflurane (5% induction, 0.5-2%

maintenance). The skull was completely cleared of all connective tissue and thoroughly

dried using alcohol. Teflon-insulated tungsten electrodes of 101.6 µm diameter were

used for LFP hippocampal CA1 recordings. For tungsten electrode placement, holes

were drilled through the skull above the dorsal hippocampus (AP, -1.8; ML, +1.12, 1.45).

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After gently cutting the dura, the electrode array was first coated with DiI and then

slowly lowered 1.4 mm (relative to Bregma) with the end target being the stratum

radiatum of the dorsal CA1 region. For 32-channel (25 μm site spacing oriented

vertically) silicon probe implantation targeting the pyramidal layer of CA1 (N=2), the

same procedure was used with slightly different coordinates (probe tip target: AP, -1.8;

ML, +1.3; DV, – 1.3). Screws placed in the bone above the frontal cortex and

cerebellum served as ground and reference, respectively. Additionally, for the tungsten

electrode surgeries, an EEG screw was placed in the skull above the hippocampus in

the contralateral hemisphere (AP, -1.8; ML, -1.35), as well as 2 EMG electrodes

consisting of stranded tungsten wires that were inserted into the neck musculature to

record postural tone. Following optic fiber, electrode, ground, and reference placement,

dental cement was applied to secure the implant permanently to the skull.

For tetrode experiments (n=3), three anchor screws were secured to the skull,

and a ground wire was positioned either above the cerebellum at midline position or the

left visual cortex. A ‘versadrive’ containing four independently movable tetrodes (Axona,

Inc) was implanted on top of the right hippocampal CA1 at the following stereotaxic

coordinates: (AP, -1.9; ML, -1.5). Tetrodes were gold-plated to lower impedances to

150-250 kΩ at 1 kHz prior to surgery. Tetrodes were lowered 0.5mm from the dorsal

surface of CA1.

2.2.3. Post-surgery habituation and electrophysiological recordings

Following 1 week of post-surgical recovery and one week of habituation to being

chronically tethered, LFP from implanted mice was recorded. All recorded signals from

implanted electrodes were amplified by the tether pre-amplifier before being digitized at

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32 KHz using a digital recording system (Neuralynx, USA). Prior to any behavioral

experiments, LFP was recorded for 4 h at the start of the light cycle as a baseline

recording.

2.2.4. Novel object place recognition procedure and behavioral analysis

Mice were tested in the novel object place recognition task, a modified version of

the novel object recognition task (Antunes and Biala, 2012). The test area consisted of

a 30.5 cm3 open-topped square container (Boyce et al., 2016). The bottom and walls of

the container were painted with white waterproof paint, and unique black patterns were

present on each of the 4 walls. The test was run on two consecutive days. On the first

day of testing (test area habituation/baseline (Day 0)), mice were placed in the empty

test area and allowed to explore freely for 10 minutes before being returned to their

home cage. For the second day of testing (Day 1), the animal received two sessions in

the behavioral apparatus. In the training session, two identical yet individually identified

objects (Object 1 and Object 2) were each placed in a randomly assigned quadrant

within the test area, and mice were again allowed to explore freely for 10 minutes before

being returned to their home cage. Upon being placed back in their home cage,

patterned light pulses were delivered via the optic fiber patchcord/implant (as described

in section 3.2.3), for light-control purposes. After the 4 h post-training period, the second

session took place. The same two identical objects were placed within the test area as

done on the session prior, with the exception that Object 2 was located in a different

quadrant relative to the training session position. Mice were given 10 minutes to explore

the test area before being returned to their home cage. Both the test area and each of

the identical objects used in this task were thoroughly washed with Peroxyguard before

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every test session. Test sessions on each day were recorded with an overhead video

camera. For analysis of novel place recognition data, the time spent exploring (sniffing

while facing the object within a 2 cm radius) each object during the test period was

measured for each mouse on the training and test sessions of Day 1. For statistical

analysis, the preference of mice for Object 2 exploration was determined by the

following equation: Object 2 recognition index, RI=(Object 2 exploration (s)−Object 1

exploration (s)) / (Object 2 exploration (s)+Object 1 exploration (s)).

Movement data for mice during each test session was completed through

analysis of video recordings from each test session using the Tracking software.

2.2.5. Passive avoidance procedure and behavioral analysis

A standard passive avoidance conditioning protocol was used (Ogren and Stiedl,

2010). Testing was completed over one week and always took place within the first 3

hours of the onset of the light cycle (8 a.m. to 11 a.m.). On the first day of testing, each

mouse underwent habituation to the apparatus, where they freely explored the entirety

of the box for 20 minutes, followed by the passive avoidance training protocol 20

minutes later. The floor of the test area consisted of a metal grid through which

electrical current could be applied, while the wall panels were composed of opaque

black Plexiglas. The apparatus was divided into two rooms by a guillotine door, one with

a roof composed of black Plexiglas and the other uncovered and illuminated with a

lamp. The mouse behavior was recorded with a video camera. During the training

session, the guillotine door was closed as soon as the animal entered the dark

compartment. The conditioning protocol consisted of two 1 second of 0.5 mA current,

spaced by a 5 second interval, and delivered through the metal grid floor as soon as the

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door was closed. The animal was returned to its homecage 10 seconds after the shock

was delivered, and patterned light pulses were delivered via the optic fiber

patchcord/implant system implant, as described in section 3-2-3, for light-control

purposes. Additional memory recall testing was done one day and one week after

training, by letting the animals freely explore the box for 10 minutes. Between each

session, the test area was cleaned thoroughly with Peroxyguard. Passive avoidance

memory performance was assessed by quantifying the latency to cross to the dark

compartment by visual inspection of the recorded videos. The R package ARTool was

used to perform a mixed ANOVA with Aligned Rank Transform of the data, since the

residuals did not follow a normal distribution. Interactions were tested using the phia R

package with a Bonferroni Holms correction (Wobbrock et al., 2011).

2.2.6. Vigilance state architecture analysis

For all recordings, EEG, CA1 LFP and EMG data were plotted, and the vigilance

state was manually scored in 5s epochs using a custom-written Matlab program.

Scoring was based on visual characteristics of the EEG, CA1 LFP, and EMG data, fast

Fourier transform analysis of each epoch scored as well as video monitoring of mouse

behavior. Wakefulness was defined by a de-synchronized low-amplitude EEG and CA1

LFP and tonic EMG activity with periods of movement-associated bursts of EMG

activity. For optimal experimental objectivity, no epochs were excluded from analysis

due to the presence of movement-related artifacts. NREM sleep was defined as

synchronized, high amplitude, low-frequency (delta, 1-4 Hz) EEG, and CA1 LFP activity

that was accompanied by reduced EMG activity relative to that observed during

wakefulness. REM sleep was defined as having reduced delta power, a prominent theta

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rhythm (4-10 Hz), and an absence of tonic muscle activity. Hypnogram analysis

(cumulative duration and average episode duration for each vigilance state) was

completed using custom scripts in Matlab.

2.2.7. Electrophysiological analysis of in vivo CA1 LFP and unit data

All analyses were performed using custom scripts written in MATLAB

(MathWorks) unless otherwise noted. Electrodes used for spectral analysis were

localized in the stratum radiatum. Only the data from one recording electrode was

analyzed per animal. Vigilance state-specific power spectral analyses were performed

with the Chronux toolbox (Bokil et al., 2010) using a moving window Fourier transform

with a window size of 5 s and step size of 1 s. For 16 channel probe analysis, current

source density (CSD) (Nicholson and Freeman, 1975) was calculated from 16-channel

silicon probe LFP recordings using the following equation:

𝐶𝑆𝐷(𝑥,𝑡)=𝜎(2(𝑥,𝑡)−(𝑥+𝛥𝑥,𝑡)−(𝑥−𝛥𝑥,𝑡))(Δx)²

where (x,t) is the potential at depth x for time point t, with Δx of 50 μm. Conductivity (σ)

was assumed constant, and units for CSD are reported as mV/ μm2.

For tetrode experiments, single units were manually isolated from 600 – 6000 Hz

filtered traces. Spike waveform thresholds were adjusted before commencing each

recording and ranged between 35-140 µV depending on unit activity. Clusters were

manually drawn using graphical cluster cutting software (Plexon, Inc) individually for

each recording session. Neurons were separated based on the peak amplitude and

principal component measures of spike waveforms with criteria similar to what has been

discussed elsewhere (Csicsvari et al., 1998). Briefly, only units that formed clusters with

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clear boundaries were used for subsequent analysis. Additional requirements for single

units included the presence of a clear refractory period of at least 2 ms between

subsequent spikes determined through inter-spike interval analysis. Cross-correlations

were used to confirm single-unit isolation.

For 32 channel probe experiments, waveform extraction and spike sorting was

conducted using SpikeDetekt and Klustakwik, followed by manual adjustment of the

waveform clusters using phy (Rossant et al., 2016), as per the requirements described

above. Once sorting was complete, timestamp and spike waveform data of isolated

units was transferred into Matlab for further analysis.

2.2.8. SWD state-specific analysis of CA1 LFP

Detection of transient CA1 LFP SWDs was done over 4 h of recordings. For each

recording, the signal was first bandpass filtered (CA1 LFP, 250- 600 Hz), and the

absolute amplitude was computed. The mean and standard deviation (SD) for the

absolute amplitude was calculated and the detection threshold was set at 10 SDs above

the mean. Values for detection threshold were selected based on accuracy of automatic

detection that was then validated by manual scoring of observed SWDs. All

automatically detected SWDs were screened manually with the help of the EEG trace.

The number of SWDs per state was then normalized by the time spent in each state.

2.2.9. Ripple analysis

Detection of transient CA1 LFP ripple activity during NREM sleep was completed

on 20s windows of continuous NREM sleep that occurred during the first hour of the

recording period. For each window, the signal was first bandpass filtered (100-250 Hz)

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and the absolute amplitude was calculated. The mean and SD for the absolute

amplitude were determined, and the detection threshold was set at 5 SD above the

mean for CA1 LFP ripple analysis, with 2SD for the edges. Values for detection

threshold were selected based on accuracy of automatic detection as compared to

manual scoring. CA1 LFP ripples were detected by taking above threshold data time

points that also passed additional criteria (minimum duration between successive

ripples = 30 ms, maximal duration =100ms).

2.2.10. Phase-amplitude coupling

For analyses of coupled oscillations, theta phase was obtained by bandpass

filtering signals between 4 and 10 Hz and performing a Hilbert transform. The theta

phase obtained was then binned in 40 bins of 9°, and low as well as high gamma power

was then averaged for each theta phase bin. The modulation index (MI) was computed

as the Kullback–Leibler distance between binned gamma amplitudes across theta

phases and a linear distribution of gamma amplitudes (Tort et al., 2010).

2.2.11. Statistical analysis

All data are presented as mean ± standard error of the mean (SEM) and statistic

test details are described in the corresponding results. The normality o the residuals

was assessed using the Shapiro–Wilk test and parametric tests were used only when

distributions were normal (non-parametric tests are described where applicable). P <

0.05 was considered statistically significant.

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2.2.12. Histological confirmation of electrode/optic fiber placement

Following completion of all in vivo experiments, mice were deeply anesthetized

with ketamine/xylazine/acepromazide (100, 16, 3 mg/kg, respectively, intraperitoneal

injection). Mice were then perfused transcardially with 4% paraformaldehyde in 1 x PBS

(PFA). The brains were then extracted and postfixed overnight in PFA at 4 º C and

subsequently cryoprotected in 15% sucrose dissolved in PBS for an additional 24 h at 4

º C. Each brain was then flash frozen and sectioned at 35 μm using a cryostat and

processed as free-floating sections. Odd sections were collected, mounted on glass

slides and coverslipped with fluoromount mounting medium containing 4′,6-diamidino-2-

phenylindole for confirmation of electrode placement. Only mice with histologically

confirmed placement of at least one electrode in CA1 stratum radiatum were used in the

present study. Additionally, only mice with histologically confirmed placement of at least

one tetrodes or probe in the CA1 pyramidal cell layer were further considered for

analysis of CA1 LFP ripple and unit activity.

2.3. Results

2.3.1. Time spent in REM, wake, and NREM does not differ in J20+ transgenic AD mice

compared to non-transgenic littermate control animals

Using EMG, EEG, and CA1 LFP recordings, we characterized the sleep

architecture in freely moving mice during 4 h recordings in their homecage. Transgenic

AD mice (J20+) and non-transgenic age-matched littermates (controls) did not

significantly differ in the amount of time spent in wake, NREM, or REM sleep (Fig. 2.1

A-B). To evaluate sleep episode fragmentation, we quantified the number of episodes

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per state (Fig. 2.1 C), during the same period and found no significant differences

between groups.

2.3.2. Shift in hippocampal theta peak frequency in AD

2.3.2.1. REM sleep

After scoring the recorded traces for sleep-wake periods, state-dependent power

spectral analysis of the hippocampal CA1 LFP was conducted to assess if any early

alteration existed in J20+ mice (Fig 2.2 A-B). First, we analyzed hippocampal activity

during REM sleep. Fig. 2.2 C shows representative raw field recordings obtained from

Fig. 2.1: Sleep Architecture in controls and J20+ mice. A. Percentage of time spent in REM,

NREM and wake in controls and J20+ mice. B. Cumulative time spent in seconds per stage. C.

Number of episodes quantified in wake, NREM and REM sleep. Data are expressed as mean ±

SEM. Control n =6, J20 n=11; two-way mixed ANOVA with Sidak’s multiple comparisons, non-

significant.

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the two groups, as well as the filtered traces at different frequencies. We then examined

power spectra during REM sleep using a moving window analysis (Fig 2.2 D). No

significant differences were observed in the spectral power of theta (4-10 Hz), low and

high gamma (30-60 and 61-120 Hz, respectively) or ripple (125-250 Hz) frequency

bands (Fig 2.2 E). Interestingly, a significant shift towards lower frequencies in the theta

band was observed in J20+, (Fig. 2.2 F; control: 7.282± 0.055 ; J20+: 6.796± 0.1374

unpaired two-tailed t-test p=0.0069) whereas other measures of theta band power

remained unaltered (Fig. 2.2 G-H). Finally, we also examined the coupling of both low

and high-frequency gamma to the different phases of theta, and although coupling

appears reduced for J20+ in the mean comodulation plots (Fig 2.2 I-K), we found no

significant differences in the mean theta-gamma MI between control and J20+ mice (Fig

2.2 J-L).

2.3.2.2. Wake

The same analysis was performed for wake periods to examine if any

deficiencies in the LFP between controls and J20+ were present (Fig 2.3.A). As in REM

sleep, no significant differences were detected in the band power of theta, low or high

gamma or ripple frequencies (Fig 2.3 B-D). However, a similar significant decrease in

theta peak frequency was observed (Fig 2.3.E, control: 6.671 ± 0.1715. J20+: 5.835

±0.1911, unpaired two-tailed t-test p=0.0129) with mean and maximum theta band

power no different from controls (Fig 2.3 F- G). Importantly and in contrast to REM

sleep, wake theta-low gamma coupling was significantly impaired in J20+ (Fig 2.3 H- I.

control: 0.78 ± 0.016, J20+: 0.725±0.017 unpaired two-tailed t-test p=0.0424). Although

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theta-high gamma coupling again appeared decreased in the comodulation plot, this

change was not significantly different (Fig. 2.3 J-k).

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*

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Fig. 2.2: Theta frequency decreased in J20+ during REM. A. Electrode recording site

located in stratum radiatum of CA1 and illustration of homecage recording during sleep.

B. Example 1 h hypnogram and representative REM sleep spectrogram. C. Example of

raw and filtered CA1 trace together with EMG during REM sleep for control and J20+

subject. D. Mean normalized spectrogram of all REM. E. Band power for theta, low

gamma, high gamma and ripple frequencies. F. Peak theta frequency. G. Mean peak

power of theta band. H Maximum peak power of the theta band. I. Low gamma

amplitude coupling to theta phase. J. Mean MI for theta-low gamma coupling. K. High

gamma amplitude coupling to theta phase. L. Mean MI for theta-high gamma coupling.

Data are expressed as mean ± SEM. Control n =6, J20 n=11; Unpaired two-tailed t-

tests, *p< 0.05

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Fig. 2 3: Theta frequency decreased in J20+ during wake and impaired theta-low

gamma coupling. A. Example 1 h hypnogram and wake spectrogram. B. Example of

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raw CA1 trace together with EMG during wake for control and J20+ subject. C. Mean

normalized spectrogram of all wake. D. Band power for theta, low gamma, high gamma

and ripple frequencies. E. Peak theta frequency. F. Mean peak power of theta band. G

Maximum peak power of the theta band. H. Low gamma amplitude coupling to theta

phase. I. Mean MI for theta-low gamma coupling. J. High gamma amplitude coupling to

theta phase. K. Mean MI for theta-high gamma coupling. Data are expressed as mean ±

SEM. Control n =6, J20 n=11; Unpaired two-tailed t-tests, *p< 0.05.

2.3.2.3 NREM

Lastly, hippocampal ripples in NREM sleep were detected in animals that had at

least one recording site in the CA1 pyramidal layer. Measurements of hippocampal

ripples, a significant memory-associated neural activity pattern, also revealed no

differences in their characteristics (Table 2.1)

Table 2.1: Analysis of CA1 cell layer LFP ripple characteristics during NREM

sleep. Ripples occurring in the first 20 minutes of the 4h period. Control: n=3; J20+; n =

6; non-significant, unpaired two-tailed t-test.

Ripple Parameter Control J20+

Number (20min) 765.3 ± 44.12 739 ± 82.86

Density (Ripples/min) 35.84 ± 1.47 31.8 ± 2.8

Ripple Duration (ms) 48.1 ± 0.61 46.45 ± 1.5

Max ripple amp (mV) 0.16 ± 0.017 0.22 ± 0.032

Ripple Interval (s) 1.83 ± 0.61 2.07 ± 0.18

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2.3.3. Hippocampal hyperactivity during sleep in AD

We next aimed to characterize aberrant hypersynchronous hippocampal activity

in J20+ mice. We detected large amplitude and short duration spike-wave discharges

(SWD) in CA1 LFP recordings (Fig 2.4. A) which have a markedly different LFP and

CSD profile compared with sharp-wave ripples (Fig 2.4 B-C). The SWD events were

absent in control animals (Fig. 2.4.D, control: 1.143± 0.553, J20+:372.6±58.25. unpaired

two-tailed t-test p=0.0001) and therefore specific to J20+.

To further characterize these interictal-like discharges, we performed a state-

dependent analysis of their incidence and features (Fig. 2.5 A). Almost no SWDs were

detected during wake periods, so the majority of subsequent comparisons were

performed in NREM and REM sleep. The width of the SWD spike was significantly

higher in REM in comparison to NREM (Fig. 2.5.B, REM: 8.69±0.808 ms, NREM:

7.33±0.631 ms, paired two-tailed t-test p=0.0467), while no differences were found in

peak amplitude. As mentioned, SWDs were preferentially detected during sleep (REM:

135.8±32.63, NREM:227.3±42.68, wake:9.63±2.509; repeated-measures one- way

ANOVA p=0.0006, Tukey’s multiple comparisons REM vs Wake: p=0.008; NREM vs

Wake: p= 0.0009) and specially during REM when normalized by the time spent per

state (Fig. 2.5.C-D, REM: 4.783±0.881, NREM: 1.755±0.3409, wake: 0.1176±0.0288;

repeated-measures one- way ANOVA p=0.0004, Tukey’s multiple comparisons REM vs

NREM: 0.013, REM vs Wake: p=0.0009; NREM vs Wake: p= 0.0014). The number of

SWDs and the length of the episode duration were strongly correlated for both sleep

stages, but especially so during REM (Fig. 2.5 E-F, REM: p<0.0001 Pearson r=.6454;

NREM: p<0.0001; r=0.4626).

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Therefore, we conducted a more in-depth episode-specific investigation where

the occurrence of SWDs was quantified according to the duration of the episode. A two-

way mixed ANOVA revealed a significant STATExDURATION interaction (p=0.0005)

and Tukey’s multiple comparison test confirmed a significant decrease in the number of

SWDs in NREM versus REM when the episodes lasted longer than 60 s (Fig. 2.5 G,

REM >60s: 0.08948±0.016, NREM >60s:0.0268±0.005; REM vs NREM >60 p=0.023).

Moreover, only during REM, the number of SWDs detected in episodes lasting longer

than 60s was significantly higher than the number detected in episodes less than 30s

(REM <30 s: 0.037±0.01, REM >60s: 0.08948±0.016, p= 0.0177).

Lastly, each episode was segmented in three position bins and SWDs were

counted at the beginning, middle or end of each episode (Fig. 2.5 H). A significant

interaction STATExSEGMENT was also found (p=0.0018) and Tukey’s multiple

comparison showed that the incidence of SWDs during the first 10 s of the episode was

significantly lower than during the rest of the episode for both REM (First 10s: 0.039 ±

Fig. 2.4: Spike-wave discharges are specific to J20+ mice. A. Example CA1 trace

during REM sleep showing the presence of SWDs. Bottom left, filtered high frequency

(250 to 750Hz) SWD. Bottom Right, example spectrogram of SWD. B. Example SWD

raw trace profile spanning CA1 with a linear probe, together with its corresponding

CSD. C. Example sharp-wave ripple raw trace profile spanning CA1 with a linear

probe, together with its corresponding CSD. D. Quantification of SWDs in a 4 h period

after onset of light cycle for both controls and J20+. Data are expressed as mean ±

SEM. control n =6, J20 n=11; Unpaired two-tailed t-tests, **p< 0.001.

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** **

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0.008, center segment:0.08 ± 0.013, last 10s: 0.106± 0.017; First 10s vs center

segment p=0.018; First 10s vs last 10s p=0.013) and less so for NREM (First 10s:

0.023± 0.005; center segment: 0.033± 0.007, last 10s: 0.033± 0.007; First 10s vs last

10s p=0.02). Interestingly, significantly lower SWD rates were also detected during the

center segment (NREM vs REM: p=0.037) and last 10s (NREM vs REM: p= 0.0118) of

NREM compared to REM episodes.

To understand in more detail how the hippocampal neural population in J20+

mice gives rise to spike-wave discharges observed in the LFP, we performed a last set

of experiments (n=5) where we recorded unit activity in the pyramidal cell layer of CA1

(Fig. 2.6). For the final population analysis, we built 1s peri-stimulus time histograms

(PSTH) with 10ms bins centered at the spike and normalized the activity of every single

Fig. 2.5: Spike-wave discharge characterization in sleep. A. Example SWD

distribution in a 4h hypnogram. B. Average peak and width of the SWD spike separated

by REM and NREM periods. C. Total number of SWDs per state. D. Total number of

SWDs normalized by time spent per state, in minutes. E. Scatterplot of episode length

in seconds vs number of SWDs in REM sleep. F. Scatterplot of episode length in

seconds vs number of SWDs in NREM sleep G. Average SWD per seconds according

to binned episode duration. H. Average SWD per second according to their timing (first

10 seconds, center of the episode or last 10 seconds) in a given episode. Data are

expressed as mean ± SEM. control n =6, J20 n=11; * p< 0.05, **p<0.001 across groups,

$ p< 0.05 within group

.

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isolated unit to that during a 1s baseline, starting 1.5s before every SWD. Multi-unit

activity (MUA) increased around the time of the spike (Fig. 2.7 A), while the firing

probability of single isolated units was heterogeneous, and increased (Fig. 2.7 B),

remained unchanged (Fig. 2.7 C), or decreased in the bin preceding the peak of the

spike.

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We then proceeded to categorize each unit depending on their activity during the

10ms just before the peak of the spike, with 2SD above the mean baseline being

classified as positively modulated (+, 66.1%), 2SD below the mean baseline as

negatively modulated (+, 27.1%), and the rest as non-modulated (~, 6.7%; Fig. 2.8 A).

PSTHs of the positively modulated units (with the firing probability normalized by

dividing by their baseline activity, so if there was no change the probability gain would

be 1) showed a significant increase in activity during the 10ms before the peak of the

spike in comparison to the 500ms time segment before, and a significant decrease in

the 500ms post-SWD (Fig 2.8B-C, Friedman test p<0,0001; Dunn’s multiple

comparisons: base vs before peak, p<0.0001; base vs post: p<0.0001). Negatively

modulated units significantly decreased their activity 10ms before the spike, at the time

of the spike, and during the 500ms post period after the spike (Fig 2.8 D-E, Friedman

test p<0,0001; Dunn’s multiple comparisons: base vs before peak, p<0.0001; base vs

peak: p<0.0001; base vs post: p<0.0001). Finally, the non-modulated neurons did not

show significant differences in firing just before, during, or after the spike (Fig 2.8 F-G).

Fig. 2.6: Unit activity in J20+ mice. A1. Unfiltered CA1 LFP. Below, filtered trace at

250-750Hz. Red arrows indicate detected SWDs. B1 Multi -unit activity. B2. Two

example isolated units. Inset shows single action potentials recorded by four channels

(different colors) of the tetrodes. Multi-unit and isolated unit examples are from same

tetrode.

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Fig. 2.7: Example PSTHs. A1. Multi-unit activity average waveform. A2. Baseline

PSTH for 35 SWDs; below, multi-unit raster-plot. A3. SWD PSTH; below, multi-unit

raster-plot. A4. Multi-unit baseline PSTH subtracted from SWD PSTH; below binned %

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change, corrected by bin size. B1. Example unit activity average waveform. B2. 200ms

autocorrelogram. B3. Baseline PSTH for the same 35 SWDs; below, unit raster-plot. B4.

SWD PSTH; below, multi-unit raster-plot. B5. unit baseline PSTH subtracted from SWD

PSTH; below binned % change, corrected by bin size. C1. Example unit activity average

waveform. C2. 200ms autocorrelogram. C3. Baseline PSTH for the same 35 SWDs;

below, unit raster-plot. C4. SWD PSTH; below, multi-unit raster-plot. Bins of PSTHs

have 5ms size. Bins of % change computed as follows: Before, -250ms to -7.5ms from

spike peak; -7.5m, from -10ms to -5ms; -2.5ms, from -5ms to 0; 2.5ms, from 0 to 5ms;

After, from 5ms to 250ms. Baselines were taken 1 s before every SWD. Examples from

same tetrode.

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ga

in

ga

in

ga

in

ga

in

** **

** ** **

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Fig. 2.8: PSTHs of all units classified by SWD response. A. Distribution of units

according to response: +, positively modulated; -, negatively modulated; ~ unmodulated.

B, D, E Positively- modulated, negatively- modulated and unmodulated unit PSTHs with

10ms bins. All units were corrected by dividing their respective baselines, a value of 1

therefore reflects no change. C,F,G. Same data around SWD with different bin size:

Base, -500ms to -10ms from spike peak; before peak, from -10ms to 0ms; peak, from 0

to 10ms; Post, from 10ms to 500ms. N=5 animals. **p< 0.001.

2.3.4. Performance impairments during behavior in AD

Our results highlight early aberrations in the hippocampal CA1 network activity of

J20+ mice during non-goal directed behavior in wake and sleep. Next, we investigated

whether these changes were associated with memory performance. Specifically, we

assessed hippocampal-dependent spatial memory in two tasks, the novel object place

recognition memory and passive avoidance tests. In the novel object place recognition

task (Fig. 2.9 A), both groups showed no preference for either object during the sample

phase and explored the objects equally. 4 hours later, the animals were reintroduced

into the open field, where one of the objects was moved to a different location. Because

mice preferentially investigate novel stimuli, if object place memory is intact, mice

should investigate the displaced object more than the non-displaced one (Antunes and

Biala, 2012). Preference for the displaced object (Recognition Index ,RI), was higher

relative to the sample phase in controls only (Fig. 2.9 B, sample: 0.485±0.014, test:

0.607±0.022; two-way mixed ANOVA , interaction p=0.0138; Sidak’s multiple

comparisons training-test: p<0.0001); J20+ did not explore the displaced object more

than during the sample phase, indicating a memory deficit (sample: 0.513±0.015, test:

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0.591±0.028). Control measures revealed no differences in locomotion or motivation

during testing between groups (Fig. 2.9 E-G).

In the passive avoidance task, the latency to cross to the dark compartment

during training was equivalent in both groups (Fig. 2.9 C-D; control:17.5±3.89 s,

J20+:12.78±4.61 s). The next day, mice were tested in the same chamber for 10min

and their latency to cross to the dark compartment was assessed. J20+ mice had

shorter latencies than controls (two-way mixed ANOVA of aligned-transformed data,

interaction p<0.001; Wilcoxon with Bonferroni-Holms adjustment p=0.0026;

control:473.9±53.83 s, J20+:108.55±28.07 s), reflecting a memory impairment. One

week later, the latency to cross to the dark compartment was still significantly lower in

the J20+ (p=0.0055; control:299.1±71.1 s, J20+:20.33±3.63 s).

Lastly, we investigated if the rate of SWDs during sleep correlates with

behavioural performance in the J20+ group. A negative correlation was found between

the #SWD/min observed during REM and the recognition index in the test phase of the

object place recognition task (Fig. 2.9 H; Pearson r=-0.62, p=0.015). This negative

correlation was also found in the same subjects when tested at the 1day mark in the

passive avoidance task (Fig. 2.9 J; Pearson r= -.5057, p=0.046). Such a relationship

was not found for the #SWD/min during NREM, suggesting that the association

between a higher number of SWD events with poorer memory performance is restricted

to REM sleep only (Fig. 2.9 I-K).

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Fig. 2.9: Spatial memory impairments in J20+ mice. A. Schematic of novel object

place memory task. Below cumulative object exploration time for both objects during

sample and test phases. B. Displaced object recognition index. C. Schematic of

passive avoidance task. D. Latency in seconds to enter the dark compartment in the

passive avoidance task. E. Time spent in quadrants during habituation to the open field.

F. Distance traveled during habituation, sample and test sessions in the open field. G.

Cumulative total object exploration time during sample and test phases. H. Scatterplot

of SWDs per minute during REM sleep versus the recognition index of the displaced

object during the test phase. I. Scatterplot of SWDs per minute during NREM sleep

versus the recognition index of the displaced object during the test phase. J. Scatterplot

of SWDs per minute during REM sleep versus the latency to the dark compartment in

the passive avoidance at the 24 h mark. K. Scatterplot of SWDs per minute during

NREM sleep versus the latency to the dark compartment in the passive avoidance at

the 24 h mark. Data are expressed as mean ± SEM; two-way mixed ANOVAs were

performed if not otherwise stated. *p< 0.05, **p< 0.001

2.4. Discussion

In this chapter, we aimed to describe early electrophysiological alterations in the

hippocampus of the J20+ model in a state-dependent manner, including a detailed

characterization of the network’s epileptiform discharges. We show that, compared to

non-transgenic littermates, subtle deficits in state-dependent hippocampal rhythms can

be detected early in the disease progression. Importantly, we found the robust presence

of abnormal large-amplitude spikes, which was associated with spatial memory

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alterations, thereby reflecting a clear hippocampal excitation-inhibition network

imbalance in our model.

2.4.1. Deficits in theta frequency and theta-gamma coupling

We first showed that in 3 to 5 month old J20+ mice, peak theta frequency was

decreased in both wake and REM sleep when compared to wild types. Considering that

amyloid beta deposition begins at approximately 6 months of age in this mouse model

(Mucke et al., 2000), it is interesting to note that the abnormality in theta frequency and

the behavioral impairments described here occurs before amyloid plaque formation is

detected. This slowing of theta is consistent with previous reports in AD patients, where

a slowing of the dominant rhythmic activity has been described (Jackson and Snyder,

2008; Jeong, 2004), as well as in AD mouse models where similar slight decreases in

peak theta frequency are seen in 5xFAD mice during home cage recordings (Siwek et

al., 2015) and in APP-PS1 mice while running (Cayzac et al., 2015). The lower theta

frequency may produce a significant slowdown of the information exchanged in the

hippocampal network, since theta cycles are suggested to serve as a temporal

reference for coordinating local computations across a more extensive network

(Buzsáki, 2002).

Secondly, we found that theta -low gamma phase- amplitude coupling was

significantly reduced during wake periods only. The coupling of gamma oscillations to

theta phase is proposed to support memory processing in rats (Scheffer-Teixeira and

Tort, 2016; Tort et al., 2009, 2008), and humans (Canolty et al., 2006; Canolty and

Knight, 2010; Daume et al., 2017; Händel and Haarmeier, 2009). Moreover, decreased

theta–gamma phase-amplitude coupling has been reported in vitro for the TgCRND8

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and J20+ (Goutagny et al., 2013; Mondragón-Rodríguez et al., 2018) as well as in vivo

in PV-J20 (Etter et al., 2019), 3xTg AD (Mably et al., 2017) and APP23 (Ittner et al.,

2014) AD mouse models, suggesting that altered phase-amplitude coupling could play a

key role in AD pathophysiology. Interestingly, gamma power was intact at the age we

recorded our J20+ mice, while in animals showing Ab plaque deposition, their

impairment in theta-gamma coupling was accompanied by a decrease in gamma power

(Etter et al., 2019; Mably et al., 2017). This may indicate that deficits in theta-gamma

coupling during wakefulness are one of the first observed perturbations in the

hippocampal network.

2.4.2. Hyperactivity during sleep and its link to memory

Next, we investigated neuronal hyperexcitability in young J20+ mice prior to

plaque deposition in the form of spike-wave discharges (SWDs). We find that SWDs

could always and reliably be detected in all J20+ we recorded from, while in controls

SWDs were mostly absent. Interestingly, SWDs occur primarily during sleep and

particularly in REM sleep, although they are also present during periods of quiet

wakefulness at a significantly reduced rate. This finding is consistent with reports in

other AD mouse models in which hyperexcitability has been described when mice are

motionless and especially in REM sleep (Born et al., 2014; Brown et al., 2018;

Gureviciene et al., 2019; Kam et al., 2016). Similarly, depth electrode recordings in AD

patients have also shown that epileptiform discharges are also detected predominantly

during sleep (Brown et al., 2018; Horváth et al., 2017; Lam et al., 2017; Vossel et al.,

2016). These findings have significant implications because sleep subserves important

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functions related to memory consolidation (Boyce et al., 2016; Wilson and McNaughton,

1994), and we hypothesize that SWDs likely disrupt this process.

Highlighting the interaction between hyperactivity and cognition, significantly

faster cognitive decline over a period of 5 years has been observed in AD patients with

recorded epileptiform activity than in those without these events (Vossel et al., 2016).

Indeed, there is reason to believe that SWDs contribute to cognitive disruption based on

previous studies of interictal spikes in epileptic rodents and patients (Kleen et al., 2013,

2010). For example, it has been shown in a rat kindling model of epilepsy that interictal

spikes occurring during a sleep epoch between the learning and test phases of a

memory task disrupts memory consolidation, likely by inducing atypical coupling

between the hippocampus and cortex (Gelinas et al., 2016). Perhaps similarly, Kam et

al have suggested that SWDs may reflect abnormal hyperactivity both locally and

across cortical and hippocampal regions, a phenomena we have also observed in our

model (data not shown). The conclusion that aberrant hyperactivity may affect memory

is also consistent with studies showing that certain anti-epileptic drugs reduce

epileptiform activity in AD mice models and in patients with mild cognitive impairment,

while also having a beneficial effect in memory (Bakker et al., 2012, 2015; Sanchez et

al., 2012a). In line with this evidence, our data shows that the rate of SWDs, specifically

during REM sleep, negatively correlates with memory performance in two hippocampal-

dependent memory tests, the novel object recognition and passive avoidance tasks.

Although on average J20+ mice are significantly deficient in these tasks when

compared to controls, those with lower memory scores tend to have higher rates of

SWDs during REM sleep. In contrast, the incidence of SWDs during NREM sleep did

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not significantly correlate with memory. Furthermore, the number and characteristics of

ripples, which are an important feature of memory consolidation during NREM sleep,

were unchanged from controls. Altogether, our results suggest that hyperactivity,

particularly during REM sleep, may contribute to the early disruption of memory

processes in our model.

In this chapter, our aim was not to describe the mechanisms underlying SWDs,

but we unexpectedly found that both the length and segment of the REM (but not

NREM) episode modulated the incidence of SWDs, with higher SWD rates detected in

longer episodes and towards the end of a given episode. This modulation hints towards

a possible interplay between mechanisms involved in SWD generation and the egress

from REM sleep (Hayashi et al., 2015; Lu et al., 2006), as changes involved in

transitioning out of REM sleep may also promote hyperactivity in networks with

handicapped excitatory/inhibitory balance. Whether a similar significant modulation of

interictal spikes by REM sleep exists in epileptic or other AD models is unknown.

Finally, our unit recording experiments shed some light on how neurons change

their firing rate before, during and after SWDs. Strikingly, the majority of isolated units

(66%) increased their activity during the 10ms before the peak of the SWD and

proceeded to be inhibited especially for the 250ms after the peak. A smaller percentage

(27%), decreased their activity during these same 10ms before the peak of the SWD,

highlighting a heterogeneity of the neural response. This heterogeneity is similar to

reports describing changes in firing rates of single units during pre-ictal activity, where

41% of principal cells increase their firing and 15% decrease it (Fujita et al., 2014;

Toyoda et al., 2015). Reduced unit activity during spikes might be an artifact because,

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during periods of high activity, spike-sorting methods are prone to false negatives

(Cymerblit-Sabba and Schiller, 2012). While acknowledging this caveat, our findings do

suggest that a large proportion of neurons are transiently and aberrantly synchronous in

sleep and also simultaneously inactive, as their firing is also briefly disrupted just after

each SWD.

To conclude, both the hyperactive and rhythmic pathophysiological phenomena

detected in J20+ mice might have detrimental effects on cognitive function. The

temporal coding within neuronal ensembles (Buzsáki, 2002) may be disrupted by the

aberrant oscillatory patterns (i.e., reduced peak theta frequency and impaired theta-

gamma coupling) observed in our AD model. In addition, the normal neuronal input-

output computation could be altered by hyperexcitability during sleep (Hall et al., 2015;

Šišková et al., 2014), likely contributing to the interference of regular memory

processes.

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Supplemental Fig 2.1

Supplemental Fig. 2.1: Unit activity in J20+ mice. A1. Unfiltered CA1 LFP. Below,

filtered trace at 250-750Hz. Red arrows indicate detected SWDs. A2 Multi -unit activity

followed by 10 isolated units from same tetrode. A3. Example view of the isolated unit

clusters plotted using the peak in two of the recording sites of the tetrode. Orange

represents MUA.

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Chapter 3: Effects of optogenetic activation of septo-hippocampal

fibers in AD.

3.1. Introduction

In the previous chapter, we described early alterations in the hippocampus of the

APP-overexpressing J20+ AD mice model. These alterations included a decrease in

theta peak frequency, a decrease of theta-low gamma coupling during wake periods,

and marked aberrant neural hyperexcitability in sleep that correlated with impaired

behavioral performance. Given that AD is the most common form of dementia, and that

current treatments do not effectively halt or reverse the disease (Huang and Mucke,

2012), methods that probe memory and cognition in AD by directly modulating the

hippocampal network are needed to guide therapeutic development. For example,

recent studies in AD mouse models have used optogenetic approaches to stimulate the

hippocampal circuit at plaque-bearing (Etter et al., 2019; Perusini et al., 2017) and pre-

plaque (Roy et al., 2016) stages of the disease, thus reversing their memory deficits and

shedding light on the mechanisms underlying this effect. In these studies, neural somas

were acutely stimulated with light during the testing phase of behaviour, thereby

influencing retrieval. In contrast, current clinical trials that employ chronic electrical

stimulation in AD patients, which could have the potential of improving cognitive

performance, have used different approaches. On one hand, these trials target the

fornix, a bundle of white fiber projections connecting the hippocampus and other

memory structures, such as the medial septum. On the other hand, treatment is not

focused to the retrieval phase of testing as patients receive constant stimulation

encompassing wake and sleep periods, and the effects on cognitive performance is

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assessed once the stimulation device is switched off (Laxton et al., 2010; Lozano et al.,

2016).

To more closely mirror these two clinical trial parameters, our present goal is

twofold: first, we aim to evaluate the effect of optogenetic stimulation on the altered

J20+ hippocampal rhythmic network activity (as described in chapter 2) by specifically

targeting medial septo-hippocampal projections at the level of the fornix. We find that

theta stimulation of the fornix increases peak power at the given stimulation frequency

and observe a significant reduction in hyperactivity during REM sleep. Second, we aim

to evaluate the effect of continuous stimulation on memory by restricting light delivery to

both sleep and non-task related awake periods. Given our findings in the J20+ showing

that hippocampal hyperactivity in the form of SWDs is higher during sleep and how their

incidence correlates with memory task performance, we focused light delivery to the

post-training period. Our results show that stimulation of the fornix after training

improved memory performance in J20+ mice.

3.2. Additional Methods

Most of the methods used in this chapter have been described in the previous

chapter. Refer to section 2.2 for details.

3.2.1. Virus-mediated targeting of opsin and eYFP expression

Adeno-associated virus (AAV) of serotype 2 containing a channelrhodopsin-2

with the E123T mutation for ultrafast control (ChETA construct) fused to eYFP and

driven by the nonspecific synapsin promoter, and control virus AAV2-hSyn-eYFP were

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obtained from the Canadian Neurophotonic Platform, at Laval University. For in vivo

electrophysiology experiments, J20+ mice were injected with AAV2-hSyn-ChETA-eYFP,

while for all other animals and tests AAV2-hSyn-eYFP was used. These viral injections

were to obtain controls for viral injection and light delivery for the optogenetic

manipulation. At ~8 weeks of age, J20+ mice and littermates were anesthetized with

isoflurane (5% induction, 1-2% maintenance). 0.5 μL were stereotaxically injected into

the medial septum (MS) (anteroposterior (AP), +0.86; mediolateral (ML), 0.0;

dorsoventral (DV), -4.5; all coordinates relative to Bregma) through a 28 G cannula at a

rate of 50 ηL/min. The injection needle was lowered through a hole drilled lateral to the

midline (AP, +0.86; ML, -0.5; DV, 4.5) at an angle of ~6.5 ° (in the ML axis) relative to

the vertical plane in order to avoid the sagittal sinus.

3.3.2. Optic fiber implantation for in vivo experiments

At ~ 12 weeks, mice injected for in vivo experiments were anesthetized with

isoflurane (5% induction, 0.5-2% maintenance). The skull was completely cleared of all

connective tissue and thoroughly dried using alcohol. To facilitate light delivery to the

transfected projections from the medial septum, a hole was drilled in the skull above the

dorsal fornix (AP, -.34; ML, -0.5). After gently cutting through the dura, an optic fiber

implant, constructed by cementing a cleaved 200 μm diameter piece of optic fiber into a

ceramic ferrule (Thor labs), was lowered through the hole at an angle of ~4.5 ° (in the

ML axis) with the optic fiber end targeted just above the dorsal fornix (AP, -0.34; ML, -

0.1; DV, -1.9). For optic fiber + LFP recordings the optic fiber was positioned as

described above; however, Teflon-insulated tungsten electrodes of 101.6 µm diameter

were also lowered in hippocampal area CA1 as described in section 2.2.2.

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3.3.3. Light delivery to the fornix

Blue (450 nm wavelength) light was delivered through a fiber optic cord using a

laser diode fiber light source (Doric Lenses, Canada). Light intensity was calibrated and

wavelength-corrected using the Power Meter Bundle with the PM100D Console and

S130C Slim Photodiode Sensorlight (Thorlabs). For each mouse, the minimum amount

of light at the tip of the optic fiber implant that was required to produce maximal effects

on the CA1 theta rhythm was estimated before behavioral experiments were started.

This estimation was done in order to minimize unwanted side-effects, including light-

induced tissue damage as well as excessive disturbance of animals. The estimated

amount of light was based on pre-surgery testing of transmittance of light transmittance

through the optic fiber patch cord and optic fiber implant and was never allowed to

exceed 20 mW. Every stimulation was performed at 50% duty cycle (100ms) with 5Hz

frequency for the duration of 4 h, the day after the baseline was recorded and starting at

the same time.

3.2.4. Immunohistochemistry and construct expression confirmation

Brain extraction and slicing were performed as described in section 2.2.12. To

confirm construct expression and for choline acetyltranferase (ChAT) quantification

experiments, double fluorescent immunohistochemistry for eYFP and ChAT were

performed. Even sections were first washed (3 x 15 min washes under agitation) in PGT

(0.45% Gelatin and 0.25% Triton in PBS). Next, slices were incubated with primary

antibodies (1:1000 chicken anti-GFP from ThermoFisher Scientific and 1:200 goat anti-

ChAT from Millipore) in PGT at 4 º C for 24 h. Following 3 x 15 min PGT washes,

sections were then incubated with secondary antibodies [1:1000 donkey anti-goat

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coupled with Alexa 647, and 1:2000 donkey anti-chicken coupled with Alexa 488 from

Jackson Immunoresearch) for 2 h, followed by PBS rinses and then mounted as

described above.

For PV staining, double fluorescent immunohistochemistry for PV and eYFP was

performed. Sections were first incubated overnight with PGT (0.45% Gelatin and 0.25%

Triton in PBS) at 4°C. Next, slices were incubated with primary antibodies (1:1000 rabbit

anti-GFP from Life Technologies, and 1:500 mouse anti-parvalbumin monoclonal IgG1

from Sigma-Aldrich) in PGT at room temperature for 2 hours. Following washes of 10,

20, and 30 min, sections were incubated with secondary antibodies [1:1000 goat anti-

rabbit coupled with Alexa 488 (Molecular Probes), and 1:500 goat anti-mouse IgG1

coupled to Alexa 555 (Life Technologies)] in PGT for 45 min. Following 10, 20, and 30

min PGT washes, sections were then mounted on glass slides and permanently

coverslipped with Fluoromount mounting medium containing 4′,6- diamidino-2-

phenylindole.

Only mice with histologically confirmed placement of at least one electrode in

CA1 stratum radiatum and optic fiber in the dorsal fornix as well as proper construct

expression in the medial septum were used in the present study. Additionally, only mice

with histologically confirmed placement of probe sites spanning the CA1 pyramidal cell

layer were further considered for analysis of unit activity.

3.3.5. In vitro patch-clamp electrophysiology.

Mice were deeply anesthetized using ketamine/xylazine/acepromazine mix, and

intracardially perfused with N-methyl- d-glutamine (NMDG) recovery solution (4°C)

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oxygenated with carbogen (5% CO2 and 95% O2). NMDG solution contains the

following (in mM): 93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES,

25 glucose, five sodium ascorbate, two thiourea, three sodium pyruvate, with the pH

adjusted with HCl to 7.4 before the addition of 10 MgSO4 and 0.5 CaCl2. Following

NMDG perfusion, brains were quickly removed and immersed for an extra 1 min in cold

NMDG recovery solution. Coronal slices (300 µm) were cut using a vibrating microtome

(Leica-VT1000S), then collected in 32°C NMDG recovery solution for 10–12 min. Slices

were then transferred to room temperature and to a bath with oxygenated artificial

cerebrospinal fluid (aCSF) containing the following (in mM): 124 NaCl, 24 NaHCO3, 2.5

KCl, 1.2 NaH2PO4, 2 MgSO4,5 HEPES, 2 CaCl2, and 12.5 glucose (pH 7.4). Patch

pipettes (3–5MΩ) were filled with internal solution, containing the following (in mM): 140

K gluconate, 2 MgCl2, 10 Hepes, 0.2 EGTA, 2 NaCl, 2 mM Na2-ATP and 0.3 mM Na2-

GTP, adjusted pH to 7.7 with KOH, adjusted osmolarity to 290. Slices were transferred

to a submerged recording chamber filled with aCSF (4 ml /min flow rate, 30 °C) that was

continuously oxygenated with carbogen (5% CO2 and 95% O2). Single whole-cell

patch-clamp recordings of septal neurons (identified by YFP fluorescence) were done

after transferring brain slices to the recording chamber. All recordings were done at 32

°C. For recordings, neurons were either tested in current-clamp mode (resting potential

between −60 and −70 mV) or maintained at −70 mV in voltage-clamp mode. For both,

the effect of different intensities of blue light (450 nm) was tested (1–20 mW), using a

laser diode fiber light source (Doric Lenses, QC, Canada). Electrophysiological signals

were amplified, using a Multiclamp 700B patch-clamp amplifier (Axon Instruments, CA,

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USA), sampled at 20 kHz, and filtered at 10 kHz. Data were analyzed using pClamp 10

(Molecular devices, CA, USA).

3.3. Results

3.3.1. Optogenetic targeting of medial septum neurons

To characterize neurons of the medial septum (MS) in mice transfected with

ChETA-eYFP, we first examined virus distribution, specificity, and co-expression with

other cell type markers within the septum. Virus expression, as indicated by eYFP

labeling of cell bodies, was distributed extensively across the MS (Fig. 3.1. A). Since

synapsin is a non-specific neuronal promoter, we quantified the extent of ChETA

expression in cholinergic and GABAergic cell populations in the MS using ChAT and

parvalbumin (PV) immunocytochemistry (Fig. 3.1. B). We found that the eYFP reporter

was widely expressed in PV neurons as 72.5 ± 2.3% of cells positive for PV also co-

expressed eYFP (number of eYFP cells counted: 2770, n = 3 mice). 21 ± 1.7% were

positive for eYFP only indicating that other cell populations were transfected. Co-

expression in cholinergic neurons was very low because only 0.5 ± 0.2% of ChAT

neurons were also positive for eYFP (Fig. 3.1. C; total number of eYFP cells counted:

2697, n = 3 mice). Therefore, the remaining ~21% eYFP labeled cells that were

negative for PV are most likely glutamatergic and non-PV GABAergic neurons, as it has

been previously shown that septal neurons can also express glutamatergic and other

GABAergic markers apart from PV (Kiss et al., 1997; Robinson et al., 2016).

We next determined whether optogenetic activation of medial septum neurons

reliably evoked firing of the transfected neurons in slice, as well as the optimal

illumination intensity to induce spikes (Fig. 3.2. A, B). Since both GABAergic and

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glutamatergic cells were transfected by the viral injection, the firing frequency of MS

cells to injected current was heterogenous, comprising fast, slow and bursting neurons

(Fig. 3.2 C, D). Voltage and current-clamp were used to characterize photocurrent and

depolarization elicited by light. We found that 100ms light pulses induced large

Fig. 3.1: Optogenetics and MS neurons. A. Coronal section showing unspecific

ChETA-eYFP expression from MS neurons and fibers. Blue/cyan: DAPI, green:

eYFP, scale bar 16um. B, ChETA-eYFP expression compared with PV and ChAT.

Scale bar 51 um. C, Quantification of eYFP, PV, ChAT and their co-localization.

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photocurrents and elicited spikes starting at 5 mW laser intensity (Fig. 3.2 E-F). We then

tested the capacity of these neurons to respond to blue light stimulation in semi-natural

firing patterns. Accordingly, we tested the fidelity of MS neural activation to the

application of 50% duty cycle blue light pulses at 5 Hz frequency. When light power was

10mW or above, the fidelity of action potential generation in MS responsive neurons

was 100% reliable on average, with more than 1 spike elicited per pulse at higher light

power intensities (a spike probability of 1 reflects 1 spike per pulse) (Fig. 3.2 G, H).

Overall, these experiments demonstrate that a reliable stimulation of MS

GABAergic/glutamatergic neurons can be achieved in a semi-naturalistic fashion.

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3.3.2 Characterizing the effect of theta stimulation of MS projections in hippocampal

rhythms

To determine whether optogenetic stimulation of the projections from MS

neurons could drive hippocampal networks at theta frequency in vivo, we activated

ChETA-expressing MS projections at the level of the fornix while recording freely

moving J20+ mice in home cage. Hippocampal oscillations were recorded both during

quiet awake and sleep states (Fig 3.3 A). Recordings consisted of 4h baseline followed

by 4h of stimulation and an additional 4h of post-stimulation, spanning the complete

duration of the light-cycle.

Fig. 3.2: In vitro characterization of AAV2-ChETA in medial septum neurons. A, In

vitro recording configuration. B, Example ChETA-eYFP MS neuron visually identified.

Scale bar: 20 μm. C, response of MS cell to injected current in current clamp. D,

Average spike frequency of identified ChETA-eYFP MS neurons in response to current

injection. E, Example photocurrent and depolarization to different light intensities in

voltage and current clamp, respectively. F, Photocurrent (left) and depolarization (right)

in response to each mW laser stimulation. G, Example depolarization in response to 5

Hz, 50% duty-cycle light stimulation at 1, 10 and 20 mW light intensity. H, Example

raster sweep of neuron in response to 5 Hz, 50% duty-cycle light stimulation at 10mW

(top) and associated spike timing histogram in response to each pulse (bottom). To the

right, spike probability for 5 Hz laser stimulation at different light intensities.

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3.3.2.1. Sleep

First, we conducted a state-dependent power analysis to assess the effect of

fornix stimulation. Starting with REM sleep, we compared hippocampal activity during

baseline, stimulation, and post-stimulation sleep periods (Fig. 3.3) using the same

measures as in chapter 2. We confirmed that optogenetic activation of the fornix was

able to drive hippocampal rhythms (Fig. 3.3.B-D). Although activation of the fornix did

not significantly affect the power of the ongoing oscillations (Fig. 3.3. E), the peak

frequency was significantly shifted to the stimulation frequency for the duration of the

stimulation (Fig. 3.3 F; Base: 6.796±0.1374 ; Stim: 5.669±0.2415 Post:6.565±0.1675; n

= 11 per group, repeated-measures one-way ANOVA p<0.0001; Dunnet’s multiple

comparisons, Baseline vs Stim: p<0.0001), and the power at the stimulating frequency

was also significantly higher than in baseline conditions (Fig. 3G; Base: 0.1588±0.02 ;

Stim: 0.4072±0.057 Post:0.1703±0.02271, repeated-measures one-way ANOVA

p<0.0001; Dunnet’s multiple comparisons, Baseline vs Stim: p<0.0001). Both peak

power and peak frequency regained their baseline levels after the stimulation. All other

measures, including the strength of theta-gamma coupling as quantified by the MI, were

unchanged (Fig. 3.3. J-G).

Additionally, we also quantified hippocampal ripples and their characteristics

during NREM in animals that had at least one recording site in the pyramidal layer of the

hippocampus. Measurements of hippocampal ripples revealed no differences in their

characteristics (Table 3-1).

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3.3.2.2. Wake

The same analysis was performed for wake periods to examine the effect of theta fornix

stimulation in the hippocampus of J20+ mice (Fig 3.4). As in REM sleep, optogenetic

activation of the fornix was able to drive hippocampal rhythms (Fig. 3.4.A-D), with no

effect on the power of ongoing oscillations (Fig. 3.4.E). Similarly, the peak frequency in

the theta band was significantly shifted to the stimulation frequency for the duration of

the stimulation (Fig. 3E; Base: 5.835±0.191 Hz; Stim: 5.307±0.167 Hz; Post:5.74±0.185

Hz; repeated-measures one-way ANOVA p<0.0127; Dunnet’s multiple comparisons,

Baseline vs Stim: p=0.0107), and the power at the stimulating frequency was also

significantly higher than in baseline conditions (Fig. 3E; Base: 0.2293±0.0205; Stim:

0.4687±0.093 Post:0.213±0.0193; repeated-measures one-way ANOVA p=0.0036;

Dunnet’s multiple comparisons, Baseline vs Stim: p=0.0077). Both peak power and

peak frequency regained their baseline levels after the stimulation. All other measures,

including the strength of theta-gamma coupling as quantified by the MI, were

unchanged (Fig. 3.4. H-J).

Finally, we investigated the effect of fornix activation in the wake-sleep cycle and

confirmed that continuous stimulation did not alter the sleep architecture of the animals

(Fig. 3.5. A-C).

Overall, these results show that rhythmically activating septal projections can

drive theta oscillations. These results confirm that optogenetic stimulation of the fornix

can modulate hippocampal network activity through septal connections.

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** **

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Table 3. 1: Analysis of CA1 cell layer LFP ripple characteristics during NREM

sleep occurring in the first 20 minutes of the 4h period. Baseline: n = 6, stimulation= 2;

non-significant, unpaired t-test).

Ripple Parameter baseline Stim

Density (Ripples/min) 31.8 ± 2.8 30.184±1.679

Ripple Duration (ms) 46.45 ± 1.5 43.109 ± 1.247

Max ripple amp (mV) 0.22 ± 0.032 0.264 ± 0.084

Ripple Interval (s) 2.07 ± 0.18 2.12 ± 0.085

Fig. 3.3: Optogenetic driving of theta frequency in the hippocampus of J20+

during REM sleep. A. Diagram and coronal sections of viral transfection in the

MS, optic fiber in the fornix and electrode placement in hippocampus. Blue: DAPI,

Green: eYFP, Red, DII. B. Example hypnogram and REM spectrogram during

baseline and B. stimulation. C. Example raw and filtered CA1 trace together with

EMG during REM sleep for baseline, stimulation and post-stimulation recordings.

D. Mean normalized spectrogram of all wake for baseline, stimulation, and post-

stimulation periods. E. Band power for theta, low gamma, high gamma and ripple

frequencies. F. Peak theta frequency. G. Maximum peak power of the theta band.

H. Mean peak power of theta band. I. Mean MI for theta-low gamma coupling. J.

Mean MI for theta-high gamma coupling. Data are expressed as mean ± SEM.

n=11 per group; repeated-measure one-way ANOVA, *p< 0.05, *p<0.001

.

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Fig. 3.4: Optogenetic driving of theta frequency in the hippocampus of J20+ mice

during wake. A. Example hypnogram and wake spectrogram during baseline and B.

stimulation. C. Example Raw CA1 trace together with EMG during wake for baseline,

stimulation and post-stimulation periods. D. Mean normalized spectrogram of all wake

for baseline, stimulation, and post-stimulation periods. E. Band power for theta, low

gamma, high gamma, and ripple frequencies. F. Peak theta frequency. G. Maximum

peak power of the theta band. H. Mean peak power of theta band. I. Mean MI for theta-

low gamma coupling. J. Mean MI for theta-high gamma coupling. Data are expressed

as mean ± SEM. n=11 per group; repeated-measure one-way ANOVA, *p< 0.05.

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3.3.3. Modulation of SWDs by fornix optogenetic activation

Next, we aimed to characterize the effect of fornix stimulation on aberrant

hypersynchronous hippocampal activity in the same cohort of J20+ mice. We employed

the same state-dependent analysis from chapter 2. Starting with REM sleep, the total

number of SWDs as well as the corrected number per time spent in REM sleep, were

Fig. 3.5: Stimulation of fornix fibers does not alter sleep architecture in J20+. A.

Percentage of time spent in REM, NREM, and wake in J20+ mice during 4h baseline,

4h stimulation, and 4h post-baseline. B. Cumulative time spent in seconds per stage. C.

Number of episodes quantified in wake, NREM, and REM sleep. Data are expressed as

mean ± SEM. N=11; two-way mixed ANOVA with Sidak’s multiple comparisons, non-

significant.

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significantly decreased during stimulation, and this effect persisted 4h post-stimulation

(fig 3.6 A-C, total number. Base: 153.7±32.63; stim: 72.09±13.07; post: 79.91±21.65,

repeated-measures one-way ANOVA, p=0.0339. Dunnett’s multiple comparisons, base

vs stim p=0.0315; base vs post =0.06; Corrected, base: 4.783±2.924; stim:2.91±0.546;

post: 3.091±0.8141; repeated-measures one-way ANOVA, p=0.024, Dunnett’s multiple

comparisons, base vs stim=0.024; base vs post =0.041). Although a decrease in the

number of SWDs in NREM was observed, this decrease was not significant (fig 3.6 D-

F). Lastly, the stimulation had no effect in the number of SWDs during wake (fig 3.6 G-

H). Interestingly, the rate of SWDs returned to baseline levels 24 hours after stimulation,

indicating that the decrease was transient (fig 3.7 A; n=5, Dunnett’s multiple

comparisons, REM base vs pos, p=0.0038, base vs post 24h, p=0.7).

Since the vast majority of SWDs occurred during sleep, we focused on further

comparing their characteristics during REM and NREM (fig 3.7). Fornix stimulation had

no effect on the amplitude or width of the spike neither during REM nor NREM periods

(fig 3.7 B-C). REM episode analysis revealed a significant difference in the number of

SWDs depending on episode duration, but not the stage of stimulation (two-way

repeated-measures ANOVA, duration: p=0.0011, stage, p=0.096). Interestingly, the

analysis of SWD distribution per episode showed a significant main difference with no

interaction for both factors, stage (Fig. 3.7 D, supplementary table 3.1 p=0.016) and

segment (Fig. 3.7 E, supplementary table 3.2 p=0.0017, two- way repeated measures

ANOVA). During the first and last 10 s of REM, the ratio of SWDs was reduced

compared to baseline levels, while the center segment was not statistically different

(First 10s, base: 0.039±0.008, stim:0.012±0.002; last 10s, base:0.106±0.017,

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stim:0.056±0.014; Sidak’s multiple comparisons first 10s: base vs stim p=0.0243; last

10s: base vs stim p=0.0103). For NREM, only an effect specific to length (p=0.0147)

and segment (p=0.0002) was observed, but the stage of stimulation was not a

significant factor (Fig. 3.7 F-G, see supplementary table 3.3-4 for multiple comparisons).

Finally, since we found a decrease in the rate of SWDs during stimulation, we

sought to determine if neurons changed their firing probability before or after these

events. We built PSTHs and classified each unit activity as described in chapter 2

(section 2.3.3). We followed the same isolated units during 4h of baseline and 4h of

stimulation by performing the spike detection and sorting in the concatenated recording

(Fig 3.8 A, C). Almost all units (98%) were found to be positively modulated during

baseline (Fig 3.8 B), with the proportion decreasing to 89% during stimulation (Fig 3.8

D). We focused on the positively modulated units and found that the overall firing

probability during the 10ms just before the peak of the SWD was significantly decreased

in the stimulation stage (matched-pair Wilcoxon with Bonferroni-Holms correction

p<0.0001, Fig 3.8 E-G).

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.

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Fig. 3.6: Effect of fornix stimulation on the number of spike-wave discharges. A.

Total number of SWDs in REM per stage (baseline, stimulation, post-stimulation). B.

Total number of SWDs normalized by time spent per REM, in minutes. C. % change of

normalized SWDs between baseline and post-stimulation and baseline-stimulation

periods during REM. D. Total number of SWDs in NREM per stage (baseline,

stimulation, post-stimulation). E. Total number of SWDs normalized by time spent per

NREM, in minutes. F. % change of normalized SWDs between baseline and post-

stimulation and baseline-stimulation periods during NREM. G. Total number of SWDs in

wake per stage (baseline, stimulation, post-stimulation). H. Total number of SWDs

normalized by time spent per wake, in minutes. Data are expressed as mean ± SEM.

n=11 * p< 0.05

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Fig. 3. 7: Effect of fornix stimulation in spike-wave discharge characteristics. A.

Total number of SWDs in REM sleep per state and per stage (baseline, post-

stimulation, post- stimulation 24 h later). B. Average peak of the SWD spike separated

by REM and NREM periods. C. Average width of the SWD spike separated by REM

and NREM periods. D. Average SWD per seconds according to binned episode

duration in REM. E. Average SWD per second according to their timing (first 10

seconds, center of the episode or last 10 seconds) in a given episode of REM. F.

Average SWD per seconds according to binned episode duration in NREM. G. Average

SWD per second according to their timing (first 10 seconds, center of the episode or

last 10 seconds) in a given episode of NREM. Data are expressed as mean ± SEM.

n=11 except A (n=5); * p< 0.05 across groups, $ p< 0.05 within group.

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3.3.4. Modulation of hippocampal memory by fornix optogenetic activation

Our results so far highlight a decrease in network hyperactivity during

optogenetic stimulation of the fornix. We next investigated whether memory

performance, which we previously showed to be impaired in J20+ mice and correlated

with SWD rate (see chapter 2) could be positively modulated by forniceal stimulation.

We assessed hippocampal-dependent spatial memory in the same two tasks (the novel

object location recognition and passive avoidance tasks), and light was delivered post-

training for 4h in homecage. In the novel object location recognition task (Fig. 3.8 A),

mice showed no preference for either of the objects during the sample phase and

explored the object equally. 4 hours later, the animals were reintroduced into the open

field, where one of the objects was moved to a different location. Preference for the

displaced object was higher relative to the sample phase in optogenetically stimulated,

J20+ ChETA mice (Fig. 3.8 B, sample: 0.509±0.013, test: 0.591±0.028; two-way mixed

ANOVA , interaction p=0.0173; Sidak’s multiple comparisons training-test: control

Fig. 3.8: Effect of fornix stimulation on hippocampal single unit activity. A.

Example raster plot and PSTH (below) of isolated unit during baseline (left) and SWD

(right), in 4h with no stimulation. B. Distribution of units according to response with no-

stimulation: +, positively modulated; -, negatively modulated; ~ unmodulated. C. Same

unit as in A, but during 4h stimulation. D. Same as in B, but during 4h stimulation. E

PSTH of all positively modulated units corrected by their respective baselines F. Same

as in E, but during 4h stimulation. PSTHs with 10ms bins. G Same data as in E ad F

with different bin size: Base, -500ms to -10ms from spike peak; before peak, from -

10ms to 0ms; peak, from 0 to 10ms; Post, from 10ms to 500ms. N=2 animals. *p<0.001

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p<0.0001, J20+ ChETA p=0.0038), reflecting a memory improvement. Control

measures revealed no evidence for differences in locomotion or motivation during

testing compared to controls (Fig. 3.8 E-G, see supplementary table 3.5-7).

In the passive avoidance task, the latency to cross to the dark compartment

during training was equivalent in the three groups (Fig. 3.8 C-D; control:17.5±3.89, J20+

eYFP:12.78±4.61, J20+ ChETA: 27.3±5.53). Light was delivered for 4 hours

immediately after training, and the next day mice were tested in the same chamber for

10min. The J20+ ChETA group had latencies comparable to controls (two-way mixed

ANOVA of aligned-transformed data, interaction p<0.001; Wilcoxon with Bonferroni-

Holms adjustment: control vs J20+ChETA p=0.93; control vs J20+ eYFP p=0.0053;

control:473.9±53.83, J20+:108.55±28.07, J20+ ChETA:429.4±64.96) indicating that the

shock was still remembered. One week later, the latency to cross to the dark

compartment was still significantly lower in the J20+ (control vs J20+ChETA p=0.19;

control vs J20+ eYFP p=0.018; control: 299.1±71.1 s, J20+:20.33±3.63 s, J20+

ChETA:107±37.84 s).

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Fig. 3. 9: Spatial memory impairments in J20+ mice. A. Schematic of novel object

place memory task. Below cumulative object exploration time for both objects during

sample and test phases. B. Displaced object recognition index. C. Schematic of

passive avoidance task. D. Latency in seconds to enter the dark compartment in the

passive avoidance task. E. Time spent in quadrants during habituation to the open field.

F. Distance traveled during habituation, sample and test sessions in the open field. G.

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Cumulative total object exploration time during sample and test phases. Data are

expressed as mean ± SEM; two-way mixed ANOVAs were performed if not otherwise

stated. *p< 0.05

3.4. Discussion

The primary purpose of the current study was to investigate if stimulation of

septo-hippocampal projections is able to rescue memory impairments by reducing early

alterations in hippocampal oscillations in an AD mouse model. To examine this, we

used optogenetics and in vivo LFP recordings to obtain evidence of state-dependent

network modulation. We first demonstrate that optogenetic activation of mainly

GABAergic fibers from the medial septum can drive hippocampal rhythms at a specific

theta frequency. At the level of the fornix, light stimulation could effectively increase

peak power at the given frequency in hippocampal CA1 without changing overall theta

power or theta-gamma coupling. Importantly, the stimulation resulted in a decrease of

hyperactivity during REM sleep, accompanied by an improvement in memory

performance in two hippocampal-dependent spatial tasks.

3.4.1. Stimulation of septo-hippocampal projections modulates hippocampal rhythms

The fornix is a white matter pathway comprising afferent and efferent

connections to and from the hippocampus and is therefore crucial in mediating

hippocampal function. Highlighting the importance of the fornix in memory, its

transection has consistently been demonstrated to impair spatial memory in rodents

(Cassel et al., 1998; Winters and Dunnett, 2004), monkeys (Gaffan, 1994; Gaffan and

Harrison, 1989) and humans (Grafman et al., 1985; Moudgil et al., 2000). Supporting

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this conclusion, forniceal electrical stimulation results in improved recollection without

affecting familiarity-based recognition, as well as enhancing spatial memory (Hamani et

al., 2008; Hao et al., 2015; Hescham et al., 2013; Sweet et al., 2014).

In the present study we used optogenetics to specifically target septo-

hippocampal projections at the level of the fornix instead of broadly stimulating afferent

and efferent hippocampal connections with current. The medial septum sends its input

to the hippocampus through the fornix, and is known to contribute prominently to

hippocampal theta rhythm generation. It has been shown that septal lesions result in a

decrease of hippocampal theta power as well as memory deficits (Winson, 1978),

whereas fornix stimulation at theta frequency reverses the observed impairments

(McNaughton et al., 2006; Shirvalkar et al., 2010). In the first part of this chapter, we

demonstrated that stimulating the fornix at 5 Hz theta frequency increased peak power

in the hippocampus at this frequency, with no other effect in power in the theta, gamma,

or ripple bands. We hypothesize this effect is mainly driven by stimulation of GABAergic

projections from the medial septum, as it has been shown that activating septal PV

projections at the level of the hippocampus also drives the network at a specific

frequency, while power remains unchanged (Bender et al., 2015). In contrast,

stimulating glutamatergic projections from the medial septum at the level of the fornix

has no effect on theta frequency or peak power in the hippocampus, and it appears their

effect on theta is mainly though local connections in the medial septum (Robinson et al.,

2016). While our virus construction used the promoter synapsin to transfect all neural

types (Schoch et al., 1996), ChAT septal neurons appeared to be minimally colocalized

with eYFP in our experiment. Thus, not only is the presence of synapsin unnecessary

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for cholinergic neurotransmitter release (Bogen et al., 2009) but also, at least in the

medial septum, synapsin is specifically absent in the majority of cholinergic neurons.

Consequently, any of our resulting effects induced by forniceal stimulation were likely

independent from direct stimulation of cholinergic projections.

Finally, in chapter 2, we had observed attenuation of theta -low gamma coupling

in J20+ during wake periods. Thus, we wanted to ascertain if fornix activation had any

effect on the modulation index. Although direct 5 Hz stimulation of principal neurons in

the hippocampus has been shown to increase theta-gamma coupling under anesthesia

(Butler et al., 2018), we did not see an equivalent theta- low gamma coupling increase

during wakefulness. Interestingly, while continuous stimulation of medial septum

neurons at gamma frequencies has been shown to increase theta-gamma coupling

during active behavior, this result is a direct consequence of increasing gamma band

power across all phases of theta, instead of enhancing coupling at a specific phase

(Etter et al., 2019). In fact, it has been proposed that local gamma- generating circuits

might not be activated at a particular theta phase, as commonly interpreted, but rather

gamma itself might be setting the phase of the local theta oscillation (Fernández-Ruiz et

al., 2017; Lopez-Madrona et al., 2018). It remains to be established if specific phase

coupling could be enhanced during freely moving behaviour without an accompanying

increase in gamma power, and if this manipulation would be sufficient to improve

memory in models of AD.

3.4.2 Stimulation of septo-hippocampal projections decreases SWDs in a state-

dependent manner

Our results show that fornix stimulation effectively reduce SWDs specifically

during REM sleep in our AD model, and this effect was accompanied by an

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improvement in memory performance. Recent studies have revealed that hyperactivity

in the form of hippocampal spike-wave discharges in AD models can be decreased with

anti-epileptic drugs, and this decrease is accompanied by an improvement in memory

performance (Gureviciene et al., 2019; Nygaard et al., 2015; Sanchez et al., 2012b; Shi

et al., 2013; Ziyatdinova et al., 2011). One such drug, levetiracetam, improves cognitive

function in human patients diagnosed with amnestic mild cognitive impairment (Bakker

et al., 2015, 2012) and AD (Cumbo and Ligori, 2010). Other drugs not employed for

epilepsy, such as the retinoid X receptor antagonist bexarotene, also exert a striking

improvement in memory in AD models that is accompanied by a reduction of

hyperactivity (Bomben et al., 2014). Similar to our stimulation effect, in these studies

once administration of the drug is stopped, hyperactivity returns to baseline levels.

In the past few years, optogenetic stimulation has been successfully used to

reduce or halt seizures in rodent models of epilepsy (Tønnesen and Kokaia,

2017). Generally, these effects have mainly been obtained by inhibiting excitatory

principal cells or by stimulating inhibitory interneurons (Krook-Magnuson et al., 2013;

Ledri et al., 2014), but the picture is likely more complex (Bui et al., 2018). In our unit

recording experiments, we showed that optogenetic stimulation of the fornix decreased

the firing probability at SWD onset, indicating that the stimulation can disrupt anomalous

synchrony by reducing the engagement of neurons just before the peak of the spike.

Although the exact mechanism is unknown, this disengagement is presumably due to

entrainment of the network at lower frequencies, which consequently disrupts higher

frequency synchronization. Indeed, others have shown that pacing the firing of either

principal cells or interneurons at low frequencies can lower ictal discharges. (Ladas et

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al., 2015; Shiri et al., 2017). Chronically pacing the activity of hippocampal interneurons

at theta frequency for 14 days also diminishes interictal spikes for the duration of the

stimulation in a model of epilepsy (Lévesque et al., 2019). From our results, we cannot

conclude that the reduction we observe in SWDs and firing probability is a consequence

of pacing the network at a particular frequency, and further experiments will be needed

to test if this effect is dependent on stimulation rate. While manipulations employing 5

Hz theta frequency stimulation effectively abate epileptiform activity and can improve

memory in epilepsy models (Kim et al., 2020; Rajdev et al., 2011; Salam et al., 2015;

Schiller and Bankirer, 2007) and patients (Koubeissi et al., 2013; Miller et al., 2015),

higher frequencies may also suppress seizures in certain conditions (Boëx et al., 2007;

Lothman and Williamson, 1993; Wyckhuys et al., 2010).

3.4.3 Post-training stimulation improves spatial memory in AD

Lastly, we demonstrated that septo-hippocampal fiber stimulation at the level of

the fornix during the post-training period restored memory performance to control levels

in the J20+. We verified this result in two hippocampal-dependent memory tasks, the

novel object-place recognition and passive avoidance tests, with similar outcomes. Our

post-training manipulation suggests that J20+ mice are able to appropriately learn the

tasks, and it is during consolidation that the memory trace is impaired. In line with our

results, it has been shown that specific re-activation of dentate gyrus engram cells,

which had been labeled during learning, improved memory in APP/PS1 mice when

activated during retrieval (Perusini et al., 2017). Moreover, Perusini et al’s co-labeling

results imply that the cells activated during context re-exposure were not the same as

those activated during memory encoding, which the authors’ interpret as a problem in

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the retrieval process. Another explanation, which our results seem to point to, is that the

encoded trace is not appropriately consolidated and, therefore, cannot be retrieved.

Memory consolidation refers to the stabilization of memory traces, possibly

including intrahippocampal synaptic reinforcement and the transfer of information

initially encoded in the hippocampal system to the neocortex for long-term storage

(Marshall and Born, 2007; Rasch and Born, 2013; Sirota et al., 2003). The consolidation

process has been proposed to occur during post-learning rest or sleep (Prince et al.,

2014; Rasch and Born, 2013; Wilson and McNaughton, 1994) via reactivation of

memory traces in short bouts of neuronal activity associated with ripple events in quiet

awake and NREM sleep (Buzsáki, 2015; Girardeau et al., 2009; Nádasdy et al., 1999)

and stabilization of these reactivated sequences during REM (Li et al., 2017; Rasch and

Born, 2013). Recently, synaptic pruning during REM was suggested to be necessary for

removing spurious neuronal connectivity and to increase the signal-to-noise ratio in

neuronal networks (Li et al., 2017). Directly linking REM to memory, inhibiting the

medial septal GABAergic population for 4h post-training during REM sleep severely

inhibits memory performance (Boyce et al., 2016). In the context of aberrant

hippocampal hyperactivity, memory consolidation deficits have been linked to abnormal

hippocampal-cortical coupling triggered by interictal epileptiform discharges (Gelinas et

al., 2016). Thus, SWDs have the potential of hijacking both the local and connected

circuits by facilitating the expression of cortical oscillations in brain states that do not

naturally exhibit these oscillations, such as during REM, resulting in suboptimal

stabilization of the memory trace (Beenhakker and Huguenard, 2009; Busche and

Konnerth, 2016; Palop and Mucke, 2016).

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Finally, while we did not investigate the effects of optogenetic stimulation on

soluble amyloid beta levels, its reduction may have contributed to the memory

improvement after stimulation (Kittelberger et al., 2012; Lesné et al., 2006; Nicole et al.,

2016), even if plaques are not observed at this time point in our model. While

stimulation of medial septum PV neurons did not have an effect in amyloid deposition

(Etter et al., 2019), direct stimulation of hippocampal PV-expressing neurons during 1h

was able to reduce cleavage of APP in APP/PS1 mice (Iaccarino et al., 2016). On the

other hand, Yamamoto and colleagues found the opposite effect, with optogenetic

manipulation resulting in an increase of amyloid beta, although the strength of the

neuronal activation might have been more intense compared to physiological

conditions, since their stimulation paradigm also resulted in seizures (Yamamoto et al.,

2015).

In conclusion, the present optogenetics study demonstrates that 1) activation of

mainly GABAergic fibers from the medial septum can drive hippocampal rhythms at a

specific theta frequency without changing overall theta power or theta-gamma coupling.

2) Fornix stimulation decreased hyperactivity in an AD mice model during REM sleep,

accompanied by an improvement in memory performance in two hippocampal-

dependent spatial tasks. Taken together these results suggest that lowering existing

hyperactivity during sleep may benefit memory processes in AD.

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3.5 Supplementary Data

Table 3.2: REM length -SWDs

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMARY

ADJUSTED P VALUE

BASELINE

<30 VS. >30 & <60 -0.01597 -0.04064 to 0.008694

No ns 0.2556

<30 VS. >60 -0.05223 -0.08814 to -0.01631

Yes ** 0.0058

>30 & <60 VS. >60 -0.03625 -0.07556 to 0.003055

No ns 0.0726

STIM

<30 VS. >30 & <60 -0.01171 -0.02884 to 0.005429

No ns 0.2193

<30 VS. >60 -0.03307 -0.06128 to -0.004856

Yes * 0.0218

>30 & <60 VS. >60 -0.02136 -0.04584 to 0.003118

No ns 0.0920

POST

<30 VS. >30 & <60 -0.01442 -0.02873 to -0.0001050

Yes * 0.0483

<30 VS. >60 -0.02904 -0.05548 to -0.002612

Yes * 0.0311

>30 & <60 VS. >60 -0.01463 -0.03914 to 0.009881

No ns 0.3153

Table 3.3: REM segment -SWDs

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMARY

ADJUSTED P VALUE

FIRST 10S

BASELINE VS. STIM 0.02692 0.003500 to 0.05033

Yes * 0.0243

BASELINE VS. POST 0.01901 -0.004621 to 0.04265

No ns 0.1268

STIM VS. POST -0.00790

4

-0.01931 to 0.003500

No ns 0.2100

CENTER SEGMENT

BASELINE VS. STIM 0.02992 -0.004043 to 0.06388

No ns 0.0885

BASELINE VS. POST 0.02842 -0.01026 to 0.06711

No ns 0.1744

STIM VS. POST -0.00149

5

-0.02300 to 0.02001

No ns 0.9964

LAST 10 S

BASELINE VS. STIM 0.05001 0.01246 to 0.08756

Yes * 0.0103

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BASELINE VS. POST 0.02171 -0.03725 to 0.08067

No ns 0.6815

STIM VS. POST -0.02830 -0.05906 to 0.002450

No ns 0.0733

BASELINE

FIRST 10S VS. CENTER SEGMENT

-0.04107 -0.06962 to -0.01252

Yes ** 0.0063

FIRST 10S VS. LAST 10 S -0.06708 -0.1111 to -0.02310

Yes ** 0.0042

CENTER SEGMENT VS. LAST 10 S

-0.02601 -0.05654 to 0.004526

No ns 0.1016

STIM

FIRST 10S VS. CENTER SEGMENT

-0.03807 -0.06560 to -0.01053

Yes ** 0.0081

FIRST 10S VS. LAST 10 S -0.04398 -0.08175 to -0.006206

Yes * 0.0227

CENTER SEGMENT VS. LAST 10 S

-0.00591

5

-0.02497 to 0.01313

No ns 0.7789

POST

FIRST 10S VS. CENTER SEGMENT

-0.03166 -0.06170 to -0.001609

Yes * 0.0386

FIRST 10S VS. LAST 10 S -0.06438 -0.1219 to -0.006845

Yes * 0.0282

CENTER SEGMENT VS. LAST 10 S

-0.03272 -0.06367 to -0.001776

Yes * 0.0379

Table 3.4: NREM length -SWDs

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMARY

ADJUSTED P VALUE

BASELINE

FIRST 10S VS. CENTER SEGMENT

-0.0099

01

-0.01909 to -0.0007169

Yes * 0.0343

FIRST 10S VS. LAST 10 S

-0.0103

0

-0.01763 to -0.002971

Yes ** 0.0073

CENTER SEGMENT VS. LAST 10 S

-0.0004

018

-0.009654 to 0.008850

No ns 0.9991

STIM

FIRST 10S VS. CENTER SEGMENT

-0.0109

7

-0.01653 to -0.005413

Yes *** 0.0006

FIRST 10S VS. LAST 10 S

-0.0059

83

-0.01092 to -0.001047

Yes * 0.0180

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CENTER SEGMENT VS. LAST 10 S

0.004990

-0.001688 to 0.01167

No ns 0.1649

POST

FIRST 10S VS. CENTER SEGMENT

-0.0059

78

-0.01157 to -0.0003837

Yes * 0.0359

FIRST 10S VS. LAST 10 S

-0.0033

89

-0.01039 to 0.003612

No ns 0.4809

CENTER SEGMENT VS. LAST 10 S

0.002589

-0.002670 to 0.007847

No ns 0.4674

Table 3.5: NREM segment -SWDs

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMAR

Y

ADJUSTED P VALUE

BASELINE

<30 VS. >30 & <60 0.008204

-0.008090 to 0.02450

No ns 0.4495

<30 VS. >60 0.01755

0.0005777 to 0.03452

Yes * 0.0424

>30 & <60 VS. >60 0.009346

-0.001128 to 0.01982

No ns 0.0838

STIM

<30 VS. >30 & <60 0.001628

-0.004548 to 0.007803

No ns 0.8497

<30 VS. >60 0.005918

-0.003620 to 0.01546

No ns 0.2863

>30 & <60 VS. >60 0.004290

-0.001217 to 0.009798

No ns 0.1426

POST

<30 VS. >30 & <60 0.006534

-0.008318 to 0.02139

No ns 0.5555

<30 VS. >60 0.01175

-0.006033 to 0.02954

No ns 0.2416

>30 & <60 VS. >60 0.005217

-0.002119 to 0.01255

No ns 0.1939

Table 3.6: Control behavioural measures

Corresponding to Fig 3.8E

SOURCE OF VARIATION

% OF TOTAL VARIATION

P VALU

E

P VALUE SUMMARY

SIGNIFICANT?

GEISSER-GREENHOUSE'S

EPSILON

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INTERACTION 20.82 0.0065

** Yes

TIME 0.9949 0.7152

ns No 0.6159

COLUMN FACTOR

0.1808 0.3312

ns No

SUBJECT 1.874 >0.9999

ns No

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMARY

ADJUSTED P VALUE

J20+ (N=9)

Q1 VS. Q2 38.54 -118.7 to 195.7

No ns 0.9992

Q1 VS. Q3 -7.889 -238.6 to 222.8

No ns >0.9999

Q1 VS. Q4 -48.33 -193.4 to 96.70

No ns 0.9801

Q2 VS. Q3 -46.43 -157.7 to 64.86

No ns 0.8892

Q2 VS. Q4 -86.87 -188.0 to 14.24

No ns 0.1120

Q3 VS. Q4 -40.44 -166.3 to 85.38

No ns 0.9857

J20- (N=9)

Q1 VS. Q2 -16.17 -71.61 to 39.28

No ns 0.9946

Q1 VS. Q3 -33.11 -145.8 to 79.62

No ns 0.9942

Q1 VS. Q4 -21.15 -89.76 to 47.46

No ns 0.9905

Q2 VS. Q3 -16.94 -117.2 to 83.33

No ns >0.9999

Q2 VS. Q4 -4.982 -80.03 to 70.06

No ns >0.9999

Q3 VS. Q4 11.96 -56.21 to 80.13

No ns >0.9999

J20+ YFP (N=9)

Q1 VS. Q2 -4.146 -70.42 to 62.13

No ns >0.9999

Q1 VS. Q3 39.88 -23.60 to 103.4

No ns 0.4081

Q1 VS. Q4 45.92 -26.44 to 118.3

No ns 0.3949

Q2 VS. Q3 44.02 -1.284 to 89.33

No ns 0.0587

Q2 VS. Q4 50.06 -16.52 to 116.6

No ns 0.2080

Q3 VS. Q4 6.042 -46.33 to 58.42

No ns >0.9999

Table 3 7: Control behavioural measures

Corresponding to Fig 3.8F

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SOURCE OF VARIATION

% OF TOTAL VARIATION

P VALU

E

P VALUE SUMMARY

SIGNIFICANT?

GEISSER-GREENHOUSE'S

EPSILON

INTERACTION 2.708 0.3230

ns No

TIME 8.693 0.0014

** Yes 0.9819

COLUMN FACTOR

20.27 0.0083

** Yes

SUBJECT 41.25 0.0005

*** Yes

SIDAK'S MULTIPLE COMPARISONS TEST

MEAN DIFF.

95.00% CI OF DIFF.

SIGNIFICANT?

SUMMARY

ADJUSTED P VALUE

HABITUATION

J20- (N=9) VS. J20+ (N=9) 126.0 -13102 to 13354

No ns >0.9999

J20- (N=9) VS. J20+ YFP (N=9)

-8993 -19433 to 1447

No ns 0.1140

SAMPLE

J20- (N=9) VS. J20+ (N=9) -4332 -17121 to 8456

No ns 0.9045

J20- (N=9) VS. J20+ YFP (N=9)

-11407 -25040 to 2227

No ns 0.1318

TEST

J20- (N=9) VS. J20+ (N=9) 4495 -4164 to 13155

No ns 0.5885

J20- (N=9) VS. J20+ YFP (N=9)

-5581 -13696 to 2534

No ns 0.2793

Table 3 8: Control behavioural measures

Corresponding to Fig 3.8G

SOURCE OF VARIATION

% OF TOTAL VARIATION

P VALUE

P VALUE SUMMARY

SIGNIFICANT?

INTERACTION 2.540 0.2409 ns No TIME 3.515 0.0520 ns No COLUMN FACTOR 13.40 0.0902 ns No SUBJECT 60.37 0.0047 ** Yes

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Chapter 4: Fornix deep brain theta-burst stimulation improves

memory performance in an Alzheimer’s disease mouse model

4.1. Introduction

In the first part of this thesis we described early electrophysiological

abnormalities in the hippocampus of an Alzheimer’s mouse model and linked these

abnormalities to spatial behavioral deficits. In this section, we investigate, using

intracranial depth electrodes (deep brain stimulation, DBS), the effect of focal electrical

stimulation of the brain on spatial memory. Recently, significant interest has grown to

use this technique to modulate the hippocampal circuit and potentially reduce negative

symptoms in AD patients.

Evidence from drug-resistant epileptic populations and in one case of morbid

obesity first gave rise to the hypothesis that stimulation of hippocampal-associated

areas may improve memory (Fell et al., 2013; Hamani et al., 2008; Koubeissi et al.,

2013; Miller et al., 2015; Oh et al., 2012; Suthana et al., 2012). Indeed, small clinical

trials in AD patients have shown that DBS of the fornix (fDBS) may potentially provide

some improvement in symptoms. In an initial Phase 1 clinical trial, six patients who were

treated with fDBS for a year showed improvement in a subset of symptoms. These

patients showed a possible slowing of cognitive decline and increased glucose

metabolism in the temporal lobe as measured with PET, together with slower rates of

hippocampal atrophy (Laxton et al., 2010; Sankar et al., 2015; Smith et al., 2012).

Similar results were found in a case study by Fontaine et al. (2013). In a subsequent

phase 2 clinical trial, a positive cognitive change was observed in patients who had

received fDBS treatment and were older than 65, while a worse cognitive score was

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seen in younger individuals when DBS was on versus off (Holroyd et al., 2015; Lozano

et al., 2016; Ponce et al., 2015).

These studies have in common the use of high-frequency DBS, which has been

most widely employed in the treatment of Parkinson’s disease, often yielding significant

reductions in symptoms (Benabid et al., 1998; Krack et al., 1997; Limousin et al., 1995).

However, the limited clinical effect of high-frequency stimulation in these AD trials partly

draws attention to the suitability of using stimulation protocols developed to alleviate

motor symptoms in Parkinson’s disease (Benabid et al., 1998; Groiss et al., 2009) which

may not be necessarily optimized to mitigate memory dysfunction in AD.

An alternative stimulation paradigm, theta-burst stimulation, has been shown to

have an effect on memory in rodent and epileptic patients (Miller et al., 2015; Shirvalkar

et al., 2010; Sweet et al., 2014; Titiz et al., 2017). Theta-burst stimulation resembles

physiological patterns of activity as it consists of temporally spaced patterns of

stimulation bursts repeated in a theta (4-10 Hz) frequency interval, and is able to

efficiently elicit long-term potentiation (LTP, (Albensi et al., 2007; Capocchi et al., 1992;

Larson et al., 1986; Nguyen and Kandel, 1997; Staubli and Lynch, 1987). The effects of

high-frequency and theta-burst stimulation of the fornix on memory have yet to be

assessed in AD mice models. Thus, in this chapter, we used high-frequency or theta-

burst stimulation in J20+ mice during the post-training period of two hippocampal-

dependent memory tasks, the passive avoidance and novel object place recognition

tests. We report that continuous theta-burst stimulation right after the training session of

the passive avoidance or novel object place recognition tasks, can significantly rescue

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memory performance, whereas under our experimental conditions high-frequency

stimulation does not.

4.2. Methods

Most of the methods used in this chapter have been described in the previous

chapter. Refer to section 2.2. and 3.2. for the details.

4.2.1. Surgical coordinates

Mice were implanted in fimbria-fornix with bipolar tungsten electrodes (AP, -0.5;

ML, 0; DV, -2.35).

4.2.3 Post-training stimulation paradigm

After the training session in each of the memory tasks, animals were returned to

their homecage and received continuous fornix theta-burst stimulation (fTBS :100μs

85µA pulses in trains of 50ms at 200 Hz, 5 times per second) or 130 Hz stimulation

(fHFS: 100μs 85µA pulses at 130 Hz) for the duration of the post-training period. These

DBS parameters have been used previously (Gallino et al., 2019; Hescham et al., 2017;

Miller et al., 2015; Sweet et al., 2014; Xia et al., 2017b). A Master-9 (AMPI) pulse

generator and stimulus isolators (WPI) were used. The sham groups were tethered but

received no stimulation. The mice were monitored for the first 2-4 hours to determine

possible adverse reactions to the stimulation (i.e. excessive rearing, grooming).

4.2.4. Passive avoidance procedure.

The standard passive avoidance conditioning protocol was used as described in

the previous chapters with the addition of a ten-minute test 5min after the training to

assess the short-term memory of all groups. Post-training stimulation started

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immediately after the short-term memory test and lasted until the 24h time mark, when

animals were tested immediately without a shock. The latency to cross to the dark

compartment was measured.

4.2.5. Novel object place recognition procedure (NOPR) procedure.

The experimental protocol was performed as previously described. After

exploring the objects in the sample phase, animals received 4 hours of fTBS, 130Hz, or

sham stimulation and were tested immediately after with a displaced object (test phase).

The recognition index (RI) of the displaced object was computed for statistical analysis:

Object 2 recognition index, RI=(Object 2 exploration (s)−Object 1 exploration (s)) /

(Object 2 exploration (s)+Object 1 exploration (s)).

4.3. Results

4.3.1. Passive avoidance memory is improved after fornix theta-burst stimulation in

J20+ mice.

To investigate the effect of fDBS in the passive avoidance test, we compared the

latency to cross to the dark compartment between controls and J20+ groups which had

received either sham (Control Sham, J20 Sham), fornix theta-burst stimulation (Control

fTBS, J20 fTBS), or fornix high frequency stimulation (fHFS: J20 130Hz) treatment

(Fig.4-1 A-B). The latency was measured during training and 5min, 1 day, and 1 week

after training (Fig.4-1 C). A mixed ANOVA of the aligned rank transformed latencies

revealed a significant interaction of GROUPxTIME (F(12,84)= 13.106, p<0.001). Holm-

adjusted interaction analysis using Chi2 test highlighted significant differences between

the J20 and Control Sham latencies in the 24h testing timepoint compared to the

training session, indicating the J20+ had poorer performance than controls 24h after

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training (Control Sham vs. J20 Sham- train vs 24h: p<0.001). Remarkably, the J20

130Hz stimulation group’s performance was similar to the J20 Sham and no rescue was

observed (Control Sham vs. J20 130Hz- train vs 24h: p<0.001). In contrast, the J20

fTBS group’s avoidance response improved at the 24h timepoint as the latency was not

significantly different from controls (Controls Sham vs. J20 fTBS- train vs 24h: p=0.226)

(Fig.4-1 D).

To investigate if the J20 130Hz group’s performance could be rescued, these

same animals were re-tested in a second experiment using an identical passive

avoidance protocol. Mice were randomly assigned to two different groups: one receiving

fHFS again (J20 130Hz re-tested, internal control) and the other fTBS (J20 fTBS re-

tested) (Fig.4-1 E). A mixed ANOVA of the aligned rank transformed latencies showed

a significant interaction of GROUPxTIME (F(6,45)=11.935, p<0.001). Furthermore, chi2

analysis showed significant differences between the J20 fTBS re-tested group and the

J20 130Hz and J20 130Hz re-tested groups, in the training vs 24h timepoints (J20

130Hz vs. J20 fTBS retested - train vs 24h: p<0.001) (Fig.4-1 F). See supplemental

materials for all statistical comparisons.

4.3.2. Novel object place recognition memory improves after fTBS in J20+ mice.

To further validate the effect of fHFS and fTBS on memory, a second

hippocampal-dependent task was used (Fig.4-1 G). A mixed-ANOVA revealed a

statistically significant interaction of GROUPxSESSION (F(4,39)=5.883, P=0.0089).

Multiple comparisons using Sidak's test highlighted significant differences only in the

test phase between the Control Sham and J20 Sham (p=0.0055), and Control Sham

and J20 130Hz groups, (p=0.0047), suggesting that the J20 Sham and J20 130Hz

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group showed impaired memory performance as there was no preference for the

displaced object during the test phase. However, performance in the J20 fTBS group

reached control levels (Fig.4-1 H).

In a second experiment, we aimed to investigate if the performance of the J20

130Hz group could be rescued with fTBS. The same J20 130Hz animals were re-tested

with different objects in different positions, this time receiving fTBS instead (Fig.4-1 I). A

mixed ANOVA revealed a statistically significant difference between groups

(F(1,9)=6.381, P=0.0324). Sidak's multiple comparisons test showed significant

differences between the J20 fTBS re-tested and J20 130Hz groups in the test phase

(sample: p=0.5071, test: p=0.0454), suggesting that fTBS but not fHFS, was able to

improve memory on the same animals (Fig.4-1.J). Controls measures revealed no

evidence for differences in locomotion or motivation during testing between groups (Fig.

4-2).

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Fig. 4.1: Memory improvements following fTBS in J20 mice. A. Electrode placement

schematic. Right: Photomicrographs illustrating fornix DBS electrode (scalebar:

500microns). B. Schematic of stimulation paradigms. C. Schematic of passive

avoidance apparatus and schedule. D. Latency to cross to dark compartment in the

passive avoidance task. Control Sham n=5, Control fTBS n=5, J20 Sham n=7, J20 fTBS

n=6, J20 130Hz n=10; *p<0.001 (statistical significance shown only compared to Control

Sham). E. Schematic of repeated design passive avoidance for the J20 130Hz group. F.

Latency to cross to dark compartment in the repeated design passive avoidance task.

J20 fTBS re-tested n=4, J20 130Hz re-tested n=4; *p<0.001 (statistical significance

shown only compared to J20 130Hz). G. Schematic of NOPR task and stimulation

delivery. Below, cumulative exploration time for both objects. H. RI of the displaced

object. I: Schematic of re-tested NOPR task design for J20 130Hz group. Below,

cumulative exploration time for both objects. J, RI of the displaced object. Control Sham

n=8, Control Treatment n=8, J20 Sham n=9, J20 Treatment n=9; J20 130Hz n=10; J20

fTBS re-tested n=6; *p<0.05.

All sample and control measures such as total object exploration time, distance

travelled, time spent in the center of the open field, distance travelled during habituation

and time spent in quadrants during habituation, showed no significant differences

between groups.

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4.4. Discussion

In the clinical trials completed to date in AD patients, the efficacy of high-

frequency fornix DBS on memory performance has been variable (Holroyd et al., 2015;

Lozano et al., 2016; Ponce et al., 2015). Recent evidence from rodent studies and

epileptic patients suggests that an alternative frequency stimulation in the form of

patterned theta-bursts may modulate memory more efficaciously. For example, after

using intra-medial septum infusions of muscimol to attenuate theta rhythm and impair

spatial memory in Long-Evans rats, Shirvalkar et al. (2010) stimulated the fimbria-

fornix using three stimulation protocols: theta (7.7 Hz), 100 Hz, and theta-burst

stimulation. The latter was the only stimulation protocol that improved spatial memory

performance. Similar results were found by Sweet et al. (2014) in a rodent model of

traumatic brain injury where theta-burst was compared to tonic low and high-

frequency fornix stimulation. Additionally, two studies with epileptic patients in which

this patterned theta burst stimulation was delivered to the fornix (Miller et al., 2015)

and entorhinal cortex (Titiz et al., 2017), indicates that the paradigm might also be

capable of improving memory in humans. The authors hypothesize that theta-burst

stimulation compared to single pulse high-frequency stimulation may enhance

Fig. 4.2: Novel object place recognition (NOPR) control measures.

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memory performance by better replicating natural physiological activity in the

hippocampus.

The present results show that fornix theta-burst stimulation, when delivered for

the duration of the post-training period, can reverse memory impairments in two

hippocampal-dependent tasks in a transgenic AD mouse model. However, under our

experimental conditions, 130 Hz high-frequency stimulation was unable to modulate

memory. The timing of stimulation delivery may be significant, since other studies have

observed enhanced memory performance with high-frequency stimulation when

delivered weeks before training (Gallino et al., 2019; Hao et al., 2015; Hescham et al.,

2017; Mann et al., 2017; Xia et al., 2017b; Zhang et al., 2015). On the other hand when

theta frequencies have been used (Hescham et al., 2013; Miller et al., 2015; Shirvalkar

et al., 2010; Sweet et al., 2014; Titiz et al., 2017) or electrodes were implanted in other

memory areas (Kádár et al., 2014; Redolar-Ripoll et al., 2002; Soriano-Mas et al., 2005;

Suthana et al., 2012; Titiz et al., 2017), stimulation was delivered during or just

before/after the task. Therefore, we cannot conclude a complete lack of effect in

memory from high-frequency stimulation, since if our timing of stimulation were different,

we might have been able to see an effect in memory performance, albeit reduced as

compared with theta-burst (Hescham et al., 2013; Shirvalkar et al., 2010; Sweet et al.,

2014).

4.4.1. Possible therapeutic mechanisms of DBS in memory circuitry

The exact mechanisms by which DBS modulates the memory circuit are not

clearly understood, and several hypotheses have been put forward.

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First, DBS studies that focus on memory performance improvements weeks after

the stimulation posit a strong involvement of neurogenesis in the process. Although not

as clear in adult humans (Snyder, 2019; Sorrells et al., 2018), neurogenesis

continuously occurs throughout life in the subventricular zone and granule cell layer of

the dentate gyrus. Moreover, adult-generated dentate gyrus cells are thought to

contribute to the formation of hippocampus-dependent memories (Deng et al., 2010). In

line with this hypothesis, DBS of the entorhinal cortex results in memory improvement

and is correlated with activity-dependent regulation of hippocampal neurogenesis in AD

(Mann et al., 2017) and unimpaired (Stone et al., 2011) mice. A similar result has been

described in a model of Rett syndrome when DBS was delivered to the fornix (Hao et

al., 2015). However another study on the effects of fDBS found no evidence for induced

neurogenesis based on numbers of BrdU/NeuN double-labeled cells in the dentate,

while memory performance still improved (Hescham et al., 2017), suggesting that

neurogenesis is not necessary for DBS to modulate memory.

Second, acute changes in circuit activity appear to be sufficient for memory

modulation. Indeed, the immediate effects we found in memory performance are in

agreement with many studies showing that changes in behavior can occur immediately

after the onset of stimulation (Deeb et al., 2019; Hamani et al., 2008; Kádár et al., 2014;

Redolar-Ripoll et al., 2002; Shirvalkar et al., 2010; Soriano-Mas et al., 2005; Sweet et

al., 2014). In accordance with these results, fDBS for 1h induced expression of c-Fos,

an immediate‐early marker of neural activation, in areas CA1 and CA3 of the

hippocampus (Gondard et al., 2015; Hescham et al., 2016). Further, fDBS can acutely

enhance acetylcholine levels in the hippocampus after 20 minutes of stimulation

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(Hescham et al., 2016) as well as increase dopaminergic and glutamatergic

transmission throughout the nucleus accumbens (Ross et al., 2016).

Moreover, fDBS has been found to rapidly enhance levels of the synaptic

markers synaptophysin and GAP-43 in the hippocampus (Gondard et al., 2015). These

molecules play a key role in axonal growth and guidance in addition to synaptic

plasticity and synaptogenesis, and are important for memory processing (Aigner et al.,

1995; Biewenga et al., 1996; Grasselli et al., 2011; Rekart et al., 2005; Um et al., 2012).

In the Gongard et al. study, BDNF was also significantly increased after fDBS. BDNF

plays an important role in neuronal differentiation, neuron survival, synapse formation,

and regulation of activity-dependent changes in synapse structure and function

(Acheson et al., 1995; Park and Poo, 2013). BDNF is also a regulator of LTP in the

hippocampus (Bramham and Messaoudi, 2005; Minichiello, 2009) and plays a crucial

role in learning and memory (Callaghan and Kelly, 2012; Furini et al., 2010; Heldt et al.,

2007). Indeed, LTP is enhanced in the hippocampus after fDBS (Hao et al., 2015).

Lastly, DBS influences amyloid beta load. Although amyloid plaques may not be

the best predictor of cognitive impairment, and their clearance does not appear to result

in cognitive improvement, as discussed elsewhere in this thesis, AD largely remains

defined by their presence (Dodart et al., 2002; Hsia et al., 1999; Jacobsen et al., 2006;

Morris et al., 2014; Mucke et al., 2000; Panza et al., 2016). In young AD transgenic

mice, entorhinal DBS is reported to reduce Aβ plaque load in the dorsal hippocampus,

prefrontal cortex, and amygdala (Xia et al., 2017b). However, in the same study, DBS

did not reduce Aβ plaque load in older AD mice, despite successful rescue of their

memory deficits. Entorhinal DBS has also been shown to induce a significant reduction

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134

in CA1 amyloid beta-42 levels along with decreased hippocampal tau (Mann et al.,

2017). fDBS was similarly able to decrease amyloidosis, as well as modulate

inflammatory response and taper neuronal loss (Leplus et al., 2019).

In conclusion, the studies mentioned here show that DBS can modulate the

hippocampal memory circuit through immediate and longer-term effects through a

myriad of mechanisms. We present evidence that theta-burst fDBS delivered post-

training is able to improve hippocampal-dependent memory in an AD mice model.

Similar to others, we found that the more physiological theta-burst pattern of stimulation

performed better than high-frequency stimulation (Hescham et al., 2013; Miller et al.,

2015; Shirvalkar et al., 2010; Sweet et al., 2014; Titiz et al., 2017). Further research will

be needed to elucidate the underlying processes by which theta-burst stimulation led to

memory improvement in our experiments.

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4.5. Supplementary Information

Contains supplementary tables 4.1, 4.2 and 4.3

Table 4.1: Passive Avoidance additional statistics.

Corresponds to data shown in Fig.4-1 D

Chisq Test:

P-value adjustment method: holm

Value Df Chisq Pr(>Chisq)

CS-JS : train-5m -41.714 1 3.9658 1.0000000

CS-CD : train-5m -30.400 1 1.8053 1.0000000

CS-JD : train-5m -48.167 1 4.9442 0.7329960

CS-J130 : train-5m -17.600 1 0.8068 1.0000000

JS-CD : train-5m 11.314 1 0.2918 1.0000000

JS-JD : train-5m -6.452 1 0.1051 1.0000000

JS-J130 : train-5m 24.114 1 1.8710 1.0000000

CD-JD : train-5m -17.767 1 0.6727 1.0000000

CD-J130 : train-5m 12.800 1 0.4267 1.0000000

JD-J130 : train-5m 30.567 1 2.7378 1.0000000

CS-JS : train-24h -127.714 1 37.1740 5.834e-08 ***

CS-CD : train-24h -43.000 1 3.6120 1.0000000

CS-JD : train-24h -58.333 1 7.2516 0.2266766

CS-J130 : train-24h -146.200 1 55.6733 5.134e-12 ***

JS-CD : train-24h 84.714 1 16.3559 0.0021522 **

JS-JD : train-24h 69.381 1 12.1524 0.0186299 *

JS-J130 : train-24h -18.486 1 1.0995 1.0000000

CD-JD : train-24h -15.333 1 0.5010 1.0000000

CD-J130 : train-24h -103.200 1 27.7404 6.937e-06 ***

JD-J130 : train-24h -87.867 1 22.6232 9.263e-05 ***

CS-JS : train-1w -63.343 1 9.1444 0.0848227 .

CS-CD : train-1w 69.400 1 9.4088 0.0783896 .

CS-JD : train-1w -50.200 1 5.3704 0.6144245

CS-J130 : train-1w -60.100 1 9.4081 0.0783896 .

JS-CD : train-1w 132.743 1 40.1590 1.288e-08 ***

JS-JD : train-1w 13.143 1 0.4361 1.0000000

JS-J130 : train-1w 3.243 1 0.0338 1.0000000

CD-JD : train-1w -119.600 1 30.4835 1.717e-06 ***

CD-J130 : train-1w -129.500 1 43.6810 2.242e-09 ***

JD-J130 : train-1w -9.900 1 0.2872 1.0000000

CS-JS : 5m-24h -86.000 1 16.8561 0.0016936 **

CS-CD : 5m-24h -12.600 1 0.3101 1.0000000

CS-JD : 5m-24h -10.167 1 0.2203 1.0000000

CS-J130 : 5m-24h -128.600 1 43.0759 3.001e-09 ***

JS-CD : 5m-24h 73.400 1 12.2787 0.0178680 *

JS-JD : 5m-24h 75.833 1 14.5178 0.0055537 **

JS-J130 : 5m-24h -42.600 1 5.8390 0.4859031

CD-JD : 5m-24h 2.433 1 0.0126 1.0000000

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136

CD-J130 : 5m-24h -116.000 1 35.0484 1.705e-07 ***

JD-J130 : 5m-24h -118.433 1 41.1011 8.095e-09 ***

CS-JS : 5m-1w -21.629 1 1.0661 1.0000000

CS-CD : 5m-1w 99.800 1 19.4570 0.0004630 ***

CS-JD : 5m-1w -2.033 1 0.0088 1.0000000

CS-J130 : 5m-1w -42.500 1 4.7047 0.7820939

JS-CD : 5m-1w 121.429 1 33.6049 3.511e-07 ***

JS-JD : 5m-1w 19.595 1 0.9694 1.0000000

JS-J130 : 5m-1w -20.871 1 1.4016 1.0000000

CD-JD : 5m-1w -101.833 1 22.0995 0.0001191 ***

CD-J130 : 5m-1w -142.300 1 52.7427 2.243e-11 ***

JD-J130 : 5m-1w -40.467 1 4.7984 0.7691095

CS-JS : 24h-1w 64.371 1 9.4438 0.0783896 .

CS-CD : 24h-1w 112.400 1 24.6801 3.316e-05 ***

CS-JD : 24h-1w 8.133 1 0.1410 1.0000000

CS-J130 : 24h-1w 86.100 1 19.3090 0.0004892 ***

JS-CD : 24h-1w 48.029 1 5.2573 0.6338043

JS-JD : 24h-1w -56.238 1 7.9844 0.1557031

JS-J130 : 24h-1w 21.729 1 1.5191 1.0000000

CD-JD : 24h-1w -104.267 1 23.1683 7.125e-05 ***

CD-J130 : 24h-1w -26.300 1 1.8016 1.0000000

JD-J130 : 24h-1w 77.967 1 17.8124 0.0010483 **

Table 4. 2: Corresponds to data shown in Fig.4-1 F.

Chisq Test:

P-value adjustment method: holm

Value Df Chisq Pr(>Chisq)

J130-J130r : train-5m 0.40 1 0.0011 1.0000000

J130-JDr : train-5m -4.35 1 0.1319 1.0000000

J130r-JDr : train-5m -4.75 1 0.1101 1.0000000

J130-J130r : train-24h -1.15 1 0.0092 1.0000000

J130-JDr : train-24h 73.35 1 37.5172 1.540e-08 ***

J130r-JDr : train-24h 74.50 1 27.0920 2.910e-06 ***

J130-J130r : train-1w -7.25 1 0.3665 1.0000000

J130-JDr : train-1w 54.25 1 20.5225 7.072e-05 ***

J130r-JDr : train-1w 61.50 1 18.4620 0.0001907 ***

J130-J130r : 5m-24h -1.55 1 0.0168 1.0000000

J130-JDr : 5m-24h 77.70 1 42.0990 1.562e-09 ***

J130r-JDr : 5m-24h 79.25 1 30.6568 4.927e-07 ***

J130-J130r : 5m-1w -7.65 1 0.4081 1.0000000

J130-JDr : 5m-1w 58.60 1 23.9456 1.387e-05 ***

J130r-JDr : 5m-1w 66.25 1 21.4240 4.786e-05 ***

J130-J130r : 24h-1w -6.10 1 0.2595 1.0000000

J130-JDr : 24h-1w -19.10 1 2.5439 1.0000000

J130r-JDr : 24h-1w -13.00 1 0.8249 1.0000000

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Table 4. 3: Novel object place recognition (NOPR) additional statistics.

Corresponds to data shown in Fig. 4-1 H

Sidak's multiple comparisons test Predicted (LS) mean diff.

95.00% CI of diff. Significant? Adjusted P Value

Sample

Control sham vs. Control fTBS 0.03389 -0.1710 to 0.2388 No 0.9888

Control sham vs. J20 Sham 0.004908 -0.1942 to 0.2041 No >0.9999

Control sham vs. J20 fTBS 0.01503 -0.1841 to 0.2142 No 0.9995

Control sham vs. J20 130hz 0.01862 -0.1758 to 0.2130 No 0.9986

Test

Control sham vs. Control fTBS -0.09338 -0.2983 to 0.1115 No 0.6816

Control sham vs. J20 Sham 0.2591 0.05999 to 0.4583 Yes 0.0055

Control sham vs. J20 fTBS -0.01191 -0.2110 to 0.1872 No 0.9998

Control sham vs. J20 130hz 0.2570 0.06256 to 0.4514 Yes 0.0047

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Chapter 5: Concluding remarks

Alzheimer’s disease (AD) is characterized by progressive cognitive deterioration,

which leads to a severely reduced quality of life. Currently, no viable therapies are able

to prevent, delay or reverse AD progression (Alzheimer’s Association, 2017). As such,

non-drug alternatives are currently being tested, including deep brain stimulation (DBS).

The rationale for using DBS in AD is that, in addition to AD being a neurodegenerative

disease, it is also a neural circuit disorder affecting several integrated cortical and

subcortical pathways, especially those involved in memory and cognition (Mirzadeh et

al., 2016). Additionally, stimulation of memory-associated areas has the potential of

improving cognitive function, which has incentivized clinical trials on DBS for AD (Chen

et al., 2014; Deeb et al., 2019; Dürschmid et al., 2017; Hamani et al., 2008; Hardenacke

et al., 2013). However, there is still no comprehensive theory explaining the effects of

DBS on AD symptoms or a consensus on which paradigms provide optimal benefits

(Benabid et al., 2002). The focus of this thesis has been to gain further insight into the

potential effects of fornix stimulation in the context of progressive memory impairment.

The work detailed here first examined early alterations in the hippocampal activity of the

J20+ AD mouse model, both during wake and sleep. Next, we explored the modulatory

effect of septo-hippocampal projection stimulation on deficient hippocampal oscillations

and memory, and finally used the more clinically relevant deep brain stimulation to

assess the effect of two distinct stimulation paradigms.

In chapter 2, we first characterized hippocampal rhythms in the J20+ in a state-

dependent manner. As discussed in previous chapters, alterations in the LFP of plaque-

bearing mice had been detected mostly in the form of decreased low gamma amplitude

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and theta- low gamma coupling during active wake (Etter et al., 2019; Mably et al.,

2017), while deficits pre-plaque seem to be more variable and inconsistent, showing no

(J20+, (Rubio et al., 2012)), slight (5xFAD (Siwek et al., 2015)) or overt (APP23 (Ittner

et al., 2014)) changes in band power. Independent from any alterations in gamma

amplitude, we found a deficit in theta- low gamma coupling during wake, indicating that

phase amplitude-coupling may be one of the first detectable alterations in AD, in line

with results from in vitro studies (Goutagny et al., 2013; Mondragón-Rodríguez et al.,

2018). Coherent oscillations are believed to take part in network communication by

allowing opportunity windows for the exchange of information (Fries, 2005; Varela et al.,

2001). The decrease in coupling specific to the low gamma band, which is thought to

mainly reflect CA3-CA1 synchrony (Colgin, 2015a, 2015b), suggest a deficit in the

precisely timed communication between these two hippocampal areas, which is strongly

associated with consolidation and retrieval processes (Buzsáki, 2015; Daumas et al.,

2005; Montgomery and Buzsáki, 2007). The peak frequency of theta was also found to

be reduced in J20+, both during wake and REM sleep, potentially contributing to this

impairment in information flow (Buzsáki, 2002). Adding to the network abnormalities,

aberrant hypersynchronous activity was highly prevalent in J20+ during sleep,

especially during periods of REM. In fact, the incidence of spike-wave discharges

appeared to be particularly modulated by REM episode duration and segment, possibly

alluding to the contribution of homeostatic processes in their rate of generation. Finally,

the frequency of SWDs in REM inversely correlated with memory performance in two

hippocampal-dependent tasks, suggesting that consolidation processes such as

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synaptic downscaling may be disrupted by these bursts of atypical hyperactivity

(Avanzini et al., 2013; Tononi and Cirelli, 2014).

In chapters 3 and 4, we demonstrated that hippocampal modulation through

fornix stimulation is able to enhance memory performance in two hippocampal tasks in

which J20+ showed early impairments. As discussed in section 4.4, the mechanisms

and brain areas recruited by deep brain stimulation, and that result in memory

enhancement or rescue are many and diverse. Through the use of optogenetics,

specific projections can be selectively stimulated and the direct effects on hippocampal

activity and memory, explored. Our results agree with other reports showing that

hippocampal theta peak power and frequency can be modulated, without altering overall

band power, by optogenetic stimulation of septo-hippocampal projections (Bender et al.,

2015; Robinson et al., 2016). Of importance for AD, this stimulation reduces the

probability of firing of single units before the peak of spike-wave discharges, as well as

the overall number of hyperactive events during REM sleep. Employing anti-epileptic

drugs also results in a similar reduction in the number of SWDs concurrent with an

improvement in memory performance (Bakker et al., 2012; Cumbo and Ligori, 2010;

Gureviciene et al., 2019; Nygaard et al., 2015; Sanchez et al., 2012b; Shi et al., 2013),

which we also observe in J20+ mice. In line with optogenetic manipulations in epileptic

models (Ladas et al., 2015; Lévesque et al., 2019; Shiri et al., 2017), our findings

suggest that artificially pacing the hippocampal network at lower frequencies may

override the occurrence of hypersynchronous events during periods important for

memory consolidation (i.e. REM sleep), thus enhancing memory performance.

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How exactly these hyperactive events contribute to the disruption of memory

consolidation in AD is currently unknown and further investigation is needed. For

example, imaging different subpopulations of neurons in the hippocampus using

calcium indicators may show if specific interneurons or subsets of principal cells

abnormally increase their firing before SWD onset. This subpopulation could then be

characterized to elucidate if early changes in their activity or connectivity are specific to

AD. Optogenetic manipulations that target specific subpopulations could also be

employed to investigate how these neurons contribute to SWD rate and memory. For

instance, by experimentally manipulating the rate of SWD (Shatskikh et al., 2006) and

distinctly increasing and decreasing their incidence in REM and NREM sleep, we could

causally probe their role in memory. In regard to stimulation effects, whether the

decrease in hippocampal hyperactivity is due to pacing the network at any frequency or

instead due to entraining the network within a specific frequency range is unknown

(Chiang et al., 2014; Lévesque et al., 2019; Shiri et al., 2017). While manipulations

employing electrical stimulation at 5 Hz theta frequency effectively abate epileptiform

activity and are able to improve memory in epilepsy models (Kim et al., 2020; Rajdev et

al., 2011; Salam et al., 2015; Schiller and Bankirer, 2007) and patients (Koubeissi et al.,

2013; Miller et al., 2015), higher frequencies may also suppress seizures in certain

conditions (Boëx et al., 2007; Lothman and Williamson, 1993; Wyckhuys et al., 2010).

Why this is the case is not clearly understood (Albensi et al., 2007; Theodore and

Fisher, 2004), as these studies employ different models, stimulate different areas and

use different measures to assess excitatory-inhibitory imbalances.

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In chapter 4, we compared the effect of high and low-frequency stimulation in

memory, by delivering electrical current to the fornix. We demonstrate that, when the

stimulation is delivered for the duration of the post-training period, low theta-burst

stimulation enhances memory performance, whereas high-frequency stimulation had no

effect. Our results differ from other studies using high frequencies, which have been

shown to improve memory, albeit at a lower level than by employing theta and theta-

burst pulses (Hescham et al., 2013; Miller et al., 2015; Shirvalkar et al., 2010; Sweet et

al., 2014; Titiz et al., 2017). One reason that may explain the difference between these

experiments is the period of stimulation. Whereas we stimulated after training, these

studies comparing low and high-frequency paradigms targeted encoding periods just

before the task and/or during the task. Therefore, we may have observed a comparable

effect of high-frequency stimulation on memory if we had focused our protocol to the

encoding phase, although some have reported that high-frequency stimulation during

the task can disrupt memory (Goyal et al., 2018; Hamani et al., 2010; Jacobs et al.,

2016; Natu et al., 2019; Talakoub et al., 2016).

One outstanding question that remains in the findings presented here is the

physiological mechanism by which these stimulation protocols differentially affect

memory. As discussed in section 4.4.1, electrical stimulation of the fornix engages many

areas and processes and disentangling which ones are essential for memory

modulation will inform future treatments. Our optogenetic experiments indicate that

stimulating septal projections is sufficient to enhance impaired memory and therefore,

concurrently modulating several other memory structures may not be necessary.

Probing this pathway using optogenetics at higher frequencies and different time points,

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for example, while recording from the hippocampus, may offer further insight on the

mechanisms underlying specific stimulation protocols. We provide evidence indicating

that increased memory performance could be linked to decreased hippocampal

hyperactivity during REM, suggesting that anti-epileptic drugs and therapeutics

specifically targeting sleep have the potential to improve quality of life in AD patients

(Cumbo and Ligori, 2010) and should be explored further. Along the same line,

stimulation delivered during specific behavioral states has the potential of being

successful, given our results connecting memory impairments to events in sleep. DBS

experiments targeting a specific phase of sleep, such as REM, would be needed to

answer if stimulation during a particular stage is sufficient to restore memory

performance. Likewise, manipulations where, for example, AD transgenic rodents

receive stimulation before, during or in between active periods of a task, with at least

two different stimulation frequencies, would be ideal to understand if the modulatory

effect on cognition is time and/or frequency-dependent.

Lastly, we also found incipient impairments in specific hippocampal rhythms, that

have been linked to memory; these may further deteriorate and become more relevant

as the disease progresses. For future studies, it will also be important to assess how

restoring deficits in hippocampal theta and gamma may affect memory. Closed-loop

approaches, where the network is regulated only when needed and/or multi-modal

approaches, where several of these deficits are targeted simultaneously, will have to be

assessed as they may be necessary in later stages of the disease when networks are

severely dysregulated.

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