Investigation of large-scale epileptic networks in rodent models of focal epilepsy

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Thesis

Reference

Investigation of large-scale epileptic networks in rodent models of focal epilepsy

QUAIRIAUX, Charles

Abstract

This Privat Docent thesis presents three of my recent studies that explore the development and the mechanisms of large-scale epileptic networks in the kainate mouse model of TLE using high density EEG combined to multiple intracerebral recordings. We reveal that a large-scale epileptic network develops and invade both hemispheres, generating epileptic patterns and altering functional connectivity at the whole brain level. We further show that low frequency synchronization and cross frequency coupling are key mechanisms at works in these large-scale pathologic networks. To introduce these works, I first discuss the fundamental, clinical and translational scientific contributions summarizing current knowledge and providing new insight on the large-scale aspects of focal epilepsies, with a particular focus on mesial temporal lobe epilepsies.

QUAIRIAUX, Charles. Investigation of large-scale epileptic networks in rodent models of focal epilepsy. Thèse de privat-docent : Univ. Genève, 2020

DOI : 10.13097/archive-ouverte/unige:150477

Available at:

http://archive-ouverte.unige.ch/unige:150477

Disclaimer: layout of this document may differ from the published version.

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Fundamental Medicine Section Department of Basic Neuroscience

"INVESTIGATION OF LARGE-SCALE EPILEPTIC NETWORKS IN RODENT MODELS OF FOCAL EPILEPSY"

for the degree of Privat-Docent by

Charles QUAIRIAUX ---

Geneva

2020

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3 2. Potential benefits of a better understanding of large-scale epileptic networks mechanisms ... 68 3. Perspective 1: Influences of the large-scale EN for the expression of epileptic activities in the focus. ... 69 4. Perspective 2: The epileptogenesis of the large-scale epileptic network ... 74 REFERENCES ... 76

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SUMMARY

Temporal lobe epilepsy (TLE) is the major type of focal epilepsy as well as the most common type of medically resistant epilepsy in general. While frequent unprovoked focal seizures are the main characteristic of the disease, TLE patients also suffer from a variety of comorbidities ranging from hippocampal associated memory disruptions to diverse cognitive and psychiatric disorders that concern areas remote from the temporal lobe such as frontal executive functions. Because of its incidence and its resistance to pharmacological treatments, TLE is the most common type of focal epilepsy treated by resective surgery. However, up to 40% of patients will bear seizures relapses within 5 years after the surgery.

The widespread brain disfunctions and the significant number of relapses after surgery that could not be attributed to insufficient focal tissue resection have led the researchers to consider larger scale networks aspects beyond the focus in TLE but also in other types of focal epilepsy. Indeed, while focal epilepsies are defined by the recurrence of seizures in the epileptic focus, abnormal activities propagate and influence remote regions of the brain during the ictal and interictal periods. Brain-wide anatomical and functional alterations at distance of the focus as well as influences of distant areas onto focus activities have been described in human patients but our knowledge about their diversity, their mechanisms and their consequences remain poor. The lack of knowledge or awareness of large-scale epileptic networks mechanisms for seizure and interictal epileptic discharges generation partially explain the weaknesses and failures of current treatments. Thus, identifying the extent and distribution of the epileptogenic processes and understanding the network mechanisms beyond the focus region is crucial in order to elucidate the pathogenesis of seizures, the cognitive comorbidities during the ictal and interictal periods and to develop efficient therapeutic strategies.

Human studies of epileptic networks face two major limitations: i) investigating the development of epileptic networks before the appearance of seizures is not feasible and record a sufficient number of neurons in several regions over large distance in the brain is rarely possible and remains highly challenging. Therefore, experimental rodent models of epilepsy are crucial in order to investigate the network and cellular mechanisms of epileptogenesis, of seizure initiation and of interictal comorbidities.

This Privat Docent thesis presents three of my recent studies that explore the development and the mechanisms of large-scale epileptic networks in the kainate mouse model of TLE using high density EEG combined to multiple intracerebral recordings. We reveal that a large-scale epileptic network develops and invade both hemispheres, generating epileptic patterns and altering functional connectivity at the whole brain level. We further show that low frequency synchronization and cross frequency coupling are key mechanisms at works in these large-scale pathologic networks. To introduce these works, I first discuss the fundamental, clinical and translational scientific contributions summarizing current knowledge and providing new insight on the large-scale aspects of focal epilepsies, with a particular focus on mesial temporal lobe epilepsies.

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INTRODUCTION

1. Focal epilepsies and the concept of large-scale epileptic networks 1.1. Introduction to the epileptic diseases

Epilepsy is a one of the most frequent chronic brain disease (Fiest et al., 2017) and is characterized by the recurrent expression of epileptic seizures. Epileptic seizures are transient occurrence of stereotyped behavioral alterations due to abnormal or excessive synchronous neuronal firing that disrupts normal brain activities and that can or not lead to loss of consciousness. Seizures can be characterized at the EEG level by typical paroxysmal discharges such as waxing and waning rhythmic activities or spike-and-waves that can last from several seconds to minutes and that can remain localized or invade the whole brain. Along with the acute effects of seizures, patients suffer from various comorbidities with cognitive deficits, increase risk of depression and shortened life expectancies. The disease burden from epilepsy (life expectancy, disability weight...) was estimated heavier than that for Alzheimer and Parkinson diseases combined (Murray et al., 2012).

As much as 5-10% of the population will experience one non-febrile seizure in his life (Hauser et al., 1996; Wilden and Cohen-Gadol, 2012), however, only a portion of persons will continue to express recurrent seizures, i.e. be diagnosed as suffering from epilepsy. Worldwide, it has been estimated that about 60 million people are suffering from epilepsy (Fisher et al., 2014; Moshé et al., 2015), mainly young infants and persons older than 65. In total, active epilepsy is considered to have a prevalence of 3-10 per 1000 (Fiest et al., 2017; Moshé et al., 2015; Ngugi et al., 2010). Medical treatments can alleviate the risk of seizures but with the cost of substantial side effects and most importantly these treatments are not efficient in about one third of patients who continue to have seizures despite any drug therapies (Kalilani et al., 2018;

Kwan et al., 2011; Wiebe and Jette, 2012).

Although it exists a lot of different types of epileptic diseases with various forms of seizures that render the classification of epilepsies a matter of long lasting debates (Blümcke, 2012), epilepsies are broadly separated in two categories based on the type of seizure onset:

generalized epilepsies, in which seizures present bisynchronous cortical onset in both hemispheres, and focal epilepsies in which the seizure onset begins in discrete to relatively distributed networks limited to a single hemisphere or a single lobe. This classification may be complexified by the facts that focal seizures may progress to generalized seizures (secondarily

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6 generalized) and that the localization of the onset cannot always be determined. Altogether, about 60% of epileptic patients suffer from focal epilepsies with a more or less well localized area of the brain producing spontaneous and recurrent seizures (Hauser et al., 1996; Nayak and Bandyopadhyay, 2020).

1.2. The case of Mesial Temporal Lobe Epilepsy

All lobes can be at the origin of a focal epilepsy, but the more epileptogenic lobe in the brain is the temporal lobe. In more than 80% of the cases, temporal lobe epilepsies (TLEs) involve the hippocampal formation and it is believed that the high epileptogenesis and the propensity of the temporal lobe to generate seizures are due to the particular neuronal network structures of the amygdala, the hippocampal formation and the entorhinal cortex (Tatum, 2012). For these reasons, focal epilepsies involving the medial region of the temporal lobe constitute a specific entity within epileptic disease define as mesial temporal lobe epilepsy (MTLE). Importantly, MTLE is the most frequent disease among pharmacoresistant epilepsies and has the worst prognosis for seizure control with anti-epileptic drugs (AED), see (Tatum, 2012; Wiebe and Jette, 2012) for a review.

The pathophysiological substrate of MTLE is predominantly hippocampal sclerosis (HS) and accordingly atrophies and gliosis in the mesial temporal lobe structures are the most commonly diagnosed lesions in adult epileptic patients requiring surgery (Blumcke et al., 2017). Those lesions are usually triggered by an initial acute event such a traumatic brain injury, infections or, predominantly, a status epilepticus (SE) or a febrile status epilepticus (Lewis et al., 2014; Milligan et al., 2009; Patterson et al., 2014). The limbic system is particularly at risk for subsequent tissue injury after an episode of status epilepticus (SE) and unilateral or bilateral lesions are often observed in neuroimaging results of epileptic patients (Tatum, 2012).

Multiple types of AEDs acting on different molecular targets can be used to control seizure occurrence. The use of AEDs however presents several inconveniences. AEDs are designed to control seizures and do not alleviate the associated comorbidities. AEDs interact not only with the epileptic networks but also with the healthy circuits and this can lead to various side effects with even worsening of the cognitive disorders in some epileptic patients (Eddy et al., 2011). Most importantly, AEDs do not always allow to suppress seizures:

pharmacoresistance occurs in up to 30% of all epileptic patients and in 50 to 90% of patients with TLE depending on the studies (Jobst and Cascino, 2015; Löscher et al., 2013; Tatum,

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7 2012; Wiebe et al., 2001). The worst prognosis is associated with abnormalities in brain imaging suggestive of HS (Wiebe and Jette, 2012). Epilepsy surgery is the major therapeutic option for drug-resistant patients diagnosed with a focal epilepsy and is especially indicated in TLE. It is admitted that when there is clear identification of a seizure-onset zone, its resection may benefit patients with intractable epilepsy despite the permanent damages induced to the brain tissue by the procedure. As for AEDs, even modern surgical therapies are not fully efficient with about 30% of operated patients not completely seizure free off drugs at 5 years after the resection (Baud et al., 2018; Kalilani et al., 2018; Mohan et al., 2018; West et al., 2019).

1.3. The concept of large-scale epileptic network in focal epilepsy

As discussed above, focal epilepsies are defined by the repetition of seizures and interictal epileptic discharges (IEDs) generated by a restricted brain region, i.e. the epileptic focus (EF). However, it is more and more accepted that large-scale neuronal networks beyond the EF are involved in the generation of seizures and IEDs as well as in the associated cognitive impairments. For example, a recent study showed that large-scale synchronization between the focus region and the thalamus and basal ganglia were modulated in the pre-ictal period (Aupy et al., 2019).

All brain regions interact with widespread networks in normal and pathological conditions and several lines of clinical observations have led the researchers to investigate brain networks beyond the focus. First, an important fraction of patients operated for a focus resection still suffer from persistent or recurrent seizures at short or long terms. The fact that all relapses cannot be attributed to insufficient focal tissue resection (Hennessy et al., 2000;

Jehi et al., 2010), suggest that more distributed epileptogenic networks beyond the initial focus might be involved. The widespread cognitive and psychiatric comorbidities, associated or not with remote anatomical abnormalities, constitute another important series of observations pleading for the importance of large-scale networks in epilepsy. In temporal lobe epilepsies, cognitive deficits are not restricted to tasks that involve memory encoding and patients can suffer from more heterogenous impairments, such as for instance frontal lobe executive dysfunctions (Keller et al., 2009; Oyegbile et al., 2004b, 2018; Ren et al., 2020; Stretton et al., 2015). One mechanism for these deficits is the interaction between IEDs and cognitive networks as demonstrated in several human and animal TLE studies showing that focal and extra focal IEDs disrupt remote areas cognitive functions (Kleen et al, 2010, 2013; Gelinas et

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8 al, 2016, Ung et al, 2017). Based on the level of synchronous activity or of causal relationships between regions as estimated using the classic surface EEG, the more invasive stereoencephalography (SSEG) or functional resonance magnetic imaging (fMRI), brain connectivity approaches in focal epilepsy have demonstrated alterations of connectivity beyond the focus in the ipsilateral and contralateral hemisphere that could correlated with the surgery outcome (Bartolomei et al., 2017, 2017; Carboni et al., 2019; Diessen et al., 2013; Englot et al., 2016; Liao et al., 2010). Finally, the developing field of connectomics applied to epilepsy also enlighten the concept that abnormal changes in the dynamic of large-scale networks might explain the emergence of seizures rather than purely local mechanisms (Engel et al., 2013;

Lopes et al., 2020; Richardson, 2012a; Terry et al., 2012).

From these multiple observations, it has been proposed that regions remote from the EF were progressively recruited along the evolution of the disease as part of a large-scale epileptic network (Goodfellow et al., 2016; Lopes et al., 2020; Richardson, 2012a; Spencer, 2002). This concept of larger epileptogenic networks may better describe the complexity of seizure dynamics and the distribution of other epileptogenic anomalies in brain networks. The relationships between the seizure onset zone, the epileptogenic areas and remotes regions of the brain are complex and further studies are needed to better understand the nature and the complexity of epileptic networks. Two complementary aspects appear to be crucial: how much the disease impacts remote brain regions and what is the importance of the potential remote epileptic network for the expression and the evolution of the disease?

In the following sections, some of the human and animal literature that led to the concept of large-scale epileptic networks in focal epilepsy will be presented.

2. Focal epilepsies as large-scale network disorders: clinical evidences

2.1. Anatomical alterations are not restricted to the focus

It is now well accepted that anatomical abnormalities in unilateral MTLE characterized by hippocampal sclerosis extend beyond the areas generating seizures to neighboring limbic structures, more distant cortical regions of the temporal pole and even far outside the temporal lobe in cortical and subcortical regions (Bell et al., 2011). A wealth of magnetic resonance imaging (MRI) studies showed that along with the identifiable sclerosis of the hippocampus, abnormalities could be detected outside of the hippocampus with in particular decreased gray matter volume in the ipsilateral amygdala, fornix, entorhinal and neocortical temporal cortices

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9 but also in the contralateral temporal cortex and in extratemporal regions, like the cingulate gyrus, the frontal and occipital lobe and in subcortical structures (Bernasconi et al., 2004;

Bonilha et al., 2010; Moran et al., 2001; Oyegbile et al., 2004a; Riederer et al., 2008;

Seidenberg et al., 2005). Structural connectivity MRI studies also reported reduction in white matter networks between the epileptogenic regions and distant brain structures as well as within these remote networks (Besson et al., 2017; Englot et al., 2016; Vaessen et al., 2012). It has been proposed that these anatomical changes have predictive values for surgical outcomes (Yasuda et al., 2010). The mechanisms underlying these widespread anatomical alterations in focal epilepsies may depend on the disease type (neocortical or mesial; congenital or acquired after a stroke, a tumour or encephalitis…), and remain not well understood however one could hypothesize that seizure propagation might have a preponderant influence (Bonilha et al., 2006).

2.2. Widespread cognitive dysfunctions in temporal lobe epilepsy

The above mentioned widespread abnormalities observed in multimodal MRI have been often correlated with cognitive impairments beyond the typical hippocampal memory functions (Seidenberg et al., 2005). Along with typical symptoms associated with damages in medial temporal structures such as verbal and nonverbal memory deficits, patients with TLE suffer from heterogeneous neurological symptoms which cannot be explained by the dysfunctional epileptic focus only: impaired verbal comprehension, visuospatial function, motor dexterity and attention as well as diverse psychiatric comorbidities such as anxiety and depression (Fisher et al., 2014; Helmstaedter et al., 2003; Hermann et al., 2008; Jones-Gotman et al., 2010; Keller et al., 2009; Oyegbile et al., 2004b, 2004b, 2018; Ren et al., 2020; Savage, 2014; Stretton et al., 2015; Tellez-Zenteno, 2005).

Thus, cognitive comorbidities in TLE may potentially affect all cognitive domains, indicating that regions outside of the seizure onset zone and propagation zones can be affected, can occur during ictal but also during the interictal periods and, importantly, appear to worsen over time along the disease suggesting that the recurrent expression of epileptic activities may gradually increases the functional changes in large-scale epileptic networks (Bell et al., 2011;

Bonilha et al., 2006; Hermann et al., 2008; Oyegbile et al., 2004a).

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10 2.3. Focal and extra focal IEDs disrupt remote cognitive functions

Propagating seizures and permanent remote structural of functional changes, either evoked by the same triggering event than the initial focal epileptic disease or by gradual changes due to the propagation of epileptic activities in the network, are not the only causes of the widespread cognitive dysfunctions in the epileptic brain. Acute interictal epileptic activities propagating to or originating from eloquent areas have also been showed to lead to various symptoms such as transient cognitive impairments (TCI).

In particular, focal IEDs disrupt memory maintenance and retrieval (Kleen et al, 2013).

The deleterious effects of focal IEDs on memory functions is not only due to local effects but also to perturbations of the temporo-parietal network involved in memory consolidation during sleep (Gelinas et al., 2016; Lambert et al., 2020). One mechanisms by which focal IEDs exert a large-scale influence has been illustrated in a study demonstrating that the hypersynchronous synaptic activity during focal IEDs induced large propagating cortical spindles in distant non- epileptic regions (Dahal et al., 2019). Furthermore, remote IEDs inducing transient cognitive impairments can also be expressed and are not infrequent as it has been estimated that the generation of IEDs in the hippocampus contralateral to the epileptic one may concern up to 50% of MTLE patients (Gollwitzer et al., 2017; Janszky et al., 2003; Kleen et al., 2013).

2.4. Large-scale functional connectivity alterations in focal epilepsies

A wealth of neuroimaging and electrophysiologic studies using data driven or model- based connectivity analyses demonstrated a progressive increase in regional and large-scale functional connectivity disturbances in focal epilepsies. Overall, patterns of increased functional connectivity in the region of the focus have been described, associated to an increase propensity to generate seizures, while decreased connectivity is often reported between the focus and distant networks as well as within distant networks, which may correlate with the risks of cognitive impairments (Englot et al., 2015, 2016; Morgan et al., 2020; Thornton et al., 2011). However, the picture appears to be more complex as increased widespread connectivity have been also demonstrated, correlating with higher risks of post-surgical seizure relapses (Carboni et al., 2019; Lagarde et al., 2018).

Functional connectivity analyses based on electrocorticography (ECoG) and SEEG signals measured during periods of background activity away from any epileptiform disturbances demonstrated in patients with focal epilepsies an increase connectivity within the seizure onset zone (SOZ) and in the propagation network of focal seizures, including in MTLE

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11 patients (Bettus et al., 2008; Englot et al., 2016; Lagarde et al., 2018; Schevon et al., 2007;

Shah et al., 2019). Similarly, fMRI studies in patients suffering from MTLE also reported enhanced connectivity within the mesial temporal structures ipsilateral to the focus region that are involved in the generation and propagation of the seizures (Haneef et al., 2014; Liao et al., 2010).

While increased connectivity is often reported within the epileptogenic regions, decreased connectivity of the affected temporal structures with diverse other brain regions have been observed using anatomical and functional MRI tools. Several studies reported decreased connectivity between the hippocampal formation and the default mode network (DMN) as well as with the prefrontal cortices, the contralateral homologous regions or the brainstem (Haneef et al., 2014; Pittau et al., 2012; Voets et al., 2012; Wirsich et al., 2016). MTLE also affects connectivity in distant regions with decreased connections within and between the frontal and parietal lobes and between the DMN areas and other regions (Liao et al., 2010). Impairments of global brain connectivity were also observed using fMRI and MEG in several focal diseases, including MTLE, and have been associated to the diffuse cortical thinning often reported in focal epilepsies (Englot et al., 2015; Luo et al., 2011). A recent study combining multimodal MRI and computational simulations uncovered extensive atrophy, microstructural disruptions and decreased thalamo-cortical connectivity in MTLE, while patients with generalized epilepsies showed only subtle structural anomalies paralleled by enhanced thalamo-cortical connectivity (Weng et al., 2020).

Hypotheses have been made to present the decreased connectivity between the epileptogenic regions and the rest of the brain as network isolation protective mechanisms against seizures (Englot et al., 2016). However, alterations of large-scale networks in focal epilepsies are not always characterized by a simple reduction in connectivity. Several fMRI studies reported both decreases and increases of functional connectivity in different brain networks (Englot et al., 2015; Haneef et al., 2014; Liao et al., 2010; Luo et al., 2011). In a fMRI connectivity mapping study lasting several years, a progressive increase in inter-hippocampal connectivity was revealed suggesting that a larger-scale epileptogenic network in which the contralateral hippocampus become a driver and a potential secondary focus may develop (Morgan et al., 2011).

Electrophysiological studies that analyzed large-scale brain networks during interictal states are not all in line with fMRI studies and often reported increased functional connectivity between the epileptogenic region and remote areas (Bartolomei et al., 2017; Bettus et al., 2008, 2011; Englot et al., 2016; Yaffe et al., 2015). It has been suggested that IEDs were influencing

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12 the activities of the same large-scale networks than those influenced by seizures activities (Pittau et al., 2013; Tousseyn et al., 2015; Vulliemoz et al., 2011). An EEG and electric source imaging study made in refractory patients before the resection surgery and that analyzed brain activities during IEDs using directed connectivity and graph analyses tools (Carboni et al., 2019) showed that patients with higher network integration at the large-scale level have a bad post-surgical outcome while those with more isolated effects of IEDs have a better outcome.

A SEEG study based on signal amplitude estimations of functional connectivity during background EEG in focal refractory epileptic patients further highlighted the importance of large-scale changes in connectivity and their consequences for the surgical outcome (Lagarde et al., 2018). This study showed that while, as expected, connectivity is higher within the SOZ than in remote regions, increased functional connectivity is also observed in the zone involved in the propagation of the focal seizures as well as further in non-involved zones, for which the focus region appears to be a driver. The level of connectivity between the regions involved in seizures and the non-involved zones correlates with poorer surgical prognosis.

On the other hand, contradictory observations have also been reported in electroencephalographic and magnetoencephalographic (MEG) studies. A SEEG study reported decreased local field potentials (LFP) synchronization between the focus and neighboring regions, suggesting a functional isolation of the focus in those patients (Warren et al., 2010). A MEG study in patients with focal epilepsy also showed widespread decreases in functional connectivity presumably reflecting the damaging effects of recurrent seizures on the long term while on the contrary enhanced regional connectivity was observed in the focus region (Englot et al., 2015). These apparent contradictions probably reflect the diversity in focal diseases origins and the variability between patients and, altogether, are concordant on the fact that patients with strong isolation of the epileptogenic network have more favorable surgical outputs.

While the SOZ influences distant networks, these networks also influence the SOZ and are important in the generation of seizures (Aupy et al., 2019). Along with associated cognitive alterations, large-scale alterations in functional connectivity could be involved in the generation of seizures. The emerging field of connectomics and brain network modelization studies the neural network mechanisms using approaches in which the focus is a node of a larger -scale network (Engel et al., 2013; Richardson, 2012b; Stacey et al., 2020; Terry et al., 2012). These computational models of epilepsy suggest that the changes in the structure of a large-scale networks, including loss of connectivity, could give rise to focal seizures (Terry 2012). One intriguing explanation could be that a loss of connections might destabilize a local

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13 network, therefore increasing his propensity to express ictal activities. Importantly, computational modelling studies that consider large-scale epileptic networks can help better describe predict seizures and surgical outcome (Goodfellow et al., 2016; Proix et al., 2017, 2018).

2.5. Various outcome after complete resection of the seizure onset zone

Surgical resection of the focus is the major therapeutic option in medically refractory patients. The ultimate goal of resective surgeries is to achieve seizure freedom on the long term, and ideally medication-free. Because of its incidence and its resistance to treatments, TLE is the most frequent type of focal epilepsy operated on, involving either the resection of the anterior temporal lobe or of more restricted resection of tissue such as in amygdalohippocampectomies. After surgery, a significant seizure reduction is obtained in above 70% of TLE patients (Baud et al., 2018; Hardy et al., 2003; Mohan et al., 2018). A wealth of long follow-up studies (about 5-10 years after the surgery) however show that 30-50

% of patients fail to become or remain completely seizure-free (Berg, 2011; Chang et al., 2015;

Hardy et al., 2003; Hemb et al., 2013; Hennessy et al., 2000; Jeha et al., 2006; Kalilani et al., 2018; Mohan et al., 2018; de Tisi et al., 2011; West et al., 2019).

In a retrospective study of 68 adults patients with persistent or recurrent seizures after resection, 20 contiguous focus could be identified but 9 had noncontiguous focus in the ipsilateral hemisphere and 14 in the contralateral hemisphere or in bilateral temporal regions (Jehi et al., 2010). The localization of the recurrent focus could not be identified in remaining patients. These observations, among others (Hennessy et al., 2000; Wada, 2005), illustrate the fact that not all surgical failures can be explained by an insufficient resection of the epileptogenic focus or a misdiagnosis of a focal disease instead of a multifocal epilepsy. A significant number of relapses are believed to originate in a more distributed than expected epileptogenic network, which would also explain the remanence of cognitive comorbidities after the surgeries. In the absence of the focus, the existence of another epileptogenic region would be revealed, for instance a hidden contralateral mesial temporal sclerosis (Thom et al., 2010). Another possibility is that remote epileptogenic areas progressively develop along the disease. Secondary epileptogenesis has been described in animal models , but demonstrations of a secondary epileptogenesis following surgeries in humans are difficult to establish and remain sparse (Gollwitzer et al., 2017; Lagarde et al., 2018; Wada, 2005). Still, the fact that the duration of the disease before the resective surgery influences the prognostic of seizure-

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14 free outcome is a strong argument in favor of secondary epileptogenesis in humans (Bjellvi et al., 2019).

Regarding the prognostic of seizure freedom, the completeness of the resection of the focus is the most important factor but the assessment of the extent of the epileptogenic zone that often outreach the apparent lesion site is often problematic. Several predictors of good surgery outcome were identified, among them the presence of unilateral hippocampal sclerosis and unilateral spikes (Ramos et al., 2009; Tonini et al., 2004). On the contrary, the presence of bilateral EEG abnormalities (Hennessy et al., 2000; Jehi et al., 2010), the occurrence of a status epilepticus (Hardy et al., 2003), the frequency of secondarily generalized seizures (Martinet et al., 2015), the functional connectivity between the focus and remote regions discussed above (Carboni et al., 2019; Lagarde et al., 2018) and the duration of the disease (Janszky et al., 2005;

Jeong et al., 1999; Yasuda et al., 2010) predicts bad postoperative outcome. These predictors are strongly suggesting the development of more extensive epileptogenic zones rather than the existence of a single spatially restricted node that would generate all epileptic activities along the entire duration of the disease.

2.6. The development of a large-scale epileptic network

The mechanisms by which regions outside of the epileptic focus are recruited within a larger-scale epileptic network are poorly understood. The associations discussed above between the duration of the disease and the recurrence of postoperative seizures as well as with the more extended cognitive and psychiatric disorders suggest that large-scale epileptic networks arise from progressive mechanisms of brain network alterations that may depend on the recurrent expression of epileptic activity patterns.

The progressive nature of cortical alterations and the correlation with the frequency occurrences of seizures is a strong argument indicating that epileptic activities can lead to large- scale neuronal damages that would affect primarily the structures receiving connections from the epileptogenic regions (Bonilha et al., 2006; Coan et al., 2009; Galovic et al., 2019). In agreement with that, human TLE studies have shown that structural damages affecting white matter and grey matter correlated with bad surgery prognosis (Yasuda et al., 2010) and were the more pronounced in the structures linked to the limbic system (Bonilha et al., 2006, 2010).

Along with the impact of focal seizures at a distance, secondary generalized seizures could also lead to the development of large-scale epileptic networks and it has been showed that secondary generalized seizures is a predictor of poor post-surgical prognosis (Janszky et al., 2005;

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15 Martinet et al., 2015). Thus, it can be hypothesized that in addition to the direct effect of the degeneration of the long-range axons from the sclerotic hippocampus, several pathologic mechanisms due to the epileptic activity itself could explain the progressive deterioration of regions distant from the focus such as a direct deleterious effect of propagating seizures onto neurons or neuronal plasticity mechanisms induced by ictal and interictal activities and influencing remote regions connected to epileptic networks.

3. Studies of large-scale epileptic networks in the kainate mouse model of temporal lobe epilepsy

Experimental rodent models of epilepsy are highly useful in order to understand the fundamental mechanisms of epileptic activities and to develop and test new therapeutic treatments. The first obvious advantage of such model is that they allowed to study the epileptogenic processes during the latent phase of the disease, i.e. before the appearance of the very first seizure, which is almost unfeasible in human as patients do not generally present to the physicians before the seizures are installed. Another important possibility offered by the model, which is crucial for the projects presented in the current thesis, is that it is possible to record local field potentials (LFPs) and action potentials simultaneously in several regions over large distance in the brain. Because of the difficulties to record brain oscillations and neurons in a sufficient number of brain regions at the same time, the reasons why seizures start, propagate and end are still only superficially understood. Thanks to the development of different types of surface and intracerebral electrodes and of new genetic tools, it is now possible to study the fine neuronal mechanisms explaining which subtype of neurons in different regions become pathologically synchronized at the origin of a seizure. However, to this date, the large-scale neuronal network level has not been specifically investigated in mouse models of focal epilepsies.

3.1. The unilateral kainate mouse model of mesial temporal lobe epilepsy

There are two main animal models of mesial temporal lobe epilepsy that use the intrahippocampal administration of chemoconvulsants, i.e. pilocarpine and kainate (kainic acid, KA). In both cases, the intracerebral injection of a small amount of chemoconvulsant induces an initial episode of long-lasting tonic-clonic seizures (status epilepticus) followed by a latent period and then the recurrence of spontaneous seizures originating from the temporal

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16 lobe during the chronic stage. Histologically, these mice develop hippocampal lesions that reproduce closely the typical hippocampal sclerosis of many MTLE human patients. These models have been extensively used in research because of their high level of histological, behavioural and electrophysiological similarities with the human disease but the kainate model is probably the most studied because it has higher survival and stronger chronic epilepsy induction rate (Arabadzisz et al., 2005; Bouilleret et al., 1999; Gröticke et al., 2007, 2008;

Langlois et al., 2010; Riban et al., 2002).

Thus, as suspected for human MTLE following a precipitating event such as a febrile seizure, intra-hippocampal kainate injections in mice induce a non-convulsive status epilepticus (SE) that lasts 10 to 17 hours (Gröticke et al., 2008; Riban et al., 2002) after which the animals develop spontaneous epileptic activities and reach the stage of chronic epilepsy within 3 to 4 weeks. The chronic phase is characterized by recurrent focal seizures that may secondarily generalize (Bui et al., 2018). During this initial latent phase of epileptogenesis, previous studies (Arabadzisz et al., 2005; Riban et al., 2002) described increasing number of spikes of increasing amplitudes developing in the injected hippocampus as well as the appearance of more and more high-frequency bursts and finally the expression of recurrent paroxysmal discharges lasting from a few seconds to a few minutes that are reminiscent of hippocampal focal seizures. Along with the seizures that define the ictal period events, several types of interictal epileptic discharges that appear during the latent stage continue to be expressed in the focus during the chronic stage: sharp transients or spike waves lasting around 50 ms, sharp waves of about 250 ms and the shorter high frequency oscillations or fast ripples (FRs) are the most frequent interictal epileptic events.

At the histological level, the unilateral kainate injection in the hippocampus induces a process of hippocampal sclerosis with a neuronal degeneration and a gliosis in the dorsal CA1, CA3 and dentate gyrus (DG) regions of the ipsilateral but not the contralateral hippocampus (Arabadzisz et al., 2005; Bouilleret et al., 1999; Heinrich et al., 2006). In both the mouse and rat intrahippocampal kainate models, similarly to what have been observed in human patients, hippocampal sclerosis is characterized by glutamatergic and GABAergic neuronal death, accompanied by aberrant mossy fiber sprouting comprising recurrent excitatory synapses, alterations in intrinsic properties and expression of receptors and strong activation of astrocytes and microglia (Bouilleret et al., 1999; Feng et al., 2019; Godale and Danzer, 2018; Goldberg and Coulter, 2013; Häussler et al., 2016; Marucci et al., 2010; Zhang et al., 2012). Some of these epileptogenic changes are believed to be the consequence of neuronal plasticity mechanisms driven by the epileptic activity itself (Ben-Ari and Dudek, 2010) and it has been

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17 proposed that new strongly interconnected neuronal assemblies are formed in the hippocampus during the initial status epilepticus and generate FRs that would sustain the development of epileptic networks further during the latent stage (Bragin et al., 2000; Li et al., 2019). A few other cytoarchitectural modifications have been described contralaterally such as a gradual increase in the surface area of the hilus (Arabadzisz et al., 2005), a weak staining of resting microglia (Zattoni et al., 2011) and a slight increase in neurogenesis (Kralic et al., 2005).

However, the expression of brain damages markers, such as the specific marker of degenerating neurons Fluoro-jade B, and the dispersion of granule cells of the DG visible during the days following the injection in the ipsilateral hippocampus is clearly absent in the contralateral hippocampus (Heinrich et al., 2006; Noè et al., 2019). Accordingly, this model has been widely used to study epilepsies of focal origin with the assumption that kainate injection does not lead to a spread pathology (Krook-Magnuson et al., 2013a; Langlois et al., 2010).

Ictal discharges during the status epilepticus begin at the level of the hilar region of the dentate gyrus, where kainate primarily acts on the high affinity KA receptors of mossy fibers (Lévesque and Avoli, 2013), and then propagate to the entorhinal cortex and more distant regions such as M1 (Lu et al., 2016) as well to the contralateral hippocampus (Noè et al., 2019).

During the chronic stage of the disease, the mechanisms of epileptic seizure generation are believed to arise from the multiple epileptogenic changes that take place at the molecular, cellular and network level rather than on a simple change in excitation/inhibition balance. A prominent hypothesis for the mechanism of epileptic seizure generation in the mouse MTLE model is that the cellular alterations lead to a breakdown in the DG gate that protects hippocampal circuits from hypersynchronous discharges (Krook-Magnuson et al., 2015).

Although seizures in the kainate mouse model are mainly focal, they occasionally generalized, evolving towards widespread seizures with behavioral manifestations such as tonic-clonic movements. A recent study demonstrated that the transition from focal to secondarily generalized seizures depends on mossy cells, a glutamatergic neuronal population that project massively to multiple layers of the ipsilateral and contralateral hippocampus (Bui et al., 2018).

3.2. Remote morphologic and physiologic abnormalities in the kainate mouse model of temporal lobe epilepsy

When kainate is injected unilaterally in one amygdala of the mouse brain, remote damages can be observed in the ipsilateral hippocampus, i.e. that become the focal region, as well as in the contralateral amygdala (Ben-Ari and Cossart, 2000). These remote damages

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18 might therefore be attributed to the propagation of epileptic activity during the status epilepticus or latter in the latent and chronic phases. However, as described above, when kainate is injected directly in the hippocampus and despite the fact that epileptic activities during the resulting status epilepticus are not confined to the injected hippocampus with intense epileptiform discharges invading the contralateral hippocampus, the induced neurodegeneration seems to be restricted to the injected hippocampus itself (Noè et al., 2019),.

The early electrographic studies in the kainate mouse model did not fully investigate the expression of epileptic activities at distance of the focus, however the observation of muscles twitches during some seizures suggested that epileptic activities could invade the motor areas (Bouilleret et al., 1999; Gröticke et al., 2008). Similar observations were made in the rat kainate model (Bragin et al., 1999). Interictal and ictal epileptic spikes were observed in the contralateral hippocampus in several rodent models of unilateral hippocampal kainate such as in mice (Arabadzisz et al., 2005), rats (Bragin et al., 2000) and guinea pigs (Noè et al., 2019), but these remote activities were not exhaustively described. Interestingly, a small but significant proportion of seizures were observed starting in the contralateral hippocampus in vivo and in vitro in the kainate rat model (Bragin et al., 1999; Khalilov et al., 2003a). These experimental evidences as well as similar clinical observations in humans (Gilmore et al., 1994;

Hennessy et al., 2000; Janszky et al., 2003; Wada, 2005) have led to the hypothesis that an independent epileptic focus, sometimes referred as a “mirror focus”, may develop in the contralateral hippocampus in certain cases, as a result of a secondary epileptogenic process driven by the focal seizures themselves (Ben-Ari and Dudek, 2010). The existence of mirror foci in MTLE is still controversial and have not been described, to our knowledge, in the kainate mouse model of MTLE.

3.3. Alterations of cognitive functions in the kainate mouse model of MTLE

Behavioral alterations in the kainate mouse model using intrahippocampal injections are less marked than those observed following systemic kainate injections or in the pilocarpine model of MTLE in mice (Gröticke et al., 2008). Cognitive deficits mostly concern memory acquisition and retention and spatial learning strategies (Gröticke et al., 2008; Van Den Herrewegen et al., 2019). These deficits are presumably attributable to the sclerosis of hippocampal tissue but interictal spikes were also shown to disrupt memory retrieval performance in the rat kainate model of MTLE (Kleen et al., 2010). In addition to the typical

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19 memory deficits, a decrease in depression-like behavior was observed in the forced swimming test in the mouse model (Gröticke et al., 2008).

3.4. Modulation of epileptic activities using interventions at distance of the focus

Deep brain stimulations (DBS) as well as chemogenetic and optogenetic tools now allow to modulate the activity of specific neuronal populations in a restricted targeted brain region and several studies have used such tools to try to decrease seizure durations or frequency in mouse models of focal epilepsies. Some of these studies demonstrated that it is possible to alter the expression of seizures by acting on neurons located at distance of the focus, further highlighting the importance of large-scale network for the generation and the modulation of epileptic activities.

Using DBS and optogenetic tools in the kainate mouse model, it has been demonstrated that large-scale subcortical circuits involving the basal ganglia and the parafascicular thalamic nuclei strongly modulate the duration and spread of seizures by activating the entorhinal cortex, the principal source of cortical projections to the hippocampus (Chen et al., 2020; Langlois et al., 2010). Similarly, optogenetic activation of the cerebellum can reduce the duration of ongoing seizures in the same mouse model (Krook-Magnuson et al., 2014). Another important study used close-loop activation of parvalbumin (PV) hippocampal interneurons to stop seizures once detected (Krook-Magnuson et al., 2013a). Crucially, this study shows that activation of PV neurons significantly reduced seizure duration in a comparable fashion when light was delivered to the hippocampus either ipsilateral to KA injection, i.e. the epileptic focus, or to the contralateral hippocampus. Along the same line, another close-loop optogenetic study in the kainate mouse model revealed that activation of contralateral mossy cells, a glutamatergic population of the DG that can drive inhibition through interneurons and that project commissural fibers to the other hippocampus, also reduced seizure durations (Bui et al., 2018). Thus, the contralateral hippocampus is not only a potential co-generator of seizures, as it had been reported in the rat kainate model (Bragin et al., 2005), but its connections with the focus are also important in ongoing seizure activity.

Close-loop optogenetic control of seizures by modulating a region remote from the focus has also been realized in another type of focal epilepsy. Following a cortical stroke, optogenetic silencing of the ventrobasal thalamic nucleus stops an ongoing electrographic seizure, illustrating again that focal epilepsies are not only a disease of a restricted region but

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20 involve diverse distributed neuronal circuits forming a large-scale epileptic network (Paz and Huguenard, 2015; Paz et al., 2013).

4. Aims of the studies presented in the thesis

Does a large-scale epileptic network (EN) develop in the kainate mouse model of MTLE? The sclerotic hippocampus is not the only region involved in the expression of the disease, however, little is known about the actual development of a large-scale EN in this model, about its extent, the type of patterns that are generated and/or propagate in the EN, the network mechanisms that drive those activities and finally, if this EN beyond the focus network itself actually exists, about its epileptogenesis mechanisms. In my lab, we therefore decided to investigate the development and the functional mechanisms of large-scale ENs in the unilateral kainate mouse model. In order to do so, we developed new recording tools allowing for simultaneous high-density EEG and multiple intracerebral recordings in awake mice. This approach allows to overcome a major obstacle in neuroscience: combine the spatial sampling of brain networks, necessary to measure the large-scale extent of epileptic activities, to the recordings of action potentials from large number of neurons, needed to understand the network mechanisms at a cellular level.

Our two main objectives were:

1. Characterize the large-scale EN in the kainate mouse model of MTLE

What kind of epileptic patterns are observed at distance of the focus along the development of the disease? Are these activities depending on the focus or are they generated independently?

Does the focal disease modify functional connectivity at the large-scale level in the brain?

These questions will be specifically addressed in:

- Article 1 “Electrophysiological Evidence for the Development of a Self-Sustained Large- Scale Epileptic Network in the Kainate Mouse Model of Temporal Lobe Epilepsy” published in The Journal of Neuroscience in 2018 (Sheybani et al., 2018).

- Article 2 “Background EEG Connectivity Captures the Time-Course of Epileptogenesis in a Mouse Model of Epilepsy” published in eNeuro in 2019 (Słowiński et al., 2019)

2. Understand the mechanisms of the generation and propagation of epileptic activities in the EN

Are the different brain regions expressing epileptic activities connected in a large-scale EN and how the different nodes of the EN interact with each other?

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21 We will address these questions in Article 3 “Large-Scale 3–5 Hz Oscillation Constrains the Expression of Neocortical Fast Ripples in a Mouse Model of Mesial Temporal Lobe Epilepsy”

published in eNeuro in 2019 (Sheybani et al., 2019).

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ARTICLE 1:

Electrophysiological Evidence for the Development of a Self-Sustained Large-Scale Epileptic Network in the Kainate Mouse Model of Temporal Lobe Epilepsy

by Sheybani, L., Birot, G., Contestabile, A., Seeck, M., Kiss, J.Z., Schaller, K., Michel, C.M., and Quairiaux, C. in The journal of Neuroscience (2018)

It has long been held that in focal epilepsies, a restricted pathological region, i.e. the epileptic focus, is responsible for triggering seizures and driving interictal activities. Yet, patients with temporal lobe epilepsy suffer from heterogeneous neurological deficits that are not explained by the dysfunctional focus, that worsen with time and that may even persist after removal of the epileptic focus. A proposed mechanism is that these deficits are consecutive to the progressive emergence along the disease of large-scale epileptic networks extending outside the focus. However, the demonstration of the existence, extent and electrophysiological signature of large-scale epileptic networks beyond the focus remain elusive. One reason for this lack of data is that longitudinal recordings including before the appearance of recurrent seizures cannot be done in human patients.

Thus, we decided to address this question using the kainate mouse model of temporal lobe epilepsy. In order to do that, we developed a new approach allowing longitudinal simultaneous surface-depth recordings in awake mice (EEG electrodes distributed over both hemispheres and multiple-site intracerebral electrodes) and video-based behavioral assessment of epileptic activity. In this first study, we demonstrate the formation of a large-scale epileptic network that emerges and evolves before the occurrence of the first seizure and that is characterized by generalized interictal epileptic discharges, or generalized spikes (GSs), inducing motor symptoms. In addition to the fast-ripples (FRs) generated in the primary focus, we further demonstrate for the first time the emergence of epileptic FRs in remote brain regions including neocortical areas. Finally, we show that while in the early stage of the disease focus- silencing rescues the epileptic large-scale network, this manipulation in the chronic stage fails to abolish hallmark epileptic activities (FRs and GS) in areas remote from the focus.

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ARTICLE 2:

Background EEG Connectivity Captures the Time-Course of Epileptogenesis in a Mouse Model of Epilepsy

by Słowiński, P., Sheybani, L., Michel, C.M., Richardson, M.P., Quairiaux, C., Terry, J.R., and Goodfellow, M. in eNeuro (2019)

As described in the introduction of this thesis, neuroimaging and electrophysiologic human studies have reported a progressive increase in regional and large-scale functional connectivity disturbances in focal epilepsies, with sometimes conflicting results regarding the extent and the direction, i.e. increase or decrease connectivity, of these modifications. Does the focal disease modify functional connectivity at the large-scale level in the kainate mouse model of temporal lobe epilepsy? We wanted to take advantage of new computer-based mathematical models of large-scale networks to describe how functional connectivity and network dynamics evolve during epileptogenesis in our mouse model.

In this second study, focusing on background EEG that allows for direct comparison of functional networks before and after the onset of the disease, we analyzed the connectivity between network nodes and we measured node ictogenicity. We observed that network dynamics inferred by means of computational modeling are different at early and later stages of epileptogenesis. While the epileptic focus is the main driving node of epileptic activities during the latent phase, the remote nodes of the large-scale EN can become major drivers as well during the chronic stage.

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ARTICLE 3:

Large-Scale 3–5 Hz Oscillation Constrains the Expression of Neocortical Fast Ripples in a Mouse Model of Mesial Temporal Lobe Epilepsy

by Sheybani, L., van Mierlo, P., Birot, G., Michel, C.M., and Quairiaux, C. in eNeuro (2019)

In the first study, we described the development of a large-scale epileptic network in the well-known mouse model of temporal lobe epilepsy. What are the neurophysiological mechanisms underlying the spread of epileptic activity outside of the epileptic focus? Diverse slow oscillations around the theta range are believed to coordinate hippocampal neural activities with neocortical regions, which is fundamental in spatial navigation and episodic memory (Buzsáki, 2005; Fujisawa and Buzsáki, 2011; Goyal et al., 2020). Recent studies further suggest that hippocampal theta oscillations are anatomically and functionally organized with distinct hippocampal networks expressing varying theta frequencies reflecting distinct behavioral functions (Goyal et al., 2020). In particular, lower theta (1-5 Hz) would be associated to hippocampal memory functions while higher theta would reflect spatial navigation processes. Slow brain waves associated with cross-frequency coupling are both operative mechanisms of communication between brain areas in physiological conditions (Lakatos, 2005; Lisman and Jensen, 2013; Varela et al., 2001).

Are such large-scale network mechanisms involved in epileptic activity propagation?

In this third study, we address this question and demonstrate in our mouse model of temporal lobe epilepsy that interictal epileptic discharges generated in the frontal cortex, i.e., at distance from the hippocampal focus, arise from large-scale network interactions. We highlight the role of a slow oscillation that starts in the epileptic focus, synchronizes distant brain regions and eventually constrains the expression of remote fast-ripples through cross-frequency coupling.

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CONCLUSION AND PERSPECTIVES

1. Main conclusions

In focal epilepsy, large-scale interactions between brain regions beyond the epileptic focus are more and more suspected to be major pathogenic factors (Carboni et al., 2019; Krook- Magnuson et al., 2013a; Lopes et al., 2020; Richardson, 2012b; Terry et al., 2012). Combining high density surface EEG and multiple site intracerebral recordings, we investigated the development of a large-scale epileptic network (EN) in the most widely used mouse model for TLE, i.e. the unilateral hippocampal kainate mouse model. From the latent phase, i.e. before the appearance of seizures, to the chronic phase of the disease, we demonstrated the progressive intensification of large-scale pathologic waveforms called generalized spikes (GS) that invade the epileptic hippocampus, the contralateral hippocampus as well as neocortical areas. These propagating activities delineate a large-scale EN and induce behavioral symptoms with the occurrence of muscular twitches that are presumably due to the propagation in the motor cortical areas. In parallel, fast ripples (FRs), a high frequency oscillation in the 200-550 Hz range believed to be evoked by transient synchronization of neuronal population firing, also rise during the latent phase in these remote areas. Crucially, pharmacological silencing of the focus in the chronic stage fails to suppress GS and FRs outside of the focus, despite seizure- freedom. These large-scale pathological activities are not seen when focus silencing is performed early in the development of the disease, indicating that the remote nodes of the EN progressively acquire their “epileptogenic” nature in terms of IEDs generation. Given this capacity to generate independent epileptic patterns one month after the status epilepticus, one could hypothesize that later in the disease, after several months of chronic recurrent seizures, some seizure might even start in the remote nodes of the EN.

In collaboration with a group of neuroscientists specialized in dynamic connectivity models of epileptic networks, we further studied the impact of the disease on large-scale functional connectivity at the whole brain level. Using a network reconstruction model based on cross-correlation matrices, we put into light in the second article a progressive asymmetry of brain networks taking place during the latent phase and that is detectable even in the background EEG, i.e. during periods without visible ictal or interictal patterns. Further analyses using network dynamics models with node resection showed that network nodes at distance of the focus could also exhibit ictogenicity. These results are in line with large-scale modifications in brain networks along the development of the disease. The fact that these widespread

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68 functional changes in neuronal networks dynamics are detected in periods devoid of ictal or interictal activity patterns background EEG give support to the clinical potential of background EEG.

We observed in our first study that a large proportion of the remote FRs were tightly linked to the GS and that those events were preceded by large-scale slow waves. We then demonstrated in our third study that the generation of pathological FRs outside of the primary focus depends on large-scale low frequency synchronization and cross-frequency coupling within the EN. It has been showed that slow frequency brain waves and cross-frequency coupling are preponderant for communication between brain areas in physiological conditions (Lakatos, 2005; Lisman and Jensen, 2013; Varela et al., 2001); we demonstrate that such large- scale network mechanisms are also acting in large-scale EN.

2. Potential benefits of a better understanding of large-scale epileptic networks mechanisms

A better understanding of large-scale epileptic networks has major potentials benefits for the future development of treatments in epilepsy. Towards this goal, animal models of epilepsy are crucial to elucidate epileptic networks mechanisms, as a direct proof of the development of large-scale EN using longitudinal recordings including before the onset of the disease and manipulation of the activity of the focus at different time-points are seemingly almost impossible in humans.

Using our animal model, we could confirm the hypothesis that focal epilepsy is not only repetition of seizures driven by the focus but is a disease of pathological networks leading to long-term changes outside of the primary focus. Time matters: a critical period exists after which controlling the focus fails to control the epileptic network. Thus, our results might explain the persistence of pathological activities, such as cognitive deficits or seizures relapses, after surgical resection of the focus in humans. Clinically, the progressive worsening of the large-scale EN and the possibility that remote nodes become able to generate pathological activities even in the absence of the focus support the concept of early-intervention in the care of epileptic patients and importantly, highlight the urge to consider distant nodes of the EN as potential targets of interventional therapeutics. Along this line, we also show that mathematical models of EN might help us to uncover the brain regions that are crucial for the pathologic dynamics in the brain, even if ictal activities could not be frequently recorded during the clinical exams.

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69 Further fundamental, theoretical and clinical studies are needed to develop new treatments for the control of focal seizures and against the large-scale consequences of epileptic diseases and secondary generalization. The identification of the neuronal circuitry involved in seizure and interictal activities and the understanding of the network mechanisms driving neuronal activities in this network are necessary for developing precise and safe interventions to control TLE. Indeed, considering the large-scale network level in focal epilepsy management will not only help to better predict surgery failures and understand the diverse cognitive comorbidities, but it will also allow to identify new targets for activity modulations aiming at controlling seizures and improve the online predictions of epileptic events for close loop modulations based on deep brain stimulations or on future cell-specific genetic tools.

3. Perspective 1: Influences of the large-scale EN for the expression of epileptic activities in the focus.

What should be done in order to further study the large-scale EN mechanisms? In light of our studies in the mouse model, a first important ensuing question that must be addressed is the impact of the large-scale epileptic network for the expression of epilepsy. In other words, does the large-scale EN nodes influence the expression of epileptic activities in the EF?

Extra-focal epileptic activity is progressively recognized as a major component of epilepsy, as it has been shown to participate in terminating seizures (Krook-Magnuson et al., 2013b), to contribute in cognitive co-morbidities (Ung et al., 2017), to trigger secondary epileptogenesis (Khalilov et al., 2003b) and to be involved in large-scale epileptic network that might explain the suboptimal seizure control after focus resection (Moseley et al., 2012). These observations suggest a bidirectional relationship between the EF and the EN: the primary focus activities affect distant brain areas, which in turn might modulate the expression of epileptic activities in the focus. In keeping with these studies, Mormann and colleagues showed that activity in regions remote from the primary epileptic focus can help predicting upcoming epileptic seizures in patients (Mormann et al., 2007). Further studies are needed to understand the network mechanisms that control the transition from normal brain activity to ictal and interictal activities (de Curtis and Gnatkovsky, 2009).

We propose to study these transitions in our mouse model, first using the numerous focal IEDs that we can detect. Could neuronal activity in regions remote from the focus anticipate focal IEDs? In other words, can focal IEDs be predicted using activity in the bi- hippocampal network? We recently made preliminary experiments indicating that the contralateral hippocampus might play a crucial causal role for the activity of the focus in the

Investigation of large-scale epileptic networks in rodent models of focal epilepsy

Thesis

Reference

Investigation of large-scale epileptic networks in rodent models of focal epilepsy

"INVESTIGATION OF LARGE-SCALE EPILEPTIC NETWORKS IN RODENT MODELS OF FOCAL EPILEPSY"

Table of contents

SUMMARY

INTRODUCTION

ARTICLE 1:

ARTICLE 2:

ARTICLE 3:

CONCLUSION AND PERSPECTIVES