Ariana Sherdil

Stephan Chabardès

Olivier David

Dr Brigitte Piallat
email: brigitte.piallat@univ-grenoble-alpes.fr

Coherence between the hippocampus and anterior thalamic nucleus as a tool to improve the effect of neurostimulation in temporal lobe epilepsy: An experimental study

Keywords: mesial temporal lobe epilepsy, deep brain stimulation, oscillation coherence, non-human primate, anterior thalamic nucleus

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Summary

Background: Although the mechanisms by which deep brain stimulation (DBS) modifies the activity of the ictal network are mostly undefined, recent studies have suggested that DBS of the anterior nucleus of the thalamus (ANT) can be an effective treatment for mesial temporal lobe epilepsy (MTLE) when resective surgery cannot be performed. In a nonhuman primate (NHP) model of MTL seizures, we showed that the ANT was actively involved during interictal and ictal periods through different patterns and that the hippocampus (HPC) and ANT synchronously oscillate in the high betaband during seizures.

Objective: Based on those findings, we evaluated whether the frequency of stimulation is an important parameter that interferes with seizures and how to adapt stimulation protocols to it.

Methods:

We investigated the effects of low-frequency (40Hz -determined as the ictal frequency of correlation between structures) and high-frequency (130Hz -as commonly used in clinic) ANT stimulation in three monkeys in which MTLE seizures were initiated.

Results: Low-frequency stimulation had a strong effect on the number of seizures and the total time spent in seizure, whereas high-frequency stimulation had no effect. The coherence of oscillations between the HPC and the ANT was significantly correlated with the success of low-frequency stimulation: the greater the coherence was, the greater the antiepileptic effect of ANT-DBS.

Conclusion:

Our results suggest that low-frequency stimulation is efficient in treating seizures in a nonhuman primate model. More importantly, the study of the coherence between the ANT and HPC during seizures can help to predict the anti-epileptic effects of ANT stimulation. Furthermore, the DBS paradigm could be customized in frequency for each patient on the basis of the coherence spectral pattern.

Highlights:

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy and is often associated with drug resistance.

For refractory MTLE, resective epilepsy surgery is sometimes not possible, and deep brain stimulation (DBS) is now considered a viable treatment option.

In a nonhuman primate model of MTLE, the activity of ANT was synchronized with the ictal activity of the hippocampal focus in low frequency bands.

Low-frequency stimulation had a good effect on the number of seizures and the total time spent in seizures, whereas high-frequency stimulation had no effect.

Tuning of the stimulation frequency could be a way to optimize the use of DBS in epilepsy.

Abbreviations:

mesial temporal lobe epilepsy (MTLE) mesial temporal lobe (MTL) hippocampus (HPC) anterior nucleus of the thalamus (ANT)

INTRODUCTION

Mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis (HS) is characterized by recurrent spontaneous focal seizures originating from mesial temporal lobe structures and often associated with aura, oral and/or gestural automatisms, loss of contact, contralateral dystonic posture and rare secondary generalization [START_REF] Téllez-Zenteno | A review of the epidemiology of temporal lobe epilepsy[END_REF]. Among these individuals, up to 30% are unsuitable for surgery due to the adverse impacts on neurocognitive or memory function, especially for patients showing bilateral lobe involvement [START_REF] Didato | Bitemporal epilepsy: A specific anatomo-electro-clinical phenotype in the temporal lobe epilepsy spectrum[END_REF], emphasizing the need for other therapeutic options.

Knowledge of the neural networks that support the expression of MTLE seizures [START_REF] Bartolomei | Neural networks involving the medial temporal structures in temporal lobe epilepsy[END_REF] is a crucial step in applying innovative therapeutic strategies. In MTLE, the well-known network supporting the seizures includes the hippocampus (HPC), the amygdala, the entorhinal cortex, and temporal pole [START_REF] Chabardès | The temporopolar cortex plays a pivotal role in temporal lobe seizures[END_REF][START_REF] Bartolomei | From mesial temporal lobe to temporoperisylvian seizures: A quantified study of temporal lobe seizure networks[END_REF]. The HPC is one of the main structures of the mesial temporal lobe projecting both directly and indirectly (Papez circuit) to the anterior thalamic nucleus (ANT). In the monkey, the overwhelming majority of direct hippocampal projections to the ANT are through the fornix [START_REF] Aggleton | Projections from the hippocampal region to the mammillary bodies in macaque monkeys[END_REF][START_REF] Aggleton | The origin, course, and termination of the hippocampothalamic projections in the macaque[END_REF]. More recently, we characterized the role of the ANT in MTLE seizures in an NHP model and showed a more complex role of the ANT with some of its neurons activated during the seizures and others inhibited [START_REF] Sherdil | Implication of Anterior Nucleus of the Thalamus in Mesial Temporal Lobe Seizures[END_REF]. Taken together, these findings show that the ANT can be considered a pivotal structure in the epileptic networks involved in the MTLE [START_REF] Cooper | Therapeutic implications of modulation of metabolism and functional activity of cerebral cortex by chronic stimulation of cerebellum and thalamus[END_REF], and the ANT has sometimes been defined as the "top of the action hierarchy" in some of these networks [START_REF] Gallistel | The organization of action: A new synthesis[END_REF].

In addition, due to its small size and well-defined boundaries, the ANT is an attractive target for neurostimulation such as deep brain stimulation (DBS). This therapeutic strategy for epilepsy had been evaluated in some animal models [START_REF] Mirski | Anticonvulsant effect of anterior thalamic high frequency electrical stimulation in the rat[END_REF][START_REF] Hamani | Bilateral Anterior Thalamic Nucleus Lesions and High-frequency Stimulation Are Protective against Pilocarpine-induced Seizures and Status Epilepticus[END_REF][START_REF] Ziai | Target-specific Catecholamine Elevation Induced by Anticonvulsant Thalamic Deep[END_REF][START_REF] Lado | Chronic bilateral stimulation of the anterior thalamus of kainate-treated rats increases seizure frequency[END_REF][START_REF] Zhang | Anticonvulsant effect of unilateral anterior thalamic high frequency electrical stimulation on amygdala-kindled seizures in rat[END_REF][START_REF] Takebayashi | The effect of electrical stimulation and lesioning of the anterior thalamic nucleus on kainic acid-induced focal cortical seizure status in rats[END_REF][START_REF] Hamani | Deep brain stimulation of the anterior nucleus of the thalamus: Effects of electrical stimulation on pilocarpine-induced seizures and status epilepticus[END_REF][START_REF] Covolan | Effects of anterior thalamic nucleus deep brain stimulation in chronic epileptic rats[END_REF][START_REF] Yang | Potential Protective Effects of Chronic Anterior Thalamic Nucleus Stimulation on Hippocampal Neurons in Epileptic Monkeys[END_REF] and showed encouraging effects. The decision to implant in the ANT was based on positive results initially found in the preliminary studies of Cooper [START_REF] Cooper | Reversibility of chronic neurologic deficits. Some effects of electrical stimulation of the thalamus and internal capsule in man[END_REF][START_REF] Upton | Evoked Metabolic Responses in the Limbic-Striate System Produced by Stimulation of Anterior Thalamic Nucleus in Man[END_REF] and in three unblinded pilot trials that had been previously conducted [22][START_REF] Hodaie | Chronic anterior thalamus stimulation for intractable epilepsy[END_REF][START_REF] Kerrigan | Electrical Stimulation of the Anterior Nucleus of the Thalamus for the Treatment of Intractable Epilepsy[END_REF]: the randomized large multicenter double-blind SANTE trial [START_REF] Fisher | Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy[END_REF] as well as the long-term trials conducted after the SANTE trial [START_REF] Salanova | Long-term efficacy and safety of thalamic stimulation for drug-resistant partial epilepsy[END_REF][START_REF] Lee | Chronic deep brain stimulation of subthalamic and anterior thalamic nuclei for controlling refractory partial epilepsy[END_REF][START_REF] Lim | Electrical stimulation of the anterior nucleus of the thalamus for intractable epilepsy: a long-term follow-up study[END_REF][START_REF] Osorio | High frequency thalamic stimulation for inoperable mesial temporal epilepsy[END_REF], which showed at 2 years, a seizure reduction of approximately 50% in 54% of stimulated patients. However, despite these promising results in patients, there is a clear need for additional translational research to better understand the MTLE network and to optimize the benefits of DBS for patients.

To evaluate the therapeutic effects of DBS in MTLE, we used an NHP model of penicillin (PNC)induced seizures (characterized in our lab), which is reproducible and that mimics human ictal semiology [START_REF] Sherdil | An on demand macaque model of mesial temporal lobe seizures induced by unilateral intra hippocampal injection of penicillin[END_REF], in which we evaluated the effects of ANT-DBS stimulation at two specific frequencies.

MATERIALS AND METHODS

Animals

This study was performed on three Macaca fascicularis (CRP, Port Louis, Mauritius), including one female (M1) and two males (M2 and M3), weighing 7-12 kg. All studies were carried out in accordance with European Communities Council Directive of 2010 (2010/63/UE) as well as the recommendations of the French National Committee (2013/113). The authorization (#00132.01) for conducting our experiments was approved by the Committee on the Ethics of Animal Experiments (#04). All experimenters received proper training and certification for animal experimentation. Every effort was made to minimize animal suffering while maximizing the data obtained. Before surgery, to minimize stress, the NHPs were progressively trained to sit in a primate chair.

Surgery and implantable device

The animals were fasted for 10 hours before surgery. The surgery was performed under general anesthesia produced by an intramuscular injection of ketamine (Imalgene, Merial laboratory, France) 0.4 mg at induction followed by 0.2 mg per hour, and xylasine (Rompun, Bayer Healthcare AG, Germany) 2%, 0.2 ml at induction followed by 0.1 ml per hour. Additional local scalp anesthesia was provided with subcutaneous injection of lidocaine chlorydrate. Prophylactic antibiotics, analgesics and anti-inflammatory drugs were delivered during the preoperative period. Surgery was performed with a stereotactic frame (David Kopf Instruments, USA) under intraoperative teleradiographic control. The HPC and the ANT were localized using bicommissural landmarks obtained by a ventriculography performed by injecting 2 ml water soluble iodine contrast medium (Bracco Imaging, France). One cannula guide was screwed into the skull in order to reach the most extended portion of dentate gyrus within the HPC to enable repeated injections of PNC (at approximately -6 mm from anterior commissure; 12 mm from midline). To monitor ictal activity, a quadrielectrode lead was implanted within the HPC close to the injection site with the lower contact (#0) located at -5 mm from the anterior commissure, at 10 mm from the midline and 30 mm below the dura. To record and stimulate the ANT, a quadrielectrode lead was implanted in the ANT, at -5 mm from the anterior commissure with an entry point in the skull at 5 mm from the midline and a latero-median angle of 10° (M1 and M3) and 30° (M2) (lead length, 40 mm; outer diameter, 1.27 mm; with contact of 1.5 mm spaced 0.5 mm apart; model #3389, Medtronic, USA) (Fig. 1A andB). All elements were affixed to the skull with fixing screws and embedded into a dental acrylic cap. Then, the back of the animal was prepared for a skin incision and a subcutaneous space was created to place the neurostimulator (Activa®PC+S, Medtronic, USA) to stimulate both targets and to record local field potentials (LFPs). At acquisition, the LFPs were bandpass filtered between 0.5 and 80 Hz and sampled at 200 Hz. Lead extensions (lead extension model 37087, 40 cm; Medtronic, USA) were tunneled through the subcutaneous space in the back and neck and connected to the implanted electrode and stimulator.

NHPs were allowed to recover and were monitored in the postoperative period. Food and water were provided ad libitum. Experimental induction of seizures was performed after at least 10 days of recovery.

Experimental design

To induce an MTLE status for several hours, PNC was injected into the HPC, at the beginning of each experiment several times with each animal (Fig. 1C; see Sherdil et al. 2018 [START_REF] Sherdil | An on demand macaque model of mesial temporal lobe seizures induced by unilateral intra hippocampal injection of penicillin[END_REF]). LFPs from the ANT and HPC were simultaneously recorded (Fig. 1D), and in some experiments, the therapeutic effect of ANT-DBS was evaluated at low (40 Hz) or high (130 Hz) frequencies of stimulation. In practice, the experiments were divided into several 20-min periods allowing 2 sessions of ANT-DBS with the same parameters during the MTLE status period (Fig. 1F). A total of 47 PNC injections, i.e., 47 experiments, were performed in the three NHPs (9 for M1, 19 for M2, and 19 for M3). Among them, 27 control experiments were performed in an alternating pattern with the DBS experiments to ensure the stability of the model throughout the study (as previously reported [START_REF] Sherdil | An on demand macaque model of mesial temporal lobe seizures induced by unilateral intra hippocampal injection of penicillin[END_REF]). Twenty DBS experiments were performed, including 7 DBS sessions for M1, 12 for M2 and 8 for M3 at 40 Hz and 12 DBS sessions (4 per NHP) at 130 Hz. The distribution of HF and LF ANT-DBS was randomized, but due to the greater severity of seizures observed at 130 Hz, the HF ANT-DBS sessions were limited to 4 per animal. In addition, due to the decrease in the PNC effect, to induce stable seizures after several sessions, no more than 7 and 8 DBS sessions could be performed in M1 and M3, respectively, compared with 12 in M2. Seizures were recorded until their extinction, which was 4.5 h/experiment on average.

Stimulation paradigms

The two contacts located in the ANT were used for the bipolar DBS mode. This corresponded to contacts 1(-)/2(+) for M1 and M3 and to contacts 2(-)/3(+) for M2. To define an alternative stimulation frequency from the one routinely used in clinical settings (130 Hz), the changes in HPC spontaneous activity amplitude in response to 10 s of ANT-DBS were measured in preliminary experiments over a range of frequencies from 2 to 100 Hz and over voltage levels from 2 to 6 volts (data not shown) according to a previously published method by Stypulkowski et al. (2011[START_REF] Stypulkowski | Brain stimulation for epilepsy -Local and remote modulation of network excitability[END_REF] .

ANT-DBS at 40 Hz showed the most significant amplitude reduction in the spontaneous activity (example in Fig. 1E). Mainly based on a double-blind clinical study [START_REF] Fisher | Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy[END_REF] and also on the observations in our animals during the preliminary setting period, which did not show any clinically relevant stimulation-induced side effects, the voltage was set at 5 volts and the pulse width was set at 450 µs for all experiments. ANT-DBS sessions consisted of 2-min ON/2-min OFF repeated 5 times for 20 min at 130 or 40 Hz. This cycling stimulation mode was chosen to facilitate the quantification of epileptic events, providing time windows without artifacts during the DBS session. The second session was performed 40 min after the first session. DBS parameters were programmed using the N'Vision programmer (Model 8840, Medtronic, US).

Coherence analysis of LFP recordings

For each NHP, time-frequency and coherence analyses were performed for 2 sets of randomly selected seizures based on the following criteria: 1) duration from 6 s to 135 s (average duration: 35.82 ± 13.71 s), 2) no artifacts throughout the recording, and 3) occurrence of the seizure before the DBS was turned ON. During the pre-DBS periods, we extracted and analyzed 31 seizures for M1, 34 seizures for M2 and 23 seizures for M3 from the 130-Hz ANT-DBS sessions, and 57 seizures for M1, 73 seizures for M2 and 24 seizures for M3 from the 40-Hz ANT-DBS sessions. The LFP signals of interest were chosen as the bipolar derivation covering the HPC and the ANT. The amplitude (squareroot of power spectral density) and coherence (normalized cross-spectral density) of oscillatory activity between 0.4 and 80 Hz were obtained using spectral density estimates based on the use on the fast Fourier transform of 0.39-Hz spectral resolution successively computed on a time window of 2.56 s duration translated every 100 ms. The first and last spikes in each discharge were used to define its onset and termination, respectively. The time window of analysis started 10 s before the seizure onset and finished 5 s after seizure termination. Time-frequency data were normalized using a standard procedure: for each frequency, the mean of the baseline (periods of at least 10 s without interictal spikes prior to each seizure) was subtracted from the data and then demeaned data were divided by the standard deviation of the baseline. Baseline was defined for every seizure as a period of 10 s without interictal spikes chosen just before seizure onset. Finally, the typical time-frequency pattern of the recorded seizure was defined as the median value over seizures of the normalized time-frequency charts. Before median averaging, time-frequency charts were resampled linearly on a time scale to normalize seizure duration to the observed average (48 s). In addition, for each animal and experiment with ANT-DBS sessions, before DBS was switched ON, a global ictal coherence score was calculated by averaging between 0.4 and 80 Hz the coherence between the ANT and HPC across seizures. Then, this score was also calculated in different frequency bands: for LF at 1-48 Hz (decomposed in delta (1-3 Hz), theta (4-8 Hz), alpha (9-13 Hz), beta (14-30 Hz) and gamma [START_REF] Sherdil | An on demand macaque model of mesial temporal lobe seizures induced by unilateral intra hippocampal injection of penicillin[END_REF][START_REF] Stypulkowski | Brain stimulation for epilepsy -Local and remote modulation of network excitability[END_REF][START_REF] Leocani | EEG Coherence in Pathological Conditions[END_REF][START_REF] Aggleton | Hippocampal-anterior thalamic pathways for memory: Uncovering a network of direct and indirect actions[END_REF][START_REF] Koubeissi | Low-frequency electrical stimulation of a fiber tract in temporal lobe epilepsy[END_REF][START_REF] Stypulkowski | Modulation of hippocampal activity with fornix Deep Brain Stimulation[END_REF][START_REF] Devergnas | The subcortical hidden side of focal motor seizures: Evidence from micro-recordings and local field potentials[END_REF][START_REF] Takebayashi | Anti-convulsant effect of electrical stimulation and lesioning of the anterior thalamic nucleus on kainic acid-induced focal limbic seizure in rats[END_REF][START_REF] Feynman | The theory of a general quantum system interacting with a linear dissipative system[END_REF][START_REF] Munari | Electro-clinical semiology of subintrant temporal lobe seizures[END_REF][START_REF] Theodore | Complex partial seizures: Clinical characteristics and differential diagnosos[END_REF][START_REF] Kramer | Clinical characteristics of complex partial seizures: A temporal versus a frontal lobe onset[END_REF](42)(43)(44)(45)(46)(47)(48) bands) and for the HF at 52-90 Hz.

Statistical analysis

For each experiment, the number of interictal spikes (per min), mean duration of a seizures (s), mean total time spent in seizure (s) and mean number of seizures were quantified and expressed as the mean ± standard error with (min-max values). Statistical tests were performed using GraphPad InStat3 software (GraphPad Software Inc., USA). A difference was considered statistically significant for a p value < 0.05. For each animal, the differences between the 3 periods of interest (pre DBS, during DBS, post DBS) were compared using repeated measures ANOVA test followed by post hoc tests. Comparison of the values obtained during the pre-DBS period (baseline) were performed using ANOVA test followed by post hoc tests. The ictal coherence score for each specific band of frequencies was calculated for each animal from a collection of seizures occurring before each type of ANT-DBS experiment (130 Hz and 40 Hz). For each animal, the analysis of the distribution of the ictal coherence score among the different frequency bands was performed using a repeated measures ANOVA followed by post hoc tests. In addition, this distribution was also analyzed between the animals with ANOVA followed by post hoc tests. For all DBS experiments, the efficacy of DBS, based on % of variation in the number of seizures during DBS compared to pre DBS, was correlated with the global ictal coherence score (0.4-80 Hz) calculated during seizures occurring prior applying DBS. Using the Spearman's correlation, the correlation coefficient (Rs) was calculated and considered significant at a two-tailed p-value < 0.05.

Results

ANT-DBS effects on HPC activity. During the epileptic status following PNC injections, interictal spike frequency and mean duration of seizure did not significantly vary during and after DBS periods in comparison to baseline (pre DBS) at 130 and 40 Hz for all NHPs (Fig. 2). On the other hand, we observed a trend for an increase in time spent in seizure in post DBS for all NHPs at 130 Hz, and the difference was significant when the data were pooled (143. Additionnally, the mean number of seizures did not significantly vary at 130 Hz (Fig. 2). In contrast, a significant decrease in the number of seizures occurred during and after DBS at 40 Hz in M1; this decrease was also significant in M2 during DBS compared to the baseline period, and a nonsignificant decrease was observed in M3. This tendency became significant when the data were pooled together (5.8 ± 0.5 [START_REF] Didato | Bitemporal epilepsy: A specific anatomo-electro-clinical phenotype in the temporal lobe epilepsy spectrum[END_REF][START_REF] Bartolomei | Neural networks involving the medial temporal structures in temporal lobe epilepsy[END_REF][START_REF] Chabardès | The temporopolar cortex plays a pivotal role in temporal lobe seizures[END_REF][START_REF] Bartolomei | From mesial temporal lobe to temporoperisylvian seizures: A quantified study of temporal lobe seizure networks[END_REF][START_REF] Aggleton | Projections from the hippocampal region to the mammillary bodies in macaque monkeys[END_REF][START_REF] Aggleton | The origin, course, and termination of the hippocampothalamic projections in the macaque[END_REF][START_REF] Sherdil | Implication of Anterior Nucleus of the Thalamus in Mesial Temporal Lobe Seizures[END_REF][START_REF] Cooper | Therapeutic implications of modulation of metabolism and functional activity of cerebral cortex by chronic stimulation of cerebellum and thalamus[END_REF][START_REF] Gallistel | The organization of action: A new synthesis[END_REF][START_REF] Mirski | Anticonvulsant effect of anterior thalamic high frequency electrical stimulation in the rat[END_REF] pre DBS vs 2.4 ± 0.4 (0-6) during DBS vs 2.6 ± 0.3 (0-8) post DBS, p < 0.0001; all NHPs pooled) (Fig. 2). In summary, in all NHPs, 40 Hz ANT-DBS induced a decrease in the number of seizures and time spent in seizure.

Coherence analysis and ANT-DBS efficacy.

Changes in LFP recordings from the ANT were time-locked with interictal spikes and seizures recorded from the HPC (Fig. 3A-B). Indeed, during the seizures, in all NHPs, significant oscillations in the LF band (0.4-48 Hz) but not in the HF band (52-80 Hz) were simultaneously identified within the HPC and the ANT and confirmed by an ictal coherence score significantly higher in LF bands than in HF bands for all NHPs (p < 0.001), with the exception of M3 in 130-Hz ANT-DBS experiments (Fig. 3C). In addition, the time frequency charts of coherence showed significant oscillations at approximately 4 Hz in M1 and at 4 and 20 Hz in M2 and also for M3 although less strong in M3. M1 exhibited a higher ictal coherence score compared to M2, and the score was higher in M2 than M3 (0.55 vs 0.51 (ns) vs 0.44 (p < 0.01), respectively), in the 130-Hz DBS experiments. This tendency was more pronounced in 40-Hz DBS experiments (0.58 vs 0.51 vs 0.46, respectively; p < 0.01). Overall, the analysis of ictal coherence scores among the LF bands showed that M1 exhibited a significantly higher levels in theta, alpha and beta bands than to the other monkeys. Interestingly, the ictal coherence score was significantly higher in M1 compared to M2 and M3 for the 40-Hz DBS experiments in delta, theta, alpha and also in gamma (30-48 Hz) bands.

Additionally, the LF-DBS was more effective in M1 than in M2 and M3 regarding the number of seizures (-88.1 ± 6.7 vs -41.6 ± 12.5 vs -42.0 ± 11.2 percentage of variation in the number of seizures DBS compared to pre DBS, respectively) (Fig. 4A). Finally, the ictal coherence score between the ANT and the HPC evaluated in pre DBS was not correlated with the percentage of variation in the number of seizures occurring during the 130-Hz DBS (Rs = -0.08; p = 0.800) but was significantly correlated with the percentage of variation in the number of seizures occurring during the 40-Hz DBS sessions (Rs = -0.46, p = 0.03) (Fig. 4B).

Localization of electrodes.

In addition to ventriculographic controls performed during surgery, lead positions were confirmed by postmortem analysis. In all NHPs, due to the mechanical lesion, post mortem studies showed slight tissue damage close to or within the ANT, allowing a reconstruction of the electrode location (Fig. 5). In M1, the contact 1 was located at the superior landmark of the ANT, whereas the contact 2 was in contact with the fornix. In M2, contacts 2 and 3 were located within the posterior part of the ANT, and in M3, active contacts were located within the anterior part of the ANT.

Discussion

It appeared that ANT-DBS at 130 Hz had no therapeutic effect on MTL seizures or interictal events, with a trend for worsening. In contrast, ANT-DBS at 40 Hz was responsible for a significant decrease in the mean number of seizures, and as the duration of the seizures remained stable, it also affected the mean total time spent in seizures. Because all NHPs presented a high rate of seizures when DBS was switched OFF, these effects are very likely to be attributed to the DBS itself and not due to a stun effect.

40-Hz stimulation versus 130-Hz stimulation.

This discrepancy between HF and LF DBS effects could be due to the involvement of different neuronal populations in the ANT in MTLE seizures. Indeed, we had previously shown that during PNCinduced seizures, some neurons in ANT were excited, whereas others were inhibited [START_REF] Sherdil | Implication of Anterior Nucleus of the Thalamus in Mesial Temporal Lobe Seizures[END_REF]. This result is not in accordance with the literature and deserves some discussion. In the previous human clinical trials [START_REF] Upton | Evoked Metabolic Responses in the Limbic-Striate System Produced by Stimulation of Anterior Thalamic Nucleus in Man[END_REF][START_REF] Hodaie | Chronic anterior thalamus stimulation for intractable epilepsy[END_REF][START_REF] Kerrigan | Electrical Stimulation of the Anterior Nucleus of the Thalamus for the Treatment of Intractable Epilepsy[END_REF][START_REF] Fisher | Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy[END_REF][START_REF] Lee | Chronic deep brain stimulation of subthalamic and anterior thalamic nuclei for controlling refractory partial epilepsy[END_REF][START_REF] Lim | Electrical stimulation of the anterior nucleus of the thalamus for intractable epilepsy: a long-term follow-up study[END_REF][START_REF] Osorio | High frequency thalamic stimulation for inoperable mesial temporal epilepsy[END_REF] showing the anti-epileptic effects of ANT-DBS at 130 Hz, patients were very heterogeneous, while we used a well-controlled model of MTLE seizures that are not strictly comparable to the more complex nature of seizures encountered in the human population. In addition, in the present experiments, only acute effects were taken into account, whereas in the SANTE trial, it has been shown that the outcome increased with the duration of stimulation (especially after 1 year). Here, we stimulated for minutes only, which may explain the lack of effect of HF DBS. Overall, the important experimental differences between our study and previous clinical trials do not make the comparison straightforward. It is clear that additional studies will be required to better understand the role of the ANT in MTLE to optimize the DBS strategy proposed to patients, because the rate of patients who were seizure-free, which is the ultimate goal of patients, was low in published studies, probably because of the empirical methodology used to define the DBS paradigm and the heterogeneity in the epileptic networks involved in the MTLE.

The model we used is known to be relatively stable [START_REF] Upton | Evoked Metabolic Responses in the Limbic-Striate System Produced by Stimulation of Anterior Thalamic Nucleus in Man[END_REF][START_REF] Hodaie | Chronic anterior thalamus stimulation for intractable epilepsy[END_REF][START_REF] Kerrigan | Electrical Stimulation of the Anterior Nucleus of the Thalamus for the Treatment of Intractable Epilepsy[END_REF][START_REF] Fisher | Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy[END_REF][START_REF] Lee | Chronic deep brain stimulation of subthalamic and anterior thalamic nuclei for controlling refractory partial epilepsy[END_REF][START_REF] Lim | Electrical stimulation of the anterior nucleus of the thalamus for intractable epilepsy: a long-term follow-up study[END_REF][START_REF] Osorio | High frequency thalamic stimulation for inoperable mesial temporal epilepsy[END_REF]. During the baseline period, we observed only in M2 that the duration of seizures was significantly higher in the 130-Hz experiments than in the 40-Hz experiments, but there were no significant differences when those data were compared to those of control experiments. This variation in the baseline, which reflects the variability of the model, was taken into account in our statistical model, as we used a paired statistical tests to compare the DBS effects over 3 successive periods. The effect of this variation can thus be considered as negligible in the reported effects.

Coherence between the ANT and the HPC is key to adapting the stimulation parameters.

A key finding in this study was that if the ANT and HPC activities were highly correlated in the lowfrequency bands (below 40 Hz), then the 40-Hz stimulation of the ANT was more effective than 130-Hz at reducing the number of seizures. During seizures, significant oscillatory activity was observed in both structures at approximatively 40 Hz, which was confirmed by the coherence analysis. In addition, a linear correlation demonstrated a significant link between the 40-Hz DBS efficiency and the coherence strength between the HPC and the ANT, a correlation that was not observed with the 130-Hz ANT-DBS. Coherence studies have been used to explore the functional connections, and it is well known that a high level of coherence reveals strong connectivity [START_REF] Leocani | EEG Coherence in Pathological Conditions[END_REF]. When we focused our analysis on the ictal coherence score, we observed that each animal had its own ictal oscillatory profile. This suggests that it might be worth quantifying ictal coherence for individual to tailor the DBS paradigm. Indeed, the best therapeutic effects were obtained for the NHP "M1", which was the animal exhibiting the highest ictal coherence score in the LF bands.

Lead location into the ANT matters.

NHP M1 was also the only one in which the lead was located more medially, allowing bilateral stimulation of the ANT and/or of the fornix. This latter structure may also play a role in HPC activity, as it includes some efferent and afferent connections between the ANT and the HPC [START_REF] Aggleton | Hippocampal-anterior thalamic pathways for memory: Uncovering a network of direct and indirect actions[END_REF]. Indeed, the fornix is an essential structure of the Papez circuit and is composed only of fibers that have an excitatory threshold inferior to that of the soma. Thus, it is likely that at 5 volts the fornix was stimulated. Only a few clinical studies have focused on the application of fornix DBS in epilepsy. Only one clinical trial resulted in published data on two epileptic patients with electrodes implanted directly into the fornix [START_REF] Koubeissi | Low-frequency electrical stimulation of a fiber tract in temporal lobe epilepsy[END_REF]. The results were interesting, showing that hippocampal spikes were decreased by fornix low-frequency stimulation (bilateral, 5 Hz, 8 mA, 0.2 ms pulse width, given in 4hour blocks) and that seizure odds (n = 7) were reduced by 92% in the 2 days after stimulation.

Additionally, it has been shown in a sheep model, that fornix-DBS produces short latency hippocampal evoked potentials. At high frequency and low voltage, an inhibition of HPC LFP was observed, although at high voltage, some pronounced bursts were elicited [START_REF] Stypulkowski | Modulation of hippocampal activity with fornix Deep Brain Stimulation[END_REF]. This differential effect can partly explain the worsening effect of HF DBS in M1 and M2. All these results lead us to think that it would be worth exploring the effect of DBS involving the fornix, but the risk of inducing memory impairment should be first carefully considered. NHP M3 did not exhibit any significant effects with DBS and presented the weakest coherence. However, it appeared that the electrode was implanted much more anteriorly than in the other two subjects, at the most anterior tip of the ANT. At this level, the ANT does not send and receive the majority of efferent and afferent fibers. Finally, M2 NHP showed intermediate results in terms of lead location, therapeutic effect and power of coherence. Overall, this result also suggests that the analysis of synchronous oscillations between the two structures could predict the therapeutic efficacy of the DBS: the stronger the correlation between the two structures in a frequency band was, greater the effect of the stimulation belonging to this frequency band. In this way, optimization of DBS parameters could be tailored to each patient.

Based on our findings, it seems that it would have been wise to test stimulation at 4 Hz for M1 and 20 Hz for M2, which were the frequencies exhibiting a strong significant coherence. Studies focusing on stimulation in the coherence frequency could be further developed and pave the road for the development of a more customized DBS strategy that could be based on the coherence analysis of the activities between structures in the epileptic network.

A correlation between two structures, the motor cortex and the subthalamic nucleus, had previously been observed in an NHP model of motor seizures [START_REF] Devergnas | The subcortical hidden side of focal motor seizures: Evidence from micro-recordings and local field potentials[END_REF]. In this model, both structures oscillated in high frequencies, generating a significant coherence in high frequency bands (> 60 Hz), and it appeared that unilateral high-frequency stimulation of the subthalamic nucleus had a therapeutic effect with a decrease in the number of seizures and of the total time spent in seizure. These data might clarify the inadequacy of the 130-Hz ANT stimulation and explain the varying effects observed across several studies [START_REF] Mirski | Anticonvulsant effect of anterior thalamic high frequency electrical stimulation in the rat[END_REF][START_REF] Hamani | Bilateral Anterior Thalamic Nucleus Lesions and High-frequency Stimulation Are Protective against Pilocarpine-induced Seizures and Status Epilepticus[END_REF][START_REF] Ziai | Target-specific Catecholamine Elevation Induced by Anticonvulsant Thalamic Deep[END_REF][START_REF] Lado | Chronic bilateral stimulation of the anterior thalamus of kainate-treated rats increases seizure frequency[END_REF][START_REF] Zhang | Anticonvulsant effect of unilateral anterior thalamic high frequency electrical stimulation on amygdala-kindled seizures in rat[END_REF][START_REF] Hamani | Deep brain stimulation of the anterior nucleus of the thalamus: Effects of electrical stimulation on pilocarpine-induced seizures and status epilepticus[END_REF][START_REF] Fisher | Electrical stimulation of the anterior nucleus of thalamus for treatment of refractory epilepsy[END_REF][START_REF] Osorio | High frequency thalamic stimulation for inoperable mesial temporal epilepsy[END_REF][START_REF] Takebayashi | Anti-convulsant effect of electrical stimulation and lesioning of the anterior thalamic nucleus on kainic acid-induced focal limbic seizure in rats[END_REF].

Destructive resonance and ANT oscillation.

The mechanism of action of 40-Hz DBS is unclear. The effect of destructive resonance could be a phenomenon that deserves to be explored. Indeed, a mechanical system responds at a high amplitude when a frequency, which is imposed on it, matches its own natural frequency of vibration (i.e., its resonance frequency) [START_REF] Feynman | The theory of a general quantum system interacting with a linear dissipative system[END_REF]. It may cause violent swaying motions and even dramatic failure (collapsed bridges, broken glass, etc.). In other words, this phenomenon has a strong effect on an oscillating system, and, as a consequence, it would be interesting to evaluate it in the context of epileptic seizures in further experiments.

Limitations of the study.

These experimental results must be taken with great caution when translating them to clinical application because we used a semiacute model of seizures induced by single injections of PNC.

Indeed, in the present model, only subintrant seizures were induced for only 4.5 hours on average, but those seizures, although mimicking clinical temporal seizures observed in humans, do not reflect the more complex nature of chronic, spontaneous temporal seizures observed in humans [START_REF] Munari | Electro-clinical semiology of subintrant temporal lobe seizures[END_REF] [START_REF] Theodore | Complex partial seizures: Clinical characteristics and differential diagnosos[END_REF][START_REF] Kramer | Clinical characteristics of complex partial seizures: A temporal versus a frontal lobe onset[END_REF]. Furthermore, one must be cautious that the mechanism of seizure induction by PNC is different from that seen in the classical MTLE (with HPC sclerosis), in posttraumatic epilepsy, or in the context of brain tumors, cortical dysplasia of chronic inflammatory processes, for example. Seizures coming from a sclerotic HPC, as in human MTLE, may share the same electroclinical features but will not share the same complex cellular organization. Nevertheless, this model remains relevant to study the dynamics of an epileptic network because, in addition to the common electricoclinical characteristics shared with human semiology, it involves similar nodes and tracts as observed in humans.

Conclusions

This study aimed to better understand the role of the ANT in MTLE to propose a suitable paradigm for ANT stimulation. We confirmed that the ANT was involved in MTLE as an active part of the network and modulation of its activity modified the expression of MTLE. The endogenous dynamics of the seizure in both the ANT and HPC (and especially the study of the coherence between them) led to an adapted ANT stimulation paradigm that significantly decreased seizure frequency and time spent in seizure. Based on this study, we suggest that the parameters of stimulation should be adjusted and personalized based on the study of ictal oscillations that are predominant in the ANT of each individual patients. Furthermore, a DBS lead implantation encompassing the fornix and the ANT should be further addressed.

  2 ± 26.4 s (84.9-201.4 s) pre DBS vs 151.4 ± 18.7 s (110.3-192.5 s) during DBS vs 238.3 ± 32.5 s (166.7-309.8 s) post DBS; p = 0.0289). Interestingly, at 40 Hz, the time spent in seizure significantly decreased during and post DBS compared to pre DBS in M1 and tended to decrease in M2 and M3. When the data from all the monkeys were pooled, the decrease was significant (108.1± 9.3 s (42.6-204.4 s) pre DBS vs 70.0 ± 14.5 s (0-228.0 s) during DBS vs 73.3 ± 12.1 s (0-248.0 s) in post DBS, p = 0.0189) (Fig. 2).

Figure 1 :

 1 Figure 1: Identification of ventriculographic landmarks (midline, anterior commissure (AC) and

Figure 2 :

 2 Figure 2: Investigation of ANT-DBS anti-epileptic effects in 12 DBS sessions at 130 Hz and in 27

Figure 3 :

 3 Figure 3: Example of local field potentials recorded, in HPC and ANT, during the occurrence of

Figure 4 :

 4 Figure 4 : DBS efficacy is expressed in percentage of change in the number of seizures with respect to

Figure 5 :

 5 Figure 5: ANT lead implantation. The location of the ANT electrode has been reconstructed based on

Acknowledgements

We wish to acknowledge Fabien Tissier for his help for image processing, Aurélien Gauffre for his help in local field potential analysis and Jacques Brocard for checking the statistical tools used in this study.

Fundings

This work was supported by Medtronic, Inc. Data were acquired on a platform of France Life Imaging network partly funded by the grant "ANR-11-INBS-0006".

Conflicts of interest

Medtronic provided financial support for the present study but the sponsor did not have any role neither in the design of the study, nor in the collection or the interpretation of the data. Pr Chabardès, and Dr Piallat have received financial support from Medtronic Europe to attend scientific meetings in the last 3 years. Pr Chabardes declares is also consultant for Medtronic and Boston Scientific. All other authors declared no conflict of interests related to this study.