Specific imbalance of excitatory/inhibitory signaling establishes ...

Articles in PresS. J Neurophysiol (April 13, 2016). doi:10.1152/jn.01128.2015 Journal of Neurophysiology JN-01128-2015 (Review)

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SPECIFIC IMBALANCE OF EXCITATORY/INHIBITORY SIGNALING

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ESTABLISHES SEIZURE ONSET PATTERN IN TEMPORAL LOBE

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EPILEPSY

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Massimo Avoli1,2,*, Marco de Curtis3, Vadym Gnatkovsky3, Jean Gotman1, Rüdiger Köhling4,

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Maxime Lévesque1, Frédéric Manseau5, Zahra Shiri1, Sylvain Williams5

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Montreal Neurological Institute and Departments of Neurology & Neurosurgery and of

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Physiology, McGill University, Montréal, H3A 2B4 Québec, Canada; 2Facoltà di Medicina e

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Odontoiatria, Sapienza Università di Roma, 00185 Roma, Italy; 3Epilepsy Unit, Fondazione

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Istituto Neurologico Carlo Besta, 20133 Milano, Italy; 4Oscar-Langendorff-Institute of

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Physiology, Rostock University Medical Center, 18057 Rostock, Germany; 5Douglas Mental

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Health University Institute, McGill University, Montréal, H4H 1R3 Québec, Canada.

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Running title: Mechanisms of seizure onset Number of characters in title and running head: 140 Number of words in abstract: 217 Number of words in body of manuscript: 3 677 Number of figures: 5 * Corresponding Author:

Massimo Avoli, M.D., Ph.D. Montreal Neurological Institute 3801 University Street, Montréal, QC Canada, H3A 2B4 Tel Canada: +1 514 998 6790 Tel Europe: +39 333 483 1060 Fax: +1 514 398 8106 E-mail: [email protected]

CONFLICTS OF INTEREST None of the authors have any conflict of interest to disclose.

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Copyright © 2016 by the American Physiological Society.

Journal of Neurophysiology JN-01128-2015 (Review)

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Abstract

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Low-voltage fast (LVF) and hypersynchronous (HYP) patterns are the seizure onset patterns

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most frequently observed in intracranial EEG recordings from mesial temporal lobe epilepsy

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(MTLE) patients. Both patterns also occur in models of MTLE in vivo and in vitro, and these

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studies have highlighted the predominant involvement of distinct neuronal network/neu-

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rotransmitter receptor signaling in each of them. First, LVF onset seizures in epileptic rodents

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can originate from several limbic structures, frequently spread, and are associated with high

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frequency oscillations (HFOs) in the ripple band (80-200 Hz), while HYP onset seizures initiate

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in the hippocampus, and tend to remain focal with predominant fast ripples (250-500 Hz).

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Second, in vitro intracellular recordings from principal cells in limbic areas indicate that

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pharmacologically induced, seizure-like discharges with LVF onset are initiated by a

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synchronous inhibitory event or by a hyperpolarizing IPSPs barrage; in contrast, HYP onset is

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associated with a progressive impairement of inhibition and concomitant unrestrained enhance-

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ment of excitation. Finally, in vitro optogenetic experiments show that under comparable

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experimental conditions (i.e., 4-aminopyridine application), the initiation of LVF or HYP onset

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seizures depends on the preponderant involvement of interneuronal or principal cell networks,

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respectively. Overall, these data may provide insight to delineate better therapeutic targets in

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the treatment of patients presenting with MTLE and, perhaps, with other epileptic disorders as

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well.

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New and noteworthy

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We review here the mechanisms leading to two seizure onset EEG patterns that occur in mesial

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temporal lobe epilepsy. Based on data obtained in epileptic patients and in animal models, we

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propose that the initiation of low-voltage fast onset and hypersynchronous onset seizures

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depend on the involvement of GABAergic interneuron and of principal (glutamatergic) networks,

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respectively, which in both cases rest on functional GABAA receptor signaling.

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Background

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The EEG activity recorded during focal seizures in patients with mesial temporal lobe epilepsy

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(MTLE) is characterized by different levels of detail, depending on the recording approach used.

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Standard scalp electrode recordings may reveal, at the onset of a seizure, flattening of the EEG

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signals localized in the temporal lobe that is at times associated with the appearance of low

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amplitude, fast rhythms (Gloor, 1975; Fisher et al., 1992; see also de Curtis and Gnatkovsky,

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2009). However, this straightforward diagnostic tool is not ideal for identifying the exact seizure

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initiation area that resides in mesial cortical structures such as the hippocampus or the

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amygdala; in addition, many seizures in MTLE do not show this pattern: some show other

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patterns, and many only show artefacts at onset. In contrast, intracranial EEG recordings

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obtained from deep limbic regions - which are the gold standard in presurgical evaluation of

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pharmacoresistant epileptic patients who are candidates for surgery - can lead to a more

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precise identification of the brain structures involved in seizure initiation while revealing in detail

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the features of discharge onset (Lieb et al., 1976; Pacia and Ebersole, 1997).

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Seizures in MTLE patients studied with intracranial electrodes have variable EEG

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features, but evidence published during the last two decades indicate that two specific seizure

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onset patterns may predominate in temporal lobe (limbic) structures (Spencer and Pappas,

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1992; Spanedda et al., 1997; Wendling et al., 2005; Ogren et al., 2009; Perucca et al., 2014;

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Singh et al., 2015). The most frequent onset pattern is characterized by “low-voltage fast” (LVF)

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activity in the gamma range, at times initiated by a single (also termed “sentinel’) spike (Fig. 1A,

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asterisk in the left panel); the second onset pattern- referred to as “hypersynchronous” (HYP) -

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is associated with an initial series of large amplitude spikes that occur at a frequeny of approx. 1

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Hz (Fig. 1A, asterisks in the right panel) and can also reappear during the initial part of the

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seizure (Fig. 1A, arrow heads in the right panel). As illustrated in Fig. 1A, LVF as well as HYP

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onset seizures evolve into a phase of sustained oscillatory activity (also defined as “tonic”) and

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then into bursting, rhythmic (“clonic”) discharges; in both cases, termination of seizure discharge

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is followed by a period of spontaneous electrical activity suppression that is termed “post-ictal

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depression”.

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The clinical evidence for two distinct seizure onset patterns has been confirmed in

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animal models of MTLE and of epileptiform synchronization in both in vivo (Fig. 1B) (Bragin et

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al., 2005; Lévesque et al., 2012; Grasse et al., 2013; Salami et al., 2015; Toyoda et al., 2015)

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and in vitro preparations (Fig. 1C) (Avoli et al., 1996; Lopantsev and avoli, 1998a and b;

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Derchansky et al., 2008; Zhang et al., 2012; Boido et al., 2014; Köhling et al., 2016; see for

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review Avoli and de Curtis, 2011). Moreover, these fundamental studies have provided evidence

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suggesting that specific neuronal networks may contribute to LVF and HYP seizure onset

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patterns. In this review, we will briefly analyze evidence obtained in patients suffering from

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MTLE; these clinical studies suggest that LVF and HYP seizure onset patterns may reflect

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different epileptic conditions along with different degrees of brain damage, at least in MTLE.

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Then, we will consider experimental data obtained in vivo from the pilocarpine model of MTLE

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as well as in normal, control rodents following acute, diverse convulsive pharmacological

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manipulations. Next, we will summarize in vitro experiments indicating the predominant

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involvement of GABAA receptor signaling in initiating LVF seizure-like discharges and the

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dynamic changes in inhibition that accompany the onset of HYP seizure-like activity. Finally, we

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will analyze recent optogenetic in vitro findings; these results strongly suggest that the initiation

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of LVF and HYP onset seizures depends on the preponderant involvement of (GABAergic)

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interneuron and of principal (glutamatergic) cell networks, respectively, which in both cases rest

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on an operational GABAA receptor signaling.

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Seizure onset patterns in the clinical context

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Long-term, intracranial depth recordings obtained during pre-surgical stereo-EEG monitoring

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have revealed that LVF seizure onset represents the most common pattern of ictal discharge

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initiation. Indeed, it occurs across several neocortical focal epilepsies and is not restricted to

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MTLE (Gotman et al., 1995; Gnatkovsky et al., 2011; Perucca et al., 2014; Wu et al., 2014;

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Singh et al., 2015). In addition, MTLE patients presenting with LVF onset seizures show diffuse

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neuronal loss that can comprise the CA2/CA4 regions (Velasco et al., 2000; Ogren et al., 2009).

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This pattern is also more frequent in patients showing amygdala atrophy in addition to

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hippocampal atrophy as well as in patients with normal mesial temporal structures (Spanedda et

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al., 1997).

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In contrast, the HYP onset pattern is most often, if not solely, observed in MTLE with

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hippocampal sclerosis and it has never been reported in neocortical focal epilepsies (Spencer et

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al., 1992; Velasco et al., 2000; Ogren et al., 2009; Perucca et al., 2014). Magnetic resonance

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imaging studies and histopathological evaluation of post-surgical specimens have also

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demonstrated that MTLE patients presenting with HYP onset seizures present with cell loss in

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all hippocampal areas with the exception of the CA2 subfield and the presubiculum (Spencer et

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al., 1992; Velasco et al., 2000). In addition, Ogren et al. (2009) have proposed that HYP onset

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seizures occur only in MTLE patients presenting with pronounced but restricted hippocampal

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atrophy. Overall, evidence obtained in epileptic patients indicate that these two seizure onset

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patterns often reflect different histopathological conditions, which in turn suggests the

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involvement of different types of neuronal networks and presumably a specific contribution of

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ligand-gated mechanisms.

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Figure 1 approx. here

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Seizure onset patterns in in vivo animal models of epilepsy and of epileptiform

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synchronization Bragin et al. (2005) were first in reporting that in the “local” kainic acid model of MTLE, rats

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generate spontaneous seizures characterized by both LVF and HYP onset patterns; in these

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experiments, EEG recordings were obtained with depth electrodes positioned in several limbic

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structures. These researchers also found that HYP onset seizures initiate most often from the

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hippocampus ipsilateral to the original kainic acid injection and that they are rarely accompanied

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by behavioral symptoms; therefore, these data suggest that these seizures remained focal. On

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the contrary, LVF onset seizures occurring in these experiments originated from the

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hippocampus and from the entorhinal cortex, and they frequently propagated to other limbic and

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extralimbic areas (Bragin et al., 2005).

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Similar results have been later obtained in the pilocarpine model of MTLE by Lévesque

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et al. (2012) who found that the majority of HYP onset seizures originated from the hippocampal

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CA3 region, while LVF onset seizures initiated in this hippocampal subfield as well as in the

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entorhinal cortex or even outside the hippocampal formation (Fig. 1B). An additional crucial

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difference between LVF and HYP seizures in the pilocarpine model rests on their association

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with specific types of pathological high frequency oscillations (HFOs) at 80-500 Hz, which have

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been arbitrarily categorized into ripples (80-200 Hz) and fast ripples (250-500 Hz) (Fig. 2A)

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according to the original proposal made by Bragin et al. (1999). HFOs - which can only be

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extracted by amplifying the appropriately filtered EEG signal - have been recorded from patients

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presenting with MTLE and in animal models mimicking this neurological condition, and they

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have been proposed to represent better biomarkers than interictal spikes for identifying seizure

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onset zones in focal epileptic disorders (Jacobs et al., 2008, 2010; see also Jacobs et al. 2012;

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Staba, 2012).

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Excitatory and inhibitory synaptic interactions along with intrinsic membrane

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oscillations, gap junctions and ephaptic coupling have been considered to underlie physiological

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(Buzsáki and Chrobak, 1995; Ylinen et al., 1995; Buzsáki, 2015) as well as pathological

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(Jefferys et al., 2012a, 2012b) HFOs, and the contribution of these mechanisms to the

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generation of HFOs is under active examination. In addition, it has been reported that HFOs

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with similar frequency ranges can differ considerably in their physiological mechanisms (Jefferys

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et al., 2012a, 2012b). However, a convenient – though reductionist and, perhaps, simplistic -

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view is that pathologic HFOs in the ripple band mainly represent population IPSPs generated by

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principal neurons that are entrained by synchronously active interneuron networks; in contrast,

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HFOs in the fast ripple band could be supported by the synchronous firing of abnormally active

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principal cells (Foffani et al., 2007), and could be independent of inhibitory neurotransmission

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(cf., Jefferys et al., 2012a). However, data obtained in some studies (e.g., D’Antuono et al.,

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2005; Bragin et al., 2011) have suggested that pathological ripples and fast ripples are both

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produced by pyramidal cell action potentials.

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As shown in Fig. 2B and C, HFO occurrence during pre-ictal and ictal periods in

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pilocarpine-treated epileptic rats markedly differ between LVF and HYP onset seizures. During

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both pre-ictal and ictal period, LVF seizures were associated with a preponderant increase in

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ripple occurrence, while HYP seizures were mostly characterized by increased occurrence of

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fast ripples (Lévesque et al., 2012). Similar results had also been previously reported in kainic

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acid- treated epileptic rats (Bragin et al., 2005). Therefore, according to what was summarized

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in the previous paragraph, evidence obtained to date suggest that distinct transmitter signaling

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(and underlying neuronal network activity) predominate during LVF and HYP seizures recorded

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from epileptic animals; specifically, LVF onset should be mirrored by increased interneuron

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(GABAergic cell) activity while excitatory (glutamatergic) cells should be the main actors in the

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initiation of HYP onset seizures (Lévesque et al., 2012).

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This hypothesis is supported by a recent study in which multiunit activity recordings

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were obtained during spontaneous seizures in awake epileptic animals treated with kainic acid

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(Grasse et al., 2013). These investigators discovered that LVF seizures are associated with

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increased interneuron activity at onset followed by intense firing generated by principal cells. No

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in vivo study has yet investigated the activity of interneurons and principal cells during HYP

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onset seizures in animal models of MTLE, although this aspect has been addressed in the feline

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neocortex in an in vivo acute preparation (Timofeev et al., 2002; Grenier et al., 2003).

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The hypothesis that the inititation of LVF onset seizures mainly rests on GABAergic

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function has been further tested in vivo by Salami et al. (2015); they induced seizures in normal,

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control rats - which were chronically implanted with depth electrodes - through the systemic

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injection of the K+ channel blocker 4-aminopyridine, which is known to enhance both

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glutamatergic and GABAergic transmission (Buckle and Haas, 1982; Rutecki et al., 1987;

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Perreault and Avoli, 1991), or the GABAA receptor antagonist picrotoxin (De Groat et al., 1972).

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These experiments have shown that 4-aminopyridine causes seizures with LVF onset in the

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majority of cases, while seizures induced by picrotoxin are consistently characterized by HYP

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onset. In addition, HFO analysis revealed that 4-aminopyridine-induced LVF seizures are

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associated with higher ripple rates compared to fast ripples, whereas picrotoxin-induced

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seizures contained significantly higher rates of fast ripples compared to ripples (Salami et al.,

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2015).

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Seizure onset patterns in in vitro models of epileptiform synchronization

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Electrograhic activity closely resembling the ictal discharges recorded in epileptic patients and in

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in vivo animal models can also be reproduced in several in vitro preparations such as the brain

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slice (Fig. 1C), the isolated hippocampus, and the guinea pig whole brain (see for review: Avoli

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and Jefferys, 2016; de Curtis et al., 2015; de Curtis and Avoli, 2016). In these studies, both LVF

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and HYP onset seizure-like discharges can be recorded from several limbic and extralimbic

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areas, and to review in detail these data is beyond the goal of this paper. However, a few

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remarks should be made. First, long-lasting periods of epileptiform synchronization, resembling

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ictal discharges, are rarely observed during pharmacological manipulations that fully antagonize

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GABAA receptor signaling, at least when employing adult brain tissue (see for review: Avoli and

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de Curtis, 2011; Avoli and Jefferys, 2016). Second, HYP onset ictal discharges are commonly

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seen when cortical tissue is perfused with medium containing low Mg2+ (Derchansky et al.,

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2008; Zhang et al., 2012) but are less frequently recorded during bath application of 4-

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aminopyridine (Lopantsev and Avoli, 1998b; Avoli et al., 2013) with the notable exception of

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experiments carried out in the perirhinal cortex (Biagini et al., 2013; Köhling et al., 2016). In fact,

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as reviewed in detail by Avoli and de Curtis (2011), this K+ channel blocker readily and most

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often induces ictal discharges that closely resemble an LVF seizure onset in several limbic and

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extralimbic structures (Figs. 1C and 3A & B).

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LVF seizure-like discharges recorded in vitro during application of 4-aminopyridine

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have been extensively studied in our laboratories, employing both rodent brain slices and the

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guinea pig whole brain preparation. When intracellularly recorded from principal (glutamatergic)

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cells of the entorhinal cortex in a brain slice preparation, the LVF onset coincides with a

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depolarization that is associated with few if any action potentials and becomes hyperpolarizing

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when the neuron membrane potential is brought to values less negative than -60 mV with

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steady injection of depolarizing current (Lopantsev and Avoli, 1998a) (Fig. 3A). Therefore, the

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LVF onset of 4-aminopyridine-induced ictal discharges appears to be associated with a robust

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synchronous inhibitory event; this view is supported by studies in which powerful interneuron

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discharges could be recorded at the onset of these ictal discharges (Ziburkus et al., 2006; de

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Curtis et al., 2015; Uva et al., 2015; Lévesque et al., 2016) as well as by the ability of GABAA

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receptor antagonists to abolish this event along with ictal discharge occurrence (Avoli et al.,

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1996; Lopantsev and Avoli, 1998a). It should be noticed that short-lasting depolarizations with

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similar pharmacological and electrophysiological (e.g., few action potentials and reversal

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potentials) characteristics are associated with the interictal-like discharges (Fig. 3A, arrow) that

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occur in vitro in the hippocampus and entorhinal cortex during 4-aminopyridine treatement (Avoli

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et al., 1996; Lopantsev and Avoli, 1998; Uva et al., 2009).

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It has also been demonstrated that the negative shift of the local field potential

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observed at the onset of an LVF seizure correlates with elevations in [K+]o (Fig. 3B) that rest on

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excessive GABAergic signaling (Avoli et al., 1996; reviewed by Avoli and de Curtis, 2011; Avoli

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et al., 2013; de Curtis and Avoli, 2016), leading to intracellular Cl- accumulation and subsequent

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activity of the KCC2 co-transporter that extrudes both Cl- and K+ from the intraneuronal

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compartment (Viitanen et al., 2010). Such increases in [K+]o should depolarize neighbouring

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neurons thus causing hyperexcitability as suggested by the occurrence of small amplitude,

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presumably ectopic spikes (Lopantsev and Avoli, 1998a; Trombin et al., 2011; Kaila et al., 2014;

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but also see Avoli et al., 1998). In addition, an elevation in [K+]o supports the emergence of

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neuronal network resonance, thus generating oscillatory patterns in the beta-gamma range

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(Bartos et al., 2007) (see also next paragraph), and should cause a positive shift of the

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membrane reversal of the GABAA receptor mediated inhibitory postsynaptic potential therefore

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weakening inhibition (Jensen et al., 1993). The notion that elevations in [K+]o increase neuronal

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excitability and cause seizure activity has been firmly established over the last few decades in

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both in vivo (Zuckermann and Glaser, 1968) and in vitro (Traynelis and Dingledine, 1988)

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preparations. Moreover, the role played by KCC2 activity in LVF onset seizure initiation and

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maintainance is supported by recent evidence showing that ictal discharges induced by 4-

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aminopyridine are abolished or facilitated by inhibiting or enhancing the activity of this co-

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transporter, respectively (Hamidi and Avoli, 2015).

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As illustrated in Fig. 3C, LVF ictal discharge in the entorhinal cortex of the isolated

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guinea pig brain can also be induced by a short lasting arterial perfusion of bicuculline, a

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pharmacological procedure that reduces inhibition efficacy only by approx. 30%, as also

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suggested by the ability of piriform or entorhinal cortical cells to generate a robust inhibitory

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post-synaptic potential following electrical activation of the lateral olfactory tract under this

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experimental condition (Fig. 3D). In this specific in vitro model of ictogenesis, LVF seizures are

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initiated by fast field oscillations in the beta-gamma range that are mirrored by intracellular

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hyperpolarizing potentials becoming of smaller amplitude as seizure progresses (Gnatkovsky et

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al., 2008) (Fig.3C). In addition, similar to what was observed in the experiments performed with

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4-aminopyridine, these LVF onset events corresponded to elevation in [K+]o along with the

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occurrence of ectopic action potentials (Gnatkovsky et al., 2008; Trombin et al., 2011).

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As already mentioned, HYP onset ictal discharges have been analyzed in cortical

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tissue bathed with medium containing low Mg2+ (Derchansky et al., 2008; Zhang et al., 2012),

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an experimental manipulation that is known to weaken GABAA receptor signaling over time

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(Whittington et al., 1995). Interestingly, maintained GABAA receptor-mediated activity was

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initially reported by Derchansky et al. (2008) during the transition to HYP seizure activity in the

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isolated immature mouse hippocampus; however, further experiments carried out in the same

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laboratory have shown that HYP seizure onset is characterized by “exhaustion of presynaptic

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release of GABA, and unopposed increase in glutamatergic excitation” (Zhang et al., 2012).

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More recently, we have reported that ictal discharges with HYP onset features can also occur in

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the perirhinal cortex in brain slices superfused with 4-aminopyridine medium (Fig. 3E) (Biagini et

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al., 2013; Köhling et al., 2015).

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By employing intracellular recordings from principal cells of the perirhinal cortex we

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have found that the recurrent field spikes typical of a HYP onset are characterized by

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intracellular depolarizations associated with action potential bursting as well as that the post-

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burst hyperpolarizations (presumably caused by activation of post-synaptic GABAA receptors)

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gradually decrease in amplitude (Fig. 3E, arrowheads), a phenomenon that is characterized by

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a gradual positive shift in their reversal potential (Köhling et al., 2015). In addition, these

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changes were accompanied by a progressive increase in the associated transient elevations in

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[K+]o (Fig. 3F, arrowheads). While these data are in line with the conclusions proposed by Zhang

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et al. (2012), who identified a progressive impairment of inhibition at HYP onset with

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concomitant unrestrained enhancement of excitation, they also reveal an additional mechanism

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of inhibition weakening that may rest on progressively larger accumulations in [K+]o due to the

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postsynaptic activation of GABAA receptors.

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Optogenetic approaches reveal the involvement of specific neuronal networks

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in different seizure onset patterns

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The pivotal role played by interneurons in the initiation of ictal discharges characterized by an

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LVF onset has recently been confirmed with optogenetic techniques in the entorhinal cortex

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during 4-aminopyridine treatment (Shiri et al., 2015a; Yekhlef et al., 2015). It was shown in

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these studies that optogenetic stimulation of parvalbumin- or somatostatin-positive interneurons

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can initiate ictal LVF onset events similar to those occurring spontaneously (Fig 4A). In addition,

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as illustrated in the expanded traces of Fig. 4Aa (arrows), the onset of an ictal discharge

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induced by optogenetic stimulation of parvalbumin-positive cells presented with the typical

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pattern consisting of one or two interictal-like transients leading to fast, beta-gamma activity

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marking the beginning of the tonic phase. Interestingly, Shiri et al. (2015a) found that during

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both spontaneous and stimulated LVF discharges, ripples rates predominated at ictal onset (Fig.

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4Ab).

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However, as shown in Fig. 4B, LVF ictal events that occurred spontaneously during 4-

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aminopyridine application virtually switched to HYP onset events when the calcium/calmodulin-

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dependent protein kinase II-positive principal cells were optogenetically stimulated in the

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entorhinal cortex (Shiri et al., 2016). Specifically, the onset of ictal discharges triggered by

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principal cell activation was characterized by repeated field spikes, thus closely resembling a

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HYP onset pattern. In addition, these optogenetically induced HYP onset ictal events were

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found to be associated with higher fast ripple rates at onset (Fig. 4Bb). Therefore, these

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optogenetic experiments demonstrate that under identical conditions (i.e., during application of

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4-aminopyridine), activation of each specific cell population can generate ictal discharges with a

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different onset pattern: LVF onset events, similar to those occurring spontaneoulsly, depended

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on the optogenetic activation of interneuronal networks while HYP onset discharges rest on the

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optogenetic activation of glutamatergic principal cells.

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Concluding remarks

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The studies reviewed here underscore the concept that the “excessive” activity of interneurons

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(and the resulting activation of postsynaptic GABAA receptors) can be sufficient to disrupt the

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excitation/inhibition balance within forebrain neuronal networks thus leading to ictal activity

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(Avoli and de Curtis 2011; de Curtis and Avoli, 2016). This conclusion is in line with in vivo data

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obtained from epileptic patients (Truccolo et al., 2011; Schevon et al., 2012) and animal models

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(Grasse et al., 2013; Toyoda et al., 2015), in which seizure onset was shown to be associated

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with sustained firing of interneurons or with depressed or consistent firing activity of principal

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cells.

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We also propose that different types of neuronal networks (i.e., interneurons and

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principal, glutamatergic cells) and thus different neurotransmitter receptor signaling,

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predominate in MTLE at the onset of LVF and HYP ictal discharges. As summarized in the

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diagrams shown in Fig. 5, two different sets of cellular events/interactions are indeed likely to be

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at work during these two focal seizure onset patterns. Moreover, in light of the evidence

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obtained in human epileptic patients (Spencer et al., 1992; Velasco et al., 2000; Ogren et al.

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2009) as well as of the structure-dependent (Köhling et al. 2015) and optogenetic (Shiri et al,

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2016) findings identified in vitro, it is reasonable to hypothesize that the preponderant

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involvement of interneuronal or principal cell networks in LVF and HYP onset seizures,

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respectively, may reflect some distinctive features in network connectivity and/or in brain state

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excitability.

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The evidence reviewed here may provide insight to delineate better therapeutic targets

351

in the treatment of patients presenting with MTLE, and suggest that (theoretically) seizure onset

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patterns and associated HFOs should be taken into consideration in order to implement optimal

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pharmacological therapies. However, this last aspect may suffer a practical drawback since both

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types of seizure onset can also coexist in the same experimental condition both in vivo

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(Lévesque et al., 2012) and in vitro (Köhling et al., 2016) as well as in patients with MTLE (B.

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Frauscher, F. Dubeau and J. Gotman, personal communication). Finally, it should be

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emphasized that the findings summarized in this review focus on determinants of seizure onset

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types (i.e., on the mechanisms underlying ictogenesis), not on epileptogenesis. Therefore, the

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impact of different seizure onset patterns on the development of MTLE remains to be defined.

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Acknowledgements

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The original work reviewed here was supported by the CIHR (Grants 8109 and 74609 to MA,

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143208 to JG, and 119340 to SW), CURE (MA), the Fondazione Banca del Monte di Lombardia

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(2014-15 to MdC), the Italian Health Ministry (Ricerca Corrente 2014; grants RF-2010-2304417

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and RF-2007-GR141 to MdC), the Savoy Foundation (MA), and the Telethon Foundation (grant

366

GGP12265 to MdC). We thank Drs. M. Barbarosie, R. Benini, G. Biagini, M. D’Antuono, P. de

367

Guzman, S. Hamidi, V. Lopantsev, J. Louvel, R. Pumain, P. Salami and L. Uva as well as Ms. I.

368

Kurcewicz for contributing to some of the original experiments that were reported in this review.

369

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Legends to figures

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Figure 1 - Electrographic features of LVF and HYP seizure onsets in humans (A) as well

561

as in in vivo (B) and in vitro (C) experimental models. Traces shown in A were obtained

562

from hippocampal recordings performed during pre-surgical intracranial monitoring with depth

563

macro-electrodes in a patient with a focal cortical dysplasia of the temporal lobe (left trace) and

564

in a patient with malformation (i.e., altered rotation) of the hippocampus that was associated

565

with hippocampal sclerosis verified on magnetic resonance and histological examinations (right

566

trace). The asterisk in the LVF sample identifies the single, “sentinel’ spike that can occur at

567

onset, while asterisks in the HYP sample highlight the initial series of large amplitude spikes at

568

approx. 1 Hz; note in this case that spikes at a similar frequency (indicated by arrow-heads)

569

reappear during the initial part of the seizure. Traces in B were recorded from the CA3 area of

570

the hippocampus in a pilocarpine-treated epileptic rat; note that both types of seizure onsets

571

could be recorded from the same animal; asterisks identify specific EEG events as described for

572

the recordings shown in panel A. Traces in C were obtained from experiments performed with

573

rat brain slices maintained in vitro and bathed in medium containing 4-aminopyridine; the LVF

574

seizure-like onset shown on the left was recorded from the medial entorhinal cortex while the

575

HYP seizure on the right was obtained from the perirhinal cortex; these in vitro recordings were

576

perfomed with DC amplifiers and thus seizure-like discharges are associated with robust

577

negative-going shifts. Traces shown in A were kindly provided by Dr. Stefano Francione and Dr.

578

Laura Tassi of the Claudio Munari Epilepsy Surgery Center, Milano, Italy.

579 580

Figure 2 - Analysis of HFOs occurring before and during LVF and HYP seizures in

581

pilocarpine-treated epileptic rats. A: The left panel shows recordings from the CA3 region of

582

a pilocarpine-treated rat during an LVF seizure. The signal is shown in the wideband, ripple (80-

27


583

200 Hz) and fast ripple (250-500 Hz) frequency ranges. Note the occurrence of a ripple (boxed

584

dotted portion of the trace) with no co-occurrence of a fast ripple. The right panel shows

585

recording from CA3 during an HYP seizure in a pilocarpine-treated animal. Note in this case the

586

occurrence of a fast ripple (boxed dotted portion of the trace). B: Temporal distribution of HFOs

587

recorded during 18 LVF seizures in pilocarpine-treated animals. Note that ripples predominate

588

over fast ripples, especially after seizure onset. C: Temporal distribution of HFOs recorded

589

during 21 HYP seizures in pilocarpine-treated animals. Note the high occurrence of fast ripples

590

compared to ripples. Rates of ripples and fast ripples were compared using non-parametric

591

Wilcoxon signed rank tests followed by Bonferroni-Holm corrections for multiple comparisons (*

592

p < 0.05). Data shown in this figure were obtained during the experiments published by

593

Lévesque et al. (2012)

594 595

Figure 3 - Field, [K+]o, and intracellular recordings obtained in vitro during LVF onset

596

seizure-like dischargers. A: Simultaneous field (Field) and intracellular (-68 mV) recordings

597

obtained from the entorhinal cortex of a rat brain slice during bath application of 4-

598

aminopyridine; in this and following panels the dotted lines indicate the resting membrane

599

potential of the neuron recorded intracellularly. Traces shown in the insert were obtained from a

600

different neuron during active depolarization with intracellular current from a resting membrane

601

potential at approx. - 62 mV. B: Field and [K+]o activities recorded during application of 4-

602

aminopyridine from the deep layers of the rat entorhnal cortex; the dotted line indicates the [K+]o

603

base level. C: Simultaneous field (Field) and intracellular (-65 mV) recordings obtained from a

604

principal cell in the entorhinal cortex during the perfusion of 50 µM bicuculline for 3 minutes in

605

the in vitro isolated guinea pig brain. The spectrogram on the top illustrates the fast activity at

606

around 20 Hz at the onset of the seizure-like event. The fast activity occurring at the onset of the

28


607

seizure-like discharge is expanded in the insert where the principal cell was depolarized by

608

intracellular injection of steady current. D: Field and intracellular recordings of the response

609

induced by lateral olfactory tract stimulation in an entorhinal cortex neuron with a resting

610

membrane potential of -60 mV. Note that the brief IPSPs that correlate to the fast activity

611

oscillations have reversal potentials similar to what is seen with the lateral olfactory tract-evoked

612

IPSP, E: Simultaneous field and intracellular recordings from a principal cell in the perirhinal

613

cortex of a rat brain slice during bath application of 4-aminopyridine; note that ictal discharge

614

onset is characterized by preictal spiking acceleration as well as that both interictal (arrow) and

615

“preictal” discharges consist of depolarizations with action potential bursts followed by a

616

hyperpolarizing potential that progressively decreases in amplitude (arrow heads) and coincides

617

with more intense action potential bursting. F: Simultaneous field and [K+]o recordings obtained

618

in the deep layers of the perirhinal cortex during 4-aminopyridine application. Note the

619

progressive increases in [K+]o that accompany the preictal spikes up to ictal discharge initiation

620

that coincides with values larger than 6.3 mM. Data shown in this figure were obtained during

621

experiments that have been published by Avoli et al. (1996, 2013), Biagini et al. (2013);

622

Gnatkovsky et al. (2008); Köhling et al. (2016); Lopantsev and Avoli (1998a and b); Trombin et

623

al. (2011); and Uva et al. (2015).

624 625

Figure 4 – Optogenetic activation of interneurons or principal cells leads to LVF or HYP

626

ictal discharges respectively. A: In a, LVF onset ictal discharges occurring spontaneously

627

during bath application of 4-aminopyridine (top) and during parvalbumin positive-interneuron

628

light stimulation (bottom); one event under each condition is further expanded to reveal the

629

onset pattern. Stimuli were 1 s long and delivered at 0.2 Hz for 30 s. Note that in both

630

spontaneous and stimulated events, the ictal discharge is preceded by one or two negative-

29


631

going interictal field potentials (arrows). In b, plots of the average rate of ripples and fast ripples

632

occurring during spontaneous (top) and optogenetically stimulated (bottom) ictal discharges (n =

633

10 events were used for both plots). Note that ripple rates are significantly higher than fast ripple

634

rates at the onset of both LVF discharges (* p < 0.01). B: In a, spontaneous LVF ictal

635

discharges occurring during bath application of 4-aminopyridine are shown in the top panel

636

while those induced by optogenetic stimulation of calcium/calmodulin-dependent protein kinase

637

II-principal cells are illustrated in the bottom; light pulses were 20 ms long and were delivered at

638

2 Hz for 30 s; note that this procedure triggers ictal discharges preceded by repeated spiking

639

(arrows), a pattern that is characteristic of HYP onset events. In b, plots of the average rate of

640

ripples and fast ripples occurring during spontaneous (top) and optogenetically stimulated

641

(bottom) ictal discharges; note that the stimulated HYP events are characterized by higher fast

642

ripple rates at onset 10 events were used for both plots (* p < 0.01). Data shown in this figure

643

were obtained from the experiments published by Shiri et al. (2015, 2016).

644 645

Figure 5 – Flow diagram of the dysfunctional mechanisms leading to LVF and HYP

646

seizure onset. Experimental evidence taken into account for providing the mechanisms

647

involved in LVF seizure onset was provided in the following studies: Avoli et al., 1996;

648

Barbarosie et al., 2000; Gnatkovsky et al., 2008; Trombin et al., 2011; Grasse et al., 2013;

649

Lévesque et al., 2016; Lopantsev et al., 1998a; Uva et al., 2015; Ziburkus et al., 2006. Data

650

identifying the mechanisms leading to HYP seizure onset originate from the following studies:

651

Lopantsev and Avoli, 1998b; Derchanski et al., 2006; Zhang et al., 2012; Köhling et al., 2016.

30