Aberrant fast spiking interneuronal activity precedes seizure transitions in humans.

There is active debate regarding how GABAergic function changes during seizure initiation and propagation, and whether interneuronal activity drives or impedes the pathophysiology. Here, we track cell-type specific firing during spontaneous human seizures to identify neocortical mechanisms of inhibitory failure. Fast-spiking interneuron activity was maximal over 1 second before equivalent excitatory increases, and showed transitions to out-of-phase firing prior to local tissue becoming incorporated into the seizure-driving territory. Using computational modeling, we linked this observation to transient saturation block as a precursor to seizure invasion, as supported by multiple lines of evidence in the patient data. We propose that transient blocking of inhibitory firing due to selective fast-spiking interneuron saturation-resulting from intense excitatory synaptic drive-is a novel mechanism that contributes to inhibitory failure, allowing seizure propagation.

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

Cell-type specific and multiscale dynamics of human focal seizures in limbic structures.

The relationship between clinically accessible epileptic biomarkers and neuronal activity underlying the transition to seizure is complex, potentially leading to imprecise delineation of epileptogenic brain areas. In particular, the pattern of interneuronal firing at seizure onset remains under debate, with some studies demonstrating increased firing and others suggesting reductions. Previous study of neocortical sites suggests that seizure recruitment occurs upon failure of inhibition, with intact feedforward inhibition in non-recruited territories. We investigated whether the same principle applies in limbic structures. We analysed simultaneous electrocorticography (ECoG) and neuronal recordings of 34 seizures in a cohort of 19 patients (10 male, 9 female) undergoing surgical evaluation for pharmacoresistant focal epilepsy. A clustering approach with five quantitative metrics computed from ECoG and multiunit data was used to distinguish three types of site-specific activity patterns during seizures, which at times co-existed within seizures. Overall, 156 single units were isolated, subclassified by cell-type and tracked through the seizure using our previously published methods to account for impacts of increased noise and single-unit waveshape changes caused by seizures. One cluster was closely associated with clinically defined seizure onset or spread. Entrainment of high-gamma activity to low-frequency ictal rhythms was the only metric that reliably identified this cluster at the level of individual seizures (P < 0.001). A second cluster demonstrated multi-unit characteristics resembling those in the first cluster, without concomitant high-gamma entrainment, suggesting feedforward effects from the seizure. The last cluster captured regions apparently unaffected by the ongoing seizure. Across all territories, the majority of both excitatory and inhibitory neurons reduced (69.2%) or ceased firing (21.8%). Transient increases in interneuronal firing rates were rare (13.5%) but showed evidence of intact feedforward inhibition, with maximal firing rate increases and waveshape deformations in territories not fully recruited but showing feedforward activity from the seizure, and a shift to burst-firing in seizure-recruited territories (P = 0.014). This study provides evidence for entrained high-gamma activity as an accurate biomarker of ictal recruitment in limbic structures. However, reduced neuronal firing suggested preserved inhibition in mesial temporal structures despite simultaneous indicators of seizure recruitment, in contrast to the inhibitory collapse scenario documented in neocortex. Further study is needed to determine if this activity is ubiquitous to hippocampal seizures or indicates a 'seizure-responsive' state in which the hippocampus is not the primary driver. If the latter, distinguishing such cases may help to refine the surgical treatment of mesial temporal lobe epilepsy.

Spatiotemporal spike-centered averaging reveals symmetry of temporal and spatial components of the spike-LFP relationship during human focal seizures.

The electrographic manifestation of neural activity can reflect the relationship between the faster action potentials of individual neurons and the slower fluctuations of the local field potential (LFP). This relationship is typically examined in the temporal domain using the spike-triggered average. In this study, we add a spatial component to this relationship. Here we first derive a theoretical model of the spike-LFP relationship across a macroelectrode. This mathematical derivation showed a special symmetry in the spike-LFP relationship wherein a sinc function in the temporal domain predicts a sinc function in the spatial domain. We show that this theoretical result is observed in a real-world system by characterizing the spike-LFP relationship using microelectrode array (MEA) recordings of human focal seizures. To do this, we present a approach, termed the spatiotemporal spike-centered average (st-SCA), that allows for visualization of the spike-LFP relationship in both the temporal and spatial domains. We applied this method to 25 MEA recordings obtained from seven patients with pharmacoresistant focal epilepsy. Of the five patients with MEAs implanted in recruited territory, three exhibited spatiotemporal patterns consistent with a sinc function, and two exhibited spatiotemporal patterns resembling deep wells of excitation. These results suggest that in some cases characterization of the spike-LFP relationship in the temporal domain is sufficient to predict the underlying spatial pattern. Finally, we discuss the biological interpretation of these findings and propose that the sinc function may reflect the role of mid-range excitatory connections during seizure activity.

Distinct signatures of loss of consciousness in focal impaired awareness versus tonic-clonic seizures.

Loss of consciousness is a hallmark of many epileptic seizures and carries risks of serious injury and sudden death. While cortical sleep-like activities accompany loss of consciousness during focal impaired awareness seizures, the mechanisms of loss of consciousness during focal to bilateral tonic-clonic seizures remain unclear. Quantifying differences in markers of cortical activation and ictal recruitment between focal impaired awareness and focal to bilateral tonic-clonic seizures may also help us to understand their different consequences for clinical outcomes and to optimize neuromodulation therapies. We quantified clinical signs of loss of consciousness and intracranial EEG activity during 129 focal impaired awareness and 50 focal to bilateral tonic-clonic from 41 patients. We characterized intracranial EEG changes both in the seizure onset zone and in areas remote from the seizure onset zone with a total of 3386 electrodes distributed across brain areas. First, we compared the dynamics of intracranial EEG sleep-like activities: slow-wave activity (1-4 Hz) and beta/delta ratio (a validated marker of cortical activation) during focal impaired awareness versus focal to bilateral tonic-clonic. Second, we quantified differences between focal to bilateral tonic-clonic and focal impaired awareness for a marker validated to detect ictal cross-frequency coupling: phase-locked high gamma (high-gamma phased-locked to low frequencies) and a marker of ictal recruitment: the epileptogenicity index. Third, we assessed changes in intracranial EEG activity preceding and accompanying behavioural generalization onset and their correlation with electromyogram channels. In addition, we analysed human cortical multi-unit activity recorded with Utah arrays during three focal to bilateral tonic-clonic seizures. Compared to focal impaired awareness, focal to bilateral tonic-clonic seizures were characterized by deeper loss of consciousness, even before generalization occurred. Unlike during focal impaired awareness, early loss of consciousness before generalization was accompanied by paradoxical decreases in slow-wave activity and by increases in high-gamma activity in parieto-occipital and temporal cortex. After generalization, when all patients displayed loss of consciousness, stronger increases in slow-wave activity were observed in parieto-occipital cortex, while more widespread increases in cortical activation (beta/delta ratio), ictal cross-frequency coupling (phase-locked high gamma) and ictal recruitment (epileptogenicity index). Behavioural generalization coincided with a whole-brain increase in high-gamma activity, which was especially synchronous in deep sources and could not be explained by EMG. Similarly, multi-unit activity analysis of focal to bilateral tonic-clonic revealed sustained increases in cortical firing rates during and after generalization onset in areas remote from the seizure onset zone. Overall, these results indicate that unlike during focal impaired awareness, the neural signatures of loss of consciousness during focal to bilateral tonic-clonic consist of paradoxical increases in cortical activation and neuronal firing found most consistently in posterior brain regions. These findings suggest differences in the mechanisms of ictal loss of consciousness between focal impaired awareness and focal to bilateral tonic-clonic and may account for the more negative prognostic consequences of focal to bilateral tonic-clonic.

Multiple sources of fast traveling waves during human seizures: resolving a controversy.

During human seizures organized waves of voltage activity rapidly sweep across the cortex. Two contradictory theories describe the source of these fast traveling waves: either a slowly advancing narrow region of multiunit activity (an ictal wavefront) or a fixed cortical location. Limited observations and different analyses prevent resolution of these incompatible theories. Here we address this disagreement by combining the methods and microelectrode array recordings (N=11 patients, 2 females, N=31 seizures) from previous human studies to analyze the traveling wave source. We find - inconsistent with both existing theories - a transient relationship between the ictal wavefront and traveling waves, and multiple stable directions of traveling waves in many seizures. Using a computational model that combines elements of both existing theories, we show that interactions between an ictal wavefront and fixed source reproduce the traveling wave dynamics observed in vivo We conclude that combining both existing theories can generate the diversity of ictal traveling waves.Significance StatementThe source of voltage discharges that propagate across cortex during human seizures remains unknown. Two candidate theories exist, each proposing a different discharge source. Support for each theory consists of observations from a small number of human subject recordings, analyzed with separately developed methods. How the different, limited data and different analysis methods impact the evidence for each theory is unclear. To resolve these differences, we combine the unique, human microelectrode array recordings collected separately for each theory and analyze these combined data with a unified approach. We show that neither existing theory adequately describes the data. We then propose a new theory that unifies existing proposals and successfully reproduces the voltage discharge dynamics observed in vivo.

Single unit analysis and wide-field imaging reveal alterations in excitatory and inhibitory neurons in glioma.

While several studies have attributed the development of tumour-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neuron in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumour-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation.

Multiscale temporal integration organizes hierarchical computation in human auditory cortex.

To derive meaning from sound, the brain must integrate information across many timescales. What computations underlie multiscale integration in human auditory cortex? Evidence suggests that auditory cortex analyses sound using both generic acoustic representations (for example, spectrotemporal modulation tuning) and category-specific computations, but the timescales over which these putatively distinct computations integrate remain unclear. To answer this question, we developed a general method to estimate sensory integration windows-the time window when stimuli alter the neural response-and applied our method to intracranial recordings from neurosurgical patients. We show that human auditory cortex integrates hierarchically across diverse timescales spanning from ~50 to 400 ms. Moreover, we find that neural populations with short and long integration windows exhibit distinct functional properties: short-integration electrodes (less than ~200 ms) show prominent spectrotemporal modulation selectivity, while long-integration electrodes (greater than ~200 ms) show prominent category selectivity. These findings reveal how multiscale integration organizes auditory computation in the human brain.

Human interictal epileptiform discharges are bidirectional traveling waves echoing ictal discharges.

Interictal epileptiform discharges (IEDs), also known as interictal spikes, are large intermittent electrophysiological events observed between seizures in patients with epilepsy. Although they occur far more often than seizures, IEDs are less studied, and their relationship to seizures remains unclear. To better understand this relationship, we examined multi-day recordings of microelectrode arrays implanted in human epilepsy patients, allowing us to precisely observe the spatiotemporal propagation of IEDs, spontaneous seizures, and how they relate. These recordings showed that the majority of IEDs are traveling waves, traversing the same path as ictal discharges during seizures, and with a fixed direction relative to seizure propagation. Moreover, the majority of IEDs, like ictal discharges, were bidirectional, with one predominant and a second, less frequent antipodal direction. These results reveal a fundamental spatiotemporal similarity between IEDs and ictal discharges. These results also imply that most IEDs arise in brain tissue outside the site of seizure onset and propagate toward it, indicating that the propagation of IEDs provides useful information for localizing the seizure focus.

Neuronal Firing and Waveform Alterations through Ictal Recruitment in Humans.

Analyzing neuronal activity during human seizures is pivotal to understanding mechanisms of seizure onset and propagation. These analyses, however, invariably using extracellular recordings, are greatly hindered by various phenomena that are well established in animal studies: changes in local ionic concentration, changes in ionic conductance, and intense, hypersynchronous firing. The first two alter the action potential waveform, whereas the third increases the "noise"; all three factors confound attempts to detect and classify single neurons. To address these analytical difficulties, we developed a novel template-matching-based spike sorting method, which enabled identification of 1239 single neurons in 27 patients (13 female) with intractable focal epilepsy, that were tracked throughout multiple seizures. These new analyses showed continued neuronal firing with widespread intense activation and stereotyped action potential alterations in tissue that was invaded by the seizure: neurons displayed increased waveform duration (p < 0.001) and reduced amplitude (p < 0.001), consistent with prior animal studies. By contrast, neurons in "penumbral" regions (those receiving intense local synaptic drive from the seizure but without neuronal evidence of local seizure invasion) showed stable waveforms. All neurons returned to their preictal waveforms after seizure termination. We conclude that the distinction between "core" territories invaded by the seizure versus "penumbral" territories is evident at the level of single neurons. Furthermore, the increased waveform duration and decreased waveform amplitude are neuron-intrinsic hallmarks of seizure invasion that impede traditional spike sorting and could be used as defining characteristics of local recruitment.SIGNIFICANCE STATEMENT Animal studies consistently show marked changes in action potential waveform during epileptic discharges, but acquiring similar evidence in humans has proven difficult. Assessing neuronal involvement in ictal events is pivotal to understanding seizure dynamics and in defining clinical localization of epileptic pathology. Using a novel method to track neuronal firing, we analyzed microelectrode array recordings of spontaneously occurring human seizures, and here report two dichotomous activity patterns. In cortex that is recruited to the seizure, neuronal firing rates increase and waveforms become longer in duration and shorter in amplitude as the neurons are recruited to the seizure, while penumbral tissue shows stable action potentials, in keeping with the "dual territory" model of seizure dynamics.

Dual mechanisms of ictal high frequency oscillations in human rhythmic onset seizures.

High frequency oscillations (HFOs) are bursts of neural activity in the range of 80 Hz or higher, recorded from intracranial electrodes during epileptiform discharges. HFOs are a proposed biomarker of epileptic brain tissue and may also be useful for seizure forecasting. Despite such clinical utility of HFOs, the spatial context and neuronal activity underlying these local field potential (LFP) events remains unclear. We sought to further understand the neuronal correlates of ictal high frequency LFPs using multielectrode array recordings in the human neocortex and mesial temporal lobe during rhythmic onset seizures. These multiscale recordings capture single cell, multiunit, and LFP activity from the human brain. We compare features of multiunit firing and high frequency LFP from microelectrodes and macroelectrodes during ictal discharges in both the seizure core and penumbra (spatial seizure domains defined by multiunit activity patterns). We report differences in spectral features, unit-local field potential coupling, and information theoretic characteristics of high frequency LFP before and after local seizure invasion. Furthermore, we tie these time-domain differences to spatial domains of seizures, showing that penumbral discharges are more broadly distributed and less useful for seizure localization. These results describe the neuronal and synaptic correlates of two types of pathological HFOs in humans and have important implications for clinical interpretation of rhythmic onset seizures.

Role of paroxysmal depolarization in focal seizure activity.

We analyze the role of inhibition in sustaining focal epileptic seizure activity. We review ongoing seizure activity at the mesoscopic scale that can be observed with microelectrode arrays as well as at the macroscale of standard clinical EEG. We provide clinical, experimental, and modeling data to support the hypothesis that paroxysmal depolarization (PD) is a critical component of the ictal machinery. We present dual-patch recordings in cortical cultures showing reduced synaptic transmission associated with presynaptic occurrence of PD, and we find that the PD threshold is cell size related. We further find evidence that optically evoked PD activity in parvalbumin neurons can promote propagation of neuronal excitation in neocortical networks in vitro. Spike sorting results from microelectrode array measurements around ictal wave propagation in human focal seizures demonstrate a strong increase in putative inhibitory firing with an approaching excitatory wave, followed by a sudden reduction of firing at passage. At the macroscopic level, we summarize evidence that this excitatory ictal wave activity is strongly correlated with oscillatory activity across a centimeter-sized cortical network. We summarize Wilson-Cowan-type modeling showing how inhibitory function is crucial for this behavior. Our findings motivated us to develop a network motif of neurons in silico, governed by a reduced version of the Hodgkin-Huxley formalism, to show how feedforward, feedback, PD, and local failure of inhibition contribute to observed dynamics across network scales. The presented multidisciplinary evidence suggests that the PD not only is a cellular marker or epiphenomenon but actively contributes to seizure activity.NEW & NOTEWORTHY We present mechanisms of ongoing focal seizures across meso- and macroscales of microelectrode array and standard clinical recordings, respectively. We find modeling, experimental, and clinical evidence for a dual role of inhibition across these scales: local failure of inhibition allows propagation of a mesoscopic ictal wave, whereas inhibition elsewhere remains intact and sustains macroscopic oscillatory activity. We present evidence for paroxysmal depolarization as a mechanism behind this dual role of inhibition in shaping ictal activity.

Multiscale recordings reveal the dynamic spatial structure of human seizures.

The cellular activity underlying human focal seizures, and its relationship to key signatures in the EEG recordings used for therapeutic purposes, has not been well characterized despite many years of investigation both in laboratory and clinical settings. The increasing use of microelectrodes in epilepsy surgery patients has made it possible to apply principles derived from laboratory research to the problem of mapping the spatiotemporal structure of human focal seizures, and characterizing the corresponding EEG signatures. In this review, we describe results from human microelectrode studies, discuss some data interpretation pitfalls, and explain the current understanding of the key mechanisms of ictogenesis and seizure spread.

The ictal wavefront is the spatiotemporal source of discharges during spontaneous human seizures.

The extensive distribution and simultaneous termination of seizures across cortical areas has led to the hypothesis that seizures are caused by large-scale coordinated networks spanning these areas. This view, however, is difficult to reconcile with most proposed mechanisms of seizure spread and termination, which operate on a cellular scale. We hypothesize that seizures evolve into self-organized structures wherein a small seizing territory projects high-intensity electrical signals over a broad cortical area. Here we investigate human seizures on both small and large electrophysiological scales. We show that the migrating edge of the seizing territory is the source of travelling waves of synaptic activity into adjacent cortical areas. As the seizure progresses, slow dynamics in induced activity from these waves indicate a weakening and eventual failure of their source. These observations support a parsimonious theory for how large-scale evolution and termination of seizures are driven from a small, migrating cortical area.

Single unit action potentials in humans and the effect of seizure activity.

Spike-sorting algorithms have been used to identify the firing patterns of isolated neurons ('single units') from implanted electrode recordings in patients undergoing assessment for epilepsy surgery, but we do not know their potential for providing helpful clinical information. It is important therefore to characterize both the stability of these recordings and also their context. A critical consideration is where the units are located with respect to the focus of the pathology. Recent analyses of neuronal spiking activity, recorded over extended spatial areas using microelectrode arrays, have demonstrated the importance of considering seizure activity in terms of two distinct spatial territories: the ictal core and penumbral territories. The pathological information in these two areas, however, is likely to be very different. We investigated, therefore, whether units could be followed reliably over prolonged periods of times in these two areas, including during seizure epochs. We isolated unit recordings from several hundred neurons from four patients undergoing video-telemetry monitoring for surgical evaluation of focal neocortical epilepsies. Unit stability could last in excess of 40 h, and across multiple seizures. A key finding was that in the penumbra, spike stereotypy was maintained even during the seizure. There was a net tendency towards increased penumbral firing during the seizure, although only a minority of units (10-20%) showed significant changes over the baseline period, and notably, these also included neurons showing significant reductions in firing. In contrast, within the ictal core territories, regions characterized by intense hypersynchronous multi-unit firing, our spike sorting algorithms failed as the units were incorporated into the seizure activity. No spike sorting was possible from that moment until the end of the seizure, but recovery of the spike shape was rapid following seizure termination: some units reappeared within tens of seconds of the end of the seizure, and over 80% reappeared within 3 min (τrecov = 104 ± 22 s). The recovery of the mean firing rate was close to pre-ictal levels also within this time frame, suggesting that the more protracted post-ictal state cannot be explained by persistent cellular neurophysiological dysfunction in either the penumbral or the core territories. These studies lay the foundation for future investigations of how these recordings may inform clinical practice.See Kimchi and Cash (doi:10.1093/awv264) for a scientific commentary on this article.

The role of inhibition in epileptic networks.

Inhibition plays many roles in cortical circuits, including coordination of network activity in different brain rhythms and neuronal clusters, gating of activity, gain control, and dictating the manner in which activity flows through the network. This latter is particularly relevant to epileptic states, when extreme hypersynchronous discharges can spread across cortical territories. We review these different physiological and pathological roles and discuss how inhibition can be compromised and why this predisposes the network to seizures.

The contribution of raised intraneuronal chloride to epileptic network activity.

Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl(-). Brief (1-10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl(-) level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002).