Pathological high-frequency oscillations (pHFOs) represent a new electrographic marker of epileptogenic tissue. Early after their discovery, pHFO analysis was introduced into the presurgical evaluation to better delineate the resection margin and to improve the outcome of surgery. pHFOs are classified according to their frequency into two main groups—ripples (80–250 Hz) and fast ripples (250–600 Hz) (1, 2). Although fast ripples are considered more specific for epileptogenic tissue than ripples, both types of pHFO can localize the epileptogenic zone or seizure onset areas in humans who undergo exploration with invasive electrodes (1, 2). Several studies have demonstrated the beneficial effect of complete resection of the pHFO generating areas on surgical outcome (3–5). The practical utilization of pHFOs in presurgical evaluation is substantially hindered by the inability to differentiate them from physiological high-frequency oscillations, such as hippocampal sharp-wave ripples (SWRs) as they display substantial frequency overlap (2, 6, 7). Currently, we do not have any effective tools, which can reliably discriminate between them. Identification of an approach to reliably discriminate between normal and pathological HFOs is a complex, but essential, issue to address if the properties of pHFOs are to be fully utilized in clinical practice.

One of the features of pHFOs is their responsiveness to antiepileptic drugs (AEDs). In the chronic pilocarpine model of temporal lobe epilepsy, pHFOs and seizure rate decrease after treatment with levetiracetam (8) or lacosamide (9). In humans, withdrawal of AEDs is associated with increased rate of pHFOs (10). A pharmacological test using AEDs could be a plausible strategy to discriminate physiological oscillations from pathological ones, providing rate of physiological HFOs is not decreased by AEDs.

In this proof-of-principle study, we explored the impact of a single dose of lacosamide or levetiracetam on the rate of SWRs—a hippocampal representative of physiological HFOs (11). SWRs play a crucial role in the process of coordinated memory reactivation and formation of long-term memory (12). We have tested the hypothesis that the application of a single therapeutic dose of levetiracetam or lacosamide does not affect SWR rate.

In total 43,011 SWRs were detected with 4,779 ± 3,125 events per animal. The average SWR rate was 16.0 events/min, which is congruent with studies focused on SWR in vivo (11, 23–25). SWR rates of all animals, after each injection, in both analyzed time windows, are shown in Figure 2. The data revealed the presence of individual variability in the SWR rate and response to the tested drugs. The crucial parameter for examination of the effect of an AED is the ratio between the SWR rate after AED injection and the SWR rate after control saline injection (Figure 3). The median ratio of the SWR rate between levetiracetam and the control was 0.91 and 0.98 for 30 min and 4.5 h after the injection, respectively (n = 9 animals). For levetiracetam, the ratios’ confidence intervals did not cross the equality margins 30 min and 4.5 h after injection (Figure 4). Therefore, SWR rates after levetiracetam and saline treatment can be considered equal within the equality margins. The Wilcoxon signed-rank test for ratios was non-significant for 30 min (p = 0.30) and 4.5 h after the injection (p = 0.57), with a post hoc power of 88%. For lacosamide, the median ratio of the SWR rate was 1.14 and 1.22 for each time epoch (n = 8 animals). The confidence intervals of the SWR rate ratios crossed the equality margins in both time windows. The Wilcoxon signed-rank test demonstrated a significant increase in SWR rate ratio 30 min after injection of lacosamide (p = 0.039). After 4.5 h the effect of lacosamide was non-significant (Wilcoxon signed-rank test; p = 0.11; power = 85%). Diazepam, which was used as a positive control, reduced the SWR rate compared to the equivalent volume of saline in all rats by >50% with a median ratio of 0.46 and 0.70 for each time window (Figure 4). However, statistical significance could not be reached due to the small number of rats (n = 3 animals; Wilcoxon signed-rank test; p = 0.25; power = 46%).

We have shown that a single dose of levetiracetam and lacosamide does not reduce the rate of SWRs—a representative of physiological HFOs. The ability to reliably differentiate between pathological and physiological HFOs represents a crucial step toward the clinical utilization of pHFOs as biomarkers of the epileptogenic zone (2, 7). Epileptic pHFOs display a spectral overlap with physiological HFOs such as SWRs or fast gamma activity. Fast ripples are considered to be exclusively of epileptic origin, but physiological activity with a frequency around ~600 Hz has been described in the neocortex. Matsumoto and colleagues described features, which discriminated task-related physiological HFOs from pathological ones (26). Pathological HFOs were characterized by a higher spectral mean, longer duration, and lower mean frequency. Other studies tried to discriminate HFOs according to their spatial distribution, relationship with sleep phases (27), background activity (28), phase relationship with slow waves (29), or using cognitive tasks (30). However, the practical implementation of these criteria is challenging. Experimental treatment with levetiracetam or lacosamide has been shown to reduce the both ripples and fast ripples in the pilocarpine model of temporal lobe epilepsy, even though the effects were region specific (8, 9). In this in vivo study, we demonstrate that levetiracetam does not decrease the rate of hippocampal SWRs. The confidence intervals were set to tolerate a 25% change in SWR rate after the treatment. Studies which explored the effect of levetiracetam and lacosamide on pHFOs showed that these drugs reduced the pathological ripple rate in hippocampal structures by an average of 57% for levetiracetam and by 43% for lacosamide (8, 9). Although we studied the effect of these AEDs only on specific subtype of physiological HFOs, these experimental results suggest that the procedure of pharmacological testing can be a plausible approach to facilitate discrimination between physiological and pathological HFOs. Both drugs are used very frequently and their withdrawal and subsequent introduction is a very common procedure in patients undergoing invasive explorations. As an alternative, the introduction of AEDs could be replaced by an intravenous application of levetiracetam at the end of monitoring. HFOs that are not altered by AEDs can be considered to be physiological HFOs.

The absence of a major effect of the AEDs on the rate of SWRs and suppression of pHFOs can be explained by the currently known mechanisms of action of the drugs and cellular mechanisms of HFOs (31, 32). SWRs are oscillations that reflect the activation of large neuronal ensembles in the hippocampal CA1 region, particularly during sleep. On the cellular level, the oscillation depends on the precise interaction between pyramidal cells and inhibitory interneurons. The fast inhibitory postsynaptic potentials on the membrane of principal neurons play a key role in the coordination of principal cell firing (11). Pyramidal cells fire heterogeneously during the SWR; some cells can fire during every successive event, while other cells can fire only occasionally (17, 33). The sequence of neuronal activity during SWRs replays the sequence of neuronal activation during behavioral tasks, and it is considered to represent a mechanism of memory reactivation and long-term memory formation (12, 34). The cellular dynamics of pHFOs in both ripple and fast ripple bands differs and is more uniform (31, 32). During each pHFO, a large population of cells generates a burst of high-frequency action potentials superimposed on a large depolarizing envelope (35–37). While high-frequency spiking depends mainly on fast sodium channel kinetics, the depolarizing envelope is associated with an increase in intracellular calcium via the opening of voltage-gated calcium channels or activation of non-NMDA and NMDA receptors. Lacosamide modifies voltage-gated sodium channel kinetics required for fast action potential firing, and it affects only neurons which are active or depolarized for prolonged periods of time and thus spares physiological functions (38). Therefore, pathological pHFOs are more susceptible to its effects than SWRs. The levetiracetam binds to SV2A, which is involved in trafficking and fusion of synaptic vesicles (39). It seems that levetiracetam reduces the vesicle release that is important for synaptic neurotransmission (40). It also partially blocks N-type calcium channels (41). However, the exact mechanisms of its actions are not well known, but we can assume that levetiracetam is capable of interfering with cellular processes involved in intense neuronal firing and pHFOs.

The absence of the effect of levetiracetam and lacosamide on the mechanisms of SWRs, in general, can also be deduced indirectly from the lack of their negative effect on cognitive functions including memory. It is well established that any drug or procedure, which has the capacity to interfere with the cellular mechanisms underlying the genesis of SWRs, also has the capacity to induce a memory deficit (42, 43). Levetiracetam is currently the drug of first choice, and lacosamide has also shown its therapeutic benefits as add-on therapy early after its introduction into epilepsy therapy. Both drugs are well tolerated by patients and demonstrate low adverse effects and an absence of a significant impact on cognition and memory (44–47). From electrophysiological perspective, these observations are supported by our experimental study, which demonstrates the absence of any suppressive effect of these drugs on the SWR generation. Moreover, lacosamide was shown to increase the speed of complex visual information processing (48), which may be in agreement with the transiently increased SWR rate observed in this study.

A possible limitation of this study is the single-dose scheme of drug administration. We might have missed the time period of SWR suppression by the drug. However, based on the known pharmacokinetics and mechanism of action of these drugs this seems unlikely (15, 16, 38, 49). Another possibility is that the drug was cleared from the body before its concentration in the brain could have reached sufficient intrathecal levels to influence the SWR rate. However, we used the same dose that was administered daily in studies exploring the effect of lacosamide and levetiracetam on pHFOs (8, 9) and pharmacokinetic studies showed that these drugs reach maximal brain concentration within 2 h after application (15, 16). Another weakness of the study is the single-dose application. We cannot exclude that prolonged or chronic application of these drugs may induce long-term changes, which would affect the properties of SWRs.

In conclusion, we have shown that levetiracetam does not change the rate of SWRs and lacosamide transiently increases it. Hence, these drugs can be considered for pharmacological testing to distinguish physiological versus pHFOs. Levetiracetam or lacosamide could be introduced toward the end of invasive exploration, and the response of HFOs to the drug introduction could be used to determine whether the HFOs are pathological or physiological. Moreover, our results are congruent with studies showing no negative effect of these drugs on cognition.

This study was carried out in accordance with the recommendations of the Animal Care and Animal Protection Law of the Czech Republic fully compatible with the guidelines of the European Union directive 2010/63/EU. The protocol was approved by the Ethics Committee of The Czech Academy of Sciences (Project License No. 71/2016).

Conceived and designed the experiments: PJ, JO, PM, and SH. Performed the experiments: JC, JK, and LK. Analyzed the data: JC, JK, LK, AP, PJ, and JO. Wrote the paper: JC, JK, PJ, KV, SW, PM, JO, and SH.

PM has received honoraria from UCB as a speaker and advisory board member. The remaining authors have no conflicts of interest. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.