Seizure development in the acute intrahippocampal epileptic focus

Currently, an epileptic seizure is considered to involve a temporary network that exists for a finite period of time. Formation of this network evolves through spread of epileptiform activity from a seizure onset zone (SOZ). Propagation of seizures evoked by kainic acid injection in hippocampus to different brain areas was analyzed at macro- and micro-intervals. The mean latency of seizure occurrence in different brain areas varied between 0.5 sec and 85 sec (mean 14.9 ± 14.5 (SD)), and it increased after each consecutive seizure in areas located contralateral to the area of injection, but not in the ipsilateral sites. We have shown that only 41% of epileptic individual events in target brain areas were driven by epileptic events generated in the SOZ once the seizure began. Fifty-nine percent of epileptiform events in target areas occurred one millisecond before or after events in the SOZ. These data illustrate that during seizure maintenance, only some individual epileptiform events in areas outside of SOZ could be consistently triggered by the SOZ; and the majority must be triggered by a driver located outside the SOZ or brain areas involved in ictal activity could be coupled to each other via an unknown mechanism such as stochastic resonance.


Results
LFP epileptiform patterns in the injection area. KA was injected when animals were awake, and their baseline electrical activity revealed prominent theta rhythm with superimposed gamma activity in most brain areas. The immediate consequence of KA in the injection area was suppression of electrical activity. In different animals, the period of suppression could last from several seconds to several minutes (Fig. 1A). After this temporal suppression, electrical activity, including theta and gamma bands, recovered (Fig. 1B). Then an increase in amplitude of 20-50 Hz (gamma frequency band) occurred, which continued to increase in amplitude for 5-30 minutes, after which the frequency suddenly dropped to 1-3 Hz activity with a combination of slow waves and fast components in the frequency band of 20 to 200 Hz. This is illustrated in Fig. 1B, where an intermittent increase of gamma events with parallel suppression of theta activity appears during the second minute after KA Propagation of recurrent epileptiform activity. Total latency descriptive data are presented in Table 1.
A total of 478 measurements of latencies from SOZ to seven recording areas (see methods) were quantified in this study. Mean propagation latency for the first seizure was 14.95 seconds, with a relatively large variation from a minimum of 0.5 seconds to a maximum of 85 seconds.

Generators of individual epileptiform events.
We had assumed that epileptiform activity in areas outside the site of injection would be driven by hypersynchronous discharges from the area of injection. Continuous calculation of wavelet magnitude-based coherence provided more comprehensive information about relationships between the SOZ and other brain areas. For all seizures we observed an initial increase in coherence in the theta frequency band after occurrence of seizures at the point of injection, followed by an increase of coherence in the gamma frequency band. Specifically, we observed significant increases of coherence amplitude in RPH&LEC On the level of micro-intervals, by measuring the time differences between individual epileptiform events recorded from the area of seizure onset and other brain areas, we observed the following: Fig. 4 illustrates superimposed LFP events during different times during the development of the seizure presented in Fig. 3. Before the spread of ictal activity (Fig. 4a) there were no visible phase-locked changes in LFPs recorded in other brain areas. At the beginning of the recruitment process there was no relation to, nor high dispersion of, LFP peaks in other brain areas with respect to the peak epileptiform event amplitude recorded in the SOZ (Fig. 4a,b). Later, as the seizure developed, the dispersion of ictal event amplitudes decreased and ictal events in other brain areas located within 3-12 mm distance from the SOZ occurred simultaneously, or even before ictal events recorded in the SOZ (Fig. 4c,d). Additional examples of coherence of ictal activity in three other rats are presented in Fig. 5 and Table 2. Figure 5A illustrates that in RAH, REC, RPir, which are remote from the area of injection, 3-7 mm ictal events occurred 7 ms earlier than at the point of injection, and in parts B,C epileptiform events occurred within 5 ms in all brain areas regardless of their location in the brain. These epileptiform LFPs are associated with an increase in synchronization of multiunit discharges (Fig. 5D) and a peak of synchronization of MUD may occur on ascending as well as on the descending phases of LFPs. Detailed analysis of MUD-LFPs relationship is a subject of another paper.
Overall, the mean latency of epileptiform events to the areas located ipsilateral to the area of injection varied from 5.0 to 5.5 ms (5.2 ± 3.8 ms), which was significantly shorter (p < 0.001) than the latency of epileptiform events to the contralateral sites (range 9.6-14.1 ms, 11.4 ± 5.9ms). Considering the physical distance between recording sites, we can approximately estimate the speed of propagation of the electrical signal between the area of injection and ipsilateral entorhinal cortex = 0.4 m/s, and between the area of injection and contralateral hippocampus and entorhinal cortex = 0.8-1.0 m/s. However, analysis of individual cases revealed that in ten animals (59%) at least one recording site had only a 1-2ms delay from the epileptiform LFP recorded in the area of injection (see Table 2, numbers in "1") and in six animals (33%) epileptiform events in areas outside the area of injection occurred 3-10ms earlier than at the site of injection (zeros outside the area of injection).

Discussion
Some properties of seizures induced by KA injection and their relation to spontaneous seizures where described in our previous publications [16][17][18] . Here we will focus our discussion on propagation of seizure activity in acute conditions.
There are two findings in this study: (1) After intrahippocampal injection of KA, ictal activity propagates from an acute epileptic focus to other brain areas within a period of several seconds to several tens of seconds, and (2) Once the seizure begins, epileptiform events in target areas are not necessarily driven by epileptiform events from SOZ.
Although the pattern of seizure spread after systemic and intrahippocampal KA injection has been described in numerous publications 19,20 (see also review Levesque and Avoli, 2013 21 ), in the current study we have performed recordings directly from the acute epileptic focus and sampled a high number of target sites during the ictal discharges. Formalized criteria were applied for measurements of latency of propagation of epileptiform activity. The latency between epileptic events in the SOZ and ipsilateral sites was 10.46 sec and to contralateral sites 19.26 sec. The latency of propagation of ictal discharges described in patients with epilepsy is in the same range and varies from 0.6 to 34 seconds 2,9,22 . Considering that a volume of rat brain is ~2 cm 3  human brain is ~1260 cm 3, 23 , we can conclude that the factor regulating propagation of ictal discharges does not necessarily depend on the distance between the SOZ and target brain areas. Moreover, such speed is too slow to explain propagation via simple synaptic or non-synaptic pathways. As indicated in several previous publications, slow propagation of ictal activity is mainly influenced by seizure-suppressing mechanisms in each individual brain area. There are several potential mechanisms opposing seizure propagation: homeostatic, ionic, and metabolic 24,25 , and so far only one of them, feedforward inhibition, has been described in detail for propagation of neocortical seizures [26][27][28][29][30][31] .
Similar studies are absent for seizures propagating between non-neocortical brain areas; however we can assume that feed-forward inhibition could be one of the mechanisms that control propagation of ictal discharges between different limbic brain areas.
Our study suggests that innate seizure-preventing mechanisms are different for ipsilateral and contralateral connections. The latency of seizures propagated to ipsilateral sites did not change for repeated seizures, while in contralateral target areas it increased for each consecutive seizure. At present, it is not clear whether the strength of feedforward inhibition between the SOZ and contralateral target areas becomes stronger after repeated seizures, or whether other properties of seizure-preventive mechanisms are involved in this process.
The occurrence of a seizure is considered to be involved in the formation of neuronal networks with specific functional connections between brain areas involved in ictal activity 24,32-37 , where recorded structures can be considered as nodes and functional connections between them as edges. There is controversy regarding how network elements functionally interact during a seizure. According to some data before and during seizure activity, elements of the seizure network become desynchronized 33,38 , while other publications describe an increase in synchronization measured by different methods 36 . One of the reasons for this controversy could be that there are different types of experimental seizures used for analysis, and different frequency bands used for assessing of coherence. In our experiments, involvement of brain areas into ictal activity was associated with an increase in spatial synchronization of electrical activity between SOZ and target areas in the theta frequency band and later in the gamma frequency band. It is logical to assume that coupling of individual ictal events contributes to increased coherence, and these individual ictal events could be driven by epileptiform events from SOZ. It would indicate that epileptiform events from SOZ are connected via morphological pathways to target areas, and we would expect dependence of the latency of epileptiform events from the SOZ to the target areas to be dependent on distance. However, as illustrated in Fig. 3 an increase in coherence between SOZ is different with different brain areas and does not depend on the distance between SOZ and recorded brain area. Moreover, in almost 60% of rats, epileptiform events in target brain areas occurred within only a 1ms delay from epileptiform events recorded in the SOZ, or even before. This may indicate the existence of a common drive for all epileptiform events during some seizures. Such a common drive could be the septum. Indirectly, this indicates an increase in coherence in the theta frequency band at the seizure onset and the septum is a main generator of theta rhythm 39,40 . Some publications have suggested the importance of septum in propagation of hippocampal seizures 41,42 ; however, none measured the temporal relationship among individual epileptiform events recorded from multiple brain areas. Direct measurements from septum and other brain areas are necessary to enable us to explore its role in driving epileptiform events during seizures. Another explanation for the near-simultaneous occurrence of ictal events in remote brain areas during a seizure would be the oscillating networks and coupling between epileptiform events in the SOZ and target brain areas due to stochastic resonance mechanism, as described in some publications [43][44][45] .

Methods
Adult Sprague-Dawley rats (male, weight 250-300 g) were used in these experiments. All procedures described in this study were approved by the Institutional Animal Care and Use Committee of the David Geffen School of Medicine and experiments were performed in accordance with the relevant guidelines and regulations of NIH. A total of 17 rats were used in these experiments, a total of 1,421 seizures were recorded, and we limited our analysis to 85 seizures, consisting of the first five seizures in each animal.
Microelectrode implantation. Animals were anesthetized with isoflurane and fixed into a stereotaxic frame. A guide cannula (200 µm OD) was implanted into the CA3 region of the right posterior hippocampus (RPH), coordinates according to 46 : anterior-posterior (AP), −5.0; medial-lateral (ML), 5.0; dorsal-ventral (DV), 5.3. The outer part of the cannula's trunk was covered by varnish while the tip was exposed to record electrical activity at the site of injection and the magnet wire was soldered to the upper part of the cannula.  Injection protocol. After one of baseline recordings, KA was injected into posterior CA3 with a 10 µl Hamilton syringe that extended to a needle that fit into the injection cannula, which was 0.5 mm longer than the implanted guide cannula. KA was injected at a concentration of 8.6 mM in a volume of 0.2 µl and a flow rate of 0.1 µl/10 s. Histological procedures. At the end of the electrophysiological experiments, rats were deeply anesthetized and perfused with 2.5% paraformaldehyde. Brains were removed and placed in 2.5% paraformaldehyde for at least 48 h before histological sectioning and Nissl staining to verify electrode placements. On the horizontal histological sections, the electrode track was traced downward until it disappeared from the section, and the last section in which each electrode's track was visible was considered the site of recording. The location of recording sites from all 17 rats is presented in Supplement Fig. 1. Calculation of the volume of kainic acid spread. In three rats, 6 injections of methylene blue (20 nmol 48 ), were injected into the posterior hippocampus with the same speed and the same location as for the kainic acid injection (see Methods). Rats were euthanized one hour after injection and perfused with Data analysis. After histological verification of recording sites, the physical distance between the area of injection and each recording area was measured using a stereotaxic atlas 46 . On the basis of these measurements, the mean distance between the area of injection and the recording brain areas was the following: RAH = 3; REC = 3 mm; rPir = 7 mm; LAH = 7 mm; LPH = 10 mm; LPir = 11 mm; LEC = 12 mm. Data analysis focused on two questions: (1) propagation of ictal activity from the area of injection, which we considered as the seizure onset zone (SOZ), estimated by measurement of the latency to seizure occurrence in other recorded brain areas; and (2) generation of ictal discharges during ictal activity, which was estimated by calculating the temporal relationship among individual epileptiform events recorded from the SOZ and other brain areas.
Analysis was performed using DataPac software. High-pass filtering (1 Hz, butterworth, rolloff 3, with zero lag compensation) was applied to the raw data to remove the slow component of electrical activity. For detection of multiunit activity raw data were filtered by a high-pass filter (300 Hz, butterworth, rolloff 3, with zero lag compensation) and a threshold of 2 SD was set to detect unit discharges. The seizure onsets at the point of injection and at other brain areas were identified at the time when the amplitude of electrical activity crossed the threshold of 1-SD of the mean amplitudes for a period longer than 10 seconds. The propagation latencies in areas outside the SOZ were computed as the time difference between the beginning of seizures in the SOZ and the beginning of ictal discharges in recorded brain areas. Means, standard deviations, and ranges for seizure latency data were computed. Analyses of variance tests (ANOVA) were performed to investigate possible relationships of seizure onset latencies to seizure numbers (SN), distances from seizure-onset-zone (dSOZ), and location of brain areas in ipsilateral or contralateral brain sites (BS). Specifically, there were five levels of SN, sorted by the occurrence of seizures, namely, seizure #1 -#5; three groups of dSOZ: short (3 mm from the SOZ), medium (7 mm from the SOZ) and long (10-12 mm from the SOZ). In addition, BS was defined as either "ipsilateral" or "contralateral" to the (dSOZ). We considered the dSOZ and the BS as two unrelated factors because both are categorical variables of description of locations. Therefore, two separate statistical analyses were performed: (1) to test the main effects of SN and dSOZ and their interactions (SN × dSOZ) in seizure latencies, and (2) to examine the effects of SN and BS and their interaction (SN × BS) in the variance of seizure latency.
Analysis of generators of ictal discharges was performed by calculation of the coherence of electrical activity between the SOZ and other brain areas, as well as by the measurement of the temporal relationships between Rat ID Areas of recording RPH-injection RAH 3 mm REC 3 mm RPir-7 mm LAH-7 mm LPH-10 mm LEC-12 mm 1 0 n/a 5 n/a n/a 5 5 2 8 n/a 1 n/a n/a n/a 0 3 10 n/a 0 0 n/a n/a n/a 4 8 n/a 1 0 n/a 24 n/a 5 0 n/a 3 1 n/a 7 n/a 6 0 n/a 3 n/a 10 8 10 7 0 5 n/a n/a 1 8 8 3 n/a 0 n/a 33 n/a n/a 9 0 15 5 n/a n/a 15 10 Mean ± SD 5.2 ± 4.6 5.0 ± 3.7 5.5 ± 6.3 14.0 ± 11 9.6 ± 6.2 10.1 ± 5.8 Table 2. The relative latency and standard deviation of epileptiform events in milliseconds in relation to the injection point after kainic acid injection. Numbers in "0" indicate brain areas where seizure events occurred first, and numbers in "1" indicate brain areas where seizure events occurred within 1ms delay. Note: in cases where epileptiform events in other brain areas occurred earlier than in the area of KA injection (rats number 2,3,4,8,10,11), the latency still was calculated as if these events were propagated from the area of injection. n/a indicate those brain areas that were no involved into a current seizure activity. individual epileptiform events recorded in the SOZ and other brain areas. The peaks of epileptiform events in the SOZ were detected using DataPac software. Then event-triggered averages of signals recorded in other channels were determined. Amplitudes of averaged events were normalized and time differences between SOZ and other brain areas were calculated based on a point at half-amplitude of each epileptiform event (see Fig. 5 for illustration).