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Written informed consent was obtained from each subject prior to the investigation and the study received approval from the Ethics Committee of Masaryk University in Brno. Intracerebral EEG measures and their mathematical analyses were taken to document the process and the ethic committee approved this consent procedure. On the behalf of children participants involved in the study the written informed consent was provided by their caretakers.
Ten patients (six males and four females) ranging in age from 17 to 41 years (with an average age of 28.9 years, std. 8.21), all with medically intractable epilepsies, participated in the study (Table 1). Depth electrodes were implanted to localize seizure origin prior to surgical treatment. Each patient received 6–15 orthogonal platinum electrodes in the temporal and/or frontal, parietal, and occipital lobes using the stereotaxic coordinate system of Talairach [40]. Bilateral and multilobar investigations were conducted for most subjects. A total of 898 intracerebral sites were electrophysiologically investigated by means of 95 multicontact depth electrodes over the subjects (49 frontal, 31 temporal, 11 parietal, and 4 occipital). Standard semiflexible depth electrodes (ALCIS) with a diameter of 0.8 mm, a contact length of 2 mm, and an intercontact interval of 1.5 mm were used for invasive EEG monitoring. The exact positions of the electrode contacts in the brain were verified using postplacement MRI with electrodes in situ. Lesional anatomical structures and epileptogenic zone structures were not included in the analysis (recording sites from these structures are not included in Table 1 - last column). All subjects were on chronic anticonvulsant medication (usually slightly reduced due to the video-EEG monitoring) and all of them had normal or corrected-to-normal vision. All the patients were able to fully understand and perform the experimental task.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0063293.t001 Patient characteristics. aT, temporal; F, frontal; P, parietal; O, occipital; R, right; L, left; HS, hippocampal sclerosis; FCD, focal cortical displasia; DNET, dysembryoplastic neuroepitelial tumour; EMC, encephalomeningocele.
Subjects were seated comfortably in a moderately lit room with a monitor screen positioned approximately 100 cm in front of their eyes. During the examination, they were requested to continuously focus their eyes on the small fixation point in the centre of the screen and to minimize blinking. A standard visual oddball task was performed: two types of stimuli (frequent and rare) were presented in the centre of the screen in random order. Clearly visible yellow capital letters O (frequent) and X (rare; approx. 50 trials) on a black background were used as experimental stimuli. The duration of stimuli exposure was constant at 500 ms; the ratio of rare to frequent stimuli was 1∶5. The interstimulus interval randomly varied between 4 and 6 s. Each subject was instructed to respond to the rare (target) stimulus as quickly and accurately as possible by pressing a microswitch button in the dominant hand.
The EEG signal was simultaneously recorded from various intracerebral structures and a limited number of midline scalp electrodes (Fz, Cz, and Pz), using the 128 channel TrueScan EEG system (Deymed Diagnostic). All recordings were monopolar, with a linked earlobe reference. All impedances were less than 5 kΩ. Eye movements were recorded from a cathode placed 1 cm laterally and 1 cm above the canthus of the left eye, and from an anode 1 cm laterally and 1 cm below the canthus of the right eye. The sampling rate was 1024 Hz. Standard anti-aliasing filters were used. Occasional eye movements and muscle artefacts were off-line rejected manually and further processing was performed with artefact-free intracerebral EEG periods.
Data processing was carried out on monopolar montages with common reference and bipolar montages in order to distinguish between far field and local field effect to signal propagation. Bipolar montages were calculated by subtracting signal recorded from adjacent contacts belonging to the same intracerebral electrode. All the following processing is the same for both monopolar and bipolar montages. Artefact-free intracerebral EEG trials with target and frequent stimuli were analyzed separately. The number of artefact-free frequent stimuli was randomly reduced to obtain the same number of trials for target and frequent stimuli for each subject. The number of analyzed trials varied from 30 to 48 depending on the subject. The EEG signal was passband filtered and analyzed in six frequency bands: δ (2–4 Hz), θ (4–8 Hz), α (8–12 Hz), β (12–20 Hz), lower γ (20–45 Hz), and upper γ (55–95 Hz). EEG signal was filtered before segmentation. Filter based on Fourier transformation was used.
The time cross-correlation of contact pairs, given by Pearson’s correlation coefficient, was computed in overlapping time window moving over the whole length of trials. The length of the time windows for correlation computing were 500, 250, 125, 80, 60, and 30 ms in frequency bands δ, θ, α, β, lower γ, and upper γ respectively. The length of the window was at least one period of the lowest frequency in the analyzed band. The shift of the moving window was tenth the window width. No mutual time shift was supposed between contacts in analyzed pairs. In this case the changes of correlation may have the origin in the change of signal shape or in time shift between channels. Within subjects, the number of cross-correlation contact pairs varied from 1,275 to 6,441.
Signal’s power envelope within each trial was evaluated by Hilbert transform demodulation in selected frequency band. The stimuli-locked signals were particularly eliminated by subtracting averaged trial from all trials before demodulation computation.
The post-stimulus changes of correlation and power corresponding to the resting phase before the stimulation were evaluated. The statistically significant changes between the mean values in the reference period 100–700 ms before stimulus and the corresponding mean values in the moving window (one-third the width of the reference period) in the time area after stimuli were determined. The non-parametric Wilcoxon test for paired samples over trials was used. Statistical significance was computed separately for each subject and each frequency band, and for target and frequent stimuli. We adjusted p value to the number of contacts in each subject by the multiple-comparison Bonferroni correction. The post-stimulus statistical significance is the basis for the following analysis. All processing was performed using ScopeWin and Matlab software.
The results of the cross-correlation analysis were given in matrices in which lines correspond to contact pairs and columns correspond to time. Matrix values have only three levels representing statistically significant increase (red color), decrease (blue color) and no significant change of correlation (transparent) relative to baseline. The level of statistical significance was set to p<0.05 after Bonferroni correction. Such matrices were computed separately for each subject and each frequency band, and for target and frequent stimuli. They provide a time overview in what latency relative to stimuli (zero in time axis) the correlation increase/decrease is significant. These matrices are used as the input data for graphic presentation (Fig. 1) and numerical analysis. Such a way of correlation representation offer clear information about time distribution. Unfortunately, the spatial localization is difficult to reach.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0063293.g001 Matrices with significant correlation changes in three subjects; three frequency bands – δ (2–4 Hz), β (12–20 Hz), and upper γ (55–95 Hz), and two different stimuli – targets vs.frequents, for monopolar and bipolar montages.The correlation increase is highlighted in red, decrease in blue. Each colored line corresponds to changes in a contact pair in time (notice all pairs of investigated subject’s contacts are represented in individual matrices for each patient). Green vertical lines define interval 250–750 ms after stimuli.
To treat a spatial interpretation of our results it is necessary to select the time interval, here 250–750 ms after stimuli was chosen. In this time window the effect of targets is expected based on previous intracranial event-related potentials, event-related synchronization/desynchronization and coupling studies [13], [17], [28], [41]. Then two approaches for graphical representation of results have been used (Fig. 2). First post-stimulus interactions in the chosen time interval after stimuli among all investigated brain sites were arranged into the individual triangular matrices (Fig. 2 D). The single value of triangular matrices was given by ratio of the length of time period that represents significant increase/decrease of correlation to the entire interval length (500 ms). Each contact pair lines (Fig. 1) were then represented by one point in triangular matrix. The relative value of increase/decrease of significant correlation changes over given time window was represented by the level of red/blue color. When the increase/decrease of correlation appeared in whole selected time window, the red/blue color was full dark. Any shorter increase/decrease was presented by lighter color, dependent on total percentage of significant points in the time window. White color expresses that no significant change of correlation occurs in given time window.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0063293.g002 An example of spatial representation of post-stimulus interactions after targets between all investigated brain sites in one subject (No.7).Correlation results are arranged in the triangular matrices into groups according to brain structures (delimited by black lines)(D) and in graphic form of “glass brains” with linked pairs of investigated electrode contacts (A – Coronal, B – Sagittal, C – Axial). Matrix values and links are colored according to the percentage of duration of the increase (red) or decrease (blue) in cross-correlations within time window 250–750 ms after stimulation. Three selected frequency bands – δ (left panel), β (middle panel), and upper γ (right panel).
The second approach was to depict three orthogonal views of a transparent “glass brain" with significant cross-correlation changes [28] (Fig. 2 A,B,C). It is the same result as in triangular matrix including blue and red color meaning, but this approach might be better used for illustrating the results of spatial group analysis. All contact pairs spatially localized with significant decrease/increase of correlation are in glass brain linked with color lines. Grand average across all subjects of cross-correlation and power changes according to stimulus (target vs. frequent) were computed (Fig. 3). Within long interval 0 to 1.5 s after stimuli and over all correlation pairs or single contact power the relative increase/decrease was calculated. This value, obtained for each subject separately, is given by dividing the area that represents significant increase/decrease by the entire area. Entire area is defined by interval length (1500 ms) and number of correlation pairs (Fig. 1) or contacts (power). A non-parametric test for paired samples was finally used to test statistical significance of relative cross-correlation and power changes between target and frequent stimulus over all subjects. Grand average was computed in six frequency bands δ, θ, α, β, lower γ, and upper γ.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0063293.g003 Relative incidences of significant post-stimulus changes in inter-areal cross-correlations (A – monopolar; B – bipolar) and post-stimulus power changes (C – monopolar; D – bipolar) across all investigated subjects.Statistical significance of target/frequent differences is indicated by asterisks.
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