In the sham sessions, electrodes were placed and triggers were set
as in the tSOS sessions, but the stimulator remained off. Post-experimental debriefing ensured that subjects were not aware of whether or not they had been stimulated. The EEG was recorded with Ag/AgCl electrodes placed at Fz, C3, Cz, C4, P3, Pz, and P4, according to the 10–20 system, all referenced to an electrode attached to the nose. The ground electrode was placed on the forehead (Fpz). Electrode impedances were < 5 kΩ. EEG signals were recorded with a Neurofax EEG-9200 (Nihon Kohden Corporation, Tokyo, Japan), and filtered between 0.05 and 30 Hz. Additionally, horizontal and vertical eye movements and a chin electromyogram were recorded for standard polysomnography and for artefact detection. All recordings were sampled at 500 Hz and stored for later offline analyses. Daporinad Sleep stages (1, 2, 3, and 4, and REM sleep), wakefulness time and movement artefacts were scored offline for 30-s intervals (Rechtschaffen
& Kales, 1968). Analyses of the acute effects of tSOS on the sleep EEG signal during the 4-min periods of stimulation were focused on the spindle frequency band (9–15 Hz). As several studies have shown that the slow oscillation has a synchronizing effect on spindles, we expected that acute effects of the stimulation would primarily show up in the spindle band frequency, although we also performed exploratory analyses for the faster beta PLX4032 concentration frequency band (15–20 Hz). Because of the strong contamination in the EEG originating from the stimulation signal, which also prevented the standard scoring of sleep stages for these periods, all activity below 4 Hz
was removed by means of a digital finite impulse response filter. This analysis was also restricted to the parietal channels (P3, Pz, and P4), because of saturation artefacts in the recorded signals of all other channels caused by the high amplitudes during stimulation. For these 4-min periods, the band-pass-filtered (5–25 Hz) EEG signal was subjected to the calculation of time–frequency plots of wavelet power in a time window ± 2 s around the sine wave peak of the tSOS signal. Additionally, we visually scored and compared arousals during the stimulation Thiamet G and sham stimulation periods by using the electromyogram, vertical and horizontal electrooculogram and EEG in Pz. For both conditions, we applied a 5-Hz high-pass filter on all four signals before scoring of arousals. In addition to changes occurring during ongoing stimulation, the effects of tSOS on sleep and EEG activity were analysed for 1-min intervals, starting 3 s after the termination of a 4-min period of tSOS or sham stimulation. The analyses concentrated on the first six of these stimulation periods, because this was the minimum number of stimulation periods applied in each subject in both conditions (number of stimulations for sham/tSOS conditions: 6/6 for n = 2; 7/7 for n = 1; 8/8 for n = 10; 7/8 for n = 1; 8/6 for n = 1).