Obtained using a discrete Fourier transformation. Analysis resulted in averaging of the time-frequency plots for all samples for each category of events. No filter was applied to the processed electrophysiological data. Statistical significance of patterns in the time-frequency plots was assessed by the method described by Zygierewicz et al [37]. Time-frequency elements with resolution of 0.250 s and 2 Hz were calculated using the corresponding mean spectral values, and the Box-Cox transformation was used to transform the values across events to approximately normal distribution. For each element, the null hypothesis of no change from a baseline period 215 to 25 s prior to the event marker was tested using ttest, assuming unequal variances (Welch t-test). CHIR-258 lactate Correction for multiple comparisons was performed by controlling false discovery rate [43] with q = 0.05 so that among all significant time-frequency elements 5 of them are false positives. Relative changes of spectral power were calculated using the ratio of the original (not transformed) mean values of the power spectral density for every time-frequency bin to the average of the valuesScoring and Event SelectionSleep staging was performed by visual inspection according to the standard criteria of Rechtshaffen and Kales [38], taking under consideration the propositions of the AASM Visual Scoring Task Force [39] as well as those of the DGSM Task Force [40], and the guidelines of the ASDA Report 18325633 [41] to identify microarousals. Scoring was further aided by the collation of a hypnospectrogram [42], that is, the whole-night FFT-based time-frequency plot for 0.05?5 Hz with a step frequency of 0.05Hz. Continuous scoring with a step of only 1 s was performed rather than epoch-based scoring in order to obtain a precise match between the derived hypnogram and the hypnospectrogram (Fig. 1). The K-complex was identified as a .500 ms well-delineated negative sharp wave usually followed by a positive phase that stands out of the EEG background (Fig. 1). In this study, singular (without another K-complex or slow wave activity immediatelySpindle Power Is Not Affected after Spontaneous KCFigure 1. Hypnogram (top) and its respective hypnospectrogram (whole-night time frequency plot of EEG power) (middle) derived from Cz for subject 2. In hypnogram green dots mark the occurrence of KCs selected for the study and vertical lines the definition of a “cycle” used in Figure 2. MA, microarousal, AW, awake, REM, rapid-eye Adriamycin movement sleep, NR1?, non-REM sleep stages 1?. Bottom part: Raw EEG of selected midline electrodes. A K-complex (A) from NREM stage II ending with a spindle (B) is seen (group KC01). Two individual sporadic spindles are also seen (C, D). D is not included in this study because of its proximity to the KC. Sleep staging for all the subjects is provided as a lasagna plot [52] in supplementary figure. doi:10.1371/journal.pone.0054343.gSpindle Power Is Not Affected after Spontaneous KCFigure 2. All graphs show Spindle Band Power developing over time: Raster images composed of individual time-frequency plots of EEG power near the frequencies of each subject’s individual spindle spectral frequency band, for 15 s before and after each event (sporadic spindles in A and KCs in B ). Average power change is shown below each raster. A1?: Spindles as reference events (at time zero). In the y-axis spindle event successive number; all averaged in A2. B1?: KCs as reference events, spindle data sorted by KC.Obtained using a discrete Fourier transformation. Analysis resulted in averaging of the time-frequency plots for all samples for each category of events. No filter was applied to the processed electrophysiological data. Statistical significance of patterns in the time-frequency plots was assessed by the method described by Zygierewicz et al [37]. Time-frequency elements with resolution of 0.250 s and 2 Hz were calculated using the corresponding mean spectral values, and the Box-Cox transformation was used to transform the values across events to approximately normal distribution. For each element, the null hypothesis of no change from a baseline period 215 to 25 s prior to the event marker was tested using ttest, assuming unequal variances (Welch t-test). Correction for multiple comparisons was performed by controlling false discovery rate [43] with q = 0.05 so that among all significant time-frequency elements 5 of them are false positives. Relative changes of spectral power were calculated using the ratio of the original (not transformed) mean values of the power spectral density for every time-frequency bin to the average of the valuesScoring and Event SelectionSleep staging was performed by visual inspection according to the standard criteria of Rechtshaffen and Kales [38], taking under consideration the propositions of the AASM Visual Scoring Task Force [39] as well as those of the DGSM Task Force [40], and the guidelines of the ASDA Report 18325633 [41] to identify microarousals. Scoring was further aided by the collation of a hypnospectrogram [42], that is, the whole-night FFT-based time-frequency plot for 0.05?5 Hz with a step frequency of 0.05Hz. Continuous scoring with a step of only 1 s was performed rather than epoch-based scoring in order to obtain a precise match between the derived hypnogram and the hypnospectrogram (Fig. 1). The K-complex was identified as a .500 ms well-delineated negative sharp wave usually followed by a positive phase that stands out of the EEG background (Fig. 1). In this study, singular (without another K-complex or slow wave activity immediatelySpindle Power Is Not Affected after Spontaneous KCFigure 1. Hypnogram (top) and its respective hypnospectrogram (whole-night time frequency plot of EEG power) (middle) derived from Cz for subject 2. In hypnogram green dots mark the occurrence of KCs selected for the study and vertical lines the definition of a “cycle” used in Figure 2. MA, microarousal, AW, awake, REM, rapid-eye movement sleep, NR1?, non-REM sleep stages 1?. Bottom part: Raw EEG of selected midline electrodes. A K-complex (A) from NREM stage II ending with a spindle (B) is seen (group KC01). Two individual sporadic spindles are also seen (C, D). D is not included in this study because of its proximity to the KC. Sleep staging for all the subjects is provided as a lasagna plot [52] in supplementary figure. doi:10.1371/journal.pone.0054343.gSpindle Power Is Not Affected after Spontaneous KCFigure 2. All graphs show Spindle Band Power developing over time: Raster images composed of individual time-frequency plots of EEG power near the frequencies of each subject’s individual spindle spectral frequency band, for 15 s before and after each event (sporadic spindles in A and KCs in B ). Average power change is shown below each raster. A1?: Spindles as reference events (at time zero). In the y-axis spindle event successive number; all averaged in A2. B1?: KCs as reference events, spindle data sorted by KC.