Eleven drug naïve RBD patients were included in the present study. All patients underwent a standardized interview using a structured sleep questionnaire, clinical neurological examinations, and single overnight PSG. The diagnosis of RBD was made based on both clinical and video-PSG as described by the second edition of the International Classification of Sleep Disorders (American Academy of Sleep Medicine, 2005; Iber and American Academy of Sleep Medicine, 2007). All patients had a typical history of DEB, as described by their spouses or family members. PSG documented RSWA in all patients (see below).

The exclusion criteria applied were; the use of medications known to affect REM sleep or tonic or phasic muscle activity (e.g., clonazepam or serotonin reuptake inhibitors), and the presence of another sleep disorder, such as, narcolepsy or moderate to severe sleep apnea (AHI >15). RBD severities were determined using the RBD severity scale (RBDSS); a simple video-PSG rating scale, proposed by Sixel-Doring et al. (2011). Eleven healthy volunteers were recruited by advertising in the local community. All control subjects completed a detailed clinical interview and a sleep questionnaire, and subsequently, those without any significant sleep problem including RBD underwent overnight PSG. Control subjects had no history of any neurologic, psychiatric, or systemic illness and no family history of a neurodegenerative disorder. All subjects provided written informed consent, and the experimental protocol was approved by the Institutional Review Board of the Korea University Medical Center.

All subjects underwent one night of PSG recording in our sleep laboratory. Sleep studies were recorded using a Somnologica (Embla, Broomfield, CO, USA). Overnight PSG was performed using a six-channel electroencephalography (EEG, F3/A2; F4/A1; C3/A2; C4/A1; O1/A2; O2/A1), a two-channel electrooculogram (EOG), an EMG (of submental, intercostal, and anterior tibialis muscles), and an electrocardiogram with surface electrodes. In order to avoid the aliasing effects, an anti-aliasing filter with a bandwidth of 70 Hz was employed before the EEG and EMG signals were sampled at 200 Hz.

A thermistor (for monitoring nasal airflow), a nasal air pressure monitor, an oximeter (for measuring oxygen saturation), piezoelectric bands (for determining thoracic and abdominal wall motion), and a body position sensor were also attached. Patients were recorded on videotape using an infrared video camera and were continuously observed by a PSG technician.

Sleep architecture was scored in 30-s epochs, and sleep staging was performed according to the standard criteria described by Rechtschaffen and Kales (1968). RSWA was scored as described in the AASM manual for the scoring of sleep and associated events (Iber and American Academy of Sleep Medicine, 2007), which include sustained muscle activity or excessive transient muscle activity. Sustained muscle activity during REM sleep was defined as REM sleep demonstrating a chin EMG amplitude greater than the minimum amplitude during non-REM sleep of duration >50% the duration of the 30-s epoch. Excessive transient muscle activity during REM sleep was defined to be present when >50% of 30-s epoch contained transient muscle activity bursts of amplitude >4 times that of background EMG activity.

The atonia index was devised by Ferri et al. (2008) to quantify the amplitude of submentalis muscle EMG during sleep, and has been used for the diagnosis of RBD. To calculate atonia index values, the rectified chin EMG signal was subdivided into 1-s mini-epochs, and the average amplitude of each of these was calculated. Then, we applied a correction method which subtracted from each mini-epoch average rectified EMG amplitude, the minimum value found in a moving window including the 60 mini-epochs surrounding it (Ferri et al., 2010).

Atonia index was defined as the ratio between the percentage of EMG mini-epochs with an average amplitude of ≤1 μV and the total mini-epochs excluding those with 1 μV < averaged EMG amplitude ≤2 μV. Possible atonia index values range from 0 to 1, where values close to 0 indicate the absence of EMG atonia and values close to 1 indicate stable EMG atonia. This index was calculated for each sleep stage.

Coherence is an extension of Pearson’s correlation coefficient in the frequency domain and is defined as the cross-spectral density normalized by auto-spectra. The coherence C_xy between two signals x and y can be calculated using;

where f represents a frequency of interest, P_xx and P_yy are the auto-spectra of x and y, respectively, and P_xy is the cross-spectral density of the two signals. This procedure returns a real number between 0 (no coherence) and 1 (perfect coherence).

In the present study, CMC values between EEGs recorded at central electrodes (C3 and C4) and EMGs acquired at leg (pretibial) and chin (submental) muscles were computed using NeuroSpec 2.0 toolbox (www.neurospec.org) implemented in MATLAB 7.1 (MathWorks Inc., USA).

Four CMC values (C3 EEG – chin EMG, C4 EEG – chin EMG, C3 EEG – right leg EMG, C4 EEG – left leg EMG) were computed for each 30-s epoch (6,000 time samples per epoch) based on the fast Fourier transform (FFT) analysis for 23 non-overlapping windows with 256 time samples. This gave a frequency resolution of approximately 0.78 Hz. To avoid influence of muscle activity during DEB on coherence measure, we excluded epochs being contaminated by sufficient amount of muscle and movement artifacts during DEB, which were identified by video monitoring, from coherence analysis. However, epochs containing either phasic EMG activity due to REM or RSWA not accompanied by DEB were included in the present study.

Before evaluating CMC values, EMG signals were rectified so as to enhance the firing rate information (Myers et al., 2003). Since beta band CMC is known to be most closely associated with motor activities (Conway et al., 1995; Halliday et al., 1998; Mima and Hallett, 1999), CMC values in beta frequency band (12–30 Hz) were averaged to obtain a single CMC value for each sleep stage.

Statistical analysis was performed using SPSS software (Version 10, SPSS, Inc.). The Mann–Whitney U test was used to compare of clinical and PSG variables between groups, and repeated measures analysis of variance (ANOVA) was used to analyze CMC and atonia index. Sleep stage (five levels: W, R, N1, N2, and N3) and muscle (two levels: chin and leg EMG) served as within-subject factors, and group (i.e., RBD patients vs. Controls) served as the between-subject factor. The Greenhouse–Geisser correction was used to evaluate F ratios to control for Type 1 error in the repeated measures design. Bonferroni’s post hoc tests were used to identify the sources found significant by ANOVA. Spearman’s rank correlation analysis was performed to evaluate correlations between CMC and clinical variables, that is, age of onset, disease duration, RBD frequency, RBDSS, and atonia index. p Values of <0.05 were considered significant.

Mean patient age was 63.8 ± 9.0 years (range, 44–73 years) and mean disease duration was 3.3 ± 2.3 years (range, 1–8 years).The frequency of RBD episodes ranged from daily to weekly in all patients (Table 1). Neurologic examinations were unremarkable in all patients. All patients showed either the presence of excessive tonic chin EMG activity during REM sleep, or excessive phasic submental or limb EMG twitching by overnight PSG. Mean RBDSS was 3.1 ± 0.9 (range, 2–4). Patients and controls were well-matched for age, sex, and BMI. Subjective sleep quality (PSQI and AIS) and excessive daytime sleepiness (defined by ESS >10) were not significantly different between patients and controls (Table 1). PSG revealed that RBD patients showed poorer sleep efficiency and a lower percentage of N2 sleep than controls (Table 2). Control subjects had a higher mean apnea–hypopnea index compared to RBD patients (7.5 ± 5.4 vs. 2.3 ± 2.6, respectively; p = 0.011). None of the patients had an AHI >10 whereas four of the controls did.

Repeated measures ANOVA revealed no significant main effect for group. However, sleep stage was found to have a significant effect (F_2.6,51.4 = 24.528, p < 0.001). The interaction between group and sleep stage was also significant (F_2.6,51.4 = 3.979, p = 0.017, Figure 1). In control subjects, the atonia index of the waking stage was significantly lower than for other sleep stages. Contrary to this, in patients, the atonia index of the waking stage was significantly lower than those of the N2 and N3 sleep only. REM sleep atonia index was similar to atonia index of waking stage in patients. RBD patients had a significantly lower mean atonia index than controls during REM sleep (0.76 ± 0.18 vs. 0.97 ± 0.02, respectively, p = 0.001). atonia index was found to be marginally correlated with RBD severity in RBD patients (r = −0.558, p = 0.057). Age of onset was not significantly correlated with REM sleep atonia index (r = −0.642, p = 0.033).

Figure 2 shows an overnight leg-CMC plot for a control subject (A) and an RBD patient (B). Repeated measures ANOVA revealed no significant main effect of group (F_1,20 = 0.571, p = 0.458) or muscle (F_1,20 = 1.283, p = 0.271). However, sleep stage was found to have a significant main effect (F_2.067,41.332 = 20.092, p < 0.001). The interaction between group and sleep stage was significant (F_2.067,41.332 = 3.438, p = 0.040; Figure 3).

In controls, CMC of waking stage was significantly higher than those of other sleep stages. In other words, CMC during REM sleep was significantly lower compared to waking stage. Contrary to this, in patients, CMC during N3 sleep was significantly lower than those of other sleep stages. REM sleep CMC value was not significantly different from that of waking stage in patients. RBD patients had a significantly higher CMC value than controls during REM sleep (0.047 ± 0.00 vs. 0.052 ± 0.00, respectively, p = 0.007).

Corticomuscular coherence during REM sleep showed significant correlation with clinical variables. However, other sleep stages did not show noteworthy correlation between CMC and clinical variables. A significant negative correlation was observed between REM sleep CMC and age of RBD onset (r = −0.642, p = 0.033). RBDSS tended to modestly correlated with REM sleep CMC (r = 0.575, p = 0.064). However, disease duration, severity, and frequency of RBD were not found to be correlated with REM sleep CMC. On the other hand, atonia index was found to be significantly correlated with REM sleep CMC (r = −0.662, p = 0.027).