Subjects

We recruited young male healthy volunteers who had no history of sleep, psychiatric, or major medical disorders. The subjects were aged 19–29 years, and each provided informed written consent prior to the enrollment in this study. The study protocol was approved by the Institutional Review Board (IRB) of Korea University Anam Hospital (IRB number: ED 12261) and was conducted in accordance with the Declaration of Helsinki. We enrolled only young healthy male subjects in order to better eliminate the impact of other factors such as age, sex, and health-related confounders.

The inclusion criteria were as follows: (i) right handedness, (ii) no current or past sleep disorders or neurocognitive disorders, (iii) no current or past medical, neurological, or psychiatric illness that may affect brain function or cognitive function, and (iv) have not taken any neurotropic or psychotropic medications for their life time. The exclusion criteria were as follows: (i) suspected of having sleep–wake cycle irregularity or circadian rhythm sleep disorders in actigraphy, (ii) suspected of having any sleep disorders in Night 1 NPSG (e.g., SE below 90%, apnea-hypopnea index >5, periodic limb movement during sleep >15, or presence of REM sleep without atonia), (iii) body mass index of >30 kg/m2, (iv) contraindication for 3T MRI (such as claustrophobia and pacemaker), and (v) structural abnormality in brain MRI.

After the initial screening of subjects’ sleeping conditions, physical health, and psychiatric health based on questionnaires, 35 subjects were interviewed face-to-face in order to evaluate their eligibility for the study by a board-certified psychiatrist (H.J.L.) majoring in sleep medicine using the semi-structured interview of DSM-IV-TR. Based on such interview, we excluded five subjects who were suspected to have a specific sleep disorder, such as obstructive sleep apnea, aberrant sleep–wake cycle, or obesity.

The participants were randomly divided into two groups and exposed to different light-intensity: 5 and 10 lux. For 1 week prior to the study, 30 participants wore an actigraph (Actiwatch-L, Mini Mitter, Bend, OR) on their non-dominant wrist in order for us to identify their sleep–wake cycles and screen the potential sleep–wake disorders and excessive LAN. We reviewed the actigraphic data and the information of the light meter for all of the participants’ 1-week sleep–wake cycles, and excluded two participants whose cycles were disturbed due to being irregular or the presence of suspected delayed-sleep-phase syndrome. One participant withdrew the consent to participate in the middle of the study. After scoring and staging the NPSG results of all participants, we further excluded four participants with a sleep efficiency of <90% or an apnea-hypopnea index of >5 in NPSG.

Finally, 23 subjects participated in our study: 11 and 12 subjects in the 5- and 10-lux groups, respectively. The fMRI analysis included 20 subjects (11 and 9 in the 5- and 10-lux groups, respectively), since 3 participants were excluded due to missing data caused by a task-button recognition error.

Study design

All of the participants were requested to sleep and be awake according to standard time schedule during a period from one week before the experiment through the end of this study, and were prohibited from napping during the daytime and consuming medicines, caffeine beverages, and alcohol. Participants were also asked to keep their routine activity and the usual brightness of lighting. NPSG was conducted for three consecutive nights (Fig. 4). During each NPSG session, the lights were turned off at 11:00 p.m. and turned on at 7:00 a.m. After the lights were turned off, participants were prohibited on the use of smartphones during the entire NPSG session. All participants underwent adaptation NPSG on the first night (Night 0) to adapt to the NPSG laboratory and to minimise the first-night effect of the NPSG. Subjects were instructed to sleep in the NPSG room without any light exposure on the second night (Night 1) and under a dim light condition of 5 or 10 lux on the third night (Night 2). The participants were asked to sleep in the supine position as much as possible and not to hide their face with a blanket. After the second and third nights, participants underwent fMRI while performing n-back tasks (0-, 1-, and 2-back tasks) in order to evaluate their cognitive function (Fig. 4).

Figure 4 Schematic of the LAN intervention procedure. To reduce the first-night effect, we have applied an additional nocturnal NPSG session on the first night (Night 0) before performing the main study on the second and third nights (Nights 1 and 2). After Nights 1 and 2, participants underwent fMRI while performing n-back tasks (0-, 1-, and 2-back tasks). Abbreviations: LAN, light at night; NPSG, nocturnal polysomnography; fMRI, functional magnetic resonance imaging. Full size image

For the whole period of Night 2, a light box producing a dim light with a daylight ‘cool white’ colour was placed on the upper part of the wall opposite to participant’s head. The box contained a light source comprising wide-spectrum light-emitting diodes with an encompassing wide wavelength (dom λ: 501.4 nm, peak λ: 463.6 nm, centre λ: 467.6 nm, centroid λ: 554.3 nm, Correlated Colour Temperature: 5779.1 K, General Colour Rendering index: 90). The luminous intensity was set to 5 or 10 lux according to the study group. Light sources were enclosed in a box, approximately 11.8 × 11.8 × 1.3 inches, with a light diffuser panel on the front. The initial light box setup was installed by an illumination expert who is affiliated with the Korea Institute of Lighting Technology. The luminous intensity was checked by an illuminometer (ANA-F11, Tokyo Photo, Japan) at the level of the participant’s eyes while supine. Participants were not informed whether the dLAN was 5 or 10 lux.

Polysomnography

The quality and quantity of subjects’ sleep were measured by NPSG for three consecutive nights (i.e., Nights 0, 1, and 2). Standard NPSG recordings were obtained using the Embla digital polysomnography and Somnologica software (Broomfield, CO, USA). The recording montage, electrode, and sensors of general parameters (i.e., electroencephalogram, electrooculogram, electromyogram, airflow signals, respiratory-effort signals, oxygen saturation, body position, and electrocardiogram) were applied according to the technical specifications recommended by the American Academy of Sleep Medicine (AASM)29. The polysomnography recordings were analysed on a computer monitor, and sleep stages and events were scored visually by a single well-trained polysomnography technologist based on the criteria of the AASM29, and all of the polysomnography data were confirmed by a sleep-specialist medical doctor (H.J.L.). Such NPSG findings on these subjects have been reported previously7.

Performing n back task during functional scanning

Participants performed a verbal version of the n-back task, which is a classical test of the WM. They were asked to monitor a series of stimuli and to respond whenever a presented stimulus (henceforth the “target” stimulus) was the same as the one previously presented n trials, where n is a pre-specified integer, usually 1, 2, or 3. Participants underwent one scanning session which is composed by six functional runs, each of which lasts 4 minutes and 47 seconds. The presentation of the stimuli was performed in a blocked design. Pre-generated random sequences of letters designed to equally distribute the targets and conditions in each runs were used30. The adopted event-related design was used to prevent the habituation to each condition (n-back conditions). Each condition (0-back, 1-back, 2-back) was equally distributed in each run. Each block was separated by a baseline control block lasting 16 seconds. Stimuli continuously appeared and the task required participants to temporarily store each stimulus in their memory for evaluation, and to discard it before the appearance of the next one. For this, three different conditions were used that varied the WM load incrementally from zero to two items. Each item was presented for 500 msec and the inter-stimulus interval was for 2500 msec. In the 0-back condition, participants responded to a single pre-specified target letter; in the 1-back condition, the target was any letter identical to the one immediately preceding it (i.e., one trial back); and in the 2-back condition, the target was any letter that was identical to the one presented two trials back. In this manner, the WM load (storage and manipulation demands) increased incrementally from the 0-back to the 2-back task (Fig. 5).

Figure 5 Sample trials of n-back tasks. The actual cues for n-back tasks were displayed in Korean. Abbreviation: ISI, interstimulus interval. Full size image

The stimuli were presented using Presentation 11.0 software (Neurobehavioral Systems, Albany, NY, USA). Responses of participants were collected using same software through fibre optic response pad (Current Designs, Philadelphia, PA).

fMRI image acquisition

Anatomical T1-weighted magnetic resonance (MR) images were acquired at Korea University Brain Imaging Center, using a 3-tesla scanner (Tim Trio, Siemens, Erlangen, Germany) which had a 32-channel sense head coil (sense reduction factor = 2). An fMRI scanning was performed during n back task for each and every participant between 9:30 a.m. and 10:30 a.m. after PSG and we made sure that the MRI room used for all participants had the same light condition of 150 lux with cool-white colour. Functional images were acquired using gradient echo planar images with BOLD contrast and the following parameters: TR = 2000 msec, TE = 30 msec, flip angle = 90 degrees, field of view = 240 × 240 mm2, slice thickness = 4 mm, and 36 interleaved slices parallel to the anterior-commissure–posterior-commissure line covering the whole brain. Each scanning sequence comprised 164 sequential volumes. After the functional scanning, a high-resolution T1-weighted anatomical scan was performed on each participant with the following parameters: three-dimensional spoiled gradient echo sequence, 162 slices, TR = 1900 msec, TE = 2.32 msec, slice thickness = 1 mm, and in-plane resolution = 1 × 1 mm2.

Data analysis: clinical and cognitive measures

The behavioral performance in the n-back task was assessed as the ratio of accurate responses and the average time to react with correct responses. The effects of LAN exposure on the 0-, 1-, and 2-back conditions were analysed based on the reaction time and response accuracy for patients allocated to 5- or 10-lux group. We compared the change in subjects’ performance after the light exposure in both 5- and 10-lux groups using either the paired t test or Wilcoxon signed-rank test and also compared differences in the exposure effect between the two groups using repeated-measures ANOVA. In case where the variance of differences in levels was not equal, the repeated-measures ANOVA was calculated using the Greenhouse and Geisser correction. All analyses were performed using SPSS for Windows, and the cutoff for statistical significance was set as p = 0.05.

Image preprocessing and statistical analysis of fMRI data

Image processing and statistical analysis of imaging were performed using SPM version 8 (Institute of Neurology, London, UK) implemented in MATLAB (MathWorks, Sherborn, MA, USA). Before the image processing, board-certified neuroradiologists reviewed the structural MR images to check if there is any structural abnormality in their brain images. The first five volumes (obtained during 10 sec) of every run were discarded in order to avoid T1 equilibration effects, and all volumes were spatially realigned. The acquired images were spatially normalised to the Montreal Neurological Institute MNI152 stereotactic standard brain template according to the 12-parameter affine transformation and 16 nonlinear iterations31. The realigned and unwarped T2*-weighted volumes were spatially transformed and resampled in 3 × 3 × 3 mm3 voxels after normalisation. All functional volumes went through a spatial smoothing with an 8-mm full-width half-maximum isotropic Gaussian kernel in order to reduce the intersubject variability.

Due to the exploratory nature of this study, we adopted the whole-brain analyses instead of the region of interest (ROI) analysis. Statistical maps were generated using a random-effect model implemented through a two-level procedure. The first level of the analysis identified participant-specific task-related activations at the baseline and after dLAN exposure, by using a factorial model consisting of three active conditions (0-, 1-, and 2-back tasks). The second level of the analysis applied separate ANOVAs within SPM to contrasts of (i) 0-, 1-, and 2-back tasks >rest and (ii) 2-back task >0- and 1-back task. In addition to these, the data were tested for the exposure group (5 and 10 lux) × time (before and after exposure) interactions (the difference of differences in before vs after exposure under 5 and 10 lux) in a mixed-model repeated-measures ANOVA within SPM. Generic activations were identified in contrasts of (i) 0-, 1-, and 2-back task >rest and (ii) 2-back task >0- and 1-back task, using one-sample t test collapsing baseline data that were set for both groups. The resulting before- and after-exposure contrast values were subtracted (before minus after) in order to obtain the amount of reduction in cerebral activity from before- to after-exposure in any given region. Specifically, a positive number represents a decrease in activation. We then performed the correlation analysis between mean BOLD signal change in the cluster with a significant decrease in the activation and the change in sleep variables (sleep efficiency and wake time after sleep onset). Data were analyzed using voxel by voxel uncorrected threshold of p = 0.001. Then we selected only the clusters with a probability of activation of p < 0.05 after FWE correction for each cluster (extent threshold) unless otherwise indicated. A post-hoc power analysis was conducted with G*Power software to determine the retrospective power of the key finding of this study32.