Subjects

Forty-six healthy adults (right-handed, 23 male, age range 21–50 years, 18–30 BMI) were recruited in response to study advertisements. They reported habitual nightly sleep durations between 6.5 h–8.5 h, habitual bedtimes between 2200h–0000h and habitual awakenings between 0600h–0900h; these reports were confirmed objectively using actigraphy. They had no evidence of habitual napping, no sleep disturbances and an absence of extreme morningness or extreme eveningness, assessed by questionnaire61. Subjects were free of acute or chronic medical and psychological conditions, as established by interviews, clinical history, questionnaires, physical examinations and blood (including a fasting blood glucose test) and urine tests. They were nonsmokers and did not participate in shift work, transmeridian travel, or irregular sleep-wake routines in the 60 days prior to the study. Enrolled subjects were monitored at home with actigraphy, sleep-wake diaries and time-stamped call-ins to assess bedtime and waketime during the week before and after the in-laboratory phase. They were not permitted to use caffeine, alcohol, tobacco and medications (except oral contraceptives) in the week before the laboratory study, as verified by urine screenings. Sleep disorders were excluded by a night of laboratory polysomnography and oximetry measurements.

The study was approved by the Institutional Review Board of the University of Pennsylvania. All subjects provided written informed consent before enrollment, which was in accordance with the Declaration of Helsinki. Subjects were compensated for participating in the study.

Experimental Design

Subjects were screened twice prior to participating in the study; training in a mock MRI scanner occurred during the second screening session. During the study, subjects remained in the laboratory at the Clinical Translational Research Center at the Hospital of the University of Pennsylvania for 5 consecutive days (4 consecutive nights). Subjects arrived at the laboratory in the afternoon and were provided 9 h time-in-bed (TIB) for their baseline sleep night (Figure 6). The first functional magnetic resonance imaging (fMRI) scan session took place the next morning (Baseline [BL] day) from 0700h–1000h. Subjects were then randomized to either a total sleep deprivation (TSD) or control condition. During the second night of the study, sleep-deprived subjects were kept awake and control subjects were allowed 8 h TIB to sleep (Figure 6). The second fMRI scan session took place the next morning (total sleep deprivation [TSD] day or control day 1 [CD1]). Sleep-deprived subjects were then allowed 12 TIB for recovery sleep and control subjects were allowed 8 h TIB to sleep (Figure 6). Each subject was scanned at the same time for each scan to avoid potential time-of-day differences between scans.

Figure 6 Protocol summary. Subjects arrived at the laboratory in the afternoon and were provided 9h time-in-bed (TIB) for their baseline sleep night. The first functional magnetic resonance imaging (fMRI) scan session took place the next morning (Baseline [BL] day) from 0700h-1000h. Subjects were then randomized to either a total sleep deprivation (TSD) or control condition. During the second night of the study, sleep-deprived subjects were kept awake and control subjects were allowed 8 h TIB to sleep. The second fMRI scan session took place the next morning from 0700h-1000h (total sleep deprivation [TSD] day or control day 1 [CD1]). Sleep-deprived subjects were then allowed 12 TIB for recovery sleep and control subjects were allowed 8 h TIB to sleep. Each subject was scanned at the same time for each scan to avoid potential time-of-day differences between scans. Subjects had ad libitum to food/drink during the study. Subjects remained in the laboratory for the duration of the study and were monitored by trained staff at all times to ensure adherence to the protocol. Full size image

Subjects were behaviorally monitored by trained staff continuously to ensure adherence and were not permitted to leave the laboratory during the study. Subjects were ambulatory and were allowed to watch television, read, play video or board games and perform other sedentary activities between test bouts (which were completed while sitting at the computer) but they were not allowed to exercise. Subjects wore a wrist actigraph throughout the study and wore ambulatory electroencephalography and electrocardiography recording equipment for 24-h intervals. The light levels were held constant at <50 lux during scheduled wakefulness and <1 lux during scheduled sleep periods. Ambient temperature was maintained between 22°–24°C. Food/drink was ad libitum throughout the protocol (caffeine was prohibited).

Measures

Subjects selected their meals/snacks by choosing from various menu options and by making requests to the monitors and study coordinator. In order to ensure that subjects were provided sufficient time to eat each day, three 30- to 45-min opportunities were specified in the protocol during the daytime and one additional 30-min opportunity to eat was specified in the protocol during the overnight period when subjects were kept awake. In addition to these specified meal times, subjects were also allowed to consume food/drink at any time during the protocol other than when they were completing neurobehavioral tests. All food was weighed and recorded prior to being provided to subjects. To enhance the measurement accuracy of each food's weight, food was provided in individual containers (for example, a dinner consisting of chicken, peas and rice was provided in three separate containers). Each day, a detailed description of the items and the amount consumed and intake time was recorded by trained monitors. Additionally, any food/drink that was leftover after each meal was weighed and recorded. The intake data were entered into The Food Processor SQL program (ESHA Research, Salem, OR), a validated62 professional nutrition analysis software and database program that provides components of food/drink intake including calories and macronutrients.

Magnetic resonance imaging was conducted in a 3T whole-body scanner (Siemens Medical Systems, Erlangen, Germany), using a standard array coil. A standard EPI sequence was used for resting-state BOLD fMRI data acquisition with the following parameters: TR = 2s, TE = 24 ms, FOV = 220 × 220 mm, matrix = 64 × 64 × 36, slices thickness = 4 mm, inter-slice gap = 4 mm. A total of 210 images were acquired for each subject. Subjects were instructed to remain still in the scanner at rest and to keep their eyes open. An eye-tracker outsider the scanner was used to monitor subjects' eyes and ensure that they did not fall asleep during the scans. After the functional scans, high-resolution (1 × 1 × 1 mm3) T1-weighted anatomic images were obtained using a standard 3D MPRAGE sequence.

Image data processing and analyses were carried out using the Statistical Parametric Mapping software (SPM8, Wellcome Department of Cognitive Neurology, UK) and the REST 2.0 toolbox (http://resting-fmri.sourceforge.net/) implemented in Matlab 14 (Math Works, Natick, MA). After head motion correction and co-registration, functional images were smoothed using an isotropic Gaussian kernel with a full-width at half-maximum (FWHM) of 8 mm and then normalized to the standard Montreal Neurological Institute (MNI) space. Linear trends were also removed. Finally, all functional volumes were band pass filtered (0.01 Hz < f < 0.08 Hz) in order to reduce low-frequency drift and physiological high-frequency respiratory and cardiac noise. Nuisance covariates including six head motion parameters, global mean signal, white matter signal and CSF signal were regressed out before the seed based functional connectivity (FC) analysis63.

The ACC seed was defined using an automated anatomical labeling region of interest library through the WFU_Pickatlas toolbox (http://fmri.wfubmc.edu/software/PickAtlas). The dACC seed was defined using MNI coordinates from reference 31 (x: ±6 y: 45 z: 9, radius: 6 mm) using Marsbar toolbox64. For each subject, the mean BOLD fMRI signal time series were extracted from the seed and used as the regressor in the FC analysis. The correlation coefficients between the time series of seed region and other brain areas were grouped into an individual FC map and transformed into z-score through a Fisher's r-to-z transformation to improve the normality of the correlation coefficients. These z-transformed individual FC maps were then entered into the second level group analysis using paired t-tests to examine connectivity differences between the scans following baseline sleep and total sleep deprivation. Threshold was defined as uncorrected p < 0.001 at voxel level and family-wise error (FWE) corrected p < 0.005 at cluster level. For ROI analyses, anatomical bilateral putamen and bilateral insula were also defined using an automated anatomical labeling region of interest library through the WFU_Pickatlas toolbox.

Statistical Analyses

Paired-samples t-tests were used to compare caloric intake and macronutrient intake during the day following baseline sleep and the day following either total sleep deprivation (sleep-deprived subjects) or the day following a night of sleep (control subjects). FC values of the dACC-Putamen and dACC-aINS were extracted and correlated with macronutrient intake on each day using Pearson's r (IBM SPSS Statistics for Windows, Version 19.0, Chicago, IL). False discovery rate correction was used to correct for multiple comparisons (protein, carbohydrate and fat) within each region for each day.