Study participants

The 49 volunteers who participated in the study were carefully screened. Physical exam, history, questionnaires, blood chemistry, urine drug screen, and breathalyzer test showed them to be healthy, not pregnant, and free of drugs. Polysomnography during the first night in the laboratory revealed no sleep disorders. Subjects had no history of learning disability; reported good habitual sleep of between 6 and 10 hours daily; and regularly woke up between 06:00 and 09:00. They reported no shift work within 3 months and no travel across time zones within 1 month of entering the study. They had normal or corrected-to-normal vision. Subjects maintained their regular sleep schedule, with no daytime naps, during the 7 days prior to admission into the laboratory. They refrained from alcohol, drugs (including tobacco) and caffeine during the 7 days before and during the laboratory study.

The study was approved by the Institutional Review Board (IRB) of Washington State University (WSU), and all subjects gave written informed consent. All study procedures conformed to those in the protocol approved by the WSU IRB.

Sleep deprivation protocol

The experiment was conducted under controlled laboratory conditions, with stable ambient temperature (21 ± 1 °C). Light levels were fixed (<100 lux) during scheduled wakefulness and lights were off during scheduled sleep. Subjects were monitored continuously throughout the experiment, and no visitors, phone calls, live radio or television, or internet access were allowed. Subjects were in the laboratory for 72 hours (4 days, 3 nights). They were randomized to a SD group or a control group (approximately 2:1). Subjects in the SD group had a 10-hour baseline sleep opportunity, were subsequently kept awake for 38 hours, and then had a 10-hour recovery sleep opportunity. Subjects in the control group had a 10-hour sleep opportunity each night. All sleep opportunities were from 22:00 until 08:00.

AX-CPT-s performance testing

The AX-CPT-s, modeled after the AX-CPT26,27, assessed flexible attentional control by measuring subjects’ ability to accurately identify valid and invalid cue–probe letter pairings. Letter pairs were presented with a 3-second delay between the first and second letters. Subjects were asked to respond by clicking the left and right mouse buttons to indicate valid and invalid cue–probe pairs, respectively. For example, if the letter “A” (valid cue) was immediately followed by the letter “X” (valid probe), subjects were to respond by clicking the left mouse button. For all other letter pairs (e.g., A–Y, B–X, B–Y, C–D), they were to respond by clicking the right mouse button. Subjects had 2 seconds to respond and were instructed to be as fast and accurate as possible (Fig. 1).

At the beginning of the task, subjects were told which cue–probe pair was the valid target. During each test session, subjects performed 14 practice trials, followed by 4 test blocks of 40 letter pairings each. Most of the pairings (70%) were valid. After the 4 test blocks, subjects were informed that the valid cue–probe pair was switching. For example, the letter “B” became the new cue, and the letter “Y” became the new valid probe. Some of the invalid cue–probe pairs included the presentation of the formerly valid cue and/or probe. After the switch, subjects performed 2 test blocks of 48 letter pairings each. Each of the cue–probe pairings were presented equally after the switch (Fig. 1).

Two different but equivalent versions of the AX-CPT-s were used, with the second version using “C” and “Z” as the initial valid cue and probe and “S” and “G” as the valid cue and probe after the switch. The order in which the two versions were administered was randomized. Performance on the AX-CPT-s was quantified based on signal detection theory14. Discriminability indices d’ were calculated for each trial block based on hit and false alarm rates for the different combinations of valid and invalid—and, after the switch, previously valid but no longer valid—cues and probes (Table 1).

The AX-CPT-s was administered at 15:30 during baseline (session 1) and again 24 hours later (session 2) while well-rested (control group) or after 31.5 hours of continuous wakefulness (SD group).

Other cognitive performance testing

The Attention Network Test (ANT)30 required subjects to correctly indicate the direction that a target stimulus is pointed under three cue conditions (no cue, alerting cue, or orienting cue) and two flanker conditions (congruent or incongruent). This task provides measures of distinct aspects of attention: alerting, orienting, and management of response conflict. We used the 10-minute version of the task31. Each test trial began with a fixation period (+), followed by either no cue or a cue (*) presented for 100 ms and positioned in the center to alert or positioned above or below the fixation point to alert and orient to where the target stimulus was going to appear. This was followed by presentation of a line of 5 left- or right-pointing arrows, centered horizontally on the screen and located either above or below the central fixation point. Subjects were asked to focus only on the direction of the center arrow, which was the target stimulus. The surrounding arrows, which served as flankers, pointed either congruently or incongruently with respect to the target stimulus. The arrows stayed on the screen for 2 seconds. Subjects were to respond by clicking the left or right mouse button corresponding to the direction of the target stimulus (center arrow). They were instructed to be as fast and accurate as possible. During each test session, subjects performed 12 practice trials, followed by 5 test blocks of 48 trials each. No-cue and center-cue trials each occurred one-third of the time; orienting-cue trials occurred one-third of the time, with above and below the central fixation point occurring equally often. Congruent and incongruent flankers occurred equally often.

Standard performance measures31 were derived for the ANT. An alerting score was calculated by subtracting mean response time (RT) for the center cue trials from mean RT for the no-cue trials. An orienting score was calculated by subtracting mean RT for the spatial (above or below) cue trials from mean RT for the center cue trials. A response conflict management score was calculated by subtracting mean RT for congruent flanker trials from mean RT for incongruent flanker trials.

The ANT was administered at 10:30 during baseline (session 1), again 24 hours later (session 2) while well-rested (control group) or after 26.5 hours of continuous wakefulness (SD group), and once more 24 hours later (session 3) after recovery sleep.

The Psychomotor Vigilance Test (PVT), a standard measure of vigilant attention2,37, required subjects to respond as quickly as possible, by pressing a button, to a simple visual stimulus that occurred at random intervals of 2 to 10 seconds for 10 minutes. Subjects were instructed to be as fast as possible without making false starts. They received feedback on their response time for 1 second after each response. For the PVT, performance was quantified by the number of lapses in vigilant attention, defined as RT ≥ 500 ms.

The PVT was practiced twice on the day of admission into the laboratory, and was then administered at 09:00, 13:00, 17:00 and 21:00 during the baseline day and at the same times of day 24 hours later while well-rested (control group) or sleep-deprived (SD group).

Genotyping

Blood samples were collected from subjects in Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) coated with K 2 EDTA to prevent clotting. Samples were immediately aliquoted and stored at −80 °C until DNA extraction. Genomic DNA was extracted from 100 µl of red cell-depleted whole blood. Samples were assayed for the dopamine receptor D2 (DRD2) gene, which codes for the D2 receptor subtype. The DRD2 gene contains a single SNP involving a cytosine (C) to thymine (T) substitution at position 957 (C957T; chromosome 11). This polymorphism does not cause a change in the amino acid sequence. Homozygosity for the C allele (C/C) is associated with decreased D2 receptor availability and decreased dopamine binding potential in the striatum.

DRD2 C957T genotypes were assayed using the Taqman SNP Genotyping Assay (ThermoFisher Scientific, Waltham, MA) per manufacturer protocol. Samples were assayed in duplicate, with inclusion of a no-DNA negative control. Following assay completion, allelic discrimination software (MJ Opticon Monitor Analysis v3.1; Bio-Rad Laboratories, Hercules, CA) was used to identify the genotypes for each subject. The DRD2 C957T genotype distributions were as follows: C/C: 8 (7 in the SD group); T/C: 24 (15 in the SD group); T/T: 17 (12 in the SD group). The overall subject sample was found to be in Hardy-Weinberg equilibrium (χ 2 1 = 0.08, p = 0.79). The sample for the SD group was in Hardy-Weinberg equilibrium (χ 2 1 = 0.34, p = 0.57), and the sample for the control group was also in Hardy-Weinberg equilibrium (χ 2 1 = 1.28, p = 0.26). Allele frequencies were consistent with those reported in the literature39,40.

Statistical analyses

Statistical testing of differences in task performance across AX-CPT-s d’ indices, test sessions and groups was performed using multivariate analysis of variance (MANOVA) with fixed effects for d’ index, group, and session, and their two-way and three-way interaction. Here d’ index and session were implemented as repeated measures. Statistical testing of differences in task performance across test sessions and between groups for specific AX-CPT-s d’ indices, specific ANT outcome measures, and PVT performance was performed using mixed-effects analysis of variance (ANOVA), with fixed effects for group and session and their interaction. For the PVT, time of day (09:00, 13:00, 17:00, 21:00) and its interactions with group and session were also included. Here session time of days were repeated measures. A random effect over subjects was placed on the intercept41. Gene analyses were performed by adding genotype as a covariate fixed effect alone and in interaction with the other effects. Head-to-head comparisons between sessions were based on a priori planned contrasts.

Data sharing

Data produced in these studies will be maintained in a secure cloud-based backup system and are available on request from any legitimate academic, scientific or governmental entity. Requests should be directed to the PI at the Sleep and Performance Research Center, Washington State University, Spokane, WA.