Participants

Twenty healthy university students participated in the study (11 males, 9 females, age: 22.0±1.7 years, age range: 19–25 years). All the participants were right-handed, non-smokers and free of any mental or somatic disorder or substance/medication use. All the participants maintained a regular sleep–wake schedule before and during the study, as documented by sleep diaries. The participants were recruited from the community and compensated for participation. The study was approved by the local Ethics Committee at the University Medical Center Freiburg and was conducted in accordance with the Declaration of Helsinki. All the participants provided written informed consent before participation.

Study design

The participants underwent a screening session and a repeated-measures electrophysiological, behavioural and molecular study protocol (Fig. 6) after one night of normal nighttime sleep at home (self-reported total sleep time 6.7±0.6 h, range 5.4–7.5 h) and one night of total sleep deprivation (T1 and T2). At screening, the participants obtained a sleep diary for 2 weeks to monitor sleep–wake behaviour across the study. T1 and T2 took place seven and 14 days, respectively, after screening. The night before T1 and T2, participants either slept about 7 h at home or underwent one night of total sleep deprivation at the Department of Psychiatry and Psychotherapy with standardized activities and continuous supervision by staff members. The order of the sleep and sleep deprivation condition was counterbalanced. Screening comprised the Composite International Diagnostic Interview to exclude any mental disorders and the Beck Depression Inventory. The Edinburgh Handedness Inventory was used to ensure that all the participants were right-handed. A TMS screening comprised questions concerning the safety of TMS. A few magnetic stimuli were administered to the primary motor cortex (M1) to ensure that TMS was applicable. The Pittsburgh Sleep Quality Index and the Epworth Sleepiness Scale were used to detect any sleep-related pathology.

Figure 6: Electrophysiological, behavioural and molecular assessments. Measurements started at 6:45 AM with the wake electroencephalography (EEG) and Psychomotor Vigilance Task (PVT). At 0800, h, the TMS protocol was started consisting of a baseline measurement followed by paired associative stimulation (PAS) and three post measurements (2, 30 and 60 min after the end of PAS). Salivary cortisol was assessed at 0600, h and 0800, h. After the TMS measurement, 15 ml of blood was taken to determine brain-derived neurotrophic factor (BDNF) plasma concentration and BDNF genotype. Last, a declarative memory task (word-pair task) was conducted. Full size image

Indices of net synaptic strength

TMS was applied using a figure-of-eight stimulation coil with an outer diameter of 90 mm that was centred tangentially on the skull over the right primary motor cortex (M1) with the handle pointing in a posterior direction and laterally at an angle of 45° away from the midline. The coil was connected to a Magstim 200 stimulator (The Magstim Company Ltd., Whitland, UK) with a monophasic current waveform. By moving the coil over M1 while administering stimuli of suprathreshold intensity at 0.25 Hz, the optimal coil position for eliciting MEPs of maximal amplitude of the left APB muscle was identified (‘hotspot’). The coil position was recorded using a stereotaxic, optically tracked navigation system to reduce experimenter bias, consisting of a camera (Polaris Vicra P6, NDI, Waterloo, Ontario, Canada), custom-made software (BrainView, Fraunhofer Institute, Stuttgart, Germany) and passive sphere markers47, and kept constant throughout the measurements. RMT was determined using a maximum-likelihood threshold-hunting paradigm48 that consisted of 16 TMS stimuli at 0.25 Hz starting at 45% MSO. The stimulation intensity was adjusted at the beginning of each condition (sleep, sleep deprivation) to elicit MEPs with peak-to-peak amplitudes of on average 600–1,400 μV (SI 1mV ) and was kept constant throughout all measurements within each condition to assess changes in mean MEP amplitudes. To adjust stimulation intensity, 20 MEPs were recorded with a stimulation intensity of 120% RMT. If the mean amplitude of the 20 MEPs was <600 μV or >1,400 μV, the stimulation intensity was increased or decreased, respectively, and another 20 MEPs were recorded until the correct intensity was determined. At each TMS measurement (baseline, post 1, post 2, post 3), 20 TMS pulses were administered at a frequency of 0.1 Hz. The MEP amplitudes were determined by measuring the two highest peaks of opposite polarity. Raw examples of individual MEP traces are presented in Fig. 7. For each TMS measurement (baseline, post 1, post 2, post 3), the mean MEP amplitude was calculated by averaging the individual peak-to-peak amplitudes of the 20 TMS pulses. To estimate cortical excitability, we compared the stimulation intensity adjusted to elicit a mean MEP amplitude of about 1 mV peak-to-peak (SI 1mV ) in each condition before PAS between the two conditions.

Figure 7: Raw examples of single motor-evoked potentials (MEPs). Single MEPs of one participant were selected to illustrate the results. MEP amplitudes increased from baseline level after paired associative stimulation (PAS) in the sleep condition, whereas MEP amplitudes decreased from baseline level after PAS in the sleep deprivation condition. Full size image

MEPs were recorded from the left APB muscle using silver/silver chloride electrodes (AMBU, Ballerup, Denmark) in a belly-tendon montage. The participants were instructed to relax the target muscle during all measurements. The overall EMG level before the application of a TMS stimulus was near zero and did not differ between the conditions. The EMG signals were band-pass filtered (20–2,000 Hz) and amplified using an Ekida DC universal amplifier (Ekida GmbH, Helmstadt, Germany), digitized at a 5 kHz sampling rate using a MICRO1401mkII data acquisition unit (Cambridge Electronic Design Ltd., Cambridge, UK) and stored on a computer for online visual display and later offline analysis using Signal Software version 3 (CED Ltd, UK). MEPs were excluded from the analysis if the muscle activity exceeded a value of 0.05 mV in a time frame 100 ms before the TMS stimulus. Very few MEPs were excluded (sleep condition: 0.75%, 12 out of 1,600; sleep deprivation condition: 0.44%, 7 out of 1,600).

The wake EEG was recorded from the electrode positions O1 and O2 referenced to Fpz according to the 10–20 system49 using a Neuroscan SynAmps amplifier (Compumedics Neuroscan, Charlotte, NC, USA). The EEG recordings were performed for a 2.5 min period of sustained wakefulness and the average spectral power in the EEG theta frequency band (3.5–8 Hz) of artifact-free 2 s epochs was calculated.

Indices of LTP-like plasticity

The PAS protocol closely followed standard procedures19. PAS consisted of 200 pairs of peripheral and cortical stimuli at a frequency of 0.25 Hz. Peripheral electrical stimulation of the median nerve at the left wrist was followed by TMS of the right M1 at the optimal site to elicit MEPs in the left APB muscle with an ISI of 25 ms. Electrical stimulation was applied through a Digitimer DS7 electrical stimulator (Digitimer Ltd., Welwyn Garden City, Hertfordshire, UK) at the optimal stimulation site at the wrist using a bipolar electrode with the cathode proximal. Stimuli were constant current square wave pulses with a duration of 1,000 μs at an intensity of 300% of the sensory perceptual threshold. TMS intensity was set at SI 1mV as determined before PAS. Taking into account that the level of attention may influence PAS effectiveness50, the participants were instructed to direct their attention to the stimulated hand and count silently the number of randomly administered electrical stimuli applied to the thumb of the stimulated hand using a second bipolar electrode (200% perceptual threshold, constant current square wave pulses, duration 200 μs, cathode proximal). Four electrical stimuli were applied at the midpoint of the interval between successive paired stimuli during the stimulation protocol. The participants were asked to report the number of stimuli after the PAS intervention. As an index of LTP-like plasticity, we analysed the change in MEP amplitude after the PAS intervention (post 1–3) compared with pre-PAS.

In the word-pair task, 46 semantically unrelated word-pairs were randomly presented on a computer screen using the Presentation software. Each word-pair was presented for 5,000 ms, separated by an ISI of 1,000 ms. Presentation of the word-pairs was immediately followed by cued recall, that is, participants were required to name the second word upon presentation of the first word. Correct and incorrect responses were noted by staff members without feedback. Three presentation/recall trials were conducted. Recall performance was assessed as the number of correctly recalled words in each trial. To control for primacy and recency effects, four additional word-pairs at the beginning and at the end were not included in the analysis. Parallel versions of the task were used for repeated measurements.

Potential modulators of synaptic plasticity

BDNF has emerged as an important modulator of synaptic plasticity51. We analysed the BDNF genotype and plasma levels. Genomic DNA was purified from 3 ml of whole blood. Fifty nanograms of genomic DNA was used to amplify a 281 bp polymerase chain reaction (PCR) product surrounding the site of the Val66Met polymorphism for subsequent direct sequencing by GATC Biotech in Konstanz, Germany. Direct sequencing was performed with 3.2 pmol of the reverse primer used for initial PCR amplification. The genotype of each participant was determined following two independent rounds of direct sequencing52. The following primers were used: 5′-CAGGTGAGAAGAGTGATGACCA-3′ (forward) and 5′-GCATCACCCTGGACGTGTAC-3′ (reversed). BDNF plasma levels were measured by E max ImmunoAssay Systems ELISAs (G7610) according to the manufacturer’s instruction.

To control for possible effects of cortisol levels, saliva specimens were collected at 0600, h (before breakfast) and 0800, h (1 h after breakfast) at T1 and T2. All the samples were stored at −20 °C until the analysis at the BioInnovationsZentrum Dresden, Germany. After thawing, salivettes were centrifuged at 3,000g for 5 min, which resulted in a clear supernatant of low viscosity. Salivary concentrations were measured using a commercially available chemiluminescence immunoassay with high sensitivity (IBL International, Hamburg, Germany). The intra- and interassay coefficients of variation for cortisol were below 8%.

To control for possible effects of vigilance, the PVT was used. The participants were instructed to press a response button as fast as possible to stop a visual millisecond (ms) counter on a computer screen starting at a variable ISI of 2,000–10,000 ms. In response to the reaction, the counter display stopped, allowing the participant to read the reaction time (RT) for 1,000 ms before restart. The total test duration was 10 min. Mean response speed (1,000 ms per RT; 100 ms ≤RT<500 ms) and number of lapses (errors of omission; RTs ≥500 ms) were analysed.

Data analysis

IBM SPSS 21 was used for statistical analysis. The data are reported as means±s.d.s, if not indicated otherwise. To test our first hypothesis of decreased inducibility of LTP-like plasticity after sleep deprivation compared with sleep, a 2 × 4 rm-ANOVA with the within-subject factors Condition (sleep, sleep deprivation) and Time (baseline, post 1, post 2, post 3) and MEP amplitudes as the primary end point was conducted. Power calculation was done for this analysis (F test with repeated measures, G*Power 3.1.9.2). To compare memory performance as a secondary index, a 2 × 3 rm-ANOVA with the within-subject factors Condition (sleep, sleep deprivation) and Session (sessions 1–3) and the number of correctly recalled word-pairs in each trial as the dependent variable was conducted. Post hoc paired-sample t-tests were conducted for significant rm-ANOVA effects. To test our second hypothesis of increased cortical excitability/net synaptic strength after sleep deprivation compared with sleep, a paired-sample t-test was used to compare TMS intensity to elicit MEPs of about 1 mV peak-to-peak (SI 1mV ) as the primary end point. To compare EEG theta power (3.5–8 Hz) as a secondary index, a paired-sample t-test was used. Partial eta square values (η p 2) were calculated as effect sizes for ANOVAs (low: <0.06; medium: ≥0.06 and <0.14; large: ≥0.14). The level of statistical significance was set at P<0.05 (two-tailed).

Data availability

The data that support the findings of this study are available from the corresponding author upon request.