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1 Supplementary Information Online for Pharmacological REM sleep suppression paradoxically improves rather than impairs skill memory Björn Rasch, Julian Pommer, Susanne Diekelmann & Jan Born 1. Supplementary Methods Subjects Thirty-two men (mean age 25.7 ± 0.7 yrs, range yrs) participated in the main experiments, investigating either effects of the SSRI fluvoxamine (n = 16) or the SNRI reboxetine (n = 16). Subjects were healthy, non-smoking, native German speaking, righthanded students. They underwent routine physical and mental health examination before participating, did not take any medication at the time of the experiments, reported a normal sleep-wake cycle and had not been on night-shifts for at least 8 weeks prior to the experiments. On experimental days, they were not allowed to take in caffeine and alcohol or to nap during daytime. The experiments were approved by the ethics committee of the University of Lübeck. Written informed consent was obtained from all subjects prior to participating. Design and procedure Each subject participated in two experimental sessions (active agent and placebo), separated by an interval of at least two weeks according to a double-blind cross-over design. The order of conditions was balanced across subjects. 1

2 To accustom subjects to sleeping under laboratory conditions, all subjects spent an adaptation night asleep in the sleep laboratory, including the placement of electrodes, before participating in the experiment. Sessions started at 20:00 h with a short interview to assure that the subjects had slept normally on the nights before. A venous catheter was inserted for blood collection and EEG electrodes were attached. During the learning phase (22:00-23:00 h) participants performed on two procedural tasks (finger sequence tapping, mirror tracing), and one declarative paired associate learning task. The order of tasks was held constant in all conditions (finger sequence tapping, paired associate learning, mirror tracing). At ~23:30 h, participants received a capsule containing either placebo or the active agent, together with a glass of water. They went to bed immediately afterwards and were allowed to sleep for 7 hours. During sleep, blood was sampled every hour. In the morning, participants went home and during the day followed their usual activities. They slept at home and came back for retrieval testing at 7:00 h the next day. Activity during this period was monitored continuously via Actigraphy (Actiwatch, Cambridge Neurotechnology). Also subjects were asked to keep a record about their activities during the period outside the lab. Before and after learning and retrieval testing, blood was collected and mood, subjective tiredness, restlessness and reaction times (to assess vigilance) were assessed. The values before and after learning and retrieval testing, respectively, were averaged for later analysis. Substance administration One group of subjects was administered either with placebo or the selective serotonin reuptake inhibitor fluvoxamine (50 mg, Neuraxpharm, Langenfeld, Germany) before sleep. The other group received placebo or the selective noradrenergic reuptake inhibitor reboxetine (2 mg; Pfizer, New York, NY). Substances were enclosed in a capsule and administered orally 30 min after the learning phase and immediately before bedtime. Plasma levels of fluvoxamine are highest 3 6 h after oral administration, with a half-life of 15 h 1. Plasma 2

3 levels of reboxetine reach a maximum 2 hours after administration, with a half-life of 13 h 2. Both substances after systemic administration have been shown to selectively increase extracellular levels of, respectively, serotonin and norepinpehrine 3,4 and to suppress REM sleep 5,6. To prevent any confounding influence of enhanced serotonergic or norepinephrinergic activity on retrieval function, retrieval testing was performed not until 32 h after substance administration, i.e., after a second night of sleep. Supplementary experiment (retrieval test after 24h) To exclude that REM sleep-associated memory processing during the second (recovery) night after learning compensated for the pharmacological suppression of this sleep stage in the first post-learning night, we conducted a supplementary experiment in twelve men (mean age 22.6 ± 1.3 yrs; range yrs) using the same procedure as in the main experiments, except that retrieval was tested after 24 h (instead of 32 h) after the learning phase, thereby omitting the recovery night. Blood was not sampled. Participants received the SNRI reboxetine or placebo according to a double-blind within-subject cross-over design. Memory tasks The mirror tracing task (adapted from 7 ) required the subject to trace several figures that he could see only in a mirror. The time needed for completion of the figures (speed) and the number of deviations from the prescribed 0.8-cm-wide path (accuracy) were recorded. Subjects were instructed to trace the lines of the figures as fast and as accurately as possible. Before tracing the four experimental figures, the subject was trained by tracing a simple starshaped figure until he could draw it in <60 s with <12 deviations. The average performance on all four experimental figures traced during learning served to indicate learning performance, and the average performance on the same four figures during retrieval testing served as measure of retrieval performance. As dependent variable, overnight changes in skill 3

4 were calculated as absolute differences of speed and accuracy between retrieval testing and training. Different sets of figures were used for the subject's two sessions. Sleep-dependent gains in mirror tracing skill have been consistently revealed, especially across periods rich of REM sleep 7,8. The finger sequence tapping task was adopted from previous studies indicating also most robust sleep-dependent improvements on this task 9. It requires the subject to tap repeatedly one of two five-element sequences ( or ) on a keyboard with the fingers of his non-dominant hand as fast and as accurately as possible for 30-s epochs interrupted by 30-s breaks. The numeric sequence was displayed on the screen at all times to keep working memory demands at a minimum. A key press resulted in a white dot in the center of the screen. Each 30-s trial was scored for speed (number of correctly completed sequences) and error rate (number of errors relative to total number of tapped sequences). After each 30-s trial, the number of correctly completed sequences and error rate was indicated to reinforce optimal performance. At learning, subjects trained on twelve 30-s trials. The average score for the last three of these trials was used to indicate learning performance. At retrieval, subjects were tested on another three trials. Overnight changes in performance were calculated as absolute differences in speed and error rate between the three trails at retrieval and the last three trials at learning. As a control for the specificity of effects on procedural memory, a declarative paired associate task was employed requiring the learning of a list of 40 pairs of semantically related words (e.g., clock church). Different word-lists were used on the subject's two experimental sessions. During the learning phase, the word-pairs were presented sequentially on a computer screen, each for 5 s, separated by interstimulus intervals of 100 ms. After presentation of the entire list, performance was tested using a cued recall procedure, i.e., the first word (cue) of each pair was presented and the subject had to name the associated second word (response). The correct response word was then displayed for 2 s, regardless of whether the response was 4

5 correct or not, to allow re-encoding of the correct word-pair. The cued recall procedure was repeated until the subject reached a criterion of 60% correct responses. Retrieval at the end of the experimental session was tested using the same cued recall procedure as during the learning phase. Absolute differences between word-pairs recalled during retrieval testing and on the criterion trial during learning served as dependent variable of overnight retention of declarative memories. Several studies showed that consolidation of word-pairs profits particularly from SWS, whereas sleep periods rich of REM sleep remained ineffective 7,10. Reaction times, mood and neuroendocrine measures Reaction times were assessed as a measure of vigilance by a standardized test that required pressing as fast as possible a button whenever a big red disc appeared on a computer screen 11. On 40 trials the subject fixated his gaze on a centrally located cross, displayed for ms on a white screen. Then, in 35 trials, a red disc appeared and, in five random no-go trails, the screen remained white. Mood, feelings of tiredness and of calmness/restlessness were assessed during the learning phase and at retrieval testing using the short form of the German version of the Multidimensional Mood Questionnaire 12. The subjects indicated on a five-point rating scale how well 12 different adjectives described their current feeling. The adjectives are assigned to one of three different bipolar dimensions, pleasant/unpleasant, alert/tired and calm/restless, adding up to values between To control for changes in blood cortisol and catecholamine concentrations possibly affecting memory consolidation 13, blood was collected during sleep via thin plastic tubes from an adjacent room without disturbing the subject's sleep. Blood was sampled before and after test performance and hourly, beginning 30 min after sleep onset. Blood was immediately centrifuged and frozen at 20 C until assay. Cortisol was determined from serum by enzyme immunoassay (Immulite, DPC Biermann, Bad Nauheim, Germany). Blood norepinephrine 5

6 levels were determined from EDTA-plasma by standard high-performance liquid chromatography (Waters, Milford, MA, USA) with electrochemical detection. EEG recordings, sleep analysis and spindle count The EEG was recorded continuously using a BrainAMP DC amplifier (Brainproducts, Munich). EEG signals were filtered between Hz and sampled with 250 Hz. Twentyseven Ag-AgCl electrodes were placed according to the international System (Fp1, Fp2, F7, F3, Fz, F4, F8, Tc5, Tc1, Tc2, Tc6, T7, C3, Cz, C4, T8, Cp5, Cp1, Cp2, Cp6, P7, P3, Pz, P4, P8, O1, O2), referenced to an electrode attached to the nose. Additionally, horizontal and vertical eye movements and the electromyogram (chin) were recorded for standard polysomnography. Sleep architecture was determined visually according to standard polysomnographic criteria using EEG recordings from C3 and C4 14. Scoring was performed independently by two experienced technicians who were blind to the assigned treatment. In case of disagreement, a third expert was consulted. For the total time in bed every 30-s epoch was scored as NonREM sleep stage 1, 2, 3, 4 or REM sleep with slow wave sleep (SWS) defined by the sum of time spent in sleep stages 3 and 4. Sleep onset was defined by the first period in stage 1 sleep followed immediately by stage 2 sleep. Sleep onset latency was determined with reference to the time lights were turned off. REM sleep latency was determined with reference to sleep onset. (We did not observe time periods with isolated features of REM sleep in the active treatment conditions, e.g. rapid eye movements without EEG desynchrony or sleep with EEG desynchrony without rapid eye movements). Spindles (counts and density) in these sleep stages was analyzed because of their wellknown relationship with overnight retention of memories Discrete spindles are a characteristic feature of sleep stage 2 and occur also in SWS, but are virtually absent during REM sleep. Slow (<13 Hz) and fast spindles (>13 Hz) were separately identified at six 6

7 selected EEG recording sites (F3, F4, C3, C4, P3, P4) during NonREM sleep stages 2, 3 and 4, based on an algorithm adopted from previous studies 18,19 : In brief, the EEG signal was filtered in the frequency bands of interest (10 13 Hz; Hz), the root mean square (RMS) of each 100-ms interval was calculated, and the events were counted for which the RMS signal exceeded an individual threshold for an interval lasting s. The detection threshold was defined by the threefold average standard deviation across all 6 EEG channels analyzed and across the subject's two experimental nights (active agent, placebo). Spindles were counted separately in each channel. Mean spindle counts were calculated by averaging spindle counts of all 6 channels. To calculate mean spindle density, mean spindle counts were divided by the number of analyzed 30-s epochs. The two separate spindle bands were chosen based on previous studies which demonstrated the presence of two kinds of spindles in humans possibly linked to different aspects of cognitive function, i.e., slow spindles that prevail over frontal cortex and show greater topographical variability than the fast spindles that concentrate over parietal cortex 20,21. Statistical analysis Data from 4 subjects of the main experiments (1 participant of the SSRI group, 3 participants of the SNRI group) were excluded from analyses because these subjects spent more than 30% of the night awake or in sleep stage 1 following intake of the SSRI and SNRI, respectively. Data were analyzed using repeated measures analyses of variance with a withinsubject factor treatment (SSRI/SNRI versus placebo) and a between-subject factor type of substance (SSRI versus SNRI). For the memory tasks, learning performance (at the end of training), retrieval performance and overnight changes (absolute differences between performance at retrieval minus performance at learning) served as dependent variable in separate analyses. Accuracy data from both procedural tasks were log transformed prior to analysis to account for deviations from normal distribution due to positive skewness. Because 7

8 data from the two placebo conditions did not differ, we report collapsed results across both placebo conditions (except in the tables listing these conditions separately). For analyses of hormone concentrations, values were averaged to obtain mean values during learning, 1 st half of sleep, 2 nd half of sleep and retrieval, respectively. A two-tailed P-value < 0.05 is considered significant. 2. Supplementary Results Sleep parameters Both the SSRI fluvoxamine and the SNRI reboxetine profoundly altered sleep architecture in the night after training, although total sleep time was comparable for treatment and placebo nights (P > 0.4; Supplementary Table 1). As expected, the two substances reduced time spent in REM sleep from 16.8 ± 1.0% during placebo nights to 12.5 ± 1.0% after SSRI, and to 2.6 ± 1.1% after SNRI administration (main effect of treatment P < ; type of substance treatment interaction P < ). Post hoc comparisons confirmed that REM sleep reductions were highly significant for both SSRI (P = 0.002) and SNRI (P < ) administration compared to placebo conditions and were stronger for SNRI as compared to SSRI nights (P < ; Supplementary Table 1). Although data from four subjects with high wake and stage 1 sleep percentages (1 after SSRI, 3 after SNRI administration) were excluded (see Supplementary Methods), percent time spent awake during the night was still significantly enhanced for both types of treatments compared to placebo nights (main effect treatment P = 0.002). Also stage 1 sleep percentage tended to be enhanced (main effect treatment P = 0.06). In nights after the SNRI reboxetine, time spent in stage 2 sleep was strongly increased (64.5 ± 1.6%) compared to the SSRI fluvoxamine (55.9 ± 1.5%; P = ) and placebo nights (55.2 ± 1.4%; P = ; type of substance treatment interaction P = 0.004, see Supplementary Table 1, for post hoc comparisons). Time in SWS remained unchanged (all P > 0.1). 8

9 Spindles characterize stage 2 sleep and usually extend into SWS. Spindles (counts and density) in these sleep stages were analyzed because of their well-known relationship with overnight retention of memories The number of fast spindles (> 13 Hz) across stage 2 and SWS was increased after administration of the SNRI, in accordance with the increased time spent in stage 2 sleep in this condition ( type of substance treatment interaction P = 0.006). Independent of the effect on NonREM sleep duration, the SNRI reboxetine also enhanced fast spindle density (spindle count/number of NonREM epochs; type of substance treatment interaction P = 0.02). Slow (< 13 Hz) spindle number or density were not affected by the treatments (all P > 0.1). In the second night after the learning phase (i.e., the night before retrieval testing), total sleep time as monitored by actigraphy did not differ between placebo, SSRI and SNRI conditions (429 ± 5 min; 428 ± 8 min and 408 ± 8 min; all P > 0.1). Because REM sleep was Supplementary Table 1: Sleep parameters Main effect Interaction Placebo SSRI Placebo SNRI Treatment Substance Treatment Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM P P Wake % 2.9 ± ± ± ± 1.1** Stage 1 % 8.8 ± ± ± ± Stage 2 % 55.1 ± ± ± ± 1.6** < SWS % 16.4 ± ± ± ± REM sleep % 16.7 ± ± 1.0** 16.8 ± ± 1.1** <0.001 <0.001 Total sleep time (min) 401 ± ± ± ± Sleep latency (min) 13 ± ± ± ± REM latency (min) 104 ± ± ± ± Slow spindles count 548 ± ± ± ± density 1.01 ± ± ± ± Fast spindles count 797 ± ± ± ± 68** density 1.46 ± ± ± ± 0.11* Sleep parameters for nights after administration of the SSRI fluvoxamine and SNRI reboxetine compared with respective nights after placebo administrations. Time periods of intermittent wakefulness (wake), stage 1 and 2 sleep, slow wave sleep (SWS) and rapid eye movement (REM) sleep are indicated as percentages of total sleep time. Total sleep time, sleep latency (with reference to light off) and REM sleep latency (with reference to sleep onset) are indicated in minutes. Spindle counts (absolute numbers during stage 2 and SWS) and density (count/30-sec epochs of stage 2 and SWS) are indicated separately for slow (<13Hz) and fast spindles (>13Hz). Means±SEMs are shown. Rightmost columns indicate P-values from analyses of variance for main effect of treatment (SSRI/SNRI versus placebo) and type of substance (SSRI versus SNRI) treatment interaction. In the case of significant main or interaction effects, post-hoc pairwise comparisons were conducted using t-tests. P < 0.1 ; *P < 0.05; **P < 0.01; for t-tests between treatment (SSRI or SNRI) and respective placebo nights; P < 0.01 for t-tests between SNRI and SSRI nights. 9

10 almost completely suppressed in the first night after the SNRI, in a subgroup of subjects (n = 4) we performed polysomnographical recordings in this second night to investigate possible REM sleep rebound effects. Despite strong REM sleep suppression during the first night in these subjects (placebo: 17.7 ± 1.5%; SNRI: 5.0 ± 1.5%; P = 0.001), no signs of a REM sleep rebound were revealed in the second night after SNRI administration (placebo: 20.1 ± 1.3% vs. SNRI: 15.7 ± 3.7%, P = 0.19). This finding is consistent with results from studies in rats indicating no REM sleep rebound effects after SSRI or SNRI administration 22. Memory performance and spindles additional analyses Supplementary Table 2 summarizes performance on the different memory tasks at learning and retrieval as well as changes in performance across the retention interval in the active treatment and placebo conditions of the main experiment. Overnight changes in mirror tracing performance or recalled word-pairs were not impaired after SSRI or SNRI administration but, unexpectedly, overnight gains in finger sequence tapping accuracy were even increased after SSRI and SNRI administration as compared to placebo conditions (P = 0.01). Re-analysis of the raw data on the basis of single trials during training and retrieval testing (instead of average scores) did also not reveal any signs of impairments after pharmacological REM sleep suppression (all P > 0.4). To further explore the role of spindles in motor memory consolidation, we correlated inter-individual differences in sleep spindle number and density with overnight gains in accuracy in the finger sequence tapping task separately for active treatment and placebo conditions. In both active treatment and placebo conditions, the number of fast spindles in the last quarter of sleep was significantly correlated with overnight gains in finger tapping accuracy (active treatment: r = 0.46; P = 0.02; placebo: r = 0.39; P = 0.05). No such correlations were observed for other sleep parameters (all P > 0.05). These results fit well previous reports of an association between, respectively, stage 2 sleep and spindle activity in 10

11 the last quarter of the night and motor skill consolidation 9,17. However, independent of acute learning preceding sleep, inter-individual differences in spindle activity are also associated with general capabilities of learning 21,23 25 which thus also contribute to this type of correlation. A causative role of spindle activity for motor skill consolidation still needs to be demonstrated. Approaches in this regard may take advantage of pharmacological agents (e.g., benzodiazepines) that more specifically enhance spindle activity 26. Supplementary Table 2: Memory performance on procedural and declarative tasks Mirror tracing Placebo SSRI Placebo SNRI Main Effect Treatment Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM P Draw time Learning 92.7 ± ± ± ± Retrieval 80.0 ± ± ± ± Change 12.7 ± ± ± ± Number of errors Learning 19.8 ± ± ± ± Retrieval 10.7 ± ± ± ± Change 9.1 ± ± ± ± Finger sequence tapping Number of Learning 18.5 ± ± ± ± sequences Retrieval 20.0 ± ± ± ± Change +1.5 ± ± ± ± Error rate Learning 7.0 ± ± ± ± Retrieval 7.4 ± ± ± ± Change +0.4 ± ± ± ± 1.0* 0.01 Word-pairs Trials to criterion Learning 2.0 ± ± ± ± Recalled word- Learning 28.5 ± ± ± ± Pairs Retrieval 27.4 ± ± ± ± Change 1.1 ± ± ± ± Means±SEMs are shown. Rightmost column indicates P-values for analysis of variance main effect treatment (active treatment vs. placebo; Substance x Treatment interactions remained non-significant; all P > 0.2). *P < 0.05, for posthoc t-tests between treatment (SSRI or SNRI) and respective placebo nights. To identify the best predictor for the difference in accuracy gains between active treatment and placebo conditions, we performed post hoc a stepwise regression analysis. As potential predictors we included the difference between active treatment and placebo nights in the percentage of REM sleep, stage 2 sleep and SWS, fast spindle number, slow spindle number, cortisol levels in the first night-half, and cortisol levels in the second night-half. The 11

12 analysis identified the single predictor number of fast spindles as best-fitting model (adjusted explained variance R 2 = 30%). Essentially the same result was obtained when spindle density (instead of number) was included in the analysis. No other predictors substantially increased the proportion of explained variance. Reaction times, mood and neuroendocrine measures At learning and retrieval testing, participants did not differ with regard to reaction time and subjective ratings of mood, wakefulness or restlessness between SSRI, SNRI and placebo conditions (all P > 0.1; Supplementary Table 3). Levels of cortisol and norepinephrine at learning and retrieval testing were comparable between the experimental conditions (all P > 0.1). However, the SNRI reboxetine strongly enhanced cortisol levels during the first half of post-learning sleep (304 ± 59 mmol/l) compared with levels after the SSRI fluvoxamine (47 ± 7 mmol/l) and placebo (44 ± 7 mmol/l; main effect treatment P < ; interaction type of substance treatment P < ; see Supplementary Table 3 for post hoc comparisons). During the second half of sleep, cortisol levels were increased after both administration of SNRI (305 ± 32 mmol/l) and SSRI (183 ± 29 mmol/l) compared to placebo (158 ± 18 mmol/l; main effect treatment P = ; Supplementary Table 3). Neither reboxetine nor fluvoxamine influenced plasma norepinephrine levels during postlearning sleep (all P > 0.2; Supplementary Table 3). The SNRI induced increase in cortisol levels during sleep after learning replicates observations in waking subjects 27. Because cortisol does not influence sleep-dependent consolidation of procedural memories 13,28, a possible confounding effect of the hormone on skill consolidation can be excluded. However, in those previous studies, increased cortisol concentrations during early SWS-rich sleep consistently impaired declarative memory consolidation, an effect not observed here after SNRI administration for the retention of word-pairs. We assume that in the present study, 12

13 simultaneously increased levels of norepinephrine compensated for impairing effects of high cortisol levels after SNRI. Supplementary Table 3: Reaction times, mood and neuroendocrine measures Main Effect Interaction Placebo SSRI Placebo SNRI Treatment Substance Treatment Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM P P RT [ms] Learning 240 ± ± ± ± Retrieval 235 ± ± ± ± Mood Learning 16.5 ± ± ± ± Retrieval 16.3 ± ± ± ± Wakefulness Learning 10.7 ± ± ± ± Retrieval 13.6 ± ± ± ± Calmness Learning 15.9 ± ± ± ± Retrieval 15.7 ± ± ± ± Cortisol Learning 182 ± ± ± ± [mmol / L] Sleep (1 st half) 45 ± 7 47 ± 7 44 ± ± 59** <0.001 <0.001 Sleep (2 nd half) 122 ± ± 29** 194 ± ± 32* < Retrieval 430 ± ± ± ± Norepinephrine Learning 1.39 ± ± ± ± [nmol / L] Sleep (1 st half) 0.44 ± ± ± ± Sleep (2 nd half) 0.51 ± ± ± ± Retrieval 1.82 ± ± ± ± Reaction times (RT, as a measure of vigilance), self-reported mood, wakefulness and calmness as well as blood cortisol and norepinephrine concentrations during learning and at retrieval testing for the SSRI (fluvoxamine) and SNRI (reboxetine) conditions, compared with respective placebo conditions. Hormonal levels were additionally measured during sleep on the first night after learning. Means±SEMs are shown. P-values from analyses of variance are indicated. In case of significant main or interaction effects, post-hoc analyses were conducted using pair-wise t-tests. *P < 0.05; **P < 0.01, for t-tests between treatment (SSRI or SNRI) and respective placebo nights; P < 0.01 for t-tests between SNRI and SSRI nights. Supplementary experiment (retrieval test after 24 h) Changes in post-learning sleep after reboxetine replicated those of the main experiment (Supplementary Table 4). The SNRI strongly reduced REM sleep (6.7 ± 1.6%) as compared to placebo (18.8 ± 1.5%; P < 0.001) and increased REM sleep latency (SNRI: 205 ± 29 min; placebo: 93 ± 11 min; P = 0.003). Conversely, time in stage 2 sleep (P = 0.05) and awake (P = 0.02) were enhanced after SNRI administration, as compared to placebo. SWS remained unaffected (P > 0.1). 13

14 Supplementary Table 4: Sleep parameters (24-h control experiment) Placebo SNRI Main effect treatment Mean ± SEM Mean ± SEM P Wake % 0.7 ± ± Stage 1 % 4.7 ± ± Stage 2 % 55.0 ± ± SWS % 20.1 ± ± REM sleep % 18.8 ± ± 1.6 <0.001 Total sleep time (min) 420 ± ± Sleep latency (min) 31 ± ± REM latency (min) 93 ± ± Means±SEMs are shown. Right column indicates P-values from pair-wise t-tests comparing the effects of SNRI vs. placebo. The effects of the SNRI on overnight memory retention (measured 24 h after learning) were strikingly similar to those observed in the main experiment where memory retention was measured after an additional recovery night (i.e., 32 h after learning). In spite of the marked REM sleep suppression, overnight changes in mirror tracing performance at the 24-h testing were entirely unchanged after SNRI, as compared with placebo (Supplementary Table 5). Draw time significantly decreased across sleep after placebo ( 13.8 ± 2.6 s; P < 0.001) as well as SNRI administration ( 15.7 ± 2.0 s; P < 0.001), and so did the number of deviations from the path (errors placebo: 8.0 ± 1.4; P < 0.001; SNRI: 9.8 ± 1.9; P < 0.001). These overnight gains in speed and accuracy were well comparable in the two experimental conditions (both P > 0.5). As in the main experiment, overnight gains in finger sequence tapping were enhanced after REM sleep suppression by SNRI administration, as compared to placebo. While tapping speed increased across sleep in both conditions (both P < 0.001), the gain in speed was significantly greater after SNRI (+5.2 ± 0.8 sequences) than placebo administration (+3.7 ± 0.6 sequences; P = 0.05). Similarly, error rate was significantly reduced only after SNRI administration ( 6.5 ± 1.4%; P < 0.001), whereas a statistical trend was observed after 14

15 placebo ( 2.4 ± 1.1%; P = 0.06; P = 0.07 for the difference in gains between reboxetine and placebo condition). Supplementary Table 5: Memory performance (retrieval test after 24h) Mirror tracing Placebo SNRI Main effect treatment Mean ± SEM Mean ± SEM P Drawing time Learning 82.5 ± ± Retrieval 68.7 ± ± Change 13.8 ± ± Number of errors Learning 18.4 ± ± Retrieval 10.4 ± ± Change 8.0 ± ± Finger sequence tapping Number of Learning 19.5 ± ± sequences Retrieval 23.2 ± ± Change +3.7 ± ± Error rate Learning 8.3 ± ± Retrieval 5.9 ± ± Change 2.4 ± ± Word-pairs Trials to criterion Learning 2.0 ± ± Recalled word- Learning 28.1 ± ± pairs Retrieval 26.5 ± ± Change 1.6 ± ± Means±SEMs are shown. Right column indicates P-values from pair-wise t-tests comparing the effects of SNRI vs. placebo. Retention of word-pairs on the declarative paired associate learning task was not affected by the SNRI (all P > 0.2; Supplementary Table 5). Retention rates averaged 1.6 ± 0.9 and 1.2 ± 0.9 word-pairs in the placebo and SNRI conditions respectively (P > 0.7). In the supplementary experiment at retrieval testing after 24 h, reaction times were slower (253 ± 7.5 ms vs 245 ± 8.6 ms; P = 0.04) and ratings of mood were lower (16.0 ± 0.7 vs 17.8 ± 0.6; P = 0.004) after SNRI than placebo administration (Supplementary Table 6). Also subjects at retrieval testing felt less alert after SNRI than placebo (13.3 ± 1.2 vs 16.4 ± 0.8; P = 0.005). Such differences were not observed in the main experiments at retrieval testing after 32 h, and might reflect immediate effects of minute amounts of the substance still circulating at this time of testing or effects secondary to altered sleep. 15

16 Supplementary Table 6: Reaction time and mood (24-h control experiment) Placebo SNRI Main effect Treatment Mean ± SEM Mean ± SEM P RT [ms] Learning 266 ± ± Retrieval 245 ± ± Mood Learning 17.7 ± ± Retrieval 17.8 ± ± Wakefulness Learning 13.8 ± ± Retrieval 16.4 ± ± Calmness Learning 15.6 ± ± Retrieval 15.2 ± ± Means±SEMs are shown. Right column indicates P-values from pair-wise t-tests comparing the effects of SNRI vs. placebo. Overall, results of this supplementary experiment confirm the main study, indicating an enhanced rather than impaired sleep-dependent gain in motor skill after administration of the SNRI reboxetine, although the substance reduced REM sleep to a minimum. The study omitting a second post-learning night (introduced in the main experiments to ensure complete recovery from immediate effects of the substance) thus safely excludes that the lacking impairment of sleep-dependent skill consolidation after pharmacological REM sleep blockade on the first post-learning night was due to compensating effects of REM sleep in the second (recovery) night. 16

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