SUPPLEMENTARY INFORMATION

Supplementary Methods and Results 1 Subjects, procedure, and memory tasks Subjects: Thirteen subjects (7 women) with a mean age of 23.8 yr (range 20 - 28 yr), free of

medication and non-smokers participated in the studies after having given informed written

consent. Subjects with, or the history of any of the following were excluded: epilepsy,

paroxysms, cognitive impairments, mental, hormonal, metabolic, circulatory disorders or

sleep disturbances. The experimental protocol was approved by the ethics committee of the

University of Lbeck.

Procedure: After an adaptation night subjects were tested in two conditions, a stimulation

condition and a sham stimulation condition, which were balanced in order across subjects (cp.

Supplementary Figure 1 for a summary of the procedure and the main findings of the paper).

The two sessions of a subject were separated by an interval of at least 1 week. Subjects

arrived at the laboratory at 19:00 h. Following preparation for slow oscillation stimulation,

EEG and polysomnographic recordings, subjects performed on tasks of declarative memory

(verbal and non-verbal paired-associate learning) and procedural memory (mirror tracing,

finger sequence tapping) between 21:00 and 22:30 h (learning period). The order of tasks was

balanced across subjects. Subjects subsequently went to bed, and polysomnographic and EEG

recordings started (lights off: 23:00 h, awakening: 06:30 h). Slow oscillation stimulation

began after the subject had attained 4 min of stable NonREM sleep for the first time after

sleep onset, i.e., after online scoring confirmed the presence of eight consecutive 30-s epochs

of sleep stage 2 or a deeper NonREM sleep stage. After 7.5 hours of sleep subjects were

awakened, whenever they were in light NonREM sleep (stages 1 or 2). About 30 min after

awakening, recall of memories was examined (retrieval period). Before learning and after

recall testing also psychometric control tests were given to assess general retrieval function,

working memory, capabilities to concentrate as well as feelings of tiredness and mood (see

Section 6).

Memory tasks and psychometric tests: To assess declarative memory, a paired-associate

learning task was used that had proven sensitive to effects of sleep previously1,2. The task

consists of the sequential presentation of 46 semantically related pairs of German nouns (see

Supplementary Information 2, Word Lists) on a monitor with a presentation rate of 1/5 s and

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an interstimulus interval of 100 ms. In addition, 4 dummy pairs of words at the beginning and

end of each list served to buffer primacy and recency effects, respectively. A different word

list was used for each of the subject's two experimental sessions. At learning before sleep,

presentation of the list was followed by a task of cued recall, i.e., the subject was to respond

by naming the second word on presentation of the first (cue) word of each pair, whereby the

46 stimulus words of the word list appeared on the screen in a different order than during the

foregoing presentation. The subject had unlimited time to recall the appropriate response

word. Immediately after word recall the correct paired-associate was revealed on the screen.

The number of correct responses was calculated immediately after presentation of all cue

words. If a minimum of 60 % correct responses was not obtained, word pairs were presented

again in a newly randomized order (to prevent serial learning) and cued recall was repeated.

At retrieval testing in the morning following sleep cue words were again displayed in a newly

randomized order, and the subject was required to recall the appropriate response words.

Overnight memory retention is represented by the difference in the number of recalled words

between morning retrieval testing and evening immediate recall. It is to note that this measure

does not allow differentiating between processes of memory stabilization (slowed decay) and

enhancement that have been proposed to underlie procedural memory consolidation (see e.g.,

ref. 3).

To test procedural memory, a finger sequence tapping task was used (adapted from ref.

4). Subjects were required to repeatedly finger-tap with the non-dominant left hand a five-

element sequence presented on a computer monitor, e.g. 4-2-3-1-4, as fast and accurately as

possible on a key board. The training period before sleep consisted of twelve 30-s intervals

with 30-s breaks between trials. Retrieval testing after sleep consisted of a practice run

followed by three 30-s test intervals. A working memory component of the task was excluded

by continuous presentation of the sequence on the screen. To prohibit interference no

feedback was given on pressing keys. Each 30-s interval was scored for the number of

correctly completed sequences and the number of errors made. Performance at learning and

retrieval testing was defined by averaged scores from the final three intervals during the

learning period, and from the three intervals of the retrieval period, respectively.

For exploratory purposes, in a subgroup of 10 subjects declarative and procedural

memory was tested additionally on a nonverbal paired-associate task and a mirror tracing

task, respectively. (Results for these tasks are presented in Supplementary Fig. 1b). On the

non-verbal paired associate task, instead of word-pairs, subjects were shown a list of 16

evenly balanced pairs of either geometric or non-geometric line-drawings (adapted from ref.

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5). During learning, presentation of the list was followed by a cued recognition task in which

subjects had unlimited time to recall the appropriate response drawing from a group of seven

simultaneously presented other drawings. Learning ended when a minimum of 10 correct

responses was reached on a run. At retrieval testing the cued recognition task was repeated

using a newly randomized order of presentation. The mirror tracing task was adopted from

previous studies indicating a beneficial effect on memory performance for this task of sleep

during the second, REM sleep dominated half of the night, but not of sleep during the first

half alone 1. In this task subjects had to trace as fast and as accurately as possible line-drawn

meaningless figures while these figures and their hand movements were visible only through a

mirror. A parallel set of figures (with 26 to 27 angles and curved corners) were used for

testing. Subjects traced each figure with an electronic stylus starting and ending at the same

point. An error consisted of moving the stylus off the line of the figure. At learning, subjects

first performed practice runs with a star-like figure until draw time was less than 1 min and

less than 12 errors were made, and then continued with 4 runs with the test figure. At retrieval

testing, after one practice run, performance on 4 runs on the test figure was examined. On

each occasion, the total time to trace a figure, the number of errors and the error time (time off

the line) were measured, and averaged across the 4 runs.

2 Slow oscillation stimulation and sleep recordings Slow oscillation stimulation: Potential fields oscillating at a frequency of 0.75 Hz (0.33 s-

on/0.33 s-off with rising and falling slopes of 0.33 s) were induced by a battery driven

constant current stimulator via stimulation electrodes (8 mm diameter) applied bilaterally at

frontolateral locations, i.e., F3 and F4 of the international 10:20 system6 and at the mastoids.

Cortical effects of transcranial electrical stimulation, although rather widespread in nature,

depend strongly on the positioning of stimulation electrodes7. Frontal-to-mastoid stimulation

was chosen since endogeneous slow oscillations are maximal and originate primarily in the

prefrontal cortex8. Electrode resistance was always < 2 kOhm. In the sham-session the

electrodes were applied as in the stimulation sessions, but the stimulator remained off. Pilot

studies assured that stimulation was not felt by the subjects even when awake.

EEG recordings: The EEG was recorded continuously using a Toennies DC/AC amplifier

(Jaeger GmbH, Germany; amplification, 1000 VV-1). EEG signals were filtered between

0.0835 Hz and sampled with 200 Hz and 16-bit precision (CED 1401 plus, Cambridge

Electronics, UK). Ag-AgCl electrodes were placed according the 10-20 System (Fz, Cz, Pz,

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C3, C4, P3, P4, F7, F8, T3, T4) and referenced to the nose. Additionally, horizontal and

vertical eye movements and the electromyogram (chin) were recorded for standard

polysomnography.

3 Analyses of sleep measures and EEG Two types of analyses were performed off-line. First, sleep structure was determined visually

based on standard polysomnographic criteria9. For the total sleep period every 30-s epoch was

scored as NonREM sleep stage 1, 2, 3, 4 or REM sleep. Slow wave sleep (SWS) was

determined as the sum of time in sleep stages 3 and 4. Intervals during acute stimulation were

not scored due to excessive signal artifacts. In the sham stimulation session corresponding

intervals (starting after the presence of eight consecutive 30-s epochs of sleep stage 2 or a

deeper NonREM sleep stage) were also not scored. Scoring for the 1-min stimulation-free

intervals (between the 5-min blocks of acute stimulation) was additionally performed for 10-s

epochs. Time spent in the different sleep stages was determined (i) for the 1-min stimulation-

free intervals and corresponding 1-min intervals of the sham stimulation condition, (ii) for a

60-min interval subsequent to the end of stimulation, and (iii) for the total nights.

In a second, more fine-grained analysis, the immediate influence of stimulation on

EEG power was investigated. Up to five non-overlapping blocks of artifact-free EEG with

2,048 data points each ( 10.2 s) were used for every 1-min stimulation-free interval and

corresponding intervals of sham-stimulation. On every 2,048-point block of EEG data, a

Hanning window was applied before calculating the power spectra using fast Fourier

transformations. Individual mean power spectra across all blocks of a stimulation-free interval

were calculated and subjected to a three-point moving average. Subsequently, mean power in

the following EEG bands was calculated: slow oscillations (0.51 Hz), low delta (11.5 Hz),

delta (14 Hz), theta (48 Hz), slow frontal spindle (812 Hz), fast parietal spindle (1215

Hz) and beta (1525 Hz). Frequency bands were selected on the basis of the mean power

spectrum and previous research: The slow oscillations show a predominant frequency of 0.7

0.8 Hz10-12. Cellular activity underlying the slow oscillation as well as the temporal dynamics

of slow oscillations across sleep episodes clearly differ from those of the faster (14 Hz) delta

oscillations10,12,13. Nevertheless due to their shape slow oscillations can affect the low

frequencies of the delta band8,11,14,15, which led us to separately explore effects on the 11.5

Hz frequency band immediately adjacent to the slow oscillation band. The separate spindle

bands refer to studies of the human EEG which have consistently demonstrated the presence

of two kinds of spindle activity, i.e., slow spindle activity that prevails over the frontal cortex

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and shows a greater topographical variability than the faster spindle activity that concentrates

over the parietal cortex16-18. A functional differentiation has been suggested whereby the slow

spindle activity appears to reflect predominant coupling among cortical networks, whereas the

fast spindle activity is presumed to be more closely associated with thalamo-cortical

coupling19. For statistical analysis mean power within a 1-min interval immediately preceding

stimulation and sham-stimulation onset was subtracted from the mean power during

stimulation-free intervals.

4 Statistical analyses Statistical analysis was based generally on analyses of variance (ANOVA), including a

within-subject factor Stimulation condition (slow oscillation stimulation versus sham

stimulation). Additional repeated measure factors were either Time (evening, morning), or for

EEG spectral analyses the five stimulation-free intervals. Where appropriate, a Greenhouse-

Geisser correction for degrees of freedom was used. Regarding behavioural measures of

memory, prior to statistical analysis difference values (between performance at retrieval after

sleep minus performance at learning before sleep) were calculated to indicate retention. For

data lacking normal distribution the Wilcoxon test was employed. A P-value < 0.05 was

considered significant.

5 Spindle counts In addition to EEG spindle power, the number of slow frontal and fast parietal discrete

spindles (spindle density for each 30-s epoch) was analysed as a further measure of spindle

activity. Detection of spindles based on an algorithm adopted from previous studies20,21: First,

the EEG signal was filtered in the corresponding frequency bands (812 Hz, 1215 Hz), then

the root mean square (RMS) of each 100-ms interval was calculated, and lastly the number of

times were counted for which the RMS signal exceeded a threshold (adapted to visually

identified templates) for 0.5-3 s. Confirming the results for EEG spindle power as described

in the main text, analyses of discrete spindle counts during the 1-minute stimulation-free

intervals revealed a significant increase with stimulation as compared with sham stimulation

for slow spindles over the frontal location (4.14 1.13 versus 2.76 0.92 spindles x 30 s-1,

F1,12 = 5.89, P < 0.05). In contrast, there was no significant difference between the stimulation

and sham conditions during these intervals in fast spindle counts over the parietal location

(2.56 0.96 versus 3.05 1.15 spindles x 30 s-1, P > 0.5).

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6 Control of unspecific cognitive and hormonal effects of stimulation In the main experiments stimulation was applied during early sleep after learning and recall

was tested the next morning. This procedure, in principle, leaves open the possibility that

changes in recall reflect a lasting effect of the stimulation on general cognition or capabilities

of retrieval that extends into the time of retrieval testing, rather than an effect on the

consolidation of respective memories. To rule out such influences, performance on several

cognitive function test and mood were assessed at retrieval testing. Additionally, two separate

control experiments were conducted and blood concentrations of several relevant hormones

were assessed.

Cognitive function tests and mood at retrieval testing: To assess the general capability to

retrieve information from long term memory, a word fluency task22 was used which required

the subject to write down within 2-min periods, respectively, as many kinds of either jobs or

hobbies, and words starting with the letter M or P. For working memory assessment, the

digit span test of the Wechsler Adult Intelligence Scale was employed, which requires to

repeat lists of orally presented digits forward and backward. Mood was assessed by the

Positive and Negative Affect Schedule (PANAS)23, and an adjective check list24 describing

the subject's current mood on various dimensions (e.g., feelings of activation, tiredness,

concentration, enthusiasm).

Performance on the word fluency task was virtually identical in both conditions (19.53

0.75 (stimulation) versus 19.87 0.88 words retrieved) indicating that slow oscillation

stimulation during sleep did not affect the subjects ability to access previously stored

knowledge on words at the time of retrieval testing (P > 0.6). Also, performance on the digit

span test was not improved, but on average was slightly lower after slow oscillation

stimulation (9.49 0.65 digits) compared with sham stimulation (10.39 0.56 digits, P >

0.15) ruling out that enhanced declarative memory recall after stimulation was a consequence

of a general improvement in prefrontal working memory function. Mood scores were also not

affected by stimulation (P > 0.2) (There were also no differences in these measures between

the two conditions at learning.)

Additional control experiments: In the main experiments stimulation started soon after

learning during the first period of NonREM sleep. If the enhanced declarative memory after

slow oscillation stimulation reflected directly a facilitatory effect on retrieval performance

rather than on consolidation this action should be enhanced by positioning the stimulation

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closer to retrieval testing. To test this possibility we performed two control experiments both

conducted according to within-subject crossover comparisons (stimulation versus sham) in

two different samples of 8 subjects (19 - 35 yr, 3 females and 5 males in each sample). In the

first experiment, the protocol was exactly identical to that in the main experiment including

stimulation and tasks except that stimulation was shifted to a period shortly before awakening,

i.e., the slow oscillation stimulation began 45 min prior to and ended 15 min prior to

awakening (at 6.30 h). Stimulation was performed independently of the presence of a certain

sleep stage and started during stage 2 NonREM sleep in 6 subjects and during REM sleep in 2

subjects. Performance on the word paired-associate task did not differ between the stimulation

and sham conditions (words recalled at learning before sleep, 36.58 1.88 (stimulation),

37.94 1.69 (sham), words recalled after sleep, 39.79 2.02 (stimulation), 40.87 1.13

(sham), P > 0.4, for all comparisons). There were also no significant effects of stimulation

conducted shortly before awakening on procedural memory for finger tapping skill

(improvement in correctly tapped sequences at retrieval after sleep with reference to

performance at learning, 2.61 0.54 (stimulation), 2.55 0.55 (sham), P > 0.5) or on

measures of vigilance at retrieval testing (P > 0.5).

Since it could be argued that non-specific cognitive effects arise from slow oscillation

stimulation only when applied during the SWS-rich early part of sleep, i.e. a brain state

particularly sensitive to this stimulation, we performed a second control experiment where

stimulation took place, as in the main experiment, during early NonREM sleep and the

retrieval period was again 30 min after awakening. However, to avoid an effect on sleep

associated processes of consolidation presumed to be strongest shortly after learning, learning

and recall were separated by 4 nights and stimulation was delayed until the fourth night, i.e.,

the last night before recall testing25. Experimental procedures were otherwise the same as in

the main experiment, except that because of the prolonged retention interval (during which

forgetting becomes prevalent) the learning criterion was set to 80 % correct responses for the

word paired-associate task. The two learning sessions were separated by an interval of at least

10 days. Also in this control experiment slow oscillation stimulation failed to improve recall

of word-pairs with on average even more words forgotten across the 4-day retention interval

in the stimulation than in the sham condition (-7.13 1.75 versus -5.38 1.38 words, P >

0.23). Also, procedural memory recall of finger tapping skill and measures of vigilance at

retrieval testing remained on average non-significantly lower after stimulation than sham

stimulation.

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Blood hormone concentrations: There is evidence that consolidation of declarative memories

is affected by hormones such as corticosteroids and catecholamines which are regulated by

sleep and appear to target specifically hippocampal function26,27. To examine whether slow

oscillation stimulation alters secretion of such hormones during sleep, the latter control

experiments included additionally the assessment of blood concentrations of cortisol, growth

hormone and norepinephrine every 15 min during the stimulation and every 30-60 min during

the remaining night. For blood sampling, a catheter was connected to a long, thin tube

enabling blood collection from an adjacent room without disturbing the subjects sleep. Blood

samples for determining cortisol and growth hormone concentrations in serum were

immediately centrifuged and the supernatant stored at 20C until assay. Cortisol and growth

hormone were measured by ELISA (Immulite, Diagnostic Products Corporation Biermann,

Bad Nauheim, Germany; cortisol: sensitivity, 0.2 gdL-1; intra- and interassay coefficients of

variation: < 8.8 and < 10 %, respectively; growth hormone: sensitivity, 0.01 ngmL-1; intra-

and interassay coefficients of variation: < 6.2 and < 6.2 %, respectively). Standard HPLC with

electrochemical detection was used for plasma norepinephrine detection (sensitivity, 15 ngL-1,

intra- and interassay coefficients of variation < 3.9 and < 5.9 %). We found no effects of

stimulation on any of the hormones measured (P > 0.5).

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Supplementary Figure and Legend

Sleep RecallLearning

a b

Declarative Declarative

Procedural Procedural

Sham Stimulation

Figu

res

-0.5

0.0

0.5

1.0

*

Dra

w t

ime

(s) -24

-16

-8

0 StimulationSham

Sleep RecallLearning

a b

Declarative DeclarativeDeclarative

Procedural Procedural

Sham Stimulation

Figu

res

-0.5

0.0

0.5

1.0

*

Dra

w t

ime

(s) -24

-16

-8

0 StimulationSham

Supplementary Figure 1 a, Stimulating the brain with slowly oscillating (0.75 Hz) potential fields during early NonREM sleep after learning of declarative and procedural memory tasks improves later recall of the declarative but not of the procedural memories. The slow oscillation stimulation leads to an immediate potentiation of endogenous slow oscillation and spindle activity (red). Thereby stimulation presumably enhances the efficacy of a slow oscillation-driven hippocampo-neocortical dialogue enabling the reprocessing and consolidation of declarative memories during sleep. b, Recall performance on two additional memory tasks introduced for exploratory purposes in a subgroup of 10 subjects. Consistent with results from word paired-associate learning (main text), slow oscillation stimulation (red) compared with sham stimulation (black bars), improved cued recognition of figures on the non-verbal declarative task (P < 0.05; top panel indicates the difference between recognition performance at retrieval testing after sleep minus performance at learning before sleep). In the stimulation condition performance improved from 11.60 0.52 figures before sleep to 12.40 0.62 figures recognized after sleep, whereas in the sham stimulation condition cued recognition performance was lower after sleep (11.10 0.67 figures) than at learning before sleep (11.30 0.42 figures). In contrast to declarative memory performance, performance on the procedural mirror-tracing task (like finger tapping, described in the main text), did not reveal significant facilitation by electrical stimulation (P > 0.2; bottom panel indicating improvement in draw time (s) with reference to performance at learning). However in both conditions the typical overnight improvement was observed (P < 0.001, stimulation condition, draw time before sleep: 94.00 14.69 s, after sleep: 73.23 11.75 s; sham condition, before sleep: 92.90 14.78 s, after sleep: 70.85 7.43 s).

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Supplementary Table 1

Table 1 Sleep during the nights of the Stimulation and Sham condition

Entire night

Stimulation (mean SEM)

Sham (mean SEM)

Awake % 1.9 0.6 3.7 2.3 S1 % 7.2 1.5 5.7 1.5 S2 % 50.6 2.5 49.7 1.7 S3 % 8.3 0.8 8.6 1.2 S4 % 2.9 1.1 3.5 1.1 SWS % 10.2 1.4 12.1 1.9 REM % 20.7 1.6 20.6 2.1 Total sleep time (min) 420.2 9.6 426.2 6.4

The hour following stimulation

Stimulation (mean SEM)

Sham (mean SEM)

Awake % 4.6 4.0 6.2 2.9 S1 % 2.4 1.0 6.6 3.4 S2 % 57.2 5.1 59.4 6.1 S3 % 13.6 1.8 11.7 2.6 S4 % 8.1 3.6 5.2 2.4 SWS % 21.7 4.9 16.9 4.1 REM % 13.6 3.2 10.5 3.5

Top panel indicates mean ( standard error of the mean) percentages (%) of total sleep time spent in different sleep stages during the entire night. Bottom panel indicates percentage of time in the different sleep stages during the hour following stimulation. Total sleep time defines the time from sleep onset until awakening. There were no significant differences between the slow oscillation stimulation and sham stimulation conditions. S1-S4, sleep stages 1-4; SWS, slow-wave sleep; REM, rapid-eye-movement sleep. Note the 30-min interval of stimulation and the corresponding interval in the sham condition were excluded from analysis of the entire nights, which decreases the proportion of SWS typically dominating this period of sleep.

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