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Participants: Ten right-handed subjects participated in the experiment for. 4 consecutive ... of sleep stage and of the time of night, pointed to a drop of transcallosal inhibition ..... left hemisphere, the less a subject is likely to report dreams; this.
Reduction of Transcallosal Inhibition upon Awakening from REM Sleep in Humans as Assessed by Transcranial Magnetic Stimulation Mario Bertini MD, PhD1; Luigi De Gennaro PhD1; Michele Ferrara PhD1,2; Giuseppe Curcio PhD1; Vincenzo Romei PhD1; Fabiana Fratello PhD1; Riccardo Cristiani PhD1; Flavia Pauri PhD3; Paolo Maria Rossini MD, PhD4,5 di Psicologia - Università di Roma “La Sapienza”; 2Dipartimento di Medicina Interna e Sanità Pubblica – Università di L’Aquila; di Neurologia Clinica, Otorinolaringoiatria, Riabilitazione Sensoria, Motoria e dei Disturbi della comunicazione - Università di Roma “La Sapienza”; 4Clinica Neurologica - Università di Roma “Campus Bio-medico”; 5AFaR - Ospedale San Giovanni Calibita Fatebenefratelli, Rome, Italy 1Dipartimento 3Dipartimento

Study Objectives: The aim of the study is to assess, in humans, transcallosal inhibition upon awakening from rapid eye movement (REM) and non-REM sleep, by paired-pulse transcranial magnetic stimulation (TMS). Design: During the daytime, a baseline session of motor evoked potentials (MEPs) was recorded. During the nighttime, the TMS sessions were administered just before sleep onset and upon awakenings from REM and stage 2 sleep, both in the early and final part of night. Setting: The sleep research laboratory at the University of Rome “La Sapienza.” Participants: Ten right-handed subjects participated in the experiment for 4 consecutive sleep-recording nights. Interventions: N/A. Measurements and Results: During the daytime, a robust transcallosal inhibition was found; the MEP amplitude reduction ranged from 35% to 40%. During the nighttime, a decrease of transcallosal inhibition from right-to-left motor cortex, as compared to that from left-to-right motor cortex, was observed. The direct assessment of MEP changes, as a function of sleep stage and of the time of night, pointed to a drop of transcallosal inhibition after awakening from REM sleep. Therefore, the inhibitory activ-

INTRODUCTION THE CEREBRAL HEMISPHERES ARE FUNCTIONALLY INTERCONNECTED BY A TONIC EXCHANGE OF IMPULSES VIA THE CORPUS CALLOSUM. Spontaneous callosal activity recorded in animal by macroelectrodes in the splenium, the genu, or both has been found to vary with the behavioral state, being far more intense during wakefulness than sleep. In fact, callosal activity progressively decreases during the electroencephalographic (EEG) synchronization process as compared to wakefulness, and a further drop of activity is observed at the onset of rapid eye movement (REM) sleep.1 These results suggest that during sleep, and in particular during stage REM, a functional disconnection of the cerebral hemispheres may occur. Data on human sleep, in line with Berlucchi’s findings in cats,1 has come from studies on spectral interhemispheric EEG coherence. During REM sleep, a higher degree of functional discon-

Disclosure Statement This research was partially supported by Ateneo 2000-2002 Grants (M.B.) and by an ESRS–SANOFI-Synthelabo Grant 2002 (M.F.). Submitted for publication October 2003 Accepted for publication March 2004 Address correspondence to: Luigi De Gennaro, PhD, Dipartimento di Psicologia - Sezione di Neuroscienze, Università degli Studi di Roma “La Sapienza”, Via dei Marsi, 78; 00185 Roma, Italy; Tel: 39 06 4991 7647; Fax: 39 06 44 51 667; E-mail: [email protected] SLEEP, Vol. 27, No. 5, 2004

ity of transcallosal fibers observed after non-REM awakening almost disappeared after REM sleep awakenings. Conclusions: The drastic drop of transcallosal inhibition after awakenings from REM sleep represents the first evidence in humans of a change of interhemispheric connectivity mediated by the corpus callosum during this sleep stage and may open new avenues for a better understanding of some aspects of sleep mechanisms (ie, dreaming function and dream mentation). Abbreviations: REM, rapid eye movement; MEP, motor evoked potentials; TMS, transcranial magnetic stimulation; EEG, electroencephalogram; ADM muscle, Abductor Digiti Minimi muscle; ISI, interstimulus intervals; MT, motor threshold; IHPP paradigm, interhemispheric paired pulse paradigm; ANOVA, analysis of variance Key Words: transcranial magnetic stimulation (TMS), transcallosal inhibition, corpus callosum, REM sleep, cerebral hemispheres Citation: Bertini M; De Gennaro L; Ferrara M et al. Reduction of transcallosal inhibition upon awakening from REM sleep in humans as assessed by transcranial magnetic stimulation. SLEEP 2004;27(5):87582.

nection of the cerebral hemispheres has been reported, compared to wakefulness.2 A decrease of EEG coherence suggesting lower than normal interhemispheric connectivity during sleep was found also after partial callosotomy3 and in corpus callosum agenesis.4 On the whole, neurophysiologic and neuropsychological data seem to converge, suggesting a decreased interhemispheric transmission during REM sleep. Unfortunately, no direct measurements of callosal function during sleep have been provided in humans, due to intrinsic methodologic difficulties in measuring callosal activity in the healthy via noninvasive techniques. However, in recent years transcranial magnetic stimulation (TMS) has become available to noninvasively assess, among other parameters, the interhemispheric connectivity. The interhemispheric paired-pulse technique consists of a TMS of the motor cortex of 1 hemisphere that inhibits electromyographic responses evoked by a magnetic stimulus given 6 to 30 milliseconds later over the opposite hemisphere.5 Ferbert et al5 first hypothesized that the inhibition is produced at cortical level via a transcallosal route. This method allows a direct evaluation of the activity of callosal fibers during REM and non-REM sleep stages. However, the acquisition of multiple responses to magnetic pulses (6-12 trials) to obtain a stable (averaged) motor evoked potential (MEP) seems incompatible with the intrinsic peculiarities of sleep because, in most cases, sleep is terminated after 1 magnetic stimulus.6 Nevertheless, it has been shown that, on 875 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

forced awakenings, a full reestablishment of the waking conditions needs some time; for instance, recovery of cerebral blood flow and metabolic waking conditions on the frontal lobes needs up to 30 minutes.7,8 These observations have confirmed the longrecognized “carry-over” effects of the preawakening cortical physiology on several behavioral variables assessed upon awakening from different stages of sleep.9-11 Within this framework, it is possible to make inferences about the sleep organization by analyzing the variables of interest immediately after provoked awakenings in the different sleep stages. By means of this approach, we have recently found that corticospinal excitability increases linearly from presleep wakefulness to REM sleep awakenings, and from the latter to stage 2 awakenings.12 The aim of the present study was to assess transcallosal inhibition upon REM and non-REM stage 2 sleep awakenings, both in the first and in the last part of the night. METHODS Subjects Ten female volunteers between the ages of 20 and 26 years (mean age = 23.30; SEM = 0.58) were selected from a university student population to participate in the study because, in a previous study, women showed higher transcallosal inhibition than did men.13 Subjects were right-handed, as assessed by a lateral preference questionnaire (scores > 0.70).14 In a clinical interview, the subjects reported the absence of epilepsy or any other neurologic condition in themselves and their family history and the absence of any other medical or psychiatric disorder. Furthermore, by a short recording session, they were selected according to the criterion of a good motor cortical representation of the target muscle (abductor digiti minimi, ADM). This selection procedure allowed us to avoid too high resting motor thresholds (MTs), as well as to reduce data variability. Further requirements for inclusion were normal sleep duration and schedule; no daytime nap habits, no excessive daytime sleepiness; and no other sleep, medical or psychiatric disorder, as assessed by a 1week sleep log and by a clinical interview. Participants were required to avoid napping throughout the experiment. An ambulatory mini motion logger actigraph (AMI Mini motionlogger, Ardsley, New York, U.S.A.), which measures wrist activity every 30 seconds and distinguishes sleep from wakefulness with a high degree of accuracy,15 was placed on the subject’s nondominant wrist. Compliance was controlled by checking the wrist activity data monitored by the actigraph. The protocol of the study was approved by the local Institutional Ethical Committee, and the subjects gave their written informed consent, according to the Declaration of Helsinki. Materials Transcranial Magnetic Stimulation The stimulators used were 2 Magstim 200 Mono Pulse devices connected to a Bistim module and to 2 figure-8 coils with external wing diameters of 9 cm (Magstim Company Limited, Whitland, Wales, U.K.). The peak magnetic field produced by such coils was 2.0 T. The MEPs of the hand muscles were recorded from the ADM muscles of both hands. Two Ag-AgCl surface cup electrodes of 9-mm diameter were used: the active electrode was placed over SLEEP, Vol. 27, No. 5, 2004

the muscle belly, while the reference electrode over the metacarpophalangeal joint of the little finger. The MEPs were recorded during complete relaxation, according to standard procedures,16,17 and were stored and analyzed with an electromyogram-dedicated software (Myto-EBNeuro, Florence, Italy). Polysomnographic Recordings A VEGA 24 polygraph (Esaote Biomedica, Florence, Italy) set at a paper speed of 10 mm per second was used for polygraphic recordings. EEG signals were high-pass filtered with a time constant of 0.3 seconds and low-pass filtered at 30 Hz. Four unipolar EEG channels (Fz-A1, Cz-A1, Pz-A1, Oz-A1) were applied using the international 10-20 system. Submental electromyography was recorded with a time constant of 0.03 seconds. Bipolar horizontal and vertical eye movements were recorded with a time constant of 1 second. Bipolar horizontal electrooculography was recorded from electrodes placed about 1 cm from the medial and lateral canthi of the dominant eye, and bipolar vertical electrooculography from electrodes located about 3 cm above and below the right eye pupil. Electrode impedance was kept below 5 kOhm. DESIGN AND PROCEDURE Procedures Transcranial Magnetic Stimulation Daytime Assessment Subjects sat on a comfortable chair, fully relaxed, with eyes open and watching a point on the wall. The effect of a conditioning stimulus delivered on the motor cortex of 1 hemisphere, on the MEP amplitude evoked in the ADM muscles by a magnetic test stimulus applied to the opposite homologous cortex, was assessed for several interstimulus intervals (ISIs) between the conditioning and test pulses. The intensity of both the conditioning and the test shock was set at 120% of the individual resting MT, measured with a standardized technique16 as the lower intensity level of stimulation able to produce at least 3 MEPs with 100 µV of amplitude (peak-to-peak) in 6 consecutive stimulations. These procedures were repeated for both hemispheres. Twelve responses per condition, both test and conditioning pulses, were collected and their peak-to-peak amplitude was measured off-line and, subsequently, averaged. The positions of the 2 coils were kept constant throughout each block of stimulations. The first step of each experiment was the definition of the MT. Thus, the most effective point on the subject’s scalp for eliciting a target muscle stimulation (“hotspot”) was localized by positioning the coil such that the junction region of the figure-8 coil was approximately over the central sulcus, moving the coil in 1cm steps. Coils were positioned tangentially to the scalp oriented in a postero-anterior direction, 45° from the midsagittal axis of the subject’s head. In this way, the induced current in the brain flowed in the anterior-posterior direction. This orientation was chosen because stronger effects of interhemispheric inhibition were found when the conditioning stimulus induced posteriorly directed currents18 and because this direction is the most effective for eliciting transcallosal inhibition.19 The baseline level of MEP responses (unconditioned responses) was measured with an independent series of test stimuli, 876 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

administered alone at 120% of the individual motor threshold. Also, for unconditioned responses, to avoid uncontrolled intervening factors, both coils were positioned on the subject’s scalp with the same orientation used to measure conditioned responses. The experimental session included 22 blocks [(10 conditioned blocks + 1 baseline) x 2 hemispheres]. The suprathreshold conditioning shock, set at 120% of MT, was delivered across the following ISIs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 milliseconds. These block sequences were balanced between subjects. The whole session lasted about 2 hours. Nighttime Assessment Due to the need to keep each TMS recording session as brief as possible, in order to maintain the specific pattern characterizing the stage in which subjects were awakened, the interhemispheric paired-pulse paradigm (IHPP) covered only the following ISIs, centered around the maximum of transcallosal inhibition effects: 8, 12, and 16 milliseconds. In fact, the major inhibition effect is usually found around 12 milliseconds.5 The identification of hotspots was carried out during presleep assessment; the definition of individual MT for each condition (presleep wake, stage 2, and REM sleep) was carried out during the first night in the laboratory, which also served as an adaptation to sleep recording. In this way, each session upon awakening was restricted to about 15 minutes, and this interval was compatible with the maintenance of the specific pattern characterizing the sleep stage in which subjects were awakened. During each recording session, 10 MEPs for each ISI and for both hemispheres were collected: the order of the 6 sessions (3 ISIs × 2 hemispheres) were partially balanced between subjects (a complete counterbalancing of the sequences would have required a larger number of subjects) in order to control for any possible sequence effect, as well as for any modification of the excitability thresholds across the testing period (about 15 minutes). The magnetic stimulus intensity was the same for conditioning and test stimulus (120% of the subject’s MT).

The second night was considered as a baseline; the subjects’ sleep was undisturbed. During the third night the effect of callosal inhibition was assessed at different times of the night: (1) during presleep wakefulness, (2) after awakening from stage 2 sleep, (3) after awakening from REM sleep, and (4) after the final awakening. During the fourth night, subjects participated in another TMS study, measuring intracortical changes upon awakening. In this case, subjects were also awakened twice in the first part of the night; these results will not be reported here. However, the final morning awakening served as a further measure of the current study (see below). The balancing of awakening sequence was obtained in the following way: in the third night, 5 subjects were first awakened in stage 2 (10 minutes after the first REM cycle) and then during the second REM cycle (5 minutes after its onset). The other 5 subjects were awakened in the second REM cycle (5 minutes after its onset) and then during the ensuing stage 2 (after 10 minutes). The final morning awakenings were from stage 2 or from REM sleep, respectively, in the third or in the fourth night; in this case, the sequence was also balanced between subjects. Each night ended after 7.5 hours of accumulated sleep. Each awakening was carried out after at least 5 consecutive minutes of the scheduled sleep stage. Immediately after each awakening, 2 experimenters entered the sleep room and, in very dim light conditions, submitted the subject to the assessment of the MTs during the first night—the transcallosal inhibition effect during the third and fourth nights.

Awakenings Schedule Each subject slept for 4 consecutive nights in a soundproof temperature-controlled room. The experimental paradigm is depicted in Figure 1. The first night was carried out to allow the subject to adapt to the experimental setting, as well as to define the MT upon awakening from different sleep stages. The MTs were assessed (1) during presleep wakefulness, 5 minutes before lights off, (2) upon stage-2 awakening, (3) upon REM-sleep awakening, and (4) upon the final morning awakening. The order of awakenings was balanced between subjects. The MT values gathered during this night were used to determine the magnetic stimulus intensity levels during the ensuing recording nights. The assessment of the MTs only during the first night was based on the knowledge that this parameters remains stable across different days20 and on the assumption that the same would be at different nights for a given sleep stage; that is, corticospinal excitability associated to a specific sleep stage should not vary as a function of consecutive nights in which any aspect of procedure is maintained stable. Moreover, the MT assessment upon each awakening would have been at odds with the need of keeping each TMS session as brief as possible. SLEEP, Vol. 27, No. 5, 2004

Figure 1—Schematic representation of the experimental paradigm. The arrows indicate the position of the transcranial magnetic stimulation recording sessions. IHPP refers to interhemispheric paired pulse; SWS, slow-wave sleep; REM, rapid eye movement sleep. 877 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

To facilitate coil placement upon awakening, the subject’s head was positioned on a special pillow. Moreover, during presleep wakefulness, the hotspot was clearly marked on the right and left scalp locations. On the whole, each testing session upon awakening lasted between 10 and 15 minutes. During this period, the polygraph was turned off to avoid the interference of the magnetic stimuli with the amplifiers. Consequently, the subject’s state of vigilance was not monitored by EEG recordings during the stimulation period. Nevertheless, each subject was awake at the end of the TMS sessions, as ascertained by both behavioral criteria (all of the subjects readily responded to the experimenters’ request to slightly raise their head to facilitate the pillow removal) and polygraphic recordings (the polygraph was immediately switched on at the end of each session, always confirming the awake EEG pattern). Two independent focal coils were applied on 2 homologous scalp areas overlying the motor cortex corresponding to the ADM muscle. Any other procedural aspect was identical to daytime assessment, excepted for the position (sitting vs lying in bed) and for the range of considered ISIs (2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 milliseconds vs 8, 12, and 16 milliseconds). Data Analysis Sleep Variables Polysomnographic recordings were scored according to the standard rules to evaluate sleep-stage distribution and to ascertain that each awakening was actually carried out from the scheduled sleep stage. Differences between nights in the main sleep variables were assessed by means of a 1-way repeated measures analysis of variance (ANOVA). Daytime TMS Assessment MEP amplitudes were measured as peak-to-peak between the 2 major ones, with a stable deflection of the opposite polarity. Changes in MEP amplitude as a consequence of conditioning pulse administration were expressed in terms of the ratio between conditioned responses (preceded by the conditioning pulse) divided by unconditioned responses (in which only the test pulse was administered—test alone). The significance of these changes was assessed by means of a 1-sample 2-tailed Student t test against the null hypothesis of a population mean ratio of 1 (conditioned responses = unconditioned responses). The ratio between MEP responses to conditioning pulses and unconditioned responses (baseline MEPs) was also calculated, and these ratios were submitted to the same 1-sample Student t test, as a further check of the specificity of effects induced by conditioned pulses. Changes in MEP amplitude were also considered as a dependent variable in a 2-way repeated measure ANOVA, Hemisphere (right, left) × ISI (2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 milliseconds) to compare the specific influence of the conditioning stimulus delivered on the left motor cortex and the conditioned on the right one (“right” condition) with the specific influence of the conditioning stimulus delivered on the right motor cortex and the conditioned on the left one (“left” condition). When interaction resulted significant, the means were compared by paired posthoc t tests.

SLEEP, Vol. 27, No. 5, 2004

Nighttime TMS Assessment A 3-way repeated measure ANOVA ISI (8, 12, 16) × Hemisphere (right, left) × State (presleep wake, intranight Stage 2, intranight REM, final Stage 2, final REM) was first carried out on changes in MEP amplitude (MEP ratios between conditioned responses divided by unconditioned responses), as an omnibus comparison of transcallosal inhibition effects across different states. The MEP ratio expressed the percentage of transcallosal inhibition. The specific effect of time of night and sleep stage on changes in MEP amplitude were assessed by a 4-way repeated measure ANOVA, ISI (8, 12, 16) × Hemisphere (right, left) × Sleep stage (stage 2, REM) × Time of Night (intranight awakening, final awakening). To disentangle any possible confusion between changes in the excitability of each motor cortex and changes in transcallosal inhibition, the same ANOVAs were repeated on the MEP amplitude values of conditioning responses (unconditioned responses), ie, MEPs in response to test pulse alone. When an interaction was significant, the means were compared by paired 2-tailed t tests. RESULTS Polysomnography As detailed in Table 1, the baseline (the second night without any experimental awakening and any TMS session) and experimental nights (the third and the fourth nights in which subjects were tested with TMS) are practically identical. No polysomnographic measure resulted in differences in the between-night Table 1—Sleep Variables Data. Variables

BSL

3RD Night

4TH Night

F(2,18) P

Sleep latency

14.00 (3.25) 128.20 (20.82) 35.60 (11.64) 270.15 (11.24) 65.65 (13.03) 102.35 (6.24) 87.52 (2.47) 473.75 (10.11) 545.45 (20.07)

10.90 (1.88) 130.05 (26.07) 34.10 (8.44) 275.60 (15.02) 60.65 (9.99) 112.40 (6.94) 89.15 (1.79) 482.75 (9.84) 544.45 (19.11)

14.30 (2.86) 111.45 (13.97) 36.25 (8.65) 265.75 (11.69) 57.45 (8.69) 115.30 (6.64) 88.55 (2.22) 474.75 (13.45) 537.10 (12.59)

0.76

0.48

0.54

0.59

0.08

0.92

0.23

0.79

0.82

0.46

2.11

0.15

0.17

0.84

0.18

0.83

0.08

0.92

REM latency Stage 1 Stage 2 SWS REM SE% TST TBT

Means and standard errors (within brackets), and ANOVA results of the sleep variables during the baseline (BSL) and the experimental nights (3RD and 4TH NIGHT), in which trascallosal inhibition was assessed. Stage durations and latencies are expressed in minutes. SWS = Slow-Wave Sleep (stages 3+4); Total Sleep Time (TST); Total Bed Time=TBT; SE = Sleep Efficiency index (Total Bed Time/Total Sleep Time x 100). 878 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

comparisons. Nocturnal awakenings were positioned after respectively 9.5 minutes (SEM = 0.6) of stage 2 sleep during the second NREM period and 5.8 minutes (SEM = 0.4) of REM sleep during the second sleep cycle. Daytime Assessment The mean motor threshold was 33.3% (SEM = 1.39) of the maximum output of the magnetic stimulator for the right and 34.1% (SEM = 1.08) for the left hemisphere. This difference was not significant (2-tailed t9 = -0.79; P = .45). The 2-tailed t tests pointed to a significant reduction of MEP amplitude at the following ISIs: 10, 12, and 14 milliseconds. The same 2-tailed t tests did not show any significant change of MEP amplitude in response to the conditioning pulses (Figure 2). In other words, the MEP inhibition at 10-, 12-, and 14-milliseconds ISIs is specific of conditioned pulses. The Hemisphere × ISI ANOVA, evaluating the specific effect of the conditioned hemisphere on MEP changes, pointed to a significant effect of ISI (F9,81 = 7.70; P < .00000001), with the 10-, 12-, and 14-millisecond ISIs being different from any other ISI, and the 12-millisecond ISI being different from 10- and 14-millisecond. No significant effect was found for Hemisphere (F9,81 = 0.84; P = .58). Nighttime Assessment The mean MT during presleep session was 32.4% (SEM = 1.3) of the maximum output of the magnetic stimulator for the right

Figure 2—Mean changes in motor evoked potentials (MEP) amplitude (expressed in percentages) of conditioned responses at different interstimulus intervals (ISI) divided by unconditioned (Test alone) responses. SEM are also reported. The 2 curves indicate MEP ratios for responses to test (filled circles) and conditioning (empty circles) pulses. Values of both hemispheres were collapsed. Asterisks point to the significance of MEP reduction, assessed by means of a 1-sample Student t test against the null hypothesis of a population mean ratio equal to 1 (conditioned responses = unconditioned responses). *** = P ≤ .0001 SLEEP, Vol. 27, No. 5, 2004

hemisphere, and 32.6% (SEM = 0.73) for the left hemisphere. This difference was not significant (2-tailed t9 = -0.23; P = .82). After REM-sleep awakenings, the MT was 34.4% (SEM = 1.48) for the right and 33.3% (SEM = 0.80) for left hemisphere; this difference was not significant (2-tailed t9 = 0.88; P = .40). Upon awakening from stage 2, the MT was 34.3% (SEM = 1.65) for the right and 35.0% (SEM = 1.16) for left hemisphere; this difference was also not significant (2-tailed t9 = 0.51; P = .62). MT changes, as a function of the different states and of time of night, have been reported elsewhere.12 Figure 3 reports changes in MEP amplitudes as a function of nighttime conditions. The ISI × Hemisphere × State ANOVA on MEP changes, expressed as the ratio between conditioned and unconditioned responses, showed a significant effect for the Hemisphere factor (F1,9 = 8.81; P = .02) with a greater inhibition when conditioned pulses were delivered to the right motor cortex (78.81% [SEM = 3.80%]) compared to the left one (90.46% [SEM = 4.50%]). No other main effects or interactions were significant. Since an effect of hemispheric difference in IHPP technique could be attributed both to differences in cortical excitability of motor cortices and to differences in transcallosal inhibition, the same ANOVA design was repeated on the MEP amplitudes of unconditioned responses. If there are basic differences also between the cortical excitability of the right and left motor cortex, a significant effect should be replicated on these values. In fact, the ANOVA yielded only a significant effect for Stage (F4,36 = 3.35; P = .02; Figure 4), indicating higher MEP amplitudes in the presleep condition [1044.22 µV (SEM =140.57)] as compared to intranight REM [697.74 µV (SEM = 113.67); P = .003], to intranight Stage 2 [810.25 µV (SEM = 132.40); P = .04], and to final REM [735.89 µV (SEM = 96.13); P = .008]. Interhemispheric differences were not significant [F1,9 = 0.97; P = .35; right = 896.29 µV (SEM =115.16); left = 789.04 µV (SEM = 143.66)].

Figure 3—Mean changes in motor evoked potentials (MEP) amplitude (expressed in percentages) of conditioned responses divided by unconditioned (test alone) responses, as a function of the different conditions. SEM are also reported. The black histogram shows the MEP changes during daytime wakefulness. It is provided only for illustrative purposes, since this condition cannot be compared to the others due to the different subject’s position (sitting vs lying in bed) in which transcranial magnetic stimulation data were collected. REM refers to rapid eye movement sleep. 879 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

Although the effect of the State factor was not significant (F4,36 = 1.88; P = .13), the changes in MEP amplitude, reported in Figure 3, clearly suggest the existence of differences in transcallosal inhibition as a function of sleep stage. In fact, the posthoc Fisher protected least significant difference (PLSD) tests on the means of this main effect point to a significant difference between the final REM awakening and both the intranight (P < .02) and the final stage 2 awakening (P < .03). The other comparisons between sleep awakenings or between any sleep awakening and presleep condition were not significant (P > .10). Therefore, intranight data were analyzed with an ISI × Hemisphere × Sleep stage (stage 2, REM) × Time of Night (intranight awakening, final awakening) ANOVA. Results confirm a significantly greater decrease of callosal inhibition (F1,9 = 4.91; P = .05) after REM awakenings (93.53% [SEM = 0.07%]) than after stage 2 awakenings (76.34% [SEM = 0.02%]). No other main effect or interaction was significant. The same ANOVA design on the MEP amplitude of unconditioned responses in each sleep condition showed no significant main effect or interaction. Once again, changes in conditioned responses seem due to the effect of the conditioning pulse, and the contribution of cortical excitability of each motor cortex could be excluded. DISCUSSION Our data show that, when a conditioning magnetic pulse is delivered during the daytime on the motor cortex of 1 hemisphere, the MEP amplitude evoked in the ADM muscles by a magnetic test stimulus applied to the opposite homologous cortex after 12 milliseconds is reduced by an amount ranging from 35% to 40%. The MEP amplitude reduction found in the current study corresponds to an inhibitory effect of transcallosal fibers5 connecting the motor cortex stimulated by the conditioning pulse to that stimulated by the test pulse. The same paired-pulse technique, considering the 8-, 12-, and 16-millisecond ISIs, was used to assess changes in callosal activity during presleep wakefulness, as well as immediately after awakenings from different sleep stages. The main finding of this study is the remarkable reduction in transcallosal inhibition after

Figure 4—Mean motor evoked potentials (MEP) amplitude (expressed in µV) of unconditioned (test alone) responses, as a function of the different conditions. SEM are also reported. REM refers to rapid eye movement sleep. SLEEP, Vol. 27, No. 5, 2004

awakenings from REM sleep in the final part of a night of sleep, with the amplitude of MEP conditioned responses close to that of unconditioned ones. This means that any effect of conditioning pulses to 1 hemisphere on the contralateral hemisphere disappears when subjects are awakened from late REM sleep. Our data also showed a clear difference between the right and left motor cortex, with larger changes in MEP amplitude when conditioned pulses were delivered to the right motor cortex. In other words, the amount of transcallosal inhibition was 21.19% for MEP responses to the stimulation of the conditioned right motor cortex, while the conditioned left motor cortex showed only a 9.54% inhibition. This difference points to a decrease of (inhibitory) activity of callosal fibers from the right to the left hemisphere (left hemisphere conditioned), since the lack of any significant hemispheric difference in MEP amplitude in response to the conditioning pulses (test alone) should allow us to exclude a specific contribution of the intrinsic excitability of each motor cortex. Although current data have been collected upon awakening from different sleep states, several findings converge in legitimating the assumption that differences can be attributed to physiology of sleep state preceding awakenings: (1) the approximately 30-minute interval to fully reestablish wake regional brain activity patterns in the anterior areas upon awakening from sleep, as assessed by H215O PET8 and by blood flow velocity7; (2) the results of studies on interhemispheric asymmetries during sleep by postawakening behavioral assessments9-11; (3) the well-known stage effect on sleep inertia measures, that is the greater negative effects on subsequent performance after SWS awakenings than REM sleep awakenings21,22; and (4) the coherent pattern of MT changes in response to magnetic stimuli delivered upon REM and non-REM sleep awakenings.12 This is indeed the first evidence in humans of a drop of transcallosal activity associated with REM sleep. The hypothesis of a functional interhemispheric disconnection in REM sleep is also consistent with the decrease of interhemispheric EEG coherence observed during this sleep stage.23 In their full-night coherence analysis, Achermann & Borbély24 also found that the REM decrease in interhemispheric coherence is larger on the anterior derivations (frontal and central sites) than on the posterior ones (parietal and occipital sites). The present direct measurement reinforces such indirect evidence. Anatomic studies on animals have shown that the motor cortex has callosal projection cells and a terminal ramification of their fibers,25-27 and a role of transcallosal information for motor execution has been suggested in humans.28,29 The interhemispheric inhibition of the opposite motor cortex should favor performance of unimanual or bimanual movements and suppress unwanted activation of the opposite hand during unimanual tasks.30,31 This motor coordination via the corpus callosum seems less functionally important during REM sleep, that is, during the sleep stage with the higher levels of cerebral metabolic rate32 and desynchronized EEG activity concomitant with a motor paralysis. The current findings of a greater functional interhemispheric disconnection during REM sleep than during non-REM sleep may open the way to a better understanding of some peculiarities of dream mentation and dream recall. In particular, they could contribute to explaining some of the qualitative differences between REM and non-REM mentation.33,34 Some characteristic 880 Reduction of Transcallosal Inhibition upon Awakening from REM—Bertini et al

features of dreams, such as the lack of insight, time distortion, amnesia, or difficulty with verbalization on awakening—which are still tentatively attributed to the relative hypoactivation of the prefrontal cortex during REM sleep35—may therefore be in part related also to the lack of interhemispheric connectivity here reported. As regards dream recall, it is reasonable to predict that the more the right hemisphere is disconnected from the linguistic left hemisphere, the less a subject is likely to report dreams; this prediction is also in line with some previous findings.9,36,37 With regard to non-REM sleep, the slight increase of transcallosal inhibition after both stage-2 awakenings (mean stage 2 MEP inhibition = 24.66%) as compared to presleep condition (mean presleep MEP inhibition = 17.55%) did not reach statistical significance (P = .16). Taking this statistical limitation into consideration, the tendency for an increased transcallosal inhibition during non-REM sleep in humans may parallel the increased transcallosally evoked inhibition observed by single-neuron recordings in the motor cortex of naturally awake and sleeping Macaca mulatta,38,39 in which motor cortex neurons display a clear-cut response peak at around 9 milliseconds during slowwave sleep (actually, it was stage 2 sleep), whereas during wakefulness there was no inhibition at intervals of 10 milliseconds or longer. In other words, in slow-wave sleep, the grouped evoked discharges were powerful enough to set into action local-circuit inhibitory interneurons and to produce sharper inhibition (Steriade, personal communication). Similarly, cortical responses evoked by callosal stimulation that were far smaller during wakefulness than during synchronized sleep were also observed in the cat.40,41 At this stage, the parallelism between the present measures of human callosal activity after awakenings from stage 2 sleep and those in the sleeping cat or monkey are indeed speculative, and only further studies will elucidate if there is actually an increased inhibition driven by callosal neurons associated with non-REM sleep, as compared to that of wakefulness. Independent of the issue of transcallosal inhibition, MEP responses to the conditioning pulse (unconditioned responses) provide a measure of corticospinal excitability across different states, since TMS during sleep activates the same corticospinal fibers as in wakefulness in all sleep stages.42 Corticospinal excitability drops after intranight awakenings from sleep stages, reaching the values corresponding to the presleep level only after the final awakening from stage 2. This reduced corticospinal excitability during early sleep is coherent with the finding, in the same group of subjects, of increased motor thresholds after awakenings from stage 2 and REM sleep in the first part of night as compared to the presleep thresholds12 and with the decreased amplitude of ADM responses after REM sleep as compared to wakefulness.6 This reduced overall excitability of the corticospinal system in early sleep may be likely explained by the hyperpolarization of thalamocortical neurons deafferenting the cortex from sensory input at sleep onset43 and by the prominent slow-wave activity (EEG power density in the 0.75- to 4.5-Hz range) in the first part of the night, declining across successive sleep cycles.44,45 In conclusion our findings may be considered as initial steps in the understanding of the functional connection, at the neurophysiologic level, between brain hemispheres during human sleep. However, some caution is warranted, since data were obtained upon awakening from different sleep stages, due to the technical limitations of using TMS during sleep. Our results may also SLEEP, Vol. 27, No. 5, 2004

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