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REM sleep selectively prunes and maintains new synapses in development and learning

Abstract

The functions and underlying mechanisms of rapid eye movement (REM) sleep remain unclear. Here we show that REM sleep prunes newly formed postsynaptic dendritic spines of layer 5 pyramidal neurons in the mouse motor cortex during development and motor learning. This REM sleep-dependent elimination of new spines facilitates subsequent spine formation during development and when a new motor task is learned, indicating a role for REM sleep in pruning to balance the number of new spines formed over time. Moreover, REM sleep also strengthens and maintains newly formed spines, which are critical for neuronal circuit development and behavioral improvement after learning. We further show that dendritic calcium spikes arising during REM sleep are important for pruning and strengthening new spines. Together, these findings indicate that REM sleep has multifaceted functions in brain development, learning and memory consolidation by selectively eliminating and maintaining newly formed synapses via dendritic calcium spike-dependent mechanisms.

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Figure 1: REM sleep prunes newly formed spines during development and after learning.
Figure 2: REM sleep-dependent spine elimination facilitates subsequent new spine formation at nearby sites.
Figure 3: REM sleep strengthens a fraction of new spines formed during development and learning.
Figure 4: REM sleep facilitates long-term maintenance of new spines during development and learning.
Figure 5: Dendritic Ca2+ spikes occurring during REM sleep are important for new spine elimination and strengthening.
Figure 6: Motor training-induced Ca2+ spikes prune and strengthen new spines in REMD mice.

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Acknowledgements

We thank J. Cichon for technical help and all the members of the Gan laboratory for comments on the manuscript. This study was supported by funding from the NIH (R01 NS047325 and R01 MH111486 to W.-B.G., R01 GM107469 and R21 AG048410 to G.Y.), by the National Natural Science Foundation of China (81100839 to W.L.) and by Shenzhen Science and Technology Innovation Funds (GJHS20120628101219327, JC201105170726A, JCYJ20160428154351820, JSGG20140703163838793, ZDSYS201504301539161 and KQTD2015032709315529 to W.L. and L.M.).

Author information

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Authors

Contributions

W.L. and L.M. contributed equally to this work. G.Y. and W.-B.G. initiated the project. W.L., L.M., G.Y. and W.-B.G. designed the experiments. W.L. and L.M. performed the experiments and analyzed the data with help from W.-B.G. W.-B.G. is a full-time faculty member at New York University School of Medicine and supervised the project as a visiting professor at Peking University Shenzhen Graduate School. W.L., L.M. and W.-B.G. prepared the manuscript with input from G.Y.

Corresponding author

Correspondence to Wen-Biao Gan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 EEG and EMG recording.

Examples of the EEG and EMG traces for the identification of various brain states and for REM sleep deprivation.

Supplementary Figure 2 REM sleep deprivation by gentle handling reduces the duration of REM sleep over 8–16 h in P21 mice.

(a) The total time of NREM and wakefulness were not significantly affected by REMD (n = 4, 4, 5 mice for ND, NREM-d, REMD, respectively). (b) The average episode duration of REM sleep was significantly reduced in REMD mice as compared to non-deprived control mice and NREM-d mice between 8-16 h (n = 4, 4, 5 mice for ND, NREM-d, REMD, respectively). (c) The episode number of REM sleep were not significantly affected by REMD (n = 4, 4, 5 mice for ND, NREM-d, REMD, respectively). (d) The number of gentle handling for REMD and NREM-d in P21 mice. To control for non-specific effect of gentle handling, the number of gentle handling for REMD was recorded each hour in REMD mice (n = 5 mice). The comparable number of gentle handling was applied to NREM-d control mice during NREM sleep (n = 4 mice). Data are presented as mean ± s.e.m. *P < 0.05, n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 3 REM sleep deprivation by gentle handling reduces the duration of REM sleep over 8–24 h in P30 mice.

(a) The total time of NREM and wakefulness were not significantly affected by REMD over 8-16 h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (b) The average episode duration of REM sleep was significantly reduced in REMD mice as compared to non-deprived control mice and NREM-d mice between 8-16h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (c) The episode number of REM sleep were not affected by REMD over 8-16 h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (d) The total time of NREM and wakefulness were not significantly affected by REMD over 16-24 h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (e) The average episode duration of REM sleep was significantly reduced in REMD mice as compared to non-deprived control mice and NREM-d mice between 16-24 h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (f) The episode number of REM sleep were not affected by REMD over 16-24h (n = 7, 5, 6 mice for ND, NREM-d, REMD, respectively). (g) The number of gentle handling for NREM-d and REMD were comparable between NREM-d and REMD mice over 8-24h (n = 5, 6 mice for NREM-d, REMD). Data are presented as mean ± s.e.m. **P < 0.01, n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 4 REM sleep deprivation for 12 h reduces the elimination of new spines formed 0–24 h after motor training.

(a) Schematic of experimental design to test whether REM sleep affects the elimination of new spines formed 0-24 h after motor training in a similar way as new spines formed 0-8 h after training (Fig.1h). After identification of learning-induced new spines, P30 mice were left either undisturbed (non-deprived control) or subjected to REM sleep deprivation. (b) The amount of REM sleep and the average duration of REM sleep were significantly reduced over 12 hours in REMD mice as compared to non-deprived control mice (n = 6 mice). (c) The elimination rate of new spines (formed between 0-24 h) was significantly higher in non-deprived control than in REMD mice in the next 12 h (n = 9 mice for ND control group and n = 6 mice for REMD group). (d) REMD did not affect the survival of existing spines over 24-36 h (n = 9, 6 mice for each group). Data are presented as mean ± s.e.m. Each point in (c) and (d) represents data from one animal. **P < 0.01, n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 5 REM sleep–dependent pruning of new spines does not affect subsequent spine formation located 6–10 μm from previously formed new spines during development and after learning.

(a) In P21 mice, the percentage of new spines formed over 16-24 h and located 6-10 μm to persistent or eliminated new spines formed over 0-8 h was comparable among non-deprived, NREM-d and REMD mice (n = 7, 5, 7 mice for ND, NREM-d, REMD, respectively). (b) In P30 mice, the percentage of new spines formed over 16-24 h after forward running and located 6-10 μm to persistent or eliminated new spines formed over 0-8 h after backward running was comparable among non-deprived, NREM-d and REMD mice (n = 6 mice for each group).(c) In P30 mice, the percentage of new spines formed 24-36 h after backward running and located 6-10 μm to persistent or eliminated new spines formed 0-8 h after forward running was comparable between non-deprived and REMD mice (n = 6 mice for each group). Data are presented as mean ± s.e.m., n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 6 REM sleep strengthens persistent new spines during development and after motor training.

(a) Distribution of size change of persistent new spines in non-deprived control, NREM-d and REMD P21 mice over 8-16 h (32 new spines from 7 mice for ND, 24 spines from 5 mice for NREM-d and 55 spines from 7 mice for REMD). (b) Repeated imaging of dendritic spines before and 24 hours after rotarod training (forward) in non-deprived control and REMD P30 mice. Arrowheads indicate new spines formed during 0-8 h after training and persisted over the next 16 hours. (c) Distribution of size change of persistent new spines induced by FW training in non-deprived control, NREM-d and REMD P30 mice over 8-16 h (22 new spines from 6 mice for ND, 37 new spines from 7 mice for NREM-d and 37 new spines from 6 mice for REMD). (d) Distribution of size change of persistent new spines induced by FW training in non-deprived control, NREM-d and REMD P30 mice over 8-24 h (54 new spines from 16 mice for ND, 31 new spines from 7 mice for NREM-d and 75 new spines from 16 mice for REMD). (e) Distribution of size change of persistent new spines induced by BW training in non-deprived control, NREM-d and REMD P30 mice over 8-16 h (23 new spines from 6 mice for ND, 23 new spines from 6 mice for NREM-d and 57 new spines from 6 mice for REMD). *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon–Mann–Whitney test.

Supplementary Figure 7 REM sleep significantly improves forward running performance.

After forward running on the rotarod at 0 h, 1-month-old mice were left either undisturbed (ND) or subjected to REMD over 8-24 h, 12 hours after REMD recovery, performance improvement was significantly lower in REMD mice as compared to non-deprived control mice over 0-36 h (n = 4 mice for each group). Data are presented as mean ± s.e.m. *P < 0.05, Wilcoxon–Mann–Whitney test.

Supplementary Figure 8 REM sleep facilitates long-term survival of new spines induced by backward running.

(a) Schematic of experimental design for measuring the survival of backward running-induced new spines. New spines were identified in mice subjected to running backward on an accelerated rotarod between 0-8 h. After undisturbed sleep, NREM-d or REMD for 8 h, the animals were subjected to forward running and the survival of new spines identified over 0-8 h was examined between 16-24 h. (b) The overall survival rate of new spines formed during backward running (0-8 h) is significantly lower in REMD mice than in non-deprived control and NREM-d mice after forward running (n = 6 mice for each group). (c) After forward running, a significant larger percentage of new spines formed during backward running (0-8 h) was eliminated in REMD mice as compared to non-deprived control mice and NREM-d mice (n = 6 mice for each group). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, Wilcoxon–Mann–Whitney test.

Supplementary Figure 9 Detection of Ca2+ spikes in apical tuft dendrites of L5 neurons under various brain states.

(a) Dendritic calcium spikes during REM sleep were quantified by measuring changes of Ca2+ fluorescence intensity along the dendritic segment (~30 micrometer) within the boxed region of interest (ROI). Ca2+ fluorescence traces of 20 active dendrites over 1-minute are shown. (b) The distribution of peak amplitudes of dendritic Ca2+ spikes recorded over a period of 1 min under various brain states (89, 124, 88, 274, 47 spikes for each group). (c) Measurements of Ca2+ fluorescence along long dendritic segments in the plane of imaging. Comparable fluorescent signals were detected across long-stretch (>30 μm) dendritic segments under running and REM sleep (n = 10 dendrites for each group). Data are presented as mean ± s.e.m. *P < 0.05, ***P < 0.001, n.s. = not significant, Wilcoxon–Mann–Whitney test in (b) and Kruskal Wallis Test in (c).

Supplementary Figure 10 The spread of fluorescence signal after repeated injection of Congo red.

(a) The spread of fluorescence signal after 3-pulse or 10-pulse injections of Congo red (to mimic MK801) into the layer 1 of the motor cortex. Green square represents the region for imaging dendritic Ca2+ spikes. (b) The fluorescence signals of Congo red spread to a region 426 ± 11 μm after 3 pulses and 612 ± 39 μm after 10 pulses in layer 1 (n = 4 mice for each group). (c) The fluorescence signals of the imaged region after 3 and 10-pulse injection of Congo red. (d) The fold change in fluorescence after 10-pulse injection of Congo red relative to 3-pulse injection. The large difference in fluorescence intensity between 3-pulse and 10-pulse injection was partially due to that the concentration of Congo red was lower at the electrode tip (~20 μm) than at the shaft. As the result, the amount of Congo red released from the first 1-2 pulse injection was less than that from the subsequent pulse injections (n = 4 mice). Data are presented as mean ± s.e.m. *P < 0.05, Wilcoxon–Mann–Whitney test.

Supplementary Figure 11 Overlap of dendritic Ca2+ spikes between forward running, pre-running REM sleep or post-running REM sleep.

Over a 1-minute recording period, the degree (~40%) of overlap between running-induced Ca2+ spikes and Ca2+ spikes occurring during pre-running REM sleep is comparable to that between running and post-running REM sleep. N = 4 mice. Data are presented as mean ± s.e.m., n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 12 The distributions of peak amplitudes of dendritic Ca2+ spikes recorded during REM sleep and retraining were comparable.

The distribution of peak amplitudes of dendritic Ca2+ spikes recorded over a period of 1 min between REM sleep and retraining was comparable (n = 274 spikes for REM and 74 spikes for retraining). n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 13 Treadmill training induces new spine formation similarly to rotarod training.

The rate of new spine formation, but not spine elimination, over 8 hours was significantly higher in mice subjected to treadmill training as compared to un-trained mice (n = 6 mice for treadmill training group and n = 4 mice for non-trained group). Data are presented as mean ± s.e.m. *P < 0.05, n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 14 Measurement of plasma corticosterone under various conditions.

Plasma corticosterone was measured with a commercially available ELISA kit. Blood was collected from 5 groups of mice. (1) Normal sleep without motor running (4 mice); (2) Forward running and normal sleep (3 mice); (3) Forward running and physical restraint (2 mice); (4) Forward running, physical restraint and then NREM sleep disturbance for 7 h (4 mice); (5) Forward running, physical restraint and then REMD for 7 h (5 mice). The level of corticosterone induced by gentle handling is comparable among NREM-d mice, REMD mice and mice with physical restraint. Data are presented as mean ± s.e.m., n.s. = not significant, Wilcoxon–Mann–Whitney test.

Supplementary Figure 15 No effect of two-photon Ca2+ imaging or injection of MK801 procedures on the patterns of NREM and REM sleep.

EEG and EMG recordings show that sleep patterns were not interrupted by the procedures of two-photon imaging (a) or injection of MK801 (b). Red lines indicate the time of two-photon scanning or pressure injection.

Supplementary information

Supplementary Text and figures

Supplementary Figures 1–15 and Supplementary Tables 1–6 (PDF 1957 kb)

Supplementary Methods Checklist (PDF 841 kb)

Ca2+ imaging of apical tuft dendrites of L5 pyramidal neurons expressing the genetically encoded calcium indicator GCaMP6s in the motor cortex under various brain states.

Two-photon imaging of dendritic Ca2+ was performed through a thinned skull window over the motor cortex. The video showed dendritic Ca2+ spikes over a period of 1 minute for each brain state (quiet, running, NREM and REM sleep) and Ca2+fluorescence traces were shown in Fig. 5b and Supplementary Fig. 9a. (AVI 4274 kb)

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Li, W., Ma, L., Yang, G. et al. REM sleep selectively prunes and maintains new synapses in development and learning. Nat Neurosci 20, 427–437 (2017). https://doi.org/10.1038/nn.4479

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