Ultrastructural effects of sleep and wake on the parallel fiber synapses of the cerebellum

  1. Sophia S Loschky
  2. Giovanna Maria Spano
  3. William Marshall
  4. Andrea Schroeder
  5. Kelsey Marie Nemec
  6. Shannon Sandra Schiereck
  7. Luisa de Vivo
  8. Michele Bellesi
  9. Sebastian Weyn Banningh
  10. Giulio Tononi
  11. Chiara Cirelli

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    This study provides compelling structural evidence on regulation of cerebellar synapses by sleep-wake states. The authors used serial block face scanning electron microscopy to obtain 3D reconstruction of more than 7,000 spines and their parallel fiber synapses in the mouse posterior vermis. The analysis shows that sleep increases the fraction of the 'naked' spines that don't carry a presynaptic partner at Purkinje cells. The authors propose that sleep promotes the pruning of branched synapses to single spines. This is an elegant and thorough study and the observations are important in light of the circuit-specific mechanisms by which sleep modulate synaptic structure and function.

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Abstract

Multiple evidence in rodents shows that the strength of excitatory synapses in the cerebral cortex and hippocampus is greater after wake than after sleep. The widespread synaptic weakening afforded by sleep is believed to keep the cost of synaptic activity under control, promote memory consolidation, and prevent synaptic saturation, thus preserving the brain’s ability to learn day after day. The cerebellum is highly plastic and the Purkinje cells, the sole output neurons of the cerebellar cortex, are endowed with a staggering number of excitatory parallel fiber synapses. However, whether these synapses are affected by sleep and wake is unknown. Here, we used serial block face scanning electron microscopy to obtain the full 3D reconstruction of more than 7000 spines and their parallel fiber synapses in the mouse posterior vermis. This analysis was done in mice whose cortical and hippocampal synapses were previously measured, revealing that average synaptic size was lower after sleep compared to wake with no major changes in synapse number. Here, instead, we find that while the average size of parallel fiber synapses does not change, the number of branched synapses is reduced in half after sleep compared to after wake, corresponding to ~16% of all spines after wake and ~8% after sleep. Branched synapses are harbored by two or more spines sharing the same neck and, as also shown here, are almost always contacted by different parallel fibers. These findings suggest that during wake, coincidences of firing over parallel fibers may translate into the formation of synapses converging on the same branched spine, which may be especially effective in driving Purkinje cells to fire. By contrast, sleep may promote the off-line pruning of branched synapses that were formed due to spurious coincidences.

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  1. Version published to 10.7554/elife.84199 on eLife
  2. eLife

    eLife assessment

    This study provides compelling structural evidence on regulation of cerebellar synapses by sleep-wake states. The authors used serial block face scanning electron microscopy to obtain 3D reconstruction of more than 7,000 spines and their parallel fiber synapses in the mouse posterior vermis. The analysis shows that sleep increases the fraction of the 'naked' spines that don't carry a presynaptic partner at Purkinje cells. The authors propose that sleep promotes the pruning of branched synapses to single spines. This is an elegant and thorough study and the observations are important in light of the circuit-specific mechanisms by which sleep modulate synaptic structure and function.

  3. eLife

    Reviewer #1 (Public Review):

    The goal of this work is to study whether sleep and wake regulate cerebellar structural plasticity. Scanning electron microscopy and 3D reconstruction were used to characterize structural changes in spines and synapses in the mouse cerebellar cortex. The net number and size of synapses did not change between sleep and wake. However, the number of small portion of spines ("naked" spines without synapses) increased during sleep, and the number of branched spines (contains more than one synapse) increased during wake.
    The methodological approach (scanning electron microscopy) is laborious but adequate. However, it cannot follow the same synapses longitudinally, and live imaging, even only in superficial regions, is an ideal approach to achieve the goals.

    The results did not find changes in the total number of spines or synapses during sleep and wake. Structural changes were found in small number of spines lacking a synapse. Whether these changes are functionally important is unclear. The results support circuit-specific, rather than global, effect of sleep on synaptic plasticity.

    The role of sleep range from cellular maintenance to memory consolidation. Multiple evidence suggests that sleep regulates synaptic plasticity. Although this work did not attempt to understand the functional role of sleep in regulating learning and memory, it discovered that sleep promotes synaptic pruning in specific circuits of the cerebellum. Future similar anatomical studies in additional brain regions, including excitatory and inhibitory circuits, combine with physiological and behavioral assays are expected to provide insights to the role of sleep in regulating synaptic plasticity, learning and memory.

  4. eLife

    Reviewer #2 (Public Review):

    This study used serial block face scanning electron in the mouse posterior vermis. The analysis showed that Purkinje cell "naked" spines, are ~5% of all spines after wakefulness but grow to ~10% of all spines after sleep. Additional analysis revealed that the observed sleep-wake difference is best explained by a change in the number of "branched" synapses, Branched spines are proposed to convert to single spines during sleep. It is speculated that sleep promotes the pruning of branched synapses. This is a beautiful study that must have taken considerable effort in addition to expertise. No such data exist in the literature and the observations are interesting in light of the prior data from cortex published by the same group.

    Major critique:

    • The abstract and in particular the second half is very difficult to follow and should be rewritten. It might be easier to follow if the authors compare to previous work in cortex
    • The figures are very well done. However, I am missing a model diagram explaining the model proposed for changes in naked spine during the sleep-wake cycle and the proposed functional consequences.
    • The authors have previously studied the effect of sleep on the ultrastructure of glial cells, astrocytes and oligoes. This might be a separate study, but it would be of interest to discuss the role of Bergmann glial cells in synaptic plasticity. One major difference is that Bergmann glia express AMPA receptors, unlike cortical astrocytes and these are important for the proximity of astrocytic processes to synapses.

  5. Version published to 10.1101/2022.10.31.514498v1 on bioRxiv