Temporally consolidated behaviors such as sleep normally occur in synchrony with endogenous circadian rhythms and both have been reported to contribute to global daily oscillations of transcription in brain. Neurons have further adapted specialized means to traffic RNA into distant dendritic and axonal arbors, where it is locally translated. Together, these mechanisms allow coordination of physiology with environmental needs.

About 6% of the forebrain transcriptome oscillates in a time-of-day–dependent manner, and it has been proposed that this oscillation is mostly driven by the sleep-wake state to enable daily changes in synaptic structure and function. In turn, such synaptic scaling is thought to form a critical feature of the sleep-wake process. Given the highly local capacity for synaptic remodeling, an essential missing link in this argument is the effects of circadian clocks and sleep pressure upon messenger RNA (mRNA) and related protein abundance at synapses themselves. To address this question, we examined daily rhythms of transcript and protein abundance in transcriptome and proteome of synapses from the mouse forebrain, using biochemically purified synaptoneurosomes isolated across the 24-hour day both at normal sleep pressure and at constant high sleep pressure.

Notably, 67% of synaptic mRNAs showed circadian oscillations, with a mean amplitude of about twofold. Further, 93% of these oscillating transcripts were exclusively rhythmic in synaptoneurosomes, suggesting an entirely posttranscriptional origin for synaptic mRNA oscillations. This observation was supported by single-molecule fluorescence in situ hybridization. Rhythmic synaptic transcripts formed two distinct waves, anticipating either dawn or dusk, and both required a functional circadian clock. These two waves showed completely different functional signatures: synaptic signaling preceded the active phase, whereas metabolism and translation preceded the resting phase. Comprehensive circadian characterization of the synaptic proteome demonstrated the functional relevance of this temporal gating for synaptic function and energy homeostasis. Overall, the oscillations of 75% of synaptic proteins were concomitant with their rhythmic transcripts, indicating a key role for local synaptic translation. Under conditions of high sleep pressure, one-fourth of mRNAs remained identically circadian, and most preserved some degree of circadian rhythmicity. In contrast, no substantial circadian rhythm could be detected in any protein when sleep pressure was constantly high.

Examining the dynamics of mRNAs in the synaptic landscape revealed the largest proportion of circadian transcripts in any tissue, cell, or organelle described to date. These synaptic oscillations are controlled posttranscriptionally and the daily dynamics of transcripts and their related proteins clearly delineate different cellular modes between sleep and wake. Our study provides insight into the connectivity between sleep and circadian rhythms and suggests an elegant paradigm whereby a molecular clock provisions synapses with mRNAs before dawn and dusk, which are later translated in response to activity-rest cycles.

(A) Workflow: Forebrain synaptoneurosomes were isolated across the day at low and high sleep pressure. (B) Synaptic transcripts can maintain circadian rhythmicity under high sleep pressure (C) but protein rhythms are completely abolished. (D) Gene ontology highlights the complete temporal segregation of predusk (top) and predawn …