Summary Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well-established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identify the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

Main The function of WAKE is conserved in mammalsWe previously identified the clock-output molecule WIDE AWAKE (WAKE) from a forward genetic screen in Drosophila 4 . WAKE modulates the activity of arousalpromoting clock neurons at night, in order to promote sleep onset and quality 4,5 . The mammalian proteome contains a single ortholog, mWAKE (also named ANKFN1/Nmf9), with 56% sequence similarity and which is enriched in the core region of the master circadian pacemaker suprachiasmatic nucleus (SCN) 4,6 ( Fig. 1a, Extended Data Fig. 1a). To investigate whether the function of WAKE is conserved in mice, we generated a putative null allele of mWAKE (mWAKE (-) ) by CRISPR/Cas9 insertion of 8 base pairs (containing a stop codon and generating a downstream frameshift) in exon 4, which is predicted to be in all splice isoforms of mWAKE ( Fig. 1b). As expected, mWAKE expression, as assessed by quantitative PCR and in situ hybridization (ISH), was markedly reduced in mWAKE (-/-) mice, likely due to nonsense-mediated decay (Fig. 1c, 1d). Given mWAKE expression in the SCN, we first examined locomotor circadian rhythms and found that mWAKE (-/-) mice exhibit a mild but non-significant decrease in circadian period length (Extended Data Fig. 1b, 1c). These results are similar to findings from fly wake mutants and mice bearing the Nmf9 mutation (a previously identified ENU-generated allele of mWAKE) 4,6 .Because we previously demonstrated that WAKE mediates circadian regulation of sleep timing and quality in fruit flies 4,5 , we next assessed sleep in mWAKE (-/-) mice via electroencephalography (EEG). Under light:dark (L:D) conditions, there was no difference in the amount of wakefulness, non-rapid eye movement (NREM), or REM sleep between mWAKE (-/-) mutants and wild-type (WT) littermate controls (Extended Data Fig. 1d). In constant darkness (D:D), there is a modest main effect of genotype on wakefulness (P<0.05) and NREM sleep (P<0.05), and a mild but significant decrease in REM sleep in mWAKE (-/-) mutants (Fig. 1e). Although the amount of wakefulness did not appreciably differ in mWAKE (-/-) mutants compared to controls, there was a change in the distribution of wakefulness at night; mutants spent more daily time in prolonged wake bouts, and some mutants exhibited dramatically long bouts of wakefulness (Extended Data Fig. 1e, 1f).

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SUMMARY The suprachiasmatic nucleus (SCN) is the central circadian clock in mammals. It is entrained by light, but resistant to temperature shifts that entrain peripheral clocks [1–5]. The SCN expresses many functionally important neuropeptides including vasoactive intestinal peptide (VIP), which drives light entrainment, synchrony, and amplitude of SCN cellular clocks, and organizes circadian behavior [5–16]. The transcription factor LHX1 drives SCN Vip expression, and cellular desynchrony in Lhx1-deficient SCN largely results from Vip loss [17,18]. LHX1 regulates many genes other than Vip, yet activity rhythms in Lhx1-deficient mice are similar to Vip−/− mice under light-dark cycles, and only somewhat worse in constant conditions. We suspected that LHX1 targets other than Vip have circadian functions overlooked in previous studies. In this study, we compared circadian sleep and temperature rhythms of Lhx1 and Vip-deficient mice, and found loss of acute light control of sleep in Lhx1 but not Vip mutants. We also found loss of circadian resistance to fever in Lhx1 but not Vip mice, which was partially recapitulated by heat application to cultured Lhx1-deficient SCN. Having identified VIP-independent functions of LHX1, we mapped the VIP-independent transcriptional network downstream of LHX1, and a largely separable VIP-dependent transcriptional network. The VIP-independent network does not affect core clock amplitude and synchrony, unlike the VIP-dependent network. . These studies identify Lhx1 as the first gene required for temperature resistance of the SCN clockworks, and demonstrate that acute light control of sleep is routed through the SCN and its immediate output regions.