Our experiments address two long-standing models for the function of the Drosophila brain circadian network: a dual oscillator model, which emphasizes the primacy of PDF-containing neurons, and a cell-autonomous model for circadian phase adjustment. We identify five different circadian (E) neurons that are a major source of rhythmicity and locomotor activity. Brief firing of PDF cells at different times of day generates a phase response curve (PRC), which mimics a light-mediated PRC and requires PDF receptor expression in the five E neurons. Firing also resembles light by causing TIM degradation in downstream neurons. Unlike light however, firing-mediated phase-shifting is CRY-independent and exploits the E3 ligase component CUL-3 in the early night to degrade TIM. Our results suggest that PDF neurons integrate light information and then modulate the phase of E cell oscillations and behavioral rhythms. The results also explain how fly brain rhythms persist in constant darkness and without CRY.
Most animals have daily rhythms of activity: some are awake during the day and asleep at night, whilst others are more active at night, or during the twilight hours around dawn and dusk. These cycles of activity are driven by an internal body clock, which is reset in response to external cues, like light and temperature, and which keeps the animal in sync with the day–night cycle.
The fruit fly Drosophila has daily—or circadian—rhythms of behavior, which are controlled by a network of genes that are switched ‘on’ or ‘off’ at different times in every 24-hr period. These circadian genes encode various proteins, including PERIOD and TIMELESS. The levels of these two proteins increase during the day and into the night, until they reach a point at which they cause their own genes to be switched off. PERIOD and TIMELESS are then destroyed each morning, and the cycle begins anew. Most of these same proteins perform similar functions in mammals.
In the fly brain, two groups of neurons express these key proteins and control the timings of activity or movement. One group, called M cells, regulates activity in the morning and also produces a small molecule called PDF. Another group, called E cells, controls evening activity, but is less well-defined.
Since M cells can maintain circadian rhythms even in total darkness, these cells were also considered key ‘pacemaker neurons’. However, Guo et al. now challenge this view by identifying five E cells that are the major source of circadian activity. Blocking the release of signaling molecules from these neurons severely disrupted movement in both the morning and the evening. The E cells are also critical for timekeeping under a normal day–night cycle.
Guo et al. found that the rhythm of the E cells is reset when the M cell neurons fire, which causes a release of PDF molecules. Further, PDF molecules reset the E cells by causing the degradation of the TIMELESS protein—which is similar to the effect of light, although light cause TIMELESS to be degraded via a different biochemical pathway.
Guo et al. suggest that under normal light–dark conditions the E cells are important for driving the flies' activity as well as for overall timekeeping. The M cells, instead, appear to function primarily to integrate information about light and reset the E cell clock. Challenges moving forward will include understanding other ways in which the firing of neurons can affect timekeeping, as well as looking if there any differences between the five E cells.