The two major prerequisites for a functional circadian system are the generation of an internal day (circadian cycle) and adjusting its length-and phase-to that of the external day (zeitgeber cycle). The generation of circadian cycles can be observed in constant conditions where organisms show a self-sustained, free-running rhythm. Their expression depends on the nature of the constant conditions (e.g., constant darkness, DD, or constant light, LL). The mechanism that synchronizes the circadian cycle length (τ) to that of the zeitgeber (T) can be explored by many experimental procedures (e.g., single light pulses), but it can only be fully understood under entertainment proper. When a clock is stably entrained, τ(LD) is, on average, equal to T, but the phase relationship between the clock (φ) and the zeitgeber (Φ) (phase of entrainment, ψ = Φ-φ) depends on the relationship between τ in constant conditions (τ(DD) or τ( LL)) and T. Phase of entrainment has traditionally been predicted by the clock's phase response curve (PRC) for a given zeitgeber stimulus and τ(DD). But there is an additional quality of the natural environment-namely, photoperiod-which is not easily incorporated into this entrainment paradigm. The authors therefore investigated phase of entrainment for 162 combinations of T, τ, and photoperiod in Neurospora crassa, which lends itself to a high-throughput approach. They entrained different strains (with long, short, and wild-type free-running periods) to different cycle lengths of the zeitgeber (16-26 h) and photoperiods (16%-84% of each cycle). These combinations produce a circadian surface with highly systematic phases of entrainment. The results suggest that the traditional entrainment paradigms using the PRC and τ(DD) have to be reevaluated.
