Figure 3. t-PDF induces complex locomotor rhythms when expressed in vivo in all circadian clock neurons of transgenic flies.

Male flies bearing UAS-t-peptide transgenes are mated to female flies bearing a tim(UAS)-GAL4 transgene to produce progeny expressing t-peptide in all circadian clock neurons. Free-running locomotor rhythms of individual male progeny entrained in 12h:12h LD conditions and then released into DD are categorized as rhythmic, complex rhythmic, or arrhythmic, and free-running periods are assigned, using Lomb-Scargle periodogram analysis. (A) t-PDF-ML is more active in vivo than t-PDF-LL, while t-PDF-SEC and t-PDF-SCR have no activity, thus recapitulating the relative in vitro activities shown in Figure 2. t-DH31-ML, while active against DH31R in vitro, does not influence free-running locomotor rhythms when expressed in vivo in circadian clock neurons. Bar graph depicts proportions of rhythmic (blue), complex rhythmic (yellow), and arrhythmic (red) flies of the indicated genotypes, with the notations in parentheses referring to specific chromosomal insertions, or combinations of two chromosomal insertions, of the UAS-t-peptide transgenes. n indicates the number of individual flies assayed, τ₀ indicates the average single free-running period of rhythmic flies, τ₁ the average shorter free-running period of complex rhythmic flies, τ₂ the average longer free-running period of complex rhythmic flies, and χ² the significance of χ² statistical comparison of the proportions for that genotype with that of tim > t-μO-MrVIA flies expressing a tethered conotoxin that has no activity in flies (***, p < 0.001). Average Lomb-Scargle periodogram powers are in parentheses following each free-running period component. (B) Representative free-running locomotor actograms of individual flies with the indicated phenotypes and genotypes. The gray bar indicates subjective day, and the black bar subjective night.