Fig. 4: PER expression in clock neurons preserves PAM neurons.

a–c Genetic rescue of per in clock neurons in per⁰ mutants. PAM neurons were counted using anti-TH staining 7 days after a control treatment with water (a) and after a 4-h treatment with 2.5% H₂O₂ performed at ZT12 in LD (b). In (c), the control group was treated with water at CT16, and 2.5% H₂O₂ treatment was performed at CT4 and CT16. Genotypes are indicated on the x-axis. a > b represents that UAS-transgene b is driven by the GAL4 driver a. n = 11–24 hemispheres (see Source Data for individual sample numbers). Rescue genotypes significantly improve the preservation of PAM neurons after the H₂O₂ treatment. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test, or Kruskal–Wallis test with Dunn’s multiple comparisons test). All the rescue genotypes in (c) exhibit a significantly higher number of PAM neurons compared to per⁰ (****p < 0.0001 for each pairwise comparison, not shown in the figure for clarity). n = 11–19 (a), n = 11–24 (b), and n = 20–39 hemispheres (c). d trans-Tango experiments using DvPdf-GAL4 and Pdf-GAL4 combined with TH immunostaining (green). Several postsynaptic targets of DvPdf neurons (magenta) were identified as PAM neurons, whereas no postsynaptic signal of Pdf neurons was found within the PAM cluster. Presynaptic signals by DvPdf-GAL4 and Pdf-GAL are not visible in these pictures. Two independent experiments. Scale bar, 20 µm. e A schematic representation of the trans-Tango labeling results. Projections of the l-LNvs are not shown for clarity. DvPdf-GAL4- positive but Pdf-GAL4-negative neurons, i.e., LNds, are presynaptic to a subset of PAM neurons. Source data are provided as a Source Data file.