Fig. 1: Rhythmic vulnerability of PAM neurons to oxidative stress.

a A schematic of the cell bodies of DA neuron clusters in the adult fly brain. PAM protocerebral anterior medial, PAL protocerebral anterior lateral, PPL1 and 2 protocerebral posterior lateral 1 and 2, PPM1, 2, and 3 protocerebral posterior medial 1, 2, and 3. b DA neuron counts in w¹¹¹⁸ flies per hemisphere in each cluster 7 days after a 4-h 10% H₂O₂ treatment performed at ZT20. The control group was treated with water only. DA neurons were detected by anti-TH immunostaining. Neurodegeneration was observed only in the PAM cluster following the H₂O₂ treatment (n = 18 hemispheres analyzed for both groups). ***p < 0.0001 (two-tailed t-test, comparing the control and H₂O₂-treated groups). In box plots in this and all following figures, box boundaries are the 25th and 75th percentiles, the horizontal line across the box is the median, and the whiskers indicate the minimum and maximum values. The dots represent all data points. c A schematic of the circadian H₂O₂ treatment protocol. d, e Quantification of the number of PAM neurons after the 4-h 10% H₂O₂ treatment performed at different times in LD (control, n = 20; ZT0, 4, 8 and 16 n = 15 each; ZT12, n = 14; ZT16, n = 17 hemispheres) (d) or DD (control, n = 16; CT0, n = 18, CT4, n = 17; CT8 and 20, n = 15 each; CT12 and 16, n = 16 each) (e). The x-axis indicates the times when H₂O₂ was applied. The control group was treated with water only at ZT20 in LD (d) and CT20 in DD (e). At all time points, PAM neuron counts in the H₂O₂ treatment group are significantly smaller than those in the control group. ****p < 0.0001 (one-way ANOVA with Tukey’s post hoc test). Within the H₂O₂-treated group, flies treated at ZT20 in LD (d) and CT4 in DD (e) showed significantly greater cell loss than the treatment at any other time. Different lowercase letters represent statistical significance by one-way ANOVA with Tukey’s HSD post hoc test. Source data are provided as a Source Data file.