Fig. 2: The clock gene per controls the magnitude and rhythms of the vulnerability of PAM neurons to oxidative stress.
From: Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila
a Representative maximum-projection images of PAM neurons in w1118 and per0 flies at indicated ages. PAM neurons were visualized by anti-TH antibodies (green) and RedStinger driven by the R58E02 driver (magenta). Approximately half of the neurons in the PAM cluster are visible. Scale bar, 20 µm. b, c The number of PAM neurons detected by anti-TH immunostaining (w1118 day1, n = 33; day7, n = 21; day14, n = 18 hemispheres. per0 day1, n = 31; day7, n = 52; day14, n = 21.) (b) and by the expression of R58E02-driven RedStinger (w1118 day1, n = 12; day7, n = 17; day14, n = 27 hemispheres. per0 day1, n = 13; day7, n = 23; day14, n = 26.) (c). per0 flies display developmental and age-dependent loss of PAM neurons. **p < 0.01 and ****p < 0.0001 (two-tailed t-test or two-tailed Mann–Whitney U test). d PAM neuron counts in Canton-S, w1118, and per0 were assessed by anti-TH staining 7 days after the treatment with different concentrations of H2O2. Treatment was performed from ZT1 for 4 h. (Canton-S control, n = 16, 2.5% H2O2, n = 12; 5%, n = 15; 10%, n = 16 hemispheres. w1118 control, n = 17; 2.5%, n = 16; 5%, n = 16; 10%, n = 17. per0 control, n = 18; 2.5%, n = 18; 5%, n = 17; 10%, n = 11.) per0 flies display increased PAM neuron susceptibility compared to control genotypes. *p < 0.05, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test). e, f PAM neuron counts in per0 flies after a 4-h 5% H2O2 treatment were performed at different time points in LD (control, n = 31; ZT0, n = 18; ZT4, n = 12; ZT8, n = 13; ZT12, n = 16; ZT16, n = 12; ZT20, n = 16 hemispheres) (e) or DD (control, n = 30; CT0, n = 15; CT4, n = 13; CT8, n = 14; CT12, n = 15; CT16, n = 14; CT20, n = 15 hemispheres) (f). The x-axis indicates the time points when H2O2 was applied. The control group was treated with water only at ZT0 in LD (e) and CT0 in DD (f). At all time points, PAM neuron counts in the H2O2 treatment group were significantly smaller than those in the control group. *p < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test). Within the H2O2-treated group, flies treated at ZT12 in LD (e) displayed a significantly greater cell loss than at any other time point. No difference was observed between time points in DD (f). Different lowercase letters represent statistical significance by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.
