(A) Schematic diagram of optogenetic stimulation paradigm for 8:16 (left), 12:12 (middle), and 16:8 (right) skeleton photoperiod entrainment. Optogenetic pulses (yellow and grey bars for nominal dawn and dusk pulses, respectively) were given twice (8 hr, 12 hr, or 16 hr apart) every 24 hr, targeting near the peak and trough (blue ticks) of PER2::LUC rhythms (black). (B) Representative double-plotted PER2::LUC bioluminescence actograms of SCN slices entrained with 15 min 10 Hz red optogenetic pulses at 8:16 (left), 12:12 (middle), or 16:8 (right) interval of 24 hr. Linear regressions of the pre-, post-entrainment cycle peaks and during-entrainment cycle half-maxes are indicated as the blue, green, and orange dashed lines, respectively. (C–E) Quantification of period during entrainment (C), phase angle of entrainment (D), period change following entrainment (E). (One-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM, n = 3, **p < 0.01). (F) Representative waveforms of PER2::LUC rhythms in SCN slices in a free-running condition (before and after entrainment), and those entrained to optogenetic 8:16, 12:12, 16:8 photoperiod entrainment. Optogenetic light pulses were given at times indicated by yellow and grey lines marking nominal dawns and dusks, respectively. (G) Fold changes in the duration of the rising phase (top) and the falling phase (bottom) during and after entrainment, compared to before entrainment. (RM two-way ANOVA with Sidak’s multiple comparisons tests, mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ****p < 0.0001).