Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit flyexhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. In this study, we re-examined the two-oscillator model for the M and E peaks ofby utilizing more than 50 Gal4 lines in conjunction with theline, which enables the restoration of the clock function in specific cells in the() null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LN) is responsible for controlling the M peak, while the other group, consisting of the 5ventrolateral neuron (5LN) and the three cryptochrome (CRY)-positive dorsolateral neurons (LN), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN) is thought to also contain M and E oscillators. In this study, we found that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, we were able to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, we demonstrate that 4 to 6 CRY-positive DNcells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, our detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescuein specific clock neurons. Drosophila melanogaster Drosophila UAS-period16period per per v v d1p1p th th
