Extended Data Fig. 6: Lim1 increases both the basal activity and the optogenetically induced calcium transients in DN1 neurons specifically during the morning.
From: Behavioral adaptation to warm conditions via Lim1-mediated acceleration of neuronal clocks
a, DN1 neurons can be activated by blue light via Cryptochrome-mediated excitation84. We used two-photon laser-scanning microscopy for calcium imaging to confirm the results obtained using regular fluorescence microscopy (Fig. 4b), which might be affected by experimental activation of Cryptochrome. Two-photon images of DN1 projections in the SMPp showing GCaMP6s fluorescence when excited at 910 nm (calcium-dependent) and 820 nm (calcium-independent). Depletion of Lim1 from DN1 neurons (DN1>lim1-RNAi + GCaMP6s) specifically abolishes the morning (ZT0-2) calcium signal compared to controls (DN1-Gal4). Each image is a maximum projection spanning ~45 µm along the anterior-posterior axis. See Methods for details. Scale bar = 5 µm. b, Left, normalized DN1 > GCaMP6s fluorescence (F910 nm/F820 nm) in the SMPp is higher in the morning (ZT0-2) than evening (ZT10-12), a difference that is abolished when Lim1 is depleted. Right, no difference was seen in the calcium-independent fluorescence (F820 nm) among different groups. n = 8 for all conditions, two-way ANOVA followed by Tukey’s multiple comparisons test. c, Optically triggered calcium transients in DN1 projections (DN1>CsChrimson + GCaMP6s) have a higher amplitude in the subjective morning (left, CT0-2) than evening (middle, CT10-12), a difference that is abolished when Lim1 is depleted from these cells (DN1>lim1-RNAi + CsChrimson + GCaMP6s). The GCaMP6s transient peaks are quantified on the right. All experiments were performed at 25 °C. n = 8 for all conditions, two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m.
