(a) Fluorescence signal changes obtained on NeuBtracker in MicroFixed configuration corresponding to the behavioral data shown in Figure 3 b,b’. The signal from the different ROIs is averaged over 4 animals with bounds of one standard deviation in dashed lines (fitted time constant: 11.9 ± 1.1 seconds.) (b) Single plane through pc (two-photon microscopy; inset shows magnification) as anatomical reference (c) Staining with the neural activity marker phosphorylated ERK (p-ERK, red), total ERK (t-ERK, green) and their ratio (p-ERK/t-ERK, cyan) from a fish imaged on NeuBtracker with ten cycles of 50 s ON/10 s OFF for 10 min. (d) Fluorescent signal changes (green traces) in the pineal complex (pc) during two cycles of light ON/OFF stimulation selected from the 10 cycles shown as average in Figure 3a from a short period in which the larva tg(gSA2AzGFF49A;UAS:GCaMP7a, 6 dpf) was relatively stationary (right) compared with a phase with swimming activity (left side) as seen from the plot of the concurrently recorded x- and y-position of the larva and its swimming speed (middle panel). The bottom panel shows as a reference the pc signal obtained from a restrained larva (embedded in agar, black trace). (e,f) To confirm the quality of image registration and control for any instrument-dependent effects on the signal changes, we performed the same experiment but using larvae expressing GFP tg(HuC:Gal4;UAS:eGFP) instead of a calcium indicator either freely swimming (e) or embedded in agar (f). The fluorescent signal traces are plotted from the ROIs indicated in the inset in (f). Scalebars represent 200 μm.