Supplementary Figure 12: Comparison of light-dependent pineal complex activity in freely moving and restrained animals. | Nature Methods

Supplementary Figure 12: Comparison of light-dependent pineal complex activity in freely moving and restrained animals.

From: NeuBtracker—imaging neurobehavioral dynamics in freely behaving fish

Supplementary Figure 12

(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.

Back to article page