Fig. 3

Fig. 3

Fig. 3. Circadian variation in cytoplasmic calcium in habenula neurons. (A-E) Long duration multiplane recording of the habenula of Et(GAL4s1011t),Tg(UAS:GCaMP6s) larvae in constant darkness. (A–D) Examples of imaging data from one fish at two different time points, ZT5 (A, B) and ZT14 (C, D), and two different focal planes, dorsal (A, C) and ventral (B, D). Scale bar = 25 μm, dorsal view with anterior to the top. The wedge shows the look up table (LUT) used to represent pixel intensities, with values ranging from 3 (blue) to 3939 (red). (E) Average level of cytoplasmic calcium in the habenula, as shown by intensity of GCaMP6s. Each coloured trace represents a different fish. The thicker black line is the mean. There is a reduction in intracellular calcium levels during the subjective night and an increase in the subjective day. The dotted brown line is a sine wave that the data was regressed to, with an R² of 0.869. (F–G) Difference in relative activity levels of habenula neurons between day (ZT3) and night (ZT15) as measured by both cytoplasmic and nuclear GCaMP6s. (F) Distribution of the mean of vectors containing z-scores of neurons with high variance in their activity. The distributions are different, as determined by Kolmogorov-Smirnov test, with p < 0.0001. (G) K-means cluster distribution for the same vectors. P < 0.0001 by Chi-squared test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)