Fig. 1

Fig. 1 Single-neuron synapse tracking across day–night cycles reveals diverse dynamics.

a, The synapse labelling construct. Zinc finger (ZF) and KRAB(A) domains limit overexpression²⁵. b, The strategy to sparsely label synapses of FoxP2.A⁺ tectal neurons (Methods). c, Example FoxP2.A:FingR(PSD95)⁺ neuron at 7 d.p.f., with the synapses (white arrowheads, left), nucleus (blue arrowheads, left) and membrane (magenta, right) co-labelled. d, Overnight time-lapse tracking of select synapses from the neuron in c. The normalized GFP intensity (shading) is shown for each synapse (rows). The complete neuron map is shown in Extended Data Fig. 2a. e, Larvae were raised on 14 h–10 h light–dark (LD) cycles (blue), constant light (LL, pink) or switched from LD to LL at 6 d.p.f. (free running (FR), green), and then imaged (arrows) (Methods). f, The average locomotor activity and 95% confidence intervals (CIs) of larvae reared under LD (blue, n = 75), clock-break LL (pink, n = 84) or FR (green, n = 98) conditions. g–j, The mean and 68% CI (column 1) and individual neuron (columns 2–4) synapse counts (g), percentage change in synapse number calculated within each neuron (h), normalized synapse intensity (i) and percentage change in synapse intensity (j) under the LD (blue), LL (pink) or FR (green) conditions. For columns 2–4, a line is shown for each neuron, collected across 8 LD, 4 LL and 4 FR independent experiments. For h, synapse number change (Δ synapse number) dynamics are different during the day from those during the night under LD conditions (*P = 0.043, repeated-measures analysis of variance (ANOVA)). Synapse number change dynamics under LD cycling are significantly different from those under LL conditions (*P = 0.015, main effect of condition, two-tailed mixed ANOVA, post hoc Benjamini–Hochberg correction; Hedge’s g = 0.761). For j, day–night dynamics are significantly different under LD from those under the other conditions (P < 0.01, repeated-measures ANOVA). Both daytime FR and LD day–night dynamics are significantly different from those under the LL condition (mixed ANOVA interaction (condition × time), P = 0.029; FR versus LL, P = 0.038, g = 0.937; LD versus LL, P = 0.027, g = 0.792; post hoc Benjamini–Hochberg correction, two-tailed). At night, LD versus FR, g = −0.538; LD versus LL, g = −0.527. The diagram in a is adapted from ref. ²⁷, CC BY 4.0, and the diagram in b is adapted from ref. ³³, CC BY 4.0. The colour key in e applies also to f–i.

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