Expression and performance of jRGECO1a, FRCaMPi, and SomaFRCaMPi in reporting neuronal activities in zebrafish larvae.

(A) Representative single plane confocal images showing fore-, mid-, and hindbrain regions of live zebrafish larva expressing jRGECO1a, FRCaMPi, and SomaFRCaMPi in neurons driven by the HuC promoter (n = 10 fish at 6 dpf for each). (B) Representative single-plane confocal image of optical tectum showing the segmentation of somatic and neuropil fluorescence (top) and plotted soma/neuropil ratio of fluorescence (bottom) (jRGECO1a: 2,597 cells from 6 fish; FRCaMPi: n = 1,826 cells from 6 fish; SomaFRCaMPi: n = 2,884 cells from 6 fish). (C) Representative max projection confocal images (top) and segmentation masks (bottom) by Cellpose (n = 14 fish for jRGECO1a, n = 16 fish for FRCaMPi, n = 23 fish for SomaFRCaMPi). (D) Number of ROIs detected per fish larva in Cellpose (n = 14 fish for jRGECO1a, n = 16 fish for FRCaMPi, n = 23 fish for SomaFRCaMPi). (E) Schematics of looming stimulation set up in which black expanding circular shadows projected in front of the larval zebrafish embedded in agarose gel. (F) Representative confocal images showing fluorescence response of optical tectum before and during looming stimulation periods (n = 3 fish each). (G) Representative single trial fluorescence traces of soma and neuropil region of red GECI-expressing neurons during looming stimulation shown as ticks (n = 18 cells from 3 fish each). (H–K) Peak ΔF/F₀ (H), peak SNR at neuronal soma (I), soma-to-neuropil peak ΔF/F₀ ratio (J), and soma-to-neuropil peak SNR ratio (K) of the fluorescence response during looming stimulation (n = 18 cells from 3 fish each). One-way ANOVA test was used, followed by a post hoc Tukey test. The Shapiro–Wilk test was used for the normal distribution test. Scale bar, 50 μm. In dot plots, dots are individual data points; the line denotes the median. Box indicates the median and 25–75th percentile range, and the whiskers represent 1.5 times the interquartile range. The quantitative data presented in this figure can be found in S1 Data. Created in BioRender, https://BioRender.com/q72h354.