Figure 4. GBT demonstrates that neural disinhibition mediated Ca²⁺ transients in mylpfa⁺ myocytes require the ryanodine receptor ryr1b in vivo.

(A) Cartoon showing approach to assay Ca²⁺ transients in zebrafish myocytes through (1) injection of p-mylpfa:GCaMP3 (Baxendale et al., 2012) at the single cell stage, (2) embedding in 1% low melt agar/20 mM pentylenetetrazole (PTZ)/5 µM (S)-(-)-blebbistatin, (3) imaging for 3 min to record transient-associated changes in myocyte GCaMP3 fluorescence at 2 days post-fertilization, and (4) Ca²⁺ transient analysis. (B–I) Static images of GCaMP3 expressing myocytes (B, F) and representative GCaMP3 time-series images showing baseline (C, G), transient peak (D, H), and recovery (E, I) in ryr1b^+/+ (C–E) and ryr1b^{mn0348Gt/mn0348Gt} (G–I) animals, respectively. Scale bar = 20 µm. (J) Representative ∆F/F₀ traces of Ca²⁺ transients from ryr1b^+/+ (black) and ryr1b^{mn0348Gt/mn0348Gt} (gray) myocytes. (K–N) Violin plots comparing transient peak ∆F/F₀ (averaged within fish) (K), Ca²⁺ transient peak-width (L), Ca²⁺ transient rise (M) and decay (N) time between ryr1b^+/+ and ryr1b^{mn0348Gt/mn0348Gt} animals. All plots show median with interquartile range. For (K) n_ryr1b+/+ = 19 animals, n_{ryr1bmn0348Gt/mn0348Gt} = 16 animals. For (L–M) n_ryr1b+/+ = 32 cells, n_{ryr1bmn0348Gt/mn0348Gt} = 16 cells. For (N) n_ryr1b+/+ = 32 cells, n_{ryr1bmn0348Gt/mn0348Gt} = 15 cells. Data are compiled from four independent experiments containing at least two animals in each group. p-values determined using the Mann-Whitney U test. Effect size (Cohen’s d)=1.829 (K) and 0.866 (M). Source data can be found in Figure 4—source data 1 (K, L, M, N) and Figure 4—source data 2 (J).

Figure 4—figure supplement 1. Ca²⁺ transients in ryr1b^+/+ myocytes have higher peak amplitude and are more frequent than in ryr1b^{mn0348Gt/mn0348Gt} myocytes.

(A) Dot plot comparing peak ∆F/F₀ responses (averaged within cell) between ryr1b^+/+ and ryr1b^{mn0348Gt/mn0348Gt} animals. (B) Dot plot representing the number of responses per cell (≥0.05 ∆F/F₀) recorded during the 3 min imaging window in ryr1b^+/+ and ryr1b^{mn0348Gt/mn0348Gt} animals. Plots show median with interquartile range. n_ryr1b+/+ = 64 cells, n_{ryr1bmn0348Gt/mn0348Gt} = 48 cells. Data were compiled from four independent experiments containing at least two animals in each group. p-values determined using the Mann-Whitney U test. Effect size (Cohen’s d)=1.445 (A) and 0.931 (B). Source data can be found in Figure 4—figure supplement 1—source data 1.