Fig. 5. Live in vivo imaging of Cac channels in BRP mutant AZs.

(A to F) Live sptPALM imaging of CacmEOS4B (Control or Con) (A and B) and CacmEOS4B;BRP^−/− (BRP^−/−) mutants in plain HL3 (B and E) or preincubated in 10-min PhTx in HL3 (C and F), imaged in 1.5 mM Ca²⁺ extracellular imaging buffer and visualized in PALM images (A to C) and trajectory maps (D to F). Scale bars, 1 μm (A to C and E to G) and 200 nm (D and H). (G and H) Live sptPALM diffusion coefficient and confinement quantification of CacmEOS4B, CacmEOS4B;BRP^−/−, and PhTx-treated CacmEOS4B;BRP^−/− NMJs. N = 17 (Con) and 13 (BRP^−/−) or 12 (BRP^−/− + PhTx) ROIs; 2192 (Con), 1644 (BRP^−/−), and 1001 (BRP^−/− + PhTx) trajectories from six animals per condition. PALM data distribution was statistically tested by a Kolmogorov-Smirnov test. Statistical significance is denoted as asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (I) Quantification of Cac channel numbers extracted from live sptPALM data further characterized within control conditions [for CacmEOS4b (Con) and CacmEOS4b; BRP^−/−] and after induction of homeostatic plasticity with a 10-min incubation of the larvae in PhTx in extracellular imaging solution. The data are from 12 to 16 NMJs from three to four animals for each condition from which 72 AZs/ROIs (Con), 80 AZs (BRP^−/−), and 26 AZs (BRP^−/− + PhTx) were analyzed. P < 0.0003 (unpaired t-test).