Figure 6.

Live imaging of Cac^sfGFP reveals Ca²⁺ channel accumulation across heterogeneous AZs during rapid homeostatic adaptation to PhTx application. A, Confocal Z-projection of Cac at AZs of a motorneuron branch in a live cac^sfGFP-N preparation immediately before and after 10-min PhTx treatment. The heat map indicates PhTx-induced Cac accumulation (color) relative to baseline Cac levels (size) at each AZ. B, Cac intensity at single AZs of the NMJ shown in A before and after PhTx reveals Cac accumulation at the majority of AZs (baseline: 75.58 ± 8.55; post-PhTx minus vehicle: 94.25 ± 10.12, n = 34 AZs from 1 NMJ branch, p < 0.0001, paired t test). C, Cac intensity at single AZs across multiple animals also reveals Ca²⁺ channel accumulation at most AZs (Baseline, 89.56 ± 3.24; post-PhTx minus vehicle, 105.7 ± 3.96, n = 496 AZs from 7 NMJs and 4 animals, p < 0.0001, Wilcoxon signed rank test). D, Cac intensity at individual AZs at baseline versus post-PhTx minus vehicle. The blue line is a least-squares fit to the data (slope = 1.16, R² = 0.94), whereas the dotted line would describe the data if there were no effect of PhTx (unity line, slope = 1), the green line would describe the data if there were an additive effect, and the orange line would describe the data if there were a multiplicative effect. E, AZs that experience either no change or a decrease in Ca²⁺ channel content after PhTx occur more frequently in lower Cac-content AZs (bottom-third, 48%; middle-third, 28%, top-third, 13%; bottom vs middle, p = 0.003, middle vs top, p = 0.02, bottom vs top, p < 0.0001, Fisher's exact test). F, AZ number is similar between control and PhTx-treated cac^sfGFP-N NMJs in fixed tissue preparations (control: 10.56 ± 0.60, n = 22 NMJs from 6 animals; PhTx: 11.73 ± 0.45, n = 23 NMJs from 6 animals, p = 0.12, Student's t test). Not significant (ns), *p < 0.05, **p < 0.01, and ****p < 0.0001.