Figure 6. Local control of AP-width at individual boutons.

(A) Image of a bouton targeted for 2P VSD imaging and 2PLU of bath applied RuBi-4AP (300 μM). APs were recorded in control and briefly following (20 ms) RuBi-4AP uncaging near the periphery of the target bouton (∼0.5 μm).

(B) AP broadening induced at boutons following 2PLU of RuBi-4AP decayed rapidly with time after uncaging (*p < 0.05).

(C) Moving the location of uncaging points lateral to the target bouton (∼2 μm; perpendicular to the axis of axon) eliminates the effect of spike broadening induced by photolysis of RuBi-4AP (*p = 0.02).

(D) APs measured iteratively in two nearby boutons on the same axon branch before and briefly following RuBi-4AP uncaging at the target bouton.

(E) Spike broadening induced by Rubi-4AP uncaging at target boutons was highly reduced in orthodromic APs measured at nearby boutons (*p = 0.009; mean distance between boutons 12.6 ± 2.2 μm). In this analysis, we used test boutons that were substantially broadened by 2PLU of Rubi-4AP (≥ 15%).

(F) APs measured in boutons before and briefly following Rubi-4AP uncaging in a background of bath applied DTX (200 nM) or TEA (500 μM).

(G) Group data comparison showing the effect of 2PLU of Rubi-4AP on AP duration in control, DTX, or TEA. 2PLU pulses without 4AP failed to induce a change in spike duration (*p < 0.05). Significance determined with one-way ANOVA followed by Tukey's multiple comparison test (B, G) or with a paired t-test (C, E). In summary graphs, mean values (± SEM), are shown in black with data from individual patches in gray.

See also Figure S7.