Figure 2.

Carbachol increases vesicle recycling and [Ca²⁺]_i. (A) FM 1–43 was loaded into vesicles of individual boutons at rest without (Rest) and with addition of carbachol (Carb), or during electrical field stimulation (Stim); unloading of the dye (arrows) was achieved by 30 s periods of field stimulation (20 Hz); ΔF in the three traces indicates the dye uptake into a releasable vesicular pool of the same bouton. (B) Plot of ΔF for 29 identical boutons in two culture dishes under conditions described in A; the differences between the columns are highly significant (∗, P < 0.001; ∗∗, P < 0.0001). (C) Changes in [Ca²⁺]_i measured after incubation of the cells in Fluo-3-AM (15 μM, >30 min); carbachol (200 μM) produced a transient Ca²⁺ rise in boutons identified by subsequent FM 1–43 loading; preincubation of the cells with BAPTA-AM (10 μM, >45 min) or atropine (10 μM, 2 min) inhibited this effect completely; average of 21 synaptic regions in 2 cultures.