Fig. 9.

Phalloidin blocks synaptic transmission after dispersal of actin at vesicle clusters by latrunculin-A. A: after pretreatment with latrunculin-A (12 μM; 30 min) a paired cell recording was made between a reticulospinal axon and a postsynaptic neuron. Synaptic transmission was not prevented by latrunculin-A pretreatment. In these recordings the presynaptic electrode contained phalloidin labeled with Alexa Fluor 488. Ai, top: 10 sequentially evoked EPSCs following presynaptic action potentials. Bottom: 10 EPSCs recorded 15 min after phalloidin injection. The remaining response is the electrical component. Aii: means of control traces and traces after phalloidin injection from Ai: postsynaptic (top) and presynaptic (bottom) action potentials. B: Alexa Fluor 488 phalloidin-labeled axons. Bi: prior to injection, only the recording microelectrode is visible. Bii: 1 min after injection, dye is visible in the axon. Biii: 10 min after injection the axon outline is visible. Biv: 20 min after injection. In no cases are presynaptic active zone vesicle clusters labeled by phalloidin, indicating that these F-actin clusters were dispersed by pretreatment with latrunculin-A. Graphs to right of each image are intensity profiles from each image from region indicated by white lines in Biv. Cortical actin is revealed after 10–20 min of phalloidin. Ci: peak EPSC amplitude before and after phalloidin injection. Cii: intensity of phalloidin Alexa Fluor 488 fluorescence was measured at the center of the axon and at its edge over time. The increase in phalloidin fluorescence at the axon border correlates well with the reduction in EPSC amplitude in Ci. D: means of EPSC amplitudes before and after phalloidin injection after treatment with latrunculin-A (n = 4), with no pretreatment (phalloidin injection, n = 5), or after latrunculin-A but with no phalloidin injection (control, n = 5) (NB: vertical axis inverted to reflect inward currents recorded).