Figure 8.

The Formin inhibitor SMIFH2 suppresses synaptic recycling. (A) Schematic of vGlut1-pHluorin experiments. SV refers to synaptic vesicle. (B) Representative panels show the fluorescence intensity change of vGlut1-pHluorin upon 600 action potential (AP) stimulation and NH₄Cl perfusion. Note that NH₄Cl alkalinizes all vesicles, revealing the total (recycling + resting) pool in these neurons. (C) Ensemble mean of vGlut1-pHluorin traces from control, 50 µM CK-666, or 30 µM SMIFH2-treated neurons (n = number of boutons). Note that although SMIFH2 attenuates neurotransmitter release and decreases synaptic vesicle endocytosis compared with control, CK-666 has no effect, quantified in C (all data normalized to total pools). (D, left) Recycling: Total pool ratio for control = 49.55 ± 3.81%; CK-666 = 45.65 ± 3.72%; and SMIFH2 = 23.84 ± 2.82%. (middle) Exocytosis rate for control = 0.017 ± 0.002; CK-666 = 0.014 ± 0.002; and SMIFH2 = 0.007 ± 0.001. (right) Endocytosis rate for control = 0.007 ± 0.002; CK-666 = 0.007 ± 0.002; and SMIFH2 = 0.001 ± 0.001. All values represent means ± SEM. (∼100–200 boutons on 7–11 coverslips were analyzed for each group from three separate batches of cultures; ***, P < 0.001; **, P < 0.01 compared with control by one-way analysis of variance followed by Dunnett’s post hoc test.)