FM1-43 unloading

The unloading protocol of fluorescence dye, FM1-43 was similar to that described previously [8]. The changes in fluorescence intensity of FM1-43 indicate synaptic vesicle recycling at presynaptic terminals. Synaptic stimulation releases the FM1-43 from presynaptic vesicles resulting in decreased fluorescence intensity of FM1-43 [30]. To load FM1-43 (Molecular Probes, Eugene, OR, USA) into synaptic vesicles, slices were exposed to high-potassium (40 mM) ACSF for 2 min with 8 µM FM1-43. To reduce nonspecific binding of the FM1-43 to tissue, slices were washed with 1 mM ADVASEP-7 for 2 min in ACSF and further perfused with 0.1 mM ADVASEP-7 in ACSF for 50 min. To unload FM1-43, electric stimulation (2 Hz) was delivered to either L1 or L3 for 2 min with glass pipettes or tungsten bipolar electrodes with 2- to 3-fold stimulus intensity for baseline EPSP amplitude. All loading and unloading procedures of FM1-43 were conducted using ACSF containing ionotropic glutamatergic blockers D-AP5 (50 µM) and DNQX (20 µM) to prevent excitotoxic damage. High-potassium ACSF was applied for 5 min at the end of experiments for the background subtraction and normalization of fluorescence signal. Fluorescence signals were acquired on the microscope for whole-cell patch-clamp recording equipped with a monochromator (Polychrome V; TILL Photonics GmbH, Gräfelfing, Germany), a long-pass filter (510 nm), and a CCD camera (Retiga-2000RV; QImaging, Surrey, BC, Canada). Wavelengths for excitation and emission of FM1-43 dye were 480 nm and 532 nm, respectively. Fluorescence images were collected at 5-sec intervals with 10–70 ms light exposure and captured using TILLvisION software (TILL Photonics).