Figure 2.
Rescue of synaptic transmission in synaptobrevin 2-deficient synapses by cellubrevin. Synaptic transmission was analyzed after infection of synaptobrevin 2-deficient hippocampal cultures with lentivirus containing either synaptobrevin 2 or cellubrevin. A, Schematic drawings compare the structure of synaptobrevin 2 and cellubrevin. TMR, Transmembrane region. B, Representative traces of spontaneous synaptic release at −70 mV in the presence of 1 μm tetrodotoxin and 100 μm picrotoxin. C, Graph depicts the average frequency and SEM of miniature EPSCs (mEPSCs). Note the markedly reduced average frequency of miniature EPSCs for synaptobrevin 2 knock-outs and its rescue by both synaptobrevin 2 and cellubrevin [n = 12 for WT; n = 6 for synaptobrevin 2 knock-out and cellubrevin infected (iCeb); n = 11 for synaptobrevin 2 infected (iSyb2)]. D, Synapses were loaded with 47 mm K+ for 90 s in the presence of 200 μm FM2-10, washed with calcium-free Tyrode's solution, and destained by applying 90 mm K+ for 60 s, which was repeated five times with a wash of 60 s after each. Averaged traces of FM2-10 fluorescence during the first round of 90 mm K+ destaining from wild-type (n = 7), Syb2 KO (n = 5), and synaptobrevin 2 knock-out synapses infected with synaptobrevin 2 (n = 7) or cellubrevin (n = 4). E, Bar graph depicts the recycling synaptic vesicle pool, which could be labeled by 47 mm K+ for 90 s and destained by five times application of 90 mm K+ for 1 min. F, Readily releasable pool recorded by addition of 500 mOsm sucrose to the bath solution. Representative traces recorded at −70 mV. G, Bar graph shows synaptic release rate estimated from the charge transfer rate [n = 4 for WT, synaptobrevin 2 KO, and synaptobrevin 2 infected (iSyb2); n = 5 for cellubrevin infected (iCeb)]. H, Representative responses to field stimulation at 1 Hz. I, Bar graphs depict the average responses to the first 60 pulses [n = 7 for WT and synaptobrevin 2 KO; n = 17 for synaptobrevin 2 infected (iSyb2); n = 5 for cellubrevin infected (iCeb)].