Figure 5

TFLLR-induced glutamate is not by vesicular exocytosis. A) To test whether Best1–shRNA affects the expression of exocytotic protein transcripts, mRNA expression levels of several endogenous vesicular machineries such as Syt4, Munc18-1, Vamp2 were analyzed from cultured astrocytes expressing Best1–shRNA or scrambled shRNA at least for 72 h. PCR cycles for Best1, Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2), and GFAP were 35, 35, 30, 30, and 25, respectively. B) Time-lapse imaging of relative CFP/YFP ratio from GluSFnR-expressing cultured astrcytes plotted as mean ± sem. Arrowhead indicates the time point of 500 μM TFLLR puff. Naïve astrocytes (Control); naïve astrocytes pretreated with 0.5 mM concanamycin (Conc.A; 2 hrs); naïve astrocytes pretreated with 10 nM tetanus toxin (TeNT; ~14 hrs); naïve astrocytes treated with hyperosmotic solution puff (Hyper). Bar graphs represent the averaged relative CFP/YFP ratio from 30 sec to 70 sec. Numbers of cells from at least two independent culture batches are indicated on the bar graph.