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. 2011 May 16;286(27):23928–23937. doi: 10.1074/jbc.M111.218032

FIGURE 3.

FIGURE 3.

Syt4 and Syt7 are important for STD DA release, whereas Syt1 is essential for terminal DA release. A, STD DA release increases with time in culture. Extracellular [3H]DA levels were measured in DA neuron cultures in the presence of 0.5 mm Ca2+. B and C, down-regulation of Syt by siRNA. Cultured DA neurons were transfected at day 17 in culture using isoform-specific siRNA. The efficiency of transfection was 95%, and its effects on RNA (B) and protein (C) levels were assessed by semiquantitative RT-PCR and immunochemistry, respectively, at different days after transfection, as indicated. B, a representative gel of each Syt mRNA levels after siRNA transfection is shown. The presence of β-actin mRNA was evaluated to confirm the presence of mRNA and as a loading control. Note that Syt7 can be expressed as three different isoforms in mouse; DA neurons preferentially express Syt7a, the smallest one. C, summary graph presenting the Syt/TH ratio measured at different days after transfection, as indicated. The cells were fixed and subjected to immunochemistry, 10 images were captured from random fields at 40× (n = 6) and quantified with Metamorph software. D, effect of Syt mRNA down-regulation on STD [3H]DA release. Transfection was performed at day 17 in culture; their effects were evaluated 4 days later. STD [3H]DA release was evaluated in the presence of 0.5 mm extracellular Ca2+. E, summary graph showing that elevation of extracellular K+ in the presence of 0.5 mm extracellular Ca2+causes a substantial increase in [Ca2+]i levels in cultured DA neurons. F, after down-regulation of Syt4/7, high potassium depolarization fails to significantly elevate extracellular DA levels in the presence of 0.5 mm extracellular Ca2+. G, the effect of Syt mRNA down-regulation on total STD [3H]DA release (STD plus terminal) was also evaluated (in the presence of 2 mm Ca2+). Syt1 down-regulation caused a large decrease in DA levels, whereas Syt7 down-regulation caused a more modest decrease. scrm, cells transfected with scrambled siRNA; GFP, cells transfected with siRNA against GFP (heterologous control); Ctl, no siRNA used during transfection. The number of experiments for each condition is indicated inside the bars. *, p < 0.05 versus 21d (A) or versus Ctl (C–G). The error bars represent S.D.