Figure 2. Copine-6 is not critical for maintaining synchronous-evoked neurotransmission. A, Bar graph represents copine-6 mRNA levels in rat primary hippocampal cultures under control condition (uninfected) or after infection with an empty (L307) or shRNA-producing lentivirus. Data are from representative experiment performed in quadruplicates. The best knockdown efficiency was observed for two lentiviruses termed KD1 and KD2, which were used throughout the study. B, Western blot analysis of rat hippocampal cultures infected with L307 virus (control) or copine-6 knockdown viruses (KD1 and KD2). Neurons from 4 coverslips were pooled together for each condition. Syx1, Syntaxin 1; Syp1, Synaptophysin 1. Both KD constructs reduced copine-6 expression substantially compared with the neurons infected with L307 virus. C, Representative IPSC traces of postsynaptic response triggered by a paired-pulse stimulation from noninfected control (Ctrl), mOrange-copine-6 (Cop6) overexpressing, empty L307 vector virus (L307) infected, and copine-6 knockdown (KD1 and KD2) hippocampal neurons in the presence of physiological Ca2+. Stimulus intervals: 1000 (1 Hz), 100 (10 Hz), 50 ms (20 Hz). D, Statistical analysis of paired-pulse ratio (P2/P1) shows no significant differences under the various conditions stated above. 1 Hz, Ctrl 0.8 ± 0.03, n = 16; and Cop6 0.8 ± 0.04, n = 14, p > 0.7, compared with Ctrl; L307, 0.8 ± 0.04, n = 12; KD1, 0.9 ± 0.06, n = 10, p1 > 0.6, KD2, 0.9 ± 0.03, n = 15, p2 > 0.6, compared with L307. 10 Hz: Ctrl, 0.7 ± 0.06, n = 23; Cop6, 0.7 ± 0.08, n = 15, p > 0.8, compared with Ctrl; L307, 0.6 ± 0.06, n = 19; KD1, 0.6 ± 0.09, n = 13, p1 > 0.5; KD2, 0.7 ± 0.06, n = 15, p2 > 0.1, compared with L307. 20 Hz: Ctrl, 0.5 ± 0.06, n = 19; and Cop6, 0.5 ± 0.06, n = 19, p > 0.9 compared with Ctrl; L307, 0.4 ± 0.06, n = 17; KD1, 0.5 ± 0.07, n = 19, p1 > 0.7; KD2, 0.5 ± 0.06, n = 15, p2 > 0.2, compared with L307. E, Representative IPSC traces during 1 Hz stimulation from control neurons and neurons infected with lentiviruses expressing Cop6, L307 control, KD1, and KD2. F, Average cumulative charge histograms from the first IPSC trace under 1 Hz stimulation depicting the synchronization of synaptic responses from cultured hippocampal neurons described above, and no statistical significance was observed among different conditions: Ctrl, n = 12; Cop6, n = 12; L307, n = 11; KD1, n = 10; KD2, n = 10.