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. 2017 Dec 8;10:402. doi: 10.3389/fnmol.2017.00402

FIGURE 6.

FIGURE 6

Single-cell genetic deletion of FRRS1L in vitro and in vivo significantly reduces AMPAR-mediated synaptic transmission. (A) Scatter plot of AMPA and NMDA EPSCs in pyramidal neurons expressing sgRNA#3 and nearby control neurons in cultured organotypic hippocampal slices. Expression of sgRNA#3 significantly reduced AMPA EPSCs (n = 10, p < 0.01, paired t-test), without affecting NMDA EPSCs (n = 10, p = 0.56, paired t-test). Scale bar, 20 pA and 100 ms. (B) The weighted decay time constant was not changed in neurons expressing sgRNA#3 (n = 10, p = 0.25, t-test). (C) There was no change of PPR in neurons expressing sgRNA#3 (n = 11, p = 0.36, t-test). Scale bar, 20 pA and 100 ms. (D) Scatter plot of AMPA and NMDA EPSCs in pyramidal neurons expressing sgRNA#1 and nearby control neurons in cultured organotypic hippocampal slices. Expression of sgRNA#1 did not change AMPA (n = 10, p = 0.98, paired t-test) and NMDA (n = 10, p = 0.88, paired t-test) EPSCs. Each open circle represents one paired recording in the scatter plots, and the solid black point is the average of all pair recordings. Scale bar, 20 pA and 100 ms. (E) The weighted decay time constant was not changed in neurons expressing sgRNA#1 (n = 10, p = 0.97, t-test). (F) There was no change of PPR in neurons expressing sgRNA#1 (n = 10, p = 0.76, t-test). Scale bar, 20 pA and 100 ms. (G) sgRNA#3 failed to reduce the expression of sgRNA resistant FRRS1L (FRRS1L∗) in HEK cells. Western blotting analysis showed that sgRNA#3 strongly reduced WT FRRS1L-Myc, but not FRRS1L-Myc∗, expression in HEK cells. Uncropped scans of Western blots were shown in Supplementary Figure 3. (H) Scatter plot of AMPA and NMDA EPSCs in pyramidal neurons expressing sgRNA#3 together with FRRS1L∗ and nearby control neurons in cultured organotypic hippocampal slices. Expression of FRRS1L∗ rescued the AMPA EPSC deficit induced by sgRNA#3 (AMPA, n = 10, p = 0.74; NMDA, n = 10, p = 0.24; paired t-test). Scale bar, 20 pA and 100 ms. (I) The weighted decay time constant was not changed in neurons expressing sgRNA#3 together with FRRS1L∗ (n = 10, p = 0.85, t-test). (J) Scatter plot of AMPA and NMDA EPSCs in CA1 neurons expressing sgRNA#3 and nearby control neurons in acute hippocampal slices from p14-16 mice that were electroporated in utero at E14.5-15.5. Expression of sgRNA#3 in vivo significantly reduced AMPA EPSCs (p < 0.05, n = 9, paired t-test with Wilcoxon test), without affecting NMDA EPSCs (n = 9, p = 0.53, paired t-test). Scale bar, 20 pA and 100 ms. (K) The weighted decay time constant was not changed in neurons expressing sgRNA#3 (n = 9, p = 0.88, t-test). (L) sgRNA#3 did not change the PPR (control and sgRNA#3: n = 9, p = 0.81, t-test). Scale bar, 20 pA and 100 ms.