Figure 6.

Scn1a-dCas9A Rescues Neuronal Excitability Defects in Cortical Mature Scn1a^+/− Interneurons

(A) Schematic drawing showing the experimental time frame for lentiviral transductions and functional analysis of Scn1a^+/+;GAD67-GFP⁺ or Scn1a^+/−;GAD67-GFP⁺ primary hippocampal neurons transduced with the two depicted lentiviruses. (B) Representative images of a patch-clamp-recorded Scn1a^+/− interneuron expressing both GFP under the GAD67 promoter and tdTomato reflecting the active Scn1a-dCas9A system (Materials and Methods). (C and D) Representative current-clamp traces of APs induced by single current steps administered to Ctrl-dCas9A-transduced WT interneurons (black trace, C), Scn1a-dCas9A-transduced Scn1a^+/+interneurons (blue trace, C), Ctrl-dCas9A-transduced Scn1a^+/− interneurons (gray trace, D), and Scn1a-dCas9A-transduced Scn1a^+/− interneurons (cyan trace, D). (E and F) Firing frequency versus injected current for Ctrl-dCas9A-transduced (E) and Scn1a-dCas9A-transduced (F) Scn1a^+/+ and Scn1a^+/− interneurons. Ctrl-dCas9A wild-type, n = 12; Ctrl-dCas9A Scn1a^+/−, n = 10; Scn1a-dCas9A wild-type, n = 10; Scn1a-dCas9A Scn1a^+/−, n = 1 (p < 0.05, 2-way ANOVA). (G and H), Histogram plots of the maximum frequency (G) and current threshold (H) reached by interneurons during the current step protocol (p = 0.02, p = 0.04, 2-way ANOVA/Bonferroni’s multiple comparisons tests). (I and J) Activity clamp analysis for the number of events during the full traces (I) and cumulative plot for AP frequency (J) (p = 0.03, 2-way ANOVA/Bonferroni’s multiple comparisons tests).