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. 2019 Sep 3;28(1):235–253. doi: 10.1016/j.ymthe.2019.08.018

Figure 4.

Figure 4

Scn1a-dCas9A Increases Neuronal Excitability in Cortical Immature Wild-Type Interneurons

(A) Schematic drawing showing the timeline of transduction with LVs expressing the dCas9A systems on primary wild-type GAD67-GFP neurons and their subsequent functional analysis. (B) Representative images of a patch-clamp-recorded interneuron expressing both GFP under the GAD67 promoter and tdTomato, reflecting the active Scn1a-dCas9A system. Scale bar, 25 μm. (C) Representative current-clamp traces of APs induced by a single current step in dCas9A (black trace, sgCtrl) or Scn1a-dCas9A interneurons (blue trace, sg1P). (D) Firing frequency versus injected current for Ctrl- and Scn1a-dCas9A-transduced interneurons (Ctrl-dCas9A, n = 11; Scn1a-dCas9A, n = 15). (E) Histogram of the maximum frequency reached by interneurons during the current step protocol (p = 0.03, Mann-Whitney U test). (F) Experimental design of activity clamp in primary neuronal cultures in the presence of 4AP (Materials and Methods). (G and H) Representative full traces (G) and magnified traces (H) for the activity clamp protocol in Ctrl-dCas9A (black trace, sgCtrl) and Scn1a-dCas9A (blue trace, sg1P) interneurons. (I) Activity clamp analysis for the number of events during the full traces (left) and cumulative plot for AP frequency (right) (Ctrl, n = 10; Scn1a-dCas9A, n = 12; p = 0.0009, unpaired Student`s t test).