Fig. 5.

scFLARE2 drives channelrhodopsin expression for neuron reactivation. (A) Schematic of reactivation of scFLARE-tagged neurons. bReaChES is a channelrhodopsin that can be activated by green 530-nm light. (B) Light- and activity-dependent expression of mCherry-p2A-bReaChES in scFLARE2-expressing rat cortical neuron cultures. The neurons were transduced with AAVs at DIV12, and stimulation (467 nm light at 60 mW/cm2, 33% duty cycle [2 s every 6 s] + 6-s electrical trains consisting of 32 1-ms 50-mA pulses at 20 Hz for a total of 15 min) was performed at DIV18. Cells were fixed and stained with anti-VP16 antibody 18 h after stimulation (scale bars, 10 μm). Additional fields of view are shown in SI Appendix, Fig. S12. (C) A quantification of the experiment in B, with 10 fields of view per condition. (D) Reactivation of scFLARE2-labeled neurons 18 h after light and electrical stimulation. (i-iv) The neurons expressing mCherry and bReaChES from the experiment in B (high Ca²⁺, light condition) were analyzed by whole-cell patch clamp electrophysiology. (v-viii) The control neurons expressing mCherry only (scFLARE2-driven expression from TRE promoter) were analyzed in the same manner. Firing could be elicited by a 350-pA depolarizing current injection in both ii and vi. However, optically induced action potentials in response to 15 ms green light (530-nm) stimulation at 16 Hz and an optically induced inward current from 1 s light stimulation were observed only in bReaChES-expressing cells iii and iv (n = 6/6) and not in control cells vii and viii (n = 3/3). Scale bars in i and v, 20 µm.