Extended Data Figure 10: bReaChES: engineering a red-shifted opsin for robust projection targeting.
From: Projections from neocortex mediate top-down control of memory retrieval
a, Schematics of ReaChR31 and bReaChES. ReaChR is a hybrid of segments from channelrhopsin-1 (blue, amino acids 1–95), Volvox channelrhodopsin-1 (red, amino acids 96–246, 279–350) and Volvox channelrhodopsin-2 (green, amino acids 247–278). The VChR1 segment contains the point mutation Leu171Ile. ReaChR was modified here for enhanced expression and membrane trafficking as well as accelerated channel kinetics, resulting in bReaChES, as follows. The first 51 amino-terminal residues were replaced by the first 11 amino-terminal residues from channelrhodopsin-2 (yellow, amino acids 1–11) and the last 5 carboxy-terminal residues were removed. Mutation of Glu 123 to Ser increases speed of channel closure. b, Spectra of C1V1TT, bReaChES and ChR2 measured between 400 and 650 nm at 0.65 mW mm−2 in cultured neurons from rat hippocampus (n = 6 each). c, Stationary photocurrents at 575 nm (C1V1TT 630 ± 109 pA (s.e.m. throughout figure), ReaChR 963 ± 113 pA, bReaChES 1,365 ± 128 pA) and 632 nm (C1V1TT 315 ± 111 pA, ReaChR 1,003 ± 95 pA, bReaChES 841 ± 102 pA). Current amplitudes were measured at −80 mV and 5 mW mm−2 light intensity, respectively. d, Speed of channel closure: τ value of mono-exponential off-kinetics (C1V1TT 79 ± 3.7 ms, n = 26; ReaChR 682 ± 86 ms, n = 6; bReaChES 49 ± 4.4 ms, n = 25; P < 0.0005). e, f, Representative current-clamp traces of ReaChR- or bReaChES-expressing cultured neurons excited with 633 nm light (5 ms, 5 mW mm−2). ReaChR kinetics were slow enough that reliable action potential generation was only possible at very low frequencies (e), while the accelerated channel closure of bReaChES allowed reliable spike generation up to 20 Hz (f). g, h, Representative voltage-clamp (g) and current-clamp (h) traces of postsynaptic cells responding to light stimulation (orange) of bReaChES-expressing presynaptic terminals. Pulse length: 5 ms. i, j, Stationary photocurrents (i) and light-evoked spike probability (j) in opsin-expressing medial PFC (mPFC) cells in acute slice (C1V1TT: n = 11, bReaChES: n = 10). k, l, Light-evoked EPSC amplitude (k) and spike probability (l) in postsynaptic cells (C1V1TT: n = 10, bReaChES: n = 18). Light wavelength 575 nm (25 nm bandwidth) and power density 5 mW mm−2. *P < 0.05, **P < 0.01, ***P < 0.001.
