ArchT, a novel high-light sensitivity optical neural silencer. (A) Screen photocurrents generated by various opsins within the archaerhodopsin class, as assessed in neuron culture (n = 5–16 neurons for each bar), measured by whole-cell voltage clamp under standardized screening illumination conditions (7.8 mW/mm², 575 ± 25 nm illumination). Abbreviations: aR-1, archaerhodopsin from Halorubrum strain aus-1; aR-2, archaerhodopsin from Halorubrum strain aus-2; aR-3, archaerhodopsin from Halorubrum sodomense (aka “Arch”); aR-4, archaerhodopsin from Halorubrum strain xz515; aR-TP009, archaerhodopsin from Halorubrum strain TP009 (aka “ArchT”); aR-BD1, archaerhodopsin from Halorubrum xingjianense. (B) Action spectrum of ArchT measured in cultured neurons by scanning illumination light wavelength through the visible spectrum (n = 8 neurons). (C) Photocurrents of ArchT and the previously published reagent Arch measured as a function of 575 ± 25 nm light irradiance (n = 4–16 neurons for each point); curves are Hill plots. *p < 0.05, t-test. (D) Rise times and fall times (measured from the time that the current takes to ramp up from 15 to 85% of its maximum value, and to decline by the same amount) for ArchT vs. Arch-mediated photocurrents in neurons. (Ei) Fluorescence image showing lentiviral ArchT-GFP expression in mouse motor cortex, ∼1 month after lentiviral injection. Scale bar, 15 μm. (Eii) Fluorescence image showing lentiviral ArchT-GFP expression in the axons of mouse infralimbic cortex neurons, here projecting into the nucleus accumbens shell, ∼1 month after lentiviral injection. Scale bar, 45 μm.

ArchT-mediated, ∼100% silencing of cortical neurons, in the awake primate brain. (A) Timeline of experiments for monkey A, indicating times of lentivirus (FCK-ArchT-GFP) injection and single-unit recording (data shown in B–G). (B) Neural activity in a representative silenced neuron in the primate brain before, during and after 1 s of green light illumination. (Bi) Action potential waveforms elicited before illumination (left), during illumination (middle), or after illumination (right); shown is the mean (black) as well as the overlay of raw waveforms (gray). (Bii) Neural activity, shown as a spike raster plot (top), and as a histogram of instantaneous firing rate averaged across trials (bottom; bin size 20 ms). (C) Histogram of instantaneous firing rate, averaged across all silenced single units recorded upon 1 s green light exposure, either using raw firing rate data (top), or using firing rate data normalized to baseline firing rate (bottom). Black line, mean; gray lines, mean ± standard error (SE); n = 45 silenced single units. (D) Histogram of peak percentage reductions in spike rate, for each individual silenced neuron. (E) Neural activity in a representative increased-activity neuron in the primate brain before, during and after 1 s of green light illumination. (Ei) Action potential waveforms elicited before illumination (left), during illumination (middle), or after illumination (right). (Eii) Neural activity, shown as a spike raster plot (top), and as a histogram of instantaneous firing rate averaged across trials (bottom; bin size 20 ms). (F) Histogram of instantaneous firing rate, averaged across all increased-activity single units recorded upon 200 ms green light exposure, either using raw firing rate data (top), or using firing rate data normalized to baseline firing rate (bottom). Black line, mean; gray lines, mean ± standard error (SE); n = 7 increased-activity single units. (G) Latencies from light onset to the beginning of quieting, for silenced neurons; plotted is median ± interquartile distance (n = 45 silenced neurons). (H) Summary histogram of all n = 45 silenced neurons, showing firing rates during 200 ms periods, either just before light onset, at the end of the light period (800–1000 ms), or at 2.5 s after light offset. ***p < 0.005, paired t-test, vs. baseline condition. (I) Summary histogram of all n = 7 increased-activity neurons, showing firing rates during 200 ms periods, either just before light onset, at the end of the light period (800–1000 ms), or at 2.5 s after light offset. (J) Fluorescence image showing lentiviral ArchT-GFP expression in monkey visual cortex, 1 month after lentiviral injection, in monkey B. Scale bar, 20 μm.