High-performance genetically targetable optical neural silencing by light-driven proton pumps

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue–green lightdrivable proton pump from the fungus Leptosphaeria maculans (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of ‘optogenetic’ voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations. We screened type I microbial opsins (see Supplementary Table 1) from archaebacteria, bacteria, plants and fungi for light-driven hyperpolarizing capability. Mammalian codon-optimized genes were synthesized, cloned into green fluorescent protein (GFP)-fusion expression vectors, and transfected into cultured neurons. We measured opsin photocurrents and cell capacitance-normalized photocurrent densities under stereotyped illumination conditions (Fig. 1a, black and grey bars, respectively), as well as opsin action spectra (photocurrent as a function of wavelength; Supplementary Table 2). From this information, we estimated the photocurrent density for each opsin at its own spectral peak (Fig. 1a, white bars). For comparison purposes, we included an earlier-characterized microbial opsin, the Natronomonas pharaonis halorhodopsin (Halo/NpHR)—a lightdriven inward chloride pump capable of modest hyperpolarizing currents. Archaerhodopsin-3 from H. sodomense (Arch/aR-3), proposed to be a proton pump, generated large photocurrents in the screen, as did two other proton pumps, the Leptosphaeria maculans opsin (Mac/LR/Ops) and cruxrhodopsin-1 (ref. 10) (albeit less than that of Arch; Fig. 1a). All light-driven chloride pumps assessed had lower screen photocurrents than these light-driven proton pumps. Arch is a yellow–green light-sensitive (Fig. 1b) opsin that seems to express well on the neural plasma membrane (Fig. 1c; see Supplementary Notes on Arch expression levels and enhancing Arch membrane trafficking). Arch-mediated currents exhibited excellent kinetics of light-activation and post-light recovery. After illumination, Arch currents rose with a 15–85% onset time of 8.86 1.8 ms (mean6 standard error (s.e.) reported throughout, unless otherwise indicated; n5 16 neurons), and after light cessation, Arch currents fell with an 85–15% offset time of 19.36 2.9 ms. Under continuous yellow illumination, Arch photocurrent declined (Fig. 1d, e), as did the photocurrents of all of the opsins in our screen. However, unlike all of the halorhodopsins we screened (including products of halorhodopsin site-directed mutagenesis aimed at improving kinetics; Supplementary Table 3), which after illumination remained inactivated for long periods of time (for example, tens of minutes, with accelerated recovery requiring more blue light), Arch spontaneously recovered function in seconds (Fig. 1d, e), more like the light-gated cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (ChR2). The magnitude of Arch-mediated photocurrents was large. At low light irradiances of 0.35 and 1.28 mW mm (Fig. 1f, left), neural Arch currents were 120 and 189 pA, respectively; at higher light powers (for example, at which Halo currents saturate), Arch currents continued to increase, approaching 900 pA at effective irradiances of 36 mW mm, well within the reach of typical in vivo experiments (Fig. 1f, right; see Methods for how effective irradiances were calculated). The high dynamic range of Arch may enable the use of light sources (for example, light-emitting diodes (LEDs), lasers) that are safe and effective for optical control in vivo. Several lines of evidence supported the idea that Arch functioned as an outward proton pump when expressed in neurons. Removing the endogenous ions that commonly subserve neural inhibition, Cl and K, from physiological solutions did not alter photocurrent magnitude (P. 0.4 comparing either K-free or Cl-free solutions to regular solutions, t-test; Fig. 2a). In solutions lacking Na, K, Cl and Ca, photocurrents were still no different from those measured in normal solutions (P. 0.4; n5 4 neurons tested without these four charge carriers). The reversal potential appeared to be less than 2120 mV (Fig. 2b), also consistent with Arch being a proton pump. We assessed the voltage swings driven by illumination of currentclamped Arch-expressing cultured neurons. As effective irradiance increased from 7.8 mW mm to 36.3 mW mm (Fig. 1f), voltageclamped neurons exhibited peak currents that increased from