High-performance genetically targetable optical neural silencing by proton pumps

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would open up the ability to investigate the causal role of specific cell classes in neural computations, behaviors, and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate very powerful, safe, multiple-color silencing of neural activity. The gene archaerhodopsin-31 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in 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. In addition, 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 channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans4 (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 vs. red light. Light-driven proton pumps thus represent a highperformance and extremely versatile class of “optogenetic” voltage and ion modulator, which will broadly empower new neuroscientific, biological, neurological, and psychiatric investigations. Users may view, print, copy, download and text and datamine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms (*)Correspondence and requests for materials should be addressed to E.S.B. (esb@media.mit.edu).. Authors' Contributions B.Y.C., X.H., and E.S.B. designed experiments, analyzed data, and wrote the paper. B.Y.C. and X.H. carried out experiments. A.S.D. assisted with electrophysiological recording. X.Q., M.L., and A.S.C. assisted with molecular biology, virus making, and transfections. M.A.H. performed Monte Carlo modelling. P.E.M., G.M.B., and Y.L. created hippocampal and cortical neural cultures. (†)These authors contributed equally to this work. Supplementary Information accompanies the paper on www.nature.com/nature GENBANK (http://www.ncbi.nlm.nih.gov/) accession numbers: mammalian codon-optimized Arch, GU045593; mammalian codonoptimized Arch fused to GFP, GU045594; mammalian codon-optimized Mac, GU045595; mammalian codon-optimized Mac fused to GFP, GU045596; ss-Prl-Arch, GU045597; ss-Arch-GFP-ER2, GU045598; ss-Prl-Arch-GFP, GU045599. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions The authors declare no competing financial interests. NIH Public Access Author Manuscript Nature. Author manuscript; available in PMC 2010 September 15. Published in final edited form as: Nature. 2010 January 7; 463(7277): 98–102. doi:10.1038/nature08652. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript We screened type I microbial opsins (see Supplementary Table 1) from archaebacteria, bacteria, plants, and fungi for light-driven hyperpolarizing capability. Mammalian codonoptimized genes were synthesized, cloned into 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 gray 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 light-driven inward chloride pump capable of modest hyperpolarizing currents5–8. Archaerhodopsin-3 from Halorubrum sodomense (Arch/aR-3), hypothesized to be a proton pump1, generated large photocurrents in the screen, as did two other proton pumps, the Leptosphaeria maculans opsin (Mac/LR/Ops)4 and cruxrhodopsin-19 (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 which appears 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. Upon illumination, Arch currents rose with a 15%– 85% onset time of 8.8 ± 1.8 ms (mean ± standard error (SE) reported throughout, unless otherwise indicated; N = 16 neurons), and after light cessation, Arch currents fell with an 85%– 15% offset time of 19.3 ± 2.9 ms. Under continuous yellow illumination, Arch photocurrent declined (Fig. 1D, 1E), 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 (e.g., tens of minutes, with accelerated recovery requiring additional blue light5,10). Arch spontaneously recovered function in seconds (Fig. 1D, 1E), more like the light-gated cation channel channelrhodopsin-2 (ChR2)2,3. The magnitude of Arch-mediated photocurrents was large. At low light irradiances of 0.35 and 1.28 mW/mm2 (Fig. 1Fi), neural Arch currents were 120 and 189 pA respectively; at higher light powers (e.g., at which Halo currents saturate), Arch currents continued to increase, approaching 900 pA at effective irradiances of 36 mW/mm2, well within the reach of typical in vivo experiments (Fig. 1Fii; see Methods for how effective irradiances were calculated). The high dynamic range of Arch may enable excellent utilization of light sources (e.g., LEDs, lasers) that are safe and effective for optical control in vivo11,12. 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 Ca2+, photocurrents were still no different from those measured in normal solutions (p > 0.4; N = 4 neurons tested without these four charge carriers). The reversal potential appeared to be less than −120 mV (Fig. 2B), also consistent with Arch being a proton pump. We assessed the voltage swings driven by illumination of current-clamped Arch-expressing cultured neurons. As effective irradiance increased from 7.8 mW/mm2 to 36.3 mW/mm2 (Fig. 1F), voltage clamped neurons exhibited peak currents that increased from 350 ± 35 pA (N = 16 neurons) to 863 ± 62 pA (N = 8 neurons) respectively. Current-clamped neurons under these two irradiance conditions were hyperpolarized by −69.6 ± 7.3 mV (N = 10) and −76.2 ± 10.1 mV (N = 8) respectively. Surprisingly, these voltage deflections, while both large, were not Chow et al. Page 2 Nature. Author manuscript; available in PMC 2010 September 15. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript significantly different from one another (p > 0.7, t-test), suggesting the existence of a rapidly activated transporter or exchanger (perhaps the Na+-dependent Cl/HCO3 − exchanger), or the opening of hyperpolarization-gated channels capable of shunting protons, which limit the effects of Arch on accumulated proton (or other charge carrier) gradients across neural membranes. This enabling of effective but not excessive silencing may make Arch safer than pumps that accumulate ions without self-regulation. We next assessed the changes in intracellular pH (pHi) driven by illumination of Archexpressing cultured neurons, using the fluorescent pH indicator carboxy-SNARF-1. Within one second of illumination with strong green light (Fig. 2C), pHi rose from 7.309 ± 0.011 to 7.431 ± 0.020, plateauing rapidly. pHi increased slightly further after 15 s of illumination, to 7.461 ± 0.024 (Fig. 1E). The fast stabilization of pHi may reflect the same self-limiting influence that limits proton-mediated voltage swings as described above, and may contribute to the safe operation of Arch in neurons by preventing large pHi swings. The changes in pHi observed here are comparable in magnitude to those observed during illumination of ChR2expressing cells13 (due to the proton currents carried by ChR23,14) and are also within the magnitudes of changes observed during normal neural activity15–18. Passive electrical properties of neurons were not affected by Arch expression (Fig. 2E–G; p > 0.6 for each measure, t-test), nor was cell death (p > 0.6, χ2 = 0.26; Fig. 2D). We estimated the tissue volumes that could be silenced, using in vitro experiments and computational modeling. In cultured neurons expressing Arch or a trafficking-improved variant of Halo, eNpHR7,8, we somatically injected brief current pulses at magnitudes chosen to mimic the current drives of neurons in the intact nervous system19–22. We exposed these neurons to periods of 575 nm yellow light (0.35, 1.28, or