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

Abstract

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 light-drivable 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.

Figure 1

Optical neural silencing via light-driven proton pumping, revealed by a cross-kingdom functional molecular screen. (A) Screen data showing outward photocurrents (left ordinate, black bars), photocurrent densities (right ordinate, gray bars), and action spectrum-normalized photocurrent densities (right ordinate, white bars), measured via whole-cell patch clamp of cultured neurons under screening illumination conditions (575±25 nm, 7.8 mW/mm² for all but Mac/LR/Ops, gPR, bPR, and Ace/AR, which were 535±25 nm, 9.4 mW/mm²; see Supplementary Table 1 for details on the molecules screened; N = 4–16 neurons for each bar). All data in this and other figures are mean ± standard error (SE) unless otherwise indicated. (B) Action spectrum of Arch measured in cultured neurons by scanning illumination light wavelength through the visible spectrum (N = 7 neurons). (C) Confocal fluorescence image of a lentivirally-infected cultured neuron expressing Arch-GFP (scale bar, 20 μm). (D) Raw current trace of a neuron lentivirally-infected with Arch, illuminated by a 15 s light pulse (575 ± 25 nm, irradiance 7.8 mW/mm²), followed by 1 s test pulses delivered starting 15, 45, 75, 105, and 135 seconds after the end of the 15 s light pulse. (E) Population data of averaged Arch photocurrents (N = 11 neurons) sampled at the times indicated by the vertical dotted lines that extend into Fig. 1D. (F) Photocurrents of Arch vs. Halo measured as a function of 575 ± 25 nm light irradiance (or effective light irradiance; see Methods for details), in patch-clamped cultured neurons (N = 4 – 16 neurons for each point), for low (i) and high (ii) light powers. The line is a single Hill fit to the data.

Figure 2

Functional properties of the light-driven proton pump Arch in neurons. (A) Photocurrent of Arch measured as a function of ionic composition (575 ± 25 nm light, 7.8 mW/mm²), showing no significant dependence of photocurrent on concentration of Cl− or K+ ions (N = 16, 8 and 7 neurons, from left to right). (B) Arch proton photocurrent vs. holding potential (N = 4 neurons). (C) Intracellular pH measurements over a one minute period of continuous illumination and simultaneous imaging (535 ± 25 nm light, 6.1 mW/mm²,) using SNARF-1 pH-sensitive ratiometric dye (N = 10 – 20 cells per datapoint). (D) Trypan blue staining of neurons lentivirally-infected with Arch vs. wild-type (WT) neurons, measured at 18 days in vitro (N = 669 Arch-expressing, 512 wild-type, neurons). (E) Membrane capacitance, (F) membrane resistance, and (G) resting potential in neurons lentivirally-infected with Arch vs. wild-type (WT) neurons, measured at 11 days in vitro (N = 7 cells each).

Figure 3

High-performance Arch-mediated optical neural silencing of neocortical regions in awake mice. (A) In vitro data showing, in cultured neurons expressing Arch or eNpHR and receiving trains of somatic current injections (15 ms pulse durations at 5 Hz), the percent reduction of spiking under varying light powers (575 ± 25 nm light) as might be encountered in vivo. *, p < 0.05; **, p < 0.01, t-test. N = 7–8 cells for each condition. (B) Fluorescence images showing Arch-GFP expression in mouse cortex ~1 month after lentiviral injection (i; scale bar, 200 μm, ii, 20 μm. (C) Representative extracellular recordings showing neurons undergoing 5-second (i, ii), 15-second (iii), and 1-minute (iv) periods of light illumination (593 nm; ~150 mW/mm² radiant flux out the fiber tip; and expected to be ~3 mW/mm² at the electrode tip ~800 um away11,12,23). (D) Neural activity in a representative neuron before, during, and after 5 seconds of yellow light illumination, shown as a spike raster plot (top), and as a histogram of instantaneous firing rate averaged across trials (bottom; bin size, 20 ms). (E) Population average of instantaneous firing rate before, during and after yellow light illumination (black line, mean; gray lines, mean ± SE; n = 13 units). (F) Average change in spike firing during 5 seconds of yellow light illumination (left) and during the 5 seconds immediately after light offset (right), for the data shown in D. (G) Histogram of percentage reductions in spike rate.

Figure 4

Multicolor silencing of two neural populations, enabled by blue- and red-light drivable ion pumps of different classes. (A) Action spectra of Mac vs. Halo; rectangles indicate filter bandwidths used for multi-color silencing in vitro. Blue light power is via a 470 ± 20 nm filter at 5.3 mW/mm², and red light power is via a 630 ± 15 nm filter at 2.1 mW/mm². (B) Membrane hyperpolarizations elicited by blue vs. red light, in cells expressing Halo or Mac (N = 5 Mac-expressing neurons, N = 6 Halo-expressing neurons). (C) Action potentials evoked by current injection into patch clamped cultured neurons transfected with Halo (i) were selectively silenced by the red light but not by the blue light, and vice-versa in neurons expressing Mac (ii). Gray boxes in the inset (iii) indicate periods of patch clamp current injection.