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 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 high-performance and extremely versatile class of “optogenetic” voltage and ion modulator, which will broadly empower new neuroscientific, biological, neurological, and psychiatric investigations.

In Vivo Application of Optogenetics for Neural Circuit Analysis

“…34 However, blue light could accelerate Halo recovery from minutes to milliseconds. 28,35 This slow recovery kinetics should be considered for experiments requiring long duration silencing. In addition, constant transport of Cl − into Halo expressing cells may change intracellular or extracellular Cl − concentration, and thus alter the reversal potential of GABA receptors, leading to neural modulation effects secondary to that produced by Halo.…”

“…Archaerhodopsins, a class of light-driven outward proton pumps, have been applied to optogenetically silence neural activities in 2010. 35 For example, Archearhodopsin-3 from Halorubrum sodomense (Arch), a green-yellow light activated proton pump, when expressed in neurons mediated rapid and reversible silencing of neural activities by pumping protons out of a cell to hyperpolarize the cell. Arch traffics well to the plasma membrane in mammalian neurons and is capable of exerting powerful inhibition in vivo.…”

“…54 However, we have never observed such artifact with hollow glass microelectrodes. 28,35,54 A few cautions have to be made with glass electrodes in optogenetic experiments. For example, if laser light reaches the Ag/AgCl wire that is in direct contact with the solution inside the glass electrode, light will induce artifact.…”

New methods for understanding systems consolidation

“…Activation or inactivation is achieved via expression of light-sensitive proteins such as channelrhodopsin (ChR2), halorhodopsin (NpHR), or archaerhodopsin (Arch) (Chow et al 2010;Yizhar et al 2011). Recent work has shown that optogenetic methods can also be combined with the tetracycline-transactivator system to control the activity of hippocampal neurons that were engaged during learning (Liu et al 2012).…”

Selective Manipulation of Neural Circuits

“…In contrast, halorhodopsin and archaerhodopsin inhibit neuronal activity. Halorhodopsin is a light-sensitive inward chloride pump that silences neuronal firing in response to yellow light (580 nm) [112]; archaerhodopsin is a proton pump responsive to yellow-green light (550 nm) [113].…”