A High-Light Sensitivity Optical Neural Silencer: Development and Application to Optogenetic Control of Non-Human Primate Cortex

Abstract

Technologies for silencing the electrical activity of genetically targeted neurons in the brain are important for assessing the contribution of specific cell types and pathways toward behaviors and pathologies. Recently we found that archaerhodopsin-3 from Halorubrum sodomense (Arch), a light-driven outward proton pump, when genetically expressed in neurons, enables them to be powerfully, transiently, and repeatedly silenced in response to pulses of light. Because of the impressive characteristics of Arch, we explored the optogenetic utility of opsins with high sequence homology to Arch, from archaea of the Halorubrum genus. We found that the archaerhodopsin from Halorubrum strain TP009, which we named ArchT, could mediate photocurrents of similar maximum amplitude to those of Arch (∼900 pA in vitro), but with a >3-fold improvement in light sensitivity over Arch, most notably in the optogenetic range of 1–10 mW/mm2, equating to >2× increase in brain tissue volume addressed by a typical single optical fiber. Upon expression in mouse or rhesus macaque cortical neurons, ArchT expressed well on neuronal membranes, including excellent trafficking for long distances down neuronal axons. The high light sensitivity prompted us to explore ArchT use in the cortex of the rhesus macaque. Optical perturbation of ArchT-expressing neurons in the brain of an awake rhesus macaque resulted in a rapid and complete (∼100%) silencing of most recorded cells, with suppressed cells achieving a median firing rate of 0 spikes/s upon illumination. A small population of neurons showed increased firing rates at long latencies following the onset of light stimulation, suggesting the existence of a mechanism of network-level neural activity balancing. The powerful net suppression of activity suggests that ArchT silencing technology might be of great use not only in the causal analysis of neural circuits, but may have therapeutic applications.

In Vivo Application of Optogenetics for Neural Circuit Analysis

“…However, we and others have observed strong voltage deflection artifact when laser light is directed onto metal electrode tips, in brain or in saline (Figure 4). 37,54,65 This effect was clearly observed when the electrode tip was positioned in the blue laser beam in saline, and was also evident in the brain with a radiant flux of 80 mW/mm 2 , an intensity often needed for in vivo optogenetic experiments, when the tip of the optical fiber is 0.5−1 mm away from the electrode tip. We have observed that the magnitude of the artifact is proportional to the power of light illumination, but varies with the wavelength of the light.…”

Reciprocal inhibition between sensory ASH and ASI neurons modulates nociception and avoidance in Caenorhabditis elegans

“…2c and Supplementary Movie 7). We employed TeTx to permanently block ASI neurotransmission, and ArchT, a lightdriven outward proton pump with high light sensitivity 34 , to optogenetically inhibit ASI neurons. Genetically silencing and optogenetically inhibiting ASIs (Fig.…”

“…The per cent changes in fluorescence intensity relative to the initial intensity Optogenetic experiments. To optogenetically inhibit the studied neurons, we used ArchT, a high light-sensitive light-driven outward proton pump, driven by neuron-specific promoters to hyperpolarize the neurons 34 . Worm strains expressing ArchT were raised on 3.5 cm NGM plates seeded with Escherichia coli OP50 and All-Trans-Retinal (Sigma, final concentration of 500 mM) or without AllTrans-Retinal as a control.…”

Basolateral amygdala projections to ventral hippocampus modulate the consolidation of footshock, but not contextual, learning in rats

“…The constructs used in inhibition experiments were CaMKIIa-eArchT3.0-GFP and its respective control CaMKIIa-GFP. This modification of the outward proton pump ArchT allows for increased trafficking to the membrane, improved expression along the axon, and more rapid activation (Han et al 2011;Mattis et al 2012). To enable sufficient time for robust opsin expression, illumination of BLA axons in the VH was conducted at least 5 wk after viral injection.…”