Abstract
Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.
Highlights
Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations
We have previously shown that the red-shifted naturally-occurring anion-conducting channelrhodopsins (nACRs) GtACR1 can induce vesicle release from thalamocortical projection neurons upon illumination of their axonal terminals in the acute brain slice[13]
Because of the high single-channel conductance, favorable photon-ion stoichiometry, and high light sensitivity of GtACR2, the light power density required for neuronal silencing with this opsin is at least one order of magnitude lower than that of other inhibitory opsins[8,44]
Summary
Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment We address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain. Anion-conducting channelrhodopsins (ACRs), a newly established set of optogenetic tools[15,16,17], are distinct from ionpumping rhodopsins in two major aspects: first, they can conduct multiple ions during each photoreaction cycle This increased photocurrent yield per photon makes channelrhodopsins superior in terms of their operational light sensitivity. We demonstrate here that stGtACR2 shows increased membrane targeting, high anion photocurrents and reduced axonal excitation, making it the most effective tool for optogenetic inhibition at the cell soma to date
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