Abstract
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
Highlights
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research
We show that among all tested variants, a combination of GtACR218 and Chrimson[12] termed BiPOLES proves most promising and allows (1) potent and reliable blue-lightmediated silencing and red-light-mediated spiking of pyramidal neurons in hippocampal slices; (2) bidirectional control of single neurons with single-photon illumination and two-photon holographic stimulation; (3) dual-color control of two distinct neuronal populations in combination with a second blue-lightsensitive ChR without cross-talk at light intensities spanning multiple orders of magnitude; (4) precise optical tuning of the membrane voltage between the chloride and cation reversal potentials; (5) bidirectional manipulations of neuronal activity in a wide range of invertebrate and vertebrate model organisms including worms, fruit flies, mice, and ferrets
Direct comparison of red-light and blue-light evoked photocurrent densities with those of ßHK-Chrimson and GtACR2 expressed alone indicated that most tandem constructs a mM Na+ 150 mM ClpH 7.2 nm nm b mM ClpH 7.2
Summary
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. Two strategies for stoichiometric expression of an inhibitory and an excitatory opsin from a single ORF were reported using either a gene fusion approach[2] or a 2A ribosomal skip sequence[3,4] In both cases, a blue-light sensitive cationconducting channel for excitation was combined with a redshifted rhodopsin pump for inhibition. The gene fusion approach was used to systematically combine the inhibitory ion pumps halorhodopsin (NpHR), bacteriorhodopsin (BR), or archaerhodopsin (Arch) with a number of channelrhodopsin-2 (ChR2) mutants to generate single tandem-proteins[2] While this strategy ensured co-localized expression of the inhibitory and excitatory opsins at a one-to-one ratio and provided important mechanistic insights into their relative ion-transport rates, membrane trafficking was not as efficient as with individually expressed opsins, limiting the potency of these fusion constructs for reliable control of neuronal activity. Dual-color control of neurons is challenging in the mammalian brain where irradiance decreases by orders of magnitude over a few millimeters in a wavelength-dependent manner[16,17]
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