Liewald, Jana F., Liu, Qiang, Zeitzschel, Nadja, Vierock, Johannes, Hegemann, Peter, Durmaz, Hilal, Gottschalk, Alexander, Wiegert, J. Simon, Bargmann, Cornelia I., Bergs, Amelie C.F.
[
International Worm Meeting,
2021]
Unlike voltage-clamp electrophysiology, optogenetics allows perturbation, but not true control of neural activity. We thus established an all-optical optogenetic voltage clamp (OVC), in which fast, live readout of the cell membrane potential is used to generate light-based feedback, which is sent to optogenetic actuators for de- or hyperpolarization to clamp the cell at a distinct potential. To this end, we combined two opposing rhodopsins with a genetically encoded voltage indicator, embedded in a software-based live closed-loop feedback system. This way, the OVC synergistically combines the non-invasive character of imaging methods with the control capabilities of electrophysiological methods in live animals. To probe the performance of the OVC, we used different excitable cell types. First, we established the approach in easily accessible muscle cells, where different configurations allowed reliable clamping of their membrane potential. Second, we turned to the spontaneously spiking pharyngeal muscle, that produces action potentials at around 4 Hz, and which the OVC could follow and clamp in a dynamic fashion. Third, we tested the OVC on cholinergic and GABAergic motor neurons, where it equally reliably allowed to clamp their membrane potential based on the voltage sensor fluorescence. Finally, we tested applicability of the OVC in the GABAergic motor neuron DVB, that activates expulsion muscle contraction during the defecation motor program, by producing action potential like activity every 45-50 s. Last, we also calibrated fluorescence signals to electrically measured membrane voltages. Live computation of acquired fluorescence data streams allowed achieving a refresh rate of up to 90 Hz, where the OVC can reliably clamp relative changes in fluorescence between -5 to 5 % deltaF/F0 in all targeted tissues. An additional version of our software enables the live selection of distinct holding values by the experimenter, facilitating an instant response to observed activities of the clamped cell. Our software works independent of the combination of optogenetic tools and hardware components, thus the OVC approach should be easily transferrable to other organisms, but also to cultured cells. Outperforming standard patch-clamp electrophysiology in terms of non-invasiveness, throughput and ease of application, the OVC paves the way for true all-optical control of individual neurons in freely behaving animals.