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Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans
(2019)
Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact C. elegans.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.