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.

13 Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive 14 fluorescence of the all-trans retinal (ATR) cofactor, while in electrochromic (eFRET) sensors, donor 15 fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. We systematically 16 assessed Arch(D95N), Archon, and QuasAr, as well as the eFRET sensors MacQ-mCitrine and QuasAr-17 mOrange, in C. elegans. ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs) 18 and the pharynx, the feeding organ, where Arch(D95N) showed ca. 125 % F/F increase per 100 mV. The ATR 19 fluorescence is very dim, however, using the retinal analog dimethylaminoretinal (DMAR), it was boosted 20 250-fold. eFRET sensors provided sensitivities of 45 % to 78 % F/F per 100 mV, induced by BWM action 21 potentials (APs). All sensors reported differences in muscle depolarization induced by a voltage-gated Ca 2+ -22 channel mutant. Optogenetically evoked de-or hyperpolarization of motor neurons increased or eliminated 23 AP activity and caused a rise or drop in BWM sensor fluorescence. Last, we could analyze voltage dynamics 24 across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior 25 and posterior parts. Our work establishes all-optical, non-invasive electrophysiology in intact C. elegans. 26

Synapses are intricately organized subcellular compartments in which molecular machines cooperate to ensure spatiotemporally precise transmission of chemical signals. Key components of this machinery are voltage-gated Ca2+-channels (VGCCs), that translate electrical signals into a trigger for fusion of synaptic vesicles (SVs) with the plasma membrane. The VGCCs and the Ca2+ microdomains they generate must be located in the right distance to the primed SV, to elicit transmitter release without delay. Rab3 interacting molecule (RIM) and RIM-binding protein (RIM-BP) were shown in different systems to contribute to the spatial organization of the active zone protein scaffold, and to localize VGCCs next to docked SVs by binding to each other and to the C-terminal region of the Cav2 VGCC α-subunit. We asked how this machinery is organized at the neuromuscular junction (NMJ) of Caenorhabditis elegans, and whether it can differentially regulate transmission in circuits composed of different neuron types. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of the UNC-2 VGCC and delaying the onset of SV fusion, while RIM deletion had much more severe defects. rimb-1 mutants caused increased cholinergic but reduced GABAergic transmission, while overall transmission at the NMJ was reduced, as shown by voltage imaging. The UNC-2 channel could further be untethered by removing its C-terminal PDZ binding motif, and this untethering could be exacerbated by combining the ΔPDZ mutant with the rimb-1 mutation. Similar phenotypes resulted from acute degradation of the UNC-2 β-subunit, indicating that destabilization of the VGCC complex causes the same phenotypes as its untethering.