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In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours (‘Quint’: ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
Locomotion circuits developed in simple animals, and circuit motifs further evolved in higher animals. To understand locomotion circuit motifs, they must be characterized in many models. The nematode Caenorhabditis elegans possesses one of the best-studied circuits for undulatory movement. Yet, for 1/6th of the cholinergic motor neurons (MNs), the AS MNs, functional information is unavailable. Ventral nerve cord (VNC) MNs coordinate undulations, in small circuits of complementary neurons innervating opposing muscles. AS MNs differ, as they innervate muscles and other MNs asymmetrically, without complementary partners. We characterized AS MNs by optogenetic, behavioral and imaging analyses. They generate asymmetric muscle activation, enabling navigation, and contribute to coordination of dorso-ventral undulation as well as anterio-posterior bending wave propagation. AS MN activity correlated with forward and backward locomotion, and they functionally connect to premotor interneurons (PINs) for both locomotion regimes. Electrical feedback from AS MNs via gap junctions may affect only backward PINs.
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.