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Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.
C. elegans is used extensively as a model system in the neurosciences due to its well defined nervous system. However, the seeming simplicity of this nervous system in anatomical structure and neuronal connectivity, at least compared to higher animals, underlies a rich diversity of behaviors. The usefulness of the worm in genome-wide mutagenesis or RNAi screens, where thousands of strains are assessed for phenotype, emphasizes the need for computational methods for automated parameterization of generated behaviors. In addition, behaviors can be modulated upon external cues like temperature, O2 and CO2 concentrations, mechanosensory and chemosensory inputs. Different machine vision tools have been developed to aid researchers in their efforts to inventory and characterize defined behavioral “outputs”. Here we aim at providing an overview of different worm-tracking packages or video analysis tools designed to quantify different aspects of locomotion such as the occurrence of directional changes (turns, omega bends), curvature of the sinusoidal shape (amplitude, body bend angles) and velocity (speed, backward or forward movement).
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.
