Bimodal activation of different neuron classes with the spectrally red-shifted channelrhodopsin chimera C1V1 in Caenorhabditis elegans

The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses

ChR2-HR/TC in GABA neurons and C1V1-ET/ET in ACh neurons enable independent bi-modal light control of either cell type. A)

<p>Innervation of muscle cells by GABAergic and cholinergic motor neurons, expressing ChR2-HR/TC and C1V1-ET/ET, respectively, in the same animal. <b>B)</b> Confocal fluorescence image of ChR2-HR/TC::YFP and C1V1-ET/ET::mCherry expressed in the ventral nerve cord motor neurons. <b>C)</b> Body length changes were measured in response to 400 or 580 nm light of the indicated intensities. Animals used expressed either ChR2-HR/TC in GABA neurons, or C1V1-ET/ET in cholinergic neurons, or both. When both proteins were expressed in the same animal, independent activation of both proteins and the respective neuron type can be achieved. Displayed are mean body length changes, relative to the initial length of the non-illuminated animals, and s.e.m. N = 12 animals of each strain were measured. Statistically significant differences were calculated by two-tailed Student’s t-test (n.s. = non significant; *p<0.05; **p<0.01; ***p<0.001).</p

(Co-)expression of ChR2-HR and C1V1-ET/ET in different cell types of the same animal.

<p><b>A)</b> ChR2-HR and C1V1-ET/ET were expressed in different cell types (GABAergic motor neurons or body wall muscle cells), either in different transgenic lines, or co-expressed in the same animal, as indicated in the pictograms. Fluorescence micrograph shows expression in the same animal of ChR2-HR::YFP in GABA neurons and C1V1-ET/ET::YFP in muscle cells. <b>B)</b> Body length changes (contraction or relaxation = elongation; given in relation to the initial body length of unstimulated animals) were measured in response to light of the indicated wavelengths and intensities. Residual contractions evoked by 400 nm light by C1V1-ET/ET in muscle could be counteracted, but not overcome, by ChR2-HR in GABA neurons. Displayed are mean body length changes and s.e.m. N = 10 animals of each strain were measured. Statistically significant differences were calculated by two-tailed Student’s t-test (n.s. = non significant; ***p<0.001).</p

Survey of various ChR variants for red-shifted excitation in <i>C. elegans</i> muscle.

<p><b>A)</b> Upper panel, fluorescence micrographs. ChR2(H134R)::YFP (ChR2-HR), C1C2 5-2::YFP and C1V1::YFP (for expression pattern, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046827#pone.0046827.s005" target="_blank">Fig. S5A</a>), were expressed in body wall muscle cells and photostimulated (lower panel; light intensities and wavelength of band pass filters used are indicated). The resulting body length changes (contractions) were measured relative to the initial length of unstimulated animals. N = 7–24 animals were used for each experiment. <b>B)</b> ChR2-HR, ChR2-TC, ChR2-HR/TC and C1V1-ET/ET were expressed in muscles cells and the resulting body length changes in response to 0.2 mW/mm² light at 5 different wavelengths were measured. N = 9–10 animals each. In A and B, absolute contractions (during light) were significantly different from the length before photostimulation for all data points, except for ChR2 and C1C2 at 560 and 568 nm. For a comparison of normalized data of the experiments in A and B, and analysis of statistically significant differences in contractions relative to those evoked by ChR2-HR, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046827#pone.0046827.s003" target="_blank">Fig. S3</a>. <b>C)</b> Expression patterns of the 4 proteins described in B, each fused to YFP, in body wall muscle cells. Scale bars are 20 µm. <b>D)</b> Light-evoked contractions of animals expressing C1V1-ET/ET or ChR2-HR/TC (individually) in body wall muscles were compared at 400 nm (0.01 mW/mm²) and 580 nm (0.2 mW/mm²). Each protein evokes strong contractions at its near-peak wavelength, but is not co-activated by the respective other wavelength. Number of animals is indicated. In A, B, D, mean body length changes and s.e.m. are displayed. In D, statistically significant differences after two-tailed Student’s t-test are indicated (***p<0.001).</p

Properties of Channelrhodopsin variants tested in this study in <i>C. elegans</i> body wall muscle cells.

<p>Summary of the results obtained with ChR::YFP fusion proteins expressed in body wall muscle (BWM) cells. Expression obtained from extrachromosomal transgenes were compared by eye, for expression level, localization to the plasma membrane and mosaic expression patterns, and classified from – (no expression) to +++ (robust, uniform and predominantly plasma membrane expression). Photo evoked contractions were deduced from work presented in the main and supplementary figures, given as relative body contraction in % of the initial length, measured at the indicated wavelength near the respective action spectrum maximum. The number of generated transgenic lines is given, as well as lines with appreciable expression, tested for light evoked reactions, and number of lines responding as by visual inspection. Each time, the best-responding line was further analyzed for quantitative data as presented throughout. ‘Rating’: A non-quantitative, arbitrary summary of the parameters tested for each protein, and from impressions obtained while experimenting with each; ranked from +++ (very well-working optogenetic tool, in <i>C. elegans</i>) to - (non-working optogenetic tool, in <i>C. elegans</i>).</p

C1V1-ET/ET and ChR2-HR evoke wavelength-dependent activity in mechanosensory neurons. A)

<p>The six gentle touch mechanoreceptor neurons, their localization and morphology are shown. <b>B)</b> The effectiveness of C1V1-ET/ET and ChR2-HR, expressed in different strains, in evoking behavioral responses in mechanoreceptor neurons (backward escape, reversals) were compared, in response to 400 and 568 nm light of different intensities, as indicated. While C1V1-ET/ET can be specifically activated without co-activation of ChR2-HR at 568 nm, significant co-activation of C1V1-ET/ET at 400 nm is apparent. Displayed is the mean probability of backing in response to blue light stimuli (and s.e.m.), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046827#s2" target="_blank">Methods</a>. Number of animals is indicated, and statistically significant differences were determined by two-tailed Student’s t-test, as indicated (***p<0.001).</p

Food Sensation Modulates Locomotion by Dopamine and Neuropeptide Signaling in a Distributed Neuronal Network

Finding food and remaining at a food source are crucial survival strategies. We show how neural circuits and signaling molecules regulate these food-related behaviors in Caenorhabditis elegans. In the absence of food, AVK interneurons release FLP-1 neuropeptides that inhibit motorneurons to regulate body posture and velocity, thereby promoting dispersal. Conversely, AVK photoinhibition promoted dwelling behavior. We identified FLP-1 receptors required for these effects in distinct motoneurons. The DVA interneuron antagonizes signaling from AVK by releasing cholecystokinin-like neuropeptides that potentiate cholinergic neurons, in response to dopaminergic neurons that sense food. Dopamine also acts directly on AVK via an inhibitory dopamine receptor. Both AVK and DVA couple to head motoneurons by electrical and chemical synapses to orchestrate either dispersal or dwelling behavior, thus integrating environmental and proprioceptive signals. Dopaminergic regulation of food-related behavior, via similar neuropeptides, may be conserved in mammals.status: Published onlin

Rhodopsin optogenetic toolbox v2.0 for light-sensitive excitation and inhibition in <i>Caenorhabditis elegans</i>

<div><p>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-<i>trans</i> 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 <i>Caenorhabditis elegans</i>. 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 <i>C</i>. <i>elegans</i> and, possibly, other systems.</p></div