Figure 1.
Expression and photoactivation of NpHR, Arch and Mac in body wall muscle cells.
A, NpHR::CFP (upper panels), Arch::GFP (middle panels) and Mac::GFP (lower panels) were expressed in body wall muscle cells (BWMs) of C. elegans using the promoter of the myo-3 gene, as shown by confocal imaging. Scale bars represent 100 µm; white arrows point to muscle arms. B, Relative body lengths of animals expressing either NpHR (upper panel, n = 10), Arch (middle panel, n = 15) or Mac (lower panel, n = 18) were monitored when challenged with 10 yellow-green (0.96 mW/mm2, 540–580 nm, Figure S2) light pulses of 1.5 s with a 1.5 s ISI. C, Body elongations of animals expressing NpHR (left panel), Arch (middle panel) or Mac (right panel) were measured upon illumination with a 2 s light pulse (0.14 mW/mm2) with different wavelengths, as indicated in the legend (Figure S3). D, Activity spectra of the three hyperpolarizers were constructed by plotting the normalized (maximal elongation is set to 1 for each strain) average body elongations for each wavelength, calculated for a 1.5 s timeframe, starting 0.5 s after the onset of the light until the end of the light pulse. Error bars display SEM values.
Figure 2.
Comparison of outward currents evoked by NpHR, Arch and Mac.
A–D, 10 yellow (3.82 mW/mm2, 590 nm, Figure S4) light pulses of 1.5 s were applied with a 1.5 s interstimulus interval on dissected worms expressing either A, NpHR, B, Arch or C, Mac in BWMs. The evoked outward currents were measured by patch clamping muscle cells. Representative traces are shown in A–C. D, Mean outward current values for the peak values and steady state are displayed for the first light pulse. Mean input resistances (GOhm) were as follows: NpHR before light: 0.68±0.22; NpHR during light: 0.23±0.08; Arch before light: 0.49±0.15; Arch during light: 0.12±0.03; Mac before light: 0.91±0.20; Mac during light: 0.38±0.12. E–H, In analogy, 10 blue (8.06 mW/mm2, 470 nm, Figure S4) light pulses were applied and evoked currents were monitored by electrophysiology. Mean input resistances (GOhm): NpHR before light: 1.05±0.53; NpHR during light: 0.40±0.13; Arch before light: 1.48±0.39; Arch during light: 0.67±0.14; Mac before light: 1.02±0.33; Mac during light: 0.33±0.09. Error bars and ± indicate SEM values; statistical significance was determined using student’s t test; *p<0.05.
Figure 3.
Inhibition of cholinergic neurons by NpHR, Arch and Mac affects locomotion.
A, Arch::YFP and B, Mac::YFP were expressed in cholinergic neurons using the promoter sequence of the unc-17 gene, as visualized by confocal imaging. Scale bars indicate 100 µm. Animals expressing C, NpHR, D, Arch or E, Mac in the cholinergic neurons were challenged with three yellow-green (0.52 mW/mm2, 540–580 nm, Figure S2) light pulses of 1 s, with a 5 s ISI. The effect on the locomotion was analyzed by automated video analysis and the resulting speed graphs indicate the mean speed with SEM values. F–H, In analogy, the same illumination protocol was performed with blue light (0.41 mW/mm2, 450–490 nm, Figure S2). I, Minimal average speeds and J, minimal achieved speeds for the individual worms are indicated for the first light pulse of the two different illumination protocols. Statistical analysis was performed using a non-parametric Kruskal-Wallis test followed by a Dunn’s post-hoc test; ***p<0.0001, *p<0.05, n.s. not significant.
Figure 4.
Evaluation of NpHR, Arch and Mac as tools to inhibit downstream signaling in the gentle touch circuit.
A, Schematic representation of a transgenic animal expressing ChR2 in the touch receptor neurons (Pmec-4::ChR2) and either Arch, Mac or NpRH in downstream command interneurons (and other cells, driven by the glr-1 promoter [28]) that coordinate forward and backward movement. The location of the six touch receptor neurons (blue) and the cells expressing glr-1 are indicated; URY is displayed in light green as it expresses glr-1 only weakly. B, Simplified wiring diagram of the gentle touch network based on [11], indicating the synaptic connections and gap junctions. The blue and green boxes indicate which neurons were photoactivated (blue) or inhibited (green). C, Using selective illumination, the AVM and ALM mechanosensory neurons were photoactivated with blue light (4.2 mW/mm2, 435–475 nm, Figure S5) for 1s by illumination of the indicated body segment. 0.5 s after the first blue light pulse, we applied a green light pulse (4.7 mW/mm2, 543–593 nm, Figure S5) of 1s on the head region of the animals; mean velocity with SEMs are indicated for different genotypes. D, Average velocities of each worm during the 1 s green light pulse is plotted. Statistical analysis was performed using a non-parametric Kruskal-Wallis test followed by a Dunn’s post-hoc test; ***p<0.0001, **p<0.005, *p<0.05, n.s. not significant. E, As a control, we only photoactivated AVM and ALM with blue light (as in C, but no green pulse was given) and plotted mean velocities with SEMs for all different genotypes. F, Average velocities of each worm for a 3 s time bin (6s–9s) after the blue light pulse is plotted. G, Animals expressing Arch or Mac in the command interneurons were challenged with the green light pulse of 1 s on the head; no ChR2 is present in these worms. Average velocity traces and SEM values (gray shade) are shown. H, Average velocities of each individual worm for three time-bins are plotted: 5 s before illumination, during the 1 s green light pulse, and 10 s after illumination. Data were further analyzed using a non-parametric repeated measures test (Friedman), followed by a Dunn’s multiple comparison test. I, In analogy, head regions of animals expressing Mac in the command cells were illuminated with a 1 s blue light pulse. J, Resulting average velocities for the three time bins as indicated in (H) are shown.
Figure 5.
One-color optogenetic analysis of the ASH circuit by ChR2 and Mac.
A, Simplified representation of the wiring diagram of the polymodal nociceptive neuron ASH with indication of the synaptic contacts and gap junctions according to [11]. B, Scheme of a worm expressing ChR2 in ASH and Mac or Arch in downstream cells, driven by the promoter of the glr-1 gene. C, Confocal imaging of the head region of animals expressing Psra-6::ChR2::YFP (expression of ChR2 in ASH and ASI, green) and Pglr-1::Mac::mCherry (expression of Mac in the command interneurons, red). The location of the terminal bulb of the pharynx is indicated by the dotted circle. D, Animals expressing ChR2 in ASH and ASI (Psra-6::ChR2::YFP, blue trace) and animals that also expressed Mac in the downstream command interneurons (Psra-6::ChR2::YFP; Pglr-1::Mac::mCherry, green trace) were challenged with blue light (0.41 mW/mm2, 450–490 nm, Figure S2) using whole field illumination for 3 s. The evoked behavior was quantified by automated video analysis and showed evoked backward movements (negative velocity) for animals expressing ChR2 in ASH and ASI. In contrast, animals that also expressed Mac in the command cells only slowed down, but did not show any reversals.
Figure 6.
Two-color optogenetic manipulation of the ASH circuit by ChR2 and Arch.
A–C, Head regions of animals expressing ChR2 in ASH (ASH::ChR2) were illuminated with blue (4.2 mW/mm2, 435–475 nm, Figure S5) light for 4 s using 5 different intensities. A, Average velocity plots are shown for three light intensities as examples. B, Minimal achieved velocity (highest speed when moving backward) for each individual animal was plotted; Kruskal-Wallis test followed by a Dunn’s post-hoc test (***p<0.0001, **p<0.005). C, Probabilities of the occurrence of a reversal or of an omega bend were plotted for each light intensity and resulting curves were fitted with Boltzmann sigmoidals. D–F, Head regions of ASH::ChR2; Pglr-1::Arch::mCherry animals were illuminated with blue light for D, 4 s, E, 2.5 s or F, 1 s. Next, the same head region was illuminated with green light (4.7 mW/mm2, 543–593 nm, Figure S5) for 1 s, 1.5 s after the start of the blue pulse. Mean velocity plots with SEMs are shown. Fractions of animals that responded with an omega bend are represented in the color-coded bar graphs.