Figure 1. Specific photodepolarization of AS MNs via ChR2 leads to body contraction, increased bending angles and reduced speed in freely moving C. elegans.

(A) ‘Subtractive’ expression and illumination strategy to achieve specific stimulation of AS MNs by optogenetic tools: (I) Silencing of optogenetic tool protein expression in the non-target subsets of MNs by dsRNA; (II) Using the Q system for conditional expression. The transcriptional activator QF binds to the QUAS sequence to induce optogenetic tool expression. The transcriptional inhibitor QS suppresses expression in unwanted cells by binding to QF; (III) Selective illumination of the VNC MNs by 470 nm blue light. The body of the worm was divided into 11 segments, of which 3 – 10 were illuminated in animals moving freely on agar plates. (B) Expression pattern of ChR2(H134R)::YFP in AS MNs by the dsRNA subtractive approach; scale bar, 20 µm. See also Figure 1—figure supplement 1. (C) Representative body postures kymograph (20 s) of normalized 2-point angles of a 100-point spine, calculated from head to tail of the animal. Positive and negative curvature is represented by blue and red color. Animal expressed ChR2 in AS MNs as in AI and was illuminated after 10 s as in AIII. Blue bar, period of 470 nm illumination. (D) Photodepolarization of AS MNs by ChR2 (in animals raised with ATR): I) Analysis of mean bending angles, before and during the blue light illumination period (as in C). (II, III) Locomotion speed: Mean ±SEM crawling speed of animals before and during blue illumination (blue bar), comparing animals expressing ChR2 in AS MNs or in all types of cholinergic MNs in the VNC, raised in the presence or absence of ATR (III: Group data of mean speed of the animals before (15–20 s) and during (21–26 s) ChR2 photoactivation); IV, (V) Mean ±SEM body length of the animals shown in I, II (V: Group data of the mean length before (15–20 s) and during (21–26 s) photoactivation). (E) Depolarization of subsets of AS MNs in body segments. (I) Scheme of anterior, midbody and posterior segmental illumination; II) Representative body posture kymographs of 2-point angles from head to tail before (20 s) and during ChR2 photoactivation by blue light in the segments of the worm body, corresponding to experiments as in E I). See also Figure 1—video 1 and Figure 1—figure supplement 2. P values *≤0.05; **≤0.01; ***≤0.001. Number of animals is indicated in D. Statistical test in D III and V: ANOVA with Tukey’s post hoc test.

Figure 1—figure supplement 1. Expression of ChR2 can be restricted to AS MNs.

(A) Expression of ChR2::YFP in the VNC MNs, either in all cholinergic neurons (upper panel), or restricted in AS MNs using the RNAi approach (middle panel), or the Q system for conditional expression (lower panel). Arrow heads point to AS MNs. Note that in the top panel, many more MN cell bodies are visible in addition to the AS MNs. Animals were photographed, then the VNCs were cut from the resulting composite images, virtually straightened and aligned at the vulva. (B) Quantification of the visible MNs in the VNC for each of the three expression approaches. P value **≤0.01. Number of animals is indicated. Statistical test: ANOVA with Tukey’s post hoc test.

Figure 1—figure supplement 2. Local AS neuron activation affects body length.

(A I, II) C I) Speed and (B I, II) (C II) body length (time traces, A, B, and group data, C) before and during photodepolarization of AS MNs or of all cholinergic MNs in the anterior, middle and posterior segments of the worm body by ChR2 (in animals raised with ATR). P value *≤0.05, **≤0.01, ***≤0.001; number of animals is indicated. Statistical test: ANOVA with Tukey’s post-hoc test.

Figure 1—figure supplement 3. Specific photodepolarization of AS MNs via Chrimson leads to body contraction, increased bending angles and reduced speed in freely moving C.  elegans.

(A) AS MNs expressing Chrimson were illuminated by 650 nm red light. (B) Representative body postures kymograph (40 s) of normalized 2-point angles of a 100-point spine, calculated from head to tail of the animal. Dorsal and ventral curvature is represented by blue and red color. The animal expressing Chrimson in AS MNs was illuminated after 20 s. Red bar, period of 650 nm illumination. Interruptions in the analysis are due to inability of the analysis script to assign periods of strong body bends when anterior and posterior body touched. (C) Photodepolarization of AS MNs by Chrimson (in animals raised with ATR): I, II) Body length and group data (II) of mean length of the animals before (15–20 s) and during (21–26 s) Chrimson photoactivation. (D) I, II) Locomotion speed: Mean ±SEM crawling speed of animals before and during blue illumination (red bar), comparing animals expressing Chrimson in AS MNs, raised in the presence or absence of ATR, and group data (II) of mean speed of the animals before (15–20 s) and during (21–26 s) Chrimson photoactivation. (E) I) Mean (±SEM) time traces of all 3-point bending angles before and during red illumination (red bar; animals raised with and without ATR). II) Group data as in I, comparing 5 s red light illumination (red bar), to the 5 s before illumination. III) Mean (±SEM) ratio of dorsal to ventral bending at the 2^nd 3-point bending angle in animals expressing AS::Chrimson during photostimulation (animals raised with and without ATR). See also Figure 1—video 2. P values **≤0.01; ***≤0.001. Number of animals is indicated. Statistical test: ANOVA with Tukey’s post hoc test.

Figure 1—video 1. Freely moving animal before and during photodepolarization of AS MNs by ChR2 (in animal raised with ATR), blue light = 470 nm, 1.8 mW/mm².

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DOI: 10.7554/eLife.34997.006

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Figure 1—video 2. Freely moving animal before and during photodepolarization of AS MNs by Chrimson (in animal raised with ATR), red light = 650 nm, 1.8 mW/mm².

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DOI: 10.7554/eLife.34997.007

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