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. 2020 Mar 27;9:e54937. doi: 10.7554/eLife.54937

Figure 7. Optogenetic suppression of swimming in larvae.

(A) Schematic of behavioural assay. (B) Opsin expression in spinal motor neurons and interneurons in a Tg(s1020t:GAL4;UAS:GtACR1-tdTomato) larva at 5 dpf. Imaging field of view corresponds to black box in (A). A, anterior; D, dorsal; P, posterior; V, ventral. Scale bar 50 μm. (C) Tg(s1020t:GAL4;UAS:GtACR1-tdTomato) larvae were positioned in individual agarose wells (left) and instantaneous swim speed was monitored by centroid tracking (right) at 50 fps (six dpf; N = 25 ± 9 fish per group, mean ± SD). 10 s light periods were delivered (459 or 617 nm, 0–2.55 mW/mm2) with a 50 s inter-stimulus interval. (D) Optogenetically-induced changes in bout rate (mean + SEM, across fish) in Tg(s1020t:GAL4) larvae expressing GtACR1 (N = 24 larvae, left) or eNpHR3.0 (N = 40 larvae, right). Horizontal grey bars indicate the time windows used to quantify behavioural changes. Each time bin corresponds to 2 s. (E,F) Normalised bout rate during the `LED On` period in larvae expressing different opsins (mean ± SEM, across fish) and in control, opsin-negative, siblings. See also Figure 7—figure supplements 1–4 and Figure 7—video 1.

Figure 7—source data 1. Data related to Figure 7.
Data provided as a XLSX file.

Figure 7.

Figure 7—figure supplement 1. Bout rate vs. time in larvae expressing different opsins in spinal neurons.

Figure 7—figure supplement 1.

(A,B) Distribution of bout rate vs. time for Tg(s1020t:GAL4) larvae (6 dpf) expressing different opsins (mean + SD, across fish). Larvae were subjected to 10 s pulses of blue (459 nm; A) or red (617 nm; B) light. Each time bin corresponds to 2 s.
Figure 7—figure supplement 2. Bout rate and speed vs. irradiance during different time periods in Tg(s1020t:GAL4) larvae.

Figure 7—figure supplement 2.

(A,B) Normalised bout rate (A) or bout speed (B) during the whole LED On period, the initial 2 s of light exposure and the `post LED` 8 s period in Tg(s1020t:GAL4) larvae (6 dpf) expressing different opsins (mean ± SEM, across fish). Control opsin-negative siblings were subjected to the same light stimuli.
Figure 7—figure supplement 3. Optogenetic suppression of swimming in Tg(mnx1:GAL4) larvae.

Figure 7—figure supplement 3.

(A) Schematics of opsin expression pattern and behavioural assay. (B) Opsin expression in spinal motor neurons in a Tg(mnx1:GAL4;UAS:GtACR1-tdTomato) larva at 5 dpf. Imaging field of view corresponds to black box in (A). A, anterior; D, dorsal; P, posterior; V, ventral. Scale bar 50 μm. (C) Background-subtracted camera field of view showing Tg(mnx1:GAL4;UAS:GtACR1-tdTomato) larvae positioned in individual agarose wells (left) and tracking of swimming speed for selected larvae (right). Behaviour was monitored at 50 fps across multiple freely-swimming larvae (6 dpf; N = 24 ± 6 fish per group, mean ± SD) while they were subjected to 10 s light periods (459 or 617 nm, 0–2.55 mW/mm2) with a 50 s inter-stimulus interval. (D) Optogenetically-induced changes in bout rate (mean + SEM, across fish) in Tg(mnx1:GAL4) larvae expressing GtACR1 (N = 29 larvae, left) or eArch3.0 (N = 23 larvae, right). Horizontal grey bars indicate the time windows used for comparative quantification of behavioural changes. Each time bin corresponds to 2 s. (E,F) Normalised bout speed during the `LED On` period in larvae expressing different opsins (mean ± SEM, across fish). Control opsin-negative siblings were subjected to the same light stimuli.
Figure 7—figure supplement 4. Bout rate and speed vs. irradiance during different time periods in Tg(mnx1:GAL4) larvae.

Figure 7—figure supplement 4.

(A–D) Normalised bout rate (A–C) or bout speed (D) during the whole `LED On` period (A), the initial 2 s of the light period (B), or the `post LED` 8 s period (C,D) in Tg(mnx1:GAL4) larvae (6 dpf) expressing different opsins (mean ± SEM, across fish). Control opsin-negative siblings were subjected to the same light stimuli.
Figure 7—video 1. Suppression of swimming upon opsin activation in larval spinal neurons.
Download video file (1.3MB, mp4)
Suppression of swimming in Tg(s1020t:GAL4;UAS:GtACR1-tdTomato) larvae (6 dpf) during 10 s of blue light (459 nm, 0.24 mW/mm2). Images were acquired at 50 frames per second and the video plays at 3 × speed. Related to Figure 7.