Larbi MC , Messa G , Jalal H , Koutsikou S .

             hindbrain development
             midbrain-hindbrain boundary development
             locomotion

	Figure 1. Midbrain lesions and design of behavioral experiments. Dorsal view of the Xenopus laevis tadpole at developmental stage 37/38. (A) Diagram of the tadpole’s anatomical features and key CNS regions. Blue dotted outline represents the area where the skin was opened to allow access to the brain. (B) The tadpole viewed under a dissection microscope with pins running across the body, through the eyes and the notochord just caudal to the obex. Blue dotted line denotes the area of open skin with the brain visible. (C) Representation of the midbrain lesions. Ctrl (Control, orange); trigeminal nerve transection at the level of the otic capsule. The trigeminal nerve transections were performed on all experimental groups. MHB (Midbrain-Hindbrain Border lesion, purple); a transverse lesion through the MHB. ML (Midline lesion, green); lesion along the midline of the midbrain. Ipsi MHB + ML (Left/Ipsilateral MHB and ML lesion, blue); combination of left MHB and ML lesions. Contra MHB + ML (Right/Contralateral MHB and ML lesion, red); combination of right MHB and ML lesions. (D) Schematic diagram of the behavioural set up with (E) a zoomed in section of the tadpole’s starting position (not drawn to scale). (F) Illustration of the tail angle measurement used in subsequent figures. Red dot indicates the level of the anus, referred to as the starting point of the tail (G) The tadpoles starting position from a dorsal view. The tadpole is positioned upright into a groove carved onto a Sylgard block. Stimulation is applied on the left side of the tadpole’s trunk skin, at the level of the anus, using a glass pipette with an attached fine rabbit hair (CUSTOM–MADE). (H) Individual frames taken from high-speed videos recorded at 420 frames per second (fps). A short poke with a single rabbit hair to trunk skin receptors on the left side (*; time = 0 ms) initiated a swim response at 129 ms. (I) Individual video frames of a tadpole’s swim trajectory captured from a 30 fps recording. In this example the control animal trial starts at stimulation (0 s) to the end of the tadpole’s swim cycle (5.6 s). A novel tracking software, FastTrack Software, was used to detect and trace the tadpole’s body using an automatic tracking algorithm. (J) Schematic showing the path taken (black), the distance (blue) and the displacement (green). Here, the displacement is described as the shortest distance from the starting position to the next frame (position), with frame intervals indicated as F1, F2 and F3, with F4 representing the final displacement. The distance here is described as the measure of the path taken from the starting to the final position. Abbreviations: Ctrl, control; MHB, midbrain-hindbrain border; ML, midbrain midline; Ipsi MHB + ML, left/ipsilateral midbrain-hindbrain border and midbrain midline; Contra MHB + ML, right/contralateral midbrain-hindbrain border, and midbrain midline.
	Figure 2. Experimental setup for fictive swimming. (A) Dorsal view of the tadpole with recording and stimulating electrodes in position. Left and right VR (ventral root) electrodes were positioned facing each other approximately at the 4th myotomal cleft. The stimulating electrode was always positioned on the right side at the level of the anus. Red dashed line denotes the area of open skin with the brain visible. Red dashed square area indicates the area presented in panel (B). (B) Picture of the trunk of a tadpole pinned to the Sylgard block during extracellular VR recordings. Right and left VR and stimulating electrodes, are positioned as described in A. (C) Example of an ipsilateral start of fictive swimming in response to electrical trunk skin stimulation (red arrowhead). The first VR burst is recorded on the right side of the body (right VR, ipsilateral; black arrowhead). (D) Example of a contralateral start of fictive swimming in response to electrical trunk skin stimulation (red arrowhead). The first VR burst is recorded on the left side of the body (contralateral, left VR; black arrowhead). In all experiments stimulation was delivered on the right side of the tadpole’s body.
	Figure 3. Midbrain lesions lead to changes in latency and side of swim initiation in freely moving animals. (A) Latency (ms) to the start of swimming in response to trunk skin stimulation. Ctrl: 104.8, 73.2–128.0 ms, MHB: 156.0, 125.9–243.5 ms, midline: 113.1, 76.79–173.8 ms, Ispi MHB + ML: 121.4, 79.76–157.7 ms, Contra MHB + ML: 126.2, 104.8–198.8 ms. (B) Percentage occurrence (% total number of trials for each experimental group) of the first behavioral motor response in relation to the side of the stimulus. Ctrl: 41% ipsilateral, 59% contralateral; MHB: 37% ipsilateral, 63% contralateral; Midline: 63% ipsilateral, 37% contralateral; Ipsi MHB + ML: 24% ipsilateral, 76% contralateral; Contra MHB + ML: 72% ipsilateral, 28% contralateral. (C) Latency to the initiation of swimming in ipsilateral vs contralateral starts. Ctrl ipsilateral 109.5, 83.3–151.2 ms, contralateral 97.6, 59.5–109.5 ms; MHB ipsilateral 191.7,150.0–251.2 ms, contralateral 145.2, 109.5–228.6 ms; Midline ipsilateral 113.1, 71.4–171.4 ms, contralateral 115.5, 84.5–180.4 ms; Ipsi MHB + ML ipsilateral 128.6, 98.8–170.2 ms, contralateral 119.1, 75.0–158.3 ms; Contra MHB + ML ipsilateral 142.9, 104.8–204.8 ms, contralateral 119.1, 92.3–189.3 ms. Ctrl: n = 10 tadpoles, trials = 32; MHB: n = 14 tadpoles, trials = 38; Midline: n = 11 tadpoles, trials = 38; Ipsi MHB + ML: n = 12 tadpoles, trials = 38; Contra MHB + ML: n = 9 tadpoles, trials = 36. For boxplot (A, C) all data are reported as median and 25–75 percentile, single data points are plotted, the middle horizontal line represents median latency, the box represents the interquartile range (IQR, 25–75 percentile), and the error bars represent the minimum and maximum values. ****p < 0.0001, **p < 0.01 for Kruskal-Wallis/Dunn’s test.
	Figure 4. Midbrain-hindbrain border (MHB) lesion leads to changes in latency and side of fictive swim initiation. (A) Latencies (ms) to the first VR burst after threshold electrical stimulus was delivered to the trunk skin in control and MHB-lesioned animals. Mann-Whitney test p = 0.0049, controls 100.9, 91.02–117.8 ms vs MHB lesioned 48.1, 36.24–61.4 ms; data reported as median, 25–75 percentile. Ctrl: n = 5 tadpoles, trials = 22; MHB: n = 7 tadpoles, trials = 29. (B) Percentage occurrence (% total number of trials for each experimental group) of the first VR burst after threshold electrical stimulation in control (orange) and MHB-lesioned animals (violet). Ctrl ipsilateral 63.6% (14/22 trials), contralateral 36.4% (8/22 trials); MHB ipsilateral 34.5% (10/29 trials), contralateral 51.7% (15/29 trials), synchrony 13.8% (4/29 trials). (C) Latencies (ms) to the first VR burst after threshold electrical stimulus delivered to the trunk skin in control (orange) and MHB-lesioned (violet) animals. Solid circles represent latencies to ipsilateral first VR burst, open circles represent latencies to contralateral first VR burst, and crosses represent VR bursts recorded simultaneously on both sides of the body. Mann-Whitney test for the ctrl group, p = 0.365; Kruskal-Wallis test for the MHB-lesioned group, p = 0.1859. Ctrl ipsilateral 101.9, 93.1–112.0 ms, contralateral 67.4, 23.6–132.8 ms; MHB ipsilateral 49.8, 39.6–64.0 ms, contralateral 50.7, 36.5–83.6 ms, synchrony 36.2, 35.9–39.8 ms. Data are reported as median, 25–75 percentiles. Ctrl: n = 5 tadpoles, trials = 22; MHB: n = 7, trials = 29. (D) Latencies (ms) to the first VR burst after suprathreshold electrical stimulus delivered to the trunk skin in control and MHB-lesioned tadpoles. Mann-Whitney test, p = 0.8249; ctrl: 27.82, 24.35–95.81 ms; MHB: 35.16, 24.28–56.94 ms. Data reported as median, 25–75 percentile. Ctrl: n = 5 tadpoles, trials = 27; MHB: n = 7, trials = 33. (E) Percentage occurrence (% total number of trials for each experimental group) of the first VR burst after suprathreshold electrical stimulation of control (orange) and MHB-lesioned animals (violet). Ctrl ipsilateral 25.9% (20/27 trials), contralateral 74.1% (7/27 trials); MHB ipsilateral 39.4% (13/33 trials), contralateral 42.4% (14/33 trials), synchrony 18.2% (6/33 trials). (F) Latencies (ms) to the first VR burst after suprathreshold electrical stimulus delivered to the trunk skin of control (orange) and MHB-lesioned animals (violet). Solid circles represent latencies to ipsilateral first VR burst, open circles represent latencies to contralateral first VR burst, and crosses represent VR bursts recorded simultaneously on both sides of the body. Mann-Whitney test for the ctrl group, p < 0.0001: ipsilateral 107.9, 97.0–120.3 ms, contralateral 25.5, 23.9–32.7 ms; Kruskal-Wallis test for MHB group, p = 0.2108: ipsilateral 36.5, 23.3–3.2 ms, contralateral 43.6, 24.8–95.7 ms, synchrony 25.8, 12.7–36.1 ms. All data are reported as median, 25–75 percentile. Ctrl: n = 5 tadpoles, trials = 27; MHB-lesioned: n = 7, trials = 33). (G) Latencies (ms) to the first alternating VR burst (i.e., the first burst indicative of the start of fictive swimming) after a threshold electrical stimulus delivered to the trunk skin of control and MHB-lesioned tadpoles. Mann-Whitney test, p = 0.0049; ctrl: 100.9, 91.0–117.8 ms; MHB: 52.1, 42.0–71.1 ms. Ctrl: n = 5 tadpoles, trials = 22; MHB: n = 7, trials = 29. (H) Latencies (ms) to the first alternating VR burst after a suprathreshold electrical stimulus delivered to the trunk skin of control and MHB-lesioned tadpoles. Mann-Whitney test, p = 0.4879; ctrl: 27.8, 24.4–95.8; MHB: 48.9, 25.1–61.1 ms. Ctrl: n = 5 tadpoles, trials = 27; MHB: n = 7, trials = 33. (I) Latency (ms) for asynchronous starts (synchrony data are omitted) after a threshold stimulation and according to the side of the first VR burst (Mann-Whitney test, p = 0.8079; ipsilateral 54.6, 42.1–63.5 ms; contralateral: 51.4, 40.6–83.5 ms; n = 7 tadpoles, trials = 29). (J) Latency to asynchronous starts after a suprathreshold stimulation and according to the side of first VR burst (Mann-Whitney test, p = 0.4654; ipsilateral 48.7, 24.8–57.6 ms, contralateral 51.6, 25.2–81.3 ms; n = 7 tadpoles, trials = 33). In all panels, data collected on controls are in orange, data collected on MHB lesioned animals are in violet. In panels (A,C,D,F–J) single data points are plotted; boxes indicate 5–95 percentile; middle horizontal line in each box represents median value; error bars indicate minimum and maximum values. In panels (B,E); filled bars: ipsilateral first VR burst; white bars: contralateral first VR burst; grey bars: synchronous VR first burst. All data reported in the figure legend are expressed as median, 25–75 percentile. **p < 0.01, ****p < 0.0001 for Kruskal-Wallis/Dunn’s test.
	Figure 5. Midbrain lesions lead to stark changes in the trajectory of tadpole swimming (A). Example swim trajectories (n = 2) for each animal group extracted from the FastTrack Software. The starting position is symbolised by the black dot. (B) Displacement time graphs of each animal trial (coloured lines) to show the tadpoles deviation from the starting position for each animal group (See section “Materials and methods” for calculation). The average is represented by the black line for each animals group. An increase in displacement refers to the tadpole swimming away from the starting position, while a decrease represents the tadpole swimming closer to the starting position. (C) Boxplot of the final displacement and total distance travelled for each animal trial within the respective animal group. For panels (B,C), Control: n = 10, trials = 17; MHB: n = 9, trials = 12; Midline: n = 8, trials = 12; Ipsi MHB + ML: n = 9, trials = 14; Contra MHB + ML: n = 10, trials = 14. For boxplot, single data points are plotted, the middle horizontal line represents the median value, the box represents the interquartile range (IQR, 25–75 percentile), and the extended vertical bars represent the minimum and maximum values.
	Figure 6. Midbrain lesions affect sustained swimming. (A) The average duration of swim episodes in control and midbrain lesioned animals. Controls swam for 3.3 ± 0.3 s after stimulation. MHB: 22.7 ± 3.8 s; ML: 11.0 ± 1.3 s; Ipsi MHB + ML: 13.5 ± 1.9 s; Contra MHB + ML: 6.3 ± 0.6 s. Reported as mean ± SEM. Single data points are plotted; the middle horizontal line represents the mean and the extended vertical bars represent the SEM. (B) Controls travelled 47.4, 41.9–63.7 mm (note the size of the arena had a radius of 45 mm). MHB: 187.5, 99.7–307.5 mm; ML: 113.7, 79.4–189.6 mm; Ipsi MHB + ML: 123.9, 97.7–195.7 mm; Contra MHB + ML: 105.8, 77.4–138.3 mm. Sample size identical to those in Figure 5. For panel (A) Results are reported as mean ± SEM; single data points are plotted, the horizontal line represents mean duration, error bars represent the SEM. For panel (B) Results are reported as median and IQR (25–75 percentile); for boxplot, single data points are plotted, the middle horizontal line represents median distance, the box represents the interquartile range (IQR, 25–75 percentile), and the extended vertical bars represent the minimum and maximum values. ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05 for ANOVA/Dunnett’s and Kruskal-Wallis/Dunn’s test.
	Figure 7. Midbrain lesions cause tail deflections. (A) The mean tail angle at rest (no stimulus applied), prior to trunk skin stimulation. Ctrl: 0.9o ± 1.1o, n = 5 tadpoles, 12 trials; MHB: 3.1o ± 3.1o, n = 6, 13 trials; ML: 4.8o ± 7.0o, n = 4, 8 trials; Ipsi MHB + ML: –5.1o ± 8.7o, n = 5, 10 trials; Contra MHB + ML: –7.8o ± 13.3o, n = 4, 8 trials. Reported as mean ± SEM. Single data points are plotted, the middle horizontal line represents the mean, and the extended vertical bars represent the SEM. ****p < 0.0001, ***p = 0.003, ANOVA/Dunnett’s. (B) A single example of the tadpole’s body posture from still images of resting tadpoles, representative of each experimental group. (C) Circular plot of the frequency distribution of the tail angle before swim starts. Each circular segment represents the number of values. Bin width is 5.8o.
	Figure 8. Summary diagram of known and proposed midbrain neural circuitry. Diagram of the brain and spinal cord of the Xenopus laevis tadpole at developmental stage 37/38, showing already known and newly proposed neuronal connections involved in the initiation of swimming in response to photic and mechanical stimuli. Mechanosensory Rohon-Beard (RB) neurons innervate the skin of the trunk and tail. The central axons of the RB neurons transmit excitation via sensory interneurons namely dorsolateral commissural (dlc) and dorsolateral ascending (dla) interneurons. Both interneurons project ascending axons to the hindbrain and midbrain. The second sensory pathway presented in this diagram involves the excitation of the pineal eye photoreceptors in response to light dimming. Axons of pineal ganglion cells form synapses on both sides of the midbrain. Midbrain diencephalic/mesencephalic descending (D/MD) neurons are excited by the pineal ganglion cells, and project caudally into the hindbrain and spinal cord where they could excite Central Pattern Generators (CPGs) directly, or via a novel hindbrain population of sensory memory neurons (hindbrain extension neurons; hexNs), which have been proposed to extend the sensory signal between the sensory pathway and reticulospinal neurons of the swimming CPG. The proposed midbrain interneurons (mINs), if different to D/MD neurons, may also project to the opposite side of the midbrain and/or directly or indirectly to hexNs and the CPG. Each neuron type population and colour coding are displayed in the list. Circles represent groups of cell bodies, single lines represent axons, and triangles represent excitatory synapses. Proposed neuronal connections are indicated by dashed lines.

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