Locomotor properties of intact spontaneous, NMDA‐induced, and ChR2‐induced fictive swimming. (a–c) Fictive swimming activity was recorded from peripheral motor nerves of intact spontaneously swimming (Intact), spinalized NMDA‐induced (NMDA), and spinalized ChR2‐induced (ChR2) Tg(vglut2a:Gal4ff)^nns20;Tg(UAS:ChR2(H134R)‐mCherry)^umn201 larval preparations (blue lines represent continuous 10 s blue light stimuli). Peripheral motor nerve voltage traces of 10 min (a), 30 s (b), and 3 s (c) are shown. Boxes indicate regions expanded in subsequent panels. (d–f) Mean values of fictive Episode Duration (d), Burst Duration (e), and Burst Frequency (f). Error bars represent SD. Asterisks indicate statistically significant differences between groups.

Robustness of ChR2‐induced fictive swimming decreases during repetitive blue light stimulation. Peripheral motor nerve voltage traces were recorded as 20 blue light stimuli were delivered at 3 min intervals. (a) Peripheral nerve voltage traces from a representative preparation during the first (t = 0) and final (t = 57 min) blue light stimuli (blue line represents continuous 10 s blue light stimulus). (b) Raster plot of burst times (vertical lines) for each of the 20 stimuli (rows) delivered to the preparation in (a). (c–g) Mean values of fictive swimming properties during repetitive 10 s stimuli: number of bursts produced during each stimulus (c), number of episodes produced during each stimulus (d), episode duration (e), burst duration (f), and within‐episode burst frequency (g). Shaded regions represent SD. Asterisks indicate significant differences between the first and final (20th) stimuli.

Comparison of robustness and organization of repetitive ChR2‐induced and continuous NMDA‐induced fictive swimming. ChR2‐induced and NMDA‐induced fictive swimming were recorded from spinalized Tg(vglut2a:Gal4ff)^nns20;Tg(UAS:ChR2(H134R)‐mCherry)^umn201 preparations. ChR2‐induced swimming was stimulated for 10 s with an inter‐stimulus period of 3 min. Corresponding 10 s‐long windows of NMDA‐induced fictive swimming were analyzed every 3 min. (a) Representative peripheral nerve voltage traces from the first (t = 0) and final (t = 57 min) timepoints of ChR2‐induced (top; blue line represents continuous 10 s blue light stimulus) and NMDA‐induced (bottom) fictive swimming. (b) Total number of bursts detected during each 10 s recording of ChR2‐induced (ChR2; black; plot also shown in Figure 2c) and NMDA‐induced (NMDA; red) fictive swimming. (c) Episodic Organization (EO) scores of ChR2‐induced (black) and NMDA‐induced (red) fictive swimming. Recordings of 10 s were grouped into bins defined by elapsed experimental time. Each bin contained 5 voltage recordings (total of 50 s/bin). Shaded regions and error bars represent SD. Asterisks indicate significant differences when comparing the first and final stimuli in b and bins in c; ns indicates that differences were not significant.

Application of exogenous dopamine reduces ChR2‐induced fictive swimming activity. Fictive swimming was induced in spinalized Tg(vglut2a:Gal4ff)^nns20;Tg(UAS:ChR2(H134R)‐mCherry)^umn201 preparations by delivering 10 s‐long blue light stimuli at 3 min intervals. Dopamine (DA) treatment preparations were perfused with extracellular saline for 10 min, 10 µM DA was added to the perfusate for 10 min, followed by wash out of DA with saline. Control preparations were continuously perfused with extracellular saline. (a) Fictive swimming activity of an untreated control preparation (top) at t = 0 (baseline) and t = 18 min (+saline) and of a DA‐treatment preparation (bottom) at t = 0 (baseline) and t = 18 min (+10 µM DA). Blue lines represent continuous 10 s blue light stimuli. (b–e) Number of bursts produced during each 10 s stimulus (b), number of swimming episodes produced during each 10 s stimulus (c), mean burst duration (d), and mean burst frequency (e) of DA‐treatment (green) and untreated control (black) preparations. Horizontal green bar indicates time of 10 µM DA application. Shaded regions represent SD. Boxes indicate final baseline (Base), DA treatment (DA), and washout (Wash) stimuli that were compared between control (plots also shown in Figure 2) and DA‐treatment groups. Asterisks indicate significant differences between groups.

Effects of repetitive blue light stimulus exposure times and frequencies on fatigue of fictive locomotor bursting activity. Ten s‐long blue light stimuli were delivered to spinalized Tg(vglut2a:Gal4ff)^nns20;Tg(UAS:ChR2(H134R)‐mCherry)^umn201 preparations with inter‐stimulus periods of 1 (60 stimuli over 59 min), 3 (20 stimuli over 57 min), and 15 min (5 stimuli over 60 min). (a) Representative peripheral nerve recordings of fictive swimming activity produced during the first (t = 0) and final 10 s blue light stimulus delivered with each inter‐stimulus period (blue lines represent continuous 10 s blue light stimuli). (b) Number of bursts (normalized to the first stimulus at t = 0) produced during 10 s blue light stimuli delivered with each inter‐stimulus period. Red circles indicate the fifth stimulus at each inter‐stimulus period. (c) Number of bursts (normalized to the first stimulus at t = 0) produced during the fifth blue light stimulus (red circles in b) for each inter‐stimulus period (expressed as time of fifth stimulus delivery). (d) Number of bursts (normalized to the first stimulus at t = 0) produced during the final blue light stimulus, expressed as the total amount of time stimulated with blue light for each inter‐stimulus period. Shaded regions and error bars represent SD. Asterisks indicate statistically significant differences from the first stimulus. Asterisks over horizontal lines indicate significant differences between groups.