Bruno JR , Udoh UG , Landen JG , Osborn PO , Asher CJ , Hunt JE , Pratt KG .

             circadian behavior
             serotonin receptor activity
             response to serotonin

	Graphical abstract
	Figure 1. Strength of preference for light displayed by developmental stage 48/49 Xenopus tadpoles oscillates over the 24 h day/night cycle (A) Schematic of the light/dark test dish. One quadrant of the test dish is light and the remaining 3/4ths is dark. Tadpoles, in groups of ten, are video-taped using a Go-Pro as they swim freely around the dish for 30 min. Preference for light is quantified by counting the number of tadpoles in the light quadrant every minute for 30 min. (B) Preference for light was tested at two time points during the day (10:00 and 16:00) and two time points during subjective night (23:00 and 05:00). Both stage 48 and 49 tadpoles displayed a stronger preference for light during the subjective day compared to night. The preference for light peaked at 16:00. Dashed line represents the % of tadpoles in the light quadrant that would be expected if no preference for, or avoidance of, light. (C) Average number of tadpoles in the light quadrant at each minute of the 30-min trial, showing the preference at 16:00 (time of day when preference for light is at its peak) and at 23:00 (when the preference for light was lowest) test times. This plot shows that the strength for the preference for light is consistent across the 30-min trial. Dashed line represents the average number of tadpoles in the light quadrant that would be expected if no preference for, or avoidance of, light. Error bars represent SEM.
	Figure 2. Pretreating tadpoles with the SSRI fluoxetine enhances their preference for light over dark (A) Experimental timeline for experiments pertaining to panels B–D: Fluoxetine (Flx) is added to tadpoles rearing solution at 16:00 and tadpoles are returned to their incubator. Tadpoles are tested 24 h later, at 16:00 on the following day. (B) Plots showing the average number of control and fluoxetine-exposed developmental stage 48 (top), and stage 49 (bottom) tadpoles that resided in the light quadrant of the test dish. Each set of connected gray data points represents the average % of tadpoles in the light quadrant for batch-matched control and fluoxetine-treated groups, and the set of black connected data points represents the overall average of all the individual experiments (∗∗∗p < 0.001 as determined by Mann-Whitney test, top: control, n = 23; Flx, n = 23. Bottom: control, n = 34; Flx, n = 34). (C) Plots showing the average number of control and fluoxetine-exposed developmental stage 48 (top) and stage 49 (bottom) tadpoles in the light quadrant at each minute of the 30-min trial. (D) Plot showing the average preferences for gray displayed by control and fluoxetine-exposed tadpoles when gray is pitted against dark. For comparison, the average preferences for light (obtained from the dark vs light test) which are shown in panels B and C are super-imposed onto this graph. Notice that the preference for gray displayed by control and fluoxetine-exposed tadpoles is weaker than the preference for light, suggesting that the strength of the preference is light intensity-dependent (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by Mann-Whitney test. Light vs. dark: Stage 48: control, n = 23; Flx, n = 23; stage 49: control, n = 34; Flx, n = 34. Gray vs. dark: stage 48: control, n = 9; Flx, n = 9; stage 49: control, n = 12; Flx, n = 12. (E, top) Experimental timeline used to test the effect of 24-h fluoxetine pre-treatment on the preference for light at 23:00: Fluoxetine (Flx) is added to tadpoles’ rearing solution at 23:00 and tadpoles are returned to their incubator. Tadpoles were tested 24 h later, at 23:00 on the following day. (Bottom) plots showing the average % of control and fluoxetine-exposed tadpoles in the light quadrant when tested at 23:00. The 16:00 and 23:00 data combined show that fluoxetine pre-treatment enhances the preference for light during the day and night (∗∗∗p < 0.001 as determined by Mann-Whitney test: stage 48, n = 10; stage 49, n = 8). All error bars represent SEM.
	Figure 3. The increased preference for light displayed by fluoxetine-treated tadpoles is manifested mainly via longer swimming bouts while in the light quadrant (A) Cumulative probability plot displaying the amount of time in seconds individual stage 48 (white dots) and 49 control (gray dots) and stage 48 (light green dots) and 49 1 μM fluoxetine exposed tadpoles (dark green dots) stay in the light from the time they enter to when they exit. Inset is a bar graph of the same data showing the overall average amount of time in seconds of the respective groups (∗p < 0.05, ∗∗∗p < 0.001 as determined by Mann-Whitney U test, Stage 48: control, n = 90; Flx, n = 90. Stage 49: control, n = 80; Flx, n = 80). (B) Cumulative probability plot displaying speed of individual stage 48 (white dots) and 49 control (gray dots) and stage 48 (light green dots) and stage 49 (dark green dots) 1 μM fluoxetine exposed tadpoles measured in mm/s from the time they enter the light to when they exit. Inset is a bar graph of the same data showing the overall average swimming speed in seconds of the respective groups (∗p < 0.05 as determined by Mann-Whitney U test, Stage 48: control, n = 25; Flx, n = 25. Stage 49: control, n = 25; Flx, n = 25). Error bars in (A) and (B) represent SEM. (C) Examples of individual tadpole swimming bouts tracked from the time they enter to when they exit the light region. The longer swimming bouts of the 1μM fluoxetine exposed tadpoles typically involve multiple U-turns at the border between the light and dark region of the dish.
	Figure 4. Carrying out the fluoxetine pre-treatment in the absence of light decreases its effectiveness (A) Experimental timeline: Fluoxetine is added to tadpoles' rearing solution at 16:00 and then tadpoles are maintained either on the regular 12:12 light/dark schedule (left time-line) or on a 24 h dark schedule (right time-line) until testing. Tadpoles are tested 24 h later, at 16:00 on the following day. (B and C) Dot plots showing the average percent of developmental stage 48 and (C) stage 49 control and fluoxetine-exposed tadpoles (green dots) when the fluoxetine exposure was carried out on a 12:12 light dark schedule and when the exposure was carried out entirely in darkness (24-h dark schedule). The total number of trials is shown in parentheses. Notice that the fluoxetine effect on preference for light is markedly attenuated when the pre-treatment is carried out in the dark, suggesting that endogenous levels of serotonin release are relatively lower in the dark. Also notice that the control tadpoles that were housed in the dark for 24 h before testing display a modest decrease in preference for light, suggesting that normal serotonin release during the day shapes the strength of preference for light (B and C, A Kruskal-Wallis non-parametric statistical test was used to determine if at least one experimental group was statistically different from the others. This was followed by a pairwise non-parametric Mann-Whitney test to compare specific groups; ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by Mann-Whitney U test). Error bars represent SEM.
	Figure 5. Inhibiting serotonin release by pretreating tadpoles with the tryptophan hydroxylase inhibitor pCPA weakens the preference for light (A) Experimental time line: pCPA (5μM) is added to tadpoles rearing solution at 16:00 and tadpoles are returned to their incubator. Tadpoles are tested 24 h later, at 16:00 on the following day. (B) Pre-treatment with pCPA decreases the average number of both stage 48 (left) and stage 49 (right) tadpoles residing in the light quadrant at any given time, suggesting that decreasing serotonin release decreases the preference for light. Each set of connected gray data points represents the average % of tadpoles in the light quadrant for batch-matched control and pCPA-treated group. The set of black connected data points represents the overall average of all the individual experiments (∗p < 0.05, ∗∗p < 0.01 as determined by Mann-Whitney U test). (C) Plots showing the average number of stage 48 (top) and stage 49 (bottom) control and pCPA-treated tadpoles in the light quadrant at each minute of the 30 min trial. Error bars represent SEM.
	Figure 6. Ethological model An ethological model suggested by the data. Normally (middle panel), during the day, tadpoles display a moderate yet significant preference for green over light, and a moderate yet significant preference for light over dark. Experimentally enhancing serotonin transmission by exposure to the SSRI fluoxetine (top panel), however, causes an abnormally strong preference for light over both dark and green wavelengths of light. In a natural setting, this could result in tadpoles residing for abnormally long periods of time in open, well-lit areas which may increase the chances of being detected by predators. Conversely, experimentally decreasing serotonin release (bottom panel) generates an abnormally weak preference for light. In a natural setting, this could impair normal exploration, foraging for food, and/or insufficient time spent in sun-warmed regions. Thus, the moderate preferences for green and light which are shaped by endogenous levels of serotonin release (middle panel) may optimize survival (Schematic by Harley Yerdon, Johnny Morris’ Wonders of Wildlife).
	Figure S1 legend Supplemental Figure 1: Fluoxetine enhances the preference for light via a non-acute mechanism which is longlasting, related to Figure 2. (A) Plot showing the average preference for light as a function of fluoxetine pre-treatment time. Notice that the preference for light gradually strengthens with increasing pre-treatment times, suggesting a nonacute mechanism of action. The dashed line represents the average control preference for light for developmental stage 48 and 49 tadpoles. (B) Plot showing the average preference for light at a function of number of days post the normal 24- hour fluoxetine exposure. Notice that the effect slowly declines over the 3 days but does not return to baseline, suggesting that 24 hours of fluoxetine exposure induces a long-lasting effect on this visually guided behavior. The dashed line represents the average control preference for light for developmental stage 48 and 49 tadpoles.

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