Generalized additive model describes locomotor response during environmental changes
To better understand the dynamics of locomotor response over time, a generalized additive model (GAM) was developed (Fig. 1D; Model, Statistical Analysis in the Methods). A GAM for each assay consisted of the linear (parametric) and smooth terms that described the response variable, locomotion. The effect of each term on locomotion was evaluated for its main effect. With all the assays, the overall variation in locomotion explained by the model ranged from 68.3–97.5% (mean = 86.6%), demonstrating a satisfactory explanatory power (Supplementary Data S1-S23). The residuals, unexplained variations, and fitted values were visualized to assess their appropriateness (Supplementary Figs. S63-S85). Post hoc pairwise analysis was performed to assess the significance of the difference in locomotion between the groups every second (Supplementary Tables S2-S66). The proportions of the significance between different assay regimens were compared to understand the effect of different durations of illumination (i.e., 4x[7.5 dark + 2-min light] regimen vs. 4x[7.5 + 7.5-min] regimen; Supplementary Figs. S8-S62; Supplementary Data. S24-S59; Supplementary Tables S67-S70).
To make the analysis process more rigorous, we used the mean of each experimental group in each assay, rather than the raw movement measurement of individual fish conventionally utilized in zebrafish behavioral studies (Response variable, Statistical Analysis in the Methods). The aggregation of the locomotor response mitigated the severe skewedness in the data.
WT larvae move more in light than in darkness during the day
To understand the changes in locomotion following illumination changes, we first established the pattern of basal locomotor activity during the day without any stimuli. In addition, we were curious whether zebrafish would move more in a lit or dark condition in the daytime. As a diurnal species, zebrafish have a circadian cycle that has evolved for high activity during the day and rest at night.49 It was not clearly established whether the larvae would move following the circadian rhythm regardless of the presence of illumination or the condition of ambient light (lit or dark) would dictate the levels of locomotion overriding the circadian cycle (called “masking”). We recorded wildtype (WT) larvae (4-, 5-, 6-, and 7-days post-fertilization [dpf]) from 10 am to 10 pm in constant illumination or darkness.
We found that illumination was a critical determinant for the larvae’s locomotion (Fig. 2Ab; Supplementary Data S1). Light had a significant positive effect on locomotion compared to the null hypothesis of no effect (light, main effect, p = 1.31e-12; Supplementary Data S1) and led to a significantly different trend over time (light spline; F = 4.937, edf = 171.881, p < 2e-16). Darkness did not result in a different trend in locomotion (dark spline; F = 0.05, edf = 0.844, p = 0.951). Larvae in light moved significantly more than their age-matched counterpart in darkness across all developmental stages over 12 hours (Fig. 2Ac). That is, 4-, 5-, 6-, and 7-dpf larvae in light moved significantly more than 4-, 5-, 6-, and 7-dpf larvae in dark at all time points (100%), respectively (Supplementary Figs. S8-S9; all p-values provided in Supplementary Tables S3-S4 and all proportions provided in Supplementary Tables S67-S68). A dark environment decreased baseline activity, masking the basal circadian activity levels in light during the day in larvae aged between 4 and 7 days.
The developmental stage was another important determinant for basal locomotion. Compared to 4-dpf larvae, 5-, 6-, and 7-dpf larvae moved significantly more (5, 6, and 7-dpf, main effect, p = 0.00099, 2.38e-11, and 6.03e-8, respectively). Activity at different ages from highest to lowest was 6-dpf fish followed by 7-, 5-, and 4-dpf fish in either light or darkness. Combined, the levels of locomotion followed the order over 12 hours: (6-dpf larvae in light > ≈ 7 dpf in light) > ≈ 5 dpf in light > (4 dpf in light ≈ 6 dpf in dark) >≈ (7 dpf in dark ≈ 5 dpf in dark) > 4 dpf in dark (Figs. 2Ab and 2Ac). 5-dpf larvae in light moved significantly more than 6-dpf in darkness for a short window of time, and moved significantly more than 7-, 5-, and 4-dpf in darkness for most of the time (Fig. 2Ac; Supplementary Figs. S8-S9; Supplementary Tables S67-S68 and S3-S4).
nr3c1 mutant larvae move less than WT siblings in light and darkness during the day
Following the study of basal locomotion in WT fish, we investigated the basal locomotion of the nr3c1ex5 fish (5 dpf) in which glucocorticoid receptor (nr3c1) is knocked out in homozygous (HM) larvae.23 Similar to the WT stock fish, illumination was a critical determinant of locomotor activity of nr3c1ex5 fish. Compared to darkness, a lit condition increased locomotion (light; main effect, p = 3.27e-6; Supplementary Data S2; Fig. 3Ab). Genotype was another determinant. nr3c1 WT and heterozygous (HT) siblings showed comparable levels of locomotion in darkness or light (HT, main effect, p = 0.683). Homozygosity decreased overall locomotion (HM, main effect, p = 0.014). WT larvae moved significantly more than HM siblings in 34.84% of the time both in darkness and illumination and the significance was concentrated between 10 am and 6 pm (Supplementary Figs. S10-S11; Supplementary Tables S6-S7 and S67-S68). Homozygosity had a sizable impact on locomotion such that HM larvae in light moved more than WT siblings in darkness only in 1.39% for the 12-h period (Fig. 3Ac; Supplementary Fig. S11; Supplementary Table S68).
mc2r mutant larvae show less activity than WT siblings only when illumination duration is shorter
After establishing that both darkness and blocking HPA axis activity decrease basal locomotion in larvae, we investigated the dynamic changes in locomotion in response to changing illumination. We previously showed that, to increase locomotor activity in post-light darkness after a brief 1-min illumination, a functioning HPA axis is essential as loss of mc2r and nr3c1 significantly decreased the locomotor response in these fish.23 Our initial hypothesis on the larval response in the dark-light repeat assay was that fish with a mutation in one of the key HPA axis receptor genes would have a deficiency in mounting an appropriate locomotor response to changing light. However, the duration of illumination is a determinant of the ensuing locomotor activity in darkness, as well as the HPA axis genes.
The ACTH receptor (mc2r) on the adrenal gland (interrenal cells in zebrafish) is a key HPA (HPI) axis receptor that initiates glucocorticoid synthesis. When mc2r was knocked out, the overall locomotion in darkness was significantly less than that of WT siblings when repeated illumination was 2 mins (HM, main effect, p = 8.007e-6) and 4 mins (main effect, p = 0.00015), but not with 7.5-min illumination (main effect, p = 0.758; Fig. 4; Supplementary Data S3-S5). The proportion of locomotion in which WT larvae moved significantly more than their HM siblings in darkness (the 3rd, 4th, and 5th dark periods) was 93.9, 96.34, and 0% in the 2-, 4-, and 7.5-min light repeat assays, respectively (Supplementary Figs. S15-S17; Supplementary Table S69). The difference in proportions was significant between the 2- and 4-min light repeat assays (two-proportions z test, \({\chi }^{2}\)=8.243, p=0.004; Supplementary Data S25), the 2- and 7.5-min light repeat assays (\({\chi }^{2}\)=2429.0, p\(\approx\)0; Supplementary Data S27), and the 4- and 7.5-min light repeat assays (\({\chi }^{2}\)=2555.1, p\(\approx\)0; Supplementary Data S29). Therefore, the mc2r HM larvae could mount an equivalent locomotion profile compared to WT siblings as the duration of illumination increased (7.5 min).
nr3c1 mutant larvae move less than WT siblings when illumination duration is shorter
nr3c1 is the canonical glucocorticoid receptor that binds to glucocorticoids (i.e., cortisol, corticosterone) and drives the various stress responses in the central nervous system and peripheral tissues. Homozygous mutant larvae in nr3c1ex5 showed significantly less locomotion in darkness in the 2-, 4-, and 7.5-min repeat assays (HM, main effect, p = 0.00054; p = 0.0029, and p = 0.0465, respectively), but not in 6-min repeat assays (main effect, p = 0.057; Fig. 5; Supplementary Data S6-S9). The proportion of locomotion where the WT fish moved significantly more than their HM siblings in darkness was 100, 87.78, 68.1, and 60.88% when 2-, 4-, 6-, and 7.5-min illumination was repeated (Supplementary Figs. S25-28; Supplementary Table S69). The difference in proportions was significant in all pairwise comparisons between 2- and 4-min (two-proportions z test, \({\chi }^{2}\)=171.48, p = 3.510e-39; Supplementary Data S31), 2- and 6-min (\({\chi }^{2}\)=507.92, p = 1.794e-112; Supplementary Data S33), 2- and 7.5-min (\({\chi }^{2}\)=647.77, p = 6.828e-143; Supplementary Data S35), 4- and 6-min (\({\chi }^{2}\)=153.23, p = 3.42e-35; Supplementary Data S37), 4- and 7.5-min (\({\chi }^{2}\)=253.14, p = 5.373e-57; Supplementary Data S39), and 6- and 7.5-min repeated illumination (\({\chi }^{2}\)=15.632, p=7.694e-5; Supplementary Data S41), showing that the proportion of the difference in locomotion between the WT and HM siblings changes as the illumination duration changes.
In another lineage of nr3c1 mutant fish where the frameshift mutation was introduced in exon 2, the effect of the duration of illumination was clearer. Homozygous mutant larvae in nr3c1ex2 moved significantly less in darkness when repeated illumination was 4 min (HM, main effect, p = 0.0071), but not when illumination was 6 min (p = 0.197) and 7.5 min (p = 0.615; Supplementary Fig. 7; Supplementary Data S21-S23). The proportion of locomotion where the WT fish moved significantly more than their HM siblings in darkness was 94.65, 71.58, and 0% when the repeated illumination was 4, 6, and 7.5 min (Supplementary Figs. S57-S59; Supplementary Table S69). The difference in overall activity levels in darkness between the WT and HM siblings decreased in both nr3c1ex5 and nr3c1ex2 mutant fish (100, 87.78, 68.1, and 60.88%; ND, 94.65, 71.58, and 0% at 2, 4, 6, and 7.5 min), respectively, as the repeated illumination increased50–52, showing that the nr3c1 HM mutants could mount increasingly similar locomotor responses in darkness as the duration of illumination increased.
Mutations in nr3c2 do not appear to have consistent effects on locomotion
Mineralocorticoid receptor (nr3c2) is another nuclear receptor that binds to glucocorticoids with higher affinity. However, we did not find a clear role in locomotor response to light changes in our previous paper.23 Similarly, again we could not identify any pattern of difference in locomotion between the WT and HM siblings (Fig. 6). The HM mutant larvae in nr3c2ex2 apparently moved less in 2-min repeated illumination at some time points (Fig. 6Ac), but there was no main effect of homozygosity compared to WT siblings (HM, main effect, p = 0.104; Supplementary Data S10). Likewise, in 4- and 7.5-min repeated illumination, the HM larvae apparently moved more at some time points, but there was no main effect of homozygosity compared to WT siblings (p = 0.0728 and p = 0.758), respectively (Supplementary Data S11-S12). Since there was no main effect of homozygosity on locomotion, the meaning of the apparent decrease or increase of locomotion amongst HM siblings at some time points is unlikely to be biologically meaningful.