Analysis of sleep/wake cycles in zebrafish larvae

Abstract

Zebrafish larvae are a model organism increasingly used in the study of the effect of neuroactive chemicals on vertebrate sleep/wake cycles. Sleep disturbances have a negative impact on mood, cognition and overall health. Here we present a protocol to assess over 24 h sleep/wake cycles in zebrafish larvae subjected to 12 h light/dark periods in 48-well plates, using video-tracking technologies. The protocol can be used to determine if the exposure to environmental pollutants or drugs can lead to sleep disturbances. The results on the effect of the tire rubber-derived 6PPD-quinone on zebrafish sleep/wake cycles presented here demonstrate the suitability of using this protocol in fish neurotoxicity studies. This protocol provides a new relevant tool to be used in the pharmacology and toxicology fields.

Keywords: Analysis of sleep/wake cycles in zebrafish larvae; Sleep disturbance; Sleep/wake cycles; Video-tracking technologies; Zebrafish larvae.

Graphical abstract

Fig. 1

Behavior battery.

Fig. 2

Dynamic table obtained from raw data. The results need to be ordered chronologically.

Fig. 3

Table obtained from reordering the columns by treatment; the rows are color-coded by period.

Fig. 4

Table with the results of Sum of cm for the CONTROL group. The results from the different plates are unified in this table. Next to it, another table should be placed with the results of immobile cumulative duration following the same display.

Fig. 5

Table with the results of immobile cumulative duration for the CONTROL larvae by each hour and period.

Fig. 6

Table with the results of immobile cumulative duration for the CONTROL group. Next to it there is the copy without data.

Fig. 7

Copy of the original table showing the results of immobile cumulative duration for the CONTROL group with the corresponding substitutions specified in the formula used.

Fig. 8

Original table, which shows the results of immobile cumulative duration with the corresponding substitutions (activity, IMM bout, -), and the copy next to it, used for calculating the start of each bout.

Fig. 9

Original table, which shows the results of immobile cumulative duration with the corresponding substitutions (activity, IMM bout, -), and the copy used for calculating the end of each bout.

Fig. 10

Bout start and Bout end tables showing the number of row in which each immobility bout starts and ends. The tables beneath them show the same results but filtering the cells that are relevant, aligning the results to be able to do the subtraction.

Fig. 11

On top, there are two tables showing the filtered results from the start and end of each immobility bout. The table showing the end of each bout has already been corrected adjusting the range of the “FILTER” formula to include the end of the bouts that start in the previous period. The table beneath shows the product of the subtraction of the tables above (following the formula explained in step 6h).

Fig. 12

Table indicating the frequency of the bouts of a specific length for each larva in every period.

Fig. 13

The table on the left there shows the results of immobile cumulative duration with the corresponding substitutions (activity, IMM bout, -). Next to it there is a table at the level of the row where the transition between periods happens. These table is used to calculate the latency between daytime and nighttime periods.

Fig. 14

Effects of 24 h of exposure to environmental concentrations of 6PPD-quinone on the wake/sleep cycle of zebrafish larvae. (A) Locomotor activity of zebrafish larvae, both control and those exposed to the three concentrations of 6PPD-quinone, entrained and tested under 12:12 h light–dark conditions. The period in which activity was recorded, from 7 to 8 dpf, corresponds to the 24 h exposure period. The bars at the bottom of the graph on the left indicate the light/dark/light periods (white bar on the left (D1): 2 pm to 8 pm / black bar in the middle (N): 8pm to 8 am / white bar on the right (D2): 8 am to 2 pm). The results are presented as the mean ± SE. (B) Total distance moved during the daytime (D1+D2) and nighttime (N) periods. Data are shown as scatter plots with the median (n=34–35; one-way ANOVA with Dunnett's multiple comparison test). (C) Time spent by the larvae in the sleep state per hour over 24 h. (D) Time spent by the larvae in the sleep state during D1 (6 h), N (12h), and D2 (6 h). Data are shown as scatter plots with the median (D1: n=31, Student's t-test; N and D2: n=31, Mann–Whitney U test). (E) Number of sleep bouts during the D1, N, and D2 periods (n=31, Student's t-test). (F) Sleep latency at night (n=30, Mann–Whitney U test).