Data Analysis see Note

1. At the end of the experiment, stop the "Swimming" macro and check the threshold to see if there are any problems. Exit the macro and Optimate.

2. Import the data file and preprocess it in Excel. The first column contains time stamps, and each succeeding column contains the measurements for one well.

3. Scan the data for blank rows that are inserted whenever the macro is restarted and delete them.

4. In new columns, calculate the averages for each treatment group or genotype.

5. Note the starting date and time, ending date and time, total number of time points, and number of columns; these are required to create CHRONO files.

6. Create a new worksheet and transform all the values in the raw file worksheet with the formula: = Round(100*value,0) + 10. This generates integers between 10 and 1000 for import into CHRONO.

7. Save the integer worksheet as a text file.

8. Import the text file into CHRONO for plotting and analysis (Fig. 3).

9. During import, fuse six time points to produce 24-min bins; enter other experimental information as directed in dialog windows.

10. Smooth the data with a 4-h centered running average (Fig. 3B).

Day of experiment

Fig. 3. Display and analysis of zebrafish activity records. All records are from the same wild-type individual. (A) Raw activity reduced to 24-min bins. (B) Activity record smoothed with a 4-h running average. (C) Actogram plot of activity peaks, plotted modulo 25.3 h. (D) Composite peak for days 5 through 7.

Phase (degrees) Phase (degrees)

Fig. 3. Display and analysis of zebrafish activity records. All records are from the same wild-type individual. (A) Raw activity reduced to 24-min bins. (B) Activity record smoothed with a 4-h running average. (C) Actogram plot of activity peaks, plotted modulo 25.3 h. (D) Composite peak for days 5 through 7.

11. To produce an actogram of activity peaks (Fig. 3C), subtract the trend calculated from a 25.6-h centered running average; make a signal ratio of the trend; set modulo t to 25.3 h or to the average free-running period of the control group.

12. Set the time limits to days 5 to 7, and plot the composite curve (Fig. 3D).

13. Measure the phase of the composite peak's center; this is reliable only if the composite curve has a single symmetrical peak with a peak-trough amplitude greater than 40 relative activity units.

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