A video method to study Drosophila sleep

Abstract

Study objectives: To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements.

Design: A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images.

Measurements and results: The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation.

Conclusions: Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep.

Figure 1

(A) Image of 2 flies in recording chambers. Positions of the flies are marked with white arrows. (B) Image of the same 2 flies captured 5 seconds after (A) fly #2 had moved and fly #1 had not. (C) The difference of (B) - (A) as grayscale values. Movement of fly #2 resulted in a white image (high grayscale values) at its previous location in the prior frame and a dark image (low grayscale values) at its new location in the current frame (marked by a white arrow). Fly #1, which had not moved, produced no white or dark pixels. The infrared beam of the Drosophila Activity Monitoring System monitor is visible in (A) and (B) as a bright spot in the lower portion of each figure in the middle of the tube. Fly #2 ends its movement into the electronic beam in (B).

Figure 2

The Drosophila Activity Monitoring System (DAMS) misses movements away from the beam. The location of a single w¹¹¹⁸ female monitored simultaneously by DAMS and video for 24 hours is shown. The infrared beam of the DAMS monitor is the reference point and equals 0 on the Y axis. The distance toward the yarn or toward the food in pixels is shown as a green bar for each 5-second video capture. The X axis is the time of day in 2-hour intervals from lights on (ZT 0). Daytime (ZT 0 to T 12) is indicated by the yellow background. Nighttime (ZT 12 to ZT 24) is indicated by the light gray background. The periods identified as sleep using video are shown as blue bars and the periods identified as sleep using DAMS are shown as red bars below the X axis. There are frequent instances, particularly during the day, in which the DAMS method considers the fly to be asleep while video does not. These periods usually correspond to times when the fly is moving in the space between the food and the beam.

Figure 3

The Drosophila Activity Monitoring System (DAMS) overestimates sleep parameters for Canton-S (CS), w¹¹¹⁸, and wRR flies of different sexes. Shown is the mean ± SD for (A) Daytime total sleep. (B) Nighttime total sleep (C) Daytime mean sleep bout duration (D) Nighttime mean sleep bout duration. Black bars = measurements by DAMS, white bar = measurements by video.

Figure 4

The Drosophila Activity Monitoring System (DAMS) overestimates both daytime and nighttime total sleep in old animals. Average ± SD Total sleep (A) and sleep bout duration (B) as determine by DAMS (black) and video (white) are shown for young (7 days) and old (45 days) w¹¹¹⁸ females.

Figure 5

Distribution of duration of sleep latency following lights on are shown for 4 groups, 2 control groups (CON) and 2 sleep-deprivation (SD) groups. Data for Canton-S females are shown as Kaplan-Meier survival curves for video (A) and Drosophila Activity Monitoring System (DAMS) (B). The Y axis is the percentage of flies still awake after lights on. The X axis is the time in minutes from ZT 0. Con 3 hr [circle] = the control group for the 3-hour deprivation experiment (n=7). Con 6 hr [triangle] = the control group for the 6-hour experiment (n = 7). 3 hr SD [square] = group deprived from ZT 20 to ZT 23 (n=7). 6 hr SD [diamond] = group deprived from ZT 17 to ZT 23 (n=8).

Figure 6

Movements within sleep bouts are considerably smaller than movements during wake bouts. Pixels moved per five second video acquisition (epoch) were normalized to the maximum pixel area of each w¹¹¹⁸ female fly (n=8). The 25%, 50%, and 75% quartiles for all single 5-second movements as a percentage of total area of the fly are shown. Wake = acquisitions on either side are also movement. Sleep = the preceding and following epochs are at least 2.5 minutes of quiescence.