The flies were collected following eclosion and placed on standard cornmeal agar media. Thirty-to-thirty-two 1–3 day post-eclosion male flies per genotype and drug group were individually collected under CO2 anesthesia and transferred into 5 mm × 65 mm polycarbonate tubes containing normal media at one end and a yarn plug at the other end. The behavioral recordings of fly sleep were collected using the Drosophila Activity Monitoring System (DAMS; TriKinetics Inc., Waltham, MA USA), which measures sleep on a 12 h light:12 h dark cycle. Drosophila activity is monitored by counting beam breaks over 5 min bins and averaged over 60 min to obtain a read-out of fly sleep/wake behavior, and sleep is defined as a 5 min period of inactivity recorded by the DAMS, as standard in the field [35 (link),36 (link),49 (link)]. Zeitgeber time (ZT) 0 is defined as the time of lights on, and ZT12 is defined as the time of lights off. Daytime sleep measures were from lights on to lights off (ZT0–ZT12), nighttime measures were from lights off to lights on (ZT12–ZT24), and total sleep measures were from the full 24 h period. Flies were placed in chambers/monitors for a total of 4–5 days. To allow time for the habituation of the flies to the chambers/monitors, analyses for daytime, nighttime, and total sleep were performed for the sleep recordings for days 3–4 for all the experimental groups.
Drosophila activity monitoring system dam
Manufactured by Trikinetics
Sourced in United States
The Drosophila Activity Monitoring System (DAMS) is a laboratory equipment designed for the continuous monitoring of locomotor activity in Drosophila (fruit flies). The system records and analyzes the movement of individual flies within their enclosures, providing precise data on their activity patterns.
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33 protocols using drosophila activity monitoring system dam
1
Drosophila Sleep Behavior Monitoring
2
Circadian Rhythm Analysis in Drosophila
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Locomotor activity was recorded using Drosophila Activity Monitoring System (DAMS, Trikinetics, Waltham) for 5 days in LD12:12 and 6 days in DD. Activity was counted every 1 min and analyzed in Excel using “Befly!” software (Department of Genetics, Leicester University). Lomb—Scargle normalized periodogram was used to determine rhythmic flies; individuals with period below 10 (confidence level 0.05) were regarded as arrhythmic. Flies, which did not survive until the end of experiments were removed from analyses. Every experiment was repeated three times, at least 60 flies in total were used.
Sleep analysis was performed on the second day of LD12:12, and sleep was recorded as at least 5 min of a fly immobility.
Sleep analysis was performed on the second day of LD12:12, and sleep was recorded as at least 5 min of a fly immobility.
3
Circadian Locomotor Activity in Flies
4
Drosophila Locomotor Behavior Assays
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5
Knockdown and Overexpression of Kinases in Drosophila
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To knock down or overexpress a kinase, timGAL4;UAS-dcr2 and UAS-dcr2;cry-GAL4-1655 (link) were crossed to RNAi lines and overexpression lines. For controls, UAS and GAL4 lines were crossed to the genetic background controls for TRIP, GD, and KK RNAi collections, w1118 and yw strains. Flies were reared on standard cornmeal–yeast–sucrose medium and kept in LD cycles at 25 °C. Locomotor activity levels of adult male flies were monitored by Drosophila Activity Monitoring System (DAMS, TriKinetics) for 7 days of LD followed by 7 days of DD.
6
Drosophila Locomotor Activity Monitoring
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Drosophila activity monitoring system (DAMS; Trikinetics, Waltham, MA) detects activity by monitoring infrared beam crossings for each animal. These data were used to calculate locomotor activity using the Drosophila Sleep Counting Macro [72 ]. Flies were anaesthetized under CO2 and loaded into DAMS tubes containing standard fly food for acclimation. After 24 hours acclimation in DAMS tubes with food, baseline activity was measured for 24 hours. Tubes were maintained in a 25°C incubator with 12:12 LD cycles.
7
Drosophila Circadian Locomotor Assay
8
Drosophila Locomotor Activity and Sleep
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Males of 1- to 2-day-old were used for this experiment. They were placed in small tubes (φ = 5 mm) to record their locomotor activity and rest cycles in monitors of the Drosophila Activity Monitoring System (DAMS) (TriKinetics, Waltham, MA, USA; Nall et al. 2016 (link)). Each monitor housed 32 tubes/flies. The tubes contained the agar–sugar medium (5% sugar and 2% agar) that is commonly used in DAMS (Nall et al. 2016 (link)) at one end and the plug at the other end. The system recorded interruptions of the infrared beam emitted by the monitors that were caused by flies walking inside the tubes. The activity of the flies was recorded for 13 days: 7 days in LD12:12 and 6 days in constant darkness (DD). During recording, the monitors were kept in incubators with set temperature, humidity, and light conditions (Rosato and Kyriacou 2006 (link)). Data were obtained in 1 min bins. Since Drosophila sleep is identified as a period of minimum 5 min of inactivity (Huber et al. 2004 (link)), for sleep examination, each hour the 5-min bins of immobility (sleep) were examined. Sleep and activity analysis was performed using ShinyR-DAMS online software (Cichewicz and Hirsh 2018 (link)). The sleep phenotypes of the flies were determined based on the second day of activity recording in LD 12:12.
9
Drosophila Locomotor Activity Assay
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Male flies around 3 to 5 days old were subjected to locomotor activity assays using the Drosophila Activity Monitoring System (DAMS) (Trikinetics, Inc.). Flies were entrained for four days at 12 hr light:12 hr dark (LD) conditions at 25°C before their free-running behavioral rhythms were assessed in total darkness (DD) for seven days. Fly activity monitoring using DAMS and data analysis using FaasX were as previously described [82 (link)]. Behavioral assays to rule out developmental effects of brm RNAi were performed at 29°C.
10
Sleep Fragmentation and Locomotion Analysis in Flies
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For locomotion and sleep analysis, newly enclosed flies were collected and raised at 25°C, 60% humidity, on a 12:12 light:dark cycle for either 5–7 or 25–27 days. Sleep fragmentation was achieved by turning on the lights once an hour for 30 min during the 12 h dark cycle. This was repeated for a total of four nights. These were considered sleep fragmented flies. Another group of flies were exposed to the same sleep fragmentation regime but afterward were allowed to recover for 4 days on a normal 12:12 h day:night cycle. This was the sleep fragmented plus recovery group. The same light was employed for both the 12 h light cycle and sleep fragmentation during the dark cycle. The light used was a full spectrum bulb at 600 lux. Locomotor activity was collected using the Drosophila activity monitoring system (DAMS; Trikinetics) for 4 days at 25°C, 60% humidity, on a 12:12 light:dark cycle. Locomotor activity was collected in 1 min bins and analyzed with PySolo (Gilestro and Cirelli, 2009 (link)). Total sleep, average sleep length and average sleep bouts was calculated based on the standard sleep definition as a period of 5 or more minutes of inactivity. Flies were allowed to adapt to the DAMS system for 24 h before analysis was performed. The assay was repeated at least three times with 30 males used for each genotype (N = 90).
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