- The Drosophila Activity Monitor, or DAM, holds centered activity tubes equipped with fly food at one end and an air-permeable closer at the other. This setup accommodates single flies during experiments lasting several days or even weeks.
Place the DAM in a controlled environment like an incubator with set temperature, humidity, and light cycle. Conduct the experiment in a dark room and, if necessary, use an infrared light source to handle the device, which unlike bright light, does not influence the fly's circadian system.
To record locomotor activity, connect the apparatus to a computer system that stores the readings from the DAM's detectors. The DAM's electronics continuously detect the integrity of an infrared beam that is emitted through the midpoint of each tube and periodically upload data to the computer system.
As the fly walks back and forth inside the tube, it crosses the infrared beam. These interruptions are counted by the DAM over time for each tube and serve as a measure of fly activity.
In the following protocol, we will use the DAM system to study circadian rhythms in Drosophila adult males.
- Prior to loading flies into activity tubes, turn on the incubators that will be used to house the activity monitors. Adjust the temperature using the incubator controls and set the light/dark regime using the DAM system light controller or the incubator's own light control system, according to the desired experimental design.
Next, anesthetize the flies with carbon dioxide. Then, using a fine paintbrush, gently transfer a single fly into an activity tube. Grab the middle of a single piece of yarn that is around half an inch with fine forceps and insert the yarn into the non-food end of the activity tube to plug the opening and prevent the fly from escaping during the experiment, while at the same time allowing airflow into the tube.
Keep the tube laid on its side until the fly awakens to prevent the fly from getting stuck to the food. Insert the tubes into the activity monitors. With the newer, more compact model of the TriKinetics monitors, it is necessary to hold the tubes in place with rubber bands to ensure that the infrared beam passes the tube at the center position.
Finally, put the activity monitors into the incubators and hook them up to the data collection system via the telephone wires. Check, using the DAM system collection software, that all the monitors are hooked up properly, and that data is being collected from each of them.
Now that the flies have been loaded into the activity tubes and the locomotor activity monitoring system is ready, the data can now be recorded. Flies are synchronized and entrained by exposing them to the desired light/dark, or LD, and temperature regime for two to five full days. The most commonly used entrainment condition is a light/dark cycle of 12 hours light/12 hours dark at 25 degrees Celsius.
For the study of circadian rhythms relative to this protocol, the time when the lights go on in the incubator is defined as zeitgeber time 0 and all other times are relative to that value. Under standard 12:12 LD conditions, wild type Drosophila melanogaster typically exhibit two bouts of activity; one centered around ZT0, termed morning peak, and another around ZT12, termed evening peak.
Free-running locomotor activity rhythms are measured under constant dark and temperature conditions after the entraining period. The setting for the light cycle can be changed anytime in the dark phase on the last day of LD, such that the subsequent day of the experiment represents the first day of DD. Seven days of DD data collection is sufficient to calculate the circadian period and amplitude or strength of rhythm of the flies.
In general, a sample size of at least 16 flies is necessary to obtain reliable free-running periods for a particular genotype. At the conclusion of the experiment, raw binary data collected using the DAM system software is downloaded onto a portable data storage device, for example, a USB key.
The raw binary data is processed using DAM Filescan102X, and summed into 15 and 30 minute bins when analyzing circadian parameters, or 1 to 5 minute bins when analyzing sleep/rest parameters. Currently, five contiguous minutes of inactivity is the standard definition of sleep/rest in Drosophila.