This video describes the Drosophila activity monitor (DAM) system used to track locomotor activity. Researchers use activity data collected from the DAM to study circadian rhythms in fruit flies. The featured protocol clip shows how to load flies in the device and record activity data for circadian experiments.
This protocol is an excerpt from Chiu et al., Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila, J. Vis. Exp. (2010).
1. Loading Flies into Activity Tubes and Locomotor Activity Monitoring System
- 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 incubators own light control system according to the desired experimental design. The time necessary to load flies into activity tubes should be sufficient for the temperature to stabilize.
- Anesthetize the flies with carbon dioxide.
- Use a fine paintbrush to 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. Alternatively, plastic caps with small holes (Trikinetics, Inc.) can be used to close the opening.
- Make sure the tubes are laid on their sides until the fly awakens, or else there is the risk of the fly getting stuck to the food.
- Insert the tubes into the activity monitors. With the newer, more compact model of the Trikinetics monitors (Trikinetics DAM2 and DAM2-7), 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.
- 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 to make sure all the monitors are hooked up properly and data is being collected from each of them. The monitor emits infrared light beam across the center of each glass activity tube. The locomotor activity of the flies are recorded as raw binary data where "one" is recorded each time the infrared beam is broken or a 'zero' is recorded in which the infrared beam is not broken.
2. Experimental Design to Record Data for Determination of Circadian Periodicity and Amplitude
- Flies are synchronized and entrained by exposing them to the desired light/dark (LD) and temperature regime for 2-5 full days. The most commonly used entrainment condition is a light/dark cycle of 12 hrs light/ 12 hrs dark (12:12 LD) at 25 °C. This generally accepted standard condition is essentially based on the thought that Drosophila originated from Afro-equatorial locations. When studying circadian rhythms there is some phraseology that one needs to become familiar with. Relevant to this protocol, the time when the lights go on in the incubator is defined as zeitgeber time 0 (ZT0) and all other times are relative to that value (e.g., in a 12:12 LD cycle, ZT12 is the time when the lights are turned off). 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 (Figure 1A). The morning and evening bouts are controlled by the endogenous clock but there are also "startle" responses that are transient bursts of activity in response to the light/dark transitions. Two days of entrainment is the minimum and could be used, for example, in large screens that are more time-consuming and are geared towards measuring free-running periods in constant darkness (see below, step 2). However, if you are interested in studying the activity patterns during a daily light-dark cycle, it is preferable to maintain the flies for 4-5 days in LD so as to obtain more data. Essentially, increasing the number of flies or the number of LD days in the final data analysis (e.g., pool data from the last two days worth of LD locomotor activity) will generate more reliable diurnal activity profiles and measurements (e.g., timing of morning or evening peak). Furthermore, the daily distribution of activity varies as a function of day-length (photoperiod) and temperature. A major reason for altering the photoperiod or temperature from the standard is if one wanted to study how daily activity patterns undergo seasonal adaptation (e.g. Chen et al., Cold Spring Harb Symp Quant Biol. (2007)). Drosophila can also be entrained to daily temperature cycles (e.g. Glaser and Stanewsky, Curr Biol. (2005); Sehadova et al., Neuron (2009)). Temperature cycles that vary by only 2-3 °C are sufficient to entrain activity rhythms.
- Free running locomotor activity rhythms are measured under constant dark and temperature conditions after the entraining period is finished (see above, step 1). 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 (e.g., power or strength of rhythm) of flies. In general, a sample size of at least 16 flies is necessary to obtain reliable free-running periods for a particular genotype. Even if one is only interested in measuring diurnal activity, it is still best to measure the flies' free-running periods in DD as changes in endogenous period can alter the daily distribution of activity in LD. For example, flies with long endogenous periods usually exhibit delayed evening peaks in LD (e.g., see Figure 2).
- At the conclusion of the experiment, raw binary data collected using the DAM System software is downloaded onto a portable data storage device, e.g. USB key.
- The raw binary data is processed using DAM Filescan102X (Trikinetics, Inc.) 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 (Hendricks et al., Neuron (2000); Ho and Sehgal, Methods Enzymol., (2005)).
- There are many different ways to analyze the data collected on the DAM System but we will only provide those methods routinely used in our lab. Microsoft Excel is used to assign genotype to different sample groups. FaasX software (M. Boudinot and F. Rouyer, Centre National de la Recherche Scientifique, Gif-sur-Yvette Cedex, France) or Insomniac (Matlab-based program; Leslie Ashmore, University of Pittsburgh, PA) are used to examine circadian (e.g. period and power) or sleep/rest (e.g. percentage sleep, mean rest bout length) parameters respectively.
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Figure 1: Eduction graphs generated using FaasX showing daily locomotor activity rhythms of rhythmic wild type flies (w per0 flies carrying a per+ transgene) (A and B) vs. arrhythmic w per0 mutants (C and D). Male flies were kept at 25 °C and entrained for 4 days in 12:12 LD (light: dark) cycles followed by seven days in DD (constant darkness). For each fly line, the locomotor activity levels of individual flies (n>32) were measured in 15-minute bins and then averaged to obtain a group profile representative for that line. A and C show the activity data generated from averaging the second and third days in light/dark cycle (LD 2-3) while B and D show the activity data generated from averaging the second and third days in constant darkness (DD 2-3). Vertical bars represent the activity (in arbitrary units) recorded in 15-minute bins during the light period (light grey) or the dark period (dark grey). Horizontal bars at the bottom of LD eduction graphs; white, lights on; black, lights off. ZT0 and ZT12 represent the start and end of the photoperiod respectively. For DD eduction graphs; CT0 and CT12 represent the start and end of the subjective day in constant dark conditions, denoted by the grey bar. In panel A, M = morning peak; E = evening peak. The arrows in panel A represent anticipatory behavior of morning and evening peaks observed in wild type flies, which are absent in w per0 arrhythmic flies. Please click here to view a larger version of this figure.
Figure 2: Double-plot actogram generated using the FaasX software illustrating locomotor activity data of flies with wild type, short, or long period. Male flies were kept at 25 °C and entrained for 4 days in 12:12 LD cycles followed by eight days in constant darkness (DD) for the calculation of the free-running period (t) using Cycle-P in FaasX. Three fly lines with wild type period [w per0; per+; per0 mutant carrying per+ transgene], long period [w per0; per(S47A); per0 mutant carrying per(S47A) transgene], and short period [w per0; per(S47D); per0 mutant carrying per(S47D) transgene] are shown here (Chiu et al. 2008). X-axis represents ZT or CT time in LD or DD respectively, and Y-axis represents activity counts (arbitrary units) summed into 15-minute bins. The red dotted lines connect the evening peaks for each day of the experiments. Note that during LD the evening peak is 'forced' to maintain synchrony with the 24-hr LD cycle, whereas in DD the free-running period can deviate from 24 hr. For example, for flies with short periods the timing of the evening activity will occur earlier on each successive day in DD (when plotted against a 24 hr time scale, as shown here), whereas a shift to the right is observed for flies with long periods. Please click here to view a larger version of this figure.
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|Drosophila activity monitor (DAM)||Trikinetics Inc.; Waltham, MA||DAM2 or DAM5||DAM2 monitors are more compact, and more can fit into a single incubator|
|Power supply interface unit (for DAM system)||Trikinetics Inc.; Waltham, MA||PSIU9||Includes PS9-1 AC Power Supply|
|Light controller||Trikinetics Inc.; Waltham, MA||LC6|
|Pyrex glass tubes||Trikinetics Inc.; Waltham, MA||PGT5, PGT7, and PGT10|
|Plastic activity tube caps||Trikinetics Inc.; Waltham, MA||CAP5||Yarn can be used instead of plastic caps.|
|DAM System data collection software||Trikinetics Inc.; Waltham, MA||Versions available for both Mac and PC|
|FaasX software||Centre National de la Recherche Scientifique||Only for Mac|
|Insomniac 2.0 software||University of Pittsburgh School of Medicine||Runs on Matlab. Can be used on both PC and Macintosh.|
|Environmental incubator with temperature and diurnal control, e.g. Percival incubator||Percival Scientific, Inc.||I-30BLL||Interior space dimension:Width: 65cm;Height: 86cm;Depth: 55cm|
|Environmental incubator with temperature and diurnal control, e.g. DigiTherm Heating/Cooling Incubator with Circadian Timed Lighting and Timed Temperature||Tritech Research, Inc.||05DT2CIRC001||Interior space dimension:Width: 36m;Height: 56m;Depth: 28cm|
|APC Smart-UPS 2200VA 120V (Emergency power backup unit)||APC||SU2200NET||Output Power Capacity of 1600 Watts|
|Bacto Agar||BD Biosciences||214010|
|TissuePrep Paraffin pellets||Fisher Scientific||T565||Melting point 56 °C-57 °C|
|Block heater||VWR international||12621-014|