We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.
Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal’s endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.
The overall design of the protocol is illustrated in Figure 1. The setup for monitoring locomotor activity using devices housed in environmentally controlled incubators located in a darkroom needs to be assembled first. Once that is completed, the system can be used in all subsequent locomotor activity rhythm measurements. For each experiment, one has to (i) prepare experimental animals, which might include generating transgenic animals or setting up necessary crosses, (ii) prepare glass activity tubes containing a food source, (iii) load flies into activity tubes and connect activity monitors to the data collection system, and (iv) record and analyze the data using different software depending on what circadian or sleep parameters one wants to examine. Herein, we define the “start” of the experiment as the time when flies in monitoring devices are first exposed to the desired light/dark conditions in environmental incubators.
1. Setting up the Locomotor Activity Monitoring System
2. Preparation of Experimental Animals
3. Preparation of Activity Tubes
4. Loading Flies into Activity Tubes and Locomotor Activity Monitoring System
5. Experimental Design to Record Data for Determination of Circadian Periodicity and Amplitude
6. Representative Results
Upon the completion of this protocol, one can use the same data set to examine both circadian and sleep parameters of the experimental animals in relation to the control animals.
Analysis of circadian parameters: Eduction graphs illustrating daily locomotor activities or average activities of flies over several days in LD or DD conditions can be generated using FaasX (Figure 3). Drosophila melanogaster generally exhibit two bouts of activity; one centered around ZT0 (or CT) termed “morning” peak and another around ZT12 (CT 12) termed “evening” peak. These two bouts of activities are controlled by the endogenous clock, and can even be observed in free-running DD conditions (Figure 3B). Changes in the timing of these activity peaks can easily be observed in eduction graphs and may indicate a change in the properties of the endogenous clock. Another property that is indicative of proper clock function is the anticipatory increase in locomotor activity observed in LD cycles that occurs prior to the actual dark-to-light or light-to-dark transitions (Figure 3A, arrows). This behavior is clearly observed in wild type flies (Figure 3A), but is absent in arrhythmic mutants such as per0 (Figure 3C) (Konopka and Benzer, PNAS, 1971). In the case of the per0 mutants, the observed “morning” and “evening” peaks in LD are purely startle responses due to abrupt changes in light/dark conditions (i.e. ‘clockless’ flies do not anticipate environmental changes but merely react to them). Loss of behavioral rhythmicity is much more pronounced in DD and generally manifests into the total loss of morning or evening peaks of locomotor activity (i.e. random bouts of activity and inactivity), as seen in per0 flies (Figure 3D). In addition to eduction graphs, locomotor activity data can be represented as double-plot actogram (FaasX), where two days of data are plotted sequentially on each line, but the last day’s profile begins the next line of two days worth of activity (Figure 4). For example, LD1 and 2 are plotted on the first line of the actogram. The next line begins with a repeat of LD2 and is followed by LD3 and so on. Following this format, the locomotor activity data spanning the entire experiment is illustrated in the actogram. Actograms can be plotted for each individual fly, or for each fly genotype. One advantage of actograms over eduction graphs is that a change in the period length of daily activity rhythms is easily observable (Figure 4). Besides generating eduction graphs and actograms, locomotor activity data from DD condition can be submitted to FaasX to calculate the period length using a number of different programs, including Cycle-P.
Analysis of sleep/rest parameters: By using the current definition of sleep/rest in Drosophila (Hendricks et al. 2000), which is five contiguous minutes of inactivity, one can analyze data recorded from locomotor activity assays and examine multiple sleep parameters using Insomniac (L. Ashmore), a Matlab-based program. The percent of time that flies spend sleeping can be calculated at different time intervals, e.g. percent sleep every hour (Figure 5A), or 12 hours (Figure 5B). Other more common sleep parameters that can be examined include mean rest bout length (Figure 5C) and wake activity count (Figure 5D). Mean sleep/rest bout length is a measure of how consolidated the sleep is and can illustrate the quality of sleep. Wake activity, as its name suggests, is a measure of the activity rate when the flies are awake. This parameter helps to differentiate between flies that are truly affected in sleep/rest behaviors vs. those that are either sick or hyperactive. For example, flies that are simply sick may seem to sleep more because they are not as mobile. For these flies, their wake activity will be lower in relation to control animals.
Figure 1: Flowchart outlining the major steps for assaying locomotor activity rhythms in Drosophila. The procedures are presented on the left while helpful comments are provided on the right. The amount of time required to perform necessary crosses and genetic manipulations to obtain flies with the right genotype for specific experiments is variable depending on the nature and design of the experiment. The two steps marked with asterisks (*) provide the time frame for when adult flies need to be seeded/mated to generate progenies of the appropriate age (1 to 5 days old) for the experiment.
Figure 2: Wiring diagram illustrating the connections between the different components for Drosophila locomotor activity data collection using the DAM System. A dedicated computer is used to record the locomotor activity counts of Drosophila. Activity monitors are housed inside incubators equipped with temperature and lighting (On/Off) control. The computer can also be used to control the timing of light On/Off in incubators if the power source of the lighting system can be hooked up to the Power supply unit (optional). Communications between the computer and activity monitors/ incubators are managed by the Power supply interface unit. The computer, Power supply unit and incubators (if the lighting control is independent of the computer) are connected to the AC power outlet via the UPC to ensure uninterrupted monitoring of activity and continuous lighting during the light phase. It is recommended to connect all the electrical appliances to the emergency backup circuits in the facility, if available.
Figure 3: 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.
Figure 4: 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.
Figure 5: Quantifying sleep parameters in Drosophila. Flies (Canton-S; CS) were exposed to standard 12:12 LD cycle at 25°C. Insomniac (L. Ashmore) was used to process the data and Microsoft Excel was used to generate the charts shown here. At least 70 flies were pooled to obtain the group averages and error bars (standard error of the mean) shown. (A) Baseline sleep calculated every hour; shown is a representative daily cycle. (B) Baseline sleep of representative daily cycle calculated every 12 hours. (C) Average length of each rest bout calculated in 12-hour increments. (D) Rate of waking activity calculated every 12 hours.
In this protocol, we described procedures for measuring Drosophila locomotor activity rhythms, a reliable behavioral output of fly circadian clocks that is used as the standard readout of clock function. This assay has been used in large-scale screens for novel clock mutants (e.g. Konopka and Benzer 1971; Dubruille et al. 2009) and is continually used to dissect and understand clock function in vivo. It has also been used to study sleep wake cycle in flies, even though recent reports suggest that video digital analysis is much more reliable in quantifying sleep than using locomotor activity rhythms (Zimmerman et al. 2008). When using locomotor activity rhythms to analyze sleep, percentage of sleep in the daytime tend to be overestimated.
To ensure the success and reproducibility of this protocol, it is critical to assay flies that are similar in age, genetic background, and reared under the same conditions, as behavioral phenotypes in fruit flies such as circadian rhythmicity and sleep/rest activity are very sensitive to all these factors. When using multiple incubators for a single experiment, it is important to make sure all incubators are at the anticipated temperature since some circadian parameters may change as a function of temperature. One word of caution when considering purchasing incubators for working with flies; not all are created equal. While we hesitate to recommend any particular unit there are many options. A good resource for finding companies that sell incubators for Drosophila work is provided at <www.flybase.org>. Some companies even sell “Drosophila circadian” incubators, wherein additional features are available, such as already wired for the Trikinetics system and temperature ramping (e.g., Tritech). Important features include the ability for diurnal light control and good temperature control in the physiological range of Drosophila (~15-30°C). Prices and sizes of incubators vary a lot but with the newer activity monitors from Trikinetics, even small incubators can accommodate quite a number of these devices. Also, although incubators with humidity control can be used, this added feature is not necessary as long as you place a small pan with water to provide humidity (50-70% is fine). Finally, although we routinely use FaasX and Insomniac for data analysis in this protocol, there are alternative programs and softwares available (Rosato and Kyriacou 2006), e.g. ClockLab (ActiMetrics), Brandeis Rhythm Package (D. Wheeler, Baylor College of Medicine, Houston), and MAZ (Zordan et al. 2007).
The authors have nothing to disclose.
This work was supported by NIH grants NIH34958 to I. E and NS061952 to J. C.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 | M. Boudinot and F. Rouyer, Centre National de la Recherche Scientifique, Gif-sur-Yvette Cedex, France | Only for Mac | ||
Insomniac 2.0 software | Leslie Ashmore, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA | 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 | |
Sucrose | Sigma | S7903 | ||
Bacto Agar | Becton Dickinson | 214010 | ||
TissuePrep Paraffin pellets | Fisher Scientific | T565 | Melting point 56°C-57°C | |
Block heater | VWR | 12621-014 |