The overall goal of the following experiment is to characterize circadian and sleep phenotypes in drosophila by assaying daily rhythms of locomotor activity. This is achieved by generating trenchgenic flies, obtaining specific mutants or setting up necessary crosses to prepare experimental animals. As a second step, glass activity tubes containing a food source need to be prepared, which will serve as the fly habitat during the experiment.
Next one to five day old flies are placed into activity tubes, which are loaded in DAM activity monitors in order to monitor locomotor activity rhythms. Using the DAM system results are obtained that showed defects and variations in circadian and sleep parameters based on analysis of daily locomotor activity rhythms using software such as far sex and insomniac. This method can help answer key questions in the field of circadian biology, such as what proteins and regulatory mechanisms are involved in generating proper rhythms, and for example, how environmental factors such as light and temperature modulate behavioral rhythms through specific molecular pathways.
The locomotor activity monitoring system involves numerous pieces of equipment such as specialty monitoring devices, environmental incubators that have the capacity for diurnal light control, data collection devices, computers, and peripheral materials such as wiring choose a well ventilated temperature control, dark room to house the locomotor activity monitoring system to avoid excessive burden on the incubators and no possible failure to maintain temperature seal the room from external light sources inside the dark room. It is not necessary to work completely in the dark as the fruit flies. Circadian system is not sensitive to infrared light.
Therefore, in cases where we need to see in the dark room, we simply use a standard flashlight that is covered with a red filter purchase, an uninterrupted power supply emergency backup unit that has enough wattage capacity to power the components of the activity monitoring system. In case of a surge spike or power failure in the building, connect the UPS emergency Backup Unit to the emergency backup circuit of the building if available. If the system controlling the lighting in the incubator is not directly regulated by the incubator, then it is sufficient to plug the incubator into the emergency power without a UPS as loss of power for a few seconds will not affect the chamber temperature.
Next, set up a computer, PC or Macintosh fully dedicated for data collection and or for like control of the incubators. Manually arrange the telephone line network neatly around the shelving of the environmentally controlled incubators to allow ease of plugging and unplugging of activity. Monitors set up multiple telephone lines in a way such that they will converge into one main line and extend outta the incubator to connect to the computer, connect the monitoring devices inside the incubators to the computer via a power supply interface unit, which serves to power the activity monitor via the telephone line.
Finally, mask possible light sources from LEDs of electronic devices or an improperly sealed incubator door with duct tape or black cloth. To ensure free running rhythms are measured in the absence of unwanted light. It is crucial to assess these phenotypes using proper control animals that are reared in the same environmental conditions and of the same age.
In addition, there is sexual dimorphism in circadian rhythmicity. The general practice is to use adult male flies for these assays that are reared at 25 degrees Celsius and between one to five days old because egg laying activity in female flies will affect true measurement of locomotor activity. When examining circadian and sleep rest parameters of specific mutant flies of interest, it is prudent to outcross the mutant stock with the wild type strain of the same genetic background.
For example, W 1118 or yw. Since there is no crossing over in drosophila males, it is better to perform the outcross by crossing mutant females with wild type males seed both the wild type control and mutant flies at the same time. In standard drosophila food for about 10 to 14 days before the locomotor activity rhythm experiment upon aclusion of the progeny, collect one to five day old male flies and set them aside to be used for the experiments.
Activity tubes represent the fly habitat. During the experiment, they are thin five millimeter glass tubes that contain food substance at one end and are plugged with yarn or a plastic plug at the other end. It is preferable to use activity tubes that are freshly made.
They're generally prepared a few days to a week ahead of starting the experiment. Since glass activity tubes can be reused multiple times the following preparation procedures describe using previously used and unclean activity tubes as the starting point. If you are using new activity tubes, simply skip to the autoclaving of the tubes as shown later.
Remove the plugs from used activity tubes and put them into large glass beaker. The tube should only fill up to half the beaker. Fill the beaker with tap water, making sure to submerge the tubes.
Microwave the beaker filled with glass tubes until the water comes to a full rapid boil. To melt the wax and agar food, use caution as the water is hot. Remove the beaker from the microwave and stir the tubes with a spatula or plastic.
10 millimeter pipette to allow trapped wax to float to the top. Then repeat the microwaving step. Remove the beaker from the microwave and wait for it to cool down.
Putting the beaker in the cold room will speed up the process. As the water cools down, the wax will collect on the surface of the water and gradually solidify. Remove the solidified wax by hand.
This should get rid of most of the wax on the tubes. Transfer the activity tubes to a new beaker with fresh tap water and repeat the microwaving and wax removing process. Since most of the wax has been removed in the previous step, it is not necessary to wait for the wax to solidify.
Simply pour the water out of the beaker and transfer the tubes into a new beaker. Use caution since the water is still hot. Repeat the microwaving process for the last time.
Pour the water outta the beaker and wait for the activity tubes to cool down. Next, load the activity tubes vertically into 250 milliliter or 500 milliliter glass beakers. Make sure they're not too tightly packed.
Sterilize them by using an autoclave with a dry cycle or simply dry the tubes in a drying oven to prepare food to load into the activity tubes. Make a solution of 5%sucrose and 2%bact to agar in distilled or tap water autoclave the solution. The autoclave food can be used immediately or stored at four degrees Celsius for an extended period of time.
Ideally, the food should be around 65 degrees Celsius filling activity tubes. Use a 10 milliliter pipette to pipette the liquid food solution along the inside wall of the glass beaker, filling the tubes until they're one third full from the bottom up. Swell the beaker around gently to make sure that all the tubes are evenly filled with food solution.
Wait for the food to solidify completely either at room temperature or four degrees Celsius. Once condensation inside the glass tubes dissipates, the tubes can be removed from the beaker. To remove the activity tubes from the beaker, push the tubes towards the bottom of the beaker and twist the tubes at the same time so that the solidified food inside the tubes and the bottom of the beaker will separate.
Take the tubes outta the beaker, preferably as a single bunch. Next, clean the tubes one by one with paper towels to remove excess food on the outer surface of the tubes. Set the tubes aside in a clean container.
Take a general laboratory block heater without the tube holder and cover the heating well carefully. With several layers of strong aluminum foil, add paraffin pellets to the aluminum lined heating well to melt, hold the tubes at the non-food end and dip the food end into the melted wax. Dip the wax portion into a glass beaker filled with cold water to speed up wax solidification.Repeat.
Once dipping the wax tubes into water will prevent the tubes from sticking together. The tubes can be used right away or stored in an airtight container at four degrees Celsius for use. Within a week, prolonged storage will lead to excessive drying of the food.
If the tubes are stored at four degrees Celsius, warm them up to ambient temperature by leaving them on the benchtop prior to use. 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 paint brush, 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 Tri Kinetics 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 Zeke GABA times zero and all other times are relative to that value. Under standard 12 to 12 LD conditions while typed roso melano gasta typically exhibit two bouts of activity one centered around ZT zero termed morning peak, and another around ZT 12 termed evening peak free running locomotor activity Rhythms are measured under constant dark and temperature conditions are the intraining 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 file scan 1 0 2 x and summed into 15 and 30 minute bins when analyzing circadian parameters or one to five minute bins when analyzing sleep.
Rest parameters. Currently five contiguous minutes of inactivity is the standard definition of sleep rest in drosophila. Upon completion of this protocol, one can use the same data set to compare the circadian and sleep parameters of the experimental and control animals for circadian parameters.
Duction graphs illustrating daily locomotor activities or average activities of flies over several days in LD or DD conditions can be generated using fast x oph melanogaster generally exhibit two bouts of activity one centered around ZT zero termed morning peak, and another around ZT 12 termed evening peak. These two bouts of activities are controlled by the endogenous clock and can be observed even in free running DD conditions. Changes in the timing of these activity peaks can easily be observed in induction 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. This behavior is clearly observed in wild type flies, but is absent and arrhythmic per zero mutants. In this mutant.
The observed morning and evening peaks in LD are purely startle responses due to abrupt changes in light dark conditions. The 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 as seen in per zero flies.
In addition to induction graphs, locomotor activity data can be represented as a double plot gram for each individual fly or fly genotype 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. For example, LD one and two are plotted on the first line of the ACTO gram. The next line begins with a repeat of LD two and is followed by LD three and so on.
Following this format, the locomotor activity data spanning the entire experiment is illustrated in the ACTO gram one advantage of ACTO grams over reduction graphs. Is that a change in the period length of daily activity? Rhythms is easily observable besides generating induction graphs and ACTO grams.
Locomotor activity data from the DD condition can be submitted to far X to calculate the period length using a number of different programs, including cycle P for analysis of sleep, rest parameters, which is defined as five contiguous minutes of inactivity. One can analyze data recorded from the locomotor activity assays and examine multiple sleep parameters using insomniac, a MATLAB based program, the percent of time that flies spend sleeping can be calculated at different time intervals. For example, percent sleep every hour or every 12 hours.
Other more common sleep parameters that can be examined include mean rest bout length, which is a measure of how consolidated the sleep is and can illustrate the quality of sleep. Another is wake activity count, which 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 versus those that are 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 relative to control animals. After watching this video, you should have a good understanding of how to characterize circadian and sleep phenotypes in drosophila by assay. Daily rhythms of locomotor activity.
