A circadian rhythm is any biological process that displays an endogenous, entrainable oscillation of about 24 hours.
These 24-hour rhythms are driven by a circadian clock. Circadian rhythms of living animals may be measured by recording their locomotor activity over time, and they are normally entrained by light intensity. Zantiks units can be used to measure the activity of animals, and also control (and change) the lighting regime. In this protocol we provide an example of how a circadian rhythm experiment with adult Drosophila could be performed in the Zantiks MWP system. This example protocol can easily be adapted to any users individual experimental needs.
Experimental set up
A multi-well plate is prepared, this can be a 24- 48- or 96- well plate depending on your experimental needs. Animals should be individually placed in each well.
- For adult Drosophila, a mixture of agar, sugar and nipigin (see reference below) is added in the base of each well. An air permeable cover is put over the top of the well plate once flies are placed inside. These allow the flies a humid environment and some available nutrition.
- Aquatic animals should be placed in each well with culture/home tank water. Use of the well plate lid is recommended to help reduce evaporation of water from the wells.
The multi-well plate is then placed onto the light stimulation plate and inserted into the chamber of the Zantiks MWP system.
Experimental procedure
The internal environment of the Zantiks MWP system, including temperature and light regime, can be fully automated and controlled in Zanscript along with the measurement of distance travelled by each individual animal providing continuous data over time.
For Drosophila: the experiment typically lasts for 6 days - during the first three days, the well plate might be exposed to 12 hours light, followed by 12 hours dark, and then during the subsequent 3 days the plate could be exposed to continuous dark.
The activity of the animals is measured as distance travelled, and is written to a data file in a time bin defined by the experimenter (e.g., 1 sec or 5 min bins). In this example protocol we will be using a 1 second time bin for data acquisition.
Results / data output
Video of Drosophila in a 96-well plate during a circadian rhythm study. Each well has been part filled with agar containing sugar and nipigin (hence the differences in background colour). This provides food and humidity for the flies for long term studies (days-weeks).
Protocol script downloads
There is one script for the circadian rhythm protocol. Ensure the correct tracking settings are adjusted to those suitable for your model organism. Adjust the headings for the data output and ensure the correct asset is loaded for your well-plate of choice. Please ensure that you understand how the timings work for this script before making any edits. Carefully read all of the annotations on this script to help you understand how the timings work. If you are unsure about editing this script, please contact us.
Assets
The asset used will depend on the well plate size chosen.
You will need to upload the asset into the Asset directory on your Zantiks Control Console & ensure the correct asset name is in the LOAD(ARENAS,"name_of_asset") command in the script.
See the Calibrating your Zantiks unit page and Asset building in the MWP page for details on how to create assets customised to your system.
