The overall goal of this experiment is to record total sleep duration in a high throughput and non-invasive manner. This method could help answer key questions in the sleep field such as total sleep duration. The main advantage of this technique is that it is high throughput, non-invasive, and easily executable.
Demonstrating the procedure will be Abigail Lemons, a post-baccalaureate from the laboratory. To begin the procedure, align a detector opposite to an emitter. Make sure the infrared beams are facing inwards and are aligned at the same height.
Next, use the provided screws to position the detectors and emitters at the desired height on the metal stand. This height should be adjusted so that the bedding of the cage is below the level of the infrared beams, but the beam is at the proper height to detect the animals'activity. This creates an internal area of 27 centimeters by 32 centimeters.
Then, connect to each detector with each emitter. Connect the emitter to the provided hub linked to the receiver. Perform this for both the X and the Y planes.
Repeat this for all setups. Afterward, connect the receiver to a computer with the provided USB hub. To set up the software, click file, then open experiment configuration to open the default experiment configuration.
Then, click experiment, properties. Subsequently click the scan tab. Change the scan rate to 10 seconds.
Next, click the activity tab. Change the activity sampling rate to 10 seconds. Then, click file, save experiment configuration as, to save these configuration settings for future experiments.
In this step singly house the mice in clean cages that are 31 centimeters long and 16.5 centimeters wide. To prevent the mice from building up the bedding and obstructing the beams, use bedding at a depth of 3 millimeters, and do not provide additional nesting material. Provide the mice with access to food and water ad libitum by means of a wire feeder that rests on the top of the cage out of the way of the beams.
If necessary, refill the food and change the water bottles when cages are changed every three to five days throughout the duration of the study. Then, align the mouse cage inside the infrared beam setup ensuring that it rests in approximately the middle of the beans for full coverage. Now, open the default configuration file.
Click file, open experiment configuration, to open the desired or default experiment configuration. Next, click experiment, then setup. Designate the location to save the data file, as well as the location to save the backup file.
Then, designate the animal ID to each sleep chamber, and enter the weight of the animal if desired. If a chamber is not being used in the experiment, unchecking the box will deactivate that chamber. Once the identification information is entered, click done.
Click file, save experiment as, to save the current experiment configuration under the desired file name. Afterward, click experiment, run, to start the recording. Wait for a 10 second epoch to make sure that all chambers are picking up activity.
As the animals have just been injected, it is highly likely that the animals will be moving enough to be detected by the infrared beams. The following day, at the time of injection, click experiment, then stop, to end the recording. Afterward, click file, export, generate subject CSV's to collect the raw data for each mouse.
Subsequently, click file, export, and sleep analysis to export the sleep file for each experiment by opening the raw. CDTA data file. Under detection parameters activity source, ensure that both the X-axis and Y-axis boxes are checked.
Next, under sleep threshold epochs, ensure that four epochs are selected. Under sleep threshold activity threshold, ensure that 0 counts are selected, and under light-dark cycle, check the appropriate time for the light-dark cycle. Under analysis window, select the desired time for analysis.
In the case of the injection studies, leave the day as default. Set the start time as ExpSTART, and the duration as 24:00 for 24 hours. Save the configuration to save time for exporting data, Then, click update.
Click generate CSV file, and save the file to the desired location. To analyze the data for each recording session, open the sleep file. To assign the subject ID, open the individual CSV file for each chamber to determine the subject ID.Record the TOT sleep, hours, minutes, and seconds, for both the light and the dark phases for all animals.
Check the individual subject CSV file for inconsistencies indicating the failure of recording. If high beam counts are detected in one plane, but no counts are observed on the other plane, this indicates beam failure. This figure shows the mice received daily IP injections of either saline or 30%cyclodextrin at 9 a.m.
in the light phase. Boxes around the arrows indicate a cage change. Post hoc T-tests suggest that sleep duration differed in the light phase on day one from other days, indicating the habituation to both the sleep setup and the IP injections.
The sleep was reduced by cage changes on days six, nine, and 13, compared to other days. Sleep duration following cyclodextrin injections was relatively stable across the days when cages were not changed, indicating that the mice habituated to the IP cyclodextrin injections. Once mastered, setup can be performed in under an hour and analysis can also performed very rapidly.
Following this procedure, other methods such as EEG can be performed in order to answer additional questions like sleep fragmentation or sleep stages.
