All the procedures were approved by the National Institute of Mental Health Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guidelines on the Care and Use of Animals.

1. Setting up the Sleep Detection Units

1. Purchase the desired number of units and software.
2. Follow the instructions to set up the monitoring systems.
   1. Align a detector opposite to an emitter. Make sure the infrared beams are facing inwards and are aligned at the same height.
   2. 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 animal's activity. This creates an internal area of 27 cm × 32 cm (10.625 in × 12.75 in).
      1. If more screws are needed, purchase more at a hardware store (6-32 ¼ in Pan Head Screw).
3. Connect 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 step for all setups.
4. Connect the receiver to a computer with the provided USB hub.

2. Software Setup

1. Install the analysis software and select the provided hardware configuration file as the default. The hardware configuration file should only be changed by the company.
2. Click File | Open Experiment Configuration. Open the default experiment configuration.
3. Click Experiment | Properties.
4. Click the Scan tab. Change the Scan Rate to 10 s.
5. Click the Activity tab. Change the activity sampling rate to 10 s.
6. Click File | Save Experiment As to save these configuration settings for future experiments.

3. Animal Setup

1. Singly house the mice in clean cages that are 31 cm (12.25 in) long and 16.5 cm (6.5 in) wide. To prevent the mouse from building up the bedding and obstructing the beams, use bedding at a depth of 3 mm. Do not provide additional nesting material for the mice.
2. Provide the mice with access to food and water ad libitum by means of a wire feeder that rests in 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 3-5 days throughout the duration of the study.
3. Align the mouse cage inside the infrared beam set up, ensuring that it rests in approximately the middle of the beams for full coverage.
4. Ensure the light dark cycle of the room is set to mirror the light dark cycle of the normal housing conditions of the mice, or change as desired based on the experiment.

4. Drug Preparation and Injections

1. Obtain sterile water and sterile cyclodextrin.
2. For all injections, use a 1 mL syringe with a 25.5-30.5 G hypodermic needle. A new sterile needle and syringe should be used for each mouse.
3. To reduce the time between injections, assemble all syringes in advance.
4. For cyclodextrin injections: prepare cyclodextrin solution by adding 3 g of cyclodextrin to 10 mL of water to make a 30% cyclodextrin solution.
5. Administer 0.3 mL of either saline or cyclodextrin via IP injection.

5. Software to Record Sleep

1. Open the default hardware configuration file.
2. Click File | Open Experiment Configuration. Open the desired or default experiment configuration.
3. Click Experiment | Setup.
   1. Designate the location to save the data file as well as a location to save the backup file. The software requires that the data file be stored in the Program Files under the specific program folder.
      NOTE: Given that some computers view the Program Files as sensitive, access to the data file may not be possible after the experiment. Therefore, it is recommended to save the backup file to another, non-sensitive area on the local computer.
   2. 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.
4. Click File | Save Experiment As to save the current experiment configuration under the desired file name.
5. Click Experiment | Run or F5 to start the recording. Wait for a 10 s epoch to make sure that all chambers are picking up activity. As the animals were just injected, it is highly likely that the animals will be moving enough to be detected by the infrared beams.
   1. If no activity is detected, try to move the mouse cage manually to ensure the beams pick up the movement. Continue troubleshooting or move to a working chamber if the activity is still not recorded.
      NOTE: Run the experiment as soon as the injections are finished.
6. Cover the computer screens with an opaque cover to block the light. Cover the flashing lights on the activity meter.
7. Run the experiment for 24 h.
   NOTE: Because of the time necessary to perform the injections, it will not be a full 24 h. The injections should be performed at the same time daily. Sleep should not be recorded during injections.
8. The following day, at the time of injections, click Experiment | Stop to end the recording. Click File | Export | Generate Subject CSVs to collect the raw data for each mouse.
   NOTE: It may take some time to export, so do this immediately and then proceed to injections while the computer is processing.
9. Click File | Export | Sleep to export the sleep file for each experiment by opening the raw .CDTA data file on any computer that has the software installed.
   1. Under Detection Parameters Activity Source, ensure that both the x-axis and y-axis boxes are checked.
   2. Under Sleep Threshold Epochs, ensure that 4 epochs are selected.
   3. Under Sleep Threshold Activity Threshold, ensure that 0 counts are selected.
   4. Under Light/Dark Cycle, check the appropriate time for the light/dark cycle.
   5. 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. Set the Duration as 24:00 for 24 h. Analysis using the software can be done for a maximum of 72 h.
   6. Save the configuration to save time for exporting data.
   7. Click Update.
   8. Click Generate CSV File and save the file to the desired location.

6. Data Analysis

1. 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 hh:mm:ss for both the light and the dark phases for all animals.
   1. 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.
      NOTE: The "% sleeping" for the light and dark phases is calculated over the total recording period, which includes both light and dark phases. For 12 h light/dark cycles, multiply this number by 2 to get the "% sleeping" in the 12 h light or dark phase. Given that the injection studies did not go for a full 24 h, the calculated "% sleeping" is not accurate. To generate the correct "% sleeping", divide the "TOT sleep hh:mm:ss" by the "Experiment elapsed time: hh:mm:ss," multiply by 2, and then multiply by 100 to get the percent. If injections were performed in the light phase, then only the light phase needs to be adjusted in this manner.
2. Since different days and phases are being compared with each other, analyze the percent sleep duration for each phase and each day.