The classical Open Field experiment.
1. Fully charge the phone battery before the experiment
2. After entering the revenue, set up the lighting condition (avoid direct light source and adopt a softer indirect light source for the video recording to circumvent the interference from light reflection).
3. Separate the mice into individual cages with the corresponding label on the cage
4. Prepare a square box for open field experiment, with a dimension of 60X60 cm in our case
5. Avoid adopting highly light-reflective for the box used in open field to prevent heterogeneous light intensity
6. Avoid adopting materials which inclined to retain smell (e.g. cartoon or wooden) to avoid interference amidst different subjects via olfactory signal
1. Turn on the computer and the phone
2. Turn on the ‘do not disturb’ mode in the phone
3. Make sure both the phone and the computer are connected to the same WLAN or WIFI network
4. Download and install the “DroidCam” app from GooglePlay store if it is not yet done
5. Open the DroidCam app in the phone and obtain the id
6. Download and install the “DroidCam Client” app from “https://www.dev47apps.com/droidcam/windows/” in the computer if it is not yet done
7. Open the DroidCam Client app in the computer and enter the corresponding id of the phone to connect the computer video input with the phone camera
8. Adjust the parameters of the phone camera (e.g. white balance, resolution) if necessary
9. In the computer, if necessary, build a folder for the experiment and build five subfolders in the experiment folder, which are ‘experiments_files’, ‘report_files’, ‘schedule_files’, ‘video_files’ and ‘zone_files’ respectively.
10. Open the SMART v3.0 software in the computer and choose an existing file (or new file with open field protocol) to work with
11. In the ‘Configuration’ tab, open the ‘Path Settings’ and set the path of various files to the corresponding path built in Step 17 if necessary.
12. In the Experiment Assistant tab, open the setting of the image source and choose DroidCam Source 2 or 3
13. Mount the phone on the phone clamp and mount the clamp on the holder scaffold
1. according to the view in the SMART Player window, adjust the position of the phone camera such that the entire field area is just included in the view
2. Adjust the position and intensity of the light source such there is no significant light reflection on the wall and at the bottom, i.e. make sure the brightness inside the box is homogeneous
3. In the calibration setting, set the calibration units to ‘Centimeters‘
4. move the two red lines such that they delimit the horizontal boundary of the field area
5. Set the horizontal calibration value to the width of the field area
6. move the two green lines such that they delimit the vertical boundary of the field area
7. Set the vertical calibration value to the height of the field area
8. Accept the calibration setting
9. Open the Zones Editor
10. Choose a preferred built-in zone definition for open field, in our experiment, the canonical ‘periphery/center’ zone definition is adopted
11. Adjust the size and position of the border of the outermost zone such that it circumscribes all the field area
12. Adjust the size and position of the inner zone (e.g. the center zone) according to the experimental design
13. Export the Zone data to the ‘zone_files’ for future reference if necessary
14. Save the zone setting and close the Zones Editor window
15. Open the ‘Time Settings’
16. Choose the pre-set time mode
17. Set a preferred duration for the latency, a timespan in which the program will wait and suspend the initiation of data acquisition after the first object movement is detected in the field. This can help to prevent the program from tracking the movement of the researcher’s hand when they place the mouse sample in the field
18. Set a preferred duration for the acquisition time, after which the data acquisition would terminate automatically
19. Open the ‘Detection Settings’
20. Click ‘Start Test’ to gauge the current detection performance
21. Click ‘Snap Shot’ to capture the empty field (without mouse subject) as the background reference for the detection process
22. After that, the image window, which is still in the ‘Start Test’ mode should exhibit a stark blank inside the outermost zone
23. Place a trial mouse in the field or simply waving hand (as a crude test) above the field to check whether the program can detect and thereby chase the moving object in the field
24. If the detection is still not satisfactory, try to fine-tune other detection parameters, ranging from threshold to erosion, from brightness to contrast, to optimize the detection performance
25. ‘Activity Detection’ can also be included to acquire extra data regarding the activity of the subject
26. Save the detection setting
27. Open the ‘Subject’ tab
28. Delete the default subject and create the subject list according to the experimental design
29. Export the subject list to the ‘schedule_files’ for future reference if necessary
30. Open the ‘Schedule’ tab
31. Delete the default trial and create the schedule according to the experimental design
32. drag and drop the corresponding subjects into experimental ‘session’ to create ‘trial’
33. Select the first subject of the experiment and click the green tick in the top menu bar to assign it as the designed subject in the ensuing data acquisition process
34. Export the schedule setting to the ‘schedule_files’ for future reference if necessary
35. In the ‘Configuration’ tab, open the ‘Event’ window, assign each behavior of interest needed to be evaluated, ranging from autoglooming to rearing, to a specific number key, and input the label of the behavior in the corresponding text area. After that, close the ‘Event’ window.
1. In the ‘View’ tab, open the ‘Event Marker’ window
2. Open the ‘Data Acquistion’ tab
3. Click ‘Start’ to initiate the data acquisition process, which nonetheless would not be activated before the detection of any object movement inside the field
4. Click the video recording button at the bottom of the image window to start recording video for future reference as well
5. Take out the first animal subject (consistent with the schedule) from its cage and release it right at the middle of the field (be cautious not to drop the subject from high altitude and also heed the handed phone meanwhile in case of any movement of the camera)
6. The program would start to track the subject and acquire data after the pre-defined initial latency. Monitor the tracking process on the screen to ensure the propriety of the tracking and acquisition processes.
7. During the data acquisition period, carefully observe the behavior of the subject, and press the corresponding key in the number keypad whenever a behavior of interest is exhibited by the subject to record the instance of that behavior. Hold the key until the termination of the behavioral exhibition to record its duration.
8. After the pre-defined acquisition duration, the tracking and data acquisition would also terminate automatically but the video recording needed to be stopped manually.
9. Save the file just in case
10. Salvage the subject out of the field (again, be mindful about the handed phone)
11. Count and record the number of feces defecated by the subject
12. Clean the feces and spray the entire field with ethanol and wait for it to evaporate
13. After the termination of data acquisition, the designated subject for the next data acquisition process would automatically change to the next ensuing subject in the schedule. If necessary, the schedule can be modified and the designated subject can also be reassigned with the aforementioned green tick in Step 33 in the Software Configuration session
14. Repeat Step 2-10 for the next subject
1. After accomplishing all the trials, close all the sub-window in SMART 3.0 and open the ‘Analysis’ tap
2. In the analysis window, select all the trial results from the left sidebar.
3. Drag and drop the results in the first row of the table in the middle, which should tabulate all the results
4. Click ‘Analyze’ to start the analysis with the default water maze summary report
5. Select and open all the available reports the analysis
6. Export all the reports in excel file to a folder (e.g. report_files)
7. Export some representative trajectory image if necessary
8. Further analyze these raw data with the appropriate statistical test
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