The classical Marris Water Maze experiment.
In this protocol, SMART v3.0 is adopted for the acquisition and analysis of the data. For detailed installation procedures and advanced configuration of the software, refer to its user manual.
1. Download the free trial version of the software from https://www.panlab.com/en/products/smart-video-tracking-software-panlab
2. Install the software to the Program Files folder
3. Install the codec in the same package for video compression/decompression
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 water maze 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 pool 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 water surface, i.e. make sure the brightness of the water surface 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 pool
5. Set the horizontal calibration value to the width of the pool
6. move the two green lines such that they delimit the vertical boundary of the pool
7. Set the vertical calibration value to the height of the pool
8. Accept the calibration setting
9. Open the Zones Editor
10. Adjust the size and position of the pool zone such that it circumscribes all the water surface in the pool
11. Adjust the size and position of the platform zone such that it circumscribes all the top surface of the platform
12. Export the Zone data to the ‘zone_files’ for future reference if necessary
13. Save the zone setting and close the Zones Editor window
14. Open the ‘Time Settings’
15. Choose the pre-set time mode
16. 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 pool zone. This can help to prevent the program from tracking the movement of the researcher’s hand when they place the mouse sample in the pool
17. Set a preferred duration for the acquisition time, after which the data acquisition would terminate automatically unless the stop condition is defined and satisfied before that
18. Set the stop condition base on the duration in which the mouse subject stays on the target zone (platform). The data acquisition would terminate once the mouse subject stays on the platform longer than the value defined in the stop condition
19. Open the ‘Detection Settings’
20. Click ‘Start Test’ to gauge the current detection performance
21. Click ‘Snap Shot’ to capture the empty pool (with platform inside but no 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 ‘pool’ zone
23. Place a trial mouse in the pool or simply waving hand (as a crude test) above the pool to check whether the program can detect and thereby chase the moving object in the ‘pool’ zone
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, for example, multiple ‘Periods’ can be built and named according to different experimental Day and multiple ‘Sessions’ can be built based on different
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
1. Open the ‘Data Acquistion’ tab
2. Click ‘Start’ to initiate the data acquisition process, which nonetheless would not be activated before the detection of any object movement inside the ‘pool’ zone
3. Click the video recording button at the bottom of the image window to start recording video for future reference as well
4. According to the experimental design, take out the first animal subject (consistent with the schedule) from its cage and release it near to the pool edge in the pre-assigned direction
5. The program would start to track the subject and acquire data after the pre-defined initial latency. Monitor the tracking process in the screen to ensure the propriety of the tracking and acquisition processes.
6. If the subject find the platform and stay there longer than the duration pre-defined in the ‘Stop Condition’, the tracking and data acquisition would be accomplished automatically, but the video recording needed to be stopped manually. Subject should then be allowed to stay on the platform for an extra 15 seconds for its memorization.
7. On the other hand, if the subject fail to find the platform before the pre-defined maximum acquisition time, the tracking and data acquisition would also terminate automatically and the video recording needed to be stopped manually. Yet, subject should then be guided toward the platform and guided to stay there for 15 seconds for training.
8. After the memorization or training, salvage the subject out of the pool
9. Wipe and dry the subject with towels and papers
10. Put back the subject to its cage and place the cage near the heater to recover the body temperature of the subject
11. Save the file just in case
12. 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
13. 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
1. Remove the impurity (including feces of the subjects) from the pool
2. the water in the pool, mixed with tempera or milk, needed to be replaced every other day. The following are the procedures.
3. connect the vacuum pump to the electric supply
4. connect the outlet of the vacuum pump to the sink via the hose
5. Place the vacuum pump at the bottom of the pool
6. Pumping out as much water as possible into the sink
7. There might be some residue remain in the pool. Heave the pool and pour the residue out into the sink if possible or wipe the residue with towels and papers
8. During the process, the position of the platform might be affected. If the camera position is not moved, the original platform position can be recovered by referring back to the saved zone definition in the screen
9. If the phone is removed from the clamp after the experiment, the camera position might be altered, which therefore necessitates the recalibration, refinement in zone definition, fine-tuning of detection setting in the next experiment session.
