Morris Water Maze - Working Memory Test

The Working Memory Morris Water Maze task places a greater demand on the memory processes of subjects, compared to the standard reference Morris maze procedure. If the subjects haven't already undergone Morris maze training, pre-training will be necessary

• The Morris maze will be set to accommodate eight platform positions. The water temperature will be maintained at 26 degrees Celsius. Prior to swimming, subjects will acclimate in the Morris maze room for 30 minutes. For this task, each session will consist of two trials, an acquisition and a test trial. The maximum duration of trials will be 60 seconds. Subjects will be tested on two sessions each day, one in the morning (around 9am) and one in the afternoon (around 3pm)

• On testing days 1-3, subjects will be tested with a 0 second Inter-Trial Interval (ITI). After twenty seconds post trial interval has elapsed, the subject will be removed from the platform and placed immediately back into the Morris maze tank at a pre-determined start position (see below).

• On testing days 4-6, subjects will be tested with a 60 second ITI following the acquisition trial. Once the subject has found the platform and 20 seconds have elapsed. The subject will be removed from the tank and put into a drying cage. As you are doing this, start the stop watch and let 1 minute elapse. Following the 1 min ITI, the subject will be removed from the drying cage and put back into the tank at a pre-determined start position.

Setting up files for the working memory Morris maze procedure

• The working memory version of the Morris maze procedure will consist of two sessions a day for a total of 6 days. Testing will commence at 9am and 3pm everyday (the interval is most important e.g. 6 hours and both sessions are in the day cycle). Each session will consist of two trails, an acquisition and a test trial. The first three days will consist of the 0 second ITI followed by three days of 60 sec ITI.

TXP file - General information

The general line format for working memory TXP files is the same as the standard Morris maze procedure

1. start position (6)

2.Platform Position (8)

3.Subject number (101)

4.day and trial number (d1t1)

Example: A line of txp text using the numbers specified above would be written like this:

6,8,1012,d1t1

• For working memory Morris maze, the platform positions are determined via a pseudorandom method. However, once a platform is selected from the randomization excel sheets on the black computer in room 10, that platform or the two adjacent platform cannon be selected again for two testing sessions.

• In order to assign start positions refer to the randomized excel sheet for WMW WM. You can find the excel sheet by going to desktop-> my documents-> study 012 Rons thesis->Morris Maze Data->Working Memory->Study012 WORKING MEMORY_Assignment.xls

Start options for platform positions

Platform	Start Options
1	7,4,10
2	9,12,6
3	11,2,8
4	12,3,9
5	1,4,10
6	2,5,11
7	4,7,1
8	6,3,9

Naming Files

Files should be named accordingly:

1.Day of testing (d1)

2.Squad number (s1)

3.Time of day (am or pm)

For example, if it the third day of testing for squad seven and you are making the afternoon files, the file name should look like this: D3s7pm. REMEMBER: File names cannot contain more than 8 characters. If your filename is more than 8 characters long, you will be making extra work for yourself or someone else later.

Both, 0 ITI and 60 sec ITI’s txps will look the same. In this form of working memory, there will be one squad per session. You will list all subjects in a row in one squad. They should look like this:

6,8,101,d1t1

9,8,101,d1t1

6,8,102,d1t1

9,8,102,d1t1

Create your files on the Microsoft notepad and save them to a floppy disk as text (.txt) files. Then you can transfer them to the HVS computer in the Morris maze room. To do this requires several simple steps:

1. Press power button on the left side of the HVS laptop in the Morris maze room.

2. Insert floppy disk

3. Double click on the My Computer icon on the desktop

4. Double click on the C Drive icon

5. Double click on the water2020 icon

6. Double click on the txp file icon

7. Now Double click on the floppy A icon

8. Drag and drop into the folder. Then exit both windows and you are ready to begin

Testing the subjects

1. Fill Morris maze tank to one inch above the platform with 26 degrees C. water. NOTE: Make sure that the red arrows located at 6 and 12 O’clock on the bottom of the external surface of the tank line up with the black arrows on the floor

2. While the tank is filling, turn on the computer and prepare for a test session

3. Click on the water 2020 icon on the desktop

4. Click on the run trial icon (looks like a white rat swimming in a blue pool).

5. On the next screen you will see six fields, enter the information as follows:

a. Field one enter: 001EXAM (no space between the numbers and letters here)

b. Field two enter: YOUR NAME

c. Field three enter: This is the filename field. Click on the Files icon on the lower left side of ::the screen. Select the appropriate text file from the left hand column of the screen. For example, if you are testing the 4th squad on the 3rd day on the morning session, the file name should look like this: d4s3am. Click Convert txt. You will see that your file has now has been moved to the central column on the screen under TXP files. Select the file you just converted. Once back at the previous screen the TXP file should be automatically added to the appropriate space.

d. Field four enter: Water

e. Field five enter: 60

f. Field six enter: 20

6. Then click Run. The computer will take you to the testing screen.

7. Plug in video camera and heat lamps. An image of the pool with the correct platform position outlined in blue should appear on the screen.

8. Measure four cups of Kid milk replacer into the Morris maze tank and stir with strainer till it is dissolved.

9. Bring the rats into the testing room. Mark their heads with black marker and make sure the correct subject numbers are printed legibly on the rats’ tail

10. Let the subjects acclimate to the testing room for thirty minutes (be sure to close the door). Place the “testing, do not disturb” sign on the door at this point.

11. Before testing make sure that: The hidden platform is in the correct position; you have removed the thermometer from the side of the pool; you have enough wyp-all towels to dry all of the subjects between trials; the stop watch is in the room AND functioning.

ZERO SECOND INTERVAL PROCEEDURE

1. Remove rat from holding cage with one hand while grasping the air clicker with the other

2. Bring the rat to the designated starting position and place it in the water. The rat’s head should be facing the wall of the tank. Release the rat and squeeze the air clicker to begin the trial. You should then quickly position yourself behind the white curtain and face the computer screen. A green crosshair should follow the rat’s movement at all times

3. When the rat finds the hidden platform press the air clicker again to end the trial so the rat can examine the visual stimuli in the room. At the conclusion of the 20 seconds, the computer will automatically set up for the next trial. (if the rat does not find the platform in the allotted time, guide the rat to the platform location and allow to sit on platform for 20 seconds.

4. After twenty seconds have elapsed, remove the rat from the platform while holding the air clicker in the other hand

5. Immediately take rat to the next starting position designated by the computer and place the rat in the water with its head facing the wall of the tank.

6. Release the rat and squeeze the clicker to begin the second trial. Then return to your designated area

7. When the rat finds the platform squeeze the clicker to end the trial. Grab a paper tower and remove the rat from the water. Dry off the rat and place it in one of the warming cages.

8. Use the strainer to remove all feces from pool and continue procedure with the next rat. Repeat this procedure until all of the rats have completed their trials.

60 Second ITI

1. Remove rat from holding cage with one hand while grasping the air clicker with the other

2. Bring the rat to the designated starting position and place it in the water. The rat’s head should be facing the wall of the tank. Release the rat and squeeze the air clicker to begin the trial. You should then quickly position yourself behind the white curtain and face the computer screen. A green crosshair should follow the rat’s movement at all times

3. When the rat finds the hidden platform press the air clicker again to end the trial so the rat can examine the visual stimuli in the room. At the conclusion of the 20 seconds, the computer will automatically set up for the next trial. (if the rat does not find the platform in the allotted time, guide the rat to the platform location and allow to sit on platform for 20 seconds).

4. After twenty seconds have elapsed, remove the rat from the platform and place in warming cage for 60 seconds

5. After the 60 seconds has elapsed, take the rat to the next starting position designated by the computer and place the rat in the water with its head facing the wall of the tank.

6. Release the rat and squeeze the clicker to begin the second trial. Then return to your designated area

7. When the rat finds the platform squeeze the clicker to end the trial. Grab a paper tower and remove the rat from the water. Dry off the rat and place it in one of the warming cages.

8. Use the strainer to remove and feces from pool and continue procedure with the next rat. Repeat this procedure until all of the rats have completed their trials.

After done with first squad for day 1

Create SUM Files

1. Click icon with green letters (right underneath the rat swimming icon)

2. Click advanced

3. Click series to summary

4. Click equi-area (under zones)

5. Counter size change to 3.0

6. Highlight first trial and last trial (i.e. d1t1 and d1t16 – squad of 4)

7. Click on summary name field and label this the same name as your txp file name

8. Click okay

Create CST Files

1. Click icon with red letters (underneath sum file icon)

2. Click on sum file that appears and hit custom

3. Then close

Saving backup HVS files

1. Exit out of program – last button on far right

2. Got to study 012 folder and within that folder, there should be subfolders for the different litters that have been tested. Go to the appropriate litter’s folder and create a subfolder labeled for each squad HVS.

3. Copy hvs data files from c:/wtr2020/hvsdata to the newly created hvs subfolder in study 012 folder.

• • HVS data files on c drive is overridden once you run your next squad you must pull these off immediately after your done with each squad.

NOW you are ready to run your next squad for the day

Room Cleaning Procedures

Rats that take part in swimming tests are sometimes prone to urinary tract and other infections. Therefore, it is crucial that we keep the water, the tank, and the testing room clean. In addition, a clean testing area promotes organization and quality research. The following is a list of procedures that should be completed every evening as well as several other items to be completed as noted.

1. Before draining tank, remove all animals from the testing room. That means, do not fire up pump while your last animals are drying in the heated cages. Let them dry for about 5 minutes before taking them to the cart in the hall.

2. Once the animals are on the cart in the hall, you may begin draining the tank. Place the pump in the tank near the twelve o’clock position. Next check the hose to be sure that there are no kinks in it and that the hose is secured between the faucet bracket on the sink. Doing this will help ensure that dirty tank water will not spill out of the sink and flood the tank room

3. Plug the pump into the outlet on the wall.

4. Return animals to colony room.

5. In a bucket add about a cup of alconox bleach to a half gallon of water. Use a sponge and/or scrubber to clean the walls of the tank. Make sure to remove all milk residue and ink spots

6. Continue to let the tank drain while you sweep any sani-chips and food pellets off the stainless steal shelves in the room. NOTE: Every two days the heated holding cages should be brought to the dishwasher. Sweep the floor everyday

Troubleshooting

Q. What should I do if I have the camera plugged in, but no picture shows up?

A. Check the connection to the laptop as that connection sometimes comes lose.

Q. What do I do if the circle where the platform is located is red instead of green?

A. You accidentally clicked on automatic stop. Exit the program restart it and there is an option on the initial squad set up screen

Q. What should I do if tracking isn’t working?

A. Again, exit the program, restart it and go to the squad set-up screen. Here you will find the “Set Detection” button. (For this you will need a practice animal as you don’t want to use an experimental animal for this as it will affect the data.) Once you press the detection button, you want to use a regular mask. Put the rat in the water at this point and click ‘freeze rat’. Next click near the rat and click the accept button. Now you should see a green crosshair on the rat. Next click confirm and let the rat swim around the tank to check if tracking is working well in the entire tank