EthoVision Set-Up:

1. Experiment Settings
a. Use either Center Point Detection or Color Marker Tracking
i. The center point is best in MWM because will detect center of animal.
Look in manual for information on marker tracking
2. Trial List
a. Look at list of possible variable choices under Show Variables
i. We used start time, duration, and three user defined: subject ID,
placement, and whether or not the platform was in the maze.
b. Add desired number or trials and fill in the variables for each
3. Arena Settings
a. Camera must be connected
b. Grab image from camera of the “arena”, which in the MWM is the circular tank
c. Define the desired “zone groups”
i. At the top of the tool bar, you will see various shapes that you may use
as a zone group
ii. We used the circle to define the tank as the arena
iii. We then added a zone group to be the quadrants using lines to divide
the tank into four regions
iv. We added another group as an approximate 6 cm area around the
platform
v. Then we added the platform zone group.
vi. Then you must add a calibration.
A. As shown below it needs to be as big as possible. We had
trouble acquiring data when we just calibrated to the 12 cm
platform. We now calibrate using the 109 cm tank.
vii. Then click Validate arena settings
viii. Then use whatever colors you would like. We used bright colors and
made the background color black.
4. Trial Control Settings
a. This section depends entirely on your protocol.
b. We added a condition for the camera to turn on after the subject was in the
arena for 1 second.
i. We but it between rule begin and action
c. Then we had two conditions for stopping the trial:
1. After a delay of 60 seconds
2. After the subject has been on the platform for a duration of 2 seconds.
d. We placed both of these condition boxes between the action (start track) and
an operator that you add
e. You must add the operator due to the two conditions for the stop track
f. The stop track was next followed by Rule End
g. You simply drag the mouse from one box to the next so that there is a line
between them all. With the two conditions, you must make sure both are
connected to the Action and Operator by a line; however, they do not have a line
joining them.
5. Detection Settings:
a. See below in the helpful hints and information for further information
b. The settings we used are below.
6. Track Smoothing Profile
a. Not used
7. Data Profiles
8. Analysis Profile
a. This is where you tell the system what information you want it to calculate
b. You choose from the list on the left hand side of the page and just click which
ones you want.
c. Make sure you have already defined your arena so that it will have the different
zone groups in the system
i. We defined variables such as the duration of time spent in each
quadrant. If you have the zone groups already in the system it will show a
list of zone groups when you click on the variable (such as the variables
under Location)
9. Acquired Trials
a. This tab is important during acquisition
b. The number in parenthesis tells you how many trials the system has recorded.
ALWAYS check the number because the system can look as if it is recording but
it is really not. (believe me)

Acquisition:

• To start a trial:
A. Click Acquisition at the top and click open acquisition
B. There should be a box that pops up with a circle that you push to start the
trial. The camera will not track until the subject is in the arena.
C. If there is not a box, which happened to me a lot. Hit Ctrl + F5 and it will start
the trial.
• During the trials there is a box at the bottom left. Pay close attention to it.
1. The box has a “Missed Sample” and “Subject Not Found”
2. These are REALLY important
3. If the calculated percentage exceeds 10% the font will go red. This is
VERY bad. The data being recorded is not usable in any way. To fix this
problem see notes below dealing with detection settings.

Data Analysis:

1. Visualize:
• If you click Visualize then plot tracks, you will see the track file from the trials
acquired.
• You can watch it in real time, sped up, or just look at the finished file. THE
SOFTWARE DOES NOT RECORD AND SAVE THE TRIALS! It just records the
samples taken as red dots.
 • At the bottom right, you will see Missing Sample Color. The system shows the
track as red dots connected with faint lines. Anytime the system misses a
sample, it will have a colored line in between the red dots. I chose a bright color
so I can easily see the number of missed samples.
2. Analyze:
• Click Calculate Statistics
o This are the ones you previously defined
• It will show you the data collected as a table
3. Export:
o Always export track data
o Export Analysis Output
o It will put all data collected into an excel file

Helpful Hints and Information:

Calibration:
• Make the calibration as big as possible
• You should expand the calibration to go from one end of the tank to the other. Not
just the size of the platform. This will ensure better stats once you clear up the
missing samples issues.

Visualize data:
• In addition to exporting the raw track data into Excel for inspection of missed
samples, you can also get an idea of your missed samples by visualizing your
tracks (Visualize menu/Plot Tracks).

Verify:
• Always verify your settings and data before conducting a full scale experiment.
This is done by confirming tracking is good/perfect via stats, exporting raw data
and visualizing the data.

Detection Settings:
• You should not use Differencing at all as that is for black and white rats which
cannot be tracked with any of the other detection methods. So it is a last resort
method if you will.
• You should first try gray scaling. This assumes your lighting is even and minimal
shadows or reflections. If it doesn’t work consistently (to get 10% or less missed
samples), then you move up to Static Subtraction. If you cannot get Static
Subtraction to work then you try Dynamic Subtraction. One of these 3 methods
will work for you. It may take some time tinkering with things. Again the goal is to
get less than 10% of missed samples. Only then are your detection settings
good/correct.
• A high sample rate can cause the PC to overload, so overall it is best to only
what you need. Since you are tracking 1 animal and you are only tracking its
center point, there is no need to use 30 samples per second (the max for that
setting). I recommend 5 or 10 samples (the max I would use) per second.
 • If you still have too many missed samples after making these changes then you
need to work on your range/contrast setting of the animal and dial that in tighter.
Not sure if you need to erode and dilate as these are ‘usually’ need for a more
challenging tracking setup (water maze is fairly standard).

Training:
• If after doing the above to your detection settings you still cannot find optimal
settings, then I suggest you purchase a training video.

EthoVision Support:
• They are very helpful and friendly. (They probably are tired of hearing from me)
• The email is support@noldus.com
• The telephone number is 1-866-860-3580
• My number is 662-316-0991, Brittany Simpson if you need anything else

EthoVision Troubleshooting (simplified):

1. Export track data to excel and should see only 10% missing
2. Make calibration to be entire diameter of the pool
3. Need to run a trial with practice mouse to ensure that the system is working with
<10% of missing data by running trial and exporting tracks and data and visualizing
(only 1% missing is optimal).
4. Change the detection settings to gray scale
-if this does not work use Static Subtraction
-if not used dynamic subtraction
5. Change the sample rat to 5 or 10 samples per second
6. If you still have too many missed samples after making these changes then you need
to work on your range/contrast setting of the animal and dial that in tighter. Not sure if
you need to erode and dilate as these are usually need for a more challenging tracking
setup (water maze is fairly standard).
7. If the timing error is given again do not keep using the PC
-reboot and turn off the camera
-If you get the error again after making these changes, please reboot the
PC and shut down the camera like you have been and let me know so I
can inform R&D. Do NOT continue to collect data while getting that error
as those track files will have to be analyzed externally to correct for the
timing and that is a huge hassle that I do not recommend.

EthoVision Settings for MWM:

1. Arena Settings:
-change the calibration to be the entire diameter of the pool (twice)
-the pool is 109 cm
2. Detection Settings:
a. Change to Gray Scaling
b. Make the range go from 36 to 142
c. Change the minimum and maximum range to 133 - 1980
d. Change the sample rate to 5.0050
e. Video Settings:
I.Video Proc Amp:
-Contrast - 731
-Brightness - 65
-Gamma - 20
II.Camera Control:
-exposure =-5
II. Video Control:
-Exposure - check and second number is 0.026565
IV. Auto Exposure
-800