Knowledge Base

Analyze DanioVision Data

Analysis Profiles and Options

Analysis Profiles in the Template Experiment

• Distance, Time & Movement - Contains the dependent variables Distance moved, Velocity and Movement.
• Path Shape - Contains the dependent variables Heading, Turn angle, Angular velocity and Meander.
• Rotation - Contains two instances of the dependent variable Rotation, to count the number of clockwise and counterclockwise rotations.

For more information on these and other dependent variables, see Dependent Variables in Detail in the EthoVision XT Help. Also see the chapter Analysis of Trial Control data in the EthoVision XT 18 - Trial and Hardware Control - Reference Manual.

Analyze Intervals

Make sure you have defined a Data profile which specifies the intervals you are interested in. Next, in the Analysis profile choose your variables (e.g. Distance moved). Then visualize the data and run analysis.

Activity Analysis

You can also use the dependent variable Activity state. This variable is only available when you selected Activity analysis in the Experiment Settings.

With Activity state you can calculate how long and how frequent your subject has been in different activity states. These states depend on the total pixel change within the arena between a sample and the previous sample. It is important that all defined arenas have exactly the same size. The number of states (two to four) and their thresholds are user-defined.

Analyze Convulsions

To analyze convulsions, you can use several variables, such as Velocity, Movement, Activity and Mobility.

Method 1 - With Movement

In this example, seizures are frequently characterized by fast zig-zag movements that last up to two seconds. To discriminate between seizure and no seizure, we use Movement, a variable based on the current (smoothed) velocity of the fish.

1. Create an instance of Movement that is active when the velocity exceeds a specific threshold value. To get an idea of what the threshold should be, plot the values of velocity with an averaging interval of 5. Play the video until a seizure occurs in one of the wells, and look at the values of velocity for that subject. In this example, velocity is higher than 15 mm/s. Check this in other wells and in a few videos and try to find an optimal cut-off value.
2. Create a Free interval that has the following settings:
   • Start Criterion: Dependent Variable; Select Variable: Movement. Set Movement with a Start velocity threshold around 1.5 cm/s and a Stop velocity threshold a bit lower (say 1.3 cm/s). Select Statistic: Current Duration of Moving and choose a value like >= 0.5 s. This way you filter out short swim bursts that are less likely to be seizures.
   • Stop Criterion: same as above, with the difference that you select Current Duration of Not Moving >= 0.5 s. The free interval ends when the fish has been Not Moving for half a second.
3. The resulting free interval captures the period of time when the velocity of the subject is higher than the threshold value.

Tip: To exclude sudden changes in velocity in the middle of a seizure, use an Averaging interval when defining Movement.

4. Calculate the necessary statistics, like the average duration of the seizures, the number of occurrences, etc.

Method 2 - With Mobility

Create a Mobility variable in your Analysis profile. Start with setting a High Mobility Threshold to a high value like 95%, and an averaging interval of 2. Similar to Movement, Mobility should only capture the periods of time when the subject is very active because of the seizure, excluding normal swimming activity. Compare the scores of Highly Mobile with those of convulsions obtained manually. Adjust the mobility thresholds until they match.

Heatmaps

To create a heatmap of the subjects' position, choose Analysis > Results > Plot Heatmaps. Choose the Data profile from the list on the toolbar to create a heatmap based on the data selected.

Notes:

• There is one scale for all the wells in the well plate. You cannot create separate scales for each well, unless you select single wells in the Data profiles (one well for each Results box).
• For DanioVision experiments, the Group Means button is not available.
• For more information on heatmaps, open the EthoVision XT Help and browse to Visualize data > Plot heatmaps.

Calculate the Statistics

Procedure

1. Choose Analysis > Analysis Profile > New.
2. Add the dependent variables of your interest. For an overview, see Dependent Variables in Detail in the EthoVision XT Help.
3. In the Trial Statistics tab, choose the statistics you want to calculate per trial for that variable. If you created groups of tracks in the Data profile, select additional statistics in the Group Statistics tab.
4. Repeat steps 2-3 to add more dependent variables.
5. Choose Analysis > Results > Statistics & Charts. Choose the Data Profile from the list on the toolbar and click the Calculate button.
6. The results appear on the screen. The Trial Statistics tab shows the statistics per trial. The Group Statistics & Charts tab shows the statistics and charts from the summarized results of all trials. See Calculate Statistics in the EthoVision XT Help for details.
7. Optional: Click the Layout button and change the layout of the results table.

Batch Calculations

It is also possible to carry out multiple analyses with different filters, data profiles and analysis profiles. To do so, click the Batch button. Make your selection from the lists under Select the profile combinations to calculate and click Add. When done, click Calculate. To view a specific analysis result, select the options from the lists on the toolbar. For more information on batch statistics calculation, see Batch statistics calculation in the EthoVision XT Help.

Source: DanioVision DVOC-0050 Reference Manual - Analyze Data, Noldus Information Technology