Knowledge Base

Carrying Out Your Trials

Protocol

Animals

In many social interaction experiments, two animals from the same treatment group are tested together and scored as a single unit. According to Lapiz-Bluhm et al. (2008), this potentially can result in an artificial exaggeration of behavioral effects, that is, a treatment that only slightly impacts social behavior may have an apparently larger and more significant effect if the social behavior of both animals is affected. Their alternative approach tests an experimental animal paired with a 'neutral' stimulus animal, whose behavior is as constant as possible. See The Sociability Cage Test on page 107.

Habituation

The experimental animals are gently handled for 2 minutes on each of 3 days prior to the social interaction test. In addition, they are individually familiarized with the testing room and social interaction arena for 5 minutes on the 3 days prior to testing. The stimulus animals are habituated to the testing room and arena for 5 minutes per day for 3 days prior to testing. The stimulus animals are pre-exposed to the test arena in pairs, with a different, unfamiliar other stimulus animal on each pre-exposure day.

When Using Color Marks

To control for the effect that the color of the marker might have on the behavior of the other animal, it is recommended to randomly assign the marker color to stimulus and experimental animals. To take into account the combinations of animal type and marker color, you can either add a user-defined variable like Marker color to the Trial List or define two Detection Settings, one for each combination. In the latter case, make sure you set the appropriate Detection Setting as current before you start data acquisition.

Social Memory Test (1-Min Confrontation)

1. Let the focal animal establish a home-cage territory (e.g. 5 days).
2. Introduce the stimulus female into the home cage of a male mouse for a 1-min interaction. At the end of the 1-min trial, remove the stimulus animal.
3. Repeat step 2 a number of times, with a 10-min inter-exposure interval.
4. "Dishabituation" trial: Introduce a different stimulus female to the same male mouse for one minute.

In EthoVision XT

• If you use color markers, alternate different colors for the same subject (e.g. half resident mice marked orange, half marked green).
• If you use different colors for the resident animal, make different detection settings in such a way that Subject 1 is always the resident animal. Before acquisition, make sure that you use the detection settings that apply to that animal.
• Mark different types of trials with an independent variable in the Trial List. For example Type, with values C1, C2 ... for confrontation with the first stimulus female and D for the second stimulus female.

Scoring Behaviors Manually

If you want to score the subject's behavior manually, define them first under Manual Scoring Settings. After you have started the trial, watch the subjects in the video and in the Manual Scoring tab at the bottom of the screen, click the button for the category that applies. As soon as the behavior of the subject changes, click the button for the corresponding category.

For more information, see Acquire Data > Score behaviors manually in the EthoVision XT Help.

Source: EthoVision XT 18 - Application Manual, Noldus Information Technology