Novel object recognition test

Novel object recognition is a highly validated test for recognition memory. It can be used to test the efficacy of memory enhancing compounds, the (negative) effects of certain other compounds on memory, the influence of genetics or age on memory, etc.

The basic idea is straightforward: the rat or mouse is exposed to two or more objects and gets to explore these for a while. Then one of the objects is replaced by another one. If memory is functioning normally, the rat or mouse will spent more time exploring this novel object than it does exploring the familiar object(s). If exploration of all objects is the same, this can be interpreted as a memory deficit. 

EthoVision XT and the novel object recognition test

Objective research

Though it seems like a straight-forward task, observing these animals and assessing objectively how much interest the test subjects actually show in the objects isn’t that easy. Manual observation is labor-intensive, and prone to both inter- and intra-observer variability. Automatic video tracking provides a solution. 

EthoVision XT is the best software to automate your novel object recognition test. It tracks the behavior of your animal, just as accurately, if not better, as a highly-trained human observer. (Cyrenne and Brown, 2011 and Benice and Raber, 2008). It does not matter which kind of arena you choose, whether you are using a straight-forward open field arena , a PhenoTyper home cage, or even a Y-maze or T-maze .  

Tracking multiple body points

Since exploration of the objects is the focus behavior in these types of studies, accurate assessment of this specific behavior – exploration of the object – is crucial. It is not simply a matter of the animal being close to the object; there is a big difference between the animals nose being close to the object, and the animal tail being close to it. Only the first can be interpreted as interest in or exploration of the object. Therefore, automatic video tracking is only useful if these body points can be accurately distinguished by the software. 

The EthoVision XT Multiple Body Points Module is able to detect and track the center point, nose point, and tail base of mice and rats. This allows for the accurate measurement of an animal’s orientation relative to certain objects in the arena or the arena itself. It also provides information about heading and rotational movement.

ethovision multiple body points
EthoVision XT detects the nose point, center point, and tail base of your animal.
ethovision xt video tracking novel object recognition
Multiple body points tracking allows for an accurate assessment of object interest. 

Exploration of objects

Of course you need your software to accurately detect animal location, movement, orientation, and behavior. You also need this data to relate to the novel and familiar objects. EthoVision XT allows you to identify these objects by drawing ‘zones’ around the object in your video image.  Exploration of the object is usually defined as the animal’s nose being in the zone of with a certain radius around the object. In that case it is practical to draw a zone with the same shape as the object, but slightly bigger depending on the radius you want to use in your study.  Of course, other methods, such as excluding the center part of the object (creating a donut shape) is also possibly, for example if you want to exclude data points in which the animal is climbing on top of the object (see Werkheiser et al., 2011).

When zones are defined, EthoVision XT automatically regi sters when the animal enters this zone, and which body points are in that zone. Parameters of interest, such as time spent, are coupled to these zones. You can also filter out data points in which the animal has both its nose and body center in the zone (e.g. indicating the animal is climbing on the object).

Another way of differentiating between interest in the object and simply passing by it is to combine the data of distance to the object and orientation towards to object. 

Automatic detection of behaviors 

Multiple Body Points detection and zone definition combined makes it possible for EthoVision XT to automatically detect the behavior of interest, in this case exploration of the object. But there are more advantages to using Multiple Body Points detection. It allows you to investigate body elongation, which indicates the stretch attend posture, giving insight into exploration and curiosity behavior. The thresholds for elongation are by default set to what is generally used in research, but since every study is unique, you can adapt this threshold yourself. This way the actual interpretation of behavior is done by researchers.  

Test set-up

The novel object recognition test can be performed in basically any arena, and as long as you can put a camera overhead to monitor the animal, you can use it in combination with EthoVision XT. Most studies use a circular or rectangular open field set-up, some choose something slightly different, such as a Y-maze (Chambon et al., 2011) or an open field with dividers (Werkheiser et al., 2011), so that object recognition is less dependent on spatial information or place preference and the animal cannot see two objects at the same time. 

In combination with EthoVision XT,  Noldus offers several types of mazes, which can be backlit with Near Infrared light. In fact, we can offer you a complete set-up, including a computer, camera, and all the other necessary hardware. Getting a complete solution from Noldus has the advantage that everything is set-up and calibrated to work together. 

Testing procedure

To get your research up and running quickly, EthoVision XT includes predefined settings (a template) for the novel object recognition test, and EthoVision XT will guide you through your research by offering help in every stage of your project, from setting up a trial list, to analyzing your data. 

For each experiment you are running, you can create a project in EthoVision XT. The Trial List function allows you to make a list of all the trials you are running, and couple independent variables for each one, such as treatment group, animal age, etc. These independent variables can be used for the selection and analysis of data later on. 

You can also connect video files to each trial. This is especially practical when you don’t have the opportunity to track live or process the videos immediately. Simply connect the video files to the matching trials in the trial list, and you can perform a batch acquisition: all the video files will be processed sequentially.  

After you fine-tune the detection settings to your research, and defined you arena (with zones of interest) you are ready to start your experiment. After habituation to the testing arena, animals are exposed to the objects.  Next, one of the objects is replaced by another, novel object. The retention time between these trials can differ, depending on which type of memory is investigated (short-term versus long-term).

A variation to this test is the novel placement test. In this case there are no novel objects, but one of the objects is moved. While novel object recognition is believed to be independent of hippocampal functioning, location recognition is not.  

Results

Obviously, the most important results include the exploration times of the objects. These are generally expressed as percentage of exploration of novel object, the preference for novelty, a ratio between familiar and novel object exploration times, or similar. In addition activity (measured as time spent moving or distance moved) is assessed to determine if less exploration can be caused by a general decrease in activity, due to the treatment of the animals. 

Examples of parameters include:

  • Time spent with only nose point in object zone 
  • Time spent with nose point in proximity of the object
  • Percentage of body elongation
  • The orientation of the animal relative to an object (direction of nose point) 

Read more

EthoVision XT is well validated in research using the novel object recognition test. See the list of publications