This is Part 1 of a two-part guide, which will explain how to set up a standard Novel Object Recognition (NOR) experiment in ANY-maze. In this first part, I will explain how to draw an apparatus map to include your objects, how to use moveable zones to counterbalance your objects and how to view the results of your tests.
In Part 2, I will cover more advanced ways of automating your experiment, such as how to automatically assign the novel object’s shape and position between animals, and how to calculate the object discrimination index.
To walk you through this setup, I’ll use a standard novel object paradigm conducted in 2 parts:
Familiarisation: Two identical objects (either two spheres or two cubes) are placed in the arena, and the animal is allowed to investigate for 5 minutes.
Testing: The following day, one of the objects is replaced with a novel object of a different shape to the familiar object, and the animal is allowed to investigate for 5 minutes. The position and shape of the novel object is counterbalanced between treatment groups to control for bias (i.e. the novel object will be either a sphere or a cube, placed either on the left or the right).
Now that we understand the paradigm, let’s start by creating a new experiment, which you can do by selecting New experiment on the File page.
Drawing the apparatus map
First we need to build the Protocol, which we’ll do using the Add item button in the ribbon bar. Let’s start by adding a Video source. Here you will select the camera or video that shows your NOR apparatus.
Then you’ll need to add an Apparatus which corresponds to your video source. It is here that you’ll draw the apparatus map, which defines both the outline of the arena and any important areas within it – in this case, the objects. As we will be using two different shaped objects in two different positions, we will need to include both shapes in the map, on both sides – see the figure below.
Note that you should draw tightly around the objects, as you’ll be adding an investigation perimeter to them in the next step.
Adding investigation zones
With the apparatus map drawn, you will then add three Zones for your objects. We’ll call them Identical objects, Novel object, and Familiar object. And because you’re interested in measuring the animal’s investigative behaviour of these objects, you’ll want to choose that these are Investigation zones.
Unlike a Standard zone, which an animal is meant to go into with his body, an Investigation zone is used to measure the animal’s exploration around an area, using his head. So, you will need to specify the distance from the object of this perimeter of exploration (the dotted line in the picture below) – the animal’s head must enter this perimeter to be counted as investigating the object.
You can also specify whether the animal must be oriented toward the object to be considered investigating it (in the image above, the cone shape protruding from the animal’s head represents the animal’s orientation). For instance, you may not consider the animal to be investigating the object if he happens to walk next to it but doesn’t actually inspect it. You can also change the angle of this orientation cone under the Orientation and movement item in the Protocol list.
One other consideration is whether you want ANY-maze to ignore instances when the centre of the animal is inside the zone (i.e., inside the solid line), in general, this means the animal is standing on top of the object.
Making the zones moveable
At this point you have your zones created for your objects, but how do we tell ANY-maze that the Identical objects zone will be two spheres for some animals and two cubes for others, or that the Novel object zone can be on the left for some animals and on the right for others? To set this up, you will make your zones into moveable zones.
From ANY-maze’s perspective, a zone that is moveable is the same zone at the end of the day, regardless of its shape or position. This means that when you go to analyse your results for investigation of the Novel object, you don’t need to consider that the novel object was the sphere on the left for one animal and the cube on right for another; ANY-maze will cope with this for you and simply display the results for the Novel object zone.
Identical objects zone
To make your zones moveable, select one of your zones (we’ll start with the Identical objects zone) and expand the drop-down menu where it says Position of the zone remains the same in all tests. Change this to The position varies within (and possibly between) the animals. This is telling ANY-maze that the objects can assume more than one position – thus, moveable zones.
Now you need to tell ANY-maze exactly where the objects can move to. Using the Add item button in the ribbon bar, let’s add a New zone position. The first position name will be Spheres. Then in the video picture, click inside both circles on your apparatus map – see figure below.
Add a second zone position for the Identical objects zone and call it Cubes. Then, click inside both squares on your apparatus map (to do this, you’ll need to click inside the circle as well as all 4 corners). In the figure below, you’ll notice the dotted line is now a square shape with rounded corners.
Novel object zone
That’s the Identical objects zone done. Now let’s move on to the Novel object zone. In the drop-down menu, select Position varies within (and possible between) the animals (the same as before), then add a new zone position for each of the four possibilities: Left sphere, Right sphere, Left cube, and Right cube. For the Left sphere position, simply select the circle on the left in the video picture, as shown below. Add the other three zone positions in the same manner.
Familiar object zone
The Familiar object will always be the opposite shape and on the opposite side than the Novel object, so we can select that the Position depends on the position of the Novel object zone.
Let’s then add a new zone position for the Left sphere, and as you’ve done previously, click on the corresponding shape and position in video picture. Then, select This zone is in this position when the Novel object zone is in the position: Right cube.
Add another zone position for the Right sphere, click the corresponding shape and position in the video picture, and choose This zone is in this position when the Novel object zone is in the position: Left cube.
Complete this for the remaining two zone positions, selecting the opposite position for each.
Your zone list should now look like this:
We’re nearly finished building our protocol. Just one more item to add!
Dividing your Experiment into Stages
The final thing to consider is the fact that our experiment is divided into two parts: Familiarisation and Testing. In ANY-maze, these different parts are called Stages.
In fact, all experiments in ANY-maze consist of at least one stage, which is why you’ll notice there’s a stage already added for you. You can rename this stage to Familiarisation, as this will come first in our experiment. Then add a second stage for Testing just beneath that.
Within each stage, you’ll specify the Test duration (I’ve used 5 minutes) and the Number of trials. In this example, we only have 1 trial per stage because the animal will only be tested once in each stage.
With your stages now set up, you’ll find listed beneath each stage are two items related to the location of your objects– see figure below.
This is ANY-maze asking where you want the two objects to be in each stage. Part 2 of this guide will explain how to have ANY-maze automatically assign the shape and location of the objects, but for now we will opt to do this manually.
First select Stages > Familiarisation > Location of the Identical objects zone during this stage and then choose the option The user will specify the location at the start of each test.
Next, select Stages > Familiarisation > Location of the Novel object zone during this stage and choose The location is irrelevant because this zone isn’t used in this stage (during Familiarisation there is no novel object!).
Moving on to the Testing stage, select Stages > Testing > Location of the Identical objects zone during this stage and choose The location is irrelevant because this zone isn’t used in this stage (the only objects in the Testing stage are the novel and familiar objects).
Finally, select Stages > Testing > Location of the Novel object zone during this stage and choose The user will specify the location at the start of each test.
That’s your protocol set up! It would be a good idea to save your protocol at this point so that you can re-use it for all of your NOR experiments moving forward.
Running the tests
Before you run your tests, the first thing you’ll need to do is tell ANY-maze about your animals and treatment groups for this particular experiment, which you’ll do on the Experiment page. Then you can proceed to the Tests page.
Tests in the Familiarisation stage
Before you begin your test, you’ll place the physical objects in the arena (being sure to place them exactly within the shapes drawn in the video image) and then tell ANY-maze where they are. As can be seen in the image below, you do this simply by clicking on the corresponding shape in the video picture and then clicking the Select the position of the next moveable zone button.
Once you’ve specified the position of the Identical objects zone, you’ll be ready to start your test! Proceed by clicking the Start test button, then place the animal in the arena and step out of the video picture.
As the test is running, you can select the Tracking indicators button in the ribbon bar to modify the indicators you’re seeing on screen, such as the head marker, the animal’s orientation cone, and zone highlighting when the animal is investigating – see figure below.
Tests in the ‘Testing’ stage
Once your animals reach the Testing stage, you’ll be prompted to select the position of the Novel object zone. After doing that, the position of the Familiar object will be filled in automatically.
The figure below shows the Test schedule from a completed experiment. You can see that the Identical objects were only used in the Familiarisation stage, and the Novel and Familiar objects were only used in the Testing stage.
Viewing your results
After performing a test, you’ll probably want to see how much time the animal spent investigating the objects. You can view the results of an individual test by selecting the test number of interest on the Test Schedule Report (the list of tests shown on the left side of the Tests page), which will bring you to the Test Details Report.
When you do this, you may not see the same results as I have listed above. This is because you need to tell ANY-maze which results you’d like to have included in your Test Details Report. Simply select the Animal and test reports link to choose the results displayed here.
You’ll also notice that the Time investigating the Identical objects zone has #N/A as the result. This is because the report I’ve chosen in this example is a test from the Testing stage, in which the Identical objects zone was irrelevant.
That’s a standard novel object recognition experiment set up in ANY-maze!
Of course, your experiment may differ slightly from this one, in which case you can adapt your protocol to fit your specific paradigm. If you have any questions about how to do that, feel free to reach out to the ANY-maze support team at [email protected] for help.
To summarize, in this guide you learned how to:
- Draw your apparatus map to include both the shapes and positions of your objects.
- Create investigation zones.
- Make the investigation zones moveable (i.e., adopt different shapes and positions in different test) so as to counterbalance between the treatment groups.
- Manually select the shape and position of the objects before each test.
- View the results of a test.
In Part 2, which will be available in February 2023, we’ll cover:
- How to have ANY-maze automatically select the positions of the objects.
- How to calculate the discrimination index.
- How to view the results of all the tests.
- How to analyse your results between your treatment groups.