Automatically scoring spontaneous alternations in the Y-maze test

Section 1

Detecting arm entries

As this guide is specifically about scoring spontaneous alternations, I won’t be describing all the steps in setting up a Y-maze protocol, but clearly we will need to know when the animal enters the various arms of the maze, therefore you will need to make sure you have set up three Zones, one for each arm, which in this guide I have called A, B and C.

Usually, in the Y-maze,  an arm entry is scored when all four paws of the animal are in the arm. If you want to use this rule then you should make sure that the Zone entry settings for each of the arms are set to use the Whole body of the animal, with an entry being scored when around 85% of the animal’s body has entered the arm – this general equates well to the four-paws-in-the-arm rule.

Section 2

Detecting a spontaneous alternation

As mentioned above, a spontaneous alternation occurs when the animal moves from, say, arm A to arm B and then to arm C, rather than back to arm A, which is where it had just come from. So what we want to do is detect a movement from zone A to zone B to zone C, which is exactly what ANY-maze Sequences are designed to do.

So you will need to add a sequence to your protocol, I recommend calling it simply ABC. You should then add a Sequence step to the sequence and specify that the animal must enter zone A in order to perform this step. Next, add another step to the sequence and specify that the animal must enter zone B in order to perform this step, and finally add a third step for zone C.

The sequence will now correctly detect movements from zones A to B to C, but there’s one small problem – in order to get from arm A to arm B, the animal will have to move through the centre of the maze. Usually this would ‘break’ the sequence, because rather than going from A to B, the animal has gone from A to Centre to B. However, if you select the sequence in the ANY-maze protocol, you will find that there is an option for Sequence is not broken by a moves to positions which are not in any step – if you select this, the sequence will correctly detect the movement from A to B to C, even though the animal will necessarily also move through the centre of the maze.

Voila! ANY-maze will now correctly detect this sequence. However, this is just one of the possible ways the animal could move between the arms and perform a spontaneous alternation, for example, it could move from arm C to B to A and that too would be a spontaneous alternation. In fact there are a total of six possible sequences that count as spontaneous alternations: ABC, ACB, BAC, BCA, CAB, and CBA.

You might spot that some of these are the just the reverse of another one, for example, the last one CBA, is just ABC but in the opposite order. Another option that’s available for a sequence is to specify that it can be performed in either direction – so if we select this option for our original ABC sequence then it will score both ABC and CBA sequences. To make this clear, you might want to rename the sequence ABC/CBA.

But what about the others? Well, you just need to set up two more sequences ACB/BCA (which, by making it bi-directional, will score both ACB and BCA) and BAC/CAB.

Section 3

Determining how many spontaneous alternations occur in the test

With the three bi-directional sequences set up, ANY-maze will score all the possible spontaneous alternations that the animal can perform. To get a total of these you would need to sum the number of ABC/CAB, ACB/BCA and BAC/CAB sequences that occur during the test.

The easiest way to do this is to add a Calculation to your protocol. You should call it Number spontaneous alternations and specify that it has zero decimal places. Then right click in the calculation area and from the menu which appears, select ABC/CBA sequence measures > Number of ABC/CBA sequences. Next, type in a + and then, from the right-click menu, select ACB/BCA sequence measures > Number of ACB/BCA sequences, and finally insert another + and add the number of BAC/CAB sequences – you should end up with something like this:

Section 4

Calculating the percentage of spontaneous alternations

The result of the above calculation will tell us how many spontaneous alternations occurred in the test, but it won’t tell us the percentage of spontaneous alternations, which is the value we really want to know. Clearly to calculate this we need to divide the number of spontaneous alternations by the number of movements the animal made between the arms of the maze and multiply by 100…

…well, almost. Imagine you ran a test in which the animal started in arm A, moved to arm B and then to arm C and then didn’t move arms again for the rest of the test. So you would have A to B to C which would be one spontaneous alternation. And you would have three arm entries, but in this test the animal couldn’t perform more than one alternation, so the percentage spontaneous alternations should be 100% but if we were to divide the number of spontaneous alternations by the number of arm entries and multiply by 100 we’d get (1 / 3) x 100 = 33%, which is wrong.

The problem here is that we really want to divide the number of spontaneous alternations by the number of opportunities the animal had to perform a spontaneous alternation, which in our example is just one, so then we’d have (1 / 1) x 100 = 100%. So what are the opportunities the animal has to perform a spontaneous alternation? The answer is simple, it’s just the number of arm entries less two. The reason is because until the animal has been into two arms it can’t perform a spontaneous alternation at all, only when it enters the third arm can it perform one. But after that every arm entry offers another opportunity. For example, if the animal goes ABCB then the move A to B to C was a spontaneous alternation; it then went from C to B, but it had come from B, so that wouldn’t be a spontaneous alternation. So in this case we had 4 arm entries and one spontaneous alternation, so the percentage would be (1 / (4 – 2)) x 100 = (1 / 2) * 100 = 50%.

To calculate the percentage spontaneous alternations in ANY-maze, you can just add another calculation to your protocol. In this case the calculation would be:

Where you just type in the parenthesis and mathematical operators, and you select the various measures from the right-click menu. Note that the first measure (Number spontaneous alternations) is the result of the first calculation that we set up.

For this calculation you should specify one decimal place and units of %.

Section 5

Conclusion

That’s it, ANY-maze will now report the Percent spontaneous alternations in all of your Y-maze tests, in just the same way as it reports other ‘built-in’ measures, such as the time spent in a zone.