B Pre training protocol for generalization and discrimination digging - michelletytong/TRGProtocols GitHub Wiki

On Day 1 (D1) and Day 2 (D2), begin by weighing the animals and their food. Remember to ]average of their free feeding weight and how much food they consume regularly. Each day, fill petri dishes with scented sand (use the reinforced odour that will be used during pretraining. We usually use 1.0Pa Limonene) and 15-20 sugar pellets, mixed in the sand. On D2, you may consider burying all 15-20 pellets. Prepare new sand and sugar each day. Carefully check for pellet in their bedding, this way you will know if they have consumed the pellets.

On D3, you begin food restricting the animals. Using the food weight you got from D1 and D2, calculate the amount of food consumed by each cage. You can start food restriction by giving 60% of the free feed amount. Monitor the weights of the mice carefully throughout food restriction, ensuring that no mouse drops below 80% of their free feeding weight (although the mice usually work quite well at 90% free feeding weight). But these steps can be done after training. On D3, training consists of habituating the animals to the testing chamber and format. Place two dishes of scented sand (the reinforced and non-reinforced odours) into the testing chamber without the centre divider. In the reinforced odour, scatter 5-10 sugar pellets (you can bury some and leave some on top). Put animals into this set up for 10 minutes each. Note if they ate the sugar or not. Most do eat the sugar within minutes and even start digging actually. I noticed if the animals are housed in multiples, the dominate males are always much more adventurous with eating the sugar than the submissive ones. Do this for two days. These are short days, because you can usually run many mice in parallel. Know your limits though, you want to always be in control and be able to give the mice enough individual attention and observation.

On D5 and D6, you’ll begin introducing them to a shorter version of the final testing format. This time, have the center divider in and the two pretraining odours with a sugar pellet on the top of the reinforced odour. Open the divider and let them into the testing chamber. Allow them to explore and retrieve the sugar pellet, then send them into the resting chamber. Do this for 10 trials and I would leave the sugar on top of the sugar for at least 5 trials, you can observe that they will be faster and faster in approaching the sugar and explore the chamber less. At this point, you’ll want to bury it deeper and deeper, but there is no need to rush the animals here. At this point, they know that odour+sand= sugar reward and they are hungry … we want to teach them that digging in the dishes is going to be a reliable source of reward. On D6, you will do the same and begin with sugar on top of the sand, but begin to bury the sugar earlier (like Trial 4?) and bury deeper and deeper. The animals should be digging for the sugar quite readily by the end of D6. With some familiarity, you MAY be able to run two animals at once, but I wouldn’t recommend it by any means, this part is very crucial, you need to be paying attention and reinforcing them reliably.

On D7, increase the trial number to 20 trials. You’ll still want to put the sugar on top for the first couple of trials. Then begin to bury the sugar very deeply. You may consider here that if they dig in the unreinforced odour first, you will usher them into the resting chamber without being rewarded. In this way, the animals will learn not to neglect investigating the odours. This day is usually pretty long.

The resting on D8-9 is both helpful and convenient. I usually let these days fall on weekends so that I only have to feed and weigh the animals and I can get a little break too. I think this part is really important. It’s at this moment where they are hungry and have just learned that there is a source of tasty food and have some practice with getting it. Letting the learning consolidate for a day or two really improves the training.

D10, 20 trials with the sugar fully buried. Restart the trial when they dig in the un-reinforced odour first. I usually also reset the trial if they have not started digging and 1 minute has passed. This can be slow, but they have generally learned by now after this, they should be ready for testing after this day!

An important thing I noticed is that mice housed in multiples actually learn much faster. I suggest doing the pretraining with these mice. You can observe the mice taking turns smelling the mouth of mice who have just undergone training. In fact, on a given training day, I’ll train one mouse per cage first, so that the others have time to check out the learned mouse. Then I’ll alternate between the cages while training.