Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

The overall goal of this experiment is to determine the extent to which rodents respond metacognitively by presenting subjects with memory tests that can be declined. This method can help answer key questions in the field of comparative psychology such as determining the extent to which rats monitor memory. The main advantage of this technique is that it is relatively easy for rats to learn as it capitalizes on rat's natural foraging behavior and primary sense of olfaction.

This technique was carefully created so that results from behavioral testing are valid and reliable. For example, we balanced the inter-trial interval with the set size from which sample odors were drawn so that there is optimal memory interference. We had the idea for this method from similar paradigms using visual stimuli in monkeys along with unrelated experiments in which rats are presented with odors to remember by digging in sand-filled cups.

The text protocol describes assembly of the testing apparatus, habituation of rats to the testing apparatus, and digging in sand cups for food rewards. Once a significant food preference is established, the rat can proceed to match-to-sample training. For this test, four different cups of scented sand should already be prepared and not more than one week old.

Store the scented sand in tightly sealed glass containers. Each match-to-sample test session consists of six trials with four-minute inter-trial intervals. For an animal to pass this training, the rat must correctly identify the hidden reward in at least three trials over two consecutive test sessions.

To set up the match-to-sample test, pour a half cup of each scented sand into four cups. Then following the randomly assigned position by the software, arrange the cups equidistant from the entrance door in the task area of the apparatus. Next, bury a full piece of reward in the pseudo randomly chosen correct scented cup.

Just don't use the same cup more than twice in a row. Also, bury a half piece of reward in an identically scented sample cup. Bury the rewards at least halfway to the bottom of the sand cup.

Start the test by presenting the sample odor to the rat. Place the rat into the sample chamber pass the intervening delay chamber. Then lower the cup containing the half reward into the chamber using a bent spatula.

As soon as the rat begins eating the reward, remove the cup. After the rat has finished eating the reward, lift the cardboard room divider to the delay chamber and the divider to the MTS task area. As soon as the rat makes a choice and retrieves a reward if present, remove the rat from the chamber and place in the transfer cage.

The rat may and likely will sniff each cup, but only allow the rat to dig into one cup. If it chooses incorrectly, remove it quickly to prevent alternate cup choices. Once in the transfer cage, start a four-minute ITI and to blind the rat from viewing the next trial setup, shield the cage.

During the rest period, vacuum up any sand that may have spilled, clean the chamber with 70%isopropyl alcohol, remove uneaten rewards, and replenish sand cups as needed. Then prepare the next pseudo randomly selected sample cup with the half reward. Set up the task area and conduct the next trial.

When the session is completed, return the rat to its home cage and start testing the next rat. After passing the MTS task advance to the Delayed Match-To-Sample or DMTS test to establish a delay criterion. This task is like the MTS, but essentially adds a retention interval between giving the rat the sample and letting the rat execute the test.

When the rats have a success rate between 40 and 70%the delay interval is sufficient to advance the rats to the forced versus choice trial. Before starting the forced versus choice trials, first conduct two trials in which the rat only finds the decline cup in the task room. The decline cup is loaded with sand of a different color and texture.

It is loaded with a one-quarter sized reward. Forced versus choice trails are like DMTS tests, but in eight out of 12 tests, the decline cup is offered on the opposite side of the chamber from the four MTS cups. Conduct 10 forced versus choice sessions per rat.

During each trial, when the rat enters the delay chamber, take note if the rat initially turns left towards the decline cup or to the right to take the MTS test. This is the perspective metamemory choice. After two sessions, calculate the rat's accuracy on the forced trials.

If the rat accuracy rises to above 70%or drops to below 40%then cease the forced versus choice testing and return to DMTS testing. Once the rat's accuracy is again between 40 and 70%on two consecutive DMTS sessions, then return to the forced versus choice trail sessions. Also, determine if over two sessions the decline choice approaches zero or 100%In this case, pause the testing and consider changing the reward size in the decline cup.

The goal is a decline choice rate between 10 and 40%Throughout the forced versus choice testing, ensure that the rat's preferences remain the same by performing preference checks once a week as described in the text protocol. Always ensure that the test chamber is cleaned with isopropanol after each trial. Depending on their criterion delay, it may be possible to test two or three rats at the same time which will make thorough cleaning especially critical.

In order to study the metacognitive responding of the subject, the proportion of correct forced trials is compared with the proportion of correct chosen trials or trials in which there is a decline option present. A positive result is indicated by a significantly higher accuracy on chosen trials compared to forced trials. If the performance is not significantly more accurate on chosen trials than on forced trials, the results are null.

This hypothetical lack of evidence for metamemory in rats is indicated by failure to use the decline test option when it would have been adaptive to do so on trials during which the sample was forgotten. After watching this video, you should have a good understanding of how to test rats for metacognitive responding using a delayed match-to-sample test with scented sand. Once mastered, a rat can be tested in a session of match-to-sample in approximately 45 minutes depending on the rat's delay if it is performed properly.

While attempting this procedure, it's important to remember that rats must be habituated to the apparatus, have a strong preference of rewards, and an individually selected and monitored retention interval.