All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Providence College.

1. Creating the Testing Apparatus

1. Use a Social Interaction Chamber (76.2 x 114.3 cm.), which is divided into 3 separate rooms.
   1. Divide the center room into a sample and delay camber using a cardboard insert that can be lifted by sliding through metal tracks attached to the side walls.
2. Use opaque contact paper to cover the clear walls to prevent visual access to other rooms.
3. On one side of the room-on-the-right (testing room; see Figure 1), cover the floor with sand granules and red contact paper. This will serve as visual and tactile a discriminative cue for the decline option.
4. On the opposite side of the testing room, cover the floor with raised stripes using strips of masking tape to serve as a visual and tactile a discriminative cue for the choice test. Cover the stripes with black contact paper. Leave the remaining middle third of the chamber unaltered.

2. Developing Preferred Food Reward Hierarchy/Preference Testing

1. Select possible food rewards for testing. Ensure one reward to be a larger reward and therefore "preferred" over the other.
   1. Use a whole piece of cereal as larger, "preferred" reward and a ¼ piece of cereal as smaller, "less preferred" reward.
      Note: Cereals can include sweetened cereal or dehydrated marshmallow. It is also possible to use two different rewards (e.g. cereal vs. standard rodent pellet (TestDiet, 45 mg)), but it was difficult to determine a reliable preference ranking within and across individuals when quality rather than quantity was the source of variation across rewards.
2. Bring rats (adult male Long Evans) into dark testing room 30 min prior to testing begins. Use red lighting to illuminate testing chamber, if rats are in the dark phase of their day's cycle. If not, test under normal lighting conditions as shown in video.
3. Place the two rewards, the whole piece of cereal and the ¼ piece of cereal, in opposite corners of the right room of the chamber (see Figure 1).
4. Place the rat on the opposite side of the chamber, facing away from the rewards, and allow the rat to eat one of the rewards.
   Note: To pass preference testing and to ensure that the larger piece of cereal is the preferred reward, the subject must eat the larger reward on 4 out of 5 trials (5 trials per session) for 2 consecutive sessions. Run two sessions per day.
   1. If, after 10 sessions, the rat has not shown a preference for the larger piece of cereal, introduce a different reward.
5. Throughout testing, to ensure that rats' preferences remain consistent, perform preference checks once a week.
   1. Run preference checks in the same manner as preference testing, with the whole and 1/4 piece of cereal on opposite corners of the chamber.
      Note: 2 trials comprise a preference check.
   2. If rats select the less preferred reward for both trials, test rat on a full preference test of 5 trials.
      1. If rat passes, it may continue testing.
      2. If rat fails, cease testing and introduce a different type or amount of reward.

3. General Methods

1. Use consistent experimenters throughout testing so rats are fully habituated to experimenter odors and handling.
   Note: It is assumed that prior to testing the rats have been handled for at least 1 week by humans, so that they are habituated to being picked up and carried by the experimenters.
2. Bring rats in their shoebox cages (home-cage) into the testing room 30 min before testing begins to acclimate them to the new room.
3. If testing is done during the dark phase of the rats’ cycle, keep the testing environment dark, with the exception of red lights to illuminate the apparatus.
4. Ensure that the scented choice cups are arranged in the proper order as determined by a randomization script that includes for counterbalancing. Bait the correct cup with the whole piece of cereal.
5. Only allow the rat to dig in one cup and immediately remove the rat from the apparatus once a cup has been selected as indicated by digging. Place the rat in home-cage where the rat can finish consuming the reward.
6. The inter-trial-interval (ITI) is fixed to 4 min. Begin timing on a stopwatch once rat is in the home-cage.
   1. During the ITI, clean the apparatus by vacuuming any spilled sand and wiping down the floor with 70% isopropyl alcohol. Discard the reward if it was not eaten during the previous trial. Also, once a week, clean out ceramic cups by discarding old sand and wiping cups with isopropyl alcohol. 
   2. Prepare the sand cups for the next trial by rearranging the order based on the randomization script, and baiting the correct cup.

4. Training to Dig in Sand Cups and Habituation to Testing Apparatus

1. Pour approximately ½ cup of play sand into ceramic cups (6.99 cm. in diameter and 8.81 cm tall).
2. Place the sand-filled cup with food rewards in each rat's home cage overnight.
   Note: Rats are required to successfully dig in the cup and eat the reward overnight for two consecutive nights.
3. Habituate rat to testing apparatus by placing the subject in apparatus for 5 min.
   1. Spread food rewards throughout the apparatus to encourage apparatus exploration and traveling through opened doors. Ensure that rats do not urinate, defecate, or freeze inside the chamber for two days in a row.
4. Transfer odorless sand-filled cup with food reward to the testing apparatus. Ensure that rats successfully dig in the sand and retrieve the reward at least two days in a row with no freezing behavior, defecation, or urination. If necessary, remove the ceramic cups and place cereals in the chamber.

5. Match-to-Sample (MTS) Training

1. Create 4 different scented piles of sand by mixing approximately 3 cups of play sand with ¼ teaspoon of scented spice (cinnamon, coffee, paprika, and thyme). To control for odor cues, crumble one piece of reward in incorrect choice cups.
   1. Remix scented sand once per week and store in glass containers so that scent strength is consistent across testing days.
2. Pour ½ cup of each scented sand into 4 cups and arrange the cups in a semi-circle on the black contact paper (MTS Task area; Figure 1A). Ensure that all cups are equidistant from the entrance door.
   1. Organize the cups by placing them in the assigned position based on the output from the programming software "R" using the sampling without replacement function.
3. Out of view of the subject, bury a full piece of reward in the correct scented cup. Bury ½ piece of reward in an identically-scented sample cup. Bury all rewards at least a ½ way down the height of the sand.
   1. Pseudo-randomly choose the correctly scented cup, the only constraint is that no one scent is the sample scent more than 2 times in a row.
4. Place the subject in the sample chamber and lower the sample cup using a bent spatula (handle-tray at a 90-degree angle). Allow subject to dig in the sample cup and retrieve the ½ cereal reward. Remove the cup as soon as the subject begins eating the reward.
5. Once the subject has finished eating the reward, lift the cardboard room divider and open the door to the testing chamber as the subject enters the delay room.
6. Allow the subject to "select" one of the cups by digging. As soon as the subject makes a choice and retrieves a reward if present, remove the subject from the chamber and place in the transfer cage. Rats may sniff each cup, but they are only allowed one choice as indicated by digging.
   1. During the 4-min ITI, clean the chamber with 70% isopropyl alcohol. Vacuum any sand that may have spilled and replenish sand cups if needed.
   2. Obtain next sample cup and bait both the sample and testing cup. If the subject selects the incorrect cup in the previous trial, remove and discard the uneaten reward.
7. Ensure that subjects receive one 6-trial MTS session per day. In order to pass MTS training, subjects must choose the correct cup three of the six trials in two consecutive sessions.
   Note: This ensures that performance is above chance but below the ceiling on the 4-choice test.

6. Delay Titration in Delayed Match to Sample (DMTS)

1. Continue training subjects on MTS as before (starting with the delay of 0 s).
   Note: Performance must remain between 40 and 70% correct, to ensure that performance is well above chance (25%), but below the ceiling. Performance in this range allows subjects to experience instances when they when remember the same and instances when they forget the sample. This creates an opportunity for use of the decline-test response to be advantageous to the subject when chosen trials are introduced.
2. If performance exceeds 70% correct for 2 consecutive 6-trial sessions, increase the delay from 0 to 30 s.
   1. In trials on which the delay is not 0 s, use a stopwatch to time each delay. After the subject has consumed the sample, raise the cardboard divider wall and allow subject to enter into the delay chamber. Close the cardboard divider and begin timing the delay on the stopwatch.
   2. After 30 s has passed, open the door to the testing room, and shut the door once the subject has entered the testing chamber.
   3. Carry on the rest of the trial as before.
3. If performance continues to remain above 70% correct for 2 consecutive 6-trial sessions, increase each subsequent delay by 1.5 times the previous delay.
4. If performance falls below 40% for 2 consecutive sessions, the delay is returned to the previous one used.
5. If performance remains between 40% and 70% for 2 consecutive sessions, set that delay as the subject-specific criterion delay.

7. Decline Use Training

1. After each session of DMTS training, place another cup of a different textured and colored sand, opposite the scented sand cups. Bait the cup with the less-preferred reward, ¼ piece of the cereal, which will be the decline cup. Remove the scented sand cups from the chamber.
2. Carry out the trial as during DMTS training, allowing the subject to sample a randomly scented cup and wait in the delay chamber for the subject-specific delay.
3. Allow subject to dig in the unscented sand cup and retrieve the reward. Then remove the subject from the chamber. This will be considered a "forced decline" trial.
4. Conduct two forced decline trials after each session of DMTS training.

8. Testing: Forced vs. Choice Trials

1. Test subjects on DMTS using the criterion delay. Test each subject on one 12-trial session per day.
   1. Randomly select 8 of the trials to be "choice" trials and place the decline cup opposite the scented cups. Use randomization script to determine the order of trial-type with the constraint that no more than 3 choice or forced trials occur in a succession.
      Note: The remaining 4 trials will be "forced," with the decline cup removed from the chamber.
   2. Depending on the subject's criterion delay, it may be possible to test 2 or 3 subjects during one rat's ITI. During 1 subject's ITI, run the second subject's trial. Ensure to clean the chamber with isopropyl alcohol after each trial.
   3. Test subjects on ten sessions.
2. During each trial, once the rat enters the delay chamber ensure that he either turn left to decline the test or turn right to take the test (see Figure 1A; top-down photograph). Record if rat travels from one side of the room to the other (e.g. "decides to take the test" and then later traverses to the decline-test side of the room).
   Note: The rat, therefore, makes a prospective metamemory choice as he chooses to take or decline the test before encountering the four choice odors (see results paper¹⁹ for thorough discussion of concurrent vs. prospective metamemory tests).
3. Calculate the accuracy on forced trials continuously after two sessions of testing and monitor decline-test use.
   1. If accuracy rises above 70% correct over the course of two sessions, cease testing and return to delay titration with a longer delay.
   2. If decline-test use approaches 0% or 100% consistently (over 2 sessions), pause testing and consider increasing or decreasing the value of the decline-test response ensuring the decline rates to be between 10% and 40%.
   3. If accuracy falls below 40% correct for 2 sessions, cease testing and return to delay titration with a shorter delay.
   4. Return to testing after accuracy on forced trials remains between 40% and 70% correct for two consecutive sessions.
   5. Throughout the testing, to ensure that rats' preferences remain the same, perform preference checks (2 trials a session) once a week. Preference checks are described above.

9. Suggested Generalization Tests

1. Generalization tests, such as those described in the results paper¹⁹ can be used to determine if metacognitive responding transfers across conditions in which no single association with external stimuli is likely to control responding.
   1. Test rats on no-sample probe trials, in which no sample phase is presented but the RI and test phase of trials is identical to normal trials. Randomly intermix 4 probe trials with 8 normal sample trials to comprise a 12-trial session. Test subjects on 10 sessions. Compare the decline rates on no sample trials with the decline rates on normal trials.
   2. Test rats on double sample trials, in which subjects are presented with the original sample scent twice to increase memory strength. Randomly intermix probe trials with normal sample trials as explained above. Compare decline rates on double sample trials with the decline rates on normal trials.
   3. Test rats with three different RIs: the criterion delay, one delay which is shorter (e.g. 0 s) than the criterion delay, and one which is longer (e.g. 4 min). For each delay within a session present subjects with four choice trials and two forced trials, such that sessions are comprised of 18 trials. Randomly intermix all trials and test rats on ten sessions. Compare the decline rates of trials on three delays, where memory strength should be decreased and increased, respectively.

10. Data Analysis

1. After testing is complete, calculate average proportion correct for forced and chosen trials for each subject.
   1. To find the proportion of correct forced trials, divide the number of correct forced trials by the total number of forced trials.
   2. To find the proportion of correct chosen trials, first subtract the trials declined from the total number of choice trials. Divide the number of correct choice trials by the total number of choice trials minus declined trials for each rat.
   3. Conduct a t-test on group data to compare the proportion of correct forced trials to the proportion of correct chosen trials (see Figure 2).
   4. If generalization tests are run, conduct t-tests on decline rates on normal trials vs. probe trials as described in 9. For mixed delay trials, as described in 9.1.3, compare forced and chosen tests with a repeated measures ANOVA with delay and trial-type as a within-subject factor. Also, conduct a one-way ANOVA on decline use across delays to determine if accuracy varied with RI.