The overall goal of this procedure is to measure cognitive flexibility as mediated by the prefrontal cortex in a mouse model. This is accomplished by first teaching a test mouse to dig a small pot to retrieve a food reward in the second step. The buried food reward is associated with a specific contextual queue such that the mouse can correctly locate and retrieve the reward in eight consecutive trials without error.
Next, through a series of queue reward pairing changes within the relevant dimension. The mouse learns to pay attention to the relevant dimension of the queue while ignoring the irrelevant dimension to locate and retrieve the reward. In the final step, the queue reward pairing is switched so that the previously irrelevant dimension is now relevant to facilitate successful location and retrieval of the reward.
Ultimately, an increase in the mean number of trials to reach the criterion between the experimental groups at the reversal stages is interpreted as cognitive inflexibility. Although this method can provide insight into the basic neuroscience of cognitive function mediated by the prefrontal cortex, it can also be applied to many other model systems such as transgenic or chronic disease models, with the benefit of excellent translational validity. Generally, individuals new to this method will struggle because the mice have not been adequately habituated or trained prior to the onset of testing.
Demonstrating the procedure will be Jillian Heisler, a recent PhD graduate from my laboratory, assisted by Lainey Ritas and Juan Morales, both research assistants in the lab Before beginning. The food restriction handle each mouse two to three minutes per day for eight days to reduce the stress associated with the handling during the testing. If multiple experimenters are to be involved in testing a set of mice, ensure that each experimenter participates in the handling process.
Weigh and record the body weights of each animal for each day of the handling. Then beginning four days prior to the start of the acclimation place two ramekins at the front of each home cage to allow the mice to acclimate to the testing pots. Add two grams of food and one piece of food reward in the ramekins each day for four days to maintain the mice at 80 to 85%of their free feeding body weight.
Then on the day before the acclimation procedure, change the home cage bedding two days prior to the start of the training and during the dark cycle under red light, spread a small amount of dirty bedding from the home cage throughout the chamber to introduce a familiar self smell and to reduce the stress of being in a new environment. Place a clean ramekin with water into the waiting area of the chamber and the two ramekins that have been used for the food restriction in the testing area with approximately 20 milligrams of food reward in each container. Then transfer the mouse to the waiting area and remove the start gate.
Allow the mouse to explore the chamber for one hour. Continuously adding cereal pieces to the empty ramekins to encourage the mice to frequently explore the testing area and the pots. Take care that the mouse can see the experimenter throughout the acclimation period as well, so that the presence of the experimenter is not an added stressor.
During the testing. Begin the training on day 15 by placing the mouse in the waiting area. Then place the empty ramekins each containing a food reward into the testing area, and lift the start gate.
Allow the mouse three minutes to retrieve both food rewards after repeating this food reward retrieval several times. Gradually add clean cage bedding to the pots in each subsequent trial. Continuing to allow the mouse three minutes to retrieve the food reward from each pot before ending the trial.
Once the mouse has retrieved the food reward from each pot, proceed to the next trial. If the mouse does not make attempts at digging, once the food reward is partially covered, hence may be provided. Continue adding bedding after each trial until the food reward is fully covered, and the mouse reliably demonstrates the ability to dig in a full pot.
To find the food reward at the time of handling on the testing day, give each mouse one food reward to avoid a lack of focus during the testing due to hunger. At the symbol discrimination stage, only one Q dimension is present. The example here is a digging substrate fully cover the food reward with media and sprinkle cereal dust over all of the pots.
Note that when odor is the relevant Q dimension, sawdust bedding is the digging substrate in both pots. Then place a mouse in the waiting area with the start gate closed and the pots on either side of the testing chamber. Now remove the gate and start the timer to begin the testing.
Allowing the mouse three minutes per trial to make a choice record whether a correct or incorrect attempt to dig or choice is made and the time until the mouse made the choice for each trial. If a mouse has not made a choice within three minutes, record this choice as incorrect as well. Once a choice has been made for either pot, remove the pot that was not chosen from the testing chamber.
If a correct choice is made felt, in this example, allow the mouse to finish the food reward. If an incorrect choice is made paper, in this example, allow the mouse to explore the pot to show that there is no food reward for that choice. Regardless of the choice.
At the end of each trial, place the mouse back in the waiting area and replace the start gate. Then in subsequent trials, using paired media and sense as shown in the table, use forceps to place a piece of food reward in the pot that indicates a correct choice following the same procedure as just demonstrated. As a note, be sure to send POTS well in advance allowing the strong initial odor to dissipate prior to testing.Testing.
In this graph, the average number of trials to meet the criterion at each stage in untreated C 57 black six J mice. Using the attentional set shifting task or a ST is shown. Differences at the simple discrimination stage are reflected as an increased number of trials to meet the criterion when odor is a relevant queue.
As illustrated here, normalization of the performance data reduces the variability associated with the learning of the initially relevant queue and enhances the visualization of the improved performance on the subsequent inter-dimensional shift stages. A significant increase in the trials required to meet the criterion during the extra dimensional shift relative to the previous inter-dimensional shift is an important control measure of the test validity. Prior to the extra dimensional shift stage, mice learned to pay attention to a single relevant Q dimension to locate the reward.
Interestingly, analysis of the intra and extra dimensional shifts within the raw data reveals a significant increase in the number of trials required to reach the criterion between the second intra and extra dimensional shifts. Further, a significant intra extra dimensional shift was apparent between both the second and third interdimensional shifts compared to the extra dimensional shift when the data was normalized to the performance at the simple discrimination stage Once mastered, this technique can be completed in less than three weeks per test mouse with each researcher testing up to two mice concurrently if it is performed properly. While attempting this procedure, it's important to remember to minimize the environmental distractions such as foot traffic, inner near the testing area, distracting odors like strong perfume or cage changes during the training and testing phases.
After watching this video, you should a good understanding of how to execute the attentional set shifting task in mice using easily attainable materials and common lab supplies.
