Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

The overall goal of this procedure is to show the simplicity of marble burying and nestle shredding as reliable paradigms to assess and demonstrate repetitive and compulsive like behaviors. In rodents for the marble burying test, a cage is prepared with 20 marbles spaced evenly on the bedding surface. The mouse is then introduced to the cage and left undisturbed with the marbles for 30 minutes.

After this time, the mouse is removed and the buried marbles are counted for the Nestle test. An intact Nestle is placed in the cage before the mouse is introduced. After 30 minutes, the mouse is removed and the Nestle is allowed to dry overnight before calculating the percent of Nestle shredded.

Ultimately, differences in marble, burying and Nestle shredding behavior can be shown among knockout and wild type mice. Demonstrating the procedures will be Denise Briggs, a graduate student, Mariana Ango Perez, a postdoc and Dean of Francescutti, a research associate from my laboratory. All animals are maintained on a 12 hour light dark cycle with free access to food and water.

For best results. Handle each mouse gently once per day for three to five days prior to testing. When the animals are ready to be tested, transport the cages to the testing room 60 minutes before starting the first test.

For the marble bearing test, use standard polycarbonate rat cages with fitted filter tops. Add fresh unscented mouse bedding material to each cage to depth of five centimeters and level the bedding surface by inserting another cage of the same size. This has the added advantage of impressing a template of parallel lines on the bedding surface.Wash.

Standard glass toy marbles in mild laboratory detergent rinse exhaustively and distilled the ionized water and dry prior to each use. When ready, gently press the marbles into the surface of the bedding. In five rows of four marbles each prepare stopwatches or timers for timing the sessions.

To begin the test, face the mouse towards the corner of the cage as far away from the marbles as possible. Place the filter top on the cage and leave the mouse undisturbed with the marbles for 30 minutes. At the end of the test period, remove the mouse and return it to its home cage.

Take extreme care not to move or dislodge the marbles in the process of removing the subject from the cage. Two to three researchers that are blind to the treatment conditions should then score the number of marbles buried. A marble is scored as buried.

If two thirds of its surface area is covered by bedding, average the scores for the number of marbles buried for each mouse. Lastly, retrieve all 20 marbles and dispose of the bedding. Use standard polycarbonate mouse cages with fitted filter tops.

For the Nestle shredding test, add fresh unscented mouse bedding material to each cage to a depth of 0.5 centimeters and pack the bedding as shown earlier. Next, weigh commercially available cotton fiber nestlets and place one Nestle on top of the bedding. In each test cage, prepare stopwatches or timers for timing the session.

When ready, place a mouse into one corner of the cage facing the wall. Place the filter top on the cage and leave the mouse undisturbed with the Nestle for 30 minutes. After this time, remove the mouse and return it to its home cage.

Remove the remaining intact nest lit material from the cage and allow it to dry overnight. The following day. Weigh the unshredded nest lit and divide this weight by the starting weight to calculate percentage of nest lit shredding.

Lastly, discard remaining shredded nest lit material and bedding. Shown here are representative cages before and after the marble burying test. Few marbles were buried or displaced from their initial location when testing wild type mice.

However, the TPH two knockout mouse buried 17 out of the 20 marbles. Remaining unburied marbles are indicated with arrows. This side view demonstrates changes in the bedding surface.

After testing, compare the relatively undisturbed surface from wild type cages with the uneven surface containing large troughs and peaks from knockout animals. Typical performance on the marble burying test is summarized here. TPH two knockout mice buried significantly more marbles when compared to wild type mice.

These photographs show representative nestlets after shredding. Here nests from wild type mice show minor amounts of shredding as compared to the nestlets from the cages of TPH two knockout mice. This graph shows the typical performance of wild type and TPH two knockout mice on the Nestle shredding test.

The TPH two knockout mice shred significantly more Nestle material than wild type mice. Once mastered, these techniques can be done in two hours if perform properly. While attempting this procedure, it's important to remember to be consistent with the criteria for defining a marble is buried, and to remove shredded pieces of Nestle from the dried and unshredded remainder.

Following this procedure, mice can be injected with drugs used to treat obsessive compulsive disorders in humans such as SSRIs, in order to determine if these drugs reduce compulsive like behaviors in mice.