Nesting behavior is a useful way of assessing activities of daily living in wildtype and transgenic mice. It's especially useful in documenting cognitive decline in neurodegeneration across the lifespan. This technic is a economical simple and elegant assessment of the daily living activities in mice.
It can also be performed in a short period of time with minimal training. In our laboratory we recommend shredded paper, as paper produces more consistent results. However, pilot studies can be conducted to determine which material best suites your goals and your budget.
In a separate testing room, set the environmental conditions so that they are consistent across trials and are identical to the housing room, and provide food, and water to the cages addlibitum. Assign each mouse a random identification number or letter. And record the original animal ID and other necessary identification in a colony record to ensure that the assistants and animal husbandry staff remain blind to the conditions.
Randomly order the cage placement in the testing room, such that the wildtype and transgenic mice are not inappropriately separated. And cover the bottom of each cage with 100 grams of dry weight of corncob bedding for the square, twist, and shredded paper trials. And 100 grams of soft cob bedding for the soft cob trials.
Then, just before introducing a mouse to a cage, place a single pressed cotton square, a single paper twist, no additional material added to the soft cob bedding, or 2.5 grams of clean shredded white printer paper cut into five to seven centimeter strips. At the same time of day, at the start of the light cycle, bring the home cage containing the mice into the testing room. And transfer each mouse into its assigned nesting cage with the material already in the cage.
When all of the mice have been placed, return the home cages to the housing room and allow the mice to complete the trial for 24 hours, undisturbed. At the end of the trial, carefully remove the lid of each cage. And when each mouse has moved off of the nest, photograph each nest capturing the assigned ID in the photograph, and minimizing the appearance of any materials outside of the cage.
When each nest has been photographed, gently transfer each mouse into individual temporary holding cages. And escort the bedding and any nesting materials from each cage. Then, replace the bedding, provide the next nesting material in the sequence.
And return the mouse to the nesting cage. When all of the trials have been completed, return the mice to their home cages and observe the mice for any potential aggressive behavior. To maintain inter-relater reliability provide baseline images for each nesting material to at least two individuals blind to the study.
And provide a series of sample nests to familiarize the scorers with each type of material. When scoring the nests, a score of one should be assigned when the shredded paper or small squares remain scattered throughout the cage, or the cotton square or twist remains untouched. A score of two is assigned when some of the material are constructed into a nest, but over 50%of the material has not been used for the nest construction.
A score of three is assigned when a noticeable nest is constructed, but several pieces are still scattered. A score of four is assigned when almost all of the material is used for the nest, but a few pieces of material remains scattered or are near the nest. A score of five is assigned when almost all of the material is used to make an unidentifiable nest.
Have the scorer's take a breaks and revisit baseline photos to avoid fatigue and bias during the scoring procedure as necessary. When all the cages have been scored, input the scores and format the data file to be in long format, such that animal scores are in separate columns. In this representative analysis across all four provided materials, wildtype mice scored higher on nest quality compared to APOE e4 mice.
With shredded paper rated significantly higher in quality then the square, bedding, or twist materials. With no differences observed between the square, bedding, and twist materials. In addition, in this experiment with htau mice, the 5.5-month-old mutant animals build a nests out of shredded paper that were significantly worst then those constructed by their age matched wildtype counterparts.
Scores should remain blind to the experimental conditions and review the scoring criteria. With the conclusions to be sound and valid, the inter and intra-rater reliabilities should also be assessed. Burrowing and Circadian wheel running are two other common ways of assessing activities of daily living.
And they may serve to compliment nesting behavior. Nesting can be used as an ancillary assessment to predict possible decline in cognition, widening the breath of behavioral assessments in conjunction with Morris water maze and circadian wheel running.
