Simple and Computer-assisted Olfactory Testing for Mice

The overall goal of the following experiment is to test innate olfactory discrimination, olfactory preference, or avoidance and olfactory sensitivity in mice. This is achieved by exposing the mouse to a novel attractant or appellant, and a neutral odor, which is water. Meanwhile, a video camera records the behavioral session.

Next, the video is analyzed in Image J using the provided macros to obtain the time spent in the odor and water parameters. The results show the preference or avoidance behavior to odors as compared to water among different genotypes based on the post hoc computer assisted analysis. The main advantage of the olfactory testing over the use of dedicated arena and olfactory is that it uses a familiar arena and it is therefore of fast completion and simple setup.

In addition, it uses Unexpensive hardware demonstrating the procedure will be Emmanuel Abry, according student in my lab. For this experiment, use three to five month old male, C 57 black, six wild type, and transgenic mice. This demonstration uses two knockout lines and litter mate controls.

For the experimental arenas, use clean sterilized mouse cages with three centimeters of bedding. Next, prepare the camera setup. Position it 58 centimeters above the cage floor on a customized tripod mount.

Make some marks on the counter to denote where the position of the cage should be. For a center image, set the camera to record a 320 by 240 pixel video at approximately 15 frames per second. To prepare the test doer have concentrates of each scent stored in solvent whenever possible.

For the preference test, make a 10%weight by volume solution of peanut butter in peanut oil. For the avoidance test, have a solution of 98%to methyl butyric acid. For the sensitivity test, collect female urine from a mouse of the same strain one to two days before the test.

Restrain a female with its belly above the cage grid and collect drops of her urine into a plastic dish. Collect samples from several females to normalize. For individual variability, pull all the samples in a 1.5 milliliter tube and store it at minus 20 degrees Celsius.

To begin habituate the animals place one in a cleaned cage and let it explore. For five minutes the environment of the test cage should be like that of the home cage. Repeat the habituation phase once on each testing day, when the habituation period is over, start the recording and pipette 60 microliters of a pleasant scent.

In this case, peanut butter onto one wall on the opposite wall, pipette the same amount of water. A neutral scent, apply the drops about 10 centimeters up each wall. Now let the animal explore for two minutes, and then stop the recording and return the mouse to its home cage.

Repeat the process with each mouse to be tested. Always using new test cages. Three to five days After performing all the preference tests, carry out the avoidance test using a new test cage.

For each mouse, perform the avoidance test. Like the attraction test, only replace the pleasant scent with the avoidance scent, which is the two methyl butyric acid. For the sensitivity test, one cage can be reused between trials for the same mouse.

So mark the water side of the cage and prepare to clean the short walls with 70%ethanol soaked paper between trials. On the day of the experiment, make four concentrations of the urine with the tenfold dilution series. Using double distilled water like the avoidance test habituate each animal to its test cage for five minutes.

Then expose each mouse to urine at the highest dilution at first, which is one to 10, 000. While recording, apply the urine dilution to the test cage and apply water to the opposite wall. As done for other tests, let the animal explore.

For two minutes. Thereafter, transfer the animal to a clean mouse cage. After all the animals have experienced the lowest concentration, clean all the test cage walls and repeat the test on each mouse using the next most concentrated urine.

Continue this process until the pure urine is tested. After collecting all the data for an experiment, open the videos in image J for adjustment and processing details of which are provided in the text protocol using the described protocols. Two different knockout lines.

Groups B and C were compared to litter made controls group A.A non-directional student's T-test with equal or unequal variance compares the time spent between the odor and water within genotypes. Compare the behavior among genotypes using a one-way inova. Both experimental groups, B and C showed no preference for the attractive odor In the avoidance test, the response toward the repulsive two methyl butyric acid odor was as expected for groups A and B, but group C showed no avoidance behavior to this odor.

In the sensitivity test, the threshold of attraction was obtained by subtracting the time spent with urine from the time spent with water, the preferred urine to water at a one to 1000 dilution. Whereas both experimental groups, B and c needed a 100 fold greater concentration to show a preference. Once master, this technique can be done in a couple of hours if it is performed properly, then following this procedure, other methods like olfactory learning and extinction can be performed to investigate olfactory plasticity and learning.