Testing for Odor Discrimination and Habituation in Mice

The overall goal of this procedure is to provide a method to investigate the ability of mice to detect habituate and dis habituate to both novel and social odors. This is accomplished by first preparing a series of novel non-social odors, such as fruit and nut-based extracts and social odors, such as cage bedding. The second step is to present the odors to the mice, starting with three novel odors and followed by two different social odors.

During each trial, the sniffing behavior of the mice is evaluated and recorded. Ultimately, the odor, discrimination and habitation test for ma olfaction is used to show that the animal can respond to a novel odor, can habituate to the odor, and then demonstrate the ability to dis habituate to the odor when presented with another novel odor or social odor. This method can be used to answer key questions in the neuroscience field, such as whether the animal is not responding to a novel animal or novel stimulus because that animal is unable to detect the odor.

Begin preparing non-social odors by placing on gloves and filling three 15 milliliter conical tubes with 10 milliliters of water. Each pipette 100 microliters of almond extract into the first tube, 100 microliters of banana extract into the second tube, and leave the third tube with just water to use as a control. Next, obtain two different cages of mice that have not been cleaned for at least three days and contain equal numbers of mice that were the same sex as the test subjects.

Use a cotton swab to wipe the bottom of the dirty cage in as zigzag fashion. Shake off any attached bedding material from the swab and place it sent side down into a large glass jar with a tight fitting lid. Repeat this process until three swabs are obtained from both cages for each mouse that will be tested.

Store the jars at room temperature and use the swabs within four to six hours. In order to maintain a strong and consistent odor, move the odor sample cages to a room outside of the testing area the day of the experiment. Do not wear scented fragrances or lotions and avoid eating anything with a strong odor.

Place the prepared social and non-social odors into the testing room, along with laboratory tape fresh cotton swabs, a waste bottle with a lid, and two timers immediately prior to the testing procedure. Put on clean gloves and weigh the first mouse. Then transfer the mouse into a clean cage with a wire lid and no water bottle.

Place the cage in an area with no unusual odors or noises and allow the mouse to acclimate to the new cage for 45 minutes. At the end of the acclimation period, move the mouse in its cage to the testing area and begin acclimating another mouse. Place the acclimated mouse in its cage on the counter at a height that is comfortable for viewing when seated.

Next, prepare one of the odors in the order described in the text protocol for the non-social odors at 100 microliters of the scent onto the tip of a fresh swab. Immediately before use. For the social odors, remove and use one of the previously prepared swabs from their respective jars.

Lift the wire top of the cage and insert the unscented end of the cotton swab into the water bottle opening from the underside of the wire lid. Avoid touching the cotton tip to anything, including fingers to avoid odor contamination. When about one inch of the scent swab remains in the cage, affix the wooden end of the swab to the underside of the cage using lab tape.

Then gently lower the lid back onto the cage. To avoid disturbing the test mouse immediately begin timing for two minutes. Using the first timer with the second stopwatch record the cumulative time the test mouse spends sniffing the odor.

Active sniffing is defined as the mouse orienting its nose towards the cotton swab while within two centimeters of the swab. When the trial is over, raise the wire lid and carefully remove the cotton swab by pulling it downward through the underside of the lid. To avoid contacting the lid with the odor containing end of the swab, seal the used swab in the waste bottle while allowing the mouse to rest for one minute, prepare the next donor swab.

Present each scent three times in a row for two minutes. When all the trials are completed, empty the waste bottle containing the used cotton swabs in a trash can outside the test room and return the mouse to its home cage. Each of the odorants were first presented to mice with the C 57 black six J background with successive presentations of each odor.

The mice spent less time investigating the odor suggesting habitation. The same pattern of habitation and discrimination can be seen with the social odors as with the non-social odors. However, the mice spent more time investigating the social odors.

When the same non-social odors were presented to a male FVB mixed background strain, a similar pattern of habitation was seen. Each new odor that was introduced was met with an increase in active sniffing, which decreased with each presentation of the same odor. However, the FVB mice showed a different pattern with the social odors as there was no significant decrease in time spent sniffing with repeat exposure.

The data suggests that the mice do not habituate to repeated presentations of the two social odors. After watching this video, you should have a good understanding of how to test the ability of a mouse to detect a new or social odor habituate to that odor, and then disa habituate to that odor when presented a new odor.