Journal of Visualized Experiments www.jove.com Video Article Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results. Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

General Health Exam
A more comprehensive treatment of general health exams can be found in Jacqueline Crawley's excellent behavioral phenotyping resource, What's Wrong with my Mouse? 1 . We first use a modified observational test battery to assess the general health of the subject. The battery includes items from Samuel Irwin's comprehensive assessment protocol 2 to quickly identify those subjects that will be unsuitable for further testing. In brief, the modified battery identifies animals that are physically abnormal (eg. severely under-or over-weight, poorly groomed, dyspneic) or that exhibit abnormal posture, locomotion or behaviors. These all could be manifestations of illness. Any initial evaluation that similarly identifies unhealthy subjects is appropriate.
It is also important to note that animals are used humanely both during the general health exam as well as during subsequent tests. No anesthesia or analgesia is used as these tests do not incorporate any potentially painful procedures.

Olfactory Sensitivity Testing
This test is designed to detect a difference in olfactory sensitivity between two groups of mice (eg. a wild type and a mutant group of animals). In this test, multiple dilutions of a scent are made. A naïve animal then investigates a single dilution of this scent and the total exploratory time (within 3 minutes) is recorded. Multiple dilutions of the scent are tested on different, naïve subjects and the total exploratory time is used to generate dose response curves for each group of mice. Two researchers should be involved in testing, with one blind to subject genotypes and/or condition.  11. If only one scent (eg. cinnamon) is to be tested, remove the first test subject from the arena. Repeat steps 6-11 for each animal, appropriately diluting the scent for each subject. If more than one scent is to be tested, however, do not remove the test subject from the arena. Instead, begin timing. 12. Appropriately dilute the next scent using the same methods as listed above. 13. After 1 minute, begin video recording. Place the filter paper square with the dilution of the next scent in the cage on the opposite end of the animal's current position. 14. Repeat steps 6-13 for each scent, noting the video file number for each trial. 15. Repeat steps 6-14 for each subject.

Figure 2. Representative results for a session of olfactory sensitivity testing.
Each mouse from either the control group (Group A) or the experimental group (Group B) was exposed to one of five different dilutions of cinnamon extract on filter paper for one, three minute session. Total time spent exploring the filter paper was assessed. Mice in Group A have a greater olfactory sensitivity than those in Group B.

Blind Researcher
The procedure for the olfactory preference testing is the same, with slight modifications of steps 2, 4 and 13. Please follow steps 1-15 in the Olfactory Sensitivity Testing Section for the blind researcher, with the following changes: For step 2, using the pre-selected scents (a combination of both attractive and aversive scents), make appropriately diluted solutions of each in conical tubes. For hydrophobic samples such as peanut butter, use oil as solvent; for all others, use distilled water. Vortex. For step 4, rather than recording the dilution to be tested, record the order of the varied scents that will be tested, randomizing the order in each trial. For step 13, place the filter paper square with the next scent in the cage on the opposite end of the animal's current position. Figure 3. Representative results for a session of olfactory preference testing. Each mouse was exposed to each of the different scents on filter paper for one, three minute session. Total time spent exploring the filter paper was assessed. Peanut butter and vanilla are attractive scents (exploratory timescent > exploratory timewater) and 2-Methylbutyric Acid is aversive (exploratory timescent < exploratory timewater).

Scoring of the Olfactory Tests
After acquiring all of the olfactory sensitivity and preference videos, it is necessary to analyze the total time spent exploring each dilution/scent. Have the blind researcher analyze all of the videos for consistency.

Discussion
The aforementioned tests allow for the quick and reliable assessment of basic olfactory function. It is not to be overlooked, however, that this reliability requires the proper selection and preparation of subjects for testing. Animals that are ill or otherwise incapacitated are often inconsistent test subjects. Also, habituation of the test subjects is key. We have found that the use of multiple cages during the habituation process allows the animals to become comfortable with experimenter handling and changing surroundings. We have also found that times shorter than those indicated for habituation are not sufficient to reduce anxiety. Anxious animals also are often unpredictable test subjects and can obscure true differences.
In the actual testing of the mice, it is important not only to be consistent when acquiring the data, but also in analyzing the subjects' behavior. We have found it useful to only count that time when the test subject's nose is <1 mm from the filter paper as part of the total exploratory time. Also, behaviors such as biting or chewing the filter paper are not included in the total exploratory time. In addition, latency time (for attractive scents) can also be a useful metric. Different mice backgrounds may require different dilutions of scents for effective discrimination between a control and experimental group. It is also important to sex-match and age-match pairs. We found it helpful to empirically derive appropriate scent dilutions (on subjects similar to the control group) before proceeding with the experimental testing of naïve mice. Finally, these tests were selected to minimize the effect of any other cognitive impairments, most notably, defects in learning and/or memory. To that end, it is critical to use naïve mice for testing.