Testing for Odor Discrimination and Habituation in Mice

Summary

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

Abstract

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal’s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning.

In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

Introduction

Mice are dependent on olfaction for navigating new environments, finding food, for recognizing other individuals, and sexual behavior^1-3. It is essential that investigators establish whether or not experimental animals have a functioning sense of smell before administering behavioral tests that involve food, social interaction, or any odors intended to elicit a response from the mouse. Anosmia or the inability to distinguish between different odors, could result in false positives or negatives in a wide variety of behavioral paradigms, so a mouse’s ability to detect and distinguish odors should be established before other types of behavioral tests are performed.

The olfactory habituation/dishabituation test was first described in the 1980s⁴. The test has been adapted for use in mice by Drs. Mu Yang and Jacqueline Crawley⁵. This is a simple and inexpensive test that allows the investigator to establish that a mouse can detect and differentiate between odors. In addition to testing olfaction, this test allows the investigator to observe the general behavior of the mouse and should be done early in a testing regimen. Qualitative observations regarding the mouse’s locomotion, signs of anxiety, level of activity, and response to social odors versus food odors may signal new areas in which testing could be carried out.

In this test, cotton swabs dipped into various odors are presented to a mouse three times in a row. With each repeated presentation of an odor, the mouse will habituate to the cotton swab, spending less time investigating it with each subsequent presentation. When a new odor is presented, dishabituation occurs, and a typical mouse will spend more time investigating the swab, indicating that it can discriminate between the current and previous odors⁵. This test is administered to one mouse at a time and includes a 45 min acclimation period followed by 45 min of testing.

Although this test is easy to carry out it may be used to investigate sophisticated questions about the mouse olfactory system. Other popular tests of olfaction, such as the buried food test, simply establish the presence or absence of anosmia. However, the olfactory discrimination and habituation test allows the investigator to determine that a mouse not only has the ability to detect odors but can discriminate between different odors. The pattern of habituation and dishabituation has been used to establish that new mutants can discriminate between odors^6,7. In a surprising study, Fadool and colleagues used complex mixtures of odors to show that mice with gene-targeted deletion of the Kv1.3 channel are “super smellers” that can discriminate very similar odors better than normal mice⁸.

When examining a new knockout mouse model it is useful to establish the presence of normal behavior for basic sensory tasks. When done early in a testing regimen, the odor discrimination and habituation test gives an investigator the opportunity to observe any unusual behaviors. These observations could prevent false positive or negative results in subsequent testing that are attributable to confounding characteristics of the mutant. As researchers continue to investigate social behavior the need to verify basic olfactory function becomes increasingly important. In addition to examining olfaction in mutant lines, this test can be used to address specific questions such as whether a pharmacological treatment specifically increases an animal’s response to social odor stimuli or if their response increases to all odor stimuli.

Protocol

The adult male mice used in the following experiment were generated and housed at Baylor University at an ambient temperature of 22 °C, with a 14 hr light and 10 hrs dark (20:00 to 6:00 hr) diurnal cycle. The mice were given ad libitum access to food and water. All procedures to the mice were in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the animal protocol was approved by Baylor University Animal Care and Use Committee. Mice were tested in the late morning/early afternoon. For all procedures below the investigator should wear gloves to eliminate odors and prevent contamination.

1. Preparation of Non-social Odors

1. Prepare fresh non-social odors each day of testing.
   Note: The investigator should use gloves to prepare the odors.
   1. Fill 3 centrifuge tubes with 10 ml of water each.
   2. Pipette 100 µl of banana extract into a 15 ml conical tube and 100 µl of almond extract into a second 15 ml conical tube for a 1:100 dilution. 
      Note: The third 15 ml tube will only contain water.

2. Preparation of Social Odors

1. Prepare fresh social odors on the morning testing will occur. Obtain social odors from two cages that have not been cleaned for at least three days and contain equal numbers of mice. Obtain social odors from mice of the same sex as the test subject.
   Note: These are to be stored at RT and should be used within 4-6 hr to maintain strong and consistent odor.
   1. Determine how many social odor cotton swabs need to be created: each mouse being tested will be presented with three swabs from social odor 1, and three from social odor 2.
   2. Prepare social odors by wiping a cotton swab in a zigzag fashion across the bottom of a dirty cage. Shake off any excess bedding clinging to the tip.
   3. Store swabs in large glass jars with lids or in beakers covered with parafilm. Designate one container for social odor 1 and a second for social odor 2.
2. Maintain odor source cages outside of the experimental testing room.

3. Acclimation of the Test Mouse

1. Put on gloves and weigh the mouse to be tested and place individually in a clean cage with a wire lid on top and the water bottle removed.
2. Allow the mouse to acclimate to the new cage for 45 min in a room other than the testing room. Ensure that there are no unusual odors or noises in the room.
3. As testing takes about 45 min, stagger the acclimation times by putting the next mouse to be tested into a clean cage to acclimate right before testing the current mouse.

4. Preparation of the Testing Room

1. Place fresh cotton swabs, laboratory tape, the bottles containing social odor swabs, the tubes of non-social odors, a micropipette, two timers, and a waste bottle with a lid in the testing room.
2. Ensure that the testing room is reasonably free of odors, loud noises, and bright light. Do not wear scented colognes or lotions.
   Note: The observer should not be chewing gum or candy during the testing period and should try to limit their exposure to strong odor foods.

5. Testing Procedure

1. Bring the acclimated mouse in its cage into the testing room and place the cage on the counter at a height that is comfortable for viewing while seated.
2. Present each odor 3 times in a row for 2 min each time. Use the presentation order that can be found in Table 1.
   1. Pipette 50 µl of odorant in the scent to be tested on to the tip of the cotton swab and re-cap the odor source. Lift the wire top of the cage and insert the unscented, wooden end of the swab into the water bottle opening, pushing the length through from the underside of the wire to the top. Avoid touching the cotton tip to anything, including fingers, so that odor contamination is avoided.
   2. Continue to draw the swab through the water bottle opening until about 1 inch of the cotton end hangs down into the cage. Affix the wooden end of the swab to the side of the cage using lab tape.
      Note: Be careful not to let the lid fall back onto the cage, causing a loud noise that would disturb the test mouse.
   3. Immediately begin timing for 2 min. Use one timer to time the 2 min presentation. Record the cumulative time spent sniffing the odor during the 2 min trial. Count the animal’s sniffing behavior using a separate stop watch. Active sniffing is defined when the animal’s nose is oriented towards the cotton tip with its nose ~2 cm or closer to the tip.
   4. After the completion of each trial, raise the wire lid and remove the cotton swab by pulling it downwards through the underside of the lid (this avoids odor contamination by preventing the cotton tip from touching the wire). Seal the used swab in a 500 ml waste bottle.
   5. Prepare and insert the next swab and begin timing. Allow approximately 1 min between each inter-trial interval. Continue until each scent has been presented three times in a row, for 2 min each time.
3. Once the mouse has completed all trials, empty the waste bottle containing the used cotton swabs in a trash can outside of the test room. Return the mouse to its home cage.

6. Analysis

1. Analyze data for each group per odor condition using two-way ANOVA followed by Newman-Keuls or a similar post hoc test to determine significant habituation (less time sniffing successive same smells), and dishabitutation (more time sniffing a new smell).

Results

Following the protocol described, results were recorded using ten adult (postnatal day 90-120) male C57BL/6J mice and eight male FVB mixed-background strain (postnatal day 50-70). Testing was carried out by a single investigator over multiple days. Each point on the graphs represents the average time spent investigating an odor.

When the cotton swab dipped in water is first introduced, the mice spend a great deal of time investigating this novel object (Figure 1: first three d...

Discussion

The results provided in this paper are optimal for mice. The mice demonstrate a strong response to a novel odor, then quickly habituate. One of the key steps in this method is the preparation of the odors. The investigator needs to take great care to isolate the odors from one another to prevent contamination. Another key aspect is the presentation of the cotton swab. The investigator needs to troubleshoot the best location of the cotton tip so the mouse can investigate the tip but does not tear at the tip. An important ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mouse cage	Allentown	Standard mouse cage
Wire lid	Allentown	BCU Mouse WBL 2500
Bedding	Harlan	7090 Sani-Chips
Cotton swabs	VWR	89031-270	6” wooden handle
Banana extract	McCormick
Almond extract	McCormick
Laboratory tape	VWR	89098-062
Stop watch	VWR	62374-000
Nitrile gloves	VWR	82026
Timing device	VWR	61161-350
15 ml conical tubes	VWR	89003-294
2 L beakers	Pyrex	1003
Parafilm	Parafilm	PM-992	4” x 250’
1 L bottle with cap	VWR	89000-240