This work is supported by a Baylor University Research Council grant and by a research grant from the Epilepsy Foundation.

The adult male mice used in the following experiment were generated and housed at Baylor University at an ambient temperature of 22 &#176;C, with a 14&#160;hr light and 10&#160;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 morn...

<ol>
	<li>Doty, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor-guided+behavior+in+mammals.">Odor-guided behavior in mammals.</a> Experientia. 42, 257-271 (1986).</li><li>Restrepo, D., Arellano, J., Oliva, A. M., Schaefer, M. L., Lin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+views+on+the+distinct+but+related+roles+of+the+main+and+accessory+olfactory+systems+in+responsiveness+to+chemosensory+signals+in+mice.">Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice.</a> Horm Behav. 46, 247-256 (2004).</li><li>Keverne, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+olfactory+and+vomeronasal+systems+for+male+sexual+function.">Importance of olfactory and vomeronasal systems for male sexual function.</a> Physiol Behav. 83, 177-187 (2004).</li><li>Gregg, B., Thiessen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+olfactory+discrimination+of+urines+for+the+Mongolian+gerbil,+Meriones+unguiculatus.">A simple method of olfactory discrimination of urines for the Mongolian gerbil, Meriones unguiculatus.</a> Physiol Behav. 26, 1133-1136 (1981).</li><li>Yang, M., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+behavioral+assessment+of+mouse+olfaction.">Simple behavioral assessment of mouse olfaction.</a> Curr Protoc Neurosci. Chapter 8 (Unit 8 24), (2009).</li><li>Pan, Y. W., Kuo, C. T., Storm, D. R., Xia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+and+targeted+deletion+of+the+ERK5+MAP+kinase+in+adult+neurogenic+regions+impairs+adult+neurogenesis+in+the+olfactory+bulb+and+several+forms+of+olfactory+behavior.">Inducible and targeted deletion of the ERK5 MAP kinase in adult neurogenic regions impairs adult neurogenesis in the olfactory bulb and several forms of olfactory behavior.</a> PLoS ONE. 7, e49622 (2012).</li><li>Wesson, D. W., Levy, E., Nixon, R. A., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+dysfunction+correlates+with+amyloid-beta+burden+in+an+Alzheimer's+disease+mouse+model.">Olfactory dysfunction correlates with amyloid-beta burden in an Alzheimer's disease mouse model.</a> J Neurosci. 30, 505-514 (2010).</li><li>Fadool, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.3+channel+gene-targeted+deletion+produces+'Super-Smeller+Mice'+with+altered+glomeruli,+interacting+scaffolding+proteins,+and+biophysics.">Kv1.3 channel gene-targeted deletion produces 'Super-Smeller Mice' with altered glomeruli, interacting scaffolding proteins, and biophysics.</a> Neuron. 41, 389-404 (2004).</li><li>Arakawa, H., Arakawa, K., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scent+marking+behavior+in+male+C57BL/6J+mice:+sexual+and+developmental+determination.">Scent marking behavior in male C57BL/6J mice: sexual and developmental determination.</a> Behav Brain Res. 182, 73-79 (2007).</li><li>Baum, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+differentiation+of+pheromone+processing:+links+to+male-typical+mating+behavior+and+partner+preference.">Sexual differentiation of pheromone processing: links to male-typical mating behavior and partner preference.</a> Horm Behav. 55, 579-588 (2009).</li><li>Ferguson, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+amnesia+in+mice+lacking+the+oxytocin+gene.">Social amnesia in mice lacking the oxytocin gene.</a> Nat Genet. 25, 284-288 (2000).</li><li>Yang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+sociability+in+BTBR+T+tf/J+mice+is+independent+of+partner+strain.">Low sociability in BTBR T+tf/J mice is independent of partner strain.</a> Physiol Behav. 107, 649-662 (2012).</li><li>Best, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-associative+learning+in+larval+zebrafish.">Non-associative learning in larval zebrafish.</a> Neuropsychopharmacology. 33, 1206-1215 (2008).</li><li>Wolman, M. A., Jain, R. A., Liss, L., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+memory+formation+in+larval+zebrafish.">Chemical modulation of memory formation in larval zebrafish.</a> Proc Natl Acad Sci U S A. , (2011).</li><li>Sorge, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+exposure+to+males,+including+men,+causes+stress+and+related+analgesia+in+rodents.">Olfactory exposure to males, including men, causes stress and related analgesia in rodents.</a> Nat Methods. 11, 629-632 (2014).</li></ol>

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 ...

Mice are dependent on olfaction for navigating new environments, finding food, for recognizing other individuals, and sexual behavior1-3. 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&#8217;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&#8217;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 odors6,7. 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 &#8220;super smellers&#8221; 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&#8217;s response to social odor stimuli or if their response increases to all odor stimuli.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse cage</td>
			<td>Allentown</td>
			<td>Standard mouse cage</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Wire lid</td>
			<td rowspan="2">Allentown</td>
			<td>BCU Mouse WBL 2500</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>Bedding</td>
			<td>Harlan</td>
			<td>7090 Sani-Chips</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>VWR</td>
			<td>89031-270</td>
			<td>6&#8221; wooden handle</td>
		</tr>
		<tr>
			<td>Banana extract</td>
			<td>McCormick</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Almond extract</td>
			<td>McCormick</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory tape</td>
			<td>VWR</td>
			<td>89098-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Stop watch</td>
			<td>VWR</td>
			<td>62374-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>VWR</td>
			<td>82026</td>
			<td></td>
		</tr>
		<tr>
			<td>Timing device</td>
			<td>VWR</td>
			<td>61161-350</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>89003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L beakers&#160;</td>
			<td>Pyrex</td>
			<td align="right">1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm</td>
			<td>PM-992</td>
			<td>4&#8221; x 250&#8217;</td>
		</tr>
		<tr>
			<td>1 L bottle with cap</td>
			<td>VWR</td>
			<td>89000-240</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing for odor discrimination and habituation in mice

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&#8217;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.

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...

Watch this Scientific Journal Video about Testing for Odor Discrimination and Habituation in Mice at JoVE.com

Testing for Odor Discrimination and Habituation in Mice

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.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

testing-for-odor-discrimination-and-habituation-in-mice

Research

JoVE Journal

Behavior

17.3K Views.  Baylor University. 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. 