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 morning/early afternoon. For all procedures below the investigator should wear gloves to eliminate odors and prevent contamination.
1. Preparation of Non-social Odors
<ol>
	<li>Prepare fresh non-social odors each day of testing. 
	Note: The investigator should use gloves to prepare the odors.
	<ol>
		<li>Fill 3 centrifuge tubes with 10 ml of water each.</li>
		<li>Pipette 100 &#181;l of banana extract into a 15 ml&#160;conical tube and 100 &#181;l&#160;of almond extract into a second 15 ml&#160;conical tube for a 1:100 dilution.&#160; 
		Note: The third 15 ml&#160;tube will only contain water.</li>
	</ol>
	</li>
</ol>
2. Preparation of Social Odors
<ol>
	<li>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.
	<ol>
		<li>Determine how many social odor cotton swabs need&#160;to be created: each mouse being tested will be presented with three swabs from social odor 1, and three from social odor 2.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Maintain odor source cages outside of the experimental testing room.</li>
</ol>
3. Acclimation of the Test Mouse
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Preparation of the Testing Room
<ol>
	<li>Place fresh cotton swabs, laboratory tape, the bottles containing social odor swabs, the tubes of non-social odors, a micropipette, two timers,&#160;and a waste bottle with a lid in the testing room.</li>
	<li>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.</li>
</ol>
5. Testing Procedure
<ol>
	<li>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.</li>
	<li>Present each odor 3 times in a row for 2 min each time. Use the presentation order that can be found in Table 1.
	<ol>
		<li>Pipette 50 &#181;l&#160;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.</li>
		<li>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.</li>
		<li>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&#8217;s sniffing behavior using a separate stop watch. Active sniffing is defined when the animal&#8217;s nose is oriented towards the cotton tip with its nose ~2 cm or closer to the tip.</li>
		<li>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&#160;waste bottle.</li>
		<li>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&#160;each time.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
6. Analysis
<ol>
	<li>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).</li>
</ol>

<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 authors have nothing to disclose.

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 ...

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17.5K 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. 

Video: Testing for Odor Discrimination and Habituation in Mice

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.