This work is funded by the Swiss National Foundation (31_138429) and Synapsis Foundation for the support of research on Alzheimer&#8217;s disease.

All the animal procedures are in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes and are approved by the local Animal Care Committee (Canton of Fribourg, Switzerland).
1. Animal Preparation
<ol>
	<li>Experimental animals
	<ol>
		<li>Perform experiments on adult male wild type and transgenic mice (C57BL/6 background) of 3-5 months of age. The three groups of mice correspond to wild type littermate controls (group A, Jagged1 flox/flox19) and two conditional KO mouse lines (group B, Jagged1ncKO and C, Jagged1gcKO).</li>
		<li>House mice under standard laboratory conditions in a ventilated room, with a 12 hr controlled dark/light cycle and provide food and water ad libitum.</li>
	</ol>
	</li>
</ol>
2. Experimental Setup
<ol>
	<li>Experimental arena
	<ol>
		<li>For the experimental arena, use a clean sterilized mouse cage (36 cm length x 20.5 cm width x 13.5 cm height) (Figure 1A).</li>
		<li>Assign each mouse to a numbered cage with fresh bedding, 3 cm high. If cages are reused, as in the odor sensitivity test, take the following measures to avoid cross-contamination between odors and mice.
		<ol>
			<li>Mark the water side.</li>
			<li>Clean the narrower walls of the cages with two tissue papers sprayed with 70% ethanol, one for each side.</li>
			<li>Pile up cages according to the genotype of the mice and store temporarily under a laminar hood.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Camera
	<ol>
		<li>Mount a camera on a customized tripod with the objective at 58 cm from the bottom of the cage (Figure 1A). Fix the position of the Tripod and the cage and delimit with marks to allow for the camera to be centered on the top of the cage.</li>
		<li>Record videos at 320 pixels x 240 pixels, 15.08 frames per second as MOV files.</li>
	</ol>
	</li>
	<li>Odors
	<ol>
		<li>Resuspend the scents, when indicated, in the solvent in which they are soluble.</li>
		<li>For the preference test use peanut butter. Resuspend the peanut butter in peanut oil (10% w/v).</li>
		<li>For the avoidance test use pure 2-Methylbutyric (2-MB) acid (98%).</li>
		<li>For the sensitivity test, use female urine from the same mouse colony and background (C57BL/6).
		<ol>
			<li>For convenience collect the urine 1-2 days prior to the olfactory test. Restrain and hold the mouse under the hood with its belly above the cage grid. Under the cage grid place a plastic petri dish to collect the drops of urine.</li>
			<li>Collect the urine from each female in a 1.5 ml tube and mix all the urine samples to normalize for variability between animals. Store at -20 &#176;C until use.</li>
			<li>On the day of the experiment, thaw the urine and perform 4 dilutions in double distilled water at a dilution factor of 10 (1:10, 1:100; 1:1,000; 1:10,000).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Olfactory Testing
Note: In this protocol odors have been deliberately chosen which are perceived as strong attractants (peanut butter and female urine) or strong repellent (2-MB acid)15. It is important to carry out the preference and sensitivity tests to pleasant odors prior to the avoidance test to eliminate the possibility of any interference with the olfactory behavior. Nevertheless, for the sake of simplicity, in this paper, preference and avoidance test will be both described under the perception test. Each behavioral session starts with a habituation phase.
<ol>
	<li>Habituation Phase
	<ol>
		<li>Place the animal in the clean assigned cage and let it explore for 5 min (Figure 1B). Since the environment of the experimental cage is familiar to the home cage, this short time is enough to allow for habituation.</li>
		<li>If the sensitivity test is completed in one day, perform habituation only once before the application of the highest diluted odor. If the sensitivity test is carried out on different days, on each day a habituation phase on a new clean cage is needed.</li>
	</ol>
	</li>
	<li>Perception Test
	<ol>
		<li>After habituation, activate the camera and immediately pipette 60 &#956;l of the pleasant scent (peanut butter) and 60 &#956;l of the neutral scent (tap water) onto the opposite walls of the cage at about 10 cm from the bottom (Figure 1C).</li>
		<li>Let the mouse explore the odors for 2 min (Figure 1D). Thereafter, switch off the camera.</li>
		<li>At this point, proceed with the next mouse starting from the habituation phase. Perform the avoidance test exactly in the same way by applying 60 &#956;l of the repellent odor (2-MB acid) and 60 &#956;l of water.</li>
	</ol>
	</li>
	<li>Sensitivity test
	<ol>
		<li>Evaluate the attraction threshold of male mice to increasing concentrations of female urine in the following order: 1:10,000; 1:1,000; 1:100; 1:10 and pure urine.</li>
		<li>After habituation, expose each mouse to the highest dilution pipetted by the experimenter as previously described in 3.2.1.</li>
		<li>Record the exploratory behavior of urine versus water, within a 2 min time frame on a video camera. After all mice cohorts are tested for the highest dilution (1:10,000), expose to a higher concentration of urine, as indicated above.</li>
	</ol>
	</li>
</ol>
4. Post-hoc Data Analysis
Note: All behavioral tests described are processed post hoc following the data analysis instructions.
<ol>
	<li>Open MOV Files in ImageJ for Windows systems
	<ol>
		<li>Install Quick Time for Java using the customized settings from http://www.apple.com/quicktime/download .</li>
		<li>Install the Quick Time plugin from the ImageJ website (http://rsb.info.nih.gov/ij/plugins/qt-capture.html).</li>
		<li>Import the QTJava.zip (C:\Program Files\QuickTime\QTSystem) into the library extension of ImageJ (.ImageJ\jre\lib\ext).</li>
		<li>Copy also the QTJava.zip in the plugins folder and rename it as QTJava.jar.</li>
		<li>Install the six scripts attached in the Macros folder (ImageJ\plugins\Macros).</li>
		<li>Open ImageJ and compile and run the Quick Time plugin, thereafter close ImageJ.</li>
		<li>Reopen ImageJ and open the MOV file using File&#62; import&#62; using Quick Time.</li>
	</ol>
	</li>
	<li>Video Adjustment
	<ol>
		<li>Once the video file is opened in ImageJ, cut the video in order to obtain a constant 2 min exploration from the time the experimenter has pipetted the odorants into the cage (T0). Identify the frame corresponding to the T0 and remove the previous frames using increments of 1 (ImageJ\Image\Stacks\ToolsSlice remover). Use the same command to delete all the frames exceeding the 2 min exploration.</li>
		<li>Make sure that the cage is centered and if necessary use the Image&#62;Transform&#62;Rotate command to align it.</li>
	</ol>
	</li>
	<li>Video Processing 
	Note: Video processing is fully computer-assisted and uses macros commands accompanying this paper.
	<ol>
		<li>In order to restrict the area onto the cage of a 127 pixels x 218 pixels size run the Step 1 macro from the Plugin&#62;Macros&#62;Run command. Move the fixed rectangle over the cage (Figure 2, Step 1).</li>
		<li>Crop the area of the cage on the region of interest (ROI) using the Step 2 macro (Figure 2, Step 2).</li>
		<li>Use the Step 3 macro to extract the mouse image from the background by assigning a threshold signal, despeckling and filtering the signal variance. The output values in the Z axis plot indicate the mean grey values, corresponding to the intensity of the mouse shadow moving within the ROI of the &#8220;water chamber&#8221; during the 2 min exploration. Copy the results into a worksheet named according to the ROI in a spreadsheet file (Figure 2, Step 3).</li>
		<li>Use the Step 4 macro to extract the mean grey values of the mouse in the ROI &#8220;odor chamber&#8221;. Copy the results into a worksheet named according to the ROI in the same spreadsheet file as in 4.3.3 (Figure 2, Step 4).</li>
		<li>In order to further restrict the analysis of the mouse movement in the ROI &#8220;water perimeter&#8221; use the Step 5 macro. Copy the result onto the worksheet named according to the ROI in the same spreadsheet file as in 4.3.3 (Figure 2, Step 5).</li>
		<li>To restrict the analysis of the mouse movement in the ROI &#8220;odor perimeter&#8221; use the Step 6 macro. Copy the result onto the worksheet named according to the ROI spreadsheet file as in 4.3.3 (Figure 2, Step 6).</li>
		<li>Process all videos and check for consistency in the number of frames per animals. Here, record all animals for 1,810 frames corresponding to a 2 min exploration session.</li>
		<li>For each animal and for each ROI sort frames with mean grey values larger than 0. Divide the number of frames by the values corresponding to 1 second and obtain the seconds spent in each ROI.</li>
	</ol>
	</li>
</ol>
5. Statistical Analysis
<ol>
	<li>For each test verify homogeneity of variance within groups/ genotypes by Bartlett&#8217;s test using the formula available on http://www.real-statistics.com/one-way-analysis-of-variance-anova/homogeneity-variances/ .</li>
	<li>In the attraction and avoidance test, carry out comparisons between the times spent with water versus odor within one group using a non-directional Student&#8217;s t-test assuming equal or unequal variances depending on the results of the Bartlett&#8217;s test. Compare the times spent with the odors subtracted by the time spent with water between the genotypes by one-way ANOVA with Bonferroni&#8217;s post-hoc test.</li>
	<li>In the sensitivity test analyze the comparisons of the time spent with the odor subtracted from the time spent with water among groups at a specific dilutions of urine by one-way ANOVA with Bonferroni&#8217;s post-hoc test. Compare the sensitivity among groups to growing odor concentrations by 2-Way ANOVA with repetitions with Bonferroni&#8217;s post-hoc test.</li>
	<li>Interaction between genotypes and treatments in the attraction and avoidance test are investigated by 2-way ANOVA with Bonferroni&#8217;s post-hoc test.</li>
</ol>

<ol>
	<li>Doty, R. L., Kamath, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influences+of+age+on+olfaction:+a+review.">The influences of age on olfaction: a review.</a> Cognitive Science. 5, 20 (2014).</li><li>Mesholam, R. I., Moberg, P. J., Mahr, R. N., 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=Olfaction+in+neurodegenerative+disease:+a+meta-analysis+of+olfactory+functioning+in+Alzheimer&#8217;s+and+Parkinson&#8217;s+diseases.">Olfaction in neurodegenerative disease: a meta-analysis of olfactory functioning in Alzheimer&#8217;s and Parkinson&#8217;s diseases.</a> Archives of Neurology. 55 (1), 84-90 (1998).</li><li>Moberg, P. J., 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+Dysfunction+in+Schizophrenia:+A+Qualitative+and+Quantitative+Review.">Olfactory Dysfunction in Schizophrenia: A Qualitative and Quantitative Review.</a> Neuropsychopharmacology. 21 (3), 325-340 (1999).</li><li>Kov&#225;cs, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+olfactory+dysfunction+in+aging+and+neurodegenerative+disorders.">Mechanisms of olfactory dysfunction in aging and neurodegenerative disorders.</a> Ageing Research Reviews. 3 (2), 215-232 (2004).</li><li>Barrios, F. 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=Olfaction+and+neurodegeneration+in+HD.">Olfaction and neurodegeneration in HD.</a> Neuroreport. 18 (1), 73-76 (2007).</li><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=Olfaction+in+Parkinson&#8217;s+disease+and+related+disorders.">Olfaction in Parkinson&#8217;s disease and related disorders.</a> Neurobiology of Disease. 46 (3), 527-552 (2012).</li><li>Doty, R. L., Shaman, P., Dann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+University+of+Pennsylvania+Smell+Identification+Test:+a+standardized+microencapsulated+test+of+olfactory+function.">Development of the University of Pennsylvania Smell Identification Test: a standardized microencapsulated test of olfactory function.</a> Physiology &amp; Behavior. 32 (3), 489-502 (1984).</li><li>Devanand, D. p., 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+Deficits+in+Patients+With+Mild+Cognitive+Impairment+Predict+Alzheimer&#8217;s+Disease+at+Follow-Up.">Olfactory Deficits in Patients With Mild Cognitive Impairment Predict Alzheimer&#8217;s Disease at Follow-Up.</a> American Journal of Psychiatry. 157 (9), 1399-1405 (2000).</li><li>Conti, M. Z., 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=Odor+Identification+Deficit+Predicts+Clinical+Conversion+from+Mild+Cognitive+Impairment+to+Dementia+Due+to+Alzheimer&#8217;s+Disease.">Odor Identification Deficit Predicts Clinical Conversion from Mild Cognitive Impairment to Dementia Due to Alzheimer&#8217;s Disease.</a> Archives of Clinical Neuropsychology. 28 (5), 391-399 (2013).</li><li>Keller, A., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+Smelling+Through+Genetics:+Mammalian+Odor+Perception.">Better Smelling Through Genetics: Mammalian Odor Perception.</a> Current opinion in neurobiology. 18 (4), 364-369 (2008).</li><li>Brai, 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=Notch1+activity+in+the+olfactory+bulb+is+odour-dependent+and+contributes+to+olfactory+behaviour.">Notch1 activity in the olfactory bulb is odour-dependent and contributes to olfactory behaviour.</a> European Journal of Neuroscience. 40 (10), 3436-3449 (2014).</li><li>Larson, J., Hoffman, J. S., Guidotti, A., Costa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+discrimination+learning+deficit+in+heterozygous+reeler+mice.">Olfactory discrimination learning deficit in heterozygous reeler mice.</a> Brain Research. 971 (1), 40-46 (2003).</li><li>Alonso, 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=Olfactory+Discrimination+Learning+Increases+the+Survival+of+Adult-Born+Neurons+in+the+Olfactory+Bulb.">Olfactory Discrimination Learning Increases the Survival of Adult-Born Neurons in the Olfactory Bulb.</a> The Journal of Neuroscience. 26 (41), 10508-10513 (2006).</li><li>Wesson, D. W., Keller, M., Douhard, Q., Baum, M. J., Bakker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+urinary+odor+discrimination+in+female+aromatase+knockout+(ArKO)+mice.">Enhanced urinary odor discrimination in female aromatase knockout (ArKO) mice.</a> Hormones and behavior. 49 (5), 580-586 (2006).</li><li>Kobayakawa, K., 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=Innate+versus+learned+odour+processing+in+the+mouse+olfactory+bulb.">Innate versus learned odour processing in the mouse olfactory bulb.</a> Nature. 450 (7169), 503-508 (2007).</li><li>Witt, R. M., Galligan, M. R., Despinoy, J., Segal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Behavioral+Testing+in+the+Adult+Mouse.">Olfactory Behavioral Testing in the Adult Mouse.</a> Journal of Visualized Experiments JoVE. (23), (2009).</li><li>Lee, A. W., Emsley, J. G., Brown, R. E., Hagg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marked+differences+in+olfactory+sensitivity+and+apparent+speed+of+forebrain+neuroblast+migration+in+three+inbred+strains+of+mice.">Marked differences in olfactory sensitivity and apparent speed of forebrain neuroblast migration in three inbred strains of mice.</a> Neuroscience. 118 (1), 263-270 (2003).</li><li>Page, D. T., 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=Computerized+assessment+of+social+approach+behavior+in+mouse.">Computerized assessment of social approach behavior in mouse.</a> Frontiers in Behavioral Neuroscience. 3, 48 (2009).</li><li>Nyfeler, Y., 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=Jagged1+signals+in+the+postnatal+subventricular+zone+are+required+for+neural+stem+cell+self-renewal.">Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal.</a> Embo J. 24 (19), 3504-3515 (2005).</li><li>Tong, M. T., Peace, S. T., Cleland, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+mechanisms+of+olfactory+learning+and+memory.">Properties and mechanisms of olfactory learning and memory.</a> Frontiers in Behavioral Neuroscience. 8, (2014).</li><li>Corthell, J., Stathopoulos, A., Watson, C., Bertram, R., Trombley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Bulb+Monoamine+Concentrations+Vary+with+Time+of+Day.">Olfactory Bulb Monoamine Concentrations Vary with Time of Day.</a> Neuroscience. 247, 234-241 (2013).</li><li>Lehmkuhl, A. M., Dirr, E. R., Fleming, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+assays+for+mouse+models+of+neurodegenerative+disease.">Olfactory assays for mouse models of neurodegenerative disease.</a> Journal of Visualized Experiments: JoVE. (90), e51804 (2014).</li></ol>

There is no conflict of interest.

The tests proposed in this protocol allow to evaluate different aspects of innate olfactory behavior in mice: perception to odors, discrimination between odors versus water and sensitivity to odors. This protocol can be applied to any odor according to the preference and avoidance scale previously shown15. Since the protocol is based on exploratory activity it is important that mice do not display any motor impairment or anxiety which may affect their movement and interfere with olfactory exploration. The test...

Olfaction is one of the most developed and important sensory functions in mammals. Any impairment in olfactory activity may affect food intake, social behavior and, in the worst case scenario, even survival. In humans, olfactory deterioration is age dependent1 and is considered a strong predictor of neurological disorders2&#8211;6. The olfactory identification test developed by the University of Pennsylvania currently represents one of the most used, non-invasive and quantifiable, diagnostic tests which can assess early neurological deficits7 and predict with high probability the progression of dementia8,9.
The accessibility of the olfactory system and the prominence of olfaction in rodents, has sparked an intense line of research addressing the mechanisms underlying olfactory functions10. We have previously shown that loss of function of the signaling receptor Notch1 affects olfactory avoidance11. In this protocol we use mice lacking the signaling ligand, Jagged1, in neurons or glia to study olfactory performance.
Innate olfaction is defined by three parameters as perception, discrimination between odors and olfactory sensitivity4. Olfactory testing in rodents can be done in a variety of ways and some behavioral studies make use of olfactometers, which provide the odor to the animal at a specific vapor concentrations and in a precise time frame12&#8211;14. Nevertheless, this instrumentation is expensive and may be available only in specialized facilities. In our work, we provide a simple, fast and reproducible olfactory testing protocol, which is carried out using volatile scents. The tests described measure perception to an attractant or a repellent odor and evaluate the discrimination between the scent and water11,15,16. Using the same setup, we also can measure the sensitivity to an odor at different concentrations16,17. The post-hoc computer-assisted video processing, inspired by the work of Page and colleagues18, provides unbiased results without the need of experimental blinding and allowing for a single person to carry out the whole experiment.
This protocol is intended to provide a starting point for studying olfactory behavior in mice.

<table border="0" cellpadding="0" cellspacing="0" style="border-collapse:
 collapse;width:192pt">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr>
			<td class="xl63">Mouse cage</td>
			<td class="xl64">Italplast (Italy)</td>
			<td class="xl64">1144B</td>
			<td class="xl64">36 cm length x 20.5 cm width x 13.5 cm height</td>
		</tr>
		<tr>
			<td class="xl63">Chipped wood bedding</td>
			<td class="xl64">Abedd (Austria)</td>
			<td class="xl64">LTE E-001</td>
			<td class="xl64">3 cm high</td>
		</tr>
		<tr>
			<td class="xl63">Peanut butter</td>
			<td class="xl64">Migros (Swizterland)</td>
			<td class="xl64">NA</td>
			<td align="right" class="xl65">1:10</td>
		</tr>
		<tr>
			<td class="xl63">2-Methylbutyric</td>
			<td class="xl64">Sigma Aldrich (Switzerland)</td>
			<td class="xl64">W269514</td>
			<td class="xl64">Pure</td>
		</tr>
		<tr>
			<td class="xl63">Female Urine from fertile females of same mouse strain</td>
			<td class="xl64">NA</td>
			<td class="xl64">NA</td>
			<td class="xl64">Dilution series</td>
		</tr>
		<tr>
			<td class="xl63">Camera</td>
			<td class="xl64">Olympus (US)</td>
			<td class="xl64">Camedia C-8080</td>
			<td class="xl64">MOV files</td>
		</tr>
		<tr>
			<td class="xl63">Quicktime for Java (Windows)</td>
			<td class="xl64">Apple (USA)</td>
			<td class="xl64">NA</td>
			<td class="xl64">video plugin for visualizing MOV files</td>
		</tr>
		<tr>
			<td class="xl63">Image J for Windows</td>
			<td class="xl64">NIH (USA)</td>
			<td class="xl64">NA</td>
			<td class="xl64">Video Processing/Analysis</td>
		</tr>
	</tbody>
</table>

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.

The perception test measures the attraction to peanut butter and avoidance to 2-MB acid. Three groups of mice are tested and the time spent in the &#8220;odor perimeter&#8221; are quantified as compared to water. In the preference test, the control group A displays significant preference to the odor as compared to water (t8 = 2.52, p &#60;0.05). On the other hand, group B does not show any significant attraction to peanut butter and spends more time with water (t6 = 3.22, p &#60;0.05). Thus, it beha...

Watch this Scientific Journal Video about Simple and Computer-assisted Olfactory Testing for Mice at JoVE.com

Simple and Computer-assisted Olfactory Testing for Mice

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 ...

simple-and-computer-assisted-olfactory-testing-for-mice

Research

JoVE Journal

Behavior

9.9K Views.  University of Fribourg. 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. 

Video: Simple and Computer-assisted Olfactory Testing for Mice

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 ...

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.

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 ...

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, ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Task Interruption and Resumption Paradigm for Testing the Activation and Pursuit of an Abstract Thinking Goal

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a higher resumption rate after a period of delay or interruption. The task interruption and resumption protocol described in this research is tailored to test the activation of cognitive goals (e.g., a goal to think more abstractly). Cognitive goals may be pursued even during the interruption period; thus, to prevent this, the protocol involves cognitive distraction. The protocol consists of several stages. Specifically, the initial stage includes the goal activation process, where the treatment (versus control) condition receives a manipulation to activate the cognitive goal being tested by the researcher. In the next stage, participants are presented with the introduction of a task that is perceived to either satisfy or not satisfy the cognitive goal of interest. Importantly, this task is interrupted a few seconds after it begins. The task interruption forces a delay period and introduces a cognitive distraction to prevent the automatic pursuit and fulfillment of the cognitive goal. After the interruption period, participants are given a choice between resuming the interrupted task and abandoning the interrupted task to complete an alternative task instead. Among participants whose cognitive goals had been activated at the earlier stage, the task resumption rate should be higher if the task was perceived as an opportunity to satisfy (versus not satisfy) the goal. Such a finding would provide empirical evidence that the cognitive goal has been activated and pursued. In previous research, this protocol has been used to test whether causal uncertainty activates an abstract thinking goal. Adapting the protocol to test the activation of other cognitive goals is also discussed.

This protocol was designed to test the activation and pursuit of cognitive goals (e.g., an abstract thinking goal) using the task interruption and resumption paradigm. The protocol is suitable for cognitive goals that are automatically pursued once activated, as the distraction procedure prevents goal pursuit during the interruption period.

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

A Simple Non-invasive Method for Temporary Knockdown of Upper Limb Proprioception

Proprioception may be the least well measured of all contributors to the neural control of movement. New precise, reliable measures of proprioception are needed for clinical diagnosis of impairment, and to measure outcomes of proprioceptive training. The purpose of this simple, non-invasive method is to temporarily knockdown upper limb proprioception in healthy adults, to an extent that would be useful in the development and testing of upper limb proprioception measures. Knockdown models have two main advantages over studying humans with impaired proprioception: participant availability and the ability to control the extent of impairment across participants. Current published methods of temporary proprioception knockdown of the upper limb, such as ischemic nerve blocks and cryotherapy, are invasive, impractical, or uncomfortable for the participant. Here, vibration over the ulnar groove was used to reduce upper limb proprioception. High frequency vibration may reduce proprioceptive acuity by inhibiting pacinian corpuscle-induced input. The effect of vibration used in this protocol was confirmed using two quantitative measures. This method was simple to administer, comfortable for participants, and practical.

The goal of this protocol is to demonstrate a practical method to temporarily interfere with proprioception in the upper limb of healthy humans.

Proprioception may be the least well measured of all contributors to the neural control of movement. New precise, reliable measures of proprioception are ...

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 ...

Computerized Adaptive Testing System of Functional Assessment of Stroke

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the upper and lower extremities, postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency. CAT, a modern measurement method, aims to provide a reliable estimate of the examinee's level of function rapidly. CAT administers only a few items whose item difficulties match an examinee's level of function and, thus, the administered items of CAT can provide sufficient information to reliably estimate the examinee's level of function in a short time. The CAT-FAS was developed through four steps: (1) determining the item bank, (2) determining the stopping rules, (3) validating the CAT-FAS, and (4) establishing a platform of online administration. The results of this study indicate that the CAT-FAS has sufficient administrative efficiency (average number of items = 8.5) and reliability (group-level Rasch reliability: 0.88 - 0.93; individual-level Rasch reliability: &#8805;70% of patients had Rasch reliability score &#8805;0.90) to simultaneously assess four functions in patients with stroke. In addition, because the CAT-FAS is a computer-based test, the CAT-FAS has three additional advantages: the automatic calculation of scores, the immediate storage of data, and the easy exporting of data. These advantages of the CAT-FAS will be beneficial to data management for clinicians and researchers.

Here, we present a protocol to develop the computerized adaptive testing system of the functional assessment of stroke (CAT-FAS). The CAT-FAS can simultaneously assess four functions (two motor functions [upper and lower extremities], postural control, and basic activities of daily living) with sufficient reliability and administrative efficiency.

The computerized adaptive testing system of the functional assessment of stroke (CAT-FAS) can simultaneously assess four functions (motor functions of the ...

Olfactory Context Dependent Memory: Direct Presentation of Odorants

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed context dependent memory (CDM). One category of cues that have been shown to effectively produce CDM effects are odors. However, it is unclear what the boundary conditions of these CDM effects are. In particular, given that olfaction has been called an implicit sense, it is possible that odors are only effective mnemonic cues when they are presented in the background. This assertion seems even more likely given that previous research has shown odors to be poor cues during paired associate memory tests, where odors are in the focus of attention as mnemonic cues for other information. In order to determine whether odors are only effective contextual mnemonic cues when presented outside the central focus of an observer, an olfactory CDM experiment was performed in which odorants were presented directly, rather than ambiently. Direct presentation was accomplished with the aid of an olfactometer. The olfactometer not only allows for direct presentation of odorants, but provides other methodological benefits, including the allowance of trial by trial manipulations of odorant presentations and, relatedly, time-specific releases of odorants. The presence of the same odor during both encoding and retrieval enhanced memory performance, regardless of whether the odor was presented ambiently or directly. This finding can serve as a basis for future olfactory CDM research which can utilize the benefits of direct presentation.

The use of an olfactometer for directly presenting odorants opens exciting opportunities for researchers of olfactory memory. The current paper discusses issues related to this methodology as related to a previously published experiment on olfactory context dependent memory.

Information is retrieved more effectively when the retrieval occurs in the same context as that in which the information was first encoded. This is termed ...