Name: simple and computer-assisted olfactory testing for mice
Uploaded: 2015-06-15T15:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Simple and Computer-assisted Olfactory Testing for Mice at JoVE.com

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>

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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 tests described are intended for adult male mice however they can be adapted to investigate olfaction also in adult females or aged mice.
Before commencing such a study investigating olfaction in mice it is important to pay attention to the following aspects: 1) perform each test at an interval of at least 3 days. Avoidance should be tested as last to minimize interference of olfactory memory20; 2) perform the experiments at the same time of the day, preferably in the late afternoon, when the mice are in their active cycle21 and use a dimmed source of light. In addition, scheduling the olfactory testing at defined times controls for possible circadian changes in olfactory functions22; 3) before starting the avoidance test, which uses repellent odorants, such as acids, bring one cage at the time in the experimental suite and keep the cage under a laminar hood. This step is important to avoid habituation to the odorant and obtain a more homogeneous response in the same group; 4) temporarily separate the mice which have been tested until all mice of the same cage are exposed to the odorant, to minimize odorant contamination; 5) use animals of the same strain, since different strains can behave in a heterogeneous manner when exposed to an odorant23; 6) the experimenter should wear a lab coat at all times and change gloves between animals to prevent odor mixing; 7) after pipetting the operator should move slowly away from the cage at a distance of 1.3 meters to prevent any confounding stimulation to the mice during the olfactory exploration; 8) mice displaying mean grey values only in one chamber should be excluded from the study, since mice are expected to explore both chambers to different degrees.
The method described offers several advantages over other protocols: it is extremely simple to set up, uses inexpensive materials, it is of fast completion and takes advantage of open source software, such as ImageJ. In addition, we provide macros that are ready to be installed and which can be custom-used and adapted to any arena and more than 2 odor perimeters. It has to be noted that only the time spent in the assigned odor perimeter is a measure of olfactory activity. Whereas the time spent in each chamber gives a readout of the exploratory activity of the mouse and is only a rough estimation of the olfactory behavior. As with other methods, statistical power can be gained by increasing the number of animals per group.
As compared to the olfactory testing using olfactometers, which can control automatically for vapor pressure and delivery time12&#8211;14, the proposed protocol is less controlled. Nevertheless, all the odors are applied in equal volumes, at defined distance and for the same time window. Thus, keeping these variables constant, in this testing an olfactometer is not required. There is another potential limitation to this protocol consisting in the time required for the adjustment and cutting of each video to obtain a fixed number of frames. Nevertheless, the same computer-assisted analysis can be used also on more sophisticated setups with odor ports delivering the odor at specific times. In this case, the video cutting could be automatically set.
As compared to other protocols using cotton pads impregnated with odor to test attraction and avoidance, the present protocol provides an additional information about the olfactory discrimination between a novel odor and a neutral odor (water)15,16 in one single experimental session. Moreover, the protocol does not require experimental blinding and can be entirely conducted by a single experimenter using the unbiased computer-assisted analysis.
These simple tests can be used to monitor the progression of neural deficits in Alzheimer&#8217;s or Parkinson&#8217;s disease mouse models and to investigate mechanisms of olfactory transmission.

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><tbody><tr><td>Mouse cage</td><td>Italplast (Italy)</td><td>1144B</td><td>36 cm length x 20.5 cm width x 13.5 cm height</td></tr><tr><td>Chipped wood bedding</td><td>Abedd (Austria)</td><td>LTE E-001</td><td>3 cm high</td></tr><tr><td>Peanut butter</td><td>Migros (Switzerland)</td><td>NA</td><td>1:10</td></tr><tr><td>2-Methylbutyric</td><td>Sigma Aldrich (Switzerland)</td><td>W269514</td><td>Pure</td></tr><tr><td>Female urine from fertile females of same mouse strain</td><td>NA</td><td>NA</td><td>Dilution series</td></tr><tr><td>Camera</td><td>Olympus (US)</td><td>Camedia C-8080</td><td>MOV files</td></tr><tr><td>Quicktime for Java (Windows)</td><td>Apple (USA)</td><td>NA</td><td>video plugin for visualizing MOV files</td></tr><tr><td>ImageJ for Windows</td><td>NIH (USA)</td><td>NA</td><td>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 behaves differently from control group A (F1,7 = 26.39, p &#60;0.005). In addition, group C shows no discrimination and spends about the same time with water and peanut butter (t8 = 0.78, p = 0.45). On the whole, the three groups behave differently (F2,9 = 19.83, p &#60;0.005) and there is a significant interaction between the genotype and treatment (peanut butter and water) (F2,1 = 4.90, p &#60;0.005) (Figure 3A).
In response to 2-MB acid the control group displays an avoidance reflex and as a result spends more time with water (t8 = 2.67, p &#60;0.05). Similarly, group B shows a pronounced avoidance reflex to 2-MB acid (t6 = 3.71, p &#60;0.01). On the other hand, group C does not discriminate between the two odors and spends comparable times with 2-MB acid and water (t8 = 2.2, p = 0.6) (Figure 3B). On the whole, comparing the avoidance response the three groups do not display a significant different behavior (F2, 9 = 0.76, p = 0.49) as a result there is no interaction between treatment and genotype (F1, 2 = 0.52, p = 0.63).
In the olfactory sensitivity test to female urine, the curve displays the preference to urine at different concentrations versus water (Preference index= time spent with urine subtracted by the time spent with water). In this test, we observe that control group A has an attraction threshold to urine at a dilution of 1:1,000 and displays increasing attraction to urine with rising concentrations. Group B and C display a 100-fold higher threshold to attraction (1:10) as compared to group A (F2,9 = 4.78, p &#60;0.05). Group B and C display comparable sensitivity curves (F1,19 = 0.36, p = 0.55). Comparing the sensitivity among groups, it appears that group A has higher sensitivity to female urine as compared to group B and C (F2,19 = 7.12, p &#60;0.01) (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/52944/52944fig1.jpg" /> 
Figure 1: Representation of the setup used to perform the olfactory tests. (A) Camera above the cage. (B) Mice are placed in a cage for a 5 minutes habituation period. (C) The odorants are pipetted on the wall of the cage. (D) The exploratory activity of an odorant versus water is tested in a 2 min window.
<img alt="Figure 2" src="/files/ftp_upload/52944/52944fig2.jpg" /> 
Figure 2: Workflow of computer-assisted video processing using macros commands in ImageJ. The example refers to a mouse from group A exposed to urine at a 1:10 dilution. <a href="https://www.jove.com/files/ftp_upload/52944/52944fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52944/52944fig3.jpg" /> 
Figure 3: Representative results of olfactory preference and avoidance tests. The mice of the three groups (n = 5 for group A, n = 4 for group B and n = 5 for group C) have been exposed to (A) peanut butter and (B) 2-MB acid for a 2 min exploration session. The total time exploring the odor (black circles) versus water (grey circles) is represented. Significant differences in olfactory behavior among groups are indicated by black horizontal bars and asterisks. Significant differences in sniffing times between the odor and water within groups are shown by grey horizontal bars and asterisks. *P &#60;0.05, **P &#60;0.01, ***P &#60;0.01 (grey horizontal bars, Student&#8217;s t-test; black horizontal bar, one-way ANOVA). Error bars are standard errors of the mean (SEM).
<img alt="Figure 4" src="/files/ftp_upload/52944/52944fig4.jpg" /> 
Figure 4: Representative results of sensitivity tests to increasing concentrations of female urine. The preference index curve, given by the exploration time with urine at different concentrations subtracted by the time spent with water, shows that group A (n = 5) has the highest sensitivity to urine as compared to group B (n = 4) and C (n = 5). *P &#60;0.05 (black horizontal bars, one-way ANOVA). Error bars are SEM.

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

Nanyang Technological University

Research

JoVE Journal

Behavior

10.0K 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, ...

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 ...

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 ...

Olfactory Behavioral Testing in the Adult Mouse

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

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Biology

Assessing Olfactory Impairment in Mice Due to Inhaled Environmental Pollutants

Studying the Effects of Inhaled Environmental Pollutants on Olfactory Function in Mice

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental pollutants exposure may impair olfaction; thereby, there is an urgent need for methods to evaluate the effects of inhaled environmental pollutants exposure on olfaction. Mice are ideal models for olfactory experiments because of their highly developed olfactory system and behavioral characteristics. To assess the effects of inhaled environmental pollutants exposure on olfactory function in mice, a detailed buried food test and social odor discrimination experiment is provided, including the experiment preparation, the selection and construction of experimental facilities, the testing process, and indexes of time. Meanwhile, timekeeping equipment, operational details, and the experimental environment are discussed to ensure the success of the assay. Zinc sulfate is used as the treatment to demonstrate the feasibility of the experimental approach. The protocol provides a simple and clear operational process for assessing the effects of inhaled environmental pollutants on olfactory function in mice.

Author Spotlight: Assessing the Olfactory Effects of Airborne Pollutants &#8212; Buried Food and Social Odor Tests

This paper provides a detailed description of the buried food test and social odor discrimination experiment to assess the effects of inhaled environmental pollutants exposure on olfactory function in mice.

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental ...

Environment

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Neuroscience

Designing a Flexible, Cost-Effective Olfactometer for Rodent Olfactory Experiments

Constructing an Olfactometer for Rodent Olfactory Behavior Studies

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These sophisticated devices allow researchers to precisely control and deliver odor stimuli, enabling the investigation of complex olfactory processes in rodents. Although commercially available olfactometers are convenient, they present challenges when technical issues arise, often requiring costly assistance and potentially disrupting research timelines. This article details the construction of a custom olfactometer specifically designed for mouse olfactory behavior experiments, providing a comprehensive list of parts and step-by-step instructions. The olfactometer is controlled through MATLAB, offering a user-friendly interface for researchers. Importantly, the open-source code allows users to modify and adapt the system, tailoring behavioral tasks to meet specific experimental needs. Building a customized olfactometer empowers users with the knowledge and capability to perform custom experimental design and troubleshooting independently, saving both time and resources. This approach not only enhances research flexibility but also fosters a deeper understanding of the equipment's functionality, ultimately leading to more robust and reliable olfactory studies in rodents.

Constructing an Olfactometer for Rodent Olfactory Behavior Studies Near-Infrared Spectroscopy Hyperscanning Study in Psychological Counseling

This protocol describes the construction of an olfactometer for go/no-go olfactory behavior experiments. Step-by-step instructions, along with images, are provided to ensure the successful construction of the olfactometer. Information for troubleshooting issues encountered during the process is also included.

The use of olfactometers to study rodent behavior and brain activity during olfactory tasks is crucial for understanding brain circuits. These ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.