The Rodin olfactory system or sense of smell has become an increasingly popular area of research. In order to test olfactory sensitivity, a mouse is allowed to habituate in a cage without bedding and is then exposed to pieces of filter paper that contain increasing concentrations of odorant. The amount of time that the mouse spends investigating the filter paper is then recorded.
Olfactory preference testing is done the exact same way. However, instead of increasing concentrations of odorant, the amount of time the mouse spends investigating different odorants is noted. Hello, I'm Rochelle Witt from Dr.Rosalyn Siegel's Laboratory in the Department of Neurobiology at Harvard Medical School.
Hi, I am Jennifer Deno, also from the Siegel Lab. Today we'll show you two procedures for olfactory testing. We use these procedures in our laboratory to study olfactory sensitivity and olfactory preference.
So let's get started. To begin the olfactory sensitivity testing procedure, obtain four clean cages all without bedding. The cages should be eight inches high by 10 inches wide by 18 inches long.
Line up the four cages in a biosafety cabinet. Next, set up partitions between each arena using opaque filter paper. Then place a black poster board across the final partition so that it lines the side and bottom of the cage.
Allow light to illuminate the arena by covering the top of the cage with a clear piece of plexiglass. Now begin habituating the subjects to do this. Place the first subject in the first cage.
After 15 minutes, move the first subject to the second cage. Clean the first cage with Quatra side tb. Next place the second subject in the first cleaned cage.
Continue this process with subsequent subjects prior to testing. Place one of the subjects in the fourth cage and habituate them for another 15 minutes. Ultimately, a subject should be habituated in each of the first three cages.
We're now ready to prepare the scent samples. The scent samples should be prepared by a different researcher who is blind to the subject, genotypes and or conditions. Start the sample preparation by making an appropriate number of two inch by two inch squares of filter paper.
Then make the appropriate dilutions of the preselected scent in conical tubes. Keep in mind that different mice backgrounds may require different dilution of sense for effective discrimination between a control and experimental group. Due to this observation, we find it helpful to empirically derive appropriate scent dilutions on subjects similar to the control group before proceeding with the experimental testing of naive mice For hydrophobic samples such as peanut butter, use oil as solvent for all others.
Use distilled water vortex to make sure the dilution are properly mixed. Next, set a digital video recording device at the far end of the fume hood such that the entire cage end is in focus. Take a preliminary video to ensure that the camera settings are adequate.
In a laboratory notebook record the order in which dilutions will be tested, taking care to randomize the order in each trial. Also record the subject's litter and identification numbers. Once the subject has been habituated for 15 minutes in the arena, begin preparation for the first trial.
Pipette the first dilution up and down in the conical tube before pipetting it onto a filter paper square. The filter paper surface should optimally be approximately 85%covered. Use a Kim wipe to make sure the filter paper is not overly saturated.
Now that the scented filter paper is prepared, we're ready to perform the first olfactory sensitivity trial. To begin the trial, start the video recording. Then place the scented filter paper in the cage on the opposite end of the animal's current position.
Begin timing. After three minutes, stop the recording and remove the scent. Next, start the timer again and prepare a second scent sample.
After one minute, begin video recording. As before, place the scent sample in the cage on the opposite end of the animal's current position. Repeat this procedure for each cent dilution and for each subject, remember to record the video file number for each trial.
Now that we've shown you olfactory sensitivity testing, proceed with the olfactory preference.Testing. For olfactory preference testing. Follow the same procedure as described for olfactory sensitivity testing except for two slight modifications.One.
Rather than preparing multiple dilution of one preselected scent, prepare both attractive and aversive scent. And two, rather than recording the dilution to be tested, record the order of the varied that will be tested randomizing the order in each trial. All other steps in the procedure are identical to the olfactory testing, including the habituation of the subjects, the use of a blind researcher, the application of sent to filter paper, and the timed video recording of each trial.
Once the olfactory preference testing is complete, proceed with scoring the olfactory tests. After requiring all of the olfactory sensitivity and preference videos, score the total time that the subject spent exploring each dilution or sent for consistency. Have the blind researcher analyze the videos to perform this analysis.
It's helpful to first preview the video and note the following. When the filter paper is placed in the arena, when the mouse approaches the filter paper, when the mouse investigates the filter paper, and when three minutes have elapsed from the time when the filter paper was initially placed in the arena, knowing when to focus on the behavior of the mouse makes it easier to accurately score the total time spent exploring upon review now, review the entire video as you review the video. Use a stopwatch to time all investigations within three minutes of placing the filter paper in the arena.
The time of investigation is defined as any duration of time when the mouse's nose is less than one millimeter from the filter paper, it is important not to include any of the time when the mouse is in close proximity to the filter paper but is not actively investigating it. Do not include bouts of chewing and licking the paper, as well as bouts of sitting on the paper in the total exploratory time. Adhere closely and consistently to these rules in order to accurately analyze olfactory behavior.
After reviewing the video record the total time spent investigating the filter paper, repeat the analysis for all the videos. Here are representative results from olfactory testing. This first chart shows the results for a session of olfactory sensitivity training.
Each mouse from either the experimental group group B or the control group group A was exposed to one of five different dilutions of cinnamon extract on filter paper for one three minute session. Total time spent exploring the filter paper was assessed, and this chart shows the results for a session of olfactory preference testing. Each mouse was exposed to each of the different scents on filter paper for one three minute session.
Total time spent exploring the filter paper was assessed. We've just shown you how to test olfactory abilities in the adult mouse when using these procedures, it's important to use naive mice to accurately test olfactory abilities and not any other abilities such as learning and memory. So that's it.
Thanks for watching and good luck with your experiments.
