An Olfactory Preference Test for Measuring Olfactory Hedonic Biases in Mouse Models of Depression

A negative bias in emotional processing is a major characteristic of depressive disorders. This bias is particularly interesting to better understand the physiopathology of the disease. Here we propose a protocol for the reliable assessment of innate odor valuation using odors associated with various valence in both male and female mouse models of depression.

Assessing hedonic bias in mouse models is essential, both to better evaluate the induced depressive-like phenotype, and also as a tool to investigate neural mechanisms behind these changes in valence assignment. Overall, this protocol provides new avenues for translational research to understand the mechanism underlying depression and treatment efficacy. In the future, we will try to decipher the brain circuits behind these disrupted emotional processing and potential sex-specific mechanisms, along with the action of antidepressants onto these pathways.

To begin, prepare the experimental setup using a two-compartment arena measuring 45 centimeters by 50 centimeters by 25 centimeters with gray plexiglass walls and a white floor. Divide the arena into two equal compartments of 25 centimeters by 45 centimeters, using a gray plexiglass wall measuring 37 centimeters by 25 centimeters. Secure perforated Petri dish with adhesive and place them at the bottom of the wall in each compartment.

Then use dual-sided adhesive or Blu Tack to fixate each Petri dish vertically onto the wall of the arena. Next, position a camera two meters above the arena to record the behavior of the animals and enable automatic tracking of their movements throughout the experiment. Now divide the mice into at least two experimental groups, the control mice and test group mice.

Transfer each mouse to the behavioral testing room 30 minutes before the experiment begins. Using a lux meter, set the lighting in the arena to approximately 40 lux and verify that the illumination is evenly distributed. Next, add a clean filter paper measuring 42.5 millimeters into each of the two Petri dishes in the arena.

On the first day, gently place all mice from the same housing cage together into the arena for 10 minutes to reduce neophobia. On the day of testing, prepare in a glass vial the odor under a chemical hood, using the correct dilution. Dilute the odorant in mineral oil or distilled water depending on its chemical properties.

Just before testing each mouse, use a micro pipette with a filter tip to collect 200 microliters of the odor solution under the hood. Dispense the solution onto the filter paper in one of the Petri dishes. Leave the second filter paper in the other Petri dish untreated.

Transfer the Petri dishes to the testing room. Then vertically fix them onto the wall of the arena. Place each mouse individually into the arena at the center between the two compartments.

Record the behavior for 10 minutes using the overhead camera. Remove the mouse from the arena after the observation period ends. Remove the odor Petri dish from the arena.

Then clean the arena using a 70%ethanol disinfectant solution and dry it thoroughly. Discard the used filter paper and prepare the next odor sample for the next mouse. Use an automatic tracking software to record the mouse's position throughout the test.

Define distinct zones within the arena for analysis. During habituation, all mice spent at least 50 seconds in both the odor and control zones, indicating adequate task engagement. Importantly, mice exposed to UCMS spent the same time in the odor zone than controls.

Although some difference could be observed in other zones. Control female mice moved significantly more than UCMS females when exposed to male urine, but not during habituation or TMT exposure. All readouts of hedonic response show significant differences or a statistical tendency between control and UCMS females.

This effect is also visible through the Global Odor Exploration Index, which is a combined measure of hedonic and locomotor response. The Odor Exploration Index positively correlated with the emotionality score for both male urine and TMT in female mice. In male mice, no differences were observed during habituation or in total distance moved through the test.

The time spent in the odor zone shows a significant reduction of female urine and TMT exploration in UCMS compared to controls. UCMS mice enter significantly less in the odor zone, or tend to, and present a significantly reduced preference index for both female urine and TMT. The Odor Exploration Index shows a negatively shifted behavioral response to female urine and TMT of UCMS mice compared to controls, which tend to be positively correlated with the animal's emotionality score.