Rodent olfactometry provides researchers with a detailed, precise tool to study sensory processing, learning, and memory through controlled odor presentations. Dr. David Díaz’s expertise in advancing these techniques showcases their potential in understanding olfactory-driven behavior and its applications in neurological research.

This video is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives (CC BY-NC-ND) license. You are free to share it for non-commercial purposes with proper attribution, but no modifications or adaptations are allowed.

1.   Why use this method?

The olfactory system is one of the most important environmental stimulus acquisition systems for rodents, and rodents are the primary test unit in research. The olfactory bulbs are the initial relay stations of the olfactory pathway, and they send and receive projections from other parts of the brain. Interestingly, drugs can be administered via the intranasal route. Drugs that can be administered through this way are not only those that affect the respiratory system but also those that affect the CNS. In addition, it is also a suitable route of administration for viruses as well as cells, which gives it a great advantage due to the simplicity of administration directly into the nostril while increasing biosafety.

There are easy olfactory tests: buried food test, short-term olfactory memory test, discrimination test, and more sophisticated with olfactometer like go / no-go paradigm olfactometer test.

2.   What you’ll need

1. Buried food test: mice/rats, cage with bedding, food pellet, camera, stopwatch, scale
2. Short-term olfactory memory: mice/rats, arena, stopwatch, odorant, glass dish or small containers with holes for airflow, scale, Y-maze additional
3. Discrimination test: mice/rats, arena, stopwatch, two different odorants, two glass dishes or small containers with holes for airflow scale, Y-maze additional, double-floor cage additional (animal cannot touch the substances) 
4. Oflactometer test:
   • mice, olfactometer, positive stimulus (S+) (i.e., an odorant as (+)-carvone), negative stimulus (S-) (i.e., an odourless substance as mineral oil or another odorant, preferably the enantiomer of S+), scale, water

3.   Step-by-step instructions 

1. Buried food test:

Ensure that food deprivation for rodents results in no more than 10% weight loss, and begin this deprivation 24 hours before the test. During this period, water should always remain available to prevent dehydration.

Preparing the Test Arena:

• Use a cage or open testing box filled with bedding material to a depth of 2-5 cm (about 1-2 inches), depending on the size of the rodent.
• Fluff up the bedding to ensure no noticeable odor trails or signs from previous animals.

Bury the Food:

• Choose a small, highly palatable food item that the rodent can smell easily (e.g., a piece of cereal, a food pellet, or peanut butter).
• Bury the food 1-2 cm under the surface of the bedding in a corner or random location within the testing cage.
• Make sure the food is well-hidden and covered so that it cannot be seen but can be found based on smell.

Place the Rodent in the Arena:

• Gently place the rodent into the testing cage or arena at a location far from the buried food.
• Ensure the rodent is calm and begins exploring its surroundings.

Start the Timer:

• Start the timer as soon as the rodent is placed in the cage.
• Monitor and observe the rodent’s behavior to determine how quickly it locates and unearths the buried food.

Monitor the Rodent’s Behavior:

• The rodent should begin exploring the environment using its sense of smell to locate the food.
• Observe the time it takes for the rodent to find and uncover the food. This is the latency to find the food.
• The typical behaviors include sniffing, digging, or rooting through the bedding.

Record the Time:

• Stop the timer once the rodent uncovers and retrieves the buried food.
• Record the total time taken (in seconds or minutes) for the rodent to find the food.

End the Trial:

• Remove the rodent from the cage once it has found and consumed the food or after a maximum test duration (usually around 5-10 minutes) if the rodent does not find the food.

Return the Rodent:

• Return the rodent to its home cage and provide normal access to food and water.
• Clean the testing cage and replace the bedding for future tests.

2. Short-term olfactory memory:

Acclimatize the Rodent:

• Allow the rodent to become familiar with the testing environment for 5-10 minutes before beginning the test. This reduces stress and ensures natural exploration behavior.

Prepare Odor Containers:

• Place small amounts of the odorants on cotton balls or inside small containers with holes for airflow. This allows the rodent to smell the odor without being able to consume or physically interact with it.
• Label the containers to keep track of the different odors used.

Test Procedure:

Begin with the familiarization phase by exposing the rodent to the solvent (mineral oil) for 5 minutes, ensuring this is done 48 hours before the test. During the test, measure the time spent exploring the odorant within a 5-minute timeframe. Afterward, allow a 15 to 30-minute break. Following the break, reintroduce the rodent to the same odorantand record the time spent exploring it again.

End the Trial:

• After the test, remove the rodent from the cage and return it to its home cage.
• Clean the test cage and replace any materials to prevent odor contamination for future tests.

3. Discrimination test:

Acclimatize the Rodent:

• Allow the rodent to become familiar with the testing environment for 5-10 minutes before beginning the test. This reduces stress and ensures natural exploration behavior.

 Prepare Odor Containers:

• Place small amounts of the odorants on cotton balls or inside small containers with holes for airflow. This allows the rodent to smell the odor without being able to consume or physically interact with it.
• Label the containers to keep track of the different odors used.

Test Procedure:

Start with the familiarization phase by exposing the rodent to two plates containing the same odorant for 5-10 minutes over the course of one or two days. Then, 24 hours before the test, change one of the odorants. During the test, measure the time spent exploring each odorant within a 5–10-minute period. In further tests, you can introduce new odorants, adjust concentrations, or create mixtures of odorants to assess the rodent’s ability to discriminate between more complex olfactory stimuli.

End the Trial:

• After the test, remove the rodent from the cage and return it to its home cage.
• Clean the test cage and replace any materials to prevent odor contamination for future tests.

4. Go / no-go paradigm olfactometer test:

Acclimatize the Rodent:

Allow the rodent to become familiar with the testing environment for 5-10 minutes before beginning the test. This reduces stress and ensures natural exploration behavior.

Training without odorant:

Start by implementing partial water deprivation until the mice reach 85-80% of their body weight to motivate them to seek the water reward. Next, train the mice to “lick” the water delivery tube in order to receive the reward (water). Gradually increase the time the mouse must keep its snout in the odor sampling port, progressing through intervals of 300 ms, 600 ms, 900 ms, 1000 ms, and finally 1200 ms. This training typically spans 3-5 days, allowing the mice to progressively learn and adapt to the olfactometer system without the use of odorants.

Training with odorant:

Introduce the relation between odorants and the reward in the water delivery tube. Use a positive stimulus (S+) with the odorant (+)-carvone [10⁻¹], and a negative stimulus (S-) with mineral oil (odorless). During each block, present 20 stimuli, including 10 S+ and 10 S- at random. Conduct 10 blocks per daily session over 4-5 training days.

• For the positive stimulus (S+), the mouse should receive water as a reward when exposed to (+)-carvone.
• For the negative stimulus (S-) associated with mineral oil, there will be no reward.

This process trains the mice to associate the odorant with the reward system.

End the Trial:

• After the trail, remove the rodent from the olfactometer and return it to its home cage.
• Clean the olfactometer 

4.   Practical tips 

Choose Odorants: Select two or more distinct odorants (e.g., vanilla, mint, or lemon essential oil). Ensure that the odors are unfamiliar to the rodent and can be easily distinguished. Research could explore how rodents perceive and respond to complex odor mixtures that better mimic real-world olfactory environments. Understanding how rodents process multiple odorants simultaneously could provide more comprehensive insights into olfactory coding and decision-making.

5.   Critical appraisal & implications for future research 

Olfactometry is an essential tool in rodent research, providing precise control over odor presentation to study sensory processing, learning, and memory. However, there are both strengths and limitations associated with its use.

Strengths:

1. Controlled Stimulus Presentation: Olfactometers offer precise control over odor concentration, duration, and timing, allowing researchers to isolate specific variables related to olfactory function and behavior. This makes it an effective tool for studying rodents’ sensory processing, discrimination, and learning.
2. High Sensitivity to Odors: Rodents have a highly developed sense of smell, making them ideal models for studying olfactory-driven behavior. Using an olfactometer allows for detailed analysis of olfactory perception, such as detecting subtle differences between odorants or changes in concentration.
3. Applications in Cognitive and Neurological Research: Olfactometry allows researchers to explore how rodents learn to associate smells with rewards, which is critical for understanding sensory learning, memory, and cognitive function. This is especially important for research on conditions like Alzheimer’s, Parkinson’s, and other neurodegenerative diseases, where olfactory dysfunction is a key early symptom.
4. Customization and Flexibility: Olfactometry protocols can be tailored to specific research questions, from simple discrimination tasks to more complex multisensory integration studies. This versatility enhances its utility across a wide range of experimental paradigms.

Limitations:

Cost and Complexity: Olfactometers can be expensive and technically complex to set up and maintain. Ensuring accurate delivery of odors and avoiding cross-contamination requires careful calibration and cleaning, which may limit its accessibility for smaller labs.

1. Training Time: Rodents require extensive training to associate odors with rewards in olfactometry tasks. Training sessions can take several days, introducing variability and adding time to experimental protocols.
2. Confounding Variables: Factors such as stress, water or food deprivation, or environmental changes can affect the rodent’s olfactory performance. This can complicate data interpretation, as it may be unclear whether behavioral changes are due to sensory deficits or other confounding factors like motivation or health.
3. Limited to Behavioral Responses: Olfactometry primarily measures behavioral outcomes, such as the time spent sniffing or the accuracy of responses to odor stimuli. While these measures are informative, they don’t provide direct insights into the underlying neural mechanisms of olfaction. For a complete understanding, olfactometry often needs to be paired with techniques like electrophysiology or imaging.

This protocol is part of the Braining project, co-founded by the European Union under the project “Collaborative learning and innovative teaching in brain drug screening” (2023-1-PL01-KA220-HED-000160284). It is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.