An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice
March 22nd, 2018
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.
The overall goal of this behavior protocol is to train mice so they associate one odor with a reward, and another odor with a punishment. This method can help answer key questions in the neuroscience field, such as learning, synaptic plasticity, and sensory discrimination. The main advantage of this technique is that once trained, mice perform hundreds of trials within hours for robust statistical sampling.
The implications of this technique extend toward therapy or diagnosis of neurodegenerative diseases, because severity of olfactory dysfunction is predictive of subsequent cognitive decline in humans. Though this method can provide insight into olfaction, it can also be applied to other systems, such as vision or audition. Visual demonstration of this method is critical, as the initial steps for both setting up the behavior chamber and using the software are difficult to learn, because of different connecting ports and training steps.
To begin, assemble a mouse chamber with chamber floor. Keep the training box in a low-traffic, dimly-lit area to avoid distractions. Then, drill each water port with a small hole on top, to allow an 18-gauge needle to dispense water inside the port.
Fill a glass vial with odorant dissolved in mineral oil, and securely tighten the cap. Next, connect an 18-gauge needle to the silicone tubing. Pierce the glass vial cap, and connect the other end of the silicone tubing to the intake of the odor ports.
After that, place each silicone tube into an odor valve. Connect the vacuum line to the odor ports. Attach two 10-milliliter syringes to a metal holding rod, and connect the tubing to the syringes.
Subsequently, connect the other side of the tubing to an 18-gauge needle. Fit the needle into the drilled hole of the nose poke port on the mouse chamber, and connect the other end of the tubing to the water valve. Fill the two 10-milliliter syringes with rodent drinking water.
Then connect an airflow meter to the air intake, and maintain airflow at three to five liters per minute. Connect the two water valves, two odor valves, two water ports, odor port, and power to the USB interface system. And lastly, connect the USB interface box to power.
Next, adjust the vacuum suction to avoid cross-contamination of odors between trials, and use odor-specific tubing to connect odorant vials to the chamber. To prepare the mice for training, begin water restriction at 40 milliliters per kilogram per day. Weigh the mice daily to ensure they are above 80%of baseline weight, and keep all environmental factors constant throughout the protocol, including temperature, noise, and stray odors.
Clean the cage before each mouse is transferred to it. Spray and wipe the chamber with 70%ethanol to minimize mouse odor distraction. For stage one training, configure the behavior box with a water port in the center, and with all side ports inaccessible.
Place a mouse into the chamber and close it. Then, begin the stage one program. Consider this stage as complete when the mouse achieves 100 trials within 60 minutes.
Remove the mouse from the chamber after 60 minutes, or 100 trials have been completed. For stage two training, configure this and every subsequent stage with two water ports on the sides and the odor port in the middle. Place a mouse into the chamber and close it.
Then, begin the stage two program. Consider this stage as complete when a minimum of 40 trials are performed in 60 minutes, with at least 25%of water rewards received within five seconds of the center port nose poke. Remove the mouse once this stage has completed.
For stage three training, connect the S-plus odor to the odor delivery controller. Place a mouse into the chamber and close it. Afterward, begin the stage three program.
Consider this stage as complete when there are greater than 60 rewards within 60 minutes, and remove the mouse once the stage has competed. For stage four-A training, set the box configuration the same as previous stage three. Then, connect both S-plus and S-minus odor to the odor delivery controller.
Place a mouse into the chamber and close it. Then, begin the stage four-A program. After the mouse completes 40 trials, switch the program to randomly deliver the odors.
Remove the mouse once the stage has completed. For stage four-B training, make this stage identical to stage four-A. However, the time-out punishment for attempting a water reward after an S-minus odor is four seconds.
Connect both S-plus and S-minus odor to the odor delivery controller. Place a mouse into the chamber. Close the mouse chamber and begin the stage four-B program.
Consider this stage complete when there are more than 100 trials within 60 minutes, with accuracy more than 85%For go/no-go assay, set the box configuration the same as previous stage four-B. Connect both S-plus and S-minus odor to the odor delivery controller. Then, place a mouse into the chamber.
Begin the stage go/no-go program and then expose the mouse that has learned the olfactory learning task to novel pairs of chemically similar odors. Once the mice have learned the olfactory learning task, they can associate novel odor pairs with reward and punishment. These trained mice normally begin with about 50%accuracy on the go/no-go task.
The percentage correct can be plotted by trial block as a learning curve for novel odor pairs. Within 10 block trials, mice are able to correctly discriminate between odors, with greater than 85%accuracy. This shows that our protocol has successfully trained wild-type mice to associate one odor with a water reward and another with a time out punishment.
Once the mice learn the task, odor pairs can be modified to increase or decrease the task difficulty. For example, decreasing the odor pair concentration increases the task difficulty. Mice are also able to remember previous learned odor pairs.
After waiting seven days since learning the task, a recall test shows that wild-type mice can quickly remember previously-learned odor associations. While attempting this procedure, it's important to remember to ensure that the desired odorants are properly connected to the behavior box. It's especially important if multiple experimenters are using different odors.
Following this procedure, other methods like odor mapping can be performed in order to answer additional questions like, How does learning influence the way sensory information is represented by the brain? After its development, this technique paved the way for researchers in the field of neuroscience to explore learning-induced neural changes in the mouse olfactory system. After watching this video, you should have a good understanding of how to teach mice to associate one odor with a reward and another odor with a punishment.
Don't forget that working with sharp needles can be extremely hazardous, and precautions such as proper disposal of sharps should always be taken while performing this procedure.
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Overview
This study investigates odor discrimination using an associative learning task in mice, focusing on learning-induced structural changes in the brain. The technique examines key neuroscience questions such as sensory discrimination and synaptic plasticity.
Key Study Components
Area of Science
- Neuroscience
- Olfaction
- Behavioral learning
Background
- Associative learning protocols are pivotal in studying cognitive functions.
- Odor discrimination plays a crucial role in mammalian sensory processing.
- Understanding olfactory behavior in mice may relate to human cognitive decline.
- This method can be adapted for other sensory systems such as vision and audition.
Purpose of Study
- To train mice to associate odors with rewards and punishments.
- To explore olfactory processing and its implications for neurodegenerative disease.
- To develop a robust behavioral assay for statistical analysis of learning.
Methods Used
- Mice are trained using a specially configured behavioral chamber.
- The training involves various stages, assessing accuracy in odor discrimination.
- No multiomics workflows mentioned.
- Critical steps include water restriction to manage motivation and stage completion criteria.
- Procedures include meticulous setup of odor delivery systems and conditioning protocols.
Main Results
- Mice successfully learn to associate different odors with rewards or punishment.
- Training allows for high statistical sampling through repeated trials.
- The accuracy in odor discrimination approaches 85% following training.
- Results suggest that olfactory learning can be effectively assessed through this protocol.
Conclusions
- The protocol demonstrates a reliable method for studying associative learning in olfaction.
- This approach may offer insights into therapeutic strategies for neurodegenerative diseases.
- Findings enhance understanding of sensory discrimination mechanisms in mammals.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables high-throughput, quantitative assessment of olfactory learning and discrimination in mice, providing a robust behavioral readout for target validation in neuroscience drug discovery. By supporting mechanistic de-risking of olfactory circuit function and plasticity, it informs early-stage hypothesis testing for compounds targeting neurodegenerative or sensory processing disorders. The assay’s reproducibility and scalability facilitate cross-functional alignment between discovery biology and translational research teams.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses related to olfactory learning, synaptic plasticity, and sensory discrimination mechanisms.
- Operational Value: Supports biological de-risking by quantifying odor association learning and memory retention in a controlled, reproducible system.
- Predictive Value: Generates quantitative behavioral data (e.g., accuracy rates, learning curves) that aid in portfolio triage and go/no-go decisions for CNS-targeted compounds.
Screening & Assay Development
- Assay Readiness: Produces validated biological systems (trained mice) capable of hundreds of automated trials daily, enabling reliable compound evaluation.
- Standardization: Ensures reproducibility through strict environmental controls, water restriction protocols, and stage-based training criteria.
- Scalability: Facilitates high statistical sampling and longitudinal studies, supporting assay adaptation for dose-response or time-course screening.
Translational & Preclinical Research
- Disease Relevance: Aligns with human olfactory dysfunction as a predictive biomarker of cognitive decline, supporting translational continuity from mouse models to clinical risk assessment.
- Mechanistic De-risking: Enables study of activity-dependent neuronal survival and plasticity, linking behavior to downstream biochemical, electrophysiological, or imaging readouts.
- Risk-Adjusted Advancement: Supports decisions on compound effects on learning-induced neural changes, reducing late-stage biological risk in sensory or neurodegenerative programs.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through preclinical validation, particularly for projects focused on olfactory processing, sensory discrimination, or neurodegenerative disease mechanisms.
- Discovery Biology: Supports hypothesis testing and pathway clarification by measuring how odor learning alters neural structure and function.
- Screening: Delivers quantitative, reproducible outputs (trial accuracy, learning curves) that enable comparison across experimental conditions or genetic models.
- Analytics: Provides statistical learning curves and discrimination thresholds that help teams assess compound effects on sensory processing and memory.
- Translational Research: Connects to preclinical validation through alignment with human olfactory biomarkers predictive of cognitive decline.
- Enterprise Reuse: Functions as a reusable behavioral platform for iterative testing across multiple compounds, genotypes, or environmental manipulations.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity in olfactory learning and plasticity pathways.
- Operational Value: Delivers standardization, reproducibility, and scalability through automated trial performance and strict procedural controls.
- Strategic Value: Improves capital efficiency by enabling early de-risking of CNS targets and reducing failure risk in later stages.
- Portfolio Impact: Supports risk-adjusted prioritization of compounds based on effects on learning, memory, and sensory discrimination.
Implementation Considerations
- Requires expertise in behavioral neuroscience, olfactory system anatomy, and operant conditioning techniques.
- Depends on specialized instrumentation including odor delivery systems, water valves, vacuum lines, and USB interface controllers.
- Necessitates cross-team standardization of environmental controls (odor, noise, temperature) and training protocols to ensure reproducibility.
- Involves adaptation considerations when transferring protocols across mouse strains, ages, or odorant physicochemical properties.
- Includes practical limitations such as setup complexity, needle hazards, and the need for consistent animal handling to avoid distraction or stress confounds.
