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.
