This protocol is adapted from previous work within our lab (Huang et al.8). All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Baylor College of Medicine. It is supported by the McNair Medical Institute, NINDS grant R01NS078294 to B.R.A., NIH IDDRC grant U54HD083092, NIDDK grant F30DK112571 to JMP, and NINDS grant F31NS092435 to CKM.

All mice were used under a protocol approved by the Baylor College of Medicine Institutional Animal Care and Use Committee in accordance with NIH standards. Mice used in this protocol were all adult mice (&#62;6 weeks of age) on C57BL6/j background and included both male and female mice.After training/staging tasks, mice are returned to their home cage.
1. Construction and General Rules for Using Operant Learning Box (Figure 1B, C)
<ol>
	<li>Assemble a mouse cha...

<ol>
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The authors declare no conflicts of interest and no competing interests.

The rodent olfactory system provides a unique model to study sensory dependent plasticity. Here we present an olfactory learning paradigm to train mice to associate odorant pairs with either a reward or punishment. Through this learning task, downstream circuit changes can be studied in subsequent experiments (electrophysiology, in vivo neuronal imaging, etc.). Upon completion, mice will learn to perform a simple odor cued task to associate a water reward with one odor, and a timeout punishment with ano...

The mouse OB, where odor information first enters the central nervous system (CNS), provides an excellent model to study experience-dependent structural changes. OB circuity continually integrates adult-born neurons in an activity-dependent manner. Adult-born neuron precursors divide off from progenitors that line the subventricular zone adjacent to the lateral ventricles1. Upon migrating into the OB, these neuronal precursors either survive, differentiate, and integrate as inhibitory granule cells, or undergo apoptosis2. Selection for cell fate is influenced by olfactory activity, including olfactory learning3,4,5,6. After integration, learning-induced synaptic changes occur in granule cells during a two-week critical period7,8. Thus, assays for olfactory learning are useful for examining how experience-dependent plasticity influences structural and functional reorganization of a mature brain circuit.
This protocol offers one approach to olfactory training by using an operant conditioning paradigm. In this task, water-deprived mice are trained to associate one odor (the &#34;Go&#34; odor) with a water reward and another odor (the &#34;No-Go&#34; odor) with a trial timeout punishment. Mice progress through a graded series of training phases over the course of 2-4 weeks. When training is complete, mice respond to the Go or No-Go odor with discrete, corresponding behaviors (seeking a water reward on Go trials and not seeking the water reward on No-Go trials) (Figure 1A). After training is complete, mice can be further challenged with chemically similar odor pairs to test discrimination or become transitioned to studies investigating how olfactory learning alters the structure or function of the OB. Although odor discrimination tasks have been previously described, most rely on subjective measurements such as number of sniffs between two odorants9,10. Furthermore, the need for human scoring of such tasks is also time-intensive. The Go/No-Go olfactory learning task described in this protocol offers an unbiased, direct measurement of odor discrimination and olfactory learning.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Glass vial</td>
			<td style="width:123px;">Qorpak</td>
			<td style="width:344px;">GLC-01016</td>
			<td style="width:576px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silicon Tubing</td>
			<td>Thermo Scientific</td>
			<td>86000030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">18 gauge needles</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
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			<td height="40" style="height:40px;">1-Butanol</td>
			<td>Sigma Aldrich</td>
			<td>437603</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Propionic Acid</td>
			<td>Sigma Aldrich</td>
			<td>402907</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mouse Chamber</td>
			<td>Med Associates</td>
			<td>ENV-307W</td>
			<td></td>
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			<td height="40" style="height:40px;">Chamber Floor</td>
			<td>Med Associates</td>
			<td>ENV-307W-GFW</td>
			<td></td>
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			<td height="40" style="height:40px;">Water Port</td>
			<td>Med Associates</td>
			<td>ENV-313W</td>
			<td>Need two</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Odor stimulus</td>
			<td>Med Associates</td>
			<td>ENV-275</td>
			<td>Contain 2 valves to gate odor delivery&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Odor Port</td>
			<td>Med Associates</td>
			<td>ENV-375W-NPP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">USB Interface</td>
			<td>Med Associates</td>
			<td>DIG-703A-USB</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Desktop Computer with Windows 2000, XP, Vista, or 7</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow meter</td>
			<td>VWR</td>
			<td>97004-952</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Behavioral software</td>
			<td>Med Associates</td>
			<td>SOF-735</td>
			<td>This software, which runs each training stage, has now been replaced with Med-PC V</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Transfer software</td>
			<td>Med Associates</td>
			<td>SOF-731</td>
			<td>This software formats the data to Excel</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Training Software</td>
			<td>Med Associates</td>
			<td>DIG-703A-USB</td>
			<td>This software is used to program each training stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Valve</td>
			<td>Neptune Research</td>
			<td>225P012-11</td>
			<td>This valve is used to gate the water delivery. Need Two</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Odor Valve</td>
			<td>Neptune Research</td>
			<td>360P012-42</td>
			<td>This valve is used to gate the odor delivery. Need Two</td>
		</tr>
	</tbody>
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an objective and reproducible test of olfactory learning and discrimination in mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Once mice have learned the olfactory learning task, they can now 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 (Figure 3A). Within 10 block trials, which take most mice less than 30 min to perform, mice are able to correctly discriminate between odors with greate...

Watch this Scientific Journal Video about An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice at JoVE.com

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

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.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

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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8.4K vistas.  Baylor College of Medicine. 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 neurode...