Name: an objective and reproducible test of olfactory learning and discrimination in mice
Uploaded: 2018-03-22T13:00:00
Description: Watch this Scientific Journal Video about An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice at JoVE.com

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>
	<li>Carleton, A., Petreanu, L. T., Lansford, R., Alvarez-Buylla, A., Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Becoming+a+new+neuron+in+the+adult+olfactory+bulb.">Becoming a new neuron in the adult olfactory bulb.</a> Nat Neurosci. 6 (5), 507-518 (2003).</li><li>Petreanu, L., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+death+of+adult-born+olfactory+bulb+granule+neurons:+role+of+olfaction.">Maturation and death of adult-born olfactory bulb granule neurons: role of olfaction.</a> J Neurosci. 22 (14), 6106-6113 (2002).</li><li>Yamaguchi, M., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+period+for+sensory+experience-dependent+survival+of+newly+generated+granule+cells+in+the+adult+mouse+olfactory+bulb.">Critical period for sensory experience-dependent survival of newly generated granule cells in the adult mouse olfactory bulb.</a> PNAS. 102 (27), 9697-9702 (2005).</li><li>Rochefort, C., Gheusi, G., Vincent, J. D., Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+odor+exposure+increases+the+number+of+newborn+neurons+in+the+adult+olfactory+bulb+and+improves+odor+memory.">Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory.</a> J Neurosci. 22 (7), 2679-2689 (2002).</li><li>Arenkiel, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+remodeling+of+olfactory+bulb+microcircuits+revealed+by+monosynaptic+tracing.">Activity-induced remodeling of olfactory bulb microcircuits revealed by monosynaptic tracing.</a> PloS one. 6 (12), 29423 (2011).</li><li>Alonso, M., Viollet, C., Gabellec, M. M., Meas-Yedid, V., Olivo-Marin, J. C., Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+discrimination+learning+increases+the+survival+of+adult-born+neurons+in+the+olfactory+bulb.">Olfactory discrimination learning increases the survival of adult-born neurons in the olfactory bulb.</a> J Neurosci. 26 (41), 10508-10513 (2006).</li><li>Quast, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+broadening+of+inhibitory+sensory+maps.">Developmental broadening of inhibitory sensory maps.</a> Nat Neurosci. 20 (2), 189 (2017).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Task+learning+promotes+plasticity+of+interneuron+connectivity+maps+in+the+olfactory+bulb.">Task learning promotes plasticity of interneuron connectivity maps in the olfactory bulb.</a> J Neurosci. 36 (34), 8856-8871 (2016).</li><li>Arbuckle, E. P., Smith, G. D., Gomez, M. C., Lugo, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+for+odor+discrimination+and+habituation+in+mice.">Testing for odor discrimination and habituation in mice.</a> J Vis Sci. (99), e52615 (2015).</li><li>Zou, J., Wang, W., Pan, Y. W., Lu, S., Xia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+measure+olfactory+behavior+in+mice.">Methods to measure olfactory behavior in mice.</a> Curr Protoc Toxicol. , 11-18 (2015).</li><li>Uchida, N., Takahashi, Y. K., Tanifuji, M., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+maps+in+the+mammalian+olfactory+bulb:+domain+organization+and+odorant+structural+features.">Odor maps in the mammalian olfactory bulb: domain organization and odorant structural features.</a> Nat Neurosci. 3 (10), 1035 (2000).</li><li>Cang, J., Isaacson, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+whole-cell+recording+of+odor-evoked+synaptic+transmission+in+the+rat+olfactory+bulb.">In vivo whole-cell recording of odor-evoked synaptic transmission in the rat olfactory bulb.</a> J Neurosci. 23 (10), 4108-4116 (2003).</li><li>Parthasarathy, K., Bhalla, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laterality+and+symmetry+in+rat+olfactory+behavior+and+in+physiology+of+olfactory+input.">Laterality and symmetry in rat olfactory behavior and in physiology of olfactory input.</a> J Neurosci. 33 (13), 5750-5760 (2013).</li><li>Rajan, R., Clement, J. P., Bhalla, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats+smell+in+stereo.">Rats smell in stereo.</a> Science. 311 (5761), 666-670 (2006).</li><li>Batista-Brito, R., Close, J., Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distinct+temporal+origins+of+olfactory+bulb+interneuron+subtypes.">The distinct temporal origins of olfactory bulb interneuron subtypes.</a> J Neurosci. 28 (15), 3966-3975 (2008).</li><li>Sakamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+postnatal+neurogenesis+contributes+to+formation+of+the+olfactory+bulb+neural+circuits+and+flexible+olfactory+associative+learning.">Continuous postnatal neurogenesis contributes to formation of the olfactory bulb neural circuits and flexible olfactory associative learning.</a> J Neurosci. 34 (17), 5788-5799 (2014).</li><li>Resendez, S. L., Jennings, J. H., Ung, R. L., Namboodiri, V. M. K., Zhou, Z. C., Otis, J. M., Stuber, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cortical,+subcortical,+and+deep+brain+neural+circuit+dynamics+during+naturalistic+mammalian+behavior+with+head-mounted+microscopes+and+chronically+implanted+lenses.">Visualization of cortical, subcortical, and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.</a> Nat Protoc. 11 (3), 566 (2016).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+optogenetics+with+multifunctional+flexible+polymer+fibers.">One-step optogenetics with multifunctional flexible polymer fibers.</a> Nat Neurosci. 20 (4), 612 (2017).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1259px;" width="1258">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1-Butanol</td>
			<td>Sigma Aldrich</td>
			<td>437603</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chamber Floor</td>
			<td>Med Associates</td>
			<td>ENV-307W-GFW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Port</td>
			<td>Med Associates</td>
			<td>ENV-313W</td>
			<td>Need two</td>
		</tr>
		<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>
</table>

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 ...

Research

JoVE Journal

Neuroscience

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...