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

Detta protokoll är anpassad från tidigare arbete inom vårt labb (Huang et al. 8). alla metoderna som beskrivs här har godkänts av djur vård och användning kommittén (ACUC) av Baylor College of Medicine. Det stöds av den McNair Medical Institute, NINDS grant R01NS078294 till B.R.A., NIH IDDRC grant U54HD083092, NIDDK grant F30DK112571 till JMP och NINDS grant F31NS092435 till CKM.

Alla möss användes enligt ett protokoll som godkänts av Baylor College av medicin institutionella djur vård och användning kommittén enligt NIH standarder. Möss som används i detta protokoll var alla vuxna möss (> 6 veckor) på C57BL6/j bakgrund och inkluderade såväl kvinnliga som manliga möss. Efter utbildning/iscensättning uppgifter returneras möss till deras hem bur.
1. konstruktion och allmänna regler för att använda Operant inlärning Box (figur 1</stro...

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Gnagare luktsinnet ger en unik modell för att studera sensoriska beroende plasticitet. Här presenterar vi en lukt learning paradigm för att utbilda möss att associera luktämnet par med antingen en belöning eller bestraffning. Genom detta lärande aktivitet, kan nedströms krets förändringar studeras i efterföljande experiment (elektrofysiologi, i vivo neuronala imaging, etc.). Efter avslutad lär möss att utföra en enkel lukt cued uppgift om du vill associera en vatten belöning med en lukt, ...

Musen OB, där lukt information först träder det centrala nervsystemet (CNS), ger en utmärkt modell för att studera erfarenhet-beroende strukturella förändringar. OB circuity integrerar ständigt vuxen-född nervceller i en aktivitet-beroende sätt. Vuxen-född neuron prekursorer dela av från stamfäder som kantar den subventrikulära zonen intill den laterala ventriklar1. Vid migrerar in OB, dessa neuronala prekursorer antingen överleva, skilja, och integrera som hämmande granule celler eller genomgå apoptos2. Urval för cell öde påverkas av lukt aktivitet, inklusive olfactory lärande3,4,5,6. Efter integration sker lärande-inducerad synaptic förändringar i granule celler under en två veckors kritiska perioden7,8. Analyser för lukt lärande är således användbar för att undersöka hur erfarenhet-beroende plasticitet influenser strukturell och funktionell omorganisation av en mogen hjärnan krets.
Detta protokoll erbjuder en strategi för lukt utbildning med hjälp av ett operant betingning paradigm. I den här uppgiften är vatten-berövade möss utbildade för att associera en lukt (”gå” lukt) med en vatten-belöning och en annan lukt (”No-Go” lukt) med en rättegång timeout bestraffning. Möss framsteg genom en graderad serie av utbildning faser under loppet av 2-4 veckor. När utbildningen är klar, möss svara på Go eller No-Go lukten med diskret, motsvarande beteenden (söker en vatten belöning på Go prövningar och inte söker vatten belöningen om No-Go prövningar) (figur 1A). Efter utbildningen är klar, möss kan utmanas ytterligare med kemiskt liknande lukt par att testa diskriminering eller bli övergått till studier utreder hur luktsinnet lärande förändrar strukturen eller funktionen hos OB. Även om lukt diskriminering uppgifter har beskrivits tidigare, lita mest på subjektiva mätningar såsom antal nosar mellan två odoranter9,10. Behovet av mänskliga poängsättning av sådana uppgifter är dessutom också tidskrävande. Olfactory lärande på Go/No-Avbryt uppgift beskrivs i detta protokoll erbjuder en opartisk, direkt mätning av lukt diskriminering och lukt lärande.

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		<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

Luktsinnets funktion är det dominerande sensorisk modalitet i möss och påverkar många viktiga beteenden, inklusive födosök, predator upptäckt, parning och föräldraskap. Ännu viktigare, kan möss tränas till att associera roman lukter med specifika beteendemässiga svar att ge insikt i lukt krets funktion. Detta protokoll Detaljer förfarandet för utbildning möss på en Go/No-Avbryt operant lärande aktivitet. I denna strategi, möss är utbildade på hundratals automatiserade prövningar dagligen under 2 – 4 veckor och kan sedan testas på roman Go/No-Avbryt lukt par för att bedöma olfactory diskriminering, eller användas för studier på hur lukt lärande förändrar strukturen eller funktionen hos den lukt krets. Dessutom har mus luktbulben (OB) pågående integrationen av vuxen-född nervceller. Intressant, ökar lukt lärande både överlevnad och synapsförbindelser av dessa vuxen-född nervceller. Detta protokoll kan därför kombineras med andra biokemiska, elektrofysiologiska och imaging tekniker för att studera lärande och aktivitet-beroende faktorer som medla neuronal överlevnad och plasticitet.

När möss har lärt dig lukt lärande uppgiften, kan de nu associera roman lukt par med belöning och straff. Dessa utbildad möss normalt börja med ca 50% noggrannhet för Go/No-Avbryt aktiviteten. Andelen rätta kan ritas av rättegång block som en inlärningskurva för romanen lukt par (figur 3A). Inom 10 block-prövningar, som tar de flesta möss som är mindre än 30 min att utföra, kan möss korrekt diskriminera lukter med mer än 8...

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

Här tränar vi möss på en associativ inlärning uppgift att testa lukt diskriminering. Detta protokoll kan också för studier på lärande-inducerad strukturella förändringar i hjärnan.

En objektiv och reproducerbara Test av lukt lärande och diskriminering i möss

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.

Research

JoVE Journal

Neuroscience

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

Låg-stress Route lärande Använda Lashley III Maze hos möss

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

Morris water maze test för inlärning och minne Brister i Alzheimers Möss sjukdomsmodell

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

T-labyrinten Påtvingad alterneringsledighet och vänster-höger uppgifter diskriminering bedöma arbets-och referens minne hos möss

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

Kartläggning Kortikala Dynamics med samtidig MEG / EEG och anatomiskt-begränsade Minsta-norm uppskattningar: en auditiv uppmärksamhet Exempel

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

Appetitive Associativ Olfactory Lärande i<em&gt; Drosophila</em&gt; Larver

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 Vuxen 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

Ex vivo Beredningar av den Intakt vomeronasala orgel och tillbehör luktbulben

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

Hela Mount Märkning av Cilia i huvud Olfactory systemet Möss

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

Elektro Metod för inspelning Intracellulärt Spännings svar av<em&gt; Drosophila</em&gt; Fotoreceptorer och Intern på ljusstimuli<em&gt; In Vivo</em

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

Hög upplösning Kvantitativ Synaptic Proteome Profilering av Regioner Mouse Brain Efter Auditory Diskriminering Learning

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