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 chamber with chamber floor. Keep the training box in a low traffic, dimly lit area to avoid distractions.</li>
	<li>Drill each water port with a small hole on top to allow an 18-gauge needle to dispense water inside the port.</li>
	<li>Fill glass vial with odorant dissolved in mineral oil (replaced weekly), and securely tighten the cap.</li>
	<li>Connect 18-gauge needle to silicone tubing. Pierce glass vial cap with 18-gauge needle, and connect other end of silicone tubing to the intake of the odor ports.</li>
	<li>Place each silicone tubing into an odor valve.</li>
	<li>Connect the vacuum line to the odor ports.</li>
	<li>Attach two 10 mL syringes to a metal holding rod, and connect tubing to the syringes. Connect the other side of the tubing to an 18 G needle. Fit the needle into the drilled hole of the nose poke port on the mouse chamber. Connect the other end of the tubing to the water valve.</li>
	<li>Fill the two 10 mL syringes with rodent drinking water.</li>
	<li>Connect an air-flow meter to the air intake and maintain air flow at 3&#8211;5 L/min.</li>
	<li>Connect the 2 water valves, 2 odor valves, 2 water ports, odor port, and power to the USB interface system. Connect all valves to &#39;Output&#39; ports, all the odor and water ports to &#39;Input&#39; ports. Connect all equipment to power outputs from the USB interface box, and lastly, connect the USB interface box to power.</li>
	<li>Adjust the vacuum suction to avoid cross contamination of odors between trials.</li>
	<li>Use odor-specific tubing to connect odorant vials to the chamber.</li>
</ol>
2. Mouse Preparation: 1&#8211;3 days
<ol>
	<li>Divide mice into 3 groups: control (no olfactory training necessary), pseudo-trained (mice who receive reward or punishment at random), and trained groups. Expose pseudo-trained mice to the training box and odor delivery, but do not provide olfactory training because the outcome of reward versus punishment is randomly associated with the odor delivered. 
	NOTE: The pseudo-trained group will go through the training paradigm under the &#34;Pseudotraining&#34; stages. Trained mice will complete all training stages. Pseudotraining is optional if the purpose of the experiment is to assay for behavioral differences in odor discrimination or learning. The protocol provided here adds this group if the experimenter wants to probe for neuronal differences before and after training. The pseudotrained group would then control for passive odor exposure and non-olfactory related training.</li>
	<li>Begin water restriction in mice to 40 mL/kg/day. Avoid bodyweight loss greater than 20% of the animals&#39; baseline weight in order to avoid distress (Figure 2A).</li>
	<li>Weigh the mice daily to ensure they are above 80% of baseline weight. If a mouse falls below this threshold, remove the mouse from the study and provide free access to water.</li>
	<li>Keep all environmental factors constant throughout the protocol including temperature, noise, and stray odors (including personal body and perfume/cologne/deodorant scents). 
	NOTE: As with all animal behavior testing, small environmental changes can greatly influence results.</li>
</ol>
3. Instructions for All Stages
<ol>
	<li>Code training software for each stage below. Run the software on the behavioral software. 
	NOTE: Coding for all stages are contained in supplemental coding files. Data for 20 trails are grouped as a single block and mouse performance is displayed across blocks. Furthermore, each stage can be repeated on a mouse for a number of days until completion criteria are met.</li>
	<li>Do not keep mice in the behavior box for more than 60 min/day.</li>
	<li>Clean mouse waste from the cage before each mouse is transferred to the cage. Spray and wipe the chamber with 70% ethanol to minimize mouse odor distraction.</li>
</ol>
4. Training Stage 1: Associating Water Reward with a Center Nose Poke, 1&#8211;3 Days
<ol>
	<li>In this stage, associate mice with a water reward upon exploration of the water port.</li>
	<li>Training Stage 1 programming instructions
	<ol>
		<li>Program this stage to only use the water delivery port. Let the mouse receive a water reward for each nose poke. 
		NOTE: The program will output the time duration of the trial and the total number of water rewards the mouse received.</li>
	</ol>
	</li>
	<li>Set the pseudotraining Stage 1 programming the same as Training Stage 1.</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Configure the behavior box with a water port in the center and with all side ports inaccessible. Place a mouse into the chamber. Close the mouse chamber and begin the Stage 1 program.</li>
	</ol>
	</li>
	<li>Consider this stage as complete when the mouse achieves 100 trials within 60 min. Remove the mouse from the chamber after 60 min or 100 trials have been completed (Figure 2B). 
	NOTE: Due to individual differences, some mice will naturally refrain from exploring the box.</li>
	<li>If encouragement is needed, manually deliver water into the water port. Repeat this stage for up to 3 days. 
	NOTE: Mice that are already trained can be repeated further to maintain parity and to keep them on fluid restriction. It is also possible to promote an entire group to stage 2 when the group average reaches 100 trials/60 min. This will allow for all mice to continue training on the same day.</li>
</ol>
5. Training Stage 2: Associating a Side Port Water Reward with Center Port Nose Poke, 1&#8211;5 Days
<ol>
	<li>In this stage, let the mice poke their nose in the center port and then immediately receive a water reward on the side ports.</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Configure this and every subsequent stage with 2 water ports on the sides and the odor port in the middle. Place a mouse into the chamber. Close the mouse chamber and begin the Stage 2 program.</li>
	</ol>
	</li>
	<li>Training Stage 2 programming instructions
	<ol>
		<li>Provide the mouse with an immediate water reward on both sides after a nose poke into the center odor delivery port. Set output parameters for this stage as the time duration of the trial, the number of trials initiated, and the number of water rewards received within 5 s of a nose poke.</li>
	</ol>
	</li>
	<li>Set the pseudotraining Stage 2 programming the same as Training Stage 2.</li>
	<li>Consider this stage as complete when a minimum of 40 trials are performed in 60 min, with at least 25% of water rewards received within 5 s of the center port nose poke. Remove the mouse once this stage has completed (Figure 2C). 
	NOTE: The training program calculates the percentage of water rewards received in a timely manner in order to determine completion of this stage. Although mice vary in how fast they complete this stage, most mice will reach completion criteria within 5 days. However, if a mouse has not completed this stage within 5 days, do not advance the mouse to the next stage. This mouse will be removed from the cohort.</li>
</ol>
6. Training Stage 3: Associating a Water Reward with a Specific Odor and Within a Specific Time Window, 1&#8211;3 Days
<ol>
	<li>In this stage, let the mice receive a Go (S+) odor upon a nose poke in the center port. Subsequently, yield a water reward upon a nose poke into the side water ports within 5 s.</li>
	<li>Training Stage 3 programming instructions
	<ol>
		<li>Deliver the S+ odor in the center odor port.</li>
		<li>Deliver the water reward if the mouse pokes the side ports within 5 s of an odor delivery. 
		NOTE: The program begins by only requiring a 100 ms nose poke in the center port to yield a water reward. Once the mouse pokes the center port for correct amount of time in 80% of trials, the time duration of a nose poke required for a water reward will increase by 50 ms up to 400 ms.</li>
		<li>Set the output parameters identical to stage 2.</li>
	</ol>
	</li>
	<li>Set the pseudotraining Stage 3 programming instructions the same as Training Stage 3. However, do not connect the S+ odor to the odor delivery controller to ensure that the S+ odor is not associated with a water reward.</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Set the same configuration at stage 2.</li>
		<li>Connect the S+ odor to the odor delivery controller. Place a mouse into the chamber. Close the mouse chamber and begin the Stage 3 program. Remove the mouse once this stage has completed.</li>
	</ol>
	</li>
	<li>Consider this stage as complete when there are greater than 60 rewards within 60 min (Figure 2D).</li>
</ol>
7. Stage 4A: Associating No-Go (S-) Odor and Time-out Punishment, 1&#8211;2 Days
<ol>
	<li>In this stage, introduce the mice to a No-Go odor (S-). Provide a time out punishment to the mice that incorrectly go to the water ports after smelling the no-go odor.</li>
	<li>Training Stage 4A programming instructions
	<ol>
		<li>Deliver only the S+ odorant in this stage in the beginning, identical to stage 3. After mice complete 40 trials, begin random delivery of the odors to include both S+ and S- odors. Program a 2-s time out punishment if the mouse attempts to seek a water reward after being presented the No-Go odor. 
		NOTE: To aid in distinguishing Go vs. No-Go, the IR side lights can be manipulated such that they are on during Go odorants and off during No-Go odorants. The lights act as a secondary cue to aid mice in initial training. Once mice are trained with the lights, they should under-go training without lights to confirm they have learned the task with odors.</li>
		<li>Set the output parameters as time duration of the trial, number of trials initiated, number of trials completed, % correct, and total number of rewards received.</li>
	</ol>
	</li>
	<li>Set the pseudotraining Stage 4A programming instructions the same as Training Stage 4S except S+ and S- odors are presented randomly from the start. Give water reward or timeout punishments at random, regardless of task performance.</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Set the box configuration the same as previous stage 3.</li>
		<li>Connect both S+ and S- odor to the odor delivery controller. Place a mouse into the chamber. Close the mouse chamber and begin the Stage 4A program. After the mice complete 40 trials, switch the program to randomly deliver the odors. Remove the mouse once this stage has completed.</li>
	</ol>
	</li>
	<li>Consider this stage as complete when there are 40 trials with &#62; 60% correct responses.</li>
</ol>
8. Stage 4B: Associating No-Go (S-) Odor and Time Out Punishment, 5&#8211;11 Days
<ol>
	<li>Training Stage 4B programming instructions
	<ol>
		<li>Make this stage identical to stage 4A, however the time out punishment for attempting a water reward after a S- odor is 4 s.</li>
	</ol>
	</li>
	<li>Set the pseudotraining Stage 4B programming instructions identical to stage 4A, however water reward and time out punishment are randomly delivered per trial.</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Set the box configuration the same as previous stage 4A.</li>
		<li>Connect both S+ and S- odor to the odor delivery controller. Place a mouse into the chamber. Close the mouse chamber and begin the Stage 4B program. Consider this stage complete when there are &#62;100 trials within 60 min with accuracy &#62;85% (Figure 2E)</li>
	</ol>
	</li>
	<li>Monitor the maximum and minimum percentage correct for each session to track each mouse&#39;s progress in this stage. Approximately 85-90% of mice reach completion criteria for this stage. Exclude mice that do not achieve completion of this stage for the Go/No-Go testing phase.</li>
	<li>(Optional) At the end of this stage, apply a reversal training, where the Go/No-Go odorants are switched on the odor vial platform. Ensure that the mice are not associating with another stimulus, such as the sound of the valves associated with the side ports.</li>
</ol>
9. Go/No-Go Assay Task: 1 Day, 20 Min per Mouse per Day
<ol>
	<li>Consider this as the final stage to determine accuracy of identifying odor associations and learning to discriminate odor pairs. 
	NOTE: Novel odorants are utilized for S+/S- to test how long animals take to learn new associations. Structurally similar odorant pairs are used to increase task difficulty. Examples include 1-Butanol vs. 1-Pentanol, Isoamyl Acetate vs. Isoamyl Butyrate, and + Carvone vs. - Carvone.</li>
	<li>Programming instructions
	<ol>
		<li>Let the training software detect the length of nose poke. Program a 300 ms nose poke in the center odor port to execute the task.</li>
		<li>Set the output parameters identical to stage 4B, except do not use any light guided cues.</li>
	</ol>
	</li>
	<li>Box configuration and mouse setup
	<ol>
		<li>Set the box configuration the same as previous stage 4B.</li>
		<li>Connect both S+ and S- odor to the odor delivery controller. Place a mouse into the chamber. Close the mouse chamber and begin the Stage Go/No-Go program. Expose the mice that have learned the olfactory learning task to novel pairs of chemically similar odors. 
		NOTE: Wildtype mice with adequate odor discrimination reach &#62;85% accuracy with novel odorants after approximately 10-20 blocks or 200-400 trials (Figure 3A).</li>
	</ol>
	</li>
</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.

<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...

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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 ...

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8.5K Views.  Baylor College of Medicine. 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.

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

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 aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

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

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

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

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

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 rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions 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 cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

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

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

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 have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

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

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

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

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

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 dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

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

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

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

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

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 discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

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