Name: An Appetitive Spatial Working Memory Task for Mice in a Semi-Automated 8-Arm Radial Maze, Reducing Fearful Memory Association in the Maze (Video) | JoVE Journal
Uploaded: 2025-07-29T12:30:00.000+00:00
Duration: 14 min 24 s
Description: Learn a semi-automated 8-arm radial maze protocol to assess spatial working memory in mice while minimizing stress. This method enables reliable cognitive research with gentle handling and video monitoring.

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5 minutes

We thank our colleagues in the Wolfson Institute for Biomedical Research for helpful discussions and David Bannerman (University of Oxford) for advice on mouse behavior and for reviewing an early draft of the paper. We also thank Matthew Grist for technical and administrative support. This research was funded by the Wellcome Trust [108726/Z/15/Z and 214286/Z/18/Z] and Gerald Choa Neuroscience Institute [PJ8425013].

All mouse experiments were pre-approved by the UCL Ethical Committee and authorized under the Animals (Scientific Procedures) Act 1986 and subsequent legislation of the UK Government.
1. Preparation of programs for the automated maze control on a software application
NOTE: The automated radial arm maze, as shown in Figure 1A-C, and the room layout in Figure 1D were used.
<ol>
	<li>Prepare an experimental workflow for door closure as described in Figure 2.</li>
	<li>Set the start of the habituation stage 1 (Figure 2A) on a software application. To do so, follow the steps below.
	<ol>
		<li>Set the start of the trial and video recordings, and close all the arms on the software. 5 s after the software recognizes the mouse in the maze&#39;s central hub, it starts tracking and opens all the doors.</li>
		<li>When the mouse goes inside an arm and is 4 cm away from the closest door of the central hub doors, close all the doors. Then, immediately after closing all the doors, open the door of the arm that the mouse selected.</li>
		<li>Once the mouse returns to the central hub, close the door and confine it to the central hub for 5 s. Repeat this for 30 min.</li>
		<li>Stop the video recording and tracking when 30 min elapse, and the mouse returns to the central hub. 
		NOTE: Let the mouse explore the maze freely at their will.</li>
	</ol>
	</li>
	<li>Habituation stage 2 (Figure 2B)
	<ol>
		<li>Set the start of the trial and video recording in the associated software and close all the doors.</li>
		<li>5 s after the software recognizes the mouse in the central hub of the maze, start its tracking and open all doors.</li>
		<li>End the trial if the mouse enters each arm at least twice or after 30 min and then returns to the central hub. Let the mouse explore the maze freely at its will.</li>
	</ol>
	</li>
	<li>Habituation stage 3 (Figure 2C) 
	NOTE: This stage is the combination of stages 1 and 2.
	<ol>
		<li>Set the software to start the trial and video recording and close all the doors.</li>
		<li>Set the software to control the maze doors 5 s after the software recognizes the mouse in the central hub. Set the start of the tracking at this point and open all the doors.</li>
		<li>Close all the doors when the mouse goes inside an arm and is 4 cm away from the closest door of the central hub doors. Then open the door of the arm.</li>
		<li>Close the door when the mouse fully enters the central hub and confine the mouse in the central hub for 3 s. Repeat this sequence for 30 min or until the mouse enters each arm at least once.</li>
		<li>Set the end of the trial after fulfilling the condition, and when the mouse enters the central hub fully. 
		NOTE: Let the mouse explore the maze freely at its will.</li>
	</ol>
	</li>
	<li>Working memory task (Figure 2D, E)
	<ol>
		<li>Set the software to start the trial and recording, and close all the doors.</li>
		<li>5 s after the software recognizes the mouse in the central hub, set the software to start the tracking.</li>
		<li>Start the forced run phase. Present pseudo-randomly chosen 4 arms one by one to the mouse.</li>
		<li>When the mouse enters the first arm and is 3 cm away from the central hub, ensure the door closes and confines it in the arm for 15 s.</li>
		<li>Let the door&#160;open and let the mouse go back to the central hub.</li>
		<li>Close the door and then open the second arm.</li>
		<li>Repeat this till the fourth arm.</li>
		<li>When the mouse returns to the central hub after the fourth arm of the forced run phase, confine it to the central hub for 5 s.</li>
		<li>Let all the doors open and start the free run phase.</li>
		<li>End the trial when the mouse returns to the central hub after collecting all the rewards/entering all the arms at least once.</li>
	</ol>
	</li>
</ol>
2. Preparing home cages for individual housing
<ol>
	<li>Set up new cages using standard nesting materials for a new experiment.
	<ol>
		<li>Put new bedding, sawdust, a paper igloo, a paper tunnel, paper nesting stripes, and a wooden bite block in new cages for each mouse for the experiment.</li>
		<li>Place the food hopper and close the lid. 
		NOTE: Mice can damage the parts for ventilation if they are without the food hopper.</li>
	</ol>
	</li>
</ol>
3. Handling, weighing, and diet restriction in the animal holding room (Day -7 to Day 0)
<ol>
	<li>Guide the mouse to the paper tunnel supplied in the home cage by hand gently.</li>
	<li>Lift the mouse from the grouped cage using the paper tunnel38,39,40 with the hand, keep it at chest height for several seconds. Alternatively, let the mouse climb onto the hand voluntarily. 
	NOTE: Handling animals with minimal stress is essential for behavior experiments. Never pick them up by the tail, which is stressful for mice. Habituating mice to the experimenter is also important. Mice need to be well-habituated and relaxed, or they will not learn well in the maze later.</li>
	<li>Always hold the mouse using the same handling method. Put the mouse on the scale for body weight monitoring and record its body weight. 
	NOTE: Do not stress the mice with fast body movement or by making loud noises. It is better to do this in a quiet environment. Otherwise, they may jump off the scale or the experimenter&#39;s hand. Also, if the mice are stressed, they can show squinted eyes, squeak, and may bite the experimenter&#39;s hand41,42.</li>
	<li>After weighing, put the mouse into the new cage prepared earlier.</li>
	<li>Put mice on a diet with a restricted amount for a week prior to the exposure to the radial arm maze. This motivates them to forage the maze for food rewards provided. The food reward during the experiment is diluted sweetened condensed milk.</li>
	<li>Put 2 ~ 3 g of standard chow in the cage and maintain the body weight between 85 ~ 90% of the starting weight throughout the experiment. 
	NOTE: C57BL/6 mice eat 3-4 g of standard chow every day43.</li>
	<li>Observe the overnight drop in the body weight as a guide to standardize the amount of food to be given the following day. From then on, give a sufficient daily amount of standard chow to maintain 85-90% of their starting body weight throughout the experiment. 
	NOTE: These animals need to lose weight slowly. Monitor their body weight and facial expressions to avoid stress.</li>
	<li>Provide 1 mL of diluted condensed milk (reward; sweetened condensed milk : water = 1 : 1) in a small plastic cup in the home cage to habituate mice until the end of the habituation stage 1. Make sure mice eat the reward. Change the reward food if the mice strain shows neophobia to the presented food. 
	NOTE: From habituation stage 2, do not provide the reward in the home cage. If the body weight of any mouse drops below 80% of the starting weight, it should be euthanized as per the IACUC and country-specific guidelines. If a mouse is not in the normal body weight range for the mouse line being used (20-30 g at 8-12 weeks old) at the first body weight check, it should not be considered in the cohort to avoid difficulty in maintaining its body weight in the required range later.</li>
</ol>
4. Bringing mice to the testing room
<ol>
	<li>Use a trolley to bring mice to the behavior testing room slowly.</li>
	<li>Leave the mice undisturbed on the trolley for 5 min.</li>
</ol>
5. Room set up
<ol>
	<li>Set the room lighting illuminating the central hub indirectly, at 80 lux with warm white light bulbs, and play white noise at 70 dB measured at the central hub. NOTE: It is known that exposure to moderate-intensity white noise leads rats to perform better in a geometrically complex maze44,45. White noise seems to do more than simply mask the unavoidable noise in the environment; 70 dB white noise increases choline uptake in several brain regions of rats, including the prefrontal cortex, hypothalamus, and hippocampus (but not in the striatum)46 and RAM working memory performance is known to depend on the integrity of cholinergic pathways among the hippocampus, frontal cortex, and forebrain32,47. It is also known that animals use both distal and proximal visual cues to navigate accurately in their environments. However, mice depend on distal visual cues more than proximal cues to navigate during spatial learning48. In this experiment, proximal cues printed on a small piece of paper were introduced to the back walls of all the arms of the 8-arm RAM, but they did not reduce the daisy-chaining behavior. Geometrically asymmetrical distal visual cues (on the ceiling and walls of the room), on the other hand, reduced daisy-chaining and were essential for spatial navigation in the RAM.</li>
</ol>
6. Habituation stage 1 - Non-rewarded (Day 1 - 2)
NOTE: No rewards are baited in the maze. This stage is to habituate mice to the maze doors and their sounds.
<ol>
	<li>Start the mouse tracking and video recording by clicking the Start button on the software and bringing the mouse to the maze without stressing it. Do this by moving slowly and changing the height slowly while the mouse is in the experimenter&#39;s hand. Place the mouse in the central hub. 
	NOTE: If mice shake on the experimenter&#39;s hand, slow down the movements. It is a sign of being scared. Mice move around during the 5 s confinement period in the central hub before starting the forced run phase. They can face any side when put in the central hub.</li>
	<li>Keep watching the mouse&#39;s behavior on the PC monitor placed far from the maze or below the maze wall level. If the mouse tries to escape from the maze by climbing on the wall, simply stand up from the chair to show the experimenter&#39;s existence and walk toward it,&#160;monitoring their perfomance. In the worst-case scenario, push the mouse back into the&#160;maze. 
	NOTE: Normally, mice go back to the maze after hearing the frictional noise of the experimenter&#39;s clothes as they approach it. This needs to be done quickly. If the experimenter continues doing this, the mice will stay in the maze after the habituation stages 1 through 3. If mice keep jumping out of the maze, it is advisable to remove them from further experiments and analysis.</li>
	<li>After the trial, collect the mouse from an arm of the maze by letting the mouse climb on the experimenter&#39;s hand voluntarily while the other hand covers the arm to prevent it from jumping off the maze.</li>
	<li>Brush off mouse feces and wipe the maze with dry tissue to remove any traces of the rewards. Then, wipe with wet tissue and dry the maze with dry tissue (about 2 min).</li>
	<li>Do this for each mouse in turn for two days. 
	NOTE: After 30 min, mice might not have visited all 8 arms of the maze. This is normal and is not a problem. Mice tend to visit arms in an unsystematic, non-sequential pattern and move cautiously, spending uneven times in each arm. Mice can climb on the maze walls and/or jump out of the maze at this stage. If this happens (e.g., the mouse tries to climb on the walls), prevent it by ensuring that the experimenter makes the mouse aware of his/her presence by approaching the maze and making some noise, such as clothes rustling. Therefore, careful observation of the mouse&#39;s behavior in the RAM and quick action might be required. After a few repetitions of this procedure, mice will stay in the maze. If, despite everything, the mouse does not stay in the RAM during habituation, remove it from the analysis.</li>
</ol>
7. Habituation stage 2 - Rewarded (Day 3 - 4)
NOTE: This stage is to encourage mice to explore the maze freely for food rewards.
<ol>
	<li>Place 70 &#956;L of the food reward in each food well in each arm.</li>
	<li>Start the mouse tracking and video recording by clicking the Start button on the software and then placing the mouse onto the maze central hub as explained in 6.1.</li>
	<li>Keep watching the mouse&#39;s behavior on the PC monitor from a distant place and write it down if necessary, e.g., climbing on the wall or jumping off the maze. End the trial when the mouse enters each arm at least twice or 30 min have elapsed.</li>
	<li>After the trial, collect the mouse from the maze in an arm by letting the mouse climb on the experimenter&#39;s hand voluntarily while the other hand covers the arm to prevent it from jumping off the maze.</li>
	<li>Clean the maze as described in 6.4 (Habituation stage 1; about 2 min).</li>
	<li>Do this for each mouse in turn twice per day for two days. 
	NOTE: At this stage, mice may display daisy-chaining behavior, but it is unnecessary to worry because the additional 4 arm entries in the working memory task may discourage this movement. In this step, mice can stop in the arm without consuming the reward. On Day 4, mice should be more trained than on Day 3 but can still stop in the middle of the arm.</li>
</ol>
8. Habituation stage 3 - Rewarded (Day 5 - 6)
NOTE: This stage is to let mice get used to the environment of the combination of stages 1 and 2.
<ol>
	<li>Bait all the food wells with 70 &#956;L of the reward.</li>
	<li>Start the mouse tracking and video recording by clicking the software&#39;s start button and placing the mouse at the maze&#39;s central hub.</li>
	<li>Keep watching the mouse&#39;s behavior on the PC monitor from a distant place, until the mouse enters each arm at least once or 30 min elapse. If the mouse tries to escape from the maze, act fast as stated in Habituation Stage 1 (6.2).</li>
	<li>After the trial, remove the mouse from the maze by letting it climb on the experimenter&#39;s hand voluntarily while the other hand covers the arm to prevent it from jumping off the maze.</li>
	<li>Clean the maze as described in 6.4 (habituation stage 1) (about 2 min).</li>
	<li>Do this for each mouse twice per day for two days. 
	NOTE: At this stage, removing any mice attempting to escape from the maze from the analysis is best.</li>
</ol>
9. Working memory task - Rewarded (Day 7 - Day 15)
<ol>
	<li>Bait all the arms with 40 &#956;L of the reward. Place the reward in each food well close to the central hub to make it difficult for mice to see from the central hub.</li>
	<li>Start the mouse tracking and recording by clicking the software&#39;s start button and then place the mouse onto the maze central hub without stressing it.</li>
	<li>During the 5 s confinement in the central hub, walk away and sit somewhere away from the maze. Monitor the mouse&#39;s behavior and write down the sequence of the arm that the mouse visits in the maze (see Supplementary file 1).</li>
	<li>Stop recording the arm visit sequence when the mouse collects all eight food rewards, which is the end of the working memory task, and then let it visit four extra arms. 
	NOTE: By doing this, mice can have some more time before being caught, which allows mice to focus on the working memory task. This step also allows mice to reinforce the fact that rewards are not replenished once collected.</li>
	<li>After the four extra arm visits, remove the mouse from the maze at the end of the last arm visit by letting it climb onto the hand while covering the arm with the other hand. 
	NOTE: By not catching mice in the central hub, mice will not associate being caught with the central hub, where mice decide which arm to go to next. Once mice become familiar with the task, including the extra arm visits, they can freely move near the last food well or spend some time in the central hub. However, they should finish the 4 extra arm visits within 3 min. If they do not finish it within 5 min, return them to the home cage.</li>
	<li>Select the next cage to adjust the arousal level by tapping the cage for the next trial.</li>
	<li>Clean the maze as described in 6.4 (habituation stage 1) (about 2 min).</li>
	<li>Move on to the next mouse and repeat this 6 times for each mouse in turn for 9 days. 
	NOTE: The average duration of the four extra arm visits should become longer in later trials, as mice learn to recognize when no further rewards are available or that there are no novel, unvisited arms to go into.</li>
</ol>
10. Scoring
<ol>
	<li>Calculate &#34;daily success rate score&#34; (%) = 4(goals) / (total arm entry number[4+Errors]) x 100. 
	NOTE: After the forced run, only four arms are filled with the reward, the goals. If they enter an arm the mouse visited in the forced run, count it as one error, as well as repeated arms&#160;in the free run.</li>
	<li>Calculate &#34;perfect trial ratio score&#34; (%) in 6 trials = [(number of perfect trials) / 6 ] x 100 
	NOTE: A perfect score means there is no working memory error.</li>
	<li>Select speed, distance, and acceleration of the mouse in the software for analysis and calculate the averages for each day. Ensure to exclude the four extra arm visits from these analyses. Also, measure the duration of the four extra arm visits by reviewing the mouse tracking videos after the experiment. 
	NOTE: Mice can make quick turns immediately after&#160;entering an arm. Do not count and score this as&#160;an entry, as it is a quick mistake correction,&#160;which means working memory is valid49. However, if the mouse crosses the midpoint of the arm or stays in the arm for more than 3 s, count and score the entry. Daily success rates scores indicate mice&#39;s learning curves, and perfect trial ratio scores indicate working memory retention efficiency.</li>
</ol>
11. Statistics
<ol>
	<li>If studying the difference between the control and a knockout mouse line, as done in the previous publication50, perform the statistics as described below.
	<ol>
		<li>Compare the resulting data of the control and experimental groups using repeated measures two-way ANOVA with a post hoc analysis (e.g., Bonferroni test) [measuring each subject at 9 different time points].</li>
		<li>If comparing groups, test at least 10 and preferably ~20 mice in each group. Where the effect of gene manipulation, making lesions, or drug treatment is obvious, the sample sizes can be smaller to conclude.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.&#160;

Working memory1 is a cognitive process that can hold a limited amount of information for a short period while it is manipulated and used in the planning and execution of a mental task, such as memorizing a phone number or a route for a destination from a different location. It is closely related to fluid intelligence, which is the ability to reason quickly and think abstractly. It is believed that working memory training can improve fluid intelligence2. Working memory, therefore, is crucial for research studies.
Most popular behavior experiments for rodents were originally devised for rats in the 1980s, and researchers today often apply those protocols to mice. However, there are consistent behavioral differences between rats and mice3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19. Especially, fear management is an important factor that can affect the results of the experiments, as mice are more prone to stress compared to rats3,4,5,6,7,10,11,12,13,14,15,16,17,18,19. Therefore, it is important to consider the differences when applying those protocols to mice. In our protocol, we tried to minimize the stress factors during the working memory task in the radial arm maze. This was done by carefully timing the door closures using the mouse tracking and maze control software. Also, mice catching at the end of the session in the central hub was avoided. This approach allowed the mice to choose their next arm without associating the location with the fear of being caught. Delaying the capture of the mice until after all rewards had been collected in the maze ensured that the last reward was not linked to the experience of being caught.
The land radial arm maze (RAM) is a popular and commonly used apparatus to assess spatial and nonspatial learning and memory, and to test working and reference memory in rodents. It is one of the most popular apparatuses for spatial learning and memory in mice20,21,22. RAM takes advantage of the natural tendency of rodents to explore the maze, find novelties, and learn spatial information on a trial-and-error basis, in addition to foraging. This tendency can be augmented by restricting their diet and rewarding them with preferred food in the maze. Nowadays, several variants of the RAM apparatus are used, e.g., with 6, 8, 12, or 16 arms and a partially enclosed or open/elevated apparatus23,24,25. Most present-day protocols are based on that of Olton et al.20,26, but use simpler (6- or 8-arm) mazes9,24,27,28,29. Testing working memory in a radial arm maze (6 or 8 arms) is more powerful than the T maze (2 arms) because animals have more options of arms to explore.
The original RAM was designed by Olton and Samuelson20 to assess spatial learning and memory in rats using food baits for positive reinforcement, unlike water mazes such as the Morris water maze and 8-arm radial water maze that use negative reinforcement, i.e., water immersion20,30,31,32. Olton and Samuelson20found that rats have excellent memories of places as they pass through the RAM. They showed that the hippocampus is involved in spatial information processing. They, therefore, introduced the concept of a&#160;dual memory system, comprising working and&#160;reference memory, in rodents. This dual&#160;memory system had been suggested&#160;previously by Honig36&#160; based on experiments&#160;with pigeons. Olton et al.&#160;found that hippocampal function is essential for working memory. This was achieved by making lesions in the entorhinal area, the body of the fimbria-fornix, anterior to the hippocampus, septum, or postcommissural fornix to disrupt each of the major extrinsic fiber connections of the hippocampal formation26,33,34,35. Fornix-fimbria lesions did not affect reference memory26.&#160;
In modern protocols, animals are habituated to the environment by allowing them to explore the maze freely for 10-15 min per day for 3 consecutive days, followed by maze training for 1-3 weeks with a preferred food at the end of all arms or fixed selected arms, testing working memory, or working memory and reference memory in the same experiment, respectively9,29,37.
Often, these protocols are not described fully, and the behavioral differences between rats and mice are not well considered. Here we present our protocol and experience in detail, especially how to avoid associating fearful experiences with places and timing based on fear management differences between rats and mice. This protocol is for researchers who want to study spatial working memory and apply it to electrophysiology, real-time imaging, and histology using mice.

<table><tbody><tr><td>Artery forceps or plier</td><td>Any</td><td>N/A</td><td>For cutting the standard chow</td></tr><tr><td>C57BL/6J mice</td><td>Jackson laboratory</td><td>000664</td><td>2~3 months old</td></tr><tr><td>Condensed milk (Carnation)</td><td>Nestle</td><td>N/A</td><td>Used as the reward in the task</td></tr><tr><td>Distilled water</td><td>Any</td><td>N/A</td><td>For cleaning and diluent of the condensed milk</td></tr><tr><td>EthovisionXT13&amp;nbsp;</td><td>Noldus</td><td>N/A</td><td>Control the maze doors and tracking mouse</td></tr><tr><td>--Multiple body point module</td><td>Noldus</td><td>N/A</td><td>For Multiple body point tracking</td></tr><tr><td>--Trial &amp;amp; Hardware Control Module</td><td>Noldus</td><td>N/A</td><td>For Trial &amp;amp; Hardware Control</td></tr><tr><td>-- I/O box</td><td>Noldus</td><td>N/A</td><td>Control the maze doors</td></tr><tr><td>Extramaze visual cues</td><td>Any</td><td>N/A</td><td>For mapping the maze by mice</td></tr><tr><td>High-resolution camera</td><td>Tracksys</td><td>N/A</td><td>For mouse tracking and filming</td></tr><tr><td>Paintbrush</td><td>Any</td><td>N/A</td><td>For cleaning</td></tr><tr><td>Paper tunnel</td><td>Any</td><td>N/A</td><td>For handling mice</td></tr><tr><td>Paper igloo</td><td>Any</td><td>N/A</td><td>For nesting</td></tr><tr><td>Paper nesting stripes</td><td>Any</td><td>N/A</td><td>For nesting</td></tr><tr><td>Pipetteman P1000&amp;nbsp;</td><td>Gilson</td><td>F144059M</td><td>For providing diluted condensed milk to the home cage</td></tr><tr><td>Pipette tip P1000</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Pipetteman P200</td><td>Gilson</td><td>F144058M</td><td>For baiting the maze</td></tr><tr><td>Pipette tip P200</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Radial arm maze</td><td>Tracksys</td><td>N/A</td><td>Custom made</td></tr><tr><td>Room lamps</td><td>Any</td><td>N/A</td><td>For even illumination in the maze</td></tr><tr><td>Small plastic cup</td><td>Any</td><td>N/A</td><td>For providing diluted condensed milk to the home cage</td></tr><tr><td>Speakers for white noise</td><td>Any</td><td>N/A</td><td>For playing white noise</td></tr><tr><td>Standard chow</td><td>Any</td><td>N/A</td><td>For diet restriction</td></tr><tr><td>tissue paper</td><td>Any</td><td>N/A</td><td>For cleaning</td></tr><tr><td>Verifocal lens</td><td>Tracksys</td><td>N/A</td><td>5-50 mm, manual focus, manual iris</td></tr><tr><td>Wooden bite block</td><td>Any</td><td>N/A</td><td>For nesting</td></tr><tr><td>50 ml centrifuge tube</td><td>Falcon</td><td>Falcon&amp;trade; 352070</td><td>For mixing the condense milk with distilled water (1:1)</td></tr></tbody></table>

an appetitive spatial working memory task for mice in a semi-automated 8-arm radial maze, reducing fearful memory association in the maze

An Appetitive Spatial Working Memory Task for Mice in a Semi-Automated 8-Arm Radial Maze, Reducing Fearful Memory Association in the Maze

This method paper details a protocol to test spatial working memory in mice using a semi-automated radial 8-arm maze (RAM). The RAM is a partially enclosed apparatus with 8 horizontal, equally spaced arms radiating from a central hub, from which access to each arm can be controlled individually by servo-controlled motorized doors. Animals start in the central hub and are allowed to explore the maze for a food reward at the end of each arm or selected arm. The RAM task was originally designed for rats, but we have adapted the protocol for mice, for example, by including more habituation steps. In our protocol, all arms are initially baited with sweetened condensed milk, and mice are admitted sequentially into four pseudo-randomly selected arms to collect the rewards ("forced run") before opening all doors together to allow the mice to run freely and find the remaining four rewards ("free run"). A 5 s delay is introduced between the forced and free runs to challenge working memory; an error is recorded if the mouse enters any previously visited arm during the free run. The task is complete when all rewards are recovered. After 6 days of habituation and 9 days of maze training, male C57BL/6 mice regularly achieve &#8805; 80% daily success rate score, defined as 4/(4+E), where E is the number of errors. This semi-automated task could, in principle, be combined with in vivo monitoring methods such as electrophysiology, multiple-photon microscopy, or calcium imaging.

This protocol was used in our previous publication, where we asked whether new oligodendrocytes and myelin sheaths are necessary for spatial working memory task performance using MyRF cKO mice and the control50. We found that the working memory task performance was improved in the control mice but not in MyRF cKO mice. The working memory task stimulated and promoted oligodendrocyte precursor cell (OPCs) proliferation and differentiation, and oligodendrocyte (OL) myelination in the brain areas related to working memory processing in wild-type mice. We also performed the T-maze rewarded alternation task to assess spatial working memory. This task was performed to detect working memory deficiency in MyRF cKO mice. However, no significant differences were observed in the histological analysis between the mouse group that had the T-maze rewarded alternation task and the home cage control group. Therefore, we concluded that the protocol presented here is more effective than the T-maze rewarded alternation task for studying working memory.
Each mouse had 6 trials of the working memory task per day that lasted for one hour in total. Thus, this protocol allowed for a maximum of 8 mice in a cohort. In the early days of habituation, mice spent uneven time in the maze arms, and their movements were slow. They stayed at the end of the arms for a long time. Until the end of day 5 of the habituation in the maze, they spent variable times in the maze arms and could return to the central hub without touching the rewards (Figure 3A). However, on the sixth day, animals entered each arm at least once within 8-15 min, spent a similar period in each arm, which indicated that the animals were habituated to the maze and were ready for the task (Figure 3A, day 6 lower row and Figure 3B).
During the habituation stages, they often stood on their hind limbs and looked up from the arms or the central hub. Similar behavior was observed during the working memory task, but they did not necessarily go around in the same direction (e.g., 2 -&gt; 3-&gt; 5 -&gt; 8 [clockwise] or 8 -&gt; 5 -&gt; 3 -&gt;2 [anti-clockwise]). They could change their direction (e.g., 5 -&gt; 4 -&gt; 6 -&gt; 1). Individual mice had no apparent communal tendency to choose the arm sequence. Mice tend to spend a little more time selecting the last rewarded arm than the first rewarded arm in the free run. This was probably because they had four options to choose from at the first choice in the free run, and only one option for the last reward. The first arm in the forced run phase was presented to mice at least 50 (15+15+15+5) s before the start of free run. Thus, this working memory task was not very easy to perform without working memory errors. Supplementary Video 1 shows a mouse undergoing a perfect trial on day 8 of the working memory task training.
Using either scoring system, we found that their performance was almost flat for the first 3 days of the 9-day RAM testing period but improved markedly after that (Figure 4A, B). After 6 days of habituation and 9 days of maze training, male C57BL/6 mice regularly achieved ≥ 80 % daily success rate score from day 8, defined as 4/(4+E), where E is the number of errors. The average distance traveled in the free runs became shorter over time, but the average speed and acceleration were fairly constant from day to day (Figure 4C-F). Mice might have developed a fear memory of being caught during the four additional arm visits, which could be one of the reasons why mice spent more time towards the end of the experiment (Figure 4G).
In this protocol, habituation steps lasted for 6 days, which could lead to adjusting the body clock for the experiment, even if the light/dark cycle was not reversed in an animal facility.
<img alt="Radial maze setup for behavioral experiments; diagram with modular arms, food well, and lab layout." class="xfigimg" src="/files/ftp_upload/66456/66456fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: RAM specifications and features. (A) Views of the maze from the ceiling-mounted CCD and from the side, and the dimensions. (B) The food well was sunk 1.5 cm below the maze floor level so that mice cannot see the contents from &gt;10 cm away. (C) Maze arena setting. The central hub and arm 1-8 are shown. (D) Schematic of the experimental room and the external visual cues with a photograph of the room. <a href="https://www.jove.com/files/ftp_upload/66456/66456fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Habituation stages diagram; mouse behavior tracking in a maze; includes tasks and conditions workflow." class="xfigimg" src="/files/ftp_upload/66456/66456fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Flow diagrams of the maze control programs. (A) Habituation stage 1 (days 1 and 2). (B) Habituation stage 2 (days 3 and 4). (C) Habituation stage 3 (days 5 and 6). (D) Working memory task (days 7-15). (E) Schema of the working memory task paradigm. Mice went through the pseudo-randomly chosen four forced run arms, were confined in the central hub for 5 s, and then went for the free run to collect the remaining rewards. <a href="https://www.jove.com/files/ftp_upload/66456/66456fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mouse habituation process; maze experiment with path tracking results and trial duration table." class="xfigimg" src="/files/ftp_upload/66456/66456fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Heat maps and bird nest maps during the 6-day habituation. (A) Heat maps of mouse trajectories during habituation days 1-6, for three mice. For habituation days 3-6, mice underwent two trials per day; the upper heatmaps show the first trials and the lower show the second trials. (B) Bird nest maps for habituation day 6. The upper three show the first trials and the lower three show the second trials of mice 1, 2, and 3. The lower Table lists trial durations for the same three mice. <a href="https://www.jove.com/files/ftp_upload/66456/66456fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Graphs analyzing mouse behavioral metrics over 9 days: success rate, cm, cm/s, cm/s², seconds." class="xfigimg" src="/files/ftp_upload/66456/66456fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: The RAM working memory task. (A) Average daily success rate scores (%) calculated by [4/(number of arm entries during the free run) x 100]. Scores from 6 trials were averaged for the day. (B) Perfect trial scores over the 9 days of the task. (C) Individual distances traveled and the average. (D) Individual running speeds of the mice in the task and the average. (E) Individual accelerations of the mice and the average. (F) Individual decelerations of the mice and the average. (G) Individual and average durations of the additional 4 arm visits. <a href="https://www.jove.com/files/ftp_upload/66456/66456fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary video 1: The mouse undergoes a perfect trial on day 8 of the working memory task training. <a href="https://www.jove.com/files/ftp_upload/66456/Supplementary%20video%20JoVE66456%20Takahiro%20Shimizu%20et%20al.avi" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Example recording sheet. Write down the sequence of the arm visits while watching the mouse's behavior. <a href="https://www.jove.com/files/ftp_upload/66456/Example%20Recording%20Sheet%20JoVE66456%20Takahiro%20Shimizu%20et%20al.pdf" target="_blank">Please click here to download this File.</a>

Most behavior experiments for rodents were originally devised for rats. Due to the behavioral differences between rats and mice, sometimes modifications are required for mice. We present a method to test spatial working memory in mice, minimizing the stress during the working memory task.

This method paper details a protocol to test spatial working memory in mice using a semi-automated radial 8-arm maze (RAM). The RAM is a partially ...

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JoVE Journal

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An Appetitive Spatial Working Memory Task for Mice in a Semi-Automated 8-Arm Radial Maze, Reducing Fearful Memory Association in the Maze

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