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

Most behavior experiments for rodents are originally devised for rats. With recent advantages in genetic engineering, researchers often perform behavior experiments with mice, using protocols established for rats. Due to the behavior differences between rats and mice, sometimes you need to modify the protocol for mice. In this video, we describe a protocol to test spacial working memory in mice. Working memory is a cognitive process that can hold a limited amount of information for a short time while it is manipulated and used in the planning and execution of a mental task. It correlates with measures of fluid intelligence in humans and is important for learning. Therefore, working memory is an important subject for study on its own right.

[Instructor] Set up new cages, using standard nesting materials for a new experiment. Put new bedding sawdust, paper paper tunnel, paper nesting stripes, and wooden bite block in the new cage. Place the hood hopper and close the lid. Guide the mouse to the paper tunnel, supplied in the home cage by hand. Gently lift the mouse from the grouped cage using the paper tunnel. Put the mouse on your hand and keep it at chest height for several seconds. Alternatively, let the mouse climb on your hand. Never pick mice up by their tails. Handling animals with minimal stress is essential for behavior experiments. Habituating mice to the experimenter is also important. Catch the mouse using the same handling method. Put the mouse on the scale for body weight monitoring and record its body weight. After weighing, put the mouse into the new cage prepared earlier. Make sure you do not make mice scared by moving fast or making big sounds while handling mice. Otherwise, mice can jump off the scale or hand. Put mice on that amount restriction for a week prior to exposure to the apparatus. Control the amount of standard chow to make them motivated to forage the maze for food rewards, which is diluted, sweetened condensed milk, during the experiment. Put two to three grams of standard chow in the cage and maintain their body weights between 85 and 90% of their starting body weights throughout the experiment. Also at this stage, provide one milliliter of diluted condensed milk in the small plastic cup in the home cage to habituate mice to it. Observe them if they eat it for a while, making sure that the mouse do not show food neophobia. Bring the mice slowly to the behavior testing room on the trolley. Then set the lighting of the room, illuminating the central hub in directory at 80 lux with warm white light bulbs and play white noise at 70 decibel, measured at the central hub. Leave the mice undisturbed for five minutes before starting the experiment. Habituation, stage one. The maze control is set to let the mouse explore the maze with doors operated by the mouse tracking software. This stage is to habitate mice to the door movements and their sounds. Start your trial and the video recording by clicking the start button on the software and then bring the mouse to the maze and place the mouse in the central hub. Keep watching the mouse behavior. There is no food reward in the maze. The mouse can visit any arm, and however many times on its will. Doors are controlled without disturbing the movements of the mouse for 30 minutes. Continue till 30 minutes elapse. After the trial, collect the mouse in an arm and put it back in the home cage. Clean the maze for the next mouse. Do this for each mouse in turn for two days. Arbitration stage two. The maze control is set to let the mouse explore the maze freely without a doors operated, and find food rewards in the food wells at the end of the arms. This stage encourage the mice to explore the maze freely for food rewards. Place 70 microliter of the reward in each hood well. Do not give it in the home cages anymore, from habitation, stage two. Start a trial and the video recording by clicking the start button on the software, and then bring the mouse to the maze. Watch the mouse's behavior. During this stage, mice move slowly, seem to be cautious, and can go back to the food well multiple times. Continue till the mouse enters each arm at least twice or 30 minutes elapse. After the trial, collect the mouse in an arm and put it back in the home cage. Clean the maze for the next mouse. Do this for each mouse in turn twice per day for two days. Habitation stage three. The maze control is set to let the mouse get habituated to the combination of stages one and two. Mice are allowed to enter each arm at least once. Bait all the food wells with 70 microliter of the reward. Start the trial and the video recording by clicking the start button on the software and then bring the mouse to the maze. Watch the mouse's behavior. Continue till the mouse enters each arm at least once or 30 minutes elapse. After the trial, collect the mouse in an arm and put it back in the home cage. Clean the maze for the next mouse. Do this for each mouse in turn, twice per day for two days. As you can see in this panel, mice visit more arms over time. On day six of habitation, mice enter each arm at least once within seven to 15 minutes and spend a similar period of time in each arm, which indicates animals are habituated to the maze and are ready for the task. This spacial working memory task consists of three parts in one trial. Mice go through the forced run, five second confinement in the central hub, and then a free run. In the forced run, four arms are pseudo-randomly chosen and presented to the mouse one by one. During the free run, mice need to remember which arms they have visited in the forced run in the trial for the most efficient collection of the remaining four rewards. The maze is set to follow the schema shown in the previous screen, and each must go through the same set of the forced arms in the trial of the round. Mice do six trials per day for nine days Before starting a trial, bait all the arms with 40 microliter 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. Start a trial and a video recording by clicking the start button on the software. Bring the mouse to the maze center hub without causing stress to the mouse. The trial will be automatically started after five seconds confinement in the central hub. During the five second confinement, walk away from the maze and have a seat somewhere not too close to the maze. Monitor the behavior and write down the arm visit sequence for scoring until the mouse collects all the rewards. Once the mouse collected all the rewards in the maze, let it visit four extra arms. By doing this, mice will not associate being caught in the central hub where it is important for mice to decide where to go next. Also, the working memory task completion and being caught will not be directly associated. Additionally, it will reinforce the fact that rewards are not replenished in previously visited arms. At the back of the fourth arm, let the mouse get on your hand while covering the arm with the other hand, and put the mouse back into the home cage. Before cleaning the maze, tap the home cage of the next mouse to level their arousal state before the next trial. To clean the maze, use a paint brush, one wet tissue, and a two dry tissue. Use a paint brush to remove mouse droppings. Wipe the maze with dry tissue first to remove any traces of condenses milk. Wipe the maze floor with wet tissue. Dry the maze with dry tissue and move on to the next trial. The daily success rate score is calculated as four divided by the number of total arm entries, and the average success rate reaches about 80% on the last day of the experiment. The perfect trial ratio score is calculated as a number of perfect trials divided by six, and reached above 60% on the last day of the experiment. Select analysis of velocity, distance, and acceleration of the mouse on the software, and analyze them. Ensure to exclude the four extra arm visits from those analysis. Also measure the duration of the four extra arm visits. The distance should become shorter with time. Velocity and acceleration should be flat, and the duration of the additional four arm visits should become longer with time if they learn their task well.

When performing this experiment, it is crucial to familiarize the mice with the experimenter and habituate the mice to the experimental room environment, which altogether should minimize the stress for the mice.

This protocol is easy to use and reliable for reproducing data. Since the maze is semi-automated, this protocol is not labor intensive and is applicable for lab imaging and electrophysiology. This protocol is a powerful tool to study spacial working memory.