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

In the Lash three maze, mice learned to navigate through a series of arms to reach a pseudo home cage environment. On repeated trials at the start of a trial, mice are placed in the Starbucks, which is separated from the maze by a door. Once the door is opened, mice enter the maze arms and are allowed to freely explore the path the mouse takes through.

The maze is recorded, including entries into dead end cul-de-sac error zones. Upon entry into the gold box, another door is closed to prevent maze reentry and directing the mouse to enter the pseudo home cage. Concluding the maze trial mazes performance is compared across age groups or strains to evaluate the role in learning and memory.

Hi, I'm Amanda Bressler. I work in the Anne Andrews laboratory here at Pennsylvania State University. I'm going to show you today how to use the Lashley three maze.

We use this maze primarily in our lab to study aging genetically modified mice. So here we go. First, let's see what the Lashley three maze looks like.

The start box maze, arms and gold box are modular to allow for cleaning and modifiability of the maze. The walls of the start box, maze, arms, and gold box are all constructed of black plexiglass to eliminate visual cues from outside and within the maze. Maze arms are 45 centimeters long, seven centimeters high and five centimeters wide.

Doors leading to consecutive arms of the mace are four centimeters by four centimeters and are situated 11 centimeters from the outer walls of the mace, the gold box is 19.5 centimeters long by seven centimeters high by five centimeters wide. The start box is eight centimeters by 9.5 centimeters by seven centimeters. Separate clear plexiglass pieces are placed on top of the Starbucks maze and gold box to allow visualization of each mouse as it progresses through the maze.

While preventing mice from escaping the maze rests on a base constructed of red plexiglass that is large enough to hold the three pieces of the maze, plus a pseudo home cage that is the same size as the animal's standard home cage. To prepare pseudo home cages prior to the first MACE trial, add fresh bedding material to individual cage bottoms with doorways cut into the sides, place wire tops on the cage, bottoms and individually number them one for each animal in a testing cohort. These cages will serve as individual pseudo home cages for each animal for the duration of the testing timeframe.

With the maze prepared, let's see how to conduct behavioral testing at least one hour. Before testing, move the animals to the behavior testing room to allow them to acclimate to the testing environment. 30 minutes Before testing, remove the food and water from all home cages.

Keep the food and water in the behavior testing room, but out of reach of the mice. The experiment is typically time so that the food and water are removed 30 minutes after the change from the light phase to the dark phase when peak feeding occurs. This ensures that animals are mostly food and water satiated prior to testing for the purposes of demonstration.

In this video, we are showing this procedure under bright light. The test is usually conducted during the dark phase of the light dark cycle. When the 30 minute pretest period has elapsed, clean the maze with 70%ethanol.

Leave the door leading from the Starbucks into the maze. Arms closed. The doors at both ends of the gold box should be left open.

Now place the pseudo home cage corresponding to the first animal. At the end of the gold box, carry the first mouse to the maze. Cupping it in your hand.

Put the mouse in the Starbucks and replace the lid. When in position to observe the maze without obstruction, open the start box door and immediately start three stopwatches. When all four paws of the mouse have left the start box, close the start box door and stop the first timer by hand.

Record the pathway. The mouse travels as it navigates the maze. On a score sheet, a mouse enters a new zone or arm of the maze When all four paws have crossed into that area.

When all four paws enter the goal box, close the door between the maze and the gold box and stop the second stopwatch. When the mouse has completely entered the pseudo home cage, close the door between the gold box and the cage. Stop the third stopwatch.

Record the times from the three stopwatches and count the number of errors the mouse has made. An error is defined as an entry into a dead end cul-de-sac zone or traveling back through an already traveled arm of the maze. Leave the animal in the pseudo home cage for one minute before returning it to its home cage.

After testing all the mice in the cohort, wait 30 minutes and then return food and water to all mice. Finally, return the animals to their colony room. Repeat this procedure on consecutive nights with one trial per testing day per animal until all mice in the cohort have reached a defined learning criteria or until a predetermined number of test trials has occurred.

A mouse is classified as having learned the maze when it can perform the task with zero to one errors on two consecutive trials. Mice are tested in the same order on successive trials. Now we'll show some representative behavioral results.

Here we see data from young two month and aging, 24 month male, C 57 black six and CR mice that were trained on the lashley maze for up to 15 trials. In figure two A, the young mice learned the maze in an average of 7.6 days. In contrast, the aging animals took 11.7 days to reach criterion.

There was a strong trend for aging C 57, black six NCR R mice to take longer to learn the mace. The learning index is the ratio of correct arm entries to total path segments traveled on a single trial when the learning index is calculated in figure two B.Both age groups have a ratio of approximately 0.50 for the first trial. Both young and aging mice show a steady increase in the learning index from trials two through four, but there were no significant differences between the two groups.Shown.

Here is data from studies examining the effect of background strain on learning behavior. Young male, C 57, black six NCR and control CD one mice four to five months were compared. The C 57 black six mice are the same mice as the young cohort in figure two.

In figure three, a CD one mice learn the lashley three maze in 4.5 days. There was no significant difference between the strains in days required to reach criteria. By contrast, examination of the learning index in figure three B shows that while all animals were navigating the maze on the first trial by chance, CD one mice learned the task more quickly.

This is illustrated by the statistically significant increase in the learning index in trials two through four in CD one mice compared to C 57 BL six and CR mice. Interestingly, if CD one mice continue to be trained in the maze beyond the point of reaching criterion, they exhibit behavior indicative of overtraining In figure four A, there's an increase in the number of errors made after day five. Day five was the day the CD one mice reached criterion.

The CD one mice were also spending more time inside the MACE after acquiring the task, which can be seen in figure four B.So we just showed you how to use the Lashley three maze to measure learning and memory and mice. When implementing this experiment in your own lab, it may be necessary to conduct some pilot work as different strains of mice will acquire the MAs at different rates and may be subject to overtrain. Thank you for watching and good luck in your future experiments.