The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

The overall goal of the following experiment is to investigate spatial working memory and rodents and assess their learning and behavioral strategy. This is achieved by first constructing a double H maze to assess learning and memory performance. As a second step, animals are habituated to the maze environment prior to behavioral training.

Next animals are trained daily in the maze using an egocentric learning paradigm in order to develop a habitual response. Alternatively, an allocentric paradigm may be utilized to develop a place response. The final step is to assess the animal's learning strategy in a probe trial in which their starting position is altered and the pattern removed.

The results provide an insight into the choice of learning strategy utilized in the maze based on the recordings of animal behavior. The main advantage of this technique over existing methods, such as the eight arm radio maze or the Morris Water maze, is that it can be rapidly acquired in as little as two days, and it allows the experimenter to direct the type of strategy that is to be learned by the animal. This method can help answer key questions in the field of behavior, such as the dynamic interplay in between the procedural and declarative memory systems, and how these systems are affected by different treatment modalities.

Demonstrating this procedure will be Dr.Richard Pinnell, a postdoc from the laboratory of neuro electronic systems. The first step is to construct the double H maze. The maze consists of a central alleyway intersected by three parallel run arms.

Each corridor is 20 centimeters wide and is surrounded by high transparent plexiglass walls. The walls are secured with special glue, for example, agri fix and screws and waterproofed at all internal angles. First, the plexiglass is cut to size and the internal joints are jammed.

The protective plastic coating is removed near the joint to be glued, and all dust particles are removed with a cleaning solvent. The two sides are placed into a 90 degree form, and a special tape is used to seal the ends to prevent leakage of the glue. During the curing process, the glue is prepared by mixing a polymerizing agent and gently stirred the Croix glue is applied using an air driven syringe.

The joint is then fixed and waterproofed and is allowed to cure. An outlet should be placed within the maze for drainage. Place the maze on a table that allows access to all sides.

Well contrasted cues should be placed on the surrounding walls. Next, label the north and south arms for orientation later during video analysis. Finally, position a camera above the maze for post-test analysis of animal behavior.

Switch the camera on to record the animal's movements before each trial. To set up for the pre-training phase, fill the maze with transparent water to an approximate height of 17 centimeters. Pre-training can happen one day before or on the same day as the training sessions.

Maintain the water temperature between 21 to 23 degrees Celsius to provide incentive for the rats to seek the escape platform. Fill the maze with water the day before testing to allow the water temperature to stabilize. Regardless of the start position, always block entry into the opposite arm with a transparent guillotine door.

When ready, release the rat from the extremity of one of the center arms. Upon reaching the escape platform, allow the rat to rest and observe their surroundings for approximately 15 seconds. Administer four consecutive trials with at least 10 seconds of rest in between trials.

To begin training, relocate the escape platform from its pre-training position to the chosen arm and add water to submerge the platform to one centimeter below the water surface. Next, render the water opaque by mixing in 250 grams of skim milk powder. First, premix the milk powder and water in a separate bottle and distribute this mixture in several locations around the maze.

Stirring will help to reach homogeneity in the maze water faster. Release the rats from a center alley as demonstrated earlier, making sure that entry to the opposite arm is blocked. Once released, observe the animal's behavior and record its performance either manually or by video camera.

If the target platform is not reached within 60 seconds, gently guide the rat to the escape platform and allow the rat to rest there and observe their surroundings. For approximately 15 seconds, the rat is then removed and fluffed dried with a towel before the next trial. This is important so that the rats do not become under cooled for the next four days.

Follow the basic training protocol. Release the rats from either the north or south start arm in an unpredictable sequence such that both arms are used twice. Remove the escape platform to perform the probe trials on day six.

Release the rat from a different arm from that used during training. For example, if released in the north arm released from the northeast arm with the southeast arm closed off, allow the rat to swim for a maximum of 60 seconds. Release the rat from the same start arm for each of four training trials daily.

On the sixth day, remove the escape platform for the probe trial. Release the rat from a different arm from where they were released during the trial. Allow the rats to swim for 60 seconds.

After testing. Review the recordings taken from the overhead camera, manually track response, latencies, initial and repetitive errors, first choices and response types. Lastly, run statistical analysis regarding response errors, latencies to reach the goal and time spent in the target arm.

In this diagram, the maze error zones are highlighted in gray, and the closed off arms are highlighted in black for both the south and north starting positions. The hatched line depicts an example swim path in which the rat visits all four error zones. Prior to reaching the escape platform, these representative swim tracks depict movement during the probe trial.

After allocentric training, after egocentric training, rats train to start from either the north or south arm display, mainly procedural responses when their start position is shifted to the left. However, rats that had their start arm rotated 180 degrees, displayed a higher amount of place responses. Responses that were either placed nor procedural responses were designated as other typical swim tracks are shown for both the sham operated and neonatal ventral hippocampal lesion groups during the training phase.

Note the difference in performance. By the fourth training trial during the probe session, the original start arm was closed off and the rats were released from the southwest arm. Once mastered, this technique can be performed in less than five minutes if it's performed properly.

While attempting this procedure, it's important to remember to change the milk water every day, otherwise it'll become rancid. Since development, this procedure has paved the way for researchers in the field of behavior to explore procedural and declarative memory systems in awake animals.