A Within-Subject Experimental Design using an Object Location Task in Rats

The Object Location Task allows us to investigate various mechanisms of the formation or retention of spatial memory by taking advantage of the spontaneous explorative behavior in rodents. This protocol allows for the repetition of an Object Location Task up to four times helping to increase statistical power and the efficiency and output of studies that involve surgical procedures. To begin obtain a square arena made of opaque, non porous hard plastic with a minimum of 60 centimeter width, and 50 centimeter height.

For successful recording of rat movements by the automated software make sure that the floor color contrasts with that of the rat. Place the arena either inside a box or on a platform that is enclosed by a curtain. To create a context, insert a second layer of insertable walls in different colors or patterns into the arena.

Hang 3D spatial cues that have distinct geometric shapes and colors high enough so that the rats cannot reach them. Attach different pairs of objects that are non porous, non chewable, and easy to clean with distinct geometric shapes and textures to the floor of the arena. Fill a non square shaped bucket with bedding material.

Randomly place five to 10 objects of different shapes and sizes, which are different from the objects that are going to be used in the experiment into the bucket. For session one, bring all home cages to the experiment room and let the rats habituate to the room and settle for at least 30 minutes. Put two to four rats from the same cage together in the bucket for about 20 minutes.

Clean the bucket by removing any fecal matter between each group of rats. Put all rats in their home cages and return them to the housing room. For session two, bring all cages to the experiment room and leave for at least 30 minutes.

Put each rat individually in the bucket for 10 minutes. Place the rat back into the home cage and clean the bucket after each rat. When finished, return all cages to the housing room.

For session four, bring all cages to the experiment room and leave for at least 30 minutes. Place two to four rats from the same cage together in the empty arena with no context or spatial cues for 20 minutes. Place all rats back into the home cage and wipe the arena with 70%ethanol after each group of rats.

Modify the empty arena to create the first context without putting the objects in the arena. For session one of context habituation bring all cages to the experiment room and leave for at least 30 minutes. Start the recorder, then place the first rat in the center of the arena and allow the rat to explore the arena for 10 minutes.

Then stop the recorder and to place the rat back into the home cage. Return all cages to the housing room when finished. For each rat, repeat this process for sessions two and three over two consecutive days, such that there are three sessions of context habituation per rat in total.

Bring all cages to the experiment room and leave for at least 30 minutes. Using the schedule prepared beforehand place the first identical pair of objects in the designated locations by using sticky mats or double-sided tape. Start the recorder and to place the first rat in the arena at an equal distance from each object facing a wall or a corner that is not occupied by any object.

For weak encoding allow the rat to explore the arena and objects for 20 minutes for a single trial. For strong encoding allow the rat to explore for five minutes with three trials for the same rat. Stop the recorder.

Place the rat back into the home cage, remove the objects and wipe the arena with ethanol after each recording. Bring all cages to the experiment room, leaving sufficient time ahead of the first test so that the rats can be left for at least 30 minutes. Place the objects in the designated locations.

Start the recorder and place the first rat in the arena facing a wall or a corner that is not occupied by any object. Allow the rat to explore the arena and objects for five minutes. Place the rat back into the home cage.

Remove the objects and wipe both the objects and the arena thoroughly with 70%ethanol. Repeat the recording for each rat then return the cages back to the housing room. For each rat, score the exploration time for each object in both the encoding and test trials.

Score encoding trials for the whole duration and to the test trials for two minutes, for best discrimination performance. Calculate the percentage of exploration for each object or the discrimination index for each rat and to calculate mean values for the groups. Use a one sample T test for detecting a significant preference above the chance level.

The strong encoding protocol led to preference for the object at the novel location with a significantly higher mean percentage exploration than the 50%chance level in tests with both one hour and 24 hour delays. The weak encoding protocol produced a significant increase in the preference for the object at the novel location, compared to chance level in tests with a one hour delay, but not a 24 hour delay. A significant advantage of this protocol is that it can be performed four times using four distinct contexts with the same cohort of rats.

The results of one possible way of using counter balancing with two experimental groups of one hour memory and 24 hour memory are demonstrated here. The two groups were counterbalanced over contexts one and two, which was repeated in two additional contexts, three and four. The results from these four contexts are presented individually where the memory for each experimental group was assessed by comparing the preference to chance level in each context.

No significant difference was detected among the four contexts. For a better representation and within subject comparison of the data, the results from two counterbalanced contexts were combined and compared using within subjects comparison, namely the paired T test. Since this task relies on curiosity that handling and habituation is crucial so that the animal is not overwhelmed or scared during the test.

Further manipulations such as pharmacological and optogenetic intervention can be implemented in this protocol, as well as in vivo imaging for studying short-term and long-term memories and for enhancing or impairing these memories.