Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

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This method can help answer key questions about memory regulation. For example, we determine the role of specific proteins in memory by testing transgenic knockout mice in these tasks. The main advantage of this technique is that it is a rapid and cost-effective way to conduct memory testing in any lab.

Demonstration of the arena construction is critical, because the solvent welding will only yield strong results if performed with care. Begin by acquiring five sheets of acrylic, acrylic cement, and a 16-gauge hypo applicator. Remove the protective paper coating from the acrylic sheets.

Then dry-fit all the materials to confirm that sizes are correct. Next, assemble and load the syringe with acrylic cement. Align the long edge of an outside wall with the top edge of the base and ensure that they are perpendicular to each other using a combination or machinist square.

Then use the syringe to apply a small and steady bead of cement directly to the corner of the two pieces being joined. Hold the two pieces in place for five minutes until they are initially set. Repeat the same steps to attach the other outside wall to the base.

Then, attach two inner walls, one at a time, the base with acrylic cement. Finally, use the syringe to apply a small and steady bead of cement directly to the corner now being formed by the outside wall and the inside wall. Begin the habituation session by dividing mice into groups of four, and house them in individual clean holding cages.

Next, bring one group of mice into the testing room and allow them to acclimate for at least 30 minutes. After acclimation, begin recording the video. Place each mouse in the arena facing the walls of the release corner, and allow the mice to explore the arenas freely for six minutes.

Then, stop recording the video, and return mice to their clean holding cages during the inter-trial interval. Clean all arenas with animal facility recommended cleaning methods, such as wiping with 70%ethanol to minimize olfactory cues. Finally, after completing a total of three habituation sessions, return all mice to their home cages.

After 24 hours, bring the same group of mice into the testing room, and allow them to acclimate to the testing room for at least 30 minutes, as done before the habituation sessions on the previous day. Conduct the training trial using two objects placed in the arena. Affix objects with double-sided tape six by six centimeters away from two non-release corners, such that they are counterbalanced in the arena.

Allow mice to investigate the arena and objects freely for 10 minutes. Then stop the video recording. Place mice back in their clean holding cages for 20 minutes, and clean the areas with ethanol.

Next, to perform the object location task OLT, move one of the objects used in the training trial to a new non-release corner, and affix the object six centimeters from each wall of that corner with double-sided tape. Repeat the same video recording of the mice in the area for 10 minutes, as previously described. Finally, to perform the novel object recognition task NORT, replace the object that was not moved during the OLT with a novel object, and affix the novel object six centimeters from the two walls of the corner with double-sided tape.

Repeat the same video recording of the mice in the area for 10 minutes, as previously described. During the OLT, memory for object location is reflected by a group of mice spending, on average, significantly more time with an object after it has moved. This can also be represented as an increase in discrimination index.

In the NORT, memory for object location is reflected by a group of mice spending, on average, significantly more than 50%of investigation time with the novel object or showing a discrimination index significantly greater than zero. Picking objects for which mice have no inherent preference is key. For example, in a pilot test with a sample size of four, mice showed a trend towards spending less than 50%of investigation time with object A when paired against object B.When object A was then used as the novel object in a NORT, with a larger sample size of 16, mice spent significantly less than 50%of investigation time with object A.These data suggest inherent aversion to object A.During this procedure, it's important to remember to thoroughly clean all objects and arenas between use.

In this method, object investigation can be scored manually by researchers or using commercial software. Software has higher upfront costs but, once calibrated, may save time and financial resources over the long term.

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Journal (Behavior)

Journal (Behavior)

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Capitoli

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