Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common co-morbidities in temporal lobe epilepsy. We will present a protocol describing how to assess memory impairment using a mouse model of chronic epilepsy. Novel object location, novel object recognition and pattern separation test are simple and short asses that can minimally stress the animals.

It makes this test optimal for evaluating memory function in epileptic mice, without additional triggers for a spontaneous recurrent seizures. To begin, acclimate the animals to the low light in their home cages, for at least 30 minutes. Prepare an open field box.

On day one of the habituation, place and illuminometer in the center of the open field box. And adjust the illuminance to 60 Lux. To evaluate the locomotor activity of each mouse, use animal behavior video tracking software.

Once the video tracking software is opened, calibrate the size of the open field box. Then gently place an experimental mouse in the open field box facing the wall. Open Smart program, click Customized and Accept.

Click Image Source. After selecting the installed camera, press the Set button. Adjust the camera resolution and press, Accept.

After our calibration click, drag the computer mouse to fit the box size. And fill in the inner box size. Click Zone Definition to save and adjust each box size, and then click Save.

The red box is the tracked range, so it fits into the inner box size, while the pink box fits the outline. Press Detection Setting. Click Snapshot, and press Start Test, to check if a specific area is tracked, then pres Stop Test, and then Accept.

Click Time Settings. After selecting preset time, set the latency time, at three seconds acquisition time to 15 minutes and press Accept. Press Subjects to set the codename.

Click Scheduler, to delete the existing details and drag the code name modified in subjects to session one. Click on Data Acquisition. Click the Record button and click Start.

Put the mouse in the box and track it for 15 minutes. After 15 minutes of recording return the mouse to its home cage. Restore the bright light.

Click Data Acquisition. And then click Active Analysis Drag code name Sham to the window next to it. Click the Report Definitions button.

Click New Report Definitions button then Summary Report and press Accept. Click Subject Name and Distance in Zone, and send it to the side. Then press the Accept button.

Click on the red check mark and click Analyze. Check the results. On day two, and day three, repeat the habituation sessions.

On day four perform the familiarization session. In the dim light, place each mouse in the empty open field for three minutes. After this brief habituation return the animal to its home cage.

Place two identical objects, rubber dolls, into the open field arena, five centimeters away from the adjoining walls. Fix the objects with double sided tape. Introduce the experimental mouse, into the open field box facing the wall farthest from the objects.

Allow free exploration for 20 minutes and manually measure the time spent exploring both objects using two stopwatches. Do not quantify as exploratory time, any behaviors, in which the animal snout does not point towards the object. Once the mouse reaches the minimum exploration time of 30 seconds, for the two objects, stop the F1 session and transfer the mouse to its home cage.

If the mouse fails to explore the objects for 30 seconds within the 20 minutes, remove the mouse from the open field box, and exclude it from further sessions. On day five, perform the novel object location testing session. Transfer the mouse to the open field area for rehabituation for three minutes.

Then return it to its home cage. Move one object to the diagonal position and place the experimental mouse facing the wall of the open field box. Allow 10 minutes of free exploration and record with a video tracking system.

Measure, the time spent exploring each object, using two stopwatches. Next, transfer the mouse to its home cage, by grabbing the tail. For three days, let the mouse rest with free access to food and water.

On day nine perform a 15 minute habituation session, for one mouse. On day 10 perform the brief rehabituation session, for three minutes. Then temporarily return it to its home cage.

Place two identical objects, 50 milliliter plastic tubes filled with 40 milliliters of water, in the open field, five centimeters away from the adjoining walls. Introduce the experimental mouse into the open field box, facing the wall furthest from the objects. As the mouse will be exposed to the two different objects in the NO Test, a 50 milliliter plastic tube filled with 40 milliliters of water, and a glass Coplin jars jar.

Counterbalance the object during the familiarization session, by presenting two identical objects, glass Coplin jars jars, for half of the animals in the group. Then with two different objects, allow free exploration for 20 minutes and manually measure the time spent exploring both objects using two stopwatches. Once the mouse reaches the minimum expiration time of 30 seconds for both objects, stop the familiarization session.

On the next day, perform the novel object recognition test session. First, transfer the mouse to the open field for rehabituation, for three minutes, and then return it to its home cage. At five centimeters away from the adjoining walls, replace the tube with a glass Coplin jars jar, so that the mice are exposed to one tube and one glass Coplin jars jar.

For half the animals that were exposed to glass Coplin jars jars during the first familiarization session, replace the glass Coplin jars jar with a 50 milliliter plastic tube filled with 40 milliliters of water. So that the mice are exposed to one tube and one glass Coplin jars jar. Allow 10 minutes of free exploration while recording it using a video tracking system.

Then grab the tail of the experimental mouse, and transfer it to its home cage. For three days and let the mouse rest with free access to food and water. For the NL analysis, open Start Program Customized and Accept.

Click Image Source. After selecting the installed camera, press the Set button. Just the camera resolution and press Accept.

Click Open videos Folder button to open the saved video. Watch the saved video for 10 minutes from the time the mouse enters and measure the time spent exploring each object. On day 15 first performed the first familiarization session for the PS Test.

That's for the mouse to the open field area for rehabituating, for three minutes. Place the floor plate with the wide grid, and two identical objects. Plastic T-flasks filled with 50 milliliters of water, in the open field box.

Then introduced the experimental mouse into the open field box facing the wall farthest from the objects. Allow free exploration for 20 minutes. And manually measure the time spend exploring both objects using two stopwatches.

Once the mouse reaches the minimum exploration time of 30 seconds for both objects, stop the first familiarization session and transfer the animal to its home cage. To counterbalance the object and pattern separation test for half of the animals place the floor plate with the wide grid and two identical objects, glass bottles for the counter balancing experiment in the open field box. Then introduce the experimental mouse to the open field box and allow free exploration for 20 minutes.

Once the mouse reaches the minimum exploration time of 30 seconds for the two objects, stop the first familiarization session and transfer the animal to its home cage. On the next day, transfer the mouse to the field area for rehabituation for three minutes. And then return it to its home cage.

Next perform the second familiarization session with two glass bottles on the narrow grid floor. For the counterbalancing experiments, perform the second familiarization session with two T-flasks filled with water on narrow grid floor. The next day perform the pattern separation testing session.

Transfer the mouse to the open field area with no object for rehabituation for three minutes. And then return it to its home cage. Next, place the T-flask filled with water and a glass bottle on the narrow grid floor.

Make the T-flasks filled with water, in novel object. For counterbalancing experiments, place each object on the narrow grid floor to make the glass bottle a novel object in this case. Then for the experimental mouse to the open field area for 10 minutes to explore.

Allow 10 minutes of free exploration and record using a video tracking system. Grab the tail of the experimental mouse and transfer it to its home cage. Measure the time spent exploring each object using two stopwatches.

And calculate the discrimination ratio equaling the difference of time spent between the novel object and the familiar object divided by the total time exploring the two objects. Six weeks after the introduction of pilocarpine induced acute seizures. Mice will subjected to the novel object location test.

Novel object recognition test and pattern separation test. In that order separated by three day rest periods between tests. It was confirmed that there was no significant preference between the two objects presented together.

The epileptic mice showed a significant reduction, in the Discrimination ratio in the three tests compared to Sham controls. Demonstrating spatial memory impairment. The measurement of locomotor activity, showed a significant increase in epileptic animals.

Whereas the motivation to explore the objects was comparable between Sham and epileptic animals. Finally hippocampal cell death was evaluated after a pilocarpine induced status epilepticus using crystal violet staining to confirm the seizure induced neuronal damage. The pilocarpine treated animals demonstrated to pyknotic cells in the and the CA3 subfield of the hippocampus, unlike the Sham controls.

Count up in NO and PS test is critical to reduce inherent bias possible in this device specific objects. In addition chronic epileptic mice open show heightened anxiety, which requires multiple habituation steps for enough acclaim nation to the experimental settings. time modifications such as the number or the location of the objects in our protocol can provide the of memory tests.

Which can be useful to elaborate complex mechanisms of epilepsy associated with cognitive decline. After watching this video, you should have a good understanding of how to evaluate different aspects of memory impairment in epilepsy.