Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Summary

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.

Abstract

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals’ basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

Introduction

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures^1,2,3. Because repetitive seizures can cause structural and functional abnormalities in the brain^1,2,3, abnormal seizure activity can contribute to cognitive dysfunction, which is one of the most common epilepsy-associated comorbidities^4,5,6. Contrary to the chronic seizure events, which are transient and momentary....

Protocol

All experimental procedures were approved by the Ethics Committee of the Catholic University of Korea and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23).

1. Novel object location test (NL)

1. Prepare epileptic C57BL/6 or transgenic mice 4–6 weeks after pilocarpine injection.
   NOTE: Acute seizures were induced by intraperitoneal (IP) pilocarpine injection, following the protocol detailed in our previous report¹⁴.
2. Transfer the epileptic mice from the breeding room to the behavior room one day befo....

Results

A general experimental schedule and setup for evaluating cognitive function are shown in Figure 1. Six weeks after the introduction of pilocarpine-induced acute seizures, mice were subjected to the NL, NO, and PS tests in that order separated by 3 day rest periods between tests (Figure 1A). For the NL test, two identical objects were placed in the open field during the familiarization session (F1), and on the next day, one object was moved to a new location. In .......

Discussion

This work describes experimental procedures for evaluating cognitive function in mice with chronic epilepsy. Many different behavioral test paradigms are used to assess learning and memory functions in mice¹⁸. The Morris water maze, radial arm maze, Y-maze, contextual fear conditioning, and object-based tests are the most frequently used behavioral tests and provide reliable results. Among them, the NL, NO, and PS tests are efficient, simple methods for evaluating learning and memory in epileptic .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 ml syringe	Sung-shim		Use with the 26 or 30 gauge needle
70% Ethanol	Duksan	UN1170	Spray to clean the box and objects
black curtain			For avoiding unnecessary visual cues
Cresyl violet	Sigma	C5042	For Cresyl violet staining
cryotome	Leica	E21040041	For tissue sectioning
double-sided sticky tape			For the firm placement of the objects
DPX mounting medium	Sigma	06522
ethanol series	Duksan	UN1170	Make 100%, 95%, 90%, 80%, 70% ethanol solutions
floor plate with narrow grid patterns	Leehyo-bio		Behavioral experiment equipment, plate size: 42.5 x 42.5 x 0.5 cm, grid size: 2.75 x 2.75 cm
floor plate with wide grid patterns	Leehyo-bio		Behavioral experiment equipment, plate size: 42.5 x 42.5 x 0.5 cm, grid size: 5.5 x 5.5 cm
illuminometer	TES Electrical Electronic Corp.	1334A	For the measurement of the room lighting (60 Lux)
Intensive care unit	Thermocare	#W-1
ketamine hydrochloride	Yuhan	7003	Use to anesthetize the mouse for transcardial perfusion
LED lamp	Lungo	P13A-0422-WW-04	Lighting for the behavioral test room
objects			Rubber doll, 50 ml plastic tube, glass Coplin jar, plastic T-flask, glass bottle
open field box	Leehyo-bio		Behavioral experiment equipment, size: 44 x 44 x 31 cm
paper towel	Yuhan-Kimberly	47201	Use to dry open field box and objects
paraformaldehyde	Merck Millipore	104005	Make 4% solution
pilocarpine hydrochloride	Sigma	P6503
ruler			Use to locate the objects in the open field box
scopolamine methyl nitrate	Sigma	S2250	Make 10X stock
Smart system 3.0	Panlab		Video tracking system
stopwatch	Junso	JS-307	For the measurement of explorative activities of mice
sucrose	Sigma	S9378	For cryoprotection of tissue sections
terbutaline hemisulfate salt	Sigma	T2528	Make 10X stock
video camera (CCD camera)	Vision	VCE56HQ-12	Place the camera directly overhead of the open field box
xylazine (Rompun)	Bayer korea	KR10381	Use to anesthetize the mouse for transcardial perfusion
xylene	Duksan	UN1307	For Cresyl violet staining