Name: assessment of memory function in pilocarpine-induced epileptic mice
Uploaded: 2020-06-04T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Memory Function in Pilocarpine-induced Epileptic Mice at JoVE.com

We thank Dr. Jae-Min Lee for his technical support. This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (NRF-2019R1A2C1003958, NRF-2019K2A9A2A08000167).

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)
<ol>
	<li>Prepare epileptic C57BL/6 or transgenic mice 4&#8211;6 weeks after pilocarpine injection. 
	NOTE: Acute seizures were induced by intraperitoneal (IP) pilocarpine injection, following the prot...

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 ...

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures1,2,3. Because repetitive seizures can cause structural and functional abnormalities in the brain1,2,3, abnormal seizure activity can contribute to cognitive dysfunction, which is one of the most common epilepsy-associated comorbidities4,5,6. Contrary to the chronic seizure events, which are transient and momentary, cognitive impairments can persist throughout epileptic patients&#8217; lives, deteriorating their quality of life. Therefore, it is important to understand the pathophysiologic mechanisms of epilepsy-associated cognitive decline.
Various experimental animal models of epilepsy have been used to demonstrate the learning and memory deficits associated with chronic epilepsy7,8,9,10,11,12. For instance, the Morris water maze, contextual fear conditioning, hole-board, novel object location (NL), and novel object recognition (NO) tests have frequently been used to assess memory dysfunction in temporal lobe epilepsy (TLE). Because the hippocampus is one of the primary regions in which TLE shows pathology, behavioral tests that can evaluate hippocampus-dependent memory function are often preferentially selected. However, given that seizures can induce aberrant hippocampal neurogenesis and contribute to epilepsy-associated cognitive decline10, behavioral paradigms for testing dentate newborn neuronal function (i.e., spatial pattern separation, PS)8,13 can also provide valuable information about the cellular mechanisms of memory impairments in epilepsy.
In this article, we demonstrate a battery of memory tests, NL, NO, and PS, for epileptic mice. The tests are simple and easily accessible and do not require a sophisticated system.

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

assessment of memory function in pilocarpine-induced epileptic mice

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&#8217; 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.

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 ...

Watch this Scientific Journal Video about Assessment of Memory Function in Pilocarpine-induced Epileptic Mice at JoVE.com

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 comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

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