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 protocol detailed in our previous report14.</li>
	<li>Transfer the epileptic mice from the breeding room to the behavior room one day before the behavioral tests begin. Allow the mice to habituate for at least 12 h overnight.</li>
	<li>In the behavior room, separate individual mice into new cages for single housing. Write the information for each animal on the cage card and keep the animals in the same cages throughout the behavioral testing. Multiple cages can be simultaneously transferred using a cart.</li>
	<li>On the next day, begin 3 days of habituation sessions (H1&#8211;H3) in the early morning. Acclimate the animals to the low light in their home cages for at least 30 min.</li>
	<li>Prepare an open field box with outside dimensions of 44 x 44 x 31 cm and inside dimensions of 43 x 43 x 30.5 cm. On day 1 of the habituation (H1), place an illuminometer in the center of the open field box and adjust the illuminance to 60 lux.</li>
	<li>Spray the floor and walls of the open field box with 70% ethanol and wipe down with a clean paper towel to remove possible olfactory cues. Then wait for at least 1 min until the residual alcohol has dried completely.</li>
	<li>Evaluate the locomotor activity of each mouse by performing an open field test.
	<ol>
		<li>To record and track the behavior of each experimental mouse, use animal behavior video tracking software (see Table of Materials).</li>
		<li>Once the video tracking software is opened, calibrate the size of the open field box. Then, set the zone for tracking. Set 3 s of latency and 15 min of acquisition time to avoid tracking an experimenter&#8217;s hands. Insert the information about each experimental mouse (group, gender, age, etc.).</li>
		<li>Then, gently place an experimental mouse in the open field box facing the wall. Do this by placing it on the cage lid to minimize handling-associated stress and anxiety. Then, release the mouse near the wall of the open field box that is the farthest from where the objects will be during the familiarization session (step 1.9.3.). 
		NOTE: Once the mouse is in the open field box, the mouse tracking software will automatically detect it and start recording. For optimal tracking of the exploration, the camera can be placed directly above the open field box.</li>
		<li>After 15 min of recording, return the animal to its home cage by placing it on the cage lid. Clean the open field box with 70% ethanol spray and wipe down with a clean paper towel between trials. Restore the bright light and measure the animal&#8217;s total distance moved using the video tracking software according to the manufacturer&#8217;s instructions.
		<ol>
			<li>Open the video tracking software and video clips. Then, click Analysis to calculate the total distance moved based on the calibration of the open field box size.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>On day 2 and day 3, perform the habituation sessions (H2, H3) by repeating steps 1.3 to 1.7.</li>
	<li>On day 4, perform the familiarization session (F1).
	<ol>
		<li>In the dim light, place each mouse in the empty open field for 3 min. After that brief rehabituation, return the animal to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects with 70% ethanol and wipe them down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Place two identical objects (rubber dolls, object A) in the open field arena 5 cm 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.</li>
		<li>Allow free exploration for 20 min and manually measure the time spent exploring both objects using two stopwatches. Once the mouse reaches the minimum exploration time (30 s) for both objects, stop the F1 session and transfer the animal to its home cage. If the mouse fails to explore the objects for 30 s within 20 min, remove the mouse from the open field box and exclude it from further sessions.</li>
		<li>After the animal is removed from the open field box, thoroughly clean the floor and walls of the box with 70% ethanol spray and wipe them down with a paper towel. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
	</ol>
	</li>
	<li>On day 5, perform the NL testing session.
	<ol>
		<li>Transfer the mouse from its home cage to the open field area for rehabituation for 3 min. Then return the animal to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects with 70% ethanol and wipe them down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Move one object (rubber doll, object A) to the diagonal position, 5 cm away from the adjoining walls. Fix the object with double-sided tape. Transfer the experimental mouse on its cage lid to the open field area and place it facing the wall of the open field box. 
		NOTE: Counterbalance the location of the object moved to reduce any potential innate preference for a certain direction. For example, change the location of the preferred object from the familiarization session for half of the experimental animals, and for the rest of the animals, move the less preferred object from the familiarization session.</li>
		<li>Allow 10 min of free exploration and record with a video tracking system. Measure the time spent exploring each object using two stopwatches and calculate the discrimination ratio as 
		<img alt="Equation 1" src="/files/ftp_upload/60751/60751equ01.jpg" /> 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
		<li>Grab the tail of the experimental mouse and place it on its cage lid for transfer to its home cage. For 3 days (days 6&#8211;8), let the mouse rest with free access to food and water.</li>
		<li>Once the animal is removed from the open field box, thoroughly clean the floor and walls of the box with 70% ethanol spray and wipe down with a paper towel.</li>
	</ol>
	</li>
</ol>
2. Novel object recognition test (NO)
<ol>
	<li>On day 9, perform a 15 min habituation session by repeating steps 1.2&#8211;1.7.</li>
	<li>On day 10, perform the familiarization session (F1).
	<ol>
		<li>In dim light, place the mouse in the empty open field for 3 min. After rehabituating the mouse to the open field area, temporarily return it to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects with 70% ethanol and wipe down with a paper towel. Wait at least 1 min for the residual alcohol to dry completely.</li>
		<li>Place two identical objects (50 mL plastic tubes filled with 40 mL of water, object B) in the open field 5 cm 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.</li>
		<li>As the animal is exposed to the two different objects (50 mL plastic tube filled with 40 mL of water, object B; glass Coplin jar, object C) in the NO test, counterbalance the object during the F1 session. For example, present two identical objects (glass Coplin jars, object C) for half of the animals in the group.</li>
		<li>Allow free exploration for 20 min and manually measure the time spent exploring both objects using two stopwatches. Once the mouse reaches the minimum exploration time (30 s) for both objects, stop the F1 session and transfer the animal to its home cage. If the mouse fails to explore the objects for 30 s within 20 min, remove it from the open field box and exclude it from further sessions.</li>
		<li>After the animal is removed from the open field box, thoroughly clean the floor and walls of the box with 70% ethanol spray and wipe them down with a paper towel. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
	</ol>
	</li>
	<li>On the next day (day 11), perform the NO testing session.
	<ol>
		<li>Transfer the mouse from its home cage to the open field for rehabituation for 3 min, and then return the animal to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects with 70% ethanol and wipe down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Replace one object (50 mL plastic tube filled with 40 mL of water, object B) with another object (glass Coplin jar, object C) 5 cm away from the adjoining walls. Fix the objects with double-sided tape. Transfer the experimental mouse on the cage lid to the open field, and place it facing the wall. Counterbalance the objects presented together during the NO test. For example, replace one glass Coplin jar (object C) with a 50 mL plastic tube filled with 40 mL of water (object B) for the mice exposed to the two glass Coplin jars (object C) during the familiarization session. 
		NOTE: Counterbalancing the location of the object replaced can be also performed to reduce the potential innate preference for a certain direction. For example, for each cohort of the animals exposed to the set of two objects (object B or object C), change the preferred object in the familiarization session for half of the experimental animals, and for the rest of the animals, replace the object less preferred in the familiarization session.</li>
		<li>Allow 10 min of free exploration and record it using a video tracking system. Measure the time spent exploring each object using two stopwatches and calculate the discrimination ratio. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
		<li>Grab the tail of the experimental mouse and place it on the cage lid for transfer to its home cage. For 3 days (days 12&#8211;14), let the mouse rest with free access to food and water.</li>
		<li>Once the animal is removed from the open field box, thoroughly clean the floor and walls of the open field box with 70% ethanol spray and wipe down with a paper towel.</li>
	</ol>
	</li>
</ol>
3. Pattern separation test (PS)
<ol>
	<li>On day 15, perform the first familiarization session (F1) for the PS test.
	<ol>
		<li>Transfer the mouse from its home cage to the open field area for rehabituation for 3 min, and then return it to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects and the gridded floor plate with 70% ethanol and wipe down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Place the floor plate (42.5 x 42.5 x 0.5 cm) with the wide grid (5.5 x 5.5 cm) in the open field box and place two identical objects (plastic T-flasks filled with 50 mL of water, object D) in the open field 5 cm 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.</li>
		<li>As the animal is exposed to two different objects (plastic T-flask filled with 50 mL of water, object D; glass bottle, object E) in the PS test, counterbalance the object during the F1 and F2 sessions. For example, present two identical objects (glass bottles, object E) on the wide grid floor for half of the animals in the group.</li>
		<li>Allow free exploration for 20 min, and manually measure the time spent exploring both objects using two stopwatches. Once the mouse reaches the minimum exploration time total (30 s) for both objects, stop the F1 session and transfer the animal to its home cage. If the mouse fails to explore the objects for 30 s within 20 min, remove it from the open field box and exclude it from further sessions. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
		<li>After completion of the first familiarization session (F1), thoroughly clean the objects and the floor plate with 70% ethanol spray and remove them from the open field box.</li>
	</ol>
	</li>
	<li>On the next day (day 16), perform the second familiarization session (F2) for the PS test.
	<ol>
		<li>Transfer the mouse from its home cage to the open field area for rehabituation for 3 min, and then return the animal to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects and gridded floor plate with 70% ethanol and wipe down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Place the floor plate (42.5 x 42.5 x 0.5 cm) with the narrow grid (2.75 x 2.75 cm) in the open field box and place two identical objects (glass bottles, object E) in the open field 5 cm 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.</li>
		<li>For counterbalancing, present two identical objects (plastic T-flasks filled with 50 mL of water, object D) on the narrow grid floor.</li>
		<li>Allow free exploration for 20 min and manually measure the time spent exploring both objects using two stopwatches. Once the mouse reaches the minimum exploration time total (30 s) for both objects, stop the F2 session and transfer the animal to its home cage. If the mouse fails to explore the objects for 30 s within 20 min, remove it from the open field box and exclude it from further sessions. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
		<li>After completion of the second familiarization session (F2), thoroughly clean the objects and floor plate with 70% ethanol spray and remove them from the open field box.</li>
	</ol>
	</li>
	<li>On the next day (day 17), perform the PS testing session.
	<ol>
		<li>Transfer the mouse from its home cage to the open field area for rehabituation for 3 min, and then return it to its home cage.</li>
		<li>During the habituation, thoroughly clean the objects and gridded floor plate with 70% ethanol and wipe down with a paper towel. Wait for at least 1 min for the residual alcohol to dry completely.</li>
		<li>Place the floor plate with the narrow grid (2.75 x 2.75 cm) in the open field box and place two different objects (plastic T-flask filled with 50 mL of water, object D; glass bottle, object E) on the floor plate 5 cm away from the adjoining walls. Fix the objects with double-sided tape. Transfer the experimental mouse on the cage lid to the open field area and place it facing the wall.</li>
		<li>Counterbalance the objects presented together during the PS test. For example, place each object (object D, object E) on the narrow grid floor to make the object E a novel object in this context. Counterbalancing the location of object D or object E (a novel object on the narrow floor pattern) can be also performed to reduce the potential for an innate preference for a certain direction. For example, replace the preferred object from the second familiarization session for half of the experimental animals, and for the rest of the animals, replace the less preferred object from the second familiarization session.</li>
		<li>Allow 10 min of free exploration and record using a video tracking system. Measure the time spent exploring each object using two stopwatches and calculate the discrimination ratio. 
		NOTE: Measure the time when the mouse touches the objects with its whiskers, snout, or front paws. Do not quantify as exploratory time any behaviors in which the animal&#8217;s snout does not point toward the object, such as sitting on the object, passing by the object, or resting with its hind end pointing at the object.</li>
		<li>Grab the tail of the experimental mouse and place it on the cage lid for transfer to its home cage.</li>
	</ol>
	</li>
</ol>
4. Cresyl violet staining
<ol>
	<li>After completing all the behavioral tests, anesthetize the animal by injecting a cocktail (4:0.5) of ketamine (50 mg/mL) and xylazine (23.3 mg/mL) dissolved in saline at a dose of 110 mL/kg body weight (IP; 1 mL syringe; 26 G needle). Check for the depth of anesthesia by the lack of a response to a toe pinch.</li>
	<li>Once the animal is deeply anesthetized, perform transcardial perfusion with 4% paraformaldehyde to fix the brain15.</li>
	<li>After the transcardial perfusion is finished, decapitate the animal with a pair of scissors15. Then, remove the skull using a pair of iris scissors to expose the brain. After the brain is isolated, postfix it in 4% paraformaldehyde overnight, followed by cryoprotection in 30% sucrose in 0.01 M phosphate-buffered saline.</li>
	<li>Make coronal sections (30 &#956;m) from the snap-frozen brain using a cryostat.</li>
	<li>Mount the brain tissues on slides and perform a series of hydration steps from 100% ethanol to tap water by washing for 3 min sequentially in 100%, 95%, 90%, 80%, 70% ethanol.</li>
	<li>Incubate the tissue slides in 0.1% cresyl violet solution for 15 min.</li>
	<li>Remove excessive stain by immersing the tissue slides in 95% ethanol/0.1% glacial acetic acid, and then dehydrate the tissues with solutions of 100% ethanol, 50% ethanol/50% xylene, and 100% xylene.</li>
	<li>Coverslip the tissue slides using a commercially available xylene mounting medium.</li>
</ol>

The authors have nothing to disclose.

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 ...

assessment-of-memory-function-in-pilocarpine-induced-epileptic-mice

Research

JoVE Journal

Behavior

8.2K Views.  The Catholic University of Korea. 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.

Video: Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.