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 ...

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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

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8.3K 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

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Neuroscience

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Medicine

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

Planarian as an Animal Model for Experimental Acute Seizure

Epilepsy is among the most prevalent neurological disorders characterized by recurring spontaneous seizures. Seizures represent a clinical manifestation of uncontrolled, excessively synchronized neural cell activity. The extent of brain damage from seizures depends on their duration and intensity. Regrettably, there is no effective remedy for epilepsy. The aim of this investigation is to assess whether the planaria worm Dugesia dorotocephala could serve as a model to aid in identifying and developing treatments for epilepsy that can target acute seizures. Currently, various models, such as marine models, are used to evaluate antiseizure medications (ASM). However, they are very expensive, and there are ethical concerns. Alternatively, invertebrate models offer a cost-effective research opportunity in the drug discovery process for ASM. Planaria belong to the flatworm family and inhabit marine freshwater and terrestrial environments. Dugesia dorotocephala&#160;is the dominant species of aquatic planaria across North America. D. dorotocephala presents as a viable invertebrate model for epilepsy studies due to its cost-effectiveness, vertebrate-like neurons, and quantifiable behaviors, unlike other invertebrates or larger animals. They have been used in various pharmacology and environmental toxicology studies related to age, memory, and regeneration. In this study, planaria were exposed to different concentrations of pilocarpine, a common chemoconvulsant to study their behavior upon exposure. Following the observation, planaria were euthanized and preserved in either formaldehyde or Golgi solution for neurohistological assessment. Six distinct behavioral phenotypes were observed in planaria: dorsal oscillations, head oscillations, tail dorsal expansion, C-shape, head flick, and tail flick. Dorsal oscillation frequencies were significantly increased among experimental groups compared to the control and exhibited dose dependence. Additionally, pilocarpine disrupted the motility of the planaria. Pilocarpine-induced seizures in planaria can serve as a model to evaluate acute seizures and antiseizure medication, which is essential in developing therapeutic interventions for human patients suffering from epilepsy.

Seizures negatively impact various functions and life quality. Planaria worms were exposed to varying concentrations of chemoconvulsants to evaluate their seizure phenotypes and disruptive motility. This study proposes using planaria worms as a model for acute seizures in humans and holds significance in drug development for epilepsy.

Epilepsy is among the most prevalent neurological disorders characterized by recurring spontaneous seizures. Seizures represent a clinical manifestation ...

Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

<a href='https://app.jove.com/v/58455/microdialysis-excitatory-amino-acids-during-eeg-recordings-freely'>Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)</a>This video demonstrates the assessment of extracellular excitatory neurotransmitter levels in rats with chronic epilepsy induction. After implanting a microdialysis probe into the brain and confirming the absence of seizures using electroencephalography (EEG), extracellular fluid is collected to evaluate elevated neurotransmitter levels in epileptic rats. A high-potassium solution is then infused to sustain neuronal excitability and trigger an increased release of neurotransmitters, with samples collected to assess the potassium-induced peak in excitatory neurotransmitters.

Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app.jove.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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