Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Podsumowanie

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.

Streszczenie

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

Wprowadzenie

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures^1,2,3. Because repetitive seizures can cause structural and functional abnormalities in the brain^1,2,3, abnormal seizure activity can contribute to cognitive dysfunction, which is one of the most common epilepsy-associated comorbidities^4,5,6. Contrary to the chronic seizure events, which are transient and momentary, cognitive impairments can persist throughout epileptic patients’ 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 epilepsy^{7,8,9,10,11,12}. 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.

Protokół

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

1. Novel object location test (NL)

1. Prepare epileptic C57BL/6 or transgenic mice 4–6 weeks after pilocarpine injection.
   NOTE: Acute seizures were induced by intraperitoneal (IP) pilocarpine injection, following the protocol detailed in our previous report¹⁴.
2. Transfer the epileptic mice from the breeding room to the behavior room one day before the behavioral tests begin. Allow the mice to habituate for at least 12 h overnight.
3. 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.
4. On the next day, begin 3 days of habituation sessions (H1–H3) in the early morning. Acclimate the animals to the low light in their home cages for at least 30 min.
5. 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.
6. 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.
7. Evaluate the locomotor activity of each mouse by performing an open field test.
   1. To record and track the behavior of each experimental mouse, use animal behavior video tracking software (see Table of Materials).
   2. 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’s hands. Insert the information about each experimental mouse (group, gender, age, etc.).
   3. 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.
   4. 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’s total distance moved using the video tracking software according to the manufacturer’s instructions.
      1. 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.
8. On day 2 and day 3, perform the habituation sessions (H2, H3) by repeating steps 1.3 to 1.7.
9. On day 4, perform the familiarization session (F1).
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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’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.
10. On day 5, perform the NL testing session.
    1. 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.
    2. 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.
    3. 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.
    4. 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
       
       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’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.
    5. Grab the tail of the experimental mouse and place it on its cage lid for transfer to its home cage. For 3 days (days 6–8), let the mouse rest with free access to food and water.
    6. 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.

2. Novel object recognition test (NO)

1. On day 9, perform a 15 min habituation session by repeating steps 1.2–1.7.
2. On day 10, perform the familiarization session (F1).
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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’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.
3. On the next day (day 11), perform the NO testing session.
   1. 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.
   2. 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.
   3. 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.
   4. 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’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.
   5. Grab the tail of the experimental mouse and place it on the cage lid for transfer to its home cage. For 3 days (days 12–14), let the mouse rest with free access to food and water.
   6. 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.

3. Pattern separation test (PS)

1. On day 15, perform the first familiarization session (F1) for the PS test.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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’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.
   6. 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.
2. On the next day (day 16), perform the second familiarization session (F2) for the PS test.
   1. 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.
   2. 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.
   3. 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.
   4. For counterbalancing, present two identical objects (plastic T-flasks filled with 50 mL of water, object D) on the narrow grid floor.
   5. 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’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.
   6. 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.
3. On the next day (day 17), perform the PS testing session.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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’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.
   6. Grab the tail of the experimental mouse and place it on the cage lid for transfer to its home cage.

4. Cresyl violet staining

1. 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.
2. Once the animal is deeply anesthetized, perform transcardial perfusion with 4% paraformaldehyde to fix the brain¹⁵.
3. 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.
4. Make coronal sections (30 μm) from the snap-frozen brain using a cryostat.
5. 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.
6. Incubate the tissue slides in 0.1% cresyl violet solution for 15 min.
7. 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.
8. Coverslip the tissue slides using a commercially available xylene mounting medium.

Wyniki

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 ...

Dyskusje

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 ...

Materiały

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