Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Summary

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Abstract

Working and reference memory are commonly assessed using the land based radial arm maze. However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

Introduction

Working memory (WM) corresponds to a critical cognitive domain required for the representation of objects or places during goal directed behavior¹. Alternatively, reference memory (RM) is required for temporally stable representations of those objects or places. Working and reference memory have long been assessed in rodents using land based radial arm maze paradigms^2,3. However, these tasks frequently require pretraining, as rats are not predisposed to spontaneous maze running. This can significantly increase the time needed to complete an experiment and can interfere with time sensitive longitudinal designs. Other limitations of the land based eight-arm maze include requirements for food deprivation and difficulty controlling changes in scent cues left on the maze after each trial. Many of these limitations have been overcome by using the Morris water maze paradigm⁴, however historically these designs have been limited to testing reference memory and spatial learning (exceptions^5-9). The eight-arm radial water maze is a modified version of the eight-arm radial land maze, which has been used to assess both reference and working memory in rats and mice^5-7. In contrast to traditional land based mazes, the eight-arm radial water maze does not require food deprivation, minimizes potential confounding scent cues and utilizes the subjects' motivation for escape as an effective means to assess working and reference learning and memory simultaneously without the need for pretraining^5-7.

One application for this paradigm is to test working memory deficits following neonatal brain injury. Research in humans has shown that preterm infants at risk for brain injury exhibit working memory deficits later in life^10,11. Neonatal brain injury can be modeled in rodents by inducing hypoxia/ischemia (HI) early in postnatal development^12-15. More importantly, spatial learning and working memory deficits found in at risk infants are paralleled in rodents using the radial arm water maze, making it a robust model for the study of such impairments^8,9. The eight-arm radial water maze also allows for simultaneous quantification of reference and working memory making it ideal for the comparison of brain injured and noninjured subjects in time sensitive models (i.e. during discrete developmental widows) or for counter balanced longitudinal designs.

In this protocol, we describe testing procedures using an eight-arm radial water maze (see Figure 1) and example data for rats with and without neonatal hypoxic-ischemic injury. Hypoxia-ischemia (HI) was induced on postnatal day 7 following cauterization of the right common carotid artery and 120 min of 8% oxygen exposure. This surgical procedure has been extensively used to model pathology of prematurity and neonatal brain injury (for details see^12,13,16,17). We show that the radial arm water maze paradigm reveals deficits in reference memory and working memory capabilities following neonatal HI injury. In the current protocol, reference memory is assessed using four specific arms of the eight-arm radial water maze, which remain constant to extra maze cues throughout testing. These arms never contain an escape platform, thus, subject entries into these arms reflect poor reference memory performance and deficits in long-term learning. The remaining four arms all contain escape platforms at the beginning of the day, however each platform is removed once the animal successfully enters an arm and escapes the water^5-7. This requires the animal to remember which arms it entered over the four successive trials, increasing the working memory demand as each additional platform is removed. Working memory is assessed by examining re-entries into arms previously used for escape within the same testing day or re-entries into reference memory arms. Additionally, this design utilizes extra-maze cues to locate the escape platforms reflecting hippocampal dependent spatial learning. This method for assessing working and reference memory errors was originally used by Jarrard et al.¹⁸ and has since been used extensively to assess rodent models of aging related neurological disorders, learning disabilities and following hormonal manipulations^5,6,7,19,20.

The primary objective of this paradigm is to assess reference and working memory, thus, the eight-arm radial water maze design should not be limited for testing deficits due to developmental brain injury. Instead, previous studies using this paradigm show that a variety of rodent models can be evaluated to assess pathology of learning and memory across many experimental contexts^{5,6,7,18,19,20}.

Protocol

All procedures were approved by the Rhode Island College Institutional Animal Care and Use Committee and adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Maze Configuration

1. Place clear, static, salient extra-maze cues (painted shapes, furniture or other stationary room features) around the maze.
   Note: Any extra-maze cues must remain in the same location for the duration of the experiment.
2. Place the hub and arms of the radial arm maze in a plastic tub (See equipment table for example).
   Note: The maze used in this protocol consists of eight removable stainless steel arms attached to eight central supports affixed to a circular stainless steel base which has been painted black (see Figure 1 for diagram and corresponding dimensions). The plastic tub exemplified in Figure 1 has an inner diameter of 122 cm and a height of 60 cm.
3. Fill the tub with water warmed to room temperature (22-26 °C)⁷.
4. Place platforms 1 cm below the water surface at the ends of four of the eight arms.
   Note 1: For each subject the platform locations are fixed for the duration of testing. Regardless of the configuration, care should be taken to avoid placement of the platforms in more than two adjacent arms (no more than two platforms should be located in adjacent arms). Do not place a platform in the starting arm.
   Note 2: The hidden platforms must be painted the same color as the maze insert to blend in with the maze background.

2. Transferring Subjects for Testing

1. Transfer subjects from the vivarium to the testing room maintaining the designated testing order.
   Note: Animals should be placed into an opaque cage to help reduce anticipatory stress and prevent viewing of testing room cues.
2. Once in the testing room, place all of the opaque cages from group 1 onto a staging bench or table maintaining the testing order.
   Note: In a large study (e.g. 30 subjects) it may be best to break the subjects into testing groups (e.g. 3 groups of 10 subjects each). This will depend on the size of your testing room and the feasibility of subject transport.

3. Digital Tablet Protocol for Recording Maze Arm Entries

1. Open a PDF writer program on the digital tablet.
2. Import all presaved data recording templates for subjects in group 1.
   Note: Each recording template should be presaved and labeled with specific subject and trial numbers (e.g. subject 1_trial 1, subject 1_trial 2, etc.) with a total of four templates for each subject (trials 1-4). The template should have a blank representation of the maze and platform locations as well as a place to record the subject number, trial number, testing day, escape platform used and trial latency (see Figure 2).
3. Open the template for the first trial of your first subject.
   Note: This system can also be used with paper templates for researchers without access to tablets or tablet based note-taking programs.

4. Radial Arm Water Maze Testing

1. If animals are pair housed, separate the subjects prior to the testing session. Transfer the first testing subject into a separate opaque warming cage.
   Note: The cage should be warmed to 37 °C using heating pad to maintain body temperature between testing trials.
2. Prior to the first trial of each subject, confirm that the subject identification number matches the subject number on the data recording sheet and ensure that platforms are correctly positioned in the eight-arm water maze.
3. Place the subject into the start arm facing the wall of the maze and start the timer.
   Note: This protocol was designed for two experimenters, one to record the data and one to handle the subjects. During this collaboration, communication between the experimenters is paramount. Confirmation of start times, subject numbers, and proper platform placement prior to each trial is essential.
4. Remain stationary during each trial in a designated position in the room to maintain cue consistency during each trial.
   Note: Care should be taken to avoid salient sent cues (e.g. perfumes) during the experiments and the experimenters should wear the same garments (lab coats) each day to maintain cue consistency.
5. Record each entry into an arm by placing a mark on the corresponding arm in the recording template on the digital tablet.
   Note: An entry occurs when a subject's shoulders break the opening plane of the arm.
6. Allow a maximum of 120 sec for the subject to locate one of the four platforms. Once the animal reaches the platform, stop the timer, the trial is complete.
   Note: To maintain consistency, a subject is said to have reached the target when its front paws are on the platform.
7. If the subject is unable to locate the platform within 120 sec, carefully guide them to the closest platform and allow them to remain on the platform for 10 sec before removal to ensure that the animal is aware of the target location.
8. On day one only, allow the subject to sit on the platform for 10 sec following each of all four trials so the subject can reference the spatial cues.
9. Following trial completion, remove the subject from the platform by gently lifting them by the body.
10. Begin the timer for the 90 sec intertrial interval.
11. Place the subject back into the individual cage over the thermoregulation pad.
12. Record the specific platform used for escape by circling the corresponding location on the data-recording template.
13. During this interval remove the escape platform used by the subject in the previous trial.
14. Open the template for the next trial and mark all platform(s) that have been used for escape by the subject, and thus removed during previous trials, with an X.
    Note: Marking all missing platforms prior to a subsequent trial is critical for defining error types (e.g. reference memory and/or working memory correct).
15. Prior to starting a subsequent trial, confirm the subject number, the trial number and the remaining platform locations.
16. After the 90 sec intertrial interval has elapsed, place the subject into the start arm facing the wall of the maze and start the timer for the next trial.
17. Repeat this process for trials 2-4, removing the escape platform reached after each trial and marking the template appropriately during and after each trial (i.e. place and X over removed platforms, indicate arms entered, and circle the platform used for escape).
18. Once all four trials for a subject have been completed, place the escape platforms back in the appropriate arms of the maze and repeat the testing process for the next subject (testing steps 4.1-4.17).
19. Once testing is completed for group 1, export the data recording sheets to a folder for that testing day (e.g. Day 1) and save it to a file storage system.
    Note: The data recording sheets should be exported prior to starting the next testing group to minimize the chance of data loss.
20. Repeat for each group (testing steps 2.1-4.19).

5. Dependent Variables

1. For each trial, count the number of first and repeat entries into any arm that previously had a platform. This will be indicated by an X over the platform on the data recording sheet. These are the working memory correct errors.
   Note: Because working memory correct errors are dependent on remembering the location of a platform that has already been used for escape and avoiding that arm, you cannot have a working memory correct error on the first trial.
2. For each trial count the number of first entries into any arm that never contained a platform from the data recording sheet. These are reference memory errors.
3. For each trial count the number of repeat entries into any arm that never contained a platform from the data recording sheet. These are working memory incorrect errors.
4. Record the latency to reach the platform for each trial.

Results

Sample data from our lab show significantly more working memory incorrect errors in HI animals as compared to shams by day eleven of testing (t = 2.124, p<0.05, (see Figure 3A)). HI animals also showed significantly more reference memory errors as compared to sham subjects by the eleventh day of testing (t = 2.303, p<0.05, (see Figure 3B)). Working memory incorrect and reference errors include the total number of cumulative errors across all four trials for each subject on day elev...

Discussion

The eight-arm radial water maze paradigm has been utilized successfully in our lab and by others to assess working and reference memory performance in rats with and without neonatal brain injury^5-7,18-20. In the current paradigm, removal of an escape platform after each trial increases working memory demand (subjects have fewer escape options) on subsequent trials. Therefore, on trial four, only one platform remains and working memory demand is at its highest, increasing the probability that an animal will re-...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plastic Tub	Gototanks http://www.gototanks.com	ARM-19455	Size can vary depending on species tested or individual lab needs.
iPad or other digital tablet	Apple Inc.	N/A	http://www.apple.com
Notability or other note taking application	Gingerlabs	N/A	http://www.gingerlabs.com Digital pdf writer or notetaking program
Digital timer	Any	N/A
Deltaphase isothermal heating pad	Braintree Scientific	ASS7D	http://www.braintreesci.com/
Stainless steel maze	Fabricated in house	N/A