The authors would like to acknowledge William Martin and the Rhode Island College Art department for assistance with maze fabrication. We would also like to acknowledge Rhode Island College students Katrina Feyerherm, Molly La Rue and Nick Lafond for work running subjects on the eight-arm water maze. This work was supported by a grant from the Rhode Island Idea Network for Biomedical Research excellence (RIINBRE) and the NIH National Center for Research Resources (Grant# P20 RR16457-12). Support was also provided by the Eunice Kennedy Shriver National Institute Of Child Health &amp; Human Development of the National Institutes of Health under Award Number R15HD077544.

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
<ol>
	<li>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.</li>
	<li>Place the hub and arms of the...

<ol>
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The authors declare that they have no competing financial interest.

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 injury5-7,18-20. 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-...

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 paradigms2,3. 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 (exceptions5-9). 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 mice5-7. 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&#39; motivation for escape as an effective means to assess working and reference learning and memory simultaneously without the need for pretraining5-7.
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 life10,11. Neonatal brain injury can be modeled in rodents by inducing hypoxia/ischemia (HI) early in postnatal development12-15. 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 impairments8,9. 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.&#160;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 see12,13,16,17). 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 water5-7. 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 manipulations5,6,7,19,20.
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 contexts5,6,7,18,19,20.

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			<td height="78" rowspan="2" style="height:79px;width:155px;">Plastic Tub</td>
			<td rowspan="2" style="width:187px;">Gototanks http://www.gototanks.com</td>
			<td rowspan="2" style="width:131px;">ARM-19455</td>
			<td rowspan="2" style="width:131px;">Size can vary depending on species tested or individual lab needs.</td>
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			<td height="42" style="height:43px;width:155px;">iPad or other digital tablet</td>
			<td style="width:187px;">Apple Inc.&#160;</td>
			<td style="width:131px;">N/A</td>
			<td style="width:131px;">http://www.apple.com</td>
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			<td height="48" style="height:48px;width:155px;">Notability or other note taking application</td>
			<td style="width:187px;">Gingerlabs&#160;</td>
			<td style="width:131px;">N/A</td>
			<td style="width:131px;">
			http://www.gingerlabs.com
			Digital pdf writer or notetaking program
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			<td height="22" style="height:22px;width:155px;">Digital timer</td>
			<td style="width:187px;">Any</td>
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			<td height="64" style="height:64px;width:155px;">Deltaphase isothermal heating pad</td>
			<td style="width:187px;">Braintree Scientific</td>
			<td style="width:131px;">ASS7D</td>
			<td style="width:131px;">http://www.braintreesci.com/</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:155px;">Stainless steel maze</td>
			<td style="width:187px;">Fabricated in house</td>
			<td style="width:131px;">N/A</td>
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use of an eight-arm radial water maze to assess working and reference memory following neonatal brain injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;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.

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&#60;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&#60;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 el...

Watch this Scientific Journal Video about Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury at JoVE.com

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

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.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

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16.1K Görüntüleme.  Rhode Island College. 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.