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 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.</li>
	<li>Fill the tub with water warmed to room temperature (22-26 &#176;C)7.</li>
	<li>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.</li>
</ol>
2. Transferring Subjects for Testing
<ol>
	<li>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.</li>
	<li>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.&#160;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.</li>
</ol>
3. Digital Tablet Protocol for Recording Maze Arm Entries
<ol>
	<li>Open a PDF writer program on the digital tablet.</li>
	<li>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.&#160;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).</li>
	<li>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.</li>
</ol>
4. Radial Arm Water Maze Testing
<ol>
	<li>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 &#176;C using heating pad to maintain body temperature between testing trials.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.&#160;perfumes) during the experiments and the experimenters should wear the same garments (lab coats) each day to maintain cue consistency.</li>
	<li>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&#39;s shoulders break the opening plane of the arm.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Following trial completion, remove the subject from the platform by gently lifting them by the body.</li>
	<li>Begin the timer for the 90 sec intertrial interval.</li>
	<li>Place the subject back into the individual cage over the thermoregulation pad.</li>
	<li>Record the specific platform used for escape by circling the corresponding location on the data-recording template.</li>
	<li>During this interval remove the escape platform used by the subject in the previous trial.</li>
	<li>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.&#160;reference memory and/or working memory correct).</li>
	<li>Prior to starting a subsequent trial, confirm the subject number, the trial number and the remaining platform locations.</li>
	<li>After the 90 sec&#160;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.</li>
	<li>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.&#160;place and X over removed platforms, indicate arms entered, and circle the platform used for escape).</li>
	<li>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).</li>
	<li>Once testing is completed for group 1, export the data recording sheets to a folder for that testing day (e.g.&#160;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.</li>
	<li>Repeat for each group (testing steps 2.1-4.19).</li>
</ol>
5. Dependent Variables
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Record the latency to reach the platform for each trial.</li>
</ol>

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<li>Fitch, R. H., Breslawski, H., Rosen, G. D., Chrobak, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+spatial+working+memory+deficits+in+rats+with+bilateral+cortical+microgyria%5BTitle%5D)+AND+%22Behav.+Brain+Funct%22%5BJournal%5D)">Persistent spatial working memory deficits in rats with bilateral cortical microgyria.</a> Behav. Brain Funct. 4, 45(2008).</li>
<li>Szalkowski, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+spatial+working+memory+deficits+in+rats+following+in+utero+RNAi+of+Dyx1c1%5BTitle%5D)+AND+%22Genes+Brain+Behav%22%5BJournal%5D)">Persistent spatial working memory deficits in rats following in utero RNAi of Dyx1c1.</a> Genes Brain Behav. 10 (2), 244-252 (2011).</li>
<li>Conklin, H. M., Salorio, C. F., Slomine, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Working+memory+performance+following+pediatric+traumatic+brain+injury%5BTitle%5D)+AND+%22Brain+Inj%22%5BJournal%5D)">Working memory performance following pediatric traumatic brain injury.</a> Brain Inj. 22 (11), 847-857 (2008).</li>
<li>Luu, T. M., Ment, L., Allan, W., Schneider, K., Vohr, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Executive+and+memory+function+in+adolescents+born+very+preterm%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Executive and memory function in adolescents born very preterm.</a> Pediatrics. 127, 639-648 (2011).</li>
<li>McClure, M. M., Threlkeld, S. W., Fitch, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+processing+and+learning%2Fmemory+following+erythropoietin+administration+in+neonatally+hypoxic-ischemic+injured+rats%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Auditory processing and learning/memory following erythropoietin administration in neonatally hypoxic-ischemic injured rats.</a> Brain Res. 1132 (1), 203-209 (2006).</li>
<li>McClure, M. M., Thrlekeld, S. W., Fitch, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+erythropoietin+on+auditory+processing+following+neonatal+hypoxic-ischemic+injury%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">The effects of erythropoietin on auditory processing following neonatal hypoxic-ischemic injury.</a> Brain Res. 1132 (1), 203-209 (2007).</li>
<li>LaPrairie, J. L., Murphy, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+impact+of+neonatal+injury+in+male+and+female+rats%3A+Sex+differences%2C+mechanisms+and+clinical+implications%5BTitle%5D)+AND+%22Front.+Neuroendocrinol%22%5BJournal%5D)">Long-term impact of neonatal injury in male and female rats: Sex differences, mechanisms and clinical implications.</a> Front. Neuroendocrinol. 31 (2), 193-202 (2010).</li>
<li>Volpe, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perinatal+brain+injury%3A+from+pathogenesis+to+neuroprotection%5BTitle%5D)+AND+%22Ment.+Retard.+Dev.+Disabil.+Res.+Rev%22%5BJournal%5D)">Perinatal brain injury: from pathogenesis to neuroprotection.</a> Ment. Retard. Dev. Disabil. Res. Rev. 7, 56-64 (2001).</li>
<li>Vannucci, R. C., Vannucci, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+model+of+perinatal+hypoxic-ischemic+brain+damage%5BTitle%5D)+AND+%22Ann.+N.+Y.+Acad.+Sci%22%5BJournal%5D)">A model of perinatal hypoxic-ischemic brain damage.</a> Ann. N. Y. Acad. Sci. 835, 234-249 (1997).</li>
<li>McClure, M. M., Peiffer, A. M., Rosen, G. D., Fitch, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+processing+deficits+in+rats+with+neonatal+hypoxic-ischemic+injury%5BTitle%5D)+AND+%22Int.+J.+Dev.+Neurosci%22%5BJournal%5D)">Auditory processing deficits in rats with neonatal hypoxic-ischemic injury.</a> Int. J. Dev. Neurosci. 23 (4), 351-362 (2005).</li>
<li>Jarrard, L., Okaichi, H., Steward, O., Goldschmidt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+role+of+hippocampal+connections+in+the+performance+of+place+and+cue+tasks%3A+comparisons+with+damage+to+the+hippocampus%5BTitle%5D)+AND+%22Behav.+Neurosci%22%5BJournal%5D)">On the role of hippocampal connections in the performance of place and cue tasks: comparisons with damage to the hippocampus.</a> Behav. Neurosci. 98, 946-954 (1984).</li>
<li>Bimonte-Nelson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testosterone%2C+but+not+nonaromatizable+dihydrotestosterone%2C+improves+working+memory+and+alters+nerve+growth+factor+levels+in+aged+male+rats%5BTitle%5D)+AND+%22Exp.+Neurology%22%5BJournal%5D)">Testosterone, but not nonaromatizable dihydrotestosterone, improves working memory and alters nerve growth factor levels in aged male rats.</a> Exp. Neurology. 181, 301-312 (2003).</li>
<li>Acosta, J. I., Hiroi, R., Camp, B. W., Talboom, J. S., Bimonte-Nelson, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+update+on+the+cognitive+impact+of+clinically-used+hormone+therapies+in+the+female+rat%3A+Models%2C+mazes+and+mechanisms%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">An update on the cognitive impact of clinically-used hormone therapies in the female rat: Models, mazes and mechanisms.</a> Brain Res. 13 (1514), 18-39 (2013).</li>
<li>O'Keefe, J., Nadel, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aO%27Keefe%2C+J+%22The+hippocampus+as+a+cognitive+map%22">The hippocampus as a cognitive map</a>. , Clarendon Press. Oxford, England. (1978).</li>
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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-enter a previously visited arm (working memory error). Since escape arms remain constant for all days of testing, reference memory can be easily analyzed by recording first entries into an arm that never contained a platform. Other significant advantages of this paradigm include no requirement for food deprivation, less risk of potential confounding scent cues and no need for pretraining unlike with traditional land based mazes5-7,18-20.
With respect to the length of testing (number of days), researchers should be aware of the cognitive limitations and requirements of the species or strain of animal being evaluated before determining the number of days required for optimal results. As with land based mazes, young rats, those used in models of neurological disease, and mice may take longer to reach asymptotic performance on the eight-arm radial water maze as compared to adult or typical rats. The data presented in the present protocol utilized eleven days of testing. Even with the observed significant effects in the present data, we recommend that testing be extended to 15-20 days in order to reduce variability when investigating deficits between experimental and control groups.
The use of a digital tablet and PDF maze templates for recording arm entries make this a practical and economical option for labs beginning to explore domains of working and reference memory. Further, this system can also be used with paper templates, making data collection more feasible for researchers without access to tablets or tablet based note-taking programs.
Finally, while we specifically used the radial arm water maze to assess working and reference memory in a rodent model of neonatal brain injury this protocol has been used to examine working and reference memory following hormone manipulation, in models of aging related cognitive decline and to examine other models of developmental disorders5-7,18-20. However, as with any testing design there are some limitations to keep in mind when using this specific water maze protocol. For example, to assess working memory with this water maze task, subjects are required to employ a win-shift strategy in the four arms with escape platforms (once an escape platform is located, it is removed for the following trial, thus testing working memory within a day)18. This is a common strategy for rodents when food reward is used in land based mazes21, but it is counter to typical behavioral tendencies of rodents in water mazes22. For this reason, the working memory component of the eight-arm radial water maze may be more challenging for some subjects to learn as compared to other maze tasks22. Additionally, it is important that researchers are aware of the physical limitations of the species or model being evaluated such as visual abilities or thermoregulation. Any known motoric deficits could also affect comparisons of trial latency so researchers should use errors as the primary dependent measure. For this reason it may also be advisable to use a visible platform escape trial to assess motor differences before initiation of this water maze protocol. Despite these minor limitations, the eight-arm radial water maze is a robust tool for examining working and reference memory across many animal models5-7,19,20.

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.

<table><tbody><tr><td>Plastic Tub</td><td>Gototanks http://www.gototanks.com</td><td>ARM-19455</td><td>Size can vary depending on species tested or individual lab needs.</td></tr><tr><td>iPad or other digital tablet</td><td>Apple Inc.</td><td>N/A</td><td>http://www.apple.com</td></tr><tr><td>Notability or other note taking application</td><td>Gingerlabs</td><td>N/A</td><td> &lt;p&gt;http://www.gingerlabs.com&lt;/p&gt; &lt;p&gt;Digital pdf writer or notetaking program&lt;/p&gt; </td></tr><tr><td>Digital timer</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Deltaphase isothermal heating pad</td><td>Braintree Scientific</td><td>ASS7D</td><td>http://www.braintreesci.com/</td></tr><tr><td>Stainless steel maze</td><td>Fabricated in house</td><td>N/A</td><td></td></tr></tbody></table>

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&lt;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&lt;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 eleven. These results support the validity of this task to assess working and reference memory in neonatal brain injury models and further support the use of this paradigm to assess other models of neurobehavioral pathology.
<img alt="Radial maze diagram with eight arms, used for spatial memory research in rodents." fo:content-width="5in" src="/files/ftp_upload/50940/50940fig1.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. The eight-arm radial water maze. A schematic diagram showing a top view of the eight-arm radial water maze with each arm number and start position labeled. The black squares indicate locations of hidden platforms within the maze. These platform locations will remain constant to extra-maze cues throughout the testing protocol. The dimensions of the maze insert and platforms are included (L = length, W = width, H = height). The diameter of the plastic tub was 122 cm and the depth of the water was 33 cm. <a href="https://www.jove.com/files/ftp_upload/50940/50940fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Morris water maze diagram; experimental setup for spatial memory testing in rodents." fo:content-width="5in" src="/files/ftp_upload/50940/50940fig2.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Sample recording template for the data collection. An example of the data sheet used to record subject arm entries and escape patterns. This file can be converted to a PDF for use with a digital tablet and PDF writer program. Data sheets should include a place to record the subject number, trial number, day of testing, which escape platform was used during the trial, and latency to reach platform. Prior to a trial any removed platforms should be marked with an X. At the end of a trial the platform used for escape should be circled to indicate the target arm for that run. Thus, a new template used on trial four would have three platforms with an X and one open target left on the template. <a href="https://www.jove.com/files/ftp_upload/50940/50940fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Bar graph comparing working memory and reference memory errors between treatment groups." fo:content-width="5in" src="/files/ftp_upload/50940/50940fig3.jpg" width="600px" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. The effect of neonatal brain injury on reference and working memory. Using the eight-arm radial arm water maze we were able to show A) a significant increase in the number of working memory incorrect errors in rodents with hypoxic-ischemic (HI) injury by the eleventh day of testing. This is indicative of a working memory deficit. Further, we found B) a significant effect of HI injury on reference memory errors as compared to shams by the eleventh day of testing. Errors include the total cumulative errors across all four trials of day 11 (* p&lt;0.05). <a href="https://www.jove.com/files/ftp_upload/50940/50940fig3highres.jpg" target="_blank">Click here to view larger image</a>.

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 ...

use-an-eight-arm-radial-water-maze-to-assess-working-reference-memory

Nanyang Technological University

Research

JoVE Journal

Behavior

16.3K Aufrufe.  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.

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

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

Quantifizierung Kognitive Dekrementiert von Cranial Strahlentherapie verursachten Schäden

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Forschung

Medizin

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.

Die Doppel-H Maze: A Robust Verhaltenstest für Lernen und Gedächtnis in Nagetiere

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 ...

Verhalten

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-Labyrinth Erzwungener Wechsel und die Links-Rechts-Discrimination Aufgaben für die Beurteilung der Arbeitsbedingungen und Referenz-Gedächtnis bei Mäusen

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Neurowissenschaften

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

Beurteilung der räumlichen Gedächtnisstörung in einem Mausmodell der traumatischen Gehirnverletzung unter Verwendung eines Radialwasserlaufstreifens

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

Erkennung von Verhaltensstörungen Defizite bei Ratten nach Schädel-Hirn-Verletzung

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Unter Verwendung der modifizierten T-Maze um Funktionsgedächtnis Ergebnisse nach Herzstillstand zu bewerten

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Motor und Hippokampus-Dependent Spatial Learning und Referenz-Gedächtnis-Bewertung in einem transgenen Mausmodell der Alzheimer-Krankheit mit Schlaganfall

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

Ein metrischer Test zur Beurteilung des räumlichen Arbeitsgedächtnisses bei erwachsenen Ratten nach traumatischer Hirnverletzung

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.

Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

Testen des räumlichen Gedächtnisses mit Hilfe von Irrgärten

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this ...

Lehre

JoVE Science Education

Psychologie

Verhaltensforschung

The Morris Water Maze

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, rodents must swim to a platform to escape from a pool of water. The platform is then hidden under the water&#8217;s surface, so that the animal is required to remember it&#8217;s location in order to escape.This simple yet powerful maze design can be used to assay cognitive function, study animal models of neurodegenerative disease, and test potential drug therapies.
This video provides an introduction to the Morris water maze and the principles surrounding its use, including a discussion of the different types of memory tested in the maze, important points to consider when designing and conducting this experiment, and the procedures for setup and running of the test. Several applications of the maze are examined, such as investigating how radiation treatment may lead to memory impairment. Finally, other types of water mazes, such as the 8-arm radial maze, are introduced to show how this paradigm can be adapted to engage different types of memory.

Das Morris-Wasserlabyrinth

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, ...