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>

<ol>
	<li>Bunge, S. A., Ochsner, K. N., Desmond, J. E., Clover, G. H., Gabrieli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+regions+involved+in+keeping+information+in+and+out+of+mind.">Prefrontal regions involved in keeping information in and out of mind.</a> Brain. 124 (Pt 10), 2074-2086 (2001).</li><li>Olton, D., Collison, C., Werz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+memory+and+radial-arm+maze+performance+of+rats.">Spatial memory and radial-arm maze performance of rats.</a> Learn Motiv. 8, 289-314 (1977).</li><li>Olton, D., Becker, J., Handelmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampus,+space,+and+memory.">Hippocampus, space, and memory.</a> Behav. Brain Sci. 2, 313-365 (1979).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> J. Neurosci. Methods. 11 (1), 47-60 (1984).</li><li>Hyde, L. A., Hoplight, B. J., Denenberg, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+version+of+the+radial-arm+maze:+learning+in+three+inbred+strains+of+mice.">Water version of the radial-arm maze: learning in three inbred strains of mice.</a> Brain Res. 785 (2), 236-244 (1998).</li><li>Hyde, L. A., Sherman, G. F., Hoplight, B. J., Denenberg, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+memory+deficits+in+BXSB+mice+with+neocortical+ectopias.">Working memory deficits in BXSB mice with neocortical ectopias.</a> Physiol. Behav. 70 (1-2), 1-5 (2000).</li><li>Hyde, H. A., Hoplight, L. A., Denenberg, B. J., H, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+two+species,+females+exhibit+superior+working+memory+and+inferior+reference+memory+on+the+water+radial-arm+maze.">In two species, females exhibit superior working memory and inferior reference memory on the water radial-arm maze.</a> Physiol. Behav. 70 (3-4), 311-317 (2000).</li><li>Fitch, R. H., Breslawski, H., Rosen, G. D., Chrobak, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+spatial+working+memory+deficits+in+rats+with+bilateral+cortical+microgyria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+spatial+working+memory+deficits+in+rats+following+in+utero+RNAi+of+Dyx1c1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+memory+performance+following+pediatric+traumatic+brain+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+and+memory+function+in+adolescents+born+very+preterm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+processing+and+learning/memory+following+erythropoietin+administration+in+neonatally+hypoxic-ischemic+injured+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+erythropoietin+on+auditory+processing+following+neonatal+hypoxic-ischemic+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impact+of+neonatal+injury+in+male+and+female+rats:+Sex+differences,+mechanisms+and+clinical+implications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+brain+injury:+from+pathogenesis+to+neuroprotection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+perinatal+hypoxic-ischemic+brain+damage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+processing+deficits+in+rats+with+neonatal+hypoxic-ischemic+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+hippocampal+connections+in+the+performance+of+place+and+cue+tasks:+comparisons+with+damage+to+the+hippocampus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone,+but+not+nonaromatizable+dihydrotestosterone,+improves+working+memory+and+alters+nerve+growth+factor+levels+in+aged+male+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+cognitive+impact+of+clinically-used+hormone+therapies+in+the+female+rat:+Models,+mazes+and+mechanisms.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The hippocampus as a cognitive map. , (1978).</li><li>Whishaw, I. Q., Pasztor, T. 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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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		<col span="2" />
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	<tbody>
		<tr height="65">
			<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>
		</tr>
		<tr height="13">
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="48">
			<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
			</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:155px;">Digital timer</td>
			<td style="width:187px;">Any</td>
			<td style="width:131px;">N/A</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="64">
			<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>
		</tr>
		<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>
			<td style="width:131px;"></td>
		</tr>
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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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16.2K Views.  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.

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

Shallow Water (Paddling) Variants of Water Maze Tests in Mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to the mouse becoming increasingly important. But researchers found that some strains of mutant mice were prone to problems like passively floating or diving when they were tested in the Morris water maze11. This was unsurprising considering their natural habitat; rats swim naturally (classically, the "sewer rat"), whereas mice evolved in the dry areas of central Asia.
To overcome these problems, it was considered whether shallow water would be a sufficient stimulus to provide escape motivation for mice. This would also avoid the problems of drying the small creatures with a towel and then putting them in a heated recovery chamber to avoid hypothermia, which is a much more serious problem than with rats; the large ratio of surface area to volume of a mouse makes it particularly vulnerable to rapid heat loss.
Another consideration was whether a more natural escape strategy could be used, to facilitate learning. Since animals that fall into water and swim away from the safety of the shore are unlikely to pass on their genes, animals have evolved a natural tendency to swim to the edge of a body of water. The Morris water maze, however, requires them to swim to a hidden platform towards the center of the maze - exactly opposite to their evolved behavior. Therefore the paddling maze should incorporate escape to the edge of the apparatus. This feature, coupled with the use of relatively non-aversive shallow water, embodies the "Refinement" aspect of the "3 Rs" of Russell and Burch8.
Various types of maze design were tried; the common feature was that the water was always shallow (2 cm deep) and escape was via a tube piercing the transparent wall of the apparatus. Other tubes ("false exits") were also placed around the walls but these were blocked off. From the inside of the maze all false exits and the single true exit looked the same. Currently a dodecagonal (12-sided) maze is in use in Oxford, with 12 true/false exits set in the corners. In a recent development a transparent paddling Y-maze has been tested successfully.

Shallow Water Paddling Variants of Water Maze Tests in Mice

Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to ...

A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that one attempts to ignore, or by interrupting information that demands attention as part of another (secondary) task goal. Both forms of external interference have been shown to detrimentally impact the ability to maintain information in working memory (WM). Emerging evidence suggests that these different types of external interference exert different effects on behavior and may be mediated by distinct neural mechanisms. Better characterizing the distinct neuro-behavioral impact of irrelevant distractions versus attended interruptions is essential for advancing an understanding of top-down attention, resolution of external interference, and how these abilities become degraded in healthy aging and in neuropsychiatric conditions. This manuscript describes a novel cognitive paradigm developed the Gazzaley lab that has now been modified into several distinct versions used to elucidate behavioral and neural correlates of interference, by to-be-ignored distractors versus to-be-attended interruptors. Details are provided on variants of this paradigm for investigating interference in visual and auditory modalities, at multiple levels of stimulus complexity, and with experimental timing optimized for electroencephalography (EEG) or functional magnetic resonance imaging (fMRI) studies. In addition, data from younger and older adult participants obtained using this paradigm is reviewed and discussed in the context of its relationship with the broader literatures on external interference and age-related neuro-behavioral changes in resolving interference in working memory.

A novel cognitive paradigm is developed to elucidate behavioral and neural correlates of interference by to-be-ignored distractors versus interference by to-be-attended interruptors during a working memory task. In this manuscript, several variants of this paradigm are detailed, and data obtained with this paradigm in younger/older adult participants is reviewed.

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &amp;#8211; Working Memory (CABC-WM)

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

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.

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

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

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.

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

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper understanding of the neurobiological causes underlying the decision-making problems. A cost-benefit based T-maze task is used for measuring decision making in which rodents choose between a high reward arm (HRA) and a low reward arm (LRA). There are two paradigms of the T-maze decision-making task, one in which the cost is a time delay and the other in which it is physical effort. Both paradigms require a tedious and labor-intensive management of experimental animals, multiple doors, pellet reward, and arm choice recordings. In the current work, we invented an apparatus based on traditional T-maze with full automation for pellet delivery, door management and choice recordings. This automated setup can be used for the evaluation of both delay- and effort-based decision making in rodents. With the protocol described here, our lab investigated the decision-making phenotypes of multiple genetically modified mice. In the representative data, we showed that the mice with ablated medial habenular showed aversions of both delay and effort and tended to choose the immediate and effortless reward. This protocol helps to decrease the variability caused by experimenter intervention and to enhance experiment efficiency. In addition, chronic silicon probe or microelectrode recording, fiber-optic imaging and/or manipulation of neural activity can be easily applied during the decision-making task using the setup described here.

This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents.

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...