This research was supported by the Institute for Translational Health Sciences pilot project grant opportunity (UL1TR000423), the University of Washington Center on Human Development and Disability, and the University of Washington Animal Behavior Core and Brain Imaging Core. We would like to acknowledge Dr. Warren Ladiges for his role in the development and dissemination of the original Radial Water Tread maze design and protocol presented here. We also thank Toby Cole for his assistance with this project.

All procedures and animal handling were conducted in accordance with the animal care guidelines issued by the National Institutes of Health and by the University of Washington Animal Care and Use Committee.
1. Surgery
<ol>
	<li>Anesthetize the mouse at 5% isoflurane in an induction box until unconscious. Confirm anesthesia by a reduction in the breathing rate and the absence of a withdrawal reflex following toe-pinch.</li>
	<li>Maintain anesthesia via nose cone at 2-2.5% throughout surgery. Monitor the breathing rate throughout surgery to ensure the mouse remains unconscious.</li>
	<li>Place the mouse prone on a heating pad and position the mouse in the stereotactic device using ear bars, ensuring that the head is secure and flat.</li>
	<li>Remove hair from the scalp using hair removal cream. Rinse the scalp thoroughly with saline.</li>
	<li>Clean the surgical site with an alternating iodine and 70% ethanol wash.</li>
	<li>Administer a subcutaneous injection of Lidocaine and Bupivacaine (1 mg/kg) at the scalp.</li>
	<li>With surgical scissors, make a longitudinal midline incision, and retract the skin to reveal the skull.</li>
	<li>Using a 5 mm trephine saw preform a craniotomy over the left frontoparietal cortex with center point at 2.5 mm behind bregma and 2.5 mm left of the midline. Carefully remove the circle of bone to expose the brain.</li>
	<li>Set the impactor device to a velocity of 6 m/s and 200 ms dwell time.</li>
	<li>Position the impactor device until the 3 mm convex impact tip is lightly touching the surface of the brain at 2.5 mm behind bregma and 2.55 mm left of the midline. Retract the impact tip, and lower by 1 mm (impact depth). When ready, fire the device, generating the desired impact.</li>
	<li>Cover the craniotomy with a sterile polypropylene disc cemented to derma with tissue adhesive, and suture the incision closed.</li>
	<li>Remove the mouse from anesthesia, and give IP injection of Buprenorphine (0.5 mg/kg).</li>
	<li>Allow the mouse to recover in a clean cage, warmed by a heating pad.&#160;The mouse should be monitored for signs of pain or distress over the next 24 h. 
	NOTE: Sham controls should receive identical treatment as above, with steps 1.8-1.9 omitted.</li>
</ol>
2. Radial Water Tread Maze Construction
<ol>
	<li>Bore 9 exit holes, large enough to accommodate 1-1/2 inch ABS DWV SPG x SJ trap adapters, at equal intervals around the circumference of a 32 inch diameter galvanized steel tub. Center these exit holes roughly 2-1/4 inches above the floor of the tub.</li>
	<li>Fit a 1-1/2 inch ABS DWV SPG x SJ trap adapter into each of the exit holes, and secure with the included ring nuts.</li>
	<li>With rubber stoppers, cap eight of the nine exits from the exterior of the apparatus. The final, uncapped exit will serve as the escape route. It does not matter which exit is designated as the escape route.</li>
	<li>Attach a 90&#176; ABS elbow to the exterior end of the remaining exit. The 90&#176; bend serves to prevent test subjects from visually determining the correct escape route from inside the maze.</li>
	<li>Construct the escape box from any opaque box capable of being sanitized and roughly 30 cm x 15 cm x 15 cm in size. Cut a hole on the side of the box, directly above the floor, large enough to accommodate a the 90&#176; ABS elbow.</li>
	<li>Attach the escape box to the terminal end of the 90&#176; ABS elbow.</li>
	<li>Slightly elevate the escape box (less than one inch) above the surface of the floor. This allows ample room for an electric heating pad, or other heating source, to be placed beneath the escape box.</li>
	<li>Print and laminate at least 5 unique, visual cues. Use simple, high contrast images that can be easily discerned from within the apparatus. Black and white clipart shapes (triangle, square, circle) are recommended.</li>
	<li>Using magnets, adhere the visual cues to the inner walls of the apparatus. Cues should be roughly equal distance apart, around the circumference of the apparatus.</li>
</ol>
3. Radial Water Tread Maze Protocol
NOTE: Water maze testing should begin only after the surgical site has healed (roughly one week post-surgery).
<ol>
	<li>Preparing for testing.
	<ol>
		<li>Allow mice to acclimate to the testing room for at least 30 min prior to commencing testing.</li>
		<li>Sanitize the apparatus using a 70% ethanol spray.</li>
		<li>Fill the apparatus with roughly 1 inch of cold (12-14 &#176;C) water.</li>
		<li>Place an electric heating pad, or other heating source, directly underneath the escape box. Keep the escape box dark and warm throughout the duration of testing.</li>
		<li>Position a bright light source over the apparatus. 
		NOTE: If using a lamp that might be visible to research animals from the apparatus itself, take care to ensure that the lamp is placed in the same position each day. The lamp itself might represent another visual cue for the mice to use to locate the escape box, and moving it drastically from day to day might complicate results.</li>
	</ol>
	</li>
	<li>Testing Protocol
	<ol>
		<li>Remove the mouse from its cage gently by the tail, and place in the center of the apparatus.</li>
		<li>As soon as the animal is in in the apparatus, begin timing.</li>
		<li>Once the animal has found the correct exit, and has located/entered the escape box, stop timing and record the number of seconds required to find the correct path.</li>
		<li>If the animal attempts to climb into a terminating hole and does not spontaneously reenter the maze after 10 s, guide the animal back into the center of the maze by hand.</li>
		<li>If the animal fails to find the correct path to the escape box within 3 min (180 s), score the trial as a failure and record as 180 s. Carefully guide the animal toward the correct path by hand.</li>
		<li>Allow the mouse to remain in the escape box for a 1 min, inter-trial rest.</li>
		<li>Once the 1 min rest has passed, remove the animal from the escape box and return to its home cage.</li>
		<li>Thoroughly sanitize the escape box and exits with a 70% ethanol spray to prevent the mouse from using olfactory cues to locate the correct escape route. This step should take no more than a few seconds.</li>
		<li>Return the mouse to the maze for the next trial.</li>
		<li>Repeat Steps 3.2.1-3.2.9 until the mouse has completed a total of three trials, and has either located or been led to the escape box three times.</li>
		<li>Following the final 1 min rest, return the mouse to its home cage.</li>
		<li>Drain and replace the water in the apparatus between animals to ensure consistent temperature throughout testing.</li>
		<li>Repeat steps 3.2.1-3.2.12 for each mouse to be tested.</li>
		<li>On the following day, repeat preparation steps 3.2.1-3.2.5. Take special care in ensuring that visual cues remain in consistent positions between testing days.</li>
		<li>Test animals using the above daily testing protocol for three trials a day for four days (training period), with a final three-trial test for memory retention on the fifth day. A sixth three-trial test (Long Term Memory retention) can be given on day twelve.</li>
		<li>Do not perform any tests between testing days five and twelve.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>If a mouse fails to complete the maze in under 180 s during any two-day period (i.e., a total of consecutive 6 trials all scored at 180 s), consider the mouse insufficiently motivated by the testing conditions, and remove from analysis.</li>
		<li>Calculate the mean latency to complete the maze for each subject by testing date by averaging their three daily trials for that day.</li>
		<li>Obtain group differences in memory retention using standard t-tests to compare groups at the fifth day test and long-term memory testing date. If more than two groups are being compared, one-way ANOVA followed by appropriate post-hoc analysis (such as Tukey&#39;s test) to follow up on any significance obtained should be employed instead.</li>
		<li>Obtain group differences in the acquisition period (days 1-4) via repeated measure analysis of variance.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Levin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+outcome+of+closed+head+injury:+Implications+for+clinical+trials.">Neurobehavioral outcome of closed head injury: Implications for clinical trials.</a> J. Neurotrauma. 12 (4), 601-610 (1995).</li><li>Schretlen, D., Shapiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+review+of+the+effects+of+traumatic+brain+injury+on+cognitive+functioning.">A quantitative review of the effects of traumatic brain injury on cognitive functioning.</a> Int Rev Psychiatry. 15 (4), 341-349 (2003).</li><li>Cline, M. M., 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=Novel+application+of+a+radial+water+tread+maze+can+distinguish+cognitive+deficits+in+mice+with+traumatic+brain+injury.">Novel application of a radial water tread maze can distinguish cognitive deficits in mice with traumatic brain injury.</a> Brain Res. 1657, 140-147 (2017).</li><li>Ellenbroek, B., Youn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+in+neuroscience+research:+Is+it+a+rat+race?.">Rodent models in neuroscience research: Is it a rat race?.</a> Dis. Model. Mech. 9 (10), 1079-1087 (2016).</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>Barnes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+deficits+associated+with+senescence:+A+neurophysiological+and+behavioral+study+in+the+rat.">Memory deficits associated with senescence: A neurophysiological and behavioral study in the rat.</a> J. Comp. Physiol. Psych. 93 (1), 74-104 (1979).</li><li>Frick, K., Stillner, E., Berger-Sweeney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+are+not+little+rats:+Species+differences+in+a+one-day+water+maze+task.">Mice are not little rats: Species differences in a one-day water maze task.</a> Neuroreport. 11 (16), 3461-3465 (2000).</li><li>Whishaw, I., Tomie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Mice+and+Mazes:+Similarities+Between+Mice+and+Rats+on+Dry+Land+But+Not+Water+Mazes.">Of Mice and Mazes: Similarities Between Mice and Rats on Dry Land But Not Water Mazes.</a> Physiol Behav. 60 (5), 1191-1197 (1995).</li><li>Francis, D., Zaharia, M., Shanks, N., Anisman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+disturbances+in+Morris+water-maze+performance:+Interstrain+variability.">Stress-induced disturbances in Morris water-maze performance: Interstrain variability.</a> Physiol Behav. 58 (1), 57-65 (1995).</li><li>Wahlsten, D., Rustay, N., Metten, P., Crabbe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+a+better+mouse+test.">In search of a better mouse test.</a> Trends Neurosci. 26 (3), 132-136 (2003).</li><li>Crawley, , 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=Behavioral+phenotypes+of+inbred+mouse+strains:+implications+and+recommendations+for+molecular+studies.">Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.</a> Psychopharmacology. (Berl). 132 (2), 107-124 (1997).</li><li>Wahlsten, D., 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=Different+data+from+different+labs:+lessons+from+studies+of+gene-environment+interaction.">Different data from different labs: lessons from studies of gene-environment interaction.</a> J. Neurobiol. 54 (1), 283-311 (2002).</li><li>Rogers, D. C., 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=Use+of+SHIRPA+and+discriminant+analysis+to+characterise+marked+differences+in+the+behavioural+phenotype+of+six+inbred+mouse+strains.">Use of SHIRPA and discriminant analysis to characterise marked differences in the behavioural phenotype of six inbred mouse strains.</a> Behav Brain Res. 105 (2), 207-217 (1999).</li><li>Wolfer, D. P., Stagljar-Bozicevic, M., Errington, M. L., Lipp, H. <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+Learning+in+Transgenic+Mice:+Fact+or+Artifact?.">Spatial Memory and Learning in Transgenic Mice: Fact or Artifact?.</a> Physiology. 13 (3), 118-123 (1998).</li><li>Koopmans, G., Blokland, A., Vannieuwenhuijzen, P., Prickaerts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+spatial+learning+abilities+of+mice+in+a+new+circular+maze.">Assessment of spatial learning abilities of mice in a new circular maze.</a> Physiol Behav. 79 (4-5), 683-693 (2003).</li><li>Deacon, R., Rawlins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+impairments+of+hippocampal-lesioned+mice+in+a+paddling+pool.">Learning impairments of hippocampal-lesioned mice in a paddling pool.</a> Behav Neurosci. 116 (3), 472-478 (2002).</li><li>Pettan-Brewer, C., 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=A+novel+radial+water+tread+maze+tracks+age-related+cognitive+decline+in+mice.">A novel radial water tread maze tracks age-related cognitive decline in mice.</a> Pathobiol Aging Age Relat Dis. 3, 1-4 (2013).</li><li>Wiley, J., Pettan-Brewer, C., Ladiges, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylbutyric+acid+reduces+amyloid+plaques+and+rescues+cognitive+behavior+in+AD+transgenic+mice.">Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice.</a> Aging Cell. 10 (3), 418-428 (2011).</li><li>Enns, L., 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=Disruption+of+Protein+Kinase+A+in+Mice+Enhances+Healthy+Aging.">Disruption of Protein Kinase A in Mice Enhances Healthy Aging.</a> PLoS ONE. 4 (6), (2009).</li><li>Ivonen, H., Nurminen, L., Harri, M., Tanila, H., Puolivali, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+in+mice+tested+in+Morris+water+maze.">Hypothermia in mice tested in Morris water maze.</a> Behav Brain Res. 141 (2), 207-213 (2003).</li><li>Shultz, S. R., 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=Granulocyte-macrophage+colony-stimulating+factor+is+neuroprotective+in+experimental+traumatic+brain+injury.">Granulocyte-macrophage colony-stimulating factor is neuroprotective in experimental traumatic brain injury.</a> J Neurotrauma. 31 (10), 976-983 (2014).</li></ol>

The authors have nothing to disclose.

The RWT maze protocol presented here successfully distinguishes between CCI-induced TBI mice and sham controls, and represents a feasible, mouse-centric, alternative to the MWM and Barnes circular maze. While the results reported here speak only to the use of the RWT maze in a TBI mouse model, this apparatus has been used successfully in aged and transgenic models where stress-induced noncompliance resulting from swim-based testing made using the MWM impractical15,17</...

Memory impairments are among the most common symptoms reported by patients following traumatic brain injury (TBI)1,2. Accurate identification and assessment of analogous memory deficits in animal models of TBI, therefore, are essential to our understanding of this condition and its management. Here, we present a protocol to test spatial memory in a mouse model of TBI using a Radial Water Tread (RWT) maze. This apparatus was previously shown to assess cognitive deficits in mouse models of controlled cortical impact (CCI)-induced TBI3, and represents a potential alternative to rat-validated, swim-based tests of cognition.
The growing diversity and availability of transgenic mouse models has led to a recent increase in the use of mice over rats in scientific research4. Despite this shift, researchers continue to rely on behavioral and cognitive tasks that were originally designed and validated for rat use. The most common tests currently used to assess cognition in mice, the Morris Water Maze (MWM) and the Barnes circular maze, were specifically designed to capitalize on instinctual behaviors found in rats5,6. Considering the genetic, neuroethological, and cognitive differences that exist between these two species4, it is unsurprising that mice consistently underperform on these tasks7,8.
Species-dependent differences in testing ability are particularly concerning in swimming-based cognitive tests, such as the MWM. While both rats and mice are proficient swimmers, researchers have identified several mouse strains that perform remarkably poorly on swimming-based cognitive tasks9,10,11,12,13. Even in wild-type animals, rats generally outperform mice7,8. While this could be interpreted as a species-specific difference in spatial memory, analogous follow-up testing using a dry-land maze revealed no species-dependent differences in cognitive performance8. A number of factors unrelated to cognition could account for this finding, including species-dependent differences in either swimming ability or search strategy. Indeed, factor analysis of mouse-specific search strategies in the MWM show that noncognitive factors (in particular, thigomotaxis and passivity [i.e., floating]) may play a more significant role in MWM performance than spatial learning14.
Here, we demonstrate the use of a cognitive test designed to capitalize on the instinctual behavior of mice, and which does not require swimming, to measure spatial memory impairment in a mouse model of CCI-induced TBI. While the RWT maze (Figure 1A-B) was conceived as a novel hybrid of the MWM and Barnes circular maze, it was specifically designed to take advantage of thigmotactic behavior instinctual to mice15,16. The apparatus consists of a 32 inch diameter galvanized steel tub in which nine evenly spaced exit holes have been bored. The holes are centered 2-1/4 inches above the floor of the tub and are sized to fit commonly available 1-1/2 inch ABS DWV SPG x SJ trap adapters. Eight of the exits are capped from the outside and blinded to a depth of 1 inch with rubber stoppers. The ninth is connected by a 90&#176; acrylonitrile butadiene styrene (ABS) elbow to an opaque plastic box from which the mouse can be easily removed after testing. Over the course of a brief acquisition period, the mouse is trained to use the unique visual cues lining the maze to locate this escape box. During testing, the maze is filled with an inch of cold water (12-14 &#176;C), sufficiently aversive enough to promote escape, but not deep enough that the mouse is required to swim.
The RWT maze represents a low-cost, low-maintenance alternative to the MWM, and has been used successfully in aged and transgenic mice15,17,18,19, and CCI-induced mouse models of TBI3. The protocol outlined here represents a simple and effective method for measuring spatial memory impairment requiring no pre-injury training, and could be easily modified to suit the particular needs of a research laboratory.

<table><tbody><tr><td>35 Gal. Hot Dipped Steel Round Tub</td><td>Home Depot</td><td>Internet #206638142</td><td>Needed: 1</td></tr><tr><td>1-1/2 in. ABS DWV SPG x SJ Trap Adapter</td><td>Home Depot</td><td>Internet #100344703, Store SKU #188956</td><td>Needed: 9</td></tr><tr><td>1-3/4 in. x 1-7/16 in. Black Rubber Stopper</td><td>Home Depot</td><td>Internet #100114974 Store SKU #755844</td><td>Needed: 8</td></tr><tr><td>1-1/2 in. ABS DWV 90 Degree Hub x Hub Elbow</td><td>Home Depot</td><td>Internet #100346663 Store SKU #188603</td><td>Needed: 1</td></tr><tr><td>HDX&lt;br/&gt; 10 Gal. Storage Tote</td><td>Home Depot</td><td>Internet #202523587 Store SKU #258804 Store SO SKU #258804</td><td>Needed: 1</td></tr><tr><td>Impact One Stereotaxic Impactor for CCI</td><td>Leica Biosystems</td><td>39463920</td><td>Needed: 1</td></tr><tr><td>Vernier Stereotaxic w/ Manual Fine Drive Stereotaxic Instrument for Small Animals</td><td>Leica Biosystems</td><td>39463001</td><td>Needed: 1</td></tr></tbody></table>

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.

The RWT maze (Figure 1) was used to investigate injury-dependent spatial memory deficits in mice randomly assigned to receive&#160;either controlled cortical impact-induced TBI or sham surgery. The injury was generated using a solenoid-driven cortical impact with a 3 mm convex tip and the following injury parameters: 6 m/s strike velocity, 1 mm depth of penetration, and 200 ms contact time. Mice received cognitive testing starting at 35 days post-surgery, and...

Watch this Scientific Journal Video about Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze at JoVE.com

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

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 ...

assessing-spatial-memory-impairment-mouse-model-traumatic-brain

Research

JoVE Journal

Behavior

7.0K Views.  University of Washington. 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.

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

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.

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 ...

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.

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

Medicine

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.

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 ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Neuroscience

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 ...

Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific research to assess the learning and memory of animals. Due to the extensive use of the MWM test, visual experimental protocols are essential for researchers. This manuscript uses the latest studies to introduce the protocol of the MWM test. Alzheimer&#8217; Disease (AD) is characterized by a progressive loss of memory and cognitive function. An alternative and complementary treatment used for AD is Manual Acupuncture (MA). To assess the learning and memory ability of AD model mice, the MWM test was conducted. The visible platform trial, hidden platform trial, probe trial, and reversal trial of MWM were used to evaluate spatial learning and memory ability. In the visible platform trial, the swimming speed and escape latency of mice in different groups was not significantly different. In the hidden platform and reversal trials, the AD group showed a long escape latency. The escape latency decreased significantly after the MA treatment. Low platform crossover number and the proportion of time in the SW quadrant in the probe trial increased after the MA treatment (p &#60; 0.05 or p &#60; 0.01). The results of the MWM tests suggest that MA can effectively improve the spatial learning and memory abilities of AD model mice. Rigorous experimental operations provided assurance of the reliability of the results.

Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer&#8217;s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific ...

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.

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.

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

rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

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

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