Huiling Tian and Ning Ding are co-first authors. Zhigang Li and Jing Jiang are co-corresponding authors. This research was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81804178, 81473774, and 81503654). The protocol and results described herein originates from the article, &#8220;Involvement of Manual Acupuncture Regulates Behavior and Cerebral Blood Flow in the SAMP8 Mouse Model of Alzheimer&#8217;s Disease&#8221; by Dr. Ning Ding et al.

This protocol was approved by the Animal Ethics Committee of Beijing University of Chinese Medicine, and it was in accordance with all guidelines for the Care and Use of Laboratory Animals of China. There was no accidental death situation during the experimental procedure, and no animals needed to be euthanized in this study.
1. Preparation
<ol>
	<li>Purchase 30 male SAMP8 mice and 10 male SAMR1 mice (age: 8 months).</li>
	<li>House the mice individually in individual ventilation cages at a temperature of 24 &#177;2 &#176;C and a 12 h dark/light cycle.</li>
	<li>Feed the mice with a standard pellet diet available ad libitum and provide sterile drinking water.</li>
	<li>Acclimate all the mice to the environment for 5 days before experimentation.</li>
</ol>
2. Grouping of animals
<ol>
	<li>Randomly divide 30 SAMP8 mice into three groups (n = 10/group): the AD group, manual acupuncture (MA) group, and medicine (M) group.</li>
	<li>Use 10 SAMR1 mice as the normal control (N) group6.</li>
</ol>
3. Administration of donepezil hydrochloride tablets
<ol>
	<li>Crush a donepezil hydrochloride tablet (5 mg/tablet) and dissolve it in 50 mL of distilled water.</li>
	<li>Deliver the medicine prepared in step 3.1 at doses of 1 mg/kg to the mice using an oral gavage once a day8 during the entire experiment, including the days when the MA treatment and MWM tests are performed.</li>
</ol>
4. Administration of manual acupuncture
<ol>
	<li>Immobilize the mice of the MA group in mouse bags.</li>
	<li>Use disposable sterile acupuncture needles (0.25 mm x 13 mm) and apply the flat thorn method of MA on Baihui (GV20) and Yintang (GV29)5 toward the nose for 20 min. Ensure that the needle depth is 0.2&#8211;0.3 cm.</li>
	<li>Bidirectionally twirl the manipulation within 90&#176; at a speed of about 180 r/min every 5 min for ~15 s each time during the entire experiment, including days when MA treatment and MWM tests are performed.</li>
</ol>
5. MWM test
NOTE: At 24 h after the 15 consecutive days of treatment, subject the mice in the four groups to the MWM test. Conduct the visible platform trial, hidden platform trial, probe trial, and reversal trial in order.
<ol>
	<li>Prepare for the MWM test.
	<ol>
		<li>Position MWM device and the signal acquisition and processing system in an experiment room designed to maintain sound insulation.</li>
		<li>Put a circular white tank (diameter = 90 cm, height = 50 cm) surrounded by an opaque cloth in the middle of the MWM device.</li>
		<li>Fix a video camera to the ceiling of the MWM device and connect it to a video recorder with an automated tracking system to collect the data.</li>
		<li>Divide the water maze tank equally into four equal regions using two mutually perpendicular lines, labeled north (N), south (S), east (E), and west (W). Divide the pool area conceptually into four quadrants of the same size (NE, NW, SW, and SE).</li>
		<li>Within the sight of the mouse, place visual cues of different shapes on the wall of each quadrant as visual references (e.g., squares, triangles, and circles). 
		NOTE: Distal cues are the animal&#8217;s navigational reference points for locating the platform. Therefore, do not move them during the test. The position of the researcher is a potential distal cue and can influence the MWM. Therefore, the researcher should stay out of the sight of the mice while waiting for the animal to perform the test.</li>
		<li>Fill the circular tank with water to a depth of 30 cm and maintain at 22 &#177; 2 &#176;C with an electric heater.</li>
		<li>Render the water opaque with about 150 g of milk powder.</li>
	</ol>
	</li>
	<li>Perform the visible platform trial.
	<ol>
		<li>Place a plastic circular platform (diameter = 9.5 cm; height = 28 cm) 1 cm above the water surface in any quadrant at random.</li>
		<li>Put a black flag on the platform.</li>
		<li>Release each mouse gently into the water at water-level from one of the four start locations facing the tank wall. Do not drop the mouse into the water.</li>
		<li>Activate the computer tracking program as soon as the mouse is released into the water.</li>
		<li>Give each mouse 60 s to search for the platform. At the end of each trial, place each mouse on the platform and allow it to stay on it for 10&#8211;30 s.</li>
		<li>Observe the swimming trajectories of the mice on the computer, record the time the mouse took to find the platform as escape latency, and analyze the swimming speed.</li>
		<li>Dry each mouse with towels and warm it with an electric heater. Be sure to use an appropriate heat source to prevent the animal from overheating.&#160; 
		NOTE: Place each mouse into the pool at each of the four different starting quadrants for four trials, moving the platform to a different location with each subsequent trial. The interval between two trials using each mouse is 15&#8211;20 min.</li>
	</ol>
	</li>
	<li>Perform the hidden platform trial/place navigation test.
	<ol>
		<li>Place the same platform without a flag in the SE quadrant.</li>
		<li>Randomly place the mouse into the pool from each of the four quadrants (NE, NW, SW, N) facing the pool wall for four trials. Use a time interval of 15&#8211;20 min between two trials.</li>
		<li>Give each mouse 60 s to search for the hidden platform.</li>
		<li>Record the escape latency of each trial after the mouse climbs up to the platform for subsequent analysis.</li>
		<li>Dry each mouse with towels and warm it with an electric heater. 
		NOTE: Conduct the hidden platform trial from days 2&#8722;6. If the mouse cannot find the platform in 60 s, lead the mouse to climb up to the platform and allow it to stay there for 10&#8211;30 s at the end of each trial. Perform four trials/day for each mouse for 5 consecutive days, with the platform and the visual cues at constant positions.</li>
	</ol>
	</li>
	<li>Perform the probe trial. 
	NOTE: Locate each mouse in the pool at a novel start position to observe the spatial exploration ability of the mouse.
	<ol>
		<li>Remove the platform.</li>
		<li>Locate each mouse facing the tank wall in the pool once for 60 s. Ensure that the starting location is the NW quadrant, which is the quadrant furthest away from the SE quadrant.</li>
		<li>Record the swimming distance, swimming speed, and the platform crossover number in the maze.</li>
		<li>Dry each mouse with towels and provide warmth after the trial.</li>
	</ol>
	</li>
	<li>Perform the reversal trial. 
	NOTE: Perform the reversal trial from days 8&#8722;11.
	<ol>
		<li>Position the platform in the middle of the NW quadrant (instead of the SE quadrant).</li>
		<li>Follow steps 5.3.2 &#8722;5.3.5 as detailed in the hidden platform trial section.</li>
	</ol>
	</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Use statistics software (e.g., SPSS 20.0) to perform the statistical analysis.</li>
</ol>

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	<li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+water+mazes+for+assessing+spatial+and+egocentric+learning+and+memory+in+rodent+basic+research+and+regulatory+studies.">Value of water mazes for assessing spatial and egocentric learning and memory in rodent basic research and regulatory studies.</a> Neurotoxicology Teratology. 45, 75-90 (2014).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: procedures for assessing spatial and related forms of learning and memory.</a> Nature Protocol. 1 (2), 848-858 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+dementia+research:+New+frontiers;+World+Alzheimer+Report+2018.">The state of the art of dementia research: New frontiers; World Alzheimer Report 2018.</a> Alzheimer's Disease International. 9, 1-46 (2018).</li><li>Vorhees, C. V., 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=Effects+of+neonatal+(+)-methamphetamine+on+path+integration+and+spatial+learning+in+rats:+effects+of+dose+and+rearing+conditions.">Effects of neonatal (+)-methamphetamine on path integration and spatial learning in rats: effects of dose and rearing conditions.</a> International Journal of Developmental Neuroscience. 26 (6), 599-610 (2008).</li><li>Ding, N., Jiang, J., Xu, A., Tang, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+acupuncture+regulates+behavior+and+cerebral+blood+flow+in+the+SAMP8+mouse+model+of+Alzheimer's+disease.">Manual acupuncture regulates behavior and cerebral blood flow in the SAMP8 mouse model of Alzheimer's disease.</a> Frontiers in Neuroscience. 13, 37 (2019).</li><li>Ding, N., 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=Manual+acupuncture+suppresses+the+expression+of+proinflammatory+proteins+associated+with+the+NLRP3+inflammasome+in+the+hippocampus+of+SAMP8+mice.">Manual acupuncture suppresses the expression of proinflammatory proteins associated with the NLRP3 inflammasome in the hippocampus of SAMP8 mice.</a> Evidence-Based Complementary and Alternative Medicine. 2017, 1-8 (2017).</li><li>Cao, J., 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+changes+and+hippocampus+glucose+metabolism+in+APP/PS1+transgenic+mice+via+electro-acupuncture+at+governor+vessel+acupoints.">Behavioral changes and hippocampus glucose metabolism in APP/PS1 transgenic mice via electro-acupuncture at governor vessel acupoints.</a> Frontiers in Aging Neuroscience. 9, 5 (2017).</li><li>Amy, 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=Effects+of+sub-chronic+donepezil+on+brain+Abeta+and+cognition+in+a+mouse+model+of+Alzheimer's+disease.">Effects of sub-chronic donepezil on brain Abeta and cognition in a mouse model of Alzheimer's disease.</a> Psychopharmacology. 230, 279-289 (2013).</li><li>Garthe, A., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+old+test+for+new+neurons:+refining+the+Morris+water+maze+to+study+the+functional+relevance+of+adult+hippocampal+neurogenesis.">An old test for new neurons: refining the Morris water maze to study the functional relevance of adult hippocampal neurogenesis.</a> Frontiers in Neuroscience. 7, 63 (2013).</li><li>Schoenfeld, R., Schiffelholz, T., Beyer, C., Leplow, B., Foreman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variants+of+the+Morris+water+maze+task+to+comparatively+assess+human+and+rodent+place+navigation.">Variants of the Morris water maze task to comparatively assess human and rodent place navigation.</a> Neurobiology of Learning and Memory. 139, 117-127 (2017).</li><li>Otnass, M. K., Brun, V. H., Moser, M., Moser, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretraining+prevents+spatial+learning+impairment+after+saturation+of+hippocampal+long-term+potentiation.">Pretraining prevents spatial learning impairment after saturation of hippocampal long-term potentiation.</a> Journal of Neuroscience. 19 (24), 49 (1999).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+spatial+learning+and+memory+in+rodents.">Assessing spatial learning and memory in rodents.</a> Ilar Journal. 55 (2), 310-332 (2014).</li><li>Vorhees, C. V., Skelton, M. R., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+effects+of+neonatal+methamphetamine+exposure+on+spatial+learning.">Age-dependent effects of neonatal methamphetamine exposure on spatial learning.</a> Behavioural Pharmacology. 18 (5-6), 549-562 (2007).</li><li>Iivonen, H., Nurminen, L., Harri, M., Tanila, H., Puoliv&#228;li, 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> Behaviour Brain Research. 141 (2), 207-213 (2003).</li><li>Lin, S. Y., 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=Ozone+inhibits+APP/A&#946;+production+and+improves+cognition+in+an+APP/PS1+transgenic+mouse+model.">Ozone inhibits APP/A&#946; production and improves cognition in an APP/PS1 transgenic mouse model.</a> Neuroscience. , (2019).</li><li>Zuo, Y., 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=Preoperative+vitamin-rich+carbohydrate+loading+alleviates+postoperative+cognitive+dysfunction+in+aged+rats.">Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats.</a> Behavioural Brain Research. 373, 112107 (2019).</li></ol>

The authors declare no potential conflict of interest.

Although many water mazes, including the Biel water maze and the Cincinnati water maze, have been around for at least a century, only the MWM has been widely used to effectively and objectively evaluate spatial learning and memory ability because it has many advantages9. Despite extensive use of the MWM, the procedure has not always been used optimally. MWM experiments generally take a long time and are influenced by many variable factors. There are some effective and reliable aspects that help detect changes in spatial learning and memory ability that should be taken into consideration.
Four different MWM trials were performed. The visible platform trial was used on day 1 of MWM. If the animals could swim directly to the platform, it indicated that the swimming ability and vision of the animals were normal10. Otnass suggested that the visible platform trial should be conducted first11. The results of the visible platform trial in this study meant that the four groups started at the same learning level. From there, the successive experiments could be started. The hidden platform trial was used to assess the ability of the mice to acquire learning and memory ability. The probe trial was conducted on day 7, 24 h after the end of the hidden platform trial, to assess working memory. Finally, the reversal trial was used to assess working memory2. The changes in the four different trials of MWM together indicated that the AD model mice had low learning and memory ability and that MA had a positive effect on AD5.
There are no specific standards for the dimensions of the pool and platform1. A 214-cm diameter pool is used in most MWM studies. Vorhees and Williams demonstrated that with identical protocols, rats learn faster in a 122-cm pool than in a 210-cm pool; the steep slope of the learning curve indicates that the 122-cm diameter pool is extremely easy for rats to navigate12. In the current protocol, considering the old age and weak stature of the AD mice, a 90-cm diameter pool and 9.5-cm diameter platform were used. The results of preliminary experiments indicated that mice had more difficulty finding the platform in a bigger diameter pool. Therefore, tests in larger pools do not represent the real difference among the groups. The experimental animals had a harder time finding the platform in a bigger pool with a smaller platform4. Therefore, the size of the pool and the platform must be optimized in preliminary experiments according to the experimental requirements and condition of the experimental animals.
Water at temperature ranging from 20&#8211;24 &#176;C is recommended for performing the MWM test4. Aged experimental animals performed poorly in cold water13, indicating a clear age-dependent loss of thermoregulation14. In this study, a thermostat was placed at the bottom of the pool to maintain the temperature of the water at 20&#8211;24 &#176;C. The study results showed no significant difference in the swimming speeds among the four groups5.
MWM is a powerful technique to assess cognitive function and is widely used in studies presently. However, there is no defined, standard, consistent equipment to perform the MWM test, including the sizes of the pool and platform15,16. Different laboratories have different specifications for MWM. Therefore, researchers choose the appropriate experimental device according to their individual experimental requirements, which may cause confusion among researchers. Preliminary experiments are also necessary. More studies need to be conducted on basic experiments like MWM. Presently, the flexibility of MWM as an experimental tool only lies in the ability to choose the basic protocols according to the study purposed. Therefore, this test can be applied to assess cognitive function in greater depth.

Currently, the MWM experiment has become the most widely used and the standard behavioral experiment to evaluate the spatial learning and memory of animals1. It was initially designed by the British psychologist Richard G. Morris and has constantly been improved. Many advantages such as minimal training, cross-species utility, insensitivity to differences in body weight, and repeated testing ability of MWM make it the best method for assessing cognitive function2. Alzheimer&#8217;s disease (AD) is a major medical problem, primarily characterized by a decline in memory processing and cognitive function3. MWM is an indispensable experimental means to evaluate the learning and memory ability of AD model animals and the effectiveness of intervention methods. MWM experiments are generally time consuming (6&#8211;11 days) and involve many variable factors4. Although there are many articles about water maze experiments, in practice, researchers lack a coherent protocol. Therefore, an intuitive and rigorous protocol process video is particularly important. Using a previous experiment as an example5, all the steps of the MWM are described. Using MWM, previous studies suggested that acupuncture could relieve the symptoms of AD model mice5,6,7.
Herein, the MWM protocol used in a recent study5 is described to provide a simple and visible method for researchers to assess the spatial learning and memory of AD model animals.

<table>
	<tbody>
		<tr>
			<td>acupuncture needles</td>
			<td>Beijing Zhongyan Taihe Medical Instrument Limited Company</td>
			<td>511526</td>
			<td></td>
		</tr>
		<tr>
			<td>desktop computer</td>
			<td>Chengdu Techman Software Limited Liability Company</td>
			<td>Lenovo T4700D</td>
			<td></td>
		</tr>
		<tr>
			<td>Donepezil Hydrochloride Tablet</td>
			<td>Eisai China</td>
			<td>H20050978</td>
			<td>Aricept</td>
		</tr>
		<tr>
			<td>mice</td>
			<td>Zhi Shan (Beijing) Academy of Medical Science</td>
			<td>SCXK2014-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirros water maze device</td>
			<td>Chengdu Techman Software Limited Liability Company</td>
			<td>WMT-100S</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse bags</td>
			<td>home-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Signal acquisition and processing system</td>
			<td>Chengdu Techman Software Limited Liability Company</td>
			<td>BL-420N</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The time axis diagram of this protocol is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60055/60055fig01.jpg" /> 
Figure 1: Time axis diagram of the study protocol. <a href="https://www.jove.com/files/ftp_upload/60055/60055fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The time axis shows that this experiment lasted for a total of 21 days. The treatment was applied to the mouse during the whole experiment and the MWM tests began after 15 days of treatment. The visible platform, hidden platform, probe, and reversal trials were conducted in order.
Previously published results from Ding et al.5 are presented as typical results of MWM Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60055/60055fig02.jpg" /> 
Figure 2: Typical results of the Morris water maze test (n = 10).&#160;(A) Changes in the escape latency and swimming speed of rats among the different groups in the visible platform trial. (B) Changes in the escape latency of rats among the different groups in the hidden platform and reversal trials. The p-values are *p &#60; 0.05 and **p &#60; 0.01 compared with the control group. The symbol ## indicates p &#60; 0.01 compared with the AD group. (C) Changes in the platform crossover number and the percentage of the time spent by the rats in the northwest quadrant among the different experimental groups in the probe trial. The results of the visible platform, hidden platform, and reversal trial in each group are shown (n = 10, mean &#177; SD). This figure has been modified from Ding et al5. <a href="https://www.jove.com/files/ftp_upload/60055/60055fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2A shows the results of the visible platform trial. No statistical differences were observed in the escape latency or swimming speed among the groups on the first day of MWM. Figure 2B shows the results of the hidden platform and reversal trial from days 2&#8722;6 and days 8&#8722;11. The escape latency of the AD group remained at a high level on each day of the test. The escape latency of the other three groups decreased gradually. The escape latency from days 3&#8722;6 and days 8&#8722;11 was longer in the AD group than in the control group (p &#60; 0.01). The escape latencies of mice in the MA and drug groups were shorter than that of mice in the AD group on days 2&#8722;6 and days 8&#8722;11, respectively (p &#60; 0.01). Figure 2C shows the results of the probe trial. The platform crossover number of mice in the AD group was statistically lower than that in the control group (p &#60; 0.01). The platform crossover number in the MA group was higher than that in the AD group (p &#60; 0.05). The proportion of time spent in the SW quadrant by mice in the AD group was significantly lower than that in the control group (p &#60; 0.01). The proportion of time spent in the SW quadrant in the MA group was higher than that in the AD group (p &#60; 0.01).

Watch this Scientific Journal Video about Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze at JoVE.com

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

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 ...

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18.8K Views.  Beijing University of Chinese Medicine. Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer’s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

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

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 ...

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 ...

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: 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, ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

An Experimental Analysis of Children's Ability to Provide a False Report about a Crime

A considerable amount of research has evaluated children's lie-telling behaviors and skills1-2; however, limitations with the tasks used for eliciting false testimonies and interviewing children have restricted the generalizability of the findings. The primary aim of the current study is to provide an easy-to-administer and ecologically valid method for measuring the veracity and quality of school-aged children's (ages 6-11) testimonies when they are asked to provide different types of true and false reports. Moreover, the methodology enables researchers to examine the social and developmental factors that could influence the credibility of a child's testimony. In the current study, children will witness a theft, and are then asked to either falsely deny the transgression, falsely accuse a researcher of the theft, or tell the truth. Afterwards, children are to be interviewed by a second researcher using a thorough and ecologically valid interview protocol that requires children to provide closed-ended and free-recall responses about the events with the instigator (E1). Coders then evaluate the length and number of theft-related details the children give throughout the interview, as well as their ability to maintain their true and false reports. The representative results indicate that the truth and lie-telling conditions elicit the intended behaviors from the children. The open-ended interview questions encouraged children to provide free-recall information about their experiences with E1. Moreover, findings from the closed-ended questions suggest that children are significantly better at maintaining their lies with age, and when producing a false denial compared to a false accusation. Results from the current study can be used to develop a greater understanding of the characteristics of children's true and false testimonies about crime, which can potentially benefit law enforcement, legal staff and professionals who interview children.

The current methodology is designed to provide an ecologically relevant approach for measuring the veracity, length and quality of children's true and false testimonies. Implications of the current methodology for future research and professionals who interview children will also be discussed.

A considerable amount of research has evaluated children's lie-telling behaviors and skills1-2; however, limitations with the tasks used for eliciting ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

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 ...

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 ...