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>

<ol>
	<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. 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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="Morris Water Maze timeline, diagram of trials, MA treatment duration, behavioral study." class="xfigimg" src="/files/ftp_upload/60055/60055fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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="Bar and line graphs analyzing mouse swimming performance; experimental trials; control and treatment group data." class="xfigimg" src="/files/ftp_upload/60055/60055fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Typical results of the Morris water maze test (n = 10). (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 &lt; 0.05 and **p &lt; 0.01 compared with the control group. The symbol ## indicates p &lt; 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 ± 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 2−6 and days 8−11. 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 3−6 and days 8−11 was longer in the AD group than in the control group (p &lt; 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 2−6 and days 8−11, respectively (p &lt; 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 &lt; 0.01). The platform crossover number in the MA group was higher than that in the AD group (p &lt; 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 &lt; 0.01). The proportion of time spent in the SW quadrant in the MA group was higher than that in the AD group (p &lt; 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 ...

analysis-learning-memory-ability-an-alzheimer-s-disease-mouse-model

Research

JoVE Journal

Behavior

18.9K 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

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Neuroscience

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, ...

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 ...

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Biology

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 ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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 ...

Medicine

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

The Morris Water Maze

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

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