Name: morris water maze test: optimization for mouse strain and testing environment
Uploaded: 2015-06-22T13:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment at JoVE.com

This work was supported by the National Institute of General Medical Sciences (Reed/Engler-Chiurazzi - U54GM104942), the National Institute for Neurological Disorders and Stroke (Ashe &#8211; R01NS33249, R01NS63249 and R01NS79374), CoBRE (Engler-Chiurazzi - P20GM109098), the Alzheimer&#39;s Association (Reed - NIRG-12-242187), a WVU Faculty Research Senate Grant (Reed), a WVU PSCOR grant (Reed), and internal funds from the WVU College of Medicine Dean&#8217;s Office (Engler-Chiurazzi). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or Alzheimer&#39;s Association.

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The authors have nothing to disclose.

The MWM task is widely used to assess spatial learning and memory. However, the robustness of this task can be influenced by many factors and requires optimization for both background strain and testing environment. As shown in Figure 4, the same training protocol and applied visual cues used in two different testing rooms (equivalent size and layout) yielded significantly different probe performance. Since many features of the testing room might contribute to spatial cues 4, it was speculated that the two rooms were significantly different in some unknown way which made the test more difficult. In modifying the extra-maze cues, we opted to effectively increase the number of visual cues. In addition, by minimizing the use of curtains, any auditory or olfactory cues that might exist may have been altered. The light level of the two testing rooms were equivalent, however the home cage light level in the new vivarium was lower, resulting in a higher light level differential when going from home cage to testing room. It has been suggested that BALB/c mice are able to perform the MWM task providing the light levels are sufficiently low 14. However, attempts to improve performance by lowering testing room light levels were unsuccessful (unpublished observations). To date, it is not known what factor(s) contributed to the decline in performance in the new testing environment, but the modified pre-handling, cues, and training protocol have resulted in a significant increase in probe scores.
If possible, it is advised to test a group of wild-type or control mice of the same background strain and age as the planned experiment to assess rate of learning and optimal probe placement for the particular testing environment. Ideally, the first probe is placed when approximately half of the mice are displaying a probable positive search bias (%-time in target&#8805;35) and the last probe when most control mice show a positive search bias and have reached performance plateau. This strategy has been used to determine probe placement for superior performers (F1 FVB/N x 129S6) and a background which learned more slowly (mixed C57BL/6 x FVB/N x 129S6), where approximately half of the mice had target quadrant occupancies of &#8805;35 after 8 trials or 18 trials respectively with a group mean of approximately 35%-time for the first probe. Subsequent probes should be higher than the first if set in this fashion, ideally with at least a 15-point difference compared to baseline of 25%-time. If comparing probe scores which are moderately low (~35%-time), ensure that this represents a significant bias for the target and hence a valid difference, by comparing the target to non-target quadrants 4,5. In addition, the minimum group mean should not be substantially lower than 25%-time in the target quadrant, which would be considered baseline or chance performance. Comparing target to non-target occupancy may help to identify if there are room biases, e.g., mice spending more time in the opposite quadrant than either of the adjacent quadrants would be an unexpected search pattern.
Some recommendations while conducting testing include limiting the number of people testing each cohort of animals to 1 individual to reduce variability in handling and testing styles between experimenters, and carrying out testing at around the same time each day. Also, it has been suggested that mice receiving 2 trials per day learn nearly as quickly as those receiving 4 trials per day 15. It is essential that animals from different strains/backgrounds/treatment groups be represented in each testing group if more than one cohort needs to be run during a day. Finally, care must be taken to ensure the mice do not become hypothermic, as hypothermia can affect performance and is sex and background strain dependent 16. Though the inter-trial interval used here (20 min) should be sufficient to prevent hypothermia, other methods include adjusting the water temperature, placing the mice in a heated holding cage between trials, or some combination of the two as needed. However, it should be noted that water temperature can influence performance in both directions. For example, proestrous rats perform better under warm conditions (33 &#176;C), whereas estrous rats perform better under cold conditions (19 &#176;C) 17. Thus, care must be taken to control for water temperatures across experiments. Should hypothermia be a concern for a particular animal, the experimenter could hand dry that animal with a paper towel in the event that they fail to groom post-swim or give any indication of hypothermic behavior. Periodic assessment of body temperature can ensure the conditions are sufficient to prevent hypothermia.
Another procedural consideration pertains to visible platform training. Younger mice or another strain may need fewer visible platform training trials. A suggested guideline is to train until the control group has reached floor of performance during the final 3-6 consecutive training trials, providing the experimental group displays equivalent performance. If the experimental design includes both young and old mice, set the visible platform training duration for the group, which requires the highest number of training trials to reach floor of performance 5. In addition, the visible platform can be effective towards acclimatizing mice to the handling and test procedure and may obviate the need for pre-handling if the mice are not very young when first tested or human handling is not excessively aversive to the strain being tested.
Broad benefits of the Morris water maze include its relative insensitivity to motivational factors as compared to food-based tasks, its validity as a measure of hippocampal-dependent spatial navigation and reference memory, and its cross-species efficacy 15. A potential limitation of the technique is that, because this protocol is tailored for a specific background strain, it may not be effective with other animals or other background strains of mice. Additionally, as part of the protocol, attempts are made to create and strategically place identifiable cues throughout the mazing room. However, it is unclear what exact height is optimal when placing cues around the room. Thus, cues that are large and are distinguishable from one another are necessary for effective training.
In summary, optimizing the MWM task for use with a particular background strain and testing environment can significantly increase the dynamic range of the task, resulting in considerable time and cost savings.

Transgenic mouse models have been instrumental in evaluating the pathophysiology of Alzheimer&#8217;s disease (AD), as well as the potential of therapeutic interventions. Cognitive tasks, such as the Morris water maze (MWM), are commonly used with these models to identify the molecular correlates of memory deficits and to assess the efficacy of pre-clinical drugs. It is crucial, however, that the dynamic range of the cognitive task be wide enough to detect subtle treatment effects. With mouse models of AD, cognitive deficits are typically age-dependent, and mice display progressive declines in performance (e.g., 1). Use of a sensitive cognitive task can allow detection of subtle differences earlier in the animal&#8217;s life, thereby reducing the costs associated with aging animals. For example, reducing the number of training trials in the hippocampal-dependent Barnes maze from 15 to 5 increased the difficulty of the task, resulting in the detection of deficits in the 3xTg model at an earlier age than previously reported 2. Earlier detection of deficits not only offers considerable time and cost savings, it also increases the likelihood that the molecular changes underlying cognitive deficits can be identified.
One factor influencing the sensitivity of cognitive tasks is the genetic background strain of the mouse model. For example, BALB/c mice exhibit superior performance in learning and memory tasks compared to other strains, such as the C57BL/6 3. The F1 FVB/N x 129S6 background is used for two of the most widely employed models of AD, the Tg2576 and rTg(TauP301L)4510 models. This strain exhibits superior learning ability in the MWM relative to other strains, including B6/SJL mice 4. Because of this superior learning ability, the use of a single probe after extensive training may mask group differences resulting from over-training. In addition, the sensitivity of probe trials may be age-dependent. We have previously shown that earlier probe trials, after limited hidden platform training, are more sensitive to differences in young Tg2576 compared to young transgene-negative littermate controls than are probe trials inserted after more extensive training 5. In contrast, probe trials following extensive training are more sensitive in older (20-25 month) Tg2576 mice compared to older littermates than are earlier probe trials 5. By interspersing probe trials throughout training, the likelihood that a sensitive trial will be identified is increased, particularly if longitudinal testing is performed and the sensitivity of a particular probe trial is age-dependent. Figure 1 shows the superior performance of F1 FVB/N x 129S6 mice under the protocol optimized for this strain as compared to mice of the B6/SJL background trained under a protocol with more extensive training.
The MWM is generally thought to provide reliable measures that are reproducible across both time and laboratories 6. For example, the primary protocol originally used by our Minnesota laboratory 1,7 was successfully implemented with minor modifications at West Virginia University 8. Similarly, equivalent levels of impairment were observed in rTg(TauP301L)4510 mice relative to control littermates if housed under pathogen-free or conventional conditions 9. However, the testing environment can influence the sensitivity of the MWM task. Factors such as room lighting, air vents, temperature gradients, and noises all contribute to environmental cues 4 that can ultimately influence performance. When our Minnesota laboratory and vivarium were moved to a new building, up to a 38% reduction in wild-type performance was observed, substantially reducing the dynamic range of the task and the ability to detect transgene-related deficits. This change in performance occurred despite designing the testing room to be of equivalent size and configuration, and using the same applied visual cues. A &#8220;re-optimizing&#8221; of the original protocol was required to increase the dynamic range of the MWM task in the new testing environment.
Here the original protocol tailored for use with the F1 FVB/N x 129S6 background 5 is described. Because some studies suggest stress is associated with poor MWM performance 10 and pre-handling can alleviate this stress-induced deficit in performance 11, a pre-handling protocol was designed to acclimate the mice to the introduction and removal of the pool prior to MWM testing. Following pre-handling, mice undergo visible platform training, in which a raised platform is marked with a flag. Visible platform training is used to identify mice with performance problems related to sensorimotor abnormalities. Using exclusion criteria described in the protocol section, performance-incompetent mice are removed from subsequent examinations of hidden platform training and probe trials. Impairments in hidden platform training and probe trials are interpreted as cognitive deficits because sensorimotor performance is factored out of the data. After completion of visible platform training, mice begin hidden platform training where the platform is submerged in water and remains in the same position relative to external cues. Trials in which the platform is removed (probe trials) are interspersed throughout hidden platform training to assess the influence of additional training. Because probe trials occur at the beginning of each day, before additional hidden platform training, probe trials measure the ability of the animal to remember the location of the platform following a 20 hr delay, considered a measure of reference memory 12. Finally, ways in which this original protocol was re-optimized when changes in the testing environment disrupted control performance are described.

<table><tbody><tr><td>Viewer Tracking software</td><td>Biobserve</td><td></td><td>This particular software is not a requirement - there are other tracking systems available</td></tr><tr><td>Pre-handling pool</td><td></td><td></td><td>Dimensions approximately 1 foot wide x 2 feet long x 1.5 feet deep</td></tr><tr><td>Plastic beaker</td><td></td><td></td><td>1 L</td></tr><tr><td>Scoop</td><td></td><td></td><td></td></tr><tr><td>Small net</td><td></td><td></td><td></td></tr><tr><td>Stopwatch</td><td></td><td></td><td></td></tr><tr><td>Non-toxic white tempera paint</td><td></td><td></td><td>Any color paint can be used; however, most tracking software programs require that the paint contrast with the color of the animal.</td></tr><tr><td>Visible platform</td><td></td><td></td><td>Color should contrast that of maze</td></tr><tr><td>Curtain rod</td><td></td><td></td><td></td></tr><tr><td>Curtains</td><td></td><td></td><td></td></tr><tr><td>Circular tub</td><td></td><td></td><td>Usually white in color; approximately 4 feet in diameter</td></tr><tr><td>Hidden platform</td><td></td><td></td><td>Painted same color as the water</td></tr></tbody></table>

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.

We have used the Morris water maze to study the effects of beta-amyloid (Tg2576 mice) and mutant P301L tau (rTg(TauP301L)4510 mice) on spatial reference memory (e.g., 1,5,7,8). Figure 3 is the representative result reported in our study examining the effect of adult-onset P301L tau expression on learning and memory 8, utilizing testing Environment A. To assess motor and visual capabilities, mice were compared across visible platform training blocks, where each training block consisted of 3 trials. Pathlength in visible platform training did not differ between controls and TauP301L mice (Figure 3A), suggesting transgene positive and negative mice exhibit comparable swimming and both groups can see the visual cue (flag) marking the platform. No mice were identified as performance-incompetent based on the exclusion criteria. Next, performance in hidden platform training block was compared, where each block consists of 1 day (4 trials) of training. As the mice learned the location of the platform, the pathlength and time to find the platform decreased. However, pathlength was significantly longer in TauP301L mice compared to controls at each training block (Figure 3B), suggesting spatial learning was impaired in TauP301L mice. Four probe trials, in which the platform was removed, were interspersed throughout hidden platform training and took place at the beginning of the day, prior to the start of hidden platform training. Thus, these probe trials measured spatial reference memory. Comparing across these four probe trials, Controls significantly improved with additional training (Figure 3C), as indicated by increased time in the target quadrant. In contrast, TauP301L mice did not improve with additional training. Thus, the greatest differences between the two groups at this age occurred at probe trial 4. These data indicate that P301L tau expression is associated with both spatial learning and spatial reference memory deficits. While the water maze task may be relatively stable to some procedural differences, the F1 FVB/N x 129S6 background strain may be particularly sensitive to certain environmental changes. The first protocol outlined was also successfully utilized in Environment B (e.g., 1,7). However, wild-type probe performance was significantly lower when the first protocol was used in a third location, Environment C. The re-optimized protocol significantly improved wild-type probe performance (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/52706/52706fig1.jpg" /> 
Figure 1: Optimization of Protocol for Background Strain. Wild-type 7-8 month old mice were trained using the same cues and testing environment. F1 FVB/N x 129S6 (N = 24) and B6/SJL (N = 16) mice first received 18 and 24 visible platform training trials respectively, delivered at 6 and 8 trials per day respectively. Both strains received 4 hidden platform training trials per day. For F1 FVB/N x 129S6 mice, probe trials were performed 20 hr following 8, 12, 16, and 24 training trials. For B6/SJL mice, probe trials were performed 20 hr following 12, 24, and 36 training trials.
<img alt="Figure 2" src="/files/ftp_upload/52706/52706fig2.jpg" /> 
Figure 2: Timeline. Mice received visible platform training for 3 days, 6 trials per day, followed by hidden platform training for 6 days, 4 trials per day. Four probe trials were performed 20 hr after 8, 12, 16, and 24 hidden training trials.
<img alt="Figure 3" src="/files/ftp_upload/52706/52706fig3.jpg" /> 
Figure 3: Representative Results for the Morris Water Maze. TauP301L mice carrying the human P301L tau gene were examined at approximately 6.5 months of age after three months of P301L tau expression (n&#160;=&#160;41 tauP301L and n&#160;=&#160;46 controls with an approximately equal number of males and females in each group). (A) Pathlength in visible platform training did not differ between controls and TauP301L mice (ps &#62; 0.05). (B) During hidden platform training, TauP301L mice demonstrated significantly longer pathlengths across all training blocks (Transgene: F(1, 83)&#160;=&#160;41.96, p &#60;0.0001; Transgene&#160;&#215;&#160;Block: F(5, 415)&#160;=&#160;0.6141, p&#160;=&#160;0.69). (C) Controls improved across the four probe trials, whereas TauP301L mice did not (Transgene: F(1, 83)&#160;=&#160;29.1, p &#60;0.0001; Transgene&#160;&#215;&#160;Trial: F(3, 270)&#160;=&#160;4.91, p&#160;=&#160;0.008). Each training block consisted of 3 trials for visible platform training or 4 trials for hidden platform training. Tukey post-hoc analyses: *p &#60;0.05; **p &#60;0.01; ***p &#60;0.001. Portions of Figure 3 reprinted from Hunsberger et al., Effect size of memory deficits in mice with adult-onset P301L tau expression, Behav Brain Res, Vol. 272, pp. 181-95. Copyright 2014, with permission from Elsevier.
<img alt="Figure 4" src="/files/ftp_upload/52706/52706fig4.jpg" /> 
Figure 4: Probe Scores Are Significantly Affected by Testing Environment and Training Protocol. Three groups of tau negative F1 FVB/N x 129S6 mice trained using various combinations of training protocol and testing environment generated significantly different probe scores following 8, 12, and 16 training trials (Group: F(2, 42)&#160;=&#160;14.89, p &#60;0.0001; Group x Trial: F(4, 84)&#160;=&#160;1.10, p&#160;=&#160;0.36). Mice trained utilizing Protocol 1 and the same set of applied cues displayed significantly lower probe scores under Environment C compared to Environment B (Environment: F(1, 25)&#160;=&#160;28.58, p &#60;0.0001; Environment x Trial: F(2, 50)&#160;=&#160;1.93, p&#160;=&#160;0.16). Mice trained under Environment C displayed significantly higher probe scores when modified cues and the re-optimized Protocol 2 were utilized compared to mice trained utilizing the original cues and Protocol 1 (Protocol: F(1, 30)&#160;=&#160;15.32, p &#60;0.001; Protocol x Trial: F(2, 60)&#160;=&#160;0.91, p&#160;=&#160;0.41). **p &#60;0.001; ***p &#60;0.0001.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="3">Day 1</td>
			<td colspan="5">Morris Water Maze - Cohort:</td>
			<td>Date: _________</td>
		</tr>
		<tr>
			<td colspan="3">Platform in SE quadrant</td>
			<td colspan="5">Tester:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Start time:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Finish time:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Water temp:</td>
			<td>1m max trial time/15s on platform/*placed on platform</td>
		</tr>
		<tr>
			<td colspan="9">Group 1</td>
		</tr>
		<tr>
			<td colspan="9">Trial 1</td>
		</tr>
		<tr>
			<td>Animal ID</td>
			<td>Marking</td>
			<td>Cage #</td>
			<td>End 
			time</td>
			<td>Release 
			point</td>
			<td>Platform 
			location</td>
			<td>Find platform?</td>
			<td>Time</td>
			<td>Notes (i.e. odd behavior)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>W</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="9">20 minute ITI</td>
		</tr>
		<tr>
			<td colspan="9">Trial 2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>E</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="9">20 minute ITI</td>
		</tr>
		<tr>
			<td colspan="9">Trial 3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>N</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="9">20 minute ITI</td>
		</tr>
		<tr>
			<td colspan="9">Trial 4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>S</td>
			<td>SE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Hidden Platform Run Sheet. An example run sheet in which the platform is located in the southeast (SE) quadrant is provided. The experimenter should write the mouse&#8217;s ID number, any identifying marks on the tail or ears, and the cage grouping. The release point is indicated on the sheet and should be pseudo-randomly determined as described in Step 3.3.2. Record whether the mouse finds the platform and the time to find the platform. Odd behavior, including floating or thigmotaxis, should noted.

Watch this Scientific Journal Video about Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment at JoVE.com

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

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-optimization-for-mouse-strain-testing

Nanyang Technological University

Research

JoVE Journal

Behavior

21.5K Views.  West Virginia University.  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. 

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

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 ...

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

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

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

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

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

Medicine

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 ...

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

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

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

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

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

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

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

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

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

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

Cognitive Improvement in Chronic Hypoxia Mice with Acupuncture

Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice rooted in traditional Chinese medicine, entails the insertion of fine needles into precise points on the body and is commonly employed for the management of diverse conditions. Recently, acupuncture has emerged as a promising therapeutic intervention for a range of neurological diseases, including anxiety and respiratory disorders. However, the potential of acupuncture in treating cognitive dysfunction induced by chronic hypoxia has not yet been explored. This paper presents a comprehensive protocol for establishing a mouse model of chronic hypoxia-induced cognitive impairment, administering mild anesthesia, performing acupuncture treatment, and assessing behavioral changes and memory abilities using open field tests and water mazes. The step-by-step protocol provides detailed instructions on accurately locating and positioning acupoints and needles for cognitive improvement. By employing this protocol, researchers can conduct systematic studies to thoroughly evaluate the therapeutic potential of acupuncture for cognitive dysfunction.

Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice ...

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 ...

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

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