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.

All experimental procedures were conducted in accordance with the standards of the Institutional Animal Care and Use Committee (IACUC) and approved by West Virginia University&#8217;s IACUC.
1. Pre-handling
<ol>
	<li>Set Up the Pool
	<ol>
		<li>Prop the pre-handling pool so it is&#160;raised to a comfortable height.</li>
		<li>Place 2 L&#160;of water (21 &#176;C) in the pre-handling pool to a level of approximately 1 cm. 
		Note: Do not add coloring.</li>
	</ol>
	</li>
	<li>Procedure
	<...

<ol>
	<li>Ramsden, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+neurofibrillary+tangle+formation,+neuron+loss,+and+memory+impairment+in+a+mouse+model+of+human+tauopathy+(P301L).">Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L).</a> The Journal of Neuroscience. 25, 10637-10647 (2005).</li><li>Attar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shortened+barnes+maze+protocol+reveals+memory+deficits+at+4-months+of+age+in+the+triple-transgenic+mouse+model+of+Alzheimer's+disease.">A shortened barnes maze protocol reveals memory deficits at 4-months of age in the triple-transgenic mouse model of Alzheimer's disease.</a> PLoS One. 8, e80355 (2013).</li><li>Johnson, J. M., Bailey, J. M., Johnson, J. E., Newland, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+BALB/c+and+C57BL/6+mice+under+an+incremental+repeated+acquisition+of+behavioral+chains+procedure.">Performance of BALB/c and C57BL/6 mice under an incremental repeated acquisition of behavioral chains procedure.</a> Behavioural Processes. 84, 705-714 (2010).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's wrong with my Mouse Behavioral phenotyping of transgenic and knockout mice. , 94-95 (2000).</li><li>Westerman, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+abeta+and+memory+in+the+Tg2576+mouse+model+of+Alzheimer's+disease.">The relationship between abeta and memory in the Tg2576 mouse model of Alzheimer's disease.</a> The Journal of Neuroscience. 22, 1858-1867 (2002).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the morris water maze in the study of learning and memory.</a> Brain Research Reviews. 36, 60-90 (2001).</li><li>Santa Cruz, K., 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=Tau+suppression+in+a+neurodegenerative+mouse+model+improves+memory+function.">Tau suppression in a neurodegenerative mouse model improves memory function.</a> Science. 309, 476-481 (2005).</li><li>Hunsberger, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+size+of+memory+deficits+in+mice+with+adult-onset+P301L+tau+expression.">Effect size of memory deficits in mice with adult-onset P301L tau expression.</a> Behavioural Brain Research. 272, 181-195 (2014).</li><li>Yue, M., Hanna, A., Wilson, J., Roder, H., Janus, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+difference+in+pathology+and+memory+decline+in+rTg4510+mouse+model+of+tauopathy.">Sex difference in pathology and memory decline in rTg4510 mouse model of tauopathy.</a> Neurobiology of Aging. 32, 590-603 (2011).</li><li>Sandi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+mechanisms+of+action+of+glucocorticoid+involvement+in+memory+storage.">The role and mechanisms of action of glucocorticoid involvement in memory storage.</a> Neural plasticity. 6, 41-52 (1998).</li><li>H&#246;lscher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+impairs+performance+in+spatial+water+maze+learning+tasks.">C. impairs performance in spatial water maze learning tasks.</a> Behavioural Brain Research. 100, 225-235 (1999).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> Journal of Neuroscience Methods. 11, 47-60 (1984).</li><li>Reed, M. N., Liu, P., Kotilinek, L. A., Ashe, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+size+of+reference+memory+deficits+in+the+morris+water+maze+in+Tg2576+mice.">Effect size of reference memory deficits in the morris water maze in Tg2576 mice.</a> Behavioural Brain Research. 212, 115-120 (2010).</li><li>Huang, Y., Zhou, W., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+lighting+conditions+during+testing+increase+thigmotaxis+and+impair+water+maze+performance+in+BALB/c+mice.">Bright lighting conditions during testing increase thigmotaxis and impair water maze performance in BALB/c mice.</a> Behavioral Brain Research. 226, 26-31 (2012).</li><li>Ivonen, H., Nurminen, L., Harri, M., Tanila, H., Puolivali, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+in+mice+tested+in+Morris+water+maze.">Hypothermia in mice tested in Morris water maze.</a> Behavioural Brain Research. 141, 207-213 (2003).</li><li>Rubinow, M. J., Arseneau, L. M., Beverly, J. L., Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+estrous+cycle+on+water+maze+acquisition+depends+on+the+temperature+of+the+water.">Effect of estrous cycle on water maze acquisition depends on the temperature of the water.</a> Behavioral Neuroscience. 118 (4), 863-868 (2004).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0">
	<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 liter</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>White circular tub</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-toxic white tempera paint</td>
			<td></td>
			<td></td>
			<td>Any color can paint can be used; must completely cover the hidden platform</td>
		</tr>
		<tr>
			<td>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>Mouse performance tracking software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circular tub</td>
			<td></td>
			<td></td>
			<td>Uusally white in color; approximately 4 feet in diamater</td>
		</tr>
		<tr>
			<td>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 bloc...

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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

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21.2K 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. 