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 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’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’s disease transgenic model mice.
1. Preparation
2. Day 1: Visible Platform
3. Days 2-5: Hidden Platform
4. Day 6: Probe Trial
5. Data analysis
6. Representative Results
We have used the Morris Water Maze test to examine hypoxia’s effect on AD pathogenesis (7) and valproic acid (VPA)’s pharmaceutical potential for AD treatment (6) in transgenic AD model mice. Figure 3 is the representative result we reported in our study on VPA’s effect on memory deficits in the APP23 AD mouse model (6). On day 1 (visible platform trials), there is no difference between the VPA treated and control groups in latency (Fig. 3A) and path length (Fig. 3A) indicating that both of the groups have similar motor and visual capabilities. From this we assume the mice are able to see the flagged-platform and the cues in surrounding environment, and can swim acceptably. For days 2-5 (Day 1 to 4 of hidden platform trials) the example shows a difference in the escape latency (Fig. 3C) and path length (Fig 3D) between the groups, suggesting that VPA treated mice performed significantly better than controls over time. The probe trail results on the last day (Day 6) show that the number of times the mice traveled into the third quadrant, where the hidden platform was previously placed, was significantly greater with VPA treatment compared to control (Fig. 3E). These data indicate that VPA treatment significantly improves the memory deficits seen in APP23 mice.
Figure 1. Equipment setup for the Morris Water Maze visible platform test day. The pool is shielded from the experimenter using room dividers. Spatial cues are located on the walls, and maybe placed on the interior of the pool, above the water surface, if desired. The pool is filled with clear water, with the platform located 1cm above the surface. A flag has been placed on the platform to enhance visibility.
Figure 2. Screen capture from the Any-Maze™ Video Tracking System demonstrating pool calibration. The pool is viewed from above by a black and white analog tracking camera with a RTV24 Digitizer. Several zones are defined within the software and the total pool is divided into 4 quadrants. A fifth, platform zone is entered which can vary across trials, with five possible locations: NW, NE, SW, SE, or Center. A calibration line (ticked line across center) is added to allow the software to convert pixel distances into physical distances.
Figure 3. Representative results for the Morris Water Maze. The 7-month APP23 transgenic mice carrying human Swedish mutant APP gene were tested after one month of daily VPA (n=30 mice) or vehicle solution (n=30 mice) injections. (A) During the first day of visible platform tests, the VPA treated and control APP23 mice exhibited a similar latency to escape onto the visible platform. P>0.05 by student’s t-test. (B) The VPA-treated and control APP23 mice had similar swimming distances before escaping onto the visible platform in the visible platform test. P>0.05 by student’s t-test. (C) In hidden platform tests, VPA treated APP23 mice showed a shorter latency to escape onto the hidden platform on the 3rd and 4th day, P<0.001 by ANOVA. (D) The VPA-treated APP23 mice had a shorter swimming length before escaping onto the hidden platform on the 3rd and 4th day, P< 0.01 by ANOVA. (E) In the probe trial on the 6th day, the VPA-treated APP23 mice traveled into the third quadrant, where the hidden platform was previously placed, significantly more times than controls. * P<0.005 by student’s t-test. (Adapted and reprinted from The Journal of Experimental Medicine 205, 2781-2789, 2008, Rockefeller University Press, Originally published in J. Exp. Med. doi:10.1084/jem.20081588.) (6).
Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | ||
Platform location | Starting Direction | Platform Location: SW Starting Location as follows: | No platform. | ||||
Trial 1 | SW | S | W | N | N | E | N |
Trial 2 | NW | N | S | W | E | S | |
Trial 3 | NE | S | N | E | W | W | |
Trial 4 | Center | E | E | W | S | E | |
Trial 5 | SE | W | S | S | N | N |
Table 1. Sample water maze protocol*
* Note how both the platform position and starting direction change on day 1, whereas on days 2-5 the platform position remains constant while the starting direction changes. On day 6, there is no platform and a single trial. The starting direction for day 6 is farthest from the previous platform location (SW) so that the mice must travel some distance before entering the previously learned platform quadrant.
Age, sex, species, and strain differences influence MWM performance (8). Studies indicate that aged mice have poor performance in the MWM, while male rodents perform better than females; additionally, floating is more pronounced in mice than rats (9, 10). Therefore, these elements should be equated across all tests. Evidence also suggests that stressed animals perform more poorly in the MWM (11), thus environmental factors which may cause stress, such as temperature, light, and noise, should be monitored and kept constant over the task.
The authors have nothing to disclose.
This work was supported by the Canadian Institutes of Health Research (CIHR), the Townsend Family, and Jack Brown and Family Alzheimer’s Research Foundation (to W. S.). W. S. is the holder of the Canada Research Chair in Alzheimer’s Disease. P.L. was supported by a NSERC Alexander Graham Bell Canada Graduate Scholarship Doctoral Research Award and a Michael Smith Foundation for Health Research Senior Graduate Studentship.
Name of the reagent | Company | Comments (optional) |
---|---|---|
AnyMaze Video Tracking System | Stoelting Company | |
Tempera Paint | Reeves & Poole Groups | White, powdered |