Sponsored by HVS Image - video tracking for Morris Water Maze
Part I General Information
Introduction to the Morris Water MazeMorris watermaze was devised by Prof Richard Morris (RGM Morris) 30 years ago. The video original tracking was carried out using high speed electronic tracking apparatus developed by Richard Baker at HVS Image. Water Maze continues to be popular; in fact we think tracking the number of publications use has increased year on year. It is about place-learning and memory. Rats are natural swimmers. They want to get out of the water, but swimming does not distress them. Two advantages of Morris water maze over others mazes are:
The rat wants to get out, so it searches - it does not wait about.
In water there are no local cues (e.g. scent trails).
The figure shows an example of the basic apparatus, the Morris water Maze Pool kindly provided by Chris Janus, Ph.D. Assistant Professor Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease (CTRND) at University of Florida College of Medicine.
The PoolThe Morris water maze consists of a round tank (pool) of water. We think that 6ft (1.8m) to 2m diameter is good. We think that a 2 ft (0.6m) deep pool is good, with 1 ft (0.3m) of water. For mice 5-6ft (1.5-1.8m) diameter is good, and the same 2ft deep. We think that is good to place the pool on a 1-2ft base, to make working more convenient. The escape platform can be 4ins (10cms) in diameter . The platform should not be too close to the side, or the rat finds it just by circling around the pool-side. For rats the platform can be 1/4 in below the water. For mice it should be very close to the surface.
We are told that a good water temperature for rats is 26 deg C ±2 deg. Colder encourages activity, but can cause hypothermia, which impairs learning.
The RodentsWe think that the best rats are hooded. These are the same as those used by Morris. Hooded rats are easy to track. The tracker detects the black hood. HVS is like other systems, it tracks contrast. For the same reason, dark-colored mice track well. Your pool and platform are white. You make the water white, e.g. with skim milk powder (this is to stop the rat from seeing thru the water). We think that hooded rats perform better than white (albino) rats, possibly because albino rats do not see as well. Another problem with albino rats: to get contrast for tracking, the pool has got to be black with clear water. Black pools cause tracking problems, because they reflect lights. But it can be done. (See set-up hints in this document). There is an (escape) platform. This is white (or clear Plexiglas) for awhite pool. During the training phase you can allow the platform to be ~ 1/2" above the water, and you can paint the side black. This trains the rats, that there is a platform, and that is how to get out of the water. This is important - they have got to know this before any place-learning can happen. When you put the rat into the water, support it in the hand and gently lower it into the water with the tail-end lower, so the head does not go under water. Dropping them in head-first stresses them, which impairs learning. Usually they are put in with the head pointing towards the pool-side, to minimize bias.
LearningAt first the rat will swim around the side. After a while it understands there is no way out by doing that. Then it searches more to the center. If it does not find the platform after approximately two minutes, lift it out & dry it. If it really does not get the point, you can gently guide it to the platform. It gets on the platform & usually rears and looks around. This is something else to learn, that there is an escape platform. You can leave it for ~ 15 seconds, to orientate. It is looking into the room.
There must be visual cues on the room walls. It needs at least 3 different cues, e.g. 3 different posters, (so it can 'triangulate'), always visible from inside the pool. These cues should be extra-maze i.e. well outside of the pool. If they are close to the poolside, then the rat could be doing association between the cue & the platform. After some training swims, the rat will go direct to the platform. After ~15 seconds you lift it off the platform and gently dry it (better - put it in a litter of tissues, so it can dry itself). Rats can do several swims per day. Now you can use a different start-point. An informed rat does not try to back-track to the old start point. It goes direct to the platform. Because it has a spatial map in its hippocampus? The training sequence and test sequence are something that you as the scientist have to decide on. Obviously you are going to use Controls and Experimental rats.
Rats can cheat!Instead of learning where the platform is, they can learn search strategies. E.g., swim around at some distance from the side, or guess where the platform is, and make a series of sweeps. A good analysis program helps you distinguish this from spatial learning. Probe trials: There is no platform. The trial ends after 1-2 mins with you rescuing the rat. Only do this on trained rats, and not too often. Done at the start, it tests for spatial bias. Done at the end of training, it tests the rat's spatial learning.
Temperament!Rats are solid citizens. They are quite willing to swim to that platform, get on it, and orientate. Mice are also natural swimmers, but they are altogether more frisky & temperamental - this is covered elsewhere in the HVS Image Mouse Notes. Black pool with clear water with thanks to Dr Hayde Bolouri, Neuroscientist, Institutionen för neurovetenskap och fysiologi, University of Gothenburg
More on Morris Water Maze
We can examine reference learning and memory in the Morris water maze. Initially the mice are trained to locate a cued, flagged platform. During this phase of the study both the cued platform position and the starting position of the mouse are randomly varied to avoid habituating the mouse to a particular area of the pool. Following this sensory motor test the cued platform is switched with a submerged platform the position of which is fixed for each mouse. As the mouse learns its position the path-lengths & latencies to locate the platform are reduced. Following the final placed session, the platform is removed and the swim-pattern of the mice is tracked for one minute. We then compare the time the mouse spends swimming in the former platform quadrant with the time in the other three. This gives a measure of memory and also allows us to see if the mice are using different strategies to find the platform. For example, instead of using the distal cues, some mice learn to swim at a fixed distance from the edge of the pool until they find the platform. An additional HVS measure one can take includes the number of platform position crossings. Again if the mouse shows good evidence of spatial memory the mouse will exhibit a significantly increased number of former platform position crossings in comparison with the other three. A further advantage of this model is that by reversing the submerged platform position, it allows us to examine flexibility of learning, i.e. how easy is it for the mouse to let go of one learned behavior and acquire the new task. Inflexibility of learning is an early feature of AD dementia.
Statistical Analysis.When looking for statistical differences in latencies, pathlengths and swim speeds one uses repeated-measures analysis of variance (ANOVA). Assuming a significant overall main effect of treatment, genotype etc. one can employ post hoc comparisons e.g. Dunnett's test. For probe data one-way ANOVA is sufficient.
Size of pool & platform?These are a matter of personal choice. One criterion, which sounds logical, is that the task should not be so easy that it can be achieved mostly by chance. I.e. the pool should not be too small, nor the platform too big. Also if the pool is too small, the platform might be close to the sidewall. In that case, just swimming around at a fixed distance from the sidewall will find the platform quickly. For adult rats a pool diameter if 1.8m (6ft) may be good, with a 10cms (4 ins) diameter platform. Depth ~ 60cms (2 ft). In the US we supply pools 4, 5 or 6ft diameter, 2 ft deep, with a 1ft water-depth. For mice a pool diameter of 1m to 1.5m is used, with platform diameter 5cms to 10 cms. Pool height ~30 cms, water depth ~ 25cms works. Mice are very aware of the room & operator, and a sidewall above water level could help block the line of sight. With all animals, it is vital that they can see the cues (on the lab walls), from the vicinity of the platform - otherwise they can't orientate. Nearly all labs use round platforms - this sounds good, as there is no preferred direction for approach. A few use square platforms - please note that HVS Water software is designed for round platforms.
Mouse / Water maze Hints
“a) Rats are natural swimmers and if handled well will cooperate easily in Water Maze. They don't have the option to float or jump - they are solid citizens who just swim for that platform and get onto it.
b) Mice are also good swimmers, and if handled well, will also take part easily. But they are more temperamental, and they do have the option to stop swimming and just float, and to jump. These notes suggest ways to reduce stress, so your mice will feel confident, and cooperate.
IntroductionThe swimming-pool (Morris Water Maze) is a delicate balance of motivation provided by stress/reward, and a difficult task. It is an unnatural for rodents, for whom sight is a minor sense. If the stress is too high, or the reward too slight, the task is not attempted.
Platform & Water1. Cover the platform with a fibrous mesh into which the mice can dig their paws and feel secure when they get there. We found a synthetic underlay (to stop carpets slipping) ; it is whitish and blends with the water (milk). Others have used pot-scouring pads.
2. Make the water just cover the platform by ~5mm. Rats are big enough to feel they are out of the water. Mice are smaller; 1-2 cms is too much.
3. Water-temperature has to be accurate to 1 deg; we use 24 deg C. A couple of degrees colder and they just climb out
Time On Platform & RescueA) Time on platform has to be sufficient for them to adjust to the feel & location, and to see where they are. However the prime reward is not to climb onto the platform, but to leap into your arms when you intervene to rescue them. So keep it to 10 seconds before you are seen or heard coming to the rescue.
B) And they do leap ! You need the pool to be sufficiently high-sided to stop them leaping out into the room. The high-side requirement is a major determinant of water level & location of cues.
C) Because rescue by you is the reward, you will never train them while you are detectable by sight or sound. The task has to clearly direct them first to the platform.
Cuesi) Must be visible, & useful to mice. They must be far enough away to require the mice to use spatial analysis, rather than association, to solve the task. But near enough & positioned appropriately, for the mice to see and use them. And spatially specific enough to solve a spatial task. Feel-good psychedelic & animal posters stuck on the far walls of a large room ,below the line of vision of mice swimming in a high sided pool, do not qualify!
ii) We use a 2m pool, sufficiently large that I only have to place the cues just outside the walls of the pool, and the platform ~0.5m in from the side. That makes the task difficult – impossible to solve by association, and the cues are maximally visible. Although it is still obvious that mice placed near the walls have to swim to the centre to get their full bearings. There are places near the wall of the pool, where they can see 1 or 2 cues, but need all 3 to triangulate the position of the platform.
iii) For a 1m pool you have to place the cues some distance from the pool, for the task to be spatial not association. I use board 5-15 cm wide, 1m long,
Cues can consist of black stripes (vertical, horizontal, triangles) of different widths.
They need to stand out to a mouse, be positioned where they are optimally visible & angled towards the centre of the pool so they appear perpendicular to the mouse. And instantly distinguishable from each other. You can see if the mice are using them; they need 3 specific cues, and diffuse cues in the room, such as sources of light, as general orientators.
Time & Intervals of Swimming1. We use 2 mins in a 2m pool with a 15 cm platform*. If your pool is as small as 1m, I would swim them for 1 min, & when they fail to find the platform, guide them to it so they climb on. Ideally you leave them there for 10 seconds, so they assess the surroundings. In practice they will make a leap for it if you let them. So as they climb on, step well-back & try to ensure they stay on for 10 seconds. If they immediately take to the water & keep swimming, intervene again to guide them back. They have to learn that rescue is dependent on staying on that platform.
2. If they leap for you before you retreat, you may as well catch them, as at least they have got the idea. You should find that on the first day your mice mostly fail to find the platform. Then on subsequent dates their efforts are greeted with increasing success. If you leave them too long at the beginning, swimming around without success, they will associate the swim with failure & panic.
3. We find that mice given 2 trials per day learn nearly as quickly as 4 times a day. If you are worried about the stamina of elderly mice*, cut back to 2 per day. Our mice look forward to their swim (they rattle the tops of their cages when my postdoc comes to collect them). We suspect they possibly enjoy it too much, and extend the swim-time because the alternative is to go back in a boring cage. Then we tried lowering the temperature of the pool by 2 deg, and they went on strike ! What I was leading-up to is that if the mice are not panicking, I suspect the swim is not traumatic, and they enjoy doing laps of the pool.
4. Intervals between swims is important. We use 30 mins, as being a good way to reinforce learning.
Handling & Acclimatizing5. We give them 2 days in the test room, playing with them in & out of their cages, so they are comfortable with us and the room, before starting swimming. They have also been handled by us intermittently since birth, so they are happy with people.
6. Smells are very important. Even subtle amounts of perfume can confuse ! "bad" smells are totally out, e.g. rats & especially cats. If you have a pet cat, it might just work if you shower & change into fresh clothes at the lab.
Part II HVS User Hints
Water maze etc - Safety hints
Suitability for use.Typical water maze video tracking equipment is for research use only, e.g. Morris water maze using rodents. It is not suitable for clinical, diagnostic or therapeutic use with human subjects. It does not comply with electrical isolation regulations for clinical equipment, and must not be attached, directly or indirectly, to human subjects. Electrical safety. There are no user-serviceable parts inside the legacy HVS Image trackers For safety reasons do not open-up the components. Do not open-up the legacy PLUS modules. Follow manufacturer’s advise regaring notebook PCs.
• In the U.S. we sometimes advise galvanized tanks for use as water maze pools. We are advising you to apply metal primer paint before painting them white or black inside, so the white & black paint do not peel off. Regarding this primer paint, a U.S. user has suggested it is toxic, though OK if painted-over. In our view, not as paint experts or toxicologists, primer paint has to be somewhat corrosive because it has to 'bite' into the metal surface to form a bond. But it should not contain any long-term toxins e.g. heavy metals. We have a statement from Hammerite paints that their Special Metals Primer gives-off fumes such as nitrogen oxides while drying, but it is Not Classified for regulatory purposes in the UK, but please don't dispose of it down drains. We recommend you get a statement from your supplier regarding the primer you use.
• Health and safety are vital issues in "water maze" as in all lab work. Users please consult your Health & Safety advisor before setting-up and using your water system. Institutions may wish to make a Risk Assessment. Disclaimer: HVS is not an advisor, but we do care about your health & safety, and here we point-out items for especial attention:
• When fixing and adjusting the overhead camera, absolutely do not climb on furniture. If your safety officer approves, use a properly secured ladder.
• Water conducts electricity - even more so the galvanized tank (pool) we supply in the US & Canada ! You are planning to put your hands in the water while standing on a wet floor. Your electricians may want to earth-connect the tank. Please show them your setup before you apply power and start working.
• Water supply & drainage pipes should not project from the side of the pool, or present a trip hazard. Drainage can be through the pool base.
• Water maze can involve repeated reaching-out and leaning-over movements. Also it can involve long periods of solitary repetitive work without daylight. These remarks apply especially to commercial labs where high throughput and experiments lasting months or years occur. In terms of reaching or bending we distrust pools of diameter greater than 6ft (1.8m) and pools resting on the floor - our best guess is that an HVS pool should be set on a base 12ins (0.3m) high.
• Feedback & safety hints from users are welcome & can be received in confidence.
The probe test: is it always a measure of spatial memory for transgenic mice?(C) 1999 Dr Roger J Morris, UK & HVS Image
Our aim, in the watermaze, is to test the capacity of rodents to learn, and retrieve, spatially-encoded information; instead we are restricted to asking the mice to solve a problem for which the ability to learn and remember a spatially-cued task will significantly enhance performance.
But is the capability for spatial learning the main variable in this test? Experience suggests that two problems in particular are worth considering, especially when dealing with transgenic mice.
The first is the ability of the animal to concentrate upon the task in hand. If the mice are too nervous, or too weak to be at their ease swimming, their performance is compromised independently of spatial learning (both effects have misled studies of spatial learning in transgenic KO mice). It is important that mice be tested in appropriate assays to determine whether they differ significantly from the control group in levels of anxiety, willingness to explore new territory, and general locomotory activity. The latter is monitored in the watermaze as swimming speed, the former given by such assays as the elevated plus maze, holeboard and open field. Our experience suggests that problems in the watermaze such as thigmotaxis and passive floating are minimized if mice first get accustomed to being removed from their home cage to solve relatively unthreatening problems, before being placed in a large tank of water with no visible means of escape. Thus both practical and theoretical considerations suggest testing the mice for exploration/anxiety prior to the watermaze, and the results may advise against using the watermaze as a test of spatial memory.
The second concerns the strategy used by the mice to locate the platform. Have they solved the problem purely by reference to the spatial cues, or has the physical process of "swimming in the general area until bumping into a submerged object" been a significant part of the strategy? In the initial phase of the watermaze in which rodents search for the hidden platform, in each trial the rodent has only to bump into the platform once to locate it and escape; is this single encounter the result of remembering how to triangulate the location from external cues, or just good luck?
Of the methods devised to eliminate a 'lucky bump' from the analysis, the probe test is considerably the least expensive and most convenient, and works very well for rats. A rat, knowing that the platform was in a certain position, will trawl repeatedly over that position looking for it. The rat is thus indicating that it knows the position independently of such tactile cues as hitting the platform, and this is real knowledge rather than a chance passage through the target area. This simple test is conducted when the animals have learnt the straight forward task of locating the hidden platform; i.e. when their elapsed time to find the platform has dropped from an initial maximum (e.g. 90 sec for the trial) to a base level where no significant improvement is occurring each day (generally around 10-25 sec, depending on the size of the pool/platform).
This test, however, depends as much upon the rodent's strategy in solving a problem as do the initial searches for the hidden platform; but in the probe test the problem is one of spatial memory if, and only if, the rodent does not confront the fact that the platform is no longer there. If the rodent realizes that the platform really is not there, that the hitherto reliable spatial analysis will not enable it to escape the water, then its subsequent behavior cannot be interpreted as driven by spatial analysis. It will resort to alternative survival-dependent problem solving. If the mutants differ from the controls in levels of anxiety, or in other less readily identifiable variables (depression, quick-wittedness, etc), their response to finding that the platform has been withdrawn will almost certainly vary from that of the controls.
We find that mice tend to be much more adaptable than rats upon finding that the platform has gone. Our mice swim immediately to the former position of the platform. A significant number then swim back to where they were put into the pool (which is randomly varied trial by trial, but they relocate this point accurately) and swim back to the former position of the platform. Some even repeat this once more. Most spend some time swimming around in the central 70% of the pool trying to locate the platform (and will again cross the former position). At some stage, many head for the side of the pool from which they will be retrieved at the end of the trial, where some float. After 90 sec, they escape the pool - confirming the non-spatial experience of the previous trials.
Since most mice return more than once to the former platform position, the groups under test will often spend significantly more time in the former quadrant, and pass over the former position of the platform, than they will in control quadrants/counters. The probe test thus works in mice as well as rats. Where a mutant group of mice differ from the control group in this test, I suspect that in some cases the source is not a defect in spatial memory, but differences in survival strategy by rodents that realize spatial memory will not help them escape from the water. The frequency with which defects are only found in the probe test, but not in the initial searching, concerns me since in very few cases are potentially confounding properties such as anxiety investigated.
The adaptability of the mice to variation of the test can be assessed if one reverses the position of the platform. On the day following the probe test, return the platform to the pool, but in a new position. Start the mouse off by placing it for 15 sec on the platform in its new position, so it knows there is again a platform to find, but (if it learns quickly) in a new position. Then run a series of trials for the mouse to find the platform in its new position, comparing not only time/path, but passages through counters and time in quadrants as simple indicators of strategy. We have found that mice of one knockout strain, on the first day of platform reversal, return immediately to the former site of the platform, and then begin a circular search pattern for the new location (which is usually successful within 90 sec); their wild type littermates tend to restrict their search mainly to the former position of the platform, and only after two futile trials do they begin to search more widely to find the platform. By trials 3/4, both groups are equally successful in finding the platform in its new position. The genotype-specific difference seen on day 1 of reversal is, I suggest, one of survival strategy, not spatial learning.
So, in summary, how the rodent solves any of the problems confronting it in the watermaze depends not just on its capacity for spatial learning, but also its strategy for solving problems; as these problems are varied (probe vs. hidden platform), it may be a difference in strategy, rather than in learning or memory, that dominates the response of knockout, compared to wild-type, mice. I must also state that this is not a common view in the field - the probe test, after all, has proved to be one of the best ways for producing a significant difference in behavior between control and mutant groups, and the temptation to attribute this difference to defects in spatial learning is rarely resisted.
It is my opinion that, given the unknown effect of most mutations upon the emotional, sensory and cognitive functions of the mice under test, the prime test of spatial memory is given by the initial search for the hidden platform, provided this is set up appropriately. That is, the initial search must be set up so that the animal realizes early on that the problem is difficult but solvable (avoiding problems of thigmotaxis and floating); the learning curve must show progressive improvements in performance over many trials (thus canceling out the "lucky bump"); and the mice should not be overtrained. And above all, the mice must be physically and emotionally able to perform the task.