Morris water maze
The Morris water maze is one of the most widely used tasks in behavioral neuroscience for studying the psychological processes and neural mechanisms of spatial learning and memory. The basic task is very simple. Animals, usually rats or mice, are placed in a large circular pool of water and required to escape from water onto a hidden platform whose location can normally be identified only using spatial memory ( Figure 1). There are no local cues indicating where the platform is located. Conceptually, the task derives from place cells that are neurons in the hippocampus which identify or represent points in space in an environment (O'Keefe, 1976).
It was developed by Richard Morris at the University of St Andrews in Scotland and first described in two publications in the early 1980s (Morris, 1981; Morris et al., 1982). Place navigation in the watermaze is now often used as a general assay of cognitive function (Brandeis et al., 1989), for example for testing the impact of various disturbances of the nervous system (e.g. animal models of stroke (Nunn et al., 1994), aging (Gallagher and Rapp, 1997), neurodegenerative disease (Hsiao et al., 1996), or the potential impact of novel therapeutic drugs (D'Hooge and De Deyn, 2001). The task has also inspired computational neuroscientists and roboticists interested in navigation (Krichmar et al., 2005).
The task was developed at the Gatty Marine Laboratory at St Andrews, once famous for its work on the neurobiology of various marine animals. Richard Morris’s laboratory was placed there upon his appointment in 1979 to this institution, the oldest Scottish University. Walking past tanks of various sea creatures every day led him to wonder if a “maze” in which rats had to swim rather than run, could be a useful way of examining spatial memory and so investigate the functional significance of the recently discovered hippocampal place cells. From this emerged the lab jargon name - “watermaze” - a name that some have objected to on the grounds that the one thing that the apparatus is not is a maze! In any event, the name stuck and is now widely used. The popularization of the task is as much due to influential studies through the 1980s by a Canadian group in Lethbridge led by Ian Whishaw, Bryan Kolb and Robert Sutherland, with a friendly rivalry between the Canadian and Scottish groups helping to push research forward.
Distinction between the apparatus and the tasks trained within it
The watermaze is sometimes described as if it were a single task. Strictly speaking, the watermaze itself is no more than a piece of apparatus in which a variety of different tasks can be trained. The simplest water escape learning task - learning to find a hidden platform in a single fixed location - is often embedded into a series of sometimes quite complicated training and testing protocols to investigate specific theoretical issues. Distinct protocols engage different mechanisms of navigation, learning and memory.
The apparatus consists of a large circular pool, generally 1.5 to 2 metres in diameter, containing water at around 25°C made opaque by adding milk or another substance (e.g. latex liquid) that helps to hide the submerged platform (Fig. 2A). The water in the pool is filled and drained daily, via an automated filling and draining system. This choice of water temperature at around 13°C below body temperature is sufficiently stressful to motivate the animals to escape, but not so stressful as to inhibit learning. There is a mild stress reaction on day 1 of training (indicated by elevated corticosterone), but this habituates over days (Sandi et al., 1997). If the pool temperature drops to 19°C, performance improves, but when temperature drops to 12°C, it gets worse - reflecting the inverse U-shaped function relating stress to cognitive function.
A video camera is placed above the centre of the pool to capture images of the swimming animal, and this connected to a video or DVD recorder, and an on-line computer system running specialized tracking software. The top surface of the hidden platform, usually about 10-15 cm in diameter and thus between 1/50th and 1/100th of the surface area of the pool, is 1.5 cm below the water surface ( Figure 1A, C, D). The pool itself should be located in laboratory room with distinctive 2- and 3-D distal cues that aid orientation, with 3-D cues being particularly helpful. Alternatively, the pool may be surrounded with hanging curtains that occlude fixed room cues, enabling moveable cues to be hung inside that can be rotated relative to the room when this degree of precise experimental control is required. Again, it is best if these are 3D rather than flat surfaces. The use of a cue-controlled environment has proved particularly helpful in studies of pattern completion (Nakazawa et al., 2002).
Spatial reference memory
Reference memory protocols are widely used in which the platform is in a fixed location relative to the room cues across days. The animals are placed into the water at and facing the sidewalls of the pool, at different start positions across trials, and they quickly learn to swim to the correct location with decreasing escape latencies and more direct swim paths ( Figure 2B). The tracking system measures the gradually declining escape latency across trials, and parameters such as path-length, swim-speed, directionality in relation to platform location, and so on. Observation of the animals reveals that, having climbed onto the escape platform, they often rear up and look around, as if trying to identify their location in space. Rearing habituates over trials, but then dishabituates if the hidden platform is moved to a new location (Sutherland and Dyck, 1984).
Probe trials to test memory
During or after training is complete, the experimenter conducts a probe trial in which the escape platform is removed from the pool and the animal allowed to swim for 60 sec. Typically, a well-trained rat will swim to the target quadrant of the pool and repeatedly across the former location of the platform until starting to search elsewhere ( Figure 2C). This spatial bias, measured in various ways, constitutes evidence for spatial memory. Rats with lesions of the hippocampus and dentate gyrus, subiculum, or combined lesions, do poorly in post-training probe tests (Morris et al., 1982b; Sutherland et al., 1983).
Impact of overtraining
If rats with hippocampal lesions are given overtraining (typically consisting of a large number of trials over many days), their performance in probe tests can be quite good (Morris et al., 1990). Even rats with hippocampal lesions can show quite localized searching ( Figure 2D), particularly if the most septal pole of the longitudinal axis of the hippocampus is spared (Moser et al., 1993).
Numerous other protocols have been developed to test specific hypotheses. Many involve cryptically moving the hidden platform. This might be a reversal procedure in which, after one location has been thoroughly trained, the platform is moved to a different quadrant of the pool. Because it is hidden, it is not apparent that anything has changed until the animal fails to find the platform in its usual place. The focus is on how the animal reacts to this change and how quickly it learns the new location. The re-learning that occurs in reversal protocol has been used in a major genetic factor analysis of the determinants of watermaze behavior across different strains of mice (Lipp and Wolfer, 1998).
The Atlantis platform
As the animals sometimes bump into the submerged platform by chance, one useful innovation is an on-demand or Atlantis platform (named after the lost city) that is initially at the bottom of the pool and only becomes available when the animal swims in its vicinity for some predetermined time (Spooner et al., 1994; Figure 3A). An automatic release system allows the platform to rise gently to near the surface of the water (but it remains hidden). This procedure results in the acquisition of a highly focused searching strategy focused on the target location during training. Reversible inactivation of the hippocampus with a drug that blocks excitatory neurotransmission after training is complete results in animals displaying localized searching at inappropriate places in the pool (Riedel et al., 1999), indicating that they retain the procedural strategy of searching during hippocampal inactivation but do not know where to search ( Figure 3B). In contrast, pharmacological inactivation of fast synaptic transmission during training results in a failure to develop this search strategy because the animals cannot learn where to execute the strategy in the pool. The accuracy of searching can also be measured using a zone-analysis that measures time spent in a virtual zone around the place where the platform is located. This is proving very useful in ongoing studies of memory consolidation and reconsolidation.
Delayed-matching-to-place and one-trial learning
In other protocols, sometimes called working memory protocols, the platform is moved to a new location each day, creating what is called the ‘delayed matching to place’ (DMP) procedure ( Figure 4A). In this procedure, the animal cannot know where the platform is hidden on trial 1 of each day. However, once it finds the platform (usually after about 60 s of searching), it can generally encode this new location in one trial. This is shown by the animal finding the platform much faster on trial 2 and subsequent trials of that day (Steele and Morris, 1999). In effect, this procedure enables the study of repeated instances of one-trial learning. The intertrial interval (ITI or memory delay) between trials 1 and 2 can then be systematically varied to explore how well 1-trial spatial memory is remembered, a procedure with some similarities to delayed matching and non-matching tasks used to examine recognition memory. Rats with complete hippocampal lesions never show rapid 1-trial learning required in the DMP task (even after extended training) and are just as poor at a short ITI between trials 1 and 2 as a long one ( Figure 4B). In contrast, treatment with an NMDA antagonist such as D-AP5 results in a selective deficit in memory at a long ITI, but the animals can remember over short memory intervals between trials. Studies of transgenic mice with regional-specific deletions of the NMDA receptor have revealed a role for area CA3 in the hippocampus in rapid learning using this DMP paradigm (Nakazawa et al., 2003).
Other procedural variants include alterations to the apparatus, such as constraining the path of the swimming animal to minimize navigational demands (e.g. an annular watermaze; Brun et al., 2002), decreasing the number of available extramaze cues between training and testing to look at pattern completion (Nakazawa et al., 2002), the use of floating platforms, and yet other manipulations. A radial-watermaze has also been introduced, combining the virtues of the radial-maze with the ease of training to escape from water. This has proved invaluable in testing transgenic mice expressing familial Alzheimer mutations (Morgan et al., 2000).
Treatments and control procedures
A wide variety of treatments have been explored including lesions, drugs and molecular-genetic alterations. These alter watermaze performance in various ways, but experimenters must be cautious as such alterations need not be specific deficits as such alterations need not be specific to spatial learning or memory processes per se. Lesions or drugs may have a direct effect upon learning mechanisms, and many seem to do so, but they may also affect an animal’s ability to see the extramaze cues (a sensory deficit), or their motivation to escape from the water, or translate knowledge into action, rather than learning per se. Factor analytic studies reveal that many molecular-genetic alterations influence the probability of mice to stay at the side-walls (thigmotaxis) instead of swimming into the centre of the pool. These performance effects are statistically independent of effects on spatial memory.
Accordingly, treatments must be accompanied by relevant control conditions. A common control protocol is to include trials in which the escape platform is made visible, the idea being that treatments which merely affect motivation to escape should impair performance in this task as well as the basic task. It is unclear how sensitive this assay really is, but it does provide a first-pass at detecting gross sensorimotor abnormalities. As blind rats have been claimed to do surprisingly well in the watermaze (except in probe trials), more taxing psychophysical techniques have been introduced offering precise control of the spatial frequency of cues that are more sensitive to subtle visual deficits (Prusky et al., 2000). The use of sham-lesioned animals, vehicle-infusion conditions, floxed mice, and other pertinent manipulations has also become widespread to ensure that any alterations in spatial learning in the experimental group are not an unintended by-product of achieving the treatment.
Strengths and limitations of the watermaze as an assay
As our understanding of the impact of various treatments on diverse aspects of cognitive function has developed, e.g. executive function, the watermaze has been subsumed into larger test batteries for investigating diverse aspects of brain function. A clear virtue of the task is that the various protocols are so sensitive to manipulations of normal brain function in many brain areas, not just the hippocampus, that these can be used almost like a litmus test of the normality of cognitive function. This brings behavioral observations of function into fields of neuroscience that have historically relied exclusively on endocrine measures (studies of stress), neuropathology (stroke research), biochemical analyses (Alzheimer’s disease), or electrophysiology (development of cognitive enhancing drugs). The limitation, or analytical weakness, is that watermaze tasks which are affected by such a wide variety of treatments are gradually being revealed as having less specificity than was once believed (Whishaw and Jarrard, 1996; D'Hooge and De Deyn, 2001). Notwithstanding these limitations, the watermaze remains a still widely used task in behavioral neuroscience.
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