Learning and Memory Assessment

The Morris water task is one of the most widely used paradigms to evaluate learning and memory behavior. This is a spatial navigation task in which the mouse swims to find a hidden platform, using visual cues to locate the platform. Escape from the water is the positive reinforcement. The task is based on the principle that mice are highly motivated to escape from a water environment by the quickest, most direct route. The procedure requires a circular plastic tank about 30 cm in diameter and about 45 cm in height filled with tap water to a depth of about 20 cm. The tank is filled the day before so water can reach room temperature the next day for testing. Milk powder is mixed into the water so as to make an opaque liquid which makes the resting platform invisible to a mouse swimming on the surface of the water. Water is changed daily, the tank disinfected, and refilled for the next day of testing. Two platforms are used, one just below the surface of the water, and thus invisible, and one just above the surface of the water. It is essential that environmental cues and surroundings remain constant throughout the duration of testing. The same experimenter must handle the mice throughout an experiment, with no intermittent visitors. The first step of the procedure is pretraining, consisting of gently introducing the mouse to the pool and platform. Generally the mouse will jump off the platform and is allowed a swim up to 15 sec, before being guided back to the platform. Up to three pretraining sessions are conducted, measuring the visual ability of the mouse to see the room cues (latency to reach the visible platform), and the motor ability to swim in the pool (swim speed). The next step is training for the hidden platform, which starts by placing the mouse in the water at the edge of the pool, allowing 60 sec to reach the platform and climb out of the water. This is repeated for a variable number of predetermined trials over 3 to 10 days. A criterion of 10 sec to reach the platform is set, so that training is continued until controls reach the platform within this time frame. At the end of the training period, each mouse is tested on a trial probe that measures the ability of the mouse to identify the spatial location that previously contained the hidden platform within a 60 sec time period. Search time spent in the trained quadrant must be greater than search time spent in the other three quadrants of the pool. Normal performance on the visible platform task but impaired performance on the hidden platform task is interpreted as a deficit in learning and memory.22

A second learning and memory task that highly complements the Morris water test is the cued and contextual conditioning test. This trial is much easier to perform in terms of less elaborate equipment needed, less time for investigator and mouse, and less laboratory space required. The test takes only 2 days with 10 min per mouse per day. Cued and contextual conditioning is based on fear conditioning in that it measures the ability of a mouse to learn and remember an association between an aversive experience and certain environmental cues. This type of fear conditioning is highly intuitive to mice as a species, because it is in their nature to freeze at the hint of danger. Conditioning training occurs on Day 1, when a mouse is placed in a chamber and allowed to explore for 2 min. After the timed exploration period, the animal is subjected to 30 sec of an auditory cue, white noise from an 80 dB broad-band auditory clicker. Following cessation of the white noise, the unconditioned aversive stimulus, a 0.35 mA footshock, is administered for 2 sec through a grid in the floor of the chamber. The measurement of unconditioned fear is the amount of time spent freezing in the test chamber on Day 1. Twenty-four hours later, testing for Day 2 commences. The mouse is returned to the same chamber and scored for bouts of freezing behavior, in which there is no movement by the mouse except for respiration. Presence or absence of freezing behavior is recorded in cycles of every 10 sec for a continuous 5 min, after which the mouse is returned to its home cage. The number of seconds spent freezing is termed the contextually conditioned fear. The second phase of testing for Day 2 starts 1 h after the end of the first phase. The mouse is again placed in a chamber, though one that is slightly different in some way to provide an altered context. Freezing behavior is scored for 3 min. Contextual discrimination of fear conditioning is calculated by comparing the number of freezing bouts in the same chamber to the number of bouts in the altered contextual environment, the slightly different chamber. At the end of the second phase of 3 min scoring, the white noise auditory cue is presented in the altered context environment. Again, freezing behavior is scored for a time period of 3 min in the presence of the white noise. Cued conditioning is quantitated by comparing the number of freezing bouts in the altered environment without the presence of the auditory cue with the number of bouts in the altered environment with the presence of the auditory cue. Impairment or improvement of one of the components measured in this examination can generate information about neuroanatomy, neurotransmitters, and genes regulating emotional components of memory.22

There are various avoidance tasks, passive and active, that determine the ability of the mouse to remember adverse stimuli and learn to avoid their presentation. Passive avoidance tasks require the mouse to refrain from entering a chamber previously associated with an aversive stimulus. Active avoidance tasks require the mouse to leave a chamber where an aversive stimulus is present. All avoidance tasks are based on electrical shocks generated from a grid and producing a painful stimulus in the footpads. There are various intensity parameters available for use, with the minimum intensity being that required to produce vocalization and flinching. Latency to move to the required area, away from or toward the shock area dependent on whether the test is passive or active, is measured in seconds and compared between normal wild-type control mice and transgenic mice. Alterations in the avoidance test include step-down avoidance where a mouse is required to step off an elevated platform, and Y-maze and T-maze avoidance in which a shock is delivered in only one run of a three-pronged runway.22

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