Jove Barnes Maze

[Meri Thilmony]

Habituationof animals to a testing apparatus is a crucial pre-experimentation condition for the proper execution of behavioral analyses in many tests. However, in certain cases, exposure of the animal to the testing apparatus prior to experimentation may significantly interfere with subsequent results. Additionally, in cases which require or benefit from pre-experimentation environmental habituation, the procedure varies on the conditions of the number and duration of exposures as well as the specific rodent model being used. Thus, care must be taken in deciding whether or not to habituate animals to a given testing apparatus and if so, under what conditions.

Primarily, habituation to a testing apparatus transforms a potentially negative and unpredictable reaction experience into a potentially positive and more predictable response. In the case of experiments which do not rely on environmental novelty (e.g. social interaction, novel object recognition, conditional maze testing) this apparatus habituation may define the difference between an inconsistent and a consistent behavioral profile. Conversely, tasks which rely on environmental novelty (e.g. open field exploration, conditioned place preference) lose significant power from any pre-experimentation exposure and are therefore not appropriate opportunities for environmental habituation.

Here, we will discuss the impacts of and protocols for apparatus habituation in both tests which rely on environmental novelty and tests for which environmental novelty is not a parameter of interest.

Tests which rely on environmental novelty are negatively impacted by environmental exposure prior to the initiation of experimental testing. Thus, animals should not be exposed to the testing apparatus prior to the experiment. However, it is important that animals acclimate to the room in which the tests will take place in order to eliminate the influence of environmental novelty (light and smell conditions, extra-apparatus markers etc.). Thus, animals should be placed in their home cages within the room in which the testing will take place for a minimum of 10 minutes in the absence of the experimenter prior to testing.[1]

However, animals should still be acclimated to the room in which the testing apparatus will be used. Pre-experimentation exposure to the environmental area using the home cage procedure noted at the beginning of this section (using a duration of 30 minutes[4]) will habituate the animal to the room and therefore eliminate confounding novelty variables. Conversely, exposure to the EPM itself, for example, will have a deleterious effect on behavioral performance. [5]

The morris water maze (MWM) is one of the most common tasks for the assessment of spatial memory. Animals use spatial cues (most often visual cues) in order to help locate a platform hidden in opaque water in which they must otherwise continuously swim. Several variables determine whether or not habituation to the MWM apparatus is appropriate prior to experimentation. First, depending on the duration of the test, many animals (especially rats) will not actively seek escape but will rather explore the swimming arena continuously. This is more common in short-duration experiments, for example, those lasting less than one minute. In this case, pre-experiment exposure to the arena with the platform exposed (i.e. un-dyed water in order to reveal the platform location) will indicate the presence of the platform, thereby ensuring that the animal understands the breadth of the task.

Additionally, a pre-experimentation acquisition exposure to the MWM arena performed in the absence of spatial cues (such as objects or wall-markings within the room which are visible from within the MWM) eliminates the confounding variable of environmental novelty in measurements such as distance moved and time spent per quadrant in the acquisition phase of the test.

Conversely, the absence of a platform may induce significant stress in animals seeking an escape and thus confounds any measures of learning acquisition with increased levels of stress and anxiety as a result of the no-platform habituation procedure. Similarly, exposure to the arena in a no-platform condition followed by a test in which the platform is present has been argued to be a form of reversal learning, rather than spatial acquisition, and thus significantly changes the interpretation of outcome results.[6]

Thus, while apparatus habituation is not commonly used for tests involving environmental novelty, exposure to the MWM in controlled conditions may be useful for achieving uniform behavioral results in certain circumstances. Researchers using short protocols (i.e. less than one minute) may pre-expose their animals to the MWM in un-dyed water in order to indicate the presence of an escape platform. Importantly, this habituation test should be done in the same environment (i.e. testing room) in which the experiment will take place, however, any spatial cues such as wall markings which are visible from within the MWM should be removed for the duration of the test. This habituation session should be performed on the day prior to the experiment.

The barnes maze is a common test of visuospatial learning comprised of an elevated platform illuminated by aversive bright lights with escape holes located on the edge allowing escape to a dark compartment. Prior to experimentation in the Barnes maze, habituation training allows rats or mice to habituate to the light intensity such that they can properly navigate the maze during the following training periods (acquisition and, if applicable, reversal training). In order to accomplish proper habituation to the Barnes Maze, experimenters should place the rodent in the arena, situated under a glass beaker or other appropriately-sized transparent holding container for a duration of 5 minutes. All intra-maze cues should be removed and if present, extra-maze cues should be blocked from view in order to prevent the animal from learning intra/extra-maze location indicators.[9]

Performance in learning and memory tasks such as novel object recognition and object placement memory is most frequently measured as an index of the duration of time which an animal spends with one object as compared to another, or as compared to the same object in a different location.[15] Thus, any pre-existing tendency for an animal to spend more time in one area of the arena over another will inherently skew the interpretation of these measures. Rats or mice must, therefore, be habituated to the testing area prior to commencing the acquisition training period of the experiment. In order to achieve environmental habituation, researchers should place the rat or mouse in the testing arena in the absence of any object. This procedure may be done once or twice on the day prior to the first phase of testing and may last between 5 and 20 minutes.[15] As a measure of proper acclimation to the environment, animals should display an approximately uniform duration of time in all areas of the arena.

Experiments which employ operant chambers, such as discrimination or choice tasks (such as the 5 choice serial reaction time task) and self-administration protocols, require habituation to the apparatus prior to experimentation. Since the desired behavior does not rely on environmental novelty, any attention given by the animal to the apparatus environment will detract from behavioral performance and therefore from the experimental results. The duration of exposure to the operant chamber varies based on the murine model and the particular protocol, but the procedure is similar to other environmental habituation protocols. The animal should be left alone in the operant chamber in the absence of the experimenter and without any cues (such as conditional lights, reward levers etc.) for an equivalent duration of the experiment which follows.

For instance, in an odor discrimination task which normally lasts 45 minutes, animals are to be left in the chamber in the absence of odors for a duration of 45 minutes on the day prior to testing.[16] For self-administration chambers, the acclimatization procedure includes both environmental and operant habituation over the course of several days. First, the animal should be exposed to the chamber for an equivalent duration to the desired experimental session which will follow. Then, the animal can be trained to understand the operant mechanism (e.g. nose-poke or lever) using either the experimental stimulus or another rewarding stimulus (such as sucrose delivery). Again, if the eventual self-administration session is intended to last for one hour, the habituation and training sessions should also be of a duration of one hour.[17]

Clearly, habituating animals to a testing apparatus or environment has significant benefits for many behavioral testing protocols. However, in certain conditions, it may be counterproductive or even harmful to the results to perform a habituation or acclimatization procedure on subject rodents prior to testing. In short, consider whether or not the test you are performing requires environmental novelty for proper behavioral performance before deciding whether or not to habituate your animals to the testing apparatus. Then, if environmental habituation is appropriate, consider the duration of the intended experiment when deciding how to perform the habituation session. In all cases, ensure that animals in all conditions to be used in a test (e.g. drug-treated vs. naive, male vs. female etc.) undergo identical habituation procedures to ensure uniform behavioral outcomes.

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