All of the tests require some method of taking notes and collecting data in real time. Whether the experimenter chooses pen and paper or a computer-based entry system depends on budget and convenience. Most of the tests also require a stopwatch. The activity and water maze tests require an overhead-mounted video camera (an inexpensive black and white security camera is sufficient). For most of the other tests, recording with a video camera (mounted either overhead or horizontally) can be helpful for scoring purposes, but is not absolutely necessary. Additionally, a computerized video tracking system (e.g., Noldus Ethovision) is extremely helpful (albeit expensive) for several of the tests (activity, water maze, zero maze, forced swim, tail suspension) but not absolutely necessary. Note that several pieces of equipment can be built rather than purchased (anything made from wood should be sealed and waterproof for easy cleaning), and that following the exact dimensions specified in this chapter is not necessary as long as consistency is maintained within and across experiments. Finally, a spray bottle of ethanol is ideal for cleaning feces and urine from equipment after each trial.

Generally, the entire process of completing a comprehensive test battery takes three weeks. After the animals have been transferred to the housing facility nearest to the behavioral testing rooms, further transportation should be limited. For the first week (generally Monday through Friday), each animal is handled (i.e., allowed to freely crawl around the hands of the experimenter) for 1 min. Tests days 1–5 are administered Monday through Friday of the second week, and the water maze is administered Monday through Friday of the third week.

Tests generally start in the morning, shortly after the housing facility’s lights cycle on. Although some have argued for testing during the dark cycle (due to the nocturnal nature of rodents), the animals are generally used to the daytime noise and caretaker interactions, and we have found that daytime testing offers the greatest degree of convenience and generalizability across experiments and laboratories. We move the animals to the testing room and allow them to rest undisturbed for about 30 min before testing begins.

Approximately 40 animals can be tested per day by one experienced experimenter with the procedures outlined. However, most tests will go a little smoother with two experimenters—one to handle and transport the animals and the other to record data and observations. Due to the relatively complicated nature of animal behavior, extensive qualitative notes should be kept on each animal. These notes often provide invaluable answers to questions that arise while analyzing data after the experiment is completed. Ideally, the same experimenters will test the same animals every day until the experiment is complete. Experimenters should be blind to treatment condition and trained in animal handling/restraining. They should also remain a consistent and low-key presence to the animals, wearing a lab coat and similar pants every day, avoiding the use of perfumes or colognes, remaining quiet (speaking softly if necessary), silencing phone ringers, staying relatively still, and moving slowly and calmly when necessary. Experimenters should consider placing a “Do Not Disturb” sign on the outside of the testing room’s door. Additionally, we run a HEPA filter during testing to create white noise (which has the additional benefit of removing some of the animals’ fur and dander from the air).

Note that the tests described are most appropriate for post-weanling animals (21 days and older). Testing short-term behavioral deficits following brain injury in pre-weanling rodents is more difficult due to their relatively limited behavioral repertoire, but can be done with certain modifications (2–4). Additionally, these tests are most appropriate for animals that have had at least a week to recover from any acute, but transient, effects of injury. However, some of the tests, especially those administered on day 1, are also appropriate for testing such acute effects (e.g., for studies of therapeutic treatments for these effects).

Because most of the tests take multiple measurements of the same variable over time, data analysis for these tests generally consists of repeated-measures analysis of variance (ANOVA) or multiple regression. Issues regarding proper adjustments to alpha levels/p-values for multiple tests (e.g., the Bonferroni method, etc.), advanced techniques such as factor analyses (determining how variables “cluster” together), and power analysis (determining how many animals are required to produce quality data—a good starting point is ten per group) are beyond the scope of this chapter and are best discussed with an experienced biostatistician. However, the benefit of running such a comprehensive test battery is that the (potentially spurious) results of any single test or measure need not be “overinterpreted,” as the wide variety of domains tested and measures recorded will provide a relatively rich profile of each group’s behavioral characteristics.

Finally, please regard the experimental parameters described here as simply useful guidelines. Depending on several factors, including species, age, injury model, and previous test experience, certain aspects of the tests may need tweaking. Thus, pilot studies should be conducted to ensure that animals can the perform tests well enough to gather good data. Generally, the parameters should be adjusted so that controls display a “medium” level of performance, giving the experimenter the opportunity to observe group performance changes in both directions (e.g., improvement or deficit).