The overall goal of this procedure is to quantify behavioral and cognitive changes induced by cranial irradiation. The first step is to test hippocampal based spatial recognition memory using the novel place recognition task. Next, anxiety levels are assessed using the elevated plus maze task, fear-based memory formation, using the hippocampus and amygdala dependent contextual fair conditioning task, and the amygdala dependent queued fear conditioning task is then measured.
The final step is to administer the Maurice Water Maze task in order to assess spatial learning, spatial memory, and reverse learning ability. Ultimately, this protocol detects changes in mood, spatial and nons spatial learning and memory, as well as any sensory motor disruptions induced by cranial irradiation. Though this method can provide insight into the effects of cranial irradiation on behavior and cognition.
It can also be used to other situations in which cognition is impaired as an unwanted side effect of other therapy, such as in the case of chemo brain or chemotherapy induced learning and memory impairments. Individuals new to this method will struggle because of the time consuming nature of setting up and optimizing the equipment. Interpretation of the findings can be difficult if experimental procedures are not tightly controlled, and variation in the data makes it difficult to detect differences between control and experimental groups.
Visual demonstration of these methodologies is critical, so the reader can appreciate the subtleties involved in organization or the experimental rooms in which the cognitive testing is conducted, as well as the specific procedures employed. In order to ensure consistent handling of the animals, Begin the familiarization phase by preparing the test environment thoroughly clean two identical plastic blocks with 70%ethanol and place them into specific positions within the arena. Place the animal into the center of the arena and allow it to explore freely.
For five minutes, exploratory behaviors are scored. Using an automated tracking system, exploration is defined as an animal's nose being within a four centimeter radius of and directed toward the blocks. When time is up, remove the animal to a holding cage for another five minutes.
During this time, clean the testing arena with 70%ethanol. Add one identical copy of the blocks to the same spatial position as during the familiarization phase and one to a novel spatial position. After the five minute delay interval, reintroduce the animal to the testing arenas for a five minute test phase and allow them to explore freely for three minutes.
If the spatial arrangement of the blocks from the familiarization phase is remembered, the rat will spend more time exploring the block in the novel spatial location. The following day, the animals are returned once more to the arena. For a 24 hour test phase, again, one block remains in a familiar position while another is in a novel.
One, place the animal into the center of the arena and record exploration for three minutes. The elevated plus maze measures levels of anxiety in rodents. Before testing, ensure that the maze is cleaned thoroughly.
To begin place the animal into the center portion of the maze facing the open arm away from the experimenter, allow the animal to explore for five minutes. The number of entries and amount of time spent in the open and closed arms is tracked using automated tracking software. If a rat falls from the maze, quickly place it back onto an open arm and record the event.
Also note any freezing behavior along with any possible causes. Fear conditioning is a behavioral paradigm in which animals learn to fear a previously neutral stimulus. By pairing it with a noxious stimulus on the first day, place the animal into a thoroughly cleaned chamber.
Begin the training trial, which consists of three phases. The first is a five minute baseline phase when no shock or tone is administered during the training phase, multiple tone shock pairings are presented over a five minute period. Finally, during the post-training phase, the animal is given another five minutes.
In the absence of a shock or tone, the percentage of time each animal spends freezing is measured from the final 60 seconds of the baseline phase and from the final 60 seconds of the post-training phase. 24 hours later, return the animal to the chamber for a five minute contextual test and record the percentage of time spent freezing one hour after testing. In the familiar context, remove the grid floor, add panels to the outside of the chamber and clean with as scented household cleaner.
To test for cued fear conditioning, place the animal into the novel context and record freezing behavior in the presence and absence of the tone that was previously paired to the noxious stimulus. The Morris Water Maze is a test of spatial learning that requires the use of distal cues to locate a submerged escape platform in a large pool of opaque water, the task is administered over eight days and consists of four phases, queue test and acquisition. Phase a 24 hour probe test, and a reversal learning test.
To begin the queued test, place a distinct black and white flag in the middle of the platform so that the escape location is obvious to the animal. For each CUD test trial, place the animal in a different predetermined start location at the perimeter of the pool, move the platform into a different quadrant. For each of the four trials, allow the animal 30 seconds to find the escape platform.
If the maximum time is reached, guide the animals there by hand. Allow the animal to remain on the platform for 20 seconds before placing them into a warmed cage. The next day, begin the acquisition phase consisting of four trials per day for five consecutive days.
During this phase, the hidden platform remains in the same location and animals learn to navigate to it. Using distal room cues, place the animal into the pool at a different predetermined start point. For each trial record the mean latency to find the platform and the path length as measures of spatial learning.
If the animal has not escaped within the maximum swim time of 60 seconds, guided to the platform manually and allow it to remain there for 20 seconds. 24 hours after the final training trial, conduct a 62nd probe trial in which the platform is removed to determine the animal's preference for the quadrant that used to contain the platform. Following this probe trial, return the platform to the same quadrant and administer three additional acquisition trials for the Reversal Learning test.
On day eight, move the platform to a new location. Introduce the animal to the pool and measure the amount of time spent searching for the platform in its previous location versus its new one here. Results from Control and Irradiated Animals In the Novel Place Recognition Task showed that the two positions were explored equally during the familiarization phase.
However, control animals showed a clear preference for the novel spatial position following the five minute and 24 hour delay intervals while irradiated animals did not. In the elevated plus maze control and irradiated animals spent less time exploring the open arms than would be expected by chance. The percentage freezing during the initial exposure to the chamber was very low compared to the post-training period the following day.
Freezing behavior was still increased within the familiar context, although much less so in irradiated animals when placed in a novel context, freezing behavior returned to baseline levels, but were raised again by the presentation of the tone in the Morris Water Maze. Task Control Animals located the Escape platform more quickly over successive trials compared to irradiated animals. Irradiated animals also took more time and a longer path to the platform in a probe trial, 24 hours later during the Reversal Learning Test, when the location of the platform is switched, normal animals gradually stop searching the old quadrant and learn the new platform location, whereas irradiated animals do not.
While conducting these tasks, it's important to take detailed notes regarding any abnormal events or behaviors, as well as any tracking issues that arise that need to be addressed before the data are analyzed. Don't forget that working with experimental animals can be hazardous and precautions such as the use of personal protective equipment should always be used when undertaking these procedures. It is also critical to obtain institutional approval and follow institutional and federal guidelines for the safe and ethical use of animals.
