The overall goal of this procedure is to examine spatial learning and memory abilities in small rodents and rats with the dry land Barnes Maze. This is accomplished by first habituating the experimental animals to the test room and Barnes maze. The next step is to place the animal in the center of the maze and begin tracking its latency to locate the escape cage tracking is stopped when the animal enters the escape cage or after 300 seconds.
The results can show disruptions in spatial learning and memory as increased latencies, increased error rates, and a failure to change searching strategies. The main advantages of this technique over existing methods such as the water maze, are that it is somewhat less stressful, easier to perform, and more ecologically relevant for terrestrial rodents. Though this method can provide insight into the toxicological effects of spatial learning and memory disruptions.
It can also be applied to other fields such as neurodegenerative disorders, aging and congenital cognitive defects. Barnes maze designs vary, but generally each has 12 or 20 potential escape holes, only one of which leads to the home or an escape cage. The escape cage may be situated either directly below the escape hole on the maze top, or built into the surrounding wall of the maze.
The cues can vary in size from approximately 16.5 centimeters, height or width within the maze to a horizontal line, 21.6 centimeters in width placed from floor to ceiling of the room wall outside the maze. Plugs or false bottoms must cover the non escape holes to prevent the animal from falling out of the maze. The size of the test room can vary, but it must have plenty of room for the maze.
A place for the animal cages room for a computer with video set up and a place for the experimenter to sit at least one and a quarter meters from the maze. It's important that the animals are exposed to uniform environmental conditions minimize any variable noises or external cues that would differentially affect an animal's performance. Assignment of the escape cage location should be balanced among treatment groups and sex between trials.
The maze is wiped clean with ethanol to remove odor cues as is the escape box. This video does not include details on specific tracking software operation as they are likely software specific. However, such a system is desirable at the consistently used testing time.
Set up the room by lighting the lights above the maze and placing. Do not enter signs outside the lab 30 minutes prior to the first trial. Bring the mice to the test room in their normal home cages to help them habituate.
Set up the tracking program. Now gently remove the first mouse from its home cage and place it in the tall covered plastic box. Habituate the animal to the escape cage for one minute.
Ensure the paper that is blocking the tube is removed from that escape hole and that all the other holes are plugged. Place the mouse in the center of the maze and cover the mouse with a plastic box. After placing the animal in the center of the maze, quietly move to the computer area and quickly initiate the recording.
While monitoring the animal's performance on the computer record the hole number, trial number, search strategy, and number of errors made. An error is defined as sniffing at an incorrect hole. Assessment of search strategy may be made live or later with the recorded tracking patterns.
A direct strategy shows the rodent going directly into the escape cage with three or fewer errors. A serial pattern involves traveling along the maze perimeter until the escape cage is located. And lastly, the pattern may be random, meaning that the maze is crossed through the center multiple times to check various holes.
Stop the tracking program when the animal has all four paws inside the escape cage. If the animal fails to escape the maze in five minutes, stop the recording. Then gently nudge the animal to the escape hole.
Allow the nudged animals to stay in the escape cage for exactly two minutes before returning them to their home cage. Now spray the maze top with 70%ethanol and wipe dry. Then return the animal to the space outside the room and tested again 30 minutes later.
Each mouse is tested twice daily. Before starting the next mouse on the maze, plug the previously correct escape hole and remove the paper plug blocking the hole from the designated escape hole for that. Next subject object setup includes centering the maze below the overhead lights, which must be on assuring that the false bottoms that block non escapee holes and prevent the animal from falling out are securely in the maze and that the escape cage is in the designated location.
For the first subject, The Barnes maze top must be sufficiently aversive to motivate the animal. To find the escape cage, we have determined that intense bright lighting directly above the maze top serves this purpose very well. Also ensure the computer and camera are ready and a stopwatch is available.
Now turn on white noise to attenuate the background noises. The chair should be about a meter from the maze and stay there throughout the testing. Prepare a timer to count down two minutes, but to not make any noises outside the room.
Place a do not enter sign. A test sheet for taking notes should be made available as well. 30 to 60 minutes Before the first rat is tested, bring the animals in their home cages to the test room to allow for habituation.
Now, position a tube for the rat to start the trial at the May Center on the tube, place a title card with the animal's ID to easily identify the video if used. Record a few seconds to capture the animal ID sheet. Remove the animal from its home and gently place it headfirst into the escape cage.
Then cover the escape cage with a false bottom and start the two minute timer. After two minutes, gently remove the animal from the escape cage and place it into the center of the tube. Return the ID sheet to the top of the tube and gently lift the assembly, freeing the rat.
Immediately start the stopwatch and take a seat in the tester's chair on the test sheet. Record the time of day for the trial. Start and wait patiently while the rat uses up to five minutes to find the escape cage.
Record the stopwatch time. When the rat finds the escape cage, then remove the animal from the escape cage and place it back into its home cage. If the rat does not find the escape cage within five minutes, note this, then gently guide it there.
After 15 seconds in the cage, return the rat to its home cage. If the rat leaves the maze mentally, note the time and quickly grab the animal. If it is captured within 10 seconds, return it to the center of the maze and continue the trial.
Otherwise abort the trial and omit it from analysis. In either case, record the time of the fall or jump on the test sheet after the trial, stop the recording and note any comments about the trial between trials. Remove any urine or feces from the maze top.
Spray it with 70%ethanol and thoroughly wipe it dry. Clean the escape cage with 70%ethanol as well. Allow it to air dry and use a second escape cage for the next trial.
Always alternate escape cages. Now repeat the process. Testing the next animal.
Test each animal just once per day. On subsequent days, run the maze with one change. Place the rats directly into the center tube from the home cage, omitting the escape cage habituation, period.
Dear mice, A polygamous species were developmentally exposed to environmentally relevant doses of BPA in a phytoestrogen free diet, or estrogen in the same diet as a positive control or a phytoestrogen free diet as a negative control, the mice were assessed using the Barnes maze. The two highest BPA doses led to disrupted spatial navigational ability, but not the lowest dose of BPA tested, demonstrated equivalent deficits in spatial learning and an inability to convert to the direct search strategy over the trial period, male. Dear Mice, exposed to higher doses of BPA showed prolonged latency relative to low dose BPA males and relative to negative control males.
The estrogen positive controls performed just as poorly, along with an increased latency came an increased error rate as well. In contrast to male mice, female mice exposed to EE two and the mid BPA dose, but not other BPA doses exhibited masculinized patterns of spatial learning and memory. This is demonstrated by their abnormal use of the direct search strategy and logically this was reflected in a decreased latency.
In contrast to polygamous deer mice, their related cousins, the monogamous male California mice were also dosed with early BPA and EE two exposure and tested in the maze. Search strategies were unaffected. Male California mice did not use the direct search strategy like male deer mice in this species, BPA and EE two did not affect search errors relative to controls.
Notice that compared to controlled deer mice control California mice did not decrease the number of errors made over the seven consecutive test days. Latency was also unaffected. The observed difference in spatial navigational disruption between deer mice and California mice are likely due to species differences in behavioral vulnerability to developmental exposure to BPA.
Regardless, it is clear that developmental dosing did not measurably alter these behaviors of California mice under the testing regime. Following this procedure, other methods such as anxiety or activity assessments can be performed in order to answer additional questions such as whether the animals exhibit altered anxiety or activity levels or other cognitive impairments. Notably, after its development, this technique paved the way for research in the field of cognition to explore space navigation in various rodent and non rodent species.
