The overall goal of the following experiment is to detect differences in short and or long-term locomotor activity between different groups of experimentally treated mice. This is achieved by video recording mouse home cage activity in real time. As a second step, the animal's locomotor activity is analyzed in real time or from a recording using video tracking software.
Next, the scored locomotor parameters are imported into a secondary software for statistical analysis. The procedure can thus elucidate the effects of treatments or conditions on short-term and long-term locomotor activity based on evaluated parameters such as distance moved, velocity of movement and duration of movement. The main advantage of this technique over existing methods, such as line crossing and emitter implantation, is that it provides a more thorough examination of mouse locomotor activity without the need for an invasive and potentially confounding surgical procedure.
Generally, individuals new to this method will struggle because setting up cages and equipment in a way that allows optimal image quality for subsequent video tracking can be tedious and take time to learn. Short term locomotor activity is tested in the home cages of isolated mice with bedding that has a high contrast to the animal's code color. Before the experiment, the animals must be acclimated to their bedding for at least 24 hours.
To prepare the testing room begin by mounting a night vision capable video camera with at least 120 gigabytes of memory above the recording area. The room should not have any ambient sound above 70 decibels. If intermittent background noise is unavoidable, use a white noise generator to drown out those noises.
It is also important that the environmental conditions of the testing room are similar to the animal's normal housing conditions. Before setting up new subjects, clean the recording area, then cover the surface with butcher or lab bench paper of similar color to the cage bedding Next position, opaque cage dividers made of half inch plastic. On the testing surface, the dividers must have a similar color to the bedding and be clean.
Now bring the mice to the testing area and set their cages between the dividers. For the ease of analysis, always position the cages in the same way. For the test, cover the cages with quarter inch thick, clear plexiglass.
The plexiglass needs at least 11 quarter inch holes to provide adequate ventilation. Next, set up brooder clamp lights with red bulbs to illuminate the cages. Now center the camera's view on the test cages do not exceed the capability of the video tracking system.
This system is limited to tracking eight mice in real time. Now, by viewing the video camera display, adjust the angle and position of the lights to remove glare or reflections from the plexiglass cage tops. Obtaining a clean view of the mice free of any visual artifacts is the most critical step to getting good results.
Always include both experimental and control subjects in each group to ensure that the potential effects of temporal differences in LMA are minimized. Just before testing, remove any nesting and and enrichment materials from the cages as they may obstruct the view of the mouse. In the video, start the recording with a slate that identifies the relevant experimental information.
To ensure the proper identification of the video, use the timer on the camera's display to track the duration of the test and stay out of the animal's line of sight. During the recording time. When the test is finished, return all of the materials to each cage.
If the animals are to be tested again, return their access to food and water between the tests. Store the video recordings from the testing session on an archival media compatible with the video tracking system such as a hard disk drive In the video tracking system, analyze the videos in real time. Initially choose the entire cage floor as the region of interest or ROI for tracking.
For advanced analyses such as assessment of Figma taxes, create multiple intra cage ROIs for high throughput studies. Analyze all cages within the video simultaneously with individual all cage floor ROIs. Set the VTS to calculate the total distance, moved the velocity of the movements and the duration of the movements after the calculations are made, export the data to a spreadsheet file for storage and additional analysis.
Long-term testing is executed just as short-term testing. However, it requires a modified cage with a new access point for food and water. First, cut a 5.5 centimeter diameter hole in the short end wall of a standard cage.
Next, cut half inch openings into quarter inch galvanized steel mesh and burnish the mesh to remove sharp edges using the same mesh, build an external hopper for food pellets that can connect to the mesh over the opening. Next, add holes for rivets. Then cover the hole in the cage with the mesh and attached hopper using aluminum rivets and stainless steel washers.
To provide hydration, construct a water bottle from a standard 50 milliliter conical and a one hole rubber stopper that is over drilled. To allow for insertion of a no drip sipper tube. Secure this water bottle to the cage.
Using Velcro tape, the sipper tube should be inserted through the mesh covered opening. Now, while the cage is covered by the plexiglass lid, the mice retain access to food and water following the same procedures outlined for the short term locomotor assay. The long-term assay usually goes for 12 hours or for the duration of the current phase of the light dark cycle.
Long-term locomotor activity testing is generally analyzed in real time without making a video recording. However, a recording can be made using a secondary camera or via a DVD recorder linked to the primary camera. The mice can be tested as long as the video tracking system capabilities can handle, and as with short term testing, only the nest lit enrichment material is removed from the cage, not the corn cob or sawdust bedding.
In the VTS, set up the video tracking system for real-time analysis. Set the tracking ROI to encompass the entire floor for each cage being recorded. As with the short term assay, set the system to calculate the total distance, moved the velocity of movement and duration of movement.
Then following the analysis, export the data to a spreadsheet for storage and analysis using repeated short-term locomotor assays. It was found that after lipopolysaccharide injection mice housed in individually ventilated caging or IVC recovered from loss of short-term LMA from the injection more quickly than mice housed in ambient environment caging or a EC.While the initial loss of locomotor activity is similar between LPS injected groups, the recovery to basal locomotor activity is more rapid. In IVC housed mice, as was the velocity of movements and the duration of movements.
In this analysis, detection of two centimeters per second movement started tracking and 1.75 centimeters per second halted tracking using the detection variables. A small test of locomotion over two consecutive 12 hour activity cycles was made. In normal mice, there is no significant difference found in distance moved or in the velocity of movements or the duration of movements.
Should the trend for no measurable difference, repeat with a large sample size, the data would suggest that the assay could be used to replace implanted transmitters used for long-term locomotor assays. When attempting this procedure, it's important to be especially mindful of pre-ex experimental housing conditions. All experimental and control animals should be in similar environmental conditions, especially with respect to light cycle, room temperature and relative humidity.
Also, their exposure to noise and odor stimuli should be minimized Following this procedure. Other methods like the four swim test can be used to answer additional questions such as whether a particular experiment's treatment results in depressive like behavior without affecting overall locomotor activity.
