Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Jason M. York; Neil A. Blevins; Leslie K. McNeil; Gregory G. Freund

doi:10.3791/50252

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Summary

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Abstract

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Protocol

1. Short-term LMA Set Up and Procedure

Short-term LMA cage set up
1. Test short-term LMA within the home cage of a single-housed mouse. To facilitate VTS-dependent image analysis, use bedding in high contrast with mouse color (i.e. dark colored bedding for white mice and light colored bedding for black mice).
2. During short-term LMA testing, cover cages with clear ¼ in Plexiglas with 11 x ¼ in diameter holes to support appropriate ventilation.
Short-term LMA testing room
1. Test mice in a clean environment in which they have been acclimated to for at least 24 hr ⁸.
2. To illuminate the testing environment, use brooder clamp lights with red bulbs (< 125 lumens). Prior to videography, adjust the angle and position of the lights to remove glare or reflections from the Plexiglas cage tops.
3. Remove or avoid room noise above 70 dB. Use a white noise generator if intermittent background noise is unavoidable during mouse testing.
4. Cover the testing surface upon which the cages are placed in the testing room with butcher or lab bench paper of similar color to the cage bedding, in case the cage floor is exposed during videography.
Short-term LMA videography
1. Center and place a ceiling-mounted night vision-capable video camera with at least 120 GB of memory above the recording area in the testing room. Do not exceed the resolution capability of the VTS used by placing too many cages within the video camera view frame (e.g. Ethovision XT 7 by Noldus Information Technology (Leesburg VA) can simultaneously track 8 mice in real-time).
Short-term LMA testing procedure
1. Test short-term LMA during the active/dark phase of the light cycle. Make sure the time relative to the light cycle is the same across all groups, as LMA follows a crepuscular pattern, with more activity during the early and later periods of the dark cycle ¹. Include both experimental and control subjects in each group of recorded mice to ensure potential effects of temporal differences in LMA are minimized.
2. Individually house and acclimate mice at least 24 hr prior to the start of LMA testing ⁸.
3. Place the cage dividers and cages (containing individual mice) directly under the camera. Remove all nesting/enrichment material from the cages, as this may obstruct the view of the mouse (e.g. if the mouse burrows/covers itself with nesting material) and interfere with VTS accuracy. Place a clear Plexiglas lid on each cage recorded.
4. When videoing several cages simultaneously, be sure that the resolution is compatible with the chosen VTS. To ease VTS analysis, position the cages identically within each divider (i.e. upper right hand corner).
5. Start recording. Begin each video with a slate that identifies all relevant experimental information to ensure proper identification of the video independent of any additional non-video documentation. Use a timer or stopwatch to track duration of LMA testing (5-10 min). If timing information is lost, recover it from the video record.
6. For repeated measures using the short-term LMA method, allow mice access to food and water during the non-measurement intervals.
7. During testing, keep any experimenters out of sight of the mice so as not to act as an external visual cue.
8. When testing is finished, return/replace nesting/enrichment material, to reduce the potential for development of anxiety-like behavior ⁸.
Short-term LMA analysis
1. Store the video recordings from the testing session to an archival media compatible with the VTS used. For Ethovision XT 7, a hard drive disk (HDD) is used.
2. Replay the videos in the VTS using its associated media player, analyzing the videos in real-time. In Figure 1, the detection variables were: 2 cm/sec begins tracking; 1.75 cm/sec halts tracking.
3. Initially, choose the entire cage floor as the region of interest (ROI) for tracking. For advanced analyses, such as assessment of thigmotaxis, create multiple intra-cage ROIs.
4. For high-throughput studies, analyze all cages within the video simultaneously with individual all-cage floor ROIs.
5. Use the VTS to calculate the total distance moved (cm), velocity of movement (cm/sec) and duration of movement (sec). For Ethovision XT 7, export the data generated into Microsoft Excel for storage and additional analysis.

2. Long-term LMA Testing Set Up and Procedure

Long-term LMA testing cage construction
1. Cut a 5.5 cm diameter hole in the short-end wall of a standard mouse cage. Create a covering for the hole with ¼ in galvanized steel mesh. Cut the mesh, creating several ½ in openings to allow mouse access to food. Burnish any cut mesh to remove sharp edges.
2. Use additional mesh to create an external hopper directly connected to the mesh used to cover the 5.5 cm opening. Connect the hopper to the mesh used to cover the hole, and attach the assembly to the cage using aluminum rivets.
3. Construct a water bottle from a standard 50 ml conical (Corning) and a 1-hole rubber stopper (Fisher) that is over-drilled to allow for insertion of no-drip sipper tube (Sta-Pure Systems). Secure the water bottle to the cage with Velcro tape (3M) and insert the sipper tube through the mesh covered opening.
4. During long-term LMA testing, cover cages with clear ¼ in Plexiglas with 11 x ¼ in diameter holes to support appropriate ventilation.
Long-term LMA testing room
1. Long-term LMA testing room set up is identical to short-term LMA. See section 1.2 for procedure.
Long-term LMA videography
1. Center and place a ceiling-mounted infrared, security-style, camera directly linked to a PC above the recording area in the testing room. Only record the number of cages at once that results in individual cage resolution within the capability of the VTS used (e.g. Ethovision XT 7 can simultaneously track 8 mice in real-time).
Long-term LMA testing procedure
1. Test long-term LMA during the active/dark phase of the light cycle. Make sure the time relative to the light cycle is the same across all groups, as LMA follows a crepuscular pattern with more activity during the early and later periods of the dark cycle ¹. Test both experimental and control subjects in each group of recorded mice to ensure minimization of potential effects of temporal differences in LMA.
2. Individually house and acclimate the test mice in the modified cages for at least 24 hr.
3. Place the cage dividers and cages (containing individual mice) directly under the camera. Remove all nesting/enrichment material from the cages to ensure an unobstructed of view of the mouse. Mouse burrowing into the nesting material will interfere with the ability of the VTS to track the mouse. Place a clear Plexiglas lid on each of the cages to be recorded.
4. When recording several cages at once, be sure the resolution is compatible with the chosen VTS. To ease VTS analysis, position cages identically within each divider (i.e. upper right hand corner).
5. Long-term LMA testing is generally analyzed in real-time, and a video record is usually not created. A video record can be recorded by using a secondary camera or via a DVD recorder linked to the primary camera. Be sure to keep detailed records of individual mouse and group information.
6. Set up the VTS for real-time analysis. The entire cage floor should encompass the ROI for tracking. Set up ROIs for all cages being recorded. Use the VTS to calculate the total distance moved (cm), velocity of movement (cm/sec) and duration of movement (sec). In Figure 2, the detection variables were: 2 cm/sec begins tracking; 1.75 cm/sec halts tracking.
7. Begin recording mouse long-term LMA. Monitor the duration of long-term LMA testing via the clock associated with the analyzing computer.
8. During testing, keep all experimenters out of sight of the mice so as not to act as an external visual cue.
9. When testing is finished, return/replace nesting/enrichment material, to reduce the potential for development of anxiety-like behavior ⁸.
Long-term LMA analysis
1. Long-term LMA analysis occurs in real-time without the generation of a video record. Export the data into Microsoft Excel for storage and analysis, if using Ethovision XT 7.
2. To generate a video record for long-term LMA, use a secondary camera. Be sure the secondary camera has ample storage capacity sufficient to store the potentially lengthy recording(s).