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The Effect of EGR3 Conditional Knockout on Mouse Locomotion
Kathy Xiang1; Adnan Hossain1; Vicki Tysseling2; Derin Birch2
1Adlai
Background
E. Stevenson High School, 2Northwestern University
Methods and Materials
Locomotor functions are known to be directed by central pattern
generators (CPGs), which are interneuronal networks in the spinal cord
that produce rhythmic patterned outputs for motor functions such as
walking and swimming. While CPGs can be generated without sensory
input, the CPG is not sufficient to reproduce normal locomotor output.
Warren G. Tourtellotte has created a new knockout mouse called Early
Growth Response 3 (EGR3), in which the EGR3 protein, critical in the
development of muscle spindles, has been conditionally knocked out
(cKo) in the cells that produce actin (i.e. skeletal muscle cells).
Muscle spindles play a crucial role in proprioception by detecting
changes in muscle length and transmitting the information to the central
nervous system. Muscle spindles are also involved in the stretch reflex
by triggering the regulation of muscle contraction when a skeletal
muscle is stretched. The contribution of proprioceptive feedback to the
coordination of locomotor activity is uncertain.
1. Anesthetize mice with isoflurane (2.5%)
2. Insert electrode into Tibialis Anterior (TA) and Lateral Gastrocnemius
(LG) muscles
3. Attach reflective markers to 6 landmarks: ASIS, PSIS, greater
trochanter, knee joint, lateral malleolus, and head of 5th metatarsal
4. Place mice onto rodent treadmill at speed 6 m/min and 9 m/min
5. Data quantified by recordings in Spike2 and Vicon
6. Data from 43 mice collected (19 wild-type, 24 cKO). 32 mice were
selected for data analysis.
7. Wilcoxon Rank Sum tests performed to determine statistical
significance (p<0.05)
Mice donated by Warren Tourtellotte’s laboratory in Pathology and
Neurology at Northwestern University
Hypothesis
Figure 3. Sample of Wild-Type mouse electromyogram (EMG) recording in Spike2
Figure 1. Reflective markers
on leg of mouse
Figure 1. Engineering of a conditional Knockout mouse
0.52
0.56
0.74
0.516
0.54
LG Duration/Step Duration
6 m/min
0.730
0.72
0.535
0.51
0.7
0.5
0.52
0.49
0.68
0.5
0.48
0.66
0.47
0.48
0.457
0.46
0.64
0.631
0.464
0.46
0.45
0.62
0.44
0.44
0.6
0.43
0.42
0.58
0.42
Wild-type
EGR3
Center of activation of LG/step duration at
speed 6 m/min was 11.4% smaller in EGR3
mice than in wild-type.
Wild-type
EGR3
Center of activation of LG/step duration
at speed 9 m/min was 13.3% smaller in
EGR3 mice than in wild-type.
Wild-type
EGR3
LG duration/step duration at speed 6
m/min was 13.6% smaller in EGR3 mice
than in wild-type mice.
Discussion
The ratio of LG activation duration to step duration at speed 6 m/min as
well as the ratio of the center of activation of LG to step length (i.e. the
time it takes for the muscle to attain its center of activation over the
duration of the step) for both speed 6 m/min and 9 m/min were smaller
in EGR3 mice than in wild type.
What is the contribution of muscle spindles to the coordination of
locomotor activity?
Figure 2. Vicon
representation of mouse on
treadmill
0.53
Center of Activation LG/Step Duration
9 m/min
The results obtained from trials at both speed 6 m/min and 9 m/min
support the hypothesis that EGR3 mice have gait ataxia in the form of a
compromised step cycle and muscle activation pattern.
Purpose
If functional muscle spindles in the skeletal muscles of mice are
eliminated as a source of proprioceptive feedback, then the locomotive
pattern of mice will degenerate and the mice will have a compromised
step cycle and muscle activation pattern.
Center of Activation LG/Step Duration
6 m/min
The LG muscle is utilized in plantar flexion of the foot, so the results
signify that the EGR3 mice are pushing off the ground for a shorter
duration per step than are the wild-type mice. This, in effect, shortens
the entire step length as well; it is thus concluded that EGR3 mice need
to take more steps than wild-type mice to travel the same distance. The
incompetency demonstrated with a loss of muscle spindles suggests
that muscle spindles help to maximize efficiency in locomotor function.
A lack of muscle spindles ensues in an unnecessary waste of energy
during locomotion. Additionally, the loss of proprioception restricts the
ability of the EGR3 mouse to explore its environment as freely as a
normal mouse; this is evident in its shorter steps, which are better for
testing the environment.
Statistically significant differences (p<.05) were only found in
measurements of LG. However, there was strong trend of smaller TA
length/step duration ratios in EGR3 mice, meaning the dorsiflexion in
EGR3 mice was shorter than in wild-type mice.
Figure 4. Sample of EGR3 mouse electromyogram (EMG) recording in Spike2
Future kinematic analyses of limb trajectories will provide a clearer
picture of the relationships between hip, knee, and ankle joints and
possibly reveal the compensation of loss of proprioception with another
locomotive process.
Acknowledgments & References
All procedures and animal protocols were approved by the Northwestern University Institutional Animal Care and Use Committee.
Mice donated by Warren Tourtellotte’s laboratory in Pathology and Neurology at Northwestern University
Akay, T., Tourtellotte, W., Arber, S., and Jessell, T. (2014). Proceedings of the National Academy of Sciences. Degradation of Mouse Locomotor Pattern in the Absence of Proprioceptive Sensory Feedback, 111.
Scholz, John P., and Suzann K. Campbell. "Muscle Spindles and the Regulation of Movement." Acta Physiologica Scandinavica 49: Physical Therapy: Journal of the American Physical Therapy Association, 30 Mar. 1980. Web. 15 July 2015.