Coordinated ambulation is fundamental to our ability work and play. As bipedals, we humans invoke our upper limbs in walking to a greater extent than we conscientiously appreciate. Coordinated movement requires sensory and proprioceptive inputs to rapidly formulate and execute the concerted motor response; loss of even a portion of an arm greatly affects lower limb posture and kinematics.
Quadrupedal gait, from which human bipedal gait has evolved, is increasingly being studied to understand not only the mechanisms that control coordinated gait but also those that contribute to loss of coordinated gait that might occur with trauma, such as brain or spinal cord injury.
Locomotion in mammals relies on a central pattern generating circuitry of spinal interneurons established during development that coordinates limb movement. However, little is known about the constraints that link left–right coordination. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left–right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left–right alternation, are lacking. Recently published studies show that mutations in Bace1 (1), DMRT3 (2), and the ablation of V0 (3) and V2a (4) neurons lead to coordination changes in mouse models.
One study (1) suggests that the smooth processing of coordinated walking is disrupted in the absence of Bace1 due to a reduction of spindle number and maturation. Bace1 processes an isoform of neuregulin-1 (IgNrg1), a growth factor important in the regulation of nervous system development and regeneration. The researchers found that Bace1 helps regulate the physiology of muscle spindles, sensory organs dispersed through the muscles that detect changes in their length. Such information is conveyed to the brain via the central nervous system to establish their “next move”. Much of mammalian movement, human and animal alike, is executed rapidly and autonomically according to the muscles’ proprioceptive inputs. Quantitative gait analysis demonstrated that animals treated with a Bace1 inhibitor lost their footing sooner than animals treated with the vehicle. Gait analysis also demonstrated that the walking pattern of the mice was aberrant after long-term inhibition with Bace1 inhibitor. Homolateral coupling deviated considerably from vehicle-treated animals. This reflects a lack of forelimb/hind limb coordination and resulted in a swaying walking pattern.
Gait analysis is employed to quantify improvements in gait coordination between subjects with gait disturbances, such as occur with Parkinson’s disease and ALS, and treatment groups. DigiGait is also an excellent motor rater, providing assesment of fine motor skills. DigiGait enables accurate and quantitative gait analysis in rodent models, such as mice and rats. The compartments of DigiGait are interchangeable between small rodents (mice) and larger animals (hamsters, rats, guinea pigs) in less than one minute. DigiGait images and quantifies limb kinematics to describe strength, balance, and coordination in laboratory animals. Video 1, for example, depicts the ventral view of a walking control mouse; arrows indicate the direction of motion of the limbs.
Video 1. Ventral view of mouse walking on DigiGait; arrows indicate direction of motion of the limbs. This healthy mouse exhibits coordinated gait, following an alternate stepping pattern, in which left fore is followed by right hind, followed by right fore, followed by left hind, repeat.
Conditional IgNrg1 mutants displayed deficits in forelimb/hind limb coordination (1). Graded reductions in IgNrg1 signal strength appears to result in graded deficits in muscle spindle formation and aberrations in coordination. The study also showed that increased expression of igNrg1b1 resulted in a huge increase in muscle spindle numbers at birth. It would be interesting to know whether such mice develop the capacity for coordinated ambulation sooner than control mice [~post natal day 14]. Several developmental disorders, such as the leukodystrophies Canavan disease and Krabbe disease, affect myelin and are associated with loss of coordination; neonatal Canavan and Krabbe disease mice (5) exhibit postural and kinematic gait disturbances. Bace1 deficiency in mice causes hypomyelination during development and impairs remyelination if injured. The DigiGait Imaging System has shown that mice are able to treadmill walk with coordination at about post natal day 14 (6) [see Video 2].
Many models of human diseases result in early morbidity and mortality in newborn pups. DigiGait provides the opportunity to evaluate gait in neonatal animals shortly after they open their eyes, usually around postnatal day 14. Gait changes almost by the day as the pups gain muscle strength and neuromuscular control. In this proprietary model, one can see that the gait in the subject in the lower panel is not as fluid and robust as its healthy sibling. DigiGait reports numerous metrics to help identify the pathogenesis of the gait disturbance.
“…better data from every mouse!”
Video 2. DigiGait side view of juvenile [post natal day 14] mouse executing coordinated stepping.
Coordinated ambulation in quadrupeds, however, comes in several forms. The baseline “normal” gait for most rodents is an alternate stepping gait, whereby recruitment of a forelimb is followed by recruitment of the contralateral hind limb, followed by the its ipsilateral forelimb, followed by its contralateral hind limb, then repeat (7). This type of gait is a “lateral” gait sequence, because the next forepaw placement is ipsilateral to the previous hind paw placement (8 Hildebrand, 1989). Interestingly, the gait of Bace -/- mice, or mice administered a Bace1 inhibitor (1), displayed more of a pacing gait, which probably should not be considered as a defective gait. The limb recruitment pattern of LF followed by LH followed by RF followed by RH does indeed appear to result in more of a swaying type walk, but is performed by healthy quadrupeds, including horses, camels, lamas, cats, guinea pigs, and mice. It would be interesting to know the characteristics of muscle spindles in limbs of quadrupeds that routinely pace, such as camels.
While the alternate step sequence is the most efficient and stable gait pattern in mammals, the ability to change to a different gait pattern may be advantageous to different situations. Indeed, even a hopping gait is prospectively chosen by many quadrupeds. The defective gait described for mice with ablation of neuregulin-1 (1) should rather, therefore, be deemed a failure to execute any coordinated ambulation [such as hopping] because the step sequence described for these animals does not sustain locomotion, making them easy prey.
Video 3. Even small differences in walking speed significantly impact gait metrics, including coordination, stride length and paw angles. Image depicts an alternate stepping sequence of a guinea pig at a walking speed of 18 cm/s, and a pacing gait at a faster walking speed of 36 cm/s. Images and analysis performed with DigiGait.
It might be interesting to know, moreover, if under conditions where loss of coordination is phenotypic is associated with changes in maintenance of muscle spindles. Disturbed gait, for example, is one of the most consistent and salient sequelae of chronic alcoholism (9). Loss of coordination is often used to describe intoxicated movement in mice (10). Certainly acute ethanol administration results in alterations in neuromuscular function. However, look here at the effects of acute ethanol on gait as seen by DigiGait. Despite postural alterations during walking, coordination per se is not affected by acute ethanol in the example shown in Video 4. The lateral view provided by the companion DigiGait clearly indicate, rather, the effect of ethanol on reduced postural muscle tone. Note, for instance, the reduction in tail height, the reduced lift of the hind paws, and the drop of the animal’s torso during walking after ethanol administration.
Video 4a and 4b. Gait of B6 mouse after i.p. saline administration (top) and ~5 minutes after ~1.5 g/kg ethanol (bottom). Despite the clear effects of ethanol on aspects of gait – note, for example, narrowed forelimb stance width and change to the hind paw placement angle- coordination per se is intact; the ethanol-treated mouse still executes an alternate step sequence pattern.
Researchers reported that mice with mutations in DMRT3 show frequent twitching limb movements, rarely observed in controls, and also have major difficulties running at higher velocities (2). Gait analysis also reveals significantly increased stride length in all limbs of mice with mutations in DMRT3. Swing times (flexion) are increased in all limbs, whereas stance time (extension) is increased in forelimbs. Moreover, propulsion time increases in all limbs, whereas brake time is decreased in hind limbs, indicating that the mutant mice may emphasize extension movements, resulting in a longer stride. DigiGait includes its patented motorized transparent treadmill belt, which is speed adjustable over the range of 1 to 100 cm/s. This allows the mice to run at different velocities and examine a range of walking patterns and coordination of limb recruitment. DigiGait reports over 40 spatial and temporal gait indices reported in spreadsheet-ready format, including stance, swing, braking, propulsion, cadence, step sequence, regularity index, and the sciatic functional index (Figure 1).
Figure 1. DigiGait user interface (top) and the spreadsheet reporting the kinematic and postural gait metrics (bottom).
Another gait analysis study recently published in Nature shows the ablation of V0 neurons led to a hopping gait in mouse models (3). V0 neurons are characterized by the early expression of the transcription factor Dbx1. V0 neurons represent a major class of commissural neurons in the ventral spinal cord, where the locomotor network is localized. Ventral plane videography enabled the researchers to observe various gait patterns, including a hopping gait.
Crone et al. (4) applied the DigiGait Imaging System to show that V2a interneurons in the lower spinal cord are responsible for maintaining left-right alternation at faster speeds of locomotion. Excitatory V2a interneurons synapse onto commissural neurons, which coordinate activity in the left and right sides of the spinal cord. The capability of DigiGait to operate at high speeds showed that loss of V2a does not significantly alter the ability of the mice to run at a wide range of speeds.
DigiGait showed that the gait pattern of wild type mice consists of strict alternation between left and right fore and hind legs at slow (25 cm/s) and fast (85 cm/s) treadmill speeds. At slow speed (25 cm/s) the gait pattern of mice lacking V2a interneurons also shows normal alternation between left and right forelimbs and the left and right hind limbs. However, at high speeds, mice lacking V2a interneurons show a “galloping” gait in which the left and right forelimbs as well as left and right hind limbs move synchronously in the same phase. These results demonstrate a speed-dependent disruption of left-right alternation of the fore and hind limbs, resulting in the expression of a novel “galloping” gait in the absence of the V2a interneurons.
In summary, gait analysis is increasing be utilized to study coordinated gait and the loss of coordination that occurs with disease, disorders, and very often as a side effect of drugs. Also, DigiGait is useful for fine motor skill assessment and is a great motor rater. DigiGait is a powerful tool to perform quantitative gait analysis in laboratory animal models. We welcome researchers to utilize our complimentary service to generate data regarding locomotion in their animals.
- Cheret et al. Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles. The EMBO Journal 2013; 32: 2015-28.
- Andersson et al. Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice. Nature 2012; 30;488:642-6.
- Talpalar et al. Dual-mode operation of neuronal networks involved in left–right alternation. Nature 2013; 500:85-89.
- Crone et al. In mice lacking V2a interneurons, gait depends on speed of locomotion. J Neurosci. 2009; 29(21): 7098–7109.
- Rafi et al. Extended Normal Life After AAVrh10-mediated Gene Therapy in the Mouse Model of Krabbe Disease. Molecular Therapy 20: 2031–2042; 2012.
- Kale et al. Ethanol’s Effects on Gait Dynamics in Mice Investigated by Ventral Plane Videography. Alcohol Clin Exp Res; 2004; 28:1839–1848.
- Hildebrand M (1989) The quadrupedal gait of vertebrates. Bioscience 39:766 –775.
- Fein G, Greenstein D. Gait and balance deficits in chronic alcoholics: no improvement from 10 weeks through 1 year abstinence. Alcohol Clin Exp Res. 2013; 37:86-95.
- Rustay et al. Influence of task parameters on rotarod performance and sensitivity to ethanol in mice. Behav Brain Res. 2003; 15:141:237-49.
- Forbes et al. Effects of intermittent binge alcohol exposure on long-term motor function in young rats. Alcohol. 2013; 47(2):95-102.