An examination of agonist and antagonist motor unit firing properties

The interactions between opposing muscle (i.e. agonist and antagonist) groups can be extremely complex, task-dependent, and are still poorly understood. To identify possible origins of the coordination between antagonistic muscle groups, the common or shared sources of neural input need to be understood. The assessment and manipulation of motor unit firing properties, such as synchronization, can provide information regarding the common inputs to opposing muscles. PURPOSE: The purpose of this study was to introduce various interventions to systematically manipulate both agonist and antagonist motor unit firing properties, and obtain a better understanding of the interactions between the two. METHODS: Muscle activity was detected from the biceps brachii ("agonist") and the triceps brachii ("antagonist") during isometric forearm flexions. The signals from these muscles were decomposed into individual motor unit action potential trains. Subsequently, various firing properties such as mean firing rate, recruitment threshold, and synchronization were calculated. On two separate visits, either the agonist or antagonist muscle was fatigued. During another two visits, either the agonist or antagonist muscle underwent 18 minutes of prolonged stretching, which has been shown to significantly desensitize proprioceptors. RESULTS: During co-activation, the antagonist demonstrated significant motor unit synchronization, but to a lesser extent when compared to the agonist. The antagonist also exhibited a substantially smaller recruitment threshold range and higher average firing rates. Fatigue of the agonist did not show any changes to antagonist motor unit firing properties, despite a significant increase in co-activation. Fatigue of the antagonists produced effects on the motor unit behavior of the agonist, such as decreased motor unit synchronization. It was suggested that group III and IV muscle afferents originating from the antagonist were responsible for the change to the agonist. The stretching interventions provided some mixed results, often providing non-uniform changes across motor unit types. For example, agonist low-threshold motor unit pairs demonstrated an increase in short-term synchronization after agonist stretching, but the high-threshold motor unit pairs exhibited a decrease in synchronization. Future studies to help answer follow-up questions were suggested

Functional characterization of murine muscle spindles

Coordinated movements require proprioceptive information, such as information about muscle tone as well as position and movement of extremities in space. The primary proprioceptive sensory receptors are muscle spindles. Muscle spindles are complex stretch-sensitive mechanoreceptors. They detect how much and how fast a muscle is lengthened. Muscle spindles consist of specialised skeletal muscle fibers, so called intrafusal fibers. In their central part, these fibers are surrounded by a proprioceptive afferent sensory neuron in an annulospiral shape. Here the speed as well as the length of the stretch is translated into action potential frequencies, which are proportional to the length change and the speed thereof. Both polar endings are innervated by efferent γ-motoneurons. Previously it was shown that AChRs are concentrated in the polar region at the contact site between intrafusal fiber and sensory neuron. To investigate the function of these AChRs, extracellular recordings from single unit proprioceptive-afferents of wildtype murine extensor digitorum longus muscles in the absence of γ-motoneuron activity was performed. I investigated the response during ramp-and-hold stretches as well as during sinusoidal vibrations in the presence and absence of the AChR inhibitors d-tubocurarine, α-bungarotoxin or of the choline reuptake inhibitor hemicholinium-3. In the presence of either drug, the resting action potential discharge frequency was not altered but the stretch-evoked action potential frequencies were increased. Additionally, the firing rate during sinusoidal vibrations at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles. These results indicate that ACh modulates muscle spindle function during stretch in the central region of intrafusal fibers by possibly fine-tuning muscle spindle sensitivity. As a second project, I investigated the morphology and function of muscle spindles from murine models of muscular dystrophies. Muscular dystrophies comprise a heterogeneous group of hereditary diseases, which are all characterised by progressive degeneration and weakness of skeletal muscles. Murine model systems for two distinct types of muscular dystrophy with very different disease etiologies, i.e. dystrophin- and dysferlin-deficient mice, were analysed. The total number and the overall structure of muscle spindles in soleus muscles of these mice appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibers outside the region of contact to the sensory neuron. Moreover, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations. I demonstrate that mouse models for muscular dystrophy have an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches. Mutant muscle spindles show a higher action potential firing rate during sinusoidal vibrations with small amplitudes and low frequencies. I observed no exacerbated phenotype in DMDmdx- dysf-/- double transgenic mice compared to either single transgenic animal. These results demonstrate that a lack of dystrophin and or dysferlin lead to a change in muscle spindle function and suggest that an impaired proprioceptive feedback might contribute to the instable gait and the frequent falls in patients with muscular dystrophy. To test the hypothesis that an increased intracellular calcium ion concentration [Ca2+] in dystrophic muscles could cause the impaired proprioceptive function, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations in the presence and absence of the AChE inhibitor neostigmine and the calcium channel blocker nifedipine. After nifedipine and neostigmine administration an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches as well as a higher action potential firing rate during sinusoidal vibrations after neostigmine administration with small amplitudes and low frequencies was observed. Overall, I show that murine models of muscular dystrophy have an impaired muscle spindle function, which could contribute to the instable gait and posture observed in patients with muscular dystrophy, that these changes could be due to an increased intracellular [Ca2+] in muscles and that the AChR in the central part of the muscle spindles negatively modulates muscle spindle responses during stretch

Functional characterization of murine muscle spindles

Coordinated movements require proprioceptive information, such as information about muscle tone as well as position and movement of extremities in space. The primary proprioceptive sensory receptors are muscle spindles. Muscle spindles are complex stretch-sensitive mechanoreceptors. They detect how much and how fast a muscle is lengthened. Muscle spindles consist of specialised skeletal muscle fibers, so called intrafusal fibers. In their central part, these fibers are surrounded by a proprioceptive afferent sensory neuron in an annulospiral shape. Here the speed as well as the length of the stretch is translated into action potential frequencies, which are proportional to the length change and the speed thereof. Both polar endings are innervated by efferent γ-motoneurons. Previously it was shown that AChRs are concentrated in the polar region at the contact site between intrafusal fiber and sensory neuron. To investigate the function of these AChRs, extracellular recordings from single unit proprioceptive-afferents of wildtype murine extensor digitorum longus muscles in the absence of γ-motoneuron activity was performed. I investigated the response during ramp-and-hold stretches as well as during sinusoidal vibrations in the presence and absence of the AChR inhibitors d-tubocurarine, α-bungarotoxin or of the choline reuptake inhibitor hemicholinium-3. In the presence of either drug, the resting action potential discharge frequency was not altered but the stretch-evoked action potential frequencies were increased. Additionally, the firing rate during sinusoidal vibrations at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles. These results indicate that ACh modulates muscle spindle function during stretch in the central region of intrafusal fibers by possibly fine-tuning muscle spindle sensitivity. As a second project, I investigated the morphology and function of muscle spindles from murine models of muscular dystrophies. Muscular dystrophies comprise a heterogeneous group of hereditary diseases, which are all characterised by progressive degeneration and weakness of skeletal muscles. Murine model systems for two distinct types of muscular dystrophy with very different disease etiologies, i.e. dystrophin- and dysferlin-deficient mice, were analysed. The total number and the overall structure of muscle spindles in soleus muscles of these mice appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibers outside the region of contact to the sensory neuron. Moreover, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations. I demonstrate that mouse models for muscular dystrophy have an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches. Mutant muscle spindles show a higher action potential firing rate during sinusoidal vibrations with small amplitudes and low frequencies. I observed no exacerbated phenotype in DMDmdx- dysf-/- double transgenic mice compared to either single transgenic animal. These results demonstrate that a lack of dystrophin and or dysferlin lead to a change in muscle spindle function and suggest that an impaired proprioceptive feedback might contribute to the instable gait and the frequent falls in patients with muscular dystrophy. To test the hypothesis that an increased intracellular calcium ion concentration [Ca2+] in dystrophic muscles could cause the impaired proprioceptive function, extracellular recordings from single units of sensory afferents from muscle spindles of the extensor digitorum longus muscle were performed during ramp-and-hold stretches, as well as during sinusoidal vibrations in the presence and absence of the AChE inhibitor neostigmine and the calcium channel blocker nifedipine. After nifedipine and neostigmine administration an increased resting discharge but no change during the dynamic or static phase of ramp-and-hold stretches as well as a higher action potential firing rate during sinusoidal vibrations after neostigmine administration with small amplitudes and low frequencies was observed. Overall, I show that murine models of muscular dystrophy have an impaired muscle spindle function, which could contribute to the instable gait and posture observed in patients with muscular dystrophy, that these changes could be due to an increased intracellular [Ca2+] in muscles and that the AChR in the central part of the muscle spindles negatively modulates muscle spindle responses during stretch

The measurement of knee joint position sense

It is commonly stated proprioception or the perception of one’s own limb position, movement and effort is reduced following an anterior cruciate ligament (ACL) injury. Therefore, this thesis begins with an analysis of all current literature on this topic in the form of a meta-analysis. It became clear that the methods used to measure knee proprioception were very inconsistent and did not appear to provide normative levels of knee proprioception making it very difficult to syntheses results. This led to the thesis main objectives. The first study provided a reliable and valid method of knee joint position sense (JPS), the static component of proprioception, based on previous JPS protocols. This method was then used in the remaining studies to consider normative values of a UK population, the effect of ACL injury in both non-athletic and elite athletic populations, the effect of knee injuries (not including ligament damage) and the effect of fatigue on knee JPS. The most appropriate clinical method of measuring knee JPS using image capture as covered in this thesis was in a sitting position, from full extension in to 60-90 degrees of flexion and from 90 degrees of flexion in to 30-60 degrees of extension. Age, mass, height, BMI, activity level, knee condition (other than ligament injury) or fatigue did not appear to significantly affect knee JPS in an uninjured population. However, both non-athletic and elite athletic populations with previous ACL injury demonstrated significantly worse knee JPS when compared to controls. In conclusion, it would appear the only knee condition that reduces joint position sense ability is ACL injury. Although, it may also be possible the method is not sensitive enough to measure subtle changes in JPS in other populations due to large measurement error values. In the future it may not be necessary to place importance on knee joint position sense as it either may not be impacted by any injury other than ACL damage, or the methods used to collect JPS are not sufficient in measuring changes during rehabilitation. Additionally, it is important researchers consider the relationship between knee JPS and functional movements

Upper limb proprioceptive sensitivity in three-dimensional space: effects of direction, posture, and exogenous neuromodulation

abstract: Proprioception is the sense of body position, movement, force, and effort. Loss of proprioception can affect planning and control of limb and body movements, negatively impacting activities of daily living and quality of life. Assessments employing planar robots have shown that proprioceptive sensitivity is directionally dependent within the horizontal plane however, few studies have looked at proprioceptive sensitivity in 3d space. In addition, the extent to which proprioceptive sensitivity is modifiable by factors such as exogenous neuromodulation is unclear. To investigate proprioceptive sensitivity in 3d we developed a novel experimental paradigm employing a 7-DoF robot arm, which enables reliable testing of arm proprioception along arbitrary paths in 3d space, including vertical motion which has previously been neglected. A participant’s right arm was coupled to a trough held by the robot that stabilized the wrist and forearm, allowing for changes in configuration only at the elbow and shoulder. Sensitivity to imposed displacements of the endpoint of the arm were evaluated using a “same/different” task, where participant’s hands were moved 1-4 cm from a previously visited reference position. A measure of sensitivity (d’) was compared across 6 movement directions and between 2 postures. For all directions, sensitivity increased monotonically as the distance from the reference location increased. Sensitivity was also shown to be anisotropic (directionally dependent) which has implications for our understanding of the planning and control of reaching movements in 3d space. The effect of neuromodulation on proprioceptive sensitivity was assessed using transcutaneous electrical nerve stimulation (TENS), which has been shown to have beneficial effects on human cognitive and sensorimotor performance in other contexts. In this pilot study the effects of two frequencies (30hz and 300hz) and three electrode configurations were examined. No effect of electrode configuration was found, however sensitivity with 30hz stimulation was significantly lower than with 300hz stimulation (which was similar to sensitivity without stimulation). Although TENS was shown to modulate proprioceptive sensitivity, additional experiments are required to determine if TENS can produce enhancement rather than depression of sensitivity which would have positive implications for rehabilitation of proprioceptive deficits arising from stroke and other disorders.Dissertation/ThesisDoctoral Dissertation Neuroscience 201

Cadence and range of motion modulate pedal force in a rat model of motorized cycling after spinal cord injury.

Motorized cycling (MC) can be utilized post-spinal cord injury (SCI) in patients who lack the strength and/or stability to participate in traditional physical exercise interventions. MC has been applied with the goal of improving locomotor function or cardiovascular health in both human and animal models of SCI. However, a discrepancy exists between the results of human and animal studies of MC, particularly regarding cardiovascular outcomes. Despite the abundance of studies in both humans and animals, the mechanism behind the improvements in cardiovascular function following MC are poorly understood. We posited that increased venous return during MC is likely due to the skeletal muscle pump, where muscle activity during MC would be triggered by stretch reflexes. As stretch reflexes are dependent on both rate and length of muscle stretch, we hypothesized that cycling cadence and crank length could modulate muscle activity and therefore hindlimb loading during cycling. Initial studies testing the development of the instrumented pedals noted spasticity that was represented in the force traces, and a filtering technique was developed to separate spastic from non-spastic forces. Results using this technique combined with EMG of a knee flexor and extensor suggest that higher cadences (≥30 RPM) increased RMS EMG and non-spastic forces, while lower cadences (≤15 RPM) increased spastic forces. Furthermore, large spastic events were associated with a decrease in BP, while high cadence cycling with limited spasticity appeared to elevate BP and HR above baseline levels. These results suggest that MC in rats may constitute a mild eccentric training regimen; clinical translation may therefore be dependent on the ability to reflexively generate muscle contraction in patients during cycling

Protection of neuromuscular sensory endings by the WldS gene

The compartmental hypothesis of neurodegeneration proposes that the neurone, long recognized to consist of morphologically and functionally distinct compartments, also houses distinct degeneration mechanisms for the soma, axon and nerve endings. Support for this hypothesis is provided by the phenomenon of the WldS (for Wallerian Degeneration, slow) mouse, a mutant in which axons survive several weeks after transection, rather than degenerating within 24-48 hours as in wild type mice, by virtue of expression of a chimeric Nmnat1/Ube4b protein. In this thesis I used the WldS-mouse to re-examine and extend the theory of compartmental neurodegeneration by focusing specifically on sensory axons and endings; and finally by considering a fourth compartment, the dendrites. The first part of this thesis reports that Ia afferent axons and their annulospiral endings are robustly protected from degeneration in WldS mice. Homozygous or heterozygous WldS mice crossbred with transgenic mice expressing fluorescent protein in neurones were sacrificed at various times after sciatic nerve transection. Fluorescence microscopy of whole mount preparations of lumbrical muscles in these mice revealed excellent preservation of annulospiral endings on muscle spindles for at least 10 days after axotomy. No significant difference was detected in the protection with age or gene copy-number in contrast to the protection of motor nerve terminals, which degenerate rapidly in heterozygote and aged homozygote WldS mice. In an attempt to explain the difference in motor and sensory protection by WldS, examination of three hypotheses was undertaken: a) differences in protein expression, tested by western blot and immunohistochemistry; b) differences in the degree of neuronal branching, tested through examination of g-motor axons and endings which have a degree of branching intermediate to motor and sensory neurons; and c) differences in the activity in the disconnected stumps, through primary culture of the saphenous and phrenic nerve, selected because they comprise largely pure sensory and motor axons respectively. The data suggest that none of these hypotheses provides a sufficient explanation for the difference between sensory and motor protection by WldS. The last part of this thesis attempts to extend the theory of compartmental degeneration. I examine a system for investigation of WldS-mediated protection of dendrites. In preliminary experiments retinal explants from transgenic mice expressing YFP in a subset of retinal ganglion-cell neurones were cultured. The dendritic arbours of these cells were shown to be amenable for repeated visualization and accessible to injury and monitoring of degeneration. Overall the data in this thesis suggest that the level of WldS -mediated protection conferred to an axon or axonal endings varies between different neuronal types. This has implications for the potential applications of WldS research to clinical problems. Specifically, the data imply that sensory neuropathies may benefit more than motor neuropathies from treatments based on the protective effects of WldS. These findings in sensory neurones also challenge some of the assumptions made about WldS- mediated protection of neurones, for example the extent of the age-effect on axonal endings. Further investigation of WldS-mediated protection in the CNS could give renewed impetus to attempts to discover targets for treatment in common neurodegenerative diseases. Finally, a system for investigation of dendritic degeneration has been piloted, suggesting that molecules involved in the degeneration of dendrites or in protection from this degeneration may be amenable to investigation in this system, prospectively extending the compartmental hypothesis of neuronal degeneration

Mechanisms Underlying Performance Impairments Following Prolonged Static Stretching Without a Comprehensive Warm-up

Whereas a variety of pre-exercise activities have been incorporated as part of a “warm-up” prior to work, combat, and athletic activities for millennia, the inclusion of static stretching (SS) within a warm-up has lost favour in the last 25 years. Research emphasised the possibility of SS-induced impairments in subsequent performance following prolonged stretching without proper dynamic warm-up activities. Proposed mechanisms underlying stretch-induced deficits include both neural (i.e. decreased voluntary activation, persistent inward current effects on motoneurone excitability) and morphological (i.e. changes in the force-length relationship, decreased Ca2+ sensitivity, alterations in parallel elastic component) factors. Psychological influences such as a mental energy deficit and nocebo effects could also adversely affect performance. However, significant practical limitations exist within published studies, e.g. long stretching durations, stretching exercises with little task specificity, lack of warm-up before/after stretching, testing performed immediately after stretch completion, and risk of investigator and participant bias. Recent research indicates that appropriate durations of static stretching performed within a full warm-up (i.e. aerobic activities before and task-specific dynamic stretching and intense physical activities after SS) have trivial effects on subsequent performance with some evidence of improved force output at longer muscle lengths. For conditions in which muscular force production is compromised by stretching, knowledge of the underlying mechanisms would aid development of mitigation strategies. However, these mechanisms are yet to be perfectly defined. More information is needed to better understand both the warm-up components and mechanisms that contribute to performance enhancements or impairments when SS is incorporated within a pre-activity warm-up