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Functional characterization of murine muscle spindles. modulatory roles of acetylcholine receptors and of the dystrophin-associated protein complex
Functional characterization of murine muscle spindles. modulatory roles of acetylcholine receptors and of the dystrophin-associated protein complex
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.
muscle spindle, dystrophin, acetylcholine receptor
Gerwin, Laura
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Gerwin, Laura (2020): Functional characterization of murine muscle spindles: modulatory roles of acetylcholine receptors and of the dystrophin-associated protein complex. Dissertation, LMU München: Faculty of Medicine
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Abstract

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.