Functional significance of motoneuronal persistent inward currents for locomotor activity

运动神经元持续内向电流对运动活动的功能意义

• 批准号: RGPIN-2014-03861
• 负责人: Gardiner, Phillip
• 金额: $ 2.91万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630521
• 关键词: Functional; significance; motoneuronal; persistent; inward

Persistent inward currents (PICs) are excitatory (depolarizing) voltage-sensitive ionic currents in neurons that, once evoked, raise cell excitability to a given stimulus, and continue to flow after cessation of the excitatory stimulus that evoked them. Through PICs, spinal motoneurons exhibit two levels of excitability (termed “bistability”), and this elevated excitability caused by PICs can be turned on through the activation of specific calcium and sodium channels. PICs originate in the dendritic region, primarily through L-type calcium channels, their behaviour is dependent primarily on the monoamines serotonin and norepinephrine, and they seem to be evoked during locomotion and other types of movement, in animals and humans. We do not know how “turning on” of these PICs influences the expression of the forces of the innervated muscle fibers, which are prone to the effects of stimulation frequency-dependent potentiation, depression, and fatigue. In addition, whether motoneurons innervating muscle fibers of different “types” (fast fatiguing, fast fatigue-resistant, and slow) express PICs of differing amplitudes and time is unknown. Finally, we do not know if chronic increase in neuromuscular activity level causes changes in PICs, in the expression of the proteins that cause them, and in the resultant force expression by the innervated muscle fibers which also change with increased activity. The proposed research will determine the functional significance of PICs in rat hindlimb motoneurons, and motor units. In phase 1, I will measure the amplitude and time course of activation/inactivation of PICs in rat motoneurons, and determine their functional impact on the forces evoked in the innervated muscle fibers in response to injection of currents into the innervating motoneuron, by recording motoneuron electrophysiological and muscle force responses simultaneously. This will be done across the spectrum of motoneurons innervating different muscle fiber “types" (fast, slow), to determine of PICs vary systematically, and their impact on muscle force generation. In phase 2, I will determine the role played by specific ion channel and receptor proteins in the PIC phenomenon in motoneurons, by selectively down-regulating individual proteins (L-type calcium channel CaV1.3, serotonin receptor 5HT-2A, -2C, and -1A, alpha-1 adrenoceptor) using short hairpin RNA sequences delivered to motoneurons via lentivirus vectors. This will tell us if different motor unit “types” have different PIC-related protein profiles, and will also allow us to estimate the characteristics of the sodium conductance in PICs when the calcium channel is eliminated. I will also determine using this technology if PICs are important for determining basic spinal locomotion patterns in a fictive locomotion model. In phase 3, I will determine if increased physical activity for 16 weeks, including daily treadmill training and voluntary exercise wheel activity, affects PICs, and changes the expression of proteins underlying PICs. The latter will be performed by sampling motoneurons in frozen spinal cord sections in order to measure gene expression in motoneurons innervating distinct hindlimb muscles that differ in fiber-type populations. The results will improve our understanding of the impact of PICs on the functional response of the target cell (in this case, muscle fibers), and of the significance of neuromuscular heterogeneity in sculpting movements using PICs. Information will help us understand how our neuromuscular system becomes "supercharged" when initiating and performing locomotor movements, what the implications are for optimization of neuromuscular performance, and shed light on what might occur in conditions such as aging and sedentarism.

持续性内向电流（PIC）是神经元中的兴奋性（去极化）电压敏感性离子电流，一旦被诱发，就会提高细胞对给定刺激的兴奋性，并在诱发它们的兴奋性刺激停止后继续流动。通过PIC，脊髓运动神经元表现出两种水平的兴奋性（称为“双稳态”），并且由PIC引起的这种升高的兴奋性可以通过激活特定的钙和钠通道来开启。PIC起源于树突区域，主要通过L型钙通道，它们的行为主要依赖于单胺5-羟色胺和去甲肾上腺素，并且它们似乎在动物和人类的运动和其他类型的运动期间被诱发。我们不知道这些PIC的“开启”如何影响受神经支配的肌纤维的力的表达，这些肌纤维容易受到刺激频率依赖性增强、抑郁和疲劳的影响。此外，是否运动神经元支配不同的“类型”的肌纤维（快速疲劳，快速抗疲劳，慢）表达不同幅度和时间的PIC是未知的。最后，我们不知道神经肌肉活动水平的慢性增加是否会引起PIC的变化，引起它们的蛋白质的表达，以及受神经支配的肌肉纤维的合力表达也会随着活动的增加而变化。这项研究将确定大鼠后肢运动神经元和运动单位中PICs的功能意义。在第一阶段，我将测量大鼠运动神经元中PIC的激活/失活的幅度和时间过程，并通过同时记录运动神经元电生理和肌肉力反应来确定它们对神经支配的肌纤维中诱发的力的功能影响。这将在支配不同肌纤维“类型”（快，慢）的运动神经元的频谱中进行，以确定系统性变化的PIC及其对肌肉力量产生的影响。在第2阶段，我将确定特定的离子通道和受体蛋白在运动神经元PIC现象中所起的作用，通过选择性下调单个蛋白（L型钙通道CaV1.3，5-羟色胺受体5 HT-2A，-2C和-1A，alpha-1肾上腺素受体），使用短发夹RNA序列通过慢病毒载体传递到运动神经元。这将告诉我们不同的运动单位“类型”是否具有不同的PIC相关蛋白质谱，并且还将使我们能够估计当钙通道被消除时PIC中钠电导的特征。我还将确定使用这种技术，如果PIC是重要的，以确定基本的脊髓运动模式在一个虚构的运动模型。在第三阶段，我将确定16周的身体活动增加，包括每日跑步机训练和自愿运动轮活动，是否会影响PIC，并改变PIC的蛋白质表达。后者将通过对冷冻脊髓切片中的运动神经元进行取样来进行，以测量支配不同后肢肌肉的运动神经元中的基因表达，所述后肢肌肉在纤维类型群体中不同。这些结果将提高我们对PIC对靶细胞（在这种情况下，肌肉纤维）功能反应的影响的理解，以及神经肌肉异质性在使用PIC塑造运动中的意义。信息将帮助我们了解我们的神经肌肉系统如何在启动和执行运动动作时变得“增压”，这对神经肌肉性能的优化有什么影响，并阐明在衰老和久坐等条件下可能发生的情况。