Functional significance of motoneuronal persistent inward currents for locomotor activity

持续内向电流（PICs）是神经元中兴奋性（去极化）电压敏感的离子电流，一旦被激发，细胞对给定刺激的兴奋性就会提高，并且在引起它们的兴奋性刺激停止后继续流动。通过PICs，脊髓运动神经元表现出两种水平的兴奋性（称为“双稳定性”），这种由PICs引起的兴奋性升高可以通过激活特定的钙和钠通道来开启。PICs起源于树突区域，主要通过l型钙通道，其行为主要依赖于单胺- 5 -羟色胺和去甲肾上腺素，它们似乎在动物和人类的运动和其他类型的运动中被唤起。我们不知道这些PICs的“开启”如何影响受神经支配的肌纤维的力的表达，这些力容易受到刺激频率依赖性增强、抑郁和疲劳的影响。此外，支配不同“类型”（快速疲劳、快速抗疲劳和缓慢）肌纤维的运动神经元是否表达不同振幅和时间的PICs尚不清楚。最后，我们不知道神经肌肉活动水平的慢性增加是否会导致PICs的变化，导致它们的蛋白质表达的变化，以及神经支配肌纤维的合力表达的变化，这些也会随着活动的增加而变化。本研究将确定PICs在大鼠后肢运动神经元和运动单元中的功能意义。在第一阶段，我将测量大鼠运动神经元中PICs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的电生理和肌肉力反应，确定它们对神经支配肌纤维响应于向支配运动神经元注射电流时所引起的力的功能影响。这将通过支配不同肌纤维“类型”（快、慢）的运动神经元谱来完成，以确定PICs的系统性变化及其对肌肉力量产生的影响。在第二阶段，我将通过慢病毒载体将短发夹RNA序列传递到运动神经元，通过选择性下调单个蛋白（l型钙通道CaV1.3、血清素受体5HT-2A、-2C和-1A、α -1肾上腺素受体），确定特定离子通道和受体蛋白在运动神经元PIC现象中所起的作用。这将告诉我们不同的运动单元“类型”是否具有不同的pic相关蛋白质谱，并且还将允许我们估计当钙通道被消除时PICs中钠电导的特性。如果PICs对于确定有效运动模型中的基本脊柱运动模式很重要，我还将决定使用该技术。在第3阶段，我将确定16周内增加的身体活动，包括每天的跑步机训练和自愿的运动轮活动，是否会影响PICs，并改变PICs基础蛋白的表达。后者将通过在冷冻脊髓切片中取样运动神经元来进行，以测量支配不同纤维类型人群的不同后肢肌肉的运动神经元的基因表达。该结果将提高我们对PICs对靶细胞（在本例中为肌纤维）功能反应的影响的理解，以及神经肌肉异质性在使用PICs雕刻运动中的重要性。这些信息将帮助我们了解我们的神经肌肉系统在启动和执行运动运动时是如何变得“增压”的，这对优化神经肌肉性能有什么影响，并阐明在衰老和久坐不动等情况下可能发生的情况。

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.