Influence of Innervation on Diaphragm Muscle Growth

描述（由申请人提供）：拟定的研究解决了关于膈运动神经元和膈肌（DIAm）纤维出生后生长的非常基本的问题。膈运动神经元和DIAm纤维的这种生长对于满足DIAm日益增加的功能多样性是必要的。以下问题是确定神经-肌肉相互作用对肌肉生长的作用的总体目标的自然延伸。通过增强囊泡释放，BDNF/TrkB信号传导可以影响其他神经源性营养因子的释放（例如，neuregulin，NRG），从而间接促进出生后DIAm纤维生长。因此，在本修订申请中，我们将研究BDNF/TrkB信号增强囊泡释放和神经肌肉传递，从而间接促进出生后DIAm纤维生长的假设。为了解决这一假设，我们提出了两个新的具体目标：具体目标1。研究BDNF/TrkB信号在DIAm出生后早期发育过程中对囊泡释放和神经肌肉传递的作用。我们将使用两种互补的方法：1）突触囊泡循环（FM 4 -64荧光摄取）的最新共聚焦成像技术，和2）在DIAm的重复刺激期间对神经肌肉传递失败的易感性。 具体目标2。确定抑制BDNF/TrkB信号传导对出生后DIAm纤维生长的影响。我们将使用最近开发的表达敲入等位基因（TrkBF 616 A）的小鼠模型，该模型允许通过膜渗透性小分子蛋白磷酸酶PP 1衍生物（例如，1 NMPP1）。 公共卫生相关性：横膈膜是哺乳动物最重要的吸气肌，从出生起就必须提供足够的力量来维持通气。与四肢肌肉相比，它是独特的，在出生后，运动行为逐渐出现。这些研究将使我们更好地了解膈肌的正常成熟过程和支配膈肌的运动神经元，并系统地探索调节膈肌正常生长和发育的因素。

DESCRIPTION (provided by applicant): The proposed studies address very basic questions about the postnatal growth of phrenic motoneurons and diaphragm muscle (DIAm) fibers. Such growth of phrenic motoneurons and DIAm fibers is necessary to meet the increasing functional diversity of the DIAm. The following issues are a natural extension of the overall goal of determining the role of nerve-muscle interactions on muscle growth. By enhancing vesicular release, BDNF/TrkB signaling could affect release of other nerve-derived trophic factors (e.g., neuregulin, NRG), thereby indirectly promoting postnatal DIAm fiber growth. Therefore, in this revision application we will examine the hypothesis that BDNF/TrkB signaling enhances vesicular release and neuromuscular transmission and, thus, indirectly promotes postnatal growth of DIAm fibers. To address this hypothesis, we propose two new specific aims: Specific Aim 1. To examine the role of BDNF/TrkB signaling on vesicular release and neuromuscular transmission during early postnatal development of the DIAm. We will use two complementary approaches:1) state of the art confocal imaging techniques of synaptic vesicle cycling (FM4-64 fluorescence uptake), and 2) susceptibility to neuromuscular transmission failure during repetitive stimulation of the DIAm. Specific Aim 2. To determine the effect of inhibiting BDNF/TrkB signaling on postnatal growth of DIAm fibers. We will use a recently developed mouse model expressing knockin alleles (TrkBF616A) that permits rapid, reversible and selective inhibition of TrkB kinase activity by membrane-permeable small-molecule protein phosphatase PP1 derivatives (e.g., 1NMPP1). PUBLIC HEALTH RELEVANCE: The diaphragm is the most important inspiratory muscle in mammals; it must provide adequate force to sustain ventilation from birth onwards. It is unique compared to limb muscles, where movement behaviors emerge more gradually after birth. The proposed studies will provide a better understanding of the normal maturation of the diaphragm muscle and the motor neurons that innervate it. In addition, factors that regulate the normal growth and development of the diaphragm will be systematically explored.