Unconventional Synaptic Modulation at the Vertebrate Neuromuscular Junction

描述（由申请人提供）：神经元通过化学突触进行交流。突触将神经元组装成神经元网络，这些网络创造了神经系统感知世界、处理信息和指导行为的非凡能力。突触还赋予神经系统随时间变化的能力，也就是学习的能力。非传统的神经调质，如一氧化氮，内源性大麻素，和肾上腺素，已经知道了很多年;然而，他们参与突触调制在脊椎动物神经肌肉接头（NMJ），其中运动神经突触到肌肉细胞，是最近的发现。尽管它表面上很简单，但NMJ采用了一系列令人惊讶的复杂信号通路来调节其活性。使用肌肉从绿色变色龙蜥蜴，这已被我的实验室表明，进行双相调制的神经递质的释放，涉及一氧化氮和内源性大麻素2-花生四烯酸甘油（2-AG），将被用来解决五个问题的机制和/或这些非传统的神经调质的作用。第一个问题将确定由2-AG介导的突触抑制是否通过在剧烈、持续活动（例如运动）期间减少ACh释放来改善神经肌肉耐力。如果是真的，这不仅揭示了内源性大麻素在NMJ的基本功能，而且可能对内源性大麻素系统调节剂（例如利莫那班）的治疗用途具有重要意义。一氧化氮（NO）是NMJ处毒蕈碱受体激活诱导的突触调节所必需的。事实上，肌营养不良症患者NMJ的NO合成中断可能会导致疲劳。第二个问题将使用基于荧光的技术来测量NO，以确定肌肉是否是NMJ处毒蕈碱受体依赖性调节所必需的NO的来源。第三个问题紧接着第二个问题，将确定谷氨酸或二肽N-乙酰基谷氨酸（NAAG）是否激活NO的合成。NAAG与许多病理学有关，包括疼痛、创伤性脑损伤、精神分裂症和中风。虽然脊椎动物运动神经元早已知道表达高水平的NAAG，但这种肽在NMJ的功能尚未得到严格的探索。最后两个问题将集中在突触周许旺细胞（PSC）的功能，神经胶质细胞密切相关的前和突触后元件的NMJ。初步研究表明前列腺素PGE 2-G参与了NMJ毒蕈碱调节的第二阶段。预期毒蕈碱型ACh受体的活化诱导PSC中环氧合酶（考克斯）的合成，然后将2-AG转化为PGE 2-G。将通过使用定量PCR测量考克斯mRNA的水平并使用荧光原位杂交确定其位置来测试该预测。这些研究，沿着对NMJ的电生理学研究，其中PSC已被补体介导的程序急性消融，将进一步我们对NMJ的神经胶质细胞在突触可塑性中的功能的了解，可能对外周和中枢神经系统的突触调节产生影响。 公共卫生相关性：利用电生理学，光学和分子遗传学的方法在脊椎动物神经肌肉接头这项研究将探讨五个基本假设的参与非常规突触可塑性的化学突触。这项研究的结果将与内源性大麻素系统调节剂（例如利莫那班）的治疗用途直接相关。它还将提供基础知识，可以更好地告知我们对肌营养不良症和由神经胶质细胞或肽N-乙酰基谷氨酸（NAAG）功能障碍引起的许多其他疾病的理解。

DESCRIPTION (provided by applicant): Neurons communicate at chemical synapses. Synapses assemble neurons into networks of neurons and these networks create the remarkable abilities of the nervous system to perceive the world, process information, and direct behavior. Synapses also endow the nervous system with the ability to change over time - that is, to learn. Unconventional neuromodulators, such as nitric oxide, endocannabinoids, and prostaglandins, have been known about for many years; however, their participation in synaptic modulation at the vertebrate neuromuscular junction (NMJ), where a motor nerve synapses onto a muscle cell, is a more recent finding. Despite its apparent simplicity, the NMJ employs a surprisingly complex array of signaling pathways that modulate its activity. Using a muscle from the green anole lizard, which has been shown by my laboratory to undergo a biphasic modulation of neurotransmitter release that involves nitric oxide and the endocannabinoid 2-Arachidonoylglycerol (2-AG), will be used to address five questions regarding the mechanisms and/or roles of these unconventional neuromodulators. The first question will determine whether the synaptic depression mediated by 2-AG improves neuromuscular endurance by reducing ACh release during periods of intense, unremitting activity (e.g. exercise). If true, this will not only reveal a basic function of endocannabinoids at the NMJ, but may also have important implications for the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). Nitric oxide (NO) is essential for synaptic modulation induced by the activation of muscarinic receptors at the NMJ. In fact, disruption of NO synthesis at the NMJs of humans with muscular dystrophy may contribute to fatigue. The second question will use a fluorescence-based technique for measuring NO to determine whether the muscle is the source of the NO that is essential for muscarinic-receptor dependent modulation at the NMJ. The third question follows closely from the second and will determine whether glutamate or the dipeptide N-acetylaspartylglutamate (NAAG) activates the synthesis of NO. NAAG has been implicated in numerous pathologies including pain, traumatic brain injury, schizophrenia and stroke. Although vertebrate motor neurons have long been known to express high levels of NAAG, the function of this peptide at the NMJ has not been rigorously explored. The final two questions will focus on the function of perisynaptic Schwann cells (PSCs), glial cells intimately associated with the pre- and postsynaptic elements of the NMJ. Preliminary work has implicated the prostaglandin PGE2-G in the second phase of muscarinic modulation at the NMJ. Activation of muscarinic ACh receptors is predicted to induce the synthesis of the enzyme cyclooxygenase (COX) in the PSCs, which will then convert 2-AG to PGE2-G. This prediction will be tested by measuring levels of COX mRNA using quantitative PCR and determining its location using fluorescence in situ hybridization. These studies, along with an electrophysiological investigation of NMJs at which the PSCs have been acutely ablated by a complement-mediated procedure, will further our knowledge of glial cell function in synaptic plasticity at the NMJ with likely implications for synaptic modulation in the peripheral and central nervous system. PUBLIC HEALTH RELEVANCE: Using electrophysiological, optical and molecular genetic approaches at the vertebrate neuromuscular junction this research will explore five fundamental hypotheses about the involvement of unconventional synaptic plasticity at the chemical synapse. The findings from this research will have direct relevance to the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). It will also provide basic knowledge that may better inform our understanding of muscular dystrophy and numerous other diseases that are caused by the dysfunction of glial cells or the peptide N-acetylaspartylglutamate (NAAG).