Unconventional Synaptic Modulation at the Vertebrate Neuromuscular Junction

描述(由申请者提供)：这是一个申请续签的项目，该项目主要涉及本科生对脊椎动物神经肌肉接头(NMJ)突触功能的研究。多年来，在NMJ(运动神经和肌肉之间的化学突触)上进行的实验为我们提供了对突触的许多基本了解。随着这些知识的增长和技术变得更加复杂，大多数神经科学家将注意力转向了中枢神经系统中的突触。只有相对较少的人继续研究这种突触，这种突触绝对是动物生存所必需的。尽管表面上看起来很简单，但NMJ使用了一系列复杂的通路来调节突触传递。由于所有这些在中枢神经系统中都有对应的调控机制，探索NMJ的调节机制不仅将扩大我们对NMJ本身的理解，而且还将提供关于突触的有用的见解。这项拨款建议的研究将探索突触调制的三个特征。首先，它试图了解内源性大麻素(ECB)在脊椎动物NMJ的卵。这一知识至关重要，因为目前正在努力开发外围活性的欧洲央行拮抗剂来治疗肥胖症及其后遗症。与许多中枢突触一样，ECB 2-花生四烯基甘油(2-AG)起着逆行信号的作用：它在突触后细胞中产生，在这种情况下是肌肉，并通过激活突触前神经末梢上的CB1受体来抑制神经递质的释放。为了更好地理解这一过程，计划在蜥蜴NMJ和老鼠NMJ进行实验，这将是第一次在哺乳动物NMJ进行这类研究。这个项目的第二个目标是为正在进行的关于NMJ是否是三方突触的辩论做出贡献。正如已知的神经胶质细胞在中枢神经系统突触的功能和可塑性中发挥关键作用(其功能障碍导致神经和精神疾病)一样，越来越多的证据表明，突触周围雪旺细胞(PSC)在NMJ扮演着类似的角色(如果不是相同的话)。使用一种新的技术来消融(即杀死)NMJ的PSCs，省去神经末梢和肌肉，我们将检验三个假设：(I)环氧合酶-2(COX-2)，它在PSCs内将2-AG转化为前列腺素E2的甘油酯，对于由M受体(MAChRs)激活触发的神经递质释放的延迟增强是必不可少的；(Ii)mAChRs的激活诱导PSC中COX-2mRNA的转录，从而增加COX-2的水平；以及(Iii)NMJ丢失其全部或部分PSCs将缺乏某些类型的突触可塑性。这项拟议的研究的最终目标是在NMJ上解开谷氨酸的谜团。虽然NMJ是胆碱能突触，但越来越多的证据表明谷氨酸起着重要的作用；然而，谷氨酸的来源及其功能仍不清楚。我们将检验这一假设，即N-乙酰天冬氨酸谷氨酸(NAAG)是谷氨酸的来源，谷氨酸受体的激活通过谷氨酸和NAAG控制一氧化氮的合成来改变突触传递。

DESCRIPTION (provided by applicant): This is an application to renew a project that significantly involves undergraduate students in research on synaptic function at the vertebrate neuromuscular junction (NMJ). For many years, experiments performed on the NMJ, the chemical synapse between a motor nerve and muscle, provided much of our basic understanding of synapses. As this knowledge grew and as techniques became more sophisticated, most neuroscientists turned their attention to synapses in the CNS. Only a relatively few have continued studying the one synapse that absolutely must function properly for an animal to survive. Despite its apparent simplicity, the NMJ uses a complicated set of pathways that modulate synaptic transmission. Since all of these have counterparts in the CNS, exploration of NMJ modulatory mechanisms will not only extend our understanding of the NMJ per se but will also provide useful insights about synapses in general. The research proposed in this grant will explore three features of synaptic modulation. First, it seeks to understand the roe of endocannabinoids (eCBS) at the vertebrate NMJ. This knowledge is critical as efforts are currently underway to develop peripherally active eCB antagonists to treat obesity and its sequelae. As at many central synapses, the eCB 2-arachidonylglycerol (2- AG) acts as a retrograde signal: it is produced in the postsynaptic cell, in this case the muscle, and inhibits neurotransmitter release by activating CB1 receptors on the presynaptic nerve terminal. Experiments are planned to better understand this process at lizard NMJs, where it was first discovered, and at the mouse NMJ, which will be the first study of its kind at a mammalian NMJ. The second goal of this project is to contribute to the ongoing debate over whether the NMJ is a tripartite synapse. Just as glial cells are known to play a crucial role in the function and plastiity of synapses in the CNS (the dysfunction of which contribute to neurological and psychiatric illness), there is growing evidence that perisynaptic Schwann cells (PSC) play an analogous if not identical role at the NMJ. Using a novel technique for ablating (i.e. killing) PSCs at the NMJ that spares the nerve terminal and muscle, we will test three hypotheses: (i) that cyclooxygenase-2 (COX-2), which converts 2- AG to a glycerol ester of prostaglandin E2 within PSCs, is essential for the delayed enhancement of neurotransmitter release triggered by the activation of muscarinic ACh receptors (mAChRs), (ii) that activation of mAChRs induces the transcription of COX-2 mRNA in PSCs and thereby increases levels of COX-2, and (iii) that NMJs missing all or some of their PSCs will lack certain types of synaptic plasticity. The final goal of the proposed research is to solve the mystery of glutamate at the NMJ. Although the NMJ is a cholinergic synapse, new evidence is accumulating suggesting that glutamate plays an important role; however, the source of glutamate and its function have remained unclear. We will test the hypothesis that N-acetylaspartylglutamate (NAAG) is the source of the glutamate and that the activation of glutamate receptors, by both glutamate and NAAG, modify synaptic transmission through their ability to control the synthesis of nitric oxide.