Ion Channels and Chemicals Controlling Synapse Stability

描述（由申请人提供）：突触是我们大脑内信息传递的主要场所，也是许多病理学的靶标，这些病理学可以折磨人类从子宫内发育到死亡。因此，主要的研究工作是了解突触的形成和稳定在整个生命。关于突触的科学文献丰富，发现了来自神经肌肉接头（NMJ）研究的基本原理。特别是，负责NMJ功能，形成和稳定性的蛋白质相对较好地理解。然而，关于这些蛋白质之间的相互作用的基本问题仍然存在。一个重要的实验模型表明，AChRs的异质性活动影响成人NMJ的稳定性。这一建议修改和扩展该模型的发展NMJ未成熟的γ和成熟的NMJ成熟的关键阶段期间的共表达产生异质性的终板活动。我们的模型表明，终板区富含AChR介导Ca 2+内流，Ca 2+内流激活共定位的一氧化氮合酶（nNOS）。产生的一氧化氮（NO）扩散到竞争运动终板的神经末梢。新的初步数据表明，NO增强成人运动神经末梢的Ca 2+电流和递质释放。因此，发育中的神经末梢激活终板位点，含有NATAChR可以通过突触前鸟苷酸环化酶的NO激活功能增强和滋养。相反，NO可能抑制竞争性神经末梢的功能和稳定性，从而激活AChR差终板灶。小鼠胸骨三角肌（TS）制备有助于我们模型的精确测试。我们的初步数据表明，从新生小鼠分离的TS制剂允许同时记录神经末梢电流和突触后事件在终板接收神经支配来自终端起源于不同的神经干。这使得前所未有的研究的功能，和NO介导的串扰之间，哺乳动物神经末梢竞争的突触后靶点。可获得的nNOS亚基和nNOS敲除小鼠，以及nNOS AChR选择性配体Waglerin- 1进一步加强实验提出测试我们的模型。额外的新的初步数据表明，胰岛素，神经元K-ATP通道的激活剂，抑制乙酰胆碱在成人NMJ的量子释放。因此，该提议的第二个目标是发现胰岛素以及葡萄糖是否影响神经末梢在发育中的NMJ处竞争的功能和最终稳定性。这将在I型糖尿病的非肥胖小鼠模型中进行探索。总的来说，这项研究是临床相关的，因为NO信号级联在杜氏肌营养不良症以及中风动物模型中显著改变。此外，NMJ AChR的功能改变是与慢通道先天性肌无力综合征相关的NMJ病理学的原因。建议的胰岛素作用的评价是新颖的，将提高对成人和青少年形式的糖尿病的神经系统后果的理解。从这项研究中获得的知识将启发未来的分子方法来治疗困扰儿童和成人的病理。

DESCRIPTION (provided by applicant): Synapses are the major locus of information transfer within our brain as well as the target of numerous pathologies which can afflict humans from development in utero to death. Therefore, major research effort is given to understanding synapse formation and stabilization throughout life. The scientific literature concerning synapses is rich with discovery of fundamental principles derived from study of the neuromuscular junction (NMJ). In particular, proteins responsible for NMJ function, formation, and stability are relatively well understood. Nevertheless, fundamental questions remain concerning interactions between these proteins. An important experimental model suggests that heterogeneous activity of AChRs influences stability of the adult NMJ. This proposal modifies and extends that model to the developing NMJ where co-expression of immature gamma and mature epsilon AChRs during the critical phase of NMJ maturation produces heterogeneity of end-plate activity. Our model suggests that end-plate areas rich in epsilon AChR mediate Ca 2+ influx which activates co-localized nitric oxide synthase (nNOS). The nitric oxide (NO) produced diffuses to nerve terminals competing for the motor end-plate. New preliminary data suggest that NO enhances Ca2+ currents and transmitter release at adult motor nerve terminals. Thus, developing nerve terminals activating end-plate loci containing the epsilon AChR may be functionally enhanced and nurtured via NO activation of presynaptic guanylyl cyclase. In contrast, NO may repress function and stability of competing nerve terminals activating epsilon AChR poor end-plate foci. The mouse Triangularis sterni (TS) preparation facilitates exact testing of our model. Our preliminary data show that the TS preparation isolated from neonatal mice allows simultaneous recording of nerve terminal currents and post-synaptic events at end-plates receiving innervation from terminals originating in distinct nerve trunks. This allows unprecedented study of the function of, and NO-mediated cross talk between, mammalian nerve terminals competing for a postsynaptic target. The availability of epsilon subunit and nNOS knock out mice, as well as the epsilon AChR selective ligand Waglerin- 1 further strengthen experiments proposed to test our model. Additional novel preliminary data suggest that insulin, an activator of the neuronal K-ATP channel, suppresses quantal release of Ach at the adult NMJ. Therefore, a second goal of this proposal is to discover if insulin, as well as glucose, effects the function, and eventual stability, of nerve terminals competing at the developing NMJ. This will be explored in a non-obese mouse model of type I diabetes. Overall, this research is clinically relevant since NO signaling cascades are significantly altered in Duchenne muscular dystrophy as well as animal models of stroke. In addition, altered function of the epsilon AChR is responsible for NMJ pathology associated with slow channel congenital myasthenic syndrome. The proposed evaluation of insulin effects is novel and will enhance understanding of the neurologic consequence of adult and juvenile forms of diabetes. The knowledge gained from this research will enlighten future molecular approaches to treating pathologies which afflict children and adults.