Ion Channels and Chemicals Controlling Synapse Stability

控制突触稳定性的离子通道和化学物质

• 批准号: 6601343
• 负责人: JOSEPH J MCARDLE
• 金额: $ 31.6万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6601343
• 关键词: acetylcholine; calcium flux; diabetes mellitus; electrophysiology; glucose; insulin; laboratory mouse; membrane channels; neuromuscular junction; neurotransmitter receptor; nitric oxide; nitric oxide synthase; synapses; tissue /cell culture

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.

描述（由申请人提供）：突触是我们大脑中信息转移的主要源头，也是许多病理的目标，这些病理可能会使人类从子宫内发育到死亡。因此，为理解突触的形成和稳定而做出了重大的研究工作。关于突触的科学文献富含从神经肌肉结（NMJ）的研究中得出的基本原理。特别是，负责NMJ功能，形成和稳定性的蛋白质相对众所周知。然而，基本问题仍然与这些蛋白质之间的相互作用有关。一个重要的实验模型表明，ACHR的异质活性会影响成年NMJ的稳定性。该建议将该模型修改并扩展到开发的NMJ，其中NMJ成熟的临界阶段中未成熟伽玛和成熟的Epsilon ACHR的共表达会产生端板活性的异质性。我们的模型表明，富含Epsilon ACHR的端板区域介导Ca 2+的流入，从而激活共定位的一氧化氮合酶（NNOS）。一氧化氮（NO）产生了与电动机端板竞争的神经末端的扩散。新的初步数据表明，在成人运动神经末端，没有增强CA2+电流和发射机释放。因此，开发包含epsilon achR的端板基因座的神经末端可以通过无激活前鸟叶尼尔环酶的激活来增强和培养。相比之下，没有抑制竞争神经终端激活Epsilon Achr较差的端板焦点的功能和稳定性。小鼠三角形胸骨（TS）制备有助于我们模型的精确测试。我们的初步数据表明，从新生儿小鼠中分离出的TS制剂允许同时记录神经终端电流和端板的突触后事件，这些底板从末端接收神经支配的神经端子，这些末端来自不同的神经躯干。这允许对哺乳动物神经末端之间的跨性别术的功能进行前所未有的研究，竞争突触后靶点。 Epsilon亚基和NNOS的可用性淘汰了小鼠，以及Epsilon Achr选择性配体Waglerin- 1进一步增强了为测试我们的模型的提议。其他新型初步数据表明，神经元K-ATP通道的激活剂胰岛素抑制了成人NMJ的ACH的量化量。因此，该提案的第二个目标是发现胰岛素是否以及葡萄糖，葡萄糖是否影响在发育中的NMJ竞争的神经终端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讨。总体而言，这项研究在临床上是相关的，因为杜钦肌营养不良以及中风的动物模型中没有信号传导级联反应发生显着改变。此外，Epsilon ACHR的功能改变是导致与慢速通道先天性肌关系综合征相关的NMJ病理。提出的对胰岛素作用的评估是新颖的，将增强对成年和幼年形式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。