Neurotransmission in well-defined networks

描述(由申请人提供)：当代神经生物学的一个主要挑战是我们对细胞间通讯所涉及的机制和可塑性的不完全理解。确定神经元如何与网络中的其他神经元通信需要关于单个神经元中存在和释放的神经递质和神经肽的完整信息。这项建议使用著名的动物模型加州海兔，其定义良好的神经元网络，以活动依赖的方式表征单个神经元中使用的一套分子，以及它们的释放。利用新的分析工具，可以分析单个神经元和神经元亚室的化学成分，结合作为海兔基因组和转录组项目一部分的数据，将从参与重要生理功能的特定已识别神经元中鉴定出几乎完整的信号分子清单。对于经典的发射机，将使用毛细管电泳法和几种选择性检测方案(从放射性核素检测到自然荧光)。特别是，重大的努力将确定不寻常的氨基酸，d-谷氨酸和d-天冬氨酸在神经传递中的作用。虽然先前的工作已经证明，在特定的神经元中，这些分子是从它们的L氨基酸对应的氨基酸合成的，运输到释放区并可能被释放，但这里将探索它们在细胞间信号传递中的功能作用的细节。此外，还将对这些神经网络中使用的全套多肽(多肽组)进行表征；将使用的方法包括单细胞基质辅助激光解吸/电离飞行时间质谱仪、各种小体积电喷雾质谱学方法和几种生物信息学方法。这项工作的结果将是一种明确的神经化学，以补充海兔神经元网络中众所周知的生理和行为。利用分离科学和质谱学的进展，我们可以在理解已知和新型细胞间信号转导化合物的合成、翻译后加工、分布、释放和功能方面取得显著进展。在描述在协调神经元网络活动中起关键作用的神经元信号的亚细胞动力学时，这项工作将有助于加深我们对神经系统的基本理解。

DESCRIPTION (provided by applicant): A major challenge of contemporary neurobiology is our incomplete understanding of the mechanisms and plasticity involved in cell-to-cell communication. Determining how neurons communicate with other neurons within a network requires complete information about the neurotransmitters and neuropeptides present in and released from individual neurons. This proposal uses the well-known animal model Aplysia californica, with its well-defined neuronal networks, to characterize the suite of molecules used in individual neurons, as well as their release, in an activity dependent manner. By taking advantage of new analytical tools that allow single neurons and neuronal subcompartments to be assayed for their chemical constituents, combined with the data becoming available as part of the Aplysia genome and transcriptome projects, a nearly complete list of signaling molecules will be characterized from specific identified neurons involved in important physiological functions. For the classical transmitters, capillary electrophoresis with several selective detection schemes (ranging from radionuclide detection to native fluorescence) will be used. In particular, significant efforts will determine the roles of the unusual amino acids, d-glutamate and d-aspartate, in neurotransmission. While prior work has demonstrated that in specific neurons, these molecules are synthesized from their L-amino acid counterparts, transported to release zones and likely released, here the details of their functional roles in cell-to-cell signaling will be explored. In addition, the complete set of peptides (the peptidome) used in these neuronal networks will be characterized; the methods to be used include single cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, a variety of small volume electrospray mass spectrometric approaches, and several bioinformatics approaches. The outcome of this work will be a well-defined neurochemistry to complement the well-known physiology and behavior in the neuronal networks of Aplysia. By using the advances in separation science and mass spectrometry, significant gains can be made in our understanding of the synthesis, posttranslational processing, distribution, release and function of known and novel cell-to-cell signaling compounds. In leading to a description of the subcellular dynamics of neuronal signaling, which plays a crucial role in coordinating neuronal network activities, this work will contribute to furthering our basic understanding of the nervous system.