Collaborative Research: CRCNS Research Proposal: Presynaptic structure-function relationships that control AP waveforms, calcium ion, entry, and transmitter release at NMJs

神经细胞使用称为动作电位的行进电脉冲相互通信。这些脉冲到达神经细胞的末端(专门用于与邻近神经细胞进行化学交流的结构，称为突触或终末)，在那里它们可以在邻近神经细胞中触发电脉冲。尽管这些通讯事件对神经系统所做的一切都至关重要，而且可能会被神经系统疾病破坏，但令人惊讶的是，我们对是什么塑造了这些突触电脉冲的有效性，以及疾病是如何改变这一过程的，知之甚少。该项目以导致肌肉收缩的神经为模型，将神经末梢的生理学和药理学测量与显微镜相结合，以确定具有重要功能的蛋白质的密度和分布。这些细节被用来开发一种新的计算机建模方法，该方法使用结构和功能信息来产生电脉冲产生的详细模型。该项目产生的新数据和模型将促进有关突触功能的基本科学知识，并增强我们对神经疾病基础机制的理解。这项拟议的工作还将对K-12教育、本科教学和培训、研究生和研究生培训、社区推广以及未被充分代表的少数族裔机构的科学培训产生广泛影响。控制突触中递质释放的突触前事件尚不完全清楚，特别是关于定位于递质释放部位(活动区)的各种离子通道的作用。我们假设，活动区离子通道之间的结构-功能关系调节健康突触内的突触前动作电位波形，并且这种关系在疾病状态下被破坏。我们将使用来自四所大学的合作研究团队来解决这些问题，方法分为四个目标：(1)电压成像来表征突触前动作电位的形状，包括疾病模型突触中的影响，(2)动作电位波形对离子电流的影响的膜片钳测量，(3)使用超分辨率成像来表征运动神经末梢中突触前离子通道的密度和分布，以及(4)使用来自先前研究的数据与本文收集的数据相结合，我们将开发一种新的建模方法，将模拟真实神经末梢环境中的离子通道激活和离子通量与电压模拟器相结合，以预测这些离子通量对突触前动作电位形状的影响。拟议的研究将推进与突触前功能相关的基础科学问题，并加强对神经肌肉疾病潜在机制的理解。我们拟议的工作也将对K-12教育、本科教学和培训、研究生和研究生培训、社区推广、在代表性不足的少数族裔机构的培训以及突触功能的基本知识产生广泛影响。这项拨款由分子和细胞生物科学部的细胞动力学和功能集群以及生物科学局的新兴前沿部门共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Nerve cells communicate with each other using travelling electrical pulses called action potentials. These pulses arrive at the end of nerve cells (at structures specialized for chemical communication with neighboring nerve cells called synapses or terminals), where they can trigger electrical pulses in neighboring nerve cells. Despite the fact that these communication events are crucial to everything that the nervous system does, and can be compromised by neural diseases, we know surprisingly little about what shapes the effectiveness of these electrical pulses at synapses, and how diseases change this process. This project uses nerves that cause muscles to contract as a model, and combines physiology and pharmacology measurements in nerve terminals with microscopy to determine the density and distribution of functionally-important proteins. These details are used to development a new computer modeling approach that uses structural and functional information to produce detailed models of electrical pulse generation. The new data and models that project produces will advance basic scientific knowledge about synapse function, and enhance our understanding of the mechanisms that underlie neural disease. The proposed work will also have a broad impact on K-12 education, undergraduate teaching and training, graduate and post-graduate training, community outreach, and science training at under-represented minority institutions.The presynaptic events that control transmitter release at synapses are incompletely understood, particularly with respect to the role of various ion channels positioned with transmitter release sites (active zones). We hypothesize that the structure-function relationships between active zone ion channels regulates the presynaptic action potential waveform within healthy synapses, and that this relationship is disrupted in disease states. We will approach these issues using a collaborative team of investigators from four universities using an approach broken into four aims: (1) voltage imaging to characterize the shape of the presynaptic action potential, including the effects in disease model synapses, (2) patch clamp measurements of the effects of action potential waveforms on ionic currents, (3) characterization of the density and distribution of presynaptic ion channels in motor nerve terminals using super-resolution imaging, and (4) using a combination of data from prior studies with those collected here, we will develop a novel modeling approach that combines modeling ion channel activation and ion flux in a realistic nerve terminal environment with a voltage simulator that predicts the effects of these ion fluxes on the shape of presynaptic action potentials. The proposed studies will advance basic science issues related to presynaptic function and also enhance understanding of the mechanisms that underlie neuromuscular diseases. Our proposed work will also have a broad impact on K-12 education, undergraduate teaching and training, graduate and postgraduate training, community outreach, training at under-represented minority institutions, and fundamental knowledge about synaptic function.This grant was cofunded by the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences, and the Division of Emerging Frontiers in the Directorate for Biological Science.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.