Multiple Site Optical Recording of Membrane Potential

电位探针是一种染料，当它与神经元、心脏和骨骼肌、腺体和其他细胞的膜结合时，就可以作为膜电位的分子指示器。这些分子的光学性质随电位呈线性变化，可以用来监测动作电位、突触电位或其他膜电压的变化，而无需使用电极。25年来，我们的实验室开创了使用电位探针的技术，并开发了用于细胞神经生理学的新光学方法，包括用于跨膜电压多位点光学记录（MSORTV）的高分辨率系统，能够同时监测多达464个位点的膜电位变化。我们将继续将这些技术应用于准2维神经系统的研究，这是唯一适合通过光学手段分析的，即豚鼠小肠的粘膜下神经丛。我们的最终目标是完整分析这个完整的哺乳动物神经网络的整体行为。首先，我们将使用电位测定染料来监测所有神经元的电活动，这些神经元可能是最接近哺乳动物简单神经系统的：粘膜下神经丛中连接神经节的环。其次，我们将采用一种新的分析工具（引力变换）以前所未有的完整性阐明完整哺乳动物神经网络的集合行为。第三，我们将使用多位点光学记录方法和引力变换，研究该网络对改变电路功能连通性和改变其行为的药物和物理干预的动态响应。第四，我们将通过使用荧光钙指示剂在持续数十分钟的时间尺度上监测电活动的时空模式，来检查神经元组装的持久性和空间一致性。

Potentiometric probes are dyes which, when bound to the membranes of neurons, cardiac and skeletal muscle, glands, and other cells, behave as molecular indicators of membrane potential. The optical properties of these molecules vary linearly with potential and may be used to monitor action potentials, synaptic potentials, or other changes in membrane voltage from a large number of sites at once, without the use of electrodes. For twenty five years our laboratory has pioneered the technology for using potentiometric probes, and developed new optical methods for use in cellular neurophysiology, including a high resolution system for Multiple Site Optical Recording of Transmembrane Voltage (MSORTV), capable of monitoring changes in membrane potential from as many as 464 loci at once. We will continue to apply these techniques to the study of a quasi-2- dimensional nervous system that is uniquely amenable to analysis by optical means, the submucous plexus of the guinea-pig small intestine. Our eventual goal is the complete analysis of the ensemble behavior of this intact mammalian neural network. First, we will use potentiometric dyes to monitor the electrical activity of all of the neurons in what are, perhaps, the closest approximations to mammalian simple nervous systems: rings of connected ganglia in the submucous plexus. Second, we will employ a novel analytical tool (the gravitational transformation) to elucidate with unprecedented completeness the ensemble behavior of an intact mammalian neural network. Third, we will use multiple site optical recording methods and the gravitational transformation, to study the dynamic response of this network to pharmacological and physical interventions that alter the functional connectivity of the circuit and modify its behavior. Fourth, we will examine the persistence and spatial coherence of neuronal assemblies by using fluorescent Ca-indicator dyes to monitor spatio-temporal patterns of electrical activity over time scales lasting tens of minutes.