Multiple site optical recording of membrane potential

描述（由申请人提供）：电位探针是一种染料，当与可兴奋细胞的膜结合时，其表现为膜电位的分子指示剂。这些分子的光学性质随电压线性变化，并且可以用于在不使用电极的情况下同时监测来自大量位点的动作电位、突触电位或其他电压变化。近三十年来，我们的实验室率先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，包括跨膜电压的多位点光学记录系统（MSORTV）。现在，我们将采用一种新的和更敏感的高分辨率相机系统和新设计的电压敏感染料，研究单个烟碱乙酰胆碱受体（nAChR）亚型在完整的血管舒张反射中的功能作用，这些反射完全包含在肠壁内，并由肠神经系统（ENS）介导。ENS是具有确定功能的自主网络，包括位于肠壁内不同平面的两个神经丛，粘膜下和肌间神经丛。由于这些神经丛的效应器官（血管、运输上皮、神经内分泌细胞、免疫元件和平滑肌）也包含在肠壁内，因此可以解剖半完整的制备物，以保留完整的反射通路。这里描述的血管扩张反射可以通过电刺激、球囊扩张或粘膜抚摸来诱发，并且需要肌间神经元的激活，肌间神经元会聚到血管扩张粘膜下神经元上，其活动触发粘膜下小动脉的扩张。将在半完整的肌肠粘膜下层制备物中研究电刺激诱发的反射。其他将在半完整平板回肠制备中进行研究。所有这些反射都表现出对六甲铵敏感的肌间和粘膜下成分。因此，通过使用MSORTV和一种新的分析方法，我们将确定单个nAChR亚型在血管舒张中的作用。这些反射通路通过肌间神经丛投射到粘膜下层，作为构建到ENS网络中的机制的示例，其协调粘膜血流与肠中的特定运动和分泌模式。

DESCRIPTION (provided by applicant): Potentiometric probes are dyes which, when bound to the membranes of excitable cells, behave as molecular indicators of membrane potential. The optical properties of these molecules vary linearly with voltage and may be used to monitor action potentials, synaptic potentials, or other voltage changes from a large number of sites at once, without the use of electrodes. For nearly thirty years our laboratory has pioneered the technology for using potentiometric probes, and developed new optical methods for use in cellular neurophysiology, including a system for Multiple Site Optical Recording of Transmembrane Voltage (MSORTV). Now we will employ a new and much more sensitive high-resolution camera system and newly designed voltage sensitive dyes, to study the functional role of individual nicotinic acetylcholine receptor (nAChR) subtypes in intact vasodilator reflexes that are fully contained within the gut wall and are mediated by the enteric nervous system (ENS). The ENS is an autonomous network with identified functions, comprising two plexuses, submucous and myenteric, located in distinct planes within the gut wall. Since the effector organs for these plexuses (blood vessels, transporting epithelium, neuroendocrine cells, immune elements, and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve intact reflex pathways. The vasodilator reflexes described here can be evoked by electrical stimulation, balloon-distension or mucosal-stroking, and require activation of myenteric neurons that converge onto vasodilator submucosal neurons whose activity triggers the dilation of submucosal arterioles. The reflex evoked by electrical stimulation will be studied in a myenteric-submucosa semi-intact preparation. The others will be studied in a semi-intact flat-sheet ileal preparation. All of these reflexes exhibit hexamethonium-sensitive myenteric and submucosal components. Thus, by using MSORTV and a novel analytical approach, we will identify the role of individual nAChR subtypes in vasodilation. These reflex pathways, that project through the myenteric plexus to the submucosa, serve as examples of the mechanisms, built into ENS networks, which coordinate mucosal blood flow with specific motor and secretory patterns in the intestine.