Obaid Ana L, Salzberg B M
Department of Neuroscience, University of Pennsylvania School of Medicine.
J Vis Exp. 2009 Dec 4(34):1631. doi: 10.3791/1631.
The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 microm in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall (1); the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes (2,7,8,9). We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) (3) that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of approximately 2.5 microm at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.
肠神经系统（ENS）是一个具有明确功能的独立网络，能够单独执行复杂行为。其神经元（直径为10至25微米）排列在局限于肠壁不同平面的神经丛中（1）；肌间神经丛位于纵行肌层和环行肌层之间，黏膜下神经丛位于环行肌层和黏膜之间。由于这些神经丛的效应系统（转运上皮、内分泌细胞、免疫成分、血管和平滑肌）也包含在肠壁内，因此可以解剖出保留不同反射通路各个组成部分的半完整标本。效应系统的行为由协同作用的黏膜下神经丛和肌间神经丛控制。因此，详细了解神经节内和神经节之间的突触相互作用以及神经丛之间的通信，对于理解正常胃肠功能至关重要。作为一个完整的神经系统，肠神经系统是一个独特的实验模型，在其中可以将分子和细胞事件与神经元网络的电行为及其生理输出联系起来。由于其神经丛的准二维组织，肠神经系统特别适合使用采用电压敏感染料的多位点光学记录技术来研究神经网络（2,7,8,9）。我们将在此说明一种相对较新的萘基苯乙烯基吡啶染料（di-4-ANEPPDHQ）（3）的用途，该染料与其前身相比具有多种优势，包括极低的光毒性、缓慢的内化速率和出色的化学稳定性。当与允许亚毫秒时间分辨率的相机结合使用时，这种染料使我们能够在100倍放大倍数下以约2.5微米的最大空间分辨率监测视野内所有神经元的电活动。在较低放大倍数（10倍或20倍）下，单细胞分辨率的牺牲通过视角的增加得到补偿，揭示了神经节间电路的复杂性。
