PDF(Pubmed)
Abstract：
The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated NaV1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.
摘要：
肠神经系统（ENS）是一个复杂的神经元网络，组织在沿着胃肠道的整个长度延伸的神经节丛中。很大程度上独立于中枢神经系统，ENS协调消化道的运动和蠕动，调节分泌和吸收，并参与免疫过程。电生理方法如膜片钳技术特别适合于研究生理和病理生理条件下神经元的功能以及基础离子通道的生物物理参数。然而,将膜片钳方法应用于ENS神经元仍然很困难,因为它们包埋在大量组织层中,这限制了这些细胞的进入和靶向操作.这里,我们提出了一个强大的逐步方案，涉及从成年小鼠中分离ENS神经元，培养细胞，用质粒DNA转染，以及随后在电流钳和电压钳记录中单个神经元的电生理表征。有了这个协议，可以准备ENS神经元，转染,并在72小时内进行电生理表征。使用分离的ENS神经元，我们证明了通过功能性过表达与遗传性感觉和自主神经病变7型（HSAN-7）相关的重组电压门控NaV1.9突变通道的方法的可行性，一种以先天性镇痛和严重便秘为特征的疾病，需要肠胃外营养。尽管我们的重点是对孤立的ENS神经元的电生理评估，提出的方法也是有用的分析分子以外的钠通道或应用替代的下游测定，包括钙成像，蛋白质组学和核酸方法，或者免疫化学.
