Department of Biology, Swarthmore College, Swarthmore, Pennsylvania.
Department of Physics, University of California San Diego, La Jolla, California.
Curr Protoc. 2023 Feb;3(2):e674. doi: 10.1002/cpz1.674.
The serine hydrolase acetylcholinesterase (AChE) is an important neuronal enzyme which catalyzes the hydrolysis of the neurotransmitter acetylcholine and other choline esters. The breakdown of acetylcholine by AChE terminates synaptic transmission and regulates neuromuscular communication. AChE inhibition is a common mode of action of various insecticides, such as carbamates and organophosphorus pesticides. Freshwater planarians, especially the species Dugesia japonica, have been shown to possess AChE activity and to be a suitable alternative model for studying the effects of pesticides in vivo. AChE activity can be quantified in homogenates using the Ellman assay. However, this biochemical assay requires specialized equipment and large numbers of planarians. Here, we present a protocol for visualizing AChE activity in individual planarians. Activity staining can be completed in several hours and can be executed using standard laboratory equipment (a fume hood, nutator, and light microscope with imaging capability). We describe the steps for preparing the reagents, and the staining and imaging of the planarians. Planarians are treated with 10% acetic acid and fixed with 4% paraformaldehyde and then incubated in a staining solution containing the substrate acetylthiocholine. After incubation in the staining solution for 3.5 hr on a nutator at 4°C, or stationary on ice, planarians are washed and mounted for imaging. Using exposure to an organophosphorus pesticide as an example, we show how AChE inhibition leads to a loss of staining. Thus, this simple method can be used to qualitatively evaluate AChE inhibition due to chemical exposure or RNA interference, providing a new tool for mechanistic studies of effects on the cholinergic system. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing the staining solution Basic Protocol 2: Fixing, staining, and imaging whole-mount planarian specimens for visualization of acetylcholinesterase activity.
丝氨酸水解酶乙酰胆碱酯酶（AChE）是一种重要的神经元酶，它催化神经递质乙酰胆碱和其他胆碱酯的水解。AChE 对乙酰胆碱的分解终止了突触传递，调节了神经肌肉通讯。AChE 抑制是各种杀虫剂（如氨基甲酸酯类和有机磷农药）的常见作用模式。淡水水螅，特别是日本水螅（Dugesia japonica），已被证明具有 AChE 活性，并且是研究体内杀虫剂效应的合适替代模型。可以使用 Ellman 测定法在匀浆物中定量测定 AChE 活性。然而，这种生化测定需要专门的设备和大量的水螅。在这里，我们提出了一种在单个水螅中可视化 AChE 活性的方案。活性染色可以在几个小时内完成，并且可以使用标准实验室设备（通风橱、振荡器和具有成像能力的显微镜）执行。我们描述了准备试剂的步骤，以及水螅的染色和成像步骤。用 10%乙酸处理水螅，然后用 4%多聚甲醛固定，然后在含有底物乙酰硫代胆碱的染色溶液中孵育。在 4°C 的振荡器上孵育 3.5 小时或在冰上静止后，用水洗涤水螅并进行成像。以暴露于有机磷农药为例，我们展示了 AChE 抑制如何导致染色丧失。因此，这种简单的方法可用于定性评估由于化学暴露或 RNA 干扰导致的 AChE 抑制，为研究胆碱能系统的影响的机制提供了一种新工具。
