Acetylcholinesterase Activity Staining in Freshwater Planarians

Abstract

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.

Keywords: AChE inhibition; acetylthiocholine; butyrylcholinesterase; enzyme activity staining; flatworms.

Figure 1

Color progression of stain after adding each component. (A) Examples of correct color progression after addition of (i) copper sulfate, (ii) potassium ferricyanide, and (iii) ATCh. (B) Examples (i, ii) of shades of correct color of the staining solution. (C) Example of an incorrect coloration of the final staining solution.

Figure 2

Coloration of 1x (left) and 10x (right) reagents. (A) copper sulfate (B) potassium ferricyanide.

Figure 3

A schematic drawing (top) and an example (bottom) of slide preparation. Nail polish is applied around the rim of the coverslip.

Figure 4

Example images of AChE staining of Dugesia japonica. (A, B) Examples of correct staining of the nervous system. Different components can show variability in the strength of staining. In (A) the brain, ventral nerve cords (VNCs), and commissures are strongly stained while in (B) the brain staining is weaker but more peripheral nervous system is stained. (C-D) show the same planarian from the (C) ventral and (D) dorsal side, respectively. The staining is more clearly seen from the ventral side. (E) If stain is improperly prepared, red-brown precipitate can adhere to the sample. Scale bar is 0.1 mm.

Figure 5

AChE staining can be used to show reduced AChE activity following chemical exposure. (A) The control group was exposed to 0.5% DMSO for 12 days. (B) Worms treated with 1 μM diazinon for 12 days had a reduction of staining. D. japonica planarians were used. Scale bar is 0.1 mm.

Figure 6

Staining outcomes for specific circumstances. (A) AChE staining on a wildtype D. japonica imaged shortly after the protocol was completed, and (B) the same sample imaged 10 months later. (C) BChE staining in Dugesia japonica using BTCh as the substrate and (D) AChE staining in Schmidtea mediterranea using the described protocol. Scale bar is 0.1 mm.