DiI Dye-Filling as a Simple and Inexpensive Tool to Visualize Ciliated Sensory Neurons in C. elegans

Summary

DiI dye-filling is a method commonly used in C. elegans to visualize a subset of the ciliated sensory neurons, allowing for the identification of genetic mutations that alter sensory neuron structure or function.

Abstract

C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 neurons within the adult hermaphrodite nervous system, 60 are classified as ciliated sensory neurons. These neurons are central to a number of C. elegans behaviors, including but not limited to chemo-, mechano-, and osmosensing, male mating, and dauer formation. For several decades now, members of the C. elegans community have used the red fluorescent lipophilic dye DiI to visualize a subset of the ciliated sensory neurons that are directly exposed to the external environment. This dye enters the ciliated ends of the neurons and distributes in a relatively uniform pattern throughout the dendrites, cell bodies, and axons. This simple and powerful method makes an excellent first-pass tool to identify genetic mutants that impart structural or functional defects in ciliated sensory neurons. Here, we present a streamlined version of this staining method to visualize the eight pairs of amphid and two pairs of phasmid neurons that are environmentally exposed in C. elegans. We discuss tips for using this inexpensive method for imaging cellular dye-filling patterns in anesthetized animals.

Introduction

Caenorhabditis elegans (C. elegans) are easy to manipulate, have fast generation times, and are low-cost to maintain. Due to these and many other advantages, C. elegans have served as a preferred model organism for studying many biological processes, especially the development and function of the nervous system. An entire issue in the Journal of Neurogenetics was recently dedicated to the historical impacts of research on this particular topic¹. They are particularly beneficial for studying the function of primary cilia, which are involved in sensing chemical and physical environmental conditions²

Protocol

1. Preparation of solutions

1. Prepare the bleach solution. Combine 2 mL of bleach, 500 µL of 10 M NaOH, and 7.5 mL of dH₂O in a 15 mL centrifuge tube.
   NOTE: Bleach solution remains stable for up to 1 week when stored at room temperature (RT).
2. Prepare the M9 buffer. Combine 3 g of KH₂PO₄, 6 g of Na₂HPO₄, and 5 g of NaCl in 1 L of sterile water and autoclave to sterilize. Once cooled completely to RT, add 1 mL of sterile 1 M MgSO₄.
3. Prepare the DiI stock solution. Add 10 mg of DiI to 5 mL of dimethylformamide (DMF) for a final concentration of 2 mg....

Results

Adult N2 worms imaged 24 h after dye-filling demonstrate clear fluorescent signal spread relatively evenly throughout the amphid neurons (Figure 1A,A') and phasmid neurons (Figure 1D,D'). In these animals, the dendritic projections and cell bodies of the amphid neurons can be easily distinguished. There are no clumps of dye in the dendritic projections, nor are there any interruptions in fluores.......

Discussion

xSuccessful dye-filling relies on careful consideration of the developmental stage and genetic background of the animals, as well as the elapsed time until imaging. Some genetic mutations disrupt the structure and/or function of the externally exposed ciliated sensory neurons, resulting in animals that are unable to dye fill properly. Therefore, dye-filling of novel C. elegans mutants can be used as a simple first-pass identifier for defects in sensory neuron structure or function. However, one limitation of thi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DiI	Biotium	60010	Not water soluble, make 2 mg/mL solution in DMF. Solution is light sensitive, cover with foil. Store at -20 °C. Solution good for many years.
Levamisole hydrochloride	Fisher	AC187870100	10 mM solution in M9 Buffer. Store at -20 °C. Solution good for many years.
M9 Buffer	IPM Scientific	11006-517	Available for purchase, but also easy to make in house following recipe in protocol.
N2 (C. elegans strain)	CGC	N2	C. elegans wild isolate
YH2125 (C. elegans strain)	n/a	n/a	Strain generated in Yoder Laboratory (Bentley-Ford et al, 2021)