Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

Summary

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

Abstract

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Introduction

The embryonic motor neuron system of Drosophila offers a powerful experimental model to analyze the mechanisms underlying the development of the central nervous system (CNS)^1,2,3. The motor neuron system is amenable to biochemical, genetic, imaging, and electrophysiological techniques. Using the techniques, genetic manipulations and functional analyses can be carried out at the level of single motor neurons^2,4,5,6.

Protocol

1. Equipment and Supplies

1. Materials for collecting embryos and training adults to lay eggs
   1. Prepare the filtration apparatus by severing a 50 mL tube and cutting open a hole in the cap to set a mesh filter with pores of 100 µm (Table of Materials) in between the tube and the cap.
      NOTE: Alternatively, cell strainers with pores of 100 µm (Table of Materials) can be used for the filtration step of embryo collection.
   2. Make agar plates with grape agar premix (Table of Materials) according to the listed instructions. Briefly, gently stir 1 packet of the powder mix into 500 ....

Results

A representative image of the aCC and RP3 motor neurons is shown in Figure 3C to demonstrate the multicolor labeling of motor neurons at 15 h AEL. Their dendritic morphologies are largely invariant between embryos. The staining pattern obtained with anti-HRP antibody is shown in gray. A small droplet of DiO or DiD was deposited on the NMJ of muscle 1 or 6/7, respectively. Figure 4 demonstrates the capability to quantitatively measure the phenoty.......

Discussion

The use of dye labeling for studying neuronal morphology has several advantages over genetic cell-labeling techniques. The dye labeling technique can minimize the amount of time needed for labeling and imaging the morphologies of motor neurons. The dye labeling process is quite fast as it takes less than 2 h and enables us to define the outline of neuronal projections. As an alternative, one can visualize the aCC motor neuron by choosing a GAL4 line that expresses the yeast GAL4 transcription factor in aCC, and crossing .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x objective lens	Nikon		Plan
40x water-immersion lens	Nikon		NIR Apo
Capillary tubing	Frederick Haer&Co	27-31-1
Confocal microscope	Andor	N/A	Dragonfly Spinning disk confocal unit
Cover glass	Corning		22x22 mm Square #1
DiD	ThermoFisher	V22886
DiI	ThermoFisher	V22888
DiO	ThermoFisher	V22887
Dissecting microscope	Nikon	N/A	SMZ-U
Double Sided Tape	Scotch	665
Dow Corning High-Vacuum Grease	Fisher Sci.	14-635-5D
Dumont #5 Forceps	Fine Science Tools	11252-20
Egg collection cage	FlyStuff	59-100
FemtoJet 5247	Eppendorf	discontinued	FemtoJet 4i (Cat No. 5252000021)
ImageJ	NIH		Image processing software
Micromanipulator	Sutter	MP-225
Micropipette beveler	Sutter	BV-10-B
Needle puller	Narishige	PC-100
Nutri-Fly Grape Agar Powder Premix Packets	FlyStuff	47-102
Nylon Net Filter	Millipore
Paraformaldehyde 16% Solution, EM grade	Electron Microscopy Sciences	15710	Any EM grades
PBS	Roche	11666789001	Sold on sigmaaldrich, boxed 10x solution
Photo-Flo 200	Kodak	146 4510	Wetting agent
Upright fluorescence microscope	Nikon	N/A	Eclipse Ci with a LED light source
Vinyl Electrical Tape	Scotch	6143
VWR Cell Strainers	VWR	10199-659
Yeast	FlyStuff	62-103	Active dry yeast (RED STAR)