Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

Summary

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

Abstract

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3^rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Introduction

Laser ablation is a preferred tool for dissecting neural circuits in a wide variety of organisms. Developed in model genetic systems like worms and flies, it has been applied across the animal kingdom to study the structure, function, and development of the nervous system^1,2,3. Here, single-neuron ablation was employed to investigate how neurons interact during circuit assembly in Drosophila. The escape system of the fly is a favorite circuit for analysis because it contains the largest neurons and the largest synapses in the adult fly, and the circuit has been well-....

Protocol

All animals used for the protocol were of the species Drosophila melanogaster. There are no ethical issues surrounding the use of this species. Ethical clearance was not necessary to carry out this work. The details of the Drosophila species, reagents, and equipment used in the study are listed in the Table of Materials.

1. Breeding Drosophila and selecting the correct larval stage

1. Choose a Gal4 driver line that drives expression in the cells that are to be ablated and recombine it with or cross it to a UAS-GFP reporter line. Raise flies on standard fly food at 25 ....

Results

This method can be used to manipulate the development of specific neuronal networks in the nervous system of Drosophila. The primary research question here was the formation of synaptic connections. Removing either the presynaptic GF or the postsynaptic TTMn enabled the investigation of reactive synaptogenesis at this central synapse and the molecular mechanisms crucial for synaptic function and development. As described in the protocol, laser cell ablation of one of the GFs or one of the TTMns was performed, an.......

Discussion

Cell ablation with a 2-photon microscope proved to be a highly successful method to manipulate neuronal circuit development in Drosophila. Since this method is non-invasive, it causes minimal damage to the animal. The data support the usefulness of this cell-specific manipulation of known circuits.

Crucial for the success of the ablation was selecting the most appropriate Gal4 driver. Since the GFS is well studied, many specific Gal4 driver lines have been described^7.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H+L)	Jaxkson ImmunoResearch	111-545-003
Anti-green fluorescent protein, rabbit	Fisher Scientific	A11122	1:500 concentration
Apo LWD 25x/1.10W Objective	Nikon	MRD77220	water immersion long working distance
Bovine Serum Albumin (BSA)	Sigma	B4287-25G
Chameleon Ti:Sapphire Vision II Laser	Coherent
Cotton Ball	Genesee Scientific	51-101
Dextra, Tetramethylrhodamine, 10,000 MW, Lysine Fixable (fluoro-Ruby)	Fisher Scientific	D1817
Drosophila saline			recipe from Gu and O'Dowd, 2006
Ethyl Ether	Fisher Scientific	E134-1	Danger, Flammable liquid
Fly food B (Bloomington recipe)	LabExpress	7001-NV
Methyl salicylate	Fisher Scientific	O3695-500
Microcentrifuge tube 1.5 mL	Eppendorf	22363204
Microscope cover-slip 18x18 #1.5	Fisher Scientific	12-541A
Neurobiotin Tracer	Vector Laboratories	SP-1120
Nikon A1R multi-photon microscope	Nikon		on an upright FN1 microsope stand
NIS Elements Advanced Research	Nikon		Acquisition and data analysis software
Paraformaldehyde (PFA)	Fisher Scientific	T353-500
PBS (Phosphate Buffered Salin)	Fisher BioReagents	BP2944-100	Tablets
R91H05-Gal4	Bloomington Drosophila Stock Center	40594
shakB(lethal)-GAl4	Bloomington Drosophila Stock Center	51633
Superfrost microscope glass slide	Fisher Scientific	12-550-143
Triton X-100	Fisher Scientific	422355000	detergent solution
UAS-10xGFP	Bloomington Drosophila Stock Center	32185