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 3rd 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 system1,2,3. 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-characterized in the past decades4. The role neuron-neuron interactions play in the assembly of the Giant Fiber circuit is a focal point of this research.
One type of interaction that has been a focal point in neuroscience since the work of Hubel and Wiesel in the 1960s is "synaptic competition"5,6. In this protocol, laser ablation was used to revisit the role of competition through single-cell ablation in the giant fiber system (GFS) of Drosophila, where the molecular underpinnings of the phenomena might be discovered.
Ablation of neurons in the developing fly has been difficult for a variety of reasons, including visualizing the target neurons, the precision of the ablation method, and the survival of the specimen. To overcome these problems in the GFS, the UAS/Gal4 system7 was used to label neurons of interest, and a two-photon microscope was used to remove the presynaptic giant fiber or the postsynaptic jump motor neuron (TTMn).
In this study, to determine the role that neighboring bilateral neurons play in adjusting synaptic connectivity and synaptic strength in the GFS, one of the bilateral pairs of neurons (either presynaptic GF or postsynaptic motor neuron) was deleted just before pupal development. At this developmental stage, GF axonogenesis has not been completed8. The GF structure and function of the synaptic circuit in the adult were then examined, with particular attention given to the output of the remaining GF.
Protocol
Representative Results
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 described7…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Experiments on the 2-photon microscope were performed in the FAU Stiles-Nicholson Brain Institute Advanced Cell Imaging Core. We would like to thank the Jupiter Life Science Initiative for financial support.
Materials
| 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 18×18 #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 |
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