Loading Drosophila Nerve Terminals with Calcium Indicators

Summary

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca²⁺-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Abstract

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca²⁺-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca²⁺ changes in nerve terminals. However, these indicators have limited sensitivity to Ca²⁺ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca²⁺ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca²⁺ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca²⁺ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca²⁺-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca²⁺ dynamics. Dextran-conjugation helps prevent Ca²⁺ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Protocol

1. Select a clean dissection dish that has not been exposed to any fixatives.
   
   
2. Dissect a wandering 3^rd instar Drosophila larva in Schneider's Drosophila Medium containing Ca²⁺ and L-glutamine, (do not cut any nerves or damage muscle fibers Nos. 7, 6, 13 or 12).
   
   
3. Select a glass filling-pipette with a 12 micron tip (internal diameter).
   
   
4. Using a syringe and tubing (to apply negative pressure to the pipette) ensure that the pipette tip is not obstructed.
   
   
5. Select a fine plastic filling-filament that can be inserted down the length of the glass pipette.
   
   
6. Draw ~ 1 cm ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Schneider’s Insect Medium	Reagent	Sigma-Aldrich	S0146	must contain L-glutamine and calcium
L-glutamic acid monosodium salt hydrate	Reagent	Sigma-Aldrich	G1626