Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

We are trying to understand the mechanisms underlying neural circuit assembly during development, and we use simple nervous systems like Drosophila to study this question. One of these mechanisms is the role of synaptic competition in circuit assembly, and the present paper is focused on this issue. One exciting new technology in neuroscience is called optogenetics.

It uses light sensitive ion channels to turn neurons on or off using light pulses. These new methods allow us to control neural circuitry in new ways and link the mutant circuits to behave. Ablation of a single neuron in the living animal has allowed us to characterize the role of competition in the assembly of simple neural circuits.

Now we can screen for the molecular machinery that underlies this widespread phenomenon. We demonstrate a competitive interaction between the two giant neurons for synaptic contact with the target motor neurons. This result has parallels throughout the animal kingdom, especially in the vertebrae visual system where it was first discovered.

This paves the way for investigating the molecules that regulate this competition in a genetically tractable organism like drosophila. We will use this new method of optogenetics to enhance our understanding of the neural circuit underlying the escape behavior. We will also use our understanding of the neural circuit to better understand how these new optogenetic tools work.

To begin, take a glass dish with a tightly fitting lid. Place a cotton ball in the dish. Under a fume hood, add three to five milliliters of ethyl ether to the cotton ball.

Immediately cover the dish with the lid. Use a paintbrush to pick up the third instar larva of drosophila from fly vials. Transfer one larva into a small open container.

Place the container in the glass dish with ethyl ether, and immediately close the dish tightly. Use a dissection microscope to check the larva for mobility every 30 seconds. Transfer the anesthetized larva onto a glass microscope slide.

Submerge the larva in a drop of insect saline. Under the dissection microscope, remove most of the saline with a paper tissue. Position the larva dorsal side up for giant fiber ablation, or the ventral side up for TTMn ablation.

Then slowly lower a glass cover slip onto the larva. Add saline to the side of the cover slip to fill in the space between the glass slide and the cover slip. For giant fiber ablation, check the positioning of the brain under high magnification on the dissection microscope.

Ensure the brain is lying level and is visible through the cuticle. To displace any fat tissue covering the brain, use forceps to apply slight pressure to the cover slip and move the cover slip from side to side. Place the sample on a multi photon microscope stage.

Use the GFP filter to locate the sample in epifluorescence mode. For giant fiber ablation, focus on the cells of interest. Then switch to two photon mode.

Set the laser to 870 nanometers and adjust the detector gain to view the GFP expressing cells with the galvano scanner. Define the area for ablation using a circular region of interest. Next, proceed to set up the ablation protocol in the software.

To do so, sequentially, set one frame of acquisition stimulation followed by another frame of acquisition. Start the stimulation laser power at a lower value and run the stimulation protocol. If the ablation was not successful and only bleach the cell, increase the laser power by increments of 5%or the number of loops one at a time, and run the protocol again.

Do this until successful cell ablation is seen. After successfully ablating the cell, remove the cover slip. With a paintbrush, gently pick up the larvae from the slide.

Then transfer the larvae into a food vial. In giant fiber ablation samples, the ablation was verified through the absence of a GFP labeled soma on the ablated side of the brain. For the TTMn ablated larvae, the absence of TTMn on one side was easily recognized due to missing soma and dendrites.