Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

The overall goal of this procedure is to culture Drosophila larval brains for multi-day time-lapse fluorescence microscopy. This method can help answer key questions in the field of Drosophila neurogenetics, such as gene expression changes that occur in specific neuronal populations. The main advantage of this technique is that it enables the imaging of fluorescent probes at single cell resolution in live brain over the course of hours to days.

When preparing the reagents for this procedure, work under a culture hood. Both an aliquot of SAM medium and of thrombin must be thawed to begin. Set aside the thrombin.

Take a 2.5 milliliter aliquot of SAM, and put the remaining SAM on ice and set it aside. Then put penicillin streptomycin in the aliquoted SAM and keep it at room temperature. For long-term imaging experiments, prepare an imagining chamber, apply autoclaved vacuum grease to the plastic regions around the well of a glass bottom dish.

Then sterilize the greased dish with UV light under the hood. Now, mix an aliquot of SAM into the fibrinogen and transfer the mixture to 37 degrees Celsius, where it won't polymerize for two hours. For the dissection, clean the forceps in two three-well glass dissection dishes with 70%ethanol.

Then load four wells with 400 microliters of DSS, and one well with 400 microliters of SAM. Keep the dissection dishes on a chilled plate. From a vial of propagating flies, scoop out some medium containing non-wandering third instar larvae, and transfer up to seven with forceps to a well of cold DSS.

Then, pass the larvae through three of the four DSS-loaded wells to clean them off. Leave them in the final well until dissected. Use the fourth well of DSS to dissect the larval brains with a pair of clean fine forceps.

Use the well established inside-out method. With proficiency each larvae can be processed in about 30 seconds. After each dissection, use a 20 microliter pipet tip pre-rinsed with DSS to transfer the brain in about three microliters of DSS to the SAM filled well.

Prepare up to seven brains for the embedding procedure. After transferring the brain into working solution, dispense the brain and medium onto the lid of a sterile petri dish. Then, add another 3.5 microliters of SAM with antibiotics, and add 3.5 microliters of warm SAM with fibrinogen.

It is important to maintain the SAM medium with fibrinogen at 37 degrees throughout the procedure. Next, mix the solution around the brain by gently pipetting with the same pipet tip and aspirate two microliters of the solution to deposit on a coverslip for a glass bottom dish. Then add 0.8 microliters of thrombin to the droplet and mix very quickly to initialize the polymerization which will appear white.

Then, use forceps to gently pick the brain and set it onto the polymerizing fibrin matrix. I think the thrombin, after the brain was transferred to the droplet, it's not recommended, as it complicates a lot of the procedure. Orient the brain as needed, and very gently push the brain as close to the coverslip as possible.

Then pick a layer of polymerized fibrin and fold it on top of the brain using small and gentle movements that do not detach the matrix. Continue to fold layers of polymerized fibrin over the brain from different parts of the clot to stabilize the brain. Then deposit 20 microliters of SAM with antibiotics over the embedded brain to halt the thrombin activity.

Using this procedure, proceed with embedding five to six brains on a glass bottom dish. For long-term imaging, immerse the embedded brains in 600 microliters of SAM with antibiotics. Then lay a piece of PTFE membrane over the medium and stick the edges of the membrane to the greased parts of the dish.

For short assays, fill the dish with two milliliters of SAM with antibiotics, immersing the brains and omit the use of the PTFE membrane. Using the described procedures, brains expressing a molecular clock reporter driven by a clock neuron driver, were entrained in light-dark then prepared as explant cultures. The long-term cultures were set up during the light phase, just prior to dark.

A SUM-stack containing the neuron of interest was created in the mean fluorescence intensity of the area corresponding to the neuron was measured after background subtraction. To determine rhythmicity of the reporter expression, both maximum entropy spectral analysis and manual inspection were used. Next, larvae of the same genotype were dissected either one hour after subjective dawn, or one hour after subjective dusk.

The brains were imaged twice within an hour to get a baseline. Then, PDF or vehicle was added to the culture medium and the brains were imaged hourly for six hours. In brains harvested shortly after dark, the PER-TDT expression profile in LNvs showed a time of day-dependent increase in fluorescence intensity upon PDF addition.

This also occurred in the DN1s and to a lesser extent on the DN2s. After watching this video, you should have a good understanding of how to make ex vivo brain culture from Drosophila larvae using a 3D matrix of fibrin. Once mastered, the whole procedure takes about 30 minutes, from dissection to embedding.

Prior to starting the dissections, prepare the imaging setup so the brain can be imaged immediately after embedding. Following this procedure, adult Drosophila brains can be also cultured for fluorescence time-lapse imaging in order to answer additional questions.