This protocol presents a method to live image, fly olfactory circuit assembly at single axon level from a developing brain. dissection to clean the tissue covering the developing brain, we can collect high quality images from the internal brain. This method provide valuable insights into the cell biological basis of circuit development.
It can be easily modified to study the development of other circuits as well. It takes some time for a person to perform stable hand control in microdissection. More practice will be helpful.
To begin, prepare the culture dish for mobilizing the explant during time-lapse imaging by laying 0.5 centimeter thick Sylgard on the bottom surface of a Petri dish and letting it cure for 48 hours at room temperature. To prepare micro pins for immobilizing the explant on this plate using forceps, stick multiple micro pins on a tape with the sharp ends aligned on one side and cut two millimeters of sharp ends with scissors. Then using the forceps, insert the cut micro pins into the Sylgard layer of a pre-made Sylgard plate.
Using a brush, collect white pupae, which form puparium within one hour and transfer them to a new vial. Incubate the pupae in the water bath tempered at 37 degrees Celsius for 40 minutes to induce sparse ORN clones from random types. After heat shock, place the vial at 25 degrees Celsius for 30 hours resulting in pupae aged 30 hours after puparium formation.
Then prepare the culture medium for explant as described in the text manuscript and store at four degree Celsius. On the day of imaging, add fetal bovine serum, human insulin stock solution, and 20 hydroxyecdysone stock solution dissolve in ethanol to a 50 milliliter vial containing 45 milliliters of Schneider's Drosophila medium. Mix well and transfer 15 milliliters of the full medium into a new 50 milliliter conical tube and store the rest of the full medium at four degrees Celsius for a week.
Then cover the tubes opening containing full medium with paraffin film and oxygenate the medium by pumping oxygen bubbles under the liquid surface through a sterile five milliliter pipette tip at the rate of one bubble per second for 20 to 30 minutes. Next, sterilize the dissection well surface and previously prepared Sylgard plate with 70%ethanol and let them dry before use. Use a brush to transfer 30 hours APF pupa to a tissue paper for drying the external surface of the pupa for five minutes.
After applying double-sided tape on a glass slide, carefully attached the dried pupa to the sticky surface of the tape with the dorsal side facing upward. Gently press the pupa with a brush to properly attach the ventral side to the tape without damaging the pupa. Insert one sharp tip of the forceps between the brown pupal case and the pupa from the lateral side and carefully break the brown pupal case through a line to the posterior end of the pupa.
Once the brown pupal case is opened, using the forceps, gently hold the pupa and transfer it to the dissection well containing one milliliter of oxygenated full medium. Submerge the pupa in the medium for sinking it to the bottom of the well. To dissect the antenna brain explant from the pupa, gently hold the pupa using forceps with one hand and using a pair of microscissors, create a small hole from the posterior side of the pupa, which releases high pressure inside the pupa.
Starting from the hole, cut through the ventral midline up to the neck, then cut through the circumference of the neck to detach the head from the body of the pupa and place the body in a different well. Next, cut the transparent cuticle covering the dorsal side of the brain to expose the fat body on top of the brain, ensuring to keep the cuticle to which the retina and antenna attach and similarly, cut the cuticle from the ventral side of the brain. Pipette the medium toward the open regions on the dorsal and ventral sides of the head using a P10 pipette to gently wash out the fat body covering the brain and antenna.
To study the interaction of bilateral ORN axons or ORN axons to PN dendrite targeting, sever the antennal nerve during the stage by carefully placing the blades of the microscissors between the cuticle and brain and sever interested antennal nerve. For transferring the dissected explant to the Sylgard plate, place approximately 200 microliters droplet of the oxygenated full medium on the Sylgard surface. Pipette the fat body derived from the dissected trunk several times through a 200 microliter pipette tip for coating the inner surface to prevent the explants from sticking to the pipette tip during the transfer from the dissection well to the medium droplet on the Sylgard plate.
Use the forceps to pin the explant on the Sylgard layer in the two optic lobes, then carefully position the Sylgard plate on the imaging station, immobilize the plate with tapes, and slowly add 10 milliliters of the oxygenated full medium to the Sylgard plate using a P1000 pipette. The ORN axons arrive at the antennal lobe between 18 hours and 36 hours APF and then navigate the antennal lobe, cross the midline and innovate the glomeruli. Before registration, the axons exhibited some lateral drifting as the brain developed, and after registration, the drifting was corrected.
The glomerular identities of the extracted ORN axons were revealed by immunostaining of a neuropil marker and cadherin. By comparing to the internal lobe map, the identity of each glomerulus could be determined. During ex vivo culture, bacteria growth usually cause a rested development of olfactory circuit.
Therefore, thoroughly sterilizing the cultural dish and pins before imaging and keep the imaging room clean and isolated is important. For achieving higher spatial temporal resolution, this explant system can be imaged using more advanced microscope, the adaptive optics lattice light microscope. Although a factory circuit development was shown here as an example, this system can potentially be expanded to studies of other circuits and developmental processes in the developing central brain.
