The overall goal of this procedure is to culture whole developing drosophila, larva and pupil brains in an ex vivo setting. This is accomplished by first identifying appropriate stage drosophila, larvae, and pupi. Next, the brains are quickly dissected out.
Then the brains are cultured for the required period of time at 25 degrees Celsius. Finally, the cultured brains are fixed, stained, mounted, and imaged. Ultimately, the results show that it is possible to mimic the in vivo development of the oph mushroom body through an ex vivo culture technique.
The main advantage of this technique over existing methods is that we can now emulate in vivo phenotypes in the drosophila mushroom body in an ex vivo culture setting, allowing for high resolution analysis of the dynamics of the developmental process To culture larval brains. After preparing fresh, complete medium with insulin and act Dyson at 500 microliters to a 12 millimeter cell culture dish with insert for drug treatment. Prepare complete medium with 100 micromolar each of Rosco battin and oli moin.
Add a small amount of medium to a dissection. Watch glass using a small spatula, pick up wandering third instar larvae and place them in the watch glass. Next, using forceps hold the posterior end of the larva.
Then with number five, forceps, carefully open the anterior end quickly dissect out the brain keeping the ventral nerve cord intact. Using pre-wet pipette tips, carefully transfer the brain into the previously prepared cell culture dish. Next, make a wet chamber and place the dish inside the chamber.
Incubate the brain at 25 degrees Celsius for up to 48 hours to culture pupil brains. Begin by marking white puy on vials. Record this stage as zero hours after ER formation or a PF at 1.5 hours.
A PF Add 500 microliters of complete medium to a 12 millimeter cell culture dish with insert. Add a small amount of prepared medium to a dissection watch glass. Next, using a small wet spatula, pick up PPE previously marked for dissection and placed them in the watch glass.
The white pupi on the left is the zero time point, and on the right is shown the 1.5 hour A PF.Then while holding the posterior end of the puy with a pair of forceps, use number five forceps to carefully open the anterior end of the pupa case. Then quickly remove the brain keeping the ventral nerve cord intact and attached. After cutting off the top of a 20 microliter plastic tip, wet the pipette tip and use it to carefully transfer the pupil brains to the dishes of medium.
Incubate them at 25 degrees Celsius for the desired length of time. After larval or pupil brains have been incubated for the desired length of time, remove the medium and add 500 microliters of 4%formaldehyde in PBST buffer. Incubate for 15 minutes, then replace with fresh buffered 4%formaldehyde and incubate for an additional 15 minutes.
Use one XPBS with 0.5%Triton X 100 to wash the brains four times for 10 minutes each. After the last wash, incubate the samples for one hour. In blocking solution.
Add primary antibodies in blocking solution and incubate at four degrees Celsius overnight. The next morning, wash the samples with PBST four times for 10 minutes each. Then incubate in secondary antibodies at room temperature for four hours.
Repeat the washing steps to mount the brains for microscopy. Begin by equilibrating them and mounting medium for one hour and mount them on glass slides to prevent squashing the samples. Place glass chips on either side.
Then apply the cover slips after sealing the cover slips with nail polish. Store the slides in the dark. This figure shows a panel of third instar larval brains from wild type flies and flies that express a dominant negative derivative of CDK five or CDK five dn, both express actin GFP in mushroom body or MB gamma neurons as described previously, there is a region in the proximal axons of wild type gamma neurons that fails to accumulate actin GFP, thus showing up as a clear zone and fascially two protein accumulates in the axons only up to the ventral border of this zone.
The dotted lines indicate the position of the MB highlighting the acting clear zone and its ventral border. In flies expressing CDK five dn. The actin clear zone is absent and fascially two or fast two immunoreactivity spreads dorsally through that region using the method presented here for ex vivo culturing wild type fly brains were treated with a combination of the pharmacological inhibitors of CDK five activity ROS cottin and ene.
Shown here is a panel of third instar larval brains of control and drug treated wild type flies similar to flies, expressing a dominant negative CDK five construct brains treated with CDK five inhibitors for 24 hours show the characteristic loss of acting clear zone and shift in fast. Two boundary white lines show the position of the wild type fast. Two boundary shown here is a representative series of drosophila mushroom bodies from zero to 10 hours.
A PF labeled with membrane targeted. MCD eight GFP brackets indicate the dendritic projections of the MB referred to as the calyx prior to metamorphosis. MB axons course dorso ventrally in thick bundles and a fluorescent signal arises from the dense dendritic arbor.
This column shows brains dissected at the time indicated. Note the pruning of dendrites in the region indicated by the brackets such that by six to eight hours a PF, the haze dendritic signal is gone. Brains in this column were dissected at zero hours A PF before the internal act.
Dyson spike and cultured x vivo at the times indicated these show no developmental pruning of dendrites. Rather, the dendritic signal persists at 10 hours a PF to circumvent this PPR marked at zero hours EPF, but dissected at 1.5 hours a PF and cultured. This column shows a representative series of these DLA mushroom bodies from zero to 10 hours.
A PF as in the non cultured samples, the dendritic signal is undetectable by eight hours a PF.After watching this video, you should have a good understanding of how to culture hold rosler brains ex vivo. You should be able to isolate brains from late stage larva or white PrepU either before or after the hormone spike that commits to metamorphosis. You should then be able to either analyze fixed material by microscopy or to use the brains as starting material for live imaging.
