Visualizing the Beating Heart in Drosophila

Two techniques will be presented here for the visualization of a beating heart in Drosophila for the dissection of the adult heart. First, the head and thorax are removed by a single cut along the indicated line. Next, the genitalia are removed and the abdominal body cavity is opened using forceps the gut, and in the case of a female fly, the ovaries are removed.

The abdominal fat body in abdominal segments three and four is carefully sucked away to expose the adult heart for the immobilization of the larval heart. A single larva placed in buffer solution is fixed by applying histo acral glue first around the larva head region. The larvae is gently stretched and more glue is applied to the posterior end, and then on the flanks of the larva body wall.

Hi, I'm GI Fler. Hi, I'm Karen er. We're both members of the Development and Aging program at the Burnham Institute for Medical Research.

Today we will show you the procedures that we've developed to visualize the beating heart of Laval and adult drosophila. We use both of these preparations as a way to visualize, examine, and quantify heartbeat parameters in the fruit fly.Drosophila. Before getting started, we first prepare an artificial hemolymph solution.

This solution contains sucrose entry, helo, which should be added to the artificial hemolymph from refrigerated stock solutions just prior to use. In order to prevent bacterial contamination, bring the artificial hemolymph to room temperature and oxygenate the solution by air bubbling for at least 15 minutes. Next, pull several fine capillaries using a standard pipette puller, which will be needed for removal of fat bodies in the fly.

Once everything has been prepared, adult flies must be anesthetized. This can be done using brief exposure to fly nap for two to five minutes, but be careful not to leave the flies in the fly nap longer than five minutes. Short term exposure to cold can also be used to anesthetize flies, but carbon dioxide is not recommended since it has longer lasting effects on heart rate and ity.

Next, the anesthetized flies are placed dorsal side down into a Petri dish coated with a thin layer of petroleum jelly. The hydrophobic cuticle in the wings and body should reversibly stick to the jelly, but the fly can be repositioned if needed. An initial cut is made using a curved pair of spring scissors.

This is done by placing the scissor blades under the legs of the fly and angled down toward the dorsal surface of the near the neck. The head ventral nerve cord and legs are removed with a single cut once cut. The preparation is then submerged in the oxygenated artificial hemo lymph solution using spring scissors.

The posterior tip of the abdomen, which is comprised of abdominal segment seven and eight, is removed with a single cut. This provides access for making lateral cuts along both edges of the abdomen and serves to sever the connection between the posterior gut and the abdominal cuticle. Insert the blades of the scissors into the small opening in the tip of the abdomen and cut the cuticle laterally.

This frees up one edge of the ventral cuticle. Grasp the flap with forceps and make a similar cut along the opposite lateral edge of the abdomen. The freed flap of ventral abdominal cuticle can now be removed.

The gut and other abdominal organs can usually be removed as a single mass using jeweler's forceps and gentle tugging. Removing the internal organs reveals the beating heart tube, which is still attached to the dorsal cuticle by small aery muscles and is surrounded by fat bodies. The pulled capillaries are inserted into plastic, one 16th inch tubing attached to a vacuum source.

The suction force is proportional to tip diameter, so pipettes with tips larger than 40 microns in diameter should not be used once set up. This suction system is used to suck off excess fat from around the heart tube. It can also be used to remove air bubbles that accumulate around the cuticle and any debris.

Extreme care must be taken to avoid touching the heart itself, and because the posterior portion of the heart is very fragile, that region should be avoided together. The adult drosophila heart tube is now exposed and should be beating if necessary. The cuticle and attached heart can be repositioned by gently lifting the cuticle out of the petroleum jelly and carefully tamping it back down again.

Avoiding any contact with the heart tissue. At this point, the solution bathing the preparation should be replaced with fresh artificial hemolymph. This preparation should be allowed to equilibrate for 20 to 30 minutes with oxygenation prior to any manipulations.

If the preparation is to be maintained for longer than 60 minutes, it should be reperfused with fresh artificial hemolymph. This preparation is now ready to be used for optical recording and can also be used for electrophysiological recording or for histological manipulations. A different technique is required for visualizing the beating heart intra larvae.

First, prepare cover slips the day before the procedure. This is done by using a toothpick to transfer about 50 to 70 microliters of Sard solution onto a 22 by 22 millimeter cover slip. Let the syl guard harden at 65 degrees celsius overnight.

Next, make the Brody and Bates buffer once prepared, aliquots can be stored at minus 20 degrees Celsius and thawed when needed. Finally, prepare the tools required to apply the histo acral glue. First, pull several fine capillaries.

Next, connect a 1.5 milliliter pipette tip with a small plastic tubing about one 32nd inches in diameter. By inserting the smaller end into the tube, insert a capillary with its wide opening to the other end. Once set up, load the capillary with histo glue by dipping the capillary into a drop of glue and gentle suction into the plastic tip.

When working with the glue, one should have a second glass capillary at hand to remove excessive glue from the tip of the glue capillary. The glue is liquid until it comes into contact with buffered saline where it will harden within a couple of seconds. However, it usually remains plastic long enough to be removed from the glue capillary tip or while applying it to the specimen.

Now to begin immobilization of the larvae, place a drop of histo acral glue close to each corner of the syl guard. Cover slips add 50 microliters of B and B buffer to the glue drops, which will harden instantly fill the space between the glued spots with b and b. The glue will keep the buffer on the slide.

Take a wandering L three larvae and place it on a piece of wet tissue paper on ice for two minutes. This will slow down its movement. Next, transfer the larvae into the buffer and orient it with the dorsal side up.

This is the side of the larvae that lacks the identical belts. Quickly apply some glue between the head region and the sill guard. The glue will harden very quickly and prevent the head of the larvae from moving.

Using forceps carefully grab the animal by the posterior sparkles and gently stretch it. Fix the larvae in this position by applying the glue along both sides of the larvae at the posterior half. To reduce further movement of the cuticle by the body wall muscles add more glue to the flanks of the animal.

The larva is now fixed in this position. The cover slip can be placed into a small Petri dish filled with artificial hemolymph or PBS heartbeats can now be recorded without interference from body wall movements. The semi intact adult drosophila heart will beat rhythmically for hours following dissection when maintained in fresh oxygenated artificial hemolymph.

A representative example is shown here and in supplemental movie one hearts from third in star larvae shown here, and young flies those one to three weeks post elo generally exhibit a regular heartbeat at rates between one to three hertz flies older than three weeks generally are more arrhythmic. Nevertheless, these hearts are still able to beat spontaneously for hours in oxygenated artificial hemolymph hearts and adult flies that are damaged as a result of the dissection procedure typically show localized regions of extreme constriction that are unable to relax and often be uncontrollably. Heartbeat rates slower than one Hertz are rarely observed in flies younger than three weeks of age.

Consequently, such preparations are considered to be damaged and are discarded. We've just demonstrated how to prepare level and adult fruit fly hearts for optical imaging. Remember, when making the semi intact preparation never touch the heart itself.

So that's it. Thank you for watching and good luck with your experiments.