Encyclopedia of Experiments: Biology
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Transcript
- Different than in vertebrates, the Drosophila heart is part of an open circulatory system. The heart of an adult fly is a tube-like structure built from cardiomyocytes that is connected to the epidermis by alary muscles and embedded in extracellular matrix. The heart chamber is on the posterior end of the fly and pumps hemolymph into the aorta from which it flows into the open body cavity.
In insects, the hemolymph serves as both blood and interstitial fluid, carrying nutrients, signaling molecules, and immune cells. Additional landmarks of the heart include ostia, which are openings along the tube, and intracardiac valves. Pericardial cells are localized along the heart tube and are part of the fly's excretory system.
In an experimental heart preparation, the artificial hemolymph mimics physiological conditions to preserve tissue function. Although natural hemolymph is not responsible for gas exchange, the artificial hemolymph must be well oxygenated to substitute for the lack of a functional tracheal system. In the following experiment, we demonstrate a semi-intact heart preparation from an adult fly for live imaging or electrophysiology.
- Before getting started, we first prepare an artificial hemolymph solution. This solution contains sucrose and trehalose, 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 rhythmicity.
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 reversably 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 thorax 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 hemolymph solution. Using spring scissors, the posterior tip of the abdomen, which is comprised of abdominal segment 7 and 8, 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 jewelers forceps and gentle tugging. Removing the internal organs reveals the beating heart tube, which is still attached to the dorsal cuticle by small alary muscles and is surrounded by fat bodies. The pulled capillaries are inserted into plastic, 1/16-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 altogether. 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 re-perfused 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.
