Preparation of Drosophila Heart: A Technique to Study Heart Physiology in the Adult Fly

Overview

This video describes the fly's adult heart, or otherwise called the Drosophila dorsal vessel. The featured protocol demonstrates a dissection procedure for live heart preparations.

Protocol

This protocol is an excerpt from Vogler and Ocorr, Visualizing the Beating Heart in Drosophila, J. Vis. Exp. (2009).

1. Before you start

1. Freshly prepare artificial hemolymph (AH) solution containing 108mM Na⁺, 5mM K+, 2mM Ca²⁺, 8mM MgCl₂, 1mM NaH₂PO₄, 4mM NaHCO₃, 10mM sucrose, 5mM trehalose, 5mM HEPES (pH 7.1, all reagents from Sigma Chemicals). The sucrose and trehalose should be added to the AH from refrigerated stock solutions just prior to use in order to prevent bacterial contamination.
2. Bring AH to room temperature and oxygenate the solution by air-bubbling for at least 15 min.
3. Pull several fine capillaries (e.g. Glass Capillaries, 100ul, VWR) required for removal of fat.

2. Semi-intact Drosophila Heart from the Adult Fly

1. Adult Drosophila flies are anesthetized with Fly Nap (Carolina Biological Supply Co.) for 2-5 minutes. Care must be taken not to leave the flies in the Fly Nap for longer exposures. Short term exposure to cold can also be used to anesthetize flies but CO₂ is not recommended as it has longer lasting effects on heart rate and rhythmicity.
2. 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 reversibly "stick" to the jelly but the fly can be repositioned if needed. This becomes especially important during subsequent manipulations.
3. An initial cut is made using a curved pair of spring scissors (Roboz #5611). The scissor blades are placed under the legs 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. The preparation is then submerged in an oxygenated, artificial hemolymph (AH) solution.
4. Using spring scissors the posterior tip of the abdomen (comprised of abdominal segments 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. The freed flap of ventral abdominal cuticle is then removed.
5. In steps 3 and 4 the anterior and posterior connections of the gut are severed so the abdominal organs are now held in place only by tracheols. The gut and other abdominal organs can usually be removed as a single mass using jeweler's forceps (Roboz, #55) and gentle tugging. Removing the internal organs reveals the beating heart tube still attached to the dorsal cuticle surrounded by fat bodies.
6. The fat bodies are quite opaque in the light microscope consequently it is often necessary to remove some of this fat in order to see the heart tube clearly. This is accomplished by a liposuction technique using finely drawn glass capillaries. Capillaries are made using a standard pipette puller (e.g. Sutter P-97 Pipette Puller) and are then inserted into plastic tubing (Tygon, 1/16") attached to a vacuum source. This system is used to suction off excess fat from around the heart tube. Suction force is proportional to tip diameter so pipettes with tips larger than 40 microns in diameter should not be used. 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.
7. 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 AHL. This preparation should be allowed to equilibrate for 20-30 minutes with oxygenation prior to any manipulations. If the preparation is to be maintained for longer than 60 minutes it should be perfused with fresh AHL.

Materials

Name	Company	Catalog Number	Comments
Micro Dissecting Spring Scissors (curved)	Roboz Surgical Instruments Co.	RS-5611	Good for gross cuts (Step 2)
Micro Dissecting Spring Scissors (straight)	Roboz Surgical Instruments Co.	RS-5620
Dumont #55 forceps	Fine Science Tools	11295-51
Dumont #5 forceps	Fine Science Tools	11295-10
Glass Capillaries, fine	Science Products	GB100T8P
Pipette Puller	Sutter Equipment	P-97	Both horizontal and vertical pipette pullers will work
Plastic tubing	Tygon	1/16" inner diameter	for 100 µl capillaries
Histoacryl® tissue adhesive	B. Braun Medical