Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.
Before you start
Adult hearts
Larval hearts
Semi-intact Drosophila Heart from the Adult Fly
Immobilizing Drosophila larvae for optical recording
Representative Results:
The semi-intact adult Drosophila heart will beat rhythmically for hours following dissection when maintained in fresh, oxygenated AH (see Supplemental Data, Ocorr, et al., 2007). A representative example is shown in Movie 1. Hearts from 3rd instar larva and young flies (1 – 3 weeks post eclosion) generally exhibit a regular heart beat at rates between 1 – 3 Hz. Flies older than 3 weeks generally are more arrhythmic; nevertheless, these hearts are still able to beat spontaneously for hours in oxygenated AHL. Hearts in adult flies that are damaged as a result of the dissection procedure typically show localized regions of extreme constriction that are unable to relax. Heartbeat rates slower than 1 Hz are rarely observed in flies younger than 3 weeks of age; consequently such preparations are considered to be damaged and are discarded.
Click here to download Movie 1.– A one week old adult wildtype fly (w1118 laboratory strain) showing the exposed abdominal heart tube (anterior to the left). Note regular, rhythmic contractions. Opaque regions in the upper right hand corner are the remaining abdominal fat bodies; round cells on either side of the heart are pericardial cells.
The Drosophila model has proven to be a powerful genetic tool that has been used to address a variety of scientific questions ranging from embryological development to learning and memory. Recently this versatile model organism has been used to investigate the genetics of heart function in the fly. A number of attempts to quantify heart physiology in adult Drosophila have relied on observations made in intact flies through the abdominal cuticle. Most of these approaches have relied on visual observation or recordings of changes in light intensity transmitter through the abdomen to quantify a single parameter, heart rate. These methods have several limitations: typically only the anterior end of the heart can be observed, what is being observed is the movement of fat bodies secondary to the heart contractions, and the nervous input to the heart is intact. The adult semi-intact preparation provides a more detailed view of a large portion of the functioning heart permitting quantification of a number of parameters in addition to heart rate. It can be used for electrophysiological or optical (see JoVE article, 1435) recording procedures. In addition, this preparation is suitable for histological manipulations (see JoVE article, 1435). It can also be extracted as a relatively clean heart tissue sample for PCR, Western blotting, microarray analysis, etc.
The adult heart in Drosophila is, for the purposes of this dissection, conveniently fixed to the dorsal cuticle of the abdomen by a network of alary muscles. Thus it is possible to dissect away the head and ventral lying nerve cord, and then to remove the internal organs without damaging the heart. However, extreme care should be taken in step 3 to cut only the very tip of the abdomen as the heart extends into abdominal segment 5/6 and a pacemaker cell or cells is undoubtedly present in this region.
Care must also be taken to avoid touching the heart during the dissection. If contact with the heart is suspected the preparation should be discarded. Moderate suction usually suffices to remove some of the fat and tracheols that surround the heart for better visualization of the heart tube but excessive suction can also damage the heart. The suction force is controlled primarily by the size of the pipette tip used for the liposuction; tips larger than approximately 40 microns should be avoided.
Unlike in the adult, the heart tube in Drosophila larva is relatively free to move around the body cavity. Thus a semi-intact larval heart preparation presents difficulties for optical recording techniques. Due to the translucency of the integument it is possible to visualize the heart in the intact larva using brightfield microscopy. The glue technique described here immobilizes larva of any developmental stage and keeps the heart in a fixed position. This permits recording of a relatively stationary heart tube. Heart contractions recorded by such immobilized larva can be subsequently analyzed by the optical recording technique described in JoVE 1435.
KO is supported by a grant from the American Heart Association.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Micro Dissecting Spring Scissors (curved) | Roboz | RS-5611 | Good for gross cuts (Step 3) | |
Micro Dissecting Spring Scissors (straight) | Roboz | RS-5620 | ||
Dumont #55 forceps | Fine Science Tools | 11295-51 | ||
Dumont #5 forceps | Fine Science Tools | 11295-10 | ||
Glass Capillaries, 100ul | VWR | 53432-921 | ||
Glass Capillaries, fine | Science Products | GB100T8P | ||
Pipette Puller | Sutter Equipment | P-97 | Both horizontal and vertical pipette pullers will work | |
Plastic tubing | Tygon R-3603 | 1/16” inner diameter | for 100μl capillaries | |
Plastic tubing | Tygon R-3603 | 1/32” inner diameter | for small capillaries | |
Histoacryl® tissue adhesive | B.Braun, Melsungen |