Drosophila are genetically attractive for investigations of the development and function of the heart and underlying pacemaker activity. This procedure shows how to make preparations of both intact and dissected larvae and how to monitor heart rate. The dissected preparation also allows recordings of extracellular or intracellular potentials to further access ion channel function.
Hi, I'm Robin Cooper, a faculty member in the Department of Biology at the University of Kentucky. Today my students, Ann Cooper, Kyla Ryman, Matt Ward, and Easter Boku will show you various procedures to monitor the heart rate in Drosophila larvae. The following unrestrained method is called ant farm because the larvae are visualized within one focal plane.
For best visualization of the larvae, use a dissecting microscope with a two x base objective and a 0.5 x tube objective for the best spatial resolution and magnification over a one centimeter by 0.5 centimeter rectangle. Photographs and videos can be recorded through a camera on a ocular mount. To begin this method sandwich a larvae with a thin layer of food between two glass slides.
The slides are held one to 1.5 millimeters apart with plastic spacers on all four sides. Tilt the preparation between 20 and 45 degrees for a few minutes to encourage the larvae to keep its head pointed downward. Adjust the microscope mirror for best contrast of the heart or the two trachea to watch the heart beat.
The glue method is a permanent mount for restraining larvae on a glass slide. To begin the glue method, place a clean cover slip on top of a glass slide. Put a small drop of super glue on one corner of the cover slip.
Next, rinse the larvae with water. To remove excess food, soak up the liquid with tissue or the corner of a paper towel. Once the larva is thoroughly rinsed, gently pick it up with clean tweezers and place on the slide away from the cover slip under the microscope, orient the larvae on its stomach.
The stomach will have faint horizontal grooves running along it with fine black hairs. The back of the larvae should face up and features two racing stripes, which are the trachea. Using a second set of tweezers dedicated strictly to handling the glue, take a small dab from the drop at the end of the slip and place it at the corner on the opposite side.
Use a tiny amount just enough to cover the head of the tweezers. Pick up the animal and place it on the glue while ensuring it remains. Stomach side down the black mouth.
Hooks should be at or near the edge of the cover slip the mouth hooks and the brown spiral. Should not touch the glue. Gently press down on the larvae to flatten it out.
To apply a test substance, it is best to dispense small droplets with a syringe near the larvas head. Observe the heart rate by counting the number of times the spirals pulse in one minute. For longer observations, larvae can be fed in place and moistened to prevent dehydration to make a dissection plate that is usable under the microscope, use a scalpel to cut a hole, two centimeters in diameter in the center of a piece of magnetic tape and stick it to a glass slide.
Use dissecting pins that have been bent and glued to paperclips for easier pinning. During the dissection, place an intact third in star larvae on the dissection plate. Dorsal side up the trachea should be visible through the cuticle.
Place two pins in the larvae on either side of the mouth hooks, and another two pins on either side of the spirals. Next, add saline to keep the larvae moist. Then make a horizontal incision by the coddle end, followed by a longitudinal cut that should continue on beside the spirals towards the head of the animal.
Lift one dorsal pin and hook it underneath the cuticle. Use the pin to spread. Open the body cavity.
Repeat with the other three pins. Once the animal has been pinned open, remove the guts being careful not to damage the trachea or heart. Some structures including the brain and some fat bodies are attached to the heart and will damage it when they are removed.
Allow the preparation to relax in an HL three saline bath for three to five minutes after dissection, intracellular and field recordings are performed in this preparation while pharmacological agents are applied directly to the heart. The total time for this dissection step is three to six minutes. To perform electrical pacing, focal electrodes first need to be made to make a focal electrode run a glass electrode over a heating element to fire polish and to make a gradual bend of about 45 degrees, mount a focal electrode filled with room temperature HL three saline into the manipulator with the tip pointing down.
Make sure that the stimulator wire is touching the saline inside the electrode and that the stimulator is off. Secure a fresh heart dissection preparation on the microscope. Place the grounding wire into the saline.
Being careful not to touch the pins or disturb the preparation. Maintain focus on the electrode while using the manipulator to move it down into the saline. Once in the saline, position the tip of the electrode with the lumen flat against the heart and lightly touching it.
Once the electrode is positioned, set the stimulator frequency at two hertz with the voltage at its lowest setting. Turn the stimulator on. Tune the frequency to approximately three hertz and slowly increase the voltage until the heartbeat speeds up to match the rhythm of the stimulator.
The rhythm can now be manipulated through the stimulator. Compounds can be added to see if they affect contraction or electrical conduction. To perform field potential recordings, fill a macro patch recording electrode with bathing medium and mount it into a manipulator on a compound microscope.
Connect the electrode to an amplifier and recording rig. Use the manipulator to lower the lumen of the electrode over the heart of a dissection prep secured to the microscope. Very lightly touch the heart with the electrode tip.
Field recordings can now be made to record field potentials from another region of the heart. Gently raise the tip of the electrode and move it to a new area with the manipulator, then relow the tip field potential recordings. Allow precise monitoring of the rate and duration of the electrical signals in the heart to measure transmembrane potentials of larval myocytes.
First, make sure that the lumen of a sharp electrode filled with three molar potassium chloride is flat against the heart at about a 45 degree angle from vertical. Once properly positioned, gently touch the heart with the electrode tip and tap the tip into a cell. The electrode can be used again if it is not broken.
The intracellular recordings allow one to measure the ionic basis of the action potential and characteristic shapes of the potentials to examine the effects of ion channel mutations or channel modulators. So we've just shown you how to do heart recordings as well as how to take intracellular and field potentials. You can use these techniques to look at heart alterations as well as pharmacological manipulations and gene alterations.
When performing this procedure, it's important not to pull on a structure related that's attached to the heart or to press too hard with the focal electrode. Also with the glue method, take care not to glue the mouth or spherical shut. So that's it.
Thanks for watching and good luck with their experiments.
