This article describes a method for imaging calcium activity in the mushroom bodies of transgenic Drosophila using fluorescence microscopy. The technique involves perfusing nicotine and potassium chloride to stimulate neuronal activity and measure bioluminescent signals.
Secure an immobilized transgenic Drosophila with its head exposed in a recording chamber on a fluorescence microscope stage.
The fly’s mushroom body comprises neurons expressing calcium-sensitive bioluminescent fusion protein, which is activated by a preloaded cofactor.
Connect a perfusion tube to the chamber. Locate the fluorescent mushroom bodies, focusing on the calyx and medial lobes. Confirm drainage with buffer.
Set imaging parameters and focus on the calyx and medial lobes. In the dark, acquire baseline fluorescence images of the mushroom bodies prior to stimulant exposure.
Perfuse nicotine in calcium-containing buffer to activate nicotinic acetylcholine receptors, triggering calcium influx and bioluminescent light emission.
Wash with buffer to restore baseline conditions.
Perfuse potassium chloride to depolarize neurons, triggering intracellular calcium release and bioluminescent signal emission.
Wash with buffer to restore baseline conditions.
Analyze images to assess spatial and temporal calcium activity in the calyx and medial lobes.
- Next, place the sample onto the mount under the microscope, and adjust the magnification to 5x. Then, insert the perfusion tube into the channel through the puncture. Set the microscope to fluorescent mode, and then center, and bring the mushroom bodies into focus. Once the mushroom bodies are in focus, change the magnification to 20x. Recenter the image and adjust the focus.
Next, position the drainage apparatus over the drainage pool, and adjust the height of the drainage shunt so that the tip is just above the drainage pool. Then, flush the sample with Ringer's solution for 30 seconds to check for adequate drainage. Finally, pull down the shield to seal off the apparatus from any outside light.
Using the automated system in the Photon Imager program, make fine adjustments to the focus, and take a reference fluorescent image of the mushroom bodies. Adjust the position of the region of interest boxes over the calyx, the cell bodies, and the medial lobes by clicking on the boxes and dragging them into position while holding Control.
Now, record baseline values for about 5 to 10 minutes. Next, flip on the switch to begin the flow of nicotine. At the same time, press Enter, and add a note to signify the flow of nicotine has begun. After one minute, stop the flow, and make another note in the log signifying that the flow has been stopped. Then, wash the sample with Ringer's solution for five minutes.
While the sample recovers, turn off the wash, set the timer for an additional five minutes, and prepare to start the flow of potassium chloride. When ready to begin, start the flow of potassium chloride, and enter 100 millimolar potassium chloride start into the sample log. After one minute, stop the flow and enter potassium chloride shut off into the sample log. Then, wash the sample with Ringer's solution for one minute and switch the microscope to fluorescent mode. Take a final fluorescent image, and then turn off the Ringer's solution and remove the sample.
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