This study demonstrates a method for imaging calcium dynamics in Drosophila brain explants using fluorescent indicators. The approach allows researchers to observe neuronal responses to neuropeptide stimulation in real-time.
Begin with an imaging chamber containing an immobilized Drosophila brain explant submerged in a buffer to maintain cellular function.
The neurons in the brain express cytoplasmic fluorescent calcium indicators, which bind to calcium ions and fluoresce.
Place the imaging chamber under a fluorescence microscope equipped with a water-immersion lens.
Lower the lens, and under bright-field illumination, focus on the brain.
Switch to fluorescence mode and capture the image.
Next, introduce a neuropeptide into the buffer and initiate time-lapse recording.
The neuropeptide binds to its receptor on neurons, triggering intracellular signaling. This opens the calcium channels and allows the calcium ion influx.
These calcium ions bind to the indicators and increase fluorescence intensity.
Recapture the image after neuropeptide stimulation.
Compare the images before and after neuropeptide stimulation.
An increase in fluorescence intensity after neuropeptide stimulation reflects the Drosophila brain's response to neuropeptide signaling.
To acquire calcium fluorescence images, place the imaging chamber containing the brain explant under the microscope. Lower the objective lens until it touches the PBS. And under brightfield illumination, position the brain and bring it into focus.
Switch to fluorescent light and adjust the focus on the GCaMP6 labeled cells. Start the acquisition at 250 milliseconds per frame at a resolution of 512 by 512 pixels in water-cooled mode. Then, adjust the exposure time to obtain the fluorescence values within the CCD camera dynamic range, but not lower than 1,000 arbitrary units with 16-bit images.
Once the imaging parameters are determined, take the images for one minute before peptide administration to detect baseline signal intensities. Subsequently, apply the test peptide directly by pipetting 100 microliters of the prepared peptide solution into the larval bath. Record the GCaMP6 emission for a few minutes.
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