Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

This method can help answer key questions in the endocrinology field, as well as the neuroscience field, such as our research to review brains responses to endocrine signals. The main advantage of this technique is that you can examine direct effects of endocrine signals on the brain separated from other tissues. To begin this procedure, use forceps to scratch the bottom center of a plastic culture dish to create a dent for easier mounting of the ventral ganglion of the larval brain after dissection.

With a toothpick, place a small drop of superglue on either side of the dent, and attach a tungsten rod to the glue. Then, using a small piece of putty-like reusable adhesive, make a circular wall surrounding the dent. To dissect the larval brain, 90 to 120 hours after egg laying collect larvae and wash them at least three times with distilled water to remove adherent food debris.

Next, place a larva on a square one and a half inch watch glass filled with ice cold PBS. Use a pair of forceps to gently grab the middle part of the larva. Use another pair of forceps to grab and pull the mouth hooks gently to separate the anterior part of the larva containing the brain from the rest of the body.

Then, hold the anterior tip with the forceps and turn the larva inside out. Remove the extraneous tissues attached to the brain, such as imaginal discs, fat bodies, and ring gland. Gently separate the brain from the mouth parts.

Afterwards, apply 200 microliters of PBS to the inside of the adhesive ring prepared earlier in the imaging chamber. Gently suck the dissected brain with PBS into a Pasteur pipette, and transfer it to the imaging chamber. In the imaging chamber, grab the muscle fibers extending from the ventral ganglia and insert the brain gently into the dent underneath the tungsten wire.

Then, pull the tungsten wire slightly up to place the brain at the correct position for imaging. 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 bright field illumination position the brain and bring it into focus.

Switch to fluorescent light and adjust the focus on the GCaMP success labeled cells. Start the acquisition at 250 ms/frame, at a resolution of 512 x 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 1000 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 GCaMP success emission for a few minutes.

To analyze the data, open the analysis software and use the first frame which was before peptide application as a reference image. Then, select Plugins, TurboReg. Choose the serial image file as the Source, and the reference image as the Target.

Next, check Rigid Body and Accurate for the processing method and quality, respectively. Subsequently, click Batch to start image processing. Select multiple regions of interest by using Analyze, Tools, ROI Manager, in image tray.

Then, measure the pixel intensity by clicking More, Multi Measure, OK, in the ROI Manager window. In this experiment, Wild type brain was exposed to CCHa2, Ghrelin, and Nociceptin, whereas CCHa2 Receptor mutant brain was exposed to CCHa2. GCaMP6 signals in insulin producing cells were detected by confocal microscopy at 250 ms/frame, and here are the still images of selected time points.

The ratio of the change of ROI intensity to baseline signal intensity at each time point was plotted. ROIs where set on cell bodies that were detected in the same focal plane. The solid lines indicate the mean of five to 10 samples, and the dotted lines mark upper and lower limits of the standard error of the mean.

The shaded areas indicate the variation of the signals in the experiments. Once mastered, this technique can be done in one hour, if it is preformed properly. While attempting this procedure, it's important to remember to prepare the brain sample quickly, to avoid damage to it.

This procedure can be combined with genetics in order to answer additional questions like on receptor specificity. The system used in this protocol is easy to prepare and reusable. Thus, this protocol will be useful for, like in screening.

After its development, this technique paved the way for researchers in the field of endocrinology and neuroscience to explore hormonal networks that control brain function in Drosophila.