Functional Characterization of Individual Pre- and Postsynaptic Partners In the Drosophila Larval Central Nervous System Using CaMPARI

Our research focuses on assessing functional synaptic connectivity between defined neural node partners in live and dissected Drosophila larvae. Conventional methods require dissecting or physical stimulation. This protocol uses optogenetics in intact larvae, reducing artifacts.

To begin, melt the cornmeal agar medium in a microwave for 8-10 seconds without boiling and allow it to cool at room temperature without solidifying. Prior to complete cooling, add all-trans-retinal, or ATR, to a concentration of 0.5 millimolar to supplement the media for flies that require csChrimson activation. Cover the vials completely with aluminum foil to prevent light exposure.

Cross virgin females carrying the CaMPARI and csChrimson genes with males carrying the GAL4 and LexA driver genes in plastic vials containing the prepared media. Prepare parallel vials without ATR as controls. Incubate the vials at 25 degrees Celsius until the larvae reach the third instar stage.

Use a paintbrush to collect a third instar larva. Rinse the larva in a three-well micro spot plate containing 0.1 molar PBS to remove any adhering food. Place a small amount of modeling clay at each corner of a clean 18 x 18 millimeter cover slip.

Place the larva in a drop of PBS on the cover slip and allow it to orient ventral side down. Place a clean microscope slide over the cover slip containing the larva. Secure the cover slip by applying modeling clay to the corners, using just enough pressure to immobilize the larva without crushing it or breaking the glass.

Place the slide containing the larva on the stage of a confocal microscope equipped with a 40x/1.2 objective lens. In the microscope software interface, click on Live. Then under the Z Stack panel, click Set First and then Set Last followed by Stop to acquire a baseline Z Stack of postsynaptic neurons, expressing CaMPARI.

Under the Channels menu, set the 488 nanometers green channel with 0.8%laser power. Next, set the 561 nanometer red channel to 0.8%laser power. Under the Z Stack menu, set the Interval value to 1 micrometer.

In the Acquisition menu, choose Presets to set the 256 x 256 pixel resolution. A line Averaging value of 2, bidirectional scanning and a scan speed of 9, allowing automatic update of pixel time. Perform all imaging in a dark environment, maintaining identical acquisition parameters throughout the experiment.

Set the 405 nanometers photoconversion light to 0.5%laser power. And the 561 nanometers optogenetic stimulation light to 0.8%laser power. Click Live, then select Best Fit under the Histogram panel.

Allow both lasers to simultaneously stimulate the larva for 30 seconds, then stop the stimulation. During post-stimulation ZStack acquisition using the same parameters as baseline, observe red CaMPARI fluorescence in laser-stimulated neurons of ATR-fed larvae with minimal photoconversion and controls. In the Fiji ImageJ software, click on File, then choose Open.

In the BioFormats import options window with default settings, click OK to open the acquired ZStack image. Under the Image menu, choose Color and then Split Channels to view the red and green channels of the acquired image. For background subtraction, click on Process, then select Subtract Background.

In the pop-up window, set the rolling ball radius to 50 pixels then click OK and select Yes in the Process Stack window. Next, under the Analyze menu, select Tools, then choose ROI Manager to open the ROI window. Use the freehand selection tool to outline regions of interest around individual cells.

In the ROI window, select Add to save the selected outline. Then click Measure to generate area, mean, standard deviation and integrated density results. Repeating the process for multiple cells of the same larva across both green and red channels separately.

After exporting the results to a spreadsheet, measure the red to green or R by G ratio of the integrated densities for each cell, and average these values to calculate the mean R by G ratio per larva for both the pre and post-stimulation images. Finally, calculate the change in fluorescence or delta R by G by subtracting the pre-stimulation R by G ratio from the post-stimulation R by G ratio to quantify post-synaptic calcium activity. The functional connectivity studies between class IV dendritic arborization or cIVda neurons and basin-4 interneurons in third instar larvae showed that the basin-4 interneurons displayed green fluorescence due to cytosolic CaMPARI expression before photoconversion and optogenic light stimulation.

No red fluorescence was detected under baseline conditions, confirming the absence of photoconversion prior to stimulation. Simultaneous photoconversion light exposure and optogenetic stimulation resulted in CaMPARI photoconversion from green to red fluorescence. The absence of red fluorescence in control indicates that the photoconversion was specifically driven by csChrimson-mediated neuronal activity.

Delta red by green fluorescence was significantly higher in experimental animals than in no ATR controls. This protocol can be used to confirm synaptic partnership by enabling the tracking of presynaptic stimulation followed by immediate post-synaptic responses in live-developing animals. The most challenging step is proper animal immobilization will require sufficient pressure to prevent movement without harming the animal.

Future studies could enable tracking, the formation, strengthening, and even elimination of synaptic connections between specific neuronal populations during a developing animal.