Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

- To perform in vivo calcium imaging in the Drosophila nervous system, target the expression of a genetically encoded calcium indicator, such as GCaMP, to the neurons of interest. When neurons fire an action potential, rapid depolarization of the membrane causes voltage-gated calcium channels to open, leading to an influx of extracellular calcium into the cell.

GCaMP is a fusion protein in which enhanced green fluorescent protein, or EGFP, is modified and fused to the M13 fragment of the myosin light chain at the N-terminus, and to the calcium-binding protein, calmodulin, at the C-terminus. Calcium binds to calmodulin, triggers conformation changes in GCaMP, causing an increase in the protein's fluorescence.

To image changes in GCaMP fluorescence as a proxy for neuronal activity in vivo, expose the region of the nervous system with anticipated activity. Then, use a fluorescent microscope that can capture GCaMP dynamics and is equipped with a stimulus delivery setup. Deliver the stimulus, for example, an odor, while recording GCaMP fluorescence in responding neurons.

In the example protocol, we will see GCaMP functional imaging being used to visualize responses in the brain's mushroom bodies during olfactory associative learning.

- For visualization of the GFP-based calcium indicators, tune the laser of a multiphoton microscope equipped with an infrared laser and a water immersion objective, installed on a vibration isolated table, to an excitation wavelength of 920 nanometers, and install a GFP bandpass filter. Using the coarse Z adjustment knob, scan through the z-axis of the brain to locate the brain region of interest. Use the crop function to focus the scanning on only the area of interest to minimize scan time, and rotate the scan view such that the anterior of the head is facing downwards. Then, adjust the frame size to 512 by 512 pixels and select the region to be scanned, taking into account the calculated scan time for each frame to achieve a frame rate of at least 4 Hertz.

For odor-evoked calcium transient visualization, initiate a pre-programmed macro package capable of linking the image acquisition software and the odor delivery program and begin the measurement in the microscope software for 6.25 seconds to establish an F0 baseline value. In the odor delivery system, deliver a 2.5 second odor stimulus, indicated here by illumination of LEDs, triggered by the opening and closing of specific odor cup valves, followed by 12.5 seconds of recording at the end of the odor offset. Then, repeat the delivery for a second and third odorant in the same manner.

To perform associative conditioning in this setup, use the computer-controlled odor delivery system to present the conditioned stimulus plus odor for 60 seconds, alongside 12 90-volt electric shocks. After a 60 second break, present the condition stimulus minus odor alone for 60 seconds without electric shock. Measure the post-training odor-evoked calcium transients again by repeating the pre-training odor stimulation protocol 3 minutes after finishing the training phase. Then, save the imaging files in an appropriate format for later image analysis.