In Vivo Calcium Imaging Using Synaptically Localized Calcium Sensors in Drosophila melanogaster

Fix a fly with its brain exposed in an imaging chamber.

The fly expresses a genetically encoded calcium indicator, or GECI localized in the dendrites of mushroom body, or MB neurons.

Connect the fly with electrical wires and position an odor-delivery needle near its antenna.

Add a buffer droplet on the exposed brain, position the fly under a microscope, and connect the needle to an odor-delivery system.

Excite the GECI and deliver an odor stimulus.

The odorant binds to the antenna's sensory neuron receptors, generating action potentials.

The action potentials propagate from the sensory neurons to the projection neurons, inducing neurotransmitter release and facilitating calcium influx and GECI fluorescence in the postsynaptic MB neurons.

Repeat with a second and third odorant.

Next, apply the first odor with an electric shock and the second without.

Finally, deliver the three odors without shocks. Record the fluorescence.

A reduced fluorescence in response to the shock-associated stimulus compared to the controls reveals learning-induced synaptic plasticity.

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 Band-pass 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 in 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 conditioned stimulus minus odor alone for 60 seconds without electric shock. Measure the post-training odor-evoked calcium transience again by repeating the pre-training odor stimulation protocol, three minutes after finishing the training phase