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

This protocol is an excerpt from Hancock et al., In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster, J. Vis. Exp. (2019).

1. In vivo calcium imaging

1. Use a multiphoton microscope equipped with an infrared laser and a water immersion objective (see Table of Materials), installed on a vibration-isolated table. For the visualization of GFP-based calcium indicators, tune the laser to an excitation wavelength of 920 nm and install a GFP band-pass filter.
2. Using the coarse Z adjustment knob, scan through the Z axis of the brain and locate the brain region of interest. Use the crop function to focus scanning on only this area to minimize scan time, and to rotate the scan view such that the anterior of the head is facing downwards.
3. Adjust the frame size to 512 x 512 px, scan speed to > 4 Hz, and scan region (in the Y dimension) so that the neurons of interest are covered.

2. Visualization of odor-evoked calcium transients through olfactory conditioning

1. Use an odor delivery system that can deliver several odor stimuli in a temporally precise manner.
   1. Use an additional computer to control the device, and to communicate with the imaging microscope software to coordinate odor stimulations with image capture during experiments.
   2. Initiate a pre-programmed macro package capable of linking the image acquisition software and odor delivery program (e.g., a VBA macro package installed in the microscope control software, see Table of Materials) that is responsive to an external input trigger provided by the initiation of an odor delivery protocol in a separate program).
2. Start the measurement by monitoring "pre-training"/naïve odor-evoked calcium transients at a resolution of 512 x 512 px and a frame rate of 4 Hz. Deliver a 2.5 s odor stimulus flanked by additional image acquisition for 6.25 s preceding odor onset (to establish an F₀ baseline value) and 12.5 s after odor offset. Repeat this with a second odorant and then with a third odorant.
3. Continue 3 min after this measurement with classical conditioning ("training") the fly.
   1. Select one of the odors presented in the "pre-training" phase to become the CS⁺ odor and another to become the CS^– odor. Present the CS⁺ odor using the computer-controlled odor-deliver system for 60 s alongside twelve 90 V electric shocks.
   2. After a 60 s break, present the CS^– odor alone for 60 s. Use as odorants 4-methylcyclohexanol and 3-octanol. Do not present the third odorant (e.g., 1-octen-3-ol) during this training as it is used as control only before (step 2.2.) and after (step 2.4) the training phase.
4. Measure the "post-training" odor-evoked calcium transients again by repeating the "pre-training" odor stimulation protocol (step 2.2.) 3 min after finishing the training phase (step 2.3).
   NOTE: The timing of this step should reflect the time of interest after memory formation (e.g., carry out this step 3-4 min after the conditioning step to investigate short-term memory). Typically, flies can survive for several hours in this preparation.
5. Save imaging files in an appropriate format (e.g., Tiff) for later image analysis.