Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.
This technique facilitates the observation in real time of synaptic calcium activity as a physiological parameter for the cellular processes underlying learning and memory formation in drosophila melanogaster. By comparing neuronal responses to odors before and after associative training, we can draw direct correlations between synaptic activity and the formation of the memory trace in individual fruit flies. To produce transgenic fruit flies in which specific neurons of interest express a genetically encoded calcium indicator cross female-virgin and male flies carrying the desired GAL4 and UAS constructs, and age the female progeny until three to six days post eclosion.
To prepare a fly for imaging, use fine forceps to place an ice anesthetized fly into a custom prepared imaging chamber. Using a dissecting microscope to position the thorax and legs in contact with the electrical wires at the bottom of the chamber, and with the head lying flat. Fix the fly in place with clear adhesive tape, and use a surgical scalpel blade to cut a window in the tape around the head, leaving the antenna covered and only the anterior most portion of the thorax exposed.
Use an insect pin held by concave-convex jaws to carefully surround the sides and back of the head with blue light curing glue. And use a blue light emitting LED lamp to set the glue. When the glue's completely set, clear any residual unhardened glue from the dorsal surface of the fly head and cover the exposed cuticle of the head with a drop of Ringer's solution.
Using a very fine-bladed stab knife, cut through the cuticle across the posterior of the head, starting at the ocelli and cutting up each side medial to the eyes to form a flap of the cuticle that can be easily torn off using forceps. Remove any excess cuticle that may block the brain region of interest and use fine forceps to carefully clear the dorsal surface of any trachea, taking care to avoid disruption of the brain tissue itself. Remove and refresh the Ringer's solution as necessary to clear the area of tissue debris.
And place a hypodermic odor delivery needle approximately one centimeter from the head of the fly, taking care there is nothing that could obstruct odor delivery to the antenna. Then connect the imaging chamber to the odor delivery system via the hypodermic odor delivery needle. And allow the fly 10 minutes to recover from the anesthesia and surgery.
For visualization of the GFP based calcium indicators, tune the laser of a multi-photon 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 course 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 four 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 LED's triggered by the opening and closing of specific odor cup valves. Followed by 12.5 seconds of recording at the end of 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. Then save the imaging files in an appropriate format for later image analysis. Here representative mushroom body output neuron images, acquired as demonstrated can be observed.
The specific compartmentalized expression of the dHomer-GCaMP3 sensor, which is not expressed in the axonal compartments of the neuron, demonstrates a punctuated signal in the dendritic compartment. Clear though lower amplitude odor responses can be observed in flies expressing dHomer-GCaMp3, compared to flies expressing cytosolic GCaMP6f. Here, representative calcium traces from one individual fly demonstrates variances in the noise level and amplitude that can be obtained between individual preparations.
This technique has opened up the possibility of visualizing at the sub-cellular level, how olfactory representations are modulated and how memory phases are formed through conditioning. Such experiments will be vital to expand our understanding of complex brain structures such as the mushroom body, and to characterize how associative memories are stored across distributed populations of neurons.
