The overall goal of this procedure is to measure physiological activity of olfactory neurons in the drosophila and antenna lobe by optical imaging of intracellular calcium changes. This is accomplished by first assembling the mounting block wire holder and antenna shield. Next, a fly expressing the fluorescent calcium sensor GCaMP is mounted and fixed to the block.
Once the fly is mounted, the cuticle on the top of the fly head is cut away to expose the antenna lobes. The final step of the procedure is to set the fly under the microscope and stimulate it with an odor. Ultimately, results can be obtained that show odor evoked activity patterns in the DLA antenna lobes through recorded changes in relative fluorescence of the calcium sensor G camp.
This Method can help reveal how odors are represented as spatial and temporal patterns of neural activity at different levels of the olfactory circuitry. The main advantage of this technique over other methods like electrophysiology is that we can record the activity of genetically identified neurons that are not accessible to recording electrodes. Though this method can provide insights into the neural processing of odors.
It can also be applied to other regions of the joof brain, such as the auditory and gassy systems. First, thread the screws through the back of the plexiglass mounting block, such that they do not extend beyond the front. Use a precision drill to make a dent in the upper border of the circular chamber for the fly by digging under the surface to reduce the thickness of the block.
Under a dissecting scope, cut a copper grid with a scalpel perpendicular and close to the bottom of the slit to create a collar for holding the fly. Make sure the two resulting flaps are level as this will aid in the insertion of the fly. With a toothpick, place a small drop of super glue onto the mounting block above the circular chamber.
For the fly, place the copper grid onto the glue and position so that the slit is centered on the block. Gently press down on the grid and remove any excess glue with a pair of tweezers. Add tiny drops of glue under the flaps on each side and fold the grid over the front of the block so that the slit is pointing straight downwards.
Make sure the top of the grid is level with the block and allow it to dry completely. To make a wire holder, cut a plastic cover slip in half and remove a central piece to make a U shape. Use beeswax to glue a piece of wire across the top of the U so that the wire is not too t.
Construct an antenna shield by using a paper hole punch to make a hole in a plastic cover slip. Trim the edges of the cover slip to 0.5 by 0.7 centimeters. Glue the pierced plastic cover slip onto sticky tape, and then place a drop of ethanol on the tape exposed through the hole.
To remove the adhesive anesthetize one to three week old flies of the appropriate genotype by cooling them on ice. Under the dissecting scope, pick up a fly by the wing joint and slide it dorsal surface first along the copper grid so that it is suspended by the neck. If necessary, rotate the fly until it is looking down.
Place a fine cactus spine in front of the fly's head to prevent it from escaping and position the spine in front of the probos to keep it from moving. Fix the cactus spine to the block using beeswax, making sure that the top of the head is level with the top of the block. Fix the head of the fly to the block with a small drop of rosin dissolved in ethanol.
Place the block in a humidified box and allow the rosin to harden for about one hour. Using forceps, pull the antenna plate forward and drop the wire holder in the cuticular fold between the antenna and head. Fix the holder to the front of the block with more beeswax.
Using the screws built into the block, gently push the wire holder forward to create some space between the antenna plate and the head. Place the antenna shield over the top of a fly's head with the whole positioned centrally. Fix the shield to the top of the mounting block with beeswax.
Using a blade splitter, cut a small hole in the tape on the shield, exposing the cuticle of the fly head behind the antenna. The hole should not extend beyond the eyes to prevent the preparation from leaking. Seal the hole between the tape and the cuticle with two component silicon.
Remove any excess silicon from the top of the fly head. Place a drop of drosophila ringer saline on the fly's head, making sure that there are no leaks and that the antennae remain completely dry. Using a sapphire blade lightly score along the cuticular fold directly behind the antenna.
Then cut along the eyes and across the selli on the back. Finally, fold the piece of cuticle over the scored edge and cut from the underside. To avoid severing the antenna nerves, carefully remove glands and trachea with fine tweezers.
Rinse repeatedly with Drosophila ringer solution, leaving a large drop on the head. Insert a cellulose pad into a two milliliter plastic syringe with clean forceps. Using a filtered pipette tip, apply 20 microliters of diluted odorant.
Plug and cap the syringe until required for imaging. Place the mounting containing the fly under the microscope. Lower the objective until it touches the ringer solution on the fly's head.
Looking through the viewer under brightfield illumination, position the preparation so that the hole in the head capsule is centered and its borders are in focus. Switch to fluorescent light and adjust the focus until you can see the labeled neurons, which should be apparent from the basal green fluorescence of GCaMP. Acquire images with a CCD camera mounted on the microscope using appropriate acquisition software and find focus on the glomeruli of interest.
Be sure to close the diaphragm in the illumination light path to limit excitation light to the imaged area. Position the exit of the olfactometer approximately 0.5 centimeters in front of the flies antennae. Here, the anal lobes are shown in a fly expressing GCaMP 1.6 under the control of an olfactory sensory neuron promoter that drives expression in several glomeruli stimulation with 1%propionic acid elicits increases in GAMP fluorescence indicating increases in intracellular calcium, in olfactory sensory neurons, innervating glomeruli, DC four and DP one L.The temporal dynamics of calcium responses of glomeruli DC four and DP one L to propionic acid are shown here.
The black traces show the median of the mean delta F over F within the region of interest across eight animals, and the gray surface indicates the range between the first and third quartiles of the distribution. The heat maps below show the responses of the individual animals. The greened magenta boxes indicate the frames averaged to calculate the peak responses of the glomeruli above baseline fluorescence levels of GCaMP and are shown quantified here.
Correcting for bleaching of the fluorescent reporter can have effects on the response. Peak sample data on the left panel can be corrected without major changes in the shape of the response.Sample. Data on the right panel is severely affected by bleach correction, likely because the signal drops and is maintained below baseline after odor stimulus termination Once mastered, this technique can be done in about 30 minutes.
If performed properly While attempting this procedure, it is important to keep the antenna completely dry or they'll be unable to detect odors. After watching this video. You should have a good understanding of how to prepare image and analyze odor.
Evoke neuronal activity in the fruit fly.
