Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Our protocol allows imaging of the whole fly brain during behavior, which is important for understanding how neurons in different brain areas interact to shape behavior. Unlike other published protocols, this was developed specifically to access the whole brain while the fly is behaving and sensing by a smell, taste or visual stimulation. To create a head slot.

After printing the holder with a 3D printer, place a piece of sticky tape rectangularly on a flat surface under a stereo microscope. And cut an approximately five millimeter by one centimeter piece. Use two scalpels fixed together in parallel to cut a 400 by 400 micrometer next slot in the middle of the longer side of the tape, and place the tape over the flatter side of the hole on the bottom of the holder.

Use forceps to push the tape approximately 500 micrometers around the hole. And use black nail polish to cover the top of the tape and the holder. After letting the nail polish dry for at least an hour use a rolled tissue to add approximately one microliter of grease to the head slot.

To prepare the body slot, cut an approximately two centimeter piece of wide tape into two pieces and cut 1.5 millimeter wide slices. Then cut out a 0.3 millimeter deep shoulder and body slot and make sure that the tape fits the holder. To fix a fly into the holder, first place a shallow container with ice under the dissecting microscope, and place the holder upside down onto a piece of laboratory tissue placed over the ice.

Aspirate a one to four days old female fruit fly from its vile, and blow the fly onto unmelted ice. When the fly stops moving use Dell forceps to grasp the fly at the base of the wings, and slide the fly into the holder with the neck inside the slot. The eyes should be equally positioned on either side of the slot.

If needed, add one microliter of grease to the top of the head to prevent the glue from reaching the back of the head. Then cover the body with a tissue and some ice to make sure the fly does not move. To secure the head, place it at an approximately 20 degree angle from a fully posterior view.

And use a rolled up tissue to apply UV glue around the head while avoiding soiling the sensory area of interest. For taste experiments, use forceps to pull out the proboscis and add glue at the proboscis base to prevent movement. If no taste experiment is planned push and glue the proboscis into the head.

Cure the glue with UV light for five seconds, and use a rolled up tissue to carefully clean the areas surrounding the head, to remove any remaining liquid glue that could stick to the legs and doors soil, sensory areas. Then use a thin strip of tape or a tissue to move the legs to the front as necessary. To position the body, remove the ice container and turn the holder around.

Remove the water around the fly and acting quickly before the fly recovers from the anesthesia place the body slot tape over the hole and gently pushed the fly's body down, taking care not to overstretch the neck. Cover any remaining large holes with tape and add approximately one microliter of grease to the back of the head, to prevent glue from sticking in that location. Use a rolled up tissue to paint UV glue around the thorax and on top of the tape and thorax, and cure the glue with UV light for approximately five seconds.

Carefully remove any grease and uncured glue with a laboratory tissue and add approximately one milliliter of saline to the top of the head. Use forceps to push aside any air bubbles and check for leaks by placing a cover slip over the saline and turning the holder around to check for saline on the front side. If saline is observed, remove the saline and fix the hole with more glue or more grease.

To dissect the head select the highest magnification and use sharpened very fine forceps to make two cuts at the base of the central dark cuticle triangle on each side of the neck. Cut around the dark triangle, and remove this part of the cuticle. Muscle 16 and the esophagus should be visible through this hole and moving rhythmically.

Use the very fine forceps to carefully pinch the top of this area to sever muscle 16 without puncturing the esophagus. If the rhythmic movement stops the muscle 16 was likely removed. When the muscle has been excised starting from the medial edge of the dark triangle region, use the forceps like a pair of scissors to carefully cut and remove the remaining cuticle in small pieces.

Then use the forceps to grasp and slowly and steadily pull away one air sac at a time. In this video a calcium probe was expressed in all of the neurons of these two fruit fly brains. And a puff of odor was presented.

In this experiment, calcium sensor expression was restricted to dopaminergic and serotonergic neurons. Notice the tight correlation between the strong synchronous activity over the brain and the flies walking behavior illustrating how the whole brain can be observed during a specific behavior. Using principle component analysis and independent component analysis different functional regions highlighted in different colors can be extracted.

The shape and localization of the functional regions allows the regions to be mapped to anatomical templates. To identify brain regions and in some cases, neuron types. Once aligned to the appropriate anatomical template fluorescence values can be averaged within specific anatomical brain regions for quantitative analysis.

For example, in this analysis we can observe that all regions are more active during walking than during grooming. Be patient. Learning this heart dissection technique is difficult.

In my lab, people need on average three to four months before they master it and get their first useful preps. Following this protocol we can record large-scale brain activity using a fluorescence microscope. Fast methods such as light field microscopy are particularly indicated.