The overall goal of this procedure is to demonstrate dissection methods for the adult leg of Drosophila. This technique removes a portion of the cuticle, exposing the underlying tissue in a way that preserves neuronal and muscle architecture. This procedure allows us to characterize the neuromuscular junction for identified motor neuron arbors, using standard immunocytic chemical techniques.

The dissection is particularly useful for the study of slow degenerative changes which occur over relatively long periods of time. The time aspect is an important consideration, because the well-characterized neuromuscular junction of Drosophila larvae is stable for approximately five to seven days prior to the onset of metamorphosis. Conversely, the adult neurons are present throughout the life of an adult fly, approximately 90 days for a wild-type, lab-reared Drosophila melanogaster.

The technique is flexible and can be applied to a range of genotypes for different disease models, and utilize a range of antibodies available for Drosophila as a model system. The overall goal of this procedure is to demonstrate dissection methods for the adult leg of Drosophila. This technique removes a portion of the cuticle, exposing the underlying tissue in a way that preserves the neuronal and muscular architecture.

Using a paint brush, transfer flies to cold methanol in a glass well or dish for approximately 30 seconds to one minute. The methanol solubilizes the cuticular hydrocarbons, and flies can now be submerged rather than float in aqueous solutions. With forceps, carefully transfer flies to PBS.

Rinse three times in ice cold PBS to remove excess methanol, and keep flies in PBS on ice until dissection and fixation. At this point, flies should be dissected and fixed within the shortest time possible, preferably less than 30 minutes. Transfer flies to a silicone elastomer dissection dish filled with cold PBS.

Remove the metathoracic legs at the coxa using two pairs of number five Dumont forceps, or cut the legs with Vannas scissors. Transfer the legs to a well in a plastic, 24 well plate filled with one mil of PBS, and keep the plate on ice until all legs are removed and transferred to wells. We typically use 20 legs per well.

Replace the PBS solution with one milliliter FA solution, and rotate on a nutator for 30 minutes. The nutator setting is to be set at a medium speed. Ensure the legs are completely submerged in the solution during this time.

To remove FA solution, wash in one mil PBS three times quickly, followed by an additional three washes for five minutes each in one mil PBS. Hold tissues in one mil PBS on ice, prior to and during the dissection steps. To provide a brief overview of Drosophila leg anatomy, the leg segments are indicated, including the coxa, trochanter, femur, tibia, and five tarsal segments.

Here we see an anterior view of the leg, recognized by the presence of sensory bristles as opposed to naked cuticle present on the posterior side. The orientation of the proximal-distal axis and dorsal-ventral axis are also indicated. This dissection procedure removes a small piece of cuticle from the proximal femur, so that axon arbors, which project dorsally, innervating the tibia levator muscle, can be studied.

Our previous work has focused on the indicated arbor, originating from presumptive neuroblast eye lineage. In this part of the procedure, we remove cuticle from the anterior side of the femur. The dissecting forceps are critical for success, and we have found that introducing a slight bend at the end of number five super fine forceps provides a bevel which allows the cuticle to be grabbed superficially, rather than be poked, which can ruin the tissue.

Transfer legs to a silicone elastomer dish in PBS for dissection. Using one pair of forceps, pin the tibia segment to the silicone elastomer dish as shown here on the right hand side. Using the other forceps held bevel side down, grab a piece of cuticle on the distal end of the femur and pull in the proximal direction towards the trochanter.

Keep methodically removing cuticle until the naked muscle is visible throughout the proximal end of the femur. Once all the legs are dissected, replace PBS with FA solution to postfix legs for 30 minutes with shaking on a nutator at medium speed. Afterwards, wash the samples in one mil PBS for three times quick, and then three times for five minutes each in PBT solution.

To block tissues, replace one mil PBT with blocking solution consisting of one mil 5%normal goat serum diluted in PBT. Incubate dissected legs for four hours at room temperature, or overnight at four degrees Celsius. Remove blocking solution and replace with primary antibody which is diluted in blocking solution.

Here, we are using anti discs large at one to 200 dilution. Put plates back on a shaker and incubate at four degrees overnight. Following primary antibody incubation, wash primary antibodies in one mil PBT for three times briefly, and then three times for 15 minutes each.

Block tissues again in one mill 5%normal goat serum for at least two hours at room temperature, or overnight at four degrees Celsius. Remove the blocking solution and add 300 microliters of the appropriate diluted fluorescent conjugated secondary antibodies. Additionally, add one to 2000 dilution of fluorescence conjugated phalloidin to label muscle.

To incubate, seal wells with laboratory sealing tape and lid. Also, wrap plates in aluminum foil to protect fluorophores from light. Incubate for six to eight hours at room temperature, or overnight at four degrees Celsius.

To remove unbound secondary antibody, perform washes with PBT in a similar manner described previously when washing primary antibody. After this step, the samples are now ready to be mounted and imaged. Arrange the legs anterior side up, and cover with additional mounting media if needed.

Gently scrape the corners of a cover slip across a clay ball. The resulting clay will serve as a spacer between the slide and the cover slip, so that the tissues are not crushed. Press down on the cover slip until it is touching the top of the legs.

Seal the edges with nail polish and store at four degrees until imaging. Image by confocal microscopy using imaging parameters described in section four of the written protocol. To help validate the dissection protocol, we first image legs by phase contrast microscopy at low magnification.

Here is an example on panel A of a good dissection on the left, and a bad dissection on the right in which muscle fibers were disrupted. Panel C shows a confocal image of a good dissection in which muscle has not been disrupted. In contrast, panel D shows a leg damaged during the dissection, in which muscle fibers are missing, as indicated by the arrow.

Here, we stained an imaged legs with anti horseradish peroxidase to detect neuronal processes, shown here in green, anti discs large, which facilitates postsynaptic glutamate receptor clustering, shown in red, and phalloidin to label muscle, shown in blue. When captured at 20X magnification with a transmitted light channel, it is easy to see the dissected area. Note that antibodies partially penetrate into undissected areas, and can be seen through the cuticle as indicated by the white arrow.

Each of the leg motor neurons make stereotyped projections when innervating muscle. Our work is focused on motor neurons innervating the tibia levator muscle, shown here as the boxed region and indicated by the white arrow. At higher magnification 60X with 2X zoom on the top right, we can effectively image boutons, shown in green, and surrounding DLG shown in red.

Increasing the zoom further, or stepping to a 100X objective, allows for the quantification of bouton size and morphology as shown in the lower right. Using this dissection technique combined with immunocytochemistry, we found that there is H-dependent bouton swelling in a sod1 mutant model of ALS, shown here in aged H71Y legs, compared to wild type controls as indicated by the arrows. Bouton sizes are quantified in the bee swarm plot to the right.

We further found a reduction in the active zone marker Bruchpilot, shown in red, within weakly stained HRP axons, shown in green, of H71Y mutants. To study neurodegeneration, ubiquitinated protein aggregates are often detected by the antibody FK2, and shown here as FK2-positive puncta, in the axons and muscles of aged sod H71 flies on the right hand side, indicated by the arrows. Finally, mitochondria can be visualized by immunocytochemistry using an anti ATP5a monoclonal antibody, as shown here in red within the tibia levator muscle.

We found that mitochondria become enlarged with age in H71Y mutants. Once mastered, the dissected leg preparation and associated antibody staining will allow you to analyze molecular changes at the adult neuromuscular junction for your favored mutant at various time points.

Résumé