Hole mount preparation of adult drosophila ventral nerve Cord for giant fiber dye injection Introduction. This video will demonstrate how to perform a hole mount preparation of the adult drosophila ventral nerve cord. With this dissection method, the entire nervous system remains in the fly, allowing access to the ventral nerve cord for electrophysiological and or anatomical approaches with minimal damage to the ventral nerve cord.
In the second part, intracellular dye, filling of the giant fiber with fluorescent dye will be demonstrated Adult dissection under a Dissection microscope. Place an anesthetized adult fly in a sill guard coated Petri dish with Venus Scissors. Remove legs, Prosti, and wings.
Use insect pens to pin the fly dorsal side up. Place two pins behind the head and stretch the neck. Submerge the preparation in saline.
Remove air surrounding the fly using a syringe, make a lateral incision in the abdomen, cut along the dorsal surface of the abdomen towards the thorax. Cut through the flight muscles precisely along the midline of the thorax. The cut should be shallow to avoid damage to the ventral nerve cord, use pins to open the thorax and pin the muscles to the side.
Remove Heart, stomach, gut, ovaries, and salivary glands. Rinse the preparation with coal saline. Remove the fat body from under the cervical connective D fill setup.
Place the open preparation under an upright microscope with fixed stage. Place an electrode with filament in 1%Lucifer yellow until the electrode tip is filled with dye. Then backfill the electrode with three molar lithium chloride using a Hamilton syringe leaving an air bubble between the dye and lithium chloride.
Orient the posterior end of the preparation towards the DFI Electrode. Insert a ground into the Saline and place the dye filled electrode above the fly. Difi procedure.
Focus on the ventral nerve cord with a 10 x air objective And center the cervical connective Switch to 40 x Dipping lens. The big axons of the giant fibers can be seen at the dorsal surface of the cervical connective Position the electrode above one giant fiber. Lower the electrode slowly down onto the giant fiber axon.
Slide the electrode into the giant fiber. Switch to An epi fluorescent light and use the Lucifer yellow filter. Inject Lucifer yellow with a hyperpolarizing current into the axon.
Inject the dye into the giant fiber terminal, is labeled possible Applications difficulties and modifications. The method can be used to visualize the morphology of giant fiber terminals between samples of the same genotype or different genotypes, the electrical connectivity between giant fibers and their post-synaptic neurons. TTM and PSI can be tested by using small dyes like Lucifer yellow or neuro biotin, which can cross cap junctions.
DI fills of the giant fiber can be combined with immuno staining for various proteins. Image stacks of the CNS can be acquired with confocal or two photon microscopy for high resolution analysis of mutant terminals. Intracellular recordings from the muscles of the giant fiber circuit can be obtained by standard procedures before giant fiber dye injections to analyze structure and function of the circuit in the same sample.
