Drosophila Adult Brain Dissection: A Method in Fly Neurobiology
Transcrição
– Perform the dissection de a dish with a buffer solution and under uniform illumination. Transfer a fly, ventral side up, into the dish and keep the preparation submerged throughout the procedure. While holding the base of the proboscis, gently pull the fly head away from the body para separate the two. The brain is located at the back, or caudal side, of the fly’s head. Therefore, hold on para the front, or rostral, parts of the cuticle during the dissection para avoid damaging the organ.
To isolate the brain, hold the medial edge of one eye and grasp and pull the proboscis de the opposite direction para create an opening into the head capsule. While holding the cuticle at that eye, grasp the medial edge of the cuticle from the other eye and slowly pull the head capsule straight apart para reveal the brain. If necessary, continue para clear residual cuticle, attached air sacs, or trachea. High quality preparations will preserve structures like the optic lobes, central brain, and SOG. In the example protocol, we will see a detailed demonstration of a Drosophila adult brain dissection used para study the mushroom bodies.
– To setup for the dissection, place the anesthetized flies of interest on a cold metal pad or de a Petri dish sitting on ice. Alternatively, use a fly pad that emits carbon dioxide. Next, place a small amount of PTN de the center of the dissection dish para create a bubble of PTN. Then, under a stereomicroscope, fill the field of view with the PTN bubble under uniform illumination. Now, manipulate flies so that they are belly up, and transfer one by the abdomen into the PTN. Completely submerge the animal de the PTN and perform the entire dissection submerged de PTN. With a second pair of forceps, grasp the proboscis and pull the head off the body.
– Occassionally, the proboscis will be removed but the head will remain attached para the body. If this occurs, grab the medial edge of one retina and apply lateral force to remove the head.
– Discard the abdomen and thorax. It is critical para keep the head de the PTN during this step. Connections from the central brain para VNC clearly cannot be analyzed using this method. Next, grasp the medial edge of the left retina at the edge of the central hole de the cuticle, and slowly pull the forceps directly apart from each other para prevent tearing of the optic lobe, which is the opaque structure covered by white, stringy trachea. As the retina dissociates, there will be a slight decrease de tension.
– In order para prevent the brain from tearing, it is critically important para grasp the medial edge of each eye and very slowly pull the forceps apart. Moving too quickly can result de altered brain morphology or damage para brain tissue.
– As shown here, grasp the cuticle with each pair of forceps and very slowly pull them de opposite directions. If done correctly, the cuticle should separate from the brain tissue, leaving the brain intact. Very sharp forceps are needed for this step. Next, carefully remove the surrounding cuticle piece by piece. While removing stubbornly attached cuticle, it can help para secure the brain by gripping the remaining VNC para avoid crushing the brain. Finally, use a P200 pipette para transfer the dissected brains into a well of a plate filled with PTN for fixation and immunostaining.
