Monitoring Electrical Signals from the Afferent Neurons in an Immobilized Zebrafish Larva
筆記録
Immobilize zebrafish larvae and transfer them into a 35-millimeter petri dish using a large-tipped transfer pipette. Remove as much of the surrounding solution as possible. Immerse larvae in 10 microliters of 0.1% alpha bungarotoxin for approximately five minutes. Wash the paralyzed larva with extracellular solution for 10 minutes. Then, use a transfer pipette to move the larva from the extracellular solution bath to the silicone-bottomed recording dish.
Under a stereomicroscope, gently position the larvae with fine-tipped forceps above the center of the silicone mat, lateral side up with the body's anterior and posterior ends running left to right. Using fine-tipped forceps, insert the etched pin orthogonally to the silicone through the dorsal notochord of the larvae, directly dorsal to the anus. Insert the second pin through the notochord near the end of the tail, and insert the third pin through the notochord dorsal of the gas bladder. Insert the fourth pin through the otic vesicle while providing slight rotation as the pin inserts into the encapsulant.
As a slight rotation is applied, watch for the tissue between the cleithrum and otic vesicle to reveal the cluster of afferent somata. Place the pinned lava under the 10 times objective on a fixed stage of the DIC microscope, and orient the myoseptal clefts of the muscle blocks parallel to the left head stage vector.
Place the ground wire into the bath solution, and ensure that it is connected to the left head stage. Fill the afferent recording electrode with 30 microliters of extracellular solution, and insert it into the right head stage pipette holder. Then, lower it into the dish solution while applying positive pressure produced by a pneumatic transducer.
Locate the electrode and bring the electrode tip over the specimen. Using a micromanipulator, lower the afferent electrode tip until it is holding position above the cleithrum. Increase the magnification to 40 times immersion and locate the intersection of the posterior lateral line nerve and cleithrum. Bring the electrode tip over the afferent ganglion, and lower the pipette until the tip contacts the epithelium. Gently, maneuver the electrode so that the entire tip circumference contacts the afferent ganglion.
