Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses
筆記録
– In zebrafish larvae, a visual stimulus can activate somatosensory neurons and trigger an escape response. There are two types of such neurons found で the larva, trigeminal and Rohon-Beard neurons. We can manipulate the activity of these neurons by modifying them に express transgenic light-sensitive ion channels. This approach is called optogenetics.
To perform this technique, mount a transgenic larva with the dorsal side facing up で agarose gel and place it under a dissecting microscope. Use a razor blade に detach a wedge-shaped portion of agarose from around the yolk and tail. Fill this area with egg water. Pull the agarose away from the trunk and tail of the larva.
役職 the tip of an optic cable near the trunk where the cell body of the Rohon-Beard neuron is located. Deliver a pulse of blue laser light. Upon exposure に the blue light, the transgenic light-sensitive ion channels で the neuron's membrane open, allowing ions に enter, triggering an action potential that elicits the escape response. Record the larva's behavior using a high-speed camera.
In the example protocol, we will mount the larvae に activate the Rohon-Beard neuron, expressing a channelrhodopsin variant and observe the behavioral response.
– Make 1.5% low-melt agarose で double-distilled water and store で a 42 degrees Celsius heat block に prevent it from solidifying. Using a glass Pasteur pipette, transfer one of the prescreen larvae into a tube of 1.5% low-melt agarose with as little blue embryo water as possible. Then transfer the larva で a drop of agarose onto a small Petri dish.
Under a dissecting microscope, position the larva dorsal side up. When the agarose is solidified, use a thin razor blade に cut away agarose from both sides of the larva. Fill the area surrounding the agarose with embryo blue water.
Next, make two diagonal cuts of both sides of the yolk, being careful not に nick the larva. Afterwards, pull the agarose away from the trunk and the tail of the larva.
Now mount the high-speed camera onto the dissecting scope and connect the camera に the computer. Then turn on the computer and the high-speed camera. Open the ビデオ imaging software and adjust the camera settings. Next, connect the optic cable, the laser, and the stimulator together. Then turn on the stimulator and set it に a maximum of 5 volts and a pulse duration of 5 milliseconds. Subsequently, turn on the laser.
Next, use the fluorescent dissecting microscope に position the tip of the optic cable near a neuron cell body with ChEF-tdTomato expression. Deliver a pulse of blue light に activate the sensory neuron. Then record the responses using a high-speed camera set at 500 or 1,000 frames per second and repeat the experiments with at least 1 minute between activations に avoid habituation.
To release the larva, pry apart the agarose with forceps and be careful not に injure the animal. Then transfer it into fresh blue embryo water. The animals can be allowed に develop further, and the procedure can be repeated at older stages. The embryo could also be remounted for high-resolution confocal imaging of the activated cell で order に correlate the behavior with cellular structure.
