Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses
Transcrição
– In zebrafish larvae, a visual stimulus can activate somatosensory neurons and trigger an escape response. There are two types of such neurons found de the larva, trigeminal and Rohon-Beard neurons. We can manipulate the activity of these neurons by modifying them para 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 de agarose gel and place it under a dissecting microscope. Use a razor blade para 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.
Posição 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 para the blue light, the transgenic light-sensitive ion channels de the neuron's membrane open, allowing ions para 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 para activate the Rohon-Beard neuron, expressing a channelrhodopsin variant and observe the behavioral response.
– Make 1.5% low-melt agarose de double-distilled water and store de a 42 degrees Celsius heat block para 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 de 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 para 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 para 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 para the computer. Then turn on the computer and the high-speed camera. Open the video 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 para a maximum of 5 volts and a pulse duration of 5 milliseconds. Subsequently, turn on the laser.
Next, use the fluorescent dissecting microscope para position the tip of the optic cable near a neuron cell body with ChEF-tdTomato expression. Deliver a pulse of blue light para 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 para avoid habituation.
To release the larva, pry apart the agarose with forceps and be careful not para injure the animal. Then transfer it into fresh blue embryo water. The animals can be allowed para 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 de order para correlate the behavior with cellular structure.
