Drosophila Optogenetics: A Method to Manipulate Neuronal Circuits
Transcrição
– In Drosophilia flies, a looming visual stimulus signaling the approach of a predator activates neuronal circuits that trigger an escape response. To manipulate the neuronal circuit that controls such a response, express a transgenic light-sensitive opsin, like channelrhodopsin-2 de specific neurons within that circuit.
Channelrhodopsin is a photo-sensitive cation channel that opens when exposed para bright blue light. The subsequent inflow of sodium ions spikes an action potential that causes the neuron para fire and relay information along the circuit. In addition para the light stimulus, the cofactor all-trans-retinal is required for channelrhodopsin para open.
Therefore, provide all-trans-retinal de the fly food, since it is usually present de very small amounts. Additionally, when investigating the cargo of neurons de a neural circuit involved de a response para a visual stimulus, use flies that are visually blind para prevent other light responses.
Finally, use a blue LED light para trigger the same behavior that is caused by the looming stimulus. In the example protocol, we will see the food preparation and light-induced behavioral response of flies expressing channelrhodopsin de optic lobe neurons.
– Begin by melting standard cornmeal fly food de a microwave, and let it cool until warm para touch. Once cooled, mix 50 microliters of previously prepared 20-millimolar all-trans-retinal into each vial. Then, let vials cool and keep protected from light until required.
First, briefly anesthetize the flies on ice. Then, place individual flies de P1000 pipette tips with the tip cut para form a pore diameter of around 2.25 millimeters, and seal each end with tape. Once the fly has awoken and is exploring the pipette tip, remove the tape, and quickly place the tip de the groove de the vertical holder. Secure the chip de place with a thumb screw, which closes the bottom of the tip.
Flies typically explore the tip for between 30 and 60 seconds. As the fly nears the top of the tip, start the camera recording just before the fly emerges onto the platform. After the fly has emerged onto the platform, wait one para two seconds, and then turn on the blue LEDs. Use a timer para manually measure the time until the fly initiates flight.
