Drosophila Optogenetics: A Method to Manipulate Neuronal Circuits
Trascrizione
– 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 di specific neurons within that circuit.
Channelrhodopsin is a photo-sensitive cation channel that opens when exposed a bright blue light. The subsequent inflow of sodium ions spikes an action potential that causes the neuron a fire and relay information along the circuit. In addition a the light stimulus, the cofactor all-trans-retinal is required for channelrhodopsin a open.
Therefore, provide all-trans-retinal di the fly food, since it is usually present di very small amounts. Additionally, when investigating the ruolo of neurons di a neural circuit involved di a response a a visual stimulus, use flies that are visually blind a prevent other light responses.
Finally, use a blue LED light a 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 di optic lobe neurons.
– Begin by melting standard cornmeal fly food di a microwave, and let it cool until warm a 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 di P1000 pipette tips with the tip cut a 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 di the groove di the vertical holder. Secure the chip di 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 a two seconds, and then turn on the blue LEDs. Use a timer a manually measure the time until the fly initiates flight.
