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