This protocol is an excerpt&#160;from&#160;de Vries&#160;and Clandinin,&#160;Optogenetic Stimulation of Escape Behavior in&#160;Drosophila melanogaster,&#160;J. Vis. Exp.&#160;(2013).
1. Generate Channelrhodopsin Flies
<ol>
	<li>Cross UAS-ChR2 flies with the Gal4 driver of your choosing, we use G105-Gal4, which is expressed in Foma-1 neurons in the optic lobe.</li>
	<li>To eliminate the possibility of a visual response to the blue light stimulation, both fly lines are in a ...

drosophila optogenetics: a method to manipulate neuronal circuits

Source: de Vries, S. E., Clandinin, T. <a href="https://www.jove.com/video/50192/" target="_blank">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster</a>. J. Vis. Exp. (2013).
Optogenetics enables the use of light to manipulate neurons that are genetically engineered to express specific opsins&#8211;light-sensitive proteins whose light activation triggers a change in the state of the neuron. Here we describe an optogenetic approach in Drosophila using the opsin Channelrhodopsin2. The example protocol features an optogenetics assay set to study the neuronal circuitry behind the fly&#39;s escape behavior.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20149/20149fig1.jpg" /> 
Figure 1: Experimental set-up showing the platform with the vertical holder and the four coolant hosing arms holding heat sinks with LED arrays affixed to them. (A) The set-up under ambient illumination. (B) The set-up when the LEDs are illuminated. (C) A close up view of a Tri-Star LED on the heat sink. (D) A close up of the Tri-Star with the tri-le...

Drosophila Optogenetics: A Method to Manipulate Neuronal Circuits

Source: de Vries, S. E., Clandinin, T. Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster. J. Vis. Exp. (2013).
Optogenetics enables ...

- 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 to bright blue light. The subsequent inflow of sodium ions spikes an action potential that causes the neuron to fire and relay information along the circuit. In addition to the light stimulus, the cofactor all-trans-retinal is required for channelrhodopsin to open. Therefore, provide all-trans-retinal in the fly food, since it is usually present in very small amounts. Additionally, when investigating the role of neurons in a neural circuit involved in a response to a visual stimulus, use flies that are visually blind to prevent other light responses. Finally, use a blue LED light to 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 to 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 to 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 to two seconds, and then turn on the blue LEDs. Use a timer to manually measure the time until the fly initiates flight.

drosophila-optogenetics-a-method-to-manipulate-neuronal-circuits

Research

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Adult

Optogenetics enables the use of light to manipulate neurons that are genetically engineered to express specific opsins&#8211;light-sensitive proteins whose light activation triggers a change in the state of the neuron. Here we describe an optogenetic approach in Drosophila using the opsin Channelrhodopsin2. The example protocol features an optogenetics assay set to study the neuronal circuitry behind the fly&#39;s escape behavior.

Overview

Protocol

<img title="figure-representative results-25" alt="figure-representative results-25" class="xfigimg" src="/files/ftp_upload/20149/20149fig1.jpg" /> 
Figure 1: Experimental set-up showing the platform with the vertical holder and the four coolant hosing arms holding heat sinks with LED arrays affixed to them. (A) The set-up under ambient illumination. (B) The set-up when the LEDs are illuminated. (C) A close up view of a Tri-Star LED on the heat sink. (D) A close up of the Tri-Star with the tri-le...

Drosophila Optogenetics: A Method to Manipulate Neuronal Circuits

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Overview

Protocol

Representative Results

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