Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Summary

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Abstract

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster¹. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO₂ avoidance, proboscis extension and giant-fiber mediated startle response^2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-β, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Introduction

A growing arsenal of genetically encoded tools have been developed to manipulate neural activity in specific cells in Drosophila melanogaster¹. These tools enable the noninvasive activation or silencing of specific neurons in the intact and freely moving animal. Among these, Channelrhodopsin2 (ChR2), a light-activated cation channel, offers key advantages, since it can be temporally controlled and quickly induced. When neurons that express ChR2 are exposed to bright blue (470 nm) light they rapidly depolarize and exhibit elevated firing rates^3-5. Such targeted activation of specific neurons in freely moving animals has revealed ....

Protocol

1. Generate Channelrhodopsin Flies

1. 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.
2. To eliminate the possibility of a visual response to the blue light stimulation, both fly lines are in a w⁺norpA background.
3. End result: w⁺norpA;G105-Gal4/UAS-ChR2 +
4. After adult flies eclose, put selected females on fresh food, supplemented with 10 μM all-trans-retinal (a co-factor required for ChR2) and protected from light, for 3 days before performing the behavioral assay.

2. Make 10 μM All-trans-r....

Results

Blind flies expressing either ChR2 or the G105 driver alone show a low rate of take-off following their illumination with bright blue light. Blind flies exhibited the same rate of take-off regardless of illumination (Figure 2), suggesting that these take-offs were spontaneous rather than due to the illumination with blue light. When the ChR2 is expressed in the Foma1 neurons, however, illumination with blue light elicits the escape response. Over 50% of the flies tested took off within 1 sec of illuminat.......

Discussion

We have demonstrated optogenetic stimulation of escape behaviors by bathing freely walking flies in bright blue light. This approach can be easily adapted to examine other behaviors in freely walking flies, and can be scaled to larger platforms by simply tiling the LED arrays we used over a larger area. Using either the inexpensive camera we describe, or other available camera systems, the user can tailor the frame rate and spatial resolution of the images acquired to suit the behavior of interest. Additionally, o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
All-trans Retinal	Advance Scientific Chemical Inc	R3041
Equipment
Heat Sink 9.2 C/W	Luxeonstar	LPD30-30B	30 mm square X 30 mm high
Carclo 18 ° Tri-Lens	Luxeonstar	10507
Blue Rebel LED on Tri-Star Base	Luxeonstar	MR-B0030-20T	470 nm, 174 lm @ 700 mA.
700 mA BuckPuck DC Driver	Luxeonstar	3021-D-E-700
Wiring Harness for BuckPuck Driver	Luxeonstar	3021-HE
Pre-cut thermal adhesive tape	Luxeonstar	LXT-S-12	20 mm Hex Base
Snap-Loc Coolant Hose, ¼" ID	McMaster-Carr	5307K49
Snap-Loc Coolant Hose Connector	McMaster-Carr	5307K39	¼" NPT Male
Laboratory Grade Switching Mode Programmable DC Power Supply	BK Precision	1698
Exilim camera	Casio	EX-FH20