Using Drosophila to demonstrate optogenetic tools

'Optogenetics' refers to techniques used to control cells in living organisms with light. The main optogenetic tools currently in use utilize light-activated channelrhodopsins from green algae to activate neurons. By expressing a channelrhodopsin in subsets of neurons, one can study the specific behaviors controlled by those neurons by shining light of a specific wavelength (the wavelength depends on the channelrhodopsin used) to open the channelrhodopsin and activate the nerves (see below).

schematic of light activating channelrhopsin, causing it to open and ions to flow into the cell.

Ilya Vilinsky (University of Cincinnati), Karen Hibbard (Janelia Research Campus), Bruce Johnson and David Deitcher (Cornell University) have developed a set of stocks and student lab exercises that demonstrate optogenetic techniques in Drosophila melanogaster. These stocks utilize the GAL4/UAS system to express a channelrhodopsin in subsets of neurons in either larval or adult flies (see schematic below for an example of how the GAL4/UAS system works). By shining either an intense red (625 nm) or blue (460 nm) light onto animals fed all-trans retinal (a co-factor necessary for making a functional channel), the channelrhodopsins open and the neurons expressing these proteins are depolarized and activated, resulting in a specific behavior.

Schematic of GAL4/UAS system used to express channelrhodopsin in subsets of cells.  GAL4 is expressed in subsets of neurons, it then binds to UAS to activate transcription of the channelrhodopsin.

Activation of channelrhodopsins in these stocks can be achieved using inexpensive handheld red light (for CsChrimson) or blue light (for H143R-ChR2) flashlights made by companies like JOYLIT or WAYLLSHINE (look for 465 nm or 620 nm tactical CREE XP-E LED flashlights - you'll want a light with at least 200 lumens). You will need to include all-trans retinal in the fly food. Karen Hibbard says "We suspend [all-trans retinal] in ethanol to make a 100 mM stock solution. You can then make vials with a 1:500 dilution in fly food for a final concentration of 0.2 mM. It doesn’t completely dissolve, just resuspend as much as possible. I would suggest making aliquots and storing in the freezer. These stock solutions should last indefinitely. The retinal is light sensitive so avoid strong white light and cover with foil when possible."

For more information on these lines and their uses, please see Vilinsky et al., 2018. For a lab utilizing two of the lines, please download the CrawFly Workshop 'Optogenetically Induced Adult Behaviors' lab instruction sheet.

If you have questions, please contact Dr. Karen Hibbard.

Stk #BehaviorDescriptionLight requiredGenotype
79595adult escape responseExpresses mVenus-tagged red-shifted channelrhodopsin (CsChrimson) in the Giant Fiber neural network. Exposing adults raised on retinal to intense 625 nm red light triggers an adult escape response behavior (see von Reyn et al., 2014, FBrf0225495).625 nm red lightw[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18; P{y[+t7.7] w[+mC]=R17A04-p65.AD}attP40; P{y[+t7.7] w[+mC]=R68A06-GAL4.DBD}attP2
79596backward walkingExpresses mVenus-tagged red-shifted channelrhodopsin (CsChrimson) in a subset of neurons in sensory and motor neuropils. Exposing adults raised on retinal to intense 625 nm red light triggers a backward walking behavior. A.K.A. the 'Moonwalker' stock.625 nm red lightw[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18; P{y[+t7.7] w[+mC]=VT050660-GAL4}attP2/TM6B, Tb[1]
79597jumpingExpresses mVenus-tagged red-shifted channelrhodopsin (CsChrimson) in subsets of cells in the brain and ventral nerve cord. Exposing adults raised on retinal to intense 625 nm red light triggers a jumping behavior. Also known as the 'Jumping GAL4' stock.625 nm red lightw[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18; P{y[+t7.7] w[+mC]=GMR42E06-GAL4}attP2
79598larval escape rollingExpresses mVenus-tagged red-shifted channelrhodopsin (CsChrimson) in peripheral sensory neurons and is required for mechanical nocioception. Exposure to intense 625 nm red light induces the larval escape response of rolling (see FBrf0210201).625 nm red lightw[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18; P{w[+mC]=ppk-GAL4.1.9}2/CyO, P{w[+mC]=2xTb[1]-RFP}CyO
79599larval escape rollingExpresses mVenus-tagged red-shifted channelrhodopsin (CsChrimson) in the sensory domain of the larval ventral nerve cord. Exposure to intense 625 nm red light induces a strong rolling phenotype (see Ohyama et al., 2015, FBrf0228257).625 nm red lightw[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18; P{y[+t7.7] w[+mC]=GMR72F11-GAL4}attP2
79600seizureCan be used to visualize muscle innervation. mCherry-tagged Channelrhodopsin-2 is expressed in motor neurons of the ventral ganglion. GFP is expressed in muscles.460 nm blue lightw[1118]; P{w[+mW.hs]=GawB}VGlut1[OK371], P{w[+mC]=UAS-H134R-ChR2}2, P{w[+mC]=Mhc-GFP.F4-453}2/CyO
79601seizureExpresses mCherry-tagged Channelrhodopsin-2 in glutamatergic neurons including motor neurons of the ventral ganglion. Exposing larvae raised on retinal to intense 460 nm of blue light results in a 'seizure' behavior.460 nm blue lightw[1118]; P{w[+mW.hs]=GawB}VGlut1[OK371], P{w[+mC]=UAS-H134R-ChR2}2; Snap25[ts]
605134spinningExpresses a tdTomato-tagged channelrhodopsin (Chrimson) in projection neurons. Exposing adults raised on retinal to intense 625 nm red light triggers a strong and easy to see spinning behavior.625 nm red lightw[1118] P{y[+t7.7] w[+mC]=10XUAS-IVS-Syn21-Chrimson-tdT-3.1}attP18; P{w[+mW.hs]=GawB}NP0225/CyO
605135walking in circlesExpresses a tdTomato-tagged channelrhodopsin (Chrimson) in the central complex, optic lobe, lamina, and medulla. Exposing adults raised on retinal to 625 nm red light triggers the flies to walk in a circle. Light that is too strong will elicit seizures.625 nm red lightw[1118] P{y[+t7.7] w[+mC]=10XUAS-IVS-Syn21-Chrimson-tdT-3.1}attP18; P{y[+t7.7] w[+mC]=GMR13G10-GAL4}attP2
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