Optogenetics is a great way to temporarily control neuronal function. It’s typically used for light-controlled activation or inhibition of neuronal activity but there are lots of cool light-activated genetic tools to manipulate cell and gene function popping up. There are lots of things we don’t immediately think about when designing and performing optogenetic experiments though. Before you start your experiments, I recommend considering some metrics such as the wavelength and intensity of light you chose and how to choose and control your flash rate.
What is optogenetics?
Optogenetics is the term used to describe the method through which light is used to activate genetically encoded targets in cells. These targets can be light-gated ion channels, which will then activate or inactivate neurons.
Here are a few resources for learning about the basics of optogenetics:
Optogenetics: Principles and Approaches is a short video from EMBL describes the basic principles and approaches to optogenetics.
Using Drosophila to Demonstrate Optogenetic Tools is a fantastic lesson on the Bloomington Drosophila Stock Center website that covers the principles of optogenetics using a set of Drosophila optogenetic tools developed by Ilya Vilinsky (University of Cincinnati), Karen Hibbard (Janelia Research Campus), Bruce Johnson and David Deitcher (Cornell University)
Optogenetics: Controlling your Genes with Light is a helpful 30 min Stuff You Should Know podcast
Re-engineering The Brain is a 18 min TED talk by Drosophila neurogeneticist Gero Miesenboeck
What optogenetic tools should I use?
There are a lot of different lines you can use to express light-specific constructs in your neurons. Typically we will drive expression of our favorite light-specific cation or anion channels using the GAL4/UAS system. Our favorites are CsChrimson (Klapoetke et al, 2014) to activate neurons and GtACR (Mohammad et al, 2017) to inhibit neurons.
What type of LEDs should I use?
It’s important to know that optogenetic channels (like CsChrimson and GtACR) are responsive to a broad array of light. For example, you can activate CsChrimson and GtACR with ambient room white light. When we choose LEDs it is typically because we are picking a wavelength of light that hits the peak excitability of the channel, or that is a wavelength that can activate your channel without accidentally activating other channels.
If you are using csChrimson flies: we typically use Red LEDs, with 680 nm wavelength Red LEDs 680 nm. Some labs also use Red-Orange LEDs 617 nm.
If you are using GtACR2 or ChR, check out Blue LEDs 470nm.
Some have reported decent optogenetic responses in larvae using Smartphones (Meloni et al, 2020) although we haven’t tried this.
How do I build an optogenetic array?
It’s relatively simple to build your own LED array for optogenetics.
Decide if the LED lights will hit the flies from above or below the arena.
Design your LED array shape. You will want to design your array shape to maximize the flies’ exposure to optogenetic lights.
Use an LED array calculator to find the best circuit array option with the number, specifications of the LEDs and the power source you want to use:
Solder your LED array on a desired shaped PCB boards.
Once your array is finished, you can add a BuckPuck or BuckBlock DC LED Driver to assure the constant current delivery in your array.
Connect your array to a power source and an Arduino to control the pulse and LED intensity.(FlyGrAM figure 1 for detail)
How can I control flash rate?
Light intensity and frequency can be achieved by specifying LED pulse-width and frequency. Modulating the pulse-width serves as an alternative way to control the intensity of light emitted from LEDs without conventional DC voltage regulation (Pulse-Width Modulation). Make sure to try different pulse-width (ms) and frequency (Hz) to achieve maximum effect.
Does the light flashing affect fly behavior independent of neuronal activation?
We’ve noticed that flashing lights of all wavelengths can impact fly behavior. This is true of green, blue, white and red LEDs. For example, you can see that different wild-type strains respond to various red light flash rates differently (Scaplen et al, 2019 Figure 5). In order to ensure the responses you are seeing are due to changes in neuronal activation and not responses of the flies independent of channel activation, you need to run a control where you are flashing lights in a genetic heterozygote control (like your background strain crossed to your UAS or your background strain crossed to your Gal4).
Do I need to perform optogenetic experiments in the dark?
Since most of the light-gated channels you will be using for optogenetic experiments are sensitive to ambient room light (albeit they are activated less intensely than bright and focused LEDs), I recommend you rear and store your flies in the dark as well as perform your experiments in the dark. To rear and store your flies in the dark, you don’t need a dark incubator or room, you just need to cover all the relevant vials in aluminum foil and keep them in the regular incubator. To perform experiments in the dark, you will want to use a dark environment chamber you built where you control the light (ie turn off the ambient light and control LEDs for optogenetics experiments. In addition I recommend to perform these experiments in a dimly lit room (ie, the behavior environment/box in which your opto-behavior apparatus is placed is in the room with the lights off). We have low red lights in our behavior rooms (like an old-school photo developing room) but we have also successfully performed these in rooms with the overhead lights off and dim light filtering in from the windows.
What controls do I use? Flashing lights affect the behavior of flies, so you have to be careful how you design your controls. You can compare the experimental line GAL4>UAS to their heterozygous controls (GAL4/+ and UAS/+) with and without the flashing light conditions. Or, you can compare your GAL4>UAS with and without all-trans-retinal (ATR). For more information on this, check out 'What controls should I use for behavior experiments?'.
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