Cort continues his Optogenetics Odyssey in this week’s installment of “Pretty Fly for a White Guy”
Hello, again. I’m Cort, the fly guy (I also do research with fruit flies) at Backyard Brains.
Since the last time I posted I’ve made significant progress in my research! IT WORKS!!!
In the above video what you’re seeing is one of the flies I’ve bred having its proboscis extension activated with an LED. Optogenetics works! Now that I have it work, I’ve been busy playing with the new “flyscope” and raising more transgenic (organism with dna put into it from another organism, in this case the light sensitive protein) flies for my experiments. In order to breed the flies that I need, I had to dive straight into a world of genetics and Fly husbandry. I spent quite a bit of time at the University of Michigan Shafer lab learning about Drosophila (fruit flies) and how to raise them.
Fly husbandry is pretty intimidating at a glance, but once you break past the complexity it really isn’t so bad. It’s common knowledge that most complex organisms undergo a process called “recombination” during meiosis (when sex cells or “gametes” are formed in the body). One of the main reasons that scientists love using fruit flies to conduct genetic experiments is that fruit fly males don’t undergo genetic recombination at any point in their life cycle. The lack of recombination makes for very predictable and precise idea of what genes fly offspring will inherit from the parent generation.
At a glance, fly crossing looks pretty tough. But as far as genetics goes, in practice, flies are very easy! Ultimately it’s just a matching game.
Basically, I raised a stock of flies that the Shafer lab had on hand. This stock consists of the different “genetic puzzle pieces” that I needed to create flies that would express red-shifted channelrhodopsin (ReaChR, my light sensitive protein) in the neurons associated with proboscis extension and courtship behaviors. Each bottle of flies that I have contain specific fly genotypes that act as the pieces to the genetic “puzzle” of creating my optogenetic flies.
These are my flies! They all live in these tubes full of food and are not exposed to the outside world until it is time to breed (otherwise we risk contaminating food).
To make a long and confusing story short, creating these optogenetic flies requires combining flies whose genes contain ReaChR on a specific chromosome with flies from a GAL4 driver line with the appropriate target neuron or cell. In the case of my proboscis extension flies, I combined flies containing ReaChR with Gr5a-Gal4 (the neurons responsible for taste) flies. This combination will cause the progeny flies to contain genes that code for ReaChR on the Gr5a neurons. When the flies mature into adults, the Gr5a flies will express ReaChR within their Gr5a neurons, translation: by shining the right kind of light on them I can activate their neurons responsible for taste and proboscis extension.
D is an example of what I’ve define as a confirmed proboscis extension and what I am trying to excite from my flies.
Here’s a picture of my rig and the flyscope. The green thing under the scope is the “altar” where we observe the fruit flies, the black box on the arm is the LED
Even though I can cross (breed) these flies to create the offspring that I need, there’s still one important step to finish before the flies are actually susceptible to light. Retinol is a light sensitive molecule which is contained in all human retinas (Aptly named, right?). When light hits the retinol in the human retina, the molecule undergoes a shape change which causes the neurons in the retina to fire and is then perceived as vision. Fruit flies in no way produce retinol naturally, so I have to feed them a special diet of all-trans retinal mixed in with their regular food. When the flies eat the retinal food, the all-trans retinal saturates the cells in the flies body including the ReaChR cells. If the all-trans retinal is in the flies body, shining light into the cells will cause the retinal to undergo a conformational shape change and if ReaChR is present, the retinal will activate the ReaChR thus inducing the behavioral response.
This is the trans-retinol we feed the flies. We cook this ourselves…. it smells “great”
As of a few days ago, my fly crosses are finally ready to go. Data collection is underway and I’m sure that in a few short weeks that I can guarantee an affordable way to conduct and view optogenetics experiments with my flies and new and improved “flyscope”. My excitement is ramping up as I gear up for the MID-SURE poster presentations later this month at Michigan State University! Wish me luck!
“We’ve got their brains now. Pretty soon we’ll know how they think, and then we can win this war.”