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Lord of the Flies-Optogenetic Fruit Fly Conditioning

You know what’s great about fruit flies? Nothing.

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fig. 1 Fruit flies suck

Nothing, that is, other than their benefit as a model organism for simple and fast transgenic experimentation — but who really cares about all that. Drosophila melanogaster are butts, so what if they could die? Well they can (vinegar and plastic-wrap), we don’t need science for that. We can go one further. What if we can make the flies loathe existence as much as the rest of all life hates them? What if we could take away the one thing that makes their nasty, brutish, and short existence bearable?  Make them lose life’s purest love? The love of…sugar?

 

Yeah, we did that.

With

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Look at this little guy, basking in the sweet, sweet 625nm rays of ghost sugar:

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fig. 2 How long can you hold out against science little guy, how long?

If you remember from my first post, red light activates the Gr5a sweet taste neuron, making the fly feel like a kid in a candy store after the adult-apocalypse. Now if we interrupt this tiny love affair with a SYRINGE OF SCIENCE and also quinine we can get the fly to associate the bliss of sugar (which flies love), with the bitter sting of quinine (which flies do not love).

Syringes-delivering Science since 1st century CE

Syringes-delivering Science since 1st century CE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

That’s classical conditioning, Kyle. After a few pairings of the two, the flies become depressed. Or I would, if I were a fly, because at that point they can’t stomach anything sweet, and don’t even respond to a fly sized glob of syrup. I’d do things to a human sized glob of syrup you’d probably try to get me arrested for, and the knowledge that science could some day deprive me of this pleasure is a sobering thought. After this I sat in a dark room for 3 days eating candy.

When I came out, the data was still there and I learned to love the bomb. Here’s a bit more detail on the way we played with fly tastes and the numbers we got:

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fig. 3 What I done do to them flies

The flies were taken off of food for 12hr before experimentation, then adhered to a foil slide with nail polish and mounted on a stand. For the conditioning test (A, purple), proboscis extension reflex (PER) was first tested via optogenetic, and then actual sugar stimulation, to establish a baseline of both the light-induced and real sugar-induced response.  For each trial, PER was optogenetically induced 3 times, and quinine applied to the extended proboscis. After 3 trials, PER was measured as a response to light, and then sugar again. A simple control used non-optogenetic flies-with the gene for the optogenetic channel, but no second gene activating its expression, and then the same opto/quinine pair trial (B, purple).  For further control trials, (C, red) quinine was applied to the proboscis sans opto- stimulation, (D, yellow) opto- stimulation was paired with water, and (E, green) opto- stimulation was used without quinine.  Though this may seem like a lot of controls experiments, we want to establish as firmly as possible that the response we are getting is solely a result of the paired conditioning experiment we are running.

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fig. 4 ALL THE DATA

These data show strong aversion to sugar after the conditioning. Only the trials where sweet taste activation is paired with bitter shows a marked decrease in response to sugar over the average indicating that the result was due to conditioning, not random chance. Future experimentation will also see pairing the light activation with a neutral taste to the flies, like salt, to see if we can condition the flies to respond to salt as they respond to sugar, as well as the possibility of optogenetically activated bitter taste activation combined with the introduction of real sugar. Optogenetics is the cutting edge of neuroscience technology, however, so whatever comes next will be exciting!


Smells like teen spirit-Moth antenna pheromone receptors

Greetings, this is Trevor coming live from Ann Arbor in a basement…

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We have one week left in the internship and things are finally starting to come together. Last time I made a post, I was without a doubt on the struggle bus in terms of getting data worthy of a poster, let alone a journal article. Since that point I have learned a hell of a lot of what not to do while recording data. First, always make sure everything is plugged in, second, make sure your specimen is alive, and last always make sure your equipment is hooked up everywhere it should be. You may think I’m weird for this, but I think my moths are pretty cute. A couple weeks ago I was having a really hard time getting clear recording from the BYB Spikerbox due to so much activity happening in the antennae, and as a result we switched to the SpikerShield to get an electroantennogram recordings. Electroantennogram recordings are low frequency, are the preferred way to record antennae stimulus and look like this:

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Before I started testing the sex pheromone bombykol on the moths, I characterized how the male moths responded when introduced to their female counterparts, and it is best described as a circling motion in a zigzag path towards the female. It happens almost every time a female is brought within a couple feet of a male, but is not present when stimulated with just any odorant, like lemon oil or peppermint.The male seems significantly less enthused about the lemon than about the female, I wonder why?

The male seems significantly less enthused about the lemon than about the female-I wonder why?

 

Once I had that complete I began trials with bombykol using a new delivery system that went through many revisions. At first we were using a valve system to deliver odors via an air stream, but we observed a change in airflow when opening valves, which meant we couldn’t be sure if the neuronal responses we got were from the change in airflow or the added odorant. To fix this problem, we inserted syringes into the piping with some T-junctions on the way to the moth, thus keeping a constant air stream.

Now I needed a way to consistently empty the syringes, so for that I added a linear actuator and a wood based housing to hold the actuator and syringes. The housing, entitled “Mr. Orange” due to its bright orange paint job and my love for Reservoir Dogs, has three ports for three syringes, to serve as an air control, other odorant positive control, and pheromone syringes all in one recording. Along the way, thanks to our fearless leader Greg I had to edit the housing from two to three syringe ports just minutes after completing the project…

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Are you happy now, Greg?

Before “Mr. Orange” was built I was able to successfully record a bombykol stimulus on the last day of my second batch of moths. Displayed below is Bombykol compared to a negative control, aka clean air. Remember they only live 5-10 to days, and have to be ordered, so my time working with them is precious! I am currently awaiting new moths so that I can do my final trials which will involve mulberry leaves, which their caterpillars live on and eat, as a positive control for both male and female moths. This will allow me to show that both males and females react similarly to one stimulus (and ensure the prep is working), but also that they have evolved such that males are much more sensitive to bombykol than females.

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Last but not least, the interns all presented last week at MID-SURE, a poster session on Michigan State University’s campus in East Lansing, and I have included my poster below for anyone curious.

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What does the Fish say? Electric fish spikes with Bailey

Hello my name is Bailey! I am a junior majoring in electrical engineering at Michigan State University and am doing an internship at Backyard Brains this summer. Sorry I missed the first blog post, I was travelling in Japan with my sister!

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Left-my sister, Right-me

I know it doesn’t look like it from the picture, but I was doing some important background research for my project.

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I mean…circuits were involved

Ok, so it was just for fun.

After getting over my jetlag it was time to get back to work. My project this summer involved mormyrid fishes. What’s so special about these fish? Mormyrid fish are awesome because they both emit and detect electric signals. Unlike an electric eel, however, their electrical discharges are too weak to harm other fish. Instead, they use electric signals to navigate their environment, which is naturally very cloudy, and communicate with each other. A lot can be learned about these fishes’ behavior and even evolutionary history just by studying their electric organ discharges (EODs).  Unfortunately the equipment used to record their EODs is quite costly, often prohibitively so-especially for those who live in the same area as the fish. This is where we come in. My goal this summer was to build an inexpensive, easy to use, and open source device that can record EODs from weakly electric fish.

Since the EODs occur at very high frequencies, a simpler microcontroller like an Arduino is not sufficiently powerful to record their EODs in real time. Enter the BeagleBone Black.

BeagleBone

Woof

The BeagleBone Black has a 1GHz processor and 4GB of storage (that can be supplemented with a micro SD card) as well as running a full Linux OS, making it perfect for collecting the EOD data.

Now I need something to convert the analog EOD signals to digital so that the BeagleBone can process them. For this project I am using the MCP3008 analog to digital converter. Initially, I started by using a cape that had the chip on it.

Analogs, prepare to be converted!

Analogs, prepare to be converted!

The cape worked well for testing the setup with low frequency sine waves, however, the inputs it normally connected to on the BeagleBone were not able to handle the high speed data collection I needed and the data was not being recorded in real time. To work around this issue, I had to individually connect each pin to the appropriate input on the BeagleBone. This lead to the setup appropriately nicknamed “The Shiva”.

Not pictured: the 6 other arms I grew to operate this setup

Not pictured: the additional arms I grew to operate this setup

This setup allowed me to access the programmable real time units (PRUs) on the BeagleBone. PRUs are essentially microprocessors within a microprocessor that sit around eagerly awaiting a program to execute. Unlike the main CPU, the PRUs do not run Linux, allowing them to collect the data in real time. Now that I had my setup I was only missing one thing-the fish! A quick trip to the Electric Fish Lab at MSU and the newest additions to the Backyard Brains Petting Zoo, Tina and Taco, were ready for some data collection.

The one on the right with the goatee is Taco. Tina is on the left

The one on the right with the goatee is Taco. Tina is on the left

I started recording by using the example code from chapter 13 on the Exploring BeagleBone website. The code filled up the PRU memory with data from the recording, and then used another program to read the data from the memory. Although this worked well, it was not what I wanted in terms of a final product. Using the example code as a basis, Stanislav Mircic, Backyard Brains’ ultra-programmer, and I modified it to continuously write the data to a circular buffer as well as simultaneously read the buffer and check if an EOD has occurred.

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Now the program will record only the EODs, which is what we are ultimately interested in, instead of all of the raw data. Here’s an example of the output:

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Now that I’ve verified what the circuit needs to be, I have to draw out the schematic so that a board can be printed in a form that can snap onto the BeagleBone.

My drawing of the circuit

My drawing of the circuit 

Official Schematic

Official Schematic

And finally the actual board.

Goodbye Shiva!  Only two arms needed for this one.

Goodbye Shiva! Only two arms needed for this one.

For the rest of the summer I will be modifying the circuit design to better suit the goal, such as altering the gain to match the type of recordings we’ll be getting, and preparing the device for field work by building a portable power source and a case that it can float in.  Soon we hope for the device to be picking up lots of fish conversations from around the world!  Stay tuned!