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Intracellular recording in Snails Midterm Update – Juan Ferrada

Hi! Juan Ferrada here from the University of Santiago again to give you an update on my project with Backyard Brains.

Main Project – Single unit recording from Snail Neurons

First mission – Isolate the Neurons

As we spoke of a month ago, we are trying to record the individual neurons of the giant pacemaker cells of the parietal ganglia of the common garden snail Helix aspersa. Our first step is to isolate this ganglia so we can visualize the famously large F1 neurons, that can reach up to a crazy big 200 um in diameter. After anesthetizing the snail with magnesium chloride, we began the preparation.

Here we can see the exposed cerebral ganglion and parietal ganglion. They are the highly white structures around the yellowish-white esophagus.

We removed the ganglion, and you can see it is surrounded by connective tissue. Using fine #5 forceps, we slowly picked away the tissue…

until, looking at the sample below a RoachScope at high mag, we see what appear to be a cluster of spheres. These, my friends, are the neurons we are looking for.

Second mission – Get an electrode close to the neurons

Now that we have the neurons in our sights, we have to get an electrode near it, not so easy when the sample is under our microscope. Luckily, we used the Backyard Brains Manipulator to move a glass pipette that we made just by holding a hollow borosilicate glass tube (part number 615000 – 1.0 mm x 0.75 mm) over a lighter and pulling it apart in the flame to make a very fine tip. Using the manipulator holding the electrode, we have just enough clearance to move between the sample and the microscope.

We can easily see the pipette tip on our smartphone looking through the RoachScope lens, and we can manipulate the electrode to come close to our neurons, attempting to insert them into the neurons. You can see a brief video of electrode movement below.

Third mission – Get a recording

We have the neurons, we have the electrode, we have the microscope, we have the manipulator. Now it is time to do the recording. This is my trial by fire, the hardest part of the whole experiment. The plan is to stab the cell with a high resistance glass electrode, then listen and record the spontaneous action potentials. Unfortunately, so far we are only getting noise, but we are slowly improving the amplifier setup, experimenting with electrode styles, reducing 50/60 Hz noise, and chasing the dragon of weak signals. We keep trying to catch it. Stay tuned!

Side-Project – Recording from Sea Anemone Tentacles
Since we are dealing with glass microelectrodes and amplifying signal in a noisy watery environment, I have also been working with the Backyard Brains team on a project they have had in mind for a long time – extracellular recordings from the tentacles of sea anemones. The lab has been caring for 9 anemones (taken from the intertidal zone near Algarrobo, Chile, an understudied organism called Anemonia alicemartinae). Over the past four months, the Backyard Brains team has been learning how to maintain a prosperous anemone colony. Since these are Humboldt current creatures, they like their water cold. So we have a trick to keep the aquarium under 20 degrees Celcius by having a fan always blowing air over the water. To further keep the anemones healthy we feed them surf clam meat every day, and clean the tank entirely, replacing and remixing the salt water, every 4-6 weeks.

We were originally using long silver wire (32 gauge) inside our pipette but it turned out to be brittle and the insulation susceptible to breaks and shorts, causing a lot of noise. We switched to flexible 30 gauge copper Minatronics wire that we threaded into a glass pipette, sucked up a tentacle, and recorded….nothing. To try to evoke a response, we touched the anemone trunk with a glass probe, but we did not register any electric activity in the tentacles.

Our next step is to try to insert an electrode near the oral disc, where we have read that more neurons are present.

Outreach

Any Backyard Brains internship has an outreach component, and I have been helping Backyard Brains teach classes in Colegio Alberto Blest Gana in San Ramón, Santiago. In the past few weeks we have been teaching the students, ranging from 11-17, how to read circuit diagrams and use broadboards. We are building electromyogram amplifiers from scratch. I have learned more about electronics in 1 month than all the combined previous months of my life!

Now we are deep in the experiments, and we will update you at the end of May. 


Breaking Into the Toy Market

STEM Ed Toys of the Future!

BYB’s adventures at Toy Fair 2018

Toy Fair is one of the largest gatherings of toy manufacturers, distributors, and buyers in the world, and in 2018, we threw our hat into the ring! We’ve been at this whole DIY Neuroscience thing in an educational space for almost 9 years, and we thought it was about time to test the waters in the consumer market, and Toy Fair was a great opportunity to do just that: we were in the room with giants like Hasbro and ThinkFun, learning how we could improve the toy factor of our science kits. Our table was situated in the “Launchpad” section of the conference where other companies new to Toy Fair were also showing off their offerings! (Will got a sneak peek at some of the hot new STEM Ed games hitting the shelves this year during his wanderings–just you wait for Killer Snails the Card Game!)

We did a lot of demos, we did presentations for press, and we did what we could to spread the good word: Neuroscience is here, it’s important, and it’s fun! A few local news stations featured us, helping amplify our voice. We demoed some new prototypes, and our stalwart Human-Human Interface was popular as usual. We were in new territory and a lot of people had never heard of us before, so it was a great opportunity to build new relationships and attract new attention.

Zach, our Development Engineer, said, “I enjoyed demoing to people who had never seen our kits before but are part of increasing the amount of STEM education tools.  We received a lot of great feedback from others in the industry about their experiences and issues that we can avoid.  It was also great to test out some of our new/updated products that we are developing.” Zach’s newest developments include the Neuron SpikerBox Pro and Muscle SpikerBox Pro, as well as the Plant SpikerBox, his little leafy baby.

His partner in crime at Toy Fair 2018 was Will, our resident Outreach Coordinator, poet, and maker of schpiels. He’s been getting people to roll their sleeves up for science for a long time now. He said of the show, “I’m pretty used to explaining our work to educators and scientists, so Toy Fair was a totally new experience. I wasn’t sure how non-scientists would react to the gear, but I guess I shouldn’t be surprised that everyone loved the kits and wanted to try them for themselves! It was exciting to show so many people, for the first time in their lives, real neuroscience experiments and recordings from their brains and nervous systems!”

Toy Fair was a big success for us. We tried on a toymaker’s hat to see if it fit, and who knows what the future will bring?


The Taste Preferences of Fruit Flies

Hello everyone! It’s a been over a month since my project began on studying the diet and attempting taste manipulation of the Drosophila melanogaster.

Before my experiments could begin I faced many software and hardware issues. The flyPAD itself is an extremely thin 0.6mm PCB board so every slight bend of it can result in a breaking of the soldered connections with each channel’s circuits.

I transfer flies by sucking them up…don’t worry there’s a cotton stopper. Human Sips.

In order to change my flies’ taste perception, the light-sensitive proteins which were inserted into their ‘sweet’ tasting neurons had to be stimulated by an intense red light. For a proper response, this light stimulation has to happen almost exactly when the flies take sips of the target food. To make this happen, a code was programmed into Bonsai so that an LED is turned on nearly instantaneously to when food is touched by a fly, triggered by a change in capacitance between the electrodes. This is how the flies’ taste neurons are activated at the same instant they sip certain foods to influence their food choice preference.

The instant the fly’s proboscis (mouth) touches food, intense red light shines

To get to the finalized rig I use today, I experienced firsthand just how much debugging and problem-solving is involved in research. Below is a pictorial formula of how I got to my final experimental setup:

Got my flyPAD

Integrate circuit and solder under a microscope… this is what was breaking every time the .6mm boar bent… whch was all the time

Creating circuit and code for LEDs to sink up with each channel

I soldered a shield for easy LED plugin

LEDs set up with flyPAD

soldered LEDs to their drivers, which increase the light intensity        

Another arduino programmed to pulse 100 Hz light to give flies recovery time of 900 ms

Painted acrylic to avoid unwanted light penetration of neighboring LEDs

The fruit!

The bonsai workflow for LED stimulation

Over many hours of adjusting my setup and learning how to insert the food, I finally got everything working so that experiments could get underway.

To see if the flies’ taste can be changed, I had to determine what their natural food preference was. I chose banana and avocado to compare. The cumulative number of sips showed banana was preferred over avocado, as avocados have almost no sugar in them.

To see if food choice preference can be altered, LEDs were activated upon contact with avocado. This fired the gr5a sweet neurons in the flies.

As predicted, the light stimulation successfully activated the flies’ sweet neurons to alter what they perceive as sweet tasting.

To verify these results, I ran a positive and negative control. The positive, showing the desired effect which is expected from the independent variable, was the proboscis extension (seen with the green arrow) from a gr5a fly upon stimulation from the 625nm red LED light.

Fly 1

Fly 2

As for the negative control, which does not produce the desired outcome of the experiment, I put gr5a flies that had not been feeding on all trans-retinal, under the same LED treatment targeted on avocado. All trans-retinal must be ingested to activate the proteins in the flies’ neurons, otherwise light stimulation will not fire the neurons. As expected, these flies still preferred the more sugary banana over the bland avocado.

Now that I know that the alteration of Drosophila taste preference is possible, I plan to study the nutrients within the flies’ diet.

It’s easy to eat sugar, but when offered your favourite sweet snack and a healthy food, it’s hard to eat what you know is better for you. I will create this scenario for my flies. Specifically, I am interested to see if the flies will eat protein after being deprived of it, even when a tasty, sugary apple is also up for grabs.


When deprived of protein, it has been found in the past that females will eat more protein when offered it again as opposed to males since they have to produce eggs. I want to see if this is still the case with natural foods rich in protein. I hypothesize that the flies will still prefer to eat a more sugary food as opposed to the nutrient they are deprived of. If this is the case, I will use optogenetics once again to see if I can make them eat the food they require the most. Are flies’ instincts to eat the nutrients they require stronger than their pickiness of taste? This is an interesting comparison to humans – as so many of us are more than willing to indulge in decadent desserts rather than eating our veggies any day of the week. Will optogenetics one day make its way into the human realm to revolutionize eating and health forever?

Thank you for taking a look at my post! I can’t wait to see what response I get out of my flies in the near future.