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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. 


Intracellular recording in Snails – Juan Ferrada

About me

Hi! I am Juan Ferrada, a biochemistry student at the lovely Universidad de Santiago in Chile. That’s me below with my girlfriend Rocia on campus. She is an important part of my project.

At the university I work with Dr. Patricio’s Rojas, a longtime colleague of Backyard Brains. Thanks to Patricio, Backyard Brains’ equipment has been to Antarctica!

In his lab, I study the temperature dependence of ion channels. I am in my last semester of studies, and in Chile every student has to do a “práctica” which is doing an internship for a clinical lab, company, or non-university research lab. Since Backyard Brains is in a sweet spot between company product development and neuroscience research, it makes doing a project with them an exciting, novel, and unorthodox práctica.

About my project

Backyard Brains started out studying neurons in cockroaches, then expanded to muscles in humans (EMG), than hearts (EKG), then brains (EEG), and then eyes (EOG). Now we return come back to the first love and dedication, the neuron, in search of the the most iconic symbol – the intracellularly recording action potential, Hodgkin-Huxley style. Teachers all the time ask Backyard Brains how to replicate, in a certain form, the famous Hodgkin Huxley intracellularly recorded action potential, and I am here to help! To begin, we will go to our backyard. Or, well, my girlfriend’s backyard (I told you Rocia would appear again).

Rocia has a garden, full of exotic and indigenous plants, from rosales to venus flytraps, but there are other things besides plants in the garden. There are things that eat my girlfriend’s plants:

 

 

 

 

 

 

 

 

 

 

 

Specifically, The scourge of my girlfriend is named Helix aspersa, also known as the pond snail. They like green leaves, but are especially of eating the tomato plant entirely, fruit and all. In France and Spain, these are the same snails that are cooked and served as elegant dishes (escargot).

 

 

 

 

 

 

 

 

 

These snails are interesting because they have very large pacemaker cells (which fire spontaneous action potentials like the ones you can see in your heart) located in the parietal ganglia (PG) involved in the chemo-mechanical sensation. Most classical intracellular recording techniques involve electrically stimulating the neuron, which can result in artifact, requires multiple electrodes, etc… Given that we will record from cells that are constantly firing spikes, we should be able to record spontaneous action potentials without the need for electrical stimulation. We want to make the preparation as simple as we can, BYB style.

Beginning the Project

To begin, we have to get the snails, which I collected during Easter Weekend. Check.

 

 

 

 

 

 

 

 

 

 

 

 

 

Now we will do some exploratory surgeries to extract the PG and try to isolate the neurons. To do this, we will anesthetize the snails with a Magnesium Chloride solution. Once we have the neurons, we will build a DIY glass electrode, mated with the original Neuron SpikerBox, to try to record the elusive intracellular action potential. Stay tuned as we begin this project. Saludos desde el Sur!

Fun facts

Chile has some a famous squid called  Dosidicus gigas. With a mantle of 4.9 ft and 60 lbs it’s one of the largest of its kind. Experiments using the giant axon of this squid kickstarted the field of biophysics in Chile.

Due to Chile’s isolation, surrounded by the Andes Mountains, the Atacama desert, and the Pacific ocean, we have a very diverse unique fauna, indigenous to the country, and we have an even greater marine fauna thanks to the Humboldt current. But, we don’t have many mammals (around 150) for a country of such North-South extent. But… we do have the smallest wild cat in the americas, Leopardus guigna.

Sources

http://www.molluscs.at/gastropoda/index.html?/gastropoda/morphology/nervous_system.html

Tiwari SK, Woodruff ML.Helix aspersa neurons maintain vigorous electrical activity when co-cultured with intact H. aspersa ganglia. Comp Biochem Physiol C. 1992;101(1):163-74.


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?