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.