Hi Everyone. Juan here! My two month tour with Backyard Brains has reached its end, and I’m really grateful to have had the opportunity to work on this project.
I had three activites during the “practica” here at Backyard Brains:
- Recording from the Ganglia of Snails
- Helping on the Anemone Project
- Assisting in Outreach.
The snail recording was my main project at 70% of my time, the anemona project occupied about 10% of my time, and the high school outreach was 20%.
Recording from Snails.
The original aim was to record the intracellular action potential of pacemaker-like cells from the parietal ganglia of Helix aspersa, using Backyard Brains hardware for the optics, acquisition and amplification. From the last blog post you can see that we had no problem with dissecting the ganglia or visualizing the neurons with hand pulled pipettes in the microscope, but we couldn’t get any recordings. Recording electrical activity from the leg of the cockroach is different from the ganglia of the snail. Because of the dry environment of the cockroach leg and the strong signal from the leg nerves, the cockroach leg nerve activity is very easy to record. Buuuuut with the snail, we have a really weak signal in a conductive aqueous salt solution, so we must take a different approach to the experiment, as repeated attempts at the BYB lab did not yield results.
Sooo we went to familiar ground (for me) and replicated the experiment with lab equipment from the Laboratorio de Neurociencias at the University of Santiago (Usach). We had divine intervention from Darwin Contreras, a PhD student who just that day had successfully defended his Ph.D. and happened to only be coming back to the lab to get his motorcycle helmet to go motor on home and relax with his growing family. Using a large Faraday cage, a dissection scope, and a high end manipulator (but yes, a Backyard Brains Neuron SpikerBox Pro), we carefully inserted an insulated blunt tip silver wire into the ganglia.
And we listened to the low background noise coming out from the speaker of the SpikerBox. But….Every now and then there was a rattle, sparse and random enough to not be an artifact, so we recorded it and to the surprise of everyone there it was, spontaneous, asynchronous action potentials. Success!
We witnessed high amplitude spiking (download the raw data if you like!).
And we also witnessed the rhythmic “neuron dying” response.
But this was only a partial success, as it was 1) our only successful recording, and 2) made extracellularly instead of intracellularly. We found a shorter electrode, to prevent the antenna effect, and a faraday cage minimized the noise profile, but we are still far away from the original goal, recording intracellularly from the large neurons in the parietal ganglion.
At the moment, we may seek another preparation for intracellular single unit recording, as the snail preparation is a bit tricky. We may go to the intracellular recordings of the muscles of the tail of the crayfish, or perhaps try another mollusk, say a “macha,” a type of clam very common along the Chilean and Peruvian coast, that we looooovvveeee to eat.
Recording from the Nervous System of Anemones
We continue in the long term project to record from sea anemones. We built a harpoon style electrode…
but the silver wire wasn’t strong enough to pierce the membrane of the oral disc of the anemone. We had heard that there are more neurons around the oral disc (which, in an anemone, serves as its mouth, anus, and reproductive orifice). We will try tungsten next, which is the classic, strong material for small metal electrodes.
But….we had the idea that maybe we could remove the tentacles, like we remove the leg of the cockroach, and attempt a recording in a more controlled environment under a microscope. To our surprise, the tentacles kept moving for an hour after we had cut them! We may be on to a new preparation, it is very fascinating to watch. Very primordial success, yet waiting, yet to come. See our video below.
We then inserted an insulated blunt tip silver wire into the open end of the tentacle and tried to see if we could measure spontaneous activity or evoked activity (when we touched the tentacle with a probe).
But….we did not get any successful neuron recordings. We are sure there is something here though, there have got to be neurons inside the tentacle. The tentacle is moving, and neurons must be talking to the muscles. The neurons in the anemone tentacle are arranged like sheets between rings of muscle, so it’s a matter of optimizing the preparation. We are always getting closer to the elusive anemone neurophysiology, stay tuned.
During the last part of May we taught the students how to build a two stage amplifier circuit from a breadboard, and the students can now recognize the logic of how to manage components like resistors, capacitors, and transistors.
During the last two classes I helped teach about the difference between reactions and reflexes using the knee and elbow.
For the last class, we did experiments measuring the difference between audio and visual reaction times. Data collected in a classroom can be noisy. Supposedly auditory reactions are faster than visual reaction times but we did not observe that difference in the students who had well tabulated data. But I always continue in my experiments. I always continue trying to have compelling data that tells an interesting story.
Bye Guys, Now I have to write my thesis! I’ll miss the late night pizza party experiment sessions with Florencia and Tim and the workshops in the Fablab at the high school Colegio Alberto Blest Gana. I will not miss the cardboard-tasting garlic bread (pizza delivery company to remain anonymous).
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.
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.
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!
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.
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.