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Intracellular recording in Snails Final Update – Juan Ferrada

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.

Outreach

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).

 


Electrophysology at the Cambridge Science Festival

This post comes to you from our friends at Biomakespace! They are biohackers and electrophysiology enthusiasts who work and hack with our kits along with inventions of their own! They recently presented and demoed their cool tech at the annual Cambridge Sci  ence Festival in Massachusetts. We asked them a couple questions about the event and their experience using and demoing the Backyard Brains SpikerShield, and they were kind enough to prepare this debrief for us which we’re sharing with you today!

The annual Cambridge Science Festival welcomes over 40,000 visitors of all ages to hundreds of events developed and run by staff and students from departments and organisations across the University and research institutions, charities and industry around Cambridge. Events include talks, interactive demonstrations, hands-on activities, film showings and debates.

Over 1200 visitors streamed through the Plant and Life Sciences Marquee where Biomakespace had an activity table, with many stopping to find out about Biomakespace and the types of equipment/activities we had on display and where people could participate. There were a lot of families with young children, but also groups of students and adults.

Roger, our resident electrophysiology expert was reading electrical signals from the gastrocnemius muscle in the lower leg and the brachioradialis muscle in the forearm using a battery powered bioamplifier and viewing them on an oscilloscope as well as creating noise from a speaker. Visitors were really interested to see how their signals varied in ‘intensity’, both amplitude and frequency, depending on the degree of effort involved in muscle contraction and also how easy it is to observe the firing of individual ‘motor units’.

Roger also demonstrated ‘reflex arcs’ by tapping the Achilles tendon in the lower leg or the distal brachioradialis tendon in the forearm. This usually requires a ‘reflex hammer’ containing an accelerometer, but he rigged up a normal hammer with a rubber-cushioned micro switch and a 9V battery attached with heat shrink to record the timing and intensity of the strike and observe the response. Reflex arcs can be unconsciously enhanced by clenching your teeth and interlocking fingers (the classic Jendrassik Maneuver) and also by imagining something that makes you really angry (mental-imagery interference), so we tried that out as well with some great results.

Next along we had a demo with the Backyard Brains SpikerShield on an Arduino Uno. This has a series of green, yellow and red LEDs indicating the response amplitude. A long line of children excitedly tried to flex their arms to get the red LEDs to light up (which wasn’t too difficult with where we’d set the thresholds!) – we explained that although Roger’s full setup is large and can be expensive, it’s now possible to do some great experiments with low-cost hardware and a mobile phone – which is the message and ethos that Biomakespace and Backyard Brains value above all else.

Technical details:

We performed EMG recordings from the gastrocnemius muscle in the lower leg and the brachioradialis muscle in the forearm using Gold ECG Electrodes (Ag/AgCl/solid adhesive; pre-gelled: TIGA-MED, Deutschland GmbH). Differential signals were amplified 1000X or 5000X, and filtered (low pass: 30 Hz – 300 Hz, high-pass: 800 Hz – 15 kHz, depending on the experiment) using a battery powered bioamplifier and viewed on a Hameg HM407-2 Analog/Digital Oscilloscope and monitored by a conventional PC audio amplifier.

Volunteers were shown how these signals varied in ‘intensity’, both amplitude and frequency, depending on the degree of effort involved in muscle contraction and also how easy it is to observe the firing of individual ‘motor units’.

Practical demonstrations of ‘reflex arcs’ were made by localised tapping of: (i) the Achilles tendon in the lower leg, and (ii) the distal brachioradialis tendon in the forearm. In the absence of a ‘reflex hammer’ containing an accelerometer, an effective alternative was achieved with a rubber-cushioned micro push-switch (RS Components 336-74) and a 9V PP3 battery attached to a light (4 Oz/114 g) hammer using heat shrink. Activation of the switch upon tapping the tendons produced a signal that was made compatible with the ‘trigger input’ of the oscilloscope by a Grass Model SIU5B Stimulus Isolator Unit.

The principle of unconscious modulatory control of such reflex arcs was shown utilising the classic Jendrassik Maneuver and also by mental-imagery interference, both having the effect of markedly enhancing the reflex-based EMG activity.

The Muscle Spikershield is a great way to get people experimenting with neurobiology in an easy to understand way and the kit gives so many opportunities for other learning as well: basic electronics, soldering skills, how an amplifier works and coding with Arduino. The Science Makers monthly meetup at Cambridge Makespace, which was instrumental in getting the Biomakespace group together, has used the Muscle SpikerShield and other Backyard Brains boards at several meetups, from constructing the kits to experimenting with muscles, worms, plants and trying to control objects. Everyone has loved it and it’s fantastic for demos – we’ve also had several people asking where they can get one of their own.

Children were really attracted to the Muscle SpikerShield activity as it allowed them to learn something about themselves through the link between number of lights lighting up and muscle activity, which isn’t something many had experienced before. Adults were interested to see how devices like the Muscle Spikershield can show similar scientific concepts to the more expensive lab equipment and also saw the value as a teaching resource. And it is a lot of fun too! Boys were very keen to show their strength and tried to light up all the lights on the SpikerShield as well as try and make most noise when their muscles were hooked up to the Analog/Digital Oscilloscope and amplifier – especially when they competed with their dads!

Teaching Electrophysiology is important because it helps people understand how the brain and the nervous system work, which is fundamental to understanding who we are as human beings. The great thing in terms of experimenting with electrophysiology is that even at a simple level, people can find out things new things for themselves and there are so many tricks that illuminate how our brains and bodies work on an subconscious level, like the reflex arc experiments Roger was carrying out in his demo. Although electrophysiology is not a new field of science, combined with new technologies, such as advanced genetic and optical techniques, it allows to gain understanding on a wide variety of scales- from single ion channel proteins to whole organs systems and whole organisms and that keeps it really exciting.

Electrophysiology is going to play a big role in our future. First off we’ll be building and collecting devices to set up the ‘electrophysiology corner’ in the workshop and after that the projects will be community-led. Some ideas that have already been bounced around include recording spontaneous activity in invertebrates, investigating neuroplasticity in snails and record currents associated with growth or movement in plants and slime moulds. Some people are very interested in combining 3D-printing with electrophysiology kit to look at customised fitting and modified control mechanisms. One group who are associated with the space and the Science Makers group have been working for over a year on a fork of the Backyard Brains plant electrophysiology board and have re recently rigged up custom controllers for electrode micromanipulators with Playstation 2 controllers – we’d love to see more of that!

Biomakespace is a non-profit organisation creating a community laboratory on the Cambridge Biomedical Campus in Cambridge, UK. We aim to build a community of scientists, engineers, technologists, entrepreneurs, teachers, artists and members of the public interested in engineering with biology. Biomakespace provides members with affordable access to a well equipped lab and prototyping space as well as to training and social events.  By encouraging and supporting project based interdisciplinary collaborations, Biomakespace aims to contribute to awareness, knowledge and innovation in engineering with biology. As the community grows, we aim to open the space to the public for events.

Visit our website (http://biomake.space) to find out more. To become a member or support us, visit (https://biomake.space/support-us)


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.