I’m doing addiction neuroscience research, but most of my work uses frog eggs, which is obviously not a brain; why?

Models in science

Translational Science tries to make biomedical discoveries in model organisms that can apply to human health/disease.

Scientists sometime like working with models that are similar to what we want to study, but easy/faster/cheaper to work with. Human neurons are difficult to obtain because people need their brains, so traditionally, neuroscientists use animal models, like rodents or primates. Primates are difficult to work with for many reasons, so scientists tend to stick with rodents. Mice/rats obviously aren’t humans, but you’d be surprised how much rodent research translates or is applicable to humans.

I spent the summer learning to record from dopaminergic (DAergic) neurons found in the Ventral Tegmental Area of the brain. They’re very difficult to identify, and in this area of the brain, you also have GABAergic and glutamatergic neurons.

Now when it comes to the brain, working with neurons is extremely difficult. For one, they’re very sensitive, so you must make sure the environment is just right (not too acidic/basic, not too much oxygen/CO2, including certain nutrients, etc). Neurons are also extremely complicated. Cells from different brain regions may have different functions, and what happens in one type may not work in other neurons. If you wanted to study one receptor type in one neuron type, you’d have to isolate tissue from a brain region, then try to identify the neuron you want to work with, then figure out whether you need to apply certain drugs/compounds that silence or “inhibit” other receptors that might interfere with your experiments. In these experiments, you would generally be recording and looking for changes in the action potential. This is what I was learning to do at The University of Illinois, Chicago in the summer of 2018.

The African clawed tree frog

Xenopus laevis oocytes (or frog eggs) have been used as a model in neuroscience since the late 70s/early 80s. The eggs are single cells and are relatively huge (1mm3 in volume). They’re also relatively easy to keep alive and quite versatile. Scientists found that if you inject DNA/RNA into the cells, they will begin expressing your gene and therefore, make your protein. For neuroscientists, this means that you can inject DNA/RNA that codes for ion channels, and in a few days, the cells will have ion channels all over the cell surface. Ion channels serve as barriers for entering a cell. Ion channels can open by chemical stimulation (i.e. applying an agonist) which allows ions into the cell and changes the membrane potential (voltage). It doesn’t matter what kind of cell you are working with; these properties will hold true.

Our lab used to have its own frog colony, and perform surgeries on frogs to extract the cells, but we now order oocytes from a company called Ecocyte Biosciences, which raises the frogs and ships the cells to us.

Two-electrode Voltage Clamp Electrophysiology

An oocyte that is clamped but not stimulated. Note the closed Ion channels (blue).

The technique I use with frog eggs is called two-electrode voltage clamp electrophysiology (TEVC), which is based off of Ohm’s law: Voltage (V) = Resistance (R) X Current (I). Simply put, I create a circuit with the cell using two electrodes. The electrodes “clamp” the membrane potential (Vm) to a voltage I set (typically -70mV like the average neuron) by injecting current into the cell. The electrodes are made so that the have a constant resistance (R). If the cell isn’t doing anything (i.e. the ion channels are closed), then there is no change (Δ ) in current. However, if I’m expressing ion channels in the cells, and I apply a drug or agonist, the ion channels open, letting ions in. Normally, this would change the membrane potential, but remember that I’ve “clamped” it, so that I inject current into the cell to keep the voltage constant. This “injected” current is the ion channels responding to the drug/agonist.

An oocyte being stimulated. The ion channels open in response to a drug or agonist, which causes ions to enter the cell. This causes a change in current (I)

So that’s why I work with frog eggs: I can very quickly and easily perform an experiment on ion channels. One other advantage is that their huge size means that you can fit very many ion channels on the cell surface, which will give you strong current responses. Some downsides are that

1. They aren’t actual neurons (or even mammalian cells) which means they might not be physiologically relevant

2. They only express what you inject, so if you’re missing a gene found in neurons, you’re not getting the full picture.

Despite these drawbacks, they’re a really useful tool! They complement brain slice studies by letting you study the receptors in isolation and are an extremely powerful tool in drug discovery.

If you’re doing oocyte two electrode voltage clamp electrophysiology experiments, check out my posts on save your eyes with digital microscopes, tips for working with mRNA, and if you’re still using paper and pen chart recorders, check out my post on a cost-effective (<$300) digitizer!