May 10

A Bright Idea: Illuminating the Brain with GCaMP

You might’ve read stories about some brain region “lighting up” in response to some stimulus. But what does “lighting up” actually mean? Oftentimes it refers to scientists using a fMRI machine that applies sophisticated technology to translate changes in blood flow into pixels on a computer screen. Sometimes though the brain can literally light up, like fireflies on a summer night.

Of course, this isn’t exactly normal.  Despite what cartoons might have led you to believe, a lightbulb doesn’t appear above your head when you have an idea, nor does your brain itself light up. In order to get the brain to twinkle like a star, scientists have developed genetically modified proteins that glow a specific color when neurons are active. To understand how these proteins work, it’s first important to know a little bit about how neurons work.

Calcium is Key

You might’ve heard about neurotransmitters, tiny chemical messengers like dopamine or serotonin that neurons release to communicate with one another. What you may not know is what causes them to be released in the first place. Neurons store their neurotransmitters inside little bubbles called vesicles. When neurotransmitters are released, these vesicles bind to the cell membrane and dump their contents outside the cell (a process called exocytosis).  When neurons aren’t active, neurotransmitter release is inhibited. This is because release requires calcium, and calcium is in short supply inside a neuron. However, when a neuron fires an action potential (that is, when a neuron is active), little channels in the cell membrane open up and allow calcium to enter into the neuron. Once inside, this calcium binds to a group of proteins called SNAREs that help bring the vesicle to the cell membrane where it can expel its complement of neurotransmitters into the synapse. Interestingly, the reason botulism toxin (Botox) is such a potent neurotoxin – and why it is also able to paralyze the skin to remove wrinkles – is that it cleaves some of these SNARE proteins, preventing neurotransmitter release (see this recent Neuwrite post for more).

gfpSo, when a neuron fires an action potential, calcium floods into the neuron and causes neurotransmitter release.  What does this have to do with brains lighting up? Well, scientists have developed genetically encoded calcium indicators which are proteins that light up when they bind to calcium. One of these is GCaMP, which is a fusion of Green Fluorescent Protein (GFP: this protein was initially found in jellyfish that glow green, but is now widely used in neuroscience), with CaMP (a protein that is highly expressed in neurons and normally binds to calcium). GCaMP was initially developed by Junichi Nakai in 2001[1], and has been continuously refined since then (we are now on the 6th version fittingly called GCaMP6).

When GCaMP binds to calcium, this induces a change in the structure of the GCaMP protein that causes it to fluoresce (or light up). Apart from being pretty, this is an incredibly useful tool for neuroscientists. When a neuron with GCaMP in it fires an action potential, it will literally light up! Thus, scientists can use GCaMP to see how and when a neuron  – or even a constellation of neurons – is active. Of course, in neuroscience as in life, things are a bit more complicated than that. Calcium serves many other functions in neurons (and in cells in general) such as helping to induce long-term potentiation and these other functions also influence GCaMP fluorescence.

Seeing is Believing

But how do neuroscientists see this fluorescence? The brain is, after all, tucked away inside the skull.  While GCaMP has previously been used to look at how cultured neurons or brain slices respond to stimuli, recent technological advances allow scientists to image neurons in living, moving animals. One of these technologies is known as “fiber photometry” and involves the implantation of a tiny optical fiber in the brain which can carry the fluorescent signal to a camera out of the brain. Another technique is the “Miniscope” [2] which is just what it sounds like. This tiny camera can be implanted inside a mouse’s brain to image its neurons, while being small and light enough to leave normal behavior intact.  One of the strengths of the miniscope (and related technologies) is that scientists can actually track how individual neurons change over time.  This is a big deal. You may have heard that the brain is “plastic”, which basically just means that the brain is able to change and adapt. A big part of this “plasticity” comes in the form of neurons changing how they fire across time, both individually and collectively.  GCaMP helps us to understand how neurons change and adapt for instance, during learning.

miniscope

Because GCaMP is a protein, genetic engineering and viral vectors (see Neuwrite posts on chemogenetics and optogenetics) can be used to express GCaMP in specific brain regions and even in specific types of neurons. Previous technologies, including electrical recordings, could not isolate genetically-defined neurons.  This was a huge problem, since many different types of neurons inhabit the same brain region. For example, in the prefrontal cortex you can have neurons which release essentially every different type of neurotransmitter, and it’s very difficult to determine which is which from electrical recordings alone. Now with GCaMP, scientists can isolate specific cell types. As an example, they could express GCaMP only in dopamine neurons in the cortex, allowing for a much more nuanced understanding of what different types of neurons are doing. There’s even more cool stuff that can be done with GCaMP.  Recently, scientists have developed RCaMP, which uses a red instead of a green fluorescent protein [3]. By combining RCaMP and GCaMP, scientists can simultaneously image from two different populations to understand how different types of neurons function and interact.

Getting a chance to see neurons literally light up is, first and foremost, incredibly cool.  But (maybe…) more importantly it’s proven to be very useful. With fluorescent calcium indicators like GCaMP now we can isolate specific types of neurons and see how the activity of these neurons changes over time in living, moving, and learning animals.

 

References

  1. Nakai, J., Ohkura, M., & Imoto, K. (2001). A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnology, 19(2), 137–141. https://doi.org/10.1038/84397
  2. Ghosh, K. K., Burns, L. D., Cocker, E. D., Nimmerjahn, A., Ziv, Y., Gamal, A. E., & Schnitzer, M. J. (2011). Miniaturized integration of a fluorescence microscope. Nature Methods, 8(10), 871–878. https://doi.org/10.1038/nmeth.1694
  3. Akerboom, J., Carreras Calderón, N., Tian, L., Wabnig, S., Prigge, M., Tolö, J., … Looger, L. L. (2013). Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Frontiers in Molecular Neuroscience, 6. https://doi.org/10.3389/fnmol.2013.00002

Images:

porteralab.neurology.ucla.edu/images/GCaMP6s_movie.gif

http://www.zmescience.com/medicine/genetic/fluorescent-proteins-feb/

twitter.com/BlakeP_Neuro/status/903435133838999555

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