by Michael Mohammadi

Pour a nice hot cup of your morning coffee and grab a recent issue of Nature , or Science , or Neuron and scan the table of contents. I’ll bet you your next cup of Joe that at least one article in that issue (if not more) have some sort of optogenetic approach to address complicated questions in neuroscience and cell signaling. “Optogenetics” is a very trendy term in science these days and for good reason. It has been described as a way for researchers to take over your brain , provide bionic vision , possibly treat epilepsy , and for the founding researchers in the field, a way to cash in on big financial prizes . With all the excitement, there is still some misunderstanding about how the technique works and exactly why it’s relevant. I hope to answer both of these questions here without getting overly technical.

“Optogenetics” refers to a relatively new field that combines molecular biology with light stimulation to allow researchers (and someday clinicians) to have precise control over the behavior of a cell, populations of cells, or even a whole animal.

In this video we see a mouse that becomes hyperactive when blue light activates Channelrhodopsin-2 in its brain:

Optogenetics: The basics

The term optogenetics comes from the joining of two fields: using genetic tools to target light-sensitive proteins to very specific regions within a cell, or to populations of specific cells within a network, and using optical stimulation (light in the UV to the IR wavelengths) to then activate those very selectively targeted proteins (or other probes). Combine the two fields (genetics, optical stimulation) and look for a catchy term for the new discipline, and there you have it…opto–genetics.

Researchers have long used genetic approaches and optical approaches to answer tough questions in biology, so why is this all of a sudden a big deal? A major way in which optogenetics is becoming a big deal is that it lets researchers control the excitation, inhibition, or signaling pathways of very specific cells or groups of excitable cells in the brain. Remember, the foundation of neurotransmission rely on the movement of positively charged ions (anions) and negatively charged ions (cations) across the cellular membrane. The balance of these ions inside and outside of a cell, and the potential of the membrane, contribute greatly to whether or not the neuron fires an action potential. Action potentials are central to communication between neurons. Therefore, if we can control the movement of those ions, we can control the cells excitability and how and when it communicates (fires action potentials) with other cells in a network.

The key here is that only the cells (such as the dopamine neurons that are dying in Parkinson’s disease) where you genetically target these proteins will be under the control of the light (leaving other cells to function normally). Even more profound is that the technique does not only apply to researchers that want to control ion channels, optogenetics is also being used with other proteins and molecules to modulate very specific signaling pathways such as those that control protein dynamics.

A very brief history of using light in life sciences

There are full length technical reviews that give the whole story about how optogenetics came about. This is the short, less technical version. The foundation for optogenetics occurred over a few decades, beginning in the 1970’s, when researchers were beginning to use light (rather than electrical stimulation) to photoactivate (in the marine mollusk Aplysia californica) or to photobleach (damage) tissue. Light was first used to photo release molecules such as adenosine-tri-phosphate (ATP) through a technique called uncaging (a technique that was the basis of my PhD thesis, but with glutamate!). This was revolutionary work done by Jack Kaplan and colleagues at the University of Pennsylvania. Essentially they designed molecules that were biologically inert due to a large photolabile (light would release it) molecule that blocked its action. When UV light was exposed to the caged-molecule, the photolabile group was then cleaved and the molecule (i.e. ATP or glutamate) was then able to be physiologically active.

For reference see: Kaplan, J.H., Forbush, B. III, and Hoffman, J.F. 1978. Rapid photolytic release of adenosine 5_-triphosphate from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17:1929-1935

Uncaging is still very relevant today and there are now a variety of “caged” compounds that can be developed by any good chemist (our glutamate was synthesized by collaborator Dr. Joseph P Kao) or can be purchased commercially. For many applications, uncaging allows for enough precision to ask questions specific to single cells or even micro-cellular projections such as dendritic spines (small protrusions on dendrites where cells talk to each other). But as science goes, there is always room for improvement.

In a 1999 Kuffler Lecture at the University of California – San Diego, Francis Crick (unbeknownst to him) made a profound statement about the need for a technique such as optogenetics. He said:

“Basically the argument is that to understand a complex biological system one must be able to interfere with it both precisely and delicately, probably at all levels, but especially at the cellular and molecular levels.”

Enter the beauty of optogenetics!

Heading into the 21st century, researchers started using light to interact with genetically targeted probes. In 2002 Boris Zemelman (now at UT-Austin) working with Gero Miesenböck (who gave one of the more interesting TED Talks I’ve seen) published a report using a molecule they made which was a the alpha subunit of the cognate heterotrimeric G protein, which they called the chARGe’d molecule. In (somewhat) less technical terms, they took co-expressed photoreceptor (light sensitive proteins) genes from a fruit fly (called Drosophila) that “encoded” arrestin-2 (a specific protein that regulates G-protein coupled receptors, GPCRs), rhodopsin (which is just an opsin protein and retinal), and the alpha subunit of the cognate heterotrimeric G protein (a specific subunit of the GPCR) into cultured brain cells (hippocampal neurons). That’s a mouthful, but what it means is that they used genetic techniques to put a light-sensitive molecule into a mammal (mouse) and then targeted light to activate their chARGe’d molecules and control action potentials. This was the first time someone used what we now call “optogenetics” in a mammalian system (though I skipped over a lot of very important work in other species).

Molecular biologists and chemists from the 1970’s through the early 21st century were spending a lot of time studying very unique proteins that were activated by various wavelengths of light. Initial work identified a rhodopsin, or light-gated ion channel in halobacteria (halorhodopsin, or npHR) that was sensitive to 570nm light (Stoeckenius and Oesterhelt, 1971). npHRs were shown to let anions (Cl-) pass through the channel thereby hyperpolarizing the cell membrane.

Nearly 20 years later, enter the algae Chlamydomonas reinhardtii and the discovery of channelrhodopsins, or ChRs. Similar to npHRs, these ChRs are light sensitive (at 460nm) rhodopsins that when activated, undergo a conformation change thereby opening the ion channel and allowing cations to pass (Na+, K+; depolarizaing the membrane). In the algae, the ChR’s function as light-sensing receptors allowing the algae to move towards the source of light. A key discovery for optogenetics was the mapping of the genome of ChR-1 by Nagel, Hegemann and other which gave physiologists the tools to explore their uses in other model systems. Without these key discoveries optogenetics would be far behind what it is today.

The work by Nagel and Hegemann, combined with the basis for using genetic expression of optical probes by Miesenböck’s group, led to the ideas developed by a group at Stanford which published the seminal paper in what we know today as optogenetics. Ingeniously, they realized that these microbial opsins could also be put into neurons in the mammalian brain (similar to the methods of the earlier work by Zemelman et al, 2002) and using specific wavelengths of light they could either excite or inhibit the cells with precise timing and spatial control. Boyden et al. (2005) were successful in expressing channelrhodopsin-2 (ChR2) into mammalian cells (cultured hippocampal neurons) and using light, were able to control the cells output (sub-threshold firing as well as action potentials). This groundbreaking work was done by Ed Boyden (then a grad student, now assistant professor at MIT) with Feng Zhang (then a grad student, now assistant professor at MIT) in the bioengineering lab of Dr. Karl Deisseroth at Stanford University.

As I write this article there are hundreds of optogenetic probes and techniques being used in neuroscience, developmental biology, biophysics and molecular biology. From ion channels (those things that are responsible for moving Na+, K+, Cl- etc across the cell’s membrane and the basis of neurotransmission, ground-breaking work done by Ed Boyden, Karl Deisseroth and others), to g-protein coupled receptors (the most abundant receptor type in the brain, recent work by Ehud Isacoffs group at Berkeley), as well as a variety of plant proteins (some awesome work being done by Jared Toettcher with Wendel Lim/Orion Weiner at UCSF using the Andor Mosaic!) and proteins that allow for visualization of membrane voltage (amazing work being done by Adam Cohen’s group at Harvard). Optogenetics is giving researchers precise control over signaling in both space and time, allowing them to ask very specific questions without significant perturbations to their model system.

Opto-therapy for clinical applications

I’ve attended a few talks by Dr. Karl Deisseroth on his work using optogenetics (like the youtube video below) for both preclinical (lab research) and clinical applications. From treatment of epilepsy and Parkinson’s disease, to therapies involving optogenetic-based pacemakers, excitement behind the technique abounds. Most researchers and clinicians agree that two major hurdles still exist though; 1) the need to genetically target the optogenetic probe (currently we use viruses to deliver the gene and the safety of some of these viruses, though stripped down to just a packaging system, is still in question for humans. And 2) the ability to very selectively target light to the cells that express the probe. I think it’s safe to say that we have some of the greatest minds in engineering, physiology and medicine working on these problems and the potential for opto-therapies is great.

The final word

Optogenetic techniques allow for very precise targeting of optical probes that are quite delicately stimulated by light that only affects those cells that have the probe (phototoxicity aside but this is a whole other topic). The technique has been adapted to address very complex questions “at all levels”, from the intracellular signaling pathway all the way to whole animal behavior. The use and utility of the technique remains to be seen (could I really control YOUR brain with light?). Some of the research (and related ethics) that is currently being published is downright scifi scary! But with accolades and excitement abound (Method of the year, Breakthroughs of the Decade, and its own journal now) I would say that optogenetics as a research technique is definitely here to stay. And as a clinical therapy I think it’s safe to say that we are just at the tip of the optical iceberg though as others have indicated, the challenges to bring optogenetics to the clinic are quite extrodinary.

Cheers!