The rise of optogenetics — where flashes of light can manipulate brain activity and behavior — have excited scientists looking for more precise ways of manipulating cells and their components in the laboratory and the clinic. Two papers published this month by University of Chicago laboratories explore new methods with great scientific potential of controlling cells through light. We’ll cover them in two installments.
Many researchers hope that the light-activated cell technologies currently being used in laboratories will eventually cross the threshold into the clinic. The ability of optogenetics to relieve anxiety in lab rats could someday inform a similar human treatment, where psychiatric symptoms are reduced through a light-delivery implant that stimulates the right brain regions and neurotransmitters on command. But most methods that tweak cell activity with light require some form of genetic manipulation to work, and researchers have so far encountered steep barriers to effectively and safely performing gene therapy in humans.
One potential workaround would be to use a method of light activation that requires no genetic tinkering: infrared light. These long-wavelength light spectra are already used for a variety of purposes, from night vision devices to weather observation to detecting fraudulent paintings. In biology, scientists found that infrared light is capable of exciting cells that discharge electricity, such as neurons or heart cells, prompting optimism about medical uses for this less complicated form of photo-activity.
“If you’re thinking about clinical applications, already the requirement of a light fiber to deliver the light stimulus is a hurdle,” said Mikhail Shapiro, a Miller Research Fellow at the University of California, Berkeley. “If you add on top of that the need for gene therapy or using caged neurotransmitters or other light-sensitive chromophore molecules, you’re adding an additional layer of complications. I think part of the appeal of the infrared approach is all you need is the light.”
However, there was one caveat underlying the promise of infrared cell excitation: nobody was exactly sure how it worked. That was the question that Shapiro set out to tackle during a postdoctoral scholarship in the laboratory of Francisco Bezanilla, Lillian Eichelberger Cannon Professor of Biochemistry and Molecular Biophysics at the University of Chicago Biological Sciences. Over several months of experiments, recently published in an article in Nature Communications, Shapiro and his colleagues searched for the mechanism by which infrared light excites cells…eventually settling on a surprising conclusion.
“I think the interesting thing here is it’s a case where the use of the technology leaps ahead of the understanding of how it works,” Shapiro said. “But if you’re implanting things into your body and stimulating and you don’t know how it works, how comfortable can you really be with that? Are you making little holes in these cells that are exciting them?”
Zapping a cell with infrared light produces an inward current, a sign that the cell is depolarizing. In a neuron, those types of inward currents are created by the opening of ion channels, allowing positive ions such as sodium to come flooding into the cell and pushing it toward the firing of an action potential. So Shapiro, Bezanilla, and collaborators Kazuaki Homma, Sebastian Villareal, and Claus-Peter Richter started their experiments by testing infrared light on Xenopus oocytes — frog egg cells that serve as a common laboratory cell model — injected with DNA for different types of ion channels. Frustratingly, the experiments did not initially provide a simple answer.
“We tested the infrared light, and found out that regardless of what you injected, it was exactly the same response,” Bezanilla said. “Then we said, maybe we don’t need to inject anything. Sure enough, if you don’t inject anything you get exactly the same response again.”
The failure to find an ion channel sent the researchers back to basics. Infrared’s long wavelengths are capable of locally exciting molecules, producing very rapid increases in temperature. So Shapiro measured the effect of the infrared laser on water held at the same distance as their cells, and found that his beams did indeed have a heating effect. What’s more, the inward current caused by infrared light correlated with the rate of this temperature change. The results indicated that the cellular component sensitive to infrared light wasn’t a complex protein, but plain old water.
“It was very challenging, because the type of responses we were getting were very unusual,” Bezanilla said. “But when we started looking at the temperature changes, that really gave us some clue what was happening here. You don’t need to put in a special molecule, because the molecule that you use to produce the effect is the water. You don’t have to genetically engineer anything, you just apply the infrared in the right place and you get the effect.”
Further experiments determined how these temperature changes could produce cell excitation.
Cell membranes separate electric charge between the inside and outside of the cell, and thus carry a capacitance. In most biological calculations, the capacitance is presumed to be constant for a given cell. But Shapiro’s experiments discovered that the rapid infrared-induced changes in temperature changed their target cell’s capacitance, and those changes could produce the “inward current” they detected.
Tests using mammalian cells and an artificial cell membrane replicated the oocyte experiment and demonstrated that the infrared effect on temperature and capacitance was a general phenomenon. The researchers also found that they could successfully push a cell to fire an action potential using infrared stimulation.
“Knowing more about the mechanism, now we can better tune how to do an experiment. We can actually excite certain type of neurons which was difficult to do before,” Bezanilla said. “Now you know what you have to do to change the membrane capacitance to initiate the action potential.”
This improved knowledge could benefit the many projects already underway to adapt infrared stimulation for clinical use. For example, Richter’s group at Northwestern University is looking at building a new cochlear implant that uses light instead of electricity to stimulate the cochlear nerve with much higher specificity. With a better handle on why infrared exerts these effects, bioengineers can create improved devices that take advantage of this fortuitous link between light and biology.
“You can only space electrodes so close together before you lose the ability to make distinct tones,” Shapiro said. “So the attraction of using light there is you can kind of aim it more precisely to stimulate smaller areas.”
Shapiro, M., Homma, K., Villarreal, S., Richter, C., & Bezanilla, F. (2012). Infrared light excites cells by changing their electrical capacitance Nature Communications, 3 DOI: 10.1038/ncomms1742