Box Extension 12.2

Optogenetics: Controlling Cells with Light

Matthew S. Kayser

The human brain is a remarkably complex organ, with billions of neurons and trillions of synapses communicating through precisely timed electrical signals. A major limitation in understanding how our brains work is that researchers have been unable to manipulate the system on the same millisecond timescale on which it normally operates. To learn how every movement, thought, and experience we have results from groups of neurons talking to each other, don’t we need a way to speak to neurons on the same timescale they use when communicating with each other? The field of optogenetics has begun to accomplish just this by combining optics (the use of light) with manipulation of genes (thus opto + genetics). Specifically, scientists have figured out how to put genes into cells that make those cells responsive to pulses of light. Unexpectedly, this technology is possible because of light-sensitive transporter proteins and ion-channel proteins first discovered in microorganisms a few decades ago. Optogenetics involves taking the genes encoding these light-sensitive transporter and channel proteins, inserting them into target cells, and then delivering light to those cells as a way of controlling their functions. For example, neurons in the mammalian brain can be targeted to express the light-sensitive channels. Then, by delivering light to those neurons (Figure A), investigators are able to exert millisecond control over neuronal firing patterns, shedding light—literally—on mysteries of neuroscience in the process. Box Extension 12.2 describes how optogenetics was developed and its many potential applications.

Figure A A mouse prepared for an optogenetics experiment. (Courtesy of Mike J. F. Robinson.)

Beginning in the 1970s and continuing through the early 2000s, researchers doing work far from the vertebrate brain began to describe transporter proteins (pumps) and channel proteins that regulate the flow of ions across the cell membrane in response to certain wavelengths of light. These proteins were found in microorganisms, and they include the pumps bacteriorhodopsin and halorhodopsinin in archaea, and the channel protein channelrhodopsin in green algae. Bacteriorhodopsin and halorhodopsin pump protons and chloride (Cl) ions, respectively, across the cell membrane, whereas channelrhodopsin is a nonselective cation channel. These naturally occurring proteins serve functions in their hosts involving energy regulation, osmotic balance, and phototaxis. How exactly do these pumps and channels modify cell function in response to light? Just like our own eyes, they depend on vitamin A. Light enters our eyes and is absorbed by the photosensitive pigment rhodopsin, resulting in conformational changes in one of the rhodopsin components, retinal, which is derived from vitamin A. A long signaling cascade ensues, ultimately resulting in neuronal hyperpolarization caused by closing of sodium (Na+) channels in the midst of unopposed potassium (K+) flux (see pages 386–388 in the book). The result is a change in neuronal firing patterns, and this change in the firing patterns of cells in the retina is ultimately interpreted in our brains as visual stimuli.

Figure B Channelrhodopsin (ChR) opens in response to blue light, allowing cations to flow and depolarize the cell.  Halorhodopsin (HR) is activated in yellow light, pumping Cl into the cell and hyperpolarizing it. Bacteriorhodopsin and proteorhodopsin (BR/PR) pump H+ ions when activated.

The microbial opsins—bacteriorhodopsin, halorhodopsin, and channelrhodopsin—work in about the same way, with one key difference: In contrast to the mammalian signaling system, microbial opsins are proteins that, in and of themselves, accomplish both light sensing and modulation of ion transport or conductance. They are “all-in-one” molecular machines. Researchers realized that the natural coupling of these two functions within single proteins could be a powerful tool. Consider, for example, channelrhodopsin. This one protein is activated by blue light and itself can cause depolarization of a neuron. The depolarization is no different from that caused by opening of our own sodium channels. Halorhodopsin is activated by yellow light and can lead to hyperpolarization by pumping chloride ions into a cell, thus silencing neuronal firing.

Collectively, the microbial opsins seemed to provide neuroscientists with an “optogenetic toolbox”—a set of proteins that would intrinsically link light exposure and changes in the function of excitable cells. Neuroscientists wondered if in fact the proteins could be used in this way. With the optogenetic toolbox, if it worked as hoped, neuroscientists might be able to use different wavelengths of light to make populations of genetically defined neurons fire or get quiet, all within the millisecond timescale on which those cells usually communicate.

Theory aside, the question remained: What would these pumps and channels actually do if put into a neuron? Many scientists worried that they would not do much; after all, what was the likelihood that a microbial “opsin” gene put into a mammalian neuron would be expressed in the first place, and then find its way to the cell membrane, still be responsive to light, and induce enough of an electrochemical change to affect how that neuron fires?

As it turns out, all these seemingly improbable steps in fact take place when microbial opsin genes are introduced into mammalian neurons. With a few minor tweaks, for example, channelrhodopsin can be successfully expressed in neurons from many different species, and just as it does in algae, the channel opens in response to blue light, leading to cation flux, neuronal depolarization, and action potentials! Putting channelrhodopsin into certain mechanosensory neurons of a nematode modifies the worm so that shining the correct wavelength light on it causes a withdrawal response, as if the nematode had been mechanically stimulated. Channelrhodopsin targeted to hypocretin-expressing neurons in a mouse hypothalamus—neurons thought to be important for determining arousal in the sleep–wake cycle—results in waking from sleep when blue light is shined deep in the brain through optic fibers in freely moving animals. Researchers have even expressed the channels in zebrafish heart, allowing light pulses to make the heart beat faster, slower, or stop altogether!

Figure C Optogenetic stimulation with light can either excite or inhibit specific neurons that express light-responsive channels (indicated by blue or yellow dots). Neurons that don't express the channels don't respond to light stimulation.

These examples are likely the tantalizing beginning to a deeper understanding of how complex networks of cells rapidly transmit information back and forth. Importantly, optogenetic work over the last 5 years in psychiatry, neurology, and many other medical disciplines stands on the shoulders of over three previous decades of research on basic membrane biology in algae! So much for pond scum.

References

Fiala, A., A. Suska, and O. M. Schlüter. 2010. Optogenetic approaches in neuroscience. Curr. Biol. 20: R897–R903.

Miesenböck, G. 2009. The optogenetic catechism. Science 326: 395–399.

Szobota, S., and E. Y. Isacoff. 2010. Optical control of neuronal activity. Annu. Rev. Biophys. 39: 329–348.

Yizhar, O., L. E. Fenno, T. J. Davidson, M. Mogri, and K. Deisseroth. 2011. Optogenetics in neural systems. Neuron 71(1): 9–34.

Zheng, F., A. M. Aravanis, A. Adamantidis, L. de Lecea, and K. Deisseroth. 2007. Optical technologies for probing neural signals and systems. Nat. Rev. Neurosci. 8: 577–581.

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