Each of our 86 billion neurons forms, on average, a thousand connections with other neurons. Electrical and chemical signals among them produce the intricate message patterns that drive sensations, thoughts, feelings, and behaviors. To elucidate how the brain runs smoothly and how it sputters to foment illness, researchers need to unravel a mind-boggling wiring puzzle.
For decades, scientists have fantasized about arousing or quelling select neurons to assess the consequences and thereby discern the cells’ normal functions. Traditional methods fall short because they act too slowly or affect all neurons in large swathes of the brain. An ideal strategy would facilitate rapid manipulation of brain circuits in alert animals with locational and cell-type precision. In 1999, Francis Crick speculated that light might lend itself to this venture, although he deemed the idea “rather far fetched.”
Pumped up by a purple membrane
In 1969, Oesterhelt went on sabbatical from the University of Munich to the late Walter Stoeckenius’s lab (University of California, San Francisco). Stoeckenius was studying the membranes that surround a microbe now known as the archaeon Halobacterium salinurum, which flourishes in high salt environments. This creature falls apart in low salt solutions to generate separable biochemical portions, including a purple membrane fraction.
Adding particular solvents made the color fade, Oesterhelt noticed. He was perplexed by this chemically confounding behavior, but when a colleague told him that frog eyes bleach from red to yellow upon overillumination, he had a preposterous and exhilarating thought. Maybe the purple material in the single-celled archaeon contained retinal, the vitamin A-based compound that joins with proteins called opsins. Retinal and opsins are essential for vision in animals.
Oesterhelt established that the sole protein in the purple membrane binds retinal, and he and Stoeckenius named it bacteriorhodopsin after its well-known vertebrate cousin rhodopsin. In 1971, they proposed that bacteriorhodopsin functions as a light sensor, or photoreceptor.
Oesterhelt, back in Germany, and Stoeckenius continued to work in parallel and as collaborators. Oesterhelt wondered if a chemical change accompanied the color shift, so he exposed membrane preparations and intact cells to light and measured the pH. It plummeted, indicating that hydrogen ions, or protons, escaped into solution. This experiment and others established that bacteriorhodopsin transports protons out of the cell.
Subsequent studies by Oesterhelt and others established that retinal plays a pivotal role. When light hits retinal, it absorbs a photon and snaps into a different shape that forces the bacteriorhodopsin protein’s innards to change their relative positions. This perturbation places a particular proton into a chemically unfriendly environment. Consequently, it jumps to a more welcoming spot, and eventually a proton winds up outside the cell. Analogous events replenish the lost proton with one from the cell’s interior, restoring bacteriorhodopsin’s original configuration. In this way, a photon instigates transfer of a single proton from inside to outside the cell.
Scientists later found a second type of retinal-containing light-activated pump in Halobacteria. This one, called halorhodopsin, conveys chloride ions into cells. Oesterhelt’s landmark biophysical, structural, and genetic studies of bacteriorhodopsin and halorhodopsin provided foundational insights that informed the eventual development of optogenetics.
Channeling ion flow: a new class of photoreceptors
In 1980, Hegemann joined Oesterhelt (by then at the Max Planck Institute for Biochemistry, Martinsried) as a PhD student. Soon afterward, some papers about the single-celled alga Chlamydomonas caught Hegemann’s eye. This organism—a eukaryote, like mammals—has an eyespot that enables it to swim toward conditions that optimally support photosynthesis, and new evidence hinted that the eyespot’s light-sensing instrument relies on retinal.
Intrigued, Hegemann decided to track down the photoreceptor’s presumptive protein component when he established his own lab at the Max Planck Institute of Biochemistry, Martinsried. In 1991, he found that ion currents into the organism rise extremely rapidly in response to light, much faster than analogous events in the visual pathway of higher animals, whose rhodopsins require other molecules to exert their effects. Bolstered by additional results several years later, he reasoned that insufficient time existed for a molecular signal to pass from the light detector to the ion channel and proposed that the two elements form a single protein.
To test his all-in-one hypothesis, he needed a way to interrogate the apparatus’s properties in a clean system. In 2000, other scientists compiled a collection of DNA sequences from Chlamydomonas. Hegemann (by this time at the University of Regensburg) identified one that encodes a protein that resembles bacteriorhodopsin—most notably at the spots that interact with retinal and compose its proton-transporting network.
Hegemann, in collaboration with Georg Nagel and Ernst Bamberg (Max Planck Institute for Biophysics, Frankfurt), put the gene into frog eggs and evaluated electrical behavior produced by the protein encoded by the introduced gene. As Hegemann had hypothesized, this protein acts as a channel that opens upon illumination and allows protons to rush in. They announced these discoveries in 2002. The following year, they reported similar data for a second light-activated algal ion channel, which admits in addition positive ions such as sodium and calcium. Furthermore, they demonstrated that it can function when placed in mammalian kidney cells. They dubbed this new class of proteins channelrhodopsins (ChRs) (Figure 1).
Micro origins, macro impact
These three proteins, whose discovery and utility gave rise to optogenetics, facilitate light-triggered ion transfer across cell membranes. The archaeon Halobacterium salinarum contains bacteriorhodopsin, which pumps protons out of cells, and halorhodopsin, which pumps chloride ions into cells. The single-celled algae Chlamydomonas reinhardtii contains channelrhodopsins; when these channels open, positive ions surge through the membrane.
Illustration: Cassio Lynm/© Amino Creative
A bright idea
These findings electrified neurobiologists, including Deisseroth. An influx of positive ions is exactly what sparks neuronal signals, and the prospect of using a single molecule to direct brain circuits was captivating. By this time, others, including Gero Miesenböck (University of Oxford), had attempted to control neurons with light, but those procedures were impractical because, for example, they required simultaneous delivery of multiple components.
Deisseroth and Stanford colleagues Edward Boyden and Feng Zhang, in collaboration with Nagel and Bamberg, engineered a harmless virus to deliver a ChR gene to neurons grown in culture dishes. Light exposure provoked ion flow that was fast, adjustable, reproducible—and strong enough to prompt neuronal firing. They published these observations in 2005, and over the following year, four other groups described similar results, including scientists from Case Western Reserve University, Humboldt University, Frankfurt’s Goethe University, Sendai’s Tohoku University, and Wayne State University. Soon afterward, many labs across the globe began using the technology.
To implement the scheme in the mammalian brain, though, multiple innovations were required, and Deisseroth spearheaded these advances. Researchers had to devise a way to deliver light that would penetrate tissue safely, for example, and no one knew whether mature mammalian brains contain the essential retinal cofactor.
In 2007, Deisseroth reported not only that the technology operated in live rodents, but also that it could command behavior (Figure 2). He delivered a ChR gene to a part of the brain that governs whisker movement. When he shot blue light through a thin optical fiber that he had implanted, their whiskers twitched. Because the rodents’ heads were held in place, the experimental setup was not of general utility, but later that year, Deisseroth showed that the same approach worked on mice that could move freely.
Many improvements followed, as researchers honed known microbial opsins for various purposes and discovered others. In 2008, Deisseroth and Hegemann collaborated to find a natural ChR that responds to yellow rather than blue light. Later, they designed ChRs that spur especially strong currents and others that remain on for long periods of time and can be switched off with a different color of light.
In 2009, Deisseroth’s student Viviana Gradinaru pioneered another impactful refinement. Because each neuron extends its tentacle-like axons throughout the brain, the projections reach disparate locations. By focusing light in a particular area, the investigators selectively controlled firing by opsin-containing neurons whose cell bodies might lie far away. The team used this method on mice with a Parkinson Disease-like condition to probe how deep brain stimulation delivers its benefits (Alim Louis Benabid and Mahlon R. DeLong, Lasker Clinical Research Award, 2014).
Flashes for insight
In the standard optogenetic strategy, scientists engineer a control region that turns on the adjacent opsin gene in certain cell types. This DNA is packaged into a benign virus, which delivers it to the brain, where opsin protein is produced only in the selected neurons. An optic fiber carries light, which activates the opsins, thus turning these specific neurons on or off and allowing researchers to assess processes of interest.
Illustration: Cassio Lynm/© Amino Creative
For many years, the only channels available to the optogenetics field turned neurons on, as they allowed positive ions such as sodium to stream in. Some of the opsin pumps transported negative ions into cells, but because only one ion moved at a time, neuronal inhibition was weak.
In 2012, Osamu Nureki (University of Tokyo) and Deisseroth, in collaboration with Hegemann, presented the first high-resolution structure of a ChR, and they saw that the ionic travel pathway is lined with amino acids that repel negative ions. Guided by this information, Deisseroth and Hegemann independently re-engineered the channel so it would transport the negative chloride ion. This new tool blocked neuronal firing; thus, it dramatically amplified the experimental power of optogenetics by enabling inhibition with channels.
Turning on and off genetically defined subsets of neurons in living animals has transformed the study of brain function, but cells of a given type don’t always act together. In 2012, Deisseroth’s team managed to control single neurons in live mice by customizing opsins for use with so-called two-photon technology. Seven years later, the investigators applied this system to manipulate single cells in an area of the mouse brain associated with rewards gained from social interactions and food consumption. For example, they restrained feeding by targeting certain social cells.
Scientists are applying optogenetics to tease apart a vast number of adaptive and maladaptive behaviors (Figure 3). By using inhibitory and stimulatory opsins, Deisseroth and colleagues stopped thirsty mice from seeking water and prodded fully hydrated animals to guzzle. Similarly, investigators are homing in on the mechanism by which leptin regulates body weight (Douglas Coleman and Jeffrey M. Friedman, Lasker Basic Research Award, 2010), identifying some neurons that compel animals to eat voraciously and others that suppress appetite.
Optogenetic has already yielded rich information about numerous behavioral and physiological phenomena in experimental animals. It is enabling scientists to determine the contribution of individual neurons to these activities.
Illustration: Cassio Lynm/© Amino Creative
The strategy can also be applied to more complex cognitive phenomena. Deisseroth optogenetically dissected anxiety into its physiological, behavioral, and emotional components, and other researchers have similarly broken down mouse parenting into a circuit that propels a mother to find her babies and another that drives her to care for them.
The synergistic discoveries of Oesterhelt, Hegemann, and Deisseroth have launched the Era of Optogenetics—an age in which re-engineered retinal-containing opsins from single-celled organisms allow researchers to peer into the brains of living animals. Scientists can now study neurons and their circuitry with stunning clarity and probe their functional underpinnings with unprecedented resolution. Optogenetics has already exposed hitherto unknown secrets and it promises to reveal additional elusive ones.
by Evelyn Strauss
Selected Publications–Discovery of Microbial Opsins
Oesterhelt, D., and Stoeckenius, W. (1971). Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat. New Biol. 233, 149-152.
Oesterhelt, D., and Stoeckenius, W. (1973). Functions of a new photoreceptor membrane. Proc. Natl. Acad. Sci. USA. 70, 2853-2857.
Bamberg, E., Tittor, J., and Oesterhelt, D. (1993). Light-driven proton or chloride pumping by halorhodopsin. Proc. Natl. Acad. Sci. USA. 90, 639-643.
Kolbe, M., Besir, H., Essen, L.-O., and Oesterhelt, D. (2000). Structure of the light-driven chloride pump halorhodopsin at 1.8 Å resolution. Science. 288, 1390-1396.
Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A.M., Bamberg, E., and Hegemann, P. (2002). Channelrhodopsin-1, a light-gated proton channel in green algae. Science. 296, 2395-2398.
Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P., and Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA. 100, 13940-13945.
Kato, H.E., Zhang, F., Yizhar, O., Ramakrishnan, C., Nishizawa, T., Hirata, K., Ito, J., Aita, Y., Tsukazaki, T., Hayashi, S., Hegemann, P., Maturana, A.D., Ishitani, R., Deisseroth, K., and Nureki, O. (2012). Crystal structure of the channelrhodopsin light-gated cation channel. Nature. 482, 369-374.
Wietek, J., Wiegert, J.S., Adeishvili, N., Schneider, F., Watanabe, H., Tsunoda, S.P., Vogt, A., Elstner, M., Oertner, T.G., and Hegemann, P. (2014). Conversion of channelrhodopsin into a light-gated chloride channel. Science. 344, 409-412.
Selected Publications–Development of Optogenetics
Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005). Millisecond- timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263- 1268.
Zhang, F., Wang, L.-P., Brauner, M., Liewald, J.F., Kay, K., Watzke, N., Wood, P.G., Bamberg, E., Nagel, G., Gottschalk, A., and Deisseroth, K. (2007). Multimodal fast optical interrogation of neural circuitry. Nature. 446, 633-639.
Aravanis, A.M., Wang, L.-P., Zhang, F., Meltzer, L.A., Mogri, M.Z., Schneider, M.B., and Deisseroth, K. (2007). An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural. Eng. doi: 10.1088/1741- 2560/4/3/S02
Gradinaru, V. Mogri, M., Thompson, K.R., Henderson, J.M., and Deisseroth, K. (2009). Optical deconstruction of Parkinsonian neural circuitry. Science. 324, 354-359.
Berndt, A., Lee, S.Y., Wietek, J., Ramakrishnan, C., Steinberg, E.E., Rashid, A.J., Kim, H., Park, S., Santoro, A., Frankland, P.W., Iyer, S.M., Pak, S., Åhrlund-Richter, S., Delp, S.L., Malenka, R.C., Josselyn, S.A., Carlén, M., Hegemann, P., and Deisseroth, K. (2016). Structural foundations of optogenetics: determinants of channelrhodopsin ion selectivity. Proc. Natl. Acad. Sci. USA. 113, 822-829.
Allen, W.E., Chen, M.Z., Pichamoorthy, N., Tien, R.H, Pachitariu, M., Luo, L., and Deisseroth, K. (2019). Thirst regulates motivated behavior through modulation of brainwide neural population dynamics. Science. doi: 10.1126/science.aav3932
Zhang, F. Vierock, J., Yizhar, O., Fenno, L.E., Tsunoda, S., Kianianmomeni, A., Prigge, M., Berndt, A., Cushman, J., Polle, J., Magnuson, J., Hegemann, P., and Deisseroth, K. (2011). The microbial opsin family of optogenetic tools. Cell. 147, 1446-1457.
Grote, M., Engelhard, M., and Hegemann, P. (2014). Of ion pumps, sensors and channels–Perspectives on microbial rhodopsins between science and history. Biochim. Biophys. Acta. 1837, 533-545.
Deisseroth, K., and Hegemann, P. (2017). The form and function of channelrhodopsin. Science. 357. doi: 10.1126/science.aan5544
Fenno, L., Yizhar, O., and Deisseroth, K. (2011). The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389-412.
Deisseroth, K. (2015). Optogenetics: 10 years of microbial opsins in neuroscience. Nat. Neurosci. 18, 1213-1225.