Improving on nature
The number of optogenetic tools, along with the diversity of their capabilities, has since expanded rapidly because of a remarkable convergence of ecology and engineering. Investigators are adding new opsins to their tool kits by scouring the natural world for novel ones; they are also applying molecular engineering to tweak the known opsins to make them even more useful for diverse experiments in a wider range of organisms.
In 2008, for instance, our genome searches led by Feng Zhang on a different algal species, Volvox carteri, revealed a third channelrhodopsin (VChR1), which responds to yellow light instead of blue as we showed together with Peter Hegemann. Using VChR1 and the other channelrhodopsins together, we can simultaneously control mixed populations of cells, with yellow light exerting one type of control over some of them and blue light sending a different command to others. And we now have found that the most potent channelrhodopsin of all is actually a hybrid of VChR1 and ChR1 (with no contribution from ChR2 at all). Our other modified opsins (created with Ofer Yizhar, Lief Fenno, Lisa Gunaydin and Hegemann and his students) now include "fast" and "slow" channelrhodopsin mutants that offer exquisite control over the timing and duration of action potentials: the former can drive action potentials more than 200 times per second, whereas the latter can push cells into or out of stable excitable states with single pulses of light. Our newest opsins can also now respond to deep red light that borders on the infrared, which stays more sharply focused, penetrates tissues more easily and is very well tolerated by subjects. Many groups are now also pushing opsin engineering forward, including those of Hiromu Yawo in Japan, Ernst Bamberg in Frankfurt and Roger Tsien in San Diego.
Many of the natural opsin genes now being discovered in various non-animal genomes encode proteins that mammalian cells do not make well. But Viviana Gradinaru in my group has developed a number of general-purpose strategies for improving their delivery and expression. For example, pieces of "trafficking" DNA can be bundled with the opsin genes to act as "zip codes" to ensure the genes are transported to the correct compartments within mammalian cells and translated properly into functional proteins. This generalizable approach has served to unlock the broad ecological repertoire of microbial opsin genes.
Molecular engineering has also extended optogenetic control beyond cells' electrical behaviors, to well-defined biochemical events. A large fraction of all approved medical drugs act on a family of membrane proteins called G-protein coupled receptors. These proteins sense extracellular signaling chemicals, such as epinephrine, and respond by changing the levels of intracellular biochemical signals, such as calcium ions, and thus the activity of the cells. By adding the light-sensing domain from a rhodopsin molecule to G-protein coupled receptors, early in 2009 Raag Airan and others in my laboratory published a set of receptors called optoXRs that respond rapidly to green light. When viruses are used to insert the single-component optoXR genes into the brains of lab rodents, the first cell type–specific fast optical control over defined biochemical pathways was enabled, working even in freely moving mammals. Optical control over defined biochemical events is now also being explored in many laboratories, and opens the door to optogenetics in essentially every cell and tissue in biology.
With fiber-optic tools we developed and published in 2006 and 2007, investigators can now deliver light for optogenetic control to any area of the brain—whether surface or deep—in freely moving mammals. And to enable simultaneous readouts of the dynamic electrical signals elicited by optogenetic control, we also have published millisecond-scale instruments that are integrated hybrids of fiber optics and electrodes ("optrodes"). A long-sought synergy can emerge between optical stimulation and electrical recording because the two can be set up to not interfere with each other. We can now, for instance, directly observe the changing electrical activity in the neural circuits involved in motor control at the same time that we are optically controlling those circuits with microbial opsins. The more rich and complex that both our optogenetic inputs and the electrical-output measures of neural circuits become, the more powerfully we can infer the computational and informational roles of neural circuits from how they transform our signals.
The round-trip back to psychiatry
The importance of optogenetics as a research tool, particularly in conjunction with other technologies, continues to grow rapidly. In addition to distributing these diverse engineered opsin genes to more than 700 laboratories worldwide (http://www.optogenetics.org), my students have worked hard over the past few years to develop and deliver optogenetics instruction. We have found that despite the unusual combination of technologies required for optogenetics, the fundamentals can be taught in focused hands-on courses in the laboratory, which accelerate the benefits of the technology. Scientists from all over the world come to practice optogenetics and return to their home institutions, where they serve as local sources of knowledge and wisdom, resulting in application to diverse settings and challenges across the globe.
One example of an unexpected class of application involves brain imaging. In recent years, neuroscience has made many advances based on the brain-scanning technique called functional magnetic resonance imaging (fMRI). These scans are usually billed as providing detailed maps of neural activity in response to various stimuli. Yet strictly speaking, fMRI shows only changes in blood-oxygen levels in different areas of the brain; those changes are just a proxy for actual neural activity. Some nagging uncertainty has therefore always surrounded the question of whether these complex fMRI signals can be triggered by increases in local excitatory neural activity. This past May, however, my laboratory used a combination of optogenetics and fMRI (dubbed ofMRI) to verify that the firing of local excitatory neurons is fully sufficient to trigger the complex signals detected by fMRI scanners. In addition, ofMRI can map working neural circuits with an exactness and completeness not previously possible with electrodes or drugs. Optogenetics is thereby helping to validate and advance a wealth of scientific literature in neuroscience and psychiatry.
Optogenetics has also been employed to control a kind of neuron (the hypocretin cells) thought to be involved in the sleep disorder narcolepsy, in the first application of optogenetics to a freely moving mammal. Specific types of electrical activity in those neurons, we have found, set off the complex transition of awakening. Optogenetics has also been employed to help determine how dopamine-making neurons may give rise to feelings of reward and pleasure. In this work with Hsing-chen Tsai, Feng Zhang, Antonello Bonci, Garrett Stuber and Luis de Lecea, we optogenetically drove well-defined dopamine neurons in the mouse in different temporal patterns during free behavior, and found parameters that were sufficient to drive reinforced behavior (for example, in the absence of any other cue or reward, healthy animals simply chose to spend more time in places where they had received particular kinds of optogenetic bursting activity in dopamine neurons). This work is relevant to hedonic (pleasure-related) pathologies involved in depression (as in my depressed patient who could no longer even enjoy seeing her grandchildren, otherwise one of the most rewarding experiences known to humankind) and in substance abuse, as well as in healthy reward processes.
The optogenetic approach has also improved our understanding of Parkinson's disease, which involves a disturbance of information processing in certain motor-control circuits of the brain. Since the 1990s some Parkinson's patients have received relief via a therapy called deep-brain stimulation, in which an implanted device similar to a pacemaker applies carefully timed, oscillating electric stimuli to certain areas far inside the brain, such as the subthalamic nucleus. Yet the promise of this technique for Parkinson's (and indeed for a variety of other conditions) is partially limited because electrodes stimulate nearby brain cells unselectively and medical understanding of what stimuli to apply is woefully incomplete. Recently, however, we have used optogenetics to study animal models of Parkinson's and gained fundamental insight into the nature of the diseased circuitry and the mechanisms of action of therapeutic interventions. For example, we have found that deep-brain stimulation may be most effective when it targets not cells but rather the connections between cells—affecting the flow of activity between brain regions. And we have worked with Anatol Kreitzer of U.C.S.F. who has functionally mapped two pathways in brain movement circuitry: one that slows movements and one that speeds them up and can counteract the parkinsonian state.
We have also learned how to prod one kind of cell, neocortical parvalbumin neurons, to modulate 40-cycles-per-second rhythms in brain activity called gamma oscillations. Science has known for some time that schizophrenic patients have altered parvalbumin cells and that gamma oscillations are abnormal in both schizophrenia and autism—but the causal meaning of these correlations (if any) was not known. Using optogenetics, Vikaas Sohal and Feng Zhang in my group (along with Li-Huei Tsai and Chris Moore at M.I.T. and our other collaborators) showed that parvalbumin cells, in cooperation with other cell types, serve to modulate gamma waves. Those waves in turn enhance the flow of information through cortical circuits. In my patients with schizophrenia I see what clearly appear to be information-processing problems, in which mundane random events are incorrectly viewed as parts of larger themes or patterns (an informational problem perhaps giving rise to paranoia and delusions). These patients may suffer from some failure of an internal "notification" mechanism that informs us when thoughts that are self-generated (an informational problem perhaps underlying the frightening phenomenon of "hearing voices"). Conversely in my patients with autism spectrum disease, rather than inappropriately broad linkages in information, I see overly restricted information processing: they miss the big picture by focusing too narrowly on just parts of objects, people, conversations and so on. These failures of information processing may lead to failures in communication and social behavior, and better understanding of brain rhythms has provided insights into these complex diseases.
As a physician, I find this work thrilling because we are bringing engineering principles and quantitative technology to bear on seemingly "fuzzy" but devastating and intractable psychiatric diseases. Optogenetics is thus helping to move psychiatry toward a network-engineering approach, in which the complex functions of the brain (and the behaviors it produces) are interpreted as properties of the neural system that emerge from the electrochemical dynamics of the component cells and circuits. It thus fundamentally changes our understanding of how electrically excitable tissues function in health and disease. We have come a long way from bacteriorhodopsin, indeed.
Bounty of the unexpected
Scientists spend a great deal of time thinking about not just their own laboratories and their own fields, but also more general questions about how science is conducted. At large and diverse scientific gatherings (such as the Society for Neuroscience annual meeting, with more than 30,000 attendees), I have occasionally heard colleagues overwhelmed by the diversity of the field take the devil's advocate position and suggest that it would be more efficient to focus tens of thousands of scientists on one massive and urgent project at a time—for example, eliminating Alzheimer's disease. Indeed, a common topic of conversation and policymaking in science considers the extent to which either diverse exploration or massively focused efforts should predominate. Even for small focused efforts, how much should funding sources and scientific organizations set the scientific agenda and guide investigators? There is no question that both directed and exploratory approaches have value, and as a practicing psychiatrist I fully appreciate the urgency for directed efforts. As they did for me, clinical need can and should drive and inspire basic science and engineering.
But how exactly should this inspiration be implemented? The story of optogenetics makes a strong argument. Even envisaging the concept of optical control of targeted neurons, as Crick did, did not predict the need for decades of pure basic research on microbial membranes. The bottom line is that there is no possible way one could have predicted the impact of archaeal or algal biology on our understanding of Parkinson's disease and its treatment. It is still too early in the exhilarating history of humanity's march toward understanding of the natural world.
The more directed and targeted research becomes, the more likely we are to slow our progress, and the more certain it is that the distant and untraveled realms, where truly disruptive ideas can arise, will be utterly cut off from our common scientific journey. Purely directed funding will adversely affect most seriously the fields most distant from human health (such as archaeal biology), but will even stultify fields clearly relevant to human health and disease, such as neuroscience. In a supreme irony, even if 30,000 scientists could be directed toward a common goal, the very act of focusing on that goal could ensure that the goal would not be reached.
The importance and urgency of these goals should not be underestimated. The benefits to our society and to global health of preserving a healthy community of undirected research will show up in many ways, ranging from insight into psychiatric disease to protections for the environment. Consider the importance of basic science—which could be so vulnerable to criticism when misrepresented—in the case of optogenetics, which could be cast in an unflattering light as money spent studying genes from pond scum. Consider the importance of preserving biodiversity, as in the harsh, barren and otherwise quite useless Saharan salt lakes from which some of the most useful opsins originated. Consider again the stigma of brain disease—so deeply tied to our lack of understanding.
In classic microbial opsin work, we find meaning for the modern world—not just for science, but also for medicine and psychiatry—that makes a strong and clear statement for environmental protection, for preservation of biodiversity and for the pure quest for understanding. And the journey of optogenetics shows that hidden within the ground we have already traveled over or passed by, there may reside the essential tools, shouldered aside by modernity, that will allow us to map our way forward. Sometimes these neglected or archaic tools are those that are most needed—the old, the rare, the small and the weak.
Some years ago (during a period of undirected reading) in the Annual Review of Biochemistry, I stumbled across Harvard biochemist Eugene Kennedy's reflections on his career—and the tension between past and future, between old age and renewal, and between the mortality of the human body and the immortality of art and science. Kennedy concluded, "The anonymity that is the fate of nearly every scientist as the work of one generation blends almost without a trace into that of the next is a small price to pay for its unending progress, the great long-march of human reason. To feel that one has contributed to this splendid enterprise, on however small a scale, is reward enough for labor at the end of a day." I would add to that magnificent sentiment only that the story of optogenetics recounted above sends a strong, clear message. In our quest to move the enterprise of science forward we should never forget that we do not know where this long march is taking us or what we will need to get there.
Optogenetics Resource Center
Minimally Invasive Brain Stimulation
Lectures on Microbial Opsin Optogenetics
Karl Deisseroth is a member of the bioengineering and psychiatry faculties at Stanford University. He is the 2010 International Nakasone Award laureate for his development of microbial opsins and optogenetics.