(Image caption: Archer1 fluorescence in a cultured rat hippocampal neuron. By monitoring changes in this fluorescence at up to a thousand frames per second, researchers can track the electrical activity of the cell. Credit: Nicholas Flytzanis, Claire Bedbrook and Viviana Gradinaru/Caltech)

Sensing Neuronal Activity With Light

For years, neuroscientists have been trying to develop tools that would allow them to clearly view the brain’s circuitry in action—from the first moment a neuron fires to the resulting behavior in a whole organism. To get this complete picture, neuroscientists are working to develop a range of new tools to study the brain. Researchers at Caltech have developed one such tool that provides a new way of mapping neural networks in a living organism.

The work—a collaboration between Viviana Gradinaru (BS ‘05), assistant professor of biology and biological engineering, and Frances Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry—was described in two separate papers published this month.

When a neuron is at rest, channels and pumps in the cell membrane maintain a cell-specific balance of positively and negatively charged ions within and outside of the cell resulting in a steady membrane voltage called the cell’s resting potential. However, if a stimulus is detected—for example, a scent or a sound—ions flood through newly open channels causing a change in membrane voltage. This voltage change is often manifested as an action potential—the neuronal impulse that sets circuit activity into motion.

The tool developed by Gradinaru and Arnold detects and serves as a marker of these voltage changes.

"Our overarching goal for this tool was to achieve sensing of neuronal activity with light rather than traditional electrophysiology, but this goal had a few prerequisites," Gradinaru says. "The sensor had to be fast, since action potentials happen in just milliseconds. Also, the sensor had to be very bright so that the signal could be detected with existing microscopy setups. And you need to be able to simultaneously study the multiple neurons that make up a neural network."

The researchers began by optimizing Archaerhodopsin (Arch), a light-sensitive protein from bacteria. In nature, opsins like Arch detect sunlight and initiate the microbes’ movement toward the light so that they can begin photosynthesis. However, researchers can also exploit the light-responsive qualities of opsins for a neuroscience method called optogenetics—in which an organism’s neurons are genetically modified to express these microbial opsins. Then, by simply shining a light on the modified neurons, the researchers can control the activity of the cells as well as their associated behaviors in the organism.

Gradinaru had previously engineered Arch for better tolerance and performance in mammalian cells as a traditional optogenetic tool used to control an organism’s behavior with light. When the modified neurons are exposed to green light, Arch acts as an inhibitor, controlling neuronal activity—and thus the associated behaviors—by preventing the neurons from firing.

However, Gradinaru and Arnold were most interested in another property of Arch: when exposed to red light, the protein acts as a voltage sensor, responding to changes in membrane voltages by producing a flash of light in the presence of an action potential. Although this property could in principle allow Arch to detect the activity of networks of neurons, the light signal marking this neuronal activity was often too dim to see.

To fix this problem, Arnold and her colleagues made the Arch protein brighter using a method called directed evolution—a technique Arnold originally pioneered in the early 1990s. The researchers introduced mutations into the Arch gene, thus encoding millions of variants of the protein. They transferred the mutated genes into E. coli cells, which produced the mutant proteins encoded by the genes. They then screened thousands of the resulting E. coli colonies for the intensities of their fluorescence. The genes for the brightest versions were isolated and subjected to further rounds of mutagenesis and screening until the bacteria produced proteins that were 20 times brighter than the original Arch protein.

A paper describing the process and the bright new protein variants that were created was published in the September 9 issue of the Proceedings of the National Academy of Science.

"This experiment demonstrates how rapidly these remarkable bacterial proteins can evolve in response to new demands. But even more exciting is what they can do in neurons, as Viviana discovered," says Arnold.

In a separate study led by Gradinaru’s graduate students Nicholas Flytzanis and Claire Bedbrook, who is also advised by Arnold, the researchers genetically incorporated the new, brighter Arch variants into rodent neurons in culture to see which of these versions was most sensitive to voltage changes—and therefore would be the best at detecting action potentials. One variant, Archer1, was not only bright and sensitive enough to mark action potentials in mammalian neurons in real time, it could also be used to identify which neurons were synaptically connected—and communicating with one another—in a circuit.

The work is described in a study published on September 15 in the journal Nature Communications.

"What was interesting is that we would see two cells over here light up, but not this one over there—because the first two are synaptically connected," Gradinaru says. "This tool gave us a way to observe a network where the perturbation of one cell affects another."

However, sensing activity in a living organism and correlating this activity with behavior remained the biggest challenge. To accomplish this goal Gradinaru’s team worked with Paul Sternberg, the Thomas Hunt Morgan Professor of Biology, to test Archer1 as a sensor in a living organism—the tiny nematode worm C. elegans. “There are a few reasons why we used the worms here: they are powerful organisms for quick genetic engineering and their tissues are nearly transparent, making it easy to see the fluorescent protein in a living animal,” she says.

After incorporating Archer1 into neurons that were a part of the worm’s olfactory system—a primary source of sensory information for C. elegans—the researchers exposed the worm to an odorant. When the odorant was present, a baseline fluorescent signal was seen, and when the odorant was removed, the researchers could see the circuit of neurons light up, meaning that these particular neurons are repressed in the presence of the stimulus and active in the absence of the stimulus. The experiment was the first time that an Arch variant had been used to observe an active circuit in a living organism.

Gradinaru next hopes to use tools like Archer1 to better understand the complex neuronal networks of mammals, using microbial opsins as sensing and actuating tools in optogenetically modified rodents.

"For the future work it’s useful that this tool is bifunctional. Although Archer1 acts as a voltage sensor under red light, with green light, it’s an inhibitor," she says. "And so now a long-term goal for our optogenetics experiments is to combine the tools with behavior-controlling properties and the tools with voltage-sensing properties. This would allow us to obtain all-optical access to neuronal circuits. But I think there is still a lot of work ahead."

One goal for the future, Gradinaru says, is to make Archer1 even brighter. Although the protein’s fluorescence can be seen through the nearly transparent tissues of the nematode worm, opaque organs such as the mammalian brain are still a challenge. More work, she says, will need to be done before Archer1 could be used to detect voltage changes in the neurons of living, behaving mammals.

And that will require further collaborations with protein engineers and biochemists like Arnold.

"As neuroscientists we often encounter experimental barriers, which open the potential for new methods. We then collaborate to generate tools through chemistry or instrumentation, then we validate them and suggest optimizations, and it just keeps going," she says. "There are a few things that we’d like to be better, and through these many iterations and hard work it can happen."

Noninvasive brain control

Optogenetics, a technology that allows scientists to control brain activity by shining light on neurons, relies on light-sensitive proteins that can suppress or stimulate electrical signals within cells. This technique requires a light source to be implanted in the brain, where it can reach the cells to be controlled.

MIT engineers have now developed the first light-sensitive molecule that enables neurons to be silenced noninvasively, using a light source outside the skull. This makes it possible to do long-term studies without an implanted light source. The protein, known as Jaws, also allows a larger volume of tissue to be influenced at once.

This noninvasive approach could pave the way to using optogenetics in human patients to treat epilepsy and other neurological disorders, the researchers say, although much more testing and development is needed. Led by Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT, the researchers described the protein in the June 29 issue of Nature Neuroscience.

Optogenetics, a technique developed over the past 15 years, has become a common laboratory tool for shutting off or stimulating specific types of neurons in the brain, allowing neuroscientists to learn much more about their functions.

The neurons to be studied must be genetically engineered to produce light-sensitive proteins known as opsins, which are channels or pumps that influence electrical activity by controlling the flow of ions in or out of cells. Researchers then insert a light source, such as an optical fiber, into the brain to control the selected neurons.

Such implants can be difficult to insert, however, and can be incompatible with many kinds of experiments, such as studies of development, during which the brain changes size, or of neurodegenerative disorders, during which the implant can interact with brain physiology. In addition, it is difficult to perform long-term studies of chronic diseases with these implants.

Mining nature’s diversity

To find a better alternative, Boyden, graduate student Amy Chuong, and colleagues turned to the natural world. Many microbes and other organisms use opsins to detect light and react to their environment. Most of the natural opsins now used for optogenetics respond best to blue or green light.

Boyden’s team had previously identified two light-sensitive chloride ion pumps that respond to red light, which can penetrate deeper into living tissue. However, these molecules, found in the bacteria Haloarcula marismortui and Haloarcula vallismortis, did not induce a strong enough photocurrent — an electric current in response to light — to be useful in controlling neuron activity.

Chuong set out to improve the photocurrent by looking for relatives of these proteins and testing their electrical activity. She then engineered one of these relatives by making many different mutants. The result of this screen, Jaws, retained its red-light sensitivity but had a much stronger photocurrent — enough to shut down neural activity.

“This exemplifies how the genomic diversity of the natural world can yield powerful reagents that can be of use in biology and neuroscience,” says Boyden, who is a member of MIT’s Media Lab and the McGovern Institute for Brain Research.

Using this opsin, the researchers were able to shut down neuronal activity in the mouse brain with a light source outside the animal’s head. The suppression occurred as deep as 3 millimeters in the brain, and was just as effective as that of existing silencers that rely on other colors of light delivered via conventional invasive illumination.

A key advantage to this opsin is that it could enable optogenetic studies of animals with larger brains, says Garret Stuber, an assistant professor of psychiatry and cell biology and physiology at the University of North Carolina at Chapel Hill.

“In animals with larger brains, people have had difficulty getting behavior effects with optogenetics, and one possible reason is that not enough of the tissue is being inhibited,” he says. “This could potentially alleviate that.”

Restoring vision

Working with researchers at the Friedrich Miescher Institute for Biomedical Research in Switzerland, the MIT team also tested Jaws’s ability to restore the light sensitivity of retinal cells called cones. In people with a disease called retinitis pigmentosa, cones slowly atrophy, eventually causing blindness.

Friedrich Miescher Institute scientists Botond Roska and Volker Busskamp have previously shown that some vision can be restored in mice by engineering those cone cells to express light-sensitive proteins. In the new paper, Roska and Busskamp tested the Jaws protein in the mouse retina and found that it more closely resembled the eye’s natural opsins and offered a greater range of light sensitivity, making it potentially more useful for treating retinitis pigmentosa.

This type of noninvasive approach to optogenetics could also represent a step toward developing optogenetic treatments for diseases such as epilepsy, which could be controlled by shutting off misfiring neurons that cause seizures, Boyden says. “Since these molecules come from species other than humans, many studies must be done to evaluate their safety and efficacy in the context of treatment,” he says.

Boyden’s lab is working with many other research groups to further test the Jaws opsin for other applications. The team is also seeking new light-sensitive proteins and is working on high-throughput screening approaches that could speed up the development of such proteins.

Shedding a light on pain: A technique developed by Stanford bioengineers could lead to new treatments

The mice in Scott Delp’s lab, unlike their human counterparts, can get pain relief from the glow of a yellow light.

Right now these mice are helping scientists to study pain – how and why it occurs and why some people feel it so intensely without any obvious injury. But Delp, a professor of bioengineering and mechanical engineering, hopes one day the work he does with these mice can also help people who are in chronic, debilitating pain.

"This is an entirely new approach to study a huge public health issue," Delp said. "It’s a completely new tool that is now available to neuroscientists everywhere." He is the senior author of a research paper published Feb. 16 in Nature Biotechnology.

A switch for pain

The mice are modified with gene therapy to have pain-sensing nerves that can be controlled by light. One color of light makes the mice more sensitive to pain. Another reduces pain. The scientists shone a light on the paws of mice through the Plexiglas bottom of the cage.

Graduate students Shrivats Iyer and Kate Montgomery, who led the study, say it opens the door to future experiments to understand the nature of pain and also touch and other sensations that are part of our daily lives but little understood.

"The fact that we can give a mouse an injection and two weeks later shine a light on its paw to change the way it senses pain is very powerful," Iyer said.

For example, increasing or decreasing the sensation of pain in these mice could help scientists understand why pain seems to continue in people after an injury has healed. Does persistent pain change those nerves in some way? If so, how can they be changed back to a state where, in the absence of an injury, they stop sending searing messages of pain to the brain?

Leaders at the National Institutes of Health agree that the work could have important implications for treating pain. “This powerful approach shows great potential for helping the millions who suffer pain from nerve damage,” said Linda Porter, the pain policy adviser at the National Institute of Neurological Disorders and Stroke and a leader of the NIH’s Pain Consortium.

"Now, with a flick of a switch, scientists may be able to rapidly test new pain-relieving medications and, one day, doctors may be able to use light to relieve pain," she said.

Accidental discovery

The researchers took advantage of a technique called optogenetics, which involves light-sensitive proteins called opsins that are inserted into the nerves. Optogenetics was developed by Delp’s colleague Karl Deisseroth, a co-author of the journal article. He has used the technique as a way of activating precise regions of the brain to better understand how the brain functions. Deisseroth is a professor of bioengineering, psychiatry and behavioral sciences.

Delp, who has an interest in muscles and movement, saw the potential for using optogenetics not just for studying the brain – interesting though those studies may be – but also for studying the many nerves outside the brain. These are the nerves that control movement, pain, touch and other sensations throughout our body, and that are involved in diseases such as amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s Disease.

A few years ago Stanford Bio-X, which encourages interdisciplinary projects such as this one, supported Delp and Deisseroth in their efforts to use optogenetics to control the nerves that excite muscles. In the process of doing that work, Delp said, his student at the time, Michael Llewellyn, occasionally found that he had placed the opsins into nerves that signal pain rather than those that control muscle.

That accident sparked a new line of research. Delp said, “We thought, ‘Wow, we’re getting pain neurons; that could be really important.’” He suggested that Montgomery and Iyer focus on those pain nerves that had been a byproduct of the muscle work.

A faster approach

A key component of the work was a new approach to quickly incorporate opsins into the nerves of mice. The researchers started with a virus that had been engineered to contain the DNA that produces the opsin. Then they injected those modified viruses directly into mouse nerves. Weeks later, only the nerves that control pain had incorporated the opsin proteins and would fire, or be less likely to fire, in response to different colors of light.

The speed of the viral approach makes it very flexible, both for this pain work and for future studies. Researchers are developing newer forms of opsins with different properties, such as responding to different colors of light. “Because we used a viral approach we could, in the future, quickly turn around and use newer opsins,” said Montgomery, who is a Stanford Bio-X fellow.

This entire project, which spans bioengineering, neuroscience and psychiatry, is one Delp says could never have happened without the environment at Stanford that supports collaboration across departments. The pain portion of the research came out of support from NeuroVentures, which was a project incubated within Bio-X to support the intersection of neuroscience and engineering or other disciplines. That project was so successful it has spun off into the Stanford Neurosciences Institute, of which Delp is now a deputy director.

Delp said that many challenges must be met before results of these experiments – either new drugs based on what they learn, or optogenetics directly – could become available to people but that he always has that as a goal.

"Developing a new therapy from the ground up would be incredibly rewarding," he said. "Most people don’t get to do that in their careers."

Delp and Deisseroth have started a company called Circuit Therapeutics to develop therapies based on optogenetics.

Optogenetic toolkit goes multicolor

Optogenetics is a technique that allows scientists to control neurons’ electrical activity with light by engineering them to express light-sensitive proteins. Within the past decade, it has become a very powerful tool for discovering the functions of different types of cells in the brain.

Most of these light-sensitive proteins, known as opsins, respond to light in the blue-green range. Now, a team led by MIT has discovered an opsin that is sensitive to red light, which allows researchers to independently control the activity of two populations of neurons at once, enabling much more complex studies of brain function.

“If you want to see how two different sets of cells interact, or how two populations of the same cell compete against each other, you need to be able to activate those populations independently,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT and a senior author of the new study.

The new opsin is one of about 60 light-sensitive proteins found in a screen of 120 species of algae. The study, which appears in the Feb. 9 online edition of Nature Methods, also yielded the fastest opsin, enabling researchers to study neuron activity patterns with millisecond timescale precision.

Boyden and Gane Ka-Shu Wong, a professor of medicine and biological sciences at the University of Alberta, are the paper’s senior authors, and the lead author is MIT postdoc Nathan Klapoetke. Researchers from the Howard Hughes Medical Institute’s Janelia Farm Research Campus, the University of Pennsylvania, the University of Cologne, and the Beijing Genomics Institute also contributed to the study.

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New study sheds light on how and when vision evolved

Opsins, the light-sensitive proteins key to vision, may have evolved earlier and undergone fewer genetic changes than previously believed, according to a new study from the National University of Ireland Maynooth and the University of Bristol published in Proceedings of the National Academy of Sciences (PNAS) .

The study, which used computer modelling to provide a detailed picture of how and when opsins evolved, sheds light on the origin of sight in animals, including humans. The evolutionary origins of vision remain hotly debated, partly due to inconsistent reports of phylogenetic relationships among the earliest opsin-possessing animals.

Dr Davide Pisani of Bristol’s School of Earth Sciences and colleagues at NUI Maynooth performed a computational analysis to test every hypothesis of opsin evolution proposed to date. The analysis incorporated all available genomic information from all relevant animal lineages, including a newly sequenced group of sponges (Oscarella carmela) and the Cnidarians, a group of animals thought to have possessed the world’s earliest eyes.

Using this information, the researchers developed a timeline with an opsin ancestor common to all groups appearing some 700 million years ago. This opsin was considered ‘blind’ yet underwent key genetic changes over the span of 11 million years that conveyed the ability to detect light.

Dr Pisani said: “The great relevance of our study is that we traced the earliest origin of vision and we found that it originated only once in animals. This is an astonishing discovery because it implies that our study uncovered, in consequence, how and when vision evolved in humans.”

(Image credit: Roland Bircher)