Research Area: Cellular & Molecular Neuroscience

​synapses, vesicles, electrophysiology, protein complexes, signaling, small molecules, disease-linked genes, dendrites, cell death, brain disorders, neurodevelopment

New way to turn genes on

Anne Trafton |

Using a gene-editing system originally developed to delete specific genes, MIT researchers have now shown that they can reliably turn on any gene of their choosing in living cells.

This new application for the CRISPR/Cas9 gene-editing system should allow scientists to more easily determine the function of individual genes, according to Feng Zhang, the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s Departments of Brain and Cognitive Sciences and Biological Engineering, and a member of the Broad Institute and MIT’s McGovern Institute for Brain Research.

This approach also enables rapid functional screens of the entire genome, allowing scientists to identify genes involved in particular diseases. In a study published in the Dec. 10 online edition of Nature, Zhang and colleagues identified several genes that help melanoma cells become resistant to a cancer drug.

Silvana Konermann, a graduate student in Zhang’s lab, and Mark Brigham, a McGovern Institute postdoc, are the paper’s lead authors.

A new function for CRISPR

The CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have previously harnessed this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

In the past two years, scientists have developed Cas9 as a tool for turning genes off or replacing them with a different version. In the new study, Zhang and colleagues engineered the Cas9 system to turn genes on, rather than knock them out. Scientists have tried to do this before using proteins that are individually engineered to target DNA at specific sites. However, these proteins are  difficult to work with. “If you use the older generation of tools, getting the technology to do what you actually want is a project on its own,” Konermann says. “It takes a lot of time and is also quite expensive.”

There have also been attempts to use CRISPR to turn on genes by inactivating the part of the Cas9 enzyme that cuts DNA and linking Cas9 to pieces of proteins called activation domains. These domains recruit the cellular machinery necessary to begin reading copying RNA from DNA, a process known as transcription.

However, these efforts have been unable to consistently turn on gene transcription. Zhang and his colleagues, Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, decided to overhaul the CRISPR-Cas9 system based on an analysis they published earlier this year of the structure formed when Cas9 binds to the guide RNA and its target DNA. “Based on knowing its 3-D shape, we can think about how to rationally improve the system,” Zhang says.

In previous efforts, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

Genome-scale activation screening

Once the researchers had shown that the system was effective at activating genes, they created a library of 70,290 guide RNAs targeting all of the more than 20,000 genes in the human genome.

They screened this library to identify genes that confer resistance to a melanoma drug called PLX-4720. Drugs of this type work well in patients whose melanoma cells have a mutation in the BRAF gene, but cancer cells that survive the treatment can grow into new tumors, allowing the cancer to recur.

To discover the genes that help cells become resistant, the researchers delivered CRISPR components to a large population of melanoma cells grown in the lab, with each cell receiving a different guide RNA targeting a different gene. After treating the cells with PLX-4720, they identified several genes that helped the cells to survive — some previously known to be involved in drug resistance, as well as several novel targets.
Studies like this could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

“You could start with a drug that targets the mutated BRAF along with combination therapy that targets genes that allow the cell to survive. If you target both of them at the same time, you could likely prevent the cells from developing resistance mechanisms that enable further growth despite drug treatment,” Konermann says.

Scientists have tried to do large-scale screens like this by delivering single genes carried by viruses, but that does not work with all genes.

“This new technique could allow you to sample a larger spectrum of genes that might be playing a role,” says Levi Garraway, an associate professor of medicine at Dana-Farber Cancer Institute who was not involved in the research. “This is really a technology development paper, but the tantalizing results from the drug resistance screen speak to the rich biological possibilities of this approach.”

Zhang’s lab also plans to use this technique to screen for genes that, when activated, could correct the effects of autism or neurodegenerative diseases such as Alzheimer’s. He also plans to make the necessary reagents available to academic labs that want to use them, through the Addgene repository.

The research was funded by the National Institute of Mental Health; the National Institute of Neurological Disorders and Stroke; the Keck, Searle Scholars, Klingenstein, Vallee, and Simons foundations; and Bob Metcalfe.

From genes to brains

by Elizabeth Dougherty |

Many brain disorders are strongly influenced by genetics, and researchers have long hoped that the identification of genetic risk factors will provide clues to the causes and possible treatments of these mysterious conditions. In the early years, progress was slow. Many claims failed to replicate, and it became clear that in order to identify the important risk genes with confidence, researchers would need to examine the genomes of very large numbers of patients.

Until recently that would have been prohibitively expensive, but genome research has been accelerating fast. Just how fast was underlined by an announcement in January from a California-based company, Illumina, that it had achieved a long-awaited milestone: sequencing an entire human genome for under $1000. Seven years ago, this task would have cost $10M and taken weeks of work. The new system does the job in a few hours, and can sequence tens of thousands of genomes per year.

In parallel with these spectacular advances, another technological revolution has been unfolding over the past several years, with the development of a new method for editing the genome of living cells. This method, known as CRISPR, allows researchers to make precise changes to a DNA sequence—an advance that is expected to transform many areas of biomedical research and may ultimately form the basis of new treatments for human genetic disease.

The CRISPR technology, which is based on a natural bacterial defense system against viruses, uses a short strand of RNA as a “search string” to locate a corresponding DNA target sequence. This RNA string can be synthesized in the lab and can be designed to recognize any desired sequence of DNA. The RNA carries with it a protein called Cas9, which cuts the target DNA at the chosen location, allowing a new sequence to be inserted—providing researchers with a fast and flexible “search-and-replace” tool for editing the genome.

One of the pioneers in this field is McGovern Investigator Feng Zhang, who along with George Church of Harvard, was the first to show that CRISPR could be used to edit the human genome in living cells. Zhang is using the technology to study human brain disorders, building on the flood of new genetic discoveries that are emerging from advances in DNA sequencing. The Broad Institute, where Zhang holds a joint appointment, is a world leader in human psychiatric genetics, and will be among the first to acquire the new Illumina sequencing machines when they reach the market later this year.

By sequencing many thousands of individuals, geneticists are identifying the rare genetic variants that contribute to risk of diseases such as autism, schizophrenia and bipolar disorder. CRISPR will allow neuroscientists to study those gene variants in cells and in animal models. The goal, says McGovern Institute director Bob Desimone, is to understand the biological roots of brain disorders. “The biggest obstacle to new treatments has been our ignorance of fundamental mechanisms. But with these new technologies, we have a real opportunity to understand what’s wrong at the level of cells and circuits, and to identify the pressure points at which therapeutic intervention may be possible.”

Culture Club

In other fields, the influence of genetic variations on disease has turned out to be surprisingly difficult to unravel, and for neuropsychiatric disease, the challenge may be even greater. The brain is the most complex organ of the body, and the underlying pathologies that lead to disease are not yet well understood. Moreover, any given disorder may show a wide variation in symptoms from patient to patient, and it may also have many different genetic causes. “There are hundreds of genes that can contribute to autism or schizophrenia,” says McGovern Investigator Guoping Feng, who is also Poitras Professor of Neuroscience.

To study these genes, Feng and collaborators at the Broad Institute’s Stanley Center for Psychiatric Research are planning to screen thousands of cultures of neurons, grown in the tiny wells of cell culture plates. The neurons, which are grown from stem cells, can be engineered using CRISPR to contain the genetic variants that are linked to neuropsychiatric disease. Each culture will contain neurons with a different variant, and these will be examined for abnormalities that might be associated with disease.

Feng and colleagues hope this high-throughput platform will allow them to identify cellular traits, or phenotypes, that may be related to disease and which can then be studied in animal models to see if they cause defects in brain function or in behavior. In the longer term, this high-throughput platform can also be used to screen for new drugs that can reverse these defects.

Animal Kingdom

Cell cultures are necessary for large-scale screens, but ultimately the results must be translated into the context of brain circuits and behavior. “That means we must study animal models too,” says Feng.

Feng has created several mouse models of human brain disease by mutating genes that are linked to these disorders and examining the behavioral and cellular defects in the mutant animals. “We have models of obsessive-compulsive disorder and autism,” he explains. “By studying these mice we want to learn what’s wrong with their brains.”

So far, Feng has focused on single-gene models, but the majority of human psychiatric disorders are triggered by multiple genes acting in combination. One advantage of the new CRISPR method is that it allows researchers to introduce several mutations in parallel, and Zhang’s lab is now working to create autistic mice with more than one gene alteration.

Perhaps the most important advantage of CRISPR is that it can be applied to any species. Currently, almost all genetic modeling of human disease is restricted to mice. But while mouse models are convenient, they are limited, especially for diseases that affect higher brain functions and for which there are no clear parallels in rodents. “We also need to study species that are closer to humans,” says Feng.

Accordingly, he and Zhang are collaborating with colleagues in Oregon and China to use CRISPR to create primate models of neuropsychiatric disorders. Earlier this year, a team in China announced that they had used CRISPR to create transgenic monkeys that will be used to study defects in metabolism and immunity.

Feng and Zhang are planning to use a similar approach to study brain disorders, but in addition to macacques, they will also work with a smaller primate species, the marmoset. These animals, with their fast breeding cycles and complex behavioral repertoires, are ideal for genetic studies of behavior and brain function. And because they are very social with highly structured communication patterns, they represent a promising new model for understanding the neural basis of social cognition and its disruption in conditions such as autism.

Given their close evolutionary relationship to humans, marmoset models could also help accelerate the development of new therapies. Many experimental drugs for brain disorders have been tested successfully in mice, only to prove ineffective in subsequent human trials. These failures, which can be enormously expensive, have led many drug companies to cut back on their neuroscience R&D programs. Better animal models could reverse this trend by allowing companies to predict more accurately which drug candidates are most promising, before investing heavily in human clinical trials.

Feng’s mouse research provides an example of how this approach can work. He previously developed a mouse model of obsessive-compulsive disorder, in which the animals engage in obsessive self-grooming, and he has now shown that this effect can be reversed when the missing gene is reintroduced, even in adulthood. Other researchers have seemed similar results with other brain disorders such as Rett Syndrome, a condition that is often accompanied by autism. “The brain is amazingly plastic,” says Feng. “At least in a mouse, we have shown that the damage can often be repaired. If we can also show this in marmosets or other primate models, that would really give us hope that something similar is possible in humans.”

Human Race

Ultimately, to understand the genetic roots of human behavior, researchers must sequence the genomes of individual subjects in parallel with measurements of those same individuals’ behavior and brain function.

Such studies typically require very large sample sizes, but the plummeting cost of sequencing is now making this feasible. In China, for instance, a project is already underway to sequence the genomes of many thousands of individuals to uncover genetic influences on cognition and intelligence.

The next step will be to link the genetics to brain activity, says McGovern Investigator John Gabrieli, who also directs the Martinos Imaging Center at MIT. “It’s a big step to go from DNA to behavioral variation or clinical diagnosis. But we know those genes must affect brain function, so neuroimaging may help us to bridge that gap.”

But brain scans can be time-consuming, given that volunteers must perform behavioral tasks in the scanner. Studies are typically limited to a few dozen subjects, not enough to detect the often subtle effects of genomic variation.

One way to enlarge these studies, says Gabrieli, is to image the brain during rest rather than in a state of prompted activity. This procedure is fast and easy to replicate from lab to lab, and patterns of resting state activity have turned out to be surprisingly reproducible; moreover, Gabrieli is finding that differences in resting activity are associated with brain disorders such as autism, and he hopes that in the future it will be possible to relate these differences to the genetic factors that are emerging from genome studies at the Broad Institute and elsewhere.

“I’m optimistic that we’re going to see dramatic advances in our understanding of neuropsychiatric disease over the next few years.” — Bob Desimone

Confirming these associations will require a “big data” approach, in which results from multiple labs are consolidated into large repositories and analyzed for significant associations. Resting state imaging lends itself to this approach, says Gabrieli. “To find the links between brain function and genetics, big data is the direction we need to go to be successful.”

How soon might this happen? “It won’t happen overnight,” cautions Desimone. “There are a lot of dots that need to be connected. But we’ve seen in the case of genome research how fast things can move once the right technologies are in place. I’m optimistic that we’re going to see equally dramatic advances in our understanding of neuropsychiatric disease over the next few years.”

Fifteen MIT scientists receive NIH BRAIN Initiative grants

Anne Trafton |

Today, the National Institutes of Health (NIH) announced their first round of BRAIN Initiative award recipients. Six teams and 15 researchers from the Massachusetts Institute of Technology were recipients.

Mriganka Sur, principal investigator at the Picower Institute for Learning and Memory and the Paul E. Newton Professor of Neuroscience in MIT’s Department of Brain and Cognitive Sciences (BCS) leads a team studying cortical circuits and information flow during memory-guided perceptual decisions. Co-principal investigators include Emery Brown, BCS professor of computational neuroscience and the Edward Hood Taplin Professor of Medical Engineering; Kwanghun Chung, Picower Institute principal investigator and assistant professor in the Department of Chemical Engineering and the Institute for Medical Engineering and Science (IMES); and Ian Wickersham, research scientist at the McGovern Institute for Brain Research and head of MIT’s Genetic Neuroengineering Group.

Elly Nedivi, Picower Institute principal investigator and professor in BCS and the Department of Biology, leads a team studying new methods for high-speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Her co-principal investigator is Peter So, professor of mechanical and biological engineering, and director of the MIT Laser Biomedical Research Center.

Ian Wickersham will lead a team looking at novel technologies for nontoxic transsynaptic tracing. His co-principal investigators include Robert Desimone, director of the McGovern Institute and the Doris and Don Berkey Professor of Neuroscience in BCS; Li-Huei Tsai, director of the Picower Institute and the Picower Professor of Neuroscience in BCS; and Kay Tye, Picower Institute principal investigator and assistant professor of neuroscience in BCS.

Robert Desimone will lead a team studying vascular interfaces for brain imaging and stimulation. Co-principal investigators include Ed Boyden, associate professor at the MIT Media Lab, McGovern Institute, and departments of BCS and Biological Engineering; head of MIT’s Synthetic Neurobiology Group, and co-director of MIT’s Center for Neurobiological Engineering; and Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology in IMES and director of the Harvard-MIT Biomedical Engineering Center. Collaborators on this project include: Rodolfo Llinas (New York University), George Church (Harvard University), Jan Rabaey (University of California at Berkeley), Pablo Blinder (Tel Aviv University), Eric Leuthardt (Washington University/St. Louis), Michel Maharbiz (Berkeley), Jose Carmena (Berkeley), Elad Alon (Berkeley), Colin Derdeyn (Washington University in St. Louis), Lowell Wood (Bill and Melinda Gates Foundation), Xue Han (Boston University), and Adam Marblestone (MIT).

Ed Boyden will be co-principal investigator with Mark Bathe, associate professor of biological engineering, and Peng Yin of Harvard on a project to study ultra-multiplexed nanoscale in situ proteomics for understanding synapse types.

Alan Jasanoff, associate professor of biological engineering and director of the MIT Center for Neurobiological Engineering, will lead a team looking at calcium sensors for molecular fMRI. Stephen Lippard, the Arthur Amos Noyes Professor of Chemistry, is co-principal investigator.

In addition, Sur and Wickersham also received BRAIN Early Concept Grants for Exploratory Research (EAGER) from the National Science Foundation (NSF). Sur will focus on massive-scale multi-area single neuron recordings to reveal circuits underlying short-term memory. Wickersham, in collaboration with Li-Huei Tsai, Kay Tye, and Robert Desimone, will develop cell-type specific optogenetics in wild-type animals. Additional information about NSF support of the BRAIN initiative can be found at NSF.gov/brain.

The BRAIN Initiative, spearheaded by President Obama in April 2013, challenges the nation’s leading scientists to advance our sophisticated understanding of the human mind and discover new ways to treat, prevent, and cure neurological disorders like Alzheimer’s, schizophrenia, autism, and traumatic brain injury. The scientific community is charged with accelerating the invention of cutting-edge technologies that can produce dynamic images of complex neural circuits and illuminate the interaction of lightning-fast brain cells. The new capabilities are expected to provide greater insights into how brain functionality is linked to behavior, learning, memory, and the underlying mechanisms of debilitating disease. BRAIN was launched with approximately $100 million in initial investments from the NIH, the National Science Foundation, and the Defense Advanced Research Projects Agency (DARPA).

BRAIN Initiative scientists are engaged in a challenging and transformative endeavor to explore how our minds instantaneously processes, store, and retrieve vast quantities of information. Their discoveries will unlock many of the remaining mysteries inherent in the brain’s billions of neurons and trillions of connections, leading to a deeper understanding of the underlying causes of many neurological and psychiatric conditions. Their findings will enable scientists and doctors to develop the groundbreaking arsenal of tools and technologies required to more effectively treat those suffering from these devastating disorders.

Noninvasive brain control

Anne Trafton |

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.

The research at MIT was funded by Jerry and Marge Burnett, the Defense Advanced Research Projects Agency, the Human Frontiers Science Program, the IET A. F. Harvey Prize, the Janet and Sheldon Razin ’59 Fellowship of the MIT McGovern Institute, the New York Stem Cell Foundation-Robertson Investigator Award, the National Institutes of Health, the National Science Foundation, and the Wallace H. Coulter Foundation.

Illuminating neuron activity in 3-D

Anne Trafton |

Researchers at MIT and the University of Vienna have created an imaging system that reveals neural activity throughout the brains of living animals. This technique, the first that can generate 3-D movies of entire brains at the millisecond timescale, could help scientists discover how neuronal networks process sensory information and generate behavior.

The team used the new system to simultaneously image the activity of every neuron in the worm Caenorhabditis elegans, as well as the entire brain of a zebrafish larva, offering a more complete picture of nervous system activity than has been previously possible.

“Looking at the activity of just one neuron in the brain doesn’t tell you how that information is being computed; for that, you need to know what upstream neurons are doing. And to understand what the activity of a given neuron means, you have to be able to see what downstream neurons are doing,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT and one of the leaders of the research team. “In short, if you want to understand how information is being integrated from sensation all the way to action, you have to see the entire brain.”

The new approach, described May 18 in Nature Methods, could also help neuroscientists learn more about the biological basis of brain disorders. “We don’t really know, for any brain disorder, the exact set of cells involved,” Boyden says. “The ability to survey activity throughout a nervous system may help pinpoint the cells or networks that are involved with a brain disorder, leading to new ideas for therapies.”

Boyden’s team developed the brain-mapping method with researchers in the lab of Alipasha Vaziri of the University of Vienna and the Research Institute of Molecular Pathology in Vienna. The paper’s lead authors are Young-Gyu Yoon, a graduate student at MIT, and Robert Prevedel, a postdoc at the University of Vienna.

High-speed 3-D imaging

Neurons encode information — sensory data, motor plans, emotional states, and thoughts — using electrical impulses called action potentials, which provoke calcium ions to stream into each cell as it fires. By engineering fluorescent proteins to glow when they bind calcium, scientists can visualize this electrical firing of neurons. However, until now there has been no way to image this neural activity over a large volume, in three dimensions, and at high speed.

Scanning the brain with a laser beam can produce 3-D images of neural activity, but it takes a long time to capture an image because each point must be scanned individually. The MIT team wanted to achieve similar 3-D imaging but accelerate the process so they could see neuronal firing, which takes only milliseconds, as it occurs.

The new method is based on a widely used technology known as light-field imaging, which creates 3-D images by measuring the angles of incoming rays of light. Ramesh Raskar, an associate professor of media arts and sciences at MIT and an author of this paper, has worked extensively on developing this type of 3-D imaging. Microscopes that perform light-field imaging have been developed previously by multiple groups. In the new paper, the MIT and Austrian researchers optimized the light-field microscope, and applied it, for the first time, to imaging neural activity.

With this kind of microscope, the light emitted by the sample being imaged is sent through an array of lenses that refracts the light in different directions. Each point of the sample generates about 400 different points of light, which can then be recombined using a computer algorithm to recreate the 3-D structure.

“If you have one light-emitting molecule in your sample, rather than just refocusing it into a single point on the camera the way regular microscopes do, these tiny lenses will project its light onto many points. From that, you can infer the three-dimensional position of where the molecule was,” says Boyden, who is a member of MIT’s Media Lab and McGovern Institute for Brain Research.

Prevedel built the microscope, and Yoon devised the computational strategies that reconstruct the 3-D images.

Aravinthan Samuel, a professor of physics at Harvard University, says this approach seems to be an “extremely promising” way to speed up 3-D imaging of living, moving animals, and to correlate their neuronal activity with their behavior. “What’s very impressive about it is that it is such an elegantly simple implementation,” says Samuel, who was not part of the research team. “I could imagine many labs adopting this.”

Neurons in action

The researchers used this technique to image neural activity in the worm C. elegans, the only organism for which the entire neural wiring diagram is known. This 1-millimeter worm has 302 neurons, each of which the researchers imaged as the worm performed natural behaviors, such as crawling. They also observed the neuronal response to sensory stimuli, such as smells.

The downside to light field microscopy, Boyden says, is that the resolution is not as good as that of techniques that slowly scan a sample. The current resolution is high enough to see activity of individual neurons, but the researchers are now working on improving it so the microscope could also be used to image parts of neurons, such as the long dendrites that branch out from neurons’ main bodies. They also hope to speed up the computing process, which currently takes a few minutes to analyze one second of imaging data.

The researchers also plan to combine this technique with optogenetics, which enables neuronal firing to be controlled by shining light on cells engineered to express light-sensitive proteins. By stimulating a neuron with light and observing the results elsewhere in the brain, scientists could determine which neurons are participating in particular tasks.

Other co-authors at MIT include Nikita Pak, a PhD student in mechanical engineering, and Gordon Wetzstein, a research scientist at the Media Lab. The work at MIT was funded by the Allen Institute for Brain Science; the National Institutes of Health; the MIT Synthetic Intelligence Project; the IET Harvey Prize; the National Science Foundation (NSF); the New York Stem Cell Foundation-Robertson Award; Google; the NSF Center for Brains, Minds, and Machines at MIT; and Jeremy and Joyce Wertheimer.

Optogenetic toolkit goes multicolor

Anne Trafton |

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, a member of the McGovern Institute for Brain Research 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.

In living color

Opsins occur naturally in many algae and bacteria, which use the light-sensitive proteins to help them respond to their environment and generate energy.

To achieve optical control of neurons, scientists engineer brain cells to express the gene for an opsin, which transports ions across the cell’s membrane to alter its voltage. Depending on the opsin used, shining light on the cell either lowers the voltage and silences neuron firing, or boosts voltage and provokes the cell to generate an electrical impulse. This effect is nearly instantaneous and easily reversible.

Using this approach, researchers can selectively turn a population of cells on or off and observe what happens in the brain. However, until now, they could activate only one population at a time, because the only opsins that responded to red light also responded to blue light, so they couldn’t be paired with other opsins to control two different cell populations.

To seek additional useful opsins, the MIT researchers worked with Wong’s team at the University of Alberta, which is sequencing the transcriptomes of 1,000 plants, including some algae. (The transcriptome is similar to the genome but includes only the genes that are expressed by a cell, not the entirety of its genetic material.)

Once the team obtained genetic sequences that appeared to code for opsins, Klapoetke tested their light-responsiveness in mammalian brain tissue, working with Martha Constantine-Paton, a professor of brain and cognitive sciences and of biology, a member of the McGovern Institute for Brain Research at MIT, and also an author of the paper. The red-light-sensitive opsin, which the researchers named Chrimson, can mediate neural activity in response to light with a 735-nanometer
wavelength.

The researchers also discovered a blue-light-driven opsin that has two highly desirable traits: It operates at high speed, and it is sensitive to very dim light. This opsin, called Chronos, can be stimulated with levels of blue light that are too weak to activate Chrimson.

“You can use short pulses of dim blue light to drive the blue one, and you can use strong red light to drive Chrimson, and that allows you to do true two-color, zero-cross-talk activation in intact brain tissue,” says Boyden, who is a member of MIT’s Media Lab and an associate professor of biological engineering and brain and cognitive sciences at MIT.

Researchers had previously tried to modify naturally occurring opsins to make them respond faster and react to dimmer light, but trying to optimize one feature often made other features worse.

“It was apparent that when trying to engineer traits like color, light sensitivity, and kinetics, there are always tradeoffs,” Klapoetke says. “We’re very lucky that something natural actually was more than several times faster and also five or six times more light-sensitive than anything else.”

Selective control

These new opsins lend themselves to several types of studies that were not possible before, Boyden says. For one, scientists could not only manipulate activity of a cell population of interest, but also control upstream cells that influence the target population by secreting neurotransmitters.

Pairing Chrimson and Chronos could also allow scientists to study the functions of different types of cells in the same microcircuit within the brain. Such cells are usually located very close together, but with the new opsins they can be controlled independently with two different colors of light.

“I think the tools described in this excellent paper represent a major advance for both basic and translational neuroscience,” says Botond Roska, a senior group leader at the Friedrich Miescher Institute for Biomedical Research in Switzerland, who was not part of the research team. “Optogenetic tools that are shifted towards the infrared range, such as Chrimson described in this paper, are much better than the more blue-shifted variants since these are less toxic, activate less the pupillary reflex, and activate less the remaining photoreceptors of patients.”

Most optogenetic studies thus far have been done in mice, but Chrimson could be used for optogenetic studies of fruit flies, a commonly used experimental organism. Researchers have had trouble using blue-light-sensitive opsins in fruit flies because the light can get into the flies’ eyes and startle them, interfering with the behavior being studied.

Vivek Jayaraman, a research group leader at Janelia Farms and an author of the paper, was able to show that this startle response does not occur when red light is used to stimulate Chrimson in fruit flies.

Because red light is less damaging to tissue than blue light, Chrimson also holds potential for eventual therapeutic use in humans, Boyden says. Animal studies with other opsins have shown promise in helping to restore vision after the loss of photoreceptor cells in the retina.

The researchers are now trying to modify Chrimson to respond to light in the infrared range. They are also working on making both Chrimson and Chronos faster and more light sensitive.

MIT’s portion of the project was funded by the National Institutes of Health, the MIT Media Lab, the National Science Foundation, the Wallace H. Coulter Foundation, the Alfred P. Sloan Foundation, a NARSAD Young Investigator Grant, the Human Frontiers Science Program, an NYSCF Robertson Neuroscience Investigator Award, the IET A.F. Harvey Prize, Janet and Sheldon Razin ’59, and the Skolkovo Institute of Science and Technology.

Controlling genes with light

Anne Trafton |

Although human cells have an estimated 20,000 genes, only a fraction of those are turned on at any given time, depending on the cell’s needs — which can change by the minute or hour. To find out what those genes are doing, researchers need tools that can manipulate their status on similarly short timescales.

That is now possible, thanks to a new technology developed at MIT and the Broad Institute that can rapidly start or halt the expression of any gene of interest simply by shining light on the cells.

The work is based on a technique known as optogenetics, which uses proteins that change their function in response to light. In this case, the researchers adapted the light-sensitive proteins to either stimulate or suppress the expression of a specific target gene almost immediately after the light comes on.

“Cells have very dynamic gene expression happening on a fairly short timescale, but so far the methods that are used to perturb gene expression don’t even get close to those dynamics. To understand the functional impact of those gene-expression changes better, we have to be able to match the naturally occurring dynamics as closely as possible,” says Silvana Konermann, an MIT graduate student in brain and cognitive sciences.

The ability to precisely control the timing and duration of gene expression should make it much easier to figure out the roles of particular genes, especially those involved in learning and memory. The new system can also be used to study epigenetic modifications — chemical alterations of the proteins that surround DNA — which are also believed to play an important role in learning and memory.

Konermann and Mark Brigham, a graduate student at Harvard University, are the lead authors of a paper describing the technique in the July 22 online edition of Nature. The paper’s senior author is Feng Zhang, the W. M. Keck Career Development Professor in Biomedical Engineering at MIT and a core member of the Broad Institute and MIT’s McGovern Institute for Brain Research.

Shining light on genes

The new system consists of several components that interact with each other to control the copying of DNA into messenger RNA (mRNA), which carries genetic instructions to the rest of the cell. The first is a DNA-binding protein known as a transcription activator-like effector (TALE). TALEs are modular proteins that can be strung together in a customized way to bind any DNA sequence.

Fused to the TALE protein is a light-sensitive protein called CRY2 that is naturally found in Arabidopsis thaliana, a small flowering plant. When light hits CRY2, it changes shape and binds to its natural partner protein, known as CIB1. To take advantage of this, the researchers engineered a form of CIB1 that is fused to another protein that can either activate or suppress gene copying.

After the genes for these components are delivered to a cell, the TALE protein finds its target DNA and wraps around it. When light shines on the cells, the CRY2 protein binds to CIB1, which is floating in the cell. CIB1 brings along a gene activator, which initiates transcription, or the copying of DNA into mRNA. Alternatively, CIB1 could carry a repressor, which shuts off the process.

A single pulse of light is enough to stimulate the protein binding and initiate DNA copying.

The researchers found that pulses of light delivered every minute or so are the most effective way to achieve continuous transcription for the desired period of time. Within 30 minutes of light delivery, the researchers detected an uptick in the amount of mRNA being produced from the target gene. Once the pulses stop, the mRNA starts to degrade within about 30 minutes.

In this study, the researchers tried targeting nearly 30 different genes, both in neurons grown in the lab and in living animals. Depending on the gene targeted and how much it is normally expressed, the researchers were able to boost transcription by a factor of two to 200.




Epigenetic modifications



An important element of gene-expression control is epigenetic modification. One major class of epigenetic effectors is chemical modification of the proteins, known as histones, that anchor chromosomal DNA and control access to the underlying genes. The researchers showed that they can also alter these epigenetic modifications by fusing TALE proteins with histone modifiers.

Epigenetic modifications are thought to play a key role in learning and forming memories, but this has not been very well explored because there are no good ways to disrupt the modifications, short of blocking histone modification of the entire genome. The new technique offers a much more precise way to interfere with modifications of individual genes.

“We want to allow people to prove the causal role of specific epigenetic modifications in the genome,” Zhang says.

So far, the researchers have demonstrated that some of the histone effector domains can be tethered to light-sensitive proteins; they are now trying to expand the types of histone modifiers they can incorporate into the system.

“It would be really useful to expand the number of epigenetic marks that we can control. At the moment we have a successful set of histone modifications, but there are a good deal more of them that we and others are going to want to be able to use this technology for,” Brigham says.

The research was funded by a Hubert Schoemaker Fellowship; a National Institutes of Health Transformative R01 Award; an NIH Director’s Pioneer Award; the Keck, McKnight, Vallee, Damon Runyon, Searle Scholars, Klingenstein and Simons foundations; and Bob Metcalfe and Jane Pauley.

Optogenetics: A Light Switch for Neurons

by Julie Pryor |

This animation illustrates optogenetics — a radical new technology for controlling brain activity with light. Ed Boyden, the co-inventor of this technology, continues to develop new technologies for controlling brain activity.

Re-creating autism, in mice

Anne Trafton |

By mutating a single gene, researchers at MIT and Duke have produced mice with two of the most common traits of autism — compulsive, repetitive behavior and avoidance of social interaction.

They further showed that this gene, which is also implicated in many cases of human autism, appears to produce autistic behavior by interfering with communication between brain cells. The finding, reported in the March 20 online edition of Nature, could help researchers find new pathways for developing drugs to treat autism, says senior author Guoping Feng, professor of brain and cognitive sciences and member of the McGovern Institute for Brain Research at MIT.

About one in 110 children in the United States has an autism spectrum disorder, which can range in severity and symptoms but usually includes difficulties with language in addition to social avoidance and repetitive behavior. There are currently no effective drugs to treat autism, but the new finding could help uncover new drug targets, Feng says.

“We now have a very robust model with a known cause for autistic-like behaviors. We can figure out the neural circuits responsible for these behaviors, which could lead to novel targets for treatment,” he says.

The new mouse model also gives researchers a new way to test potential autism drugs before trying them in human patients.

A genetic disorder

In the past 10 years, large-scale genetic studies have identified hundreds of gene mutations that occur more frequently in autistic patients than in the general population. However, each patient has only one or a handful of those mutations, making it difficult to develop drugs against the disease.

In this study, the researchers focused on one of the most common of those genes, known as Shank3. The protein encoded by Shank3 is found in synapses — the junctions between brain cells that allow them to communicate with each other. Feng, who joined MIT and the McGovern Institute last year, began studying Shank3 a few years ago because he thought that synaptic proteins might contribute to autism and similar brain disorders, such as obsessive compulsive disorder.

At a synapse, one cell sends messages by releasing chemicals called neurotransmitters, which interact with the cell receiving the signal (known as the postsynaptic cell). This signal provokes the postsynaptic cell to alter its activity in some way — for example, turning a gene on or off. Shank3 is a “scaffold” protein, meaning that it helps to organize the hundreds of other proteins clustered on the postsynaptic cell membrane, which are necessary to coordinate the cell’s response to synaptic signals.

Feng targeted Shank3 because it is found primarily in a part of the brain called the striatum, which is involved in motor activity, decision-making and the emotional aspects of behavior. Malfunctions in the striatum are associated with several brain disorders, including autism and OCD. Feng theorized that those disorders might be caused by faulty synapses.

In a 2007 study, Feng showed that another postsynaptic protein found in the striatum, Sapap3, can cause OCD-like behavior in mice when mutated.

Communication problems

In the new Nature study, Feng and his colleagues found that Shank3 mutant mice showed compulsive behavior (specifically, excessive grooming) and avoidance of social interaction. “They’re just not interested in interacting with other mice,” he says.

The study, funded in part by the Simons Foundation Autism Research Initiative, offers the first direct evidence that mutations in Shank3 produce autistic-like behavior.

Guy Rouleau, professor of medicine at the University of Montreal, says the mouse model should give autism researchers a chance to understand the disease better and potentially develop new treatments. “It looks like this is going to be a good model that will be used to explore, more deeply, the physiology of the disorder,” says Rouleau, who was not involved in this research.

Even though only a small percentage of autistic patients have mutations in Shank3, Feng believes that many other cases may be caused by disruptions of other synaptic proteins. He is now doing a study, with researchers from the Broad Institute, to determine whether mutations in a group of other synaptic genes are associated with autism in human patients.

If that turns out to be the case, it should be possible to develop treatments that restore synaptic function, regardless of which particular synaptic protein is defective in the individual patient, Feng says.

Feng performed some of the research while at Duke, and several of his former Duke colleagues are authors on the Nature paper, including lead author Joao Peca and Professor Christopher Lascola.

Researchers find new actions of neurochemicals

by Catherine Delude |

Although the tiny roundworm Caenorhabditis elegans has only 302 neurons in its entire nervous system, studies of this simple animal have significantly advanced our understanding of human brain function because it shares many genes and neurochemical signaling molecules with humans. Now MIT researchers have found novel C. elegans neurochemical receptors, the discovery of which could lead to new therapeutic targets for psychiatric disorders if similar receptors are found in humans.

Dopamine and serotonin are members of a class of neurochemicals called biogenic amines, which function in neuronal circuitry throughout the brain. Many drugs used to treat psychiatric disorders, including depression and schizophrenia, target these signaling systems, as do cocaine and other drugs of abuse. Scientists have long known of a class of biogenic-amine receptors that are G protein-coupled receptors (GPCRs) and that, when activated, trigger a slow but long-lasting cascade of intracellular events that modulate nervous system activity.

A study in the July 3 issue of Science has found that in C. elegans these chemicals also act on receptors of a fundamentally different type. These receptors are chloride channels that open and close quickly in response to the binding of a neurochemical messenger. By allowing the passage of negatively charged chloride ions across the cell membrane, chloride channels can rapidly inactivate nerve cells.

“These results underscore the importance of determining whether, as in the C. elegans nervous system, a diversity of biogenic amine-gated chloride channels function in the human brain,” said H. Robert Horvitz of the McGovern Institute for Brain Research at MIT and senior author of the study. “If so, such channels might define novel therapeutic targets for neuropsychiatric disorders, such as depression and schizophrenia.”

In 2000, Horvitz’s group discovered that serotonin activates a chloride channel they called MOD-1, which inhibits neuronal activity in C. elegans.

In the current study, Niels Ringstad and Namiko Abe, a postdoctoral researcher and an undergraduate in Horvitz’s laboratory, respectively, looked for other ion channels that could be receptors for biogenic amines. Using both in vitro and in vivo methods, they surveyed the functions of 26 ion channels similar to MOD-1 and found three additional ion channels with an affinity for biogenic amines: dopamine activates one, serotonin another, and tyramine (the role of which in the human brain is unknown) a third. All three were chloride channels, like MOD-1.

“We now have four members of a family of chloride channels that can act as receptors for biogenic amines in the worm,” Ringstad said. “That these neurochemicals activate both GPCRs and ion channels means that they can have very complex actions in the nervous system, both as slow-acting neuromodulators and as fast-acting inhibitory neurotransmitters.”

It is unknown as yet whether an equivalent to this new class of worm receptor exists in the human brain, but Horvitz points out that worms have proved remarkably informative for providing insights into human biology. In 2002, Horvitz shared the Nobel Prize in Physiology or Medicine for the discovery based on studies of C. elegans of the mechanism of programmed cell death, a central feature of some neurodegenerative diseases and many other disorders in humans.

“Historically, studies of C. elegans have delineated mechanisms of neurotransmission that subsequently proved to be conserved in humans,” says Horvitz, the David H. Koch Professor of Biology at MIT and a Howard Hughes Medical Institute Investigator. “The next step is to look for chloride channels controlled by biogenic amines in mammalian neurons.”

This study was supported by the NIH, the Howard Hughes Medical Institute, the Life Sciences Research Foundation, and The Medical Foundation.