Caltech Researchers Receive NIH BRAIN Funding

On September 30, the National Institutes of Health (NIH) announced its first round of funding in furtherance of President Obama's "Brain Research through Advancing Innovative Neurotechnology"—or BRAIN—Initiative. Included among the 58 funded projects—all of which, according to the NIH, are geared toward the development of "new tools and technologies to understand neural circuit function and capture a dynamic view of the brain in action"—are six projects either led or co-led by Caltech researchers.

The Caltech projects are:

"Dissecting human brain circuits in vivo using ultrasonic neuromodulation"

Doris Tsao, assistant professor of biology
Mikhail Shapiro, assistant professor of chemical engineering

Tsao and Shapiro are teaming up to develop a new technology that both uses ultrasound to map and determine the function of interconnected brain networks and, ultimately, to change neural activity deep within the brain. "This would open new horizons for understanding human brain function and connectivity, and create completely new options for the noninvasive treatment of brain diseases such as intractable epilepsy, depression, and Parkinson's disease," Tsao says. "The key," Shapiro adds, "is to gain a precise understanding of the various mechanisms by which sound waves interact with neurons in the brain so we can use ultrasound to produce very specific neurological effects. We will be able to do this across the full spectrum, from molecules up to large model organisms."

"Modular nanophotonic probes for dense neural recording at single-cell resolution"

Michael Roukes, Robert M. Abbey Professor of Physics, Applied Physics, and Bioengineering
Thanos Siapas, professor of computation and neural systems

Roukes, Siapas, and their colleagues at Columbia University and Baylor College of Medicine propose to build ultra-dense arrays of miniature light-emitting and light-sensing probes using advanced silicon "chip" technology that permits their production en masse. These probes open the new field of integrated neurophotonics, Roukes says, and will permit simultaneous recording of the electrical activity of hundreds of thousands to, ultimately, millions of neurons, with single-cell resolution, in any given region of the brain. "The instrumentation we'll develop will enable us to observe the trafficking of information, in vivo, in brain circuits on an unprecedented scale, and to correlate this activity with behavior," he says.

"Time-Reversal Optical Focusing for Noninvasive Optogenetics"

Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering
Viviana Gradinaru, assistant professor of biology

Deep-brain stimulation has been used successfully for nearly two decades for the treatment of epilepsy, Parkinson's disease, chronic pain, depression, and other disorders. Current systems rely on electrodes implanted deep within the brain to modify the firing pattern of specific clusters of neurons, bringing them back into a more normal pattern. Yang and Gradinaru are working together on a method that would use only light waves to noninvasively activate light-sensitive molecules and precisely guide the firing of nerves. Biological tissues are opaque due to the scattering of light waves, and that scattering makes it impossible to finely focus a laser beam deep into brain tissue. The researchers hope to use an optical "time-reversal" trick previously developed by Yang to counteract the scattering, allowing light beams to be targeted to specific locations within the brain. "The technology to be developed in this project has the potential for wide-ranging applications, including noninvasive deep brain stimulation and precise incisionless laser surgery," he says.

"Integrative Functional Mapping of Sensory-Motor Pathways"

Michael H. Dickinson, Esther M. and Abe M. Zarem Professor of Bioengineering

As in other animals, locomotion in the fruit fly is a complicated process involving the interplay of sensory systems and motor circuits in the brain. Dickinson and his colleagues hope to decipher just how the brain uses sensory information to guide movements by developing a system to record the activity of large numbers of individual neurons from across the brains of fruit flies, as the flies fly in flight simulator or walk on a treadmill and are simultaneously exposed to various sights and sounds. Understanding sensory–motor integration, he says, should lead to a better understanding of human disorders, including Parkinson's disease, stroke, and spinal cord injury, and aid in the design and optimization of robotic prosthetic limbs and prosthetic devices that restore sight and other senses.

"Establishing a Comprehensive and Standardized Cell Type Characterization Platform"

David J. Anderson, Seymour Benzer Professor of Biology; Investigator, Howard Hughes Medical Institute (co-PI)

In collaboration with Hongkui Zeng and colleagues at the Allen Institute for Brain Science in Seattle, Anderson will help to develop a detailed, publicly available database characterizing the genetic, physiological, and morphological features of the various cell types in the brain that are involved in circuits controlling sensations and emotions. Understanding the cellular building blocks of brain circuits, the researchers say, is crucial for figuring out how those circuits can malfunction in disease. In particular, Anderson's lab will focus on the cells of the brain's hypothalamus and amygdala—structures that are vital to emotions and behavior, and involved in human psychiatric disorders such as post-traumatic stress disorder, anxiety, and depression. "This project will serve as a model for hub-and-spoke collaborations between academic laboratories and the Allen Institute, permitting access to their valuable resources and technologies while advancing the field more broadly," Anderson says.

"Vertically integrated approach to visual neuroscience: microcircuits to behavior"

Markus Meister, Lawrence A. Hanson, Jr. Professor of Biology (co-PI)

This project, led by Hyunjune Sebastian Seung of Princeton University, will use genetic, electrophysiological, and imaging tools to identify and map the neural connections of the retina, the light-sensing tissue in the eye, and determine their roles in visual perception and behavior. "Here we are shooting for a vertically integrated understanding of a neural system," Meister says. "The retina offers such a fantastic degree of experimental access that one can hope to bridge all scales of organization, from molecules to cells to microcircuits to behavior. We hope that success here can eventually serve as a blueprint for understanding other parts of the brain." Knowing the neural mechanisms for vision can also influence technological applications, such as new algorithms for computer vision, or the development of retinal prostheses for the treatment of blindness.

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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."

The work published in both papers was supported with grants from the National Institutes of Health (NIH), including an NIH/National Institute of Neurological Disorders and Stroke New Innovator Award to Gradinaru; Beckman Institute funding for the BIONIC center; grants from the U.S. Army Research Office as well as a Caltech Biology Division Training Grant and startup funds from Caltech's President and Provost, and the Division of Biology and Biological Engineering; and other financial support from the Shurl and Kay Curci Foundation and the Life Sciences Research Foundation.

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Wednesday, September 24, 2014
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Seeing Protein Synthesis in the Field

Caltech researchers have developed a novel way to visualize proteins generated by microorganisms in their natural environment—including the murky waters of Caltech's lily pond, as in this image created by Professor of Geobiology Victoria Orphan and her colleagues. The method could give scientists insights to how uncultured microbes (organisms that may not easily be grown in the lab) react and adapt to environmental stimuli over space and time.

The visualization technique, dubbed BONCAT (for "bioorthogonal non-canonical amino-acid tagging"), was developed by David Tirrell, Caltech's Ross McCollum–William H. Corcoran Professor and professor of chemistry and chemical engineering. BONCAT uses "non-canonical" amino acids—synthetic molecules that do not normally occur in proteins found in nature and that carry particular chemical tags that can attach (or "click") onto a fluorescent dye. When these artificial amino acids are incubated with environmental samples, like lily-pond water, they are taken up by microorganisms and incorporated into newly formed proteins. Adding the fluorescent dye to the mix allows these proteins to be visualized within the cell.

For example, in the image, the entire microbial community in the pond water is stained blue with a DNA dye; freshwater gammaproteobacteria are labeled with a fluorescently tagged short-chain ribosomal RNA probe, in red; and newly created proteins are dyed green by BONCAT. The cells colored green and orange in the composite image, then, show those bacteria—gammaproteobacteria and other rod-shaped cells—that are actively making proteins.

"You could apply BONCAT to almost any type of sample," Orphan says. "When you have an environmental sample, you don't know which microorganisms are active. So, assume you're interested in looking at organisms that respond to methane. You could take a sample, provide methane, add the synthetic amino acid, and ask which cells over time showed activity—made new proteins—in the presence of methane relative to samples without methane. Then you can start to sort those organisms out, and possibly use this to determine protein turnover times. These questions are not typically tractable with uncultured organisms in the environment." Orphan's lab is also now using BONCAT on samples of deep-sea sediment in which mixed groups of bacteria and archaea catalyze the anaerobic oxidation of methane.

Why sample the Caltech lily pond? Roland Hatzenpichler, a postdoctoral scholar in Orphan's lab, explains: "When I started applying BONCAT on environmental samples, I wanted to try this new approach on samples that are both interesting from a microbiological standpoint, as well as easily accessible. Samples from the lily pond fit those criteria." Hatzenpichler is lead author of a study describing BONCAT that appeared as the cover story of the August issue of the journal Environmental Microbiology.

The work is supported by the Gordon and Betty Moore Foundation Marine Microbiology Initiative.

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Wednesday, September 10, 2014
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Checking the First Data from OCO-2

On July 2, NASA successfully launched its first satellite dedicated to measuring carbon dioxide in Earth's atmosphere. The Orbiting Carbon Observatory-2 (OCO-2) mission—operated by NASA's Jet Propulsion Laboratory—will soon provide atmospheric carbon dioxide measurements from thousands of points all over the planet. Last week, the satellite reached its proper orbit—meaning that it is now beginning to return its first data to Earth.

Data from the satellite will be used to help researchers understand the anthropogenic and natural sources of CO2, and how changing levels of the greenhouse gas may affect Earth's climate. But before OCO-2 provides scientists with such a global picture of the carbon cycle—where carbon is being produced and absorbed on Earth—researchers have to convert raw satellite data into a CO2 reading and then, just as importantly, make sure that the reading is accurate. A team of Caltech researchers is playing an instrumental role in this effort.

As it orbits, OCO-2 provides data about levels of atmospheric CO2 by measuring the sunlight that reflects off Earth, below. "OCO-2 measures something that is related to the CO2 measurement we want but it's not directly what we want. So from the reflected light, we have to extract the information about CO2," says Yuk Yung, the Smits Family Professor of Planetary Science.

The process begins with the satellite's instrument, a set of high-resolution spectrometers that measure the intensity of sunlight at different wavelengths, or colors, after it has passed twice through the atmosphere—once from the sun to the surface, and then back from the surface to space. As the satellite orbits, systematically slicing over sections of Earth's atmosphere, it will collect millions of these measurements.

"OCO-2 will provide the measurements of this light at different wavelengths in millions of what we call spectra, but spectra aren't what we really want—what we really want is to know how much carbon dioxide is in the atmosphere," Yung says. "But to get the CO2 information from the spectra, we have to do what's called data retrieval—and that's one of my jobs."

The data retrieval method that Yung and his colleagues designed for OCO-2 compares the light spectra collected by the satellite to a model of how light spectra would look—based on the laws of physics and knowledge of how efficiently CO2 absorbs sunlight. This knowledge, in turn, is derived from laboratory measurements made by Caltech professor of chemical physics Mitchio Okumura and his colleagues at JPL and the National Institute of Standards and Technology.

"To make scientifically meaningful measurements, OCO-2 has to detect CO2 with better than 0.3 percent precision, and that has meant going back to the lab and measuring the spectral properties with extraordinarily high precision," Okumura says. From this retrieval, the researchers determine the amount of CO2 in the atmosphere above each of OCO-2's sampling points.

However, when OCO-2 sends its first CO2 measurements back to Earth for analysis, they'll still have to go through one more check, says Paul Wennberg, the R. Stanton Avery Professor of Atmospheric Chemistry and Environmental Science and Engineering.

"Although the OCO-2 retrieval will calculate the amount of carbon dioxide above the point where the spectrometers pointed, we know that these initial numbers will be wrong until the data are calibrated," Wennberg says. Wennberg and his team provide this calibration with their Total Carbon Column Observing Network (TCCON), a ground-based network of instruments that measure atmospheric CO2 from approximately 20 locations around the world.

TCCON and OCO-2 provide the same type of CO2 measurement—what is called a column average of CO2. This measurement provides the average abundance of CO2 in a column from the ground all the way up through Earth's atmosphere.

About once per day, the OCO-2 instrument will be commanded to point at one of TCCON's stations continuously as it passes overhead. By comparing the Earth-based and space-based measurements, researchers will evaluate the data that they receive from the satellite and improve the retrieval method.

The complete, high-quality information OCO-2 provides about global CO2 levels will be important for researchers and policymakers to determine how human activity influences the carbon cycle—and how these activities contribute to our changing planet.

"A lot of the very first satellites were developed to study astronomy and planets far away. But there has been a shift. Our changing climate means that we now have a big need to study Earth," and the information OCO-2 provides about our atmosphere will be an important part of filling that need, says Yung.

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Study of Aerosols Stands to Improve Climate Models

Aerosols, tiny particles in the atmosphere, play a significant role in Earth's climate, scattering and absorbing incoming sunlight and affecting the formation and properties of clouds. Currently, the effect that these aerosols have on clouds represents the largest uncertainty among all influences on climate change.

But now researchers from Caltech and the Jet Propulsion Laboratory have provided a global observational study of the effect that changes in aerosol levels have on low-level marine clouds—the clouds that have the largest impact on the amount of incoming sunlight that Earth reflects back into space. The findings appear in the advance online version of the journal Nature Geoscience.

Changes in aerosol levels have two main effects—they alter the amount of clouds in the atmosphere and they change the internal properties of those clouds. Using measurements from several of NASA's Earth-monitoring satellites from August 2006 through April 2011, the researchers quantified for the first time these two effects from 7.3 million individual data points.

"If you combine these two effects, you get an aerosol influence almost twice that estimated in the latest report from the Intergovernmental Panel on Climate Change," says John Seinfeld, the Louis E. Nohl Professor and professor of chemical engineering at Caltech. "These results offer unique guidance on how warm cloud processes should be incorporated in climate models with changing aerosol levels."

The lead author of the paper, "Satellite-based estimate of global aerosol-cloud radiative forcing by marine warm clouds," is Yi-Chun Chen (Ph.D. '13), a NASA postdoctoral fellow at JPL. Additional coauthors are Matthew W. Christensen of JPL and Colorado State University and Graeme L. Stephens, director of the Center for Climate Sciences at JPL. The work was supported by funding from NASA and the Office of Naval Research.

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