Tuesday, October 7, 2014
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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

A chance to meet Pasadena Unified School District Leadership

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
Avery Dining Hall – Avery House

RESCHEDULED to Sept 24th: A chance to meet Pasadena Unified School District Leadership

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.

Kimm Fesenmaier
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Friday, October 10, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

Course Ombudsperson Training

Wednesday, January 7, 2015
Center for Student Services 360 (Workshop Space) – Center for Student Services

Head TA Network

Thursday, September 25, 2014

Head TA Network


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