Improving The View Through Tissues and Organs

On Saturday, October 18, hundreds of undergraduate students will share the results of their projects during SURF Seminar Day. The event, which is open to the public, is an opportunity for students to discuss and explain their research to individuals with a wide-range of expertise and interests.

This summer, several undergraduate students at Caltech had the opportunity to help optimize a promising technique that can make tissues and organs—even entire organisms—transparent for study. As part of the Summer Undergraduate Research Fellowship (SURF) program, these students worked in the lab of Assistant Professor of Biology Viviana Gradinaru, where researchers are developing such so-called clearing techniques that make it possible to peer straight through normally opaque tissues rather than seeing them only as thinly sectioned slices that have been pieced back together.

Gradinaru's group recently published a paper in the journal Cell describing a new approach to tissue clearing. The method they have created builds on a technique called CLARITY that Gradinaru helped develop while she was a research associate at Stanford. CLARITY allowed researchers to, for the first time, create a transparent whole-brain specimen that could then be imaged with its structural and genetic information intact.

CLARITY was specifically developed for studying the brain. But the new approach developed in Gradinaru's lab, which the team has dubbed PARS (perfusion-assisted agent release in situ), can also clear other organs, such as the kidney, as well as tissue samples, such as tumor biopsies. It can even be applied to entire organisms.

Like CLARITY, PARS involves removing the light-scattering lipids in the tissue to make samples transparent without losing the structural integrity that lipids typically provide. First the sample is infused with acrylamide monomers that are then polymerized into a hydrogel that provides structural support. Next, this tissue–hydrogel hybrid is immersed in a detergent that removes the lipids. Then the sample can be stained, often with antibodies that specifically mark cells of interest, and then immersed in RIMS (refractive index matching solution) for imaging using various optical techniques such as confocal or lightsheet microscopy.

Over the summer, Sam Wie, a junior biology major at Caltech, spent 10 weeks in the Gradinaru lab working to find a polymer that would perform better than acrylamide, which has been used in the CLARITY hydrogel. "One of the limitations of CLARITY is that when you put the hydrogel tissue into the detergent, the higher solute concentration in the tissue causes liquid to rush into the cell. That causes the sample to swell, which could potentially damage the structure of the tissue," Wie explains. "So I tried different polymers to try to limit that swelling."

Wie was able to identify a polymer that produces, over a similar amount of time, about one-sixth of the swelling in the tissue.

"The SURF experience has been very rewarding," Wie says. "I've learned a lot of new techniques, and it's really exciting to be part of, and to try to improve, CLARITY, a method that will probably change the way that we image tissues from now on."

At another bench in Gradinaru's lab, sophomore bioengineering major Andy Kim spent the summer focusing on a different aspect of the PARS technique. While antibodies have been the most common markers used to tag cells of interest within cleared tissues, they are too large for some studies—for example, those that aim to image deeper parts of the brain, requiring them to cross the blood–brain barrier. Kim's project involved identifying smaller proteins, such as nanobodies, which target and bind to specific parts of proteins in tissues.

"While PARS is a huge improvement over CLARITY, using antibodies to stain is very expensive," Kim says. "However, some of these nanobodies can be produced easily, so if we can get them to work, it would not only help image the interior of the brain, it would also be a lot less costly."

During his SURF, Kim worked with others in the lab to identify about 30 of these smaller candidate binding proteins and tested them on PARS-cleared samples.

While Wie and Kim worked on improving the PARS technique itself, Donghun Ryu, a third SURFer in Gradinaru's lab, investigated different methods for imaging the cleared samples. Ryu is a senior electrical engineering and computer science major at the Gwangju Institute of Science and Technology (GIST) in the Republic of Korea.

Last summer Ryu completed a SURF as part of the Caltech–GIST Summer Undergraduate Research Exchange Program in the lab of Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering at Caltech. While completing that project, Ryu became interested in optogenetics, the use of light to control genes. Since optogenetics is one of Gradinaru's specialties, Yang suggested that he try a SURF in Gradinaru's lab.

This summer, Ryu was able to work with both Yang and Gradinaru, investigating a technique called Talbot microscopy to see whether it would be better for imaging thick, cleared tissues than more common techniques. Ryu was able to work on the optical system in Yang's lab while testing the samples cleared in Gradinaru's lab.

"It was a wonderful experience," Ryu says. "It was special to have the opportunity to work for two labs this summer. I remember one day when I had a meeting with both Professor Yang and Professor Gradinaru; it was really amazing to get to meet with two Caltech professors."

Gradinaru says that the SURF projects provided a learning opportunity not only for the participating students but also for her lab. "For example," she says, "Ryu strengthened the collaboration that we have with the Yang group for the BRAIN Initiative. And my lab members benefited from the chance to serve as mentors—to see what works and what can be improved when transferring scientific knowledge. These are very important skills in addition to the experimental know-how that they master."  

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Sensors to Simplify Diabetes Management

For many patients diagnosed with diabetes, treating the disease can mean a burdensome and uncomfortable lifelong routine of monitoring blood sugar levels and injecting the insulin that their bodies don't naturally produce. But, as part of their Summer Undergraduate Research Fellowship (SURF) projects at Caltech, several engineering students have contributed to the development of tiny biosensors that could one day eliminate the need for these manual blood sugar tests.

Because certain patients with diabetes are unable to make their own insulin—a hormone that helps transfer glucose, or sugar, from the blood into muscle and other tissues—they need to monitor frequently their blood glucose, manually injecting insulin when sugar levels surge after a meal. Most glucose monitors require that patients prick their fingertips to collect a drop of blood, sometimes up to 10 times a day for the rest of their lives.

In their SURF projects, the students, all from Caltech's Division of Engineering and Applied Science, looked for different ways to do these same tests but painlessly and automatically.

Mehmet SencanSenior applied physics major Mehmet Sencan has approached the problem with a tiny chip that can be implanted under the skin. The sensor, a square just 1.4 millimeters on each side, is designed to detect glucose levels from the interstitial fluid (fluid found in the spaces between cells) that is just under the skin. The glucose levels in this fluid directly relate to the blood glucose concentration.

Sencan has been involved in optimizing the electrochemical method that the chip will use to detect glucose levels. Much like a traditional finger-stick glucose meter, the chip uses glucose oxidase, an enzyme that reacts in the presence of glucose, to create an electrical current. Higher levels of glucose result in a stronger current, allowing the device to measure glucose levels based on the charge that passes through the fluid.

Once the glucose level is detected, the information is wirelessly transmitted via a radio wave frequency to a reader that uses the same frequency to power the device itself. Ultimately an external display will let the patient know if their levels are within range.

Sencan, who works in the laboratory of Axel Scherer, the Bernard Neches Professor of Electrical Engineering, Applied Physics, and Physics, and who is co-mentored by postdoctoral researcher Muhammad Mujeeb-U-Rahman, started this project three years ago during his very first SURF.

"When I started, we were just thinking about what kind of chemistry the sensor would use, and now we have a sensor that is actually designed to do that," he says. Over the summer, he implanted the sensors in rat models, and he will continue the study over the fall and spring terms using both rat and mouse models—a first step in determining if the design is a clinically viable option.

Sith DomrongkitchaipornJunior electrical engineering major Sith Domrongkitchaiporn from the Scherer laboratory, also co-mentored by Mujeeb-U-Rahman, took a different approach to glucose detection, making tiny biosensors that are inconspicuously wearable on the surface of a contact lens. "It's an interesting concept because instead of having to do a procedure to place something under the skin, you can use a less invasive method, placing a sensor on the eye to get the same information," he says.

He used the method optimized by Mehmet to determine blood glucose levels from interstitial fluid and adapted the chemistry to measure glucose in the eyes' tears. This summer, he will be attempting to fabricate the lens itself and improve upon the process whereby radio waves are used to power the sensor and then transmit data from the sensor to an external computer.

Jennifer Chih-Wen LinSURF student and sophomore electrical engineering major Jennifer Chih-Wen Lin wanted to incorporate a different kind of glucose sensor into a contact lens. "The concept—determining glucose readings from tears—is very similar to Sith's, but the method is very different," she says.

Instead of determining the glucose level based on the amount of electrical current that passes through a sample, Lin, who works in the laboratory of Hyuck Choo, assistant professor of electrical engineering, worked on a sensor that detects glucose levels from the interaction between light and molecules.

In her SURF project, she began optimizing the characterization of glucose molecules in a sample of glucose solution using a technique called Raman spectroscopy. When molecules encounter light, they vibrate differently based on their symmetry and the types of bonds that hold their atoms together. This vibrational information provides a unique fingerprint for each type of molecule, which is represented as peaks on the Raman spectrum—and the intensity of these peaks correlates to the concentration of that molecule within the sample.

"This step is important because once I can determine the relationship between peak intensities and glucose concentrations, our sensor can just compare that known spectrum to the reading from a sample of tears to determine the amount of glucose in the sample," she says.

Lin's project is in the very beginning stages, but if it is successful, it could provide a more accurate glucose measurement, and from a smaller volume of liquid, than is possible with the finger-stick method. Perhaps more importantly for patients, it can provide that measurement painlessly.

Sophia ChenAlso in Choo's laboratory, sophomore electrical engineering major Sophia Chen's SURF project involves a new way to power devices like these tiny sensors and other medical implants, using the vibrations from a patient's vocal cords. These vibrations produce the sound of our voice, and also create vibrations in the skull.

"We're using these devices called energy harvesters that can extract energy from vibrations at specific frequencies. When the vibrations go from the vocal folds to the skull, a structure in the energy harvester vibrates at the same frequency, generating energy—energy that can be used to power batteries or charge things," Chen says.

Chen's goal is to determine the frequency of these vibrations—and if the energy that they produce is actually enough to power a tiny device. The hope is that one day these vibrations could power, or at least supplement the power of, medical devices that need to be implanted near the head and that presently run on batteries with finite lifetimes.

Chen and the other students acknowledge that health-monitoring sensors powered by the human body might be years away from entering the clinic. However, this opportunity to apply classroom knowledge to a real-life challenge—such as diabetes treatment—is an important part of their training as tomorrow's scientists and engineers.

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Orchestrating the Healing Process in a Damaged Cornea

It is safe to say that the eye is an amazing biological system. One reason is its keratocyte cells—specialized cells that make up the bulk of the cornea. Unlike most of the other cells in our body, those in the cornea are transparent, making sight possible. Should something happen to make the cornea opaque, blindness results.

Sadly, injuries to the cornea do occur, sometimes in the simplest of ways, such as getting sand in one's eyes and scratching the cornea. The scar tissue that then grows to heal the cornea may have the unwanted side effect of being opaque. This does not happen, however, if the cornea and the tissue around it heal in a very orderly fashion. The question, then: Is it possible to encourage this orderly healing after an injury, thus preserving vision?

Professor of Chemical Engineering Julia Kornfield and graduate student Amy Fu are very much hoping that this is the case. To find out, they have assigned a few students, including Caltech senior and recent SURF fellow Jacqueline Masehi-Lano, to experiment with various growth factors that might inhibit the formation of scar tissue and promote orderly wound healing.

"We chose three growth factors to test because Amy Fu and I read several papers on growth factors that have been able to suppress some types of scar tissue," Masehi-Lano says. "In particular, we want to inhibit the formation of alpha smooth muscle actin, the type of stress fiber that creates opaque scars over corneal wounds. So far, the experiments I've done with cell cultures have worked pretty well, so it looks promising."

Eventually, the researchers hope to encapsulate the growth factors in a hydrogel that is reminiscent of the native cornea. "Our hydrogel starts out as a liquid and gels in situ on the eye," explains Masehi-Lano.

Masehi-Lano is enthusiastic about her experience with the SURF program. This past summer was her second in Kornfield's lab, and last year she was a recipient of an Amgen scholarship. "I'm really grateful that my mentor and my co-mentor have entrusted me with my own project and have allowed me to conduct my own experiments. And since it was my second summer in this lab, I was able to take up a leadership role by training a new SURF student," she says. "For me, SURF has gone beyond research. I've been able to improve my ability to present my research to the general public, which I think is extremely important." Indeed, Masehi-Lano was awarded the Caltech Doris S. Perpall SURF Speaking Competition for delivering the most outstanding oral research presentation.

Masehi-Lano plans to continue in bioengineering and is contemplating an MD/PhD program. "I've always been interested in the medical field, and though I'm committed to doing research," she explains, "I'd like to be able to do clinical trials and directly apply new medical technologies to people."

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A New Way to Prevent the Spread of Devastating Diseases

For decades, researchers have tried to develop broadly effective vaccines to prevent the spread of illnesses such as HIV, malaria, and tuberculosis. While limited progress has been made along these lines, there are still no licensed vaccinations available that can protect most people from these devastating diseases.

So what are immunologists to do when vaccines just aren't working?

At Caltech, Nobel Laureate David Baltimore and his colleagues have approached the problem in a different way. Whereas vaccines introduce substances such as antigens into the body hoping to illicit an appropriate immune response—the generation of either antibodies that might block an infection or T cells capable of attacking infected cells—the Caltech team thought: Why not provide the body with step-by-step instructions for producing specific antibodies that have been shown to neutralize a particular disease?

The method they developed—originally to trigger an immune response to HIV—is called vectored immunoprophylaxis, or VIP. The technique was so successful that it has since been applied to a number of other infectious diseases, including influenza, malaria, and hepatitis C.

"It is enormously gratifying to us that this technique can have potentially widespread use for the most difficult diseases that are faced particularly by the less developed world," says Baltimore, president emeritus and the Robert Andrews Millikan Professor of Biology at Caltech.

VIP relies on the prior identification of one or more antibodies that are able to prevent infection in laboratory tests by a wide range of isolated samples of a particular pathogen. Once that has been done, researchers can incorporate the genes that encode those antibodies into an adeno-associated virus (AAV), a small, harmless virus that has been useful in gene-therapy trials. When the AAV is injected into muscle tissue, the genes instruct the muscle tissue to generate the specified antibodies, which can then enter the circulation and protect against infection.

In 2011, the Baltimore group reported in Nature that they had used the technique to deliver antibodies that effectively protected mice from HIV infection. Alejandro Balazs was lead author on that paper and was a postdoctoral scholar in the Baltimore lab at the time.

"We expected that at some dose, the antibodies would fail to protect the mice, but it never did—even when we gave mice 100 times more HIV than would be needed to infect seven out of eight mice," said Balazs, now at the Ragon Institute of MGH, MIT and Harvard. "All of the exposures in this work were significantly larger than a human being would be likely to encounter."

At the time, the researchers noted that the leap from mice to humans is large but said they were encouraged by the high levels of antibodies the mice were able to produce after a single injection and how effectively the mice were protected from HIV infection for months on end. Baltimore's team is now working with a manufacturer to produce the materials needed for human clinical trials that will be conducted by the Vaccine Research Center at the National Institutes of Health.

Moving on from HIV, the Baltimore lab's next goal was protection against influenza A. Although reasonably effective influenza vaccines exist, each year more than 20,000 deaths, on average, are the result of seasonal flu epidemics in the United States. We are encouraged to get flu shots every fall because the influenza virus is something of a moving target—it evolves to avoid resistance. There are also many different strains of influenza A (e.g. H1N1 and H3N2), each incorporating a different combination of the various forms of the proteins hemagglutinin (H) and neuraminidase (N). To chase this target, the vaccine is reformulated each year, but sometimes it fails to prevent the spread of the strains that are prevalent that year.

But about five years ago, researchers began identifying a new class of anti-influenza antibodies that are able to prevent infection by many, many strains of the virus. Instead of binding to the head of the influenza virus, as most flu-fighting antibodies do, these new antibodies target the stalk that holds up the head. And while the head is highly adaptable—meaning that even when mutations occur there, the virus can often remain functional—the stalk must basically remain the same in order for the virus to survive. So these stalk antibodies are very hard for the virus to mutate against.

In 2013, the Baltimore group stitched the genes for two of these new antibodies into an AAV and showed that mice injected with the vector were protected against multiple flu strains, including all H1, H2, and H5 influenza strains tested. This was even true of older mice and those without a properly functioning immune system—a particularly important finding considering that most deaths from the flu occur in the elderly and immunocompromised populations. The group reported its results in the journal Nature Biotechnology.

"We have shown that we can protect mice completely against flu using a kind of antibody that doesn't need to be changed every year," says Baltimore. "It is important to note that this has not been tested in humans, so we do not yet know what concentration of antibody can be produced by VIP in humans. However, if it works as well as it does in mice, VIP may provide a plausible approach to protect even the most vulnerable patients against epidemic and pandemic influenza."

Now that the Baltimore lab has shown VIP to be so effective, other groups from around the country have adopted the Caltech-developed technique to try to ward off malaria, hepatitis C, and tuberculosis.

In August, a team led by Johns Hopkins Bloomberg School of Public Health reported in the Proceedings of the National Academy of Sciences (PNAS) that as many as 70 percent of mice that they had injected by the VIP procedure were protected from infection with malaria by Plasmodium falciparum, the parasite that carries the most lethal of the four types of the disease. A subset of mice in the study produced particularly high levels of the disease-fighting antibodies. In those mice, the immunization was 100 percent effective.

"This is also just a first-generation antibody," says Baltimore, who was a coauthor on the PNAS study. "Knowing now that you can get this kind of protection, it's worth trying to get much better antibodies, and I trust that people in the malaria field will do that."

Most recently, a group led by researchers from The Rockefeller University showed that three hepatitis-C-fighting antibodies delivered using VIP were able to protect mice efficiently from the virus. The results were published in the September 17 issue of the journal Science Translational Medicine. The researchers also found that the treatment was able to temporarily clear the virus from mice that had already been infected. Additional work is needed to determine how to prevent the disease from relapsing. Interestingly, though, the work suggests that the antibodies that are effective against hepatitis C, once it has taken root in the liver, may work by protecting uninfected liver cells from infection while allowing already infected cells to be cleared from the body.    

An additional project is currently evaluating the use of VIP for the prevention of tuberculosis—a particular challenge given the lack of proven tuberculosis-neutralizing antibodies.

"When we started this work, we imagined that it might be possible to use VIP to fight other diseases, so it has been very exciting to see other groups adopting the technique for that purpose," Baltimore says. "If we can get positive clinical results in humans with HIV, we think that would really encourage people to think about using VIP for these other diseases."

Baltimore's work is supported by funding from the National Institute of Allergy and Infectious Disease, the Bill and Melinda Gates Foundation, the Caltech-UCLA Joint Center for Translational Medicine, and a Caltech Translational Innovation Partnership Award.

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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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Emotions in the Brain: An Interview with David Anderson

This year has been a busy one for biologist David Anderson, Caltech's Seymour Benzer Professor of Biology. In 2014 alone, Anderson's lab has reported finding neurons in the male fly brain that promote fighting and, in the mouse brain, identified a "seesaw" circuit that controls the transition between social and asocial behaviors, neurons that control aggressive behavior, a neural circuit that controls anxiety, and a network of cells that switches appetite on and off.

The flurry of discoveries, made possible using state-of-the-art neurobiology techniques such as optogenetics (a technique that uses light to control neural activity), is the result of years of research by the lab to understand emotions and how they are encoded in the brain. We recently spoke to Anderson about this work, his goals, and how the interdisciplinary collaborations he is building at Caltech are helping to spur a revolution in neuroscience.

 

How would you define an "emotion"?

There has been ongoing debate for decades about what "emotion" means, and there is no generally accepted definition. In an article that Ralph Adolphs [Bren Professor of Psychology and Neuroscience and Professor of Biology] and I recently wrote, we put forth the view that emotions are a type of internal brain state with certain general properties that can exist independently of subjective, conscious experience. That means we can study such brain states in animal models like flies or mice without worrying about whether they are consciously aware or not. We use the behaviors that express those states as a readout. For example, behaviors that express the emotion state we call "fear" are freezing and flight. Behaviors that express "anger" include various forms of aggression.

 

So you study these behaviors to get at the underlying emotion and its neural circuitry?

Ultimately, yes. We use genetically based techniques that have been developed over the last 10 years or so—including but not limited to optogenetics, imaging of brain activity, and mapping of neuronal connections—to try to identify specific populations of neurons in the brain that control these "emotional" behaviors. Are there specific populations of neurons in the brain that control aggression, for example? If so, where are those neurons located in the brain? How do they function? Do they only control behaviors, or do they encode internal states as well?

 

Do you know any of these answers yet?

We have identified, in fruit flies and in mice, small populations of neurons that control aggression. In flies, we have identified a population of as few as three to five neurons that, when activated, are sufficient to make a fly fight.

In the mouse, we have identified an analogous population in a deep brain structure called the hypothalamus. There are about 2,000 of those neurons. Activating these neurons is sufficient to promote aggression, and inhibiting these neurons can stop a fight dead in it tracks.

 

Do you think similar populations of "aggression" neurons are found in humans? Could they be related to problems with violence in people?

We're studying these problems because they are fundamental to understanding how the brain works, but certainly it doesn't escape our attention that violence is a pervasive public health problem. My feeling is that we need to understand the basic brain circuitry that controls aggression if we are ever going to understand abnormal forms of aggression, such as sexual violence.

In that respect, it's interesting that we have discovered, in both flies and mice, small populations of neurons that control both aggression and mating (reproductive) behavior. So in a male mouse, for example, if you optogenetically stimulate these neurons at a lower light intensity, the animal will try to mate instead of fight. At a higher stimulation intensity, the animal switches from mounting to attack. It's amazing to watch.

A really important objective over the next several years is to try to figure out how the brain can keep sex and violence separated if the neurons are so intimately related to each other, starting with the question of whether they are the same or different neurons. Obviously that could have implications for sexual violence, for example. It could be that there are people who, as it were, have their wires crossed in these regions of the brain, and that causes them to express violent behavior inappropriately.

 

With regard to your recent study that identified neurons that function as a "brake" on appetite, could that same kind of mis-wiring contribute to eating disorders?

It could. I think the field as a whole—meaning the field of psychiatry—is moving away from the popular idea that psychiatric disorders are due to chemical imbalances in the brain, as if the brain were a bag of soup flavored with dopamine and serotonin, to the idea that psychiatric disorders are due to dysfunctions of brain circuitry as well as chemistry.

 

You've found a "seesaw circuit" in the amygdala that tips between social behavior and self-directed behavior depending on which of two populations of neurons is active. Did you expect the brain to be wired this way?

No. It was also completely unexpected that these two populations segregate according to the most basic distinction between neurons in the brain: inhibitory neurons and excitatory neurons. Inhibitory neurons control the social behaviors. Excitatory neurons control the self-grooming behaviors. It did not have to be that way.

 

Could the proportion of these neurons explain something like personality—whether a person is introverted or extroverted?

That is a fascinating question—whether differences in the behavior of individuals might reflect differences in the relative numbers of different types of neurons. We're trying to see if that is true in different strains of laboratory mice that show different levels of aggression. It is a new direction of research in my lab.

 

Does the discovery of these kinds of circuits suggest possible treatments for human disorders? Could you alter a circuit to change behavior?

It might be possible that, if you found the right population of neurons, you could override the effect of a gene mutation to promote autism or some other psychiatric disorder by pushing the activity of the circuits in a different direction.

 

Tip the balance of the seesaw . . .

Tip the balance of the seesaw in the other direction.

However, this is very far in the future.

But to take a step back to the 35,000-foot level: All of this is happening in the context of a field-wide revolution in neuroscience, a revolution in technology for understanding the brain at the neural circuit level. When I was on the advisory committee for the Obama BRAIN project, we decided that it should focus on supporting the development of this kind of technology.

The technology—in optics and nanotechnology and molecular biology and genetics—allows us to identify populations of neurons that control behaviors, map their connections, measure their activity during behaviors, and manipulate their function, turning them on and off, with a laser-like precision that we could never do before.

If you think of specific populations as a needle in a haystack, these technologies allow us to see and touch and manipulate the needle separately from the haystack. That doesn't mean it won't affect the haystack, but at least we know what we're doing.

 

Your lab's focus changed as a result of the advent of these new methods. Can you tell us about that?

Around the early 2000s, I decided that this area of neuroscience was going to be ripe for new discoveries, although much of this new technology didn't exist then. Caltech helped me to completely retool my laboratory, to move from the study of brain development and stem cell biology to the study of neural circuits and behavior—a major transition from both the intellectual and technical standpoint. It was sort of like turning a sailboat into a motorboat without stopping moving.

 

Do you have a vision of how the field will develop in the future?

This work is increasingly interdisciplinary. It needs molecular biology. It needs optical physics. It needs nanotechnology. It needs modeling, theory, computer science, and electrical engineering. No one laboratory can be competent in all of these different areas.

What has kept me here at Caltech is the ability to collaborate with people from different disciplinary backgrounds. What I am excited about, going forward, is to try to develop a new style of research here in which several laboratories devote their collective energies toward solving a challenging problem in a collaborative way, that they couldn't do if they just stayed in their silos and did their own thing.

 

Have you already set up some of these kinds of collaborations?

Yes. For example, I've been working since 2009 with Pietro Perona [Allen E. Puckett Professor of Electrical Engineering], who has applied his skills in machine vision and machine learning to figure out how to automatically measure aggressive behaviors in flies. We are trying to develop similar technology for the mouse as well. It is not only enormously labor-saving but opens a new, more quantitative approach to describing behavior. And there is also my collaboration on emotion theory with Ralph Adolphs in the Division of the Humanities and Social Sciences.

One of the strong recommendations of the BRAIN committee was to promote these kinds of interdisciplinary, cross-laboratory projects. I think it is important for Caltech to recognize that because of its strength in computer science, applied physics and engineering, and its strength in neuroscience, psychology and social sciences, it is ideally poised to promote and facilitate collaborations between physical scientists and neuroscientists.

 

Interdisciplinary work is something that Caltech does very well.

It is. But in this area of interdisciplinary neuroscience, we have particularly exciting opportunities to engage faculty in multiple divisions across campus. I think this is an ideal moment for us to seize the opportunities identified by the BRAIN initiative, and take advantage of what we do best.

 

When you say "what we do best," what do you mean?

Nimble, interdisciplinary and creative collaborations between labs, which would be harder to implement at larger institutions. Caltech is perfectly positioned to exploit the revolution in neuroscience, in its own unique and interdisciplinary way—exploiting our growing strength in neuroscience and our traditional strengths in genetics, the physical sciences, and engineering—to solve the enormous challenge of how the brain works.

 

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Slimy Fish and the Origins of Brain Development

Lamprey—slimy, eel-like parasitic fish with tooth-riddled, jawless sucking mouths—are rather disgusting to look at, but thanks to their important position on the vertebrate family tree, they can offer important insights about the evolutionary history of our own brain development, a recent study suggests.

The work appears in a paper in the September 14 advance online issue of the journal Nature.

"Lamprey are one of the most primitive vertebrates alive on Earth today, and by closely studying their genes and developmental characteristics, researchers can learn more about the evolutionary origins of modern vertebrates—like jawed fishes, frogs, and even humans," says paper coauthor Marianne Bronner, the Albert Billings Ruddock Professor of Biology and director of Caltech's unique Zebrafish/Xenopus/Lamprey facility, where the study was done.

The facility is one of the few places in the world where lampreys can be studied in captivity. Although the parasitic lamprey are an invasive pest in the Great Lakes, they are difficult to study under controlled conditions; their lifecycle takes up to 10 years and they only spawn for a few short weeks in the summer before they die.

Each summer, Bronner and her colleagues receive shipments of wild lamprey from Michigan just before the prime of breeding season. When the lamprey arrive, they are placed in tanks where the temperature of the water is adjusted to extend the breeding season from around three weeks to up to two months. In those extra weeks, the lamprey produce tens of thousands of additional eggs and sperm, which, via in vitro fertilization, generate tens of thousands of additional embryos for study. During this time, scientists from all over the world come to Caltech to perform experiments with the developing lamprey embryos.

In the current study, Bronner and her collaborators—who traveled to Caltech from Stower's Institute for Medical Research in Kansas City, Missouri—studied the origins of the vertebrate hindbrain.

The hindbrain is a part of the central nervous system common to chordates—or organisms that have a nerve cord like our spinal cord. During the development of vertebrates—a subtype of chordates that have backbones—the hindbrain is compartmentalized into eight segments, each of which becomes uniquely patterned to establish networks of neuronal circuits. These segments eventually give rise to adult brain regions like the cerebellum, which is important for motor control, and the medulla oblongata, which is necessary for breathing and other involuntary functions.

However, this segmentation is not present in so-called "invertebrate chordates"—a grouping of chordates that lack a backbone, such as sea squirts and lancelets.

"The interesting thing about lampreys is that they occupy an intermediate evolutionary position between the invertebrate chordates and the jawed vertebrates," says Hugo Parker, a postdoc at Stower's Institute and first author on the study. "By investigating aspects of lamprey embryology, we can get a picture of how vertebrate traits might have evolved."

In the vertebrates, segmental patterning genes called Hox genes help to determine the animal's head-to-tail body plan—and those same Hox genes also control the segmentation of the hindbrain. Although invertebrate chordates also have Hox genes, these animals don't have segmented hindbrains. Because lampreys are centered between these two types of organisms on the evolutionary tree, the researchers wanted to know whether or not Hox genes are involved in patterning of the lamprey hindbrain.

To their surprise, the researchers discovered that the lamprey hindbrain was not only segmented during development but the process also involved Hox genes—just like in its jawed vertebrate cousins.

"When we started, we thought that the situation was different, and the Hox genes were not really integrated into the process of segmentation as they are in jawed vertebrates," Parker says. "But in actually doing this project, we discovered the way that lamprey Hox genes are expressed and regulated is very similar to what we see in jawed vertebrates." This means that hindbrain segmentation—and the role of Hox genes in this segmentation—happened earlier on in evolution than was once thought, he says.

Parker, who has been spending his summers at Caltech studying lampreys since 2008, is next hoping to pinpoint other aspects of the lamprey hindbrain that may be conserved in modern vertebrates—information that will help contribute to a fundamental understanding of vertebrate development. And although those investigations will probably mean following the lamprey for a few more summers at Caltech, Parker says his time in the lamprey facility continually offers a one-of-a-kind experience.

"The lamprey system here is unique in the world—and it's not just the water tanks and how we've learned to maintain the animals. It's the small nucleus of people who have particular skills, people who come in from all over the world to work together, share protocols, and develop the field together," he says. "That's one of the things I've liked ever since I first came here. I really felt like I was a part of something very special.

These results were published in a paper titled "A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates." Robb Krumlauf, a scientific director at the Stower's Institute and professor at the Kansas University Medical Center, was also a coauthor on the study. The Zebrafish/Xenopus/Lamprey facility at Caltech is a Beckman Institute facility.

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Tipping the Balance of Behavior

Humans with autism often show a reduced frequency of social interactions and an increased tendency to engage in repetitive solitary behaviors. Autism has also been linked to dysfunction of the amygdala, a brain structure involved in processing emotions. Now Caltech researchers have discovered antagonistic neuron populations in the mouse amygdala that control whether the animal engages in social behaviors or asocial repetitive self-grooming. This discovery may have implications for understanding neural circuit dysfunctions that underlie autism in humans.

This discovery, which is like a "seesaw circuit," was led by postdoctoral scholar Weizhe Hong in the laboratory of David J. Anderson, the Seymour Benzer Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute. The work was published online on September 11 in the journal Cell

"We know that there is some hierarchy of behaviors, and they interact with each other because the animal can't exhibit both social and asocial behaviors at the same time. In this study, we wanted to figure out how the brain does that," Anderson says.

Anderson and his colleagues discovered two intermingled but distinct populations of neurons in the amygdala, a part of the brain that is involved in innate social behaviors. One population promotes social behaviors, such as mating, fighting, or social grooming, while the other population controls repetitive self-grooming—an asocial behavior.

Interestingly, these two populations are distinguished according to the most fundamental subdivision of neuron subtypes in the brain: the "social neurons" are inhibitory neurons (which release the neurotransmitter GABA, or gamma-aminobutyric acid), while the "self-grooming neurons" are excitatory neurons (which release the neurotransmitter glutamate, an amino acid).

To study the relationship between these two cell types and their associated behaviors, the researchers used a technique called optogenetics. In optogenetics, neurons are genetically altered so that they express light-sensitive proteins from microbial organisms. Then, by shining a light on these modified neurons via a tiny fiber optic cable inserted into the brain, researchers can control the activity of the cells as well as their associated behaviors.

Using this optogenetic approach, Anderson's team was able to selectively switch on the neurons associated with social behaviors and those linked with asocial behaviors.

With the social neurons, the behavior that was elicited depended upon the intensity of the light signal. That is, when high-intensity light was used, the mice became aggressive in the presence of an intruder mouse. When lower-intensity light was used, the mice no longer attacked, although they were still socially engaged with the intruder—either initiating mating behavior or attempting to engage in social grooming.

When the neurons associated with asocial behavior were turned on, the mouse began self-grooming behaviors such as paw licking and face grooming while completely ignoring all intruders. The self-grooming behavior was repetitive and lasted for minutes even after the light was turned off.

The researchers could also use the light-activated neurons to stop the mice from engaging in particular behaviors. For example, if a lone mouse began spontaneously self-grooming, the researchers could halt this behavior through the optogenetic activation of the social neurons. Once the light was turned off and the activation stopped, the mouse would return to its self-grooming behavior.

Surprisingly, these two groups of neurons appear to interfere with each other's function: the activation of social neurons inhibits self-grooming behavior, while the activation of self-grooming neurons inhibits social behavior. Thus these two groups of neurons seem to function like a seesaw, one that controls whether mice interact with others or instead focus on themselves. It was completely unexpected that the two groups of neurons could be distinguished by whether they were excitatory or inhibitory. "If there was ever an experiment that 'carves nature at its joints,'" says Anderson, "this is it."

This seesaw circuit, Anderson and his colleagues say, may have some relevance to human behavioral disorders such as autism.

"In autism," Anderson says, "there is a decrease in social interactions, and there is often an increase in repetitive, sometimes asocial or self-oriented, behaviors"—a phenomenon known as perseveration. "Here, by stimulating a particular set of neurons, we are both inhibiting social interactions and promoting these perseverative, persistent behaviors."

Studies from other laboratories have shown that disruptions in genes implicated in autism show a similar decrease in social interaction and increase in repetitive self-grooming behavior in mice, Anderson says. However, the current study helps to provide a needed link between gene activity, brain activity, and social behaviors, "and if you don't understand the circuitry, you are never going to understand how the gene mutation affects the behavior." Going forward, he says, such a complete understanding will be necessary for the development of future therapies.

But could this concept ever actually be used to modify a human behavior?

"All of this is very far away, but if you found the right population of neurons, it might be possible to override the genetic component of a behavioral disorder like autism, by just changing the activity of the circuits—tipping the balance of the see-saw in the other direction," he says.

The work was funded by the Simons Foundation, the National Institutes of Health and the Howard Hughes Medical Institute. Caltech coauthors on the paper include Hong, who was the lead author, and graduate student Dong-Wook Kim.

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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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Biology Made Simpler With "Clear" Tissues

In general, our knowledge of biology—and much of science in general—is limited by our ability to actually see things. Researchers who study developmental problems and disease, in particular, are often limited by their inability to look inside an organism to figure out exactly what went wrong and when.

Now, thanks to techniques developed at Caltech, scientists can see through tissues, organs, and even an entire body. The techniques offer new insight into the cell-by-cell makeup of organisms—and the promise of novel diagnostic medical applications.

"Large volumes of tissue are not optically transparent—you can't see through them," says Viviana Gradinaru (BS '05), an assistant professor of biology at Caltech and the principal investigator whose team has developed the new techniques, which are explained in a paper appearing in the journal Cell. Lipids throughout cells provide structural support, but they also prevent light from passing through the cells. "So, if we need to see individual cells within a large volume of tissue"—within a mouse kidney, for example, or a human tumor biopsy—"we have to slice the tissue very thin, separately image each slice with a microscope, and put all of the images back together with a computer. It's a very time-consuming process and it is error prone, especially if you look to map long axons or sparse cell populations such as stem cells or tumor cells," she says.

The researchers came up with a way to circumvent this long process by making an organism's entire body clear, so that it can be peered through—in 3-D—using standard optical methods such as confocal microscopy.

The new approach builds off a technique known as CLARITY that was previously developed by Gradinaru and her collaborators to create a transparent whole-brain specimen. With the CLARITY method, a rodent brain is infused with a solution of lipid-dissolving detergents and hydrogel—a water-based polymer gel that provides structural support—thus "clearing" the tissue but leaving its three-dimensional architecture intact for study.

The refined technique optimizes the CLARITY concept so that it can be used to clear other organs besides the brain, and even whole organisms. By making clever use of an organism's own network of blood vessels, Gradinaru and her colleagues—including scientific researcher Bin Yang and postdoctoral scholar Jennifer Treweek, coauthors on the paper—can quickly deliver the lipid-dissolving hydrogel and chemical solution throughout the body.

Gradinaru and her colleagues have dubbed this new technique PARS, or perfusion-assisted agent release in situ.

Once an organ or whole body has been made transparent, standard microscopy techniques can be used to easily look through a thick mass of tissue to view single cells that are genetically marked with fluorescent proteins. Even without such genetically introduced fluorescent proteins, however, the PARS technique can be used to deliver stains and dyes to individual cell types of interest. When whole-body clearing is not necessary the method works just as well on individual organs by using a technique called PACT, short for passive clarity technique.

To find out if stripping the lipids from cells also removes other potential molecules of interest—such as proteins, DNA, and RNA—Gradinaru and her team collaborated with Long Cai, an assistant professor of chemistry at Caltech, and his lab. The two groups found that strands of RNA are indeed still present and can be detected with single-molecule resolution in the cells of the transparent organisms.

The Cell paper focuses on the use of PACT and PARS as research tools for studying disease and development in research organisms. However, Gradinaru and her UCLA collaborator Rajan Kulkarni, have already found a diagnostic medical application for the methods. Using the techniques on a biopsy from a human skin tumor, the researchers were able to view the distribution of individual tumor cells within a tissue mass. In the future, Gradinaru says, the methods could be used in the clinic for the rapid detection of cancer cells in biopsy samples.

The ability to make an entire organism transparent while retaining its structural and genetic integrity has broad-ranging applications, Gradinaru says. For example, the neurons of the peripheral nervous system could be mapped throughout a whole body, as could the distribution of viruses, such as HIV, in an animal model.

Gradinaru also leads Caltech's Beckman Institute BIONIC center for optogenetics and tissue clearing and plans to offer training sessions to researchers interested in learning how to use PACT and PARS in their own labs.

"I think these new techniques are very practical for many fields in biology," she says. "When you can just look through an organism for the exact cells or fine axons you want to see—without slicing and realigning individual sections—it frees up the time of the researcher. That means there is more time to the answer big questions, rather than spending time on menial jobs."

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