Remembering Ray D. Owen (1915–2014)

Immunology pioneer Ray D. Owen, professor of biology, emeritus, at Caltech, passed away on Sunday, September 21 at the Californian-Pasadena Convalescent Hospital in Pasadena, California. He was 98.

Owen's major scientific contribution was his discovery, in 1945, of immunological tolerance in twin cattle. Using blood typing, he recognized that one of a set of fraternal twin cattle had no immune response to the foreign antigens (substances that provoke an immune response) introduced from their twins. The finding paved the way for the experimental induction of tolerance through immune suppression and for early tissue grafting—which initiated the era of organ transplantation—by Frank Macfarlane Burnet and Peter Brian Medawar, who received the Nobel Prize for the work in 1960. "In fact, Owen was the first to postulate that immunosuppressive treatments such as x-irradiation might allow incompatible transplants, and participated in the experiments in which bone-marrow transplants to irradiated recipients were first successfully demonstrated," says Elliot Meyerowitz, Caltech's George W. Beadle Professor of Biology.

Owen's later work included studies on human antibodies, blood-group antigens, the evolution of immune systems, and the genetic analysis of the major histocompatibility complex—a large family of genes that plays an important role in the immune system and autoimmunity—of the mouse. "He was, perhaps, the most outstanding immunologist of his generation," wrote Leroy Hood (BS '60, PhD '68), cofounder of the Institute for Systems Biology in Seattle, inventor of the automated DNA sequencer, and a former student—and later colleague—of Owen's at Caltech.

"Ray promoted and loved genetics, as much or even more so than immunology," says Mitch Kronenberg (PhD '83), president and chief scientific officer at the La Jolla Institute for Allergy and Immunology, Hood's former grad student and postdoc and a self-described "trainee" of Owen's. "In a sense, he was a pioneer in perceiving the importance of genetic variability as a determinant of biologic complexity, long before the advent of next-generation DNA sequencing and the concept of personalized medicine.

"He was amazing in that he never lost his interest in the progress of research," Kronenberg adds. "On occasion he would drop me a congratulatory note after reading a paper from my lab—what a thrill for me—even when he was well into his eighties. In an interview at age 95, he disputed the notion that everything important would soon be known, but instead strongly expressed his excitement about the frontiers of science."

Owen was born October 30, 1915, on a dairy farm in Genesee, Wisconsin. In 1937, he received a BS from Carroll College in Waukesha, Wisconsin—where he met June, his wife of 74 years, from whom he was inseparable; in 1941, he received a PhD in genetics from the University of Wisconsin. After working for two years as a postdoctoral researcher at the University of Wisconsin and as an assistant professor at the same institution, Owen took a position as an associate professor at Caltech in 1947; he was promoted to full professor in 1953 and became professor emeritus in 1983.

At Caltech, Owen also was noted for his dedicated teaching—he received an award for teaching excellence from the Associated
 Students of the California Institute of Technology (ASCIT); for his extraordinary commitment to mentoring young scientists; and for his administrative roles. He served as chairman of the Division of Biology from 1961 to 1968 and as vice president for student affairs and dean of students from 1975 to 1980.

He chaired the ad hoc "Committee on the Freshman Year" that recommended the pass/fail grading system for freshmen (designed to make the transition to Caltech less "traumatic," Owen once noted), adopted in 1964, and the introduction of electives into the previously rigid freshman curriculum. Under Owen's leadership, the committee also spearheaded the effort to admit female undergraduate students to Caltech; in 1970, the first female undergraduates enrolled at the Institute.

Many of his former students and colleagues recall that Owen did not just help open the doors to female students, he actively assisted and nurtured them, both professionally and personally. As one of those first undergrads later described it, "However well women were mainstreamed into the biological sciences, women undergrads were definitely minorities at Caltech. We were beset by a constant stream of fellow undergrads, grad students, TAs, postdocs and professors who appeared, called, wrote, popped into our dorm rooms, sent notes, flowers and gifts, solicited dates, proposed marriage, pledged undying love and devotion and everything in between! Then, we were trotted out to render the female perspective to faculty, alumni, parents' groups, news media, potential students or donors, trustees, and other luminaries. We often suffered from too much attention. Ray's calming presence was an antidote for those stresses. His maturity and his giving, caring attitude, gave all of his students a restful haven in which to develop their science craft."

Over more than six decades at Caltech, Owen was a beloved mentor not just to those first female students and subsequent generations of male and female undergrads, but also to graduate students, postdocs, and young faculty.

"I believe that much of the wonderful scientific atmosphere I have the privilege of enjoying at Caltech is due, in large part, to the efforts of Ray Owen," says Pamela Bjorkman, Caltech's Max Delbrück Professor of Biology.

"Dr. Owen's belief in the genderlessness and color-blindness of intelligence and creativity has encouraged men and women to excel in their chosen fields," wrote Leonore Herzenberg, professor of genetics at the Stanford School of Medicine, in a letter recommending Owen for a lifetime mentoring award. "The success of this mentoring can be measured in terms of the contributions made by his students and many others who came in contact with him. In addition, it can be measured by the way in which those people for whom Dr. Owen served as a mentor have tended, like him, to tithe a portion of their time to help others achieve academic excellence."

Noted Roger Perlmutter, executive vice president and president of Merck Research Laboratories and a senior research fellow at Caltech in the early 1980s: "Ray was then, and had been for many years, the very heart and soul of the Caltech biology division. His office in the basement of Kerckhoff, decorated with trophies courageously secured and lovingly forwarded by admiring former trainees, and masses of postcards from students and friends, served as an informal counseling suite. Ray's door was always open, tea and coffee were always available, and there was a steady stream of students who stopped by to discuss results, to seek advice, or simply to chat . . . Ray had time for everyone."

"Ray was a true gentleman," says Kronenberg. "Although he could be critical about a scientific approach or finding, his comments would be tinged with gentle humor or light sarcasm. He did not gossip, it was never a personal matter for him, and he never expressed disdain or a lack of respect for anyone. He seemed untouched by envy or enmity; these were emotions he just did not express."

Owen, who coauthored General Genetics—the most widely used genetics textbook of its time—received the Thomas Hunt Morgan Medal from the Genetics Society of America, given for lifetime achievement in the field of genetics, in 1993. He was awarded the Mendel Medal of the Czechoslovak Academy of Sciences in 1966, earned honorary degrees from Carroll College and the University of the Pacific, and was a member of the National Academy of Sciences (NAS), the American Academy of Arts and Sciences, and the American Philosophical Society, among others.

In addition, Owen was president of the Genetics Society of America in 1962, a member of the Genetics Study Section of the National Institutes of Health (NIH) from 1958 to 1961 and its chairman from 1961 to 1963, a member of the Immunobiology Study Section of the NIH from 1966 to 1967 and its chairman from 1967 to 1970, chairman of the Genetics Section of the NAS from 1969 to 1972, and a scientist-member of the three-person President's Cancer Panel from 1972 to 1975, where he served as an advisor to Presidents Nixon and Ford.

In his personal life, Owen professed of a love of his family; his home, located a short walk from the Caltech campus, where he often conducted evening classes for students with his wife June serving cookies; his garden (camellias and chrysanthemums were his specialty); his travels and his friends in the international community of scientists; his research; his teaching; and his students.

"I think, as I look back at it," said Owen, in a 1983 interview for the Caltech Oral History Project, "I've had a very fortunate and satisfying life. But when you get a letter from a student or get some word back about somebody who's gone out into the world, and it appears that you have done something to influence a young person's life or made a difference in his life for the good—I think that's the most ego-rewarding aspect of one's life. And I've had a good many opportunities along those lines."

Owen was predeceased by his wife, June, in 2013, who also passed away at the Californian-Pasadena Convalescent Hospital, and by a son, Griffith Hugh, who died in a car accident in 1970. He is survived by his son David.

A memorial service honoring both Ray and June is being planned by the Division of Biology and Biological Engineering. The details will be announced.

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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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Wednesday, September 24, 2014
Annenberg Lecture Hall

A chance to meet Pasadena Unified School District Leadership

Wednesday, September 10, 2014
Avery Dining Hall

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

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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Friday, October 10, 2014
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