Caltech Researchers Obtain First Brain Recordings from Behaving Fruit Flies

Research opens a new avenue for linking genes to behavior

PASADENA, Calif.—Researchers at the California Institute of Technology (Caltech) have obtained the first recordings of brain-cell activity in an actively flying fruit fly. 

The work—by Michael Dickinson, the Esther M. and Abe M. Zarem Professor of Bioengineering, with postdoctoral scholars Gaby Maimon and Andrew Straw—suggests that at least part of the brain of the fruit fly (Drosophila melanogaster) "is in a different and more sensitive state during flight than when the fly is quiescent," Dickinson says. 

A paper describing the research appeared February 14 in the advance online edition of Nature Neuroscience

"Prior work on fruit flies has led to many important breakthroughs in biology. For example, the fact that genes reside on chromosomes and our understanding of how genes control development both emerged from experiments on fruit flies," Maimon says. "New research hopes to use these tiny insects to help determine how neurons give rise to complex behavior. This effort is helped by the fact that it is easy to manipulate the genes of fruit flies, but one problem remains: these insects are really, really tiny, which means it is very difficult to record from their brain during active behaviors such as flight."

"Researchers have recorded the neural-cell activity of fruit flies before, but only in restrained preparations—animals that had been stuck or glued down," Dickinson explains. "Gaby was able to develop a preparation where the animal is tethered"—its head clamped into place—"but free to flap its wings." By slicing off a patch of the hard cuticle covering the brain, "we were able to target our electrodes onto genetically marked neurons," he says. 

A puff of air was used to spur the flies into flapping their wings, while electrodes measured the activity of the marked neurons and high-speed digital cameras simultaneously recorded the flies' behavior. (View a video at http://images.caltech.edu/podcasts/research_news/FlyFlying_wCell.mov )

In particular, the researchers focused on those neurons in the fly's visual system that keep the animal flying stably. "These cells basically help the fly detect when its body posture changes," Dickinson says. "The signals from these cells are thought to control tiny steering muscles that then change the pattern of wing motion and bring the animal back into equilibrium." 

In their experiments, the researchers discovered that when the animals began to fly, the visual cells immediately ramped up their activity. "The neurons' responses to visual motion roughly double when the flies begin to fly, which suggests that the system is more sensitive during flight," Dickinson says. "The increase is very abrupt. It's not at all a subtle change, and so we suspect that there is a neurochemical quickly released during flight that sets the animal's brain in this different state."

Previous studies in locusts—which are far bigger and thus far easier to study—had suggested the existence of this effect. However, the genetics of locusts are not nearly as well understood as those of Drosophila, which has made it impossible to pinpoint the genetic basis for the phenomenon.

In Drosophila, Dickinson says, it now should be possible to "figure out specifically what causes the change in sensitivity. Is the system turned off when the fly is on the ground? What neurochemicals are involved? Now we can start to use the genetic tricks that are available in fruit flies to get a better idea of what is going on." 

Maimon adds "Our work on Drosophila is of general interest because sensory neurons in many species—including birds, rodents, and primates—change their response strength depending on the behavioral state of the animal, but why these changes in sensitivity take place is not entirely clear."

In addition, the researchers plan to use their tethered-flight system to record the activity of other types of cells, including olfactory and motor cells, to determine if these also behave differently during flight and when flies are at rest.

"The question is, 'Is the entire brain completely different in flight?'" Dickinson says. "We suspect that this phenomenon is not unique to the visual cells we have studied. Most cells care whether the animal is flying or not."

The work in the paper, "Active flight increases the gain of visual motion processing in Drosophila," was supported by the National Science Foundation and a Caltech Della Martin Fellowship.

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Caltech Researchers Revise Long-Held Theory of Fruit-Fly Development

Research shows that the influence of a key transcription factor is less widespread than thought, and varies over time

PASADENA, Calif.—For decades, science texts have told a simple and straightforward story about a particular protein—a transcription factor—that helps the embryo of the fruit fly, Drosophila melanogaster, pattern tissues in a manner that depends on the levels of this factor within individual cells.

"For 20 years, this system of patterning has been used in textbooks as a paradigm for patterning in embryos, controlled by transcription factors," says Angelike Stathopoulos, assistant professor of biology at the California Institute of Technology (Caltech).

Now Stathopoulos and her Caltech colleagues, reporting in the online edition of the Proceedings of the National Academy of Sciences (PNAS), have called that paradigm into question, revealing a tale that is both more complicated and potentially more interesting than the one previously described.

The football-shaped embryo of the fruit fly has a dorsal (back/top) side and a ventral (front/bottom) side. During development, the cells in each of these regions begin to differentiate and take on specific, specialized roles.

Those decisions are influenced, at least in part, by chemical signals in the cells' environment, including signals called transcription factors—proteins that, by promoting the transcription of particular DNA sequences, regulate whether specific genes are turned on or off.

In Drosophila, the textbooks said, decisions in the early embryo are made by a transcription factor called Dorsal (which, confusingly, is found primarily in the cells in the ventral part of the embryo, and is absent in those in the dorsal part). Dorsal was said to be the key determinant of the ultimate fate of the cells in which it is present—as long as it is present in high enough concentrations to be noticed by the nuclei.

"There's a threshold," says Caltech postdoctoral scholar Greg Reeves. "Depending on the level of the signal, the decision of whether to differentiate one way or another is made."

And the strength of the signal the nuclei are exposed to, he says, is determined at least in part by their position; the signal changes on a gradient along the dorsal-ventral axis of the embryo that goes from high to nearly nonexistent levels of the factor.

"The gradient sets up boundaries of gene expression," explains Reeves. "It's like a radio-tower signal; you can tell how far away you are from the tower by how clear a signal you receive. At some distance, you won't be able to hear the signal at all."

But if you look closely at the patterning that occurs in the Drosophila embryo, Stathopoulos notes, this theory that Dorsal is the main determinant of patterning falls short of explaining the whole process. That's because, as their study showed, some of the nuclei in the embryo are responding to a signal they shouldn't be able to hear.

"These are places where the levels of the factor flatline," she points out, "and yet you still have patterns forming there."

Why hadn't this disparity been seen before? Because previous measurements in the Drosophila embryo had looked at overall levels of Dorsal—at its levels not only in the nuclei, where the transcription factor does its work, but in the cytoplasm as well. Because cytoplasmic levels of Dorsal rise when nuclear levels fall, cells with little to no working transcription factor in the nucleus may still show significant levels of the factor overall, due to its presence in the cytoplasm.  Thus, researchers have always believed that Dorsal signaling is critical in a large portion of the embryo.

The Caltech team—which included Stathopoulos, Reeves, and former Caltech graduate student Louisa Liberman, now at Duke University—showed in their PNAS paper that the actual signaling from nuclear Dorsal works over quite a short range. "We think, now, that it's only controlling half the patterning that goes on in these embryos," Stathopoulos says.

In their paper, the Caltech team looked not only at the nuclear levels of the transcription factor, but at how those levels change throughout the early stages of embryonic development. They found that Dorsal provides constantly changing positional information to the nuclei, raising the question of how such a dynamic signal could be interpreted.

"We've shown that, instead of a steady amount of signal telling the cells what to do, the signal changes over time," says Reeves.

Still, the researchers say that Dorsal clearly is critical for patterning in parts of the embryo. "It's just not controlling all of the domains all of the time," says Stathopoulos.

The researchers propose that the key to understanding patterning in Drosophila embryos is to identify other transcription factors that work with Dorsal to control patterning in dorsal regions. Even if the levels of Dorsal flatline here, these other factors may provide the necessary additional information cells need to decipher their relative positions.  The group is actively searching for such transcription factors.

The work described in the PNAS paper, "Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila," was supported by grants from the National Institute of Health's National Institute of General Medical Sciences and the Jane Coffin Childs Memorial Fund for Medical Research.

The paper's abstract can be found at http://www.pnas.org/content/early/2009/12/15/0906227106.abstract.

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Lori Oliwenstein
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Caltech, UCLA Launch Joint Center for Translational Medicine

Broad Foundation gift will help guide new research toward clinical applications

Pasadena and Westwood, Calif.-The California Institute of Technology (Caltech) and the University of California, Los Angeles (UCLA), have announced the establishment of the Joint Center for Translational Medicine (JCTM), which will advance experimental research into clinical applications, including the diagnosis and therapy of diseases such as cancer.

Initial funding for the new center comes from a two-year, $5 million gift from The Eli and Edythe Broad Foundation.

"The strengths of both institutions will be brought together in this new center to help move discoveries from research into clinical practice," says David Baltimore, Nobel laureate and the Robert Andrews Millikan Professor of Biology at Caltech. Baltimore will be the center's director.

Owen Witte, a Howard Hughes Medical Institute investigator and director of the Broad Stem Cell Research Center at UCLA, will serve as deputy director of the new center. The center will build upon UCLA's strength and international reputation in conducting translational research, including development of the molecularly targeted drugs Herceptin, Gleevec, Avastin, and Sprycel. The program, Witte says, will take the best science from the laboratories at Caltech and UCLA and will transform it into new and more effective therapies for debilitating diseases.

"The move to combine the expertise and experience at these two premier research institutions will set the standard for the Los Angeles area," he adds. "This new center is the natural evolution of several research collaborations between UCLA and Caltech and will result, we hope, in many new options for people with a host of diseases."

The first project of the center will be the investigation into a potentially revolutionary treatment for late-stage melanoma, in which the body's killer immune cells are programmed to recognize and destroy tumor cells. This work started in 2006 as joint research between Caltech and UCLA and led to the idea for the center. The melanoma research has already advanced to ongoing clinical trials involving a half-dozen patients.

"We saw the success of the melanoma research program and asked ourselves, 'Is there something else we can do for other diseases?'" says Baltimore. "The Broad Foundation has always looked for programs that elevate the quality of research in Los Angeles, and this new center will go a long way toward enhancing the region's reputation for medical research."

"We have a great deal of admiration and respect for Dr. Baltimore and Dr. Witte, and this new center brings together some of the brightest minds in science and medicine," says Eli Broad, founder of The Eli and Edythe Broad Foundation and a major funder of the Broad Center for the Biological Sciences at Caltech, the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at the University of California, San Francisco, and the Broad Institute for Biomedical Research in partnership with Harvard and MIT.  

In addition to Baltimore's research, the work of Caltech's Mark Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering, and James Heath, the Elizabeth W. Gilloon Professor and professor of chemistry, is already generating joint translational opportunities with UCLA. Researchers at UCLA involved in joint efforts with the three Caltech labs include Antoni Ribas, associate professor of hematology/oncology; Caius Radu, assistant professor of molecular and medical pharmacology; and Michael Phelps, the Norton Simon Professor, chair of the molecular and medical pharmacology department, and inventor of positron emission tomography scanning.

"The hope is to bring in additional researchers who will use the center as a vehicle for moving their work from a research phase to one with clinical potential," says Baltimore.

To help foster that process, the center will immediately begin a Translational Acceleration Grants (TAG) program, which will offer seed grants for work by Caltech and UCLA faculty members. The awards will support highly competitive research proposals that have the potential to move toward clinical applications. Each TAG will consist of an initial one-year award of up to $100,000 to support direct research costs, with the possibility of a one-year extension.

An advisory board will assist in the grant review process and in making policy decisions for the new center. Six members, three from each institution, sit on the board. The Caltech board members are Peter Dervan, the Bren Professor of Chemistry; Ray Deshaies, professor of biology; and Scott Fraser, the Anna L. Rosen Professor of Biology and professor of bioengineering, and director of the Donna and Benjamin M. Rosen Bioengineering Center. The UCLA board members are Judith C. Gasson, director of UCLA's Jonsson Comprehensive Cancer Center; Donald B. Kohn, director of the Human Gene Medicine Program; and Bruce Dunn, the Nippon Sheet Glass Professor of Materials Science and Engineering.

"We expect this new center to strengthen our current work on both campuses and identify future translational work," says Baltimore, "as well as create stronger ties between Caltech and UCLA." 

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Caltech Scientists Discover Aggression-Promoting Pheromone in Flies

Study, published in Nature, also identifies pheromone-detecting neurons in fly's antenna

PASADENA, Calif.—Have you ever found yourself struggling to get your order taken at a crowded bar or lunch counter, only to walk away in disgust as more aggressive customers elbow their way to the front? It turns out that flies do much the same thing, according to biologists from the California Institute of Technology (Caltech).

Reporting in the advance online edition of the journal Nature, the scientists say they have identified an aggression-promoting pheromone that controls such behaviors, and have pinpointed the neurons in the fly's antenna that detect this pheromone and relay the information to the brain to elicit aggression. Their results provide an important first step toward unraveling the mystery of how aggression—an innate (unlearned) behavior—is hardwired into the brain by an animal's genes.

Pheromones—specific chemicals used by a particular species to communicate and to control their behavior—have been identified in the scent glands of other insect species, such as ants and beetles, and have been shown to elicit aggressive behavior when presented in synthetic form to the insects. It has been difficult, however, to prove that the insects normally use these pheromones to control their aggressive behavior, notes study coauthor David Anderson, Caltech's Seymour Benzer Professor of Biology and a Howard Hughes Medical Institute investigator.

"Obtaining such proof required the ability to experimentally interfere with the insects' capacity to sense the pheromone," he explains. "And that, in turn, necessitated identification of the receptor molecules that detect aggression pheromones, and of the olfactory sensory neurons that express these receptors."

As it turns out, the only insect in which these conditions could be met was the vinegar fly, Drosophila melanogaster, explains Liming Wang, a graduate student in Anderson's lab and the Nature paper's first author. "The genetic/molecular architecture of the olfactory system in Drosophila is well understood," Wang explains. "Thus, one can easily test whether a specific olfactory receptor, and the sensory neurons expressing it, are involved in a given behavior."

Wang discovered that 11-cis-vaccenylacetate (cVA)—a pheromone present in the male fly's cuticle—"robustly promotes aggression in pairs of male flies," Anderson says.

Aggressive behavior in Drosophila consists of brief "lunges" in which one fly rears up on its hind legs and snaps down with its forelegs on its opponent.

When Wang and Anderson added synthetic cVA to an "arena" in which combatant flies were tested, the frequency of lunges was dramatically increased. Building upon earlier work from other laboratories that had identified the receptors for this pheromone, Wang next showed that silencing the neurons in the fly's antenna that contain these specific receptors could block the ability of synthetic cVA to promote aggression.

These findings allowed Wang and Anderson to test whether flies can actually detect the release of this pheromone from other flies—and whether such detection promotes aggression.

To do this test, they trapped between 20 and 100 "donor" male flies—so called because they "donate" the volatile pheromones into the surrounding environment—in a tiny cage surrounded by a fine mesh screen. The screen allowed pheromones to escape, but kept the donor flies inside.

The researchers then measured the effect these donor flies had on the aggressiveness of a pair of "tester" male flies placed on top of the cage. The tester flies were close enough to sense the pheromone, but were prevented from coming into contact with the donor males by the mesh screen. "Remarkably," says Anderson, "the presence of the caged donor flies strongly increased aggression between the tester flies, and this aggression-promoting effect increased with a higher number of donor male flies."

Most importantly, the effect of the donor flies on the aggressiveness of the tester flies could be blocked by inactivating, in the tester flies' antennae, the neurons that sense the aggression pheromone.

"These experiments suggested that the presence of high densities of male flies in a local environment can indeed promote aggression through their release of cVA and its detection by other flies," Wang explains.

Based on these findings, Wang and Anderson began to speculate whether this pheromone might play a role in limiting the population density of male flies in a given environment. Normally, male flies are attracted to food in order to feed and because it gives them the opportunity to mate with feeding female flies. If the density of male flies on a food resource is too high, however, the competition between the flies might prevent feeding and mating. Since aggressive flies tend to chase away their competitors, an aggression-promoting pheromone should tend to keep the density of flies from becoming too high. 

Wang tested this hypothesis by allowing a small number of flies to compete for a limited supply of food, while genetically manipulating their cVA-receptor neurons to make those neurons hyperactive.

Surprisingly, says Anderson, the flies with the hyperactive neurons quickly dispersed, leaving the food resource behind. "They fought one another until a dominant fly became 'king of the hill' and drove the other flies away," he explains.

"In contrast," Anderson adds, "flies whose genes weren't manipulated in this way ate happily together, like cows grazing placidly on an alpine meadow."

According to Wang and Anderson, these results suggest that when the population of male flies reaches a high-enough density, the concentration of cVA rises to a level that promotes aggression, forcing some of the flies off the food. The departure of those flies causes the ambient concentration of the pheromone to decrease, thereby decreasing aggression. "Once this occurs," says Wang, "the population becomes stabilized at an optimal density until more flies become attracted to the food, and the cycle repeats itself."

Although their observations of this behavior were made under artificial laboratory conditions, the researchers believe that it should be possible to test their hypothesis in the wild.

The discovery of the fly's response to an aggression pheromone raises a number of intriguing questions, such as whether this fly pheromone might be sensed by humans. This is very unlikely, says Anderson, as pheromones have evolved as a "private" chemical communication channel within a given species.

But that does not mean humans lack aggression pheromones altogether, he notes. After all, aggression-promoting pheromones have been discovered in mice, which are evolutionarily closer to humans than flies. It is possible, therefore, that humans have their own aggression-promoting pheromones.

"Do these pheromones keep the lines from getting too long at a crowded lunch counter, as irate patrons jockey for position in the queue and some walk away in frustration?" Anderson asks. "Only time will tell."

The work described in the Nature paper, "Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila," was funded by grants from the National Science Foundation and the Howard Hughes Medical Institute.

After publication, the paper's abstract can be accessed on the Nature website

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Caltech Scientists Show How Ubiquitin Chains are Added to Cell-Cycle Proteins

Findings could one day lead to the development of targeted cancer therapies

PASADENA, Calif.—Researchers from the California Institute of Technology (Caltech) have been able to view in detail, and for the first time, the previously mysterious process by which long chains of a protein called ubiquitin are added by enzymes called ubiquitin ligases to proteins that control the cell cycle. Ubiquitin chains tag target proteins for destruction by protein-degrading complexes in the cell.

"We found that ubiquitin ligases build ubiquitin chains very rapidly by transferring ubiquitins one at a time," says Raymond Deshaies, professor of biology at Caltech and Howard Hughes Medical Institute investigator.

Their findings, and the innovative process by which they were obtained, are described in this week's issue of the journal Nature.

Ubiquitin is one of nature's most unusual proteins. Unlike most of its protein brethren, ubiquitin has to be physically attached to other proteins to do its job.

“As its name implies, ubiquitin is found in essentially every kind of eukaryotic cell," says Caltech graduate student Nathan Pierce, the Nature paper's lead author.

In their Nature paper, the Caltech team looked at the process of ubiquitylation, the method by which ubiquitin and ubiquitin chains are added to target proteins. The target proteins used in the study, cyclin E and β-Catenin, are both involved in controlling the cell cycle.

It was already known, Pierce explains, that the addition of a chain of four or more ubiquitins to a target protein marks that protein for annihilation.

The destruction of cyclin E is critical for the accurate replication of DNA, while the degradation of β-Catenin keeps cells from dividing during development at the wrong time. If β-Catenin is not degraded, cells proliferate excessively and become predisposed to tumorigenesis. Meanwhile, cells that don't degrade cyclin E accumulate DNA damage and mutations, which can help fuel the unchecked growth of a tumor.

It was also already known that ubiquitin chains are added to the protein using three different enzymes, dubbed E1, E2, and E3. Simply put, E1 activates ubiquitin for transfer, then passes it over to E2. E3 then gets into the act. A form of E3 called a RING ligase (RING stands for "really interesting new gene") plays a key role in the tagging of cyclin E and β-Catenin; according to Pierce, the RING ligase  "simultaneously binds to E2 and the target protein (like cyclin E), and then causes E2 to transfer the ubiquitin to the target protein."

Despite all of this knowledge, one question has remained: is the chain transferred to the protein in an already assembled form, or are the ubiquitins moved over one at a time?

"The process is so complicated and so fast," Pierce notes, "that we weren't able to see how the chain is actually built."

To address that issue, Pierce created a sort of biological stop-motion animation that allowed the Caltech team to watch every step in the transfer of ubiquitin from E2 onto the cyclin E protein substrate.

"We devised methods to take snapshots of ubiquitin ligase reactions at a rate of up to 100 'pictures' every second," says Deshaies. "This enables us to see things that would normally evade detection. "

Previous studies had looked at the reaction on the scale of seconds or minutes, Pierce adds. But through an innovative use of a laboratory tool called a quench-flow machine—a machine that allows for extreme precision in the stopping, or "quenching," of a reaction—the team was able to look at what was going on over intervals of just 10 milliseconds in both yeast and human proteins.

"Prior methods did not have sufficient time resolution to see what was going on," says Deshaies. "It's as if you gave an ice-cream cone to a kid and took pictures every minute. You would see the ice cream disappear from the first photo to the next, but since the pictures are too far apart in time, you would have no idea whether the child ate the ice cream one bite at a time, or swallowed the entire scoop in one gulp."

The new method revealed the biological equivalent of small, single bites of ice cream. "Using our approach," Deshaies says, "we could see that our ubiquitin ligase builds ubiquitin chains one ubiquitin at a time."

"Once we knew what the steps were, we calculated the rates at which they occur," adds Pierce. "And from those rates, we were able to really describe the biology of how this system works."

The quest doesn't stop there, of course. "One thing we have to understand now is, how do ubiquitin ligases achieve the speeds that they do?" asks Deshaies. "What special mechanisms do they have to enable them to build chains rapidly? And the flip side of the coin: What sets the speed limit? Why can't our ubiquitin ligase work even faster?"

A recent paper published in the journal Cell by Gary Kleiger, a postdoctoral scholar in the Deshaies lab, answered some of these speed-related questions. By measuring the rates at which E2 and E3 interacted with one another, Kleiger was able to demonstrate their unusually fast association—faster than predicted for normal proteins. E2 and E3 use oppositely-charged surfaces to attract each other, thereby speeding up the formation of a functional complex of the two proteins. This helps explain how the rapid sequential additions of ubiquitin described in the Nature paper are possible.

Gaining these kinds of insights into the ubiquitin system is important, Deshaies says, because ubiquitin ligases play a critical role in a number of human diseases, including cancer, due to their role in the regulation of the cell cycle.

"Once we understand these aspects of how ubiquitin ligases work, and what limits their speed, we will be in an excellent position to think about how we might develop drugs that attack the ligase's Achilles' heel, to make its slowest step even slower," he says. "If we can slow down ubiquitin ligases enough, they may become too slow to get their job done—to build chains—in the time available to them to do so. Being able to develop drugs to block their function would open up a new frontier in medicine."

"We were able to invent HIV therapeutics because we understand how reverse transcriptase works," adds Pierce. "The same applies here. We need to understand how these enzymes work if we're ever going to be able to target them with therapeutics."

In addition to Pierce and Deshaies, other researchers involved in the study included Kleiger and Shu-ou Shan, assistant professor of chemistry at Caltech.

The work described in the Nature paper, "Detection of Sequential Polyubiquitylation on a Millisecond Time-Scale," was funded by a Gordon Ross Fellowship, National Institutes of Health training and research grants, and the Howard Hughes Medical Institute.

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Lori Oliwenstein
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Caltech Scientists Find Emotion-like Behaviors, Regulated by Dopamine, in Fruit Flies

Finding may provide new insights into the neurological basis of ADHD, learning deficits, and more

PASADENA, Calif.—Scientists at the California Institute of Technology (Caltech) have uncovered evidence of a primitive emotion-like behavior in the fruit fly, Drosophila melanogaster.

Their findings, which may be relevant to the relationship between the neurotransmitter dopamine and attention deficit hyperactivity disorder (ADHD), are described in the December issue of the journal Neuron.

The Drosophila brain contains only about 20,000 neurons and has long been considered a powerful system with which to study the genetic basis of behaviors such as learning and courtship, as well as memory and circadian rhythms. What hasn't been clear is whether the Drosophila brain also could be used to study the genetic basis of "emotional" behaviors.

"Such studies are important," says David Anderson, Caltech's Seymour Benzer Professor of Biology and a Howard Hughes Medical Institute investigator, "because it's believed that abnormalities in these types of behaviors may underlie many psychiatric disorders."

Most of the genes found in the fruit fly—more accurately referred to as the vinegar fly—are found in humans as well, including those neurons that produce brain chemicals like dopamine and serotonin, which have been implicated in psychiatric disorders.

In their Neuron paper, the Caltech team—led by postdoctoral fellow Tim Lebestky—found that a series of brief but brisk air puffs, delivered in rapid succession, caused flies to run around their test chamber in what Anderson calls a "frantic manner." This behavior persisted for several minutes after the last of the puffs.

"Even after the flies had 'calmed down,'" he adds, "they remained hypersensitive to a single air puff."

To quantify the flies' behavior, Anderson's group collaborated with Pietro Perona, the Allen E. Puckett Professor of Electrical Engineering at Caltech. Together with his students, Perona developed an automated machine-vision-based system to track the movement of the flies, and derived a simple mathematical model to fit the movement data and to extract metrics that described various aspects of the flies' responses under different conditions.

The researchers used this test to search for flies with an abnormally exaggerated hyperactivity response; genetic studies of these flies revealed that a mutation in a dopamine receptor (a mutation that eliminates the receptor) produced the aberrant behavior. Flies with this dopamine-receptor mutation were hypersensitive to the air puffs, and took much longer to calm down than did "normal" flies without the mutation.

What is surprising about this result, notes Lebestky, “is that previous studies in both flies and vertebrates had suggested that dopamine promotes activity, but our experiments uncovered a function of dopamine in the opposite direction."  Because removing the receptor causes hypersensitivity to the air puffs, these results “suggested that dopamine actively inhibits the hyperactivity response,” Lebestky says.

This observation suggested a possible link to ADHD, a behavioral disorder characterized by impulsivity, hyperactivity, and short attention span. Humans with the disorder often take drugs, such as Ritalin, that increase levels of brain dopamine in order to reduce hyperactivity.

The ways the mutant flies respond to the air puffs is, moreover, "reminiscent of the way in which individuals with ADHD display hypersensitivity to environmental stimuli and are more easily aroused by such influences," says Anderson. Importantly, ADHD has been genetically linked to abnormalities of the dopamine system in humans, further strengthening the analogy between the mutant flies and this psychiatric disorder.

There is also another possible link: some individuals with ADHD display learning disabilities. Similarly, researchers from Pennsylvania State University—who collaborated on the Neuron study—have shown previously that flies with the same dopamine receptor mutation are unable to learn to associate a particular odor with an electric shock, and do not avoid the odor when subsequently tested. (Flies without the mutation quickly learn to make the association.)

It is often assumed that because individuals with ADHD are hyperactive and easily distracted, they have difficulty learning. The Caltech group showed, however, that hyperactivity and learning disabilities are not causally related in flies bearing the dopamine receptor mutation, thereby disproving the theory—at least in flies.

"We could separately 'rescue' the hyperactivity and learning deficits in a completely independent manner," says Anderson, "by genetically restoring the dopamine receptor to different regions of the fly's brain."

Thus, in dopamine-receptor-mutant flies, hyperactivity does not seem to cause learning deficits. Instead, these two "symptoms" reflect independent effects of the mutation that manifest themselves in distinct brain regions. "Being able to observe and manipulate the different neural substrates for learning and arousal will hopefully give us a unique method for identifying new molecular pathways that could be investigated and validated in higher organisms," says Lebestky.

This finding in flies, notes Anderson, raises the possibility that hyperactivity and learning deficits also may not be causally linked in humans with ADHD. If so, he says, it ultimately may prove more effective to develop drugs to treat these two symptoms separately, rather than trying to cure them both with the broad-spectrum pharmaceuticals currently available, which have many undesirable side effects.

"The finding that flies exhibit emotion-like behaviors that are controlled by some of the same brain chemicals as in humans opens up the possibility of applying the powerful genetics of this 'model organism' to understanding how these chemicals influence behavior through their actions on specific brain circuits," says Anderson. "While the specific details of where and how this occurs are likely to be different in flies and in humans, the basic principles are likely to be evolutionarily conserved, and may aid in our understanding of what goes wrong in disorders such as ADHD."

In addition to Lebestky, Anderson, and Perona, other researchers on the Neuron paper, "Two Different Forms of Arousal in Drosophila are Oppositely Regulated by the Dopamine D1 Receptor Ortholog DopR via Distinct Neural Circuits," are Jung-Sook Chang, Heiko Dankert, and Lihi Zelnik from Caltech; Young-Cho Kim and Kyung-An Han from Pennsylvania State University; and Fred Wolf from the University of California, San Francisco. Their work was funded by grants from the National Science Foundation and the Howard Hughes Medical Institute.

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Lori Oliwenstein
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Two Caltech Researchers Receive DARPA Young Faculty Awards

PASADENA, Calif.—The Defense Advanced Research Projects Agency (DARPA) has selected two researchers from the California Institute of Technology (Caltech) to participate in its Young Faculty Award (YFA) program.

Julia R. Greer, assistant professor of materials science, and Doris Tsao, assistant professor of biology, are among the 33 "rising stars" from 24 U.S. universities who each will receive grants of approximately $300,000 to develop and validate their research ideas over the next 24 months.

Greer joined the Caltech faculty in the Division of Engineering and Applied Science (EAS) in 2007 after receiving her PhD from Stanford University in 2005. In 2008, Greer made Technology Review's list of the world's top innovators under the age of 35 for her work with materials at the nanoscale level. In 2008, she received a Faculty Early Career Development award from the National Science Foundation.

Greer's YFA project is aimed at understanding and subsequently mimicking the superior mechanical robustness and strength of naturally occurring protective layers—such as nacre, or mother of pearl, a composite produced by some mollusks to line their inner shell—to create strong, ductile, damage-tolerant materials that maintain a relatively low density.

"Drawing inspiration from hard biological systems will allow us to gain insight into new physical phenomena operating in these materials, and to subsequently create innovative material systems with greatly amplified mechanical properties dictated by the choice of individual components, specific geometries, and microstructure in a truly across-scales fashion," says Greer.

One key objective of the work will be to fabricate a "brick-and-mortar" architecture using tiny plates of a metallic glass and ultrafine-grained ductile metal with nanoscale dimensions; this hierarchical architecture could then be used to fabricate new engineering composites with amplified strength and ductility.

"Greer's nature-inspired work exemplifies the cutting-edge research being carried out in the division," says Ares Rosakis, chair of EAS and Theodore von Kármán Professor of Aeronautics and Mechanical Engineering.

Tsao received her PhD from Harvard University in 2002, and came to Caltech in 2009 from the University of Bremen in Germany. She was named on Technology Review's 2007 list of top young innovators; in 2009, she became a John Merck Scholar, a Searle Scholar, and a Klingenstein Scholar.

Tsao uses functional magnetic resonance imaging, electrical recordings from single neurons, anatomical measurements, and mathematical modeling to understand how the brain identifies objects and reconstructs the three-dimensional world. Specifically, her proposed work will attempt to decipher the neural machinery underlying spatial navigation. 

"Navigation, which is the purposeful movement through space guided by sensory feedback and memory, is a defining behavior in all animals," says Tsao. Understanding the brain mechanisms responsible for navigation, she says, "constitutes a critical step toward designing artificial systems capable of human-like autonomous navigation. Such systems may be used to explore dangerous terrain and to perform tasks, such as clearing land mines, that could risk the loss of human life."

The objective of the DARPA YFA program is to identify and engage rising research stars in junior faculty positions in academia.  The YFA program provides funding mentoring, and industry and Department of Defense (DOD) contacts to these faculty members early in their careers, so that they can develop their research ideas in the context of the needs of the DOD. DARPA's long-term goal for this program is to develop the next generation of academic scientists, engineers, and mathematicians in key disciplines who will focus a significant portion of their careers on DOD and National Security issues.

The YFA awardees were chosen though a competitive selection process. Applicants were required to be untenured faculty at U.S. institutions within six years of appointment to a tenure-track position. Nearly 300 proposals were reviewed for the 2009 awards.

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Caltech Researchers Show Efficacy of Gene Therapy in Mouse Models of Huntington's Disease

Antibodies against intracellular targets can reduce symptoms, lengthen life span

Pasadena, Calif.—Researchers at the California Institute of Technology (Caltech) have shown that a highly specific intrabody (an antibody fragment that works against a target inside a cell) is capable of stalling the development of Huntington's disease in a variety of mouse models.

"Gene therapy in these models successfully attenuated the symptoms of Huntington's disease and increased life span," notes Paul Patterson, the Anne P. and Benjamin F. Biaggini Professor of Biological Sciences.

Patterson is the senior investigator on the study, which was published in the October 28 issue of the Journal of Neuroscience.

Huntington's disease is a neurodegenerative disorder with a genetic basis. The disorder has its roots in a mutation in a protein called huntingtin, or Htt. (The gene itself is also referred to as the huntingtin gene.)

All versions of the Htt gene have repeats of a particular trio of nucleotides—specifically, C, A, and G, which together code for the amino acid glutamine. In most people, that trio is repeated between 10 and 35 times. But in people who develop Huntington's disease, that genetic stutter goes on and on; they will have anywhere from 36 to upwards of 120 repeats.

The result of all these repeats? An abnormally long version of the Htt protein, which gets chopped up into smaller, toxic pieces and accumulates in nerve cells, debilitating them.

Enter Patterson group members Amber Southwell and Jan Ko, who began to look at the efficacy of two different intrabodies that had been shown, in cell cultures and fruit-fly models, to reduce the accumulation of toxic Htt protein. To see whether those effects would hold true in mammalian systems as well, the team tested the intrabodies in a series of five different mouse models of Huntington's.

One of the two intrabodies had some negative results, actually increasing Huntington's-related mortality in one model.

But the other intrabody—called Happ1—was an unqualified success, restoring motor and cognitive function to the mice, and reducing neuron loss as well as toxic protein accumulation. And in one model, it increased both body weight and life span.

Happ1 targets an amino-acid sequence unique to the Htt protein that is rich in the amino acid proline. Because of this, the action of Happ1 is expected to be extremely specific. "Our studies show that the use of intrabodies can block the parts of mutant huntingtin that cause its toxicity without affecting the wildtype, or normal, huntingtin—or any other proteins," says Patterson. In other words, he says, this has the potential to be the kind of "silver-bullet therapy" that many medical researchers look for.

This sort of research is of particular importance in the treatment of Huntington's disease, says Patterson. Despite the fact that this disorder has a single-gene origin, current treatments tend to address the symptoms of the disease, not its cause. That means it is currently impossible to prevent the disease from doing significant damage in the first place.

What's the next step in pursuit of this goal? "We need to improve the efficacy of the intrabody," Patterson says, "and we need to build a viral vector that can be controlled—induced and turned off—in case of unexpected side effects. This is a general goal shared by all types of experimental gene therapies."

The research described in the Journal of Neuroscience paper, "Intrabody Gene Therapy Ameliorates Motor, Cognitive, and Neuropathological Symptoms in Multiple Mouse Models of Huntington's Disease," was funded by the Hereditary Disease Foundation and the National Institute of Neurological Disorders and Stroke.

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Cells in developing tissue consider their signaling exposure history to determine location

Pasadena, Calif.—Researchers at the California Institute of Technology (Caltech) have proposed a novel model that differs from a widely held hypothesis about the mechanisms by which developing animals pattern their tissues and structures.

Cells in a developing animal require information about their position with respect to other cells so that they can adopt specific patterns of gene expression and function correctly. The most accepted paradigm is that this positional information comes in the form of chemical signals called morphogens; morphogens are differentially distributed across the developing field, with cells acquiring the information about their position relative to their neighbors by "measuring" and interpreting the local concentrations of the morphogen.

Despite the identification of several families of morphogens in many organisms, the hypothesis that cells differentially respond to morphogen concentrations generally hasn't been directly tested.

The Caltech researchers, led by assistant professor of biology Angelike Stathopoulos, used an approach that combines mathematical modeling and developmental genetics experiments to examine the mechanisms underlying patterning of the developing wing in the fruit fly, Drosophila melanogaster. They found that cells cannot adopt multiple patterns of gene expression solely by measuring the local concentration of a morphogen.

"During metamorphosis, imaginal disc tissues need to form structures that contribute to the adult body plan," explains Stathopoulos. Imaginal discs are parts of the insect larva that will become structures that contribute to the adult body plan. "This imaginal disc tissue is plastic, in that the cells are still making decisions about what part of the organ they should develop into. A decision has been made that these cells must make 'body part X,' but how do they determine whether to make the proximal or distal portion, or decide which is facing up or down? This is where morphogens come in."

In fruit flies, a regulatory molecule called Hedgehog provides such information, along the anterior-posterior axis (from base to tip) of the developing wing. For the wing to properly form, cells along this axis need to "act" by turning on the Hedgehog signaling pathway, although not all of the cells do so, Stathopoulos says.

"We found that in the developing wing of the fruit fly, cells do not acquire positional information by only measuring the concentration of the Hedgehog morphogen at a given time, but instead require information about their history of exposure," says Marcos Nahmad, a graduate student in control and dynamical systems at Caltech, jointly supervised by Stathopoulos and John Doyle, the John G. Braun Professor of Control and Dynamical Systems, Electrical Engineering, and Bioengineering. Nahmad is the first author of a paper about the research, coauthored by Stathopoulos, that appears in the September 29 issue of the open-access online journal PLoS Biology.

In their experiments, the researchers found evidence that certain cells receive the Hedgehog signal only for a short period of time, and this transient exposure causes them to adopt a gene expression pattern that is different from that of other cells that receive the Hedgehog signal constantly, and from those that never received it at all.

In a sense, says Stathopoulos, the cells are able to "remember" that they've been exposed to the morphogen. "What I mean is they remember having 'seen' a morphogen concentration that activates a signal within them. Even if the concentration of the morphogen decreases subsequently, cells still retain the ability to activate the pathway.

"An exciting outcome of our model is that the ability of cells to respond to the history of morphogen exposure is wired in the gene network architecture that controls patterning of the developing wing. As the Hedgehog pathway architecture is widely conserved from flies to humans, this mechanism of patterning may explain how cells in other developing systems acquire positional information," Nahmad says.

"As developmental biologists, we want to understand how the body plan is specified, and how different animals exhibit different shapes and patterns. What we've shown here is that it's important to consider the temporal sequences of events," Stathopoulos says.

"The dynamics of the system are instructional. In the past, for the most part, this has been ignored, because it's just too complicated. People have formulated models by looking at the endpoint. We believe that even more insights into patterning have likely been missed for this reason," she adds.

The work in the paper, "Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc," was funded by the National Institutes of Health and the Searle Scholar Program.

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Caltech Scientists Get Detailed Glimpse of Chemoreceptor Architecture in Bacterial Cells

Findings show structure is same throughout bacterial kingdom; may provide insight into more complex signaling pathways

PASADENA, Calif.—Using state-of-the-art electron microscopy techniques, a team led by researchers from the California Institute of Technology (Caltech) has for the first time visualized and described the precise arrangement of chemoreceptors—the receptors that sense and respond to chemical stimuli—in bacteria. In addition, they have found that this specific architecture is the same throughout a wide variety of bacterial species, which means that this is a stable, universal structure that has been conserved over evolutionary time.

Their research, which was published this week in the online early edition of the Proceedings of the National Academy of Sciences (PNAS), may help scientists better understand the complex signaling pathways that are at the core of many biological processes.

Bacteria swim using flagella to propel themselves. But it's not as simple as that, explains Grant Jensen, associate professor of biology at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI), who led the team. After all, they need to decide where to swim. "They tend to swim toward a favorable environment, and away from a harsh environment," Jensen says.

How do they know which is which? Enter the chemoreceptors, tiny protein molecules found at the front of the bacterium, near the flagella. "It's like a protein antenna that protrudes from the bacterial cell body, through the membrane, and out onto the surface," says Jensen. "It binds to nutrients and other chemical stimuli."

While swimming in a single direction, a bacterium such as Escherichia coli may encounter some nutrients, which then bind to the chemoreceptors. "This transmits a signal to the inside of the cell saying that things are good," says Jensen. "So the bacterium will keep swimming in the same direction. But if there are no good nutrients, the cell will do something called 'tumbling,' in which it stops and randomly flips over in the fluid, then starts swimming again in a random direction in a search for better conditions."

"One of the remarkable things about this system," says Jensen, "is that the chemoreceptors are exquisitely sensitive to changes in the concentrations of positive and negative stimuli. 

Scientists believe that this sensitivity is due to the way the hundreds of chemoreceptors cluster together in the bacterial cell. "It is known that if one receptor binds a stimulus molecule, it turns on other receptors around it as well to amplify the signal," Jensen explains. "The whole system also adapts to changing conditions, dynamically adjusting the range of concentrations that it responds to. 

To fully understand just what is happening in these cells, Jensen says, it is thus important to figure out the ways in which these receptors interact with one another, which in turn depends on understanding precisely how they are situated in relation to one another.

In other words, scientists need to be able to "see" the internal architecture of the bacterial cell and, in particular, how its chemoreceptors are arrayed. 

Jensen and his team were able to get just such a glimpse at the chemoreceptor architecture at the macromolecular level, thanks to a state-of-the-art electron cryomicroscope that was purchased with a gift from the Gordon and Betty Moore Foundation.

"The electron cryomicroscope allows us to see the arrangement of individual proteins inside cells in a lifelike state," says Jensen. "To do this, living cells are quickly frozen so that all the proteins are frozen in place—in the same places they were in the living state."

The high-tech microscope allowed the researchers to take 3-D images of intact cells through a technique called electron cryotomography. The researchers looked at some 700 tomographic images—or tomograms—of bacterial cells, says Caltech postdoctoral scholar Ariane Briegel, the first author on the PNAS paper, and an HHMI associate. "This is the first time such a large number of tomograms was used to answer a biological question," she notes. "And it was made possible by the combination of a state-of-the-art electron microscope and fully automated data collection."

What they saw when they glimpsed the insides of these quick-frozen bacteria were chemoreceptors arranged in a regular, repeating lattice of hexagons—a structure with six sides and six corners or vertices—that are 12 nanometers apart, center to center. At each of the vertices sit six chemoreceptors, arranged in what scientists call "trimers of dimers," which means there are three sets of two paired receptors in each grouping. The two receptors in each dimer twine around one another, and those dimers then cluster together at one vertex of the hexagon to form a trimer.

A model of the hexagonal arrangement of the chemoreceptors and their groupings in what are known as "trimers of dimers."
Credit: Molecular Microbiology/Ariane Briegel, Caltech

"One beauty of this is that we've shown that the receptors cluster in cells in the same way they did in the crystal structure," says Jensen. "In the past, we didn't know if that was an artifact of the crystallography. Now we can see how the pieces fit together in real cells."

The paper also showed that this particular architecture is no single-species fluke. "We looked at 13 different species that cover the whole bacterial kingdom," Jensen says. "The arrays were all the same. This shows us that this structure has been universally conserved, that it's a universal architecture."

And that's important to know, he adds, because it gives scientists a basis for trying to figure out how this sort of architecture leads to the bacteria's sensitivity to chemical cues in its environment and establishes that work using key model systems such as E. coli will be generally applicable.

"Bacterial chemotaxis consists of only a few key components, making it an important model system for all cell signaling pathways," says Briegel. "We need to understand this system first before we can hope to fully understand the more complex eukaryotic signaling systems. Chemotaxis also plays an important role in the first steps of host invasion for pathogenic bacteria. Understanding it might help in the development of new antimicrobial agents."

In addition to Jensen and Briegel, other authors on the PNAS paper, "Universal architecture of bacterial chemoreceptor arrays," include Caltech's Elitza Tocheva, Zhuo Li, Songye Chen, Axel Müller, Cristina Iancu, Gavin Murphy, and Megan Dobro; Davi Ortega and Kristin Wuichet of the University of Tennessee; and Igor Zhulin of the University of Tennessee and of Oak Ridge National Laboratory.

Their work was funded by grants from the National Institutes of Health, the Howard Hughes Medical Institute, the Beckman Institute at Caltech, and gifts to Caltech from the Gordon and Betty Moore Foundation and the Agouron Institute.

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Lori Oliwenstein
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