News this Week

Science  15 Nov 1996:
Vol. 274, Issue 5290, pp. 1071
  1. Science Policy

    A Postelection Vote for Consensus

    1. Andrew Lawler
    1. With additional reporting by Jeffrey Mervis.

    Democrats and Republicans call for a truce in the bruising ideological fights of the last 2 years. That respite could help rescue science from the wholesale cuts needed to balance the federal budget

    If the incoming 105th Congress takes a more bipartisan approach to R&D issues next year, give some of the credit to the chef at the Monocle, a popular Capitol Hill restaurant. Representatives James Sensenbrenner (R-WI), the heir apparent to the chair of the House Science Committee, and George Brown (D-CA), the panel's ranking minority member, spent a pleasant evening there earlier this fall discussing how to end the partisan bickering that has dogged the committee's work for the past 2 years. The meal may have set the stage for a more united front for science and technology.

    Their search for common ground reflects a new trend following last week's elections that gave President Clinton a second term and granted Republicans continued control of Congress. With each party capable of foiling the other's proposals, politicians are scrambling to show that they can avoid legislative gridlock by shedding some of their ideological armor. White House officials, lawmakers, their staffs, and science lobbyists say that such a rapprochement could be crucial to prevent science and technology programs from being savaged by the overall federal budget cuts both parties now support to erase the deficit. But both sides will have to overcome a legacy of distrust from the ideological battles of the last Congress.

    Brown, who won another squeaker—this one by less than 1000 votes—to return for his 17th term, is optimistic: “The House will be loaded with moderates who will want to cooperate and achieve some kind of change,” he says. “The name of the game will be to seek areas of cooperation, and I don't see why R&D shouldn't be one of those areas.” The retirement of committee chair Robert Walker (R-PA), with whom Brown clashed repeatedly in the past 2 years, will help lower the temperature on the committee. While the vacationing Sensenbrenner could not be reached for comment, Representative Steve Schiff (R-NM), who will remain as chair of the science panel's basic research subcommittee, seconds Brown's wish: “I hope that we can be as nonpartisan as possible. I don't think that science needs to be a partisan issue.”

    Such words hearten those in the science community who witnessed bitter fights in the previous Congress over applied technology efforts, global-warming research, and environmental regulations. “If the talk of the last couple of days about bipartisanship is sustained by both sides, then it will greatly help science,” says Jack Crowley, who directs the Washington office of the Massachusetts Institute of Technology.

    Much will hinge on who holds the reins of power in the committees, federal agencies, and the White House. The two parties will caucus next week to distribute committee assignments, while reshuffling in the Administration is already under way. Meanwhile, the White House is wrapping up work on a 1998 budget request to be submitted in February, and presidential science adviser Jack Gibbons is arranging a series of meetings with congressional leaders to discuss R&D spending.

    Calmer heads.

    Agency heads testifying at a 1995 Science Committee hearing hope to escape partisan wrangling that plagued panel under Walker (bottom, with Brown)


    Turning talk of cooperation into something concrete will not be easy. Republicans say they will continue criticizing the Commerce Department's Advanced Technology Program for being too favorable to big business and NASA's Mission to Planet Earth for being too costly. And not long after his dinner with Sensenbrenner, Brown blasted Republicans for making a mockery of the scientific process by allowing a parade of skeptical researchers to dominate environmental hearings. A moderate tack in the House also seems likely to collide with a more conservative Senate.

    The first experiment in R&D bipartisanship could come at the space summit planned for January between White House and congressional leaders. “It will test our ability to forge a bipartisan commitment to science and technology spending,” says Rick Borchelt, press secretary at the White House Office of Science and Technology Policy.

    It is not clear yet who will be around in the Administration to help forge those bipartisan policies. Gibbons, a charter member of the Administration, says he hopes to remain the president's top science official for the time being. He told Science recently that a second term would be “a very exciting time,” adding that he has renewed the lease on his Washington apartment. Many of his staff, however, have left or are planning to do so in the coming months.

    Across the R&D agencies, the biggest change will be the departure of Energy Secretary Hazel O'Leary, who has gained notoriety for her extensive travel and has made powerful enemies on Capitol Hill as well as within the White House. But three senior science managers with solid reputations—National Institutes of Health (NIH) director Harold Varmus, National Science Foundation (NSF) director Neal Lane, and NASA Administrator Daniel Goldin—are likely to stay.

    In the House, Sensenbrenner will take over from Walker. During a fall campaign visit, House Speaker Newt Gingrich (R-GA) introduced the Wisconsin lawmaker as the next Science Committee chair—quieting rumors that the Republican caucus would abolish the panel in a quest to streamline the House. “He shares the same overall goals as Walker, but he won't shove those down [the Democrats'] throats,” says one Republican staffer. Adds Brown: “He's eager to have a collegial relationship, laments the lack of civility, and wants to change that.”

    He is also on good terms with Representative Jerry Lewis (R-CA), who heads the appropriations panel that funds NASA, NSF, and the Environmental Protection Agency. Lewis told Science that he and Sensenbrenner hope to capitalize on Gingrich's support of basic research. He adds that they “need to do a lot of educating” of both new and old colleagues, and he pledges to work cooperatively with the Science Committee.

    Meanwhile, Representative Joe Knollenberg (R-MI), whose R&D track record is thin, is in the running to lead the appropriations panel that oversees Department of Energy (DOE) funding after the retirement of Representative John Myers (R-IN). In the Senate, John McCain (R-AZ) likely will take over as chair of the Commerce, Science, and Transportation Committee following the defeat of Senator Larry Pressler (R-SD). McCain has opposed some technology funding and has been a critic of the Energy and Commerce departments. Senator Pete Domenici (R-NM), however, remains in a strong position to defend DOE and the two DOE labs in his state as chair of the Budget Committee and of the energy spending panel. Senator Dan Coats (R-IN), another relative unknown in science circles, hopes to chair the Labor and Human Resources Committee, which oversees NIH, after the retirement of Senator Nancy Kassebaum (R-KS).

    The first challenge to R&D supporters will be to ensure that the House and Senate Budget Committees divide up the spending pie without sacrificing large chunks of science and technology spending. And Brown, despite his antipathy toward Walker, says that Walker's absence from the House budget panel will create a power vacuum that could allow panel members from both parties to wield their fiscal axes on science and technology. That, in turn, could set the tone for appropriators.

    Ultimately, the bipartisan desire to end the federal deficit may prove an overwhelming force. “We shouldn't have any illusions—the push for a balanced budget will have an effect on R&D,” says Erich Bloch, a former NSF director and now a distinguished fellow at the Council on Competitiveness. And a little streamlining might do more good than harm, he adds. But R&D advocates are hoping that the example of Sensenbrenner and Brown's quiet dinner—for which the future chair graciously paid—is a sign that legislators will have the appetite to fight together for continued support of science.

  2. Science Policy

    California Bans Affirmative Action

    1. Marcia Barinaga

    Programs to boost the number of women and minority scientists have been part of the science education landscape for years, but in the wake of election results from California, educators nationwide are soberly pondering the future of their efforts. Last week, California voters passed an anti-affirmative action initiative, Proposition 209, that outlaws race or gender preferences in state employment, education, and contracting.

    The law kills state-funded minority graduate fellowships, forces rapid changes in undergraduate admissions at the University of California (UC), and raises questions about the continued success of model programs that bring young minorities and women into science. It is seen by many as a bellwether of what may come in other states, or even at the federal level. “This will lead to more efforts to get [similar laws] on the ballot, or to get cases taken before the courts,” says Shirley McBay, president of the Quality Education for Minorities Network in Washington, D.C.

    Some groups have filed lawsuits challenging 209, but state universities must prepare to comply immediately. That means “significant declines” in the number of minority students at the most competitive UC campuses, warns Tom Lifka, assistant vice chancellor at UC Los Angeles (UCLA). And although the law doesn't specifically target science, shrinking the already small minority pipeline will reduce the numbers of those who choose science and engineering, says physicist Stan Prussin, director of the minority-targeted Professional Development Program at UC Berkeley. “It has been a very hard fight” to increase ethnic diversity in science programs, he says, “and I have great fear of what they are going to look like in the next year.”

    The chilly climate for affirmative action began in California even before the elections, when the UC regents last year ordered the removal of race and gender from admissions and hiring criteria (Science, 9 February, p. 752). The rules on undergraduate admissions were to be postponed until 1998—but Proposition 209 forces them to take effect immediately, erasing any hope for a reversal of the regents' decision. The greatest impact will be at top UC campuses that accept only a small fraction of applicants and have relied on affirmative action to boost the numbers of minority students. At UCLA and Berkeley, the percentage of black, Hispanic, and American Indian students in future freshman classes is expected to be halved (see table of estimates).


    View this table:

    Also likely to suffer are minority outreach programs that target primary- and secondary-school students, such as the statewide Mathematics, Engineering, and Science Achievement (MESA) program, which has been lauded nationwide as one of a handful of programs that really work (Science, 13 November 1992, p. 1190). MESA focuses on minority students who are already “doing OK” in school, says program director Mike Aldaco, and offers extra preparation to help them succeed in the competitive UC atmosphere. Aldaco can't say yet just how Proposition 209 will change MESA. “I'm hoping that we will not end up with a policy that looks solely at socioeconomic status as the measure of disadvantage,” he says. If so, many of MESA's best prospects—minority children from blue-collar working families—will be missed.

    Indeed, says UCLA's Lifka, 209 will force programs to include students regardless of race—and as a result they will reach fewer minorities. “To capture every one of the [minority] students we were serving previously, you are probably talking about a 10-fold increase in budget,” he says. “And you're not going to get that.”

    Meanwhile, minority graduate students will lose special state-funded fellowships; UCLA alone will have to stop awarding $4 million to $5 million in such fellowships, says Lifka. Federal fellowships that mandate minority preference will be exempt from 209, but may be up for change themselves. Recent court decisions have restricted affirmative action, and last year the National Science Foundation compiled a list of 24 programs—with a total annual budget of roughly $100 million—that target by race or gender and so may be at risk. But officials note that a 1980 law orders NSF to try to boost diversity in the scientific work force, which may protect those programs from legal challenge.

    For now, no one can fully predict whether the rest of the country will follow California's lead. But science educators nationwide will be watching anxiously as Proposition 209 takes effect.


  3. Intellectual Property

    Database Access Fight Heats Up

    1. Andrew Lawler

    Science officials in Washington are stepping up their efforts to head off what they see as a threat to scientists' ready access to databases. They are trying to persuade U.S. trade negotiators to press for clear exemptions for scientific research in proposed new international rules protecting private databases from piracy (Science, 25 October, p. 494).

    The U.S. government hopes to clarify its position by 2 December, when talks are scheduled to begin in Geneva on a draft treaty of the World Intellectual Property Organization that would give industry added protection against unauthorized use of commercial databases. Researchers don't dispute the industry's need for protection, but they fear that what has been proposed could make it harder and more expensive to access data on everything from the human genome to global weather. Industry officials say that fear is exaggerated, and that all they want are rules updated to fit today's information society.

    The Clinton Administration is divided over the issue, and officials say they have made little progress in finding a middle ground. “It's a clash of cultures, and I'm not sure we can come up with a united stand,” says one Administration source. Some officials speculate that this dispute, combined with a host of unrelated concerns shared by several countries and constituencies, may even delay the start of negotiations until next summer.

    In the meantime, scientific forces rallied their troops last week at a meeting in Washington under the auspices of the American Association for the Advancement of Science (which publishes Science). “The fact is there has been no opportunity for dialogue,” says William Wulf, the interim president of the National Academy of Engineering (NAE), which last month raised the alarm in a letter to Commerce Secretary Mickey Kantor. “We're saying let's slow things down.” Tempers at one point in the meeting got a bit frayed: When an industry representative argued that attempts by European nations to establish a new regime make it essential for the United States to do the same, Wulf snapped, “I don't like that kind of threat.”

    This week Patent and Trademark Office chief Bruce Lehman was slated to meet with library and education representatives to hear their concerns, and PTO is accepting comments on the proposed treaty through 22 November. So far, however, neither side appears willing to compromise.

    In an attempt to influence the debate, the National Research Council—the operating arm of the NAE and the National Academy of Sciences—next week will take the unusual step of releasing a portion of an upcoming report that opponents of the draft treaty see as bolstering their case. The report, called “Bits of Power,” concludes that market forces are not enough to protect the public good associated with open access for scientists and educators, according to panel members who spoke on the condition of anonymity. Excerpts of the document, 2 years in the making, will be released on 22 November in conjunction with a symposium on database protection.

  4. Scientific Literacy

    Global Interest High, Knowledge Low

    1. Dennis Normile

    TOKYO—Public understanding of science and technology lags well behind public interest in these fields throughout the major industrial countries, according to a pair of new studies released here last week. The studies, commissioned by the Organization for Economic Cooperation and Development, sparked a lively discussion at an OECD-sponsored Symposium on Public Understanding of Science and Technology.

    The more broadly based of the two was a comparative analysis of public understanding of science and technology in 14 OECD countries done by Jon Miller, vice president of the Chicago Academy of Sciences. After synthesizing results from three separate national surveys and one Europe-wide survey done between 1989 and 1995, Miller found that self-described interest in science and technology among the general public remains high in virtually all industrial nations (see table). But only about one in 10 is also well informed, as measured by responses to specific questions about aspects of science and the scientific process. It is only those people who consider themselves well informed—Miller calls them the “attentive public”—who are likely to get involved in public-policy debates, he says. Miller argues that the subset should be larger. “It is essential that there be a sufficient number of citizens who are attentive to an area and who are able to comprehend the debates among [policy-makers] on the issue,” he says.

    Curiosity and competence.

    According to several surveys, in most countries a higher percentage of adults follow science than can answer specific questions about the natural world.


    The second study explored attitudes toward science and technology of young people in a cross section of OECD countries. Helen Connell, a Paris-based educational consultant who reviewed a large number of previous studies, concluded that there has been no recent detectable drop-off among students in either personal interest in science or selection of university science majors. She did find a shift away from the physical sciences and toward the life and information sciences, however. Echoing Miller's concerns, Connell says the larger question is “whether the level of learning [of science and technology] is adequate” to hold down a job and perform civic duties.

    Most of those attending the conference seemed to agree that the OECD countries need to improve public understanding of science and technology. “Increasingly, a wide range of careers and professions will require a good knowledge of science and technology,” said William Blanpied, senior international analyst for the U.S. National Science Foundation, in summing up the presentations. The purpose of boosting greater public understanding, added Yoichiro Murakami, a science historian at International Christian University in Tokyo, is not just to ensure bigger research budgets but to “cultivate the sound common sense” needed to debate “the purposes of science and technology.”

  5. Space Science

    Flotilla Is Heading to Mars Seeking End to Data Drought

    1. Andrew Lawler
    1. With additional reporting by Gretchen Vogel.

    A Zen-like patience is a prerequisite for planetary scientists. Next summer, if all goes well, the first Mars data in 15 years will begin streaming back to Earth thanks to three missions launched within a 4-week period this month and next. The spacecraft—NASA's Global Surveyor, launched last week; Russia's Mars '96, scheduled for 16 November; and NASA's Mars Pathfinder, planned for 2 December—herald the start of an impressive flotilla of U.S., Japanese, and Russian spacecraft headed for Mars in the next decade. If they succeed, they will also help ease the bad memories of ill-fated U.S. and Russian probes.

    A new era of exploration is, however, far from assured. NASA managers are betting that possible evidence of life in a Martian meteorite will protect the U.S. missions from a budget knife hanging over the space agency. Their hopes are also riding on innovative technology that is intended to prevent a repeat of the 1992 explosion that destroyed the $500 million Mars Observer shortly before its arrival and shook the confidence of politicians and the research community.

    For Russian space scientists, the Mars '96 probe could revitalize their flagging field and reverse a long string of unsuccessful missions to Mars. The last was a 1988 flight to Mars's companion, Phobos, that ended abruptly when one satellite was lost due to a ground-control error, and a second went dead within sight of the moon. But the pressure is on: Russia has already abandoned plans for a 1998 mission, and there is no funding yet for one proposed in 2001, says Lev Mukhin, a planetary scientist who serves as senior science and technology counselor at the Russian embassy in Washington. Last week, a Russian Academy of Sciences panel discussed the 2001 Russian mission and the possibilities for conducting it jointly with the United States.

    Japan, meanwhile, intends to send one modest satellite to the Red Planet in 1998, while cash-strapped Europe has abandoned a proposal to build a network of sensors on Mars to relay scientific data back to Earth. Instead, European researchers are putting a handful of their own instruments aboard U.S. and Russian spacecraft.

    The three missions blasting off this fall will seek data on Martian climate changes and the planet's dramatic geology. “These are not missions designed to look for life,” says Wes Huntress, NASA's space science chief. “We want to ask broad, general questions up front until we learn enough to ask more specific questions.” Once the satellites reach their target, an extensive array of instruments—in Martian orbit, on ground-based landers, and attached to tiny rovers—will probe, pick at, and penetrate the planet. A second wave is slated for 1998, when the Earth and Mars are again favorably aligned, followed by 2001 and 2003 launches culminating in an effort to bring Martian rocks and soil back to Earth for analysis.

    NASA is still debating the details of returning samples. Huntress says one option is a 2003 mission to gather rocks in one place, followed 2 years later by a ship to bring them back to Earth. But researchers insist there is no rush, citing the importance of pinpointing which rocks would yield the most clues to possible Martian life. “We have a lot of science to do before we bring back a sample,” says NASA Administrator Dan Goldin.

    The quantity and quality of that science hinge on the success of the landers, orbiters, and small rovers that will gather the data in coming years. Their success, in turn, could pave the way for human flights—“if we do the right things and figure out how people could go to Mars cheaply,” says Goldin. A NASA document presented recently to the agency's advisory council suggests such a landing in 2011.

    In the short term, however, modest successes in delivering the current batch of robots to Mars would go a long way to lifting the curse of the Red Planet that has plagued would-be Martian explorers since the 1976 launch of the Viking mission.


    View this table:


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  6. Child Development

    Small Refugees Suffer the Effects of Early Neglect

    1. Constance Holden

    The moment Thais Tepper laid eyes on little Drue, she knew he was “in deep trouble.” It was 1991, and she and her husband had traveled from their home in Pittsburgh to a courthouse in Arad, Romania, for a first meeting with the child they were adopting. He had been described to them as a “healthy little boy who's living with his mother.” But as she found out later, the 18-month-old had spent most of his life at a state-run maternity hospital. He couldn't sit up, and his head was flattened on one side from curling up next to the bottle that had been attached to his crib as if he were “a hamster,” she says. Back in the United States, child development experts assured her that Drue would be fine. But a year later, he was still scarcely making a sound. Even after 2 years, he remained so withdrawn that when he fell down and hurt himself, he wouldn't cry. He had also become disturbingly aggressive, flying into arbitrary rages.

    Drue is one of a wave of Eastern European children who in the wake of the breakdown of the Soviet bloc have found adoptive homes in Western Europe and North America. The bleak conditions in which many of these children spent their early days have left them with a host of behavioral and physical problems for their new parents to cope with. And these small refugees have also attracted interest from scientists who study how early deprivation can alter brain chemistry and behavior.

    Romanian orphans in particular present a huge, tragic experiment in early deprivation. Many spent months or years in orphanages where they were fed and diapered on military-style schedules, with little or no playing, cuddling, or talk. After the regime of dictator Nicolai Ceausescu was overthrown in 1989, the world was gripped by stories of children who stared blankly at visitors, who were uncannily silent because they had learned that crying brought no response. The orphans are “by far the biggest group of deprived babies we've [been able to study] so far,” says child psychiatrist Michael Rutter of the London Institute of Psychiatry.

    With tools ranging from brain scans to intelligence quotient (IQ) tests, researchers are trying to understand both biochemically and behaviorally the toll taken by deprivation and the extent to which the damage can be undone. Much of the work is still preliminary, and the results are endlessly confounded by such factors as maternal drinking and smoking and the heavy industrial pollution in Eastern Europe. Nonetheless, researchers hope to get more precise answers than ever before.

    Scientists have a rich store of animal studies to help guide them as they seek to understand how early neglect can shape the developing brain. They include Harry Harlow's landmark studies, conducted during the 1950s at the University of Wisconsin, of monkeys who never matured socially after being reared without mothers, and studies of rats showing that lack of maternal stimulation disrupts endocrine functioning. Scientists suspect that all this can happen to deprived young humans too. Indeed, neurobiologist Mary Carlson of Harvard University, who worked in Harlow's lab as a student, relates that many of the orphans she saw in Romania were withdrawn and engaged in rocking and other stereotyped behaviors that reminded her of Harlow's socially deprived primates.

    Carlson and her husband, Harvard psychiatrist Felton Earls, have been exploring stress hormones in these children. The rat studies had suggested that early neglect can permanently alter the functioning of the hypothalamic-pituitary-adrenal (HPA) axis, a system that regulates stress, sex, and growth hormones. One such hormone is the stress hormone cortisol, which, in humans, has been linked to attentional problems and attachment disorders, both of which are often seen in institutionalized children. So Carlson and Earls compared cortisol levels in saliva from institutionalized toddlers who had been thoroughly neglected and from children who had spent 13 months in an “enriched environment” with more caretakers. But the results were inconclusive: Both groups had unusually high cortisol levels.

    Neurologist Harry Chugani of Wayne State University Children's Hospital in Detroit did find neurological differences when he looked at a handful of Romanian adoptees with positron emission tomography (PET): Their brains seemed to use glucose, the brain's basic fuel, at an unusually high rate. Chugani did PET scans of four adoptees and compared them with data from age-matched controls. “The scans [from the adoptees] are all quite abnormal,” says Chugani. Resting glucose metabolism values are about 50% higher than normal in every area of the brain. Also, glucose metabolism in the children's frontal lobes was lower than in other parts of their brains—a pattern that Chugani says is associated with attention-deficit disorder.

    Chugani is taking blood samples from the children for future cortisol testing, and he intends to do PET scans on several more of the adoptees, who are being brought to Michigan by ABC-TV, which is airing a special about them on the 19 December Turning Point. He allows that his results are still preliminary, but suggests that because all the children he has tested have lived with their adoptive families for at least 2 years, the effects he is seeing are probably more than transient.

    Some researchers are skeptical, however, that physiological tests on such a mixed group of children—including many abandoned because of retardation or medical problems—will yield meaningful results. Neurobiologist Seymour Levine, professor emeritus at Stanford University, says “These are interesting results. … [But] the studies are lacking a lot of very critical controls.” Cortisol is especially dicey: Because levels change rapidly and are highly responsive to circumstances, says Levine, “one of the hardest things to do is to get a basal level.” He also points out that small changes in the timing of stressful events can make a big difference in outcomes. “If you subject a 3-day-old rat to maternal deprivation, it will hypersecrete stress hormones later in life.” But if you deprive a rat when it's 11 days old, “then the HPA system becomes hyporesponsive.” Says Levine: “A human infant's age at the time of institutionalization could be just as crucial.”

    Another neurobiologist, who prefers not to be quoted, also argues that some measures may be too blunt to say anything about the effects of early stress or deprivation. For instance, he says, high glucose metabolism is commonly seen in people who are mentally retarded or doing unfamiliar tasks—as would be true of many of the adoptees.

    But as these scientists point out, it's not these children's chemistry but their behavior—and the extent to which they can recover from neglect—that really matters. One study of adoptees, led by psychologist Elinor Ames of Simon Fraser University in British Columbia, suggests that the severity of impairment is proportional to the length of institutionalization. The researchers have been comparing three groups of children: Romanian adoptees who spent 8 months or more in an institution; Romanian children who were adopted by the age of 4 months; and age- and sex-matched controls of British Columbian children living with their own families. On measures of attachment and socialization, the early-adopted group resembled the controls. The late-adopted group, by contrast, was more withdrawn and more likely to engage in stereotyped behaviors, such as rocking. They also had more eating problems, including refusing solid food and eating excessively.

    Follow-up testing 3 years after adoption, when children were between 4 1/2 and 8, suggests that while early problems fade, they don't go away. “The longer a child had spent in an orphanage, the more behavior problems he or she had 3 years later,” Ames says. Parents of the late-adopted children reported some improvement in the children's “attachment security,” as measured by willingness to explore independently, among other behaviors. But many still were “indiscriminately friendly,” a common aftereffect of institutionalization. Numbed by neglect, they are often unable to form more than superficial attachments, but at the same time their need for attention is so strong that they will accept it from anyone. Many late adoptees also still had symptoms of depression or withdrawal, including a tendency to stare blankly. But, like little Drue Tepper, “their main behavior problems [had become] poor control of temper, fighting, or demanding attention,” says Ames.

    Still, findings from two other adoption studies suggest that most children have a remarkable ability to recover from the effects of early neglect. Psychologist Susan Goldberg of Toronto Children's Hospital has studied 56 Romanian children, aged 2 1/2 to 5 years, 19 of whom had been institutionalized for up to 4 years. While some still showed the indiscriminately friendly behavior familiar to orphan-watchers, “when we looked at them as a group, the really striking thing was how well most of these kids had done,” she says. All had formed some kind of attachment, and their English language skills were within the normal range.

    The largest scale adoption study is being conducted by Michael Rutter in London, who has been tracking 166 children adopted from Romania into the United Kingdom. Rutter calls them an “extraordinarily deprived group,” many of whom were institutionalized for more than a year and about half of whom were in the bottom third percentile in height, weight, and other bodily measurements. Rutter tested the children at two time points—ages 4 and 6. In language development, physical growth, and ability to make emotional attachments, the children have made a “spectacular recovery,” he says. They are still lagging behind, though, in IQ—by about 10 points—and in social behavior as shown by problems in submitting to school discipline and in “picking up social cues.”

    Even though mixed and tentative, the results from these studies are reaching an eager audience—the thousands of parents who adopted Eastern European children. “We've been contacted by 3000 people,” says Tepper, who has launched a national parents' group. She and neuropsychologist Ronald Federici of Alexandria, Virginia, have organized a meeting for parents and experts to be held next weekend in Arlington, Virginia. And although scientists may never be able to quantify the effects of early deprivation, the studies are driving home one clear message, says a scientist: “You need to be nice to people, and especially developing people.”

  7. Breast Cancer

    Activists Vote $14 Million for Research

    1. Eliot Marshall

    During a tense meeting on 7 November, breast cancer activists took an extraordinary step: They rejected an attempt to build a small fiefdom on their behalf in the U.S. Department of Health and Human Services (HHS), arguing that the money should be spent instead on peer-reviewed research. As a result, the National Cancer Institute (NCI) is likely to get an extra $14 million for research in 1997. This shows, as one activist said, that “we don't want to be perceived as just another special-interest group.”

    The activists' move was an embarrassing defeat for Susan Blumenthal, director of HHS's Office on Women's Health. She is in charge of administering the National Action Plan on Breast Cancer, a strategic plan that Congress directed HHS to develop after breast cancer activists lobbied for it. Congress earmarked $10 million of NCI's budget for the plan in 1995 and $14.75 million in 1996. The Administration, with Blumenthal making the case, sought $20 million for 1997, and Congress again approved $14.75 million. But activists have grown unhappy with Blumenthal's strategy to inflate the plan and make it a permanent adjunct to her office.

    Dissatisfaction surfaced this summer in letters from Frances Visco, the Philadelphia attorney who presides over the National Breast Cancer Coalition, to Senator Arlen Specter (R-PA), chair of a subcommittee that drafts the HHS and NCI budgets. On 23 July, Visco wrote to say her influential group wanted to see breast cancer research at NCI expanded, and to avoid diverting money from “quality” research, she asked that no more than $4 million be set aside for the Action Plan in 1997. Specter, however, sought $14.75 million. An aide says it seemed the least controversial thing to do. But on 10 October, after the bill passed, Visco wrote Specter, reminding him that she represents 350 organizations and insisting that the earmark was “too much money.”

    The disagreement came to a head on 7 November when the steering committee of the National Action Plan—co-chaired by Blumenthal and Visco—met at a Washington, D.C., hotel to vote on how the $14.75 million should be used. Blumenthal was pushing what she calls a “broad program” to sponsor not just research, but education, mammography for the poor, new treatment initiatives, and other “crosscutting” agency activities. The panel was not persuaded. At one point, panel member Kay Dickersin, a University of Maryland epidemiologist and member of Visco's coalition, asked: “Is there anyone on this committee who agrees [with Blumenthal's position]?” Apparently no one did. The committee voted 13-0, with four abstentions, to send all but $750,000 of the money to NCI for peer-reviewed research. (The plan would have about $4 million available in unspent money from last year.)

    Blumenthal's reaction: “It represents a genuine difference of philosophy.” She concludes that the steering committee was so tight with NCI that it had “a vested interest in holding onto that money” for research. “I see it as a missed opportunity” to launch new prevention programs, she adds. Visco responds: “It may be a missed opportunity for Susan Blumenthal, but not for breast cancer research.”

    Now it's up to HHS Secretary Donna Shalala to decide what to do with the $14.75 million. A spokesperson says Shalala is “giving very careful consideration” to the steering panel's recommendation that the money be used for research. Her decision will be announced early next year.

  8. Astronomy

    Dust Grains Bring Long-Lost Stars Into the Laboratory

    1. James Glanz

    ST. LOUIS—Slice open a hailstone, and you will see the signature of the thunderstorm that spawned it: a bull's-eye of concentric ice layers laid down by wind currents and temperature gradients within the storm. Much the same goes for tiny grains of “stardust” born in the stormy atmospheres of long-vanished stars, a research team here at Washington University has found. By picking apart these bits of grit—which formed billions of years ago around old, bloated stars and supernovas, then wafted through space and fell to Earth in meteorites—the team is reading a story of stellar turbulence in unprecedented detail.

    That's just one of the ways in which these grains are bringing far-off stars into the laboratory. Only a few years ago, says Roberto Gallino of the University of Torino in Italy, researchers were still marveling at their discovery that these micrometer-sized interlopers in meteorites have isotopic compositions marking them as fossils from long-dead stars. Today, as Gallino and more than 100 other meteoriticists, astrophysicists, and astronomers discussed at a recent meeting * here, new analytical techniques have turned stardust into a bona fide astrophysical probe. Says David Arnett of the University of Arizona: “What we have here is an entirely new sort of connection between stars and us.”

    Aside from the light they shed on stellar atmospheres, grains spewed out by old, long-lived stars are being put to work as a new measure of the Milky Way's age. The precise balance of isotopes in the grains, reflecting how the parent stars churned and burned their nuclear fuel, has also hinted at unsuspected circulation patterns inside stars—a finding with implications for efforts to infer how elements were made in stars and even in the big bang. It may even be possible to use stardust as a probe of conditions in the nascent solar system. “It's a new science that is exploding these last 5 years,” says Gallino.

    Nitty gritty of stars.

    Sliced open, a graphite grain reveals a tiny “seed” crystal of titanium carbide, implying unexpectedly high densities in the parent star's atmosphere.


    The entire field of stardust studies, says Thomas Bernatowicz of Washington University, rests on the premise of “the solar system as a big Cuisinart” that didn't quite finish the job. Like cooks heaping ingredients into a food processor, winds from nearby stars, debris flung from supernovas, and the general flotsam and jetsam of the interstellar medium all contributed dust and gas to the giant cloud that eventually formed the solar system. Ingredients from each source had their own patterns of elements and isotopes. But once the nebula collapsed into a warm, whirling disk, those patterns blended together to produce averaged abundances that, today, are about the same no matter where in the solar system we look. Here and there, though, the original ingredients didn't get perfectly homogenized.

    The unmixed lumps take the form of tiny grains of silicon carbide, graphite, aluminum oxide, and other materials, now found in certain rocky meteorites. The grains betray their exotic origin with isotopic compositions that are wildly out of line with the usual solar system values (Science, 26 July 1991, p. 380). As geophysicist G. J. Wasserburg of the California Institute of Technology (Caltech) puts it, each bit of anomalous material “is a grain made around a star.”

    Painstaking analysis of these tiny bits of grit (see sidebar) reveals which kinds of stars they come from. Certain graphite and silicon carbide grains are laced with calcium-44, a decay product of radioactive titanium-44, which Donald Clayton of Clemson University in Clemson, South Carolina, has shown must have formed in the flurry of nuclear reactions in a supernova. Some of the graphite grains that Bernatowicz and his colleagues have studied contain unusually large amounts of silicon-28 and high ratios of carbon-12 to carbon-13—two other signs of a supernova origin. Others carry the anomalously low carbon ratios characteristic of winds from the old, bloated, low-mass stars called asymptotic giant-branch (AGB) stars.

    Bernatowicz, who organized the conference along with stardust pioneers Robert Walker and Ernst Zinner of the McDonnell Center for the Space Sciences at Washington University, is going beyond identifying stars to probing conditions inside them. By cutting the grains with a microtome and examining the slices with an electron microscope, he discovered even smaller particles at their center—crystals of titanium carbide, for example, that looked like they were engulfed by the graphite as it condensed, layer by layer. Says Bernatowicz, “You're pretty safe in the assumption that the titanium carbide was there first” when the graphite condensed, like dust particles caught in growing hailstones. The team realized that this sequence of formation—along with the near absence of silicon carbide, even though silicon is abundant in AGB stars—was a clue to conditions in the stellar atmosphere.

    Graphite condenses at about the same temperature regardless of pressure, for example, but titanium carbide condenses more readily as the pressure goes up. Calculations by team member Katharina Lodders and others showed that for the titanium carbide to have formed first, the density of the stellar atmosphere had to be extraordinarily high—at least 100 times higher than astronomers had estimated. At those densities, AGB stars would shed mass far too quickly in the high-speed winds that astronomers have clocked blowing outward from them, unless the high densities are limited to narrow jets or knots of turbulence.

    Bernatowicz says that the grains bearing the isotopic signature of supernovas are flecked with such inclusions as well, implying that supernova atmospheres also have pockets of high density. “It's really a beautiful result,” says Arizona's Arnett. Recent computer models of supernovas did imply lumpy atmospheres, but “now we have a way to test [such predictions] in the laboratory.”

    Washington University's Larry Nittler and Caltech's Wasserburg and Gary Huss, meanwhile, are finding that another kind of grain may offer a deeper look into stars. Examining particles of aluminum oxide that seem to come from red giant stars, the researchers found that one oxygen isotope, oxygen-18, was unexpectedly scarce. The depletion suggested that before the grains condensed, the gases that formed them had been churned deep into the stars, through layers hot enough to “cook” that isotope into heavier elements. The same process would also deplete the surface gases of helium-3—an isotope that traces element-forming processes in the big bang. Such stellar churning, if it's common enough, could explain why the cosmos as a whole seems to contain less helium-3 than some scenarios of element formation predict (Science, 7 June, p. 1429).

    Another set of oxide grains might help settle a different cosmic conundrum: the age of the universe. Various measures of cosmic age point to wildly different figures—anywhere between 8 billion and 15 billion years—so astronomers would welcome an extra indicator. The first step in extracting an age from the oxide grains is to add the 4.6-billion-year age of the solar system to the likely age—just short of 6 billion years—of the red giant stars that contributed the grains to the presolar nebula. The next is to determine how old the galaxy was when the stars formed, and a complex comparison of three different oxygen isotopes in the grains points to an answer: slightly under 4 billion years. The rough total is 14 billion years. Because the universe as a whole has to be at least that old, the result—although uncertain—is squarely on the side of an old universe.

    The grains have plenty of other secrets to reveal, participants at the conference emphasized. Besides offering glimpses of their stellar birthplaces, they may also hold clues to conditions in the primordial solar nebula. Patrick Cassen of the NASA Ames Research Center, for example, presented model calculations he did with his colleague Kenneth Chick showing that meteorites formed in different parts of the nebula might contain different amounts of stardust, depending on the amount of heating and mixing their ingredients had undergone. As a result, stardust counts might be used to map out the workings of the primordial Cuisinart. For astronomers facing the blizzard of data from such studies, said Zinner in summing up the conference, it all adds up to “a much harder life, but I hope a much more enjoyable life.”

    • * “The Astrophysical Implications of the Laboratory Study of Presolar Materials,” held at Washington University from 31 October to 2 November.

  9. Astronomy

    Picking Out the True Grit of Stars

    1. James Glanz

    The explosion of stardust studies wouldn't be possible without technologies that can identify genuine bits of stars in grit extracted from meteorites—technologies like the device built by Washington University's Larry Nittler. Just across a parking lot and up a hill from the stardust conference, Nittler showed off a riot of knobs, flanges, vacuum pumps, coaxial cables, and computer screens, all designed to map the isotopic compositions of dozens of grains at once. The apparatus lets him pluck the one bit of true stardust from mounds of ordinary material.

    Nittler explains that the device plays a broad beam of ions over meteorite grains spread out on a piece of gold foil. The beam dislodges secondary ions from the grains, and the instrument analyzes the ions to determine the grains' composition and to produce maps of isotope abundances and ratios. “I can find the one grain out of 1000 that is isotopically anomalous”—a likely bit of stardust—says Nittler.

    That kind of discrimination is crucial, for example, in identifying stellar grains made of oxide minerals. Oxide grains are among the most informative kinds of stardust (see main text), but they are hard to pick out from the ordinary oxides that make up meteorites. With the system, which builds on work by Washington University's Ernst Zinner and Robert Walker and by Peter Hoppe (now at the University of Bern in Switzerland), Nittler has now found 79 of the 92 presolar oxide grains identified since Caltech's Gary Huss discovered the first one in 1992.

    The advance over standard grain-by-grain techniques for identifying and analyzing stardust is so huge, says Clemson University's Donald Clayton, that Nittler's forthcoming report in the Astrophysical Journal “is one of the great science papers of the last few years. It's going to open up a whole new realm.”

  10. Data Storage

    Tiny Abacus Points to New Devices

    1. Robert F. Service

    Talk about coming full circle. This week, a team of researchers at IBM's Zurich Research Laboratory reported crafting the first nanoscale abacus, some 2000 years after its macroscopic cousin first came into widespread use. Unlike a traditional abacus, a counting device consisting of sets of beads threaded on parallel wires, the IBM version is composed of spherical carbon molecules, called buckyballs, lined up along a steplike edge on a copper surface. Although the new abacus may not have quite the impact of its predecessor, it could lead to data-storage devices capable of holding vastly greater amounts of information than today's computer chips and disk drives can hold.

    “The work is certainly a landmark experiment,” says Stephen Minne, an electrical engineer at Stanford University in Palo Alto, California. Indeed, it represents the first time researchers have succeeded in using individual molecules at room temperature to store numerical information. The IBM group used their abacus to store numbers from 1 to 10 by sliding the buckyballs along the edge one by one, with an ultrasharp tip of a scanning tunneling microscope (STM). Still, the scientists have a long way to go before their work can be applied to real-world computing, say Minne and others. For starters, an STM tip can only move one buckyball at a time; for the technique to be useful for reading and writing huge amounts of data, researchers would need to figure out how to manipulate thousands or millions of the buckyballs in concert.

    The new work, which is described in the 11 November issue of Applied Physics Letters, builds on plenty of previous research. Another team of IBMers, led by physicist Donald Eigler at the Almaden Research Center in California, was the first to push atoms around on a metal surface with an STM tip. But in that experiment, conducted in 1990, the researchers had to chill their sample down to just a few degrees above absolute zero to keep the surface atoms from skittering around. Earlier this year, the Zurich-based IBM team—led by physicist James Gimzewski—constructed a new member of a class of organic molecules known as porphyrins that readily adhered to metal surfaces at room temperature. But these molecules were difficult to control when pushed by an STM tip, so the researchers couldn't use them to represent numerical information (Science, 12 January, p. 181).

    For the current experiment, Gimzewski and his colleagues Maria Teresa Cuberes and Reto Schlittler tried soccerball-shaped buckyballs on copper. Researchers have known for some time that metal surfaces are rarely perfectly flat; rather they resemble a series of flat terraces separated by atomic-scale steps. Researchers have also noted that buckyballs cling to metal surfaces, and they preferentially line up along the steps, where they share the most attractive electronic interactions with neighboring metal atoms. The team decided to try to use one of these steps to keep the buckyballs in line, much as an abacus's wires hold the beads in place.

    And it worked. After depositing buckyballs on a copper sample, the researchers used the STM to take a look at the surface. As they had hoped, they found a row of buckyballs lined up along the step between two terraces. Then they pushed the buckyballs along the step with the STM tip, one at a time, much like one would push abacus beads. After moving each buckyball, they used the atomic-imaging tip to take a new picture of the surface. Finally, they pieced together the 10 images into one composite image.

    While the current demonstration doesn't store numbers as computer-friendly binary 1s and 0s, it's easy to imagine how to change the setup to make binary data storage possible, says Cuberes. One approach would be to create tiny grooves in the copper surface, just wide enough for one buckyball to fit inside and only long enough for it to move back and forth when pushed by an STM tip. One side of the groove would be the 0 position; the other side, the 1. The researchers are nowhere near accomplishing this, Cuberes acknowledges. But if they can pull it off, this nanoscale device, like the original abacus, could create a bit of history of its own.

  11. Extinctions

    A Shocking View of the Permo-Triassic

    1. Richard A. Kerr

    DENVER—The great whodunit of the dinosaur extinctions has a likely suspect—an asteroid impact. But what of the largest mass extinction of all time, 250 million years ago at the Permo-Triassic boundary? This great dying marked the end of the 300-million-year reign of the “old life” of the Paleozoic era—typified by the last of the trilobites—and made way for more diverse and predatory life, including the dinosaurs. Its cause has long been a mystery, with theories ranging from anoxia in the oceans to massive volcanic eruptions on land (Science, 1 December 1995, p. 1441).

    Now, at the annual meeting here of the Geological Society of America, paleontologist Gregory Retallack of the University of Oregon has presented pictures of microscopic quartz grains that he claims are the “first unequivocal evidence of an impact,” implicating a comet or asteroid in this extinction too. The hallways buzzed with paleontologists and geologists exchanging opinions on Retallack's photos, which purportedly showed faint bands of glass-filled fractures within the grains. Retallack thinks the fractures formed in the shock of a massive impact and notes that similar grains have been linked to the Cretaceous-Tertiary extinction. The hallway buzz was more cautious. “There may be something there,” says petrologist Glen Izett of The College of William and Mary in Williamsburg, Virginia, “but photographs don't show what your eye does through a microscope.”

    If Retallack and his Oregon colleagues Abbas Seyedolali and David Krinsley are right, then the Permo-Triassic extinction will have not only a new cause but also a new time scale. Most paleontologists have seen the crisis as a protracted “event” or even as two separate pulses of extinction. But an impact extinction would have happened in a geologic instant. It is a crucial question, notes paleontologist Douglas Erwin of the National Museum of Natural History, because “if the Permo-Triassic extinction hadn't happened the way it did, you would find a whole different bunch of beasts” alive today.

    Although intrigued, many of Retallack's colleagues are not yet convinced. The quartz grains are old and fractured by more recent, mundane stresses, notes Philippe Claeys of Berlin's Museum of Natural History, making it difficult to see the faint traces that might have been left by an impact. Truly shocked quartz is riddled with thin, straight, parallel planar structures called planar deformation features (PDFs), which form sets that intersect at predictable angles depending on the crystal structure of the quartz. Photomicrographs of the Oregon group's grains, which come from the Permo-Triassic boundary near Sydney, Australia, and from the Transantarctic Mountains in Antarctica, reveal one set of possible PDFs, says Claeys. But he argues that several sets intersecting at the correct angles would be required for conclusive proof.

    Shock effect?

    Permo-Triassic quartz grain (above) is fractured, but the banding characteristic of an impact doesn't show as clearly as in a truly shocked grain (top).


    Retallack counters that Claeys and others haven't yet seen all there is to see. Under the microscope, where the full depth of a quartz grain can be viewed by changing the depth of focus, all the grains can be seen to have at least three sets of PDFs, he says; one has seven.

    If other claims of shocked quartz are any guide, it may take a while to convince the community. Researchers have searched the rock record from one end of geologic time to the other for signs of impacts coinciding with biological crises, and the only success so far has been at the end of the Cretaceous. Some claims of shocked quartz have been summarily rejected, while others, such as possible shocked quartz from the Jurassic-Triassic boundary 202 million years ago, have interested but not yet persuaded researchers (Science, 11 January 1991, p. 161).

    Ideally, experts would like their own three-dimensional look at Retallack's grains. Barring that, they want numbers: more quantitative data, such as the refractive index of the grains, which is altered by shock, as well as the orientations of PDFs. The Oregon group says they are gathering those data in cooperation with colleagues. They are also examining their samples for iridium—another telltale sign of an impact, abundant at the Cretaceous-Tertiary boundary. Better pictures, specifically transmission electron microscopic (TEM) images that can identify the shock-generated glass unique to PDFs, would help too. Retallack “has got a fairly good chance,” says Claeys, “but he's got to do the TEM.” Otherwise, his data may not be so shocking after all.


  12. Microbiology

    Quick-Change Pathogens Gain an Evolutionary Edge

    1. Denise Grady
    1. Denise Grady is a free-lance writer in Edina, Minnesota.

    Over the past few months, a particularly nasty bug called Escherichia coli 0157:H7 has shaken up health authorities on two continents. Earlier this year, it sickened thousands of people in Japan who ate uncooked radish sprouts, and during the past month, it felled more than 60 Americans who consumed fruit drinks containing unpasteurized apple juice, killing one child. The same bacterium had struck before: It killed three children in the United States in 1993 and caused serious illness in 600 other people who ate contaminated hamburger meat.

    Such outbreaks, caused by a strain that was not even known before 1982, are humbling reminders of the prodigious abilities of bacteria to reinvent themselves, speedily adapting to new hosts, new conditions, and new antibiotic countermeasures. Now, a group of researchers at the Food and Drug Administration (FDA) has uncovered what may be one of the secrets of that versatility, at least in E. coli and another pathogen, Salmonella enterica.

    By studying strains from different outbreaks, the researchers, led by Thomas Cebula, found an exceptionally high percentage of “mutators”—microbes with a genetic flaw that makes them a prolific source of new variants. As the group reports on page 1208, these mutators can't repair errors in their own DNA, and they readily take up DNA from other bacteria. It's a “double whammy” speeding up their evolution, Cebula says. And because the findings may apply to other pathogenic bacteria as well, says Philip Hanawalt, an expert on DNA repair at Stanford University, “this work has very far-reaching implications and is even a bit ominous.”

    Researchers already knew that bacteria have a seemingly limitless capacity to alter their genes by trading bits of DNA between strains. But to Cebula, who studies foodborne pathogens, those mechanisms by themselves didn't seem enough to account for the swift pace of change and the high variability of microorganisms like E. coli and S. enterica. In just the past few years, for instance, strains of E. coli have emerged that can thrive in salted foods like sausage or acidic foods such as apple juice, and certain strains of Salmonella have developed the ability to resist food-processing temperatures that kill other organisms. Moreover, antibiotic resistance has become commonplace.

    Cebula wondered if this swift evolution is being driven by a subset of microbes capable of much faster than normal variation. To test the hypothesis, he turned to a rogues' gallery of organisms: “We [had] a collection of all the outbreak strains we've collected over the years,” he says. “We thought, there's probably nothing here, but let's screen some of these strains and see if we find a mutator.” He and his colleagues cultured the strains and then used antibiotics to screen for “hypermutable” strains that quickly developed drug resistance. “We were surprised at how often we found mutators,” Cebula says.

    Researchers had expected that, because most mutations are deleterious, mutators would tend to die out, limiting them to no more than 0.01% of a bacterial population. But Cebula and his colleagues found that the frequency of mutators in the E. coli and S. enterica isolates was far higher than that: more than 1%, and exceeding 5% and 6% in some strains.

    What's more, says Cebula, studies of the mutators showed that they were defective in genes needed for methyl-directed mismatch repair—the ability to correct mismatches between a newly synthesized DNA strand and the template it was copied from. Because of this flaw, such bacteria would not only alter their own genes faster than normal; they should also take in DNA from other bacteria more readily. As Miroslav Radman of the Institut Jacques Monod in Paris suggested in 1989, defects in mismatch repair open the genetic door between bacterial species: They allow recombinations that the system would normally abort when the foreign DNA creates mismatches.

    Because the genes for antibiotic resistance often travel between bacteria on foreign DNA, says Hanawalt, the FDA group's findings could account for the rapid spread of resistance among Salmonella and E. coli strains. “This can actually explain why resistance-transfer factors can get an edge in these cells,” he says. The same goes for genes that can make a bacterium more virulent, for example by helping it penetrate epithelial cells, says Paul Sniegowski, who worked on mutators at Michigan State University and is moving to the University of Pennsylvania: “There are chunks of DNA called pathogenicity islands that are quite similar to one another between otherwise different bacterial strains, and Cebula offers a route whereby those chunks could be more easily passed between the species.”

    These effects could explain why mutators are so common among the pathogenic strains of E. coli and Salmonella, Cebula and his colleagues say. Radman agrees. “Mutators win races much of the time, both in the lab and in vivo,” he says. “The mutator phenotype allows a much faster, 100- to 1000-fold faster, evolution in terms of adapting to the host.” To Radman, “The interesting question, then, is why are not all bacteria mutators?”

    Radman suggests that despite their advantages, mutators are ultimately less fit than organisms with more stable genomes. The isolates from disease outbreaks that Cebula studied, for instance, were most likely in the process of adapting to new niches and under pressure to escape immune surveillance and pharmaceutical assaults. Without that pressure, he says, the mutator frequency would drop. Cebula speculates that there may even be subpopulations of mutators that can actually regulate their mutation rates in response to their living conditions, keeping their mutational abilities in reserve for times of stress.

    The findings sound a warning note about food processing, says Douglas Archer, a food scientist at the University of Florida, Gainesville: “We have to think about different approaches, about assuring lethality, or building in multiple barriers that can't be overcome by microbes.” Indeed, in response to the latest E. coli outbreak in the United States, the government is now considering a requirement that fruit juices be pasteurized.

    But to Radman, the work also raises a hopeful possibility. “One could think seriously about devising a bactericidal drug or treatment that would be particularly effective in eliminating mutator bacteria,” he says. “But I would never promise people that even if we do find a trick to kill the mutators, the bacteria won't find another trick to avoid it. And then we'll have to find another. And so on. That's life.”

  13. Developmental Biology

    Tracing Backbone Evolution Through a Tunicate's Lost Tail

    1. Elizabeth Pennisi

    For years, researchers studying the evolutionary origins of the backbone have bypassed the world's furred, feathered, and finned creatures in favor of leathery little bundles of tissue called tunicates. Adult tunicates are sedentary sea-dwellers with no sign of a backbone, and they live like mussels, attached to a shell or rock and filtering food through chimneylike siphons. But larval tunicates are something else entirely: They're tadpoles, complete with a dorsal nerve, a flexible rod of support cells called a notochord, a tail, and skeletal muscles that power them through shallow tidal flats. As they develop into adults, the larvae shed these vertebratelike traits, reverting back to the ancient ways of the invertebrates—and in effect turning back the evolutionary clock.

    Now, as reported on page 1205, it seems that a single gene may guide the development of the tunicate larvae's suite of vertebratelike characters. Developmental biologists William Jeffery and Billie J. Swalla of the University of California studied a tunicate species that lacks even its embryonic backbone and traced this loss to the disruption of a single gene, named Manx after a tailless cat. Without Manx, the tunicate larva “seems to lose the whole [group] of characters at once,” says Swalla, now at Vanderbilt University in Nashville, Tennessee.

    It's an impressive result, biologists say. “I can't think of a single case when such a dramatic phenotypic difference can be traced to one gene,” marvels developmental biologist Sean Carroll at the University of Wisconsin, Madison. Manx “is going to strike people as a very powerful master regulatory gene … probably something very basic [to] setting up vertebrate architecture,” agrees developmental biologist Rudolf Raff of Indiana University in Bloomington.

    Evolutionary biologists are intrigued as well. By turning Manx off in specially bred tailed embryos, the team has experimentally re-created an evolutionary change that likely led to the tailless tunicates—a landmark achievement for researchers trying to piece together evolutionary history, says Raff. It's even possible that Manx was central to the evolution of chordates, the umbrella phylum of organisms that includes both vertebrates and tunicates and is defined by the presence of a notochord, spinal cord, and other characters.

    Jeffery and Swalla, then a postdoctoral fellow, began probing this problem almost a decade ago, theorizing that if they could find two closely related tunicate species with tailed and tailless larvae, the hybrids could serve as an excellent experimental system for identifying genes crucial to tail development. They found that there are about a dozen tailless species among the some 3000 tunicate species, and that taillessness arose independently five times. But only one tailless species, Mogula occulta, found off the city of Roscoff, France, turned out to have a close enough tailed relative, Mogula oculata, that the two could interbreed.

    In 1990, Jeffery and Swalla found that hybrid embryos of these two species looked identical to the tailed embryos, although their tails were somewhat shorter. That suggested that one or more genes were lost or disrupted in the tailless species, and that tails could be partially restored with only one copy of the gene. And in 1993, Jeffery, Swalla, and Niriyuki Satoh and Kazuhiro Makabe, now at the University of Kyoto in Japan, described a candidate gene—Manx—that was active in both the hybrid and in the tailed species but not in the tailless one. Suspicions that Manx was the crucial gene were further bolstered by the fact that Manx RNA shows up early in the development of cells destined to become notochord, tail muscle, and dorsal nerve cord.

    Turning tail.

    Embryos of tailed tunicate species (top), tailless species (middle), and hybrids (bottom) differ in expression of the Manx gene.


    Now Jeffery and Swalla have taken the next crucial step, demonstrating experimentally that Manx plays a vital role in the formation of tails and other chordate traits, and that it was altered in the evolution of M. occulta. They treated hybrid embryos with antisense DNA, which binds to Manx RNA and prevents the production of Manx protein. In the hybrids in which Manx was effectively turned off by antisense, the tail never developed. (They tried this experiment in embryos of the tailed species too, but the antisense could not block the maternal Manx protein in the egg, and so Manx activity could not be completely stopped.)

    It appears that Manx may act by turning on a cascade of other genes that control many chordate traits. Once the notochord formed in the hybrids, for example, it apparently coaxed nearby tissues to develop into chordate features such as skeletal muscles and a sensory organ in the head. And the team found that the gene's sequence contains a region that codes for a “zinc finger”—a feature of proteins that bind to DNA and act as transcription factors, regulating other genes. Another DNA segment codes for a nuclear localization signal, which helps get the Manx protein into the nucleus, says Jeffery.

    Those findings leave researchers wondering what other genes interact with Manx. “Manx is just a dumb transcription factor. It's being regulated upstream by something,” Carroll says. “What are the other things in the Manx pathway?” Jeffery adds that he and Swalla haven't ruled out the possibility that other pathways must also be disrupted to create tailless tunicates: “We need to find out if [Manx is] sufficient as well as required.” He suspects that the answer may be yes, because taillessness has arisen independently so many times. “This sort of suggests there may be a simple switch,” he says.

    He and Swalla are now trying to re-create the details of the genetic events that could have flipped that switch and led to the evolution of M. occulta. For example, as they sequenced Manx, they noticed that a seemingly noncoding segment of the gene actually contains a second gene, which codes for a protein called p68, whose precise function is unknown; this protein is found in much larger amounts in the tailless species than in the tailed one. Swalla speculates that taillessness arose when a mutation changed the splicing of the Manx gene, so that cells in the tailless embryo make p68 when they should be making Manx protein. Once the tailless, sessile larvae appeared, natural selection might have favored these mutants in some environments, for example in areas where sand flats are patchy and mobile larvae might be swept into deep water.

    Meanwhile, everyone is interested in whether Manx played a role much earlier in evolution, 550 million years ago when the chordates first evolved. Many biologists think developmental genes may have been involved in this event, for mutations in such genes offer a plausible way to dramatically alter an organism's body plan, and certainly Manx is now a leading candidate. Raff and Carroll expect researchers to start scrambling to find Manx equivalents in model species such as mice and frogs, to see whether the gene governs notochord and tail development in these true vertebrates.

    If so, then it's possible that one of the classical divides in animal life could be traced back to a single genetic change. Of course, “what we don't know, and what [our] studies cannot tell us, is what the true genetic event was,” says Nipam H. Patel, developmental biologist at the University of Chicago. “You can't run the tape of time backward, so all you can do is make good guesses.”

  14. Paleontology

    Early Birds Rise From China Fossil Beds

    1. Ann Gibbons

    Paleontologists have been arguing about the ancestry of modern birds ever since 1861, with the discovery of the first indisputable bird, Archaeopteryx, in Bavaria. With conspicuous teeth, a lizardlike tail, and feathers draped over a dinosaurlike body, this 150-million-year-old fossil has been touted as a “missing link” between birds and dinosaurs. But the bones of modern birds look different, and researchers have been unable to agree on whether Archaeopteryx or its close cousins did indeed lead to modern birds; some ornithologists even doubt that modern birds descended from dinosaurs. On page 1164, Chinese and American scientists present new fossils sure to take the debate to new heights.

    The bones represent what may be the oldest modern-looking bird, or ornithurine. If the dating is confirmed, it lived at the same time as a primitive, Archaeopteryx-like bird—and just an instant, geologically speaking, after Archaeopteryx itself. That could shove Archaeopteryx and another large group of primitive birds off the evolutionary branch that led to modern birds; it could even imply an earlier origin for all birds, perhaps before their putative dinosaurian ancestors. “It shows that there was a dichotomy, and that Archaeopteryx and most of the other early birds were a side line of avian evolution,” says University of North Carolina ornithologist Alan Feduccia, co-author of the paper with paleontologists Lianhai Hou and Zhonghe Zhou of the Chinese Academy of Sciences, and Larry Martin of the University of Kansas.

    But other paleontologists think Feduccia is out on a limb. Although few argue that Archaeopteryx itself led directly to modern birds, many think that a related primitive group called the opposite birds are close kin to modern birds. What's more, says Yale University paleontologist John Ostrom, “the dating [of the Chinese fossils] is controversial.” If the new fossils are substantially younger than Archaeopteryx, as other dating work suggests, then they may offer little new insight into the origins of birds, says paleontologist Luis Chiappe of the American Museum of Natural History in New York.

    Reconstructing the bird family tree has been difficult because of a dearth of fossils after Archaeopteryx. But new finds in the past 5 years have revealed unexpected diversity in early birds starting at about 135 million years ago. Most of these, the dominant birds of the Mesozoic, were enantiornithurines, or “opposite” birds, so named because three bones of their feet are partially fused from the top down, rather than from the bottom up as in modern birds. In contrast, the first few fragmentary remains that might be ornithurines didn't appear in the fossil record until later, about 120 million years ago.

    Until recently, many paleontologists thought that Archaeopteryx itself gave rise to opposite birds, which in turn gradually evolved into modern birds. That view has faded, but Chiappe and others still hold that opposite and modern birds are closely related sister taxa, with a recent common ancestor that lived at about the time of Archaeopteryx or a bit earlier; this scenario allows enough time for birds to descend from Jurassic dinosaurs.

    Feduccia and his colleagues now challenge that view with fossils of a bird the size of a sparrow, called Liaoningornis. The specimen, unearthed by a farmer in the Yixian formation in northeastern China's Liaoning Province, lacks a skull but includes a nearly complete skeleton with foot bones and a keeled sternum that resemble those of modern birds. Yet the Chinese scientists cite radiometric dates of 137 million to 142 million years for the volcanic rock of the Yixian formation, which would make the bird almost as old as Archaeopteryx. And the same beds also yielded a magpie-sized primitive bird called Confuciusornis, which shares many traits with both Archaeopteryx and opposite birds. Indeed, these rich beds also produced a controversial “feathered” dinosaur (Science, 1 November 1996, p. 720).

    According to Feduccia and Martin, the discoveries imply that by the time of Archaeopteryx, birds had already diverged into two lineages and had a rich history that is missing from the fossil record. One lineage led to modern birds. Another led to Archaeopteryx and the opposite birds, which they view as sister taxa, closely related to each other but distinct from the line that led to modern birds. And both these bird lineages must have descended from a much earlier ancestral bird. Feduccia reckons that the first bird must have lived about 76 million years before the birdlike dinosaurs of the Cretaceous—a fact that he says raises questions about the dinosaurian origins of birds.

    However, not everyone agrees with those dates. New argon-argon dates on the volcanic rock and sediment in the Yixian formation, presented at the recent Society of Vertebrate Paleontology meetings in New York, date the birds at only about 121 million years, says University of Toronto physicist Derek York, a co-author on the poster at the meeting. If so, then it's possible that Chiappe is right: Some unknown bird that lived about the time of Archaeopteryx underwent rapid evolution and gave rise to both opposite and modern birds, as represented by Confuciusornis and Liaoningornis.

    Feduccia's co-author Martin questions the new argon-argon date, noting that arthropods and pollen in the Yixian formation are characteristic of the Late Jurassic about 140 million years ago. Feduccia simply says that the dating controversy is overblown: “Whatever the date is, we're getting both types of birds shortly after Archaeopteryx”—making the stunningly successful modern birds early birds indeed.


  15. Developmental Neurobiology: Guiding Neurons to the Cortex

    1. Marcia Barinaga

    Mutations that disrupt cortical development are helping researchers identify the biochemical signals that neurons use to find their final destinations in the brain's cortex

    The woman whose brain image appeared on the cover of last January's issue of Neuron is outwardly normal. A recently married college graduate with a high-powered job, she complains only of occasional epileptic seizures. But inside her brain, the magnetic resonance image had revealed a striking abnormality. During embryonic development, large numbers of neurons had never made it to the woman's cerebral cortex, the neuron-rich outer layers of the brain, but instead remained jumbled in the neuron nurseries deep in the brain where they were born. This woman has one of several genetic mutations that seem to derail neurons on their journey to their proper locations in the highly ordered cortex. But she is one of the lucky ones. Most people with such mutations not only have epilepsy, but are mentally retarded as well.

    Until recently, neurobiologists knew little about the molecular causes of these rare conditions. But that has begun to change, and as researchers have gained insight into these human tragedies, they are also finding clues to a fundamental question about normal brain development: What molecular signals guide immature neurons in their complex migrations from their place of birth to their proper locations in the cortex?

    In the past 3 years, researchers who study these human conditions as well as several similar genetic mutations in mice have isolated two genes apparently at fault in the cortical malformations, and have mapped the chromosomal locations of three more. While they do not yet know what the proteins encoded by these genes do, they hope that the genes will ultimately help them piece together the signaling pathways that help brain neurons find their way. “Having a gene allows you to jump into a genetic pathway,” says neuroscientist Chris Walsh of Harvard Medical School, who studies two of the human conditions. The promise of this approach, says neuroscientist Verne Caviness, also of Harvard Medical School, means “people are going to work on this like a dog on a bone. They aren't going to let it go. It is too interesting.”

    Researchers have marveled for decades over the orderly migrations of young cortical neurons that give rise to the neatly layered gray matter of the cortex. By tracking the neurons in animal embryos, they found that they travel from the areas deep in the brain where they are born, following roadways formed by support cells called radial glia.

    The radial glia lead to a layer of cells near the outer surface of the developing brain called the preplate which the migrating neurons enter, arranging themselves in the middle “like the filling in a sandwich,” says developmental neurobiologist Carla Shatz of the University of California, Berkeley. That simple sandwich eventually becomes a multilayer club special, as subsequent generations of young neurons continue to stream in from below, passing through the layers of neurons already in place and arranging themselves in overlying layers, until there are six in all.


    A lissencephalic brain (bottom) appears smooth compared to the normal brain above.


    A jumbled brain

    Despite all they had learned about the neurons' migration, researchers still didn't know the identity of the protein components of the molecular machinery that gets them to their destination. The best entree to those proteins would be through their genes, which when mutated should cause the process to go awry. The first such mutation came along nearly 50 years ago, when D. S. Falconer, of the Institute of Animal Genetics in Edinburgh, U.K., described a mutant strain of mice known as reeler for their reeling gait, whose brains lack the normal cortical layers and have a jumbled-up collection of neurons instead. As researchers learned more about brain development, they discovered that normal cortical layers don't form in reeler because the migrating neurons never enter the preplate, but pile up below it. “This gene seemed to be indispensable to the conduct of the migration process,” says Caviness.

    But to learn what the reeler gene actually does, researchers needed to find the gene and its protein product. Finally, in the spring of 1995, that goal was achieved with two remarkable findings that re-energized the field: A research group led by Masaharu Ogawa at Kochi Medical School in Japan reported that it had made antibodies to the reeler protein and used them to identify the cells that express the gene, and a team led by Tom Curran, then at the Roche Institute of Molecular Biology in New Jersey, cloned the reeler gene itself.

    The Ogawa team made its antibodies by immunizing reeler mutant mice with the brains of normal mice. The idea was that because the mutants don't make the normal protein, they would react to it as foreign and make antibodies to it. But says Curran, now at St. Jude's Children's Hospital in Memphis, Tennessee, while such an experiment makes sense on paper, no one expected that it would work in reality: “What they did was really amazing.”

    The Ogawa team's next result was also “a big surprise,” says Caviness. Using their antibodies, the researchers identified the cells that make Reelin: the so-called Cajal-Retzius (C-R) cells, which are found in the part of the preplate that becomes the marginal zone, the top layer of the ever-growing club sandwich. Because loss of Reelin interferes with the migration of the cortical neurons up the radial glia, Caviness and others expected that the protein would be made by the cortical cells themselves, or perhaps by the glia.

    No less amazing was the Curran group's serendipitous cloning of reeler, while they were studying a different gene, called fos. They had introduced a mutant form of fos into mice to study its function. But one of their transgenic mouse strains “looked crazy,” says Curran. “[The animals] kept falling over. When we looked at their brains, my colleague Richard Smeyne said ‘That looks just like reeler.’” Sure enough, the introduced fos gene had accidentally disrupted and inactivated the reeler gene. Using molecular probes for fos, postdoc Gabriela D'Arcangelo and graduate student Graham Miao pulled out the reeler gene, which codes for a large (400-kilodalton) protein they named Reelin.

    Based on Reelin's structure, Curran predicts that the protein is secreted by the C-R cells and sticks to the molecular matrix surrounding the cells. This extracellular location suggests, he says, that Reelin may be signaling the migrating neurons to insert between the marginal zone at the top of the sandwich and the so-called subplate neurons at the bottom.

    But the nature of that signal is anyone's guess. Kazunori Nakajima, a neuroscientist at RIKEN in Tsukuba, Japan, who as a graduate student conceived of and worked on the antibody experiment with Ogawa, proposes that Reelin acts as a stop signal for each wave of arriving cortical neurons, telling them to get off the radial glia fibers and develop into a layer of mature neurons just under the marginal zone. But there are other possibilities, Curran notes: “Does [Reelin] allow the insertion because it creates a space, by repelling the subplate neurons? Or does it provide an attractive signal to the migrating cortical neurons?”

    One way to test those possibilities is to track down the receptor that allows cells to respond to Reelin and see which cells wear it on their surfaces. Curran's group and Nakajima are working hard to find proteins that bind Reelin and might therefore be its receptor. And there is the possibility of even more serendipity: Researchers at the Jackson Laboratories in Bar Harbor, Maine, have identified a new mutant mouse called scrambler, which has characteristics similar to reeler. Curran's group, in collaboration with Dan Goldowitz of the University of Tennessee, found that the scrambler mutation doesn't affect the Reelin protein. That, says Curran, means the scrambler protein “could potentially be a receptor” for Reelin.

    Another newly created mutant mouse may also help researchers tease out the Reelin signaling pathway. In the 1 October Proceedings of the National Academy of Sciences, a research group led by Ashok Kulkarni at the National Institute of Dental Research reported that it had made a mutant mouse lacking the gene for the catalytic subunit of a neuron-specific kinase called cyclin-dependent kinase type 5 (Cdk5). Although these animals suffer more serious defects—unlike reeler mice, they die before birth—their brains seem to lack cortical layers, as those of reeler mice do. Developmental neurobiologist Karl Herrup, of Case Western Reserve University in Cleveland, is now studying the mutant mice in collaboration with Kulkarni's group, to see how close their resemblance to reeler mice really is. It may be that Cdk5 is involved in the intracellular signaling triggered by Reelin, he says. But he cautions that the apparent similarity may also “turn out to be a red herring.”

    On the edge.

    Reelin (bright areas) is made in the marginal zone of the developing mouse brain.


    Missing ridges and valleys

    Mutant mice aren't the only guides to the genes that orchestrate cortical neuron migration. Human mutations that disrupt cortical development are offering more candidates. “What is really exciting” about the human conditions, says Berkeley's Shatz, “is that you see these abnormal cells, and they are reminiscent of reeler.” That suggests that the genes responsible for the human conditions may be involved in the very same neuronal migration process.

    The most severe of the human conditions is lissencephaly, which means “smooth brain,” so named because those stricken with it have a cerebral cortex that lacks the ridges and valleys characteristic of a normal human brain. Lissencephalic children are severely retarded; many can't respond to the world or communicate at all, and most suffer from seizures and die in the first few years of life. Their brains show abnormal cortical layering, reminiscent of that in reeler mice, says Orly Reiner, who studies lissencephaly at the Weizmann Institute in Israel.

    Three years ago, Reiner cloned the gene mutated in lissencephaly, and it turned out to code for a subunit of an enzyme that inactivates a phospholipid signaling molecule called platelet-activating factor. At present, researchers have no idea about how the enzyme might influence neuronal migration.

    To try to resolve the mystery, Reiner and her colleagues have cloned the same gene from mice and found that it is expressed in the migrating cortical neurons and in the newly forming cortical layers. That, she says, suggests that it is indeed involved in cortical development. Her group is making conditional knockouts of the gene so that she can turn the gene off in developing mice and study its effect on development.

    Even less is known about the genes mutated in periventricular heterotopia (PH), the condition that afflicts the woman whose brain scan appeared on the cover of Neuron, and in a disorder called double cortex (DC), whose symptoms—epilepsy, usually with mental retardation—resemble those of PH. Both genes are located on the X chromosome. As a result, virtually all PH and DC patients are women because males, who have only one X, are so severely affected if they inherit the mutant genes that they die before birth or at an early age.

    Like the lissencephaly mutation, those in PH and DC seem to disturb cortical neuron migration, says Harvard's Walsh, who is trying to clone the genes. Women with DC have a thinner than normal cortex with the correct layering system, and underneath it a wide band of cortical neurons that Walsh says appear to have “left the region where they were formed, migrated about halfway [to their destination beneath the marginal zone], and then stopped dead right there.” PH “is very similar to double cortex,” says Walsh, except that the affected neurons never leave the brain's ventricles, where they were born.

    Walsh cautions that although DC and PH look like migration defects, his group hasn't proven that is true. To do that will require neuron-tracing experiments that can't be done in humans. As soon as the human genes are cloned, says Walsh, the next task will be to clone their counterparts in mice, make mouse mutants, and trace the paths of the mutant neurons as the rodents' brains develop.

    These won't be the last genes for researchers in this field to add to their list; at last year's Society for Neuroscience meeting in San Diego, Kevin Lee of the University of Virginia reported his discovery of a mutant rat with a double cortex similar to that of human DC patients. The as-yet-uncloned gene appears different from the gene that causes the human disorder. With genes accumulating at this rate, the mechanisms that guide newborn neurons are starting to look almost as intricate as the cortical architecture their journeys create.

  16. The Neuromuscular Junction: The Supple Synapse: An Affair That Remembers

    1. Wade Roush

    As in a rocky marriage, the link between a muscle fiber and its controlling nerve endings is perpetually in flux, alternately gaining and giving up strength in the face of shifting demands. Some of these couplings must grow in size and power to keep up with muscle growth in young animals, for example, while others naturally shrivel and disappear when no longer needed. But while the sources of marital ups and downs among humans are all too obvious, scientists are still struggling to make out the ingredients of the remarkable—and advantageous—plasticity of the neuromuscular synapse. “There have to be very tight molecular controls” behind the synapse's ability to wax and wane in strength, says University of California, Berkeley, neuroscientist Graeme Davis, who studies the phenomenon in fruit flies. “We're doing our best to understand that.”

    Understanding the ups and downs of the neuromuscular synapse, after all, could lead to a bigger prize: insight into the workings of our own brains. Neuroscientists believe that our long-term memories are deposited in the brain in the form of physical connections between neurons that—like the neuromuscular junctions in fruit flies—wax and wane in strength. And strong behavioral and anatomical similarities between fruit fly synapses and nerve connections in the mammalian brain have led some investigators to believe that decoding plasticity in the fly could lead to a better understanding of learning and memory. As Berkeley developmental neuroscientist Corey Goodman puts it, “We think of the fly neuromuscular junction as more or less like the central synapses of the vertebrate brain.”

    In a package of recent experiments described in last month's issue of the journal Neuron, his group and others have now identified some of the genes and proteins involved in remodeling fly synapses—and shed light on broader puzzles in synaptic plasticity. One set of experiments, by Davis and colleagues in Goodman's laboratory, shows that synapse remodeling in molting fly larvae is highly reminiscent of the early development of the nervous system. Both make use, for example, of a protein called fasciclin II (Fas II), which helps hold bundles of growing nerve axons together until they reach their final destinations in the embryo. Neuroscientists have long speculated that postembryonic plasticity is an extension of developmental plasticity. But, says neurobiologist Mu-Ming Poo of the University of California, San Diego, “it's very exciting to be able to find out some of the molecular bases for these processes.”

    The Goodman team may also have come up with a solution to one of the central mysteries of plasticity: how changes in gene expression in a neuron that has many axons and many synaptic connections can alter the strength of only some of its synapses. Their studies suggest that the neuron nucleus oversees the assembly of the synaptic machinery while local factors in individual axons determine where in the nerve cell this machinery gets installed. And other researchers have shown how the cells on the other side of the synapses—in this case, muscle cells—help complete synaptic renovations.

    Free to flourish.

    In muscles from flies with decreased Fas II protein (bottom), enlarged synapses bear many more boutons (brown dots) than do normal synapses (top).


    FAScinating rhythm. Goodman and his colleagues took their first steps toward the current work when they were using the fruit fly (Drosophila melanogaster) to unravel a different puzzle: how the nervous system is wired up during development. In work begun about 9 years ago, they had shown that Fas II, an adhesive protein, is needed to keep long-distance axons stuck together in bundles, or “fascicles,” while they grow toward their target muscles. But in 1993, Columbia University neuroscientist Eric Kandel and co-workers made an observation that suggested Fas II might also come in handy during synapse remodeling.

    In studies of the sea snail Aplysia begun more than 2 decades earlier, Kandel's lab showed that changes in electrical activity can lead to long-term structural changes in the neuromuscular synapse. Kandel and colleague Sam Schacher then found they could re-create those activity-dependent changes in a tissue-culture dish by combining the neurons and muscle cells that bring about the gill retraction reflex in Aplysia and exposing them to tiny puffs of serotonin, the neurotransmitter that activates the reflex. Multiple puffs, they found, caused the neurons to sprout new axons and form more synapses with the muscle cells. This strengthening of neuronal connections, known as “sensitization,” is a crude form of learning and memory.

    The serotonin puffs, Kandel and co-workers later showed, work by activating an intracellular messenger in the neurons called cyclic AMP (cAMP). This, they found, has both local and general effects on a membrane-bound protein called Aplysia cell adhesion molecule, or apCAM. At active synapses, one of cAMP's jobs is to cause the cell to retrieve the protein from its membrane. Immediately after this change, the neurons sprout new axons and form more synapses. The timing suggests, Kandel says, that “these CAMs usually serve to inhibit synapse growth by zipping [nerve extensions] together. One of the prerequisites for growth may be to allow them to come apart.”

    That idea intrigued Goodman, because Fas II is apCAM's structural equivalent in the fruit fly. If Fas II was also apCAM's functional equivalent in regulating synapse formation, he hypothesized, then decreased production of Fas II or its removal from the cell surface would strengthen synaptic connections.

    But where Kandel's studies had merely shown a correlation between reduced cell adhesion and sprouting, the Berkeley researchers could use Drosophila's powerful genetics to show direct causation. Goodman and Neuron co-authors Davis, Christoph Schuster, and Richard Fetter compared the synapses of normal fruit flies to those of a mutant strain that produces Fas II at only half the normal level. The mutant fly, they found, had about a 50% increase in the number of “boutons,” the nodules containing neurotransmitters and their release machinery, at the synaptic connections with a body-wall muscle. This indicates that the decreased production of the protein did in fact foster synapse growth. In contrast, engineering the fly genome to express Fas II at high levels caused a sharp reduction in bouton number. These results “provided the strongest evidence yet that changes in the level of an adhesion molecule at the synapse really are regulating growth and remodeling,” says Goodman.

    But another mutant strain called dunce, which has abnormally high cAMP levels, suggested there was more to the story than just Fas II. When the Berkeley group examined the neuromuscular synapses in the dunce mutants, they found—as Kandel's studies predict—that like the Fas II mutants, they bore less Fas II than normal and had more boutons. But the researchers also found a change not seen in the Fas II mutants: The dunce synapses were functionally stronger, putting out more neurotransmitter per bouton when stimulated. In contrast, the average neurotransmitter output of the boutons in Fas II mutants was actually lower than normal. “We were a little surprised at that point,” says Goodman, for the result seemed to show that a reduction in Fas II could accomplish only part of the job of synaptic strengthening. One possibility was that cAMP strengthens synapses by affecting not just Fas II but another protein as well.

    And that hypothesis led Goodman and his colleagues to yet another protein: the cAMP response element-binding protein (CREB), a Janus-faced molecule that reacts to increased cAMP levels by either repressing or activating the expression of certain genes in the nucleus. Work in Kandel's lab had already shown that CREB is essential for long-term changes in synapse structure in Aplysia. Moreover, behavioral geneticists Tim Tully and Jerry Yin at Cold Spring Harbor Laboratory on Long Island had shown that doping fly neurons with CREB's activator form greatly increases the speed with which flies learn a simple task. Davis, Schuster, and Goodman wondered whether CREB's role in plasticity might be to strengthen synapses by carrying cAMP's second signal to the nucleus and triggering the construction of new neurotransmitter release machinery.

    To test the new hypothesis, the researchers created transgenic fly larvae carrying both the flawed Fas II gene and an extra CREB gene that they could turn on artificially. The results were “beautiful,” says Goodman. “When we put the two together—increased CREB activator at the nucleus and decreased Fas II at the synapse—we got a bigger, stronger synapse that was indistinguishable from those of the dunce mutants.” The researchers had reconstituted two of the major biochemical pathways that strengthen a synapse. Increased neuronal activity activates cAMP, which triggers both the removal of Fas II at the synapse—allowing physical expansion—and CREB-mediated construction of new neurotransmitter machinery, providing extra firepower.

    As Kandel and Columbia colleague Kelsey Martin argue in a review article in the same issue of Neuron, the Berkeley group's findings also suggest a possible solution to a problem that has puzzled neuroscientists ever since the early experiments on sensitization, cAMP, and CREB. In the vertebrate brain, a single neuron may make synaptic contact with as many as 1000 other neurons, but the changes in synapse size and strength that encode any particular long-term memory may occur in only a few of these synapses. If changes in gene expression are crucial to learning and memory, how can these changes affect only a few of a neuron's many synapses? Kandel proposes that the synapse enlargement and other changes triggered by the down-regulation of Fas II in specific axons pave the way for the installation of new neurotransmitter release machinery produced at the direction of the nucleus. Axons still gummy with Fas II, on the other hand, have no room for such reinforcements. That way, the cellwide effects of the changes in gene expression can be targeted to just a few synapses.

    Axon addition.

    In the Goodman group's new model, neural activity both reduces cell adhesion locally (C) and increases production of neurotransmitter release machinery (D), creating bigger, stronger synapses (E).


    Care to dance? Intriguing as they are, though, the Berkeley group's findings tell only one side of the plasticity story. Added neurotransmitter output by neurons counts for nothing unless muscle cells are equipped with enough neurotransmitter receptors to detect this amplified signal, and results described in the October Neuron by another team point to an important role for another fly protein, called discs-large (DLG), in ensuring that there is enough acreage in the muscle membrane around each synaptic bouton for the needed neurotransmitter receptors.

    DLG is made by the neurons, but it crosses the synapse and accumulates mainly in the subsynaptic reticulum (SSR), a convoluted part of the muscle cell membrane that surrounds each bouton and harbors the receptors. Flies in which the DLG gene is mutated have shrunken SSRs, and that plus DLG's cellular location led neuroscientists Vivian Budnik, Michael Gorczyca, and co-workers at the University of Massachusetts, Amherst, to suspect that the protein might be involved in synapse development and plasticity. Consistent with that idea, Budnik's group discovered that in the mutant flies, in what may be an effort to compensate for that deficiency, the electrical output of each axon is much greater than normal.

    What finally persuaded the Massachusetts team that DLG is the neurons' tool for coordinating SSR size—a muscle cell's “receptive area”—with the neurotransmitter output of the adjacent neuron was their finding that artificially expressing the protein in mutant neurons both restored the electrical output of the boutons to normal and corrected the SSR defects. In both development and plasticity, says Budnik, “there has to be some sort of adjustment between the pre- and postsynaptic sites. Perhaps DLG is involved in that matching.”

    While the Budnik and Goodman studies have concentrated on the neuron's role in plasticity, a third team's findings show that the neuron doesn't always lead in its complicated dance with its muscle-cell partner. Neurobiologists Michael Bate, Andreas Prokop, and colleagues at Cambridge University studied mutant fly larvae whose embryonic muscle tissue never differentiates into individual cells. They report in October's Neuron that axons in these mutants find their way to the correct target muscle precursors, but they then mistakenly form synapses facing supporting tissues, body fluids, and each other—but never muscle. The message, says Bate, is that “you actually need something from the muscle for the axon's synaptic apparatus to be properly localized”—an as-yet-unknown molecule that's missing from muscle tissue whose development is derailed by the mutation.

    With all the new proteins and genes they have seen taking part in synapse formation and remodeling, neuroscientists now have a number of leads they can follow as they attempt to solve the molecular mystery of plasticity at the neuron-muscle junction. And those investigations, in turn, promise to take them closer to their ultimate goal—the brain. The only surprise, say Goodman and other researchers, would be if the fine-tuning of neuromuscular synaptic connections and mechanisms for learning didn't have molecular steps in common. Says Kandel: “Whenever you convert a short-term change in activity to a long-term change involving growth, it may require a similar program. This may be only the tip of the iceberg.”

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