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Trials, and a series of errors, in the brain lab

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The myth of modern science, that it proceeds carefully, rationally, incrementally, building bit by bit from rock-solid foundations to impregnable fortresses of fact, comes unraveled in contemporary neuroscience. Fortresses, entire kingdoms of neuroscience have been built on what turn out to be frail premises that get swept away entirely when the next new thing comes along.

A few years ago, a huge amount of effort was spent researching the then-thought marvelous qualities of a humble molecule called nitric oxide. This molecule, better-known in the broader world as the key element in laughing gas, was celebrated as a vital actor in human memory and cognition.

Science Magazine, as if honoring a rock star or president, put the thing on its cover and declared it Molecule of the Year.
FOR THE RECORD:
Chasing memory: An article in Monday's Section A about UC Irvine neuroscientist Gary Lynch's research into memory said nitric oxide was a key element in laughing gas. It is not; nitrous oxide is what makes up laughing gas.

By the end of the next year, nitric oxide had fallen off the end of the Earth. Little of what had been claimed on its behalf turned out to be true. This was but one example in a long, sad tradition of a science, as if gripped by mass hysteria, going off the deep end and pretending it knew how to swim.

There was no guarantee, neuroscientist Gary Lynch liked to say, that something was important just because you happened to study it.

"You always imagine those animals out in a herd, the wildebeests -- they're running along, and a lion jumps up and takes out this guy named Clyde," Lynch said. And the world proceeds as if Clyde never happened. "They don't talk about Clyde anymore. It's just not good form to talk about him."

Lynch, who runs a lab at UC Irvine, has spent three decades studying a phenomenon known within neuroscience as long-term potentiation, or LTP, which can be very loosely defined as a process in which electrical stimulation strengthens connections between brain cells. Lynch had taken up the study of LTP because it had characteristics strikingly similar to human learning and memory. It seemed to take place in parts of the brain where memory was thought to occur, and like memory, it occurred in an instant and could last a lifetime.

The practical reality of memory -- that human beings, from very young ages on, learn and store information -- had been established and studied for millenniums. How it happened, however, remained a dark continent yet to be mapped.

When people, even scientists, talked about memory, they likened it to objects or concepts in everyday life. They talked about filing cabinets, and photographs, and videotape replays. They almost never talked about what memories really, physically were. Why? They didn't know. LTP seemed an excellent candidate to be that physical, molecular underpinning of memory.

Beyond the pure scientific intrigue of it, memory research has grown more important as medical advances allow more people to live into old age. With longevity has come an epidemic of memory failure among the aged. Alzheimer's disease and other forms of crippling dementia threaten to make living longer less a blessing than a curse.

Things can go radically wrong inside an old brain, and unless you understood how the physical processes of memory worked, Lynch thought, you'd never be able to fix it when it broke.

Brain scientists generally agreed that networks of neurons somehow wired together in the brain were fundamental elements of memory. LTP was hypothesized as the means by which that wiring occurred.

Lynch had bet his career that he could work out the details of LTP, and that what he found would matter -- that it would turn out to be something beyond "an interesting little bit of biology." There were too many similarities between LTP and memory, he thought. The gods were unpredictable, he said, but seldom that cruel.

LTP had been discovered in 1973 not as a naturally occurring phenomenon, but in the artificial and arbitrary conditions of a laboratory experiment. Because of this, not everyone was convinced LTP had significance outside the lab. As one of Lynch's rivals, Nobel laureate Eric Kandel of Columbia University, said: "You know what LTP is? It's an artificial way of stimulating your brain. Who knows if this is what happens in learning and memory?"

That no one had figured out LTP was due largely to the inherent complexity of brain biology. Seth Grant, a neuroscientist at the Sanger Institute outside London, has counted more than 1,000 proteins thought to be involved in memory. If even half of that number actually were involved, isolating and understanding the behavior of each would be a Herculean undertaking.

Lynch was more prosaic. "It's a bitch and two-thirds," he said. "And stupid too."

Other scientists had moved on. "The boys," as Lynch routinely referred to the neuroscience establishment, turned en masse to the exploration of what genes might be involved. That they were able to find such genes almost at will was read by gene proponents as a reason to stay and look for more. To Lynch, it made no sense. "It's like trying to understand a computer by studying it a transistor at a time. Not only will it take forever, it will never work. You'll never get there unless you understand the programming."

"I asked myself: 'Why am I following you down this alley?' So I didn't."

Lynch instead rode out the LTP bet. In January 2005, after decades of studying and teasing out its details, he was in the midst of an experiment to determine once and for all whether LTP was a mere laboratory curiosity or the real thing -- the means by which neurons were wired together to form memories. Lynch, in other words, was about to find out whether he was a candidate to stand Nobel shoulder to Nobel shoulder with Kandel. The alternative? He was Clyde.

Lynch had long ago proposed that the end result of LTP was a micro-scale physical remodeling of neurons that allowed them to communicate better with one another. The lab had just developed a new technique that Lynch thought would allow researchers to visualize this remodeling, in fact, to see the physical trace of memories.

This new technique promised to answer conclusively what had been supposition, and to answer it in such a way you would literally see the result.

Neuroscience comprises many distinct disciplines, or tribes, as Lynch called them, ranging from mathematicians to evolutionary biologists. Eniko Kramar, a senior scientist in the lab, was actually going to run the experiment. Kramar was a neurophysiologist, meaning she studied the function of brain cells. Physiologists, generally, can be thought of as engineers. They're practical people, interested in how stuff works.

At the moment, the stuff in question was synapses in a rat brain. Most brain research labs used animals in their work, the main reason being the lack of human subjects willing to have their brains dissected. Lynch Lab used rats almost exclusively.

Other labs used mice or simpler creatures -- fruit flies and sea slugs. Genes that performed certain known functions in fruit flies did the same or similar work in humans; the genes were conserved, scientists say -- natural selection winners passed up the evolutionary chain. Use of these animal models is a daily expression of unquestioned trust in evolution as a central fact of human history. Even as debates might rage in broader society over the idea that human beings are descended from apes, there was a strong conclusion in biology labs that human antecedents go back way past the apes to the flies and beyond.

Still, Lynch wondered about the practicality of studying memory in nonmammals that, in human terms, didn't have any. "Memory is an emergent phenomenon," he said. "Steam is an emergent phenomenon. If you want to study steam, you better study hot water. You ain't going to get steam out of mud."

Visualizing success

Kramar's experiment began with the death of a rat, which she accomplished using a small guillotine. It took her less than five minutes to decapitate -- or, as she put it, sacrifice -- the animal, cut open its skull, remove the brain and separate the hippocampus, a portion of the temporal lobe thought to be involved in memory, from the rest of its cortex. She then sliced the hippocampus, which in a rat is about the size of a clipping from a thick thumbnail, into five very thin sections.

The slices were transferred to a small, circular Plexiglas chamber centered on a workbench under a microscope. The chamber was fed by separate lines carrying a nutrient-rich warm liquid and oxygen, which together kept the brain alive and in some sense functioning.

The top of the chamber had cutouts that allowed electric probes to be placed into the brain slices, one for stimulating and one for recording. The stimulating electrode could be set to deliver currents of precise timing and duration. Lynch and colleagues had discovered decades before that LTP was optimized when initiated by electric currents that mimicked a naturally occurring rhythm within the human nervous system known as theta rhythm. This coincidence -- that the best way to obtain LTP in the lab was to mimic actual real-world biology -- had, more than anything, convinced Lynch that LTP was real.

"That day -- the day we found theta -- our mouths fell open," said John Larson, who worked with Lynch on the discovery.

Kramar's chamber was situated on a table equipped with shock absorbers to prevent the rumble of a truck or car outside from disturbing the queasy equilibrium of the experiment. Because the electrical measurements needed to be precise, any equipment that might interfere with them was grounded, and connections were shielded with aluminum foil to prevent stray signals from intervening.

With all the foil and electrical tape and ground wires, the whole apparatus, which the scientists referred to as a rig, had a kind of jerry-built, Rube Goldberg quality.

After the slice was stimulated to mimic the theta rhythm, the current would pass along previously identified pathways, setting off biochemical reactions. The recording electrode measured the current as it exited the slice. The data were fed automatically into a computer program that translated and graphed the results. If LTP occurred -- that is, if more neurons were wired together -- more current would move through the slice after the stimulation than before.

Then, by using chemicals to block the actions of different molecules within the brain slice, the scientists could tell whether those molecules were essential to LTP. By laborious process of elimination, they ought to be able to unveil the entire process. The essential parts of that process would be the fundamental building blocks of memory. They had done a great deal of the work already, identifying what they thought were the key steps. They hoped their new visualization technique would allow them to actually see some of those steps.

Kramar was an exacting person, naturally fastidious in setting up the experiment. She knew what was at stake. She didn't need Lynch leaning over her shoulder to tell her this was important. Theirs was a fraught relationship. In one important way they were complementary. Lynch was given to big-picture conceptualization, while Kramar lived at the level of the brush stroke. Their temperaments were so utterly different as to be nearly opposed.

Kramar punched a key on her computer, initiated the electric pulse and waited. The recording electrode was picking up interference from elsewhere, overwhelming the readings. Kramar tried, patiently at first, to isolate and banish the interference. She spent hours looking, but never found the source. The brain tissue died in the chamber.

The day's work ended before it ever got underway. Lynch wouldn't be happy and Kramar knew it, but she was more upset that she had killed a rat to no good end.

The seemingly random interference was the kind of thing that drove the scientists crazy. It wasn't enough that they were opposed, they felt, every step of the way by the complexity of the biology. They had to fight their way to even get to the biology. Kramar had run slices in other experiments in this rig for months without anything like this ever happening.

"You do the same thing every day for a year, and then one day, for no reason at all, you can't do it," she said. "It makes no sense, but you just have to come back and do it again."

The next day, the interference was gone, a ghost vanished. Kramar ran the experiment, stimulating the slices, taking her readings, then infusing the slices with a dye that would stain only the portions of the neurons that had been changed in the experiment. They would show whether Lynch's hypothesized remodeling had occurred.

Afterward, she packed the slices on ice and took them to a nearby lab, where they would be prepped and mounted on slides.

Two days later, Kramar, with her own and Lynch's great anticipation, got the mounted brain slices back. They were worthless. Either the experiment had failed to produce any effect on the slices -- unlikely -- or the slices had been improperly mounted. She would have to start over.

Lynch said: "You're always surprised or horrified or pleased or something. It's not what you expected. It's always a bunch of crap."

A whimper, not a bang

The next day, Lynch said: "Several years ago, I sent a student out and said, 'Your job is to find out what the boys know about assembly.' That's what grad students are for. They're the cannon fodder of science. You throw them at problems that have no chance of being solved. One day, the student came back and said a new thing -- integrins."

Integrins tie cells down to a particular place. They fix, for example, blood cells into place so that a cut will clot. Think of them as a kind of cellular thread that stitches cells into place.

As is typical in biology, molecules that perform a specific function in one place often perform some variation of the same function elsewhere. So Lynch presumed with integrins. He made them a key part of his investigation, and the lab had since reported that integrins in the brain fastened neurons in place, locking in the changes LTP created.

"The only thing that keeps the neurons in your brain from rolling out your nose is the fact that they're stuck together at adhesion junctions. The adhesion junctions are actually the synapses," Lynch said. "It's the boring biology of wound-healing, of blood platelets clotting. . . . That's what I love about it, you know. It's like the T.S. Eliot thing -- when it's all over it'll be a whimper, it won't be a bang. It won't be a magic protein, it won't be a special gene. It won't be any of that crap. It'll be watching a cell crawl across the dish."

Just then, Ted Yanagihara, a gifted undergrad who was working with Kramar, poked his head inside Lynch's office. It was a mark of the meritocratic nature of the lab that Yanagihara, just a kid, really, was entrusted with such work.

"Bad news," he said. "I have a result, and it's not a good result."

Kramar, in addition to the visualization experiment, was working with Yanagihara trying to gather further evidence that integrin molecules were one of the building blocks of LTP. They used chemicals, called antibodies, to stop integrins from having any effect. If they did, they hypothesized, they would block the final stage of LTP.

Kramar was doing one version of this test in her slice experiments. Yanagihara was doing another working with single cells. Everybody was on edge. Some days the methods failed and they couldn't gather results. On days their methods worked, the data were wrong or confusing. Sometimes the integrins were blocked, sometimes they weren't. This continued for two weeks.

Said Lynch: "That's the trouble with biology: There are just too damned many variables."

"It's a wonder anything ever gets done," Yanagihara said.

Lynch said: "If you want clean results, go be a physicist."

Lynch accepted the repeated failures with surprising equanimity. He'd been through worse droughts before. One experiment in the early 1980s took two years. It had turned an entire cohort of grad students into a contemporary legion of the damned, but the legion kept marching and eventually the experiment succeeded.

Lynch amused himself. He had been shopping for a new car, his first since a 1987 Ford Mustang that was now falling apart, day by day, piece by piece, in the parking lot. Lynch had been a drag-racer as a kid, and cars, along with single-malt Scotch whiskey and good books, were among the very few possessions he cared much about.

He wanted a brand-new Chevrolet Corvette convertible, a formidable machine, but he couldn't find the model he wanted nearby. While everything was falling apart in the lab, he found a dealership in San Jose that had it. He hopped a flight, wrote a check and drove the car home that night.

Kramar took the setbacks more personally. By the end of the month, she was exhausted and did the unthinkable -- she took a weekend off. "I thought, I'm not even going to show my face. When I get like that, I have to back away from everything. I went out and bought books, then sat home and read them."

The mood in the lab had grown very dark. One day, Yanagihara said to Kramar: "This is a nervous moment."

Kramar replied: "You're nervous? It's my career at stake."

She laughed dryly; no one laughed along. The integrins were a crucial part of the hypothesis, and apart from what would be the acute embarrassment of having to retract previously published conclusions, Lynch had no alternative explanation. His entire research program would be a shambles.

It was remarkable that so much could go so wrong all at once. Some days the electric probes were too noisy to produce reliable results. One day a computer melted, smoke rising from its innards. Programs crashed. A day's work was halted when a grad student couldn't make it to the lab.

"The answer is sitting there waiting for you, and you can't do anything about it because your graduate student got his car impounded," Lynch said, then went off on a long rant about the torture of academic biology. "If I never go to another meeting, get involved in another symposium, I'll be happy. I don't care if I ever train another graduate student. Don't get me wrong. I'm pleased with the way they've turned out. Lots of them have gone on to do interesting things. But I want to be done. Done. Over."

There was an occasional ray of hope.

Yanagihara one day, finally, working with the brains of young rats, got his experiment to work right, and found the result he was expecting to find. "If we get it tomorrow in middle-aged rats, it's great," he said.

"If you see a garbage can flying out of the lab onto the hedge, you'll know we didn't," Kramar said.

The next day, the trash cans remained inside, but only because nobody had the energy to throw them out the window. The experiment had failed again.

terry.mcdermott@latimes.com Copyright © 2014, Los Angeles Times
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