October 01, 2007
A biochemistry-biophysics collaboration produces insights into the formation of long-term memory.
Few things about aging are more terrifying than losing our memory. Perhaps it’s no surprise, then, that figuring out how memories are made is currently one of the hottest topics in scientific research.
At Lewis & Clark, two professors have focused their microscopes on neural proteins to learn more about how the brain locks in memories for the long haul. Biochemist Janis Lochner, Dr. Robert B. Pamplin Jr. Professor of Science, and biophysicist Bethe Scalettar, professor of physics, are examining the sequence of molecular events that occur at the synapse, the space separating two neurons where signals between the two nerve cells are transmitted.
They hope to piece together a section of the brain’s larger learning-and-memory puzzle: work that may someday contribute to treatments for neurological diseases such as Alzheimer’s and Huntington’s. Already, by focusing on a pivotal family of proteins, and by being the first to detail the traffic patterns of these proteins in individual cells, the pair has won accolades usually reserved for scientists at large biomedical research labs:
In June, Lochner spoke on a panel at a conference in Stockholm sponsored by the Nobel Committee on Chemistry; joining her on the dais were scientists representing high-powered labs at Harvard University, Rockefeller University, the University of Cambridge, and Switzerland’s Friedrich Miescher Institute.
Aided by four Lewis & Clark student coauthors, Lochner and Scalettar authored the Journal of Neurobiology’s May 2006 cover story. Their experiments showed for the first time that the protein tissue plasminogen activator, or tPA, was released from a neuron into the synaptic divide.
Earlier this year, a committee of peers ranked Lochner and Scalettar’s two most recent grant proposals to the National Institutes of Health in the top 1 percent of all applications in their discipline.
By combining their expertise in molecular biology (Lochner) and fluorescence microscopy (Scalettar), the two longtime colleagues are making a name for themselves in a highly relevant and highly competitive field. Together they’ve been able to attract grant monies that would otherwise be out of reach. “Our ability to survive in the memory field,” Scalettar says, “is due to our strong, long-lasting collaboration.”
What’s Known About Memory
Since the late 1950s, scientists have known that memories are inscribed on a small, seahorse-shaped part of the brain called the hippocampus. Memories form when the brain’s nerve cells, known as neurons, communicate by swapping molecules at places between the neurons called synapses. (Think of the synapse as a kind of “Memory Lane” between neurons.)
Also, it’s long been known that to form long-lasting memories, rather than fleeting ones, the neuron must produce specific proteins. But the breakthroughs most relevant to Lochner and Scalettar’s work came in the 1980s and 1990s, when Columbia University neuroscientist Eric Kandel identified these new proteins and showed that they enable long-term memories by strengthening the synaptic connection between two neurons. (To continue the analogy, it’s as if the proteins don hard hats to widen and repave Memory Lane.)
Since Kandel’s discoveries, various researchers have been sorting out which genes activate which proteins, and trying to determine the precise role of each of the dozens of proteins implicated in the formation of a long-term memory. Most of these researchers study neurotransmitters, chemicals that act as messengers from the transmitting neuron to the receiving neuron. Lochner and Scalettar, however, study different proteins, known as neuromodulators, which enhance or inhibit the transmission of nerve impulses. Three neuromodulators involved in the long-term memory process–tPA, brain derived neurotrophic factor (BDNF), and plasminogen–are thought to be released into the synapse by the receiving neuron.
“Kandel identified several proteins essential for synaptic strengthening,” explains Lochner, “and we’re trying to understand how they’re released and what types of stimuli trigger optimal release.”
Making the Protein Glow
How do you locate and track a protein in a single nerve cell? When Lochner and Scalettar first joined forces on a project, in the mid-1990s, they won a grant from a National Science Foundation program that encourages collaborative research at undergraduate institutions. They proposed to determine the role of a certain family of proteins, called proteases, in still-developing nerve cells using a brand-new research tool.
It turns out there’s a protein in a species of Northwest jellyfish that radiates an emerald green color, and when you append this jellyfish protein to another protein, “it acts like a little light bulb that glows green,” Scalettar explains. Scientists cloned the so-called green fluorescent protein (GFP) in the 1990s, and soon engineered variations that give off different hues, including royal blue and cherry red.
Lochner and Scalettar first used GFP to track their proteases, one of which was tPA. Later, Kandel identified tPA as critical to memory and learning, and the Lewis & Clark pair segued into the more interesting, grant-rich field of neuromodulators–with the neon jellyfish gene still playing a leading role in their research.
In their current work, one of Lochner’s jobs is to combine the various-colored glowing genes with the genes that trigger the production of the three neuromodulators. Using tiny test tubes in her first-floor laboratory in the Olin Center for Physics and Chemistry, she links and sequences the genes to create a hybrid that will express the target protein in the right place and with the right characteristics.
Then she’ll take the hybrid gene to nearby Oregon Health & Science University (OHSU) and introduce the hybrid gene into rat hippocampal neurons maintained in the laboratory of Gary Banker, director of OHSU’s cellular neurobiology program. Banker is credited with pioneering the techniques to isolate and maintain hippocampal neurons.
Lochner spent a year’s sabbatical with Banker in 1998-99 to learn how to work with these delicate and demanding cells; Scalettar is spending her sabbatical in 2007-08 there as well. Both say their relationship with a large, nearby research institution provides significant value, mostly through access to additional scientists, resources, and techniques.
The same advantages extend to Lewis & Clark undergraduates, who, by rubbing shoulders with graduate and postdoc students in Banker’s lab, get a taste of what’s it like to work in a university bioresearch laboratory. Banker says he and his staff appreciate the interaction with the Lewis & Clark professors and students, too.
“I think most people here who’ve had a chance to talk with [Lochner and Scalettar] are pretty blown away, especially given that they’re at an undergraduate institution like Lewis & Clark,” Banker says, citing the Journal of Neurobiology cover story as one example. “It’s unusual for faculty at a small liberal arts school to be able to publish in a top-flight journal in a field that [requires as much grant money] as biomedical research.”
Through the Microscope
Once researchers have inserted the genes into the neuron, they repeatedly stimulate the cell using electricity or chemicals to mimic the memory-formation process. Then Scalettar takes over. “I’m a microscopy person,” she explains, “and the microscope is useful in studying transport and distribution and release, looking at how tPA and the other proteins get from inside to outside the cell.”
Scalettar examines the glowing proteins using a fluorescence microscope, a sophisticated, camera-equipped device that–because of its six-figure price tag–is rarely found at liberal arts colleges. Scalettar uses a reliable DeltaVision model developed by former colleagues at the University of California at San Francisco. “I think we’re lucky to have it here,” she says.
Using precise “micro-stepper” motors, which change the plane of focus in incredibly small increments, and sophisticated image-deblurring software, Scalettar can zero in on different layers of the cell and take amazingly sharp images of the neuron’s protein traffic. The images have confirmed the pair’s unprecedented findings: that tPA and the other neuromodulators are exiting the receiving neuron at sites where long-term memories are formed.
“Not many people have been able to look at that release at a level this fine,” says Lochner. “A lot of people have shown when a culture of a million neurons is stimulated in a manner similar to invoking a long-term memory, these proteins come out. But we study release from individual synapses. That’s what is technically hard to do.”
Overcoming that challenge is what gives Lochner and Scalettar the most satisfaction. While they like the fact that their research might someday help produce a cure for dementia, to them the true payoff is helping decipher one of the brain’s most complex, vital, and–to scientists, at least –enigmatic functions.
“Bethe and I,” Lochner says, “think there is a sophisticated regulatory mechanism for releasing these proteins.” Stay tuned: they aim to discover it.
Dan Sadowsky is a freelance writer in Portland.