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Cells say the darndest things

In an important early step toward understanding the chemistry of human thought, Stanford chemists have managed for the first time to read the individual chemical messages that cells exchange.

These messages account for nearly all of the communication that takes place in the brain, but previous efforts to record them one at a time have been thwarted by their extremely small size. More than a billion vesicles -- the microscopic membrane-bound pouches that carry the chemical signals -- could fit into an average-sized drop of water.

"We really are seeing a new era dawning in which people are trying to understand the chemistry of the brain and of the central nervous system, with the possibilities of amazing consequences, from treating mental disease to improving mental powers," says Richard N. Zare, who headed the research effort.

Zare, the Marguerite Blake Wilbur Professor of Chemistry, and his colleagues published the technique that allowed them to read out the contents of individual vesicles in the journal Science earlier this year.

Cells use vesicles to signal each other in a variety of ways. Such messages include mail to the brain that says "ouch!" from a bumped funny bone. Other messages regulate the female reproductive cycle.

By analyzing large numbers of vesicles together, scientists had learned a great deal about the vocabulary of the cell's language. But this method can deduce only average messages. It is as if researchers knew that in 1,000 letters, the word "food" appeared 1,800 times, but they could not discern how many times (if at all) "food" showed up in any one letter.

Zare suspected the average message might not be a good representation of any single vesicle's contents. "Could it be that the real signals are being hidden and confused by looking at only averages?" he wondered. "I thought likely so, and indeed our work shows that to be the case."

The signal from a single vesicle is important because cells send and receive vesicles one at a time. "The contents of one vesicle are enough to stimulate a cell," says Richard Scheller, professor of molecular and cellular physiology at Stanford and a Howard Hughes Medical Institute investigator. He and Zare have been working toward analyzing single cellular packages for several years.

"Almost all [communication] within our brains is the result of vesicles being released one at a time," Scheller says. "Different responses are elicited from vesicles with different contents. So it's very important to know the contents of the individual vesicles."

But the vesicles that transmit messages between brain neurons are too tiny to manipulate and examine. Instead, Zare and Scheller chose to work with the vesicles that regulate egg-laying in sea slugs. These vesicles are about 1,000 times larger than those the brain uses ­ large enough to analyze with Zare's new technique. "Snails don't have many cells, but the cells they have are large and juicy," Zare explains.

Daniel Chiu, a graduate student, and Sheri Lillard, a postdoc in Zare's lab, developed the technique, which captures one vesicle at a time, pops it open and analyzes its contents.

The first step in this process is to trap a single vesicle and introduce it into a tiny chamber, where Chiu and Lillard can manipulate it. This step is not as simple as it may sound. Because the vesicle is so small, the researchers have to use delicate procedures.

The chemists trap the tiny package using a laser. The vesicle moves to the most intense area of the laser beam and stops there. Once the vesicle is immobilized, the researchers move a minuscule tube next to the vesicle. A slight electric current slides the vesicle into the tube.

Next, the researchers add chemicals that tear open the package and label its contents with a dye. This dye, which attaches only to specific molecules, glows when light hits it. The dye transforms the vesicle's chemicals into beacons.

The dye fastens to several different molecules ­ perhaps all the nouns in the cellular message ­ but the researchers want to know how much of each compound was in the vesicle. So Chiu and Lillard separate the mixture into its components by forcing the chemicals to move along the tiny tube that contains them.

Because each different chemical moves at a characteristic speed, the various compounds arrive at a detecting station at different times. The detecting station watches for the lights to pass, a brighter glow indicating a larger amount of one chemical, a dimmer light meaning a smaller quantity. In this way, Lillard and Chiu measure the relative amounts of the various chemicals.

The chemists then identify each chemical compound in collaboration with researchers led by two former postdoctoral researchers from Zare's lab: Owe Orwar, a chemist at Chalmers University in Göteborg, Sweden, and Evan R. Williams, a chemistry professor at the University of California-Berkeley. The researchers can thereby read the cellular mail.

Using this technique, the researchers have found that individual vesicles have varying contents, even though the vesicles came from the same gland. "One vesicle would have one component but it would be completely absent from another vesicle," Lillard says. "We knew that in a population, both components were present, but when we got down to the level of a single vesicle, there were clear differences."

But what does this variability mean for the sea slug? "I can summarize that in three words," says Zare. "I don't know. Not yet. Is it the difference between mature and immature vesicles? Is it the difference of some control? I don't know."

It's one thing to read individual words in cellular mail; it's another problem altogether to translate the language. But simply reading the chemical codes that the vesicles contain is an important step toward understanding cellular communication, the researchers maintain.

Eventually, Zare and Scheller hope to extend the technique to look at the contents of smaller vesicles, such as those released by neurons in the brain.

Zare says, "I think we are showing the way, developing it step by step, so that we can apply this to smaller systems and, indeed, learn about the chemical basis of thought."

The research was funded in part by the National Institute on Drug Abuse and the National Science Foundation.


By Lila Guterman

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