This article first appeared in the Fall 1995 issue of LabNotes, an MBL publication.

In the years following Young's 1936 paper, "very suitable" turned out to be an understatement, and squid axons soon became one of the classic texts studied by biologists. By 1939, scientists at the MBL and in England were putting electrodes inside the axon to measure the electrical activity associated with nerve impulses. By 1949 they were working out a detailed picture of the cell's electrical activity by measuring the flow of charged particles back and forth across the axon's membrane. With the foundations for modern neuroscience in place, interest in the squid giant axon did not diminish, but rather increased.

By the 1960s, more than 50 scientists, most of them neurobiologists, were coming to the MBL every year to study the squid axon, and collecting crews were scouring the Vineyard Sound for 10,000 squid - a catch that yielded more than a mile of nerve fiber every summer.

Those numbers have remained more or less constant over the last thirty years - scores of scientists, tens of thousands of squid, mile upon mile of nerve fiber. When you think of it, the attention being paid to this one cell is remarkable. How much more can there be to learn about a single cell?

A lot, according to Yale's Walter Boron, one of the army of investigators who came to Woods Hole to study squid axons in 1995.

For one thing, squid nerve cells turn out to be a good place to start figuring out how human kidney cells maintain the body's pH level - the acid/base balance in the blood.

Kidney cells regulate pH by secreting varying amounts of acid into the urine. When the blood begins getting too acidic, the kidney cells pump out more acid. When the blood is too alkaline (basic), they secrete less.

The triggers for acid secretion seem to be blood levels of either bicarbonate (HCO3-) or carbon dioxide (CO2), which enter and leave the cell through transporters in the cell membrane. It is not at all clear whether the pH key is bicarbonate or CO2, or both, and the question is difficult to study directly in kidney cells, which are too small to accommodate the micro-hardware needed to explore the problem.

Fortunately, J. Z. Young's suitable squid axon also has bicarbonate and carbon dioxide transporters, and it can accommodate the collection of electrodes and sensors Boron and his colleagues use to study the pH-balancing mechanisms embedded in cell membranes.

Boron is a 49-year-old cell physiologist whose interests in pH and cell membranes intersect with a number of medical questions. His 10-person research lab at Yale currently includes four working physicians - two kidney specialists, a gastroenterologist, and a critical care pediatrician. The pediatrician is exploring the question of how pH controls the flow of blood to the brain. The gastroenterologist is exploring the permeability of colon cell membranes while the nephrologists are trying to unravel the acid/base regulating mechanism in kidney cells. None of the physicians are marine biologists - or neurobiologists - but over the course of the summer each comes to Woods Hole to spend some time working on the same squid axon J. Z. Young touted in the long-ago summer of 1936.

Boron has been doing the work related to kidney pH regulation over the past three summers with Dr. Jinhua Zhao, a Beijing physician. Zhao wanted to know whether carbon dioxide or bicarbonate serves as the signal for kidney cells to step up their acid secretion. That sounds like an easy enough question to answer: simply bathe one set of kidney cells in bicarbonate and another in CO2, and see which cells increase their acid secretion.

Unfortunately, bicarbonate and CO2 are generally found in equilibrium - which is the chemist's way of saying that in nature you don't find solutions rich in one and lacking the other. If you make a solution with a lot of bicarbonate and no CO2 (or vice versa) , some of the bicarbonate is quickly converted to CO2. Unable to set up an experimental situation in which they had one without the other, Boron and Zhao were unable to figure out which was the trigger.

Then in 1993 Boron and his colleagues at Yale thought up a way around the equilibrium problem - a solution so simple that it seemed obvious in retrospect. They devised a way of mixing solutions that would be either bicarbonate-free or CO2-free and flowing them past a cell less than one second after the solution was mixed - before nature has time to convert more than a trace of bicarbonate to CO2 (or vice versa).

"A lot of times we come here to Woods Hole to do things and we don't know if they're going to work or not," Boron said. "But the rapid mixing technique had to work - it was just a matter of working out the plumbing."

"In theory we could have tried it on any cell," Boron said, "but the squid giant axon is the perfect cell for us for two reasons. First, we were already studying a bicarbonate transporter in the squid axon membrane. And second, the axon is just so big. The diameter is about 550 microns - so the axon is about as thick as the kidney tubule is long.

"The rapid mixing technique," Boron concluded, "is the pate fois gras, and the giant axon is the cracker we put it on."

With the ability to manipulate bicarbonate and carbon dioxide levels for the first time, Boron and his colleagues soon discovered in squid a previously unknown bicarbonate transporter, which they described in a 1994 Nature paper that also announced the rapid mixing technique. Over the past two summers, they have continued to study this newly-identified transporter.

While the Boron team has continued the work in squid, the rapid mixing technique should also be useful in studying medically important questions in mammalian models.

"There are many physiological effects in humans caused by CO2 or bicarbonate," Boron explained. "CO2 or bicarbonate or acid in the brain stimulates breathing. When there's too much CO2 in your blood, somehow that causes the nerves in the brain stem to cause you to breath faster. Nobody really knows how that happens. CO2 and/or bicarbonate triggers various other physiological changes in the body, but up until now it was impossible to know if it was the CO2 or the bicarbonate. Now we should be able to figure that out."

The first medically important question Boron's group is tackling is the kidney question. Back at Yale, Zhao has been applying the rapid mixing technique, worked out in Woods Hole on squid axons, to rabbit kidney tubules. And what is she finding? Is CO2 the trigger that steps up acid secretion in kidneys, or is it bicarbonate?

"We're not finished yet," Boron said, "but the preliminary data suggest it's both, which is a surprise. We were thinking it would be one or the other. On grant applications, I've always been careful to say CO2 and/or bicarbonate, but I never really believed the and part. If we hadn't learned to manipulate them, forever on we would have been thinking it was one or the other."

While Boron's team explores questions far afield from the original questions about how axons generate nerve impulses, other biologists are looking to axons for a more and more detailed understanding of how nerve cells perform their special function - communication. NYU's Rodolfo Llinas has been coming to the MBL since the 1960s to study synaptic transmission - the process by which nerve cells release molecules that carry information across gaps (synapses) to neighboring nerve cells.

And how important is the giant axon to biologists who study synaptic transmission?

"Its importance is not astronomic," Llinas says, "it is galactic."

It is, Llinas says, as if nature presented neurobiologists with a cell in which they could learn everything they want to know about synaptic transmission.

The synapse between two large squid axons was the site where it was demonstrated, in the late 1960s and early 1970s, that calcium ions are the signal for nerve cells to release transmitters - the molecules that carry information across the synapse. The squid axon was the cell in which neurobiologists more recently discovered and came to understand the channels through which calcium enters, and the changes it causes inside the cell that result in the release of transmitters. And the squid axon is the cell in which biologists today are studying the proteins that regulate the release of neurotransmitters.

The importance of this line of research, Llinas says, goes beyond the question of how nerve cells work. Synaptic transmission, after all, is just one of the ways cells modify their membranes in response to the world outside the cell. Proteins similar or identical to the ones that regulate the release of neurotransmitters are found in a variety of cell types, where they regulate, for instance, the Golgi apparatus, a membrane-like structure inside cells that stores, modifies, and packages substances the cell secretes. Other proteins discovered in studies of synaptic transmission regulate the cell's release of hormones and growth factors.

Today, long after the original questions about the electrical activity of nerve cells were answered, the molecular mechanisms of neurotransmitter release are shedding light on the question of how cell size is regulated and how cell membranes are modified in response to internal and external signals.

Nine presidents have been have come and gone since J. Z. Young suggested squid axons might be suitable for study. The price of men's suits has risen by an order of magnitude and the discovery of the chemical structure of genes is now a part of science history - and the end of the information biologists hope to extract from J. Z. Young's suitable cell still is not in sight.