Microelectrode arrays reveal spatial variations in cell signaling

Nerve cells communicate with one another by releasing chemical messengers across cell–cell gaps called synapses. Electrochemical analysis has been particularly valuable in studying this communication process, called exocytosis, in which small quantities of these easily oxidized neurotransmitters are released from intracellular vesicles. But single microelectrodes miss spatial heterogeneity, which has emerged as an important factor in nerve cell communication, says Andrew Ewing of Pennsylvania State University and Göteborg University (Sweden).

(a) Schematic diagram and (b) SEM images of carbon microelectrode arrays composed of two, three, and seven individually addressable electrodes. Each electrode tip measures ~5 µm in diameter.

“We’ve been looking at exocytosis from [model] PC12 cells since 1994,” says Ewing. “We wanted to be able to record from more than one site, but we didn’t have the capability we needed.” As Ewing and colleagues report in their latest paper in AC (2008, 80, 1394–1400), they found a solution in carbon microelectrode arrays (MEAs).

“You can get a lot of information about single-cell secretion processes with a traditional, single carbon fiber electrode,” says Bo Zhang, first author on the paper. “But you can’t get spatial information from a single electrode, and exocytosis is actually a very dynamic and spatially heterogeneous process.” Multiple discrete carbon microelectrodes are very difficult to manipulate. Existing microchip-based MEAs are too large—each electrode tip is 10–20 µm in diameter, about the same size as a PC12 cell. The challenge, says Zhang, “was to find smaller electrodes—and a way to put them together.”

Zhang turned to carbon fiber electrodes. Typically, a single carbon fiber would be threaded through a glass capillary, which is then pulled into a fine, 5–10 µm diam tip. Zhang says, “We thought we might be able to use a multibarreled capillary and make a carbon fiber array” by threading each of the conjoined barrels with a single, 5 µm diam carbon fiber before pulling. Zhang reasoned if he could produce seven 5 µm diam electrodes with 10–20 µm diam tips, isolated from one another by the intervening glass, he would have the resolution needed for simultaneous recording of exocytosis at distinct sites around a PC12 cell.

The approach worked—eventually. Zhang quickly found that the fabrication wasn’t going to be trivial. “We had to do quite a bit of work to get it to function,” says Ewing. A major challenge, according to Zhang, was learning to handle the 5 µm diam carbon fibers. It initially took him most of an afternoon to thread a seven-barrel capillary, but Zhang says he can now produce a dozen or more seven-electrode arrays in a day.

The team worked to establish and model electrode–cell connections and successfully monitored changes in the rate and location of exocytosis from a stimulated PC12 cell. “There was quite a bit of collective-brain collaboration to decide how to use it,” Ewing recalls. “But it was Bo’s spark that got it started, and his effort that got it to work.”

The persistence paid off. Using MEAs with two, three, and seven discrete, individually addressable electrodes, the group was able to follow cell secretions over time and to collect detailed records of exocytosis “hot” and “cold” spots—areas of heightened or decreased activity. “Bo’s data shows that hot and cold spots can move around,” says Ewing, “and I don’t think that’s been shown clearly before.”

The biological significance of the observation is still unknown, but Ewing would like to find out whether there’s a link to neuronal plasticity. “My feeling, though there isn’t strong evidence yet, is that cells can control the rate and place of individual exocytosis events, and that may be part of memory and learning,” he says. That intriguing idea, however, awaits even smaller electrodes, capable of probing within active synaptic gaps.

Zhang is continuing to develop the carbon MEAs, including modified arrays that would allow simultaneous recording from two or more cells in a network. He plans to develop multifunctional arrays, in which each electrode would be capable of sensing a distinct analyte, such as glucose or oxygen, or pH. In the meantime, says Ewing, plenty can be learned with the new MEAs by recording exocytosis dynamics under treatment with estrogen or other hormones, for example. “We’re just trying to see where these events are happening, and when, so that we can figure out how the cell works,” says Ewing. “It’s very basic cell biology, but we had to develop a new analytical technology to be able to do it.”

—Thomas Hayden