How to ‘Film’ Firing Neurons

(Inside Science) -- On June 15, 1878, in front of a crowd of reporters, British photographer Eadweard Muybridge deployed an elaborate apparatus of trip wires and cameras to snap 12 rapid-fire pictures of a horse as it trotted down a California track. The series of images, captured in less than half a second, proved a much-debated theory of the time that the animal does indeed have all four feet simultaneously off the ground midstride.

Muybridge’s high-speed, stop-action photographs -- of which the horse is just one famous example -- captured detailed motions of humans and animals that the human eye alone could not observe. More than 130 years later, scientists are using a similar approach to reveal new insights about life at an even faster and much tinier scale: firing neurons.

Shigeki Watanabe, a cell biologist at the Johns Hopkins School of Medicine in Baltimore, and colleagues have developed a method to take flipbook-like images of brain cells in action.

The scientists start by cultivating modified mouse neurons that have been designed to fire in response to light. When the researchers hit the cells with a flash of light, it acts like a starter gun, sending electrical signals shooting down the neurons like runners in a race. In about one-thousandth of a second the “runners” reach the end of the cells, where they trigger the release of chemicals called neurotransmitters that pass the signal to other cells. After a set period -- ranging from milliseconds to seconds -- a high-pressure cooling system quickly douses the neurons with liquid nitrogen, literally freezing the moment in time.

The cold bath kills the cells, so the researchers can’t capture the continuous action of any individual neuron. But by looking with an electron microscope at thousands of cells frozen at various times, they can piece together key steps in the signaling process. They can see cell components that are hundreds of times smaller than a speck of dust and movements that happen faster than the blink of an eye.

A similar freezing experiment was performed with frog nerves in 1979 by lead scientists John Heuser and Thomas Reese. They dropped the tissue past an electrical switch that stimulated the nerve, then slammed it into an ultra-cold metal block to freeze it. The approach was both simple and effective, but Watanabe and his colleagues’ new method is far more flexible, said Graeme Davis, a neuroscientist at the University of California, San Francisco.

Comparing the experimental apparatuses, “Heuser and Reese had the Model-T, and Shigeki’s driving the Tesla,” he said.

Watanabe and his colleagues have been using their technique to study what happens at the synapse, or junction between neurons. The cells store neurotransmitters near the synapse in membrane-enclosed containers called vesicles. The vesicles merge with the outer membrane of the neuron to release their contents, but because there’s a limited number of vesicles at each synapse, the cell needs to regenerate the containers locally to communicate for longer than a few seconds.

Image

A “flipbook” of images taken with an electron microscope shows a vesicle fusing into the outer membrane of a neuron (1-4), then the membrane folding back into the cell (5-12), all within one-tenth of a second. The colors are added for emphasis.

Media credits

Images courtesy of Shigeki Watanabe. Color and animation by Abigail Malate.

The flipbook images have already illustrated one major discovery: a new, ultrafast way that neurons recycle the vesicles. Less than one-tenth of a second after the vesicles merge with the outer cell membrane, the membrane folds back in on itself, creating a large container that is later divided into multiple new vesicles. The process is similar to recycling beer bottles by melting the glass, Watanabe said. “[The neurons] are re-making the vesicles, but they are doing it in bulk, so that’s why it’s much faster,” he said.

The results are an excellent example of how new technology often drives new discoveries, said Alberto Pereda, a neuroscientist at the Albert Einstein College of Medicine in New York who was not involved in the study.

Most recently, the team has identified key proteins that make the fast recycling possible, and whose absence may be linked to neurological diseases. Watanabe presented the technique and the new findings at a meeting of the American Crystallographic Association in late May in New Orleans, in a session devoted in part to cryo-electron microscopy, an increasingly popular approach to studying biological materials by freezing them and examining them with an electron microscope.

Traditionally, electron microscopy was best suited to seeing membranes in cells, but scientists are now figuring out ways to use the technique to see proteins -- the cellular machines that “make things happen,” Davis said. Watanabe’s technique can add exquisite time resolution to the detailed static images that electron microscopy provides, he said.

Davis is so enthusiastic about Watanabe’s methods, in fact, that the two scientists recently started a collaboration to use the flash-and-freeze technique to study how neurons can work steadily for decades, even as all their component parts are replaced over time.

For anyone who’s interested in how life works inside of cells, “this is going to be one very powerful way to go forward,” he said.