‘Light beads’ method images activity across the mouse brain

The new technique can record hundreds of thousands of neurons firing nearly simultaneously across big swaths of brain tissue in living mice.

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A new imaging technique captures neurons firing nearly simultaneously across big swaths of brain tissue in living mice. The method could help researchers understand how wide-ranging networks of neurons communicate and how these patterns differ between wildtype mice and mouse models of autism. According to one theory of autism, unusual patterns of signaling between distant regions of the brain underlie the condition’s traits.

Researchers often monitor brain activity in mice by using a technique called two-photon calcium imaging, adding fluorescent protein tags to the calcium ions that flood into cells when neurons fire. They can excite the proteins with a laser and detect the fluorescence with a microscope to observe cells in action.

This method typically limits researchers to one small area for study. To image a larger portion of the brain, they often have to scan section by section, which is too slow to capture the interactions of distant neurons. Researchers can speed up the process by illuminating neighboring regions in the brain with a series of sequential laser beams, each slightly delayed from the previous one to avoid muddying signals. But splitting a laser and delaying each beam usually requires complex equipment, making it difficult to scale up. Scientists have been able to capture the activity of only 12,000 neurons using this approach.

The new method enables researchers to track the activity of more than 1 million individual neurons in about 16 cubic millimeters of mouse brain — more than 10 times the volume imaged using previous techniques. “That is definitely by far the largest volume and the largest number of neurons that anyone has captured simultaneously,” says Alipasha Vaziri, professor of neurotechnology and biophysics at Rockefeller University in New York City. He led the new work, which is described in August in Nature Methods.

The technique — called light beads microscopy — involves splitting a laser beam into 30 beams, each one exciting a spot at a slightly different depth in the brain. This split creates a column of “light beads” that measures about 500 microns in depth, enabling researchers to scan an entire block of the brain in the same time it normally takes to scan a small 2D slice with conventional approaches.

Activity tracker: The firing patterns of a subset of neurons (bright dots) correlate with the timing of a mouse’s spontaneous behaviors.

Splitting light:

To form the light beads, Vaziri and his colleagues created a special mirror-lined cavity. When a pulse of light enters the cavity, it bounces around until it hits a partially reflective mirror: Some of the light passes through the mirror toward a microscope, while the rest is reflected back into the cavity for another round trip. Each trip shifts the beam’s focus to a slightly shallower depth and delays the beam by a few nanoseconds. Researchers can then assign fluorescence detected at different points in time to specific spots in the brain.

The team tested the technique by imaging the brains of awake mice whose neurons express a protein that fluoresces in the presence of calcium. They placed the mice on a treadmill and fixed the animals’ heads in place. In one experiment, the researchers recorded neurons firing in both brain hemispheres. In another test, they recorded from one hemisphere as the mouse was exposed to repeated stimuli, such as brushing the whiskers or presenting a moving image of black and white lines. The team also recorded movements in the animals’ limbs.

When tracking one hemisphere, the team captured more than 200,000 neurons firing in different brain regions. By analyzing correlations between the timing of neuronal activity and the onset of stimuli or movements, the researchers also identified groups of neurons tuned to distinct stimuli or spontaneous behavior.

As expected, neurons within brain regions known to be involved in processing sensory or visual information responded to whisker or visual stimulation, the researchers reported. More surprising was that neurons in many other cortical regions also lit up in response to these stimuli. And when whisker and visual stimulation were combined, the additional stimulus modulated the activity of some neurons tuned to the other stimulus. Individual neurons’ firing patterns also varied with each presentation of the stimuli.

These results highlight the importance of capturing wide-ranging brain activity at high speed to help untangle the complexity of neural networks, the researchers say.