MIT Research News' Journal

New method visualizes groups of neurons as they compute

Using a fluorescent probe that lights up when brain cells are electrically active, MIT and Boston University researchers have shown that they can image the activity of many neurons at once, in the brains of mice.

This technique, which can be performed using a simple light microscope, could allow neuroscientists to visualize the activity of circuits within the brain and link them to specific behaviors, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering and of brain and cognitive sciences at MIT.

“If you want to study a behavior, or a disease, you need to image the activity of populations of neurons because they work together in a network,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research.

Using this voltage-sensing molecule, the researchers showed that they could record electrical activity from many more neurons than has been possible with any existing, fully genetically encoded, fluorescent voltage probe.

Boyden and Xue Han, an associate professor of biomedical engineering at Boston University, are the senior authors of the study, which appears in the Oct. 9 online edition of Nature. The lead authors of the paper are MIT postdoc Kiryl Piatkevich, BU graduate student Seth Bensussen, and BU research scientist Hua-an Tseng.

Seeing connections

Neurons compute using rapid electrical impulses, which underlie our thoughts, behavior, and perception of the world. Traditional methods for measuring this electrical activity require inserting an electrode into the brain, a process that is labor-intensive and usually allows researchers to record from only one neuron at a time. Multielectrode arrays allow the monitoring of electrical activity from many neurons at once, but they don’t sample densely enough to get all the neurons within a given volume. Calcium imaging does allow such dense sampling, but it measures calcium, an indirect and slow measure of neural electrical activity.

In 2018, Boyden’s team developed an alternative way to monitor electrical activity by labeling neurons with a fluorescent probe. Using a technique known as directed protein evolution, his group engineered a molecule called Archon1 that can be genetically inserted into neurons, where it becomes embedded in the cell membrane. When a neuron’s electrical activity increases, the molecule becomes brighter, and this fluorescence can be seen with a standard light microscope.

In the 2018 paper, Boyden and his colleagues showed that they could use the molecule to image electrical activity in the brains of transparent worms and zebrafish embryos, and also in mouse brain slices. In the new study, they wanted to try to use it in living, awake mice as they engaged in a specific behavior.

To do that, the researchers had to modify the probe so that it would go to a subregion of the neuron membrane. They found that when the molecule inserts itself throughout the entire cell membrane, the resulting images are blurry because the axons and dendrites that extend from neurons also fluoresce. To overcome that, the researchers attached a small peptide that guides the probe specifically to membranes of the cell bodies of neurons. They called this modified protein SomArchon.

“With SomArchon, you can see each cell as a distinct sphere,” Boyden says. “Rather than having one cell’s light blurring all its neighbors, each cell can speak by itself loudly and clearly, uncontaminated by its neighbors.”

The researchers used this probe to image activity in a part of the brain called the striatum, which is involved in planning movement, as mice ran on a ball. They were able to monitor activity in several neurons simultaneously and correlate each one’s activity with the mice’s movement. Some neurons’ activity went up when the mice were running, some went down, and others showed no significant change.

“Over the years, my lab has tried many different versions of voltage sensors, and none of them have worked in living mammalian brains until this one,” Han says.

Using this fluorescent probe, the researchers were able to obtain measurements similar to those recorded by an electrical probe, which can pick up activity on a very rapid timescale. This makes the measurements more informative than existing techniques such as imaging calcium, which neuroscientists often use as a proxy for electrical activity.

“We want to record electrical activity on a millisecond timescale,” Han says. “The timescale and activity patterns that we get from calcium imaging are very different. We really don’t know exactly how these calcium changes are related to electrical dynamics.”

With the new voltage sensor, it is also possible to measure very small fluctuations in activity that occur even when a neuron is not firing a spike. This could help neuroscientists study how small fluctuations impact a neuron’s overall behavior, which has previously been very difficult in living brains, Han says.

The study “introduces a new and powerful genetic tool” for imaging voltage in the brains of awake mice, says Adam Cohen, a professor of chemistry, chemical biology, and physics at Harvard University.

“Previously, researchers had to impale neurons with fine glass capillaries to make electrical recordings, and it was only possible to record from one or two cells at a time. The Boyden team recorded from about 10 cells at a time. That’s a lot of cells,” says Cohen, who was not involved in the research. “These tools open new possibilities to study the statistical structure of neural activity. But a mouse brain contains about 75 million neurons, so we still have a long way to go.”

Mapping circuits

The researchers also showed that this imaging technique can be combined with optogenetics — a technique developed by the Boyden lab and collaborators that allows researchers to turn neurons on and off with light by engineering them to express light-sensitive proteins. In this case, the researchers activated certain neurons with light and then measured the resulting electrical activity in these neurons.

This imaging technology could also be combined with expansion microscopy, a technique that Boyden’s lab developed to expand brain tissue before imaging it, make it easier to see the anatomical connections between neurons in high resolution.

“One of my dream experiments is to image all the activity in a brain, and then use expansion microscopy to find the wiring between those neurons,” Boyden says. “Then can we predict how neural computations emerge from the wiring.”

Such wiring diagrams could allow researchers to pinpoint circuit abnormalities that underlie brain disorders, and may also help researchers to design artificial intelligence that more closely mimics the human brain, Boyden says.

The MIT portion of the research was funded by Edward and Kay Poitras, the National Institutes of Health, including a Director’s Pioneer Award, Charles Hieken, John Doerr, the National Science Foundation, the HHMI-Simons Faculty Scholars Program, the Human Frontier Science Program, and the U.S. Army Research Office.

Engineers put Leonardo da Vinci’s bridge design to the test

In 1502 A.D., Sultan Bayezid II sent out the Renaissance equivalent of a government RFP (request for proposals), seeking a design for a bridge to connect Istanbul with its neighbor city Galata. Leonardo da Vinci, already a well-known artist and inventor, came up with a novel bridge design that he described in a letter to the Sultan and sketched in a small drawing in his notebook.

He didn’t get the job. But 500 years after his death, the design for what would have been the world’s longest bridge span of its time intrigued researchers at MIT, who wondered how thought-through Leonardo’s concept was and whether it really would have worked.

Spoiler alert: Leonardo knew what he was doing.

To study the question, recent graduate student Karly Bast MEng ’19, working with professor of architecture and of civil and environmental engineering John Ochsendorf and undergraduate Michelle Xie, tackled the problem by analyzing the available documents, the possible materials and construction methods that were available at the time, and the geological conditions at the proposed site, which was a river estuary called the Golden Horn. Ultimately, the team built a detailed scale model to test the structure’s ability to stand and support weight, and even to withstand settlement of its foundations.

The results of the study were presented in Barcelona this week at the conference of the International Association for Shell and Spatial Structures. They will also be featured in a talk at Draper in Cambridge, Massachusetts, later this month and in an episode of the PBS program NOVA, set to air on Nov. 13.

A flattened arch

In Leonardo’s time, most masonry bridge supports were made in the form of conventional semicircular arches, which would have required 10 or more piers along the span to support such a long bridge. Leonardo’s bridge concept was dramatically different — a flattened arch that would be tall enough to allow a sailboat to pass underneath with its mast in place, as illustrated in his sketch, but that would cross the wide span with a single enormous arch.

The bridge would have been about 280 meters long (though Leonardo himself was using a different measurement system, since the metric system was still a few centuries off), making it the longest span in the world at that time, had it been built. “It’s incredibly ambitious,” Bast says. “It was about 10 times longer than typical bridges of that time.”

The design also featured an unusual way of stabilizing the span against lateral motions — something that has resulted in the collapse of many bridges over the centuries. To combat that, Leonardo proposed abutments that splayed outward on either side, like a standing subway rider widening her stance to balance in a swaying car.

In his notebooks and letter to the Sultan, Leonardo provided no details about the materials that would be used or the method of construction. Bast and the team analyzed the materials available at the time and concluded that the bridge could only have been made of stone, because wood or brick could not have carried the loads of such a long span. And they concluded that, as in classical masonry bridges such as those built by the Romans, the bridge would stand on its own under the force of gravity, without any fasteners or mortar to hold the stone together.

To prove that, they had to build a model and demonstrate its stability. That required figuring out how to slice up the complex shape into individual blocks that could be assembled into the final structure. While the full-scale bridge would have been made up of thousands of stone blocks, they decided on a design with 126 blocks for their model, which was built at a scale of 1 to 500 (making it about 32 inches long). Then the individual blocks were made on a 3D printer, taking about six hours per block to produce.

“It was time-consuming, but 3D printing allowed us to accurately recreate this very complex geometry,” Bast says.

Testing the design’s feasibility

This is not the first attempt to reproduce Leonardo’s basic bridge design in physical form. Others, including a pedestrian bridge in Norway, have been inspired by his design, but in that case modern materials — steel and concrete — were used, so that construction provided no information about the practicality of Leonardo’s engineering.

“That was not a test to see if his design would work with the technology from his time,” Bast says. But because of the nature of gravity-supported masonry, the faithful scale model, albeit made of a different material, would provide such a test.

“It’s all held together by compression only,” she says. “We wanted to really show that the forces are all being transferred within the structure,” which is key to ensuring that the bridge would stand solidly and not topple.

As with actual masonry arch bridge construction, the “stones” were supported by a scaffolding structure as they were assembled, and only after they were all in place could the scaffolding be removed to allow the structure to support itself. Then it came time to insert the final piece in the structure, the keystone at the very top of the arch.

“When we put it in, we had to squeeze it in. That was the critical moment when we first put the bridge together. I had a lot of doubts” as to whether it would all work, Bast recalls. But “when I put the keystone in, I thought, ‘this is going to work.’ And after that, we took the scaffolding out, and it stood up.”

“It’s the power of geometry” that makes it work, she says. “This is a strong concept. It was well thought out.” Score another victory for Leonardo.

“Was this sketch just freehanded, something he did in 50 seconds, or is it something he really sat down and thought deeply about? It’s difficult to know” from the available historical material, she says. But proving the effectiveness of the design suggests that Leonardo really did work it out carefully and thoughtfully, she says. “He knew how the physical world works.”

He also apparently understood that the region was prone to earthquakes, and incorporated features such as the spread footings that would provide extra stability. To test the structure’s resilience, Bast and Xie built the bridge on two movable platforms and then moved one away from the other to simulate the foundation movements that might result from weak soil. The bridge showed resilience to the horizontal movement, only deforming slightly until being stretched to the point of complete collapse.

The design may not have practical implications for modern bridge designers, Bast says, since today’s materials and methods provide many more options for lighter, stronger designs. But the proof of the feasibility of this design sheds more light on what ambitious construction projects might have been possible using only the materials and methods of the early Renaissance. And it once again underscores the brilliance of one of the world’s most prolific inventors.

It also demonstrates, Bast says, that “you don’t necessarily need fancy technology to come up with the best ideas.”