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Laser beams on a microscope

To neuroscientist Valentin Nägerl, the brain is a biological Rubin’s vase, defined both by the contours that comprise it and those that constrain it. Between the structures of well-studied brain cells are the outer contours, the space Nägerl calls the field’s next frontier.

He’s building new tools and techniques that can help create a reverse profile of brain structures. He aims to visualize the crevices between the cells. Called the extracellular space, it’s more than vast emptiness: It’s a convoluted network of sheets, tubes and scaffolding where microglia crawl and dendritic spines connect with axonal boutons.

A headshot of a man.
“Imaging has this incredible dynamic range, both in space and in time to capture the brain,” says Valentin Nägerl. “It just opens up the horizon.”

To see them in action, scientists need new microscopy techniques that can image down to roughly 20 nanometers — without damaging surrounding cells.

“It’s a very different experimental challenge,” Nägerl said.

He currently leads a group at the University of Bordeaux’s Interdisciplinary Institute for Neuroscience. The team is urgently focused on exploring the extracellular space with super-resolution microscopy. He believes discoveries there will shed light on higher brain functions such as memory, learning and sleep — and eventually build new avenues for treating brain diseases.

“Right now, there’s all kinds of theory that says, ‘Yes, extracellular space should be important,’” he said. “But experimentally, there’s next to nothing. There is so much to be discovered.”

Nägerl’s work is powered in part by funding from the Human Frontier Science Program and the European Research Council. He will also share it at the interdisciplinary Molecular Neurobiology Workshop on the Greek island of Crete in May.

Hosted by the European Molecular Biology Organization and co-sponsored by the Institute for Protein Innovation, the workshop was founded to convene experts from different specialties who are working in and around neuroscience. As a microscopist, Nägerl will bring knowledge of sharp imaging tools and visual data from an oft-overlooked region of the brain.

Microscopists have helped lay the groundwork for such neurosynergy for decades. Going forward, Nägerl hopes to leverage it in his quest to resolve tradeoffs that have long stymied the field’s leaders.

Bringing the frontier into focus

Despite Nägerl’s tech-centered research program, his work still tries to balance the strengths and drawbacks of microscopes. Conventional light microscopy can image live samples, illuminating the dynamic organization of proteins on cells’ surfaces, but is limited in spatial resolution to about a quarter of a micron. Electron microscopy can zoom in all the way, revealing cellular ultrastructure at the nanoscale, but not in live cells.

In 2000, microscopist Stefan Hell shattered the diffraction barrier of light microscopy, which had so far kept scientists from imaging the nanoworld of living cells. That milestone enabled stimulated emission depletion (STED) microscopy, which spawned the field of super-resolution fluorescence microscopy. With transformational implications for neurobiology, Hell’s innovations were recognized by the Nobel Prize in 2014.

A neuron illuminated in yellow against a black background.
A neuron imaged in a living brain slice using stimulated emission depletion (STED) microscopy. STED was the first microscopy technique researchers could use to visualize the interiors of living cells. Image: “Nanoscale imaging of the functional anatomy of the brain” by Arizono, M. et al.

It was a boon for neuroscientists, but one subject to limitations. Bombarding fluorophores with intense light eventually bleaches them. No longer excitable, they won’t produce an image. At the same time, imaging the extracellular space calls for still-higher resolutions than STED can achieve.

Then, in 2018, Nägerl and his team invented super-resolution shadow imaging, which they dubbed SUSHI. It marked the first application of STED to imaging the extracellular space.

Instead of labeling a cellular structure, the technique involves labeling of the extracellular fluid surrounding it with a diffusible fluorophore. It does not permeate cell membranes, uniquely enabling scientists to illuminate the space surrounding cells with a single gush of dye. The resulting images are high-contrast, high-resolution negative imprints of living cells.

“The fact that the dye is everywhere, we see the whole structure completely. We’re not missing anything.” said Nägerl, who worked with Hell at the Max Planck Institute for Biophysical Chemistry in 2007.

Because cells do not take up the dye, they and their contents are immune to the bleaching that plagues traditional STED microscopy.

“With classic labeling, you take a few images and then things start to bleach or become phototoxic,” Nägerl said. “We can take hundreds of images, nonstop almost.”

A microscope image of a neuron.
This neuron, orange, is surrounded by a network of interwoven nerve fibers. The neuron’s organelles, illuminated here by SUSHI, give the cell its textured appearance. Image: “Super resolution imaging of the extracellular space in living brain tissue” by Tønnesen, J., Inavalli, K., and Nägerl, UV.

In a paper published in Cell, his team first demonstrated SUSHI’s merit using mouse hippocampal slices. The scientists discovered that more than 80% of the extracellular spaces there are larger than 100 nanometers — well within SUSHI’s resolving power — and are filled with a snaking web of scaffolding.

To try to understand what was going on within that architecture, the team used SUSHI to visualize the workings of live cells. The researchers first subjected brain cells to osmotic challenges, basically pouring sodium chloride into the extracellular space. The compartment ballooned, nearly doubling in size. But it shrunk back when the salt was removed, showing it was a dynamic entity.

To test those dynamics and whether SUSHI could capture them, Nägerl’s team induced epileptiform discharges in neural cells by adding picrotoxin, which blocks GABAA receptors. They could measure the change in voltage, but also saw that discharges caused a drop in fluorescence intensity, indicating a significant decrease in the spaces’ volume.

Neurons modeled in different colors, layered on top of one another.
Cell structures revealed by SUSHI images can inform three-dimensional reconstructions of how the brain’s neurons and nerve fibers layer over one another. Model: Image: “Super resolution imaging of the extracellular space in living brain tissue” by Tønnesen, J., Inavalli, K., and Nägerl, UV.

The team then deployed video to monitor cell migration through the space. The scientists first created lesions that activated microglial cells and then watched their ameboid movement around and through cell bodies.

Finally, the scientists imaged the labeled extracellular space containing unlabeled neurons, their dendritic spines making connections with axon boutons. The team also labeled astrocytes and visualized the neuropils around them, revealing tripartite synapses and silhouettes of intracellular structures like the endoplasmic reticulum and Golgi apparatus.

In the future, Nägerl and his team hope to develop new ways to measure how synapses change in response to their environments. Applying advanced technology like adaptive optics and image processing based on machine learning can also help scientists reveal “information that is still hiding in the blur,” Nägerl said.

At the Neurobiology Workshop, he will present some of those tools and technologies.

“We’re just at the very, very beginning of a system that’s ultra-complex. It doesn’t take much really to see that the brain does amazing things, but we have so little idea how it all works.”

Writer: Halle Marchese,
Source: U. Valentin Nägerl,

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