Neural Cartography

15-09-2020

The ESRF can image dense neuronal wiring in the brain with a resolution never seen before, as a paper published today in Nature Neuroscience shows. The Extremely Brilliant Source (EBS), the new machine at the ESRF, will enable scientists to accelerate imaging speed and further improve resolving power, opening new avenues in X-ray neuroimaging.    

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One of the grand quests in neuroscience is to build precise maps of the brain, charting all its neurons and the connections between them. Such wiring diagrams, called connectomes, promise to shed light on how a collection of cells can together give rise to thoughts, memories and behaviours, and to help uncover the pathways of neurological diseases. “Mammalian brains contain tens of millions to billions of neurons, so studying neural circuits is a monumental challenge that requires to develop new techniques”, says Alexandra Pacureanu, researcher at the ESRF and co-corresponding author of the paper.

Today, researchers led by the ESRF and Harvard Medical School and Boston Children’s Hospital (US), demonstrate in Nature Neuroscience a cryogenic X-ray microscopy approach to image dense neuronal wiring in millimeter-sized samples. The technique, developed at the nano-imaging beamline ID16A of ESRF, and dubbed X-ray holographic nano-tomography (XNH), achieves unprecedented resolving power in thick brain tissue specimens.

Combined with artificial intelligence-driven image analysis methods developed in collaboration with scientists at Janelia Research Campus, the team reconstructed neuronal processes in 3D, comprehensively cataloging neurons and even tracing individual connections from muscles to the central nervous system in fruit flies.

“XNH imaging allows us to resolve the individual wires, but at the same time cover enough volume to see the big picture. This can fundamentally change how we understand brain circuits, to fully appreciate how populations of intricately interconnected neurons give rise to nervous function and ultimately cognition”, says Aaron Kuan, researcher at Harvard Medical School.

The authors imaged over two days a fruit fly leg and its connections to the central nervous system, a structure that extends over a millimetre and is difficult to section and study with Electron Microscopy (EM). They were able to map all of the motor neurons extending from the fly equivalent of a spinal cord into a leg, as well as the sensory neurons that relay signals to the central nervous system. "I am amazed at how much I have learned from this technique. My mental picture of the musculature, tendons, joints, and neural control is practically complete now", explains Anthony Azevedo, neuroscientist working with fruit flies at Washington University in Seattle (US).

They also studied a mouse brain, focusing on an area of the cortex involved in integrating sensory stimuli and perceptual decision-making. Previous EM studies have noted interesting structural characteristics of so-called pyramidal neurons in this area but have been limited to sample sizes of around 20 neurons. Using XNH, the researchers scanned over 3200 cells in this area. Combined with aligned EM data, the team characterised the structure and connectivity of hundreds of pyramidal neurons, which revealed distinct structural properties—such as strong and spatially compressed inhibitory inputs onto certain neurite areas—that suggest previously undescribed functional properties.

Throughout recent years, scientists have made remarkable progress in imaging neural circuits, primarily using a painstaking approach: taking serial slices of brains, each a thousand times thinner than a human hair, imaging the slices with EM and stitching the images together for analysis. This can be prohibitively costly in terms of time and resources, requiring large numbers of EM images, which have a narrow field of view, and intense effort to reconstruct even small neural circuits. With XNH, there is no need to section or ablate the tissue and data collection times may be reduced from months or years to days. “XNH has transformed how we think about mapping brain wiring. It may be an exciting new route to mammalian whole-brain connectomes”, explains Wei-Chung Allen Lee, co-corresponding author.

The new ESRF source, EBS, will enable to accelerate throughput and it will improve imaging resolution. “We strive to further push resolving power and, at the same time, extend the tissue coverage capabilities. By harnessing the benefits of EBS, we hope to identify synapses connecting neurons and investigate neural circuits in multiple samples to discover how connectomes evolve, for example, in the context of neurological disorders”, concludes Pacureanu, who leads the X-ray neuroimaging at nanoscale research unit at ESRF.

The X-ray neuroimaging at nanoscale research unit is supported by an ERC starting grant of 1.43 million euros awarded to Pacureanu for a project entitled ‘Bright, coherent and focused light to resolve neural circuits’, which will exploit the capabilities of the new EBS.

Reference:

Kuan, A.T., et al, Nature Neuroscience, 14 September, 2020. https://doi.org/10.1038/s41593-020-0704-9.

Top image: The nerves in a fruit fly's leg. Credit: A. Pacureanu.