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Imaging Technology Illustrates the Migration Path of Neurons in Rett Syndrome

Utilizing organoids to model early development, scientists employed a developing microscopy technology to observe that new neurons struggled to attain their developmental goal.

Imaging Technology Illustrates the Migration Path of Neurons in Rett Syndrome.

Image Credit: The Picower Institute.

Researchers at The Picower Institute for Learning and Memory at MIT noticed how newborn neurons struggle to attain their appropriate places in developed human brain tissue models of Rett syndrome using an advanced microscopy method, thereby generating new insight into how developmental deficits seen in the patients’ brains with the disturbing disorder may appear.

Mutations in the gene MECP2 cause Rett syndrome — a syndrome defined by symptoms that include severe intellectual incapacity and impaired social behavior. To achieve a new understanding of how the mutation impacts the early stages of human brain growth, scientists in the lab Mriganka Sur, Newton Professor of Neuroscience in MIT’s Department of Brain and Cognitive Sciences, cultivated 3D cell cultures called cerebral organoids, which are otherwise called minibrains.

This was done with cells from people with MECP2 mutations and associated them with otherwise similar cultures without the mutations. Then, the team headed by postdoc Murat Yildirim assessed the growth of each type of minibrain with the use of an advanced imaging technology called third harmonic generation (THG) three-photon microscopy.

THG, which Yildirim has helped to pioneer in Sur’s lab in association with MIT mechanical engineering Professor Peter So, enables very high-resolution imaging deep into live and intact tissues without needing to add any chemicals to label cells.

The new study was recently published in eLife. It was the first study to employ THG to image organoids while leaving them virtually undisturbed, Yildirim stated. Earlier organoid imaging studies required such technologies that cannot image throughout the 3D tissue or methods that require the killing of the cultures: either by chemically clearing and labeling them or slicing them into thin sections.

Three-photon microscopy employs a laser, but Yildirim and So custom engineered the lab’s microscope to apply no more power to the tissue than a cat toy laser pointer (less than 5 milliwatts).

You should make sure you are not changing or affecting the neuronal physiology in any adverse way. You should really keep everything intact and make sure you are not bringing something external that could be damaging. That’s why we are so careful about power (and chemical labeling).

Peter So, Professor, Mechanical Engineering, Massachusetts Institute of Technology

They gained sufficient signal to attain label-free, intact imaging of fixed and live organoids even at low power. To verify that, they compared their THG images with images achieved through more conventional chemical labeling methods.

The THG system enabled them to track the newborn neurons’ migration as they moved from the rim surrounding open spaces in the minibrains (known as ventricles) to the outer edge, which is directly analogous to the brain’s cortex.

They observed that the emerging neurons in the minibrains modeling Rett syndrome gradually moved and in meandering paths compared to the quicker motion in straighter lines shown by the identical cell types in minibrains without MECP2 mutation. Sur concluded that the consequences of those migration deficits are consistent with what researchers, along with those in his lab, have theorized to happen in Rett syndrome fetuses.

We know from postmortem brains and brain imaging methods that things go awry during brain development in Rett syndrome, but it has been astonishingly difficult to figure out what and why. This method has enabled us to directly visualize a key contributor.

Mriganka Sur, Newton Professor of Neuroscience, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology

He is also the director of Simons Center for the Social Brain at MIT.

Tissues are imaged without labels by THG because it is very sensitive to modifications in the materials’ refractive index, Yildirim stated. Therefore, it resolves boundaries among biological structures, like cell membranes, blood vessels, and extracellular spaces.

As neural shapes vary during their growth, the team could also see the delineation between the ventricular zone (the area surrounding the ventricles where the newborn neurons develop) and the cortical plate (the area where the mature neurons settle into). It was also very easy to resolve different ventricles and section them into separate regions.

Such properties enabled the scientists to see that in Rett syndrome organoids, the ventricles were bigger and more in number and that the ventricular zones — where the rims surrounding the ventricles where neurons emerged — were thinner. They could track some of the neurons moving toward the cortex after a few days in live organoids, clicking a new picture every 20 minutes, similar to what neurons in real growing brains also try to do.

Researchers observed that Rett syndrome neurons attained only around two-thirds the pace of non-mutated neurons. The Rett neuron paths were also considerably more wiggly. The two differences together meant that the Rett cells did not get half as far.

We now want to know how MECP2 influences genes and molecules that influence neuronal migration. By screening Rett syndrome organoids, we have some good guesses, which we are eager to test.

Mriganka Sur, Newton Professor of Neuroscience, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology

Yildirim, who will be launching his own lab as an assistant professor at the Cleveland Clinic’s Lerner Research Institute in September, stated that he has new queries based on the results. He wishes to image the later stages of organoid growth to track the results of the sinuous migration. He also wishes to know more about whether particular cell types find it hard to migrate more or less, which could change how cortical circuits function.

Also, Yildirim said he hopes to continue developing THG three-photon microscopy, which he believes has the capacity for fine-grained imaging in humans. It could be a key benefit in people, especially as the imaging approach can penetrate deep into living tissue with no need for artificial labels.

Along with Yildirim, Sur, and So, the other authors of the paper are Chloe Delepine, Danielle Feldman, Vincent Pham, Stephanie Chou, Jacque Pak Kan Ip, Alexi Nott, Li-Huei Tsai, and Guo-li Ming.

The National Institutes of Health, The National Science Foundation, the JPB Foundation, and the Massachusetts Life Sciences Initiative supported this research.

Advanced imaging of Rett syndrome organoids

An overview of THG imaging and how it enabled new findings about early brain development in Rett syndrome. Video Credit: The Picower Institute.

Journal Reference:

Yildirim, M., et al., (2022) Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome. eLife.


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