Advanced imaging finds entangled neuronal migration in lab model of Rett . syndrome

Using innovative microscopy methods, scientists at The Picower Institute for Learning and Memory at MIT observed how nascent neurons struggle to reach their rightful places in a sophisticated human brain network model of Rett syndrome, yielding new insights into how developmental deficits are observed in humans. patient’s brain. with devastating interference may arise.

Rett syndrome, which is characterized by symptoms including severe intellectual disability and impaired social behavior, is caused by a mutation in the MECP2 gene. To gain new insight into how mutations affect the early stages of human brain development, researchers in the lab of Mriganka Sur, Newton Professor of Neuroscience in MIT’s Department of Brain and Cognitive Sciences, grew 3D cell cultures called cerebral organoids, or miniature brains, using cells from humans. -people with the MECP2 mutation and compared them with identical cultures without the mutation. Then a team led by postdoc Murat Yildirim examined the development of each type of miniature brain using an advanced imaging technology called a third harmonic generation three photon microscope (THG).

THG, which Yildirim has helped to pioneer in Sur’s laboratory working with MIT mechanical engineering Professor Peter So, allows for very high resolution imaging deep into living, intact tissue without having to add any chemicals to label cells. The new study, published in eLife, is the first to use THG to image organoids, leaving them virtually undisturbed, Yildirim said. Previous organoid imaging studies have required the use of technology that cannot 3D image all tissue, or methods that require killing cultures: either slicing them into thin sections or cleaning and chemically labeling them.

Three-photon microscopes use lasers, but Yildirim and So engineered a laboratory microscope to apply no more power to tissue than a cat toy laser pointer (less than 5 milliwatts).

“You have to make sure that you don’t alter or affect neurophysiology in a detrimental way,” Yildirim says. “You really have to keep everything intact and make sure you don’t bring anything from the outside that could damage. That’s why we’re very careful about strength (and labeling of chemicals).”

Even at low power, they achieve sufficient signal to achieve label-free and intact imaging of fixed and living organoids. To validate that they compared their THG images with images created via more traditional chemical labeling methods.

The THG system allowed them to track the migration of nascent neurons as they traveled from the periphery around the open spaces in the minibrain (called the ventricles) to the outer edge, which is directly analogous to the cerebral cortex. They saw that nascent neurons in the modeling of the Rett syndrome minibrain moved slowly and in tortuous pathways compared to the faster movement in straight lines exhibited by the same cell types in the minibrain without the MECP2 mutation. Sur said the consequences of such a migratory deficit are consistent with the hypothesis that scientists, including in his lab, have hypothesized to occur in Rett syndrome fetuses.

“We know from postmortem brain and brain imaging methods that something goes wrong during brain development in Rett syndrome, but it’s very difficult to know what and why,” said Sur, who directs the Simons Center for the Social Brain at MIT. “This method allows us to directly visualize the main contributors.” THG imaged tissue without labels because it is very sensitive to changes in the material’s refractive index, Yildirim said. It therefore resolves the boundaries between biological structures, such as blood vessels, cell membranes and extracellular spaces. Because the shape of the nerve changes during its development, the team was also able to clearly see the delineation between the ventricular zone (the area around the ventricle where newborn neurons emerge) and the cortical plate (the area where adult neurons settle). It is also very easy to complete the various ventricles and divide them into different regions.

These properties allowed the researchers to see that in Rett syndrome organoids, the ventricles are larger and more numerous and that the ventricular zone – the rim around the ventricle where neurons are born – is thinner. In living organoids, they can track some of the neurons going to the cortex over several days, taking a new image every 20 minutes, as neurons in the developing brain are also trying to do. They saw that Rett syndrome neurons only reached about two-thirds the speed of non-mutated neurons. Rett’s neuron pathways were also significantly more wobbly. The two differences combined mean that Rett cells barely make up for half.

“We now want to know how MECP2 affects the genes and molecules that influence neural migration,” Sur said. “By screening for Rett syndrome organoids, we have some good guesses, which we want to test.” Yildirim, who will launch his own lab as an assistant professor at the Cleveland Clinic’s Lerner Research Institute in September, said he had new questions based on the findings. He wanted images later in organoid development to trace the consequences of tortuous migration. He also wanted to know more about whether certain types of cells struggle to migrate more or less, which could change how cortical circuits work.

Yildirim also said he hopes to continue advancing the three-photon THG microscope, which he sees has potential for fine-grained imaging in humans. This can be an important advantage in people especially that the imaging method can penetrate deep into living tissue without the need for artificial labels.

Besides 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 provided funding for this research.

Reference:

1. Murat Yildirim, Chloe Delepine, Danielle Feldman, Vincent A Pham, Stephanie Chou, Jacque Ip, Alexi Nott, Li-Huei Tsai, Guo-Li Ming, Peter TC So, Mriganka Sur. Label-free three-photon imaging of intact human cerebral organoids to track early events in brain development and deficits in Rett syndrome. eLife, 2022; 11 DOI: 10.7554/eLife.78079

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