Using an innovative microscopy technique, scientists at MIT’s Pickover Institute for Learning and Memory observed how newborn neurons struggle to reach their proper locations in advanced models of human Rett syndrome brain tissue, creating new insights into how developmental deficits are seen in the brains of patients with Rett syndrome. . a destructive disorder can occur.
Rett syndrome, characterized by symptoms including severe intellectual disability and impaired social behavior, is caused by mutations in the MECP2 gene. To gain new insight into how the mutation affects the early stages of human brain development, researchers in the lab of Mriganka Sur, a professor of neuroscience in MIT’s Department of Brain and Cognitive Sciences, grew 3D cultures of cells called cerebral organoids, or minibrains, using cells from of people with MECP2 mutations and compared them to identical cultures without the mutations. The team, led by postdoc Murat Yıldırım, then studied the development of each type of minibrain using an advanced imaging technology called three-photon third harmonic generation (THG) microscopy.
THG, which Yildirim helped pioneer in Sur’s lab in collaboration with MIT mechanical engineering professor Peter Soh, allows for very high-resolution imaging deep into living, intact tissue without the need to add any chemicals to label the cells. A new study published in eLife, is the first to use THG to image organoids, leaving them virtually intact, Yıldırım said. Previous studies of organoid imaging have required the use of technologies that do not allow imaging all the way through 3D tissue, or methods that require the destruction of cultures: either by slicing them into thin pieces, or by chemical clearing and labeling.
Three-photon microscopy uses a laser, but Yildirim and So specifically designed the lab microscope to add no more energy to the tissue than a cat laser pointer (less than 5 milliwatts).
“You have to make sure that you don’t change or affect the physiology of the neurons in any negative way,” Yıldırım said. “You really have to keep everything safe and sound and make sure you’re not bringing anything outside that could cause harm. That’s why we’re so careful about energy (and chemical labelling).”
Even at low power, they achieved adequate signal to obtain undamaged images of unlabeled fixed and live organelles. To confirm, they compared their THG images to images taken using more traditional chemical labeling techniques.
The THG system allowed them to track the migration of newborn neurons as they made their way from the edge around the open spaces in the mini-brains (called ventricles) to the outer edge, which is a direct analogue of the cerebral cortex. They saw that nascent neurons in minibrains that modeled Rett syndrome moved slowly and in tortuous paths, compared to the faster movement in straighter lines exhibited by the same cell types in minibrains without the MECP2 mutation. Suhr said the effects of such a migration deficit are consistent with what scientists, including those in his lab, have hypothesized is what happens in a fetus with Rett syndrome.
“We know from postmortem brains and brain imaging that things go wrong during brain development in Rett syndrome, but it’s been surprisingly difficult to understand what and why,” said Suhr, who directs the Simons Center for the Social Brain at MIT. “This method allowed us to directly visualize a key participant.” THG images fabrics without labels because it is very sensitive to changes in the refractive index of materials, Yıldırım said. Thus, it resolves the boundaries between biological structures such as blood vessels, cell membranes, and the extracellular space. Because neurons change shape during their development, the team was also able to clearly see the demarcation between the ventricular zone (the area around the ventricles where newborn neurons appear) and the cortical plate (the area where mature neurons are located). It was also very easy to separate the different ventricles and segment them into different regions.
These properties allowed the researchers to see that in organoids of Rett syndrome the ventricles are larger and more numerous, and the ventricular zones – the rims around the ventricles where neurons are born – are thinner. In living organoids, they were able to track some of the neurons making their way to the cortex over several days, taking a new snapshot every 20 minutes, just as neurons in a real developing brain try to do. They saw that neurons with Rett syndrome only reached about two-thirds the speed of neurons without the mutation. The pathways of Rett neurons were also much more tortuous. The two differences combined meant that Rett cells barely made it through half as many.
“Now we want to know how MECP2 affects genes and molecules that affect neuronal migration,” Sur said. “By screening Rett syndrome organelles, we’ve got some good ideas that we really want to test.” Yıldırım, who will open his own lab in September as an assistant professor at Cleveland Clinic’s Lerner Research Institute, said he has new questions based on the findings. He wants to image later in the organoid’s development to trace the effects of tortuous migration. He also wants to learn more about whether certain types of cells struggle with more or less migration, which could change how cortical circuits work.
Yıldırım also said he hopes to continue developing three-photon THG microscopy, which he believes has the potential to produce fine-grained imaging in humans. This could be an important advantage for humans, especially since the imaging method can penetrate deep into living tissue without the need for artificial labels.
In addition to Yıldırım, Sur and Soh, the paper’s other contributors are Chloe Deliapin, Danielle Feldman, Vincent Pham, Stephanie Chou, Jacques Pak Kang Yip, Alexi Noth, Li-Huei Tsai and Gu-Li Ming.
The National Institutes of Health, the National Science Foundation, the JPB Foundation, and the Massachusetts Life Sciences Initiative funded the study.