Conventional light microscopy couldn’t take Mark Schnitzer where he wanted to go: beyond the surface. He wanted to see cells deep within the brain and watch them over time.
So the HHMI investigator at Stanford University designed a technique that works in mice. His team uses micro-endoscopes to reach into areas such as the hippocampus, which supports certain forms of learning and memory. Most recently, his group has made the device more portable so that a mouse can go about its day while images are being captured.
|Hear Schnitzer describe his new technique.|
He is able to image deep-brain neurons and monitor them over days or months. “We can return to the same subcellular processes, the same dendrites,” Schnitzer says. “This gives us the capability of watching how neurons and other cell types may change over the long term.” They can observe changes in the brain during normal events, such as learning, and when things go wrong, such as during disease progression.
Postdocs Alessio Attardo and Yaniv Ziv work side by side at the lab bench, each with an anesthetized mouse under a microscope. They implant a thin glass tube—half as thin as a grain of rice—into each mouse’s brain. The tube will help guide the insertion of a needle-like probe adjacent to the mouse’s hippocampus. After the delicate surgery, the mice return to their cages until the researchers are ready to take images.
For the imaging, the researchers take the mice to a room with a fluorescence microscope. The mice have been engineered to express fluorescent proteins in their hippocampal neurons. A microendoscopic probe, coupled to the microscope and inserted through the guide tube, shines a laser on the neurons and detects emitted fluorescence. The researchers can collect images of the neurons repeatedly over time, with the guide tubes ensuring that they observe the same cells each time.
In work published February 2011 in Nature Medicine, Schnitzer and colleagues used this technique to see whether particular neurons in the mouse hippocampus change their branching pattern over time. They monitored CA1 pyramidal neurons, critical for forming long-term memory, over a seven-week period. Those neurons “are only two synapses away from the dentate gyrus, where there’s ongoing birth of neurons throughout adult life,” Schnitzer says. “We had hypothesized that these neurons might show some dendritic turnover.” Not so, according to their results. The dendrites formed by those hippocampal neurons stayed quite stable. “This raises some interesting questions about how the circuit may accommodate the new inputs,” he says.
Schnitzer’s group has also used the technique to study mouse models of glioma, the most common malignant brain tumor in humans. Aggressive gliomas usually occur deep in the brain, but previous studies in animals had examined tumors only on the brain’s surface, where conventional light microscopes work well.
They measured blood flow to deep tumors as well as growth of blood vessels. They found growth patterns similar to those in surface tumors, and, he says, demonstrated that the technique can be used to follow disease progression.
|Take a 3-D journey through a living mouse’s brain. Videos: Yaniv Ziv and the Schnitzer Lab|
Schnitzer is planning with other research groups to disseminate the technology. “We think this ability to track on a microscopic scale the features of brain disease should be a broadly applicable approach to many different brain disorders in animal models,” he says.
For the previous studies, the mice had to be immobilized, but Schnitzer also wanted to image the brain cells of active mice. So he and his team designed a microscope that a mouse can wear—a tiny device with miniature lenses, filters, and other optics that weighs just a couple of grams. The mouse wears the miniature microscope on its head, like a hat, snapped into a base plate implanted in its skull. The mouse moves freely, tethered only by a fiber optic cable to the laser-light source.
Schnitzer opens his laptop to show a video of a mouse wearing the mini-microscope. On one half of the screen, a camera-wearing mouse explores its enclosure. The other side shows a network image of neurons fluorescing green.
“Putting it all together can be a challenge at the systems level, integrating mechanical features, optical features, and making the whole system work at a miniaturized scale.” Schnitzer says. His group includes mechanical engineers, electrical engineers, and applied physicists, all working closely with biologists and neuroscientists.
Down the road, Schnitzer plans to build on the group’s experience and develop grid-like microscopes to do “massively parallel brain imaging” on fruit flies. Such microscopes could dramatically increase the throughput of imaging for the systematic study of mutations that affect neural activity.
The ultimate goal is to connect behavior to what’s happening on a cellular level. “One thing that I’m keenly interested in is understanding how concerted activity at the level of hundreds to thousands of neurons influences behavior,” Schnitzer says.