Peering into the Brain

In the middle of a pitch-black room, a very special mouse lies motionless on a microscope stage. Months before, the mouse had two small holes cut out of its skull, revealing the dura–the brain’s outer membrane–and the blood vessels below. The holes have been permanently covered with clear glass so that scientists can peer directly into the mouse’s brain, where some of its neurons glow green under the laser light of a microscope.

For images of this brain-watching lab, click here.

As the anesthetized mouse sleeps, Wei-Chung Lee, a postdoc at MIT’s Picower Center for Learning and Memo­ry, snaps a series of pictures of a single neuron. He will combine these photos into a 3-D image of the neuron and compare it with an image of the same cell created a week ago, to determine how it has changed over time.

The technique has given MIT scientists an unprecedented look at the surprising growth of neurons in the course of an adult mouse’s everyday life. “You see the full range of types of growth you see during development, such as growth spurts, extensions, retractions, or new additions,” says Elly Nedivi, the associate professor of neurobiology at MIT who heads the research. “This is what the brain is doing on a day-to-day basis.” Nedivi helped develop the new imaging procedure in collaboration with Peter So, an imaging expert in MIT’s bioengineering department, hoping to better explore changes in complex webs of branchlike projections that relay messages between neurons. What she has found is helping change the scientific picture of the brain.

Once upon a time, scientists thought that the adult brain was mostly static in structure–that after the neural growth spurts of infancy and adolescence, connections between neurons were permanently laid out, like a network of paved roads. But a growing body of evidence suggests that the adult brain has a surprising capacity to reorganize. Nedivi and her team have provided new support for this idea, furnishing the first proof in living animals that neurons’ information-exchanging projections can grow and retract in adulthood, in ways that are qualitatively similar to what they do early in life.

Neuroscientists knew that some neural changes must take place in the adult brain, because we continue to learn throughout life. “But ­people didn’t know if that plasticity was accompanied by structural changes,” says David Kleinfeld, a neuroscientist at the University of California, San Diego. Nedivi and her collaborators, he says, have shown that neurons of at least one particular type “continue to grow and evolve in the adult mouse.”

The key to Nedivi’s discovery was the ability to look at the same neuron in a living animal week after week. Most previous research on neuroplasticity–the brain’s ability to form new neural connections–examined brain slices, sections of brain that are kept alive for a short time. Although such in vitro studies allow scientists to test how neural connections are affected by specific factors, such as jolts of electricity or different types of drugs, they can’t show what happens to neurons in the living brain as an animal grows old, or develops a disease, or is raised in isolation.

Looking at the same cell for a period of weeks can reveal the slow growth of neurons in the brain. Nedivi hopes to use the process to determine what goes awry in the brains of mice engineered to model Alzheimer’s and schizophrenia. Such studies will offer both new information about the human diseases and a way to test novel therapies.

The researchers also hope to determine the best ways to encourage brain cells to grow. If neurons could be coaxed to grow new projections in specific parts of the brain or spinal cord, they might be able to compensate for the damage caused by spinal-cord injury or stroke.

A Blossoming Tree

To watch neurons in the living brain, Nedivi and her collaborators put windows in the skulls of mice genetically engineered to produce a fluorescent dye in a few randomly selected brain cells. Through the windows, they take pictures of the fluorescent neurons using a two-photon microscope, an instrument that creates very high-resolution images.

An ultrafast titanium-sapphire laser sends packets of light through a complex series of lenses and mirrors, which directs the light onto individual cells in the brain of the anesthetized mouse. The fluorescent dye in the selected neurons glows only if two photons hit a dye molecule at exactly the same time, allowing for more precise imaging of the cells. (This is why the room must be pitch black: any extraneous light would be picked up by the microscope’s photon detector, muddying the resulting image. Researchers wear head lamps in case they need to adjust equipment.)

Neurons consist of a central cell body and a series of branching projections that extend into different parts of the brain to send and receive electrical signals. To capture the entire structure of a given neuron, the laser scans it in horizontal cross sections, plunging deeper into the brain with each sweep. Researchers go through the images–which resemble ­Jackson Pollock paintings–and select the shapes that correspond to the projections. A computer program then stitches the images together to create a 3-D model.

To record how the neurons change over time, the MIT researchers take pictures of the same neuron every week for several weeks, using nearby blood vessels to help locate it. In a paper published earlier this year in Public Library of Science ­Biology, the team showed that dendrites–the projections that neurons use to receive information from other brain cells–could grow and twist and bend, sending out new shoots like a blossoming tree. “It’s very powerful–you can actually see the changes,” says Lee. And because the images are collected from a living animal, he says, they capture the brain’s behavior much more accurately than images of a brain slice, in which many of the neural connections are severed.

This type of growth had never been seen before in studies of living animals. Previous studies using two-­photon imaging found small structural changes in dendritic spines, tiny bumps on the surfaces of dendrites. But these studies reconstructed only small sections of each neuron. By modeling entire cells, ­Nedivi and colleagues were able to see larger-scale changes that may previously have gone unnoticed. “What is most exciting about their work is that it shows an unanticipated amount of dynamism in neurons,” says Josh Sanes, a ­Harvard University neuroscientist whose lab engineered the mice used in the study.

In addition, Nedivi’s team found that only a certain type of neuron undergoes these changes. Previous studies focused on excitatory neurons, which send electrical signals that cause other neurons to fire. Inhibitory neurons, on the other hand, release chemicals that stop other neurons from firing. It is these neurons that can extend and retract new projections. Inhibitory neurons are less prevalent in the brain than their excitatory cousins and are less well studied.

Growth Factors

Nedivi’s first experiments focused on mice living a standard lab life, but now that she and her team have defined the normal amount of adult neural plasticity, they can examine how different environmental or genetic factors affect the growth of the brain. Previous research, for ex­ample, showed that giving young rodents toys or raising them in a varied landscape spurs the birth of new brain cells. Two-photon imaging will allow researchers to explore how living in a complex environment influences the neural organization of the brain. Lee has recently started studying how visual deprivation affects neural plas­ticity in the visual cortex. These projects will help researchers figure out whether different environments make the animals’ neurons grow more rapidly or rearrange more frequently, and whether those changes eventually lead to differences in behavior.

Meanwhile, Nedivi says, she has been flooded with requests from scientists studying diseases such as Alzheimer’s and schizophrenia. “Once we characterize the problem with each different disease–maybe fewer projections are growing or only a certain kind of neuron is affected–then we can tailor treatments to that problem,” she says. “We could also use this technology as a platform to screen for therapies.”

Of course, each experiment will require months at the microscope. It takes hours to image each neuron, and days to construct a three-­dimensional model from the two-dimensional images. In addition, scientists will need to meticulously compare neurons from many animals to get a sense of the differences between the behavior of diseased cells and that of healthy ones. Unfortunately, time is something Nedivi currently does not have; the lab uses a customized microscope in So’s lab. Once a week, her students box up their mice and take them to the lab, imaging as many neurons as time allows. Soon Nedivi hopes to get the $500,000 necessary to set up an instrument in her own lab, which would give her researchers unlimited time to watch the brain in action.