Scientists at MIT and Stanford say they have developed a technique named FLARE to label neurons when they become active to provide a view of their activity at a moment in time. The method could offer important new insights into neuronal function by offering greater temporal precision than current cell-labeling techniques, which capture activity across time windows of hours or days, according to the investigators.
“A thought or a cognitive function usually lasts 30 seconds or a minute. That's the range of what we're hoping to be able to capture,” says Kay Tye, Ph.D., an assistant professor in the department of brain and cognitive sciences at MIT, a member of the Picower Institute for Learning and Memory. She is one of the senior authors of the study (“A Light- and Calcium-Gated Transcription Factor for Imaging and Manipulating Activated Neurons”), which appears in Nature Biotechnology.
“…we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window,” write the researchers. “FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene.”
Dr. Tye believes that the novel tool could be used to help decipher the neural circuits involved in learning and memory, among many other possibilities. She developed the technology with former MIT professor Alice Ting, Ph.D., who is now a professor of genetics and biology at Stanford and is also a senior author of the paper. The paper's lead author is Wenjing Wang, Ph.D., a Stanford postdoc.
Existing tools allow researchers to engineer cells so that when neurons turn on a gene called cfos, which helps cells respond to new information, they also turn on an artificially introduced gene for a fluorescent protein or another tagging molecule. The system is designed so that this labeling takes place only when animals are exposed to a drug that activates the system, giving scientists control over the timing. However, the control is not very precise.
“Those activity-dependent tools have been hugely impactful, but those tools really only work on the timescale of a couple of days,” Dr. Tye says. “If you think about the speed of the neural code, it's operating more at the pace of milliseconds. What I wanted was a tool that we could use to take a snapshot of activity at a given moment.”
The team designed their tool to respond to calcium, because neurons experience a calcium ion flux every time they fire an electrical impulse. However, the neurons are only labeled if this calcium flux occurs while the cell is also exposed to a beam of blue light delivered by the researchers.
This combination of light exposure and calcium activity triggers the activation of a transcription factor that turns on a target gene that the researchers have engineered into the cells' genome. This gene could encode a fluorescent protein or anything else that could be used to label or manipulate neurons.
In this study, the researchers demonstrated how FLARE works by turning on a red fluorescent protein called mCherry in the motor cortex neurons of mice as they ran on a treadmill.
This method could also be used to label cells with light-sensitive proteins that would allow the targeted neurons to be controlled by optogenetics, or new proteins called DREADDS that allow neurons to be controlled using small-molecule drugs. Because all of the tool components can be delivered using viral vectors, the system could be used in any model organism, note the scientists.
Being able to label and then manipulate sets of neurons that are active during specific tasks opens up a wide range of studies that have been previously impossible, according to Dr. Tye. For example, researchers could investigate what happens as the brain makes quick decisions, responds to stimuli associated with strong emotions, or determines which behaviors are appropriate for the current situation.
For this kind of study, it's particularly important to have a tool that works quickly because the same neuron may be involved in different tasks at different times. The current version of the technique can label neurons within a few minutes.
“This is just a first-generation tool, but we're already able to get very tight labeling,” explains Dr. Tye. “Now we have something that we can work with. We're within striking range of the temporal precision of neural activity.”
The technique could also be useful for studying and treating diseases, continues Dr. Tye. For example, researchers could use it to identify diseased neurons that cause Alzheimer's disease, potentially allowing them to pinpoint the neurons that need to be treated while leaving nearby healthy neurons alone, she says.