New Fluorescence Imaging Method Reveals Neural Circuits Altered By Learning

Carnegie Mellon University researchers have developed a new fluorescence-based method for detecting synaptic connections between specific types of neurons, allowing them to detect and quantify subtle structural changes that take place during learning. Using molecular genetic tools for cell-type specific labeling of synapses, the researchers could identify which connections were changing as mice learned a sensory association task. The research was published in the Journal of Neuroscience.

"There are trillions of synapses in the brain, and finding ones that have been modified during learning is a classic needle-in-the-haystack problem," said Alison Barth, the Maxwell H. and Gloria C. Connan Professor of Biological Sciences and member of Carnegie Mellon's Neuroscience Institute. "This new method allows us to detect very subtle shifts in the distribution of synapse size to see how specific synapses are modified as animals are mastering a task."

The brain changes with learning and experience, an ability known as plasticity. Much of that change happens at synapses, the small gaps between neurons where they pass messages to communicate. Although it has long been appreciated that synaptic plasticity is critical for learning, figuring out which synapses are modified has been a daunting task.

Neuroscientists typically study synaptic plasticity by measuring neurons' electrical response to a stimulus like a touch or sound, which can indicate that a synaptic connection has been strengthened or weakened. While existing electrophysiological recording techniques are good at indicating when a neuron fires and how big the response is, they are not good at showing where the response occurred. The where is critical.

"For a very long time, people have been studying how synapses are changing, but they had no idea how to relate synapse dynamics to information flow without knowing the presynaptic partner," Barth said. "A synapse is a conversation between partners — listening to half of the conversation can't tell you what is being discussed. It really matters where these synapses come from and where they are going to." 

In the current study, Barth and her colleagues examined how a specific neural circuit — a series of connected neurons that send signals from one part of the brain to another — changes as mice learn to associate a whisker stimulus with a water reward. The researchers modified existing multicolor, fluorescence-based tags to label the components of the circuit: axons from neurons originating in the thalamus (a part of the brain sensitive to context and rewards), dendrites of neurons in the somatosensory cortex (which processes sensations from the body), and the synapses where the two types of neurons meet and communicate.

The research team, led by postdoctoral associate Ajit Ray, has spent significant time recording from these neurons, so they knew some of the synapses were getting larger in response to learning. With their fluorescent imaging method, the team was able to expand their scope to visualize tens of thousands of synapses, which is orders of magnitude more synapses than can be measured with current recording techniques. The new approach enabled them to see small differences that otherwise may go unnoticed.

The researchers discovered that these synapses are modified in response to learning, but not where they expected. Barth said they had good reason to think that the neurons in layer 1 of the somatosensory cortex were changing, but it turned out that they weren't.

"They were actually changing deeper in the brain," Barth said. "But nobody had ever looked before because there wasn't any way to see them."

The researchers also discovered that the synaptic changes are temporary. Once the animal becomes an expert at the task, the modified synapses revert to their original state, suggesting that there is a transient enhancement of information processing in the cerebral cortex while learning is happening.

"There's a whole cottage industry of people who are trying to figure out where in the brain memories lie. If you have a visual or a touch memory, the obvious place to look is in the part of the brain that encodes vision or touch. And yet, if those changes are just very brief, there's a glimmer of change and then they disappear, then they may be stored somewhere else," Barth said. "I think what we are finding is that the somatosensory cortex helps you learn it, but it doesn't necessarily help you remember it later."

Barth, Ajit and the research team are using their imaging technique now to look at other neural pathways.

"We think this technique can be broadly applied to many types of synapses and to other pathways that might be altered during learning as well as pathways that might be altered in different disease states," said Barth.

She acknowledges that while their fluorescence approach doesn't offer the type of high-resolution imaging that electron microscopy provides, it does quickly generate huge amounts of fine-scale anatomical data that offers insight into how the brain works.

"Our tool isn't perfect, but it's still good enough to tell us things we didn't know before. And it is a very democratic method," Barth said. "There are thousands of labs across the country that have what they need to be able to do it. The bottom line is that we can use it to make new discoveries, so that's a huge advance."

Additional study authors include: Joseph Christian, Matthew Mosso, and Eunsol Park at Carnegie Mellon, and Waja Wegner and Katrin Willig at the Center for Nanoscale Microscopy and Molecular Physiology of the Brain at the University Medical Center Göttingen and the Max Planck Institute for Multidisciplinary Sciences in Germany. The study was supported by the Carnegie Mellon Neuroscience Institute-Indian Institute of Science Fellowship Program, the National Institutes of Health and the Max Planck Institute for Multidisciplinary Sciences.