Scientists led by a team at the University of Minnesota Twin Cities say they have developed a novel method that allows scientists and engineers to visualize mRNA molecules in the brains of living mice—reportedly for the first time. The study reveals new insights into how memories are formed and stored in the brain and could provide scientists with new information about diseases such as Alzheimer’s, according to the researchers who published their work “Real-time visualization of mRNA synthesis during memory formation in live mice” in Proceedings of the National Academy of Sciences (PNAS).

It is well known that mRNA is produced during the process of forming and storing memories, but the technology for studying this process on the cellular level has been limited. Previous studies have often involved dissecting mice in order to examine their brains. The new technique gives scientists a window into RNA synthesis in the brain of a mouse while it is still alive.

“Memories are thought to be encoded in populations of neurons called memory trace or engram cells. However, little is known about the dynamics of these cells because of the difficulty in real-time monitoring of them over long periods of time in vivo. To overcome this limitation, we present a genetically encoded RNA indicator (GERI) mouse for intravital chronic imaging of endogenous Arc messenger RNA (mRNA)—a popular marker for memory trace cells,” write the investigators.

“We used our GERI to identify Arc-positive neurons in real time without the delay associated with reporter protein expression in conventional approaches. We found that the Arc-positive neuronal populations rapidly turned over within 2 d in the hippocampal CA1 region, whereas ∼4% of neurons in the retrosplenial cortex consistently expressed Arc following contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and a calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the population of neurons expressing Arc during both encoding and retrieval exhibited relatively high calcium activity in a context-specific manner.

“This in vivo RNA-imaging approach opens the possibility of unraveling the dynamics of the neuronal population underlying various learning and memory processes.”

“We still know little about memories in the brain,” explained Hye Yoon Park, PhD, an associate professor in the University of Minnesota department of electrical and computer engineering and the study’s lead author. “It’s well known that mRNA synthesis is important for memory, but it was never possible to image this in a live brain. Our work is an important contribution to this field. We now have this new technology that neurobiologists can use for various different experiments and memory tests in the future.”

Process involved a number of critical steps

The process involved genetic engineering, two-photon excitation microscopy, and optimized image processing software. By genetically modifying a mouse so that it produced mRNA labeled with green fluorescent proteins, the researchers were able to see when and where the mouse’s brain generated Arc mRNA, the specific type of molecule they were looking for.

Because the mouse is alive, the scientists could study it for longer periods of time. Using this new process, the researchers performed two experiments on the mouse in which they were able to see in real time over a month what the neurons were doing as the mouse was forming and storing memories.

Historically, neuroscientists have theorized that certain groups of neurons in the brain fire when a memory is formed, and that those same cells fire again when that moment or event is remembered. However, in both experiments, the researchers found that different groups of neurons fired each day they triggered the memory in the mouse.

Over the course of several days after the mouse created this memory, they were able to locate a small group of cells that overlapped, or consistently generated the Arc mRNA each day, in the retrosplenial cortex (RSC) region of the brain, a group which they believe is responsible for the long-term storage of that memory.

“Our research is about memory generation and retrieval,” Park said. “If we can understand how this happens, it will be helpful for us in understanding Alzheimer’s disease and other memory-related diseases. Maybe people with Alzheimer’s disease still store the memories somewhere—they just can’t retrieve them. So in the very long-term, perhaps this research can help us overcome these diseases.”

Scientists from Seoul National University and the Korea Institute of Science and Technology were also involved in this research.

Watch a 3D video visualizing the hippocampus region of a live mouse brain.