Researchers at the Mark and Mary Stevens Neuroimaging and Informatics Institute (Stevens INI) at the Keck School of Medicine of USC have uncovered a previously unrecognized organizational pattern in one of the brain’s key regions for learning and memory. According to findings reported in Nature Communications, the CA1 section of a mouse’s hippocampus contains four separate layers of specialized cell types. The hippocampus plays an essential role in forming memories, guiding spatial navigation, and influencing emotions, and the discovery of these layers offers new insight into how information moves through this part of the brain. It also provides clues about why some cell types are especially vulnerable in conditions such as Alzheimer’s disease and epilepsy.

"Researchers have long suspected that different parts of the hippocampus’ CA1 region handle different aspects of learning and memory, but it wasn’t clear how the underlying cells were arranged," said Michael S. Bienkowski, PhD, senior author of the study and assistant professor of physiology and neuroscience and of biomedical engineering.

"Our study shows that CA1 neurons are organized into four thin, continuous bands, each representing a different neuron type defined by a unique molecular signature. These layers aren’t fixed in place; instead, they subtly shift and change in thickness along the length of the hippocampus. This shifting pattern means that each part of CA1 contains its own mix of neuron types, which helps explain why different regions support different behaviors. This may also clarify why certain CA1 neurons are more vulnerable in conditions like Alzheimer’s disease and epilepsy: if a disease targets one layer’s cell type, the effects will vary depending on where in CA1 that layer is most prominent."

High-resolution RNA imaging reveals cellular distinctions

To examine this structure, the research team used an RNA labeling technique called RNAscope together with high-resolution microscopy. This approach allowed them to observe single-molecule gene expression inside mouse CA1 tissue and identify individual neuron types based on their active genes. From 58.065 CA1 pyramidal cells, the scientists recorded more than 330,000 RNA molecules, which represent the genetic instructions that indicate when and where genes are expressed. By mapping these gene activity patterns, they produced a detailed cellular atlas outlining the boundaries between distinct nerve cell types across the CA1 region.

Their results showed that CA1 contains four continuous layers of nerve cells, each distinguished by its own pattern of active genes. When viewed in three dimensions, these layers form sheet-like structures that vary in thickness and shape along the hippocampus. This well-defined arrangement clarifies earlier studies that had described CA1 as a more blended or mosaic mixture of cell types.

Hidden "stripes" highlight internal brain architecture

"When we visualized gene RNA patterns at single-cell resolution, we could see clear stripes, like geological layers in rock, each representing a distinct neuron type," said Maricarmen Pachicano, doctoral researcher at the Stevens INI’s Center for Integrative Connectomics and co-first author of the paper. "It’s like lifting a veil on the brain’s internal architecture. These hidden layers may explain differences in how hippocampal circuits support learning and memory."

Because the hippocampus is one of the first regions affected in Alzheimer’s disease and is involved in epilepsy, depression, and other neurological conditions, identifying the CA1’s layered structure offers a promising guide for determining which neuron types may be most at risk as these disorders progress.

Advancing brain mapping with modern imaging and data science

"Discoveries like this exemplify how modern imaging and data science can transform our view of brain anatomy," said Arthur W. Toga, PhD, director of the Stevens INI and the Ghada Irani Chair in Neuroscience at the Keck School of Medicine of USC. "This work builds on the Stevens INI’s long tradition of mapping the brain at every scale, from molecules to whole networks, and will inform both basic neuroscience and translational studies targeting memory and cognition."

A new CA1 cell-type atlas available to researchers

The team compiled its findings into a new CA1 cell-type atlas using data from the Hippocampus Gene Expression Atlas (HGEA). This resource is freely available to scientists worldwide and includes interactive 3D visualizations accessible through the Schol-AR augmented-reality app developed at the Stevens INI. The tool allows researchers to explore the layered structure of the hippocampus in great detail.

Because this layered pattern in mice resembles similar arrangements seen in primates and humans, including comparable variations in CA1 thickness, the researchers believe the organization may be shared across many mammalian species. Further work is needed to determine how closely this structure in humans matches what has been observed in mice, but the findings create a strong starting point for future studies examining how hippocampal architecture supports memory and cognition.

"Understanding how these layers connect to behavior is the next frontier," Bienkowski said. "We now have a framework to study how specific neuron layers contribute to such different functions like memory, navigation, and emotion, and how their disruption may lead to disease."

About the study

In addition to Bienkowski and Pachicano, the study’s other authors include Shrey Mehta, Angela Hurtado, Tyler Ard, Jim Stanis, and Bayla Breningstall.

This work was supported by the National Institutes of Health/National Institute of Aging (K01AG066847, R36AG087310-01, supplement P30-AG066530-03S1), National Science Foundation (grant 2121164), and funding from the USC Center for Neuronal Longevity. Research data reported in this publication was supported by the Office of the Director, National Institutes of Health under award number S10OD032285.