Every day, our brains continuously integrate information about the world around us with our goals, emotions, and past experiences to guide our thoughts and actions. One region that plays a central role in this process is the dorsal anterior cingulate cortex (dACC), a small but highly interconnected area of the brain involved in cognitive control, attention, reward processing, pain perception, and emotional regulation. Altered dACC function has also been implicated in numerous neuropsychiatric disorders, including depression, schizophrenia, post-traumatic stress disorder, and chronic pain.
Unlike more extensively studied regions of the cerebral cortex, the human dACC has lacked the detailed molecular maps needed to understand how its specialized architecture supports its unique functions. Researchers at the Lieber Institute for Brain Development and Johns Hopkins University have now addressed this gap by generating the first molecular map of the human dACC. Combining spatial transcriptomics with single-nucleus RNA sequencing in postmortem human brain tissue, the team identified the molecular signatures of individual cell types and mapped their precise spatial organization across the region. The resulting resource provides an important foundation for understanding how the dACC is organized and for investigating how its molecular architecture may be altered in neuropsychiatric and neurological disorders.
Keri Martinowich, Chief Scientific Officer at the Lieber Institute for Brain Development and senior author of the study, said: “The dACC has fascinated neuroscientists for decades because it plays such a central role in integrating cognition and emotion, yet we still know relatively little about how this region is organized at the cellular and molecular level. By creating this map, we’ve established a resource that helps us better understand the biology underlying this region’s unique functions.”
Building a Molecular Map of the Human dACC
The researchers studied postmortem brain tissue from ten neurotypical adult donors, combining two complementary genomic technologies to examine the dACC at high molecular resolution.
Using spatial transcriptomics, they measured gene activity while preserving where those genes were expressed within the tissue. This allowed them to determine how distinct molecular features were organized across the cortical layers of the dACC. They paired these data with single-nucleus RNA sequencing, which identifies the genes expressed by individual cell types.
Integrating these datasets allowed the researchers to determine not only which cell types were present, but also where they were located within the dACC. Because the same donors had previously been profiled in the dorsolateral prefrontal cortex (dlPFC), the team was also able to directly compare the molecular organization of these neighboring brain regions while minimizing differences attributable to individual donors.
Together, these approaches produced the first comprehensive molecular map of the human dACC, providing a resource that links cellular identity with anatomical organization across one of the brain’s most important regions for cognitive and emotional function.
Revealing the dACC's Unique Architecture
Most areas of the cerebral cortex are organized into six distinct layers, each containing characteristic populations of neurons. The dACC, however, lacks a well-defined fourth layer, a feature recognized by neuroanatomists for more than a century but never before characterized in molecular detail across the entire region.
The new molecular map provides compelling evidence for this unique agranular organization. By examining patterns of gene expression across the dACC, the researchers identified seven distinct spatial domains corresponding to the cortical layers and white matter. Notably, they found no molecular evidence for a discrete Layer 4.
The study also revealed previously unrecognized molecular differences within the deepest layers of the dACC. Rather than a single Layer 6, the researchers identified two distinct molecular sublayers, each with unique patterns of gene expression and cellular composition. Together, these results provide the first comprehensive molecular framework for understanding the cytoarchitecture of the human dACC and establish a foundation for comparing this region with other areas of the cerebral cortex.
Identifying One of the Brain's Most Unusual Neuron
Among the most distinctive features of the dACC is the presence of von Economo neurons (VENs), a rare type of large, spindle-shaped neuron found primarily in humans and great apes. Unlike most cortical neurons, VENs are present in only a small number of brain regions and have long been hypothesized to support rapid communication across widely distributed brain networks involved in complex cognitive and social behaviors.
Although VENs have attracted considerable interest because of their potential roles in disorders such as frontotemporal dementia, schizophrenia, and autism spectrum disorder, their molecular biology has remained poorly understood.
Using their integrated molecular map, the researchers identified a gene expression program that was highly enriched in neurons located deep within Layer 5 of the dACC. These cells expressed multiple established molecular markers of VENs and were largely absent from the neighboring dorsolateral prefrontal cortex, highlighting an important feature that distinguishes the dACC from other cortical regions.
The study also provides new clues about the potential connectivity of these specialized neurons. By integrating their findings with previously published mouse connectivity datasets, the researchers predicted that VENs are associated with projection pathways linking the dACC to subcortical brain regions involved in emotion, motivation, and behavioral responses. While these predicted connections will require further experimental validation, the findings provide a framework for investigating how these uniquely specialized neurons contribute to human brain function.
A Resource for Understanding Brain Disorders
The dACC has been implicated in a wide range of neurological and psychiatric disorders, but understanding how disease affects this region has been limited by the lack of detailed molecular reference maps. This new molecular map provides that foundation.
Dr. Martinowich added: “One of the strengths of this study is that it provides a reference for the broader neuroscience community. As researchers generate dACC datasets from individuals with psychiatric and neurological disorders, this molecular map will help identify which cell types, circuits, and biological pathways are linked to disease.”
By integrating their data with large genetic studies of human disease, the researchers identified specific cell types and cortical layers associated with the genetic architecture of several neuropsychiatric and neurological conditions. They also compared the molecular signatures identified in the healthy dACC with previously published gene expression data from depression, post-traumatic stress disorder, and chronic pain, highlighting the cell populations and biological pathways that may be involved in these conditions.
Looking Ahead
Beyond the discoveries reported in this study, the molecular map of the human dACC represents a valuable resource for the neuroscience community. The researchers have made the data and interactive analysis tools available to other researchers, enabling scientists to build upon these findings.
As spatial genomics technologies continue to advance, resources like this one will become increasingly important for understanding how the human brain is organized in health and how that organization is disrupted in disease. By establishing a detailed molecular reference for the dACC, this work provides a foundation for future studies investigating the mechanisms underlying neuropsychiatric and neurological disorders and for comparing this region across development, aging, and disease.
Understanding the human brain requires more than cataloging the genes expressed by individual cells. It also requires knowing where those cells are located, how they are organized, and how they work together. This study represents an important step toward that goal.