Single-neuron projectomes in macaque prefrontal cortex reveal primate-specific connectivity principle

In a study published in Cell on July 10, a team from the Center for Excellence in Brain Science and Intelligence Technology (CEBSIT) of the Chinese Academy of Sciences, along with a team from the HUST-Suzhou Institute for Brainsmatics, reported the first comprehensive study of whole-brain projectomes in the macaque prefrontal cortex (PFC) at the single-neuron level and revealed the organization of macaque PFC connectivity. By comparing macaque and mouse PFC single-neuron projectomes, they revealed highly refined axon targeting and arborization in primates.

The PFC in primates, including humans, has dramatically expanded over the course of evolution, which is believed to be the structural basis for higher cognitive functions. Previous studies of PFC connectivity in non-human primates have mainly relied on population-level viral tracing and functional magnetic resonance imaging (fMRI), which generally lack single-cell resolution to examine projection diversity. However, capturing whole-brain images that trace individual axons in the primate brain produces extremely large datasets, creating significant challenges for data storage and analysis.

In response to this problem, Dr. YAN Jun's team from CEBSIT developed the Fast Neurite Tracer (FNT)—a software tool for reconstructing single-neuron projectomes from large-scale brain imaging datasets. Originally designed to reconstruct single-neuron projectomes in mouse PFC from terabyte-sized optical imaging datasets, FNT has now evolved into a high-throughput single-neuron reconstruction system called Gapr, which integrates petabyte-scale data processing, AI-algorithm-based automatic reconstruction, and large-scale collaborative proofreading, making it possible to map single-neuron projectomes in primate brains.

In this study, the researchers performed sparse labeling of single neurons in 19 injection sites of the prefrontal cortex in macaques (Macaca fascicularis) by viral injection and conducted fluorescence micro-optical sectioning tomography (fMOST) imaging. Using Gapr, they achieved large-scale three-dimensional (3D) reconstruction of whole-brain axon projections, yielding 2,231 single-neuron projectomes of the macaque PFC. 

Based on axonal morphology in the whole brain, these neurons were classified into 32 projection subtypes, each exhibiting distinct projection patterns targeting different brain regions including the parietal and temporal cortices, contralateral hemisphere, striatum, thalamus, midbrain, and pons. Further AI-based functional predictions suggested that these projection subtypes were closely associated with sensory, motor, emotional, cognitive, and memory-related biological functions.

Through in-depth analysis of single-neuron projectomes in the macaque PFC, the researchers uncovered a set of organizational principles. Distinct PFC neuron subtypes project to either the parietal or temporal lobes, and neurons in different PFC subregions project to different subregions within these targets. The macaque PFC exhibits a modular network of intra-PFC connections, which may provide a structural basis for working memory. 

Moreover, patchy terminal arbors were found in the PFC projections to the striatum and contralateral cortex in macaques but not in mice, highlighting a more spatially refined innervation pattern in the primate brain. Macaque PFC neurons with bilateral projections show a stronger preference for contralateral targeting compared to those in mice, suggesting functional specialization of neurons projecting to the contralateral hemisphere in primates. The PFC neurons projecting to subcortical areas display a topographic relationship between their somata and targets, suggesting differential downstream innervations across different prefrontal regions.

Through a systematic comparison between the single-neuron projectomes in the macaque and mouse PFC, the researchers revealed that macaque PFC neurons share similar target specificity but exhibit distinct morphological features, including longer axon trunks, fewer collateral branches, and relatively smaller axon terminal arbors. These findings show that, compared to rodent neurons, primate neurons possess simpler structures, more restricted projection targets, and a more spatially refined innervation pattern. Such highly modular and selective connectivity of the primate PFC may provide the structural foundation for the emergence of advanced cognitive and executive functions in primate brains.

This study reveals the structural basis for the evolution of higher cognitive functions in primate brains, provides important clues for exploring the neural origins of psychiatric disorders in the human brain, and may inspire new designs in artificial intelligence.