Whole-brain spatial organization of hippocampal single-neuron projectomes

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons. Editor’s summary Neuronal connectivity is an essential ingredient for understanding and interpreting neuronal operations. The hippocampus plays a major role in learning and memory and other crucial brain functions, so an understanding of intrahippocampal connectivity is of paramount importance. Qiu et al. reconstructed axonal arborization of 10,100 neurons in the mouse hippocampus. Spatial transcriptomic analysis of hippocampal sections was also performed to determine the correlations between soma location, transcriptome profile, and axonal projection. The results revealed some previously unknown organization principles of hippocampal axon projections. —Mattia Maroso Single-cell projectome analysis elucidates axonal arborization of more than 10,000 neurons in the mouse hippocampus. INTRODUCTION In the brain circuitry, a single neuron could broadcast output signals to other neurons located in nearby or distant areas. Therefore, understanding the spatial organization of axon projections at the single-cell level is crucial for elucidating the neural circuitry underlying various brain functions. As a brain structure essential for learning, memory, cognition, stress responses and emotional behaviors, it is known that the hippocampus (HIP) is widely connected with various brain areas, including the cortex, thalamus, hypothalamus, olfactory areas, and amygdala. However, it remains unclear how single HIP neurons project to brain-wide target areas and how a single-neuron projectome can be specified by the soma location within the HIP. RATIONALE To reconstruct single-neuron projectomes of the mouse HIP, we combined sparse-labeling methods with fluorescence micro-optical sectioning tomography and reconstructed the soma, axon arbors, and dendrites of individual neurons. We used a wide range of analytic tools for the projectome analysis and created a state-of-art web interface to visualize the projectome data. RESULTS We have created an open and comprehensive database with 10,100 single-neuron projectomes throughout the HIP, which allows interactive query, visualization, and analysis of reconstructed brain-wide projectomes. We classified HIP neurons into 341 projection patterns, and then 43 projectome subtypes, based on their axon morphology and brain-wide target areas. Our study revealed previously unknown axon projection patterns, target-dependent soma distribution within HIP subdomains, a general rule for bihemispheric projections, axon orientation rules for mossy fibers and Schaffer collaterals, and topographic correlation between axon arbors and soma location along HIP axes. CONCLUSION Single-neuron projectome analyses have provided unprecedented information on axon projection patterns at the single-cell resolution and elucidated the organizational principles of whole-brain connectivity of HIP neurons. Such knowledge could be further combined with gene expression data to define HIP neuron subtypes and serve as the structural basis for understanding their diverse and coordinated functions. Spatial organization principles of HIP single-neuron projectomes. A comprehensive database for single-neuron projectomes was created by a large-scale reconstruction of brain-wide axon projections from single neurons throughout the HIP. Single-neuron projectome analysis revealed spatial organization principles of brain-wide axon projections from HIP neurons.