Whole-brain spatial organization of hippocampal single-neuron projectomes

Mapping hippocampal single-neuron projections is essential for understanding brain-wide circuit organization and diverse functions of the hippocampus, a brain structure underlying episodic memory and cognition. Here, we reconstructed 10,100 single-neuron projectomes of the mouse hippocampus, identified rostral and caudal axon pathways that preferentially innervated cortical vs. subcortical areas, and classified 43 projectome subtypes with distinct axon targeting patterns. Notably, the soma locations along hippocampal longitudinal and transverse axes determined the number of their target areas and the spatial distribution and complexity of their axon arbors within the targets. We defined selective hippocampal subdomains based on spatial transcriptomic profiles and found that many projectome subtypes were enriched in specific subdomains. Next, we defined the wiring diagram for hippocampal neurons exclusively projecting to hippocampal formation (HPF) and those projecting to both intra- and extra-HPF targets with coordinated projection strengths. Furthermore, bi-hemispheric projecting hippocampal 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 diverse but coordinated functions of hippocampal neurons.

Methods
In brief, male mice (C57BL/6J, 11 weeks old) were perfused with artificial cerebrospinal fluid at -4℃. Brain tissue blocks were cut from left brain hemisphere, embedded in OCT (4583#, Sakura), frozen with dry ice and stored in -80℃ fridge. Brain sections (10-μm thick) for Stereo-seq were cut from brain tissue blocks in a pre-cooled cryochamber at -20℃ (Thermo Fisher Cryostar NX50) at various bregma coordinates (100-μm interval). The sections were carefully placed on a pre-cooled Stereo-seq chip (-20℃) and moved from one end to the other with a finger gently pressed under the chip. The temperature of the finger kept the section close to the chip, reducing bubbles and wrinkles. The RNA RIN value of total RNA extracted from adjacent brain sections were above 9, indicating qualified brain sections for spatial transcriptome experiments. Brain sections covering hippocampus (Bregma -0.9 – -4.4 mm) were laid on Stereo-seq chips for mRNA collection and followed by gene sequencing.