ROLE OF HETEROGENEITY AND DEVELOPMENT IN HIPPOCAMPAL CA3 CIRCUIT ARCHITECTURE

The hippocampal CA3 subregion is composed of diverse pyramidal neuron (PN) populations defined by their morphology, function, and transcriptome. This diversity likely arises from a combination of genetic and developmental factors, resulting in complex wiring rules in the CA3 circuit, though the underlying mechanisms remain unresolved. To address how genetic factors shape neuronal diversity in the CA3 circuit, we generated a Cre knock-in mouse labelling a genetically-defined subset of CA3 PNs. Cre+ neurons were predominantly found deep in the CA3 pyramidal layer, and through patch-clamp recordings we observed that they functionally align with previously studied deep CA3 PNs (Watson et al., 2025, Cell Reports). To determine how this diversity arises from development, we targeted cells originating at specific embryonic time points using a combination of BrdU staining and genetics-based developmental labelling (Ngn2-CreER mouse). We confirm that deep CA3 PNs are preferentially early-born, and that early-born neurons are found radially deeper than later-born counterparts, yet genetic labelling produced more defined anatomical and functional properties than developmental labelling. Finally, we examined the wiring rules of genetically-identified Cre+ CA3 PNs in the hippocampus using Cre-dependent viral tracing. Contrary to the classical broad projection pattern of CA3 PNs, the labeled PN population predominantly projects longitudinally on the dorso-ventral axis within ipsilateral CA3, with a defined projection within stratum radiatum. Together, we dissect genetic and developmental contribution generating hippocampal PN diversity, demonstrate distinct properties and wiring of PN subpopulations, and highlight the importance of understanding cellular heterogeneity and its impact on wiring and brain function