CA3 cFOS Quantification

Trans-synaptic adhesion molecules are proposed to confer specificity to neural circuits, yet how they function in vivo and how disease-associated variants perturb their activity remain unclear. Here, we define how the homophilic adhesion molecule Kirrel3 shapes synapse formation and circuit function in the mouse hippocampus. Using conditional genetics, we show that Kirrel3 is absolutely required in both presynaptic dentate gyrus neurons and postsynaptic GABAergic interneurons to drive formation of mossy fiber filopodial synapses and constrain CA3 activity, providing in vivo evidence for a trans-synaptic mechanism. Despite this strict requirement, partial loss of Kirrel3 produces only modest synaptic deficits without altering circuit activity, revealing that this pathway is robust to reduced gene dosage. In contrast, expression of a disease-associated, adhesion-deficient Kirrel3 variant dominantly disrupts synapse formation, cell adhesion, and elevates CA3 activity, whereas a binding adhesion-competent variant likely behaves as a loss-of-function allele. These results advance the understanding of how cell adhesion molecules function in vivo and indicate why one Kirrel3 missense variant allele could contribute to disease.