Dentate gyrus and CA3 activity mediates light-tone second-order conditioning expression in mice

Second-order conditioning (SOC) enables animals to form predictions about their environment without direct reinforcement. Although the neural basis of first-order conditioning (FOC) is well characterized, the circuits supporting SOC recall remain unclear. Here, we investigated the brain regions and cell types involved in SOC retrieval in mice and addressed the challenge of quantifying brain-wide neural activity. Using a light-tone SOC paradigm in TRAP2:Ai14 mice, we tagged neurons active during SOC recall by expressing tdTomato fluorescent protein. We quantified the fluorescent cells in brain slices with CellRake, a self-developed Python package. We applied generalized linear models that revealed that activity in the dentate gyrus (DG) and cornu ammonis 3 (CA3) subfields of the dorsal hippocampus was associated with SOC-related behavioral responses. Chemogenetic inhibition of CaMKII⁺ neurons in these regions reduced SOC recall, supporting a causal contribution of DG/CA3 circuits. These findings identify the dorsal hippocampus as a key substrate for retrieving indirectly learned associations.