Pinho, J. S. | Search bioRxiv

bioRxiv2025

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

Second-order conditioning (SOC) enables animals to form complex predictions about their environment, even in the absence of direct experience. While the neural mechanisms underlying first-order conditioning (FOC) are well characterized, the circuits supporting SOC expression remain poorly understood. To address this gap, we investigated the brain regions and cell types involved in SOC recall in mice and tackled the technical challenges of quantifying brain-wide neural activity. We employed a light-tone SOC paradigm in TRAP2:Ai14 mice, which allowed us to tag neurons active during SOC recall via tdTomato expression. Applying generalized linear models, we identified that the activity in the dentate gyrus (DG) and CA3 regions of the dorsal hippocampus significantly associated with SOC-related behavioral responses. To test their functional relevance, we used chemogenetic inhibition of CaMKII+ neurons in these regions, which confirmed a causal role for DG/CA3 circuits in SOC recall. Together, our results highlight the dorsal hippocampus as a critical substrate for retrieving indirectly learned associations.

neuroscience↗

bioRxiv2025

Dorsal hippocampus mediates light-tone sensory preconditioning task in mice

Daily choices are often influenced by environmental cues that are not directly associated with reinforcers. This phenomenon, known as higher-order conditioning, can be studied using sensory preconditioning tasks in rodents. This behavioral paradigm involves the repeated pairing of two innocuous stimuli, such as a light and a tone, followed by a devaluation phase in which one stimulus is associated with an unconditioned stimulus, such as a mild foot-shock. The result is a conditioned response (e.g., freezing) to both the conditioned stimulus (direct learning) and the non-conditioned stimulus (mediated learning). In our study, we successfully established a light-tone sensory preconditioning task specifically in male mice, as sex differences were observed in both control experimental groups and in sensory preconditioning responses. We employed in vivo, freely moving fiber photometry to monitor neural activity in the dorsal and ventral subregions of the hippocampus in male mice during the formation of associations between innocuous stimuli and reinforcers. Additionally, we combined our sensory preconditioning task with chemogenetic approaches to investigate the roles of these hippocampal subregions in sensory preconditioning. Our results indicate that dorsal, but not ventral, CaMKII-positive neurons are involved in encoding innocuous stimuli during the preconditioning phase. Overall, we developed a novel light-tone sensory preconditioning protocol in male mice, enabling the detection of sex differences and furthering our understanding of how specific hippocampal subregions and cell types regulate complex cognitive processes.

neuroscience↗