Higher-Order Olfactory Threat Associations in the Amygdala-Posterior Piriform Cortex Network

Fear, while crucial for survival, is a component of a myriad of psychiatric illnesses in its extreme. Persistent fear memories can form through processes such as second-order conditioning (SOC), during which a second-order conditioned stimulus (CS2) acquires significance by associating with a first-order conditioned stimulus (CS1). The neural circuitry underlying SOC, particularly the roles of sensory cortices, remains poorly understood. We explored the mechanisms of olfactory SOC in rats, focusing on the basolateral amygdala (BLA) and posterior piriform cortex (pPC). Our results demonstrate that NMDAR-dependent plasticity in both regions is essential for SOC. The BLA mediates the CS2-CS1 association, while the pPC, receiving inputs from the locus coeruleus and BLA, is critical for memory encoding and retrieval. Transcriptomic analysis of Fos+ ensembles in the two structures shows distinct gene activation in excitatory neurons, highlighting key pathways involved in learning and memory. These findings highlight the pPC’s role in integrating sensory and affective information, essential for long-term threat memory consolidation. Employing a multi-omics approach, including single-nucleus RNA sequencing (sn-RNAseq) and assay for transposase-accessible chromatin sequencing (sn-ATACseq), we analyzed BLA and PC tissues from animals subjected to SOC. A total of 12 rats were used in this experiment, with three rats per group (paired or unpaired) at each time point (early consolidation or memory retrieval). Tissue samples were harvested from the BLA and pPC at two time points following SOC: 90 minutes post-conditioning and 90 minutes post-retrieval test. BLA tissue was extracted from frozen sections in the cryostat using a 1 mm diameter biopsy punch, while pPC tissue was collected using a 0.5 mm diameter punch at three lateral-to-medial locations. Single-nucleus suspensions were prepared following the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics, User Guide Rev F, CG000338).