Multimodal mapping of cell types and projections in the central nucleus of the amygdala

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of similar to 30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Automated summary

Researchers used single-cell RNA sequencing and fluorescence in situ hybridization to classify cell types in the central nucleus of the amygdala (CEA) and determine their spatial distribution and connectivity. They discovered a spatiomolecular organization in the CEA, with medial and lateral CEA associated with distinct cell families. They also found a long-range axon projection network from the CEA, which interacts with multiple molecularly defined cell types. This study provides important marker genes and axon-projection types for studying the diverse functions of the CEA.