This study presents a comprehensive molecular, spatial, and functional single-cell atlas of the mouse hypothalamic preoptic region. Using a combination of single-cell RNA sequencing (scRNA-seq) and multiplexed error-robust fluorescence in situ hybridization (MERFISH), the researchers identified ~70 distinct neuronal populations, characterized by unique molecular signatures and spatial distributions. The study reveals the molecular identity, spatial organization, and functional roles of neurons in the preoptic region, which is critical for regulating essential social behaviors and homeostatic functions. The scRNA-seq analysis identified major cell classes, including inhibitory and excitatory neurons, microglia, astrocytes, and oligodendrocytes, along with their subpopulations. MERFISH provided high-resolution spatial information, revealing the distribution of these cell types within specific nuclei of the preoptic region. The study also identified specific neuronal populations activated during social behaviors such as parenting, aggression, and mating in both male and female mice. The findings highlight the diversity of neurons in the preoptic region, with many populations defined by combinations of genes. The study also identified clusters enriched in genes associated with specific functions, such as Galanin, Tyrosine Hydroxylase, and Adcyap1. These clusters were further validated using MERFISH, which allowed for the spatial mapping of gene expression and the identification of cell populations activated by specific behaviors. The study demonstrates the power of combining scRNA-seq and MERFISH to create a detailed map of cell types and their organization in the brain. This approach provides insights into the molecular and spatial organization of neurons, their functional roles in diverse behaviors, and the underlying mechanisms of neural circuits. The identification of marker genes and spatial locations of neuronal populations in the preoptic region provides tools for the precise targeting and study of these neurons. The study also highlights the importance of spatial context in understanding neural function and the potential for future research into the molecular and cellular mechanisms underlying social and homeostatic behaviors.This study presents a comprehensive molecular, spatial, and functional single-cell atlas of the mouse hypothalamic preoptic region. Using a combination of single-cell RNA sequencing (scRNA-seq) and multiplexed error-robust fluorescence in situ hybridization (MERFISH), the researchers identified ~70 distinct neuronal populations, characterized by unique molecular signatures and spatial distributions. The study reveals the molecular identity, spatial organization, and functional roles of neurons in the preoptic region, which is critical for regulating essential social behaviors and homeostatic functions. The scRNA-seq analysis identified major cell classes, including inhibitory and excitatory neurons, microglia, astrocytes, and oligodendrocytes, along with their subpopulations. MERFISH provided high-resolution spatial information, revealing the distribution of these cell types within specific nuclei of the preoptic region. The study also identified specific neuronal populations activated during social behaviors such as parenting, aggression, and mating in both male and female mice. The findings highlight the diversity of neurons in the preoptic region, with many populations defined by combinations of genes. The study also identified clusters enriched in genes associated with specific functions, such as Galanin, Tyrosine Hydroxylase, and Adcyap1. These clusters were further validated using MERFISH, which allowed for the spatial mapping of gene expression and the identification of cell populations activated by specific behaviors. The study demonstrates the power of combining scRNA-seq and MERFISH to create a detailed map of cell types and their organization in the brain. This approach provides insights into the molecular and spatial organization of neurons, their functional roles in diverse behaviors, and the underlying mechanisms of neural circuits. The identification of marker genes and spatial locations of neuronal populations in the preoptic region provides tools for the precise targeting and study of these neurons. The study also highlights the importance of spatial context in understanding neural function and the potential for future research into the molecular and cellular mechanisms underlying social and homeostatic behaviors.