Molecular, spatial and functional single-cell profiling of the hypothalamic preoptic region
2018
INTRODUCTION A mechanistic understanding of brain function requires the identification of distinct cell types in the brain at a molecular, spatial, and functional level. The preoptic region of the hypothalamus comprises multiple nuclei and controls many social behaviors and homeostatic functions. Discrete neuronal types within the preoptic region have been associated with specific hypothalamic behaviors and homeostatic controls, yet the organizational principles of the underlying circuits remain elusive. Further progress requires methods that can identify molecularly distinct cell types and map their spatial and functional organization in the tissue. RATIONALE Single-cell RNA sequencing (scRNA-seq) has revolutionized the understanding of many tissues by allowing a systematic, genome-wide molecular identification of cell types. However, scRNA-seq requires cell dissociation, leading to a loss of spatial context that is essential to understand the cellular architecture of brain circuits. Image-based approaches to single-cell transcriptomics enables gene expression profiling of individual cells within their native tissue and offers opportunities for simultaneous in situ cell-type identification and spatial mapping, as well as functional characterization when combined with activity marker imaging. The combination of these complementary techniques would allow us to generate a molecular inventory of neuronal types while mapping their spatial and functional organization. RESULTS We combined scRNA-seq and multiplexed error robust fluorescence in situ hybridization (MERFISH), a single-cell transcriptome imaging method, to investigate the molecular, spatial, and functional organization of the mouse hypothalamic preoptic region. We profiled ~31,000 cells using scRNA-seq and imaged ~1.1 million cells within intact tissues using MERFISH. Our data revealed a remarkable diversity of neurons in this region, comprising ~70 different neuronal populations, many of which were previously unknown. These neuronal types exhibited distinct neuromodulatory signatures and revealed a striking heterogeneity within cell populations that were previously thought to be functionally unitary. MERFISH measurements further allowed us to map the spatial organization of these neuronal types, determine the cellular composition of distinct nuclei, and provide insights into the functional organization of neuron populations, including topographical relationships that underlie sex hormone signaling. Last, we combined MERFISH with immediate-early-gene expression imaging to identify specific neuronal populations activated by social behaviors, including parenting, mating, and aggression. Several neuronal populations were selectively activated in each of these behaviors, supporting the notion that transcriptionally distinct neuronal types control specific hypothalamic functions. We identified a core neuronal population activated in all animals that exhibit parenting, as well as cell populations differentially activated in mothers and fathers, providing insights into how physiological state may affect parental behavior. Moreover, we identified cells associated with sexual behavior in males and females as well as male aggression toward infants and conspecific males. CONCLUSION By combining MERFISH with scRNA-seq, we have revealed the molecular, spatial, and functional organization of neurons within the hypothalamic preoptic region. These results provide a framework for mechanistic investigation of behavior circuits with high molecular and spatial resolution and opens avenues for identifying and mapping cell types in a diverse range of tissues and organisms.
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