Connect-seq to superimpose molecular on anatomical neural circuit maps

Significance Single-cell transcriptomics has emerged as a powerful means to define the molecular heterogeneity of brain neurons. However, which of the neurons with known transcriptomes interact with each other in specific neural circuits is largely unknown. Here, we devised a strategy, termed “Connect-seq,” which combines retrograde viral tracing and single-cell transcriptomics to determine the molecular identities of individual upstream neurons in a defined circuit. Using Connect-seq, we uncovered a large variety of signaling molecules expressed in neurons upstream of hypothalamic neurons that control physiological responses to stress. Information obtained by Connect-seq can be used to overlay molecular maps on anatomical neural circuit maps and generate molecular tools for probing the functions of individual circuit components. The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed “Connect-seq,” which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.