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.
