SUN-LB55 Connect-seq to Superimpose Molecular on Anatomical Neural Circuit Maps

Animals exhibit instinctive behavioral and physiological responses to a variety of stressors to overcome danger and restore homeostasis. The physiological response to stress is governed by hypothalamic corticotropin-releasing hormone (CRH) neurons which regulate the hypothalamic-pituitary-adrenal axis to control blood levels of stress hormones. At present, the neural circuits and signaling mechanisms through which different stress signals are transmitted to CRH neurons are poorly understood. Here, we devised a new method, termed “Connect-Seq,” which couples single-cell transcriptomics and retrograde viral tracing to define the molecular identities of individual neurons in neural circuits. As a proof of concept, using Connect-Seq, we profiled single-cell transcriptomes of 124 brain neurons upstream of CRH neurons and identified subpopulations that are likely to communicate stress-related signals to CRH neurons. Analyses of single-cell transcriptomes for ‘fast-acting’ neurotransmitters revealed subsets of upstream neurons that expressed markers of inhibitory GABAergic neurons or excitatory glutamatergic neurons. Further analyses showed a number of other neuromodulators/neurotransmitters in upstream neurons, including acetylcholine, dopamine, histamine, and 43 different neuropeptides, each expressed in individual neurons or subsets of neurons. These findings reveal extreme molecular heterogeneity among upstream neurons and suggest the upstream neurons use diverse neurochemical messengers to transmit signals to CRH neurons. Many neurons coexpressed different neurotransmitters/neuromodulators, suggesting the co-release of neurochemical messengers. Dual labeling of brain sections verified expression of specific neuromodulators in virus-infected neurons upstream of CRH neurons in selected brain areas. Our results indicate that Connect-Seq can be applied to genetically dissect neural circuits and uncover molecular identities of neurons upstream of specific neuronal types of known function. Molecular markers identified in those neurons lay a foundation for the application of cell-specific genetic tools to investigate the functions and physiological significance of diverse neuronal subsets within complex neural circuits.