A spatial and projection-based transcriptomic atlas of paraventricular hypothalamic cell types (Drop-Seq)

The paraventricular hypothalamus (PVH) controls many behavioral and physiologic processes, including appetite, social behavior, autonomic outflow, and pituitary hormone secretion. However, molecular markers for functionally-specific PVH neuron projections remain largely undefined, and a complete census of PVH cell types has not been established. Therefore, we performed extensive single-cell/nucleus RNA sequencing to identify unique neuronal subtypes and multiplexed error-robust fluorescence in situ hybridization (MERFISH) to validate and map them spatially. Our spatial transcriptomic atlas resolved 26 Sim1+ and 29 GABAergic neuron populations from the PVH and surrounding areas, revealing multiple subtypes not described previously and distinct transcriptional programs between neuroendocrine and centrally-projecting neurons. Additionally, projection-based profiling determined neuronal subtypes that project to the parabrachial region (PB) and spinal cord, helping to identify PVH populations that regulate satiety and sympathetic nervous system activity, respectively. Notably, PB-projecting PVH neurons expressing bombesin-like receptor 3 (Brs3) induce satiety, and their silencing causes obesity. Together, this atlas fills a critical gap by contributing high-resolution PVH spatial and circuit-based gene expression profiles, representing a valuable resource for the field of homeostasis. Overall design: Single cells and single nuclei were isolated from dissections of mouse PVH tissue from 1mm-thick coronal brain slices. The transcriptional profile of individual cells and nuclei were then measured using a combination of Drop-seq, DroNc-seq, and 10X Chromium v3 assays. Data were collected from both male and female mice aged 6-12 weeks across 11 batches (Drop-seq = 7 batches, DroNc-seq = 2 batches, and 10X Chromium-v3 = 2 batches). Additionally, we completed targeted projection-specific sequencing of PVH nuclei fluorescently-labeled with retrograde AAV-cre injections into either the spinal cord or parabrachial region of H2B-TRAP mice. Fluorescently-labeled nuclei were then sequenced with either 10X Chromium v3 or Smart-Seq2 sequencing technologies.