Unveiling Hypothalamic Molecular Signatures via Retrograde Viral Tracing and Single-Cell Transcriptomics

Abstract

Despite the importance of hypothalamic neurocircuits in regulating homeostatic and survival-related behaviors, our understanding of the intrinsic molecular identities of neural components involved in these complex multi-synaptic interactions remains limited. In this study, we constructed a Cre recombinase-dependent pseudorabies virus (PRVs) capable of crossing synapses, coupled with transcriptome analysis of single upstream neurons post-infection. By utilizing this retrograde nuclear Connect-seq (nuConnect-seq) approach, we generated a single nuclei RNA-seq (snRNA-seq) dataset of 1,533 cells derived from the hypothalamus of CRH-IRES-Cre (CRH-Cre) mice. To ensure the technical validity of our nuConnect-seq dataset, we employed a label transfer technique against an integrated reference dataset of postnatal mouse hypothalamus comprising 152,524 QC-passed cells. The uniqueness of our approach lies in the integration of diverse datasets for validation, providing a more nuanced diversity of hypothalamic cell types. The presented validated dataset may deepen our understanding of hypothalamic neurocircuits and underscore the essential role of comprehensive integrated transcriptomic data for technical validity.

Molecular Connectomics Reveals a Glucagon-Like Peptide 1 Sensitive Neural Circuit for Satiety

“…Viruses other than rabies can be used to trans-synaptically label afferent neurons for molecular classification. For instance, previous studies have used pseudorabies virus (PRV) to label afferents to Crh-expressing neurons of the paraventricular hypothalamus, with methods called Connect-seq (dissociated cells) 69 and nuConnect-seq (cell nuclei) 70 . Like RAMPANT, nuConnect-seq labels afferents to a genetically defined neuron population with a retrograde transsynaptic virus, profiles the labeled cells by single-nucleus RNA-seq and identifies them by transferring cell-type labels from a reference dataset of uninfected cells.…”

“…Another key difference is that nuConnect-Seq captures more non-neuronal cells (i.e., glia and endothelial cells) than neurons. This may be due to the spread of PRV into neighboring, unconnected cells 70 , raising the possibility that PRV could similarly infect bystander neurons and so confound interpretation of the results. In contrast, our RAMPANT study of AgRP neurons afferents detected only neurons, consistent with previous studies showing that G-deficient rabies spreads specifically between connected neurons 20,72 .…”