Intersectional genetic targeting of molecularly distinct melanin-concentrating hormone neuron subpopulations reveals distinct and overlapping subcircuit architecture

Abstract

The lateral hypothalamic area (LHA) coordinates crucial and innate behaviors through heterogeneous yet poorly understood neuronal populations and circuits. Neurons that produce melanin-concentrating hormone (MCH; encoded by the Pmch gene) are primarily localized in the LHA and contribute to feeding, sleep-wake regulation, memory, and reward-associated behaviors. Building on foundational anatomical studies, our single-cell transcriptomic analysis of the LHA identified markers distinguishing at least two transcriptionally distinct MCH subpopulations. These subpopulations are distinguished by the selective enrichment of the Tacr3 and Cartpt transcripts, with each comprising approximately half of the total MCH population. However, these subpopulations remain poorly understood owing to limited genetic targeting tools. We hypothesized that MCH neurons operate through functionally distinct, parallel subcircuits that exhibit unique neuroanatomical projections. In this study, we leveraged Pmch and Tacr3 expression to intersectionally dissociate these neurons into Type I MCH (Pmch+/Tacr3+) and Type II MCH (Pmch+/Tacr3-) subpopulations. We first validated a Tacr3-Flpo mouse line for the genetic targeting of the Type I MCH subpopulation. We then generated an intersectional Type I MCH neuron reporter line to visualize Type I axonal projections. In parallel, we injected a dual recombinase mouse line with the intersectional genetic targeting viruses, Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), and identified overlapping and distinct projections by Type I and Type II MCH neurons using fluorescence microscopy, super-resolution imaging, and high-spatial resolution fiber mapping. Overall, these data establish the first subpopulation-resolved anatomical framework for molecularly distinct Type I and Type II MCH subcircuits, thus establishing a foundation for future circuit-level functional studies.