729: Molecular Architecture of Mouse Spinoparabrachial Projection Neurons

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Omics (Proteomics/Metabolomics/Transcriptomics/Genomics)

Background and Aims: The anterolateral system (ALS) transmits pain, itch, and temperature information from the spinal cord to supraspinal centres and is essential for somatosensory perception. Despite its central importance, the cellular composition of this system remains poorly defined. Anatomical and functional studies indicate substantial heterogeneity among spinal projection neurons, yet the molecular identity of many cell types is unknown. Previous single-nucleus transcriptomic work from our group focused on Phox2a‑expressing projection neurons, identifying major molecular classes within the ALS (1). However, it remains unclear how these populations relate to the broader diversity of projection neurons. Here, we apply a similar methodology to characterise the entire spinoparabrachial tract in mice, enabling an unbiased analysis of projection neuron diversity in the dorsal horn.

Methods: Projection neurons were labelled in adult mice by retrograde delivery of AAV11‑H2B‑GFP into the lateral parabrachial nucleus. Spinal cord sections adjacent to those processed for sequencing were immunostained to verify labelling patterns, which were consistent with previously reported spinoparabrachial distributions. GFP‑labelled neuronal nuclei were isolated from the spinal cord and fluorescence‑activated cell sorting was used to deposit single nuclei into 384‑well plates. Single‑nucleus RNA sequencing was performed using the Smart‑seq3xpress protocol. Downstream analyses included clustering, marker gene identification, and label transfer from previously defined Phox2a populations. Complementary in situ hybridisation using RNAscope was performed for cluster‑specific markers to resolve laminar localisation and validate transcriptomic findings.

Results: A total of 1,018 nuclei passed quality control and were retained for analysis. Unsupervised clustering identified 20 initial clusters, which segregated into two major groups based on expression of the dorsal horn marker Lmx1b, consistent with a dI5 lineage. RNAscope confirmed Lmx1b expression exclusively within dorsal horn projection neurons, and subsequent analyses focused on this population. Within these cells, 11 transcriptionally distinct projection neuron subtypes were resolved, each defined by unique marker gene combinations. Several clusters mapped to previously described Phox2a populations via label transfer, while additional novel ALS neuron subtypes were identified, including molecularly distinct lamina I and lateral spinal nucleus populations. RNAscope validation demonstrated discrete and stereotyped laminar distributions for multiple cluster types.

Conclusions: Unbiased transcriptomic profiling of the parabrachial‑projecting ALS reveals substantial and previously unrecognised cellular diversity among spinal projection neurons. Phox2a‑expressing neurons represent major molecular classes within the ALS and are transcriptionally distinct from other identified projection neuron subtypes. These findings establish a refined molecular framework for the ALS and provide a foundation for future studies addressing the functional roles of newly identified projection neuron populations and their contributions to chronic pain processing.

References: 1. Bell et al., ‘Deep Sequencing of Phox2a Nuclei Reveals Five Classes of Anterolateral System Neurons’. PNAS, 2024

Presenting Author: Allen C. Dickie
Submitting Author: Allen Dickie

Senior Author: Andrew M. Bell

IASP 2026 Global Year theme of Neuropathic Pain: No

• Mansi Yadav - University of Glasgow
• Allen C. Dickie - University of Glasgow
• Wenhui Ma - University of Glasgow
• Andrew J. Todd - University of Glasgow
• Andrew M. Bell - University of Glasgow