Selective loss and transcriptional reprogramming of Nox4(+) GABAergic neurons in the trigeminal nucleus caudalis of NTG-induced chronic migraine model.

BACKGROUND: Chronic migraine is a disabling neurological disorder with complex mechanisms. The trigeminal nucleus caudalis (TNC) is a critical relay in migraine pathogenesis, yet its cellular and molecular underpinnings remain unclear. METHODS: We applied single-nucleus RNA sequencing (snRNA-seq) to the TNC of a nitroglycerin (NTG)-induced chronic migraine mouse model and controls. Major neuronal classes were annotated, followed by subclustering of GABAergic neurons. We assessed cell-type abundance changes, reconstructed transcriptional trajectories, and applied network- and pathway-level analyses including high-dimensional weighted gene co-expression network analysis (hdWGCNA), pseudotime modeling, and CellChat-based intercellular communication profiling. RESULTS: We identified a previously unrecognized GABAergic subpopulation characterized by high Nox4 expression (GABA_Nox4). This subpopulation was significantly reduced in NTG-treated mice compared with controls (p = 0.018), a finding further validated by immunofluorescence staining. hdWGCNA identified a migraine-associated co-expression module enriched in GABA_Nox4 neurons, which was further supported by disease enrichment analysis. Pseudotime analysis showed that GABA_Nox4 neurons diverged into distinct transcriptional states under NTG treatment. Intercellular communication analysis revealed enhanced crosstalk of GABA_Nox4 neurons with astrocytes, endothelial cells, and OPCs in the NTG group, mediated by NTG-enriched ligand–receptor pairs such as Agrn–Dag1, Ncam1–Ncam2, and endothelial-derived Ncam1–L1cam, whereas VEH-specific interactions such as Pdgfa–Pdgfra and Pdgfa–Pdgfrb were diminished. CONCLUSIONS: Our integrative single-cell analysis identifies GABA_Nox4 neurons as a vulnerable and communication-active GABAergic subpopulation in the TNC that is selectively reduced and transcriptionally reprogrammed in chronic migraine. The disruption of inhibitory signaling and rewiring of ligand–receptor networks highlight this population as a potential cellular driver of migraine pathophysiology and a candidate target for therapeutic intervention. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10194-025-02203-z.