A Recurrent Slc17a6-High Glutamatergic Microdomain in the Parabrachial Region Following Paclitaxel Exposure: GPR12-Associated Delayed Neuronal Remodeling Across Mouse Spatial and Human Time-Resolved Transcriptomics

BackgroundBuilding on this spatial observation, we asked whether a receptor-associated program could identify a molecular entry point into thepersistent state. GPR12, an orphan G protein-coupled receptor with constitutive Gs/cAMP activity and established neuronalexpression, emerged as a candidate associated with paclitaxel-linked remodeling.Paclitaxel is a widely used antineoplastic agent whose therapeutic utility is limited by persistent neurological adverse effects,including chemotherapy-induced peripheral neuropathy (CIPN). Most mechanistic studies of paclitaxel neurotoxicity have focused onperipheral sensory neurons, dorsal root ganglia, axonal transport, mitochondrial dysfunction, neuroimmune signaling, and spinalsensitization. Increasing evidence, however, indicates that chemotherapy also reorganizes supraspinal neural systems involved innociceptive processing. Whether paclitaxel exposure is associated with reproducible, spatially localized molecular states withinspecific brain regions remains poorly understood. MethodsVersion 2 retained the original GSE325554 spatial analysis and integrated receptor-focused secondary analyses. Candidatereceptors were evaluated in local PB windows with Slc17a6 expression and Slc17a6-high membership withheld from receptor-statediscovery where feasible and used only at the final phenotype-prioritization stage. Untreated mouse parabrachial-region single-cellRNA-seq (GSE207708) was used to characterize the normal GPR12-positive neuronal substrate. Reciprocal PTX1-to-PTX2 andPTX2-to-PTX1 analyses were used to identify a replicated GPR12-associated remodeling core. An independent time-resolvedhuman iPSC-derived sensory-neuron paclitaxel dataset (GSE312881) was then reanalyzed to test whether GPR12 also occupied adelayed component of the paclitaxel response outside the mouse PB system.We performed an exploratory secondary analysis of publicly available whole-brain spatial transcriptomic data frompaclitaxel-exposed and control mouse brain sections (GSE325554). Spatial gene-expression matrices were integrated with tissuecoordinates and mapped computationally to the Allen Mouse Brain Common Coordinate Framework. Candidate molecular stateswere initially examined across genes associated with glutamatergic transmission, somatostatin signaling, MAPK signaling,serotonergic systems, neuronal activity, and pain-related pathways. A spatial Gate score incorporating Sst, Slc17a6, and Mapk14initially identified localized candidate domains. Component decomposition subsequently demonstrated that Slc17a6, encodingvesicular glutamate transporter 2 (VGLUT2), was the most reproducible component within the parabrachial region (PB), whereasPB-specific Sst enrichment was absent and Mapk14 enrichment was inconsistent across paclitaxel sections. We therefore refinedthe analysis around a Slc17a6-high PB spatial state. Spatial reproducibility was quantified between independent paclitaxel sectionsusing Jaccard and Dice overlap statistics. To determine whether the observed overlap exceeded that expected from intrinsic PBgeometry, we generated 10,000 PB-constrained spatial-null configurations using rotation and translation of the microdomain whileapproximately preserving its spatial extent. ResultsReceptor-focused analysis repeatedly nominated GPR12 within the PTX-remodeled PB environment. In untreated PBN single-celldata, GPR12-positive neurons formed a pre-existing substrate and were predominantly glutamatergic. However, a stringent normalGPR12-positive excitatory-neuron signature did not reproducibly project into both PTX Slc17a6-high microdomains, arguing againstsimple expansion of a baseline GPR12-positive state. Reciprocal PTX state discovery identified a 34-gene GPR12-associated coreshared across PTX sections. In the independent human sensory-neuron time course, GPR12 was nearly unchanged at 2-6 h andshowed a delayed positive shift at 48-72 h, although these individual transcript-level changes did not meet FDR < 0.05. Thus, thehuman analysis provides temporal triangulation rather than direct PB replication.Whole-brain spatial transcriptomic reanalysis identified a recurrent, spatially organized Slc17a6-high glutamatergic microdomainwithin the parabrachial region after paclitaxel exposure. The two independently analyzed paclitaxel sections exhibited substantialspatial concordance of the Slc17a6-high PB microdomain, with a Jaccard index of 0.506 and a Dice coefficient of 0.672. Under a PBconstrained spatial-null model, the mean null Jaccard index was 0.158, with 95th and 99th percentiles of 1DOI:0.400 and 0.481, respectively. The observed Jaccard overlap exceeded the 99th percentile of the spatial- null distribution (P =0.0044). Dice-based testing produced the same spatial-null probability (P = 0.0044). Comparison with the evaluable control sectionsuggested greater PTX-PTX than PTX-control spatial concordance. PTX1-Ctrl2 and PTX2-Ctrl2 Jaccard indices were 0.235 andDOI: 10.5281/zenodo.22477920DOI: 10.5281/zenodo.2247792010.5281/zenodo.2247792020.182, respectively, compared with 0.506 between PTX1 and PTX2. The resulting ∆Jaccard was +0.298. Barcode-level bootstrapanalysis supported the direction of this effect in 94.7% of bootstrap iterations, although its 95% bootstrap confidence interval crossedzero (-0.037 to 0.402), indicating that treatment-level specificity remains incompletely established. Atlas-based refinementconsistently assigned the recurrent state to the parabrachial region in both paclitaxel sections. The available two-dimensional atlascoordinates did not retain sufficient information for definitive assignment to individual PB subnuclei. ConclusionTogether, the spatial and receptor-centered analyses support a unified model in which paclitaxel exposure is associated withrecurrent central molecular-spatial organization and delayed GPR12-linked neuronal remodeling. GPR12 is a testable molecularentry point into this persistent PB state, but causal and therapeutic claims require direct perturbation.These findings identify a reproducible spatial organization of a Slc17a6-high glutamatergic state within the parabrachial regionfollowing paclitaxel exposure. The result extends previous evidence implicating glutamatergic PB circuitry in neuropathic andnociplastic pain by demonstrating a spatially recurrent molecular architecture in paclitaxel-exposed brain tissue. Because the sourcedataset was generated to investigate long-term paclitaxel-associated brain changes rather than painful neuropathy, and because nopain behavior was linked directly to the analyzed sections, the identified PB microdomain should not yet be interpreted as a causalsubstrate of CIPN. Instead, it represents a spatially defined candidate central node for experimental investigation of persistentchemotherapy-associated neural states.