A Fluorescence Imaging-Based 3D Analysis Pipeline for Mouse Trigeminal Ganglion Neurons

As the primary peripheral relay station for vibrissal tactile information, the trigeminal ganglion (TG) features heterogeneous three-dimensional (3D) cytoarchitecture that eludes full characterization using conventional two-dimensional methodologies. A high-resolution 3D imaging and reconstruction pipeline is thus required to unveil TG structural organization and define the spatial framework of target-related sensory neurons. Herein, we established a fluorescence micro-optical sectioning tomography (fMOST)-based workflow for 3D cytoarchitectural mapping of TG anatomy and validated its utility for profiling the distributions of TG neurons innervating vibrissae via single-axon tracing. fMOST imaging coupled with propidium iodide (PI) staining was applied to acquire whole-head anatomical data encompassing the vibrissae and the TG at cellular resolution. Based on clearly resolved cellular morphology and the spatial distribution of neuronal somata, we delineated the soma distribution of TG neurons and revealed a spatially heterogeneous 3D organization pattern, from which we operationally defined two anatomically distinct subdomains: the neuronal soma-rich region (NSRR) and the fiber-rich region (FRR). Furthermore, with retrograde viral/genetic labeling combined with neuronal tracing, TG neurons innervating the C2, D3, and δ vibrissae were observed in both NSRR and FRR, showing partially overlapping yet spatially biased distributions consistent with previous population-level observations of vibrissa-row-dependent topography. Notably, TG neurons innervating the δ vibrissa occupied a comparatively broader spatial extent along the anteroposterior plane in our dataset. Overall, this study facilitates an in-depth mechanistic and anatomical understanding of TG cytoarchitectural organization and underlying functional mechanisms.