ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the central canal

The spatial location of cerebrospinal fluid contacting (CSF-c) neurons enables important regulatory homeostatic functions regarding pH and motion control. Their intricate organization, facing the central canal and extending across the spinal cord, in relation to specific subtypes is poorly understood. This calls for imaging methods with a high spatial resolution (5-10 nm) to resolve the synaptic and ciliary compartments of each individual cell to elucidate their signalling pathways and enough throughput to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine and somatostatin CSF-c neuronal types. The number of somatostatin-containing dense core vesicles, resolved by STED microscopy, was shown to be markedly reduced upon each exposure to alkaline or acidic pH inhibiting any movement as part of a homeostatic response. Their cilia symmetry was unravelled by ExSTED as sensory in contrast with the motile one found in the dopaminergic ph insensitive neurons. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell type classification.