Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barre syndrome in children) and analgesic drug screening.

Automated summary

In this study, a unique morphological feature of 50B11 nociceptive sensory neurons in in vitro culture was revealed, which was related to forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was used to observe increased neurite arborization and elongation. Second harmonic generation microscopy allowed for monitoring microtubule organization and cellular multi-photon autofluorescence. This work is the first to examine neuronal cell lines growing and differentiating in an upside-down orientation, providing potential improvements for the study of sensory neurons in peripheral nervous system disease and analgesic drug screening.