Structural and molecular differentiation of cultured human neurons is accompanied by alterations of spontaneous and...

During development, neuronal precursors transform from a pluripotent state into specialized neurons. While much research has been conducted into morphological and molecular changes, there is a pressing need to define accompanying functional alterations. We used immunofluorescence microscopy and live imaging in SH-SY5Y-derived human neurons to elucidate the relationship between structural and molecular differentiation with evoked and spontaneous Ca²⁺ dynamics. In the undifferentiated state expressing trace amounts of neuronal markers, SH-SY5Y cells maintain spontaneous high-amplitude slow Ca²⁺ oscillations, with their stimulation by carbochol activating low-amplitude Ca²⁺ transients. Driving SH-SY5Y cells into the 2CL state by retinoic acid facilitated the outgrowth of neurites and expression of neuron-specific proteins. These changes are accompanied by the abolition of Ca²⁺ oscillations. Differentiating SH-SY5Y cells into definitive neurons by a cocktail of retinoic acid and BDNF induced their polarization and enrichment with specific neuronal markers, accompanied by a resurgence of spontaneous Ca²⁺ oscillations but with faster kinetics. The carbachol-induced rise of Ca²⁺ in these cells showed a higher peak and biphasic decay. At all developmental stages, Ca²⁺ transients in response to ionomycin were indistinguishable. These findings lead us to conclude that a switch of Ca²⁺ dynamics accompanies structural and molecular differentiation of SH-SY5Y cell-derived human neurons, contributing to the developmental process.