Structural and Molecular Differentiation of Neuroblastoma-Derived Human Neurons is Associated with Alterations in Spontaneous and Evoked Calcium Dynamics

Abstract

During development, neuronal precursors transform from non-differentiated pluripotent state into specialized neurons. Much research has been conducted into morphological and molecular alterations during this transition, with underlying functional dynamics and mechanisms waiting to be elucidated. We combined structural and molecular imaging studies of neuroblastoma-derived developing neurons with functional characterization of evoked and spontaneous Ca²⁺ dynamics. In a non-differentiated state, we detected trace amounts of neuronal markers, with live imaging showing high-amplitude slow spontaneous Ca²⁺ oscillations. Application of carbochol induced monophasic low-amplitude Ca²⁺ transients. Differentiation of cells into the next 2CL stage, using retinoic acid, has promoted the enrichment of cells with neuron-specific proteins and mild morphological polarization with neurite outgrowth. These changes were associated with strong suppression of Ca²⁺ oscillations, while evoked Ca²⁺ transients remained unchanged. Converting cells further into morphologically differentiated neurons by combining BDNF and retinoic acid treatment promoted extensive polarization of cells with a strong enrichment with neuron-specific markers. These changes were accompanied by a rebound of spontaneous oscillation of Ca²⁺ but of lower amplitude and higher frequency variance. The evoked by carbachol Ca²⁺ transients at this stage were enhanced and showed a bi-phasis decay. At all differentiation stages, ionomycin-induced Ca²⁺ transients were indistinguishable. These findings led us to conclude that the structural and molecular transmutation of neuroblastoma-derived human neurons is associated with extensive adjustments in Ca²⁺ dynamics, likely contributing to their differentiation.