Retinoic acid induces neuron-like voltage-gated ionic currents in a human neuroectodermal cell line

Introduction TB cells are a human cell line isolated from the cerebrospinal fluid (CSF) specimen of a patient with clinical diagnosis of primary leptomeningeal melanomatosis and characterized by immunological and ultrastructural analysis because of their ability to morphologically differentiate towards a neuronal-like phenotype when cultured with 10 µM retinoic acid (RA) [1]. Aims The main aim of this work is to study the electrophysiological properties of TB cells by whole cell patch clamp technique to understand if, together with morphological and biochemical modifications, these cells also undergo functional changes after RA-treatment. Materials and Method TB cells were grown in DMEM medium, to which 15% FBS and 1% pen/strep were added. Then, cells were seeded on plastic coverslips in Petri dishes. After 24 hours in vitro, 10 µM RA was added to induce the differentiation. TB cells without RA were also prepared (control). After 7 days of RA-treatment, whole cell patch clamp recordings were performed by means of glass capillaries filled with different intracellular solutions in order to detect membrane whole currents or alternatively only Ca2+ currents. Electrophysiological recordings were performed by submerging TB cells in extracellular solution, whose composition was similar to artificial cerebrospinal fluid (aCSF). To avoid cell detachment, aCSF flow rate was kept very low (< 1 mL/min). Results After 7 days, RA-treatment upregulates a fast-inactivating voltage-gated inward current. The reversal potential of this current, as well as experiments by TTX and lidocaine, prove that it is voltage-dependent Na+ fast current. A depolarizing voltage step elicites the presence, only in a subset of RA-treated cells, of a late-onset inward current that was completely blocked by Cd2+, a broad spectrum blocker of Ca2+ currents. Therefore, while the fast component is due to sodium entry, the slow component is due to calcium. However, TB cells are not able to produce spikes. Discussion and Conclusion The treatment with RA drives the differentiation of TB cells in the direction of a neuronal-like phenotype. In particular, RA primarily modulates expression or functionality of Na+ and Ca2+ currents in TB cells. However, the insufficient increase of the Na+ conductance and the non-regulation of the K+ one are probably the main reason for the inability of treated-TB in producing spikes. From these considerations, we have to conclude that while the RA-treatment can produce a fully morphological differentiation of these cells in a neuronal-like phenotype [1], the same does not hold from the functional point of view.