Role of Low Voltage Activated Calcium Channels in Neuritogenesis and Active Migration of Embryonic Neural Progenitor Cells

Abstract

The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

Transient Receptor Potential Channels and Their Role in Modulating Radial Glial–Neuronal Interaction: A Signaling Pathway Involving mGluR5

“…1A). The expression pattern was similar in cells from the FVB mice strain (data not shown) used in previous studies [20,26]. Using RT-qPCR (normalized against proliferating cells), the mRNA expression for Trpc1 and Trpc3 was analyzed in cells from 3/6 DKO and 1/4/5 TKO mice.…”

“…Our previous studies have shown a cellular phenotype distribution with regard to the distance from the mothersphere [20,23,26]. The neuronal marker MAP-2 dominates at the outer regions, while the radial glial marker GLAST dominates close to the neurosphere as a dense network.…”

“…NPCs were isolated from the walls of the lateral ventricles of embryonic day 14 FVB mice and TRPC-deficient mice (3/ 6 DKO and 1/4/5 TKO mice) and their respective wild-type controls, as described previously [26]. Briefly, cells were grown as free-floating aggregates, known as neurospheres, in Dulbecco's Modified Eagle's Medium:Ham's Nutrient Mixture F-12 media (1:1) containing B27 supplement (both from Gibco, Life Technologies Ltd.), 2 mM l-glutamine, 15 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 100 U/mL penicillin, and 100 U/mL streptomycin (all from Sigma-Aldrich), in the presence of 10 ng/mL fibroblast growth factor and 20 ng/mL epidermal growth factor (both from PeproTech EC Ltd.), in a 5% CO 2 humidified incubator at 37°C.…”

“…Calcium imaging was performed as described previously [22,23,26]. For the experiments, 20-30 neurospheres were plated on poly-dl-ornithine-coated 25-mm cover glasses and differentiated for the indicated time periods.…”

Expression and Role of T-type Calcium Channels during Neuroendocrine Differentiation

“…The role of T-type calcium channels in neurite formation has been investigated in embryonic neural progenitor cells [78]. From the inhibition of calcium signals induced mainly by T-type channels inhibitors (mibefradil, NNC-550396), it was concluded in this study that T-type calcium channels are expressed during the early stages of neuronal differentiation (one day).…”