Role of Low Voltage Activated Calcium Channels in Neuritogenesis and Active Migration of Embryonic Neural Progenitor Cells Lauri M. Louhivuori, 1 Verna Louhivuori, 1 Henna-Kaisa Wigren, 1 Elina Hakala, 2 Linda C. Jansson, 1 Tommy Nordstro ¨ m, 1 Maija L. Castre ´ n, 1 and Karl E. A ˚ kerman 1 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 configu- ration 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 Ca 2 + instead of 10 mM Ba 2 + 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. Introduction E arly electrical activity triggering spatial and tempo- ral calcium transients is of significance in neuronal mi- gration and differentiation. Calcium influx via voltage-gated calcium channels (VGCC) is thought to have a central role in activity-dependent regulation of embryonic development [1,2]. In vertebrate neurons, 2 main types of calcium con- ductance are well distinguished on the basis of their voltage- dependent activation: high-voltage-activated (HVA; L-, N-, and P/Q-type; Ca v 1.x, Ca v 2.x) and low-voltage-activated (LVA; T-type; Ca v 3.x) calcium channels. There is evidence that calcium influx through VGCC, in particular L- and N-type calcium channels, results in signaling that affects the expression of genes involved in cell proliferation, pro- grammed cell death, and neuronal differentiation [1,3–6]. Several laboratories have shown a simple pattern of VGCC expression with a correlation between neuronal markers and L-type calcium channel expression [7–10]. Studies on VGCC in central nervous system development have usually been performed on postnatal neurons such as young Purkinje cells. These studies have been paramount in furthering our understanding of the changes in cellular functions during neuronal maturation. However, despite the clear role of VGCC in neural development [2,11,12], the ex- pression and function of these channels during embryonic neurogenesis are less well investigated. There have been few reported studies investigating functional VGCC in neural progenitor/stem cells. Voltage- gated calcium channels have previously been reported to be functionally expressed in differentiated postnatal neural progenitor cells (NPCs), with mainly L-type VGCC appear- ing after differentiation for 3 days [10]. Studies that have focused on characterizing VGCC have usually directed their measurements of NPCs after several days to several weeks of in vitro culturing. Further, the cells have usually been plated as monolayers, often overlooking the possible correlation between migration and VGCC channel function, in which the neurosphere-plating method provides, since cell migration with reference to the neurosphere can be used as a measur- ing parameter. As specific classes of cells come to reside in specific layers of the neocortex, migration reflects a program 1 Biomedicum Helsinki, Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland. 2 Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland. STEM CELLS AND DEVELOPMENT Volume 22, Number 8, 2013 Ó Mary Ann Liebert, Inc. DOI: 10.1089/scd.2012.0234 1206