Using varying concentrations of KCl in undifferentiated M17 cells (Fig…

Using varying concentrations of KCl in undifferentiated M17 cells (Figure 7A); a strong increase in the uptake of radiolabeled 45Ca2+ was observed with RA differentiation of M17 cells with a maximum opening of voltage-gated Ca2+ channels at 25 mM KCl. The presence of both N and P/Q type Ca2+ channels was indicated by the 50 ?60 reduction in Ca2+ uptake when conotoxin GVIA (N-type blocker) (Figure 7D) or agatoxin IVA (P/Q blocker) (Figure 7E) were applied to the culture. Using NNC 55?396, only a small amount of T-type Ca2+ channels were detected (Figure 7C); however, the assay may not be sensitive enough to pick up the small change in intracellular Ca2+ concentration due to the small unitary conductance of T-type Ca2+ channels. Since neuroexocytosis is Ca2+ dependent, the lack of functional voltage-gated Ca2+ channels in undifferentiated M17 cells is detrimental for its use as a cell model for neurotoxicity research. The treatment of M17 with RA for a minimum of 72 hrs may be essential for functional neuronal cultures. It has been postulated that neuronal functions are cell maturation dependent [17]. In toxicity studies, it is important to look at both morphological and functional changes to determine toxicological mechanisms. It has been shown here as well as in other studies that maturation of neuronal cultures is very important when studying the effects of toxicants [17]. It is known that intracellular Ca2+ is highly regulated and involved in normal cell functions and in toxicological mechanisms. The lack of voltage-gated Ca2+ channel expression in undifferentiated M17 cells could limit their use as a neurotoxicity model. This is supported by our observation that differentiation of M17 cells with RA was required to see the changes in [Ca2+]i following exposure to CG. The [Ca2+]i decrease due to CG is a toxicant response in neuronal cells that can lead to apoptosis and death of neurons [39,40]. Acquisition in voltage-gated Ca2+ channels in differentiated neurons may be a prerequisite for studying neurotoxicity due to chemicals other than CG.Andres et al. BMC Neuroscience 2013, 14:49 http://www.biomedcentral.com/1471-2202/14/Page 11 ofConclusion The results reported here show that the human neuroblastoma BE(2)-M17 cells need to be treated with RA to become differentiated into mature neurons and to exhibit functional neuroexocytosis. Differentiation with RA induces M17 cells to undergo morphological differentiation and synaptic Nelfinavir (Mesylate) maturation. The apparent formation of neural networks, the presence and function of SNARE proteins and voltage-gated Ca2+ channels are necessary for functional neuroexocytosis. Our results showing the presence of these characteristics supports the use of differentiated M17 cells as a cell model for neurobiology and/or neurotoxicity research. Additional fileAdditional file 1: Figure S1. Split confocal image of synapsin-1/2 and 3-tubulin expression in RA-induced M17 cells at 120 h. M17 neuroblastoma cells were grown on cover slips. Cells were fixed, stained, and immunofluorescent images were taken (63X). Synapsin-1/2 (green), 3-tubulin (red) and nuclei (blue). Split panels diffuse synapsin expression in cell body; with punctuate expression PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 apparent in elongated neurites.Disclaimer The views expressed in this article are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. Received: 15 November 2012 Accepted: 9 April 2013 Published:.