Indicates that the RA differentiated M17 cells are not cholinergic but would most likely be involved in post- and pre-synaptic excitation in the brain and not post-ganglion nerves in the CNS or exocrine glands [37,38].The differential expression of other neuronal proteins than those previously described, expression of voltagegated Ca2+ channels and ionotropic receptors, which ultimately lead to an increase in neurotransmitter release, can be used to confirm neuronal characteristics and neuroexocytosis. The presence of SNAP-25 and synapsin are indicative of the potential to Staurosporine form functioning pre-synaptic compartments that mediate synaptic vesicle fusion with the pre-synaptic membrane and neurotransmitter release under depolarizing conditions. Although immunoblot demonstrated that overall synapsin expression in M17 cells does not significantly change after differentiation with RA (Figure 4B), synapsin-1/2 becomes distributed along processes, withAndres et al. BMC Neuroscience 2013, 14:49 http://www.biomedcentral.com/1471-2202/14/Page 10 ofFigure 8 The effects of a Ca2+ ionophore and phosgene (CG) on intracellular Ca2+ changes in differentiated vs. undifferentiated M17 cells. M17 cells were cultured in Transwell inserts without and with RA differentiation to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25751659 80 confluency. They were then exposed to 5 M Ca2+ ionophore, A23187 or 0 and 16 ppm CG. Intracellular free Ca2+ levels were monitored using Fluo-4 Ca2+ indicator assay. (A) Change in intracellular Ca2+ following A23187 exposure (N=3); (B) Change in intracellular Ca2+ following either 0 ppm (air) or 16 ppm CG exposure for 8 min (N=13). *p<0.05 when compared to treated (A23187 or CG) cells.a punctuate appearance (Figure 2 and supporting Additional file 1: Figure S1) and within the growth cone during neuritogenesis (Figure 3). SNAP-25 is a major component of the SNARE complex that is required for the fusion of vesicle to the cell membrane for the exocytosis of neurotransmitters. A two fold increase in the level of SNAP-25 (Figure 4A) was observed. This increase in SNAP-25 level may correlate with the significant (p<0.01) increase in KCl stimulated [3H] glycine release seen in differentiated M17 cells (Figure 6). The increase in the stimulated release as shown in Figure 6 doesn't look impressive; however, it is quite marked because we are comparing the fraction of the total pool of [3H] glycine that is released in differentiated cells vs. undifferentiated cells. As mentioned earlier, we studied [3H] glycine release because this assay has been utilized successfully in assessment of neurotoxicity in cell culture models [15-18]. Others have studied glutamate release and glutamate induced excitotoxicity in M17 cells [12]and as such these cells could be suitable to study glutamate neurotoxicity. Since M17 cells have been reported to have a poor GABAergic property [13] these cells might not be a suitable model for GABA studies. In this report we demonstrated that a representative neurotransmitter function is enhanced in differentiated M17 cells compared to immature cells. For functional neuroexocytosis, neurons need both the ability to form the SNARE complex and to have functional voltage-gated Ca2+ channels. The ability of Ca2+ and other ions to move across the cell membrane is necessary for excitation and signal transmission between neurons. Therefore, we studied the uptake of Ca2+ in both undifferentiated and differentiated M17 cells. There was no increase in the uptake of radiolabeled 45Ca2+.