CMP

Croscopic observations (Figure 1) and immunofluorescence staining (Figure 2B - D). Although differentiated M17 cells demonstrated evidence of maturing neuronal organization and properties, the functional verification of synaptic activity remains to be done. Besides morphological characteristics, we also observed differences in expression of several neuron specific proteins between undifferentiated and differentiated M17 cells (Figure 4). The presence or levels of certain proteins can vary between immature and mature neurons. One such protein is neuron specific enolase (NSE) which is responsible for generating phosphopyruvate hydratase that participates in glycolosis/gluconeogenesis; the levels of NSE increase as the neurons mature. While undifferentiated M17 cells do express NSE, its level increases due to differentiation. The formation of neuritelike processes as a part of synaptic organization and activity can be further characterized with the differential expression of the neurofilament proteins, NF-M, and that help form the neurofibrils within axons [33]. Developing neurons generally do not express either of these neurofilament proteins until they become post-mitotic, which is fairly late in development. Another neurofilament subunit, vimentin, decreases as neurons mature [34]. We were able to detect vimentin (Figure 4C) and the neurofilament proteins -M as well as -H (data not shown). We observed a decreased level of vimentin, whereas neurofilaments H and M increased due to differentiation. This might be an indication that under the conditions used, M17 cells could be in an early stage of maturation. This hypothesis is supported by the wide-spread expression of the immature neuronal marker 3-tubulin and the accumulation of synapsin-1/2 at the tip of the growth cone (Figure 3). The presence of synapsin within the growth cone is consistent with studies suggesting an axonogenic role during neurite extension and branching, which is a early aspect of neuronal maturation [35,36]. The levels and localization of theseAndres et al. BMC Neuroscience 2013, 14:49 http://www.biomedcentral.com/1471-2202/14/Page 9 ofFigure 7 Effect of 10 M RA on the expression of functional voltage-sensitive. Ca2+ channels in M17 cells. M17 cells were treated with 1 mCi/ml 3H-Valine 24 hours prior to each experiment. M17 cells were either (A) undifferentiated cells stimulated for 4 minutes with KCL, or differentiated cells (B) stimulated for 4 minutes with KCl, (C) KCl + 10 M NNC 55-0396/KCl, (D) KCl + 1 mM w conotoxin, GVIA, or (E) KCl + 300 nM w agatoxin IVA. Each of these KCl solutions contained 1 mCi/ml PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 of 45Ca2+. The ratio of 45Ca2+/3H was then used to calculate the percentage difference of Ca2+ channel activity. of control was calculated by dividing the experimental ratio of 45Ca2+/3H by the ratio of 45Ca2+/3H generated Lenalidomide with 5.9 mM KCl alone. n=4 *p<0.05 when compared to 5.9 mM KCl. **p<0.01 when compared to 5.9 mM KCl.developmentally staged proteins are anticipated to further change during prolonged culture in the presence of RA. Since M17 cells are multipotential with regard to neuronal enzyme expression, we looked at the effects of RA differentiation on the expression of the main isoforms of acetylcholine (ACh) receptors (M1 mAChR, nAChR ?7) and choline acetyltransferase (ChAT) to determine the type of neurons that RA differentiated M17 cells could be. In Figure 5, nAChR-7 was the only one of the mentioned proteins that was able to be detected. This.