Besides traditional methodologies for investigations on neuronal function, such as electrophysiology and neurochemistry, in-vivo living imaging has been recently used as a reliable method for first-level-of-analysis of the functionality of neural systems. The aims of our research are focused on the expression of molecular markers of plasticity together with the evaluation of responses to different stimuli applications in neuronal primary culture. The experimental strategy is therefore based on set up in non-neuronal cells, and then in neuronal cell culture, calcium imaging and immunofluorescence assays, respectively applied in subsequent manner. First step was the development of assays in a non-neuronal cell line (mouse pheochromocytoma cells, PC12). Undifferentiated PC12 cells kept for 4-5 days in growing medium, were placed on glass slide and differentiated with 50 ng/ml NGF-enriched medium for 5-6 days. Calcium imaging experiments were performed by applying 10, 25 or 55mM KCl on cells previously incubated with 5uM Fura2/AM. PC12 differentiated into neuron-like cells responded to 10, 25 or 55mM KCl in a dose-dependent manner. Secondly, brain cortex was dissected from rat Sprague-Dawley at embryo stage E20, and then dissociated cells were cultured for 3 weeks in order to get primary neuronal cell cultures. Calcium imaging experiments were performed applying at different time-points 55 mM KCl or glutamatergic agonists, after incubation with 5µM Fura2/AM. Primary cultured cortical neurons responded to 55 mM KCl and to glutamatergic agonists. On same cultures, immunofluorescence was then performed by using antibodies raised against microtubule associated protein (MAP2), glial fibrillary acid protein (GFAP), synaptophysine, postsynaptic marker (PSD-95) and glutamatergic receptors (GluR2, NR1). Immunofluorescence analysis by confocal microscopy highlighted glia cells positive for GFAP and neurons positive for the specific MAP2; moreover same neurons were also positive for synaptophysine, PSD95, and NR1, GluR2 glutamate receptors. Calcium imaging assay was successfully set-up on PC12 cell line and effectively applied to rat primary neuronal cortical cultured cells. A combined protocol of calcium imaging and immunofluorescence would allow to have a concomitant evaluation of cortical neurons culture function and morphology, in order to correlate calcium related responses to expression of molecular markers of plasticity.
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