engleski

P19 NEURONS –A MODEL FOR STUDY GLUTAMATE EXCITOTOXICITY AND NEUROPROTECTIVE EFFECTS OF GABAERGIC DRUGS

Excessive increase in synaptic glutamate level, the major fast excitatory neurotransmitter in the mammalian central nervous system, is toxic to neurons and triggers the process of neuronal cell death commonly referred to as glutamate excitotoxicity. Glutamate excitotoxicity contributes to ischemia-induced brain damage, epilepsy, and various chronic neurodegenerative diseases. While primary neuronal cell cultures have been used to investigate excitotoxicity, development of cell lines exhibiting glutamate receptor-mediated death is desirable. The aim of this study was to establish the culture of P19 neurons and to determine if it can serve as a pharmacological model for the investigation of glutamate-induced excitotoxic signaling pathways, as well as a model for studying a potential neuroprotective effect of drugs that act through the GABA-A receptor complex. P19 mouse embryonal carcinoma cells, when exposed to all-trans retinoic acid, differentiated into a mixed population of neurons, fibroblasts and astrocytes, the number of the latter being reduced by exposure to cytosine arabinofuranoside. Under the phase-contrast microscope, it was visible that cell bodies of P19 neurons characteristically clumped together into tight aggregates, while visually detectable networks of extensive processes connected clusters of cell bodies. Immunofluorescence staining against neuron specific protein β-III tubulin showed the expression of β-III tubulin in P19 neurons thus suggesting a complete neuronal maturation of these cells. A trypan blue exclusion assay was performed to assess viability of P19 neurons after exposure to 300 microM glutamate (3h, 37°C). The survival rate of these neurons was significantly reduced 24 hours after glutamate-treatment (54.6 ± 8.2%). Radioligand binding studies were conducted to confirm the expression of GABA-A receptors on P19 neurons. Scatchard analysis of [3H]flunitrazepam binding data revealed that P19-neurons possessed a single type of binding sites for [3H]flunitrazepam on GABA-A receptors with a Kd of 2.13 ± 0.36 nM and a Bmax value of 0.209 ± 0.020 pmol/mg proteins. The obtained results suggest that differentiated culture of P19 neurons provide a genetically open system useful for studying glutamate receptor-mediated phenomena at a molecular level and may serve for screening of potential neuroprotective agents that act as modulators of GABA-A receptor complex.

P19 neurons; glutamate excitotoxicity; neuroprotective effects; GABAergic drugs

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