engleski

P19 NEURONS – A MODEL FOR PHARMACOLOGICAL STUDIES

Introduction: 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. 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 and for research of possible neuroprotective effects of drugs that acts after the binding to GABA-A receptor complex. Material and Methods: Mouse P19 embryonal carcinoma cells were exposed to 1 microM all-trans retinoic acid in the DMEM medium supplemented with 5% foetal bovine serum (FBS) in the bacterial grade Petri-dishes for 4 days. P19-cells were then plated onto tissue culture plates and grown in DMEM medium supplemented with 10% FBS for two days. After that, P19 neurons were grown in the DMEM medium supplemented with 5 microg/mL insulin, 30 microg/mL transferrin, 20 microM ethanolamine and 30 nM sodium selenite for additional 72 hours (first 48 hours a mitotic inhibitor cytosine arabinofuranoside was added to inhibit the proliferation of non-neuronal cells). Level of differentiation 9 days after induction was monitored by immunofluorescence staining with antibody for neuron specific protein β-III tubulin. A Trypan Blue exclusion assay was performed to assess P19-N cell viability after exposure to 300 microM glutamate (3h, 37°C). Radioligand binding studies with [3H]flunitrazepam were conducted to confirm the expression of GABA-A receptors on P19 neurons. Results: 4-days treatment with retinoic acid induced differentiation of P19 precursor cells 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 is 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. P19 neurons express neuron specific marker β-III tubulin, which means that neuronal maturation was fully completed. Scatchard analysis of [3H]flunitrazepam binding data revealed that differentiated P19-neurons posses 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. P19 neurons are vulnerable when exposed to 300 microM glutamate (3h, 37°C), and the survival rate of these neurons was significantly reduced 24 hours after the glutamate-exposure (54.6 ± 8.2%). Conclusions: The obtained results suggest that differentiated culture of P19 neurons is a suitable model system for studying a glutamate- receptor dependent excitotoxic signaling pathways and may serve for rapid screening of potential neuroprotective agents that act as modulators of GABA-A receptor complex.

P19 neurons; glutamate excitotoxicity; neuroprotection; GABA-A receptor modulators

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