Changes in the morphofunctional development of the neuronal network in a dissociated cell culture of rat cerebral cortical neurons

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Abstract

Introduction. Study of the morphofunctional neuronal development in a dissociated cerebrocortical cell culture, using modern cell technologies, is a priority in experimental neurology, which is required for successful in vitro modelling of acute and chronic forms of cerebral pathology.

Aim. A morphofunctional study of the in vitro changes in neuronal differentiation of rat cerebral cortical neurons, using a range of analysis methods, including immunohistochemistry, fluorescence, and electrophysiology.

Materials and methods. We investigated the degree of culture differentiation on day 3–4 and day 10–11 of in vitro cultivation, measured by the intensity of the PSA-NCAM protein expression and the level of neuronal glutamate-induced calcium overload. That was then compared with the functional activity of the neuronal network cultivated on a microelectrode array, and with changes of the neuronal network’s activity in response to glutamate receptor overstimulation.

Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 10–11 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 3–4 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 10–11, with had no effect on the generation of single action potentials. At the same time, kainate, the exogenous selective agonist of the one of the glutamate subtypes, completely blocked spontaneous activity of the mature neurons.

Conclusion. Neocortical rat neurons reach the differentiation level necessary for the modelling of the cerebral pathologies by day 10–11 of in vitro cultivation. At this point, the process of disruption of the microelectrode array cultivated neuronal network by the glutamate receptor overactivation, has become multilayered: excitotoxic glutamate-induced damage produces selective disruption of neuronal burst activity, and with the greater cytotoxicity caused by kainate, spontaneous bioelectrical activity is completely blocked.

About the authors

Elizaveta E. Genrikhs

Research Center of Neurology

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Olga P. Aleksandrova

Research Center of Neurology

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Elena V. Stelmashuk

Research Center of Neurology

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Svetlana V. Novikova

Research Center of Neurology

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Dmitriy N. Voronkov

Research Center of Neurology

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Nikolay K. Isaev

Research Center of Neurology;M.V.Lomonosov Moscow State University

Email: khaspekleon@mail.ru
Russian Federation, Moscow

Leonid G. Khaspekov

Research Center of Neurology

Author for correspondence.
Email: khaspekleon@mail.ru
Russian Federation, Moscow

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Copyright (c) 2019 Genrikhs E.E., Aleksandrova O.P., Stelmashuk E.V., Novikova S.V., Voronkov D.N., Isaev N.K., Khaspekov L.G.

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