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

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 34 and day 1011 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 networks activity in response to glutamate receptor overstimulation. Results. A significant glutamate-induced increase of the intracellular calcium concentration was typical for mature neurons (day 1011 of cultivation), along with a lack of PSA-NCAM paranuclear accumulation, which was only found in immature cells (day 34 of cultivation). There was a glutamate suppression of the neuronal network burst activity, formed in vitro by day 1011, 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 1011 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.

Keywords

• cultivated neurons
• psa-ncam protein
• glutamate receptors
• intracellular calcium concentration
• multielectrode system
• spontaneous bioelectrical activity