Russian Journal of PhysiologyRussian Journal of PhysiologyРоссийский физиологический журнал им. И.М. Сеченова0869-81392658-655XThe Russian Academy of Sciences65175310.31857/S0869813924090142AIROIJEXPERIMENTAL ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИResearch ArticleElastic properties of the cell surface and metabolic profile of an embryonic primary mixed culture of hippocampal neurons under conditions of P2X3 receptor blockadeУпруго-эластические свойства клеточной поверхности и метаболический профиль эмбриональной первичной смешанной культуры нейронов гиппокампа в условиях блокады Р2Х3-рецептораZelentsovaA. S.ЗеленцоваА. С.
Russian Federation
marinaskorkina0077@gmail.comShmigerovaV. S.ШмигероваВ. С.
Russian Federation
marinaskorkina0077@gmail.comStepenkoY. V.СтепенкоЮ. В.
Russian Federation
marinaskorkina0077@gmail.comSkorkinaM. Yu.СкоркинаМ. Ю.
Russian Federation
marinaskorkina0077@gmail.comDeykinA. V.ДейкинА. В.
Russian Federation
marinaskorkina0077@gmail.comBelgorod State National Research UniversityБелгородский государственный национальный исследовательский университет1509202411091475148702022025Copyright ©; 2024, Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0869-8139/article/view/651753

P2X3-receptors localized in the hippocampus participate in the transmission of excitation and the formation of synaptic plasticity underlying learning and memory. P2X3-receptors are of great importance in the occurrence of neuropathic pain in epilepsy, acute and inflammatory pain of various genesis and localization as well as in the activation and growth of nerves after traumatic brain injury. The aim of the study was to study the elastic properties of the surface and the metabolic profile of neurons in an embryonic primary mixed hippocampal culture under P2X3-receptor blockade. The study was performed on a primary mixed culture of hippocampal neurons obtained from CD1 mice on the 18th day of gestation (E18). The highly selective blocker 5-(5-iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2.4-diamine monochloride salt was selected as a P2X3-receptor blocker. To assess the elastic properties of neurons Young's modulus that characterizes the rigidity of the cell surface was measured. Measurements on an atomic force microscope applying a load in 25 local areas of the cell surface were performed. At each point, the force curves of the cantilever approach and retraction were recorded with subsequent calculation of Young's modulus. The metabolic profile of the neuroglial culture in Energy Phenotype test on a Seahorse HS mini cell metabolism analyzer (USA) was studied. The Young's modulus of the cell surface of neurons in the control was in the range from 6.8 ± 0.1 to 9.7 ± 0.2 kPa, and under the action of the P2X3-receptor blocker in the range from 3.1 ± 0.1 kPa to 8.5 ± 0.1 kPa. Under the conditions of P2X3-receptor blockade on the 5th day of differentiation the Young's modulus of the cell surface was reduced by 62% (p < 0.05), on the 8th day it increased by 22% (p < 0.05) and by the 11th day it decreased by 16.7% (p < 0.05) compared to the control. Aerobic respiration was characteristic of the embryonic hippocampal culture both in the control and with the P2X3-receptor blockade. Consequently, the blockade of the P2X3-receptor did not affect the metabolic profile of the E18 hippocampal culture. The obtained data indicate the direct participation of the P2X3-receptor in the formation of biomechanical properties of the cell surface in the processes of differentiation and signal transduction. It is possible, that the blockade of the P2X3-receptor will be one of the promising molecular targets that can reduce neuronal damage in brain injuries, neuroinflammation, hypoxia, and epilepsy. In addition, the study of the P2X3-receptor blockade can expand the fundamental understanding of the role of the purinergic signaling system in the formation of complex neuronal morphology at early stages of embryonic development under conditions of rapid excitatory signal transmission mediated by the ATP molecule.

Р2Х3-рецепторы, локализованные в гиппокампе, принимают участие в передаче возбуждения и формировании синаптической пластичности, лежащей в основе обучения и памяти. Важное значение Р2Х3-рецепторы имеют в возникновении нейропатической боли при эпилепсии, острой и воспалительной боли различного генеза и локализации, а также в активации и росте нервов после черепно-мозговой травмы. Целью выполненного исследования явилось изучить упруго-эластические свойства поверхности и метаболический профиль нейронов эмбриональной первичной смешанной культуры гиппокампа в условиях блокады Р2Х3-рецептора. Исследование выполнено на первичной смешанной культуре нейронов гиппокампа, полученной от мышей линии CD1 на 18-й день гестации (Е18). В качестве блокатора Р2Х3-рецептора выбран высокоселективный блокатор 5-(5-йод-2-изопропил-4-метоксифенокси)пиримидин-2.4-диамин монохлоридная соль. Для оценки упруго-эластических свойств нейронов измеряли модуль Юнга, характеризующий жесткость клеточной поверхности. Измерения проводили на атомно-силовом микроскопе, накладывая нагрузку в 25 локальных участках клеточной поверхности. В каждой точке регистрировали силовые кривые подвода и отвода кантилевера с последующим расчетом модуля Юнга. Метаболический профиль нейроглиальной культуры изучали в тестах Energy Phenotype на анализаторе клеточного метаболизма Seahorse HS mini (США). Модуль Юнга клеточной поверхности нейронов в контроле находился в пределах от 6.8 ± 0.1 до 9.7 ± 0.2 кПА, а под действием блокатора Р2Х3-рецептора в диапазоне от 3.1 ± 0.1 до 8.5 ± 0.1 кПА. В условиях блокады Р2Х3-рецептора на 5-е сутки дифференцировки модуль Юнга клеточной поверхности был снижен на 62% (p < 0.05), на 8-е сутки увеличился на 22% (р < 0.05), а к 11-м суткам – снизился на 16.7% (р < 0.05) по сравнению с контролем. Для эмбриональной культуры гиппокампа был характерен аэробный тип дыхания как в контроле, так и при блокаде Р2Х3-рецептора. Следовательно, блокада Р2Х3-рецептора не оказывала влияния на метаболический профиль культуры гиппокампа Е18. Полученные данные указывают на непосредственное участие Р2Х3-рецептора в формировании биомеханических свойств клеточной поверхности в процессах дифференцировки и сигнальной трансдукции. Не исключено, что блокада Р2Х3-рецептора может быть одной из перспективных молекулярных мишеней, позволяющих снизить повреждение нейронов при травмах головного мозга, нейровоспалении, эпилепсии. Кроме того, изучение последствий блокады Р2Х3-рецептора может расширить фундаментальные представления о роли пуринергической сигнальной системы в формировании сложной морфологии нейронов на ранних стадиях развития эмбрионов.

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