Elastic properties of the cell surface and metabolic profile of an embryonic primary mixed culture of hippocampal neurons under conditions of P2X3 receptor blockade

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Аннотация

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.

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Авторлар туралы

A. Zelentsova

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Ресей, Belgorod

V. Shmigerova

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Ресей, Belgorod

Y. Stepenko

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Ресей, Belgorod

M. Skorkina

Belgorod State National Research University

Хат алмасуға жауапты Автор.
Email: marinaskorkina0077@gmail.com
Ресей, Belgorod

A. Deykin

Belgorod State National Research University

Email: marinaskorkina0077@gmail.com
Ресей, Belgorod

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2. Fig. 1. Atomic force spectroscopy procedure of the neuron cell surface on the first day of differentiation in culture: (a) – schematic diagram of sample scanning: cell on a substrate; cantilever that moves along the cell surface; laser beam is tuned to a flexible cantilever console that deflects when moving along the surface and transmits information to a 4-section photodiode, (b) – neuron image obtained by scanning in Nova software with load (1 nN) applied at 25 points on the surface, (c) – approach (blue) and retraction (red) curves at each load application point, based on which the software constructs local elasticity maps, (d) – local elasticity map of the neuron surface (dark areas of the map indicate high values ​​of Young's modulus, and therefore higher surface rigidity).

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3. Fig. 2. Young's modulus of the cell surface of the primary mixed culture of E18 neurons under conditions of P2X3 receptor blockade. * significant differences at p < 0.05 according to Student's criterion.

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4. Fig. 3. Metabolic map of the energy phenotype of the primary mixed neuronal culture: the following respiration regions are indicated: Aerobic – aerobic respiration; Quiescence – respiratory rest; Energy – cellular metabolism based on mixed respiration (oxidative phosphorylation and glycolysis); Glycolytic – anaerobic respiration. The control group is shown in gray, the experimental group (Ro-4 blocker) in black. Filled squares indicate respiration before the blockade of the electron transport chain, unfilled squares indicate respiration after the blockade of the electron transport chain by stressors.

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