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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
The Effects of Caloric Restriction and Western Diet on Astrocytes
A. Lebedeva*, A. Plata, V. Tovpyga, P. Denisov, S. Makovkin, A. Semyanov
Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia. * Presenting e-mail: lebedeva@neuro.nnov.ru
The study of the processes involved in aging has ample importance in neurobiology. Life expectancy has augmented in developed countries with the consequent increase in the incidence of neurodegenerative diseases. Preliminary studies have shown that caloric restriction diet can improve memory in the elderly. Furthermore, high-fatty (Western) diet can contribute to the development of neurodegenerative disorders such as Alzheimer's disease. Most of the recent works have involved different mechanisms of neural networks in aging and neuropathological processes. However, the role of neuroglia has been less frequently studied. In order to elucidate the role of astrocytes in aging, we studied how low caloric and high fatty regimens alter astroglia activity over time. We used male mice C57/BL/6 of different postnatal days (P), slices were taken at day 1-3, 5, 7, 14 and 30 after the beginning of a specific diet. Caloric restriction diet consists of lowering to 70% the standard ration of the animals. Western diet contained 50% more fat than in the standard menu. The control mice receive food ad libitum in compliance with the standards stipulated for laboratory animals. Currents from astrocytes of hippocampal slices were measured by patch clamp technique in physiological stimulation and subsequently under pharmacological blockade of the excitatory amino acid transporters (EAATs) with TBOA (50 |oM).
Acknowledgements
This work was supported by the Russian Science Foundation (project 16-14-00201).
GDNF Influence on the Morpho-Structural Integrity of Hippocampal Neural Network in Hypoxia Modeling In Vitro
T.V. Shishkina12 *, T.A. Mishchenko2,1, E.V. Mitroshina2,1, I.V. Mukhina2A, V.B. Kazantsev1, M.V. Vedunova12
1 The Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;
2 Molecular and cell technologies group, Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia. * Presenting e-mail: schischkina.tatiana2012@yandex.ru
The preservation of neural networks' functional integrity under the influence of various stress factors is one of the topical issues in modern neurobiology. Neural networks are regarded as a minimal functional unit of the central nervous system, responsible for the transmission and information storage processes. Stressogenic factors, such as hypoxia, stimulate not only the death of the functionally important neurons, but reorganize the synaptic plasticity processes, which significantly affects the neural network functional structure. Nowadays, a substantial question, concerning the exploration of substances, able to maintain the viability and functional activity of brain cells in stressful conditions, remains open. Particular attention is given to investigation of neurotrophic factors, especially Glial cell line-derived neurotrophic factor (GDNF), which regulate various cellular processes during development and in mature brain. The one of the unique properties, that distinguishes this protein from other neurotrophins, is its ability to affect the synaptic plasticity by influencing on promoter activity of GluR2-subunit of AMPA-receptors.
In this regard, the aim of the investigation is to study the GDNF influence on viability and spontaneous bioelectrical activity of neural networks in primary hippocampal cultures during hypoxia modeling in vitro.
Materials and Methods
Dissociated hippocampal cells were taken from C57Bl6 mice embryos (E18) and cultured during 14 days in vitro according to the previously developed protocol on multielectrode arrays (Alpha Med Science, Japan) or coverslips. Hypoxia modeling was performed on DIV14 by replacing the normoxic cultural medium with a medium containing low oxygen for 10 min. The main parameters of spontaneous bioelectrical activity such as the number of bursts, the number of spikes in a burst, the burst duration were established. In addition, the method of correlation graphs was used. We also conducted the cell viability detection. Moreover, SmartFlareTM RNA Detection Probes (Merck Milli-pore, SFC-534, France) were applied for intravital detection of mRNA GluR2 in primary hippocampal cultures. The carried out experiments revealed an acute increase in the number of dead cells (4,5 times) in primary hippocam-
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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
pal cultures and, as a consequence, a complete inhibition of spontaneous bioelectrical activity of neural networks up to day 7 of the posthypoxic period. Preventive GDNF 1ng/ml application eliminates the negative hypoxic effects by increasing cellular viability as well as by maintaining the functional characteristics of neural networks.
Evaluation of the network structural changes by using the method of correlation analysis showed that hypoxia leads to simplification of the internal structure of neural networks in primary hippocampal cultures: there was a significant decrease in the number of active electrodes as well as in the average number of connections per electrodes (before: 4,22 ± 0,48; after 2,69 ± 0,41) on day 3 after hypoxia modeling. Moreover, the time of signal transmission from electrode to electrode was increased in 4,6 times (p<0,05, ANOVA).
Changes in the functional structure of network burst towards a simplification could be explained by loss of the part of functionally important neurons. Preventive GDNF application contributes to maintaining a complexity of network architecture: at constant/minor increase of the number of active electrodes an average number of connections on the electrodes is significantly reduced, a large value of electrodes with a low number of connections is detected, the amount of hubbs is decreased whereas the time of signal transmission is significantly higher than in the intact group (p <0,05 ANOVA).
To evaluate the possible molecular mechanisms of GDNF neuroprotective action, the GDNF influence on the expression of mRNA GluR2 subunit of AMPA-receptors was investigated. It was shown that hypoxia reduced the expression of mRNA GluR2 in primary hippocampal cultures whereas a preventive GDNF 1ng/ml application negates this effect, contributing to the increase in the number of mRNA GluR2 positive cells. Therefore, GDNF is able to influence on synaptic plasticity under stress conditions.
Acknowledgements
The research was supported by grants of the Russian Foundation for Basic Research 16-34-00301, 16-04-00245 and prepared as a part of the state project "Provision Scientific Research".
Network Ca*- Cell Activity Field CA3 Hippocampal Slices of Rat Early and Late Postnatal Development
Y.I. Mitaeva1 *, A.M. Mozherov1,I.V. Mukhina1'2
1 Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;
2 Novgorod State Medical Academy, Nizhny Novgorod, Russia. * Presenting e-mail: yasya13@mail.ru
Hippocampus - the structure of the central nervous system, which is involved in the mechanisms of emotion and memory consolidation. The hippocampus has a certain topology distribution of cellular elements, which provides the many cellular networks. One of them is the network of neurons in the CA3 field. This network receives inputs from cells of the entorhinal cortex and the dentate gyrus, in addition CA3 pyramidal neurons form the connection between themselves and interneurons, forming a closed network that operates in conditions of acute slice and generates spontaneous Ca2+ activity. Neuronal network interacts with the glial network, the main manifestation of activity which is Ca2+ oscillations. Therefore, to estimate the age dependence of Ca2+ activity in the cells were investigated Ca2+ oscillations in neuronal and glial networks and the interactions between them. In this work, we investigated changes in the characteristics of Ca2+ oscillations cells of rat hippocampal CA3 field in early (P5-8, P14-16) and late (P21-25), postnatal development. Also shown the effect of temperature of perfusion solution on cells Ca2+ activity of CA3 field hippocampal slices of rats in different postnatal periods. Besides in the study was valued role of network activity in the formation of spontaneous Ca2+ oscillations cells of rat hippocampal CA3 field in early and late stages of postnatal development.. Experiments were carried out on acute hippocampal slices from rats. Was used laser scanning confocal microscope Carl Zeiss LSM 510 Duoscan (Germany). Recording fluorescence kinetics were carried out in full frame (field of view of 400x400 mm), with a resolution of 256x256 pixels digital and scanning frequency of 1 Hz. Fluorescence indicators recorded in the range 500-530 nm (Oregon Green488 BAPTA-1 AM) and 650-710 nm (Sulfor-hodamine 101). The fluorescence intensity (s.u.) shows the dependence of the concentration of [Ca2+]i in time, indicating the metabolic activity of cells. Method of cross - correlation analysis was used to evaluate synchrony of Ca2+ oscillations cells of CA3 field of rat hippocampus. We chose the time interval size in 3 seconds and within this interval were found synchronous Ca2+ oscillations in all possible pairs of cells. Further, the number of synchronously occurring Ca2+ oscillations were normalized to the minimum number of Ca2+ oscillations in one of the cells analyzed pairs. The studies have shown that the parameters of cell Ca2+ oscillations field CA3 of hippocampal slices vary depending on
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