Event Abstract

Glial cell line-derived neurotrophic factor as a new approach for functional neural networks reorganization in oxygen stress conditions

  • 1 Lobachevsky State University of Nizhny Novgorod, The Institute of Biology and Biomedicine, Russia
  • 2 Russian Academy of Sciences, Federal Scientific Research Centre "Crystallography and Photonics, Russia

Motivation Investigation of functional neural networks reorganization in stress conditions as well as searching for a new neuroprotective substances is a topical issue in neurobiology and biomedicine. Hypoxia considered as one of the most significant pathogenetic factors playing a key role in a variety of diseases. Hypoxia-induced damages of neural networks activity are associated not only with the death of the significant functional neurons, but also with changes in synaptic weights of connections in the network. Glial cell line-derived neurotrophic factor (GDNF) is one of key factors of neuronal survival, promoting the differentiation and maintenance the viability of various cell populations in the central and peripheral nervous systems. It was shown that GDNF exhibits neuroptotective effect in hypoxic conditions and contribute to maintaining neural network activity in the posthypoxic period. However, a question concerning the role of GDNF as an endogenous factor of synaptic plasticity of neural networks in acute stress conditions, such as hypoxia, remains open. Material and Methods Dissociated hippocampal cells were obtained from CBA mice embryos (E18) and cultured during 14 days in vitro (DIV) according to the previously developed protocol on multielectrode arrays (MED64, 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 were registered: the number of bursts, the number of spikes in a burst, the burst duration. The detection of small network bursts was performed by calculating the total spiking rate (TSR), accounting the total number of spikes from all electrodes within 50 ms time bins. The criterion of small network burst is the rapid appearance of a large number of spikes over 4 electrodes within a small (50 ms) time bin. Cross-correlation method and graphs were used for neural network's interior structure studies. Dataset, obtained from spontaneous bioelectrical network activity recordings, was represented as a raster plot. To assess a degree of synchronization between all pairs of cells taking into account axonal delays the proportion of transmitted spikes was calculated. This measure is an analog of the cross-correlation coefficient for the continuous signals. Then we selected the largest 5% of Cij coefficients and defined a set of indices – hubs of cells with a maximum number of functionally active connections (Fig. 1). In addition, the functional metabolic calcium activity of neural networks was conducted with the calcium-sensitive dye Oregon Green 488 BAPTA-1 (Invitrogen, O-6807, USA) and a confocal laser-scanning microscope (Zeiss LSM 510, Germany). SmartFlareTM RNA Detection Probes (Merck Millipore, SFC-534, France) were applied for intravital detection of mRNA GluR2 in primary hippocampal cultures. Moreover, the viability of cells was evaluated by using propidium iodide/ bisBenzimide test. Results and discussion Investigation of interior structure of neural network by cross-correlation method revealed that neural networks development is accompanied by the increase of graph structure complexity, growth of the intersections amount and insignificant decrease in the delay time. The reduction of neural networks activity was observed during 7 days of the posthypoxic period. There was a significant decrease in the number of spikes in a burst (ð<0,05) and a large number of dead cells in primary hippocampal cultures (35,55±3,72). The internal structure of a burst is simplified and characterized by reduced the number of connections between the electrodes and increased the time of signal transmission from electrode to electrode. Preventive GDNF 1 ng/ml application led to the cellular viability preservation and to the decrease the negative effect of hypoxia exerted on spontaneous neural network activity. It was shown that GDNF affects on the network structure in the posthypoxic period. The number of spikes and bursts remained distinguishable from baseline on day 7 after hypoxia modeling. Cross-correlation analysis revealed that the number of connections between network elements became stable, but the excitation transmission time was significantly increased (ð<0,05). The structure of burst under GDNF application remains in baseline. Thus, GDNF provides the neuroprotective effect in hypoxic conditions and influences on synaptic plasticity. However, the mechanisms of GDNF action on neural network activity are not identified. Activation of impermeable for Ca2+ ions postsynaptic AMPA-receptor GluR2 subunits synthesis is considered a key mechanism of neural network activity maintaining in hypoxia and excitotoxicity as well as a possible mechanism of GDNF neuroprotective action. GDNF is able to stimulate promoter activity of GluR2-subunits of AMPA-receptors, playing an important role in synaptogenesis and neural network formation. Our studies revealed the increased number of mRNA GluR2 positive cells in primary hippocampal cultures after GDNF (1 ng/ml) application in normoxic conditions (Sham - 58,94±10,23%; GDNF - 78,68±10,72%, p<0,05). Hypoxia reduced the amount of these cells more than 1,5 times. Preventive GDNF (1 ng/ml) addition contributed to maintaining mRNA GluR2 positive cells in primary cultures even under conditions, characterizing by low energy substrates and protein synthesis capacity of cells (Hypoxia – 32,85± 6,71%; Hypoxia + GDNF - 68,8±13,12%, p<0,05,). Conclusion Glial cell line-derived neurotrophic factor has a strong neuroprotective action and affects on the synaptic plasticity in normoxia and in the posthypoxic period via increasing of mRNA GluR2 synthesis. Figure Legend Figure 1. Example of spontaneous bioelectrical activity analysis by cross correlation method and graphs

Figure 1

Acknowledgements

The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014–2020” of the Ministry of Education and Science of Russia (Project ID RFMEFI60715X0117).

Keywords: Neuroprotection, hypoxia, neural networks, multielectrode arrays, gdnf

Conference: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.

Presentation Type: Poster Presentation

Topic: MEA Meeting 2016

Citation: Vedunova M, Mishchenko TA, Shishkina T, Mitroshina E, Shirokova O, Pimashkin A, Kastalskiy I and Kazantsev V (2016). Glial cell line-derived neurotrophic factor as a new approach for functional neural networks reorganization in oxygen stress conditions. Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00020

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Received: 22 Jun 2016; Published Online: 24 Jun 2016.

* Correspondence: Dr. Maria Vedunova, Lobachevsky State University of Nizhny Novgorod, The Institute of Biology and Biomedicine, Nizhny Novgorod, Russia, mvedunova@yandex.ru