Features of exogenous influence of the Brain-derived neurotrophic factor and blockade of TrkB receptor on the neural network activity in primary hippocampal cultures
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1
N. I. Lobachevsky State University of Nizhny Novgorod, The Institute of Biology and Biomedicine, Russia
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2
Privolzhsky Research Medical University (PIMU), Molecular and cell technologies group, Central Research Laboratory, Russia
Motivation
According to modern concepts, the minimal functional unit of the nervous system is the neural network. At the neural network level, the processing and transmission of information, memory consolidation, the formation of emotions and behavioural reactions occur [1]. It is considered that neural networks activity in the brain depends not only on the number of synapses, but also on the strength of individual synaptic contacts. Brain-derived neurotrophic factor (BDNF) regarded as one of the substances able to influence the synaptic strength and the synaptic transmission. Binding with high-affinity tyrosine-kinase receptor B (TrkB), BDNF initiates signaling mechanisms determining synaptogenesis, neuronal differentiation, maturation and cell viability.
The aim of this study was to investigate the features of spontaneous bioelectrical activity of neural network in primary hippocampal cultures on the background of chronic application of BDNF and TrkB-receptors blocker (ANA-12) during their cultivation in vitro.
Materials and methods
Long-term cultivation of primary hippocampal cells obtained from C57BL/6 embryos (E18) was performed according to the previously developed protocol [2]. The cells were seeded on multielectrode arrays MEA 60 (Multichannel systems, Germany) at approximate initial density - 9000 cells/mm2. BDNF (1ng/ml) and a selective blocker of TrkB receptors - ANA-12 (1 μM) and their combination were daily added into culture medium beginning with day 3 of culture development in vitro (DIV). For registration and subsequent analysis of spontaneous bioelectrical activity of primary hippocampal cultures, a MC Rack multielectrode system software and a “Meaman” original package of algorithms developed in the custom-made software (MATLAB®) were used. 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. The main parameters of spontaneous bioelectrical activity were analyzed: the number of bursts, the number of spikes in a burst, the burst duration. In addition, to investigate the features of neural network interior structure cross-correlation method and graphs were used according to [3,4].
Next, the features of functional metabolic activity of primary hippocampal cell cultures were investigated using a method of functional calcium imaging. Detection of calcium events was performed by using a specific calcium-sensitive dye Oregon Green 488 BAPTA-1 АМ (Invitrogen, USA) and a confocal laser-scanning microscope Zeiss LSM 510 (Zeiss, Germany). Using an “Astroscanner” original software we analyzed the following parameters: the duration of calcium oscillations (s), the frequency of calcium oscillations (the number of oscillations per minute), and the percentage of cells exhibited calcium activity.
Results
Carried out experiments revealed that chronic BDNF and ANA-12 application modulate the spontaneous bioelectrical activity of primary hippocampal cultures mainly by changes in internal functional structure of neural networks. In the “BDNF” and “ANA-12” group there was a significant increase in the number of spikes formed the network burst in the late stages of culture development in vitro (BDNF - 565±20.7, ANA-12 - 670±18.9) (figure 1). The neural network profile in the “ANA-12 + BDNF” group was amounted to 365 ± 16.3, which also exceeded the values of the “Sham” group – 291 ± 18.3.
Figure 1. Characteristic profile of network burst forming spontaneous bioelectrical activity of primary hippocampal cultures on DIV7 and DIV14: A – Sham, B – BDNF, C – ANA-12, D – ANA-12 + BDNF
Daily BDNF application promotes an increase in the functional calcium activity of the primary hippocampal cells cultures during their development in vitro. The observed effect manifested in significant increase in the number of cells exhibiting calcium activity (14 DIV: Sham – 62.77±3.84%; BDNF - 79.39±2.52%), and the frequency of network calcium events (10 DIV: Sham – 0.92±0.11 osc/min; BDNF – 1.31±0.15 osc/min). Blockade of TrkB receptors leads to an irreversible inhibition of all parameters of the spontaneous calcium activity of neural networks beginning with DIV10.
Conclusion
Chronic influence of exogenous BDNF and TrkB-receptor blocker leads to modulation of the spontaneous bioelectrical and calcium activity of neural networks in primary hippocampal cultures during their cultivation in vitro.
Acknowledgements
The reported study was funded by RFBR according to the research projects No.18-015-00391, 17-04-01128, 16-04-00245 and Grant of the President of the Russian Federation MD-2634.2017.4. This publication has been also prepared as a part of the state projects “Provision of scientific research” No.6.6379.2017/8.9, 17.3335.2017/4.6, and 6.6659.2017/6.7.
References
1. Yuste R., "From the neuron doctrine to neural networks", Nat Rev Neurosc, vol. 16 (8), pp. 487–497, 2015
2. Vedunova M.V., Mishchenko T.A., Mitroshina E.V., Mukhina I.V., “TrkB-mediated neuroprotective and antihypoxic properties of brain-derived neurotrophic factor”. Oxid Med Cell Longev, vol. 2015, no 453901, https://doi.org/10.1155/2015/453901, 2015
3. Pimashkin A, Kastalskiy I, Simonov A, Koryagina E, Mukhina I, Kazantsev V., “Spiking signatures of spontaneous activity bursts in hippocampal cultures”, Front Comput Neurosci. vol. 5 (46), doi: 10.3389/fncom.2011.00046. 2011.
4. Shishkina T.V., Mishchenko T.A., Mitroshina E.V., Shirokova O.M., Pimashkin A.S., Kastalskiy I.A., Mukhina I.V., Kazantsev V.B., Vedunova M.V., “Glial cell line-derived neurotrophic factor (GDNF) counteracts hypoxic damage to hippocampal neural network function in vitro”, Brain research. vol. 1678, pp. 310–321, doi: 10.1016/j.brainres.2017.10.023, 2018.
Keywords:
BDNF,
Trk-B receptor,
neural networks,
spontaneous bioelectrical activity,
calcium imaging,
primary hippocampal culture
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Neural Networks
Citation:
Mishchenko
TA,
Usenko
AV,
Voronova
NV,
Mitroshina
EV and
Vedunova
MV
(2019). Features of exogenous influence of the Brain-derived neurotrophic factor and blockade of TrkB receptor on the neural network activity in primary hippocampal cultures.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00059
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
PhD. Tatiana A Mishchenko, N. I. Lobachevsky State University of Nizhny Novgorod, The Institute of Biology and Biomedicine, Nizhny Novgorod, Russia, SaHarnova87@mail.ru