Induction of localized synaptic plasticity in cultured neural network grown in microfluidic device
-
1
Saint-Petersburg Academic University, Nanotechnology Research and Education Centre of the RAS, Russia
-
2
Lobachevsky State University of Nizhny Novgorod, Department of Neuroengineering, Center of Translational Technologies, Russia
-
3
Nizhny Novgorod State Medical Academy, Cell Technology Department, Russia
Abstract
Synaptic plasticity in neural networks remains one of the key questions in the research of fundamental mechanisms of the brain including information coding, learning and memory. In this study we investigate synaptic plasticity using microfluidic device with special shaped channels in order to guide axons and construct neural circuits with two subnetworks coupled by directed synaptic pathways. We applied high-frequency electrical stimulation to isolated neural connections which induced potentiation or depression of synaptic pathways.
Motivation
Cultures of dissociated neuronal cells are widely used model to study cellular and molecular mechanisms of synaptic plasticity. The model allows long term monitoring of bioelectrical functional activity and morphological changes. Many studies were done with stimulus induced plasticity in the networks of dissociated neuronal cells but provide highly variable results. Connections between the neurons in such networks are formed randomly which obstructs application of precise plasticity induction protocols. Recently developedt microfluidic methods provide guided neuritis growth and construct cultures with predefined functional connections [1,2]. Neural network consist of two sub-cultures coupled by microchannels in which axons grow dominantly in one direction. Artificially designed morphology of the network provide easy access to the presynaptic and postsynaptic neurons in order to induce localazed synaptic plasticity.
Materials and methods
PDMS microfluidic chips containing an array of microchannels between two chambers A and B were fabricated by two layer lithography and PDMS molding techniques. Channel design determined axonal growth dominantly from A chamber to B (see [2] for more details).
Hippocampal neural cells from mice embryos at 18-th prenatal day were plated on 60-electrode arrays (Multichannel systems, Germany) with diameter of 30 µm and 200 µm interelectrode distance. In order to investigate spike propagation inside the microchannels, each PDMS chip was positioned and mounted onto the surface of a planar microelectrode array (MEA, Multichannel systems, Germany) with 60 electrodes of 30 µm in diameter, so as to locate several electrodes in the microchannels. Before cell plating the device was covered with polyethyleneimine. Dissociated hippocampal neurons were plated into separate subcompartments (chambers). Electrical activity was recorded after by USB-MEA system (Multichannel systems, Germany).
Low frequency stimulation (60 pulses per each of 7 selected electrodes (3 in A, 3 in B, 1 in channel) with 3 s interval) was implied to induce network response. High frequency stimulation (±800mV, 260µs per phase, 20 stimulus with 100ms interval repeated 150 times with 6s interval (Wagenaar, 2003) of two subpopulations with 10ms delay was implied to induce network plasticity. Scheme of the protocol is shown in figure 2.
Results
We growed cultures with directed archticeture of the neural network up to 30 days in vitro. To analyze spike propagation between chambers with neurons we used low-frequency electrical stimulation applied to single electrodes in various locations of the chambers. Number of stimuli that evoked a network burst response was calculated separately for two chambers. We estimated a percent of the stimuli that evoked responses in both chambers which was defined as Propagation probability (PPAB). This parameter indicated a strength between pre- and postsynaptic pathways of neuronal subpopulations. Next we induced synaptic plasticity of the connections in the microchannels by high-frequency electrical stimulation. After the stimulation the responses to low-frequency stimuli were potentiated in B chamber of the cultured network by 6.61% PPAB increase in contrast to 0.37% increase in control stimulation. Opposite effect (depression) was observed in other three cultures.
Discussion and Conclusion
In this study we investigated synaptic plasticity in hippocampal cultures with unidirectional architecture of the network grown in microfuidic chip. Such device composed of two chambers connected by specific microchannels which guided axon growth between chambers in one-way direction provided certain control over synaptic pathways. The results show that burst propagation probability can be modified (potentiated or depressed) by application of high-frequency stimulation applied to the synaptic pathways in the microchannels.
References
[1] Le Feber J., Postma W., de Weerd E., Weusthof M., RuttenW. L. C. Barbed channels enhance unidirectional connectivity between neuronal networks cultured on multi electrode arrays // Front. Neurosci. 2015. Ò. 9. ¹ NOV. Ñ. 1–10.
[2] Malishev E., Pimashkin A., Gladkov A., Pigareva Y., Bukatin A., Kazantsev V., Mukhina I., Dubina M. (2015). Microfluidic device for unidirectional axon growth. Journal of Physics: Conference Series, 643, 012025
[3] Wagenaar D. a, Pine J., Potter S.M. Searching for plasticity in dissociated cortical cultures on multi-electrode arrays. // J. Negat. Results Biomed. 2006. 5. 16.
Figure Legend
Fig.1. Microfluidic device for unidirectional axon growth A. 60-electrode array with PDMS microfluidic chip. B. Schematic view of microchannel for unidirectional axon growth. C. Microscope photo of microchannels with neuritis.
Fig.2. Effects of the high frequency stimulation A. Scheme of experimental protocol. B. Firing rate (Hz) represented spikes during the evoked bursts in presynaptic chamber (blue) and postsynaptic chamber (green) in two control and post-tetanic stimulation. Values of PPAB indicated a percent of evoked responses propagated from presynaptic to postsynaptic sub-network.
Acknowledgements
This research was supported by the Russian Science Foundation (¹ 14-19-01381).
Keywords:
synaptic plasticity,
Microfluidic Device,
neural cultures,
MEA,
Microchannel
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:
Malishev
E,
Gladkov
A,
Bukatin
A,
Pigareva
Y,
Kolpakov
V,
Kazantsev
V,
Mukhina
I and
Pimashkin
A
(2016). Induction of localized synaptic plasticity in cultured neural network grown in microfluidic device.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00055
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Eugene Malishev, Saint-Petersburg Academic University, Nanotechnology Research and Education Centre of the RAS, Saint-Petersburg, Russia, lazarevich@neuro.nnov.ru