Neuroengineering isolated and localized unidirectional synaptic connectivity between multiple neuronal networks
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N. I. Lobachevsky State University of Nizhny Novgorod, Center of Translational Technologies, Russia
Information processing, development of a memory and complex cognitive functions remain a key fundamental problems in Neuroscience. A reductionist approach to uncover basic mechanisms is using a cultured neuronal networks which consisted of few thousands neurons. The cultures after few week of development form randomly connected networks with electrophysiological spiking activity. However, the disordered spontaneously formed architecture of the cultured network prevents functional maturation which in vivo conditions developed by neurogenesis. The solution can be found using neural engineering methods. Cell patterning using adhesion molecules allowed to grow the cultures with various heterogeneous structure. Recent progress in microfludics allows more precise network structuring – a separation of cell cultures into clusters and the formation of synaptic connections between them with a known direction of signal propagation. It's possible to grow complex networks with architecture similar to the neural circuits in a real ordered brain structures (cortex, hippocampus, etc.).
In this study we present a new methods for engineering the network morphology using microfluidics, which allows to: 1) define a location of pre- and postsynaptic neurons; 2) formation of precise location of the synapses from one axon to the dendrites of several isolated postsynaptic neurons; 3) 4) simultaneous non-invasive recording and stimulation of electrical activity on several axons and dendrites.
Methods
Hippocampal neural cells from mice embryos at the 18-th prenatal day were cultured within microfluidic chips combined with 60-electrode arrays (Multichannel systems, Germany). The initial density was approximately 8,000-9,000 cells / mm2. Microfluidic chips were made from a biocompatible polydimethylsiloxane (PDMS). The chips consisted of two chambers for the cells and microchannels between the chambers. The cultured subnetworks were defined as Presynaptic and the Postsynaptic networks, according to the predefined direction of axon growth. The chambers were connected by the microchannels with grid like structure (Fig. 1). The microchannels had a size of 7 x 5 um in cross section which prevented to flow in a cell soma and allowed to grow only the neurities inside.
Results
We designed a chip which shaped the network architecture by synaptically coupling the axons of the neurons from one culture with individual dendrites of the second culture. After plating the dissociated neurons in two chambers the neurities grew through microchannels from the both sites simultaneously and met each other on several cross sides of the microchannels (Fig. 1). Note, that from the Presynaptic site the length of microchannel from the cell some to the first cross section was 400 um which was longer than a maximum dendrite length and allowed only to long axons to grow until that point. On the other side, the length between the cell soma in Postsynaptic site to and first cross section was 200 um which allowed to grow the axons as well as the dendrites. Thus, the cross sections potentially were the most probable places for formation of the synapses between the cells. After DIV 6 the neurities filled the microchannels and the cross sections in designed manner. After DIV 14 the cultures started to generate spontaneous spiking activity and we tested the synaptic structure of the network by application of short electrical stimulus to the axons of Presynaptic site and the dendrites of Posynaptic site. We founs that axon depolaristion induced spike along side the microchannel follwed by synaptically induced spikes (>5 ms delay) in meighbouring microchannels with axons and dendrites from Postsynaptic site. Then we excitatory synaptic activity in Presynaptic chamber by application AMPA/NMDA antagonists with APV and CPP. Spontaneous activity within that chamber significantly reduced as well as evked synaptic activity during the stimulation. However, the synaptically evoked spikes were still visible on the electrodes in the cross sections where the axons grew from the Postsynaptic site and no spikes were found in the axons of Presynaptic site. It suggests that the stimulus evoked the spike on presynaptic axons, then synaptically evoked EPSP on the dendrite of the posynaptic neuron in the microchannel, the neuron generated the spike on the axon which propagated back to the microchannel.
Discussion
Presented approach allowed to form a multiple synaptic connection sites between two populations of the neurons with precise location of the synapses on a scale of tens of micrometers with general axo-dendritic tree. Growing cultured networks with precise location of several synapses with isolated soma of pre- and postsynaptic neurons opens up a new perspective method to study long-term effects of synaptic and heterosynaptic plasticity, effects of local synaptic changes on the rest of the network and test vaious hypothesises of information coding and memory formation.
Such approach will allow to change individual connections between several cellular layers, which determines the function of processing the input pattern of activity evoked by the electrical stimulus to the output pattern of activity of the network response. It allows to reliably study the complex principles of information processing in the brain, to construct a new theory and to test them with neuroengineering methods. It will allow to develop the methods of "programming" the architecture of inter-neural connections which will model functional development similar to in vivo conditions.
Following a global trend in the development of the Lab-on-chip approach, which may replace many diagnostic and clinical methods in medicine, the proposed research concept will allow to form yet unique direction of "brain-on-a-chip" technology.
The results also can be applied to the development of new technologies in tissue engineering - artificial three-dimensional bioengineering structures (scaffolds) for neurotransplantation. Such approach allows to implant neuronal cells for functional regeneration. However, this approach lacks of the technology of appropriate functional integration of the cells into the heterogeneous architecture of the network at the injury site. Our methods of network engineering can be used for scaffold structure design that determines the correct architecture of the implantable cellular network.
Figure 1. Experimentla design. A. Scheme of microfluidic chip. B. Example of axon and dendrite cross each other. C. Example of Axon growing through the microchannel. D. Mean firing rate on MEA before (left) and after(right) application of CPP/APV.
Acknowledgements
Acknowledgment
This research was supported by the grant of the president of Russian Federation MК-6795.2018.4
References
1.Le Feber, J., Postma, W., de Weerd, E., Weusthof, M. & Rutten, W. L. C. Barbed channels enhance unidirectional connectivity between neuronal networks cultured on multi electrode arrays. Front. Neurosci. 9, 412, https://doi.org/10.3389/fnins.2015.00412 (2015).
2.Aebersold M. J. et al. “Brains on a chip”: Towards engineered neural networks //TrAC Trends in Analytical Chemistry. – 2016. – Т. 78. – С. 60-69.
3. Li Y. et al. Application of hierarchical dissociated neural network in closed-loop hybrid system integrating biological and mechanical intelligence //PloS one. – 2015. – Т. 10. – №. 5. – С. e0127452.
4.Poli D. et al. Sparse and specific coding during information transmission between co-cultured dentate gyrus and CA3 hippocampal networks //Frontiers in Neural Circuits. – 2017. – Т. 11.
5.Anne M Taylor, Mathew Blurton-Jones, Seog Woo Rhee, David H Cribbs, Carl W Cotman & Noo Li Jeon. A microfluidic culture platform for CNS axonal injury, regeneration and transport // Nature Methods. (2005) V.2, P. 599–605. doi:10.1038/nmeth777 https://www.nature.com/articles/nmeth777
6.Gladkov, A., Pigareva, Y., Kutyina, D., Kolpakov, V., Bukatin, A., Mukhina, I., … Pimashkin, A. (2017). Design of Cultured Neuron Networks in vitro with Predefined Connectivity Using Asymmetric Microfluidic Channels. Scientific Reports, 7(1), 15625. http://doi.org/10.1038/s41598-017-15506-2
Keywords:
Neural Engineering,
microfluidic microchannel,
Axon navigation,
Synaptic connectivity,
microelectrode arrays,
Microfluidic chip,
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:
Pimashkin
A,
Gladkov
AA,
Kolpakov
V,
Pigareva
Y,
Antipova
O,
Mukhina
I and
Kazantsev
VB
(2019). Neuroengineering isolated and localized unidirectional synaptic connectivity between multiple neuronal networks.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00087
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Alexey Pimashkin, N. I. Lobachevsky State University of Nizhny Novgorod, Center of Translational Technologies, Nizhny Novgorod, Nizhny Novgorod, 603950, Russia, pimashkin@neuro.nnov.ru