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Integration of Micro Electrode Array with compartimentalized microfluidics for the analysis of reconstructed neuronal junctions

Benoit Charlot1*, Eve Moutaux2, Fabrice Bardin1, 3, Eugene Malyshev1, Frederic Saudou2, 4 and Maxime Cazorla2
  • 1 UMR5214 Institut d'Electronique et des Systèmes (IES), France
  • 2 INSERM U1216 Grenoble Institut des Neurosciences (GIN), France
  • 3 University of Nîmes, France
  • 4 Centre Hospitalier Universitaire de Grenoble, France

Compartmentalized circuits are the most employed microfluidic devices in neuroscience. These circuits allow the in-vitro reconstitution of neuronal circuits using primary cultures of different neuronal population. These systems can be used to build thousands of specific neuronal junctions and to study intracellular dynamics using selective markers and live fluorescent reporters. In this work we added a dedicated Micro Electrode Array that fits the microfluidics design to record and control electrical activity of pre- and postsynaptic neurons while simultaneously monitoring intracellular dynamics. Because microfluidics are fluidically isolated, each compartment can be independently manipulated to determine their contribution to network functions. Microfluidic circuit are based on the design of Taylor et al. 2010 (3) for the reconstruction of cortico-cortical networks in which each compartment (presynaptic, synaptic and postsynaptic) is identified (space compartmentalization) and in which the progression from axonal growth to synapse regulation is controlled (time compartmentalization). The device shown in Figure 1(a,b,c) consists of two opposite neuronal chambers that communicate via an intermediate synaptic chamber through long microchannels on one side (to select axons only) and short ones on the other side (to allow dendrites to reach the synaptic chamber). This configuration allows the reconstruction of physiological axodendritic contacts between two cortical populations such as those found between cortical layers in vivo. In order to extend the functional analysis of reconstructed cortico-cortical networks, we developed a specific Micro Electrode Array (MEA) substrate that fits to the microfluidic geometry to stimulate and record pre- and post-synaptic neurons (Figure1(c)). Presynaptic electrodes were disposed at the entrance of axonal channels in order to stimulate the axon initiation segment that will generate physiological action potentials and trigger release of neurotransmitter in the synaptic chamber. Postsynaptic electrodes are located under the cell bodies to concomitantly stimulate or record electrical activity of target neurons. Combined with high-resolution fluorescence videomicroscopy and fast calcium imaging (Figure1(e)), this system allows monitoring intracellular dynamics in response to different patterns of neuronal activity. The MEA have been produced specifically to be included in microfluidic circuit. They are built in clean room with simple, efficient and robust microfabrication process that emphasis on repetability. The microfabrication process for building the MEA chips (Figure1(a)) are made of a two-step lithographic process on top of 4.9 x 4.9cm, 170µm thin glass substrates. The first step consists in the deposition of micro electrodes by a lift-off of 10/200nm Ti/Pt metal over an AZ 2020 patterned photoresist. Next, a 500nm thick Silicon Nitride (Si3N4) passivation layer is deposited by Plasma Enhanced Chemical Vapor Deposition (PECVD) process at 280°C. A second photolithographic step is done to define openings in the Si3N4 passivation layer on the microelectrodes and connexion pads. These openings are made by Reactive Ion etching with a mix of CHF3 and 02 plasma. This process has been stabilized in order to produces hundreds of devices with a fabrication yield of more than 80%. The microfluidic circuit is made of PDMS using a standard dual thickness SU8 on silicon process that is aligned on top of the MEA and bonded by oxygen plasma activation of the surface. Microelectrodes show electrical impedances of 235 and 165kΩ (for 50 and 30µm diameter) in PBS 1X medium. The MEA-microfluidic platform is connected to a 60-channels MEA workstation (MultiChannel Systems) and is then mounted on an inverted confocal spinning-disc videomicroscope (CSU-W1, Yokogawa). Rat primary neurons are cultured into their respective chambers and are infected with lentiviruses to express markers of intracellular dynamics such as the fast calcium indicator GCaMP6f. Spontaneous electrical activity and calcium dynamics in pre- and post-synaptic neurons can be followed throughout network maturation (Day 4 to 21). Specific stimulation protocols mimicking different patterns of neuronal activity can then be applied to study calcium dynamics in presynaptic axons and in postsynaptic dendrites. Synaptic transmission between the two populations can also be assessed by applying selective drugs in the synaptic compartment (Figure 2(d,e)). By combining space-time compartmentalization of neuronal populations, electrophysiological stimulation and recording, and high-resolution videomicroscopy, our integrated platform allows to decipher the cellular events that are involved in synaptic transmission and plasticity within neuronal networks. This device can be applied to virtually any type of neuronal circuits, in normal and pathological conditions.

Figure 1
Image

Acknowledgements

This work is funded by grants from Agence Nationale pour la Recherche (ANR-12-PDOC-0004-01-BATMAN, M. C.), and Labex NUMEV (ANR-10-LABX-20, B.C.)

References

[1] J.M. Jang, J. Lee, H. Kim, N.L. Jeon, W. Jung, "One-photon and two-photon stimulation of neu-rons in a microfluidic culture system", Lab Chip 16, 1684-1690 (2016).
[2] M. Peyrin et al., "Axon diodes for the reconstruction of oriented neuronal networks in microflu-idic chambers", Lab Chip 11, 3663-3673 (2011).
[3] A.M. Taylor, D.C. Dieterich, H.T. Ito, S.A. Kim, E.M. Schuman,"Microfluidic local perfusion chambers for the visualization and manipulation of synapses", Neuron 66, 57-68 (2010).

Keywords: Microfluidics, Micro electrode arrays (MEAs), calcium imaging, Electrophysiology, microfabrication

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Oral Presentation

Topic: Microelectrode Array Technology

Citation: Charlot B, Moutaux E, Bardin F, Malyshev E, Saudou F and Cazorla M (2019). Integration of Micro Electrode Array with compartimentalized microfluidics for the analysis of reconstructed neuronal junctions. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00039

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Received: 18 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Dr. Benoit Charlot, UMR5214 Institut d'Electronique et des Systèmes (IES), Montpellier, France, benoit.charlot@umontpellier.fr