Non-invasive long-term recording of microchannel-confined axonal activity
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1
Italian Institute of Technology (IIT), Dept. of Neuroscience and Brain Technologies, Italy
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2
Italian Institute of Technology (IIT), Dept. of Pattern Analysis & Computer Vision , Italy
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3
Italian Institute of Technology (IIT), Dept. of Nanophysics, Italy
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4
Italian Institute of Technology (IIT), Dept. of Neuroscience and Brain Technologies, Italy
1 Motivation
The exact timing of action potential propagation is critical for neuron-neuron communication, which is mediated by delicate axonal projections (1). Because of their dimensions, the biophysical features of axons are difficult to study by patch clamp recording, especially over periods of several days in vitro (DIVs) (2). On the other hand, without any auxiliary amplification mechanism, planar MEA electrodes are usually not able to detect the weak extracellular potentials from complex axonal branches that are located in their vicinity (3). We demonstrate that polydimethylsiloxane (PDMS) microchannels help in not only isolating, guiding and coupling axonal projections to MEA electrodes, but allow for the amplification and non-invasive recording of their extracellular activity over weeks.
2 Material and methods
The thin and flexible PDMS devices were fabricated by pouring PDMS (pre-polymer and catalyst; 10:1; Sylgard 184, Dow Corning) on bi-level SU-8 5/50 (MicroChem) microchannel templates. Their overall thickness was leveled to 200 μm by using a plastic film. A device included two small reservoirs for accommodating cell somata (h=100 μm, w=400 μm, l=1800 μm). They were connected by eight microchannels (h=5 μm, w=30 μm, l=1000 μm). A neurite cavity with 50 μm and 5 μm height was designed between reservoir and microchannels (Fig. 1 A and C). Bigger cell loading reservoirs (r = 2 mm) were punched at the two opposite corners of each small reservoir. MEAs (200/10iR-ITO, MCS) were coated with 0.1 mg/ml poly-D-lysine (PDL) and 0.05 mg/ml laminin (Sigma). Rat cortical neurons (E18, Charles River) were seeded into one of the open reservoirs (5 μl, 5000 to 7000 cells/μl). Activity was recorded between 13 DIV and 35 DIV.
3 Results
The growth dynamics of the isolated axonal branches inside the microchannels were monitored by light microscopy (Fig. 1 C and D). The neurite cavity prevented cell body and dendrite penetration into the proximal microchannels. The microchannel geometry amplified the extracellular activity recorded from elongating axons (40 to 1200 μV; Fig. 1 B). Axons growing within long microchannels (1000 μm) without making synaptic connections at their terminals showed rich activity for more than 5 weeks (Fig. 1 F and G). The stable cellular microenvironment in the PDMS microdevices helped to keep small-world cortical networks (<3000 neuron/device) functionally alive over the entire period. Activity in axonal segments increased over time. It was correlated with a corresponding activity increase in the somal compartment (Fig. 1 F and G). By recording from subsequent points of the same axonal projections in one microchannel, it was possible to study the biophysical properties of propagating spikes (Fig. 1 E). The mean time-latency between subsequent electrode pairs could be extracted by cross correlation analysis, which provided information about the direction of the propagating wave, its velocity and its decay over the entire microchannel length (Fig. 1 E).
4 Discussion
The flexible PDMS microchannel device that had been aligned over the MEA electrodes allowed for a simple cell loading procedure and provided a thin and transparent cell culture chamber. In addition to the clear optical access, the small device geometries stabilized the cellular microenvironment. In our previous setup, a similar PDMS microchannel device was used to keep low-density cortical and hippocampal networks functionally alive for more than 45 DIVs after axonal laser microdissection (4).The described microchannel environment is fully compatible with axonal (electro)physiology. Axonal morphology remains intact even without establishing any synaptic connection to neurons in the counterpart reservoir.
5 Conclusion
By combining PDMS microchannel devices and MEAs, multidimensional optical and electrophysiological long-term datasets can be collected from individual axons. The system can easily be adapted to different MEA configurations and experimental paradigms that may include pharmacological and molecular studies on axons.
7 References
1 Debanne D. (2004). Nat Rev Neurosci 5(4): 304-316.
2 Hu W, Shu Y. (2012). Neurosci Bull 28(4):342-50.
3 Pan L, Alagapan S, Franca E, Brewer GJ, Wheeler BC (2011). J Neural Eng 8(4):046031.
4 Habibey R, Golabchi A, Latifi S, Difato F, Blau A. (2015). Lab Chip 15(24):4578-90.
***
Fig. 1. Microchannel device for the long-term recording of axonal electrophysiology with MEAs. A) Schematic topside view of the PDMS device aligned on MEA electrodes (black circles): the cortical network resides in the reservoir (orange circles) and its axonal projections in the microchannels. B) Long- (left) and short-term (right) view of recorded activity from five electrodes in one microchannel. C) Exemplary cortical network (3 DIV) in a PDMS device aligned with MEA electrodes. D) The magnified view of the green rectangle in B shows how axons grow into the neurite cavity and into the proximal microchannel (yellow arrows). E) Cross correlogram of the activity recorded from five subsequent electrodes in one microchannel. The first electrode acted as the reference electrode and the remaining four as target electrodes (Xmin=10 ms, Xmax=10 ms, bin = 40 μs). F) Average activity recorded from somata in the reservoir collected from three MEAs at different DIVs. G) Average activity recorded from axons in the microchannels collected from three MEAs at different DIVs.
Acknowledgements
IIT intramural funds and access to infrastructure in support of this research are highly appreciated.
Keywords:
Axon,
Microelectrode Array (MEA),
Polydimethylsiloxane (PDMS),
Microchannel device,
long-term electophysiology
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:
Habibey
R,
Mousavi
H,
Pesce
M,
Nanni
M and
Blau
A
(2016). Non-invasive long-term recording of microchannel-confined axonal activity.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00047
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Axel Blau, Italian Institute of Technology (IIT), Dept. of Neuroscience and Brain Technologies, Genoa, Italy, axel.blau@outlook.de