Axon Conduction Changes by Partial Blockade of Na+ Channels Revealed with Microtunnel-Coupled MEA
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1
The University of Tokyo, Japan
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2
Tokyo Institute of Technology, Japan
Central nervous system is composed of neurons interconnected with their axons. In conventional view, axon is regarded as a cable for conducting digital impulse, and no modulation occur during the conduction. Recently, propagating action potential was reported to be analogue signal (Sasaki et al., 2011). After the finding, there is great interest in signal modulation during axon conduction. Recent progress in microfabrication makes it possible to guide axon elongation and record propagating action potential along axon. We developed a culture device composed of microtunnels and a microelectrode array (MEA) for recording action potentials propagating along axons, and found that recording axonal conduction is useful for evaluating neuronal properties at various levels (Shimba et al., 2015). However, since microtunnel structure covers axons in the previous device, it is impossible to treat axon with pharmacological reagent during recording. Here, we aim to improve our previous device and achieve selective pharmacological treatment to axons for elucidating the contribution of Na+ channels on axonal conduction.
For realizing selective treatment to axons, an axon chamber was made over the microtunnels. A microtunnel structure with the width of 3 micrometers was employed for guiding a small number of axons. Two electrodes are aligned on the bottom of a microtunnel with a 600 micrometer interval. The axon chamber allows to treat axons with pharmacological reagents. For fabricating the culture device, a master mold was made of SU-8 photoresist. Then, polydimethylsiloxane (PDMS) pre-polymer and hardening reagent was mixed and poured onto the mold, and hardened in an oven. The PDMS chamber was aligned on an MEA which was coated with polyethyleneimine. Finally, the culture device was filled with a culture medium. Cortical neurons were dissected from E19 Wistar rat embryos and seeded into the culture device. Extracellular potential was recorded as previously described (Jimbo et al., 2003). Spikes were detected with a threshold set at the five times the standard deviation of recorded signal, and then spike sorting was performed to separate mixed signals into individual axons. Axon conduction was detected by calculating time delay between spike trains recorded with two electrodes set at a microtunnel.
Figure 1 shows a fluorescent image of axons around an entrance of microtunnel, which was antidromically stained with calcein AM dye from the axon chamber. A few number of axons (up to 10 axons) passed a microtunnel. Axon conduction was detected from 10 days after seeding. The conduction delay was 1.8 ± 0.5 ms at 20 DIV. Since the distance between two electrodes is 600 micrometers, the conduction velocity was 0.26-0.50 m/s, which is consistent with unmyelinated axons previously reported (Soleng et al., 2003). Then, the axons were treated with various concentrations of tetrodotoxin (TTX), a sodium channel blocker, for testifying if the device is suitable to evaluate axon conduction changes during a pharmacological treatment. Figure 2 shows the conduction change after TTX treatment. Gray and red thin traces at left panels show activity recorded from left and right electrodes (Figure 2A), respectively. The waveforms were superimposed by triggering the spike times at the left electrode, and the bold lines show the averaged waveforms. The waveforms recorded from the right electrode shifted to right direction with a concentration dependent manner, indicating that partial blockade of sodium ion channel by TTX increased the conduction delay. Figure 2B shows the results of conduction delay under various concentration of TTX. While no significant change was observed between control and 1 nM of TTX (p > 0.5, Paired t test), the conduction delay significantly increased with more than 5 nM of TTX (p < 0.001, Paired t test). No conduction was detected after treatment with 100 nM of TTX. These results suggest that axon conduction velocity decreases during partial blockade of sodium ion channels.
In this study, we developed a culture device for selective treatment of axons with pharmacological reagents. Axons elongated into microtunnels and propagating action potentials were detected. Axons in microtunnels were then treated with various concentrations of TTX. As a result, conduction delay increased with a concentration dependent manner. These results show that our device is a feasible to study changes in conduction properties of axons during pharmacological treatment.
References
Jimbo Y., N. Kasai, K. Torimitsu, T. Tateno and H. P. Robinson (2003) A system for MEA-based multisite stimulation. IEEE Trans Biomed Eng, 50(2), 241-248.
Sasaki T., N. Matsuki and Y. Ikegaya (2011) Action-potential modulation during axonal conduction. Science, 331(6017), 599-601.
Shimba K., K. Sakai, T. Isomura, K. Kotani and Y. Jimbo (2015) Axonal conduction slowing induced by spontaneous bursting activity in cortical neurons cultured in a microtunnel device. Integr Biol (Camb), 7(1), 64-72.
Soleng A. F., K. Chiu and M. Raastad (2003) Unmyelinated axons in the rat hippocampus hyperpolarize and activate an H current when spike frequency exceeds 1 Hz. J Physiol, 552(Pt 2), 459-470.
Keywords:
Axon,
Sodium channel,
cortical neuron,
Pharmacology,
microfabrication
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Microphysiological systems
Citation:
Shimba
K,
Sakai
K,
Kotani
K,
Yagi
T and
Jimbo
Y
(2019). Axon Conduction Changes by Partial Blockade of Na+ Channels Revealed with Microtunnel-Coupled MEA.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00001
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Dr. Kenta Shimba, The University of Tokyo, Bunkyō, Tokyo, Japan, shimba@neuron.t.u-tokyo.ac.jp