Studying extracellular action potential waveforms using HD MEAs

• ¹ ETH Zurich, Department of Biosystems Science and Engineering, Switzerland
• ² ETH Zurich, Department of Biosystems Science and Engineering, Switzerland

Motivation For a thorough understanding of neural signal integration, action potential generation and transmission, the sub-cellular distribution of ion channels is of critical importance. Imaging and immunolabeling approaches provide valuable information on the patterns of ion channel expression in different cellular compartments (1), and electrophysiology tools, such as whole cell patch clamp, inside- and outside-out patches are routinely used to study biophysical properties and functional distribution of ion channels. However, patch clamp techniques are invasive and can compromise the physiological function of ion channels (2). Moreover, it is not always possible to perform conventional electrophysiological recordings from thin neuronal processes, which requires recording from the axonal blebs and causes inevitable damage to neurons. Non-invasive extracellular recording techniques were not considered a useful tool to study ion channel expression for decades due to poor spatial resolution and the inability to record from single cells. Recent advances in microelectrode array (MEA) technology helped to overcome this issue. Novel high-density arrays (HD MEAs) not only allowed for recording from single cells, but revealed a variety of extracellular action potential (EAP) waveforms in different subcellular compartments (3). The biophysical origin of these waveforms is, however, still unknown, and the way that particular membrane ion currents define the shape of the EAPs remains to be determined. Materials and Methods Here, we simultaneously measure the intracellular and extracellular potentials of single neurons in cultures of cortical neurons. To this end, we use a whole-cell patch clamp setup on top of a HD MEA featuring 11,011 electrodes at 17.8 µm pitch within an area of 1.8 × 2.0 mm2 (4). The high electrode density allows for sampling the complete 2-dimensional extracellular voltage signal of the neuron. Results The profiles of the extracellular action potentials and of the negative first derivative of the intracellular action potentials (-dV/dt IAP) were qualitatively similar, but also featured noticeable differences. The negative spikes of the EAPs were narrower than that of IAP derivatives. The negative peak of the first derivative of the IAP, resembling the fastest rising phase of the AP, coincided with the EAPs as recorded from the electrodes proximal to the cell soma. However, the largest extracellular spike in the majority of the cells was typically localized ~20-30 µm away from the soma and preceded the peak of the IAP derivative by 100-200 µs. Somatic and AIS extracellular spikes always contained two major components: a large narrow negative spike, presumably caused by Na+ currents, followed by a wider positive peak. Application of 5 mM 4-AP (blocker of potassium A-type channels) reduced the amplitude of the positive peak by approximately 25% and increased its full width at half maximum by 30%, suggesting that the positive peak of EAP is only partially caused by potassium currents during the repolarization phase of the action potential. Stimulation of the cell in voltage clamp mode evoked voltage-gated sodium and potassium transient currents, which could be recorded extracellularly with the MEA. The shape of the extracellular spikes, evoked by voltage pulse stimulation, was similar to those produced by the IAPs. Blockage of the voltage-gated sodium channels allowed for isolating potassium currents in the voltage clamp recordings. Discussion We found evidence that the EAP does not fully match the first derivative of the IAP, as was predicted from published simulation data (5). In most of the cases, the FWHM of the negative peak of the IAP was noticeably wider than in extracellular recordings. It is clear from the data that depolarization of the cellular membrane does not happen simultaneously in all the compartments. Intracellular recordings do not allow for discriminating between different cell compartments and yield signals integrated over all central cell compartments. The fact that the depolarization of the different cell compartments happens at different times leads to a widening of the resulting intracellular waveform. The slowly-rising positive peak of the extracellular potential is thought to be driven by repolarizing potassium currents. According to our observation it is only partially affected by potassium blockers and is completely inhibited by sodium channel blockers suggesting a strong sodium current component in the positive peak of the EAP. Conclusions High-density MEAs are suitable to detect the differences in EAPs evoked in different cellular compartments. Analysis of the EAP waveforms can give valuable information on ion-channel expression in subcellular neuronal compartments. Filtering properties of the MEA circuitry need to be taken into account in the analysis of EAP waveforms obtained from HD MEAs. References 1. Nusser, Z. Variability in the subcellular distribution of ion channels increases neuronal diversity. Trends Neurosci. 32, 267–274 (2009). 2. Suh, B.-C. & Hille, B. PIP2 is a necessary cofactor for ion channel function: how and why? Annu. Rev. Biophys. 37, 175–195 (2008). 3. Müller, J. et al. High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab. Chip 15, 2767–2780 (2015). 4. Frey, U. et al. Cell recordings with a CMOS high-density microelectrode array. Conf. Proc. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. IEEE Eng. Med. Biol. Soc. Annu. Conf. 2007, 167–170 (2007). 5. Gold, C., Henze, D. A., Koch, C. & Buzsáki, G. On the origin of the extracellular action potential waveform: A modeling study. J. Neurophysiol. 95, 3113–3128 (2006). Figure Legend Figure 1. HD MEAs allow for detecting differences in the EAP waveforms produced by different subcellular compartments (A). The negative peak of –dV/dt of the IAP (red trace, fig. B) coincides with EAPs recorded at the cell soma (green traces fig. B, green dots fig. A). Application of 4-AP reduces the amplitude of the positive peak of the EAP (C). Voltage clamp stimulation allows for recording an EAP footprint and reveals the changes of the EAP waveform after blockage of A-type potassium channels (D).