Feb 01, 2024

Public workspaceElectrophysiology with iPSC-derived neurons

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7pebkgwz/v1
  • Minee-Liane Choi1
  • 1UCL Institute of Neurology
Minee L Choi
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7pebkgwz/v1
Protocol Citation: Minee-Liane Choi 2024. Electrophysiology with iPSC-derived neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pebkgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working. This protocol was originally provided by Olga Kopach.
Created: February 01, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94531
Keywords: ASAPCRN, electrophysiology with ipsc, clamp recordings of ipsc, derived neuron, ipsc, clamp recording, neurons this protocol, electrophysiology, neuron
Abstract
This protocol describes the method to perform patch-clamp recordings of iPSC-derived neurons.
Troubleshooting
Patch-clamp recordings of iPSC-derived neurons are performed using an infrared differential interference contrast (DIC) imaging system on an Olympus BX51WI upright microscope (Olympus, Japan) coupled with a Multipatch 700B amplifier under the control of pClamp 10.2 software package (Molecular Devices, USA)
For the recordings, a neuronal culture or co-culture is plated on glass coverslips, placed in a recording chamber mounted on the microscope stage and constantly perfused with a physiological buffer medium.
Whole-cell recordings are performed using glass pipettes with a resistance of 3.5-6 MΩ when filled with the intracellular solution.

Note
Perfusion medium (in mM):
126 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 D-glucose
The medium is continuously bubbled with. Concentration95 % volume O2 and Concentration5 % volume CO2 (Ph7.4 ) and maintained at Temperature30 °C - Temperature33 °C .


Whole-cell recordings are performed using glass pipettes with a resistance of 3.5-6 MΩ when filled with the intracellular solution.
Note
Intracellular solution (in mM):
126 K-gluconate, 4 KCl, 4 MgCl2, 2 BAPTA, 4 Mg-ATP, 0.4 Na-ATP (Ph7.2 , osmolarity ~295 mOsmol)


In the whole-cell (immediately after membrane breakthrough), iPSC-derived neurons are recorded for the resting membrane potential (Vrest), membrane capacitance (Cm), the membrane time constant (τm), and input resistance (Rin)
To induce neuronal firing, a series of sub- and supra-threshold rectangular current pulses are applied with a stepwise-increased stimulus intensity at the Vhold set at −60 mV to −70 mV.

Note
The second protocol tested is a slow-ramp current injection, ramped up with a 100–200 pA/s slope.

The analysis of the AP waveform was performed for the first AP only to quantify the threshold value, the spike amplitude, overshoot, the spike width (duration at half-maximal amplitude), the rates of depolarisation and re-polarisation phase.