Oct 05, 2022
Electrophysiology
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. Electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l274mjg1y/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
Created: October 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70867
Keywords: ASAPCRN
Abstract
Electrophysiology of iPSC-derived mDA neurons
Electrophysiology
Electrophysiology
Visualized patch-clamp recordings from cell cultures were performed using an infrared differential interference contrast imaging system and a Multipatch 700B amplifier controlled by pClamp 10.2 software package (Molecular Devices, USA).
For the recordings, a neuronal culture on a glass coverslip was placed in a recording chamber mounted on the stage of an Olympus BX51WI upright microscope (Olympus, Japan).
The perfusion solution contained the following (in mM): 119 NaCl, 2.5 KCl, 1.3 Na2SO4, 2.5 CaCl2, 26.2 NaHCO3, 1 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 glucose (or 22 in some recordings) and was continuously bubbled with 95% O2 and 5% CO2, at a pH of 7.4.
Whole-cell recordings were performed at 32-34 °C ; the patch-clamp pipette resistance was 3-7 MΩ depending on particular experimental conditions.
Series resistance was monitored throughout experiments using a +5 mV step command, cells with very high series resistance (above 25 MΩ) or unstable holding current were rejected.
The intracellular pipette solution for voltage-clamp experiments contained (in mM): 120.5 CsCl, 10 KOH-HEPES, 2 EGTA, 8 NaCl, 5 QX-314 Br- salt, 2 Na-ATP, 0.3 Na-GTP.
For current-clamp experiments, the intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 5 HEPES, 15 glucose, 1 K2SO4×7 H2O, 2 BAPTA, 3 Na-ATP. The pH was adjusted to 7.2 and osmolarity adjusted to 295 mOsm.
To isolate response of NMDA receptors we added to a perfusion solution: 50 mM picrotoxin, 20 mM NBQX, 1 mM strychnine, 1 mM CGP-55845, 100 mM MCPG, with zero Mg2+.
To isolate response of GABAAreceptors, we added 50 mM APV, 20 mM NBQX, 1 mM strychnine, 1 mM CGP-55845, 100 mM MCPG. All chemicals were purchased from Tocris Bioscience.
In the whole-cell (immediately after membrane breakthrough), iPSC-derived neurons were recorded for the resting membrane potential (Vrest), membrane capacitance (Cm), the membrane time constant (τm), and input resistance (Rin), measured from the hyperpolarizing squire current pulse steps in current mode
To assess the firing capability of the cells, a series of sub- and supra-threshold rectangular current pulses were applied to elicit neuronal firing, with a stepwise-increased stimulus intensity (an increment of 5–10 pA).
The Vrest was set at −60 mV to −70 mV, by injecting a hyperpolarizing bias current where required.
The analysis of the AP waveform was performed for the first AP only.
The parameters of individual APs recorded were: the spike amplitude (measured from the threshold to the peak), the threshold value, overshoot and the spike width (duration at half-maximal amplitude), the rates of depolarisation and repolarisation phases.