Feb 16, 2026

Public workspaceEx Vivo Whole-Cell Patch Clamp Recording and Pharmacological Isolation of Light-Evoked GABAA Currents

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx82odv8j/v1
  • Nicola Berretta1,
  • Ezzia Guatteo1,2,
  • Nicola Biagio Mercuri1,3
  • 1Experimental Neurology Laboratory, IRCCS Santa Lucia Foundation, Rome, Italy;
  • 2Department of Medical, Human Movement and Well-Being Sciences, University of Naples ‘Parthenope’, Naples, Italy;
  • 3Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
Cristian González-Cabrera
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx82odv8j/v1
Protocol Citation: Nicola Berretta, Ezzia Guatteo, Nicola Biagio Mercuri 2026. Ex Vivo Whole-Cell Patch Clamp Recording and Pharmacological Isolation of Light-Evoked GABAA Currents. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx82odv8j/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: February 16, 2026
Last Modified: February 17, 2026
Protocol Integer ID: 243380
Keywords: cell patch clamp recordings from vta neuron, evoked gabaa current, gabaa receptor mediation, evoked synaptic current, cell patch clamp recording, vta neuron, pharmacological isolation of light, gabaa currents this protocol, synaptic current, picrotoxin sensitivity
Abstract
This protocol describes whole-cell patch clamp recordings from VTA neurons in acute horizontal slices. Light-evoked synaptic currents are recorded at -60 mV and pharmacologically isolated. GABAA receptor mediation is confirmed by picrotoxin sensitivity.
Materials
**Recording aCSF (mM): NaCl 126, NaHCO3 24, glucose 10, KCl 2.5, CaCl2 2.4, NaH2PO4 1.2, MgCl2 1.2. Equilibrate with 95% O2 - 5% CO2, pH 7.3. Maintain at 32 C.

**Internal Solution (mM): 125 KCl, 10 HEPES, 10 EGTA, 5.2 CaCl2, 4 ATP-Mg2, 0.3 GTP-Na3, 10 phosphocreatine-Na2. Adjust pH to 7.3 with KOH.
Troubleshooting
Step-by-Step Procedure
Transfer one recovered slice to the recording chamber and perfuse with recording aCSF at 32 C and 2.5-3.0 ml/min.
Identify VTA dopamine neurons based on anatomical location.
Verify slow or absent spontaneous firing (< 5 Hz) in cell-attached mode.
After whole-cell access, confirm presence of Ih using hyperpolarizing voltage steps.
Use 4-6 MOhm pipettes filled with internal solution and hold the cell at -60 mV.
Add CNQX 10 µM and D-AP5 50 µM to block glutamatergic transmission.
Add TTX 0.5 µM and 4-AP 100 µM to isolate monosynaptic release.
Deliver TTL-triggered light pulses (480 or 560 nm) using CoolLED system.
Record light-evoked inward synaptic currents.
Apply picrotoxin 100 µM to confirm GABAA receptor mediation.
Filter signals at 3-4 kHz and digitize at 10 kHz.