Feb 16, 2026
Acute Midbrain Slice Preparation
- Nicola Berretta1,
- Ezia Guatteo1,2,
- Nicola Biagio Mercuri3,4
- 1Experimental Neurology Laboratory, IRCCS Santa Lucia Foundation, Rome, Italy;
- 2Department of Medical, Human Movement and Well-Being Sciences, University of Naples ‘Parthenope’, Naples, Italy;
- 3Fondazione Santa Lucia IRCSS;
- 4Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
Protocol Citation: Nicola Berretta, Ezia Guatteo, Nicola Biagio Mercuri 2026. Acute Midbrain Slice Preparation . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g776dkgwz/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 16, 2026
Protocol Integer ID: 243379
Keywords: acute midbrain slice preparation, μm horizontal midbrain slice, using chilled nmdg, chilled nmdg, protective cutting solution, graded sodium reintroduction, based protective cutting solution, sodium reintroduction, preparation, electrophysiological recording, storage in hepe
Abstract
This protocol describes preparation of 250 μm horizontal midbrain slices containing the VTA using chilled NMDG-based protective cutting solution, followed by graded sodium reintroduction and storage in HEPES-based aCSF prior to electrophysiological recording.
Materials
**NMDG-Based Cutting Solution (mM)**
92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 10 MgSO4. Equilibrate with 95% O2 - 5% CO2, pH 7.3. Keep chilled.
**HEPES-Based Storage Solution (mM)**
92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 2 CaCl2, 2 MgSO4. Equilibrate with 95% O2 - 5% CO2, pH 7.3. Store at room temperature.
Troubleshooting
Step-by-Step Procedure
Anesthetize the mouse with halothane and decapitate rapidly.
Remove the brain and immediately place it into chilled carbogenated NMDG solution.
Mount the brain for horizontal slicing.
Cut 250 μm horizontal slices containing the midbrain using a vibratome.
Transfer slices to NMDG solution at 33.0 C and incubate for 15 min.
Gradually reintroduce sodium by adding increasing volumes of 2 M NaCl solution every 5 min for 40 min total.
Transfer slices to HEPES-based aCSF at room temperature.
Allow at least 1 h recovery before recording.