Jul 10, 2024
Mouse brain slice electrophysiology
- 1Duke University
Document Citation: Shiyi Wang 2024. Mouse brain slice electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp44nl5b/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: July 10, 2024
Document Integer ID: 82335
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Abstract
Mouse brain slice electrophysiology
1. For whole-cell patch-clamp recordings, 3-4 mice of each genotype and condition were used for miniature excitatory postsynaptic current (mEPSC) and miniature inhibitory postsynaptic current (mIPSC) measurements.
2. WT and LRRK2 G2019Ski/ki mice of both sexes were anesthetized with 200 mg/kg tribromoethanol (avertin) and decapitated.
3. After decapitation, the brains were immersed in ice-cold artificial cerebrospinal fluid (aCSF, in mM): 125 NaCl, 2.5 KCl, 3 mM MgCl2, 0.1 mM CaCl2, 10 glucose, 25 NaHCO3, 1.25 NaHPO4, 0.4 L-ascorbic acid, and 2 Na-pyruvate, pH 7.3-7.4 (310 mOsmol).
4. 350 μm thick coronal slices containing the ACC were obtained using a vibrating tissue slicer (Leica VT1200; Leica Biosystems).
5. Slices were immediately transferred to standard aCSF (33°C, continuously bubbled with 95% O2– 5% CO2) containing the same as the low-calcium aCSF but with 1 mM MgCl2and 1-2 mM CaCl2.
6. After 30-minute incubation at 33°C, slices were transferred to a holding chamber with the same extracellular buffer at room temperature (RT: ∼25°C).
7. Brain slices were visualized by an upright microscope (BX61WI, Olympus) through a 40× water-immersion objective equipped with infrared-differential interference contrast optics in combination with a digital camera (ODA-IR2000WCTRL).
8. Patch-clamp recordings were performed by using an EPC 10 patch-clamp amplifier, controlled by Patchmaster Software (HEKA). Data were acquired at a sampling rate of 50 kHz and low-pass filtered at 6 kHz.
9. To measure mEPSCs, the internal solution contained the following (in mM): 125 K-gluconate, 10 NaCl, 10 HEPES, 0.2 EGTA, 4.5 MgATP, 0.3 NaGTP, and 10 Na-phosphocreatine, pH adjusted to 7.2 – 7.4 with KOH and osmolality set to ∼ 300 mOsmol.
10. mEPSCs were measured in the aCSF bath solution containing 1 µM tetrodotoxin and 50 µM Picrotoxin at -70 mV in voltage-clamp mode.
11. To measure mIPSCs, the internal solution contained the following (in mM): 77 K-gluconate, 77 KCl, 10 HEPES, 0.2 EGTA, 4.5 MgATP, 0.3 NaGTP, and 10 Na-phosphocreatine, pH adjusted to 7.2 – 7.4 with KOH and osmolality set to ∼ 300 mOsmol.
12. mIPSCs were measured in the aCSF bath solution containing 1 µM tetrodotoxin and 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 50 µM D-2-amino-5-phosphonopentanoate (D-AP5) at -70 mV in voltage-clamp mode.
13. mEPSCs and mIPSCs recorded at -70 mV were detected using Minhee Analysis software (https://github.com/parkgilbong/Minhee_Analysis_Pack).
14. To analyze the frequency, events were counted over 5 minutes of recording.
15. To obtain the average events for each cell, at least 100 non-overlapping events were detected and averaged. The peak amplitude of the average mEPSCs was measured relative to the baseline current.