Jul 11, 2024
Mouse brain slice electrophysiology
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Mouse brain slice electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj7y8lx1/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: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103202
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Mouse brain slice electrophysiology
**Animal Handling**
- Anesthetize 3-4 mice of each genotype (WT and LRRK2 G2019Ski/ki) and condition using 200 mg/kg tribromoethanol (Avertin).
- Ensure proper anesthesia depth and confirm absence of reflexes before proceeding.
**Brain Extraction and Slicing**
- Decapitate the mice and immediately immerse the brains in ice-cold artificial cerebrospinal fluid (aCSF) containing (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).
- Use a vibrating tissue slicer (Leica VT1200) to obtain 350 μm thick coronal brain slices containing the anterior cingulate cortex (ACC).
**Slice Incubation**
- Transfer slices immediately to standard aCSF maintained at 33°C and continuously bubbled with 95% O2 – 5% CO2.
- Incubate slices for 30 minutes at 33°C to recover.
**Transfer to Holding Chamber**
- After incubation, transfer slices to a holding chamber at room temperature (approximately 25°C) with the same extracellular buffer.
**Microscopy Setup**
- Visualize brain slices using an upright microscope (BX61WI, Olympus) equipped with a 40x water-immersion objective and infrared-differential interference contrast optics.
- Use a digital camera (ODA-IR2000WCTRL) for image acquisition.
**Patch-Clamp Setup**
- Perform patch-clamp recordings using an EPC 10 patch-clamp amplifier controlled by Patchmaster Software (HEKA).
- Set data acquisition at a sampling rate of 50 kHz and low-pass filter at 6 kHz.
**Recording Configuration**
- Use aCSF bath solution containing: For mEPSCs: 1 µM tetrodotoxin and 50 µM Picrotoxin, held at -70 mV in voltage-clamp mode; For mIPSCs: 1 µM tetrodotoxin, 10 µM CNQX, and 50 µM D-AP5, held at -70 mV in voltage-clamp mode.
**Internal Solution Preparation**
- Prepare internal solutions for patch pipettes: For mEPSCs: 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, osmolality ~300 mOsmol. For mIPSCs: 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, osmolality ~300 mOsmol.
**Data Acquisition and Analysis**
- Record mEPSCs and mIPSCs using Minhee Analysis software.
- Analyze frequency by counting events over 5 minutes of recording.
- Calculate average events per cell based on at least 100 non-overlapping events.
- Measure peak amplitude of average mEPSCs relative to baseline current.