Mar 29, 2024
Ex vivo electrophysiology
- Katerina Rademacher1,
- Ken Nakamura1
- 1Gladstone Institute of Neurological Disease
Protocol Citation: Katerina Rademacher, Ken Nakamura 2024. Ex vivo electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedqn7v47/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: March 07, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96306
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020529
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol describes steps for ex vivo electrophysiology in mouse brain slices. This protocol also includes instructions for clozapine N-oxide (CNO) testing in DREADD-expressing neurons.
Materials
- Isoflurane
- Rodent guillotine
- Vibratome (Campden Instruments, 7000smz-2)
- aCSF solution containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose saturated with 95% O2—5% CO2
- 3 – 5 MOhm pipettes containing (in mM): 123 K-gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, and 0.3 Na3GTP, pH 7.2, osmolarity adjusted to 275. Biocytin (0.1%, Sigma) is included in the internal solution to identify neurons after recordings where desired.
- Axio Examiner A1 equipped with Dodt and IR optics
- Zeiss Axiocam 506 mono
- Neurolucida 2023 software
- Sutter IPA and SutterPatch v2.3.1 software (Sutter Instruments)
- Clozapine N-oxide (for DREADD-expressing neurons; Tocris)
- 4% formaldehyde in PBS
Tissue Preparation
Tissue Preparation
Deeply anesthetize the mouse with isofluorane, decapitate, and remove the brain.
Using a vibratome, cut 150mm horizontal slices containing the region of interest in ice-cold aCSF solution and allow to recover at 33˚ C in aCSF for at least one hour.
Recording
Recording
For fluorescent imaging, visualize slices under an Axio Examiner A1 equipped with Dodt and IR optics using a Zeiss Axiocam 506 mono and Neruolucida 2023 software.
Whole-cell patch-clamp recordings are made at 33˚ C using 3 – 5 MOhm pipettes. Recordings are made using Sutter IPA and SutterPatch v2.3.1 software (Sutter Instruments), filtered at 5 kHz and collected at 10 kHz.
For Ih: voltage clamp cells at -60mV and step to -40, -50, -70, -80, -90, -100, -110, and -120 mV. Ih magnitude is quantified as the difference between the initial steady-state response to the -120mV step and the asymptote of the slow current sag.
For spontaneous firing rates: record in current-clamp mode (I= 0 pA). Spontaneous firing rate is measured as the mean firing rate during the first 2 min of whole-cell recording.
Action potential (AP) waveform measurements are made from averages across at least 8 APs from the first 2 min of recording.
For input resistance: Apply a brief hyperpolarizing pulse once every 10 sec and average across the measurements made during the first 2 min of recording.
For CNO testing in DREADD-expressing neurons: spontaneous firing rate or resting membrane potential are monitored until a stable baseline is observed for at least 5 min. Then switch the perfusion solution to 1mM CNO for 5 min.
When recordings are complete, drop fix slices in 4% formaldehyde in PBS for at least 2 hr.
Complete statistical analyses in R, first testing whether data meet the criteria for parametric statistical evaluation.