Oct 08, 2025

Public workspaceACUTE HIPPOCAMPAL SLICE EPHYS PROTOCOL

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6pr9g5b/v1
  • Carmen Perez De Nanclares1
  • 1University of Minnesota
  • Team Lee
Jane Balster
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6pr9g5b/v1
Protocol Citation: Carmen Perez De Nanclares 2025. ACUTE HIPPOCAMPAL SLICE EPHYS PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6pr9g5b/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 20, 2025
Last Modified: October 10, 2025
Protocol Integer ID: 224259
Keywords: Hippocampal, Tetrodotoxin, Morphology, acute hippocampal slice ephys protocol, acute hippocampal slice ephys protocol this protocol, electrophysiological recording of mouse ca1 hippocampal neuron, mouse ca1 hippocampal neuron, cell spontaneous miniature excitatory, electrophysiological recording, spontaneous miniature excitatory, term potentiation
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol details the Electrophysiological recording of mouse CA1 hippocampal neurons from ex vivo thick slice preparations to analyze whole-cell spontaneous miniature excitatory postsynaptic current and long-term potentiation.
Materials
  • Vibratome (Leica VT 1200S)
Equipment
Leica VT1200 S Fully automated vibrating blade microtome
NAME
Vibratome
TYPE
Leica
BRAND
VT1200S
SKU
https://www.leicabiosystems.com/us/research/vibratomes/leica-vt1200-s/
LINK
Sucrose 189, glucose 10, NaHCO3 26, KCl 3, MgSO4 5, CaCl2 0.1, NaH2PO4 1.25

AB
Sucrose solution (in mM)
Sucrose189
Glucose10
NaHCO326
KCl3
MgSO45
CaCl20.1
NaH2PO41.25
NaCl 124, KCl 2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, ascorbic acid 0.4, and glucose 10

AB
Artificial cerebrospinal fluid (ACSF) (in mM)
NaCl124
KCl2.69
KH2PO41.25
MgSO42
NaHCO326
CaCl22
Ascorbic acid0.4
Glucose10
  • Olympus BX50WI microscope
Cesium gluconate 117, HEPES 20, EGTA 0.4, NaCl 2.8, TEA-Cl 5, ATP 2, GTP 0.3 (pH 7.3)

AB
Internal solution (in mM)
Cesium gluconate117
HEPES20
EGTA0.4
NaCl2.8
TEA-Cl5
ATP2
GTP0.3 (pH 7.3)
  • PC-ONE amplifier (Dagan Instruments; Minneapolis, MN, USA)
  • Digidata 1440A digitizer (Molecular Devices; San Jose, CA, USA)
  • pCLAMP 10.4 (Axon Instruments, Molecular Devices; San Jose, CA, USA)
  • Temperature control system (Warner Instruments electric TC-324C)
Troubleshooting
Obtain acute dorsolateral hippocampal coronal slices (Thikness350 µm thick) from 2-3 months old mice using a vibratome (Leica VT 1200S).
Briefly, remove the brain quickly after decapitation and placed in ice-cold high sucrose solution of the following composition (in mM): sucrose 189, glucose 10, NaHCO3 26, KCl 3, MgSO4 5, CaCl2 0.1, NaH2PO4 1.25.
Afterward, incubate slices for at least Duration01:00:00 at TemperatureRoom temperature (Temperature21 °C –Temperature24 °C ) in artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 124, KCl 2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, ascorbic acid 0.4, and glucose 10, and continuously bubbled with carbogen (95% O2 and 5% CO2) (Ph7.4 ).
1h
Incubation
Transfer slices to an immersion recording chamber and superfused at Amount2 µL with gassed ACSF at Temperature30 °C -Temperature32 °C and visualized under an Olympus BX50WI microscope (Olympus Optical; Japan).
Imaging
Identify CA1 hippocampal neurons visually based on morphology and location in the pyramidal layer.
To study excitatory postsynaptic currents (EPSCs) and add picrotoxin (Concentration50 micromolar (µM) ) and CGP54626 (Concentration1 micromolar (µM) ) are to the solution to block the GABAA and GABAB receptors, respectively.
Obtain Whole-cell electrophysiological recordings from CA1 pyramidal neurons using patch electrodes (3–10 MΩ) filled with an internal solution containing in mM: cesium gluconate 117, HEPES 20, EGTA 0.4, NaCl 2.8, TEA-Cl 5, ATP 2, GTP 0.3 (Ph7.3 ).
Obtain recordings with a PC-ONE amplifier (Dagan Instruments; Minneapolis, MN, USA).
Membrane potentials are held at −70 mV unless otherwise stated.
Filter signals at 1 kHz, acquired at a 10 kHz sampling rate, and fed to a Digidata 1440A digitizer (Molecular Devices; San Jose, CA, USA).
Monitor series and input resistances throughout the experiment using a -5 mV pulse.
Discard Cells when series and input resistances changed >20%.
pCLAMP 10.4 (Axon Instruments, Molecular Devices; San Jose, CA, USA) is used for stimulus generation, data display, data acquisition, and data storage.
To record evoked EPSCs, theta capillaries filled with ACSF are used for bipolar stimulation and place in the stratum radiatum to stimulate the Schaffer collaterals.
Obtain Input–output curves of EPSCs by increasing stimulus intensities from 0 to 100 μA.
Acquire Paired-pulse facilitation by applying paired pulses (2 ms duration) with 25, 50, 75, 100, 200, 300, and 500 ms inter-pulse intervals.
Calculate the paired-pulse ratio by dividing the amplitude of the second EPSC by the first (PPR=EPSC-2/EPSC-1).
Obtain AMPA currents at a holding potential of -70 mV.
Obtain NMDA currents at a holding potential of +40 mV.
To ascertain the AMPA to NMDA receptor current ratio, the NMDA component measure 50 msec after the stimulus, when the AMPA component had decayed.
Recordings of miniature EPSCs (mEPSCs) and slow inward currents (SICs) are made in the presence of tetrodotoxin (TTX; Concentration1 micromolar (µM) ).
Define SICs as currents with a rise time >5 ms, Τon >5 and decay time >10 ms.
For long-term potentiation (LTP), CA1 pyramidal neurons first underwent a baseline recording of Duration00:10:00 followed by theta-burst tetanic stimulation in the Schaffer collaterals (4 trains at 100 Hz for Duration00:00:01 ; Duration00:00:30 intervals).
10m 31s
After LTP induction, record neurons for Duration01:00:00 .
1h
Determine differences in LTP by comparing the average EPSC amplitudes from the last Duration00:10:00 of post-stimulus recordings with the average EPSC amplitudes from the Duration00:10:00 baseline recordings.
20m
Perform all experiments at Temperature30 °C -Temperature32 °C using a temperature control system (Warner Instruments electric TC-324C).