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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Whole-cell Patch-clamp Recordings in Brain Slices

Published: June 15th, 2016

DOI:

10.3791/54024

1Department of Psychiatry, University of Texas Southwestern Medical Center

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

The patch-clamp technique, an electrophysiological technique that has been developed in the late 1970s1,2, is a primary tool for studying single or multiple ion channel functions in live tissue. Among the different patch configurations that can be achieved, whole-cell patch-clamp recordings allow the study of the electrical behavior of a substantial part of the neuron. Classically, this technique is performed in vitro either on brain slices, freshly dissociated neurons, or on cell culture models3. When performed on neurons in brain slices, this technique presents several advantages. In particular: (i) neurons are recorded in relatively p....

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All experiments were carried out in accordance with protocols approved by the UT Southwestern Institutional Animal Care and Use Committee, and were chosen so as to minimize stress, discomfort, and pain experienced by the experimental animals.

1. Solutions

Note: Prepare micropipette internal solutions in advance. For most basic experimental purposes, two kinds of solutions should suffice: Cs+-based and K+-based solutions.

  1. Use Cs+-based solutions (e.g., Cs.......

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Temperature, a factor that is easily controlled by the experimenter, influences the biophysical properties of ion channels and receptors, and thereby the waveform of post-synaptic currents (PSCs) (EPSC and IPSCs) and the capability of neurons to elicit spikes. Figure 3 and Figure 4 show the effect of temperature on neuronal firing and the slope of evoked EPSCs (eEPSCs) respectively. The firing pattern (Figure 3) (i.e., latency to.......

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This protocol describes the basic procedure for performing whole-cell patch-clamp experiments on neurons in brain slices. However, the complexity, potential and sensitivity of this technique cannot be fully described in this article. Here, we have tried to delineate the most basic steps and underscore important parameters that must be controlled for achieving successful and rigorous whole-cell recordings. For further theoretical learning, many books and articles have been published on both whole-cell patch-clamp recordin.......

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This research was supported by UT Southwestern startup funds (SK).

....

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Name Company Catalog Number Comments
Isolated pulse stimulus generator A.M.P.I Master-8
Isolation unit (ISO-Flex) A.M.P.I ISO-Flex
Computer controlled Amplifier  Molecular Devices Multiclamp 700B
Digital Acquisition system Molecular Devices Digidata 1500
Microscope Olympus BX-51
Micromanipulator Sutter Instruments MPC-200
Chamber and in-line Heater Warner Instruments TC-344B
Vibratome Slicer Leica  VT1000 S
Micropipette Puller Narishige PC-10
Imaging Camera Q Imaging QIClick-F-M-12
Narishige pipette puller PC-10 Narishige PC-10
Glass capillaries WPI TW150F-3
Slice hold-down (harp) Warner Instruments 64-0255
Slice Chamber Warner Instruments RC-26
Nonmetallic syringe needle World Precision Instruments MF28G67-5
Syringe filters Nalgene 176-0045
Glue Gun Home Depot various
Gas dispersion tube Ace Glass Inc. various
Decapitation scissors Home Depot 100649198
Scalpel Handle #3 World Precision Instruments 500236
Small straight sharp tips scissors World Precision Instruments 14218
Vessel canulation forceps  World Precision Instruments 500453
Curved hemostatic forceps World Precision Instruments 501288
Economy Tweezers #3 World Precision Instruments 501976-6
Spatula Fisher Scientific 14357Q
Scooping spatula Fisher Scientific 14-357Q
Petri dish Fisher Scientific 08-747B
Filter paper Lab Depot CFP1-110
Name of Material/ Equipment Company Catalog Number Comments
Solutions
Cs-Gluconate internal solution (pH 7.2–7.3, 280–290 mOsm)
D-gluconic acid 50%  Sigma Aldrich/various G1951
Cesium-OH (CsOH) 50%  Sigma Aldrich/various 232041
NaCl, 2.8 mM Sigma Aldrich/various S7653
HEPES, 20 mM Sigma Aldrich/various H3375
EGTA, 0.4 mM Sigma Aldrich/various E4378
tetraethylammonium-Cl, 5 mM Sigma Aldrich/various T2265
Na2GTP, 0.3 mM Sigma Aldrich/various G8877
MgATP, 2 mM Sigma Aldrich/various A9187
Name of Material/ Equipment Company Catalog Number Comments
K-Gluconate internal solution (pH 7.2–7.3, 280–290 mOsm)
K D-gluconate, 120 mM Sigma Aldrich/various G4500
KCl, 20 mM Sigma Aldrich/various P3911
HEPES, 10 mM Sigma Aldrich/various H3375
EGTA, 0.2 mM Sigma Aldrich/various E4378
MgCl2 Sigma Aldrich/various M8266
Na2GTP, 0.3 mM Sigma Aldrich/various G8877
MgATP, 2 mM Sigma Aldrich/various A9187
Name of Material/ Equipment Company Catalog Number Comments
Standard artificial cerebrospinal fluid (ACSF, osmolarity ≈ 300-310 mOsm)
KCl, 2.5 mM Sigma Aldrich/various P3911
NaCl, 119 mM Sigma Aldrich/various S7653
NaH2PO4-H20, 1 mM Sigma Aldrich/various S9638
NaHCO3, 26.2 mM Sigma Aldrich/various S8875
Glucose, 11 mM Sigma Aldrich/various G8270
MgSO4-7H2O, 1.3 mM Sigma Aldrich/various 230391
CaCl2-2H20, 2.5 mM Sigma Aldrich/various C3881
Name of Material/ Equipment Company Catalog Number Comments
Additional compounds used for solutions preparation
KOH various
Kynurenic acid Sigma Aldrich/various K3375

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