Synaptic current patch-clamp recording in brain slices
Materials Required
Principle
Whole-cell patch-clamp recording in acute brain slices measures membrane current from visually identified neurons while preserving part of the local synaptic circuit; in voltage clamp, postsynaptic currents are generated by synaptic receptor-channel activation and are recorded as inward or outward currents at a defined holding potential[1][2][3]. Miniature synaptic currents are recorded during action-potential blockade with tetrodotoxin, whereas evoked synaptic currents are generated by pathway stimulation and isolated pharmacologically as EPSCs or IPSCs[4][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use oxygenated ACSF for slicing, recovery, perfusion, and recording; published slice protocols use ACSF-like extracellular solution continuously bubbled with 95% O2/5% CO2, and adult-slice methods report protective NMDG-based ACSF for improved adult mouse slice quality[1][2].
• Use intracellular pipette solution for whole-cell access; use tetrodotoxin for miniature event recordings, bicuculline or picrotoxin to block GABA_A receptors during EPSC recording, and CNQX/APV or equivalent glutamate receptor antagonists to block excitatory transmission during IPSC recording when these isolations are required[4][5].
• Fluorescence labels may be used only when the experimental design requires targeted recording from labeled neurons, as reported in slice patch-clamp protocols[2][6].
• Use a vibrating tissue slicer to cut acute brain slices, an upright microscope with visual guidance such as infrared-DIC optics for identifying neurons in living slices, a patch-clamp amplifier, digitizer/acquisition software, micromanipulators, borosilicate patch pipettes, perfusion chamber, temperature control when required, gas supply for oxygenation, and a stimulating electrode for evoked synaptic currents[1][3][6][7].
• Use intracellular pipette solution for whole-cell access; use tetrodotoxin for miniature event recordings, bicuculline or picrotoxin to block GABA_A receptors during EPSC recording, and CNQX/APV or equivalent glutamate receptor antagonists to block excitatory transmission during IPSC recording when these isolations are required[4][5].
• Fluorescence labels may be used only when the experimental design requires targeted recording from labeled neurons, as reported in slice patch-clamp protocols[2][6].
• Use a vibrating tissue slicer to cut acute brain slices, an upright microscope with visual guidance such as infrared-DIC optics for identifying neurons in living slices, a patch-clamp amplifier, digitizer/acquisition software, micromanipulators, borosilicate patch pipettes, perfusion chamber, temperature control when required, gas supply for oxygenation, and a stimulating electrode for evoked synaptic currents[1][3][6][7].
Experimental Procedure
• Prepare acute brain slices in oxygenated extracellular solution using a vibrating slicer; thin-slice patch-clamp methods established that individual CNS neurons can be visually identified in slices and accessed for high-resistance seal formation after localized cleaning of the soma[3].
• For adult or aging mouse tissue, use the published protective recovery approach with NMDG-based ACSF when the goal is improved targeted patch-clamp success in mature slices[2].
• Prepare the recording chamber with continuous perfusion of oxygenated ACSF and transfer one recovered slice to the chamber before recording[1][2].
• Pull patch pipettes suitable for whole-cell recording, fill them with intracellular solution, approach the visually identified neuron under positive pressure, form a high-resistance seal, and rupture the patch membrane to establish the whole-cell configuration[1][3][7].
• For spontaneous EPSCs or IPSCs, hold the cell in voltage clamp and record synaptic events under the receptor-blocking condition needed to isolate the current type[4][5].
• For miniature EPSCs or IPSCs, add tetrodotoxin to block action-potential-dependent release and record remaining quantal events[4][5].
• For evoked EPSCs or IPSCs, place a stimulating electrode in the relevant afferent pathway and deliver electrical stimuli while pharmacologically isolating the desired synaptic current[4][5].
• Monitor recording quality during acquisition, including seal stability, access resistance, and holding current; exclude recordings that show unstable access or large drift because these artifacts directly affect voltage-clamp control and current amplitude interpretation[1][7].
• Analyze synaptic-current traces by event frequency, amplitude, rise time, decay kinetics, charge transfer, and evoked peak amplitude when applicable; miniature event frequency is commonly interpreted as reflecting presynaptic release or synapse number, whereas miniature event amplitude and kinetics are commonly interpreted as postsynaptic receptor or synaptic response properties[4][5].
• Include pharmacological negative controls by applying receptor antagonists that abolish the isolated current, and compare groups using cells and animals as biological units rather than treating repeated events from one cell as independent biological replicates[4][5].
• For adult or aging mouse tissue, use the published protective recovery approach with NMDG-based ACSF when the goal is improved targeted patch-clamp success in mature slices[2].
• Prepare the recording chamber with continuous perfusion of oxygenated ACSF and transfer one recovered slice to the chamber before recording[1][2].
• Pull patch pipettes suitable for whole-cell recording, fill them with intracellular solution, approach the visually identified neuron under positive pressure, form a high-resistance seal, and rupture the patch membrane to establish the whole-cell configuration[1][3][7].
• For spontaneous EPSCs or IPSCs, hold the cell in voltage clamp and record synaptic events under the receptor-blocking condition needed to isolate the current type[4][5].
• For miniature EPSCs or IPSCs, add tetrodotoxin to block action-potential-dependent release and record remaining quantal events[4][5].
• For evoked EPSCs or IPSCs, place a stimulating electrode in the relevant afferent pathway and deliver electrical stimuli while pharmacologically isolating the desired synaptic current[4][5].
• Monitor recording quality during acquisition, including seal stability, access resistance, and holding current; exclude recordings that show unstable access or large drift because these artifacts directly affect voltage-clamp control and current amplitude interpretation[1][7].
• Analyze synaptic-current traces by event frequency, amplitude, rise time, decay kinetics, charge transfer, and evoked peak amplitude when applicable; miniature event frequency is commonly interpreted as reflecting presynaptic release or synapse number, whereas miniature event amplitude and kinetics are commonly interpreted as postsynaptic receptor or synaptic response properties[4][5].
• Include pharmacological negative controls by applying receptor antagonists that abolish the isolated current, and compare groups using cells and animals as biological units rather than treating repeated events from one cell as independent biological replicates[4][5].
Troubleshooting
Failure to obtain stable whole-cell access.
Possible Cause:The cell surface is not sufficiently clean or the slice surface is damaged
Literature-supported Solution:
Select visually healthy neurons below the slice surface, use visual guidance, and gently clean the soma before seal formation as described in thin-slice patch-clamp methods[3][6].
Poor recording success in adult brain slices.
Possible Cause:Adult tissue is more vulnerable to slicing-related swelling and superficial damage
Literature-supported Solution:
Use the adult-slice protective recovery method with NMDG-based ACSF when recording from mature adult or aging mouse tissue[2].
Miniature events are contaminated by network-driven synaptic activity.
Possible Cause:Action potentials are not blocked
Literature-supported Solution:
Add tetrodotoxin during miniature EPSC or IPSC recordings[4][5].
EPSCs or IPSCs are not pharmacologically isolated.
Possible Cause:Opposing synaptic receptor classes remain active
Literature-supported Solution:
Record EPSCs with GABA_A receptor blockade and record IPSCs with ionotropic glutamate receptor blockade, matching the antagonist condition to the current being measured[4][5].
参考文献:
- [1]. Segev A, et al. Whole-cell patch-clamp recordings in brain slices. J Vis Exp. 2016;(112):54024. [Content Brief]
- [2]. Ting JT, et al. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol Biol. 2014;1183:221-242. [Content Brief]
- [3]. Edwards FA, et al. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch. 1989;414(5):600-612. [Content Brief]
- [4]. Hollrigel GS, et al. The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats. Neuroscience. 1998;84(1):71-79. [Content Brief]
- [5]. Jonas P, et al. Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus. J Physiol. 1993;472:615-663. [Content Brief]
- [6]. Dodt HU, et al. Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res. 1990;537(1-2):333-336. [Content Brief]
- [7]. Hamill OP, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391(2):85-100. [Content Brief]
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