Patch Clamp Technology
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Introduction
Ion channels in neurons play an important role in neuronal signal transduction, neuronal cell excitation, electrolyte transport, and muscle contraction. Patch clamp technology is considered the gold standard in ion channel research and has been used in various laboratories to characterize the ion channel properties of various cells. Since ion channels are the ultimate functional markers of mature cells, such as neurons and muscle cells, patch clamp technology has recently been used to verify the maturity of differentiated cells from stem cells. Recent advancements in sensor proteins, actuator proteins, and optical tools have enabled “all-optical” approaches to electrophysiology, which utilize fluorescent voltage sensors, calcium sensors, or calcium sensors.
Patch Clamp Configurations
Initially, "patch clamp" referred to voltage clamp recording current through a single ion channel from an isolated small piece of membrane. The first step is to get the electrodes close to the cell. Apply mild positive pressure inside the electrode. When the tip of the electrode comes into contact with the outer membrane, the second step begins by gently applying negative pressure to the inside of the electrode. This draws the film tightly to the edge of the electrode tip, which induces the formation of a gigaseal. This configuration is called “cell-attached”.
Fig.1 Operational modalities of patch-clamp techniques for high-resolution electrophysiological recording.1
Patch Clamp Recordings In Vivo
The first successful in vivo whole-cell patch clamp recordings were demonstrated in the visual cortex of live, anesthetized cats. Several years later, it was demonstrated that whole-cell patch clamp recordings can be obtained from awake, head-fixed, and even freely-moving rodents, establishing the patch clamp technique as an invaluable tool for correlating single neuron activity to higher order brain functions.
- Blind Patch Clamp Recordings
The first in vivo whole-cell patch clamp recordings were performed in a “blind” fashion. The advantages of blind paste techniques include (in theory) unrestricted recording depth (because the target depth is not limited by optical constraints) and a relatively large working area above the artifact.
- Image-guided Patch Clamp Recordings
In vivo two-photon laser scanning microscopy, which enables imaging of fluorescence signals relatively deep in the intact brain, has been integrated with patch clamping. Image-guided patches have proven to be very valuable for cell-type-specific features of neurons in the intact brain.
Patch Clamp Technology
- Conventional Patch Clamp Technique
Traditional patch clamp experiments, including manual electrophysiology methods, use glass microelectrodes to compress the cell surface and form a tight interaction with the giga-Ohm (GΩ) sealing resistance between the cell membrane and the edge of the glass microelectrode. The manual patch clamp has long being considered the gold standard for investigations of ion channel properties and compound activities because it usually generates high-quality data, but the experimental procedures are complicated and time-consuming.
- High Throughput Patch Clamp Technique
Currently available automated electrophysiological techniques can be divided into three categories: (A) glass pipetting-based automated patch clamps; (B) microstructured planar electrode patch clamp; (C) automated TEVC on Xenopus oocytes. The automation of patch clamp system greatly simplifies the procedure of electrophysiological experiments and significantly improves the flux of compound screening. This automation has broad applications in the ion channel drug discovery process.
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Reference
- Faria, R.X.; et al. The Mystery of P2X7 Ionotropic Receptor: From a Small Conductance Channel to a Large Conductance Channel. Neurosci.-Deal. Front. 2012. Distributed under Open Access License CC BY 3.0 without modification.
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