Over the last 20 years, the membrane clamp technique has become one of the main research tools in modern electrophysiology. Initially used only to measure single-channel currents, it has now become a powerful tool to study cellular excitability: the functional and pharmacological effects of ion channels and the mechanisms of ion channel regulation by different metabolic factors. The different recording modes of the membrane clamp technique allow us to study not only macroscopic whole-cell currents, but also single-channel currents on microscopic membrane slices. An outstanding advantage of this technique is that experiments can be performed with control of the voltage and solution composition on both sides of the membrane and these conditions can be changed during the experiment.
Operation method
Experiments in diaphragm clamp technology
Materials and Instruments
HEPES-NaOH buffer EGTA BAPTA Move 1.Drawing electrode The invention of the program-controlled puller greatly simplifies the process of making glass electrodes. One only needs to choose the appropriate drawing program, put the glass capillary, and then two identical glass electrodes will be drawn. However, it is quite difficult to program different pulling schemes because different parameters affect the shape of the tip and the opening diameter of the glass electrode. The procedure for preparing a drawing program can usually be found in the manual of the device. It is important to realize that the shape of the glass electrode is the main factor determining the resistance of the electrode. Modern microprocessor-controlled equipment is capable of drawing glass electrodes in several steps. In the first step, the capillary glass tube is thinned down to a 7 to 100_-section, which is then drawn into an electrode in the next few steps, and the diameter of the tip opening is determined. Standard diaphragm electrodes are often drawn in 2?6 steps. In general, increasing the number of drawing steps increases the angle of the tip cone, resulting in a smaller electrode resistance for the same tip opening diameter. Another advantage of the programmable puller is the repeatability of the drawn diaphragm electrode. Note: The impedance of glass electrodes for whole-cell recording is usually 1-5 MΩ while that of single-channel electrodes for research is 5-30 MΩ. 2. Apply Sylgard To reduce the capacitance of the recording electrode, the surface near the opening of its tip can be covered with an insulator. This not only reduces background noise but also reduces the capacitive current that masks the recorded signal. This coating is more important for single-channel recordings such as cell-adherent, inside-out, and outside-in; it is not necessary for whole-cell experiments, where currents are higher and the cell membrane capacitance is much greater than that of the sheet electrode. In general, the coating operation is important for obtaining giga-seals. Sylgard is a two-component mixture that is widely used to make glass electrode coatings. The resin and catalyst are mixed together and pre-cured at room temperature for 2-3 h. Pre-cured Sylgard in syringes can be stored in a refrigerator (_18°F) for several months. Sylgard is applied to the surface of the tip of the glass electrode by rotating the electrode under a microscope (X10-20) with a fine needle (Figure 6-3A). Although the coating should be applied as close as possible to the tip of the electrode, care should be taken to avoid covering the opening of the tip, and since only clean glass can form a seal with the cell membrane, Sylgard should not be applied closer than 10-50 fxm to the tip; the length of the coating in most cases is 5-7 mm. The applied Sylgard needs to be cured as quickly as possible, which can be accomplished by placing the tip of the glass electrode in the presence of a stream of hot air [Figure 6-3A (X10-20)]. This can be accomplished by placing the glass electrode tip in a hot air stream [Figure 6-3A(b)] or in a heating coil. NOTE: If a giga-seal is not obtained using a coated glass electrode, try an uncoated glass electrode. If the coating is the cause of the failed seal, try increasing the coating thickness of the Sylgard by extending the pre-cure time and/or increasing the length of the uncoated area at the tip of the glass electrode. In this case, glass electrodes do not form stable seals because (i) they have sharp and uneven tip opening edges and (ii) despite the care taken during coating, uncured Sylgard diffuses into the glass electrode tip openings and forms a thin film that covers them. Fire polishing smoothed the opening edges and removed the Sylgard film. 3. Polishing the tip The glass electrode is polished with the Polishing Instrument (Figure 6-3B) under visual control. The tip of the glass electrode is placed 10 to 20 um from the platinum wire that is heated to produce a dark red color. The polishing process lasts only a few seconds, and a thickening of the glass wall and smoothing of the edges of the tip of the glass electrode can be observed. Note: Polishing determines the final diameter of the glass electrode tip opening. Strong polishing can greatly reduce the diameter of the tip opening. Polishing is often done after the glass electrode has been coated and before recording begins. Polished electrodes should be protected from dust. 4.Filling electrode Electrodes containing glass capillaries can be filled from the end with a syringe with a needle of suitable diameter, whereas electrodes without built-in glass capillaries require a two-step filling process, the first step being to fill the glass electrode tip. The first step is to fill the glass electrode tip. The tip is immersed in a small beaker containing the electrode liquid, after which negative pressure is applied at the end of the electrode through an IOml syringe connected to a silicone tube to draw the electrode liquid into the electrode head. Depending on the diameter of the tip opening, tip filling takes from a few seconds to I~2 min, and suction must be released before the glass electrode tip leaves the liquid surface. The second step is the standard tail filling process using a syringe. Any air bubbles left in the tip of the glass electrode after filling can be removed by gently flicking the glass electrode rod. The glass electrode should not be filled with more than 5?10 mm of liquid; too much liquid will increase the capacitance and electrical noise of the glass electrode. Note: The liquid used to fill the glass electrode is determined by the type of membrane clamp experiment. Cell-adherent and inside-out recordings are filled with external fluid, while whole-cell and outside-out recordings are filled with internal fluid. A filter (0.2 Mm) is inserted between the needle and the syringe when filling the glass electrode from the tail and adding the electrode liquid to the beaker. If the glass electrode tip is filled in a beaker, a slight positive pressure is applied to the glass electrode tip as it passes through the gas-liquid and liquid-gas interfaces, as this helps to avoid contamination of the glass electrode tip by liquid surface dust. EGTA-containing liquids can chemically react with the metal needle of the syringe used to fill the glass electrode, so in this case the metal needle needs to be replaced with a thinned plastic tube. Another possible method is to push an appropriate amount of liquid out of the syringe before filling the electrode. 5. Install the filled glass electrode on the electrode holder and fix it with a nut. Note that the rubber ring used to hold the glass electrode must be adjusted to fit tightly. If this is not the case, there will be air leakage inside the system, which will affect the formation of the seal. The air pressure applied to the inside of the system is given by means of a silicone tube connected to a special outlet on the holder, either by mouth or by means of a U-shaped water manometer. To prevent contamination of the glass electrode tip, a positive pressure is applied to the system so that liquid flows out of the glass electrode tip as it passes through the gas-liquid interface and as it moves toward the cell. After the glass electrode enters the bath liquid, but before it contacts the cell membrane, two operations are accomplished: measuring the glass electrode resistance and compensating for the offset potential. The amplifier was set in voltage clamp mode with the clamp voltage (Vh) set at 〇 mV. A 50-ms rectangular depolarizing command pulse (l~2 mV,IHz) was applied to Vc (Fig. 6-4A) to observe the leakage current change (Fig. 6-4B) 〇Because the liquid in the glass electrode exhibits linear resistance, the current response is also rectangular (the transient current from the electrode capacitance bowing I can be ignored), with a magnitude of Δ I. 6. The electrode resistance (RP) can be calculated by dividing (Vc-Vh) by ΔI. The experiment in Figure 64B shows an RP value of approximately 5 MΩ. 7. The offset potential between the recording electrode and the bath electrode can be adjusted to 0 by adjusting the amplifier's offset compensation circuitry; this step is important because uncompensated offset potentials will be superimposed on Vh. 8. gigabit seal formation. When the glass electrode contacts the cell membrane (usually a decrease in AI amplitude is seen), the positive pressure is released and the negative pressure is gently applied (suction). As the tip of the glass electrode forms a seal with the membrane, the contact resistance rises, resulting in a significant decrease in ΔI (Fig. 6-4B, giga-seal). Although gigabit seals sometimes form spontaneously, in most cases suction is necessary for seal formation. The resistance of gigabit seals is typically in the range of 1-100 GΩ. Gigabit seal formation may take Is to l~2 min, and is often accompanied by a reduction in background noise. Note: The membrane electrode can only be used once. If a giga-high resistance seal to the cell membrane is not formed, another glass electrode should be used and steps 1 through 8 must be repeated. If the seal is not successful, try: (1) checking the glass electrode holder for air leaks; (2) using an uncoated electrode; (3) preparing a new solution; (4) replacing the specimen with a new one; and (5) changing the glass to one covered with fire-polished platinum filaments. Steps 1 to 8 are the same for all membrane clamp recording modes. The next 5 steps (Steps 9 through 13) describe the acquisition of the different recording modes for the patch clamp technique. 9. At the end of step 8, the cell attachment pattern is essentially formed (Figure 6-4B, Cell Attachment Pattern). In addition, the corresponding circuitry of the membrane clamp amplifier needs to be adjusted to compensate for the fast capacitance current and to adjust the gain to the value required for single-channel recording. Note: For cell-attached recording, setting Vh at OmV is a reasonable choice. Assuming a resting potential of Vr=-80mV, the voltage drop across the membrane is Vr-VrH=-80mVt5 In order to depolarize the membrane, e.g., to -30mV, a command pulse of Vc=-50mV needs to be applied. at the end of the experiment, the membrane is broken by suction, and VR can be accurately measured in the current clamp mode. if, for any reason, Vr cannot be measured at the end of the experiment, consider reporting the membrane potential with an unknown Vr. If, for any reason, Vr cannot be measured at the end of the experiment, it may be considered to report the membrane potential with an unknown Vr. 10. After establishing the gigabit seal (step 8), first set Vh and Vc to the appropriate values. Vh is usually placed at -80mV, corresponding to the expected resting membrane potential value. The command pulse Vc = -30 mV. The fast component of the transient current generated by the electrode capacitance is eliminated. Giving a short pumping pulse breaks the membrane sheet and results in a whole-cell mode, in which a large slow transient current occurs and both leakage current and electronic noise increase (Fig. 6-4B, whole-cell mode). These changes are due to the increase in the clamped diaphragm area when transitioning from cell-attached to whole-cell mode. Another sign of successful formation of the whole-cell mode on neurons is the recording of voltage-activated Na+ currents. At this point, the slow transient current should be compensated for by the appropriate circuitry of the diaphragm clamp amplifier. When compensating, the cell should be stimulated with a small hyperpolarizing pulse, as this does not activate the fast conductance. Note: Two problems are encountered when recording whole cells: 11. Current clamp recording. The whole-cell mode can also be used for current-clamp recording, where the clamp is on the membrane current and the recording is on the change in membrane potential. Current clamp mode can be used to measure resting membrane potentials as well as postsynaptic and action potentials: to switch between current clamp and voltage clamp, simply select the appropriate button on the membrane clamp amplifier. 12. The outside-out recording is made from the whole-cell pattern (step 100, before compensation for the slow component of the capacitive current). Vh is set at -80 mV, and the command pulse is adjusted to Vc = -30 mV. The glass electrode is slowly pulled away from the cell until the connection is lost. The formation of the outward-facing outward pattern was accompanied by a progressive decrease in Na+ current, disappearance of the slow component of the capacitive current, and a significant decrease in background noise. After the formation of the outside-out style, the ionic current could only be seen when the amplification gain was increased to the level of single-channel recording. The fast component of the transient current needs to be compensated for.'' Note: If attempts to pull into the outside-out style diaphragm are unsuccessful, try to go more slowly when pulling the other diaphragm. It is recommended to pull the glass electrode tip several millimeters away from the cell to ensure that it is no longer attached to the cell membrane. 13. After establishing the gigabit seal (step 8), first set Vh and Vc to the appropriate values. In the inside-out style, the electrode potential is applied to the outer surface of the cell membrane, while the inner surface of the membrane has the same potential as the bath electrode. Thus, if Vh is set to +80mV, the voltage across the membrane is the physiological potential. The command pulse Vc can be set to +30 mV. Afterwards, the glass electrode is carefully pulled until it is no longer connected to the cell. There are two ways to obtain an inside-out diaphragm: (1) Sometimes, pulling the glass electrode can directly result in the formation of an inside-out diaphragm (Fig. 6-1B). (2) However, in most cases small vesicles are formed (Fig. 6-1B). In this case, briefly exposing the glass electrode tip to air can also destroy the outer membrane of the vesicle. Compensate for the fast transient current due to charging of the glass electrode capacitance after forming the inside-out style, and the amplification gain should be adjusted to be suitable for single-channel recording. Problems can arise when performing inside-out experiments in baths containing experimental specimens such as cultured cells or brain slices. In this case, the bath is often perfused with a high K+ internal fluid, which can depolarize the cell membrane potential to OmV. prolonged exposure to the internal fluid can cause many cells to die or become difficult to seal. Therefore, the dish or brain slice must be discarded after each internal-to-external recording, even if the membrane slice does not contain the channel of interest or does not survive to complete the entire experimental procedure. This is very frustrating in experiments because: the number of brain slices or petri dishes is limited; several inner-facing outward-facing diaphragms need to be obtained from the same -society; and preparing the cells for sealing requires additional time consuming tasks such as cleaning connective tissues on the brain slices. The use of a multichannel perfusion miscellaneous allows for endotransmission and exotransmission recordings without the use of a labeled sleeve (Yellen 1982). Perfusion channels made of capillary glass tubing are connected via silicone tubing to syringes containing different solutions (Fig. 6-6A). The solutions flow slowly downward due to gravity, and the flow rate of the solutions is adjusted so that the liquid does not 雛ナ祕所娜 at the outlet of the channel. Na have fine Xiao external type break ___ pole tip frontier age inserted when the liquid at the exit of the channel. Can be turned: move g] glass electrode tip turn J another - channel towel while replacing the diaphragm cell miscellaneous (fiber. Both the inside-out and the outside-in approaches can be used for this type of multifaceted perfusion, but the system has one drawback: 1) placement of the multifaceted perfusion system takes up more space in the recording bath. For example, in brain slice experiments, the water-immersion objective already takes up most of the space in the bath, and it is not desirable to increase the volume of the bath because it will reduce the intensity of the external fluid perfusion to the brain slice. ② Internal fluid flowing out of the channel can contaminate the bath fluid. Inside-out recording can also be performed in an additional small bath (Fig. 6-6B, Safronov and Vogel 1995). This bath (containing the internal solution) is 0.2 to 0.5 mm from the main bath and is electrically connected to the main bath by a standard agar bridge. The two baths are filled so that the solution forms a high raised level. The principle of the inside-out diaphragm formation is very similar to that of Fig. 6-IB. First, in the main bath the glass electrode forms a seal with the cell membrane, and a slow pull on the glass electrode leads to vesicle formation. Then, the glass electrode is rapidly moved (jumped) to an additional bath, and the vesicle ruptures due to brief exposure to air; the jumping process is performed under a dissecting microscope ^ The main steps are: (1) Focus the microscope on the boundary between the two baths. To facilitate focusing, a dot can be drawn or engraved on or near the boundary with a waterproof marker; (2) Raise the microscope 300 to 400 yards; (3) Move the glass electrode slowly towards the boundary between the two baths so that the tip of the electrode is in focus near the boundary. Be careful to maintain the stability of the liquid level in both baths; (4) Accelerate the movement of the glass electrode so that the glass electrode reaches its maximum rate as it leaves the main bath. Typically, the diaphragm is exposed to air during the jumping process for a shorter period of time than is required for the conventional glass electrode to be pulled out and then inserted back into the bath. This method allows us to obtain up to 20 inside-out diaphragms from the same cell. Below are the results obtained in our lab using this system: (2) If the outer membrane of the vesicle is not broken during the jump, the jump can be repeated several times. (3) Both coated and uncoated glass electrodes can be jumped. (4) During single-channel recording, an additional bath can be filled with different liquids. For this purpose, two cannulas are added to the bath for suction and addition of liquids. (5) Sometimes, if the electrode voltage is lowered from +80mV to +60~+40mV, the success rate will be improved, which may be due to the lower membrane stress effect of the low potential pair. After the jump, the glass electrode potential is then reset to +80mV. The addition of a bath for inside-out recording has the following advantages: An additional bath requires less bath liquid (200/J), which is beneficial when using valuable reagents. One bath is separated so that the wood in the main bath can be contaminated with internal liquids. Note: The material used to make the baths is critical to the shape and stability of the surface bumps. Choosing a hydrophobic material avoids mixing of the liquids in the two baths when jumping. teflon is a good material for making baths that can form a stable raised liquid surface. Unfortunately, it is a rather soft material and it requires a high degree of skill to create narrow borders between baths. An excellent alternative to Teflon is ddrin (paraformaldehyde, DuPont, France). Compared to Teflon, ddrin has a higher mechanical hardness and is therefore easier to fabricate by hand, but it is slightly less hydrophobic and therefore the stability of the raised liquid surface in baths made of it is slightly lower. However, this can be remedied by applying a thin layer of petroleum jelly to the surface of the ddrin bath. To do this, the petroleum jelly is first applied to the surface of the bath and then removed with dry cotton paper until it is no longer visible and the remaining thin layer of petroleum jelly is still sufficient to maintain the stability of the raised liquid level. The bath can be used for several months with one application of petroleum jelly, and the bath should be washed with distilled water after each experiment. For more product details, please visit Aladdin Scientific website.
Inverted Microscope Diaphragm Clamp Amplifier AD-DA Converter Faraday Cage
II. Diaphragm clamp experiment
IV. Whole-cell
VI. Inside-out type
(1) The success rate of vesicle-containing jumps is high (~90%). However, the success rate decreases if an inner face outward pattern is already formed in the main bath.
X. Results 
