Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices - PubMed (original) (raw)
Review
Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices
Katherine L Perkins. J Neurosci Methods. 2006.
Abstract
Cell-attached recording provides a way to record the activity of - and to stimulate - neurons in brain slices without rupturing the cell membrane. This review uses theory and experimental data to address the proper application of this technique and the correct interpretation of the data. Voltage-clamp mode is best-suited for recording cell firing activity, and current-clamp mode is best-suited for recording resting membrane potential and synaptic potentials. The magnitude of the seal resistance determines what types of experiments can be accomplished with a cell-attached recording: a loose seal is adequate for recording action potential currents, and a tight seal is required for evoking action potentials in the attached cell and for recording resting and synaptic potentials. When recording action potential currents, if the researcher does not want to change the firing activity of the cell, then it is important that no current passes from the amplifier through the patch resistance. In order to accomplish this condition, the recording pipette should be held at the potential that gives a holding current of 0. An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.
Figures
Fig. 1. Circuit involved in cell-attached current clamp recording
(A) Arrow indicates direction of current flow. The voltage is measured at point Vx. The battery represents the resting membrane potential of the cell. The current flows through the three resistors in series. The largest voltage drop will occur across the largest resistor. If Rseal is infinitely large, then all of the voltage drop will occur across Rseal, and the voltage at Vx will be equal to −70 mV. If Rseal ≪ Rpatch, then all of the voltage drop will occur before the point labeled Vx, and Vx will be 0 mV. (B) Schematic showing the cell, the patch electrode, and the placement of the different resistances. (C) Adding an electrode and an amplifier to the circuit shown in A does not change the voltage at Vx. The impedance of the amplifier is so great that no current flows through the electrode to the amplifier, and no voltage drop occurs between the point labeled Vx and the amplifier. The voltage measured by the amplifier is equal to Vx. (D) When the electrode solution and bath solution are not the same, the liquid junction potential (Elj) between the electrode solution and the bath solution must be considered. When the electrode is in solution, before a seal is attempted, Elj is offset by applying an equal and opposite voltage at the amplifier E*lj. As long as Rseal ≪ Rpatch +cell, Elj and E*lj cancel each other out, and the amplifier measures 0 mV. As Rseal increases, the amplifier senses less of Elj but all of E*lj, so that they no longer cancel each other out. In all conditions, the voltage measured by the amplifier = E*lj + the weighted average of Elj and Em (see equation 2). (Relec is left out of D for simplicity, since it does not affect measured voltage.)
Fig. 2. The relative resistances of Rseal and Rpatch + cell determine the voltage measured by the amplifier in cell-attached current-clamp mode
(A) Graph of measured voltage (V0) vs. resistance ratio for a theoretical recording. Plot was generated using equation 4, with a resting membrane potential (Em) of −70 mV. As the ratio of Rseal to Rpatch +cell increases, the measured voltage approaches −70 mV. Note the log scale of the x-axis. (B) Graph of V0/Em vs. resistance ratio for a theoretical recording. V0/Em approaches 1 at large ratios of Rseal to Rpatch + cell. Plot was generated using equation 5. Note the log scale of the x-axis.
Fig. 3. Channels opening and closing in the membrane patch will change the voltage measured by the amplifier. The effect is much smaller with a large resistance ratio
(A) Channels opening in the membrane patch will change the voltage measured by the amplifier to a value closer to actual membrane potential. Plot of mV change in V0 with a doubling of gpatch. Peak change in V0 (in mV) with a doubling of patch conductance occurs with an original resistance ratio near 0.72. Values used to generate graphs were a constant Rcell of 100 MΩ, constant Em of −70 mV, gpatch = 500 pS (Rpatch = 2 GΩ) doubling to gpatch = 1 nS (Rpatch = 1 GΩ). Equation 4 was used to calculate V0 before and after a theoretical doubling of gpatch. (B) Resistance ratio affects stability of measured voltage. Recorded from a CA3 pyramidal cell in cell-attached current-clamp mode (1st pyramidal cell listed in Table 1). Resistance ratio increased as the recording progressed. Top trace is earlier in the recording when measured resting potential (V0) varied widely from −20 – −40 mV. Bottom trace was recorded several minutes later after Rseal had increased; note the more stable V0. (Single channel openings and closings, which would cause a varying Rpatch, were apparent in voltage-clamp mode.)
Fig. 4. Synaptic potentials can be recorded in tight-seal cell-attached current clamp mode
CA1 pyramidal cell (2nd pyramidal cell listed in Table 1) recording. (A) Synaptic potentials recorded in 4-aminopyridine (4-AP) with both glutamatergic and GABAergic transmission intact. Top row is cell-attached current-clamp. Bottom row is a simultaneous field recording. (B) Same recording after the addition of ionotropic glutamate antagonists CNQX and D-AP5. The large hyperpolarizing synaptic potential in the cell-attached current-clamp recording is a giant GABA-mediated postsynaptic potential (GPSP). The depolarizing potentials present in the 4-AP-alone condition shown in A were blocked by the glutamate antagonists, indicating that they were glutamate-mediated synaptic potentials.
Fig. 5. Action potential currents recorded using cell-attached voltage-clamp
(A) Cell-attached voltage-clamp recording from a neuron in the CA1 pyramidal cell layer (3rd pyramidal cell listed in Table 1). Recording pipette is voltage-clamped at 0 mV; baseline Iamp is +15 pA (therefore cell is depolarized – see Fig. 8). (B) One action potential current from A shown on an expanded time scale, illustrating the characteristic negative-then-positive shape. Extracellular saline included 4-AP, CNQX, and D-AP5.
Fig. 6. Circuit involved in cell-attached voltage clamp recording
In this case, Rseal is parallel to Rpatch and Rcell, and Rpatch and Rcell are in series. The current Iamp will split between the two available pathways to ground. The pathway with least resistance will have the greater current; therefore if Rseal is larger than Rpatch + Rcell, a greater proportion of Iamp will travel across Rpatch and Rcell than across Rseal.
Fig. 7. The voltage measured in cell-attached current-clamp mode is equal to the Vp associated with zero current in voltage clamp mode
Amplifier was switched from current clamp (CC) mode to voltage clamp (VC) mode by flipping the switch on the amplifier. Iamp and Vp were recorded simultaneously. Note that the amplifier measures −60 mV in CC mode and that clamping at −60 mV in voltage-clamp mode gives 0 current; whereas, clamping at 0 mV in VC mode gives +10 pA. (Current transients caused by flipping between modes and current transient at beginning of voltage steps have been clipped.) Recording is from a cell in the CA1 pyramidal cell layer (6th pyramidal cell in Table 1). No drugs were in the extracellular saline.
Fig. 8. Command potential affects firing rate of the attached cell. Cells can be stimulated to fire action potentials by commanding a depolarizing step with a tight-seal cell-attached recording
(A) Tight-seal cell-attached voltage-clamp recording from interneuron in CA1 str. oriens (1st interneuron listed in Table 1). Current measured by the amplifier (Iamp) is shown in the upper panel. Commanded pipette voltage (Vp) is shown below. Cell did not fire at rest (−70 mV). Commanding increasingly depolarized potentials caused the amplifier to pass positive current (seen as a positive shift in baseline current). Current passed across the patch into the cell and depolarized the cell. The resulting action potentials were recorded as action potential currents. Recorded in the presence of 4-AP, CNQX, and D-AP5. (B) Tight-seal cell-attached recording from an interneuron in CA1 str. lacunosum-moleculare (2nd interneuron listed in Table 1). A commanded voltage step from the zero-current potential of −60 mV to +30 mV passed current into the cell and caused it to fire action potentials. Recorded in the presence of 4-AP, CNQX, and D-AP5. (C) Tight-seal cell-attached recording from an interneuron in CA3 str. lacunosum-moleculare (5th interneuron listed in Table 1). Firing frequency increased as Vp was made more positive. No drugs were in the extracellular saline. (Current transients at beginning and end of voltage steps in A, B, and C have been clipped.)
Fig. 9. Increasing the ratio Rseal/Rpatch + cell increases the proportion of Iamp that travels across the patch
Graph of Ipatch/Iamp vs. resistance ratio for a theoretical recording. Plot was generated using equation 13. Note the log scale of the x-axis.
Fig. 10. Magnitude of Ipatch depends upon Vp, Rseal, and Rpatch+cell
Graph of Ipatch vs. Vp for 3 different values of Rseal. Larger Rseal and more positive Vp give a larger Ipatch. Larger Rpatch+cell gives a smaller Ipatch. The equation used to generate the plot is Ipatch = [(Vp − V0) − (Iamp × Relec)]/Rpatch +cell, which is equation 9. Equation 8 was used to calculate Iamp. Relec value used was 5 MΩ. Rseal and Rpatch + cell values for the different plots are listed in the box. A larger value of Rpatch+cell was used when Rseal < 1GΔ in order to reflect the actual experimental situation (see Part II).
Fig. 11. A positive Iamp depolarizes the cell. The magnitude of the change in membrane potential depends upon Rseal and Rcell
(A) Theoretical graph of change in membrane potential vs. Iamp. The change in membrane potential is larger with a larger Rseal because a greater proportion of Iamp travels across the patch. Note that a positive Iamp causes a depolarization and a negative Iamp causes a hyperpolarization. Equation 11 was used to calculate Ipatch. Rpatch was constant at 2GΩ, and Rcell was 200 MΩ. (B) Theoretical graph of change in membrane potential vs. Rcell. The change in membrane potential for a given Vp is greater for a larger Rcell, up to a point. The change in membrane potential peaks at Rcell = 3.4 GΩ. Iamp was calculated using equation 8 with Vp = +30 mV. Ipatch was calculated using equation 11. Change in membrane potential = Ipatch × Rcell.
Fig. 12. Tight-seal cell-attached current-clamp recording is best-suited for recording synaptic events whereas voltage-clamp recording is best-suited for recording action potentials
Tight-seal cell-attached recording from CA3 pyramidal cell (5th pyramidal cell listed in Table 1). Extracellular saline includes 4-AP, CNQX, D-AP5, and the GABAB antagonist CGP 55845. Top row is cell-attached. Bottom row is a simultaneous field recording. Left panel is current clamp (CC). Right panel is voltage clamp (VC). Two successive GPSPs were recorded, one in current clamp and one in voltage clamp. Slash marks in field recording indicate a time gap of approximately 65 s. Recording alternately in CC and VC modes along with a simultaneous field recording allows us to see that the action potential currents recorded in voltage-clamp mode are caused by a large depolarization like the one measured in current-clamp mode. Notice that in the current-clamp recording, the action potentials have been greatly filtered by the patch capacitance and that in the voltage-clamp recording, the synaptic event is not evident. (A recording similar to the right panel but from a different cell is shown in Kantrowitz et al. 2005).
Fig. 13. The circuit involved when the cell membrane potential is changing
The membrane patch is represented by a resistor and capacitor in parallel. In cell-attached current-clamp mode, the charging of the capacitor slows down the apparent kinetics of fast membrane potential changes such as action potentials. In addition, the apparent magnitude of fast changes in membrane potential is reduced; for example, apparent action potential magnitude is profoundly reduced due to the filtering effect of the patch of membrane. In cell-attached voltage-clamp mode, the capacitor provides a low impedance pathway through the patch for fast events such as action potentials, resulting in the measurement of a current with an approximately 10-fold larger magnitude than would be measured at steady state for the same magnitude change in membrane potential.
Fig. 14. Loose seal is adequate for recording action potential currents in cell-attached voltage-clamp mode
Recording is from an interneuron in CA1 str. oriens. The left side of the trace shows the current response to a 5 mV step. The action potential current (negative deflection of 74 pA followed by smaller positive deflection) appears on the right side of the trace. The 5 mV step caused a ΔI of 302 pA. Using Ohm’s law gives an Rtot of 16.6 MΩ. With an electrode resistance of approximately 5 MΩ, Rseal calculates to roughly 11 MΩ, which is a loose seal. No drugs were added to the extracellular saline.
Fig. 15. The magnitude and kinetics of the measured change in potential are affected by Rseal and the membrane patch, but the polarity of the ΔVm is accurately depicted
Response to a theoretical 80 mV square step of Vm from −70 mV to +10 mV. Curved black lines show the voltage measured by the amplifier for three different theoretical Rseal values. Time 0 is the moment of the immediate 80 mV step in cell membrane potential. A smaller Rseal results in a less negative beginning V0, a less positive ending V0 and a smaller ΔV0. A smaller Rseal also results in a longer time for Vp to reach its new steady-state level. Note that Vp takes several ms to reach its new steady-state level even with an infinite Rseal. Importantly, the magnitude of Rseal does not affect the measurement of the direction of the −Vm, i.e. hyperpolarizing v. depolarizing. Equations 15–17 were used to generate the plots. Values used were Rpatch = 2 GΩ and Rcell = 100 MΩ.
References
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