In discontinuous single-electrode voltage-clamp (dSEVC), the tasks of voltage recording and current passing are allocated to the same micropipette. Time-sharing techniques are used to prevent interactions between the two tasks (Fig. 1).
Figure 1. Circuit drawing of the discontinuous single electrode voltage-clamp.
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ME1 = Single micropipette
Vm = Instantaneous membrane potential Vp = Voltage recorded A1 = Unity gain headstage SH1 = Sample and hold circuit A2 = Differential amplifier Vms = Recorded membrane potential GƮ = Transconductance circuit gain |
CCS = Controlled-current source
Io = Output current Ɛ = Steady stage error S1 = Voltage recording / current passing switch S2 = Votlage clamp / current clamp switch Vcmd = Command voltage Icmd = Command current |
A single micropipette (ME1) penetrates the cell and the voltage recorded (Vp) is buffered by a unity-gain headstage (A1). Assume that Vp is exactly equal to the instantaneous membrane potential (Vm). A sample-and-hold circuit (SH1) samples Vm and holds the recorded value (Vms) for the rest of the cycle.
Vms is compared with a command voltage (Vcmd) in a differential amplifier (A2). The output of this amplifier becomes the input of a controlled-current source (CCS) if the switch S1 is in the current-passing position. The CCS injects a current into the micropipette that is directly proportional to the voltage at the input of the CCS irrespective of the resistance of the micropipette. The gain of this transconductance circuit is GƮ.
The period of current injection is illustrated at the start of the timing waveform.
Figure 2. Timing waves of the discontinuous single electrode voltage-clamp.
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T = Timing waveform
S1 = Current passing/voltage recording switchl Vp = Voltage recorded ʈp = Micropipette time constants Vm = Instanteous membrane potential Vcmd = Command voltage Vms = Recorded membrane potential
Vms(avg) = Average recorded membrane potential
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S1 is shown in the current-passing position during which a square pulse of current is injected into the micropipette, causing a rise in Vp. The rate of the rise is limited by the parasitic effects of the capacitance through the wall of the glass micropipette to the solution and the capacitance at the input of the buffer amplifier. The final value of Vp mostly consists of the IR voltage drop across the micropipette due to the passage of current Io through the micropipette resistance Rp. Only a tiny fraction of Vp consists of the membrane potential (Vm) is recorded at the tip.
S1 then switches to the voltage-recording position. When the input of the CCS is 0 volts, its output current is zero and Vp passively decays. During the voltage-recording period, Vp decays asymptotically towards Vm. Sufficient time must be allowed for Vp to reach within a millivolt or less of Vm. This requires a period of up to nine micropipette time constants (ʈp). At the end of the voltage-recording period, a new sample of Vm is taken and a new cycle begins. The actual voltage used for recording purposes is Vms.
As illustrated in the bottom timing waveform, Vms moves in small increments about the average value. The difference between Vms(avg) and Vcmd is the steady-state error (Ɛ) of the clamp that arises because the gain (GƮ) of the CCS is finite. The error becomes progressively smaller as GƮ is increased.
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