Abstract
Intracellular recordings using sharp microelectrodes often rely on a technique called discontinuous current-clamp (DCC) to accurately record the membrane potential while injecting current through the same microelectrode. It is well known that a poor choice of DCC switching rate can lead to underestimation or overestimation of the cell potential; however, its effect on the cell firing is rarely discussed. Here, we show that suboptimal switching rates lead to an overestimation of cell excitability. We performed intracellular recordings of mouse spinal motoneurons and recorded their firing in response to pulses and ramps of current in Bridge and DCC mode at various switching rates. We demonstrate that using an incorrect (too low) DCC frequency leads not only to an underestimation of the input resistance, but also, paradoxically, to an artificial overestimation of the firing of these cells: neurons fire at lower current, and at higher frequencies than at higher DCC rates, or than the same neuron recorded in Bridge mode. These effects are dependent on the membrane time constant of the recorded cell, and special care needs to be taken in large cells with very short time constants. Our work highlights the importance of choosing an appropriate DCC switching rate to obtain not only accurate membrane potential readings but also an accurate representation of the firing of the cell.
Highlights
Neurons, by virtue of their plasma membrane and the numerous ion channels that can be found therein, behave, to a first approximation, like RC circuits
We demonstrate that using an incorrect discontinuous currentclamp (DCC) frequency leads to an underestimation of the input resistance, and, paradoxically, to an artificial overestimation of the firing of these cells: neurons fire at lower current, and at higher frequencies than at higher DCC rates, or than the same neuron recorded in Bridge mode
It is usually difficult to estimate the resistance of the cell in Bridge mode, as the IR drop through the electrode might change depending on the intensity of the injected current and cannot be perfectly compensated by the Bridge balance circuit
Summary
By virtue of their plasma membrane and the numerous ion channels that can be found therein, behave, to a first approximation, like RC circuits. The electrode itself, because of its very small tip, acts as an additional RC circuit, and experiences an IR drop when current is applied In these conditions, it is essential to be able to separate the physiological response of the cell from a change of voltage caused by the resistance of the very electrode used to perform the recording. The first one, the so-called “Bridge” mode, consists (broadly speaking) in subtracting the voltage drop caused by the current injection through a variable resistor set to a value close to the estimated electrode resistance from the voltage measured by the electrode. We demonstrate that using a suboptimal (too low) DCC frequency lead to an underestimation of the cell resistance, and, paradoxically, to an artificial overestimation of the firing of these cells: neurons fire at lower current, and at higher frequencies than at higher DCC rates, or than the same neuron recorded in Bridge mode
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