Determination and compensation of series resistances during whole-cell patch-clamp recordings using an active bridge circuit and the phase-sensitive technique.

We present a technique which combines two methods in order to measure the series resistance (R S) during whole-cell patch-clamp recordings from excitable and non-excitable cells. R S is determined in the amplifier's current-clamp mode by means of an active bridge circuit. The correct neutralization of the electrode capacitance and the adjustment of the bridge circuit is achieved by the so-called phase-sensitive method: Short sine wave currents with frequencies between 3 and 7 kHz are injected into the cells. Complete capacitance neutralization is indicated by the disappearance of the phase lag between current and voltage, and correct bridge balance is indicated by a minimized voltage response to the sine wave current. The R S value determined in the current-clamp mode then provides the basis for R S compensation in the voltage-clamp recording mode. The accuracy of the procedure has been confirmed on single-compartment cell models where the error amounted to 2-3 %. Similar errors were observed during dual patch clamp recordings from single neocortical layer 5 pyramidal cells where one electrode was connected to the bridge amplifier and the other one to a time-sharing, single-electrode current- and voltage-clamp amplifier with negligible R S. The technique presented here allows R S compensation for up to 80-90 %, even in cells with low input resistances (e.g., astrocytes). In addition, the present study underlines the importance of correct R S compensation by showing that significant series resistances directly affect the determination of membrane conductances as well as the kinetic properties of spontaneous synaptic currents with small amplitudes.

Single-cell juxtacellular transfection and recording technique.

Genetic modifications and pharmacological studies enable the analysis of protein function in living cells. While many of these studies investigate the effect of proteins by bulk administration or withdrawal of the protein in complex cellular networks, understanding the more subtle mechanisms of protein function requires fine-tuned changes on a single-cell level without affecting the balance of the system. In order to analyse the consequences of protein modification at the single-cell level, we have developed a single-cell transfection method in the loose patch configuration, which allows juxtacellular recordings of neuronal cells prior to juxtacellular transfection. CA1 pyramidal neurons were selected based on morphological and electrophysiological criteria. Using a patch clamp amplifier which allows sensitive recordings of action currents in the loose seal mode as well as electroporation with high-voltage electrical stimulation the identified neurons were transfected with a combination of specific nucleotides, e.g. siRNA and a plasmid coding for GFP for later cell retrieval. Two days after transfection, whole-cell patch clamp recordings of transfected cells were performed to analyse electrophysiological properties. Action potential firing and synaptic transmission of single electroporated CA1 pyramidal cells were comparable to untransfected cells. Our study presents a method which enables identification of neurons by juxtacellular recording prior to single-cell juxtacellular transfection, allowing subsequent analysis of morphological and electrophysiological parameters several days after the genetic modification.

Voltage-clamp-controlled current-clamp recordings from neurons: an electrophysiological technique enabling the detection of fast potential changes at preset holding potentials.

Investigations of the properties of fast, transient potential changes (e.g. receptor potentials or synaptic potentials) in excitable cells by means of current-clamp recording techniques require the exact adjustment and control of membrane potentials. Usually, the desired membrane potential values are set by current injection via the recording electrode and are controlled manually by regulating the current strength necessary to maintain a constant potential. However, this technique is associated with a number of disadvantages. A single-electrode current- and voltage-clamp amplifier was therefore modified to compensate for slow membrane potential changes without affecting faster voltage responses. Basically, low-pass filters with selectable time constants were incorporated into the voltage-clamp feedback circuit to control the amplifier's response speed. In addition, the amplifier's electronic circuits were altered to enable current pulse injection into the cells. Thus, while recording at preset and controlled membrane potentials, it was possible to monitor the cell's input resistance or current/voltage relationship. This new recording technique has been designated "voltage-clamp-controlled current clamp" (VCcCC) and its performance was tested by intracellular recordings from neocortical and neostriatal neurons in vitro using either conventional microelectrodes or patch-clamp electrodes.