Current Clamp protocol:

To understand how a neuron can be excited, or to study the behavior of membrane potential, a current clamp is used. Current-clamp is a method of intracellular recording involving measurement of the voltage difference across the cellular membrane while injecting constant positive or negative current (as "square" d.c. pulses) into the cell. Voltage recording without current injection or other perturbation will usually tell the researcher only what the membrane potential is (usually around -60 to -80 mV in resting neurons). However, by injecting repetitive constant current pulses (or steps) into the cell and using appropriate "bridge" methods to balance out the resistive influence of the recording micropipette, the electrophysiologist can obtain from the voltage response a relative measure of the resistance (or, inversely, of conductance: g) of the membrane.

Ohm's law (E = IR) can be applied here to obtain a simple relationship between the current injected (I), the voltage recorded (E), and the "input resistance" (R) of the membrane. If a drug or transmitter is then applied to the cell, a change in the size of the voltage response to the current pulse indicates a change in ionic conductance (g = 1/R). By incrementally varying the amplitudes of the current steps over a wide range (typically from 0.1 to 1 nA in mammalian CNS neurons), a family of voltage responses can be obtained for construction of a voltage-current (V-I) curve (where voltage is typically plotted as a function of injected current. This curve reveals much about the "macroscopic" currents (that is, the aggregate currents flowing through many ionic channels) passing through the neuronal membrane at different membrane potentials. Any drug treatment that alters ionic conductance will also alter the slope and shape of the V-I curve. Thus, a reduction in the slope of the V-I curve indicates increased ionic conductance, whereas a steeper slope indicates decreased conductance.

In practice, current-clamp recording is usually performed by inserting a single sharp micropipette into a neuron while recording voltage and injecting current through the same pipette. Penetration of the cell is signaled by an abrupt transition to a large negative voltage (about -70 mV), accompanied by an increase in input resistance (as typically reflected in the voltage deflection produced by a current pulse). After successful settling ("sealing") of the pipette into the membrane, control V-I curves and synaptic activations can be generated (usually nowadays by sophisticated computer methods); drug administration by superfusion or pipette application is then followed by repeated V-I and synaptic measures for statistical comparison to the control measures. Reversal of any drug effects by washout with the vehicle (artificial cerebrospinal fluid) alone assures the researcher that any changes are not merely the result of a rundown (e.g., slow death) of the cell or a slowly improving penetration "seal." The usual measures taken in current clamp include: resting membrane potential, input resistance, I-V curves, and voltage responses (excitatory postsynaptic potentials or inhibitory postsynaptic potentials) to activation of inhibitory or excitatory synaptic afferents. In addition, much information about membrane and drug properties can be obtained from the rebound voltage responses (so-called "anodal break" depolarizations, due to activation of several possible currents) immediately following strong hyperpolarizing current steps, or from the prolonged hyperpolarizations [after hyperpolarizations (AHPs) due to Ca2+-dependent K+ conductances] following the burst of spikes evoked by strong depolarizing current steps. Many neurotransmitters have been shown to potently alter the latter measure.