4.1 Patch-Clamp Electrophysiology
Overview of Cellular Methods
Since we have learned that neurons contain channels, receptors and transporters in Unit 1, one of the most useful parameters that neuroscientists examine involves determining the movement of ions and the resultant modulation of neuronal membrane potential. Specifically, electrophysiological techniques used in excitable tissues rely on the ionic conductance of ion-channels and how these influence changes in the membrane potential of the cell being examined. Various electrophysiology techniques have been developed to detect and manipulate ion-channel function and/or action potential generation. Determining when to use each electrophysiological technique depends on many different factors; these include the biophysical properties of the recorded cell, the type of tissue being examined, the use of current- and/or voltage-clamp, whether the intra- and/or extracellular environments will be modulated in the experiments, and most importantly whether a single channel or several ion channels will be recorded.
Patch-clamp Electrophysiology
The most common method used to assess ion-channel function is known as the patch-clamp electrophysiological technique that was developed in the 1970s by Nobel Prize Laureates Erwin Neher and Bert Sakmann. The patch-clamp technique allows a researcher to measure the biophysical properties of ion-channels on millisecond timescales. Patch-clamp requires the initial formation of a Giga-ohm (GΩ) seal between the plasma membrane and the blunt tip (0.5–2 μm in diameter) of a heat-polished glass or quartz micropipette (electrode). Once a Giga-ohm seal has been created, this cell-attached configuration (Figure 1.) maintains the integrity of the plasma membrane (i.e. the membrane is not ruptured) preventing the intracellular solution inside the micropipette from dialyzing into the cell. However, this also restricts electrical access to the cell intracellular space resulting in an inability to control the membrane potential of the cell. In this configuration, only the patch membrane potential relative to the cell’s resting potential can be directly controlled. Through the alteration of the magnitude of the seal resistance (a loose seal vs. tight seal) and/or whether the recording electrode is current- vs. voltage-clamped, the cell-attached configuration can be used to measure single-channel currents, spontaneous neuronal cell firing and synaptic potentials as well as evoked action potentials within the cell. Although the configuration is limited as described above, a major advantage is that this configuration is the starting point for the majority of the types of patch-clamp recordings.
To increase electrical access to the cell interior, two different methods are used. First, the internal pipette solution contains antibiotics or antifungal agents (e.g., nystatin, gramicidin, amphotericin-B), these agents form small, monovalent ion-permeable pores that ‘perforate’ (Figure