Recording a neuron with patch clamp

Patch clamp records the electrical activity of a single cell through a glass pipette with a tip about a micrometre across. The tip is pressed gently against the cell membrane and suction is applied until the glass and the membrane seal together with an electrical resistance of more than a gigaohm (a gigaseal). Neher and Sakmann first recorded the tiny currents that flow through single ion channels in a patch of membrane under a pipette pressed against a muscle fibre (Neher and Sakmann, 1976); their seals were looser than a gigaohm, and the tighter gigaseal came a few years later. If the patch under the tip is then broken, the pipette is connected to the inside of the whole cell: this is the whole-cell configuration.

In whole-cell mode the amplifier works in one of two ways. In current clamp it injects a chosen current and records the membrane potential, including action potentials. In voltage clamp it holds the membrane potential at chosen values and records the current needed to do so, which is the current flowing through the ion channels. Most of the skill lies in the steps before the first recording: a clean pipette of the right resistance, a careful approach, a good seal and a clean break-in.

The cell is a teaching model. Its membrane follows the Hodgkin–Huxley description of the squid giant axon at 6.3 °C, the temperature of their experiments (Hodgkin and Huxley, 1952). Hodgkin and Huxley used a wire electrode inside the axon, not a patch pipette, and squid giant axons are not recorded by whole-cell patch clamp, so the model is a teaching hybrid: a squid membrane in a small cell, recorded as a mammalian neuron would be. Their equations describe membrane current per unit area, in microamperes per square centimetre (µA/cm²). Here that membrane is placed in a small spherical cell about 18 µm across, with a surface area of approximately 1000 µm² and therefore a membrane capacitance of about 10 pF. For this cell, 1 µA/cm² corresponds to a whole-cell current of 10 pA, and 1 mA/cm² to 10 nA. Because the membrane has 1 µF of capacitance per cm², a current density in µA/cm² has the same number as the current divided by the cell capacitance in pA/pF, the unit most laboratories report.

The pipette resistance, the approach, the seal, the break-in, the access resistance and the time course of drug wash-in and wash-out are simplified illustrations, because the Hodgkin–Huxley model has no pipette; they are labelled as such. The recordings in current clamp and voltage clamp, and the effects of the drugs on them, come from the Hodgkin–Huxley equations. A few behaviours belong to the model rather than to real neurons, for example spontaneous firing when most potassium channels are blocked; these are noted where they occur. Mammalian neurons have many more types of ion channel and are usually recorded nearer body temperature, so their firing differs in detail.

Learning objectives

• Name the parts of a patch-clamp rig and say what each one does
• Choose the internal solution and pipette resistance for a whole-cell recording, and measure the pipette resistance from a test pulse with Ohm's law
• Approach a cell with positive pressure, recognise contact, form a gigaseal and break in to whole-cell mode, reading each step from the test-pulse current
• Measure access resistance, membrane capacitance, input resistance and resting potential, and decide by criteria set in advance whether to keep a cell
• Record firing in current clamp and ionic currents in voltage clamp, find the rheobase and the reversal potential, and convert between current density (µA/cm², pA/pF) and whole-cell current (pA, nA)
• Apply and wash out tetrodotoxin and tetraethylammonium, with the cell as its own control, and explain their effects

Ethics and safety

Neurons for patch clamp usually come from acute brain slices or cultures prepared from animals that are humanely killed by trained and authorised staff, under approval by an Institutional Animal Ethics Committee; in India such work is governed by the CCSEA under the Prevention of Cruelty to Animals Act 1960, with equivalent frameworks in the European Union (Directive 2010/63/EU), the United States (Animal Welfare Act and PHS Policy) and the United Kingdom (Animals (Scientific Procedures) Act 1986). The 3Rs apply: many cells are recorded from each animal and each slice (Reduction), and cultured or stem-cell-derived neurons can replace animals for some questions (Replacement). Recordings are reported with the number of cells and of animals, and with the quality criteria used to include or exclude cells, set before the data are seen. Tetrodotoxin is extremely toxic and tetraethylammonium is harmful: both are handled under a local risk assessment, with stock solutions labelled and kept secure. Glass capillaries and pipettes are sharp, and the rig is mains-powered equipment used near salt solutions.

Key terms

• Patch clamp: Recording the current or voltage of a cell through a glass pipette sealed onto its membrane. See patch clamp.
• ACSF and internal solution: Artificial cerebrospinal fluid (ACSF) bathes the cell and resembles the fluid outside cells: high in sodium, low in potassium. The internal solution fills the pipette and resembles the cytoplasm: high in potassium, low in sodium.
• Pipette resistance: The electrical resistance of the filled pipette in the bath, in megaohms (MΩ); it is set mainly by the size of the tip opening.
• Test pulse: A small, repeated voltage step (here 10 mV) applied by the amplifier; the current it drives shows the resistance at the tip, by Ohm's law R = V / I.
• Gigaseal: A seal between glass and membrane with a resistance above 1 gigaohm (1 GΩ = 1000 MΩ), so that almost no current leaks around the tip.
• Whole-cell configuration: The recording mode after the membrane patch under the tip is broken: the pipette solution is continuous with the cytoplasm.
• Access resistance: Also called series resistance: the resistance between the pipette wire and the inside of the cell, mainly the tip. It slows and distorts the voltage clamp, so it is kept low (often below about 20 to 25 MΩ).
• Membrane capacitance: The charge stored by the membrane per unit voltage, about 1 µF per cm² of membrane; it gives the size of the cell (here about 10 pF).
• Current clamp: The amplifier injects a set current and records the membrane potential. See current clamp.
• Voltage clamp: The amplifier holds the membrane potential at a set value and records the current needed. See voltage clamp.
• Holding potential: The membrane potential at which the cell is held in voltage clamp between steps, for example −70 mV.
• Rheobase: The smallest current step that evokes an action potential. See rheobase.
• Current density: Current divided by membrane area (µA/cm²) or by capacitance (pA/pF). It allows cells of different sizes to be compared.
• TTX and TEA: Tetrodotoxin (TTX) blocks voltage-gated sodium channels; tetraethylammonium (TEA) blocks many potassium channels.