Electrophysiology: general

Principle

Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage change or electrical current flow by electrodes in various systems, from single ion channel proteins to single neurons (particularly action potentials) and whole tissues like the heart.

Classical electrophysiological techniques

The principal types of electrodes are:

1) metal discs (singles or arrays),

2) metal tracings on printed circuit boards.

2) needles (singles or arrays), diameter μm scale, inserted into or just outside a single cell.

4) small glass pipettes, also for extra- or intracellular recording.

The principal preparations include:

· living organisms,

· excised tissue (acute or cultured),

· dissociated cells from excised tissue (acute or cultured),

· artificially grown cells or tissues,

· hybrids of the above.

The pipette techniques has various versions:

· With a low impedance pipette an extracellular placement may pick up the activity of several nearby cells simultaneously, and this is termed multi-unit recording (also with a metal microelectrode).

· With the electrode tip (tip size ca. 1 μm, resistance some MΩ) more closely to the neuron, the recording is termed single unit recording (also with metal microelectrodes, resistance some 100 kΩ).

· With the pipette tip pressed against the cell membrane, to which it tightly adheres. This is a semi-intracellular recording with spikes of some 5-10 mV. (pipette resistance a few tens of MΩ).

· The electrolyte within the pipette may be brought into fluid continuity with the cytoplasm by delivering a pulse of pressure to the electrolyte in order to rupture the small patch of membrane encircled by the pipette rim (intracellular or whole cell recording, resistance some 50 MΩ). The electrolyte may contain a drug. When the pipette contains a tracer (e.g. HRP) then the resistance can be about 200 MΩ.

· Alternatively, ionic continuity may be established by "perforating" the patch by allowing exogenous ion channels within the electrolyte to insert themselves into the patch (perforated patch recording).

· The patch may be left intact (patch recording).

· Finally, the patch is so small that it comprises only one channel: a single channel recording.

As electrode size increases, the resolving power decreases. Larger electrodes are sensitive only to the net activity of many cells, the so called local field potentials (see More Info). Still larger electrodes, such as non insulated needles and surface electrodes (discs) used by clinical (EEG, and surgical neurophysiologists, are sensitive only to certain types of synchronous activity within populations of cells numbering in the millions.

Optical electrophysiological techniques

Optical electrophysiological techniques were created to overcome the limitations that electrical activity is recorded at approximately a single point within a volume of tissue. Interest in the spatial distribution of bioelectric activity prompted development of molecules capable of emitting light in response to their electrical or chemical environment. Examples are voltage sensitive dyes and fluorescent proteins.

With one or more such compounds administrated via perfusion, injection or gene expression, the distribution of electrical activity may be observed and recorded.

Application

Many particular electrophysiological techniques and recordings have abundant clinical application:

Electrocardiography (ECG), for the heart;

Electroencephalography (EEG), for the brain and Electrocorticography for the cerebral cortex;

Electromyography (EMG) for the muscles

Electro-oculography, for the eyes

Electroretinography,for the retina

Electro-olfactography for the olfactory receptors;

Evoked potentials for auditory, visual, somatosensory assessment.

Bioelectric Recognition Assay (BERA)

BERA is a novel method for measuring changes in the membrane potential E_m of cells immobilized in a gel matrix. Apart from the increased stability of the electrode-cell interface, immobilization preserves the viability and physiological functions of the cells. BERA is primary used in biosensor applications in order to assay analytes which can change E_m in a characteristic, ‘signature-like’ way. BERA has been used for the detection for human viruses, veterinary disease agents and plants in a highly specific, rapid (1-2 minutes), reproducible and cost-efficient fashion. The method has also been used for the detection of environmental toxins, such as herbicides and the determination of very low concentrations of superoxide anion in clinical samples. A recent development of the BERA technology is the technique called Molecular Identification through Membrane Engineering (MIME). This technique allows for building cells with absolutely defined specificity against virtually any molecule of interest, by embedding thousand of artificial receptors into the cell membrane.

More info

Extracellular recording

A microelectrode (tip size ca. 1 μm), into the brain, nerve or sensory tissue (e.g. retina) of a living animal will usually detect the activity of one neuron or axon. The spikes of a "single unit" recording are very like the intracellular spikes, but they are much smaller (typically 0.1-1 mV).

Multi-unit recordings are often used in conscious animals to record changes in the activity of a discrete brain area during some behavior of the animal. Recordings from one or more such electrodes which are closely spaced can be used to identify the number of cells around it as well as which of the spikes come from which cell. This process is called spike sorting and is suitable in areas where there are identified types of cells with well defined spike characteristics.

If the electrode tip is bigger still, generally the activity of individual neurons cannot be distinguished but the electrode will still be able to record a field potential generated by the activity of many cells. Such an electrode inserted in or near the auditory nerve can record a compound action potential.

Intracellular recording

Intracellular recording involves measuring voltage and/or current across the membrane of a cell with (generally) a glass micropipette (< 1μm and a resistance of tens of MΩ) filled with an electrolyte and connected with a metal wire (generally an Ag wire coated with AgCl), to make a connection between the electrolyte and the amplifier. The coating reduces the galvanic potential between electrolyte and silver wire. This is necessary since the galvanic potential is very large (+1.98 V). Moreover, it reduces electrode noise which is higher the smaller the contact surface.

The pipette must be inserted inside the cell, so that the membrane potential can be measured (in rest −60 to −80 mV). Micropipettes are filled with a solution that has a similar ionic composition to the intracellular fluid of the cell. The voltage measured by the electrode is compared to the voltage of a reference electrode, usually an Ag-AgCl wire in contact with the extracellular fluid somewhere in the tissue.

Pipette impedance

In general, the smaller the electrode tip, the higher its electrical resistance so an electrode is a compromise between size (small enough to penetrate with minimum damage) and resistance (low enough so that small neuronal signals can be discerned from thermal noise in the electrode tip, see below). Pipettes have also a capacitance, as does the electrode cable and the amplifier has an input capacitance. With a capacitance poor cable (also short, and with a driven guard, i.e. the shield has the amplifier input voltage by using a buffer amplifier) and capacitance poor amplifier, these parallel capacitances together can be brought down to about 10 pF.

The pipette restistance R_pip is:

R_pipette = R_shaft + R_tip + R_medium,

R_pipette = r_electrolyteL _shaft/(¼πd_shaft²) + (2/d_tip─ 2/d_shaft)r_electrolyteL_tip/(½πd_shaft) + r_medium/(2πd_tip), (1)

where d is diameter and L is length. With r_electrolyte = 75 Ωcm, = r_medium = 750 Ωcm, L _shaft = 5 cm. L_tip = 0.5 cm, d_shaft = 0.08 cm and d_tip = 5 10^─5 cm, then we obtain:

R_pipette = 75 kΩ + 11.9 MΩ + 2.4 MΩ.

The far majority of R_pipette is due to the tip geometry. With d_tip fixed, R_pipette can only be reduced by reducing L_tip. With an electrode resistance 100 MΩ the resulting low pass first order filter (see Linear first order system) has a cut off frequency of 160 Hz (time constant 1 ms), hardly enough to record intracellular spikes. Since the pipette capacitance is determined by the thickness (reciprocally) and the surface (linear) of the glass wall (the dielectricum), the geometry of the tip is of great importance.

Sharp electrode technique

For recording the potential inside the cell membrane with minimal effect on the ionic constitution of the intracellular fluid a sharp electrode can be used. They look like patch clamp pipettes but the pore is much smaller so that there is very little ion exchange between the intracellular fluid and the electrolyte in the pipette. The resistance of the electrode is tens to hundreds of MΩ. Often the tip of the electrode is filled with various kinds of voltage sensitive dyes like Lucifer yellow to be injected by Electrophoresis. A positive or negative, DC or pulsed voltage is applied to the electrodes depending on the polarity of the dye. Later, this enables identification of the cell under a microscope.

Field potentials

Fig. 1 A schematic diagram of a field potential recording from a rat hippocampus. When the synapse releases glutamate the postsynaptic membrane opens ionotropic glutamate receptor channels. The net flow of current is inward, so a current sink is generated. A nearby electrode (#2) detects this as negativity. An intracellular electrode placed inside the cell body (#1) records the change in membrane potential that the incoming current causes.

Extracellular field potential

Extracellular field potentials are local current sinks or sources that are generated by the collective activity of many cells. Usually a field potential is generated by the simultaneous activation of many neurons by synaptic transmission. Fig. 1 to the right shows a hippocampal synaptic field potential. The lower trace shows a negative wave that corresponds to a current sink caused by positive charges entering cells through postsynaptic glutamate receptors, while the upper trace shows a positive wave that is generated by the current that leaves the cell (at the cell body) to complete the circuit.

Local field potential

A local field potential (LFP) is recorded using a low impedance extracellular microelectrode, placed sufficiently far from individual local neurons to prevent any particular cell from dominating the signal. The unfiltered signal reflects the sum of action potentials from cells within approximately 50-350 μm from the tip of the electrode and slower ionic events from within 0.5-3 mm from the tip of the electrode. This signal is then low-pass filtered, cut off at ~100 Hz, to obtain the LFP. The low impedance and positioning of the electrode allows the activity of a very large number of neurons to contribute to the signal. The low-pass filter removes the spikes and the LFP remains.

The amplifier measures the electrical potential difference between the microelectrode and a reference electrode, placed somewhere else in the body or tissue with a similar extracellular medium (to cancel the both electrochemical polarization effects at the interface of both metal electrodes (or the Ag/AgCl wires in the pipette).

Synchronized Input

The LPF is believed to represent the synchronized input into the observed area. The quick fluctuations, caused by the short inward and outward currents of the action potential, are filtered out, leaving only the slower fluctuations. Therefore the spike plays no part in the LFP, which thus comprises the more sustained currents in the tissue, typical of somato-dendritic origin. The major slow current is the PSP. It was thought until recently that EPSP’s and IPSP’s were the exclusive constituents of LFP’s. However, phenomena unrelated to synaptic events have been found to contribute to the LFP.

Geometrical Arrangement

Cells which contribute to the slow field variations are determined by the geometric configuration of the cells themselves (as with the pyramidal cells.)

When there is simultaneous activation of the dendrites a strong dipole is produced. His may even give rise to a signal recordable with EEG. In cells where the dendrites are arranged more radially, in a plane, the potentials between individual dendrites and the soma tend to cancel.

Thermal noise

The thermal noise generated in a conductor (e.g.a resistor), V_n (amplitude, measured as the effective (rms) value, in Volt) is:

V_n = 2(kTR∙Δf)^0.5, (2)

where k is Boltzmann’s constant, T the absolute temperature (K), R the resistance (Ω), and Δf the bandwidth of the amplifier.

For extracellular recording V_n hardly plays a role since R is low. As example: R = 2.5 MΩ, T = 300 K, and Δf is 300 Hz, then V_n = 3.5 μV, whereas spikes are generally larger than 10 μV. For intracellular recordings the same holds. With R = 250 MΩ, V_n = 35 μV, just small enough to find the spikes extracellular before impaling the cell. However, in addition there are other noise sources: amplifier noise; with a digital output the noise in the analog-to-digital converter; noise generated at the interface of the metal electrode (or the Ag/AgCl wires in the pipette); noise generated by the opening of an ion channel caused by the net flow of ions into the cell from the extracellular medium, or out of the cell into the extracellular medium. Which type of noise dominates depends on the precise conditions of the recording.