Principles of microiontophoresis

Microiontophoresis is the technique whereby ions and charged molecules can be ejected in very small amounts from solutions contained in glass micropipettes. Microiontophoresis is most often used for : (1) deposition of dyes and neural transport tracers for histological examination or (2) for administration of neuroactive compounds (e.g. transmitters, modulators, drugs or hormones) by microiontophoresis to examine their effects on firing parameters of single neurons in vivo.

Microiontophoretic ejection is accomplished by applying a voltage across the micropipette (electrode) and causing it to become polarized. If a voltage is applied to a solution, ions and charged molecules will migrate toward and away from the source of the imposed electrical field depending upon the sign of their net charge. If the pipette is positioned close to a neuron, drugs may be ejected and their pharmacological effects inferred by resulting changes in the rate and/or pattern of firing. Typically this neuropharmacological technique is used to determine the effects of various substances upon firing parameters of neurons. A chief advantage of the microiontophoretic method is that it is possible to examine the effects of drugs upon single neurons without affecting the whole of the nervous system such as may occur when drugs are administered systemically.

The basic principle of microiontophoresis is illustrated on the schematic. The figure shows direction of current necessary to eject positively charged particles (cations). In practical applications, multibarrel micropipette assemblies are used. They are constructed of heat-fused or glued-together glass pipettes (usually 5 or 7) having radially situated drug barrels and a centrally located pipette channel used for recording of cellular unit discharges (combination electrodes). The necessary microiontophoretic current is generated by precision constant current sources (iontophoresis pumps). Multibarrel micropipettes permit the experimenter to test several compounds in the same neuron and, if the iontophoresis pump is controlled by a computer, in a preprogrammed fashion. See a possible experimental design for computer-controlled iontophoresis and extracellular single unit recording.

Deposition of dyes and tracer substances

A dye or tracer substance can be ejected by iontophoresis into the cytoplasm of a cell (intracellular iontophoresis) or into the intercellular space (extracellular iontophoresis). Intracellular deposition of a dye or tracer by microiontophoresis is made to mark the cell for subsequent histological examination. In extracellular studies, iontophoresis is used to mark brain sites where recordings have been made. Neuronal projections are also studied by microiontophoretic application of tracers into specific brain areas. In such experiments, tracer compounds iontophoresed into the intercellular space are taken up by axonterminals (retrograde tracing) or by dendrites and somata (anterograde tracing) and are then intracellularly transported across the whole cell.

Intracellular deposition of a dye or tracer by microiontophoresis is made to mark the cell for subsequent histological examination. The frequently-used markers for intracellular iontophoresis include Lucifer Yellow and horseradish peroxidase (HRP). Intracellular deposition is usually accomplished by applying iontophoretic currents of several nanoamperes for several minutes.

In extracellular studies, Phaseolus vulgaris leucoagglutinin is a popular tract-tracer substance. An important application of extracellular deposition of dyes (e.g. Pontamine Sky Blue or Fast Green) is to mark the site of recording/iontophoresis in extracellular electrophysiology. Typically, these compounds are ejected at 1-10 µA of applied for 1-45 min.

See our Dyes & Tracers page for iontophoretic values (pipette concentration, ejection time and polarity) for selected neural sitemarking or track-tracing compounds.

Testing effects of neuroactive compounds by microiontophoresis

Iontophoretic ejection from multibarrel pipettes into the vicinity of a neuron allows the screening of a wide variety of compounds for pharmacological activity at cells of particular brain regions. Comparisons of the potencies of those substances which affect the rates of firing of these neurons are often made, and there are attempts to seek out fundamental similarities between the nature of the drug-elicited actions and the properties of synaptically-evoked effects. Antagonists may also be tested upon the drug elicited and synaptic responses. Iontophoretic values (pipette concentration, pH, ejection polarity) for a variety of neuroactive substances are provided on our Neuroactive substances page. To perform such "micropharmacology" by microiontophoresis, extremely small amounts are ejected using iontophoretic currents between 10 and 100 nA for 5-120 seconds.

Microelectrodes for combined extracellular recording and iontophoresis

Multibarrel iontophoresis assemblies are manufactured from borosilicate glass capillary tubing. For the commonly used five- or seven-barreled multibarrel assemblies several pieces of tubing are fused or glued together before pulling. In extracellular studies, microiontophoresis is most often used in conjunction with extracellular recording of neuronal firing. To accomplish this, the center barrel of a multibarrel pipette is filled with a suitable electrolyte solution such as sodium chloride. Alternatively, a multibarrel micropipette can be combined with a conventional single unit recording electrode such as tungsten. Recently, small diameter (5-8 µm) carbon fibers have been introduced as conductive elements in iontophoresis/recording combination electrodes. They provide excellent signal-to-noise ratio recording. If you would like to try our Carbostar-7 carbon fiber containing combination electrode (pictured) contact kations@aol.com for free sample. Our Carbostar series microelectrodes are for sale, see Kation Scientific's Order and Price list page.

Extracellular spike recording

Signals picked up by extracellular electrodes are in the microvolt range and they need to be amplified to be able to be processed in more conventional electronic devices such as oscilloscopes, analyzers or computers. Our ExAmp-20KB is a battery powered AC/DC differential amplifier designed for low-noise extracellular recording from nerve cells with carbon fiber microelectrodes like the Carbostar series. It works equally well with tungsten or other solid-conductor microelectrodes. The unique headstage probe design puts first stage of amplification at microelectrode interface permitting less external interference noise pickup. It can be used in a number of research or teaching applications. The ExAmp-20KB is for sale, see Kation Scientific's Order and Price list page.

Microiontophoretic current sources

The current passed through the iontophoretic pipette (electrode) can be calculated from Ohm's Law: I=V/R, where I is the current in amperes; V is the potential difference in volts; and R is the resistance of the electrode in ohms . Most micropipette barrels have resistances of between 1 and 50 Megohms when filled with drug solution. During an experiment the resistance may fluctuate for a variety of reasons. This requires a precision constant current source (or iontophoresis pump as it is popularly called) which automatically maintains a constant current flow through the barrel, independently of the electrode (tip) resistance. An iontophoresis pump, in compensation to fluctuations in tip resistance, automatically changes the voltage applied to the iontophoresis pipette.

Microiontophoretic pumps come in two basic varieties: (1) pumps which deliver currents in the 1-10 µA range for extracellular deposition of dyes and tracers and (2) pumps which deliver currents in the 0-100 nA range for intracellular deposition or to determine the effects of various substances upon firing parameters of neurons. In the latter case, extremely small amounts of neuroactive substances (transmitters, modulators, hormones, drugs) ejected to study pharmacological responses of single nerve cells.

Here, we introduce an iontophoresis pump specifically designed to deliver currents in the rage of 1-20 µA for extracellular deposition of dyes and tracer substances. Three modes of operation can be selected. In continuous mode, iontophoresis current is continuously generated when the polarity switch is in "Positive" or "Negative" position. In external mode, the output current can be gated through its "Input" BNC jack by any logic pulse generator or computer. If you would like to buy one, see Kation Scientific's Order and Price list page.