Introduction to the Art 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. 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-coupled 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. (In the picture: ExAmp-20KB
extracellular amplifier)
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
(see our BAB-501 model) 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 (see our Union-40 model).
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. (In the picture: BAB-501
microampere iontophoresis pump)
