Healthcare and Medicine Reference
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The study of neural tissue development is unique
because the cells possess diverse electrical properties. These
properties result from two essential components. First, the
neuron must produce batteries by selectively pumping ions
from one side of the membrane to the other. Second, the
neuron must produce switches, commonly referred to as
voltage- and ligand-gated channels, that allow the batteries
to discharge (i.e., ionic currents flow due to an electrochem-
ical gradient) across the membrane. To determine how
pumps and channels operate, one must be able to record
from a single neuron (or a portion of it), and to control the
neurons environment. The most important parameters that
must be controlled include ionic composition, voltage
across the membrane, and the presence of ligands. The tech-
nical challenges presented by these requirements have
largely been overcome in the past two decades, providing
some fundamental discoveries about developing neurons.
To study the voltage-gated channels, one must be able
to move the membrane potential to different holding volt-
ages (voltage-clamp), and then observe whether current
flows across the membrane. Thus, if one depolarizes an
axon, voltage-gated sodium channels will open at some
criterion voltage, termed threshold , and Na + will enter the
cell (i.e., inward current). A novel set of recording tech-
niques, called patch-clamping , was introduced to fully
characterize different types of channels. Patch-clamp elec-
trodes can form high-resistance seals (“giga-seals”) with
small areas of membrane, and these patches of membrane
can then be excised from the cell (Hamill et al., 1981). This
approach has several advantages. Small patches of mem-
brane often contain single channels, they are relatively
easy to voltage-clamp, and either side of the membrane
may be exposed to the defined media. These techniques
allow one to determine a channel's characteristic proper-
ties: the voltage at which activation and inactivation occur,
the mean channel open time, the mean current amplitude,
the relative permeability to different ions, and the pharma-
cological profile. Finally, when the excised patches of tissue
contain a known class of neurotransmitter receptors, then
the recording pipet may be used to detect the release of
neurotransmitter (“sniffer pipets”). This approach has led
to the discovery that growth cones release transmitter
(Young and Poo, 1983; Hume et al., 1983).
It is also possible to form a giga-seal with the neuron
of interest, and then rupture the membrane, forming a
whole-cell recording configuration. Although this tech-
nique is qualitatively similar to a standard sharp electrode
intracellular recording, there are added benefits. The tip
of the recording electrode is much larger than that of the
sharp electrode, both improving the signal-to-noise ratio
and allowing for relatively large current injections. The
large tip diameter translates into a large hole in the mem-
brane through which the patch pipet solution travels
quite easily, allowing the intracellular composition to be
controlled within a matter of minutes. In a more elegant
form of this technique, a perfusion system is added to the
recording pipet so that intracellular composition can be
altered during a recording session (Chen et al., 1990).
Although the patch-clamp techniques offer rigid bio-
physical measures, they seldom allow one to evaluate the
movement of a single type of ion. One common strategy
requires the use of several antagonists to block the contri-
bution of contaminating ions (e.g., magnesium ions block
the flow of calcium). A second approach makes use of a
novel group of electrodes, each of which is responsive to
changes in the concentration of a specific ion, such as
potassium (Syková, 1992). The tips of these electrodes are
filled with a liquid membrane that is selectively perme-
able to one species of ion, so that local changes in concen-
tration result in the net movement of that ion across the
membrane, resulting in a detectable potential difference.
When employed in the central nervous system, these elec-
trodes reveal substantial developmental changes in the
regulation of extracellular potassium and pH (Connors et
al., 1982; Davis et al., 1987; Jendelova and Syková, 1991).
The fields of electrophysiology and image processing
have found a productive relationship in the area of mem-
brane channels. The introduction of ion-sensitive fluores-
cent dyes has provided a noninvasive means of assessing
functional properties, while providing a high degree of
spatial resolution. Each of these dyes emits light at a spe-
cific wavelength when activated with a beam of light at a
different exciting wavelength. The amount of emitted
light is proportional to the free concentration of a specific
species of ion. That is because a dye's absorption or emis-
sion properties is altered when it binds to the ion. Selec-
tive indicator dyes now exist for a wide range of ions
including Na + , Ca 2+ , Cl - , and H + . The indicator dye, fluo-
3, has been used to demonstrate an elevation of Ca 2+
immediately following contact between growth cone and
target cell (Dai and Peng, 1993). A novel variation of this
technology makes use of compounds that exist in a
“caged” configuration and that only become activated
when exposed to light of a specific wavelength. In this
manner, one may elevate the concentration of a specific
substance with great temporal and spatial resolution.
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