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The Journal of Neuroscience, February 15, 1998, 18(4):1449-1464
The Differential Expression of Low-Threshold Sustained Potassium
Current Contributes to the Distinct Firing Patterns in Embryonic
Central Vestibular Neurons
Georgi
Gamkrelidze,
Christian
Giaume, and
Kenna D.
Peusner
Department of Anatomy and Cell Biology, and Neuroscience Program,
George Washington University Medical Center, Washington, DC 20037
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ABSTRACT |
The principal cells of the chick tangential nucleus are
second-order sensory neurons that participate in the three-neuron vestibulo-ocular and vestibulocollic reflexes. In postnatal animals, second-order vestibular neurons fire repetitively on depolarization. Previous studies have shown that, although this is an important feature
for normal reflex function, it is only acquired gradually during
embryonic development. Whereas at 13 embryonic days (E13) the principal
cells accommodate after firing a single spike, at E16 a few principal
cells repetitively can fire multiple action potentials on
depolarization. Finally, in the hatchling, the vast majority of
principal cells is capable of nonaccommodating firing on
depolarization. As a first step in understanding the mechanisms underlying developmental change in excitability of these second-order vestibular neurons, we analyzed the outward potassium currents and
their role in accommodation, using brainstem slices at E16. The
principal cells exhibited transient and sustained potassium currents,
with both of these containing calcium-dependent components. Further,
both high- and low-threshold sustained potassium currents have been
distinguished. The low-threshold dendrotoxin-sensitive sustained
potassium current (IDS) is associated
with principal cells that accommodate and is not expressed in those
that fire repetitively. Finally, blocking of
IDS transforms accommodating cells into
neurons capable of firing trains of action potentials on
depolarization. These findings indicate that suppression of IDS during development is sufficient to
transform accommodating principal cells into nonaccommodating firing
neurons and suggests that developmental regulation of this current is
necessary for the establishment of normal vestibular function.
Key words:
dendrotoxin; potassium current; central vestibular
neurons; chick embryo; excitability; firing pattern
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INTRODUCTION |
The three-neuron vestibulo-ocular
and vestibulocollic reflexes represent the most direct vestibular
pathways involved in the stabilization of gaze during head and body
movements (Kelly, 1991 ). The chick tangential nucleus, located in the
lateral part of medulla oblongata, participates in the central segment
of this circuit (Cox and Peusner, 1990). The bodies of the major cell
population of the tangential nucleus, the principal cells (80%),
receive large calyx-like terminals from the largest-diameter primary
vestibular fibers, the colossal fibers (see Fig. 1A)
(Peusner and Morest, 1977). The axons of the principal cells innervate
motor neurons situated at cervical levels of the spinal cord (Gross and
Oppenheim, 1985; Cox and Peusner, 1990) and also in brainstem
oculomotor nuclei, including the oculomotor (Wold, 1978), trochlear
(Labandeira-Garcia et al., 1989), and abducens nuclei (Cox and Peusner,
1990). Thus, these second-order vestibular neurons relay signals in
both the vestibulo-ocular and the vestibulocollic reflex pathways.
From studies of brainstem slices of postnatal chicks, it appears
that second-order vestibular neurons are capable of sustained, nonaccommodating firing of trains of action potentials on
depolarization (du Lac and Lisberger, 1995a,b; Peusner and Giaume,
1997). During vestibulo-ocular and vestibulocollic behavior these
vestibular neurons appear to encode information on firing rate (Chubb
et al., 1984; Lisberger et al., 1994; Ris et al., 1995; Boyle et al.,
1996; Phillips et al., 1996). Apparently, the ability of nonaccommodating firing of action potentials by second-order vestibular neurons is essential for the normal function of these reflexes (du Lac
and Lisberger, 1995a). Previously, it has been shown (Peusner and
Giaume, 1997) that the principal cells gradually acquire the ability of
nonaccommodating firing of action potentials during chick embryonic
development. At embryonic day 13 (E13) the principal cells accommodate
after firing a single action potential, whereas E15-E16 appears to
represent a turning point when only a few principal cells exist that
can fire multiple action potentials. Finally, in the hatchling, the
vast majority of principal cells fires repetitively on depolarization
(Peusner and Giaume, 1997).
In various nuclei of the central auditory pathway, it has been found
that potassium currents play an essential role in determining whether
these neurons are capable of firing trains of action potentials or of
accommodating (Manis and Marx, 1991; Banks and Smith, 1992; Forsythe
and Barnes-Davies, 1993; Brew and Forsythe, 1995). As a first step in
understanding the mechanisms underlying developmental changes in the
excitability of second-order vestibular neurons, we have analyzed
outward potassium currents and their role in accommodation of the
principal cells at E16. We have found that the principal cells possess
transient and sustained potassium currents and that both of these
currents exhibit calcium-dependent components. Further, both high- and
low-threshold sustained potassium currents are present in these cells.
The low-threshold, dendrotoxin-sensitive sustained potassium current
(IDS) is associated with accommodating principal cells and is not expressed in those principal cells that fire
multiple spikes on depolarization. Finally, block of IDS transforms neurons firing a single spike
into those capable of nonaccommodating firing of action potentials.
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MATERIALS AND METHODS |
Preparation and solutions. All observations were made
on 16-d-old white Leghorn chick embryos (Gallus gallus)
obtained from Truslow farms (Chestertown, MD) as eggs that were
incubated in the laboratory until the desired age. The age of the
embryos was determined by reference to the staging criteria of
Hamburger and Hamilton (1951).
The basic techniques for the preparation of brain slices of the
vestibular nuclei were followed as already described (Peusner and
Giaume, 1989, 1994). The protocols applied to animals were approved by
the Institutional Animal Care and Use Committee of the George
Washington University. Embryos were cooled to room temperature, removed
from the egg, and decapitated. The medulla was sectioned transversely
at 300 µm thickness in chilled (5°C) artificial CSF (ACSF) with a
microslicer (DSK, model 3000, Ted Pella, Redding, CA). ACSF solution
(in mM) contained 124 NaCl, 5 KCl, 2.2 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose. The pH of this solution was 7.3-7.4 after saturation with 95% O2/5% CO2 at 30-31°C.
Slice perfusion and microscopy. Slices containing the
tangential nucleus were submerged in a small glass-bottom recording chamber (volume, 250 µl; Warner Instrument, Hamden, CT) and held in
place by nylon threads glued to a U-shaped, flattened platinum wire.
The slices were perfused with heated ACSF (30-31°C) at a rate 1-2
ml/min. After the slices were incubated in the chamber, they were
allowed to equilibrate for at least 1 hr before recording was started.
The slices were viewed with the aid of a fixed-stage upright microscope
(Axioskop FS, Zeiss Instruments, Jena, Germany) equipped with
differential interference contrast (DIC) Nomarski optics and a 40×
water immersion lens (0.75 numerical aperture) with a 1.9 mm working
distance (Zeiss Instruments). Visualization of the recorded neuron and
pipette movement was achieved by using an infrared light source (filter
lmax = 770 nm), which was detected by an
infrared-sensitive camera (Vidicon C2400-01; Hamamatsu, Hamamatsu City,
Japan) and observed on a monitor (Sony, Tokyo, Japan). The microscope
image was magnified further by a 4× lens placed between the microscope
and the camera. Image contrast and shading were adjusted with a camera
controller (Hamamatsu C2400), and prints were made on a videographic
printer (Sony UP-890MD).
In experiments using 2 mM CoCl2,
CaCl2 was removed to assist in blocking the calcium
currents, and NaH2PO4 was removed to prevent
its precipitation with cobalt. In experiments using 300 µM CdCl2, both
NaH2PO4 and MgSO4 were removed to
avoid their precipitation with cadmium. Finally, in solutions
containing 0 mM Ca, 5 mM MgCl2 was
added. When >2 mM changes were made in the total
concentration of the solution, NaCl was adjusted in equinormal amounts.
All of the drugs were prepared daily by dissolving them in ACSF and then adding them to the bath to achieve their final concentrations. Tetraethylammonium chloride (TEA; Sigma, St. Louis, MO) was applied at
final concentrations of 1, 3, and 10 mM (Spigelman et al., 1992), and 4-aminopyridine (4-AP; Sigma) was applied at a final concentration of 100 µM (Halliwell et al., 1986).
Tetrodotoxin (TTX) and dendrotoxin (DTX; Research Biochemicals, Natick,
MA) were applied at final concentrations of 1 µM and 100, 200, and 400 nM (Halliwell et al., 1986), respectively.
Muscarin (Sigma) was applied at a final concentration of 20 µM (Halliwell and Adams, 1982). TTX (1 µM)
was used routinely in the voltage-clamp experiments to block
voltage-dependent Na+ currents, unless otherwise
stated.
Electrophysiology. Recording pipettes were pulled from 1.5 mm (external diameter) thin-walled borosilicate glass tubing (World Precision Instruments, Sarasota, FL) with a horizontal pipette puller
(model P-87, Sutter Instruments, Novato, CA). Recording pipettes (1-3
M ) contained the following solution (in mM): 140 KCl,
1.1 EGTA, 10 HEPES, 0.1 CaCl2, and 2 Mg-ATP. The pH
of the solution was adjusted to 7.2 with KOH (8.0N), resulting in a
final intrapipette [K+] of ~148 mM;
the osmolarity was 270-280 mOsm. The K+ equilibrium
potential (Ek) was calculated from the
Nernst equation to be  88 mV, assuming the complete exchange between
the intracellular and intrapipette contents. In experiments in which
Ca2+ was buffered and
Ca2+-dependent currents were blocked, EGTA was
increased to 10 mM, KCl was decreased to 130 mM, and CaCl2 was removed from the recording pipette solution. Before recordings were performed, the pipette tips
were covered with Sigmacote (Sigma) and allowed to air dry for a few
seconds, which helped to decrease the pipette capacitance.
The Axopatch 1D amplifier (Axon Instruments, Foster City, CA) was
used for recording in whole-cell voltage-clamp and in current-clamp modes, as already described (Sakmann and Stuart, 1995). To obtain a
high-resistance (G) seal, we advanced the recording pipettes through
the slice under positive pressure, using a piezoelectric patch-clamp
manipulator (model PCS-250, Burleigh, Fishers, NY). The pipette was
clamped onto the soma of the neuron at an ~45° angle under visual
guidance. Then, negative pressure was applied to form a cell-electrode
seal (>3 G); finally, further suction was applied to break through
the membrane. The formation of the seal and breaking through the
membrane were monitored on an oscilloscope (model PM3350A, Philips
Electronic, Eindhoven, The Netherlands) or on a computer (Gateway 2000, Pentium 100, N. Sioux City, SD) display by observing the current
response to a hyperpolarizing voltage step of 1-2 mV. After the
whole-cell recording mode was established, the resting membrane
potential of the cell was recorded. The series resistance was
calculated by adjusting the Cs (cell capacitance) and Rs (series resistance)
potentiometers on the amplifier (Jackson, 1992). The series resistance
was 7.8 ± 0.2 M (n = 50). The
Rs compensation was set at 80% (lag, 10 µsec). During whole-cell recordings the series resistance was
monitored frequently and kept as stable as possible.
Characteristically, for whole-cell recordings in brain slices the
voltage could be clamped effectively only in the somata and in the
proximal parts of the dendrites and axon, so we assume that some
distortion in the recorded currents was caused by the absence of good
space clamp. However, this restriction does not affect the main
conclusions of this article, which emphasize qualitative distinctions
between the conductance types.
In voltage-clamp experiments the holding potential was 60 mV. No leak
current was subtracted from the current traces. The input resistance of
the neurons was measured in voltage-clamp experiments by stepping the
membrane potential between 70 and 80 mV, with 5 mV increments,
where the leak current is predominant. In current-clamp experiments in
which the threshold for the generation of action potentials was
measured, the resting membrane potential of the tested neurons was
between 64 and 69 mV, including the liquid junction potential. In
those experiments in which the neurons were depolarized, they were held
in this range with hyperpolarizing currents. However, those neurons
with resting membrane potentials more positive than 53 mV, including
the liquid junction potential, were excluded from the analysis. The
threshold for action potential generation was considered to be the
membrane potential at which at least one spike was generated during
gradual depolarization, using 0.1 or 0.3 nA (300 msec) current steps.
The membrane potential was measured at the end of the current step
where it was most stable. In current-clamp experiments the bridge
circuit was adjusted and checked frequently. Grounding was performed
with an Ag-AgCl reference electrode, or a 1% agar/ACSF bridge was
used to complete the circuit between ACSF and the reference
electrode.
Dye injections and histological processing. The morphology
of the recorded neuron was verified by allowing 0.1-0.2% Lucifer yellow (Sigma) to diffuse through the pipette into the cell. The slices
with Lucifer yellow-filled cells were fixed in 4% paraformaldehyde in
0.1 M PBS, pH 7.4, and refrigerated overnight before being transferred to a solution of 0.1 M PBS for 1 d. The
slices were cleared of water by using the solvent dimethyl sulfoxide
(DMSO; Sigma), as previously described (Grace and Llinás, 1985).
Stained neurons were observed and photographed on a Nikon Optiphot
light microscope equipped with fluorescent attachments and a Nikon 35 mm camera.
Data acquisition and analysis. No correction was
applied to the voltage offset observed after withdrawing the pipette
from the cell ( 2 mV). The correction for the liquid junction
potential between the intrapipette and external solutions was obtained
by placing the recording pipette in ACSF, zeroing the voltage, and measuring the potential shift produced after replacing the bath ACSF
with the pipette solution. A saturated KCl reference electrode was used
in this procedure to eliminate the change in reference electrode
potential (Spigelman et al., 1992). No correction was applied to the
data for the +3 mV potential shifts, unless stated otherwise. Decay
time constants ( ) were determined by fitting (using a simplex
algorithm) current recordings with single, double, or triple
exponential functions of the form I = A0 + A1
exp(t/1) + A2
exp(t/2) + A3
exp(t/3), where
A0 to A3 are amplitude coefficients and 1, 2, and
3 are the time constants. Reversal potentials were
determined by the analysis of the tail currents. The tail currents were
fit to the exponential functions with one or two time constants. The
first 1 msec of the trace, which contains the capacitative charging
transient, was omitted from the fits. Then the instantaneous outward
current at the start of the step was determined by extrapolation of the
exponential function to the origin (Manis and Marx, 1991). The
steady-state activation or steady-state inactivation plot was fit to
the Boltzmann equation in the form of
G/Gmax = {1 + exp[(V1/2 V)/K]} 1, where
G is the conductance calculated by dividing the measured peak current by the driving force (V Ek) and is normalized to its maximum
value, Gmax; V is the step
potential; V1/2 is the membrane potential at
half-maximal conductance; and K is a constant describing the
steepness of activation or inactivation. Continuous lines were fit to
the data points by a Marquart-Levenberg algorithm (Spigelman et al.,
1992). All of the data were stored and analyzed by pCLAMP (program
6.0.3; Axon Instruments) and Sigmaplot 3.0 software (Jandel Scientific,
San Rafael, CA). Current-clamp data were digitized at 10 kHz and
filtered at 5 kHz, whereas the voltage-clamp data were digitized at
1-5 kHz and filtered at 1-2 kHz. All of the data values are given as
the mean ± SEM. Group differences were analyzed with a Student's
t test for independent samples and considered to be
significant with p < 0.05.
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RESULTS |
The data presented in this paper were obtained from 89 cells,
clearly identified as principal cells of the tangential nucleus by
visualizing with an infrared camera their oval somata, which were
situated between the primary vestibular fibers (Fig.
1B). Further
identification was made by examining Lucifer yellow-filled principal
cells. From this and previous work using Lucifer yellow and biocytin
(Peusner and Giaume, 1989, 1994), it is known that the principal cells
have different dendritic branching patterns. Some cells exhibit a
single lateral dendrite (Fig. 1C), whereas most have
multiple dendrites radiating in at least four directions. In this study
the axons were always observed to course in a medial direction (Fig.
1C).
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Figure 1.
A, Diagram of chick tangential
nucleus and peripheral vestibular apparatus. Somata of first-order
vestibular neurons are located in the vestibular ganglia
(VG) and relay impulses from hair cells in the
vestibular epithelium (VE) to second-order vestibular
neurons located in the tangential nucleus (TN).
The largest vestibular afferents, the colossal fibers
(CF), form large calyx-like spoon terminals on
the principal cells (PC) of the nucleus. The elongate cells (EC) compose <20% of the tangential neurons and
receive input only from the small vestibular afferents
(SVA). Vc, Voltage command.
B, Infrared camera image of a principal cell
(PC) during a recording. A single arrow
indicates the patch electrode, and the double arrows
indicate a colossal fiber. Calibration bar, 15 µm. C,
A principal cell after Lucifer yellow injection. Different cells are
shown in B and C. D,
Dendrite; Ax, axon. Calibration bar, 30 µm.
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The criteria applied to select principal cells for analysis included a
stable resting membrane potential of at least 53 mV, including the
liquid junction potential. The resting membrane potential of the
principal cells after adjusting the liquid junction potential was
66 ± 0.6 mV (n = 89), and the input resistance (Rn) was 74 ± 4 M
(n = 30). None of the principal cells fired spontaneously. When depolarizing current steps were applied, three subpopulations of principal cells were distinguished in this study. The
large majority (70%, 62 of 89) responded to depolarizing current injections by firing a single spike (accommodating principal cells) (Fig. 2A). The
threshold for spike initiation was 37 ± 2 mV (n = 20), including the liquid junction potential. In 13% (12 of 89) of
the principal cells, an action potential could not be initiated from
the resting membrane potential, even after large depolarizations (silent principal cells; Fig. 2B). In contrast, 17%
(15 of 89) of the principal cells were capable of firing multiple
spikes in response to depolarizing current injections (nonaccommodating principal cells; Fig. 2C), and their threshold (48 ± 2 mV, including the liquid junction potential; n = 10)
for activation of the action potential was significantly
(p < 0.05) lower than that observed for the
principal cells firing a single spike. From Lucifer yellow injections
no significant differences in the morphology of these three
physiologically distinctive populations of principal cells were
observed. Furthermore, these subpopulations of principal cells
exhibited no significant differences in either their resting membrane
potentials or input resistances. Because the vast majority of principal
cells at E16 belongs to the subclass of cells firing only one spike in
response to depolarizing current, this study is focused on identifying
the membrane currents underlying this distinctive firing pattern.
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Figure 2.
Current-clamp recordings of the responses to
depolarization pulses of physiologically different subpopulations of
principal cells. A, The vast majority of principal cells
responds to current injection (bottom traces) by firing
a single action potential, followed by accommodation (top
traces). B, Silent principal cells do not fire
action potentials from resting membrane potential, even after a large
depolarization. C, Some principal cells can fire
multiple action potentials on depolarization. Calibration bars apply to
A-C.
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Separation of the outward and inward currents
In voltage-clamp experiments the depolarization of the
accommodating (n = 6) (Fig.
3A1) and nonaccommodating
principal cells (n = 4; data not shown) induced a
large, inward transient current on the order of 2 nA, which could be
blocked by 1 µM TTX (Fig. 3B). In addition, in
current-clamp experiments TTX blocked the generation of action
potentials in accommodating principal cells (n = 3;
data not shown). These experiments indicate that sodium channels are
responsible for the generation of action potentials in embryonic
principal cells. In those principal cells that did not generate action
potentials on depolarization after voltage steps more positive than
40 mV, these cells exhibited an inward deflection in the outward
current (Fig. 3A2), which was blocked by 1 µM
TTX (n = 2). The TTX effect suggests that the silent
principal cells also express sodium channels, which are insufficient to generate action potentials. In accommodating principal cells exposed to
TTX, the outward transient current was decreased significantly (Fig.
3B, inset), suggesting that principal cells possess a
Na+-dependent K+ current, as
described in avian brainstem neurons and peripheral ganglia (Bader et
al., 1985; Dryer et al., 1989). However, the presence of a space-clamp
problem in this preparation prevents drawing a definitive conclusion
(Dryer et al., 1989).
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Figure 3.
Whole-cell patch-clamp recordings of the inward
and outward currents. Current traces are shown at the same scale,
whereas the time base was varied, as indicated in the calibration bars. A1, A2, Current traces (bottom
traces) from accommodating (A1) and silent
principal cells (A2) elicited by 5 mV (20 msec) voltage steps from 35 to 20 mV (top traces), performed in
control ACSF. B, Current traces in accommodating
principal cell (bottom traces) elicited by 10 mV (80 msec) voltage steps from 130 mV to 0 voltage (top
traces in inset), performed in ACSF containing 1 µM TTX. Inset, Current-voltage curve of
peak current in accommodating principal cells, measured between 3 and 6 msec after onset of voltage steps in control ACSF ( )
(n = 4) and in the presence of 1 µM
TTX ( ) (n = 4). There was a significant
difference (p < 0.05) in outward current
amplitude from 30 to 0 mV before and after TTX application. This
suggests the presence of a Na-dependent outward current. All of the
subsequent recordings were performed in the presence of TTX.
C, Current traces (bottom traces)
elicited by using 600 msec voltage steps from 60 to 10 mV, with 10 mV increments preceded by a 200 msec prestep to 120 mV (top
traces). Transient and sustained components can be observed in
the current traces in addition to spontaneous EPSCs in
C-E. C-E are shown at the same time
base. D, Current traces (bottom traces)
elicited by using 600 msec voltage steps from 60 to 10 mV, with 10 mV increments. The steps are preceded by a 200 msec prestep to 40 mV
(top traces). The transient current was inactivated
entirely by the prestep. E, Transient current obtained
by subtraction of D from C.
F, Instantaneous current-voltage curve () obtained by extrapolating the tail current at the onset of the testing voltage
step, and the steady-state current-voltage curve () obtained by
measuring the current amplitude at the end of testing voltage steps.
The intersection of the two curves indicates the
reversal potential at 75 mV (78 mV after adjustment of the liquid
junction potential). Inset, Reversal of the tail
currents with voltage (bottom traces). The total current
was activated by a 6 msec voltage step to +20 mV, which was preceded by
a 200 msec prestep to 120 mV (top traces). Reversal of
the tail currents was analyzed by using 50 msec test potentials from
60 to 130 mV, with 10 mV increments.
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Some principal cells demonstrated inward rectification on
hyperpolarization, which activated at potentials more negative than 80 mV (Fig. 3B). Leak conductance was predominant between
60 and 80 mV. We considered a cell to be damaged if the amplitude of the leak current induced by stepping the membrane potential from
70 to 80 mV exceeded 0.3 nA, and these cells were excluded from
further analysis.
All of the principal cells exhibited outward currents composed of both
transient and sustained components in the presence of TTX (Fig.
3B,C). The separation of the outward transient and sustained
currents was achieved with a standard subtraction procedure that was
based on their different voltage dependencies (Spigelman et al., 1992;
Klee et al., 1995). After 200 or 800 msec presteps at potentials near
40 mV (Fig. 3D), only sustained currents were elicited,
and they were subtracted from the total currents activated after a 200 msec prestep to 120 mV (Fig. 3C) or after an 800 msec
prestep to 100 mV (n = 15). The isolated transient
currents are shown in Figure 3E. Because of run-down of the
calcium current and, subsequently, the calcium-dependent outward
sustained currents, a fraction of the sustained currents was present in
the remaining currents obtained by the subtraction procedure. Indeed,
in the presence of calcium channel blockers (Cd2+
and Co2+) or in 0 calcium solution, the sustained
currents were not observed in the isolated currents when 200 msec
presteps were used for separation (n = 5).
However, when 800 msec presteps were used, part of the DTX-sensitive
sustained current (IDS) inactivated as
well.
To characterize the ionic basis of the outward currents, we
investigated their reversal potential
(Er). An outward current, composed of
both transient and sustained components, was evoked by a 6 msec test
pulse to +20 mV, which was preceded by a prestep to 120 mV. Then the
cell was repolarized to various potentials (Fig. 3F,
inset). The value of Er was
determined from a plot of the current-voltage relationship by
identifying the membrane potential at which the instantaneous and
steady-state currents intersect (Fig. 3F) (Manis and
Marx, 1991). The instantaneous current-voltage relation is not linear,
which may be attributed to an underestimation of the currents at
negative potentials, where they decline rapidly (Fig.
3F). The reversal potential of the outward currents
of accommodating principal cells is 72 ± 2 mV, including the
liquid junction potential (n = 8). In two cases the
reversal potentials of the sustained currents were calculated. In these
cases a longer test step (150 msec) was applied, during which the
transient currents inactivated. The calculated values of
Er for the sustained currents were 75 and 68
mV, including the liquid junction potential, which are in good
agreement with the Er value for the total
outward currents (72 mV). The equilibrium potential for chloride
(ECl) in our recording solution was +1.3
mV. Thus, the calculated reversal potential for the outward currents is
near the theoretical equilibrium potential for potassium
(EK) of 88 mV. To investigate further the ionic species carrying the outward currents, we raised the extracellular potassium concentration from 5 to 50 mM. This
procedure shifted EK to 28 mV, and the outward
current Er shifted to 40 ± 3 mV,
including the liquid junction potential (n = 3). The
Er value of the outward currents in normal ACSF
and its shift in direction to the new EK in ACSF
containing a high potassium concentration suggest that at least part of
the outward current is carried by potassium ions in accommodating
principal cells. Also, when we tested the reversal potential of the
outward current in silent (n = 2) and in
nonaccommodating principal cells (n = 3), we observed no significant differences in the Er between
these subpopulations and the accommodating principal cells.
Transient outward current kinetics and its role in
spike generation
To investigate the role of the transient outward current in
accommodation and the ability of second-order vestibular neurons to
generate action potentials, we analyzed steady-state activation, inactivation, and the removal of inactivation from this current in the
different subpopulations of principal cells. The decay rate of the
transient current at 0 potential in every tested principal cell
(n = 13) was best fit by two or sometimes three
exponentials, which suggests that more than one type of channel is
responsible for the transient current (Hille, 1992). The slowest time
constant () was placed between 30 and 180 msec (n = 13). Steady-state activation of the transient current was investigated
by isolating this current from the total current, as already described
(Fig. 3C-E). Normalized conductance values for steady-state
activation of the transient current from accommodating principal cells
are shown in Figure 4A.
Steady-state inactivation of the transient current was investigated by
evoking currents with a fixed depolarizing command to +10 mV, which was
preceded by hyperpolarizing prepulses to various potentials (Fig.
4B, inset). Normalized conductance values
for steady-state inactivation of the transient current from
accommodating principal cells is shown in Figure 4B.
The steady-state activation and inactivation data obtained from the various subpopulations of principal cells were fit by the Boltzmann equation, and the values for V1/2 and
K have been calculated (Table 1). To test the removal of inactivation
from the transient current in accommodating and nonaccommodating
principal cells, we held the neurons for 200 msec at 40 mV to
inactivate the transient currents completely. Then hyperpolarizing
voltage commands of varying durations were applied, which were followed
by a fixed test pulse to +10 mV (Fig. 4C, inset).
Near-complete activation of the transient current was observed with
prepulse durations >10 msec. The time constant for removal of the
inactivation was obtained by fitting the data points with an
exponential function in the form (I Is)/(Imax Is) = [1 exp(t/a)], where (I Is)/(Imax Is) is the transient current amplitude
normalized to its maximum value (Imax Is), Is is the
sustained current amplitude, t is the prepulse duration, and
a is the time constant for removal of inactivation (Fig.
4C). In those principal cells that fire either single or
multiple action potentials, the mean time constant for removal of
inactivation was 3.3 ± 0.6 msec (n = 5) or
3.1 ± 0.6 msec (n = 3), respectively. No
significant difference was found among the kinetics of steady-state
inactivation, activation, or removal of inactivation of the transient
currents from these different subpopulations of principal cells.
Further, in the different subpopulations we have not observed
significant differences in the amplitude of the transient current,
which could vary from 0.6 to 3 nA. The similarities in the kinetics of
the transient currents observed in the different subpopulations of the
principal cells suggest that this current does not contribute to the
accommodation response observed in most of the principal cells. In
fact, in current-clamp experiments the depolarization of the principal cells with current steps (from +0.1 to +1.6 nA), which were preceded by
300-800 msec presteps to 40 mV that entirely inactivated the transient current, did not alter the accommodation response for these
cells. They were still capable of firing only a single action potential
(n = 4), and we did not observe a change in their
firing threshold. However, in the case of the silent cells, after
presteps to 40 mV, depolarization could trigger a single action
potential (Fig. 5A,B)
(n = 3). These experiments strongly suggest that the transient current is not responsible for the accommodation observed in
the majority of principal cells. However, a small group of principal
cells, the silent ones, may be prevented from firing a single action
potential by the transient current.
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Figure 4.
A, Steady-state activation of the
transient current from an accommodating principal cell, plotted as a
function of the membrane potential. Averaged, normalized conductance
values were fit with the Boltzmann equation. Conductance
(G) values were calculated by dividing the
measured peak transient current by the driving force
(V Ek). The
dotted line indicates V1/2 = 29 ± 3 mV. B, Steady-state inactivation of the
transient current. Averaged, normalized conductance values were fit
with the Boltzmann equation. Normalized conductance values
for the transient current were obtained by measuring the peak amplitude
of the total current (I), subtracting the
sustained current amplitude (Is), and
then dividing by the total current maximum amplitude
(Imax) minus the sustained current (Is). The dotted line
indicates V1/2 = 58 ± 1.2 mV.
Inset, Current traces (bottom traces)
elicited by voltage steps to + 10 mV, which were preceded by 200 msec voltage presteps from 30 to 100 mV, with 10 mV increments
(top traces). C, Plot of averaged,
normalized peak transient current versus the duration of the
hyperpolarizing prepulse, fit with an exponential function.
Inset, Transient current traces (bottom
traces) elicited by stepping the membrane potential to +10,
preceded by different duration hyperpolarizing presteps to 120 mV,
which were preceded by a 200 msec prepotential to 40 mV. The
normalized transient current value was calculated from the total peak
current (I) by subtracting the sustained
current (Is) and dividing by the
total current maximum value (Imax)
minus the sustained current
(Is).
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Figure 5.
Current-clamp recordings of the effect of
transient current inactivation on action potential generation in a
silent principal cell. A, Depolarization of the silent
principal cell (top trace) elicited by 300 msec current
injection (2 nA; bottom trace) from its
resting membrane potential (67 mV, including the liquid junction potential) to approximately 10 mV did not generate an action potential. B, After a 300 msec prestep close to 40 mV
(induced by 0.28 nA holding current), the same depolarization could
trigger a single action potential.
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Sustained outward currents
To determine whether the sustained outward current contributes to
the accommodation observed in most principal cells, we compared the
amplitudes of sustained currents obtained from accommodating and
nonaccommodating principal cells. Although there was no significant difference in current values obtained at voltages more negative than
60 mV, significant differences (p < 0.05)
were observed in the amplitudes of the sustained outward currents that
activated at potentials between 60 and 0 mV (Fig.
6). This suggests that these two groups
of principal cells express different sustained outward currents, which
could contribute to their distinctive firing patterns. Accordingly,
further identification of the sustained outward currents was
performed.
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Figure 6.
Current-voltage curves of the sustained currents
observed for repetitive firing (, n = 8) and
accommodating principal cells (, n = 5). The
sustained current amplitude was measured 150 msec after the onset of
the voltage steps from the 60 mV holding potential (see
inset). The current amplitudes between 60 and 0 mV
potentials were significantly different (p < 0.05). An asterisk indicates points of significant
difference. Inset, An example of current recordings
(bottom traces) from an accommodating principal cell used to draw the I-V curve (an arrow
indicates the point at which the current amplitude was measured). The
currents were induced by stepping the membrane potential from 120 mV
to 0 potential with 10 mV increments (top trace).
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Calcium-dependent potassium currents
The presence of a calcium-dependent outward potassium
current was investigated in accommodating principal cells by using ACSF solution containing (1) 300 µM CdCl2
(n = 5), (2) 0 Ca2+ and 2 mM cobalt (n = 3), or (3) 0 Ca2+ (n = 5). In all of these
modified ACSF solutions both the sustained and the transient currents
were reduced in amplitude (Fig. 7). The
plot of the normalized amplitude of the sustained current in
accommodating principal cells before and after exposure to modified
ACSF (Fig. 7B) suggests that this subpopulation of principal cells expresses a calcium-dependent sustained potassium current, which
activates at potentials more positive than 20 mV. To rule out the
possibility that the apparent decrease of the sustained current
amplitude in modified ACSF was attributable to a shift in its
steady-state activation curve along the voltage axis, we plotted
normalized conductances versus membrane potential in normal and
modified ACSF (Fig. 7B, inset). When the
Boltzmann equation was fit to the data (V1/2 = 28.7 ± 3 mV, K = 13.4 ± 0.4 in normal ACSF; V1/2 = 26.4 ± 2 mV,
K = 14.3 ± 0.6 mV in modified ACSF), we did not
observe a significant shift of the steady-state activation curve. This
finding further supports the conclusion that the decrease of the
sustained current amplitude in modified ACSF was attributable to
blocking a calcium-dependent sustained current. In contrast, the
decrease of the transient current amplitude in the presence of modified
ACSF solution, containing the divalent cations Cd2+
or Co2+, was associated with shifts in both the
steady-state activation and inactivation curves (n = 5), which may reflect the interaction of these divalent cations with
the transient current channels (Mayer and Sugiyama, 1988). However, the
0 Ca2+-modified ACSF did not cause a shift in either
the steady-state activation or inactivation curves but did produce a
decrease in the transient current amplitude (Fig. 7C),
suggesting that part of this current is calcium-dependent. Finally, the
absence of good space clamp in this preparation makes it difficult to
draw a definite conclusion (Rudy, 1988). Using calcium-free and
cobalt-containing ACSF, we also identified a calcium-dependent
sustained potassium current in the silent (n = 2) and
nonaccommodating (n = 2) principal cells (data not
shown).
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Figure 7.
Ca2+-dependent outward currents
in accommodating principal cells. A, Current traces
(bottom traces) elicited in normal
(Control), 0 Ca2+ ACSF, and
wash-out solution, using 200 msec voltage steps to +10 mV, preceded by
a 200 msec voltage prestep to 100 mV (top traces).
B, Averaged, normalized sustained current amplitude in 0 Ca+2 ACSF (n = 5), 3 mM CoCl2 ACSF (n = 5), or
300 µM CdCl2 ACSF (, n = 3) and in normal ACSF (, n = 13) versus
membrane potential. The currents were measured at the end of 600 msec
voltage steps applied with 10 mV increments from 70 to +20 mV,
preceded by a 800 msec prestep to 100 mV. There were significant
differences (p < 0.05) between the current
amplitudes started at 10 mV and continuing through +20 mV.
X indicates points of significant difference. Inset, Plot of averaged, normalized conductances versus
membrane potential for the same principal cells as shown in the
I-V curves in normal () and modified ACSF (). The
Boltzmann equation was fit to the data (V1/2 = 28.7 ± 3 mV, K = 13.4 ± 0.4 in
normal ACSF and V1/2 = 26.4 ± 2 mV,
K = 14.3 ± 0.6 mV) in modified ACSF. The
liquid junction potential was included. The current-amplitude measurements necessary for the conductance calculations were the same
ones used to draw the I-V curves.
C1-C3, Transient current traces (bottom
traces) from the same cell as shown in A,
induced by stepping the membrane potential to +10 mV and preceded
by 200 msec presteps to 60, 70, and 80 mV (top
traces); C1, performed in normal ACSF
(Control); C2, in 0 Ca2+ ACSF; C3, in control
solution (Wash out).
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To investigate the role of calcium-dependent potassium currents
in the accommodation of the principal cells, we examined action potentials firing on depolarization in calcium-free ACSF or in calcium-free and cobalt-containing ACSF. The EGTA concentration in the
recording pipette was increased from 1.1 to 10 mM to
enhance the calcium buffering inside the cells. During recording in
normal ACSF with pipettes containing a high concentration of EGTA,
accommodation was still observed (n = 10). Furthermore,
long exposures (20 min) to modified ACSF did not alter the response to
depolarization of accommodating or silent principal cells. For this
purpose, recordings were performed in the absence of external calcium
for both classes of cells (silent cells, n = 4;
accommodating cells, n = 3) and by substituting
Co2+ for Ca2+ in ACSF for
accommodating cells (n = 3). Together, these
observations indicate that the calcium-dependent potassium currents
were not involved in the observed accommodation and were not
responsible for the silence of some principal cells.
Subsequent analyses of the sustained currents were performed in
calcium-free ACSF with recording pipettes containing 10 mM EGTA to avoid interference from run-down of the calcium-dependent currents and also to block synaptic transmission.
High-threshold sustained potassium current
A high-threshold, calcium-independent sustained current
was identified in the principal cells by using 1-3 mM TEA.
In those principal cells that fire either a single spike
(n = 6) or none at all (n = 4), TEA
blocked reversibly a slowly activating, noninactivating portion of the
outward potassium current (Fig.
8A-C). Even during a
16 sec voltage step the TEA-sensitive current, activated by stepping
the membrane potential to 10 mV, did notdisplay significant inactivation (n = 2; data not shown). In accommodating
and silent principal cells, TEA blocked similar portions of the
current. Therefore, the data for steady-state activation from these two subpopulations have been combined (Fig. 8D). The
high-threshold, TEA-sensitive current activated at po-tentials between
45 and 35 mV; the V1/2 for activation was
19 ± 2.2 mV, including the liquid junction potential, and
K was 5.3 ± 0.5 mV (n = 10). Also, we
have observed high-threshold, TEA-sensitive current in nonaccommodating principal cells (n = 2).
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Figure 8.
Effect of tetraethylammonium (TEA)
on the outward currents in accommodating and silent principal cells.
Shown are outward currents (bottom traces) in 0 Ca
2+ ACSF (A, Control)
and in 0 Ca 2+ ACSF containing 3 mM TEA
(B). The currents were induced by using 600 msec
voltage steps from 80 to 10 mV, with 10 mV increments preceded by
an 800 msec prestep to 100 mV (top traces).
C, Current traces obtained by subtraction of
B from A. D, Steady-state
activation of averaged (n = 10) and normalized
TEA-sensitive current as a function of membrane potential. The
Boltzmann equation was fit to the data. The TEA-sensitive current
amplitude, necessary for the conductance calculation, was measured 300 msec from the onset of the voltage steps. The dotted
line indicates V1/2 = 16 ± 2.2 mV.
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In current-clamp experiments the exposure of the accommodating
(n = 3) or silent principal cells (n = 3) to calcium-free, cobalt-containing or to a calcium-free ACSF that
also contained 3 mM TEA did not alter their responses to
depolarization. The continued accommodation of these principal cells
treated with modified ACSF and TEA was not attributable to damage,
because in the presence of 200 nM DTX these neurons could
fire trains of action potentials on depolarization (n = 2; data not shown). Thus, the current-clamp data suggest that
calcium-dependent and calcium-independent TEA-sensitive currents do not
play a major role in accommodation of the principal cells.
Low-threshold potassium current
In different slice preparations DTX in nanomolar concentrations
has been demonstrated to block specifically a low-threshold potassium
current (Halliwell et al., 1986; Brew and Forsythe, 1995). In the
present study the results obtained by the application of various
concentrations (100, 200, and 400 nM) of DTX (synthesized  -DTX) indicate that both accommodating (n = 10) and
silent principal cells (n = 4) possess a DTX-sensitive,
low-threshold potassium current. Indeed, DTX blocked part of the
sustained and a small portion of the transient current in these cells
(Fig. 9A-C), suggesting that
possibly more than one type of DTX-sensitive channel is present in
these neurons. In all cases the effect of DTX was irreversible, as
reported in other preparations (Halliwell et al., 1986; Brew and
Forsythe, 1995). Further analysis of the DTX-sensitive current in
silent and accommodating principal cells indicated that there was no
significant difference (p > 0.05) in the effect
of 200 versus 400 nM concentrations. Moreover, at 30 mV,
200 and 400 nM DTX blocked 45.2 ± 3.7% of the
sustained current (measured 300 msec after the start of the step;
n = 10) in these cell groups. Although the 100 nM DTX seemed less effective, we were unable to measure
reliably the amplitude of the blocked current because of the relatively
long latency of this response at the low DTX dose (n = 3). At 40 and 50 mV, DTX reduced the sustained currents almost to
the level of the leak current (Figs. 9B,
10B), which suggests that the DTX-sensitive current
predominates at these potentials. The DTX-sensitive sustained potassium
current (IDS) activated between 55 and
65 mV (Fig. 9D). The kinetics of steady-state activation
of IDS observed in the accommodating and silent
principal cells were similar, and the data are summarized together
(Fig. 9D). The V1/2 for activation of
the IDS was 41.0 ± 2.1 mV, including the
liquid junction potential, and K was 8.1 ± 0.5 mV
(n = 10). The IDS was
inactivated only partly (63 ± 5.3%, n = 5)
during a 16 sec voltage steps to 30 mV (Fig. 9E). The
inactivation rate was fit well with a single exponential curve, and the
inactivation time constant was = 1.8 ± 0.2 sec
(n = 5; fit after the first 300 msec to exclude
interference from the fast transient current). To analyze the
steady-state inactivation of the IDS, we
partly inactivated the current by stepping the membrane potential to 30 mV for 8 sec. After inactivation the membrane potential was stepped from 40 to 110 mV with 10 mV increments for 1200 msec, which was followed by a 400 msec test voltage step to 30 mV (Fig. 9F). The inactivated sustained current was sensitive
to DTX, which established its identity as the
IDS current (n = 3). The
V1/2 for inactivation of
IDS was 69 ± 3 mV, including the liquid
junction potential, and K was 4.4 ± 1.8 mV (Fig.
9G) (n = 4). To avoid an incorrect estimate
of V1/2 caused by the partial inactivation of
the current, we fit an alternative inactivation curve, which assumed
that the IDS could achieve full inactivation
(Brew and Forsythe, 1995). We used the amplitude of the current after
exposure to 200 nM DTX as a hypothetical measure of the
fully inactivated IDS and fit the curve through
this point and the previous inactivation data. Current values were
included only after inactivating pulses more negative than 50 mV.
From this, the V1/2 for inactivation was
determined to be 63 ± 1 mV, including the liquid junction potential (n = 3). Therefore, it is likely that the
true V1/2 for inactivation is located between
63 and 69 mV, which suggests that ~50% of this current is
available at the resting membrane potential of the principal cells
(66 ± 0.6 mV). The removal of inactivation from
IDS appears to be a relatively slow process, because 200 msec hyperpolarizing voltage steps to 100 mV were not
sufficient to produce significant recovery from inactivation (n = 3). On the other hand, 1200 msec voltage steps
seemed sufficient for the full recovery of the
IDS from inactivation. Thus, it is likely that
the time constant of inactivation removal is located between 200 and
1200 msec.
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Figure 9.
Effect of dendrotoxin on the outward potassium
current in accommodating and silent principal cells. Shown is outward
potassium current (bottom traces) in 0 Ca2+ ACSF (A) and in 0 Ca2+ ACSF containing 200 nM DTX
(B). The currents were induced by using 600 msec
voltage steps from 80 to 20 mV, with 10 mV increments preceded by
an 800 msec prestep to 100 mV (top traces).
C, DTX-sensitive current obtained by the subtraction of
B from A. D, Steady-state activation of averaged and normalized IDS as
a function of membrane potential. The IDS
amplitude necessary for the conductance calculation was measured 300 msec from the onset of the voltage steps. The Boltzmann equation was
fit to the data. The dotted line indicates V1/2 = 38 ± 2.1 mV.
E, The DTX-sensitive current (bottom
trace) elicited by 16 sec voltage steps to 30 mV and preceded
by 800 msec presteps to 100 mV. The DTX-sensitive current was
obtained by subtracting the current traces obtained in the presence of 200 nM DTX from control traces recorded in 0 Ca2+ ACSF. F, To study steady-state
inactivation of IDS, we first kept
the membrane potential for 8 sec at 30 mV, which was followed by 1200 msec steps from 40 to 110 mV, with 10 mV increments. The current
amplitude (bottom traces) was tested by stepping for 400 msec to 30 mV (top trace). G, Averaged
(n = 4) and normalized steady-state inactivation of
the DTX-sensitive current as a function of membrane potential. The
current amplitudes (I and
Imax, maximum amplitude) were measured 300 msec from the voltage step onset, and the amplitude of the
noninactivating current (Inonin) was subtracted. The dotted line indicates
V1/2 = 66 ± 3 mV.
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Besides DTX, IDS also could be blocked in the
presence of 100 µM 4-AP in the accommodating principal
cells (n = 3; data not shown). In addition, when high
concentrations of TEA (10 mM) were applied, both high- and
low-threshold potassium currents were blocked (n = 3;
data not shown). However, in the presence of 200 nM DTX,
neither 4-AP (n = 2) nor 10 mM TEA
(n = 2) could reduce further the low-threshold current,
indicating that they act on the same type of channel as DTX (data not
shown). Exposure of the nonaccommodating principal cells to DTX (200 nM, n = 6; 400 nM,
n = 2) blocked only the small, transient portion of the
outward potassium current, which almost entirely inactivated during 300 msec (Fig. 10A). This
suggests that these principal cells do not possess
IDS. Furthermore, after exposure of
accommodating or silent principal cells to DTX, the amplitudes of their
sustained currents were not significantly different from firing
principal cells (Fig. 10B). This further suggests
that the differential possession of IDS is
likely to contribute to the distinct firing phenotype expressed in
these subpopulations of second-order vestibular neurons.
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Figure 10.
Effect of dendrotoxin on the outward currents in
principal cells firing multiple action potentials. A,
Current traces (bottom traces) induced by stepping the
membrane potential to 30 mV for 600 msec, which was preceded by an
800 msec prestep to 100 mV (top traces) in 0 Ca2+ ACSF (Control) and in 0 Ca2+ ACSF containing 200 nM DTX.
B, Current-voltage curve of the sustained currents from
(1) averaged, accommodating, and silent cell (n = 12) in control, 0 Ca2+ ACSF (); (2) averaged,
accommodating, and silent cell (n = 12) in 0 Ca2+ ACSF containing 200 nM DTX ( );
and (3) firing principal cells in control, 0 Ca2+
ACSF (). An asterisk indicates points of significant
difference between the combined data from the silent and accommodating
() versus nonaccommodating () principal cells. X
indicates points of significant difference for combined data from the
silent and accommodating principal cells before () and after ()
exposure to DTX. There was no significant difference in the currents
between the silent and accommodating principal cells exposed to DTX
() and the nonaccommodating cells (). The currents were elicited by using 600 msec voltage steps from 80 to 10 mV, with 10 mV increments, which were preceded by an 800 msec prestep to 100 mV. The
sustained current amplitude was measured 300 msec after the onset of
the voltage step.
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To address this question further, in current-clamp experiments we
investigated the effect of DTX on the firing pattern of principal
cells. The experiments were performed in ACSF containing normal
concentrations of calcium, and the recording pipettes contained 1.1 mM EGTA. Exposure of the accommodating (n = 6) or silent principal cells (n = 4) to 200 or 400 nM DTX transformed both classes of cells into neurons
firing multiple action potentials on depolarization, with frequencies
as high as 100 Hz in response to 1 nA currents (Fig.
11A-C). These action
potentials were blocked in the presence of 1 µM TTX
(n = 4), indicating that sodium channels were
responsible for the trains of spikes on depolarization in these
DTX-exposed principal cells. After exposure of the accommodating and
silent principal cells to DTX, the threshold for spike initiation was 50 ± 3 mV (n = 8), which is similar to that of
nonaccommodating principal cells (48 ± 2 mV; n = 8). Together, these observations indicate that the differential
presence in the principal cells of IDS is at
least partly responsible for the difference in the firing patterns of
this special class of developing vestibular neurons in the 16-d-old
chick embryo.
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Figure 11.
Dendrotoxin transforms accommodating and silent
principal cells into neurons capable of firing trains of spikes. Shown
are current-clamp recordings of an accommodating principal cell before (A) and after (B) 200 nM DTX exposure in normal ACSF. C, A plot of
the number of spikes in 300 msec versus injected current amplitude for
accommodating principal cells (, n = 5), firing
principal cells (, n = 5), accommodating
principal cells after exposure to DTX (, n = 5),
and silent principal cells ( , n=3) after exposure to
DTX.
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Finally, in some neurons, an M-current has been found to be responsible
for their accommodation response (Brown and Constanti, 1980). To test
for the presence of an M-current, we obtained voltage-clamp recordings
from accommodating principal cells exposed to 20 µM muscarin, which blocks the M-current in hippocampal slices (Halliwell and Adams, 1982). Applying the same voltage waveform used to test DTX
(Fig. 9A), we found that exposure of accommodating principal cells to muscarin (in calcium-free ACSF) did not produce a measurable change in the outward current amplitude (n = 3). This
finding suggests that the principal cells do not possess an
M-current.
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DISCUSSION |
The present work is a first attempt to analyze membrane currents
and their role in the excitability of embryonic second-order vestibular
neurons, using whole-cell voltage-clamp recordings. Previous analyses
of membrane currents in this class of vestibular neurons were performed
on postnatal animal preparations, using current-clamp recordings
(Serafin et al., 1991b; Johnston et al., 1994; Dutia et al., 1995; du
Lac, 1996) (for review, see Darlington et al., 1995). Our study
confirms a previous report (Peusner and Giaume, 1997) on the presence
of two distinct subpopulations of embryonic tangential principal cells
that fire either single or multiple action potentials on
depolarization. In addition, we have identified a third subpopulation
of silent principal cells, which on depolarization do not fire spikes
from resting membrane potential.
In principal cells the outward currents could be separated into
transient and sustained currents, which may exhibit both high- and
low-threshold components. Most important, we found that a low-threshold, DTX-sensitive sustained potassium current
(IDS) is expressed only in the subclasses
of silent and accommodating principal cells and that this current is
absent in principal cells capable of firing multiple spikes on
depolarization. We propose that the differential expression of
IDS contributes to the regulation of
excitability in the tangential principal cells by controlling their
firing rate in response to depolarization.
DTX-sensitive currents in other preparations
DTX-sensitive currents have been identified in isolated
embryonic neuron cultures prepared from rat hippocampus and chick ciliary ganglion (Wu and Barish, 1992; Wisgirda and Dryer, 1993). Like
the A-like current in the principal cells, the transient currents in
hippocampal neurons have both DTX-sensitive (1 µM) and
DTX-insensitive components (Wu and Barish, 1992). In neurons from the
chick ciliary ganglion, DTX (260-580 nM) suppressed
transient and sustained outward currents, which were not tested for
their effects on excitability (Wisgirda and Dryer, 1993). The present work is the first report on DTX-sensitive currents in embryonic neurons
studied in brain slice preparations. Neurons investigated from brain
slices are thought to be more similar to those studied in intact brain,
because slices retain considerably more tissue integrity than isolated
neuron cell cultures.
In postnatal animal preparations DTX-sensitive currents have been
characterized extensively. A low-threshold, DTX-sensitive sustained
current, also sensitive to a low concentration of 4-AP, was first
described in rat nodose ganglion (Stansfeld et al., 1986) and later in
neurons from slice preparations of the medial nucleus of the trapezoid
body (Forsythe and Barnes-Davies, 1993; Brew and Forsythe, 1995). In
both systems the DTX-sensitive current was found to block repetitive
firing on depolarization. In hippocampal slices a slowly inactivating
4-AP and DTX-sensitive current was responsible for a delayed spike
discharge on depolarization (Halliwell et al., 1986; Storm, 1988).
IDS identified in tangential principal cells was
similar in function and steady-state activation kinetics to those
DTX-sensitive currents, but it exhibited a partial and slower
inactivation. In fact, there was little inactivation during a 500 msec
voltage steps of IDS in principal cells, as
observed in dorsal root ganglion neurons (Penner et al., 1986). From
studies performed on rat neostriatum it was found that those neurons
possess a DTX-sensitive current responsible for a long latency to spike discharge (Nisenbaum et al., 1994). In neurons from the nucleus of the
lateral lemniscus (Wen Fu et al., 1996) and the ciliary ganglion
(Wisgirda and Dryer, 1993) high-threshold, DTX-sensitive currents were
identified, which probably play roles in the repolarization of action
potentials (Wen Fu et al., 1996). Finally, it has been found that
potassium channel proteins from the Shaker family, including
subunits Kv1.1 and Kv1.2, express high sensitivity to DTX (Grissmer et
al., 1994; Hopkins et al., 1994). From this, we suggest that the
presence of Kv1.1 and Kv1.2 in embryonic second-order vestibular
neurons may be responsible for generating DTX-sensitive currents.
Transient and high-threshold sustained currents in
other preparations
This is the first study reporting a direct identification in
second-order vestibular neurons of a transient A-like current, the
presence of which has been proposed from current-clamp recordings on
medial vestibular nucleus neurons in postnatal animals (Serafin et al.,
1991a,b; Johnston et al., 1994). In tangential principal cells the
A-like current possesses at least two components, as described in other
embryonic and postnatal systems (Zbicz and Weight, 1985; Storm, 1986;
Wu and Barish, 1992) (for review, see Rudy, 1988). The A-like current
may be responsible for the fast afterhyperpolarization observed in
postnatal vestibular neurons (Serafin et al., 1991a,b; Johnston et al.,
1994) and in our preparation. However, we cannot rule out a possible
role for sodium and calcium-dependent outward potassium currents in
this function. In hippocampal pyramidal and sympathetic ganglion cells,
calcium-dependent potassium currents have been shown to be responsible
for accommodation (Madison and Nicoll, 1982; Lancaster and Nicoll,
1987; Sacchi et al., 1995). However, calcium-dependent and
TEA-sensitive sustained potassium currents apparently do not play a
significant role in the accommodation of tangential principal cells,
perhaps because of their high thresholds of activation. We suggest that
the TEA-sensitive sustained current, the calcium-dependent potassium
currents, and the A-like current may all be involved in the
repolarization of the action potential, as already proposed in
postnatal neurons of the medial vestibular nucleus (Johnston et al.,
1994).
Role of A-like and IDS in
spike generation
In the majority of embryonic principal cells the A-like current
does not appear to play an important role in setting the action potential threshold or in accommodation, probably because of its relatively positive V1/2 for activation and its
fast inactivation. However, in the silent principal cells the
inactivation of the transient current and possibly a small portion of
the IDS by a depolarizing prestep could trigger
an action potential. So far, we cannot rule out that the silent
principal cells represent a developmentally less mature cell type,
which possesses sodium channels that are incapable of producing
sufficient inward current to overcome the A-like and
IDS and generate action potentials. In contrast
to the A-like current, IDS exhibits a more
negative V1/2, a steeper slope of
activation, and slower inactivation, which allow
IDS to regulate action potential threshold and
the firing of multiple spikes. This conclusion is supported further by
the finding that the nonaccommodating principal cells do not possess an
IDS. Although a contribution from inward
currents cannot be excluded, the difference in the expression of
IDS is sufficient to explain the distinction in
action potential threshold and in firing pattern between the
accommodating and nonaccommodating principal cells. The absence of
IDS from the firing principal cells is not only
sufficient but may be necessary for the acquisition of a repetitive
firing pattern. Indeed, it has been found that the expression of even
small amounts of a low-threshold sustained potassium current could
silence rhythmically discharging R15 neurons from mature
Aplysia (Zhao et al., 1994). Furthermore, from mathematical neuron models it appears that the presence or absence of the
low-threshold sustained potassium current is essential in determining
whether neurons exhibit accommodation or repetitive discharge to
depolarization (Del Negro and Chandler, 1997).
IDS and signal processing in the central
vestibular system
In the 16-d-old chick embryo the firing of multiple spikes on
depolarization by the vast majority of principal cells is actively blocked, suggesting that this property could be undesirable for these
developing second-order vestibular neurons at this stage. For example,
in Xenopus neurons of the neural plate it has been shown
that electrical activity plays a critical role in their normal
differentiation (Jones and Ribera, 1994). As shown for central auditory
neurons (Brew and Forsythe, 1995), IDS could allow tangential principal cells to follow closely the signals generated by incoming synaptic inputs, especially the large EPSPs generated by the spoon terminals, which could be important for establishing a preliminary connection between the peripheral vestibular receptor and these cells. Further, the simultaneous existence at one
age of three subpopulations of principal cells may be important for the
function of the vestibular system in the embryo (Rogers, 1996).
Finally, the few principal cells firing multiple action potentials on
depolarization at E16 resemble the majority of principal cells in young
hatchling chickens (Peusner and Giaume, 1997). This suggests that the
nonaccommodating principal cells at E16 are more mature developmentally
than the other two classes. We have found that the suppression of
IDS is sufficient and possibly necessary for the
transformation of accommodating and silent principal cells into the
more developmentally mature cells that fire repetitively. This implies
that IDS is regulated developmentally. In fact,
our preliminary data from hatchling chickens indicate that principal cells firing multiple spikes on depolarization lack
IDS (Gamkrelidze et al., 1997). In conclusion,
our data suggest that the developmental regulation of
IDS is probably essential for the establishment of normal function in the vestibular reflex pathways of the
chicken.
| |
FOOTNOTES |
Received Aug. 26, 1997; revised Nov. 17, 1997; accepted Nov. 20, 1997.
This work has been supported by the National Institute on Deafness and
Other Communication Disorders, National Institutes of Health, Grant RO1
DC00970.
Correspondence should be addressed to Dr. Kenna D. Peusner, Department
of Anatomy and Cell Biology, and Neuroscience Program, George
Washington University Medical Center, 2300 I Street, NW, Washington, DC
20037.
Dr. Giaume's present address: Institut National de la Santé et
de la Recherche Médicale (INSERM) U114, College de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France.
| |
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Top
Abstract
Introduction
