1Department of Biomedical Engineering,
2Department of Neuroscience, and
3Department of OtolaryngologyHead and Neck
Surgery, The Center for Hearing and Balance, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Molitor, Scott C. and
Paul B. Manis.
Voltage-gated Ca2+ conductances in acutely isolated guinea
pig dorsal cochlear nucleus neurons. Although it is known that
voltage-gated Ca2+ conductances (VGCCs) contribute to the
responses of dorsal cochlear nucleus (DCN) neurons, little is known
about the properties of VGCCs in the DCN. In this study, the whole cell
voltage-clamp technique was used to examine the pharmacology and
voltage dependence of VGCCs in unidentified DCN neurons acutely
isolated from guinea pig brain stem. The majority of cells responded to
depolarization with sustained inward currents that were enhanced when
Ca2+ was replaced by Ba2+, were blocked
partially by Ni2+ (100 µM), and were blocked almost
completely by Cd2+ (50 µM). Experiments using nifedipine
(10 µM), Aga IVA (100 nM) and
CTX GVIA (500 nM) demonstrated
that a variety of VGCC subtypes contributed to the Ba2+
current in most cells, including the L, N, and P/Q types and antagonist-insensitive R type. Although a large depolarization from
rest was required to activate VGCCs in DCN neurons, VGCC activation was
rapid at depolarized levels, having time constants <1 ms at 22°C. No
fast low-threshold inactivation was observed, and a slow high-threshold
inactivation was observed at voltages more positive than
20 mV,
indicating that Ba2+ currents were carried by high-voltage
activated VGCCs. The VGCC subtypes contributing to the overall
Ba2+ current had similar voltage-dependent properties, with
the exception of the antagonist-insensitive R-type component, which had
a slower activation and a more pronounced inactivation than the other
components. These data suggest that a variety of VGCCs is present in
DCN neurons, and these conductances generate a rapid Ca2+
influx in response to depolarizing stimuli.
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INTRODUCTION |
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Postsynaptic Ca2+ plays an important
role in many neuronal processes, including the activation of ionic
conductances (McManus 1991; Sah 1996
) and
synaptic plasticity (Linden 1994
; Nicoll and Malenka 1995
). Voltage-gated Ca2+ conductances
(VGCCs) provide a major source of postsynaptic Ca2+ influx
in response to synaptic input (Eilers et al. 1995
;
Jaffe et al. 1994
; Miyakawa et al. 1992
)
and during action potentials (Jaffe et al. 1992
;
Lev-Ram et al. 1992
; Markram et al.
1995
). Ca2+ imaging experiments in dorsal cochlear
nucleus (DCN) have revealed that action potentials elicit a
VGCC-mediated Ca2+ influx in the soma and proximal
dendrites of two major populations of DCN neurons (Manis and
Molitor 1996
; Molitor and Manis 1996
). VGCCs
also contribute to the electrogenic responses that determine the
discharge pattern of neurons during depolarization (Llinás 1988
) and may be involved in generating the intrinsic discharge patterns of DCN neurons. A slow depolarization resulting from VGCC
activation may be responsible for the complex spiking behavior of
cartwheel cells (Agar et al. 1996
; Manis et al.
1994
; Waller and Godfrey 1994
; Zhang and
Oertel 1993
). In addition, K+ channels activated by
a Ca2+ influx through VGCCs may be responsible for the slow
afterhyperpolarization observed in pyramidal cells after sustained
depolarization (Hirsch and Oertel 1988
; Manis
1990
). However, no studies have been performed that investigate
the biophysical and pharmacological properties of VGCCs that contribute
to Ca2+ influx and discharge patterns in DCN neurons.
Mammalian neurons possess a variety of VGCC subtypes that can be
distinguished by their voltage dependence and their sensitivity to
various pharmacological agents. VGCCs can be classified broadly in
terms of the voltage-dependent properties they exhibit: low-voltage activated (LVA), which activate near the resting potential and exhibit
a rapid inactivation over this voltage range (Huguenard 1996), and high-voltage activated (HVA), which activate at more depolarized voltages and exhibit a slow inactivation at these depolarized voltages (Bean 1989
; Tsien et al.
1988
). At the present, at least five HVA subtypes have been
found in the CNS: the dihydropyridine-sensitive L type, the
CTX
GVIA-sensitive N type (Fox et al. 1987
), the
Aga IVA
and
CTX MVIIC-sensitive P and Q types (Hillyard et al. 1992
; Llinás et al. 1989
; Mintz et
al. 1992b
), and a dihydropyridine- and peptide
toxin-insensitive R type, which may be a collection of one or more
additional VGCC subtypes (Randall and Tsien 1995
; Tottene et al. 1996
). The existence of multiple VGCC
subtypes within a single neuron could generate differential responses
at the synaptic level, having differences in their voltage dependence (Forti et al. 1994
; Fox et al. 1987
) or
in their responses to neurotransmitter modulation (Bean
1989
).
The present study addresses two objectives: to survey the VGCC subtypes present in DCN neurons and to estimate the types of stimuli that will generate a Ca2+ influx through VGCCs. To this end, we have investigated the pharmacology and voltage dependence of VGCCs in acutely isolated guinea pig DCN neurons using the whole cell voltage-clamp technique. Our results indicate that various HVA VGCCs are present in DCN neurons, and these conductances are capable of generating a large Ca2+ influx in response to action potentials. The Ca2+ influx and discharge patterns produced by VGCCs may play important roles in the neuronal processing that occurs in the DCN.
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METHODS |
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Acute cell isolation
Pigmented guinea pigs weighing 150-300 g were anesthetized with
pentobarbital (35-40 mg/kg), decapitated, and the brainstem was
removed quickly and placed into an oxygenated piperazine-N, N'-bis-(2-ethanesulfonic acid) (PIPES)-buffered dissection
solution at 30°C (see composition in Solutions). Using an
oscillating tissue slicer, the cochlear nuclei were cut along the
strial axis, parallel to the orientation of the parallel fibers, into
400-µm-thick slices. The DCN was isolated from the remainder of the
brain stem using Castroviejo scissors. DCN slices were placed in a
spinner flask containing oxygenated dissection solution with 0.67 mg/ml
of bovine pancreatic trypsin (Sigma, type XI) and 0.5 mg/ml bovine
serum albumin (BSA, Sigma, A7638) and were spun slowly (90 rpm) for 30 min. Enzymatically treated slices were thoroughly rinsed in enzyme-free
dissection solution with BSA and allowed to incubate for 1 h in this
solution. Before recording, two or three slices were triturated gently
in 0.3-0.5 ml of the dissection/BSA solution with a sequence of three
or four fire-polished pipettes having gradually decreasing diameters
(2-0.5 mm). Cells were plated on 35-mm culture dishes coated with 10 µg/ml of poly-D-lysine to promote cell adherence
(Yavin and Yavin 1974
). After waiting 10-15 min for
cells to adhere, a continuous flow of a
N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)-buffered recording solution (0.5 ml/min) was established to clear away debris and for the exchange of extracellular fluids. Fluid flow was maintained with gravity-fed lines and solution exchange
was performed using solenoid valves. Isolated cells were maintained at
room temperature (~22°C), and were viable for
2-3 h after plating.
Solutions
The dissection solution contained (in mM) 110 NaCl, 5 KCl, 25 glucose, 0.2 CaCl2, 4 MgCl2, and 20 PIPES, pH
7.0 with 5 M NaOH. A Na+/K+ recording solution
contained (in mM) 130 NaCl, 5 KCl, 25 glucose, 2.5 CaCl2,
1.3 MgCl2, and 10 HEPES, pH 7.35 with 5 M NaOH. To isolate
VGCCs pharmacologically from voltage-gated Na+ and
K+ conductances, a Ca2+ recording solution
containing (in mM) 120 choline Cl, 20 tetraethylammonium chloride (TEA
Cl), 4 4-aminopyridine (4-AP), 25 glucose, 2.5 CaCl2, 1.3 MgCl2, and 10 HEPES, pH 7.35 with 1 M TEA OH, and a
Ba2+ recording solution containing (in mM) 115 choline Cl,
20 TEA Cl, 4 4-AP, 25 glucose, 10 BaCl2, 1.3 MgCl2, and 10 HEPES, pH 7.35 with 1 M TEA OH, were used. In
some experiments, the choline Cl was replaced with NaCl and 1 µM TTX
was added to block Na+ conductances. Currents are referred
to as Ca2+ or Ba2+ currents to indicate which
recording solution was used. The VGCC antagonists NiCl2
(100 µM), CdCl2 (50 µM), nifedipine (10 µM), Aga
IVA (100 nM), and
CTX GVIA (500 nM) were diluted from concentrated stock solutions and added to small aliquots of the Ba2+
recording solution before recording. A Cs+ electrode
solution containing (in mM) 130 CsCl, 4 NaCl, 11 EGTA, and 10 HEPES, pH
7.20 with 1 M CsOH and a Tris electrode solution containing (in mM) 90 Tris PO4, 108 Tris base, 20 TEA Cl, 11 EGTA, and 10 sucrose, pH 7.20 with 2 M Tris base were used to provide further block
of outward K+ conductances. In addition, 2 mM MgATP, 100 µM leupeptin, 100 µM GTP, 10 mM creatine phosphate, and 100 U/ml
creatine phosphokinase were added to the electrode solutions to promote
the stability of whole cell recordings and to retard rundown of VGCCs
(Horn and Korn 1992
).
Aga IVA was a generous gift of
Pfizer, and
CTX GVIA was obtained from Alomone Labs; the remaining
chemicals were obtained from Sigma or Aldrich.
Whole cell recording
Dissociated cells were visualized on an inverted scope (IM-35,
Carl Zeiss) with Hoffman modulation contrast optics using ×25, 0.45 N. A. or ×40, 0.65 N. A. objectives. Electrodes were pulled from borosilicate glass capillaries (TW150F-4 glass, World Precision Instruments), fire-polished, and coated with silicone elastomer (Sylgard, Dow Corning) and had a resistance of 3-5 M with either the Cs+ or the Tris electrode solution. Before seal
formation, a small amount of enzyme-free electrode solution was sucked
into the tip of the pipette to facilitate gigohm seal formation. The
whole cell recording configuration (Hamill et al. 1981
)
was obtained in the Na+/K+ recording solution.
Junction potentials for both the Cs+ and Tris electrode
solutions were measured to be <2 mV in all recording solutions and
were not included in any voltage measures. There was a significant
increase in series resistance when using the Tris electrode solution
[12.5 ± 1.6 (SE) M
in Cs+,
n = 18; compared with 24.3 ± 1.2 M
in Tris,
n = 85]; however, the Tris solution generally produced
more stable recordings and provided a better block of K+
currents at depolarized potentials. Recordings in either the Ba2+ or Ca2+ recording solution were obtained
if a rapidly inactivating choline- or TTX-sensitive current was
observed on depolarization above
45 mV in the
Na+/K+ recording solution. Whole cell
recordings were obtained using an Axopatch 200 amplifier (Axon
Instruments), filtered at 1-50 kHz and digitized at 1-100 kHz with a
12 bit A/D converter (Digidata 1200, Axon Instruments).
Data analysis
Currents elicited over a range of voltages were used to assess
the voltage-dependent properties of VGCCs. Averages of four current
traces were obtained at a given command step, which were presented
sequentially in 5- or 10-mV intervals ranging from 150 to +50 mV. The
rate at which command steps were presented was dependent on their
duration: 10-ms steps were presented every 250 ms, 100-ms steps were
presented every 1 s, and 2- to 4-s steps were presented every
10-15 s. Command steps were presented directly from the holding
potential or from a prepulse step; tail currents were elicited by
repolarizing steps immediately after the command step. Leakage and
residual capacitive currents were eliminated by subtracting scaled
versions of the average current generated by a sequence of
hyperpolarizing command steps. Current-voltage relationships were
constructed by plotting the average or peak current magnitudes against
corresponding transmembrane voltage values (see APPENDIX)
over specified time windows. Time constants were obtained by performing
exponential fits of current traces using a Marquardt-Levenberg
minimization algorithm. Exponential fits were limited so that the
transmembrane voltage did not deviate >5 mV from the steady-state
voltage over the duration of the fit window. Although the same command
potentials were used, the transmembrane voltage varied between cells,
and a uniform voltage scale was needed to average current magnitudes
and time constants across experiments. Therefore the measured currents
and time constants were interpolated linearly onto a standardized
voltage scale before averaging data across experiments.
VGCC subtype-specific antagonists were used to assess the contribution
of VGCC subtypes to the overall current. Currents were elicited with
100-ms steps to 10 mV once every 10 s during exposure to various
VGCC antagonists. Despite the inclusion of ATP and an ATP-regenerating
system in the pipette solution, a slow rundown of VGCCs was observed
that could produce time-dependent errors in estimating the effects of a
particular antagonist. To minimize the effects of rundown, an
exponential was fit to the time course of the current magnitudes before
bath infusion of any antagonists (typically 3-5 min), and the
resulting fit was extrapolated over the duration of the experiment
(Mintz et al. 1992a
). Estimates of antagonist
sensitivity were obtained by averaging steady-state current magnitudes
over the last minute of antagonist application and normalizing these
averages to the extrapolated exponential fit over the same period. This
procedure assumes that the rate of rundown was equal among all VGCC
subtypes; this is unlikely to be a valid assumption. If one subtype has
a faster rundown rate, its contribution would be underestimated and the
contribution of the remaining subtypes would be overestimated.
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RESULTS |
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General properties
The vast majority of isolated DCN neurons respond to
depolarization with sustained inward currents in the presence of
voltage-gated Na+ and K+ channel antagonists.
During recording in either the Ca2+ or the Ba2+
solution, depolarizing command steps elicited measurable inward currents in 97 of 103 neurons (Fig.
1A). Peak inward currents averaged 286 ± 35 pA (n = 25) in the
Ca2+ recording solution and
550 ± 54 pA
(n = 74) in the Ba2+ recording solution.
When normalized to membrane capacitance, peak Ca2+ current
densities averaged
22.8 ± 3.0 pA/pF and peak Ba2+
current densities averaged
48.7 ± 4.4 pA/pF. Ba2+
currents initially activated between
50 and
40 mV, and the largest
inward currents were evoked with command steps between
15 and 0 mV
(Fig. 1A). After the activation of inward currents with
depolarization, large and rapidly deactivating inward tail currents
were observed immediately on repolarization after the command step
(arrowhead in Fig. 1A). The magnitude of these tail currents
increased monotonically with command step voltage and reached maximal
amplitudes with command steps between +20 and +35 mV.
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The deviation of the transmembrane voltage from the desired command
potential complicates the interpretation of voltage-clamp data. During
Ba2+ current activation, the transmembrane voltage time
course is determined by the flow of current across the uncompensated
series resistance. Current flow into the pipette due to VGCC activation (Fig. 1A) produces a voltage drop across the finite series
resistance, resulting in transmembrane voltages (Fig. 1B,
) that are positive relative to the command voltage (Fig.
1B, - - -). Although the difference between the command
and transmembrane voltage is usually a few millivolts once steady state
is attained (0.5-4 mV in Fig. 1B), the delay to steady
state can be
1 ms, depending on the membrane properties and
the magnitude of the Ba2+ currents. The voltage errors
during Ba2+ current deactivation are more pronounced
(arrowhead in Fig. 1B; see also Figs. 5B and
6B), partially due to the large magnitude of the tail
currents elicited during repolarization. The rapid time course of these
tail currents augments these voltage errors: the voltage errors due to
current flow across the uncompensated series resistance are
superimposed on the errors due to the filtering of the repolarizing
step by the uncompensated membrane capacitance. Because the dependence
of activating and deactivating Ba2+ current magnitudes on
the transmembrane voltage is a critical measure of VGCC function, a
method for estimating the transmembrane voltage was developed to more
accurately assess the biophysical properties of VGCCs (see
APPENDIX).
Divalent ion pharmacology
The divalent ion pharmacology of the Ca2+ and Ba2+ currents recorded from isolated DCN neurons suggests that these inward currents were generated by VGCCs. Peak inward currents recorded with Ca2+ (2.5 mM) as the charge carrier were 73.5 ± 3.5% (n = 6) smaller than the corresponding peak inward currents recorded when Ba2+ (10 mM) was the charge carrier (Fig. 2A). Adding Ni2+ (100 µM) to the Ba2+ solution reduced the peak inward current by 55.6 ± 6.5% (n = 6), whereas Cd2+ (50 µM) reduced the peak Ba2+ current by 98.4 ± 0.4% (n = 4). Although Ca2+ is the primary charge carrier through VGCCs in vivo, Ba2+ was used in the majority of these experiments. The substitution of Ba2+ for Ca2+ increased current magnitudes, allowing for a more accurate characterization of VGCC properties in most cells. In addition, Ba2+ blocked residual outward currents, providing a more complete isolation of currents carried by VGCCs. However, with the exception of the outward currents in the Ca2+ solution at depolarized voltages, the steady-state voltage dependence of the inward currents was not altered when Ba2+ replaced Ca2+ as the primary charge carrier (Fig. 2B). Therefore the remaining data were obtained in the Ba2+ recording solution to assess the pharmacological and voltage-dependent properties of VGCCs.
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VGCC subtypes
The Ba2+ currents recorded from acutely isolated DCN
neurons were carried by a variety of VGCC subtypes. The VGCC
subtype-specific antagonists nifedipine (10 µM), Aga IVA (100 nM),
and
CTX GVIA (500 nM) were used to assay for the presence of known
VGCC subtypes. The L-type antagonist nifedipine produced a consistent
reduction in Ba2+ current magnitude in all cells tested
(Fig. 3, A-D). The P/Q-type antagonist
Aga IVA blocked a significant portion of VGCCs in some
neurons (Fig. 3, A and C) but had little or no
effect in others (Fig. 3, B and D). Similarly,
the N-type antagonist
CTX GVIA blocked a substantial fraction of the
Ba2+ current in some cells (Fig. 3, B and
C) and had little or no effect in others (Fig. 3,
A and D). In most cells, a portion of the
Ba2+ current was resistant to nifedipine,
Aga IVA, and
CTX GVIA but could be blocked by 50 µM Cd2+ (Fig. 3,
A-D). Thus the Ba2+ currents in most isolated
DCN neurons were carried by three or more known HVA VGCC subtypes.
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With the exception of the component insensitive to nifedipine, Aga
IVA, and
CTX GVIA, VGCC subtypes contributing to the overall
Ba2+ current appeared to possess similar biophysical
properties. Current traces were averaged before and during exposure to
a particular antagonist and were subtracted to estimate the portion of
the Ba2+ current blocked by that antagonist (Fig. 3,
A-D, insets). Although the rundown of VGCCs could confound
these results, similarities were observed between difference traces
from cells in which rundown was not prominent (Figs. 3, A
and C, insets). Measures of activation rates and
inactivation extent were obtained from difference currents to quantify
the biophysical properties of the individual VGCC subtypes. The average
activation time constants were 1.4 ± 0.2 ms for the
Aga
IVA-sensitive component (n = 10), 1.6 ± 0.2 ms for the
CTX GVIA-sensitive component (n = 10), and
1.5 ± 0.2 ms for the nifedipine-sensitive component
(n = 10). The antagonist-insensitive component had an
average activation time constant of 2.7 ± 0.4 ms
(n = 10) and was significantly slower than the other
three components (unpaired Student's t-test,
P < 0.02 for all comparisons). The magnitude of
inactivation was 6.8 ± 2.0% for the
Aga IVA-sensitive component (n = 11), 6.0 ± 1.5% for the
CTX
GVIA-sensitive component (n = 12), and 0.1 ± 0.1% for the nifedipine-sensitive component (n = 13).
The antagonist-insensitive component exhibited 16.5 ± 2.9%
inactivation (n = 13), which was significantly larger
than the other three components (unpaired Student's t-test,
P < 0.02 for all comparisons). The
nifedipine-sensitive component also had significantly less inactivation
than the
Aga IVA- and
CTX GVIA-sensitive components (unpaired
Student's t-test, P < 0.001 for both comparisons).
The order in which antagonists were applied was varied to minimize the
effects of rundown in determining the relative contributions of VGCC
subtypes to the overall Ba2+ current. Two different
antagonist application sequences were developed under the assumption
that the effects of nifedipine, but not those of Aga IVA or
CTX
GVIA, were reversible over the time course of these experiments. The
first sequence consisted of applying nifedipine after the peptide
toxins
Aga IVA and
CTX GVIA had been applied (Fig.
3A); the second sequence consisted of applying the peptide
toxins during a continuous application of nifedipine (Fig. 3,
B-D). No statistically significant differences could be
detected in the relative proportions of VGCC subtypes observed in cells
in which nifedipine was presented before and during presentation of the
peptide toxins (Fig. 4A) when
compared with cells in which nifedipine was presented after
Aga IVA
and
CTX GVIA had been applied (Fig. 4B). Across all
experiments, nifedipine blocked 32.5 ± 3.6% (n = 13) of the Ba2+ current,
Aga IVA blocked 15.5 ± 4.2% (n = 11), and
CTX GVIA blocked 23.2 ± 6.3% (n = 12). Some of the Ba2+ current
was insensitive to nifedipine,
Aga IVA, and
CTX GVIA but still
could be blocked by Cd2+ (23.3 ± 4.1%,
n = 11), whereas a small fraction of the overall Ba2+ current was not blocked by any antagonists, including
Cd2+ (5.5 ± 1.0%, n = 12).
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Voltage dependence of Ba2+ currents
Open-channel Ba2+ current-voltage relationships were
constructed by measuring the magnitudes of tail currents elicited on
return to various repolarizing voltage steps after the activation of VGCCs with a command step to +50 mV. Under the conditions of these experiments, there are two problems with the interpretation of these
data. First, the transmembrane voltage (Fig.
5B, ) does not follow the
repolarizing step potential (Fig. 5B, ···) over the
duration of the tail current. Large differences between the repolarizing step potential and the estimated transmembrane voltage could be observed at the tail current peak (estimated transmembrane voltages at the tail current peak are indicated in Fig. 5B,
- - -). To minimize errors due to the difference between repolarizing and transmembrane voltages, tail current magnitudes
(Itail in Fig. 5A) were plotted as a
function of the estimated transmembrane voltage at the tail current
peak (Vtail in Fig. 5B). Second, tail current peaks are delayed relative to the onset of the repolarizing step (peak times indicated in Fig. 5A, - - -). A
significant number of channels could close during the delay to the tail
current peak, resulting in an underestimation of the open-channel
current (Taylor 1988
). Unfortunately, accurate
exponential fits of tail current decay could not be obtained to correct
for the underestimation of open-channel current magnitudes. The decay
of larger tail currents did not follow an exponential time course,
presumably due to a changing transmembrane voltage throughout the
duration of the tail current (Fig. 5B). Despite the likely
underestimation of open-channel currents, large inward open-channel
Ba2+ currents were observed at hyperpolarized voltages
(Fig. 5C). Open-channel Ba2+ current magnitudes
decreased monotonically with voltage; an inward rectification at
depolarized voltages prevented the reversal to an outward current.
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Similar to the open-channel Ba2+ current, the voltage
dependence of steady-state VGCC open probability could be estimated by measuring the voltage dependence of tail current magnitudes. Tail currents were elicited at a repolarizing potential of 80 or
55 mV
after the activation of VGCCs with command steps ranging from
100 to
+50 mV. Steady-state open probability then was estimated by plotting
tail current magnitudes (Itail in Fig.
6A) as a function of the
steady-state transmembrane voltage (Vss in Fig.
6B). However, the magnitudes of these tail currents are also
a function of the transmembrane voltage at each tail current peak
(Vtail in Fig. 6B). Therefore the
transmembrane voltage at the tail current peak Vtail must be held constant to obtain an
accurate estimate of the dependence of the open probability on the
steady-state voltage Vss. Because of variations
in the transmembrane voltage at each tail current peak (Fig.
6B), an alternative method for estimating the steady-state
open probability was required.
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To correct for variations in the transmembrane voltages at each tail
current peak, the steady-state VGCC open probability was estimated as a
ratio of tail currents elicited at the same transmembrane voltage.
Assuming that the VGCC open probability is maximal at +50 mV, the
steady-state open probability
Po(Vss) was calculated by
comparing the magnitude of a tail current elicited after a step to
Vss to the magnitude of a tail elicited after a
step to +50 mV
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The voltage dependence of steady-state Ba2+ current
magnitudes was described accurately by a product of the open-channel
Ba2+ current and the steady-state open probability.
Steady-state current-voltage relationships were constructed by plotting
the average current during the last 1 ms of a 10-ms command step
(Iss in Fig. 6A) against the average
transmembrane voltage over the same period (Vss
in Fig. 6B). Measurable inward currents initially were
observed at transmembrane voltages above 50 mV, and the largest
inward currents were obtained around
8 mV (Fig. 6D,
).
Outward currents were sometimes observed around or above +50 mV;
however, the lack of intracellular Ba2+ requires that any
outward currents be carried by other ions present in the intracellular
solution. Steady-state Ba2+ current magnitudes were
reconstructed as a product of the steady-state open probability (Fig.
6C) and open-channel Ba2+ current (Fig.
5C) to confirm the accuracy of these estimates (Fig.
6D,
). The reconstructed current magnitudes compare
favorably to the measured values over a wide voltage range. Differences between the measured and reconstructed values at hyperpolarized voltages can be attributed to slightly positive steady-state open probability values and large inward open-channel current magnitudes over this voltage range.
Ba2+ currents activated rapidly over the range of voltages
at which these currents could be elicited. The time course of VGCC activation in response to a voltage step will contain multiple components that can be described by a sum of exponentials. However, an
imperfect voltage clamp can prevent the accurate measurement of these
components: if there is any uncompensated series resistance, changes in
current magnitude will produce a corresponding change in transmembrane
voltage. Because the time course of VGCC activation is voltage
dependent, changes in transmembrane voltage subsequently will alter the
current time course. Rather than extending over the entire duration of
the 10-ms command step, exponential fits were limited so that the
transmembrane voltage at the beginning of the fit did not deviate >5
mV from steady-state values (Fig. 7A, thick lines). This
procedure precludes the use of higher-order kinetic models: only the
slowest components of VGCC activation could be measured; faster
components would have decayed before the beginning of the fit window.
The time constants from these exponential fits had a bell-shaped
voltage dependence, with the slowest time constants occurring around
20 mV, and the fastest values occurring at the most depolarized
voltages (Fig. 7B). It is not possible to correct for the
filtering of membrane currents by any remaining uncompensated membrane
time constant (Sigworth 1983
), which can distort the
time course of faster currents (Armstrong and Gilly
1992
). It is likely that the time course of VGCC activation is
affected by this filtering and would occur more rapidly under an ideal
voltage clamp.
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On longer time scales, a slower component of VGCC activation could be
observed in some Ba2+ current traces. This slow activation
sometimes was present during the first 100 ms of the 2- to 4-s command
steps used to investigate the time course of inactivation (Fig.
8B, ). In addition, a
slowly developing inward current could be elicited by 100-ms command steps (
CTX-sensitive difference traces in Fig. 3, B and
C; nifedipine-sensitive difference traces in Fig. 3,
B and D). In most cases, a slowly decaying tail
current was associated with the slowly developing inward current. This
slow component of VGCC activation and deactivation may correspond to
the "mode 2" gating augmented by dihydropyridine agonists during
single-channel recordings of L-type VGCCs (Tsien et al.
1986
). However, the conditions required to evoke this slow component were not investigated in this study, and it is not clear if
this component would be prominent under physiological conditions.
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A slow inactivation was observed at depolarized voltages, suggesting
that a majority of VGCCs recorded from acutely isolated DCN neurons
were of the HVA subtype. Unlike LVA VGCCs, the inward current did not
appear to have any significant low-threshold inactivation. No
significant changes in the peak current magnitude elicited by a 100-ms
command step to 0 mV were observed with 50-ms precommand steps to
either 100 or
40 mV from the holding potential of
70 mV (Fig.
8A). Across experiments, 50-ms precommand steps to
100 mV
resulted in peak current magnitudes that were 99.6 ± 2.0%
(n = 8) of the peak current magnitudes elicited
directly from the holding potential of
70 mV; and 50-ms precommand
steps to
40 mV resulted in peak current magnitudes that were
92.2 ± 4.2% (n = 8) relative to currents
elicited directly from
70 mV. Consistent with the behavior of HVA
VGCCs, Ba2+ currents elicited by 2- to 4-s command steps
from a holding potential of
70 mV showed a slow inactivation at
voltages higher than
25 mV (Fig. 8B). Although this
inactivation was faster with more depolarized voltages, inactivation
time constants typically remained >1 s at most voltages (Fig.
8C), which is more than three orders of magnitude larger
than the activation time constants at similar voltages (Fig.
7B).
It is possible that individual VGCC subtypes exhibited different
voltage-dependent properties so that the voltage dependence of the
overall Ba2+ current represented the average behavior of
these individual subtypes. Comparison of the difference traces obtained
during exposure to various antagonists (Fig. 3) suggests that the
individual VGCC subtypes shared similar biophysical properties.
However, these currents were elicited at a single voltage, and these
similarities may not be present over a range of voltages. To compare
the biophysical properties of VGCC subtypes over a wide voltage range,
Ba2+ currents were elicited by a range of 10-ms command
steps before and during the application of 10 µM nifedipine. The
nifedipine-sensitive portion of the current was estimated by
subtracting the currents obtained during exposure to nifedipine from
the currents obtained before nifedipine had been applied. Some
differences between nifedipine-sensitive and -insensitive VGCCs were
found: nifedipine-insensitive VGCCs had more steeply voltage-dependent
open-channel Ba2+ currents (Fig.
9A, ) and activated more
rapidly below
15 mV (Fig. 9D,
) when compared with
nifedipine-sensitive currents (Fig. 9, A and D,
). However, the steady-state open probability (Fig. 9B)
and steady-state current magnitudes (Fig. 9C) were similar over a wide voltage range as were the activation time constants above
10 mV (Fig. 9D). Although the antagonism of VGCCs by some dihydropyridine compounds is modulated by voltage at physiological pH
(Sanguinetti and Kass 1984
), the saturating
concentration of nifedipine used in these experiments should provide a
complete block of L-type VGCCs regardless of voltage. Unfortunately, it was not possible to compare voltage dependence of VGCC subtypes in
other cells or with additional pharmacological agents; the combination
of pharmacological effects and rundown usually resulted in currents
that were too small to obtain accurate measures over the voltage range
of interest.
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DISCUSSION |
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The results of this study show that the majority of neurons isolated from guinea pig DCN possess VGCCs. There are many neuronal populations residing within the guinea pig DCN, and our results indicate that a variety of VGCC subtypes are present in these neurons. Given these conditions, it is reasonable to expect VGCCs to exhibit a diverse range of biophysical properties, and the apparent uniformity of VGCC properties in isolated DCN neurons is an intriguing result. In all neurons from which Ba2+ currents were observed, VGCCs had similar steady-state voltage dependence, activated and deactivated rapidly, and exhibited no voltage-dependent inactivation on physiologically relevant time scales. Regardless of subtype, the VGCCs present in isolated DCN neurons are poised to produce a rapid Ca2+ influx in response to a large depolarization.
The use of the isolated cell preparation is advantageous for the
characterization of voltage-gated conductances in adult mammalian neurons (Kay and Wong 1986); however, the acute
isolation procedure poses some potential problems for the
interpretation of the results obtained in the present study. First, the
trauma resulting from the enzymatic and mechanical dispersion of
neuronal tissue could alter the expression of proteins contributing to
the electrophysiologic properties of neurons, resulting in patterns of
conductances that would not be observed in recordings from intact
preparations. Acutely isolated ventral cochlear nucleus neurons produce
responses to depolarizing current injection that are similar to
responses obtained from in vitro slice preparations (Manis and
Marx 1991
), indicating that these neurons do not express
different sets of conductances as a result of the acute isolation
procedure. Second, the acute isolation procedure may modify the
properties of existing membrane proteins, including voltage-gated
conductances. Trypsin, the enzyme used in the dissociation procedure,
is known to inactivate N-methyl-D-aspartate
receptors (Allen et al. 1988
). However, the use of
trypsin in previous studies did not appear to adversely affect VGCCs
(Kay and Wong 1987
; Thompson and Wong
1991
). In addition, the biophysical and pharmacological
properties of VGCCs in the present study are similar to those found in
neurons dissociated without the use of trypsin (Regan
1991
; Zidanic and Fuchs 1995
). Therefore the use
of the acute dissociation procedure is not likely to adversely affect
VGCCs in the present study.
VGCC subtypes
Acutely isolated guinea pig DCN neurons possess a variety of HVA
VGCC subtypes. The divalent ion pharmacology is more typical of HVA
rather than LVA VGCCs: unlike their LVA counterparts, HVA VGCCs are
usually more selective for Ba2+ than for Ca2+
and are blocked more potently by Cd2+ than by
Ni2+ (Bean 1989; Huguenard
1996
). Responses to subtype-specific antagonists also confirmed
the presence of HVA VGCCs. All neurons tested contained a significant
L-type component; only some neurons contained N- and P/Q-type
components, and these appeared in varying amounts. The observation of a
consistent nifedipine-sensitive component and variable
Aga IVA- and
CTX GVIA-sensitive components could be explained by the differential
localization of VGCC subtypes within a single cell. In hippocampal
pyramidal cells, L-type VGCCs cluster in the proximal dendrites
(Westenbroek et al. 1990
); whereas N-type VGCCs are
distributed over the entire dendritic arbor (Westenbroek et al.
1992
). Because the acute cell isolation procedure produces neurons that only possess a cell body and proximal processes, it is
expected that such a distribution of VGCCs would result in a consistent
nifedipine-sensitive component, and a reduced, if not erratic,
CTX GVIA-sensitive component.
Most neurons possess a significant inward current that remains in the
presence of nifedipine after exposure to Aga IVA and
CTX GVIA or
during a simultaneous exposure to all three antagonists. The majority
of this remaining inward current is blocked by 50 µM Cd2+
and may correspond to R-type VGCCs (Ellinor et al. 1993
;
Zhang et al. 1993
). In the present study, the
antagonist-insensitive component exhibits a slower activation and a
more pronounced inactivation than the L-, N-, and P/Q-type components.
This is consistent with the voltage-dependent properties of R-type
VGCCs, which exhibit many similarities to LVA VGCCs (Bourinet et
al. 1996
; Ellinor et al. 1993
; Soong et
al. 1993
). It is possible that this antagonist-insensitive component could consist of LVA VGCCs as well; however, the high sensitivity of residual currents to Cd2+ and the
insensitivity of the overall current to a hyperpolarized prepulse
voltage suggest that these channels belong to the HVA family. It is
also possible that Q-type VGCCs could contribute to this
antagonist-insensitive component. Although the concentrations of the
antagonists used should provide a complete block of L-, N-, and P-type
VGCCs, the concentration of
Aga IVA used in these experiments was
only slightly greater than the IC50 for Q-type channels
(Randall and Tsien 1995
). In some neurons, it is
possible that additional
Aga IVA would have blocked a larger portion
of the antagonist-insensitive current by providing a more complete block of Q-type channels. However, several neurons exhibited little or
no response to 100 nM
Aga IVA, and it is unlikely that additional
Aga IVA would have provided a further block of the remaining current
in these cells.
The results of the study do not provide any information about the
distribution of VGCCs throughout the DCN. Various cell populations reside within the guinea pig DCN (Hackney et al. 1990),
and the use of the isolated cell preparation does not permit positive morphological identification of cell types. Without morphological identification, there is no way to determine whether the various VGCC
subtypes observed in the present study are distributed differentially among the individual DCN cell populations. In addition, the results of
this study do not provide any information about the distribution of
VGCC subtypes throughout the dendritic arbors of the various cell
types. A VGCC-mediated Ca2+ influx into the dendrites of
DCN cartwheel and pyramidal cells can be elicited by somatic action
potentials (Manis and Molitor 1996
; Molitor and
Manis 1996
); however, these dendritic processes inevitably are
destroyed by the cell-isolation procedure. Recordings from hippocampal
pyramidal and cerebellar Purkinje neurons have shown that the relative
levels of VGCC subtypes differed between the soma and the dendrites of
these neurons (Kavalali et al. 1997
; Mouginot et
al. 1997
). Therefore it is possible that the dendrites of DCN
neurons possess VGCCs that differ from those characterized in this study.
Voltage-dependent properties
The voltage-dependent properties of VGCCs suggest that these
conductances are suited to respond to action potentials. A large depolarization from rest is required to activate VGCCs in isolated DCN
neurons: <10% of maximal activation is achieved below 40 mV,
whereas >90% of maximal activation is achieved above +10 mV. An
action potential would maximally activate VGCCs, whereas a subthreshold
synaptic depolarization would result in little or no VGCC activation.
In addition, the activation time course is rapid enough at depolarized
voltages to reach steady-state levels within the duration of an action
potential. The lack of any low-threshold inactivation should prevent
previous neuronal activity from altering the response of VGCCs to
action potentials; the sustained depolarization required to produce any
voltage-dependent inactivation is not likely to occur during periods of
normal neuronal activity. However, VGCCs are known to inactivate due to
elevated internal Ca2+ (de Leon et al. 1995
;
Imredy and Yue 1994
). It is not clear whether elevated
intracellular Ca2+ inactivates VGCCs in DCN neurons; the
use of Ba2+ as the charge carrier minimized this effect so
that voltage-dependent inactivation could be investigated in isolation.
Once activated, VGCCs are capable of producing a large Ca2+
influx. An asymmetric distribution of Ba2+ in these
experiments (10 mM Ba2+ outside, 11 mM EGTA inside) results
in large inward currents at hyperpolarized voltages and produces an
inward rectification at depolarized voltages, which prevents the
reversal to an outward current. A similar asymmetric Ca2+
distribution exists in vivo, and the voltage dependence of the Ca2+ influx should exhibit similar properties under
physiological conditions. The voltage dependence of open-channel
currents suggests that the bulk of the Ca2+ influx will
occur after the action potential peak during the repolarizing phase
before VGCCs deactivate in response to hyperpolarized voltage levels
(Llinás et al. 1981). However, imaging studies at
the parallel fiber-stellate cell synapse in the rat cerebellum indicate
that the Ca2+ influx in presynaptic terminals becomes more
prominent during the rising phase of parallel fiber-mediated action
potentials at physiological temperatures (Sabatini and Regehr
1996
). It is possible that VGCCs in DCN neurons behave in a
similar fashion at physiological temperatures, resulting in a
Ca2+ influx that occurs more rapidly with respect to action
potential initiation.
Functional implications
VGCCs may be involved in producing different discharge patterns in
DCN neurons. VGCCs are likely to be involved in the evoked responses of
cartwheel cells, which are a major population of inhibitory
interneurons in the superficial DCN. Cartwheel cells respond to
depolarizing current injection with a burst of fast action potentials
superimposed on a slow depolarization that has been attributed to VGCCs
(Agar et al. 1996; Manis et al. 1994
; Zhang and Oertel 1993
). In contrast, VGCCs may
contribute indirectly to the evoked responses of pyramidal cells, which
form the main projection pathway from the DCN to the inferior
colliculus. Pyramidal cells are only capable of generating
Ca2+-dependent action potentials in the presence of
voltage-gated Na+ and K+ conductance
antagonists (Hirsch and Oertel 1988
), and models incorporating only voltage-gated Na+ and K+
conductances are capable of reproducing many pyramidal cell responses to intracellular current injection (Hewitt and Meddis
1995
; Kim et al. 1994
). However, a slow
afterhyperpolarization observed in pyramidal cells after a sustained
discharge of action potentials (Hirsch and Oertel 1988
;
Manis 1990
) may be attributed to
Ca2+-activated K+ conductances, which would be
activated by the Ca2+ influx through VGCCs. Thus VGCCs may
play different roles in determining the patterns of evoked responses
across neuronal populations of the DCN.
VGCCs are also capable of regulating neuronal responses through the
actions of Ca2+ as an intracellular second messenger.
Despite the differential involvement of VGCCs in the generation of
evoked responses, somatically evoked action potentials result in a
VGCC-mediated Ca2+ influx into the soma and proximal
dendrites of both pyramidal and cartwheel cells (Manis and
Molitor 1996; Molitor and Manis 1996
). Because
Ca2+ is an important intracellular second messenger
(Tsien and Tsien 1990
), an evoked Ca2+
influx through VGCCs could play a role in regulating neuronal responses. Ca2+ can interact with and regulate other
signaling pathways in the DCN, such as protein kinase C, which
regulates the strength of synaptic transmission at parallel fiber
synapses (Francis and Manis 1995
; Scott and Manis
1992
); and intracellular Ca2+ release through
inositol 1,4,5-trisphosphate receptors (Mignery et al.
1989
; Ryugo et al. 1995
), which may be activated
through metabotropic glutamate receptors present at parallel fiber and auditory nerve synapses (Molitor and Manis 1997
). A
Ca2+ influx through VGCCs could act on many different time
scales, from the short-term activation of ionic conductances (such as Ca2+-activated K+ conductances), to the
long-term regulation of synaptic responses. Regardless of the
contribution of VGCCs to discharge patterns, an influx of
Ca2+ through VGCCs may be an important indicator of
neuronal activity and may have significant implications for information
processing in this nucleus.
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APPENDIX |
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The study of voltage-dependent conductances requires the accurate control of transmembrane voltage. In many circumstances, the single-electrode, whole cell voltage-clamp technique can be used to provide this control. However, the experimental conditions in the present study resulted in deviations of the transmembrane voltage from the presented command potential. This discrepancy arises from two sources: the voltage drop due to the flow of ionic current across a finite series resistance and the filtering of the command waveform by the membrane capacitance and series resistance. Therefore accurate estimates of the transmembrane voltage are needed to assess the voltage dependence of the Ba2+ currents presented in this study.
A method for calculating the transmembrane voltage can be derived from
a simple circuit used to model the isolated neurons in these
experiments. The cell is modeled as a membrane resistance Rm in parallel with a membrane capacitance
Cm and a current source Ix(t); an additional resistance
Rs is added in series to represent the access
between the pipette tip and cell interior. Under the conditions of
these experiments, it is assumed that Rm
Rs, which effectively eliminates
Rm and reduces the model circuit to
Rs in series with Cm and
Ix(t). The solution to the
differential equation governing this reduced circuit can be expressed
in the Laplace domain as a function of complex frequency (s)
![]() |
(A1) |
The properties of the membrane filter must be known to calculate the
transmembrane voltage. The values of Rs and
m can be obtained from currents elicited by command
steps that do not activate ionic currents. In the absence of any
on-line amplifier compensation or off-line capacitive current
subtraction, the response of the model circuit to a hyperpolarizing
command step of magnitude
V will be a decaying
exponential with amplitude
V/Rs
and time constant
m. For the majority of neurons used in
the present study, the capacitive currents evoked by hyperpolarizing
command steps were more accurately described by a sum of two
exponentials
![]() |
(A2) |
The true command potential also must be known to calculate the
transmembrane voltage. Many voltage-clamp amplifiers provide compensation circuitry that use estimates of Rs
and Cm to modify the command potential so that
the transmembrane voltage will more faithfully replicate the desired
command potential. The Axopatch 200 amplifier used in these experiments
provides two separate compensation pathways by which the command
potential is modified: prediction and correction compensation.
Prediction compensation reduces low-pass filtering of the membrane by
boosting the high-frequency components of the command potential
![]() |
(A3) |
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(A4) |
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ACKNOWLEDGMENTS |
---|
We thank Dr. Nicholas Saccomano of Pfizer for the generous
gift of Aga IVA.
This work was supported by National Institute of Deafness and Other Communication Disorders Grants RO1 DC-00425 and K04 DC-00048 to P. B. Manis, a National Science Foundation predoctoral fellowship to S. C. Molitor, and a grant from the W. M. Keck Foundation.
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FOOTNOTES |
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Address for reprint requests: S. C. Molitor, The Johns Hopkins University School of Medicine, 420 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 June 1998; accepted in final form 30 October 1998.
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REFERENCES |
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