Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bal, Ramazan and
Donata Oertel.
Hyperpolarization-Activated, Mixed-Cation Current
(Ih) in Octopus Cells of the Mammalian
Cochlear Nucleus.
J. Neurophysiol. 84: 806-817, 2000.
Octopus cells in the posteroventral cochlear
nucleus of mammals detect the coincidence of synchronous firing in
populations of auditory nerve fibers and convey the timing of that
coincidence with great temporal precision. Earlier recordings in
current clamp have shown that two conductances contribute to the low
input resistance and therefore to the ability of octopus cells to
encode timing precisely, a low-threshold K+ conductance and
a hyperpolarization-activated mixed-cation conductance, gh. The present experiments describe the
properties of gh in octopus cells as they
are revealed under voltage clamp with whole-cell, patch recordings. The
hyperpolarization-activated current, Ih, was
blocked by extracellular Cs+ (5 mM) and
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (50-100 nM) but not by extracellular
Ba2+ (2 mM). The reversal potential for
Ih in octopus cells under normal
physiological conditions was 38 mV. Increasing the extracellular potassium concentration from 3 to 12 mM shifted the reversal potential to
26 mV; lowering extracellular sodium concentration from 138 to 10 mM shifted the reversal potential to
77 mV. These pharmacological and
ion substitution experiments show that Ih in
octopus cells is a mixed-cation current that resembles
Ih in other neurons and in heart muscle
cells. Under control conditions when cells were perfused
intracellularly with ATP and GTP, Ih had an
activation threshold between about
35 to
40 mV and became fully
activated at
110 mV. The maximum conductance associated with
hyperpolarizing voltage steps to
112 mV ranged from 87 to 212 nS
[150 ± 30 (SD) nS, n = 36]. The voltage
dependence of gh obtained from peak tail currents is fit by a Boltzmann function with a half-activation potential of
65 ± 3 mV and a slope factor of 7.7 ± 0.7. This relationship reveals that gh was
activated 41% at the mean resting potential of octopus cells,
62 mV,
and that at rest Ih contributes a steady
inward current of between 0.9 and 2.1 nA. The voltage dependence of
gh was unaffected by the extracellular
application of dibutyryl cAMP but was shifted in hyperpolarizing
direction, independent of the presence or absence of dibutyryl cAMP, by
the removal of intracellular ATP and GTP.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In all mammals,
octopus cells occupy a distinct region of the dorsal and caudal
posteroventral cochlear nucleus, a region in which there is no apparent
intermingling with the other cells and which is particularly prominent
in humans (Adams 1997; Brawer et al.
1974
; Disterhoft 1980
; Golding et al.
1995
, 1999
; Hackney et al. 1990
; Morest
et al. 1990
; Oertel et al. 1990
; Osen
1969
; Wickesberg et al. 1991
, 1994
;
Willott and Bross 1990
). In mice there are ~200
octopus cells in each cochlear nucleus (Willott and Bross
1990
). Each octopus cell has several large, scantily branched
dendrites that emerge from the rostral side of the cell body
(Osen 1969
). The dendrites spread perpendicularly across the paths of auditory nerve fibers in the dorsocaudal posteroventral cochlear nucleus where the tonotopic array of fibers is closely bundled
(Golding et al. 1995
; Hackney et al.
1990
; Oertel et al. 1990
; Willott and
Bross 1990
). A relatively large number of auditory nerve
fibers, more than 60 in mice, excite octopus cells through small,
bouton terminals (Golding et al. 1995
; Willott
and Bross 1990
). Octopus cells form one of the major ascending
pathways from the ventral cochlear nucleus, projecting through the
intermediate acoustic stria to terminate contralaterally in the
superior paraolivary nucleus and in the ventral nucleus of the lateral
lemniscus (Adams 1997
; Schofield 1995
;
Schofield and Cant 1997
). The projection to spherical
bushy neurons in the ventral nucleus of the lateral lemniscus from
octopus cells is through large, calyceal terminals (Adams
1997
; Schofield and Cant 1997
; Smith et
al. 1993
; Vater et al. 1997
; Warr
1969
).
As the anatomical organization suggests, octopus cells are broadly
tuned and are relatively insensitive to pure tones in vivo (Godfrey et al. 1975; Rhode and Smith
1986
). Octopus cells respond with sharply timed action
potentials to clicks, to the onset of tones, and to periodic sounds
(Friauf and Ostwald 1988
; Godfrey et al.
1975
; Rhode and Smith 1986
; Rhode et al.
1983
; Smith et al. 1993
). Timing of the action
potential at the onset of the stimulus is sharper than that of any
other units in the auditory system (Rhode and Smith
1986
). In response to click trains or loud tones at low
frequencies, octopus cells can follow with one action potential at
every cycle
1 kHz (Rhode and Smith 1986
).
The unusual biophysical characteristics of octopus cells enable them to
detect coincident activation of auditory nerve fibers and convey the
timing of that coincidence with temporal fidelity. Current-clamp
experiments revealed that two conductances,
gh and a low-threshold potassium
conductance, gK(L), each of which is partly activated at the resting potential, help to determine how octopus cells fire in response to synaptic activation of the auditory nerve (Golding et al. 1995, 1999
). These conductances
contribute to the resting low input resistance, 6.7 M
, which gives
octopus cells short time constants (about 200 µs) and short
integration times (~1 ms). They also prevent firing in response to
asynchronous synaptic inputs (Ferragamo and Oertel
1998
). In the present study we describe
gh quantitatively under voltage clamp.
The results show that gh in octopus
cells generally resembles gh in other cells in its biophysical characteristics but that the maximum conductance is unusually large.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular whole-cell patch recordings were made from coronal
slices of the brain stem containing the posteroventral cochlear nucleus
from mice (ICR strain) of between 18 and 24 days after birth.
Immediately after decapitation, the head was immersed in "normal"
physiological saline containing (in mM) 130 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, and 10 glucose, saturated with
95% O2-5% CO2, pH 7.4, between 25 and 31°C (Golding et al. 1995, 1999
). The
whole brain was removed and cut coronally at the level of the inferior
colliculi. The specimen was mounted, with the colliculus end down, with
a cyanoacrylate glue (Superglue) onto a Teflon block. This block was
fixed in a bath, which was filled with saline that was continuously
oxygenated. Slices 180-µm thick were then cut using an oscillating
tissue slicer (Frederick Haer, Newbrunswick, ME). The one to two slices
containing the octopus cell area were transferred to a storage chamber
containing fresh, oxygenated saline. For recording, a slice was
transferred to a recording chamber with 0.3 ml volume in which it was
continuously perfused at a rate of about 8 ml/min with saline whose
temperature was maintained by a feedback-controlled heater at 33°C.
In many of the voltage-clamp experiments, the extracellular solution
was modified to allow Ih to be
isolated. To block voltage-sensitive sodium, potassium, and
synaptically activated currents, slices were bathed in a "control"
solution containing (in mM) 138 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgCl2, 10 HEPES,
10 glucose, 2 4-aminopyridine (4-AP), 0.001 tetrodotoxin (TTX), and
0.04 6,7-dinitroquinoxaline-2,3-dione (DNQX). In some
experiments, 5 mM Cs+, 50 nM
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288; Tocris Cookson, UK), -dendrotoxin (DTX)
(Alomone Labs, Israel), or dibutyryl cAMP were added to the
control solution. Tetraethylammonium chloride (TEA) at 10 mM was made
by equimolar substitution for NaCl. Low-sodium solution (10 mM) was
made by equimolar substitution of choline chloride for NaCl.
High-potassium solution (12 mM) was made by substitution of 9 mM KCl
for NaCl. Test solutions were added to the chamber by redirecting the
flow of liquid through a system of tubing and valves. Except where
stated otherwise, all chemicals were obtained from Sigma.
Patch electrodes (3-8 M) were pulled from borosilicate glass (1.2 mm OD). The pipette solution contained (in mM) 108 potassium gluconate,
9 HEPES, 9 EGTA, 4.5 MgCl2, 14 phosphocreatinine
(Tris salt), 4 ATP (Na-salt), and 0.3 GTP (Tris salt); pH was adjusted to 7.4 with KOH (Forcher and Oxford 1985
). The results
have been corrected for a junction potential of
12 mV. The addition
of ATP, GTP, and phosphocreatine made recordings from octopus cells stable for several hours; recordings in an earlier study using pipettes
without these high-energy phosphates lasted for about 30 min
(Golding et al. 1999
). To assess modulation of
gh, a modified pipette solution was
used in which ATP and GTP were eliminated.
Octopus cells were recorded under visual control. They form bright
holes among fascicles of heavily myelinated fibers when visualized
using a Zeiss Photoskop with a ×63 water-immersion lens under
bright-field illumination when the field diaphragm was almost
completely closed. Current- and voltage-clamp data were obtained with
standard whole-cell patch-clamp techniques using an Axopatch-200A
amplifier. Analog records were low-pass-filtered at 5-10 kHz. Current
and voltage records were sampled at 10-40 kHz using a Digidata 1200 interface (Axon Instruments, Foster City, CA), fed to a chart recorder,
and stored on a IBM-compatible personal computer for further analysis.
Stimulus generation, data acquisition, and off-line analysis of
digitized data were performed using pClamp software (version 6.03; Axon
Instruments). High-resistance seals (>1 G) were obtained before
going to the whole-cell configuration. Series resistance and
capacitance compensation were applied on-line. Series resistance was
always compensated to
96%; in most recordings series resistance was
compensated between 98 and 99%.
Numerical results are given as means ± SD, with n being the number of cells on which the measurement was made. Significant differences between the groups were evaluated using a paired Student's t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initial observations
The results presented here are based on patch-clamp recordings
from 184 octopus cells. The octopus cell area, which in mice contains
no other cell types, can be visualized in coronal sections in the most
caudal and dorsal portion of the ventral cochlear nucleus amid the
bundles of myelinated auditory nerve fibers. The identity of octopus
cells was confirmed by their characteristic biophysical features
(Golding et al. 1995, 1999
). The cells had a mean
resting potential of
61.8 ± 1.7 mV (n = 135).
From a randomly chosen sample of these cells, measurements were made of
input resistance, duration, and amplitude of action potential. The mean input resistance, calculated from a linear fit of the negative section
of the current-voltage relationship of the peak voltage responses near
rest, was found to be 6.7 ± 2.0 M
(n = 56).
The input resistance measured as the slope of the steady-state voltage changes as a function of current was 3.8 ± 1.8 M
(n = 25). The mean time constant for hyperpolarizing
responses close to the resting potential was 0.21 ± 0.07 ms
(n = 20). The amplitude of action potentials was
between 20 and 35 mV with a mean 26 ± 4 mV (n = 20). The mean duration of action potentials measured halfway between
the peak and the inflection point was 0.34 ± 0.07 ms
(n = 20). The small differences between the properties
reported here and those from the same cells in an earlier report
(Golding et al. 1999
) may have arisen from the inclusion
of phosphocreatine and nucleotide triphosphates in the pipette solution.
All the octopus cells tested with the injection of hyperpolarizing
current exhibited a depolarizing sag of the membrane potential toward
the resting value. The amplitude of the sag increased with the
amplitude of hyperpolarizing current. This sag was blocked by the
extracellular application of 5 mM Cs+ or 50 nM
ZD7288 (Fig. 1). Moreover, both blockers
caused the resting potential to become hyperpolarized
(Cs+ by 4.6 ± 1.2 mV, n = 5; ZD7288 by 10.3 ± 3.1 mV, n = 12), indicating that the conductance associated with the sag is active at rest and
contributes to the normal resting potential. In the presence of ZD7288,
the shape of the action potential was altered. The rise of action
potentials occurred more slowly and the height of action potentials
measured from the inflection to the peak was higher by ~25%. The
width of action potentials at their base increased from 0.31 ± 0.05 to 0.87 ± 0.7 ms. This observation also indicates that the
reversal potential for this current is more positive than the resting
potential, as expected from a mixed cationic
hyperpolarization-activated current,
Ih (Golding et al. 1999). The effects of Cs+ on
Ih were reversible but those of ZD7288
were not. These observations demonstrate that the pharmacological
characteristics of Ih in octopus cells
match those of mixed-cation, hyperpolarization-activated currents
in other types of neurons and muscle cells (Chen 1997
; DiFrancesco 1993
; Fu et al. 1997
;
Khakh and Henderson 1998
; Maccaferri and
McBain 1996
; Mo and Davis 1997
; Pape
1996
).
|
Biophysical properties under voltage clamp
All octopus cells tested under voltage clamp (n = 159) displayed a large, time- and voltage-dependent current when they were hyperpolarized, a current that is the manifestation in voltage clamp of the sag that was described in the preceding text in current clamp. Figure 2A illustrates a typical family of inward currents in response to hyperpolarizing voltage pulses. These inward currents had two components. Immediately after the capacitative transient, an "instantaneous" change in current, (IINS) was detected. This was followed by a slowly developing inward current whose amplitude and rate of rise depended on the size of the voltage step. Figure 2B is a plot of the amplitude of instantaneous and steady-state currents as a function of voltage. The slope of the instantaneous current-voltage relationship (119 nS) reflected the conductance of the cell at the holding potential immediately before the voltage steps and was strongly dependent on the holding potential. The plot of the steady-state current near the end of the voltage steps was not linear and reflected the activation of Ih. The difference between IINS and ISS (Fig. 2B) reflects the current that was activated by the hyperpolarizing step. In octopus cells, ISS showed no sign of inactivation even with very long voltage pulses (Fig. 2C).
|
The total current activated by hyperpolarization was consistently large
in octopus cells. Its amplitude ranged from 6.5 to
15.7 nA,
averaging 11.1 ± 2.2 nA (n = 36) at
112 mV.
Some rundown of Ih (~10%) was
observed over the course of experiments that typically ran ~1 h.
Isolation of Ih
To study Ih, this current first
had to be isolated from other voltage-dependent ionic currents. Earlier
studies have revealed the presence of voltage-dependent
Na+, Ca2+, and
K+ currents (Golding et al. 1995,
1999
). In the present experiments, 0.1 µM TTX and 2 mM 4-AP
were added to block Na+ and
K+ currents, respectively. In some experiments
which involved depolarizations more than 5 mV from the resting
potential, 6 mM 4-AP, 10 mM TEA, and also 0.2 mM
Cd2+ were used to block low-threshold
IK(L) and high-threshold
IK(H) K+
currents and Ca2+ current, respectively.
At the resting potential and in the cell's physiological voltage
range, both Ih and
IK(L) are partially activated
(Ferragamo and Oertel 1998; Golding et al.
1999
). It was crucial therefore to block
IK(L), whose voltage sensitivity
overlaps that of Ih near the resting
potential and to test whether Ih is
affected by those blockers. Two agents have been reported to block
IK(L), 4-AP and dendrotoxin
(Ferragamo and Oertel 1998
; Forsythe and Barnes-Davies 1993
; Manis and Marx 1991
;
Rathouz and Trussell 1998
). To test whether these drugs
affect Ih, octopus cells were subjected to large, hyperpolarizing voltage pulses (
80 to
100 mV),
conditions under which Ih would be
expected to be strongly activated and
IK(L) and other
depolarization-sensitive currents would be expected to be minimally
activated. Figure 3, A and
B, shows that while 2 mM 4-AP did not significantly affect
hyperpolarization-activated currents, 6 mM 4-AP and 100 nM DTX
suppressed Ih by ~10%. The current-voltage relationships show that the block of
Ih by these agents is
voltage-independent. In the following experiments therefore, estimates
of the amplitude of Ih were made in
the presence of only 2 mM 4-AP under conditions in which
Ih was not measurably suppressed. For
studying the voltage-dependence of Ih,
which required not only hyperpolarization but also depolarization to
20 mV from rest, it was more important to avoid contamination by
depolarization-sensitive K+ currents than to
have currents of maximum amplitude. For these experiments, 10 mM TEA
was added to the control solution to block IK(H), a higher concentration of 4-AP
(5-6 mM) was added to suppress IK(L),
and Cd2+ was added to suppress the inward
Ca2+ current. Although the amplitude of
Ih was reduced somewhat by both
TEA and 4-AP at these concentrations, the voltage-dependence was not
measurably affected.
|
Pharmacological identification of Ih
To characterize the hyperpolarization-activated current pharmacologically, its sensitivity was tested to extracellularly applied Ba2+, Cs+, and ZD7288. These drugs differentiate two types of hyperpolarization-activated inward current: Cs+ and Ba2+ but not ZD7288 block inward rectifiers, IKIR, that are highly selective for potassium ions and that activate relatively quickly. Cs+ and ZD7288 but not Ba2+ have been shown to block Ih, a current that reflects a hyperpolarization-activated permeability to sodium and potassium ions. Figure 4 shows the results of such tests.
|
Application of 5 mM Cs+ blocked most of the current activated by hyperpolarization (91.5 ± 1.8%, n = 7; Fig. 4A). Increasing the concentration to 10 mM Cs+ resulted in a block of 94% (n = 2). The effects on Ih were largely reversible on removal of Cs+.
A bradicardiac agent, ZD7288, has also been reported to block
Ih selectively in many types of
neurons (Harris and Constanti 1995; Khakh and
Henderson 1998
; Luthi et al. 1998
;
Maccaferri and McBain 1996
). Figure 4B shows
the result of externally applied 50 nM ZD7288 on
hyperpolarization-activated currents. There appeared to be a release
from block during hyperpolarization manifested by a gradual increase of
inward current. Such effects have been also reported in hippocampal
neurons (Maccaferri and McBain 1996
). ZD7288 blocked
inward currents, measured near the beginning of the pulse, by 91 ± 4% (n = 3). In octopus cells, as in other cells, the blocking effect developed slowly, taking ~10-20 min to become maximal, and could not be reversed with washout periods
50 min (Harris and Constanti 1995
; Maccaferri and McBain
1996
).
These experiments raise the question whether the block by 5 mM Cs+ and ZD7288 is incomplete or whether a portion of the hyperpolarization-activated current was mediated through a different type of conductance. Extracellular application of 2 mM Ba2+ produced a 13 ± 4% inhibition of Ih (n = 4; Fig. 4C). To determine whether the Cs+-insensitive current and Ba2+-sensitive currents were different, the sensitivity to Ba2+ of the residual currents in the presence of Cs+ was tested. Three experiments showed that the 5 mM Cs+-insensitive, hyperpolarization-activated current was blocked by Ba2+ by only 4% (n = 3), indicating that the block by Ba2+ in these experiments was nonspecific and that <1% of the hyperpolarization-activated current could have been IKIR. The interpretation that the residual current in the presence of 5 mM Cs+ represents incomplete blocking of Ih is strengthened by the finding that a larger proportion (94%) of the hyperpolarization-activated current is blocked by a larger concentration of Cs+ (10 mM). We conclude that the hyperpolarization-activated current can be attributed to Ih.
Kinetics of activation and deactivation
The kinetics of activation of Ih
was examined by fitting the activation phase of the current with
exponentials. The first 7 ms of the current trace at the beginning and
after the end of a voltage pulse can include the capacitative transient
and was therefore ignored for the exponential fitting (Solomon
and Nerbonne 1993a). The rise of the current after this period
was well described by double-exponential processes (Fig. 5,
A and B). Both the
fast and slow time constants of activation,
fast and
slow, were
voltage dependent with
fast = 44 ± 6 ms
and
slow = 181 ± 39 ms at
77 mV and decreasing to
fast = 16 ± 3 ms
and
slow = 84 ± 20 ms at
107 mV
(n = 8). This decrease in time constants at large step potentials was statistically significant (P
0.001).
Figure 5B shows the mean values of activation time
constants,
fast and
slow, plotted as a function of voltage.
|
The rate of deactivation of the inward current was studied by
depolarizing the membrane from a hyperpolarized holding potential, 102 mV (Fig. 5C). The current traces were best fitted with
a single-exponential function, which varied as a function of voltage. In six experiments, the time constants were 126 ± 15 ms at
62 mV and 178 ± 33 ms at
87 mV.
Voltage-dependence of activation
To examine the voltage dependence of
Ih required that the current be
isolated not only at hyperpolarized potentials but also in the voltage
range depolarized from rest. Ih was
therefore measured under conditions when depolarization-sensitive
currents were blocked by 10 mM TEA, 5 mM 4-AP, 250 µM
Cd2+, and 1 µM TTX but under which
Ih was also slightly blocked.
Ih was isolated by its
Cs+ sensitivity (data not shown). This
measurement revealed that Ih has an
activation threshold between about 45 and
50 mV, and that it is
maximally activated at voltages more hyperpolarized than about
90 mV
(n = 10).
A second method for determining the voltage-dependence of
Ih comes from measuring the peak tail
currents at a voltage that is as much as possible within the activation
range of Ih but outside the activation
range of other voltage-sensitive currents. This method is illustrated
in Fig. 6. The cell's voltage was
stepped from 62 mV to a range of voltages between
25 and
115 and
then repolarized to
77 mV. The amplitude of the tail current when the
voltage was stepped to
77 mV reflected the relative magnitude of the
conductance change caused by the previous step to variable voltages.
This tail current reflects a deactivation when the voltage is stepped
from more hyperpolarizing potentials to
77 mV and activation of
Ih when the voltage is stepped from
more depolarizing potentials to
77 mV. The family of tail currents
shown in Fig. 6A shows clusters of traces superimposed both
at the top and bottom. Those at the
top represent the activation of
Ih at
77 mV from voltage steps more
depolarized than the threshold of Ih;
from these potentials activation of Ih
with hyperpolarization to
77 mV activated a current of constant
magnitude. The cluster of current traces at the bottom
represents the deactivation of currents from hyperpolarizations that
produce maximal activation. The relative amplitude of tail currents,
measured immediately after the relaxation of the capacitative
transient, are plotted in Fig. 6B. The amplitude of tail
currents was normalized to the maximum current levels obtained after
the most negative prepulse,
115 and then plotted as a function of
step potential. This plot shows the voltage-sensitivity of
gh as the fraction of maximal
activation. This relationship is well fitted by the Boltzmann
relationship
![]() |
|
The average maximum conductance associated with
Ih can be derived. From the
measurement of currents in responses to voltage pulses to 112 mV, the
maximum conductances were calculated to range from 87 to 212 nS and
averaged 150 ± 30 nS (n = 36).
From these results it is possible to determine
gh and
Ih at the resting potential of octopus
cells. At 62 mV, the mean resting potential of octopus cells, 41% of
the maximum conductance is activated (Fig. 6B). Therefore at
rest gh ranged from 36 to 87 nS, with
an average of 62 ± 12 nS. With a reversal potential of
38 mV,
Ih contributes a steady inward current
of between 0.9 and 2.1 nA at the resting potential.
Reversal potential of Ih
The reversal potential for Ih was
calculated from currents measured below 60 mV, in a voltage range in
which depolarization-sensitive currents are minimally activated
(Banks et al. 1993
). Chord conductances were plotted
from measurements of the instantaneous current. The slopes of the
current-voltage relationships of the instantaneous current reflect the
ohmic relationship of the conductance of the cell at the previous
holding potential. The slopes of the plots thus vary, depending on the
degree of activation of Ih at the holding potential, and intersect at the reversal potential of Ih where there is no driving force. An
underlying assumption for these experiments is that
Ih is the sole or major conductance activated at the three holding potentials under the present conditions. Two observations support that assumption. The first is that the plots
are linear; the second is that all three plots intersect at the same
point. For the traces in Figs.
7A, the intersection of the
chord conductances that reflects the reversal potential of
Ih was
39 mV (Fig. 7B).
The mean reversal potential of Ih measured in five cells was
38 ± 2 mV.
|
Ionic nature of Ih
The measured reversal potential for
Ih, 38 mV, is consistent with the
conclusion that this is a mixed-cation current. We tested this
conclusion directly by varying extracellular concentrations of
K+ and Na+, ions that have
been shown to contribute to Ih in
other preparations.
Increasing the extracellular K+ concentration
from 3 to 12 mM increased the amplitude of
Ih in all cells tested (Fig.
8A). The reversal potential
for Ih shifted in the depolarizing
direction to 25.9 ± 1.2 mV (n = 3; Fig.
8C). The increase in amplitude of
Ih resulted from an increase in the
driving force through the conductance that underlies
Ih. The shift in the reversal
potential was 20 mV/10-fold change in extracellular
K+ concentration. Reduction of the extracellular
Na+ concentration from 138 to 10 mM caused
Ih to decrease (Fig. 8B). The reversal potential of Ih shifted
in the hyperpolarizing direction to
77 ± 3.5 mV
(n = 4; Fig. 8D). The hyperpolarizing shift
of the reversal potential caused the decrease in
Ih by decreasing the driving force
that acts on this conductance. The shift in reversal potential was 35 mV/10-fold change extracellular Na+
concentration. These results indicate that Na+
ions as well as K+ ions are the charge carriers
for Ih in octopus cells.
|
Assessment of the voltage clamp
In a class of neurons with low input resistance, the possibility
that voltage is not well controlled in portions of the cell at
locations distant from the electrode must be considered. In the present
series of experiments, two observations suggest that space-clamping is
adequate for the study of Ih. One
observation is that it was possible to use instantaneous currents to
measure reversal potentials from the convergence of chord conductances at a single point (Figs. 7B and 8, C and
D). If voltage pulses produced voltage changes that were
much smaller in the dendrites than at the cell body, chord conductances
measured from instantaneous currents would be expected to deviate from
linearity and not to convergence at a single point. The observations
illustrated in Figs. 7 and 8 show that the chord conductances are
linear and that they do converge at a single value. The second piece of
evidence that space clamping is adequate comes from the comparison of
reversal potentials and permeability ratios under varying conditions.
If space clamping is adequate, the measured reversal potential would be
expected to be a consistent function of the concentration gradients of
permeable ions. We tested this condition by comparing permeability ratios assuming that only Na+ and
K+ are involved, that their movement is
independent and that the electric field in the membrane is constant,
using the Goldman-Hodgkin-Katz equation. The permeability ratio,
PNa/PK,
was calculated under control conditions, when the extracellular
K+ concentration was high, and when the
extracellular Na+ concentration was low using the
equation:
![]() |
Modulation of the voltage sensitivity
Modulation of gh through
G-protein-coupled receptors and cAMP is characteristic of this type of
conductance (Pape 1996). We tested whether
gh in octopus cells was modulated by
cAMP by comparing the voltage-sensitivity measured from the tail
currents before and after the extracellular application of dibutyryl
cAMP in four cells. No significant difference was observed. When no ATP
or GTP was included in the intracellular pipette solution, the average Vhalf was shifted in the
hyperpolarizing direction. In comparison with measurements in the
presence of ATP and GTP where Vhalf
was
66 mV, in the absence of ATP and GTP,
Vhalf was
72 mV and shifted further
with time; Vhalf shifted to
81.6 mV
over 12 min (n = 6). In the absence of ATP, the
amplitude of Ih gradually decreased. Rundown was on average 40%/h (n = 8). The rundown and
shift was similar in cells bathed in 1 mM dibutyryl cAMP
(n = 6) and in the absence of a cAMP analogue
(n = 2). The rundown and hyperpolarizing shift in the
activation curve are therefore independent of cAMP.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present experiments have provided a description of the
hyperpolarization-activated mixed-cation conductance of octopus cells
in the posteroventral cochlear nucleus. This study shows that while the
gh in octopus cells resembles
gh in other types of cells in its
biophysical and pharmacological properties, two characteristics of
gh in octopus cells are notable: the
maximum conductance activated by hyperpolarization is large, ranging
between 87 and 212 nS, averaging 150 ± 30 nS, and a large
proportion of the maximum conductance is activated at rest. At the
average resting potential of octopus cells, 62 mV, 41% of the
maximum conductance is activated. Therefore at rest
gh contributed from 35 to 85 nS, with
a mean of 62 nS, to the input conductance of the cell.
A rough estimate can be made of the proportion of the resting input
conductance that can be attributed to
gh. The present study shows that the
input resistance measured from peak voltage responses to
hyperpolarizing current pulses is 6.7 M or 149 nS. [This value of
input resistance measured from peak responses presumably reflects the
input resistance at rest more closely than the slope of the
steady-state relationship of voltage responses to current pulses that
was reported earlier (Golding et al. 1999
).] On
average, 61.5 of the 149 nS or 41% of the resting input conductance is gh. The present experiments thus
confirm the conclusion that gh contributes to the exceptionally low input resistance that enables octopus cells to respond quickly and with temporal fidelity to coincident activation of auditory nerve fibers (Golding et al. 1995
, 1999
). Blocking Ih with
Cs+ causes action potentials (Golding et
al. 1999
) and synaptic responses (Golding et al.
1995
) to be larger and broader. The present experiments confirm
that gh affects signaling in the
physiological voltage range. Blocking
Ih with ZD7288 made action potentials
slower, higher, and broader.
The hyperpolarization-activated gh is
involved in setting the resting potential and the cell's excitability
(Lamas 1998; Pape 1996
). The present
results show that when gh was blocked,
the resting potential of octopus cells became hyperpolarized by 4-10 mV. At the average resting potential of octopus cells,
61.8 ± 1.7 mV (n = 135), 41% of the maximum conductance
specifically associated with Ih, 150 nS, on average 61.5 nS, was activated at rest. With a reversal
potential of
38 mV, such a conductance leads to an average, steady
inward current, Ih, of 1.47 nA at the
resting potential. The existence of this large steady current raises
the question how this current is balanced in the resting, steady-state
condition. A low-threshold, depolarization-sensitive K+ current
(IK(L)), plays an important role in
the functioning of these cells (Ferragamo and Oertel
1998
; Golding et al. 1995
, 1999
). Blocking this
4-AP- and DTX-sensitive current depolarizes cells, indicating that the
current is partially activated at rest.
Ih, an inward current, balances this
outward current and regulates the degree of its activation by
controlling the resting potential. Although
Ih has been called the "pacemaker
current" (Bal and McCormick 1997
;
Maccaferri and McBain 1996
; McCormick and Pape
1990b
; Pape 1996
), that is not the role of
Ih in octopus cells, which are characterized by their inability to fire rhythmically (Golding et al. 1995
, 1999
). The interplay between
IK(L) and
Ih would, however, be expected to
shape synaptic responses to activation of the auditory nerve.
Ih contributes to regulating the
"gain" of synaptic responses in octopus cells. Octopus cells detect
synchronous firing among its inputs (Golding et al.
1995). Individual auditory nerve inputs contribute a synaptic
current; the amplitude of the response to that synaptic current is
determined by the input conductance of octopus cells. The present
results show that gh contributes
~41% of the input conductance. In addition to its contribution to
the input conductance of octopus cells,
gh also affects the activation of
gK(L) by affecting the membrane
potential near rest.
In hippocampal neurons, the greater density and lower voltage range of
activation of Ih in the dendrites
relative to the soma affects both the temporal summation of synaptic
inputs and also the effectiveness of the back-propagation of voltage
into dendrites (Magee 1998). It is not known whether
gh is expressed nonuniformly in
octopus cells, but the possibility that
gh affects the pattern of temporal and
spatial summation of auditory nerve inputs in octopus cells is an
intriguing one in cells that encode the timing of auditory nerve inputs
with precision (Godfrey et al. 1975
; Golding et
al. 1995
; Rhode and Smith 1986
; Rhode et
al. 1983
).
Two classes of hyperpolarization-sensitive conductances
Two distinct classes of hyperpolarization-sensitive conductances
have been described and cloned. One underlies a rectifying K+ current and has been called
IKIR; the other underlies a
mixed-cation current and has been called
Ih,
If in the heart, or the "pacemaker current" on the basis of the role it has been shown to play in the
heart and in thalamic and cortical neurons. These conductances are
mediated through two distinct classes of ion channels. The subunits of
ion channels that underlie IKIR are
distantly related to the family of depolarization-sensitive
K+ channels; their rectification arises from a
voltage-dependent block by polyamines and Mg2+
(Kubo et al. 1993; Nichols and Lopatin
1997
; Nichols et al. 1994
). Ih arises through ion channels whose
molecular structure resembles depolarization-sensitive
K+ channels more closely (Biel et al.
1999
; Ishii et al. 1999
; Ludwig et al.
1998
; Santoro et al. 1998
; Seifert et al.
1999
). This current is characterized by its sensitivity to
hyperpolarization, by being carried by both K+
and Na+, and by being regulated by cyclic
nucleotides (Pape 1996
). Four families of ion channels
have been described that mediate Ih
with differing rates of activation and differing sensitivity to cyclic nucleotides, termed HCN (for hyperpolarization-activated and
cyclic nucleotide-gated channels) (Clapham
1998
; Ludwig et al. 1998
, 1999
; Santoro
and Tibbs 1999
; Santoro et al. 1998
;
Seifert et al. 1999
). The two classes of
hyperpolarization-sensitive currents, Ih and
IKIR, can be distinguished
pharmacologically. While IKIR is
sensitive to extracellular cations including
Cs+ and Ba2+ (Kubo
et al. 1993
), Ih is sensitive
to the bradicardiac agent, ZD7288, but relatively insensitive to
Ba2+ (BoSmith et al. 1993
;
Gasparini and DiFrancesco 1997
; Khakh and Henderson 1998
; Lüthi et al. 1998
;
Pape 1994
; Santoro et al. 1998
;
Spain et al. 1987
; van Ginneken and Giles
1991
; Womble and Moises 1993
).
Hyperpolarization-activated, mixed-cation currents have been described
in many neuronal and nonneuronal cells (Pape 1996). In
general, the properties of Ih in
octopus cells are similar to those of
Ih recorded in other cells including
primary auditory neurons (Chen 1997
; Mo
and Davis 1997
) brain stem auditory neurons (Banks et
al. 1993
; Fu et al. 1997
), nonauditory neurons
(Doan and Kunze 1999
; Ingram and Williams
1996
; Khakh and Henderson 1998
;
Maccaferri and McBain 1996
; Magee 1998
;
Pape 1996
; Vargas and Lucero 1999
) as
well as cardiac muscle (DiFrancesco 1993
; Pape
1996
).
Reversal potential and ionic basis of Ih
The reversal potential measured for
Ih in octopus cells was 38 mV under
control conditions and depended on the extracellular concentrations of
both Na+ and K+, indicating
that Ih in octopus cells is carried by
both cations. These findings confirm the conclusion based on
current-clamp experiments (Golding et al. 1999
). The
present findings are comparable to those reported in other cells.
Measurements of reversal potentials of
Ih in other cells measured under
similar but not identical ionic conditions were similar, ranging from
30 to
44 mV (Banks et al. 1993
; Chen
1997
; Maccaferri and McBain 1996
;
McCormick and Pape 1990b
; Mo and Davis
1997
). The permeability ratio,
PNa/PK, for Ih in octopus cells was estimated
from the Goldman-Hodgkin-Katz equation to be between 0.16 and 0.29, depending on the ionic conditions. These values lie within the range
measured in other types of cells between 0.2 and 0.4 (Hestrin
1987
; Magee 1998
; Maricq and Korenbrot 1990
; Solomon and Nerbonne 1993b
).
Voltage sensitivity of Ih
Under control physiological conditions, the activation curve for
gh derived from tail current
measurements in octopus cells revealed an activation threshold of about
35 to
40 mV and full activation at voltages more hyperpolarized
than
100 mV. Fitting the steady-state activation curve with the
Boltzmann relationship showed that the
Vhalf and slope factor, k,
of
65.2 ± 3.17 and 7.7 ± 0.7 mV, respectively. A
comparison of Vhalf in some of the cells in which it has been measured is given in Fig.
9. This comparison reveals that
Vhalf in octopus cells lies at a
relatively depolarized potential relative to other cells in which it
has been measured. It is the large amplitude of the maximal
gh together with the relatively
depolarized voltage range of activation that leads to the large
gh in the physiological voltage range
of octopus cells.
|
The measurement of voltage sensitivity depends on the conditions under
which the experiments were done. It is characteristic of
gh that its voltage sensitivity is
regulated by neurotransmitters through intracellular messengers
(Cathala and Paupardin-Tritsch 1999;
DiFrancesco and Tortora 1991
; Ingram and Williams
1996
; Ludwig et al. 1998
; McCormick and
Pape 1990b
; Pape 1996
; Pape and McCormick
1989
; Santoro and Tibbs 1999
; Santoro et
al. 1998
; Tokimasa and Akasu 1990
; Vargas
and Lucero 1999
; Zaza et al. 1996
). In most
cells, cAMP shifted Vhalf in the
depolarizing direction (Banks et al. 1993
; Chen
1997
; DiFrancesco and Tortora 1991
;
Tokimasa and Akasu 1990
; van Ginneken and Giles
1991
). Perhaps it is not surprising that in octopus cells,
where Vhalf lies at a relatively depolarized level even in the absence of the application of cAMP and
its analogues, dibutyryl cAMP had no measurable effect. The shift of
Vhalf in the hyperpolarizing direction
by the removal of intracellular nucleotide triphosphates, independent
of cAMP and accompanied by rundown that was observed in octopus cells, has also been reported in the heart and in bullfrog sympathetic neurons
(Di Francesco and Mangoni 1994
; Tokimasa and
Akasu 1990
).
The question arises to what extent the activation values measured with
patch-clamping and intracellular perfusion reflect the function of
gh in octopus cells in situ. Two lines
of evidence indicate that gh is
strongly activated at rest under physiological conditions. The first is
that the prominence of gh has been
documented in recordings made with sharp microelectrodes
(Golding et al. 1995). These recordings reveal
properties that are similar to those recorded in current clamp in the
present recordings. The second line of evidence is that the properties
of octopus cells revealed both in sharp-electrode recordings and
whole-cell patch recordings are consistent with their responses to
sound (Godfrey et al. 1975
; Golding et al.
1995
; Rhode and Smith 1986
).
Amplitude of Ih
In octopus cells, gh and
Ih are larger than in other neuronal
cell types in which they have been measured. To make a comparison of
amplitude of gh across different types
of cells, gh at 100 mV and the
voltage at which the conductance is activated half-maximally are
graphed in Fig. 9. At
100 mV gh is
99% activated in octopus cells, averaging 150 nS. It is two orders of
magnitude larger than in many cells and it is at least twice as large
as the largest conductance measured in any other group of cells. The
combination of a relatively large conductance and relatively
depolarized voltage range of activation gives octopus cells an
unusually large gh in the
physiological voltage range. Interestingly,
gh is also particularly large and the
half-maximal voltage of activation is relatively depolarized in another
auditory brain stem nucleus, the medial nucleus of the trapezoid body
(Banks et al. 1993
). Comparison between cells is
necessarily crude because measurements were made under conditions that
differed in the temperature and the composition of extracellular and
intracellular solutions as well as in the species in which they have
been recorded. It is likely that the states of modulation also differed
in different studies. Comparisons in amplitude and voltage of
half-maximal activation are further hampered by the fact that the rates
of activation of Ih are in some cases
so slow that it is not practical to measure activation in the steady
state. Crude though the comparison may be, it shows that
gh in octopus cells is unusually large.
Rate of activation
The activation phase of Ih in
octopus cells was best described by the sum of two voltage-dependent
exponentials in the tens and hundreds of milliseconds, respectively.
The rate of deactivation was described by a single exponential with a
time constant in the hundreds of milliseconds (Fig. 5). These rates are
comparable to the fastest rates of activation and deactivation observed
in other types of cells. The rates of activation that have been
described vary over a wide range. While in some cells, including most
neurons, Ih is fully activated within
1 s, in other cells, including thalamic neurons and cardiac cells,
Ih is not fully activated even after several seconds (Ludwig et al. 1999; Santoro and
Tibbs 1999
; Seifert et al. 1999
; Solomon
et al. 1993
). These differences probably reflect differences in
the molecular composition of the ion channels. Four members of the HCN
gene family have been cloned, one of which (HCN4) has particularly slow
rates of activation when measured in an expression system and is
strongly expressed in the heart and thalamus (Ludwig et al.
1999
; Santoro and Tibbs 1999
). Our results
suggest that the slow forms HCN2 and HCN4 are probably not strongly
expressed in octopus cells, but at present it is not possible to know
which of the other classes of subunits would be expected to be prominent.
![]() |
ACKNOWLEDGMENTS |
---|
We thank I. Siggelkow and J. Meister whose technical support was crucial to the success of these experiments. We also thank S. Gardner whose instruction and attention to details made experiments work. This study built on and was enlivened by discussions with N. Golding, M. Ferragamo, R. Fettiplace, and L. Trussell for which we are grateful.
This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-00176.
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. Oertel, Dept. of Physiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706 (E-mail: oertel{at}physiology.wisc.edu).
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 18 January 2000; accepted in final form 1 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|