School of Biomedical Sciences, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia
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ABSTRACT |
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Hogg, R. C.,
A. A. Harper, and
D. J. Adams.
Developmental Changes in Hyperpolarization-Activated Currents
Ih and IK(IR) in
Isolated Rat Intracardiac Neurons.
J. Neurophysiol. 86: 312-320, 2001.
The hyperpolarization-activated nonselective cation
current, Ih, was investigated in
neonatal and adult rat intracardiac neurons. Ih was observed in all neurons studied
and displayed slow time-dependent rectification.
Ih was isolated by blockade with
external Cs+ (2 mM) and was inhibited
irreversibly by the bradycardic agent, ZD 7288. Current density of
Ih was approximately twofold greater in neurons from neonatal (4.1 pA/pF at
130 mV) as compared with adult (
2.3 pA/pF) rats; however, the reversal potential and
activation parameters were unchanged. The reversal potential and
amplitude of Ih was sensitive to
changes in external Na+ and
K+ concentrations. An inwardly rectifying
K+ current,
IK(IR), was also present in
intracardiac neurons from adult but not neonatal rats and was blocked
by extracellular Ba2+.
IK(IR) was present in approximately
one-third of the adult intracardiac neurons studied, with a current
density of
0.6 pA/pF at
130 mV.
IK(IR) displayed rapid activation
kinetics and no time-dependent rectification consistent with the
rapidly activating, inward K+ rectifier described
in other mammalian autonomic neurons.
IK(IR) was sensitive to changes in
external K+, whereby raising the external
K+ concentration from 3 to 15 mM shifted the
reversal potential by approximately +36 mV. Substitution of external
Na+ had no effect on the reversal potential or
amplitude of IK(IR). IK(IR) density increases as a function
of postnatal development in a population of rat intracardiac neurons,
which together with a concomitant decrease in
Ih may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.
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INTRODUCTION |
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Autonomic control of heart
rate changes during early postnatal development in the rat. Direct
vagal nerve stimulation studies reveal significant decreases with
postnatal age in the intrinsic heart rate and maximal parasympathetic
control of heart rate (Quigley et al. 1996). The
electrical properties of autonomic ganglion neurons from immature
animals have been reported to be different from those of mature, adult
animals (e.g., Gottmann et al. 1988
; Hirst and
McLachlan 1984
). The intrinsic membrane properties of a neuron
are not static but may alter during development by the up or
downregulation of ion channel expression or as a result of modulation
of the existing complement of ion channel subunits. Membrane
hyperpolarization can activate two inwardly rectifying conductances in
autonomic neurons: a nonselective cation current (Ih), with Na+
and K+ as the charge carriers, and a
K+-selective current
(IK(IR)).
Electrophysiological studies have revealed that
Ih is expressed in all neonatal
(Cuevas et al. 1997) and adult (Xi-Moy and Dunn
1995
) rat intracardiac neurons.
Ih was originally identified in mouse
primary afferent neurons (Mayer and Westbrook 1983
), and
is analogous to If in the cardiac
conduction system (DiFrancesco et al. 1986
), which
contributes to the generation of the cardiac rhythm. In guinea pig
intracardiac ganglia, the population of neurons possessing
Ih frequently display an inherent
repetitive spontaneous discharge and have been proposed to serve a
sensory role (Edwards et al. 1995
).
Inwardly rectifying K+ channels open on membrane
hyperpolarization and close with membrane depolarization. The inwardly
rectifying K+ current, first characterized in
rabbit sympathetic ganglion neurons as a time-independent rectification
of hyperpolarizing electrotonic potentials (Christ and Nishi
1973), has been identified in a variety of mammalian autonomic
neurons (see Adams and Harper 1995
). Whereas both
Ih and
IK(IR) are activated by
hyperpolarization and are sensitive to the extracellular
K+ concentration, they exhibit different voltage
dependence, kinetics, and pharmacological sensitivity.
IK(IR) activation is voltage dependent
with a threshold of approximately
85 mV in physiological extracellular K+ concentrations and effectively
instantaneous kinetics. In contrast, Ih activates slowly, typically with a
time constant of tens of milliseconds at voltages negative to
60 mV,
and the time course of activation is voltage sensitive.
Ih is reversibly blocked by external
Cs+ (
1 mM) but not by
Ba2+, distinguishing it from
IK(IR), which is blocked by low
micromolar Ba2+ concentrations (Adams and
Nonner 1990
). The reversal potential of
Ih is positive to the resting membrane
potential (Em), ranging between
50
and
20 mV (Pape 1996
), whereas the reversal potential of IK(IR) follows the
K+ equilibrium potential
(EK). These characteristics suggest
that Ih and
IK(IR) may be suited to modulate the
subthreshold resting potential.
The expression of Ih and
IK(IR) has been reported to vary
significantly in adult rat dorsal root ganglion (DRG) neurons of different size (Petruska et al. 2000; Scroggs et al.
1994
), and regional differences in the distribution of the
hyperpolarization-activated currents,
Ih and
IK(IR), have been demonstrated in rat
primary afferent neurons (Wang et al. 1997
).
Ih was largely confined to the soma,
whereas IK(IR) was less frequently
found in the soma than in the growth cone. This inhomogeneous
distribution of these ion channels is consistent with the differential
functions of these currents. The aim of this present study was to
determine whether the expression and properties of
Ih and
IK(IR) are related to the stage of
postnatal development in rat intracardiac neurons and subsequently to
determine whether these currents are involved in control of firing
behavior. Preliminary reports of some of these results have been
presented to the Physiological Society (Harper et al.
1998
; Hogg et al. 1999
).
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METHODS |
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Preparation
The isolation and culture of parasympathetic neurons from
neonatal rat intracardiac ganglia has been described previously (Xu and Adams 1992). Briefly, neonatal rats (2-8 days
old) were killed by decapitation in accordance with the guidelines of
the University of Queensland Animal Experimentation Ethics Committee. The heart was excised and placed in a saline solution containing (in
mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose, and 10 histidine (pH 7.2).
Atria were separated and the medial region containing the pulmonary
veins and superior vena cava was identified, isolated, and incubated in
a saline solution containing collagenase (1 mg/ml,
Worthington-Biochemical Type 2, specific activity ~100 units/mg) at
37°C for 60 min. Cross sections of ganglia were dissected from the
epicardial ganglion plexus and dispersed by trituration in a high
glucose culture medium (Dulbecco's modified Eagle medium), containing
10% fetal calf serum, 100 units/ml penicillin and 0.1 mg/ml
streptomycin. The dissociated neurons were plated on to laminin-coated
glass coverslips and incubated at 37°C under a 95% air-5%
CO2 atmosphere for 24-60 h. In a series of
experiments, intracardiac neurons were isolated from neonatal rats
using trypsin (0.2 mg/ml) in the dissociation procedure and the same
enzymatic conditions used for obtaining adult neurons.
Young adult female Wistar rats (5-6 wk, 160-180 g) were killed by
stunning and cervical dislocation, the hearts excised, atria isolated
and placed in cold saline solution. The intracardiac ganglia were
identified and dissected from the fat pads of the dorsal surfaces of
the atria. Intracardiac neurons were isolated using a combination of
enzymatic and mechanical dispersion using a protocol adapted from that
described previously (Jeong and Wurster 1997). The
ganglia were incubated in a saline solution containing 1.2 mg/ml
collagenase (specific activity ~1,000 units/mg; Sigma Type 1A) and
0.1-0.2 mg/ml trypsin from bovine pancreas (~13,000 units/mg; Sigma)
at 37°C for 60 min. The ganglia were dispersed by trituration and
washed twice with culture medium. The protocol for plating and
incubation followed that described for neonatal neurons.
Electrophysiological recording
Neurons plated on glass coverslips were transferred to a
recording chamber (volume 0.5 ml) mounted on an inverted phase contrast microscope (×400 magnification), allowing isolated neurons to be
identified. Membrane voltage and current were recorded using the
perforated patch whole cell recording configuration. The perforated patch recording configuration was used to preserve intracellular regulatory systems and reduce "rundown" or loss of membrane
currents through cell dialysis. Our previous studies have demonstrated that some ionic currents including Ih
are absent in dialyzed intracardiac neurons (Cuevas et al.
1997). A final concentration of 240 µg/ml amphotericin B in
0.4% DMSO was used in the pipette solution. Patch electrodes were
pulled from thin-walled borosilicate glass (GC150TF; Harvard Apparatus
Ltd., Edenbridge, Kent, UK) and after fire polishing had resistances of
~1 M
. Access resistances using the perforated patch configuration
were routinely
2 M
following series resistance compensation.
Membrane current and voltage were recorded using an Axopatch 200A
patch-clamp amplifier (Axon Instruments, Foster City, CA) and were
filtered at 10 and 3 kHz, respectively, then digitized at 10-50 kHz
(Digidata 1200A interface, Axon Instruments) and stored on the hard
disk of a PC for viewing and analysis. Voltage and current protocols
were applied using pClamp software (Version 6.1.3, Axon Instruments).
Exponential curve fits were made using the Chebyshev fitting routine in
pClamp (Axon Instruments), Boltzmann fits were made using a
least-squares nonlinear curve fitting routine in Microcal Origin 5.0 (Microcal Software, Northampton, MA), and straight line fits were by
linear regression. No corrections were made for liquid junction
potentials. Data are presented as means ± SE and are compared
using paired or unpaired t-tests.
Solutions
The control external solution for perforated-patch whole cell recordings was physiological saline solution (PSS) containing (in mM) 140 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose, and 10 HEPES-NaOH buffered to pH 7.2. The pipette solution contained (in mM), 75 K2SO4, 55 KCl, 5 MgSO4, and 10 HEPES (titrated to pH 7.2 with N-methyl-D-glucamine). Alterations in extracellular K+ concentration were made by equimolar substitution of K+ for Na+. Changes in extracellular Na+ were made by replacing extracellular Na+ with either N-methyl-D-glucamine (NMDG) or arginine. The osmolarity of the extracellular and pipette solutions was monitored by a vapor pressure osmometer (Wescor 5500, Logan, UT) and were in the range 280-290 mmol/kg. The temperature of the superfusing solutions was controlled by a Peltier thermoelectric device and monitored by an independent thermistor probe in the recording chamber. The recording chamber was continuously superfused with a maximum deviation of 1°C in any individual procedure. Pharmacological agents were bath applied at the concentrations indicated. All chemicals used were of analytical grade. The following drugs were used: amphotericin B (Sigma Chemical, St. Louis, MO), N-ethyl-1,6-dihydro-1,2-dimethyl-6-(methylamino)-N-phenyl-4-pyrimidinamine hydrochloride (ZD 7288; Tocris Cookson, Bristol, UK).
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RESULTS |
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Passive electrical properties of intracardiac neurons from neonatal and adult rats
Hyperpolarization-activated currents were studied in a total of 25 neonatal and 16 adult rat intracardiac neurons. Neurons isolated from neonatal rats were on average smaller in size than those from adults having a mean cell capacitance of 19.0 ± 3.4 pF (n = 12) compared with 54.3 ± 8.5 pF (n = 8) for adult neurons. The mean cell input resistance was higher in neonatal than adult neurons, and there was no significant difference in the resting membrane potential (see Table 1).
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Current-clamp recording of Ih and IK(IR)
Ih was evident in current-clamp
records as a characteristic sag in the voltage response to a
hyperpolarizing current step (Fig.
1A). Peak and steady-state
values for the voltage response in an adult rat intracardiac neuron are
plotted in Fig. 1D for control ( and
) and in the
presence of 10 µM Ba2+ (
and
) to inhibit
IK(IR). External
Ba2+ (10 µM) slightly increased the membrane
resistance and caused a leftward shift in the current-voltage
(I-V) relationship of a similar magnitude for both peak and
steady-state voltage responses to hyperpolarizing currents (Fig.
1D).
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External Cs+ (2 mM) reversibly inhibited the sag
in the voltage response (Fig. 1C). The effect of 2 mM
Cs+ on the steady-state I-V
relationship is shown in Fig. 1E. External Cs+ caused an increase in the membrane resistance
in the activation range of Ih. Block
of Ih with 2 mM
Cs+ produced a small, but significant,
hyperpolarization of membrane potential at both 22°C (52.9 ± 2.0 mV control, mean ± SE;
54.7 ± 1.8 mV,
Cs+, n = 6, P = 0.04) and 37°C (
50.0 ± 2.9 mV control;
52.6 ± 3.1 mV,
Cs+, n = 5, P = 0.01). Under the same conditions, Cs+ lengthened
the duration between action potentials within bursts evoked by a
sustained depolarizing current pulse and lengthened the
afterhyperpolarization (AHP) following single action potentials evoked
by a short depolarizing pulse (500 pA, 2.5 ms). The frequency of action
potential discharge in response to depolarizing currents was slowed by
12% in the presence of external Cs+ at 37°C;
the interval between the first and second action potential being
20.1 ± 1.7 ms (control) and 22.6 ± 1.7 ms
(Cs+; n = 4) but was not affected
at 22°C (Fig. 2). Similarly, whereas the time for recovery of the action potential AHP (AHP duration to 80%
recovery) at 37°C significantly increased from 22.1 ± 5.9 ms
(control) to 28.2 ± 6.6 ms (n = 3;
P = 0.03) in the presence of Cs+,
no change in AHP duration was observed at 22°C. Temperature-dependent effects of Cs+ on membrane potential, spike
interval, and the AHP were similar in intracardiac neurons isolated
from adult and neonatal rats.
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Pharmacological isolation of Ih
Ih was isolated under
voltage-clamp conditions in neurons from neonatal and adult rats as a
slowly activating inward current in response to hyperpolarizing voltage
steps negative to 60 mV. Ih was
reversibly inhibited by the addition of 2 mM CsCl to the bathing
solution and could be isolated by subtraction of currents recorded in
the presence of Cs+ from those obtained in
control conditions (Fig. 3A).
The bradycardic agent ZD 7288 (10-100 µM), which has been shown to
block If and Ih (BoSmith et al.
1993
; Harris and Constanti 1995
) also inhibited Ih in rat intracardiac neurons in a
concentration-dependent and irreversible manner (Fig. 3B).
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Several distinct protocols for determining the I-V
relationship of Ih have been
described, and the measurement of the reversal potential with the
protocol used (Wang et al. 1997) was in agreement with
that calculated using the other methods (Jafri and Weinrich 1998
; Lamas 1998
). Figure 3C shows an
I-V relationship for Ih in
a neonatal neuron determined by subtraction of the total current I-V relationship in control conditions from that in the
presence of 2 mM Cs+. Figure 3D shows
the difference I-V relationship in an adult neuron obtained
in the absence and presence of 100 µM ZD 7288. There was no
significant difference in the reversal potential of
Ih obtained in the presence of either
Cs+ or ZD 7288 (see Table
2).
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The current density of Ih was twofold greater in neonatal than in adult rat intracardiac neurons (Fig. 4, A and B; see Table 2). The relationship between steady-state current and cell size (pF) was well described by a linear regression for each stage of development (R = 0.93 and 0.89 for neonatal and adult neurons, respectively). Representative traces of Ih obtained from neonatal (23 pF) and adult (63 pF) neurons are shown in Fig. 4, A and B.
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Ih has been reported to be
absent in neurons dissociated from neonatal rat cerebral cortex
immediately following cell isolation and reappears after 1-2 days in
culture (Budde et al. 1994). The disappearance of
Ih was attributed to the use of
trypsin in the dissociation procedure, and
Ih was also sensitive to proteolysis following superfusion of the preparation with trypsin. In a series of
experiments, collagenase and trypsin were used to dissociate neonatal
intracardiac neurons. Ih was present
in all neonatal neurons dissociated with trypsin and collagenase with a
current density of
5.6 ± 1.2 pA/pF (n = 6),
which was not significantly different (P < 0.05) from
the mean value obtained for neonatal neurons dissociated with
collagenase only (cf. Table 2).
Characterization of IK(IR) in adult neurons
A rapidly activating inwardly rectifying current,
IK(IR) was present in approximately
one-third (6 of 16 cells) of the adult rat intracardiac neurons studied
but not in neurons obtained from neonatal rats (n = 25). IK(IR) was blocked by bath
application of solutions containing 10 µM Ba2+.
IK(IR) displayed rapid activation
kinetics but did not exhibit time-dependent rectification consistent
with the rapidly activating, inward K+ rectifier
described in other mammalian autonomic neurons (see Adams and
Harper 1995). Under current-clamp conditions, 10 µM Ba2+ reduced both the peak and steady-state
voltage response to hyperpolarizing current pulses to a similar degree
(Fig. 1). The Ba2+-sensitive component was
isolated by subtraction of currents obtained in the presence of 10 µM
Ba2+ from the total current. Figure
5A shows that the
Ba2+-sensitive current,
IK(IR), was not affected by the
Ih inhibitor, ZD 7288 (100 µM).
IK(IR) was sensitive to changes in
external K+ but not Na+.
Raising external K+ from 3 to 15 mM shifted the
reversal potential by approximately +36 mV (Fig. 5C) similar
to that predicted by the Nernst equation for a
K+-selective electrode.
IK(IR) had a current density of
0.61 ± 0.26 pA/pF (n = 6) at
130 mV in 3 mM
extracellular K+.
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Activation characteristics of Ih
The steady-state I-V relationship for
Ih was determined from tail current
amplitude following maximal current activation and was linear in both
neonatal and adult neurons (Fig. 6,
A and B). The reversal (zero-current) potential
was determined by extrapolation of the I-V relationship for
tail currents. There was no significant difference in the reversal
potentials obtained for Ih in neonatal (22.2 ± 7.0 mV, n = 3) and adult (
23.6 ± 3.6 mV, n = 7) rat intracardiac neurons,
respectively. The mean slope conductance was 42.3 ± 8.4 pS/pF
(n = 3) for neonates and 22.2 ± 3.0 pS/pF (n = 7) for adults.
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The steady-state activation characteristics of
Ih were compared in voltage-clamped
neonatal and adult intracardiac neurons. Neurons were stepped from test
potentials between 60 and
120 mV to
50 mV (Fig. 6C).
Tail current amplitudes were normalized to the maximum current measured
at
120 mV. The relationship between the prepulse potential and the
normalized current amplitude was fit by a Boltzmann equation (Fig.
6D). There was no significant difference in the slope or
midpoint of the activation curves of Ih from neonatal or adult neurons (see
Table 2).
The activation kinetics of Ih were
compared in neonatal and adult neurons, and the activation time course
was best fit by a single exponential. The time course of activation was
voltage dependent, becoming more rapid with increasing
membrane hyperpolarization (Fig.
7A). The time constant of
activation () for Ih between
130
and
100 mV is plotted against membrane potential as shown in Fig.
7B.
was significantly faster in intracardiac neurons from adult (89 ± 16 ms, n = 11) as compared with
neonatal rats (263 ± 32 ms, n = 8) at
130 mV
and 22°C and exhibited an e-fold change per 27.0 mV in
adult and per 28.5 mV in neonatal rat neurons.
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Effect of temperature on Ih
The properties of Ih in
neurons from adult and neonatal rats were examined at 22 and 37°C
under voltage-clamp conditions. The time constant of activation
decreased exponentially with increasing hyperpolarization (Fig.
8A) and exhibited an
e-fold change per 24 mV at 22°C and 18 mV at 37°C.
Raising the temperature from 22 to 37°C significantly reduced the
time constant of activation at 130 mV in neurons from neonatal rats
(Fig. 8B) from 263 ± 32 ms (n = 3) to
46 ± 7 ms (n = 3) giving a
Q10 of 3.1. The current density of
Ih measured at 22°C was
significantly greater in neurons from neonatal (
4.1 pA/pF at
130
mV) as compared with adult (
2.3 pA/pF) rats. The current density of
Ih in neurons from neonatal rats
increased to
6.3 pA/pF at 37°C and exhibited a
Q10 of 1.3.
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Ionic basis of Ih
Ih was sensitive to changes in the extracellular K+ concentration, whereby elevating external K+ from 3 to 15 mM caused an approximate threefold increase in the amplitude of the inward current in response to hyperpolarizing voltage steps. Raising external K+ had no effect on the time course of activation (Fig. 9A). The fully activated and steady-state I-V relationships were determined for adult neurons in 3 and 15 mM extracellular K+ (Fig. 9, B and C). A fivefold increase in the extracellular K+ concentration caused a four- to fivefold increase in the slope conductance without a significant shift of the reversal potential (Fig. 9B). The calculated PNa/PK ratio obtained in the presence of 3 mM K+ and 15 mM K+ was 0.42 and 0.35, respectively. Replacement of extracellular Na+ with either NMDG or arginine shifted the I-V relationship and reversal potential to more negative membrane potentials, indicating that Na+ contributes to Ih in these neurons (n = 5, data not shown). Elevating the extracellular K+ concentration did not affect the steady-state activation characteristics of Ih. Steady-state activation curves obtained from adult rat intracardiac neurons are shown in Fig. 9D.
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DISCUSSION |
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We have analyzed two distinct hyperpolarization-activated currents
in rat intracardiac neurons, Ih and
IK(IR).
Ih was identified on the basis of its
pharmacological and biophysical properties that are similar to a
nonselective cation current Ih
described in central and peripheral neurons (see Pape
1996) and was present in intracardiac neurons from neonatal and
adult rats. Another inwardly rectifying
K+-selective current,
IK(IR), was observed in intracardiac
neurons from adult rats only. Neonatal and adult intracardiac neurons differed in size with the mean cell capacitance of adult neurons more
than twofold higher than neonatal. Ih
current density was significantly lower in adult rat intracardiac
neurons, which coincided with the appearance of
IK(IR) in some neurons from adult
rats. In previous experiments using the conventional dialyzed whole cell recording configuration, Ih was
not observed, indicating that a diffusable intracellular factor may be
required (Xu and Adams 1992
). The lower current density
of Ih in neurons from adult rats was
not due to disappearance of Ih caused
by the use of trypsin during dissociation (cf. Budde et al.
1994
) as intracardiac neurons isolated from neonatal rats using
collagenase and trypsin did not exhibit a significantly lower density
of Ih than for neurons isolated using
collagenase only. Changes in expression of
Ih with development have been reported
previously whereby an increase in the number of cells expressing
Ih increases during development in
embryonic quail ganglion neurons (Schlichter et al.
1991
). Age-related changes in
Ih have also been described in rat
hypoglossal motoneurons (Bayliss et al. 1994
), and in
rabbit sinoatrial node cells the slope conductance of
If is greater in newborn than in adult
rabbits (Accili et al. 1997
).
Although Ih has been reported
previously in intracardiac neurons from both neonatal (Cuevas et
al. 1997) and adult rats (Xi-Moy and Dun 1995
),
the reversal potential, ionic selectivity, and activation
characteristics of Ih have not been
described. In vitro studies of autonomic neurons are often carried out
at room temperature (20-22°C); however, the temperature dependence
of ionic currents is often nonlinear, and different ionic conductances
can have distinct temperature sensitivities. This results in
conductances having differing effects on neuronal firing at 22 and
37°C. The effect of temperature on
Ih indicates that the kinetics of
Ih activation is highly temperature
dependent, whereas current density was, in contrast, less sensitive to
temperature change. The increased AHP and interval between action
potentials at 37°C but not 22°C following block of
Ih with Cs+
suggest that Ih contributes to the
control of neuronal excitability primarily at 37°C.
Our studies have shown that the firing behavior of adult intracardiac
neurons is different from that observed in neonatal neurons. Adaptive
firing observed at 37°C in intracardiac neurons from neonatal rats
has been shown to be regulated by the muscarine-sensitive K+ current (IM)
(Cuevas et al. 1997), whereas the firing behavior of
neurons from adult rats under similar conditions is not altered in the
presence of ACh (Hogg and Adams, unpublished observation). Differences
in current density and activation kinetics of
Ih may contribute to this change in
firing properties. Ih is likely to be
activated during the AHP of the action potential, and, given that the
reversal potential is positive to the
Em, it will act to depolarize the
cell, limit the AHP duration, and shorten the interval between action
potentials to promote multiple discharge. The effects of external
Cs+ on the Em
and action potential discharge at 37°C suggest that Ih may play a role in regulating
action potential firing in intracardiac neurons.
Ih has been reported to contribute to
the Em in rat superior cervical
ganglion sympathetic neurons (Lamas 1998
) and contribute to the resting and active properties of sensory (Jafri and
Weinrich 1998
) and central neurons (Maccaferri et al.
1993
; McCormick and Pape 1990
; Solomon
and Nerbonne 1993
; Womble and Moises 1993
). Ih is subject to modulation by
neurotransmitters, neuropeptides, and inflammatory mediators that
converge to act via adenyl cyclase (Pape 1996
). The
membrane permeable analogue of cAMP, 8-bromo cAMP, increased the
amplitude of Ih in neonatal rat
intracardiac neurons (Hogg and Adams, unpublished observations). It is
possible that Ih plays a more
significant role in resting and active properties in intracardiac
neurons in vitro due to neuromodulation than is apparent in isolated neurons.
IK(IR) activates instantaneously, does
not exhibit time-dependent rectification, and is blocked by externally
applied Ba2+ (10 µM) similar to the inward
rectifying K+ current recorded in numerous neuron
types (see Adams and Harper 1995; Rudy
1988
). The precise role of
IK(IR) in adult intracardiac neurons
remains unclear. The activation range of
IK(IR) would confine its activation to
membrane potentials negative to Em, such as would occur during the action potential AHP. The differences in
firing behavior in neonatal and adult rat intracardiac neurons are
likely due to changes in the contribution of both
Ih and
IK(IR).
The ion channel inhibitors, Cs+ and
Ba2+, have been shown to differentially affect
the vagally induced pacemaker response in anesthetized dogs, whereby
BaCl2 attenuated the vagally induced bradycardia
without affecting other components of the response. In contrast, CsCl
had no effect on the initial vagal slowing of atrial rate but abolished
the acceleratory portion of the response (Wallick et al.
1997). Local arterial infusion of barium chloride to the right
atrial ganglionated plexus has also been shown to directly modulate the
electrical activity of canine intracardiac neurons in situ
(Thompson et al. 2000
). The inhibition of
Ih and IK(IR) by Cs+
and Ba2+, respectively, may contribute to
the observed changes in neuronal excitability of mammalian intrinsic
cardiac ganglia. Furthermore, the changes in the functional expression
of Ih and
IK(IR) with postnatal development
suggests that different ionic mechanisms may contribute to modulating
neuronal excitability neonatal and adult intracardiac ganglion neurons.
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ACKNOWLEDGMENTS |
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This work was supported by National Health and Medical Research Council of Australia and Australian Research Council grants to D. J. Adams and a Travel Research Award from The Royal Society and The Carnegie Trust for the Universities of Scotland to A. A. Harper.
Present address of A. A. Harper: Div. of Molecular Physiology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, UK.
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FOOTNOTES |
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Address for reprint requests: D. J. Adams (E-mail: dadams{at}plpk.uq.edu.au).
Received 22 December 2000; accepted in final form 14 March 2001.
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REFERENCES |
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