Department of Physiology, Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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ABSTRACT |
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Martin-Caraballo, Miguel and John J. Greer. Development of Potassium Conductances in Perinatal Rat Phrenic Motoneurons. J. Neurophysiol. 83: 3497-3508, 2000. Prior to the inception of inspiratory synaptic drive transmission from medullary respiratory centers, rat phrenic motoneurons (PMNs) have action potential and repetitive firing characteristics typical of immature embryonic motoneurons. During the period spanning from when respiratory bulbospinal and segmental afferent synaptic connections are formed at embryonic day 17 (E17) through to birth (gestational period is ~21 days), a pronounced transformation of PMN electrophysiological properties occurs. In this study, we test the hypothesis that the elaboration of action potential afterpotentials and the resulting changes in repetitive firing properties are due in large part to developmental changes in PMN potassium conductances. Ionic conductances were measured via whole cell patch recordings using a cervical slice-phrenic nerve preparation isolated from perinatal rats. Voltage- and current-clamp recordings revealed that PMNs expressed outward rectifier (IKV) and A-type potassium currents that regulated PMN action potential and repetitive firing properties throughout the perinatal period. There was an age-dependent leftward shift in the activation voltage and a decrease in the time-to-peak of IKV during the period from E16 through to birth. The most dramatic change during the perinatal period was the increase in calcium-activated potassium currents after the inception of inspiratory drive transmission at E17. Block of the maxi-type calcium-dependent potassium conductance caused a significant increase in action potential duration and a suppression of the fast afterhyperpolarizing potential. Block of the small conductance calcium-dependent potassium channels resulted in a marked suppression of the medium afterhyperpolarizing potential and an increase in the repetitive firing frequency. In conclusion, the increase in calcium-mediated potassium conductances are in large part responsible for the marked transformation in action potential shape and firing properties of PMNs from the time between the inception of fetal respiratory drive transmission and birth.
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INTRODUCTION |
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Phrenic motoneurons (PMNs) commence
functioning prenatally to drive the diaphragm for the generation of
fetal breathing movements. Prior to the inception of inspiratory
synaptic drive transmission from medullary respiratory centers, rat
PMNs have action potential and repetitive firing characteristics
typical of immature embryonic motoneurons (Martin-Caraballo and
Greer 1999). However, during the period spanning from when
respiratory bulbospinal and segmental afferent synaptic connections are
formed at embryonic day 17 (E17) (Allan
and Greer 1997a
,b
) through to birth (gestational period is
~21 days), a pronounced transformation of PMN electrophysiological properties occurs. The changes include a 50% reduction in action potential duration, a ~10-mV increase in action potential amplitude, a twofold increase in repetitive firing rates, and the expression of
well-developed afterpotentials (Martin-Caraballo and Greer 1999
). The underlying changes in ionic conductances responsible for these marked transformations in PMN properties were previously unknown. In this study, we test the hypothesis that the elaboration of
action potential afterpotentials and the resulting changes in
repetitive firing properties are due in large part to developmental changes in PMN potassium conductances.
Potassium conductances influence the shaping of action potentials,
neuronal repetitive firing patterns, and the summation of synaptic
inputs in neural cells (reviewed by McLarnor 1995). Several types of potassium channels have been identified based on their
electrophysiological and pharmacological properties (Rudy 1988
). The most widely distributed potassium channels are the delayed outward rectifier (IKV),
transient A-type (IA), and
calcium-activated potassium (IKCa)
conductances. Other potassium conductances that have a more limited
expression in neuronal systems include the inward rectifier,
muscarine-activated, and ATP-sensitive potassium channels. The delayed
rectifier and A-type potassium conductances regulate the timing of
action potential formation and the repetitive firing pattern of
neuronal cells (Dekin and Getting 1987
; Spigelman et al. 1992
). Calcium-activated potassium conductances are
important in generating afterhyperpolarizing potentials that ultimately influence neuronal firing properties. The expression pattern of these
various classes of potassium conductances are developmentally regulated
(Gao and Ziskind-Conhaim 1998
; McCobb et al.
1990
; Mienville and Barker 1997
; O'Dowd
et al. 1988
; Spigelman et al. 1992
). At very
early stages of neuronal development, there are pronounced increases in
the expression of delayed rectifier and A-type potassium conductances
(McCobb et al. 1990
; O'Dowd et al. 1988
;
Spigelman et al. 1992
). In this study, we found those
conductances to be well established in PMNs prior to the inception of
fetal respiratory synaptic drive and to change little during the latter
stages of embryonic development. However, we found that
calcium-activated potassium channel expression increased dramatically
after the inception of respiratory synaptic drive. Further, the
potassium currents associated with these channels underlie many of the
changes in PMN action potential characteristics and firing properties observed perinatally. A preliminary account of this work has appeared previously (Martin-Caraballo and Greer 1997
,
1998
).
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METHODS |
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Electrophysiological experiments were carried out using a
cervical spinal slice-phrenic nerve preparation as previously described (Martin-Caraballo and Greer 1999). Embryos
(E16-E18) were delivered from timed-pregnant Sprague-Dawley
rats anesthetized with halothane (1.2-1.5% delivered in 95%
O2-5% CO2) and maintained
at 37°C by radiant heat, following procedures approved by the Animal
Welfare Committee at the University of Alberta. To determine the timing of pregnancy, the day in which a morning test revealed the appearance of sperm plugs was labeled as E0. Fetal age was confirmed by
comparing the crown-rump length of the embryos with previously
published values by Angulo y González (1932)
.
Newborn rats [postnatal day 0 to 1 (P0-P1)] were anesthetized by inhalation of metofane
(2-3%). Embryos and newborns were decerebrated, and the brain
stem-spinal cord with the phrenic nerve attached was dissected in
artificial cerebrospinal fluid (ACSF) at 27 ± 1°C. The spinal
segment was cut with a vibratome (Pelco, Redding, CA) into a single
slice containing the C4 segment (E18,
P0-P1) or C3-C4
segment (E16) with the phrenic nerve and the dorsal root
ganglia attached (
750 µm thick). The dorsal roots were then cut to
prevent reflex-mediated synaptic activation of PMNs in response to
antidromic stimulation of the phrenic nerve used for identification of
PMNs. The spinal cord slice was transferred to a silicone elastomer
(Sylgard)-coated recording chamber and pinned down (at the dorsal
border of the white matter) and continuously perfused with ACSF
solution at 27 ± 1°C (perfusion rate 2 ml/s, volume of the
chamber 1.5 ml). The slice was left to equilibrate for at least 1 h before recording, and data were typically acquired for up to 5 h
after slice preparation.
Whole cell recordings
Recording electrodes were fabricated from thin wall borosilicate
glass (1.5 mm external and 1.12 mm internal diameter purchased from A-M
Systems, Everett, WA). The pipette resistances were between 3 and 4 M. To decrease capacitative transients, pipette tips were coated
with Sigmacote (Sigma Chemical, St. Louis, MO) and the level of fluid
submerging the slice was minimized during recordings. The electrode was
advanced with a stepping motor (PMC 100, Newport; Irvine, CA) into the
PMN pool located in the medial zone of the ventral horn close to the
border between the white and gray matter. To avoid clogging of the
recording electrode, positive pressure (
30 mmHg) was applied while
entering the tissue to a depth of at least 100 µm from the slice
surface. Pressure was then removed while advancing within the PMN pool.
Once the pipette made contact with a cell, negative pressure was used
to form a gigaohm (>1 G
) cell-pipette seal and gentle suction then
applied to rupture the patch membrane. Seal formation and membrane
breakthrough were monitored by observing the response to a
hyperpolarizing current step (0.3 nA). Whole cell recordings were
initially established in the ACSF solution and performed with an
AxoClamp 2B amplifier (Axon Instruments; Foster City, CA). The liquid
junction potential between the pipette and external solutions was ~10
mV. The appropriate correction was applied to the reversal potential of
the fast and medium duration afterhyperpolarizing potentials (fAHP and
mAHP, respectively). Data were filtered at 30 kHz, digitized with an A/D interface, and analyzed with the use of pCLAMP (Axon Instruments) and Origin (Northampton, MA) software.
Seal formation and whole cell recordings were carried out in
current-clamp mode. Once the whole cell configuration was established, motoneurons were identified as belonging to the PMN pool by antidromic stimulation of the phrenic nerve via a suction electrode. Rectangular pulses of 0.5 ms duration and 0.5 Hz frequency were delivered with a
pulse generator (Master 8, AMPI, Jerusalem, Israel) while pulse
amplitude was manually controlled with a stimulus isolation unit
(Iso-Flex, AMPI; Jerusalem, Israel). Stimulation amplitudes were 0.2
mA. To minimize current spread, the ground wire for antidromic
stimulation was wrapped around the tip of the suction electrode.
Antidromic action potentials were recorded on tape with a video
cassette recorder (Sony, Tokyo) or captured with pCLAMP software for
subsequent analysis. The anatomical location of antidromically
activated PMNs within the ventromedial zone of the ventral horn
(Allan and Greer 1997a
) was also confirmed with
successful intracellular fills with Lucifer yellow in ~31% of PMNs
analyzed. Neurons not responding to antidromic stimulation were not
analyzed. Presumptive glial cells, characterized by having resting
membrane potentials more negative than
70 mV and incapable of firing
action potentials in response to antidromic or orthodromic stimulation,
were also encountered but not analyzed.
Electrophysiological properties of PMNs were studied in E16 (n = 48), E18 (n = 50), and P0-P1 (n = 58) PMNs. Action potentials and firing patterns were recorded at varying holding membrane potentials in the current-clamp configuration. Independent action potentials were evoked by antidromic stimulation of the phrenic nerve or by orthodromic injection of depolarizing step of current (0.5 ms duration). Antidromic or orthodromic action potential duration was measured at half-maximal amplitude. The duration of the afterhyperpolarizing potential (AHP) was measured from the falling phase of the action potential to the point in the mAHP membrane potential trajectory that returned to the holding membrane potential. The mAHP amplitude was measured at the point of maximum hyperpolarized voltage deflection relative to the holding membrane potential. The fAHP amplitude was measured from the resting membrane potential to the negative deflection between the repolarizing phase of the action potential and the following afterdepolarizing potential. Repetitive firing properties were investigated following injection of 1-s-long depolarizing pulses of increasing amplitude. Firing frequency was determined as the number of action potentials per 1-s stimulus at the lowest intensity of current necessary for evoking repetitive firing. The effects of various drugs on firing frequency were studied at the lowest stimulation current (threshold current) necessary to elicit repetitive firing. All data values are presented as means ± SE. The n value represents the number of PMNs from which a particular measurement was made. Significant differences between values before and after a pharmacological treatment within a given age were calculated by using paired Student's t-test, whereas differences among various age groups were tested using ANOVA (with a Student-Newman-Keuls post hoc test).
On completing the examination of PMN action potential and repetitive
firing properties in current-clamp mode, we switched to the single
electrode voltage-clamp configuration (discontinuous mode) to record
potassium currents. The switching rate was <40 kHz, the output
bandwidth was set at 1 kHz, and the clamp gain was ~0.8 nA/mV. Head
stage output was monitored with a separate oscilloscope to monitor the
voltage transients prior to sampling. Outward rectifier and A-type
potassium conductances were measured in calcium-free solution in the
presence of TTX (0.5-1 µM) to eliminate calcium- and sodium-mediated
conductances, respectively. For recording outward rectifier potassium
currents, a 200-ms depolarizing prepulse (to 40 mV) was applied from
a holding potential of
70 mV, whereas a 200-ms hyperpolarizing
prepulse (to
110 mV) was applied to maximally activate A-type
potassium currents. Leak and capacitative currents were subtracted from
the scaled control currents by a P/4 protocol.
The steady-state activation and inactivation of the transient component
and the activation of the noninactivating component were plotted as a
function of membrane potential. For the inactivation plots, the current
at a given membrane potential was normalized to maximal current. For
activation, conductances (G) were calculated from currents
as G = I/(Vc Vr) where
Vc is the command potential and
Vr is the calculated reversal
potential for potassium (
97 mV). The plots of normalized current or
conductance versus membrane potential were fitted with a Boltzman
function in the form I/Imax (or G/Gmax) = 1/ [1 + exp(V1/2
V)/k], where V is the step potential, V1/2 is the potential at half-maximal
normalized value, and k characterizes the steepness of the
activation or inactivation curves.
Intracellular and extracellular solutions
ACSF contained (in mM) 128 NaCl, 3 KCl, 0.5 NaHPO4, 1.5 CaCl2, 1 MgCl2, 23.5 NaHCO3, and 30 glucose, pH 7.4 when bubbling with 95%O2-5% CO2. In the calcium-free solution, calcium ions were replaced by an equimolar concentration of cadmium or cobalt chloride, and NaHPO4 was removed to avoid precipitation. The standard pipette solution contained (in mM) 130 potassium gluconate, 10 NaCl, 1 CaCl2, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 10 HEPES, 5 Mg ATP, 0.3 NaGTP, pH 7.3 with KOH. A solution containing lower levels of the calcium chelator BAPTA was used when examining PMN afterpotentials to maximize the underlying calcium-mediated potassium conductances. The composition of the pipette solution with a lower calcium buffer capacity was similar to that of the standard solution except for the following changes: BAPTA was decreased to 0.1 mM, calcium was not added, potassium gluconate was 126 mM, and phosphocreatine (10 mM) was added. The pipette solution utilized for blocking potassium conductances contained (in mM) 110 cesium methanesulphonate, 30 tetraethylammonium chloride (TEACl), 10 BAPTA, 10 HEPES, 5 Mg ATP, and 0.3 NaGTP, pH 7.3 with tetraethylammonium hydroxide (TEAOH).
The osmolarity of all the external and internal solutions were ~325 and ~315 mOsm, respectively, as measured with a freezing point osmometer (Advanced Instruments; Needham, MA).
Drugs
Stock solutions of drugs were prepared as 100-1,000 times
concentrates. All drugs were added into the perfusate by switching to
reservoirs containing the appropriate test solution. A waiting period
of 5 min was used to allow for equilibration before data were
collected. The following drugs were used: TTX, TEACl, apamin, 4-aminopyridine (4-AP), cytochrome-c (Sigma), iberotoxin,
lidocaine-ethyl bromide (QX 314, RBI, Natick, MA);
-agatoxin
(Peninsula Laboratories, Belmont, CA); and
-conotoxin GVIA (Alomone
Laboratories, Jerusalem, Israel). The perfusion medium of the spinal
slice with
-conotoxin GVIA or
-agatoxin also contained
cytochrome-c (0.05%) to avoid nonspecific binding to plastic tubing.
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RESULTS |
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Developmental changes in spike properties of phrenic motoneurons
Phrenic motoneurons undergo considerable changes in their passive
and active properties during the E16-P1 period
(Martin-Caraballo and Greer 1999). As shown in Fig.
1, these include obvious changes in
action potential duration, amplitude, and afterpotentials. In
E16 PMNs, the main spike was followed by a slowly
decrementing afterdepolarization (ADP) that is transformed to a
hump-ADP by birth. The fAHP and mAHP, which were absent at
E16, developed by birth. As described below, a combination
of current- and voltage-clamp experiments in conjunction with
pharmacological channel blockers were used to dissect out the role of
various potassium conductances during this period of PMN
electrophysiological transformation.
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Effects of blocking multiple potassium conductances
The role of potassium conductances in regulating action potential
duration was initially studied using TEA and cesium-filled recording
electrodes to block multiple potassium conductances. Upon general
blockade of potassium conductances, a long-duration spike was evident
at all ages studied (Fig. 2A).
The conductances underlying the long-duration component were examined
by selectively blocking sodium and calcium conductances with
intracellular QX 314 (1 mM) or following incubation in calcium-free
medium, respectively. As shown in Fig. 2B (left
panel), stimulation with 10 consecutive depolarizing pulses
resulted in the disappearance of the fast, sodium-dependent spike due
to the use-dependent blockade of sodium channels with QX 314 (Connors and Prince 1982). The long-duration component,
which remained after sodium current blockade, was attenuated following
incubation in calcium-free medium (Fig. 2B, right panel). The duration of the calcium-dependent long-duration component decreased
dramatically with age. The times for the plateau to attenuate to
half-maximal amplitude were as follows: E16 = 320 ± 45 ms (mean ± SE, n = 8); E18 = 126 ± 23 ms (n = 6, P
0.05 vs. E16); P0 = 41 ± 10 ms
(n = 7, P
0.05 vs. E16).
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Effects of blocking potassium currents with tetraethylammonium (TEA)
The maturation of outward potassium conductances play an important
role in shaping the action potential waveform and repetitive firing
patterns in several differentiating neuronal systems (McCobb et
al. 1990; Spigelman et al. 1992
). Externally
applied TEA (10 mM) was used to block outward potassium conductances to
evaluate their role in the spike repolarization of PMNs. At all ages
tested, TEA significantly increased the action potential duration
(Table 1). However, the effect of TEA on
prolonging the action potential decreased significantly with age (Table
1). This is consistent with the reduction of action potential duration
with age found after inhibition of all potassium conductances with
intracellular TEA and Cs+ (see above). The effect
of TEA on the regulation of the repetitive firing of PMNs was more
difficult to quantify since motoneurons became very excitable. In
E16 and E18 PMNs, TEA treatment evoked complex
spikes, such as plateau potentials, whereas in neonatal PMNs, TEA
application increased the firing frequency by ~28% (11 ± 1 vs.
15 ± 1 Hz, n = 4; Fig.
3).
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Effects of blocking A-type potassium conductances with 4-AP
The effects of TEA on the action potential duration were
less pronounced at hyperpolarized holding potentials (Table 1). Thus we
tested whether this was due to the presence of an A-type potassium
current that has a transient character and is inactivated at
depolarized potentials (McCobb et al. 1990;
Spigelman et al. 1992
). Bath application of the A-type
current inhibitor 4-AP (1-3 mM) prolonged the duration of the spike
without significantly changing action potential amplitude at all ages
tested (Fig. 4A, Table 1).
Following 4-AP treatment, the action potential duration tended to
increase with age; however, this increase was not statistically significant (Table 1). Further, the prolongation of action potential duration by 4-AP was enhanced when action potentials were elicited from
hyperpolarized holding potentials (Fig. 4A).
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In addition to its contribution to action potential repolarization, the
4-AP-sensitive potassium conductance was involved in regulating the
firing behavior of PMN at all ages studied. When PMNs fired from
hyperpolarized holding potentials, a delayed excitation was observed
(Fig. 4B). The delay excitation was defined as a lag between
the onset of depolarization and the onset of firing. Delayed
excitations were observed in 47% of E16 (8 of 17), 54% of
E18 (13 of 24), and 21% (4 of 19) of neonatal PMNs. The
ramplike depolarization characteristic of delayed excitation was
blocked by 4-AP (1 mM; Fig. 4B) and insensitive to TTX
(0.5-1 µM, n = 6; not shown) or incubation in
calcium-free medium (n = 6; not shown). Despite the
removal of the delayed excitation by 4-AP, the firing frequency of PMNs
was reduced. This is likely explained by the concomitant increase in
action potential duration caused by 4-AP (Fig. 4B).
Role of calcium-activated potassium conductances in controlling firing properties and afterpotential formation
Previous results indicate that calcium ions contribute to shape
the action potential in developing PMNs (Martin-Caraballo and
Greer 1999). In particular, incubation in calcium-free medium eliminates the mAHP in E18 and neonatal motoneurons and
results in the widening of action potentials postnatally. Thus we
investigated whether these calcium-dependent effects were mediated by
calcium-activated potassium conductances.
To investigate the role of calcium-activated potassium conductances in
the regulation of repetitive firing properties, we tested the
effect of calcium-free medium (Figs. 5
and 6). The diminution of calcium
conductances had little effect on the repetitive firing properties of
PMNs at E16. However, the removal of calcium from the medium
had pronounced effects on the steady-state firing frequency of PMNs at
E18 and P0. When measured at the threshold current required to elicit repetitive firing, the change in
steady-state frequency in control versus calcium-free medium were as
follows: E16, 10 ± 1 versus 11 ± 2 (n = 4); E18, 8 ± 1 versus 19 ± 1 (n = 7, P 0.05 vs. control);
P0-P1, 15 ± 1 versus 21 ± 1 (n = 6, P
0.05 vs. control). Further, the
spike-frequency adaptation, which starts to develop by E18
and becomes quite pronounced by P0, is reduced when
calcium-activated potassium conductances are diminished (Fig. 6,
bottom panel).
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ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL PROPERTIES OF THE
mAHP.
The expression of a mAHP in PMNs is first observed in a minority of
E18 motoneurons (Martin-Caraballo and Greer
1999). By birth, 80% (16 of 20) of PMNs express a clear mAHP
(Fig. 7A). Thus we restricted
our analyses of the pharmacological and electrophysiological properties
of mAHPs to neonatal PMNs.
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ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL PROPERTIES OF
THE fAHP.
A fast AHP was not observed in E16 or E18 PMNs.
However, by P0-P1, 29% (8 of 29) of PMNs had a clear fast
AHP (Fig. 8A). The amplitude
of the fAHP was enhanced by hyperpolarizing holding potentials.
Further, the membrane potential reached by the peak fAHP varied with
the holding potential (Fig. 8A). The amplitude of the fAHP,
as a function of voltage, was fitted by a linear regression, and
the reversal potential of the fAHP was estimated by extrapolation to be
50 ± 2 mV (n = 8; Fig. 8B).
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Voltage-clamp analysis of voltage-dependent potassium currents
To further characterize potassium-mediated conductances in
perinatal PMNs, recordings were made in voltage-clamp mode.
Voltage-clamp experiments revealed that by E16 outward
potassium currents expressed two voltage-dependent components: a
transient, fast inactivating and a noninactivating conductance (Fig.
10A). Based on previous results in other developing neurons (McCobb et al. 1990;
Spigelman et al. 1992
), the noninactivating and
transient components of potassium currents were classified as being the
delayed outward rectifier (IKv) and
A-type (IA) potassium conductances,
respectively. The transient component was revealed when potassium
currents were recorded following a hyperpolarizing prepulse (to
110
mV for 200 ms; Fig. 10Aa). Holding the membrane potential at
more depolarized potentials (at
40 mV for 200 ms) resulted in the
inactivation of the transient component (Fig. 10Ab) and the
generation of a noninactivating potassium conductance. The absolute
value of the transient component was isolated by subtraction of the
noninactivating current generated by a depolarizing prepulse from total
current generated by the hyperpolarizing prepulse (Fig. 10,
Aa and Ab). Activation and inactivation of the
transient components were plotted as a function of voltage and fitted
to a Boltzman equation (Fig. 10C).
|
It was evident from the kinetics of the current recordings that there
were space-clamp problems that effected the accuracy of current
measurements. These problems were particularly pronounced in neonatal
PMNs that have elaborate dendritic trees and increased soma diameter
relative to embryonic PMNs (Allan and Greer 1997b). For
instance, the slow time-to-peak (19.0 ± 2.6 ms; n = 16) and the more depolarized activation
V1/2 value (
5 ± 3 mV;
n = 13) of the IA
conductance at P0 compared with data from other motoneurons (McCobb et al. 1990
; Safronov and Vogel
1995
) likely resulted from space-clamp problems. Thus these
limitations precluded us from systematically quantifying age-dependent
changes in the density or kinetics of potassium-mediated conductances.
Further, we were unable to reliably record calcium-activated potassium
currents in P0-P1 PMNs. However, there was a striking
change in the time-to-peak for the nonactivating component that could
not be attributed to an artifact of the space-clamp problem. Between
E16 and P0-P1, there was an ~55% decrease in
the time-to-peak (133 ± 26, n = 23 vs. 57 ± 21 ms, n = 26) and an ~8-mV leftward shift in the voltage for half-maximal activation
(V1/2, 19 ± 1, n = 10, P
0.05, vs. 11 ± 2 mV, n = 11) without any significant change in the steepness of the activation
curve (k, 14 ± 1 vs. 15 ± 1/mV; Fig.
11). If anything, these age-dependent
differences in IKV kinetics would have
been underestimated due to space-clamp problems.
|
At all ages tested, the IKV and IA had similar pharmacological sensitivities. The transient A-type potassium conductance was completely eliminated following incubation with 3 mM of 4-AP without significant changes in the outward rectifier potassium conductance (not shown). Treatment of the noninactivating steady current with 10 mM TEA greatly diminished the expression of IKV in PMNs (not shown).
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DISCUSSION |
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The present findings demonstrate that the outward rectifier and A-type potassium currents are involved in regulating PMN action potential and repetitive firing properties throughout the perinatal period spanning from E16 through to P0-P1. The most profound change in potassium conductances during this period resulted from the marked increase in calcium-activated potassium channel expression following the inception of inspiratory drive transmission. The emergence of these conductances appear to be in large part responsible for the marked transformation in action potential shape and firing properties of PMNs prior to birth.
Calcium-dependent potassium conductances
PMN action potential shape and duration were not affected by calcium-dependent potassium conductances until after the inception of inspiratory drive transmission (i.e., post E17). This was despite the fact that there was a substantial calcium conductance during the action potential in E16 PMNs. Thus there is likely an age-dependent increase in the expression of calcium-activated potassium channels in PMNs during the perinatal period.
MAXI-TYPE CALCIUM-DEPENDENT CURRENT.
Block of the maxi-type calcium-dependent potassium conductance with
iberotoxin caused a significant increase in action potential duration
in neonatal PMNs, implicating a calcium-dependent potassium conductance
in spike repolarization. A similar conductance has been implicated in
spike repolarization in neonatal lumbar and hypoglossal motoneurons
(Takahashi 1990; Viana et al. 1993
) and the shaping of the locomotion pattern in Xenopus tadpoles
(Sun and Dale 1998
). Functionally, besides decreasing
action potential duration, the presence of calcium-activated potassium
conductances will prevent the large accumulation of calcium ions during
alternating bursts of activity such as respiration and locomotion.
SMALL-CONDUCTANCE CALCIUM-DEPENDENT CURRENT.
Block of the small-conductance calcium-dependent potassium channels in
PMNs with apamin did not have any effect on spike repolarization. However, there was a marked suppression of the mAHP and an increase in
the repetitive firing frequency of PMNs. The activation of small-conductance potassium conductances has also been implicated in
spike frequency adaptation in other mammalian motoneurons (Gao and Ziskind-Conhaim 1998; Schwindt and Crill
1981
; Viana et al. 1993
; Walton and
Fulton 1986
). A detailed study of the ontogeny of calcium
conductances in PMNs is currently under investigation and will provide
information regarding the interaction between calcium and potassium
conductances. However, the data from the current study demonstrates
that the calcium ions responsible for generating the mAHP in PMNs
entered via
-conotoxin GVIA-sensitive calcium channels, suggesting
that the small-conductance calcium-activated potassium channels are
localized near the N-type calcium channels in PMNs. This is similar to
the findings from studies of hypoglossal motoneurons (Viana et
al. 1993
). However, given that the ADP was not blocked by
-conotoxin GVIA, it is likely that the calcium influx necessary for
generating the ADP and mAHP are separate.
Outward rectifier and A-type potassium conductances
Transient, 4-AP-sensitive
IA and slowly activating,
noninactivating TEA-sensitive IKV
conductances are functional in PMNs as early as E16 and
continue to be expressed into the postnatal period. The actions of
IKV and
IA for regulating action potential duration and repetitive firing properties of PMNs were examined. Blockade of all potassium conductances with intracellular TEA and
Cs+ or externally applied TEA resulted in a
prolongation of action potential duration, the prolonged influx of
calcium, and the generation of plateau potentials that were
particularly pronounced in embryonic PMNs. This is likely due to the
significant role of calcium entry in shaping the action potential in
embryonic PMNs. We observed an age-dependent leftward shift in the
activation voltage and a decrease in the time-to-peak of
IKV similar to those found for Xenopus spinal neurons (Burger and Ribera
1996; Gurantz et al. 1996
). The leftward shift
in the activation of IKV combined with the fact that the action potential in neonatal PMNs can reach a larger
overshooting potential than in embryonic neurons, will result in a
faster repolarization of the action potential as delayed rectifier
potassium channels activate faster and at lower voltages (Martin-Caraballo and Greer 1999
). Repetitive firing
properties were affected by a delayed excitation mediated by a
IA conductance. The degree to which
the IA-mediated delayed excitation
influences PMN firing during the inspiratory phase of the respiratory
cycle would vary depending on the magnitude of the membrane
hyperpolarization during the preceding expiratory phase. Thus although
IA conductances are expressed in PMNs
throughout the perinatal period, the
IA-mediated effects on firing will
change as a function of inhibitory synaptic drive development
(Gao and Ziskind-Conhaim 1995
; Singer et al. 1998
; Wu et al. 1992
) and changes in resting
membrane potential [becomes more hyperpolarized with age (Liu
and Feldman 1992
; Martin-Caraballo and Greer
1999
)].
FUNCTIONAL SIGNIFICANCE.
Collectively, the changes in potassium conductances result in a
reduction in PMN action potential duration, resulting in increases in
the rate and range of repetitive firing frequencies attainable at
birth. There are also parallel changes in the contractile properties of
the diaphragmatic musculature (Martin-Caraballo et al.
2000). Prenatally, the kinetics of muscle twitches are slow,
and thus fused tetanic contractions are achieved at a relatively low
frequency. Diaphragmatic muscle twitch contractions are considerably
faster and the tetanic frequency higher by birth. The net result of the concomitant age-dependent changes in PMN and diaphragmatic muscle properties is that the full-range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Peter Smith, David Bennett, and Calvin Wong for helpful comments on the manuscript. J. J. Greer is a Senior Scholar of the Alberta Heritage Foundation for Medical Research (AHFMR) and M. Martin-Caraballo is a recipient of AHFMR and Neuroscience Canada Foundation Studentships.
This work was funded by the Alberta Lung Association and the Medical Research Council of Canada (MRC).
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FOOTNOTES |
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Address for reprint requests: J. J. Greer, Dept. of Physiology, 513 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
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 October 1999; accepted in final form 6 March 2000.
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REFERENCES |
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