1Department of Neuroscience and 2Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 3Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201
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ABSTRACT |
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Cameron, William E.,
Pedro A. Núñez-Abades,
Ilan A. Kerman, and
Tracy
M. Hodgson.
Role of Potassium Conductances in Determining Input Resistance of
Developing Brain Stem Motoneurons.
J. Neurophysiol. 84: 2330-2339, 2000.
The role of potassium
conductances in determining input resistance was studied in 166 genioglossal (GG) motoneurons using sharp electrode recording in brain
stem slices of the rats aged 5-7 days, 13-15 days, and 19-24 days
postnatal (P). A high magnesium (Mg2+;
6 mM) perfusate was used to block calcium-mediated synaptic release
while intracellular or extracellular cesium (Cs+)
and/or extracellular tetraethylammonium (TEA) or barium
(Ba2+) were used to block potassium conductances.
In all cases, the addition of TEA to the high
Mg2+ perfusate generated a larger increase in
both input resistance (Rn) and the
first membrane time constant (0) than did high
Mg2+ alone indicating a substantial nonsynaptic
contribution to input resistance. With intracellular injection of
Cs+, GG motoneurons with lower resistance (<40
M
), on the average, showed a larger percent increase in
Rn than cells with higher resistance
(>40 M
). There was also a significant increase in the effect of
internal Cs+ on
Rn and
0 with
age. The largest percent increase (67%) in the
0 due to intracellular
Cs+ occurred at P13-15, a
developmental stage characterized by a large reduction in specific
membrane resistance. Addition of external Cs+
blocked conductances (further increasing
Rn and
0)
beyond those blocked by the TEA perfusate. Substitution of external
calcium with 2 mM barium chloride produced a significant increase in
both Rn and
0
at all ages studied. The addition of either intracellular Cs+ or extracellular Ba2+
created a depolarization shift of the membrane potential. The amount of
injected current required to maintain the membrane potential was
negatively correlated with the control
Rn of the cell at most ages. Thus low
resistance cells had, on the average, more Cs+-
and Ba2+-sensitive channels than their high
resistance counterparts. There was also a disproportionately larger
percent increase in
0 as compared with
Rn for both internal
Cs+ and external Ba2+.
Based on a model by Redman and colleagues, it might be
suggested that the majority of these potassium conductances underlying
membrane resistance are initially located in the distal dendrites but
become more uniformly distributed over the motoneuron surface in the oldest animals.
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INTRODUCTION |
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A critical event in the
differentiation of mammalian motoneurons is the decrease in input
resistance associated with the motoneurons innervating fast twitch
muscle fibers during postnatal development (Navarrete and
Vrbová 1993). In the first paper, we investigated the
role of synaptic input in determining the membrane resistance. It was
found that synaptic inputs accounted for a significant portion of the
resting conductance of developing brain stem motoneurons. More
specifically, glycine/GABA-mediated conductances were found to increase
with age. There was a significant increase in the percent change of
input resistance between the first and second week of postnatal life
after synaptic blockade with either tetrodotoxin (TTX), high magnesium,
or receptor blockers. However, the magnitude of this synaptic effect
cannot account for the halving of the Rn observed during this time period.
One alternative source of this decreased resistance would be
nonsynaptic potassium channels.
There is a substantial body of evidence to suggest that specific
membrane resistance is modulated by many voltage-sensitive channels, in
addition to tonic synaptic activity (Rall et al. 1992).
A variety of potassium channels including the delayed rectifier, inward
rectifier, and A- and leak channels have been implicated in
establishing the membrane resistance of mammalian motoneurons (Binder et al. 1996
; Crill and Schwindt
1983
). These channels are sensitive to a diversity of
substances including internal and/or external tetraethyl ammonium
(TEA), cesium, and barium in a wide variety of neurons (Hille
1992
). Internal cesium reduces resting conductance in cat
spinal motoneurons (Puil and Werman 1981
), while
external TEA has a similar effect in rat vagal motoneurons (Yarom et al. 1985
). External TEA also prolongs the
duration of the action potential by reducing the voltage-sensitive
potassium conductances underlying the fast afterhyperpolarization (AHP) and the spike repolarization in cat lumbar (Schwindt and Crill 1980a
) and rat hypoglossal motoneurons (Viana et al.
1993
). In addition to depressing the fast voltage-sensitive
potassium conductance, external barium decreases the potassium leak
conductance (Schwindt and Crill 1980b
). This
barium-sensitive component of potassium leak conductance is modulated
in rat hypoglossal motoneurons by thyrotropin-releasing hormone
(Bayliss et al. 1992
) and norepinephrine (Parkis
et al. 1995
) to change both motoneuron excitability and repetitive firing characteristics.
It has been proposed that the properties of the motoneuron membrane are
not uniform. There are data to suggest that there is a difference in
the specific membrane resistance between fast and slow motoneurons
(Burke 1987; Burke et al. 1982
). In
addition, it has been suggested that there is a difference in the
membrane resistivity between the cell body and dendrites of the same
cell. The decreased resistance of the soma (somatic shunt) was first postulated by the Redman laboratory (Iansek and Redman
1973
) in their study of cat lumbosacral motoneurons. More
recently, work from the laboratory of P. K. Rose (Campbell
and Rose 1997
) has intracellularly injected cesium to assess
the contribution of potassium channels to this somatic shunt in
cervical motoneurons studied in vivo. These authors found an increase
in the somatic time constant with injection of intracellular cesium
while only a small decrease in the dendritic time constant. They
concluded that the distribution of cesium-sensitive channels was
concentrated in the somatic and proximal dendritic membrane. In
contrast, studies of barium-sensitive conductances in lumbar
sympathetic ganglion cells (Redman et al. 1987
) have
suggested a more distal dendritic location for the blocked potassium
conductances. It would be interesting to know when the adult
distribution of voltage-sensitive channels influencing membrane
resistance is established during development and whether the patterns
seen in these other neuron populations also applies to developing brain
stem motoneurons. Thus we have examined the contribution of various
potassium conductances to the membrane properties of input resistance
and time constant to gain a better understanding of the changes
occurring in the distribution of voltage-sensitive channels during the
period when motoneurons differentiate. Some of these data have been
presented in abstract form (Cameron 1998
; Cameron
et al. 1996
).
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METHODS |
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Brain stem slices were prepared from male Sprague Dawley rats from three postnatal (P) age groups (P5-7, P13-15, and P19-24) as described in the preceding paper. In brief, rat pups were deeply anesthetized with halothane, tracheotomized, and ventilated with 100% O2. Anesthetized pups were transcardially perfused with cold (4°C) sucrose-artificial cerebral spinal fluid (ACSF) and quickly decapitated. The brain stem was removed and sectioned at 300 µm with a Vibraslice. The composition of sucrose-ACSF was as follows (in mM): 240 sucrose, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 10 glucose, 5 MgSO4, and 1 CaCl2. All slices were incubated in a holding chamber in normal ACSF consisting of (in mM) 126 NaCl, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 20 glucose, 2 MgSO4, and 2 CaCl2, at room temperature, bubbled with 95% O2-5% CO2 (pH 7.35). In experiments in which 6 mM MgCl2, 20 mM tetraethylammonium chloride (TEA), or 5 mM CsCl (Sigma) was added to the bath solution, ionic strength was maintained by an equivalent decrease in the concentration of NaCl. In experiments in which 2 mM barium chloride was substituted for calcium chloride, 10 mM HEPES was substituted for both phosphate and bicarbonate buffers, and solution was bubbled with 100% O2. This alternate buffer system was chosen to prevent barium from precipitating in the presence of phosphate buffer. The flow rates of normal and modified ACSF were kept constant at 1-2 ml/min using a perfusion pump. All membrane properties were measured at room temperature (21 ± 1oC).
Motoneurons were recorded from the ventromedial portion of the
hypoglossal nucleus, a region shown to contain genioglossal (GG)
motoneurons (Mazza et al. 1992). The criteria for a
healthy cell and the protocols for measuring the membrane properties
were described in the preceding paper. There were three separate
protocols employed to block potassium conductances. In the first series of experiments, the role of synaptic inputs and nonsynaptic potassium conductances in determining membrane resistance was tested. Input resistance (Rn), first membrane time
constant (
0), rheobase
(Irh), and repetitive firing was
measured in normal ACSF. The validity of using a long, hyperpolarizing
current pulse (500 ms duration) to measure
Rn and
0 in
cells exhibiting an inward rectifying or "sag" current has been
discussed in the first paper of this series. In a few cells, the sag
current could be blocked by the application of external cesium
(Cs+, Fig. 1) with
only a moderate change in the two membrane properties. However, in the
majority of cases, the sag current was only partially blocked by
external Cs+, and there was evidence that
additional currents were being effected (see Fig. 8). Given this
inconsistency, it was determined that exposure to external cesium would
not provide any more consistent control value for measurement of
Rn and
0 than
the measurements potentially contaminated by the sag current.
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After making the control measurements in the first protocol, the calcium-dependent neurotransmitter release was blocked by the addition of 6 mM (high) magnesium to the perfusate. The membrane properties were measured at 5 and 10 min after the start of the perfusate with blocker. Following 10 min of high Mg2+, the perfusate was switched to one containing the high Mg2+ plus 20 mM TEA, and the measurements were repeated. Similar measurements were made after 5 and 10 min in the Mg2+/TEA perfusate and after 10, 20, and 30 min of wash out. In a separate protocol, 5 mM Cs+ was added to the Mg2+/TEA perfusate to assess the contribution of potassium channels that were Cs+ sensitive and TEA insensitive.
In the second protocol examining the effect of internal
Cs+, electrodes were filled with 3 M cesium
acetate. A series of control measurements of the action potential
Rn and 0 were
made immediately after impalement. Then Cs+ was
injected into the cell using a 50-ms, 5-Hz positive pulse with an
amplitude sufficient to evoke several action potentials (0.2-1 nA;
duration, 2-4 min). Measurements were made at 2 and 4 min of injection
and 10-40 min after injection was stopped.
In all protocols other than internal Cs+, the
membrane potentials of developing GG motoneurons were recorded with
glass micropipettes filled with a 3:1 mix of 3 M potassium acetate and
3 M potassium chloride (resistance, 60-100 M). To avoid activating
other voltage-sensitive conductances, the initial membrane potential
was maintained by injecting a constant (bias) current to offset any
depolarizing shift of the membrane. This value of bias current required
to oppose the depolarization generated by the block of potassium conductances was recorded for further analysis. The level of membrane hyperpolarization at which the time constants were calculated was
insufficient to activate voltage-sensitive currents (inward rectification) noted with larger hyperpolarizations in some cells (Fig.
1B, preceding paper).
In the final protocol, the slices were exposed to external barium. Due to the spontaneous firing associated with external Ba2+, control measurements were made both before and after the addition 1 µM tetrodotoxin (TTX) to the normal ACSF. After 10 min in TTX, an ACSF solution containing 2 mM BaCl was introduced into the bath and measurements made at 5 and 10 min and after 20-30 min of wash out. The measurement after 10 min in TTX (devoid of evoked synaptic release) was used as the control value for comparisons with the barium data. In most instances, cells returned to their control (TTX) values for input resistance and membrane time constant after 30 min of wash out.
The values of Rn and
0 are presented in the text and tables as
means ± SE. A two-way ANOVA was employed to determine whether there were any significant interactions between the levels of treatment
and levels of postnatal age. A pair-wise multiple comparison procedure
(Tukey test) was performed to test the differences between means. If
significant differences were indicated from the Tukey test, then a
one-way ANOVA with repeated measures or a paired t-test was
performed to determine the differences between age groups. Statistical
significance was defined as P < 0.05.
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RESULTS |
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Recordings were made from genioglossal (GG) motoneurons located in
the ventromedial portion of the hypoglossal nucleus. A total of 166 motoneurons met our acceptance criteria and fell into one of the
following postnatal age groups: P5-7 (n = 34); P13-15 (n = 68); P19-24
(n = 64). Throughout the postnatal period studied, no
differences were found in the mean resting membrane potential or action
potential amplitude for these motoneurons. The mean membrane potential
was 64.4 ± 0.2 mV.
High magnesium and TEA blockade
The goal of this experimental series was to determine what proportion of the resting conductance was mediated by potassium conductances as compared with that mediated by synaptic input examined in the previous paper. Like the previous study, high (6 mM) Mg2+ was added to the perfusate to inhibit calcium-mediated synaptic transmission. Potassium conductances were inhibited by either TEA or Cs+. Figure 2 shows one P20 motoneuron from the first experimental series illustrating the effect external Mg2+ and TEA on membrane properties. The top panel presents three actions potentials evoked by a depolarizing current pulse (50 ms, 1 Hz) in the control bath solution, and in solutions containing high Mg2+ and high Mg2+ plus 20 mM TEA. The bottom panel shows the membrane response to a series of six hyperpolarizing current pulses. High Mg2+ media abolished the afterdepolarization (ADP; arrow) of the action potential observed in the control and increased Rn. With the addition of TEA, the repolarization phase of the action potential was slowed and a more substantial increase in Rn was observed than that seen in Mg2+ alone. However, neither high Mg2+ nor Mg2+/TEA had any major effect on the depolarizing sag produced by the inward rectification, as seen in the traces in the bottom panel.
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Figure 3 shows the effects of blockade of
calcium-mediated synaptic activity and potassium conductances on
Rn for two earlier periods of
development (P7 and P15). The control recording
from a P7 animal was punctuated by significant spontaneous
synaptic activity that was nearly abolished by the addition of high
Mg2+ solution. These two cells were selected
because they demonstrated a clear incremental effect of the addition of
external TEA to the high Mg2+ perfusate; however,
the magnitude of the change in Rn was
not representative of the larger sample. Table
1 summarizes the group data for 38 GG
motoneurons analyzed in the first series of experiments. The two-way
ANOVA showed that there were no interactions between age and treatment
for either Rn or
0 and that there were significant differences
among treatments but not among the different ages. Further statistical
analyses revealed significant differences in
Rn between high
Mg2+ and Mg2+/TEA for each
age group. Similar analyses for
0 revealed a
significant difference between control and high
Mg2+ only at the youngest age (P5-7),
while the addition of TEA to the Mg2+ containing
bath generated significantly larger values of
0 at all ages. Over the developmental period
studied, the absolute magnitudes and relative percents of change in
Rn and
0 with
the addition of TEA to the high Mg2+ perfusate
were at least twofold greater than that produced by Mg2+ alone.
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Internal cesium blockade
The contribution of potassium conductances in determining
Rn and 0 were
also assessed before and after an intracellular injection of cesium
(Cs+). Internal Cs+ had a
more profound effect on the shape of the action potential than did
extracellular TEA. Figure 4 shows the
effect of internal Cs+ on the action potential
and Rn of a GG motoneuron from a
P15 animal. The maximum spike half-width was achieved after
2 min of injection and showed little change at 4 min. In contrast to the spike width, the Rn continued to
increase between 2 and 4 min of injection. The staggering in the
further reduction of potassium conductance implies that the potassium
channels governing repolarization of the somatic action potential are
not the same as those governing the resistance of the cell. This
conclusion is supported by the time course of blockade demonstrated in
Fig. 5. A GG motoneuron from a 22-day-old
rat was injected with Cs+ for 6 min. The action
potential exhibited a slow repolarization and multiple spikes. Twenty
minutes after the cessation of Cs+ injection, the
action potential was narrowing and, by 40 min, it has reached its
control width (showing an ADP, arrow). The membrane resistance of this
cell continued to increase even after the injection had halted. With
recovery of the somatic potassium channels governing repolarization
(delayed rectifier, Ikv), another subset of potassium channels must be responsible for the increased resistance.
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The one-way ANOVA with repeated measures revealed a significant
difference between control and cesium treatments. When individual age
groups were analyzed, intracellular cesium produced a significant increase in both Rn and
0 at all ages studied (Table
2). No age-dependent changes were found
in absolute values of either membrane property; however, when expressed
as a percent change from control, there was a developmental change (see
Fig. 7).
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The percent change from control values of
Rn varied greatly among the 65 motoneurons studied in this protocol and plotted in Fig.
6. We found a negative linear relation
between control input resistance and percent change in
Rn with intracellular
Cs+ (r = 0.58,
P < 0.001). A vertical line arbitrarily divides the motoneurons into a low resistance (<40 M
) and a high resistance (>40 M
) group. When divided in such a fashion, with few exceptions, low resistance cells tend to show larger percent increases (>30%) in
Rn with intracellular
Cs+ than most high resistance cells (<30%).
Thus Cs+-sensitive conductance is greater in the
lower resistance than the higher resistance cells. No equivalent
relationship was found for the Mg2+-sensitive
component of Rn in the preceding study
(data not shown).
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The magnitude of the Cs+ effect on both
Rn and 0
varied with postnatal age. Figure 7
summarizes the effects of internal Cs+ on
Rn and
0 for
the three age groups studied. The effect of Cs+
injection on Rn increased with each
succeeding week, while the effect on
0 was
characterized by a prominent increase in membrane time constant between
the first and second week of postnatal life. If we assume that the
specific capacitance of the cell (Cm)
remains constant during the Cs+ injection and
m can be approximated by
0, then the changes in
0 reflect changes in the specific membrane
resistance (Rm, where
m = Rm × Cm). Thus the decreased resistance in
GG motoneurons at 2 wk of age is due, in large part, to the
proliferation of Cs+-sensitive channels.
Alternatively, as the animal matures, the distribution of
cesium-sensitive potassium channels may shift from a distal
distribution to a more uniform distribution or near the soma. The
contribution of this subset of potassium channels to specific membrane
resistance (
0) appears to be maximal at 2 wk
of age, while the contribution to Rn
was greatest at 3 wk. Given the doubling of membrane surface area
between 2 and 3 wk (Núñez-Abades and Cameron
1995
), the increased numbers of cesium-sensitive channels at 3 wk responsible for the Rn are
distributed over a larger surface area. As a result of this growth, the
cesium-sensitive component of
0 at 3 wk was
reduced. These development trends were also evident in the absolute
changes in Rn and
0 (Table 2). This interpretation may be overly
simplistic, and an alternative will be presented in the discussion.
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External cesium blockade
We studied the effect of adding external Cs+
to determine what, if any, increases may be seen in
Rn and 0
above that generated by external TEA. One effect of external
Cs+ was to reduce the inward rectification in
some cells (Fig. 1). In the P24 GG motoneuron shown in Fig.
8, external Cs+
greatly increased both Rn and
0 above that achieved with external TEA. In
the experimental protocol, the high Mg2+ blocked
the ADP present in the control action potential and increased Rn slightly. With the addition of TEA,
the action potential broadened, and there was a further increase
Rn. Neither of these treatments greatly affected the inward rectification. Finally, with the addition of external Cs+, there was a further increase in
Rn and enhanced broadening of the
action potential. This pattern occurred in five of five experiments. Within 60 min of wash out, the action potential narrowed to control width and the ADP returned; however,
Rn failed to return to control levels.
For 13 cells tested at P20-24, the mean percent change by
external Cs+ was 61.4 ± 18.9% (mean ± SE) and 71.5 ± 25.3% for Rn
and
0, respectively. Similar values of
61.0 ± 29.3% and 69.0 ± 7.0% were measured for
Rn and
0 for
two cells at P5-7. This large increase in
Rn and
0 was
observed in GG motoneurons irrespective of whether the motoneurons
exhibited an inward current or not.
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External barium blockade
External barium blocks some potassium conductances and the leak
current in spinal motoneurons (Schwindt and Crill
1980b). Because barium increased the spontaneous activity of
the slice, it was necessary to block spontaneously generated action
potentials with TTX if the membrane properties were to be measured.
Figure 9 shows the effect of
Ba2+ on the membrane responses of a P7
and a P14 GG motoneuron. At each age, there was a
significant increase in Rn and
0 of the TTX-treated cells after perfusion
with 2 mM barium chloride. The effect of external
Ba2+ on 40 GG motoneurons is summarized in Table
3. The magnitude of this effect on
0 was relatively consistent between the
different ages. There was a larger absolute change in
Rn at P5-6 than at the two
older ages. Based on the internal cesium data, one might predict that
the magnitude of the barium-sensitive component would be larger at
P13-15, but this was not the case. A plot of the percent
change in resistance with Ba2+ as a function of
the control Rn yielded no correlation
(data not shown).
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With the addition of barium to perfusate or cesium to the intracellular compartment, almost all cells were found to depolarize. To avoid activating any voltage-sensitive channels, a bias current was applied to counteract this drift of the membrane potential. Figure 10 presents a plot of the bias (injected) current as a function of the control Rn for intracellular cesium and extracellular barium. There is a negative correlation between injected current and input resistance at P5-6 and P13-15 and for the pooled data for cesium. A similar negative (P < 0.02) correlation was found in cells at P19-23 for external Ba2+. These correlations may reflect that there are more cesium- and barium-sensitive channels controlling resting membrane potential in the low resistance motoneurons as compared with the high resistance cells. Alternatively, the lack of a strong correlation in some age groups was a result of the wider range of injected currents associated with low resistance than high resistance cells.
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Distribution of cesium- and barium-sensitive channels
It was evident from Fig. 7 that the mean percent change in
Rn and 0 with
intracellular cesium was not equivalent. Similar to the first paper, we
have applied the analysis of Redman and colleagues (Redman et
al. 1987
) to our data on cesium and barium blockade. Starting
with a simple model of a small spherical ganglion cell, the model would
predict a proportional change in Rn
and
0 in response to the actions of barium on
resting conductance. When expressed as a ratio of change in
0 to Rn, the
ratio would equal 1.0. With the addition of a dendrite to the model,
changes in specific resistance of the dendrite are not reflected by a proportionate change in the Rn result
in larger changes in
0 than
Rn and a ratio <1.0. Figure
11 plots the percent change in
0 as a function of the percent change in
Rn. Points lying above a unity line
indicate a larger change in
0 than
Rn. Most of the cells from both
treatments (Cs+ and Ba2+)
are found above the line. In the context of the Redman model, this
outcome is interpreted to mean that most of the blocked conductances reside in the dendritic tree and not at the cell body. This conclusion is supported by the earlier observation (Fig. 5) that suggested that
the cesium-sensitive component of Rn
was located in the distal dendrites.
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On closer inspection, there is a developmental trend in the data for
both cesium and barium blockade. One alternative to the scatter plot is
to quantitate the proportion of change in
Rn as compared with that in
0 by calculating the ratio. A proportionate change in the two membrane properties would yield a ratio of 1.0, while
values exceeding 1.0 would predict a more distal dendritic distribution. When the means and standard deviations were calculated at
each age in response to intracellular cesium and external barium (Table
4), the oldest age group demonstrated the
smallest mean and standard deviation. Because of the large standard
deviations, none of these differences reached statistical significance;
however, there was a trend for the ratios to decrease (approaching 1.0) with age. This reduction suggests that the conductances that were predominantly in distal dendrites at the younger ages actually become
more uniformly distributed by the oldest age.
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DISCUSSION |
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This is the first study to examine the role of potassium
conductances in determining the input resistance of developing
mammalian motoneurons. The input resistance is the critical parameter
in establishing the order of motoneuron recruitment in a spontaneous motor behavior (Cameron et al. 1991). During postnatal
development, the pattern of activity in the cat phrenic motor nucleus
was dramatically altered after the mean input resistance (and specific
membrane resistance by inference) was reduced by half. Similar to the
cat motoneurons, the rat genioglossal (GG) motoneurons studied in vitro
undergo similar developmental changes in both their electrophysiology (Núñez-Abades et al. 1993
) and anatomy
(Núñez-Abades and Cameron 1995
;
Núñez-Abades et al. 1994
). The major
findings of this study on GG motoneurons is that increases in the
Cs+-sensitive conductances make the substantial
contribution to the decrease in Rn and
0 found during postnatal development. From data in the preceding paper, synaptic blockade generated a 21 and 36%
increase in Rn and a 29 and 38%
increase in
0 at 1 and 2 wk of age,
respectively. At these same ages, internal cesium blockade of potassium
channels produced a comparable 22 and 31% increase in
Rn but a larger 30 and 67% increase
in
0. In general, the cells having lower input
resistance tend to exhibit more Cs+- and
Ba2+-sensitive conductances than their higher
resistance counterparts. Initially, these potassium conductances are
predicted to be located in the distal dendrites but become more
homogeneously distributed with time.
Extracellular TEA blockade
The various contributions of synaptic inputs and potassium
currents have been dissected using a variety of ionic/pharmacological manipulations. In the previous paper, almost all of the synaptically mediated conductances (evoked and spontaneous) were blocked by high
magnesium. By applying the high magnesium prior to the application of
external TEA, we wanted to assess the role of nonsynaptically mediated
potassium conductances. The size of the external TEA response after
high Mg2+ was taken as evidence for a large role
for voltage-sensitive potassium conductances. Extracellular TEA
increases motoneuron input resistance in rat vagal motoneurons
(Yarom et al. 1985) while it specifically suppress both
the delayed-rectifier and A-currents in spinal motoneurons
(Safronov and Vogel 1995
). In rat hypoglossal
motoneurons, external TEA effectively blocks the other inward
rectifying channels but not the Ih
(sag) current (Bayliss et al. 1994
), the
Ih being more sensitive to
Cs+ than either Ba2+ or TEA.
It has been proposed that the Ih
current in rat hypoglossal motoneurons is active at membrane potentials
more negative than 65 mV (Bayliss et al. 1994
) and,
thereby, contributes to the membrane resistance. In fact, the
current-voltage (I-V) plots from this report demonstrate
little Ih current at potentials more positive than
80 mV. In the present study, small current steps were
used for the measurement of time constant to avoid activating the
inward rectifier. Based on the linearity of the semi-log plots at small
(
0.05 nA) current steps, there was little apparent sag contamination.
Bayliss and colleagues also described a 10-fold increase in the
amplitude of this Ih current between
P2 and P65. Given an order of magnitude increase
with postnatal development in this current, it is not surprising that
little sag was detectable in our youngest age group. In the present
study, the sag current was sensitive to external cesium, but, in many
instances, the value of input resistance was only minimally effected by
blockade of the sag current (Fig. 1). When sag was detected, the
voltage was measured at the peak response prior to the initiation of
the inward current. As a result, we do not believe our measurements of
Rn to be significantly impacted by the
sag current.
In the first series of experiments, high Mg2+ was
relatively effective at depressing the calcium-mediated potassium
current underlying the medium AHP. However, this channel was not
apparently the source of the decrease resistance. The delayed rectifier
can be blocked by either external TEA or cesium (Hille
1992; Schwindt and Crill 1981
). It is
interesting to note that there was a synergistic effect between
external TEA and cesium on the delayed rectifier as evidenced by the
increase in the duration of the action potential repolarization. This
depression of the delayed rectifier was accompanied by an increase in
the calculated Rn. Given the
generalized nature of our blockers, the increment in resistivity
resulting from the addition of external cesium cannot be attributed to
a known set of potassium channels. It is interesting to note that
combination of magnesium and TEA lead to an approximately 40% increase
in Rn and a 70% increase in
0. After adding cesium to the external solution, input resistance increased by 60-70%, and the membrane time
constant increased by 60-70%. According to the Redman model, the TEA-
and Cs+-sensitive potasium channels have
different patterns of distribution over the motoneuronal membrane of
these developing brain stem motoneurons.
Intracellular cesium
In cat lumbosacral motoneurons, intracellular cesium resulted in a
prolongation of the falling phase of the action potential, a large
reduction in the amplitude of the AHP, and a reduction in resting
membrane conductance, up to half its original value (Puil and
Werman 1981). Recovery of the action potentials from the cesium
was dose dependent and could take from 4 to 35 min. However, changes in
conductances were not, in most instances, reversible, especially with
large injections. We also observed that there was a partial recovery
from intracellular cesium. Figure 4 demonstrates that intracellular
cesium blocked the delayed rectifier in the present experiments.
However, as the cesium effect lessened in the cell body (presumably due
to the diffusion of cesium into the dendrites), the somatic action
potential recovered while the Rn
remained elevated (Fig. 5). This persistent, reduced conductance suggests that either the delayed rectifier is not involved in establishing resting conductance and/or the resistance of a cell is
determined predominantly by the resistivity of the dendrites.
The effects of intracellular cesium were recently studied in cat
cervical motoneurons (Campbell and Rose 1997). These
investigators concluded that the increased conductance of the soma
(somatic shunt) is due to tonic activation of voltage-dependent
potassium channels located on or near the soma. The present study
suggests a less uniform distribution for cesium-sensitive channels at
the younger ages for brain stem motoneurons that is subject to change during development. Based on a model by Redman and colleagues (Redman et al. 1987
), the percent change in
Rn and
0
would be equal if the channels were uniformly spread over the
motoneuron membrane. The larger change in
0
relative to Rn in the present study
with intracellular cesium mimics the response in sympathetic ganglion
cells when exposed to external barium. Our observations at the younger
ages are consistent with the conclusion of Redman and colleagues that
the blocked conductances reside in the more distant compartments of the dendrites.
The interpretation of the present data set is not trivial. During
development, the size of the motoneuron (especially between weeks 2 and
3), the number of voltage-dependent channels, and the distribution of
these channels are all changing. The changes can interact to produce
complex effects on Rn and
0. This problem is most evident in the summary
of data presented in Fig. 7. One major conclusion from these data are
that cesium-sensitive channels constitute a major component of
the reduction in Rn occurring between
1 and 2 wk after birth. However, the doubling of membrane surface area
between 2 and 3 postnatal weeks (Núñez-Abades and Cameron 1995
) should produce a 50% reduction in
Rn, assuming that all other membrane
characteristics remain the same. This is not the case; in fact, the
Rn is approximately the same at both
ages (Núñez-Abades et al. 1993
). One
possible explanation as to why the anticipated decrease in
Rn did not occur is a simultaneous increase in specific membrane resistivity. However, assuming that the
measurement of the slowest time constant approximates the specific
resistivity, there is no evidence for such an increase. The situation
becomes more complicated when it becomes apparent that the distribution
of the voltage-dependent channels may be changing (Fig. 11). In the
oldest animals, these data points are closer to the unity line than at
earlier stages of development, suggesting a more uniform distribution
(Table 4). It is not clear, without extensive modeling, how this
redistribution of channels might impact the measurements of
Rn and
0 in
the present study.
Given the potential limits of the analyses, there is one interesting
interpretation of our data. Based on the greater
Cs+ sensitivity of the low resistance
motoneurons, we propose that there is a differential proliferation of
Cs+-sensitive potassium channels in the
motoneurons innervating fast- versus those innervating slow-twitch
muscle fiber types. This differential in
Cs+-sensitive channels might be more easily
demonstrable in a muscle with a more diverse fiber type composition
than GG muscle like the diaphragm (Brozanski et al.
1993). This selective proliferation of channels roughly
coincides with the elimination of polyneuronal innervation
(Redfern 1970
) and may result from an induction signal derived from the maturing muscle.
Barium-sensitive conductances
When iontophoresed onto a cat lumbosacral motoneuron,
extracellular barium depressed the delayed rectifier and leak
conductance (Schwindt and Crill 1980b). In neostriatal
cells, barium-sensitive conductances were primarily associated with the
linear conductances making up the somatic shunt while cesium-sensitive
conductances preferentially acted on the inward rectifier (Reyes
et al. 1998
). We applied extracellular barium onto developing
motoneurons in vitro to assess what role that the leak conductance, in
particular, played in producing the decrease in membrane resistance
during postnatal development. If part of the reduced resistance of the second week of development was a result of a proliferation of the leak
conductance, then we would expect that external barium would produce a
larger increase in the resistance of motoneurons at 13-15 days.
Statistical analyses failed to demonstrate any difference between age
groups in absolute or percent change of either membrane property.
However, like the response to intracellular cesium, there was a larger
percent increase in
0 associated with external
Ba2+ at P13-15 than that noted for
Rn.
There are three conclusions about barium-sensitive channels that
paralleled those for cesium-sensitive channels. First, there was a
proportionately larger increase in 0 as
compared with Rn, suggesting a distal
dendritic location for these conductances. Second, the ratio of percent
change in
0 to
Rn approached 1.0 with increasing age,
suggesting that the distribution of barium-sensitive channels was more
uniform in the older animals. Third, there was a negative correlation
between control Rn and current
injected to maintain membrane potential constant. The block of the
barium-sensitive leak conductance generated a larger depolarizing shift
in cells with low Rn. Thus GG
motoneurons with a low resistance were found to have more
Ba2+- and Cs+-sensitive
leak conductances than cells with high resistance. We would propose
that the proliferation of leak channels in low resistance cells may be
responsible, in part, for the lower specific membrane resistance found
at P13-15.
A recent study (Talley et al. 2000) presented anatomical
evidence in support of a differential distribution of leak channels in
brain stem and spinal cord motor nuclei of the adult rat including the
hypoglossal nucleus. These authors measured the relative expression levels of TASK-1, a two-pore domain potassium channel possessing properties that fit the behavior of a leak channel. They used in situ
hybridization to demonstrate that TASK-1 mRNA was localized to the soma
and proximal dendrites of hypoglossal motoneurons. These data revealed
that some hypoglossal motoneurons were more heavily labeled than
others, a pattern evident in other motor nuclei as well. Finally, these
authors noted a lower density of labeling in brain stem and spinal cord
motoneurons of younger animals (P7) than found in the adult.
Thus this report supports the ideas that leak channels are
proliferating with age and that some adult motoneurons (presumptive low
resistance) have more expression of these channels than others
(presumptive high resistance).
The barium-sensitive component of membrane resistivity is modulated by
thyrotropin-releasing hormone (Bayliss et al. 1993, 1997
), norepinephrine (Parkis et al.
1995
), and serotonin (Hsiao et al. 1997
) through
a G-protein-coupled mechanism (Bayliss et al. 1997
).
These studies suggest that the modulation of the leak channel is
important for altering the excitability of brain stem motoneurons and
lowering the threshold for their repetitive firing. If all leak
channels are under the regulation of the neuromodulators, then low
resistance cells, with their greater number of leak channels, would
show greater changes in excitability as compared with high resistance
cells. This hypothesis requires more rigorous testing.
Based on the data from these two companion papers, we would propose that there is both a synaptically mediated and a nonsynaptically mediated component contributing to the changes occurring in membrane resistance during postnatal development. A major part of this nonsynaptically mediated conductance is mediated by potassium currents. Although all the specific potassium channels involved are not known, it is clear that there is a substantial contribution of the cesium-sensitive conductances to the developmental process of motoneuron differentiation. Future studies will be necessary to determine 1) what signals identify a motoneuron as predestined to become a low or high resistance cell, 2) what factors direct the formation of synaptic connections to specific regions of the motoneuron membrane, and 3) what processes determine the number of potassium channels will be expressed, where the channels will be inserted into the membrane, and how this distribution may be altered during development.
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ACKNOWLEDGMENTS |
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The authors thank Dr. David Robinson and the reviewers for comments on the manuscript.
This work was supported by National Institute of Child Health and Human Development Grant HD-22703 and a local Sudden Infant Death Syndrome research foundation, Megan's Run, Wilsonville, Oregon.
Present address of P. A. Núñez-Abades: Dept. of Animal Physiology and Biology, Faculty of Pharmacy, University of Seville, calle Tramontana, 41012 Seville, Spain.
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FOOTNOTES |
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Address for reprint requests: W. E. Cameron, Dept. of Physiology and Pharmacology, L-334, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201 (E-mail: cameronw{at}ohsu.edu).
Received 26 July 1999; accepted in final form 28 July 2000.
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REFERENCES |
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